Microbial metabolites in skin barrier development

Mercedes Gomez de Agüero

Institute of Systems Immunology. Max Planck Research Group. University of Würzburg

From birth, neonates require a fully functional skin barrier to protect against threats such as pathogens, harmful substances, and mechanical stress, while maintaining dynamic interactions with the environment. The development of this sophisticated barrier occurs during embryogenesis and is vital for postnatal survival. While the molecular mechanisms of epidermal barrier formation are well characterised, the specific regulatory factors that control this process-and could be targeted therapeutically-remain unidentified. Postnatally, commensal microbiota is known to support epidermal barrier maturation; however, their influence during prenatal development has not yet been addressed. In our previous study, we demonstrated that sterile metabolites derived from the maternal microbiota can cross the placenta and reach the embryo, where they may influence epidermal morphogenesis and promote the formation of a functional neonatal skin barrier.
To address this hypothesis, pregnant germ-free mice were colonized either with auxotrophic Escherichia coli HA107 for a reversible microbial exposure or with complex microbial communities to model a more physiological condition. Late-embryonic offspring from gestational colonized dams exhibited skin characterised by a well-organised basal layer of undifferentiated and stem cells, overlaid by multiple layers of differentiated keratinocytes. In contrast, embryos from germ-free dams developed an under-structured epidermis composed of several layers of undifferentiated and proliferative keratinocytes. Notably, keratinocytes within hair follicles of offspring from gestational colonized dams were also more differentiated, consistent with enhanced follicular development and suggesting an effect on a common progenitor. Furthermore, the transition of ectodermal cells into primary keratinocyte progenitors – as indicated by increased p63 expression – was amplified in these embryos, highlighting sa regulatory role of maternal microbiota in governing keratinocyte stemness.
To gain mechanistic insight, we 13C-tracing of the maternal microbial metabolites and found that key compounds involved in epidermal differentiation and skin barrier development – including vitamins, nicotinamide, tryptophan and derivatives – accumulated in embryonic skin after gestational colonization. Using epidermal 3D-organoid models from primary keratinocytes, we demonstrated a cell-intrinsic regulation driven aryl hydrocarbon receptor (AHR) ligands originated from the maternal microbiota. These AHR ligands modulated the differentiation capacity of keratinocyte stem cells by the downregulating glycolysis. Notably, pre-gestational or postnatal microbial metabolite exposure failed to compensate for their absence during gestation.
Embryos lacking maternal microbial metabolite exposure developed a dysfunctional skin barrier, as evidenced by increased transepidermal water loss, enhanced chemical penetration, accumulation of pathobiomes, and impaired wound healing in neonatal skin.
In summary, our results demonstrated that maternal microbial metabolites shape embryonic epidermal morphogenesis and are essential for establishing a functional skin barrier at birth.

Gnotobiology as a Window into Host-Microbe Interactions

Marijana Basic

Hannover Medical School, Institute for Laboratory Animals Science, Carl-Neuberg Str. 1, 30625 Hannover, Germany

The intestinal microbiota forms a complex ecosystem that plays a crucial role in host physiology while also being implicated in various diseases. Our research group investigates gut microbial communities to elucidate the dynamic interactions between the microbiota and the host. We focus on identifying microbial factors that influence host health, with a particular emphasis on how alterations in the gut microbiota contribute to the development and modulation of intestinal inflammation. A key tool in our research is the use of gnotobiotic mice, which are raised in germ-free or selectively colonized environments. Although studies in germ-free models are necessary to confirm microbiota-induced phenotype, studies in models with defined microbial composition are particularly appealing, as here, beneficial or detrimental effects of individual microbes or microbial communities can be assessed. This model allows precise manipulation of microbial communities, providing critical insights into the specific roles of individual microbes and their collective impact on host physiology. Understanding these mechanisms is essential for developing targeted microbiome-based strategies to promote host health and mitigate inflammation-related diseases.

Food, Microbes, and Viral Encephalitis: Rewiring Brain Immunity via the Gut-Brain Axis

Anirban Basu

National Brain Research Center, Manesar, Haryana-122052, India

The dynamic interplay between food, microbes, and immunity is central to understanding host defense mechanisms and disease outcomes, particularly in neurotropic viral infections. At the heart of this interface lies the gut-brain axis—a complex bidirectional communication network linking the gastrointestinal tract, its resident microbiota, the immune system, and the central nervous system (CNS). Disruptions in this axis have profound implications for neuroinflammation and neuronal health, especially during infections caused by viruses such as the Japanese Encephalitis Virus (JEV).
Japanese Encephalitis (JE) remains a significant public health challenge in Asia, characterized by acute CNS inflammation, high mortality, and frequent long-term neurological sequelae. Recent research highlights the role of the gut microbiome as a key modulator of immune responses during JEV infection. Alterations in gut microbial composition—triggered by the infection itself or pre-existing dietary and environmental factors—can influence systemic and CNS immune activation, potentially affecting disease severity and recovery.
In this talk, we will present emerging evidence from our laboratory and others on how JEV infection disrupts gut microbial communities, impairs intestinal barrier integrity, and contributes to a pro-inflammatory immune milieu that exacerbates CNS pathology. We will also discuss how the gut microbiota shapes innate and adaptive immune responses during the course of infection, influencing viral persistence and neuroinflammation.
Understanding these mechanisms opens new avenues for microbiome-informed preventive and therapeutic strategies, including the possibility of using dietary modulation or microbiota preservation as supportive measures in JE management. While direct clinical applications remain in development, the gut-brain-immune axis provides a compelling framework to reinterpret host-pathogen interactions in JE from a systems biology perspective.
This integrative approach underscores the need to consider the microbial and nutritional context of the host when addressing neurotropic viral infections. By situating the discussion of JEV within the broader theme of Food, Microbe, and Immunity, this presentation aims to foster interdisciplinary dialogue and pave the way for innovative strategies to mitigate the burden of viral encephalitis through microbiome-centered interventions.
By unlocking the therapeutic potential of the gut-brain connection, we aim to revolutionize treatment paradigms for viral diseases which impacting the brain, and other neuro degenerative disorders.

Diet-microbial interactions underlying abdominal pain

Přemysl Berčík

Faculty of Health Sciences, McMaster University, Hamilton, Canada

Accumulating evidence suggests that microbiota, apart from shaping the intestinal immune system, plays an important role in determining gastrointestinal motility and visceral sensitivity, and also affects distant organs, including the brain. Gem-free mice have altered intestinal transit, permeability, pain perception and behavior, all of which normalize after bacterial colonization. Despite significant advances in microbiota-host interactions, including signaling through the immune and neural systems, the underlying mechanisms are still not fully understood.

Gut microbiota composition and function differ between healthy individuals and patients with disorders of gut-brain interaction, such as Irritable Bowel Syndrome (IBS), although a specific microbial signature has not been identified. Considering that the gut microbiota produces myriads of immunomodulatory and neuroactive molecules, it is likely that multiple mechanisms lead to genesis of IBS symptoms, including pain. Recent preclinical and clinical studies have identified bacterial histamine, produced by conversion of dietary histidine by bacterial histidine decarboxylase, as a key mediator of abdominal pain in a subset of patients with IBS. Additional mediators underlying visceral hyperalgesia include bacterial bioactive phospholipids, generated by microbial metabolism of dietary phosphatidylcholine, and bacterial proteases. Inhibition of bacterial enzymes involved in the genesis of these pronociceptive mediators or use of novel probiotics metabolizing these mediators, together with dietary interventions may be an effective strategy for effective management of patients with chronic abdominal pain.

Mucosal microbiota alterations in primary sclerosis cholangitis

Alena Bohdanecka, Monika Cahova and Petra Polakovicova

Institute for Clinical and Experimental Medicine, Prague, The Czech Republic

Background
Primary sclerosing cholangitis (PSC) is a progressive bile duct disease that often leads to orthotopic liver transplantation in advanced stages. Genetic factors account for only about 10% of PSC cases, highlighting the role of environmental factors such as gut microbiota composition. Approximately 70% of PSC patients also have idiopathic bowel disease (IBD), particularly ulcerative colitis (UC), with a unique clinical phenotype that differs from Crohn’s disease. While characteristic fecal microbiota features have been identified in PSC, data on mucosal microbiota—potentially more relevant to disease pathophysiology—are scarce and derived from short amplicon 16S rRNA sequencing. This study utilized a large colonoscopy cohort of PSC and UC patients at IKEM to address these knowledge gaps through the whole 16S rRNA sequencing.
Methods
Intestinal mucosa biopsies from the terminal ileum, ascending colon, and descending/sigmoid colon were analyzed from PSC (n=36), UC (n=38), and healthy controls (n=54). Microbial DNA was isolated, amplified using primers for the complete 16S rRNA gene, and purified. Sequencing was conducted using the PacBio Revio System.
Results
PSC-IBD patients exhibited lower alpha diversity compared to healthy controls, with altered microbiota in the terminal ileum and colon. Machine learning models accurately distinguished PSC patients from healthy controls, although differentiating PSC from UC was less effective. The mucosal microbiota in PSC was dominated by Pseudomonas, Veillonella, and Klebsiella, with a notable underrepresentation of some common commensals.
Conclusions
Revio sequencing enables more precise identification of gut microbiome composition in different gut regions, correlated with varying inflammatory activities in PSC-IBD patients.
Support
The study was supported by grant MH CR no. NW24-05-00168
Key words: biopsy, PSC, IBD, gut microbiome, whole 16S gene sequencing

A microbial perspective on animal development and behaviour

Thomas C. G. Bosch

Zoological Institute, University of Kiel, Kiel, Germany

Animals have evolved within the framework of the microbes and are constantly exposed to diverse microbiota. This dominance of the microbial world is forcing all fields of biology to question some of their most basic premises, with developmental biology being no exception. While animals under laboratory conditions can develop and live without microbes, they are far from normal, and would not survive under natural conditions, where their fitness would be strongly compromised. Since much of the undescribed biodiversity on Earth is microbial, any consideration of animal development in the absence of the recognition of microbes will be incomplete. Here, using the ancestral metazoan Hydra as model, I will show that animal development and behaviour may never have been autonomous, rather it requires transient or persistent interactions with the microbial world. I propose that to formulate a comprehensive understanding of embryogenesis and post-embryonic development, we must recognize that symbiotic microbes provide important developmental signals and contribute in significant ways to phenotype production. By integrating the microbiome perspective into our understanding of evolution and function, we gain richer insights into the dynamic interplay that drives health, adaptation, and the very fabric of life itself.

Of Bowels, Brain and Behaviour: The Microbiota-Gut-Brain Axis in Psychiatry and Beyond

Gerard Clarke^1,2

1 Department of Psychiatry and Neurobehavioural Science, University College Cork, Cork, Ireland
2 APC Microbiome Ireland, University College Cork, Cork, Ireland

Understanding the relationship between the gut microbiota and host physiology has taken important steps forward over the past few decades, particularly in the context of microbial regulation of brain function and behaviour via the gut-brain axis. This axis provides the framework for continuous bidirectional communication between the gut and the brain which is facilitated by neuronal, endocrine, metabolic, and immune pathways. The gut microbiota influences these signalling pathways via several mechanisms and the immune system in particular represents a key pathway of communication along this axis with the microbiome implicated in neuroinflammation in health and disease. A variety of preclinical experimental approaches have documented the important of gut microbes for a range of stress-related behaviours relevant to both psychiatry and neurogastrotenterology. These observations are supported by clinical studies have shown compositional and functional alterations in the gut microbiota in stress-related psychiatric disorders as well as in irritable bowel syndrome (IBS), a gut-brain axis disorder sharing high levels of psychiatric comorbidity including both anxiety and depression. There are also now examples of successful, partial and unsuccessful translation of potential therapeutic options from bench to bedside. Developments in in silico analyses have enhanced our understanding of the gut microbiome and have moved the field towards pinpointing the host-microbe interactions most important for optimal gut-brain axis function. Advancing microbiome-gut-brain axis science further requires systematic, rational and translational approaches to bridge the critical knowledge gaps currently preventing full exploitation of the gut microbiome as a tractable therapeutic target for gastrointestinal and brain health.

The role of probiotics in supporting dietary treatment in gluten-dependent diseases

Bożena Cukrowska

The Children’s Memorial Health Institute, Warsaw

Gluten-dependent diseases (celiac disease -CD, allergy, non-celiac gluten sensitivity -NCGS) occur with a frequency reaching even 8-10% of the general population. In the initiation of these diseases, especially CD, immunoreactive gluten peptides rich in proline, which human enzymes present in the digestive tract are unable to degrade, activate immunological processes leading to autoaggression and, as a result to atrophy of intestinal villi. Studies indicate that peptidases produced by intestinal microbiota may play a role in the digestion of these peptides. Although the phenotype of intestinal dysbiosis (composition of microbiota) has not been clearly established in gluten-dependent diseases, functional analysis of the microbiome revealed that microbial-derived peptidases hydrolyzing gluten peptides rich in proline bonds are altered in CD, NCGS vs controls (Pryor et al., Gut Microbes, 2025). Therefore, we are looking for probiotic strains producing peptidases that digest immunoreactive gluten peptides. The aim of this study was to evaluate gliadin hydrolysis capacity of 14 probiotic strains and to identify peptidase-encoding genes in the genomes of the most efficient strains (Leszczyńska et al., Nutrients, 2024). Residual gliadin immunoreactivity was measured after one- or two-step hydrolysis using commercial enzymes and bacterial peptidase preparations by G12 and R5 immunoenzymatic assays. Peptidase preparations from Lacticaseibacillus (L.) casei LC130, L. paracasei LPC100 and Streptococcus (S.) thermophilus ST250 strains significantly reduced the immunoreactivity of gliadin peptides, including 33-mer, and this effect was markedly higher when a mixture of these strains was used. In silico genome analyses of L. casei LC130 and L. paracasei LPC100 revealed the presence of genes encoding different types of peptidases with the potential to hydrolyze bonds in proline-rich peptides. This suggests that L. casei LC130, L. paracasei LPC100 and S. thermophilus ST250, especially when used as a mixture, have the ability to hydrolyze immunoreactive gliadin peptides and could be administered to patients on a restricted gluten-free diet to help treat gluten-related diseases. In addition, L. casei LC300 and L. paracasei LPC100 exhibit pathways for producing bacteriocins, short-chain fatty acids (acetic acid, butyric acid), possess all the genes necessary for efficient lactose metabolism, and are lactic acid producers (Szczepankowska et al. Microbiol Resour Announc 2024). The features described are important in the case of patients, who, despite following a gluten-free diet, still have chronic inflammation, impaired calcium and iron absorption, and secondary lactose intolerance. We are currently planning to conduct a clinical trial to assess the efficacy of selected probiotic strains in patients with CD who, despite following a gluten-free diet, still have clinical symptoms.

Unconventional Antigen Presenting Cells in Orchestrating Adaptive Immune Responses against Pathobionts

Iva Pacáková¹, Katarína Kováčová¹, Tomáš Brabec¹, Anna Jelínková², Martin Schwarzer², Jan Dobeš¹

1 Department of Cell Biology, Faculty of Science, Charles University, Prague, Czechia

2 Laboratory of Gnotobiology, Institute of Microbiology of the Czech Academy of Sciences, Nový Hrádek, Czechia

Segmented filamentous bacteria (SFB) are anaerobic, spore-forming, Clostridia-like microbes. Their adhesion to the intestinal epithelium triggers a strong immune response, characterized by the accumulation of Th17 cells and increased IgA secretion by plasma cells. Recently, we reported that helper T cells reactive to SFB can also differentiate into induced intraepithelial lymphocytes and begin to express cytotoxic molecules.
Traditionally, the helper versus cytotoxic role of T cells was believed to be hardwired. However, the mechanisms enabling this transition are only now being uncovered. Using genetic models, we identified the antigen-presenting cells and molecular pathways responsible for driving the shift from helper T cells to cytotoxic intraepithelial lymphocytes, as well as their functional implications in gut physiology.
These findings advance our understanding of host–gut microbiota interactions and reveal the cellular mechanisms underlying the unconventional transition of CD4⁺ T cells into cytotoxic intraepithelial lymphocytes. Understanding these processes provides valuable insights into gut barrier regulation and may offer new avenues for manipulating intraepithelial lymphocytes to benefit host health.

Safeguarding Early Microbiota: The Role of RORγt⁺ Tregs

Dominik Filipp

Laboratory of Immunobiology, Institute of Molecular Genetics of the Czech Academy of Sciences, Prague, Czech Republic

A balanced immune system relies on FoxP3+ regulatory T cells (Tregs) to prevent excessive immune responses. While thymus-derived Tregs are well known, a unique subset of microbiota-specific peripherally induced RORγt+ Tregs, has emerged as a critical regulator of intestinal immunity. Our data has revealed that RORγt+ Tregs originate in mesenteric lymph nodes (mLNs) during early-life microbiota colonization and, remarkably, can persist long-term even in the absence of microbial antigens. This rigidity of weaning-derived RORγt+Tregs, reflected in their long-term persistence and limited adaptability to microbial changes, is further supported by our discovery that most of these cells express Sca-1 which enables precise tracking and characterization of this memory subset. Sca-1⁺ RORγt⁺ Tregs make up to 85% of the stable pTreg population in mLNs, whereas RORγt⁺ Tregs generated in adulthood are largely Sca-1⁻. These findings suggest that post-weaning development of RORγt⁺ Tregs in adulthood has a limited capacity to replace the original weaning-derived RORγt⁺ Treg population in dmLNs. Thus, our data points to a fundamental developmental distinction between early- and late-arising RORγt⁺ Tregs. The presence of Sca-1 may indicate previously unrecognized properties of this subset, particularly their long-term memory stem cell characteristics. This suggests a role in safeguarding an imprint of early-established beneficial primary microbiota, which is crucial for maintaining intestinal immune homeostasis. Targeting RORγt⁺ Tregs to modulate microbiota composition could present new therapeutic opportunities for dysbiosis-related diseases.

Diet determines IgA induction and repertoire characteristics in the GALT independently of the microbiota

Catherine Mooser *^1,2, Francesca Ronchi *^1,2,3, Julien P. Limenitakis *^1,2, Cristina Kalbermatter^1,2, Sandro Christensen^1,2, Mercedes Gomez de Agüero^1,2, Tobias Fuhrer⁴, Uwe Sauer⁴, Andrew J. Macpherson^1,2, Stephanie C. Ganal-Vonarburg^1,2

1 Department of Visceral Surgery and Medicine, Inselspital, Bern University Hospital, University of Bern, Switzerland
2 Department for BioMedical Research, Visceral Surgery and Medicine, University of Bern, Switzerland
3 Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health (BIH), Berlin, Germany: Institute of Microbiology, Infectious Diseases and Immunology (I-MIDI)
4 Institute of Molecular Systems Biology, Swiss Federal Institute of Technology (ETH) Zurich, 8093 Zürich, Switzerland

A hallmark of the main secreted antibody immunoglobulin-A (IgA) is its mutational load that accumulates throughout life. Although this is mainly interpreted in terms of continuing microbial induction, we show that dietary composition during early life can promote IgA induction, its repertoire and mutational diversification independently of microbial exposure. Using germ-free and colonized mice fed different diets formulated with proprietary grain-based processing or from purified chemicals with different principal macronutrient calorie sources, we found that dietary lipopolysaccharide contamination led to TLR4 signaling and promoted germinal center activity in the intestinal immune compartment. Effects of lipopolysaccharide on mucosal immune induction were phenocopied only when presented within colloidal liposomes rather than in dispersed solution. These findings indicate that dietary composition and its formulation can leave a durable impression on the resultant IgA repertoire.

Pros and cons of using mouse models for studying diet, microbiota, and disease expression.

Axel Kornerup Hansen

Department of Veterinary and Animal Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Ridebanevej 9, DK-1870 Frederiksberg C, Denmark

Mice are a convenient and cost-effective tool for preclinical studies under well-controlled conditions prior to more complex and costly trials in human patients because they are cheap, allow for proper statistical analysis and, there is a wealth of literature demonstrating the efficacy of various food and medical components. Laboratory mice live under specific pathogen-free (SPF) conditions, and therefore, their microbiota is less diverse than in humans, and they are unexposed to juvenile and current infections. So, their immune system is more comparable to that of new-borns rather than adult humans. Furthermore, their microbiota responds to other oligosaccharides than a human microbiota. Although, so-called ‘dirty’ mice harbouring all the previously eradicated pathogens have a higher translational value within certain research areas, reintroducing pathogens would lead to the uncontrolled spread of infections and associated diseases, so research facilities need safer alternatives. Also, it is challenging to supply mice with a full human microbiota or just some single human bacteria. On the other hand, studies have shown that mice to a certain extent can have their immune system matured artificially, and that this may have a beneficial impact on their function as model within certain research areas. Even their different responses to human food components can be used to create translational information.

Prevention of Colitis by Probiotic Escherichia coli O83:K24:H31: Modulation of Proportional and Functional Characteristics of Leukocyte Subsets

Jiří Hrdý

Institute of Clinical Immunology and Allergology, First Faculty of Medicine Charles University and General University Hospital in Prague

Early postnatal administration of the probiotic strain Escherichia coli O83:K24:H31 (EcO83) has previously been demonstrated to exert beneficial immunomodulatory effects in promotion of maturation of immature immune system of neonates, including a reduction in allergy incidence. Early postantal EcO83 supplementation promoted immunoregulatory pathways (increased function of Tregs and generally elevated IL-10 in sera of peripheral blood) that are critical for establishing and maintaining immune homeostasis during early life. These findings underscore its preventive and/or therapeutic potential in possible amelioration of inflammatory bowel diseases (IBD).
The aim of the present study was to investigate the protective and therapeutic effects of E. coli O83:K24:H31 in several experimental mouse models of intestinal inflammation, in order to evaluate its potential beneficial effect together with identification of modes of actions in vivo. Using flow cytometry, we have characterized immune cell subpopulations in mesenteric lymph nodes and intestinal tissues, focusing on the proportional and functional capacity of specific subsets of T cells, with special attention to regulatory T cells (Tregs) and type 3 innate lymphoid cells (ILC3s). Gene expression of selected pro-inflammatory and immunoregulatory markers, as well as proteins involved in maintaining gut barrier integrity, was assessed using quantitative real-time PCR. To study colonization and persistence of EcO83 in the murine gut, we engineered bioluminescent and fluorescent strains of E. coli O83 (EcO83) and visualized them using in vivo bioimaging (IVIS Spectrum).
Early postnatal supplementation with EcO83 significantly reduced the severity of colitis induced in adulthood. Surprisingly, preventive administration of EcO83 in adulthood had minor protective effect compared to early-life supplementation. EcO83 supplemented mice showed decreased expression of pro-inflammatory markers (Il6, Tnfa, Cxcl2). Interestingly, expression of key immunoregulatory cytokine Il10 was highest in mice experiencing the most severe form of colitis. Gene expression of tight junction proteins (Tjp, Zo, Cldn, Ocln) associated with barrier function was elevated in EcO83-supplemented mice, suggesting improved barrier function. EcO83 supplementation enhanced both the proportion and functional capacity (measured by intracellular presence of IL-10) of Tregs. Furthermore, levels of IL-22 promoting antimicrobial peptide secretion and maintenance of gut homeostasis were increased in EcO83-treated mice, both on the level of gene expression in gut and intracellular presence in ILC3. Bioluminescent EcO83 was detectable in neonatal mice several days upon administration, exhibiting distinct colonization pattern and dynamics compared to EcO83 supplemented adult mice.
Our findings provide new insights into the potential mechanisms by which EcO83 mediates its beneficial effects in preventing or reducing the severity of colitis in mouse models. Specifically, we have identified the capacity of EcO83 to gut homeostasis via induction gene expression of tight junction proteins promoting gut barrier function, antimicrobial peptides and IL-22. EcO83 promoted both proportional and functional capacity of Tregs. This work supports the continued investigation of E. coli O83:K24:H31 as a promising candidate for the prevention or adjunct treatment of chronic intestinal inflammation.
This study was supported by project EI22_002/0000879 and Cooperatio IMMU207032.

The Microbiome in the First 1000 Days of life

Omry Koren

Bar-Ilan University, Safed, Israel

The initial 1000 days of life, encompassing gestation through early childhood, are pivotal for the establishment of the human microbiome, which in turn influences long-term health outcomes. Research into the intricate interactions between maternal and infant microbiomes during this critical period has demonstrated that pregnancy induces significant shifts in the maternal gut microbiome, characterized by decreased diversity and increased abundance of Proteobacteria, which are essential for fetal development.
Further investigations reveal that hormonal changes, particularly elevated progesterone levels in late pregnancy, selectively enhance the relative abundance of beneficial Bifidobacterium species, potentially priming the neonatal gut for optimal colonization. However, perturbations such as antibiotic exposure during the perinatal period can disrupt microbial succession, leading to long-term consequences including impaired growth trajectories in children and behavioral changes.
Alterations in gut microbiota composition have been linked to the development of gestational metabolic conditions, including gestational diabetes mellitus (GDM), with inflammation mediated by microbial dysbiosis preceding GDM diagnosis by several months. Interventional studies in murine models further suggest that dietary modulation of the microbiome can ameliorate GDM symptoms, underscoring the therapeutic potential of targeting microbial communities.
Collectively, these findings underscore the profound impact of early-life microbiome composition on both maternal and child health and advocate for strategies aimed at preserving or restoring beneficial microbial populations during pregnancy and infancy to promote favorable health outcomes.

The levels of serum biomarkers and therapy-related adverse effects presence in patients with multiple myeloma

Katarina Krempaska, Lucie Dlouha, Johana Rehakova, Marek Trneny, Klara Kostovcikova

1 Laboratory of Cellular and Molecular Immunology, Institute of Microbiology, Czech Academy of Sciences, Prague, Czech Republic
2 1^st Faculty of Medicine, Charles University and 1^st Department of Internal Medicine – Department of Hematology, General University Hospital, Prague, Czech Republic

Significant changes in gut microbiome have been associated with chemotherapy treatment in the patients with multiple myeloma. Here, in the patients undergoing autologous stem cell transplantation, we followed the levels of serum biomarkers that were related to gut barrier integrity, inflammation and microbiota translocation and correlate them with a presence/absence of adverse effects.

We collected serum samples before transplantation and evaluated the toxicity between 7 to 10 days after the transplantation. We focused on four different types of toxicity, especially hemorrhage, oral inflammation, diarrhea and nausea.

We detected the levels of IL- 18, I-FABP (intestinal fatty acid-binding protein), CD14, LBP (lipopolysaccharide-binding protein), MBL (mannose-binding lectin), IGFII (insulin-like growth factor-II), S100A8/A9 by ELISA and the data was distributed according to presence or absence of adverse effects and analyzed by non-parametric Mann-Whitneyi U-test. In the patients with hemorrhagic complications, we observed non-significant but increasing trend for IL-18 (p=0.057) and a significant increase of LBP (p=0.004). Patients with oral toxicity had significantly increased levels of CD14 (p=0.047) and decreased levels of LBP (p=0.004). We found that the presence of diarrhea was associated with a significant decrease of IL- 18 (p=0.032), OPG (p= 0.042) and IGFII (p=0.042). Although we did not observe any significant changes in the patients with nausea, an increasing trend was present for CD14, OPG, IGFII.

There are changes in the levels of serum biomarkers even before the treatment and development of adverse effects. Further analysis of serum biomarkers may help to improve personalized treatment of patients with multiple myeloma.

Supported by the Ministry of Health of the Czech Republic in cooperation with the Czech Health Research Council under project No. NU22-03-00370.

Immunomodulatory effects of antimicrobials in cancer treatment

Miloslav Kverka

Institute of Microbiology of the Czech Academy of Sciences, Prague, Czech Republic

The gut microbiota modulates the development and regulation of the immune system and influences both directly and indirectly the hallmarks of cancer. Gut dysbiosis plays an important role in the response to cancer immunotherapy, and antibiotic treatment is usually associated with a poorer response to immunotherapy. However, our understanding of the underlying mechanisms is still incomplete. Therefore, we investigated how different antibiotics affect the function of the immune system and the tumor microenvironment (TME) and how this affects the outcome of anti-PD1 cancer therapy. Thus, we used a mouse model of cancer allografts with different cancer cells (MC-38 colon adenocarcinoma or RENCA renal adenocarcinoma) treated with different oral antibiotics (streptomycin, vancomycin, colistin or metronidazole) or their mixture with or without anti-PD1 treatment. We found that the RENCA tumors did not respond to any antibiotic or to anti-PD1 treatment, while MC-38 showed an unexpected pattern. Streptomycin increased the mortality of the mice without altering tumor growth, but both colistin and vancomycin or a mixture of all antibiotics reduced tumor growth. The effect of the colistin synergized with the anti–PD1 treatment, was undetectable in germ-free mice andcould be transferred to naïve recipients with the microbiota. The addition of colistin to anti-PD1 treatment increased the production of IFN-γ in the gut and TME and IL-17A in the TME, and the TME contained a significant NK cell and CD8+ T cell signature on RNAseq. Depletion of IL-17A did not alter the effect of colistin, but the effect was lost in IFN-γ-deficient mice or when the cell exit was blocked with FTY720. This suggests that some antibiotics such as colistin may actually enhance the therapeutic effect of anti-PD1 in MC-38 adenocarcinomas in some tumors by increasing the antitumor response in the tumor microenvironment. This effect is driven by IFN-γ, but not IL-17A, and requires unimpeded migration of T cells.
Supported by the Czech Academy of Sciences (LQ200202105) and the Ministry of Education, Youth and Sports of the Czech Republic (CZ.02.01.01/00/22_008/0004597).

Impact of Diet and Gut Microbiota on the Development of Ulcerative Colitis

Kazuyo Moro

1 Laboratory for Innate Immune Systems, Graduate School of Medicine & IFReC, Osaka University, Japan
2 Laboratory for Innate Immune Systems, RIKEN-IMS, Japan

Ulcerative colitis (UC) is an inflammatory bowel disease (IBD) with an increasing global prevalence. Although biologics such as anti-TNF-α antibodies have expanded UC treatment options, a definitive cure remains elusive. Epidemiological studies have indicated that individuals who undergo appendectomy before the age of 20 have a reduced lifetime risk of developing UC. This correlation has been consistently observed across multiple cohort studies worldwide. Despite this clinical observation, the underlying mechanisms have remained unexplored in basic research for over two decades. To address this gap, we initiated a project aimed at uncovering the mechanism by which appendectomy reduces the risk of colitis, with the ultimate goal of developing novel therapeutic strategies.

Given that mice lack an appendix, we utilized a cecal resection (CR) model to investigate whether the protective effect observed in humans could be replicated. In CR mice, dextran sulfate sodium (DSS)-induced colitis was markedly attenuated, mirroring the reduced IL-1β and IL-6 production, decreased intestinal atrophy, and mitigated weight loss seen in human cohort studies. Mechanistically, CR led to a significant increase in group 2 innate lymphoid cells (ILC2), which in turn promoted IL-13 production, thus protecting the intestinal mucosa by enhancing mucin secretion. Upstream of ILC2 activation, CR-induced changes in the gut microbiota increased IL-25 production from tuft cells, a type of intestinal epithelial cell. Importantly, the protective effect of CR was abolished in IL-25 knockout mice, ILC2 knockout mice, and IL-13 knockout mice, underscoring the critical role of this pathway in maintaining mucosal integrity and preventing colitis.
While most IBD research has focused on factors that exacerbate colitis, our study shifts the focus toward mechanisms that inhibit its development. We hope that this approach will pave the way for new preventive strategies against IBD.

Depletion of Myeloid Cells Reduces Tumorigenesis Driven by a High-Protein Diet in a Colitis-Associated Cancer Model

Veronika Motúzová, Janaina L S Donadio, Michal Kraus, Štěpán Coufal, Maliha Rizwan, Miloslav Kverka

Laboratory of Cellular and Molecular Immunology, Institute of Microbiology of the Czech Academy of Sciences, Prague, Czech Republic

Diet shapes the tumor microenvironment in colorectal cancer by modulating the reactivity of the immune system. We investigated the immunomodulatory properties of increased dietary protein intake in the early phase of tumorigenesis in a mouse model of colitis-associated cancer.

BALB/c mice were fed a synthetic control diet (CD) or a high-protein diet (HPD) and subsequently treated with azoxymethane and dextran sodium sulfate to induce colitis-associated cancer. The involvement of macrophages in tumorigenesis was examined by depleting them with clodronate-loaded liposomes. Immunophenotyping was performed by flow cytometry, and cytokine production in Peyer’s patches (PPs) was measured by ELISA. Colonoscopic screening was performed to assess tumor characteristics.

HPD-fed mice developed more numerous and larger tumors and had a lower proportion of systemic CD8+ T cells. However, clodronate-mediated depletion of macrophages during early tumorigenesis significantly reduced tumor incidence and restored CD8+ T cells. In the spleens of clodronate-treated mice, a proinflammatory macrophage subpopulation was partially depleted, along with a significant reduction in neutrophils, while NK, NKT and DC cells increased. In addition, HPD-fed mice tended to have elevated proinflammatory TNFα, IL-17, IL-1β and S100A8 in PPs, with TNFα and IL-17 even higher in clodronate-untreated mice.

Our findings demonstrated that HPD contributes to intestinal inflammation and exacerbates tumor development driven either by macrophage or neutrophil stimulation. This study was supported by the Czech Academy of Sciences (LQ200202105) and the Ministry of Education, Youth and Sports of the Czech Republic (CZ.02.01.01/00/22_008/0004597).

Impact of selected Bifidobacteria on juvenile host growth upon malnutrition in a gnotobiotic mouse model

Tereza Novotná, Veronika Drgoňová, Umesh Kumar Gautam, Anna Jelínková, Tereza Horníková, Dagmar Šrůtková, Martin Schwarzer

Laboratory of Gnotobiology, Institute of Microbiology of the Czech Academy of Sciences, Doly 183, 549 22 Nový Hrádek, Czech Republic

Introduction: The ability of Lactiplantibacillus plantarum WJL bacteria to support the growth of gnotobiotic invertebrate and vertebrate hosts in models of chronic malnutrition is known and well-established (Storelli et al, 2011, Schwarzer et al., 2016). Whether other bacteria from different genera exert the growth-promoting capability is currently unknown. We selected two different bacterial strains from two different Bifidobacteria species and tested them in a gnotobiotic mouse model of chronic undernutrition.
Methods: Parental generation of germ-free (GF) C57BL/6J mice was monocolonized with one of these four bifidobacterial strains: Bifidobacterium longum ssp. longum (Bl367 and Bl372) and Bifidobacterium adolescentis (Bad368 and Bad373). Male mice from the filial generation were weaned at day 21 after birth on an experimental low-protein, low-fat (MAL) diet. Their growth was monitored weekly by measuring body length and weight for 5 weeks.
Results: Mice monocolonized with Bifidobacterium longum (Bl) strains showed improved systemic growth compared to mice monocolonized with Bifidobacterium adolescentis (Bad) strains or the control GF group. Monocolonization with Bl372 strain resulted in significantly enhanced weight and length gains. This was accompanied by elevated levels of IGF-1 in the serum. Histological analysis of the small intestine revealed significantly longer intestinal villi of Bl372 monocolonized mice. Bacterial load in the gut was reduced in all mice on MAL diet compared to breeding diet-fed controls. This was accompanied by a change in the shape of bacterial cells, probably caused by nutritional stress.
Conclusion: Our results show that selected Bifidobacterium longum 372 strain has the ability to support juvenile growth upon nutritional challenge. The ability to support growth differs among Bifidobacteria and is species- as well as strain-specific.
Acknowledgments: The work was supported by grant GAČR 21-19640M.
References:
Storelli G, Defaye A, Erkosar B, Hols P, Royet J, Leulier F. Lactobacillus plantarum promotes Drosophila systemic growth by modulating hormonal signals through TOR-dependent nutrient sensing. Cell Metab. 2011 Sep 7;14(3):403-14. doi: 10.1016/j.cmet.2011.07.012. PMID: 21907145.
Schwarzer M, Makki K, Storelli G, Machuca-Gayet I, Srutkova D, Hermanova P, Martino ME, Balmand S, Hudcovic T, Heddi A, Rieusset J, Kozakova H, Vidal H, Leulier F: Lactobacillus plantarum strain maintains growth of infant mice during chronic undernutrition. Science 2016, 351(6275):854-857.

Immune resistance to viruses

Cliona O'Farrelly

Trinity College, Dublin, Ireland

N/A

Unravelling a causal role of the gut microbiota in experimental colitis

Claire Pearson

Kennedy Institute of Rheumatology, University of Oxford, Roosevelt Drive, Headington, Oxford OX3 7FY. UK

The increasing prevalence of inflammatory bowel diseases (IBD) across the world reinforces the need to understand the mechanisms that lead to the onset of inflammation in the gut. The gut microbiota is altered in IBD, tending to become less diverse and with an increase in species that are more pathogenic. However it’s not currently known whether this is a cause of disease or a downstream consequence of other processes. In order to understand bacteria-bacteria and bacteria-host interactions that might underlie disease we have utilised a bacterially-driven colitis model within different microbiota complexities including specific pathogen free, colonisation with a 12 member synthetic microbial community, monocolonisation and germ free conditions. Colonisation with the pathobiont Helicobacter hepaticus combined with a defect in immune regulation driven by blockade of the IL-10 pathway results in colitis under SPF conditions, but under less complex microbiota conditions the picture is highly dependent on the individual species present and may be therapeutically targetable. Unravelling the role of specific bacteria in different parts of the disease process is helping to better understand the underlying causes of IBD

Dietary Habits and Healthy Ageing traced by epigenomic and gut microbiota profiling in a long-lived Blue Zone population

Gianni Pes

University of Sassari, Italy

In 2024, the Epigenos Project was launched on the Mediterranean island of Sardinia to investigate the complex interplay between biological age, aging, and the exposome in a population renowned for exceptional longevity. The study focuses on the island’s Blue Zone (BZ), a mountainous area where both men and women exhibit an equal probability of reaching 100 years of age, indicating the absence of a gender-based survival gap.
Using an integrated multi-omic approach that combines next-generation epigenetic clock analyses with shotgun metagenomic profiling of the gut microbiota (GM), the project aims to identify associations between microbial patterns, epigenetic age acceleration or deceleration, and health-related variables.
A stratified cohort analysis involving the entire adult population of the village of Seulo is ongoing. Kinship relationships were meticulously mapped through the reconstruction of the village’s complete genealogical tree. To date, nearly 200 participants aged ≥50 years have been recruited, providing blood samples for epigenomic profiling and stool samples for GM characterization. Health assessments included comorbidity and cognitive function, evaluated using the Cumulative Illness Rating Scale (CIRS) and Mini-Mental State Examination (MMSE), respectively, while dietary habits were assessed via the Italian version of the 276-item EPIC questionnaire.
Preliminary results show no significant sex-based differences in alpha diversity. When age was modeled as a continuous variable, alpha diversity declined between ages 50 and 90, followed by an increase in older individuals. DNA methylation data indicated a trend toward greater epigenetic age acceleration in males compared to females, although this was not statistically significant (Kruskal-Wallis, p = 0.159), despite demographic data suggesting equal longevity across sexes.
Across multiple epigenetic clocks, an increased abundance of Gammaproteobacteria was consistently associated with accelerated biological aging. Notably, several positive and negative correlations were observed between Gammaproteobacteria and inflammation-related epigenetic surrogate protein markers, as well as between alpha diversity and both the CIRS severity index and MMSE scores.
Cluster analysis of dietary intake and GM abundance revealed a positive correlation within a group comprising both "slow food" and "fast food" consumers. One latent dietary factor—characterized by fresh fruit, meat, and raw vegetables—was associated with increased Escherichia coli abundance, potentially reflecting inadequate washing of produce, particularly fruit and raw vegetables.
In conclusion, these early findings underscore the complex associations between gut microbiota composition and biological age acceleration. Ongoing research, including more refined functional characterization of the oldest residents and enhanced epigenomic profiling, particularly in relation to traditional dietary practices, will be essential to unravel the roles of the microbiome and epigenome in promoting the exceptional longevity of this unique population.

Algae and health

Ondřej Prášil, Richard Lhotský, Karolína Štěrbová, Pavel Hrouzek, Miloslav Kverka

Institute of Microbiology of the Czech Academy of Sciences, Center Algatech, Třeboň, Czech Republic

The talk will start with an introduction to algal biotechnology and historical overview of the medicinal use of algae. We will present short history of the algal biotech research at the Center Algatech of the Institute of Microbiology in Třeboň and some of its achievements related to the use of algae in human and veterinary medicine. These included the development of veterinary immunostimulants, wound-healing ointments, vaginal globules, dental extracts, and ulcer-healing powders. We will present our recent results regarding the effects of algal diet on the gut immune response. Our experiments have shown that heterotrophically grown chlorella biomass and so called ”chlorella growth factor” prevents DSS-induced colitis and reduced numbers of pro-inflammatory lymphocytes. Motivated by these preliminary experiments, our institute has joined large international consortium of research institutes, hospitals and small SMEs to provide the solutions for prevention and treatment of inflammatory bowel disease and ulcerative colitis by developing food supplements containing algal biomass. Large screening of algal organic extracts has confirmed that both microalgae and macroalgae are beneficial source of compounds with wide array of compounds possessing anti-inflammatory, analgetic and antioxidant properties. Currently the selected algae and their organic extract undergo validation in vivo which decide on their final application in the food supplements. Since our results show positive effects of algae on gut, we collaborate with companies that produce algae-containing food in order to improve the health benefits. For example, the Dutch company Phycom produces Chlorella kessleri strain HY-1, which is rich in lutein, and the regional MPM Bakery incorporates heterotrophic Chlorella sorokiniana into its bread products. Finally, we have just launched a new project Bio2AgroFood investigating the prebiotic and gut-stimulatory effects of algae in both humans and animals.

Microbial and metabolomic alterations in eating disorders

Petra Procházková¹, Radka Roubalová¹, Janet Ježková^1,2, Hana Papezová^2,3

1 Laboratory of Cellular and Molecular Immunology, Institute of Microbiology of the Czech Academy of Sciences, Prague, Czech Republic

2 First Faculty of Medicine, Charles University Prague, Czech Republic

3 Department of Psychiatry, First Faculty of Medicine, Charles University and General University Hospital in Prague, Prague, Czech Republic

Abstract
The gut microbiota can communicate with the brain via different pathways and this communication can play an important role in various neurological or psychiatric disorders. People with eating disorders such as anorexia nervosa often have a dysbiotic gut microbiome, which can affect their mental health.
Anorexia nervosa (AN), an eating disorder, is associated with marked changes in the microbiome and metabolic profile of those affected. In this study, we compared the diversity and composition of the gut microbiota and the differences in various parameters between patients with acute AN, severe and enduring AN (SEAN), and healthy controls. Both patient groups differed in the assessment of eating behaviors, depression symptoms, stressful events in adulthood, and use of antidepressants. The SEAN group showed elevated markers of gut damage that correlated negatively with BMI. Both patient groups showed lower alpha diversity and compositional differences compared to healthy controls, with the SEAN group showing the largest inter-individual variation.
Certain bacterial taxa, such as Faecalibacterium, Fusicatenbacter, Lachnospiraceae, and CAG-56, were less abundant in the patients' microbiomes, while Erysipelatoclostridium and UBA1819 were more abundant, but with no difference between patient groups. Functional prediction of the microbiome revealed differences in metabolic pathways, particularly in amino acid metabolism and oxidative stress responses, which were more pronounced in patients with SEAN. Certain bacteria such as Christensenellaceae, Ruminococcaceae, and Escherichia-Shigella negatively affect GABA metabolism, as evidenced by the lower serum and fecal concentrations in both patient groups compared to healthy women. Members of the Christensenellaceae affect microbial fermentation, resulting in significant differences in acetic, propionic, and butyric acid levels in stool and serum samples from AN patients. These findings highlight the complex interplay between the gut microbiota and metabolic changes in AN patients and provide insights into potential microbial biomarkers and therapeutic targets for this disease. The gut microbiota and its impact on mental health may be the missing element in understanding the etiology of eating disorders.
Acknowledgements
The study was supported by the Ministry of Health of the Czech Republic under grant nr. NU22-04-00010 and NU23-04-00381 and by the Ministry of Education, Youth, and Sports of the Czech Republic under grant Talking microbes - understanding microbial interactions within One Health framework (CZ.02.01.01/00/22_008/0004597).

Leveraging Human Microbiomes for Disease Prediction and Treatment

Tor C. Savidge

Department of Pathology and Immunology, Baylor College of Medicine, Houston, TX
Texas Children’s Microbiome Center, Department of Pathology, Texas Children’s Hospital, Houston, TX

The human microbiome comprises a diverse array of microorganisms that inhabit various anatomical sites and play a critical role in maintaining health. As their importance in human physiology and disease becomes increasingly evident, there is growing interest in harnessing individual microbiome profiles for early disease detection, prevention, and prediction of therapeutic efficacy. Despite accumulating evidence linking disease symptomatology to gut dysbiosis, microbiome-informed diagnostic tools remain in their infancy. Integration into clinical practice has been hindered by the inherent complexity of microbiome data and inconsistent findings across studies, often compounded by demographic and technological biases that limit the development of reliable disease classifiers.
This presentation will highlight our recent progress in developing a point-of-care platform for the rapid and quantitative detection of key gut microbial taxa. Our aim is to enhance the clinical utility of microbiome data for precision diagnostics and personalized therapeutic strategies. Specifically, we seek to enable real-time risk stratification by identifying baseline microbiome signatures predictive of differential treatment responses, for example, to dietary interventions such as the low FODMAP diet in individuals with irritable bowel syndrome. Additionally, the presentation will address key challenges in establishing microbiome-disease causality, such as distinguishing Clostridioides difficile colonization from active infection, which is critical for ensuring accurate diagnostics and effective treatment of complex diseases.

Bacterial extracellular vesicles: Cargo composition, host communication, and promise in allergy treatment

Irma Schabussova

Institute of Specific Prophylaxis and Tropical Medicine, Centre for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria

Extracellular vesicles (EVs) are key mediators of bacterial communication and adaptation to environmental stress, with their size, cargo, and charge shaped by environmental conditions, bacterial physiology, and isolation methods. These characteristics are also highly strain-specific, influencing how EVs interact with host cells and contribute to health or disease. In this study, we explored the compositional and functional diversity of bacterial EVs from two probiotic strains—Escherichia coli O83 (EcO83) and Lactiplantibacillus plantarum NCIMB 8826 (L. plantarum)—focusing on their role in environmental adaptation, host communication, and potential in allergy treatment.
EVs from EcO83 (EcO83-EVs) were isolated by ultracentrifugation and shown to be spherical nanoparticles (~110 nm) enriched in 1120 proteins, 136 of which were significantly elevated compared to their parent bacteria. These EVs carried lipopolysaccharide (LPS), DNA, RNA, and lipids, and activated the pattern recognition receptors TLR2/4/5 as well as NOD1 and NOD2. In a mouse model, intranasally administered EcO83-EVs targeted the nasal-associated lymphoid tissue, were internalised by airway macrophages, and stimulated neutrophil recruitment. Mechanistically, EcO83-EVs activated the NF-κB pathway, leading to nitric oxide production. These immunomodulatory effects translated into reduced airway hyperresponsiveness, eosinophilia, Th2 cytokine production, and mucus secretion in a mouse model of ovalbumin-induced allergic airway inflammation, highlighting their promise as a postbiotic alternative to live bacteria for allergy prevention and mucosal immunotherapy.
To examine how environmental factors influence bacterial EV properties, we investigated EVs released by L. plantarum (LpEVs) under bile stress as a model for gut-associated conditions. Using ultracentrifugation followed by size-exclusion chromatography (SEC), we obtained highly pure LpEVs (~70 nm) and characterised them according to MISEV guidelines. SEC purification efficiently removed proteins and peptidoglycan contaminants, refining the EV composition. Compared to the parent cells, purified LpEVs displayed distinct surface lipid profiles and zeta potential, along with remarkable stability across a range of pH values, salt concentrations, and detergent exposures. Notably, bile exposure led to the production of larger LpEVs enriched in proteins involved in bile metabolism, suggesting an adaptive mechanism. Fourier-transform infrared (FTIR) spectroscopy confirmed bile-induced molecular changes in LpEVs, distinct from those in the bacterial cells themselves. These findings illustrate the dynamic responsiveness of bacterial EVs to environmental stimuli and underscore their potential in early host-microbe interactions, possibly preceding overt bacterial adaptation.
Together, these studies demonstrate that bacterial EVs serve as highly adaptable vehicles for molecular communication between microbes and host tissues. Their cargo composition reflects both bacterial origin and environmental pressures, and they can exert immunomodulatory effects that may be harnessed therapeutically. EVs from both probiotics strains show promise as next-generation biologics for modulating immune responses, especially in the context of allergic diseases. Further studies are warranted to optimise their production and evaluate their clinical potential as safe, effective tools for allergy prophylaxis and therapy.
The Government of Lower Austria project Danube-Allergy Research Cluster (P17); The FWF project P 34867; MSCA-PF project LactoVES (No. 101066450), OEAD: CZ 04/2024, CZ 07/2023, CZ 15/2023, RS 08/2022

Overcoming undernutrition related stunting: Bacteria in mammalian host juvenile growth

Martin Schwarzer

Laboratory of Gnotobiology, Institute of Microbiology, Czech Academy of Sciences, Novy Hradek, Czechia

We have shown that the gut microbiota and the specific bacterium Lactiplantibacillus plantarum WJL support the growth of Drosophila and mouse hosts during chronic malnutrition (Schwarzer et al., 2016, Schwarzer et al., 2023). We hypothesised that administration of LpWJL during the refeeding process following malnutrition-induced stunting could improve growth of juvenile mice. Stunting was induced in male C57BL/6 mice by feeding a low protein/low fat diet (isocaloric to the control diet AIN93) from day (D) 21 to D28. On day 28, the stunted mice received the full AIN93 diet and placebo or LpWJL (2*10^8 CFU/mouse) 5 times per week. Mice fed the AIN93 diet from D21 onwards served as controls for normal growth. Mice were weighed and measured weekly. Oral and intraperitoneal glucose tolerance tests (GTT) were performed and samples were taken at D84 for further analyses. Our results show that mice fed LpWJL showed improvements in length, weight, internal organ weight and bone length compared to the placebo-fed group. The GTT revealed that LpWJL-fed mice had better glucose clearance. Analysis of the histology of the jejunum of the LpWJL-fed mice showed longer villi compared to the placebo-fed mice. Overall, our results suggest that supplementation with LpWJL in combination with re-feeding strategies has the potential to alleviate persistent stunting.
Supported by the Czech Science Foundation grant No. 21-19640M.
References:
Schwarzer M, ..., Leulier F: Lactobacillus plantarum strain maintains growth of infant mice during chronic undernutrition. Science 2016, doi: 10.1126/science.aad8588
Schwarzer M, …, Leulier F: Microbe-mediated intestinal NOD2 stimulation improves linear growth of undernourished infant mice. Science 2023, doi: 10.1126/science.ade9767

Asymptomatic chronic infections - the power of intestinal bacteria

Ulrich Steinhoff

Philipps University of Marburg, Marburg, Germany

The intestinal microbiota fundamentally guides the development of a normal intestinal physiology, the education and functioning of the mucosal immune system. The C. rodentium-carrier model is suitable to study a blunted host-pathogen interaction in the absence of the gut microbiota. Here, we describe that colonization of adult carrier mice with 14 selected commensal microbes (OMM¹²+MC²) was sufficient to reestablish the host immune response to enteric pathogens; this conversion was rendered possible by maturation and activation of the intestinal blood vessel system and the step-and timewise stimulation of innate and adaptive immunity. While the immature colon of C. rodentium-infected GF mice did not allow sufficient extravasation of neutrophils into the gut lumen, colonization with OMM¹²+MC² commensals initiated the expansion and activation of the visceral vascular system enabling granulocyte transmigration into the gut lumen for effective pathogen elimination. Consortium modeling revealed that the addition of two facultative anaerobes to the OMM¹² community was essential to further progress the intestinal development. This study demonstrates the therapeutic value of as few as 14 commensal bacteria in promoting intestinal maturation and immunity even in adult organisms.

Some viruses can modulate immune tolerance

Marek Sinkora, Katerina Stepanova, Jana Sinkorova

Laboratory of Gnotobiology, Institute of Microbiology, Czech Academy of
Sciences, Novy Hradek, Czech Republic

Porcine reproductive and respiratory virus (PRRSV) is a prototypic pathogen that has evolved a mechanism to induce immune tolerance to its structures while maintaining the host's ability to defend itself against other antigens and pathogens. Recent research has shown that PRRSV infects the thymus and alters the T cell receptor repertoire during T lymphocyte development. The developing thymocytes are affected during negative selection when they transition from the triple-negative to the double-positive stage just before they enter the medulla at the corticomedullary junction. The "hole" in the T cell repertoire allows the acceptance of PRRSV-neutralizing epitopes as self-antigens. Without cognate T cells, the selection of B cells capable of producing neutralizing antibodies with high affinity is impaired, as is the generation of specific cytotoxic T cells. Interestingly, PRRSV affects the T cell repertoire specifically for its strain and each individual host, based on aberrant selection of T cell receptors by MHC-I and MHC-II molecules. This prevents the development of an effective vaccine. The lack of an effective immune response against the critical viral structures tolerated as self-antigens and the inability to combat the virus with vaccines allow the virus to escape, survive, persist, and continue to spread. Research supported by the Ministry of Education, Youth and Sports of the Czech Republic grant CZ.02.01.01/00/22_008/0004597.

Host-microbiome interplay in epicutaneous sensitization and food allergy development

Šrůtková D.¹, Horníková T.¹, Jelínková A. ¹, Thon T. ², Poloučková A. ³, Kopelentová E. ³, Šedivá A.³, Tlaskalová-Hogenová H. ², Schwarzer M. ¹, Jirásková Zákostelská Z. ²

1 Laboratory of Gnotobiology, Institute of Microbiology, Academy of Sciences of the Czech Republic

2 Laboratory of Cellular and Molecular Immunology, Institute of Microbiology, Academy of Sciences of the Czech Republic

3 Department of Immunology, 2nd Faculty of Medicine, Charles University in Prague, and Motol University Hospital

Introduction: Atopic dermatitis (AD) is often accompanied by the subsequent development of food allergy (FA) due to epicutaneous sensitization (EC) by food allergens. Genetic predisposition and environmental factors such as skin and intestinal microbial dysbiosis contribute to its development. We aimed to assess the role of host genetic background as well as skin and intestinal microbiota in the development of the AD and FA symptoms in experimental mouse model.

Material & Methods: Specific-pathogen-free BALB/c and C57BL/6 and germ-free (GF) BALB/c mice were epicutaneously sensitized by three one-week exposures of ovalbumin (OVA) applied as a patch to shaved and tape stripped skin continued with intragastric OVA challenge. Further, 3-weeks old GF mice were colonized by fecal and skin microbiota from healthy infant or infant with manifested AD symptoms, subsequently EC sensitized and FA symptoms were induced by repeated intragastric OVA challenge. The development of food allergy symptoms (anaphylactic hypothermia and diarhea), the level of Th2-related cytokines, antibodies as well as the mast cell immune response and differences in the skin and gut microbiota were investigated.
Results: BALB/c mice, but not C57BL/6 mice, showed severe clinical signs of FA (hypothermia, diarrhea) as well as stronger Th2 cytokine, antibody and mast cell response. The 16S rRNA sequencing analysis revealed lower abundance of short-chain fatty acids producing bacteria in the gut microbiome of OVA-treated BALB/c mice, which correlated with more severe food allergy symptoms. Changes in the β-diversity of the gut microbiome reflect both the genetic background as well as the OVA treatment of experimental mice. Compared to SPF mice, GF mice developed more severe anaphylactic hypothermia but no diarrhea, although they had a higher mast cell count. Further, the colonization of GF mice by fecal together with skin microbiota from pediatric AD patient have impact on intestinal morphology as well as number of mast cells in mice after the EC sensitization and FA induction. Anaphylactic hypothermia determined as the drop of body temperature after each gavage, elevated number of mast cells in jejunum as well as allergen-specific antibodies in sera were more apparent in mice colonized with patient microbiota than with those from healthy control. Microbiome analysis showed the dominance of family Lachnospiraceae, followed by Enterobacteriaceae, Enterococcaceae and Veillonelaceae in fecal microbiome and elevated abundance of Cutibacterium and Staphylococcus in skin samples of mice colonized by patient’s microbiota. Mice colonized by microbiota from healthy donor revealed improvement of food allergy symptoms such as anaphylactic hypothermia or infiltration of mast cells in jejunum. Microbiome analysis showed high abundancy of Bifidobacteriaceae, Streptococcaceae and Lachnospiraceae in the gut and common commensals such as Streptococcus gallolyticus , Staphylococcus epidermidis or genus Cutibacterium on the skin of mice with healthy control microbiota.
Conclusion: Our results highlight the significant influence of genetic background and microbiota on susceptibility to epicutaneous sensitization and food allergy and emphasize the complex interplay of these factors in the allergic response.
Acknowledments: Supported by Czech Health Research Council (NU20-05-00038) and Youth and Sports of the Czech Republic grant Talking microbes - understanding microbial interactions within One Health framework (CZ.02.01.01/00/22_008/0004597).

Targeting the gut microbiome in cancer immunotherapy

Giorgio Trinchieri

Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA

In recent years, evidence has shown that the gut microbiome significantly influences responses to immunotherapy. This has sparked interest in targeting it to improve therapy outcomes and predictions of response and toxicity. The gut microbiome composition has been leveraged for its power to predict clinical outcomes using machine learning, and surprisingly, its predictive capability is comparable to that of other described multi-biomarker clinical scores. The bacterial taxa affecting positively or negatively immunotherapy responses are context dependent and different taxa and mechanisms may be involved in different types of immunotherapy or even tumor types. While a clear consensus has yet to emerge on the optimal species for therapeutic use, altering the gut microbiome of immunotherapy-refractory patients by fecal microbiome transplantation or possibly diet has provided proof of concept that it is possible to target it for improving cancer immunotherapy response.

Bacterial pathogenesis in celiac disease

Elena Verdú

Farncombe Family Digestive Health Research Institute, McMaster University, Hamilton, Canada.

Celiac disease is a common, chronic inflammatory condition that develops in individuals that express the autoimmune risk genes HLA-DQ2 and, or DQ8. The current prevalence is on the rise and affects 1% of most ethnic populations worldwide. The main environmental trigger is gluten, a complex group of related proteins that are proteolytic resistant and are found in wheat, rye, and barley. It is now well established that 2 years after initiating the gluten-free diet, which currently is the only management strategy for celiac disease, 70% of patients continue to experience inflammation of the small intestinal mucosa. This parallels with nutritional deficiencies and complications, which include osteoporosis and higher risk of fractures and incidence of autoimmune conditions such as type 1 diabetes (particularly in women) and some cancers. Thus, adjuvant therapies to the gluten-free diet constitute an unmet need in celiac disease. Until recently, gluten was considered the sole driver in celiac disease, even though only a minority of genetically susceptible individuals develop the disease. However, small intestinal microbes can differentially modify the immunogenicity of gluten. Using mice expressing celiac risk genes and generated in germ-free conditions colonized with Pseudomonas aeruginosa-producing elastase isolated from patients, demonstrate that bacterial elastase increases gluten antigenicity and uptake of gluten peptides through the mucosa and enhances immune reactivity of gluten specific T cells in celiac patients. However, the combined presence of commensal bacteria such as lactobacillus aids in the full digestion of gluten peptides, reducing their immunogenicity. Independently from gluten, duodenal microbes can influence inflammation in CeD through metabolism of the essential amino acid, tryptophan. This aminoacid is poorly metabolized by gut microbes present in the duodenum of celiac patients, leading to impaired activation of the aryl hydrocarbon receptor in the intestinal mucosa and inflammation. Ongoing studies in CeD patients non-responsive to the gluten free diet will be discussed.

Human Milk Immunoglobulin A Binding of Early-Life Microbiota: Identifying Commensals Highly Targeted by IgA

Christine Bäuerl, Michela Amato, Raúl Cabrera-Rubio, Isabel Campillo, Nuria Ruiz-Costa, Gaspar Pérez Martínez, Maria Carmen Collado

Institute of Agrochemistry and Food Technology/Spanish National Research Council (IATA-CSIC), Department of Biotechnology, Valencia, Spain

Although the origins of secretory IgA (sIgA) in human milk and its role to clear pathogens are well established, its interactions with early-life gut commensal microbiota - and the resulting impact on immune and microbiota maturation - are less well understood. In particular, it still remains unclear why certain bacteria are targeted over others and the functional outcome for bacterial fitness.

Our objective was to study the recognition of early-life commensals by sIgA. Therefore, we assessed the binding capacity of various Bifidobacterium isolates, obtained from fecal samples of healthy infants, to sIgA purified from human colostrum. Among them, B. bifidum and B. pseudocatenulatum were found to be highly targeted. Furthermore, in a proof-of-concept longitudinal study in 10 mother-infant pairs (5 breastfed and 5 formula-fed infants), we evaluated the influence of lactation on sIgA binding to bacteria over the first year of life. We isolated IgA-coated (IgA⁺) bacteria using magnetic beads (Dynabeads®) from fecal samples at one, six and 12 months of age and subjected IgA⁺/IgA^- fractions to 16S rRNA amplicon sequencing. Additionally, the proportion of IgA-coated bacteria and free available IgA in fecal water was determined.

Our results showed that breastfed infants had significantly higher microbial diversity (Shannon index) in the IgA^- fraction (p = 0.032). At one month of age, both IgA bacterial coating and free fecal IgA were detected in formula-fed infants, indicating that at this age infants could produce their own IgA. Interestingly, B. bifidum was significantly enriched in the IgA⁺ fraction in samples from both formula and breastfed infants, supporting the in vitro findings from binding studies using purified sIgA. RNA-seq analysis further showed that sIgA binding to B. bifidum upregulated several glycosyl hydrolase genes, indicating a possible role of microbial glycan recognition. Further research will delve deeper into the molecular determinants responsible for IgA binding to B. bifidum.

Role of the virome and immune imprinting on physiological trade-offs during undernutrition

Lucie Bernard-Raichon, Lucas Rebiffe, François Leulier

Institut de génomique fonctionnelle de Lyon

Nearly half of all deaths in children under five are linked to undernutrition, which causes developmental issues and increased susceptibility to infections and intestinal inflammation that are not fully corrected by traditional nutrition strategies. Early exposure to commensal bacteria shapes immune trajectories, influencing susceptibility to infections and inflammatory diseases both early in life and in the long-term. Research from our group has demonstrated that commensal bacteria contribute to host development, particularly under conditions of undernutrition. Similarly, commensal viruses (aka the virome) can modulate immune responses. Infants, especially in regions with high rates of undernutrition, are highly exposed to these viruses. However, the virome’s impact on immune development and its contribution to the effects of undernutrition remains largely unexplored. This projects investigates how early immune imprinting by the virome alters host-development trajectories —including growth, metabolism, and sexual maturation—and affects long-term susceptibility to infections and inflammatory diseases in male and female mice. It uses a combined approached of hypothesis-driven research (focusing on the homeostatic role of type I interferons) and a RNA sequencing-based discovery, to uncover unknown physiological functions of intestinal immune cells that are critical in challenging environments like undernutrition. The findings from this study will provide new insights into how understudied factors—such as the virome, sex, and undernutrition—affect immune function, including host-defenses and physiological functions. This knowledge could lead to innovative preventive and therapeutic strategies for undernourished children, such as microbiota-targeted food and immune-based interventions, tailored specifically for boys and girls.

The extent of cecal dilatation is influenced by the genetic background in germ-free mice

Silvia Bolsega ¹, Anna Smoczek ¹, Chen Meng ², Karin Kleigrewe ², Tim Scheele ¹, Sebastian Meller ³, Silke Glage ¹, Holger A. Volk ^3,4, André Bleich ¹, and Marijana Basic ¹

1 Institute for Laboratory Animal Science, Hannover Medical School, Germany
2 Bavarian Center for Biomolecular Mass Spectrometry, TUM School of Life Sciences, Technical University Munich, Germany
3 Department of Small Animal Medicine and Surgery, University of Veterinary Medicine Hannover, Germany
4 Center for Systems Neuroscience Hannover, Germany

Germ-free (GF) rodents have become a valuable tool for studying the role of intestinal microbes on the host physiology. The major characteristic of GF rodents is an enlarged cecum. The accumulation of mucopolysaccharides, digestion enzymes and water in the intestinal lumen drives this phenotype. Microbial colonization normalizes the cecum size in ex-GF animals. However, whether strain genetics influences the cecal enlargement is unknown. In this study, we investigated the impact of mouse genetic background on the cecal size in five GF strains frequently used in biomedical research. The cecal weight of GF mice on B6 background (B6J and B6N) represented up to 20% of total body weight. GF NMRI and BALBc mice showed an intermediate phenotype of 5–10%, and those on the C3H background of up to 5%. Reduced cecal size in GF C3H mice correlated with decreased water content, increased expression of water transporters, and reduced production of acidic mucins, but was independent of the level of digestive enzymes in the lumen. In contrast, GF B6J mice with greatly enlarged cecum showed increased water content and a distinct metabolic profile characterized by altered amino acid and bile acid metabolism, and increased acidic mucin production. Together, our results show that genetic background influences the cecal enlargement in GF mice by regulating the water transport, production of acidic mucins, and metabolic profiles.

Vegan diet signature from a multi-omics study on different European populations is related to favorable metabolic outcomes

Monika Cahová¹, Anna Ouřadová², Giulio Ferrero³, Marek Kuzma⁴, Filip Tichánek¹, Jan Gojda², Barbara Pardini⁵, Alessio Nacarrati⁵

1 Institute for Clinical and Experimental Medicine, Prague, CR ;
2 Department of Internal Medicine, Kralovske Vinohrady University Hospital and Third Faculty of Medicine, Charles University, Prague, CR;
3 Dept. of Clinical and Biological Sciences, University of Turin, Turin, Italy;
4 Institute of Microbiology of the Czech Academy of Sciences, Prague, CR;
5 Italian Institute for Genomic Medicine, Turin, Italy

Plant-based diets are gaining popularity due to their lower environmental footprint and potential to mitigate the risks of non-communicable diseases (NCDs). This cross-sectional, binational observational study explored the specific signature of a vegan diet across microbiome, metabolome, and lipidome profiles. The study included 98 healthy vegan participants and 73 omnivores from the Czech Republic and Italy. To ensure the generalizability of the findings, results were validated in an independent cohort of 142 subjects. We identified a vegan diet-associated, country-independent multi-omics profile comprising 88 lipid metabolites, 20 non-lipid metabolites, and 17 bacteria. Most of the identified markers convey protective effects against NCDs. While circulating lipids and certain non-lipid metabolites were directly related to diet composition, the gut microbiota also shifted toward a health-promoting profile. Using a machine learning approach, we accurately classified vegans and omnivores based on individual omics, achieving AUCs of 0.95 (lipids), 0.89 (metabolites), and 0.87 (microbial species). Functional differences were observed in metagenomes: vegans exhibited enrichment in amino acid biosynthesis, inositol degradation, and the pentose phosphate pathway, whereas omnivores showed enhanced amino acid fermentation, fatty acid biosynthesis, and propanoate metabolism. These findings provide insight into host-microbiota interactions and highlight pathways modulated by diet.

Ocrelizumab causes transient changes in the composition of intestinal bacteria and modulates the immune response depending on the response to treatment

Stepan Coufal¹, Zuzana Jiraskova Zakostelska¹, Tomas Thon¹, Radka Roubalova¹, Dominika Kadleckova², Martina Salakova², Ruth Tachezy², Tomas Hrncir³, Miloslav Kverka¹, Veronika Ticha⁴, Miluse Pavelcova⁴, Pavlina Kleinova⁴, Jana Lizrova Preiningerova⁴, Ivana Kovarova⁴, Jakub Kreisinger⁵, Helena Tlaskalova-Hogenova¹ and Eva Kubala Havrdova⁴

1 Laboratory of Cellular and Molecular Immunology, Institute of Microbiology of the Czech Academy of Sciences, Prague, Czech Republic
2 Department of Genetics and Microbiology, Faculty of Science, Charles University, BIOCEV, Vestec, Czech Republic
3 Laboratory of Gnotobiology, Institute of Microbiology of the Czech Academy of Sciences, Novy Hradek, Czech Republic
4 Department of Neurology and Centre of Clinical Neuroscience, First Medical Faculty, Charles University and General Medical Hospital in Prague, Prague, Czech Republic.
5 Laboratory of Animal Evolutionary Biology, Faculty of Science, Department of Zoology, Charles University, Prague, Czech Republic.

Multiple sclerosis (MS) is an autoimmune disease that leads to the loss of myelin and atrophy of the central nervous system. The role of gut microbiota dysbiosis has been implicated in MS pathogenesis and may also influence treatment outcomes.

In our study, we included 25 newly diagnosed persons with MS (PwMS) with clinically isolated syndrome (CIS), which were treatment-naïve and 9 PwMS on IFN-β who had relapsed and were indicated for treatment with ocrelizumab. The eighty-one healthy control subjects were also recruited. Stool and serum in study groups were collected before the treatment and every 3 months for a minimum of 12 months.

We identified changes in the gut microbiota that are already present in CIS persons who are naive to MS treatment. While treatment responders showed an increasing trend in alpha diversity after 12 months to the point that they approached values similar in healthy controls, non-responders showed a significant decrease in gut microbiota alpha diversity. The main changes were seen in bacteria, including Parabacteroides spp., which are recognized or putative producers of short-chain fatty acids, which support gut barrier functions and have anti-inflammatory potential. We found altered levels of gut barrier biomarkers and antibodies against ten common gut commensal bacteria in the sera of PwMS compared to healthy controls, we also found that this biomarker profile in PwMS was altered by anti-CD20 treatment. Additionally, we found significant decreases in lipopolysaccharide-binding protein and mannose-binding lectin but only in sera of responders during treatment; no changes occurred in non-responders.

These results suggest a link between intestinal barrier damage and subsequent immune responses associated with microbial translocation and MS pathogenesis and treatment.

This research was supported by grants from the Ministry of Health of the Czech Republic (NU20-04-00077), the Ministry of Education, Youth and Sports of the Czech Republic grant Talking microbes-understanding microbial interactions within One Health framework (CZ.02.01.01/00/22_008/0004597), the National Institute of Virology and Bacteriology Project (EXCELES Program, LX22NPO5103), funded by the European Union, Next Generation EU, and the Academy of Sciences of the Czech Republic (LQ200202105).

Western Diet Disrupts Anti-Tumor Immunity and Promotes Melanoma Progression Through Myeloid and T Cell Modulation

Janaina L S Donadio, Anna Pavlicova, Stepan Coufal, Veronika Motuzova, Michal Kraus, Miloslav Kverka

Institute of Microbiology of the Czech Academy of Sciences

Melanoma incidence is higher in countries where Western-type diets (WD) are common. While WD is known to promote a tumor-supporting environment, the immune mechanisms behind this — and how they might involve the gut microbiota — remain poorly understood.

In this study, we investigated how WD shapes anti-tumor immunity using a syngeneic melanoma model. Female C57BL/6 mice were fed either WD or control diet (CT) for 15 days, followed by subcutaneous implantation of B16-F10 melanoma cells. We conducted two parallel experiments:
(1) a survival study, where tumor growth and survival were monitored over time; and
(2) an immune profiling study, where tumors, spleens, and mesenteric lymph nodes (mLN) were analyzed by flow cytometry at day 21 post-implantation.

In the survival study, WD-fed mice showed larger tumors and reduced overall survival compared to CT-fed mice. Interestingly, in the follow-up immune profiling study, although tumor sizes were similar between groups, immune cell patterns were different. WD-fed mice had an increased accumulation of monocytic myeloid-derived suppressor cells (MO-MDSCs) and non-antigen-presenting myeloid cells within the tumors, suggesting enhanced local immunosuppression and impaired CD4⁺ T helper cell activation. In the mLN, T regulatory cells (Tregs, CD25⁻FoxP3⁺) were reduced, indicating disrupted mucosal immune regulation. In the spleen, there was an increase in Th17 cells, pointing to a shift toward inflammatory T cell responses.

These findings highlight that dietary interventions reshape anti-tumor immunity in complex ways: WD appears to drive systemic inflammation while maintaining a less immunogenic tumor. These effects suggest an alteration in gut microbiota composition, known to modulate Th17, Tregs, and myeloid populations. We propose that dietary patterns induce changes in the microbiota and immune system, creating different environments across immune tissues, potentially modulating how the body responds to tumors

Bifidobacterium longum ssp. longum CCM 7952-derived peptidoglycan alleviates allergic inflammation through TLR2-dependent signaling in a mouse model of airway allergy

Tereza Hornikova¹, Katarzyna Pacyga-Prus², Katarzyna Leszczyńska-Nowak², Sabina Górska², Sudhanshu Shekhar¹, Umesh Kumar Gautam¹, Martin Schwarzer¹, Dagmar Srutkova^1*

1 Laboratory of Gnotobiology, Institute of Microbiology, Czech Academy of Sciences, Novy Hradek, Czechia
2 Laboratory of Microbiome Immunobiology, Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroclaw, Poland

Previously, we have shown that intranasal administration of heat-killed Bifidobacterium longum ssp. longum CCM 7952 (Bl7952), similar to live bacteria, retains the ability to suppress airway inflammation and alleviate allergy in ovalbumin (OVA)-sensitized and challenged mice (Pyclik et al., 2021). This finding prompted us to isolate specific Bl7952 cell wall antigens to identify those responsible for the anti-allergic effect.
Mice were sensitized three times intraperitoneally with ovalbumin emulsified with the adjuvant alum. Four hours before each sensitization and before each of the four intranasal ovalbumin challenges, isolated Bl7952 antigens (slime exopolysaccharides, cell wall polysaccharides, surface proteins or peptidoglycan (PG)) were applied intranasally.
We show that of the antigens tested, only PG was able to reduce the total cell count and eosinophil count in bronchoalveolar lavage (BAL) and lower the lung histopathology score compared to the PBS/OVA group. In addition, PG administration reduced the levels of OVA-specific IgE antibodies in both serum and BAL, serum levels of total IgE antibodies and levels of Th2-related cytokines in splenocyte cultures. In vitro, PG was able to signal via TLR2 and NOD2 receptors in the HEK293 cell reporter system. To determine whether either of these receptors is necessary for the observed allergy-reducing effect, we used NOD2 and MyD88 whole-body knockout (KO) mice. The anti-allergic effect of PG was partially maintained in NOD2 KO mice, but not in MyD88 knockout mice. This indicates that the PG effect is mediated by the MyD88 adaptor molecule suggesting the signalization through TLR2 receptor.
These results suggest that Bl7952 PG has anti-allergic properties and may be a promising postbiotic candidate for further investigation.
Supported by the Czech Science Foundation No. 23-04050L and EMBO installation grant (M. Schwarzer).
References:
Pyclik MJ et al: Viability Status-Dependent Effect of Bifidobacterium longum ssp. longum CCM 7952 on Prevention of Allergic Inflammation in Mouse Model. Frontiers in Immunology 2021, doi: 10.3389/fimmu.2021.707728

Microbiota metabolite indirectly regulates T cell fate through CD11c+ innate cells

D. Chang, A. Bertocchi, C. Pearson and F. Powrie

Kennedy Institute of Rheumatology, University of Oxford, Oxford, UK

Trillions of microbiota live inside the gut, partially due to the millions of microbiota we eat in daily. Gut microbiota, together with the metabolites they produced, play essential roles in helping the immune system against pathogens. The imbalance between microbiota and immune system may cause inflammation or even barrier damage. ADP-heptose, a gram-negative bacteria metabolite, was originally shown to induce inflammatory response in human epithelial cells in vitro. However, we found ADP-heptose can suppress LPS-induced pro-inflammatory cytokines production in dendritic cells (DCs), which could shape T cell differentiation in vitro. By using Helicobacter hepaticus (Hh) infection model and Alpk1-/- (Alpha kinase 1, Pattern Recognition Receptor (PRR) of ADP-heptose) mice, we further confirm the protective role of ADP-heptose against infection. Mechanically, ADP-heptose-Alpk1 compete with LPS-TLR4 pathway for MAPK/NF-kB activation. However, whether other Alpk1-expressing myeloid cells or ILC3s are involved in this process is still unknown. We aim to understand how innate cells educate T cells development and maintain immune homeostasis, also shed light on new direction by using metabolite for microbiota-related disease treatment.

Ruminococcus gnavus in Spondyloarthritis, story of a commensal killer

Jacoutot M^1,2, Dias Munhoz D^1,2, Beaufrère M^1,2,³, Karpman P^1,2, Frison E^1,2, Robert V ⁴, Said-Nahal R^1,2,³, Bellais S⁵, Daillere R⁵, Gautreau G⁶, Langella P⁴, Chatel JM⁴, Glatigny S^1,2, Breban M^1,2,³

1 UMR1173 INSERM, Université Paris-Saclay, Montigny-le-Bretonneux, France;
2 INFLAMEX, Laboratoire d’Excellence, Université Paris Diderot, Sorbonne Paris Cité, Paris, France;
3 Ambroise-Paré Hospital, Rheumatology Department, AP-HP, Boulogne-Billancourt, France;
4 UMR1319 MICALIS, INRAe, AgroParisTech, Université Paris-Saclay, Jouy-en-Josas, France;
5 Bioaster,
6 Université Paris-Saclay, INRAE, MaIAGE, Jouy-en-Josas, France

Introduction. Spondylarthritis (SpA) is a group of chronic inflammatory disorders characterized by osteoarticular and extra-articular manifestations, including inflammatory bowel disease (IBD). It is well-established that intestinal microbiota dysbiosis plays a role in IBD pathophysiology. We recently evidenced an intestinal dysbiosis in SpA patients characterized by a decreased bacterial diversity associated to an increased relative abundance of the bacterium Ruminococcus gnavus. In this study, our goal was first to determine if specific R. gnavus strains are present during SpA. Second, we tested if R. gnavus strains isolated from SpA patients were more toxic and proinflammatory than those from healthy controls (HC).
Methods. R. gnavus colonies were isolated from colonic biopsies and/or stools from SpA patients and HCs. Isolated R. gnavus strains were sequenced by next generation sequencing (NGS). Functional assays were conducted by stimulating monocytes isolated from blood of SpA patients with the isolated strains (or their components) to assess toxicity and tumor necrosis factor (TNF) production.
Results. A total of 118 R. gnavus colonies were isolated and sequenced (68 from SpA patients and 39 from HC), with no common strains between groups. Phylogenetic analysis revealed clustering of strains isolated from SpA patients with high disease activity. Functional studies showed that R. gnavus strains isolated from SpA patients were enriched for toxic and proinflammatory functions. Identification of the bacterial feature(s) responsible for the proinflammatory and toxic effects is under way.
Conclusions. Our work demonstrates a broad R. gnavus diversity in stools and biopsies from SpA patients and HCs. Furthermore, an enhanced toxic and proinflammatory potential of R. gnavus strains isolated from SpA patients was evidenced. Further research should investigate the molecular nature of these proinflammatory components to clarify their contribution to disease progression.

Microbes and their role in appetite regulation in Binge eating disorder

Janet Ježková ^1,2, Enrico Patrono¹, Gabriela Kubišová¹, Petra Procházková ¹, Helena Tlaskalová-Hogenová ¹, Hana Papežová ³, and Radka Roubalová ¹

1 Laboratory of Cellular and Molecular Immunology, Institute of Microbiology of the Czech Academy of Sciences, Prague, Czechia
2 First Faculty of Medicine, Charles University, Prague, Czechia
3 Department of Psychiatry, First Faculty of Medicine, Charles University and General University Hospital in Prague, Prague, Czechia

The gut microbiota plays seemingly an important role in appetite regulation through the microbiome-gut-brain axis. Emerging evidence suggests that microbiota-derived metabolites influence key processes such as reward signaling, emotional regulation, and feeding behavior. Understanding these interactions could prove valuable in the context of Binge Eating Disorder (BED), a prevalent psychiatric condition characterized by recurrent episodes of excessive food intake, often triggered by emotional distress rather than hunger. BED is strongly associated with obesity and mood disorders. The underlying biological mechanisms of this disorder remain poorly understood.
In this study, we successfully implemented a previously validated binge eating mouse model that mimics key features of the disorder observed in humans. Our results show altered expression of genes important for appetite regulation and stress control in the hypothalami of experimental mice, suggesting dysregulation of neural pathways that may promote compulsive overeating. Specifically, we found lower expression of adra1a, drd2, 5ht2c, gad1 and npy and higher expression of gabbr1 in mice exhibiting binge eating disorder compared to control mice. We also observed changes in microbial composition during the final phase of the experiment, which coincided with the onset of compulsive eating behavior, suggesting a potential link between microbiome alterations and binge eating.
By elucidating the role of gut microbes in BED onset and persistence, this research aims to identify microbial targets for potential therapeutic interventions. A deeper understanding of how microbes influence eating behaviors and stress resilience could pave the way for novel microbiome-based strategies to treat BED.
This study was supported by the Ministry of Health of the Czech Republic, grant nr. NU23-04-00381.

Microbiota related changes influencing the response to the therapy in patients with lymphoma

Klara Kostovcikova ¹, Marketa Tenglerova ¹, Stepan Coufal ¹, Lucie Dlouha ², Michaela Brichova ³, Katerina Benesova ³, Petra Svozilkova ³, Jarmila Heissigerova ³ and Marek Trneny ²

1 Institute of Microbiology of the Czech Academy of Sciences, Prague, Czech Republic
2 Department of Medicine, 1st Faculty of Medicine, Charles University and General University Hospital in Prague, Czech Republic
3 Department of Ophthalmology, 1st Faculty of Medicine, Charles University and General University Hospital in Prague, Czech Republic

Recently, we have observed that patients with lymphoma harbor significantly different gut and mouth microbiota when compared to healthy individuals. Here, in the pilot study, we analyzed serum biomarkers related to microbial translocation and mouth swab and fecal microbiota in patients with Diffuse Large B-Cell Lymphoma (DLBCL) and Lymphoma of Immune-Privileged Sites (LIPS) regarding to the response to therapy.
We collected 26 mouth swabs and 15 fecal samples from patients with DLBCL and 18 mouth swabs and 9 fecal samples from patients with LIPS before chemotherapy initiation and processed them for sequencing by Illumina MiSeq approach. In addition, we analyzed the levels of serum biomarkers by ELISA.
We found that mouth microbiota of patients with DLBCL responding to the therapy (DLBCL-R) was characterized by significant reduction of genus Streptococcus compared to the non-responders (DLBCL-NR). In the fecal microbiota of the responders to the therapy, we observed significant enrichment of Faecalibacterium and reduction of Akkermansia in patients with LIPS (LIPS-R) whereas Methanobrevibacter decline specifically characterized DLBCL-R. In the analysis of serum biomarkers, we observed lower levels of i-FABP (p=0.081), MBL (p=0.092) and soluble CD14 (p=0.011) in DLBCL-R when compared to the DLBCL-NR. The principal component analysis evaluating the contribution of all measured factors showed that DLBCL-R were associated with higher abundance of Lactobacillus and higher levels of S100A8/9 whereas DLBCL-NR showed association with the increased abundance of Veillonella. Higher abundance of Lachnoclostridium, Bacteroides and Subdoligranulum and increased production of IL-18 characterized LIPS-R. On the other hand, higher abundance of Escherichia/Shigella was linked to non-responders among the patients with LIPS.
Our results suggest that gut microbiota composition influences the integrity of the gut barrier in patients with lymphoma and together may affect their response to the therapy. The effect of microbiota is more pronounced in patients with DLBCL.
Supported by the Ministry of Health of the Czech Republic in cooperation with the Czech Health Research Council under project No. NU22-03-00370.

Gut microbiota protects mice from acute colitis by improving intestinal barrier function

Michal Kraus ^1,3, Zuzana Jacková ^1,2, Eliška Krčmářová ^2,3, Štěpán Coufal ¹, Tomas Thon ^1,2, Lenka Súkeníková ^2,3, Jiří Hrdý ³, and Miloslav Kverka ¹

1 Czech Academy of Sciences, Institute of Microbiology, Vídeňská 1083, 142 20 Prague 4, Czech Republic
2 Charles University, Faculty of Science, Albertov 6, 128 00 Prague 2, Czech Republic
3 Charles University, First Faculty of Medicine, Kateřinská 32, 121 08 Prague 2, Czech Republic

The gut microbiota drives inflammation by shaping the function of the intestinal barrier and stimulating the immune system of intestinal mucosa. Our aim was to analyze the mechanisms by which microbiotas from two different animal facilities influence the development of acute experimental colitis.

We induced acute colitis in conventional BALB/c mice originating from the animal facility of the Institute of Microbiology (IMIC) or the First Faculty of Medicine (FoM), and in ex-germ-free mice colonized with intestinal contents from either facility. Colitis was induced by intrarectal administration of 2,4,6-trinitrobenzenesulfonic acid (TNBS) dissolved in 50% ethanol, with (hapten-type) or without (toxic-type colitis) skin presensitization. The severity of colitis was assessed using Wallace's score and colon length measurement. Intestinal microbiota was analyzed by 16S rRNA sequencing, gut barrier function was evaluated using a fluorescein isothiocyanate dextran (FITC-Dx) permeability assay, and immune system activity was measured by flow cytometry in cells from the spleen and mesenteric lymph nodes.

IMIC mice were significantly more resistant to both types of TNBS colitis and had a higher abundance of bacteria from the Verrucomicrobiae and Campylobacteria classes in their feces. IMIC mice also had a significantly less permeable gut barrier, and significantly fewer effector memory T cells and lower percentages of IFNγ-, IL-17- and TNFα-producing CD4+ T cells in both the spleen and mesenteric lymph nodes even before the colitis induction. While microbial consortia in the colonized ex-germ-free mice resembled those of the conventional mice, there were no significant differences in either sensitivity to colitis or T cells subsets.

We conclude that early-life colonization with members of the Verrucomicrobiae and Campylobacteria classes promotes development of the gut barrier and reduces immune system reactivity, resulting in resistance to acute intestinal inflammation in mice.

Impact of Antidepressants on Beneficial Gut Bacteria: Insights from In Vitro Studies

Tiziana Maria Mahayri ¹, Kateřina Olša Fliegerová ¹, Lea Jakob ^2,3, Kristina Hakenová ^2,3, Isis Koutrouli ^2,3, Jiří Killer ¹, Filip Španiel ^2,3, Tomáš Paleníček ^2,3

1 Institute of Animal Physiology and Genetics, Czech Academy of Sciences, Prague, Czech Republic

2 National Institute of Mental Health, Klecany, Czech Republic

3 Charles University, Third Faculty of Medicine, Prague, Czech Republic

Major depressive disorder is a common mental health condition primarily treated with antidepressants; however, their impact on the gut microbiome remains poorly understood. This study aimed to examine direct drug-bacteria interactions between an antidepressant and beneficial gut bacteria, and to determine whether probiotic supplementation should be considered alongside antidepressant use.
Bacterial strains of Lactiplantibacillus and Bifidobacterium were cultured on TPY agar under anaerobic conditions (three replicates per strain). A central well was created in each plate. Wells were filled with: (1) vortioxetine (Brintellix) at 800 μg/mL diluted in phosphate buffer, (2) phosphate buffer (negative control), or (3) an antibiotic mixture (positive control). Plates were incubated at 37°C for 24 hours. Inhibition zone diameters were measured as twice the radius from the well edge to the edge of the clear zone (excluding the well), using millimeter-scale paper strips. The Kruskal–Wallis test was used to compare results (P < 0.05).
Results demonstrated variable inhibition effects ranging from strong to none. Bifidobacterium species showed greater inhibition than Lactiplantibacillus, with Bifidobacterium bifidum CNN 3762 exhibiting a mean inhibition zone of 12 mm (P = 0.02) and Bifidobacterium pseudolongum DSMZ 2073 showing 14 mm (P = 0.02). In contrast, Lactiplantibacillus bulgaricus and Lactobacillus rhamnosus exhibited smaller zones (4.7 mm, P = 0.03 and 3.3 mm, P = 0.03, respectively). Several strains, including Lactiplantibacillus plantarum b5 (2) L 189/87 and Bifidobacterium dentinum DSM 20436T, displayed no inhibition.

RORγt⁺ Regulatory T Cells Form a Clonally Stable Compartment Suggesting a Long-term Footprint of the Primordial Microbiota

David Machač ^1,2,3, Martin Schwarzer ³, Domink Filipp ¹

1. Laboratory of Immunobiology, Institute of Molecular Genetics of the Czech Academy of Sciences, Prague, Czech Republic,
2. Charles University, Faculty of Science, Department of Cell Biology, Prague, Czech Republic,
3. Laboratory of Gnotobiology, Institute of Microbiology of the Czech Academy of Sciences, Nový Hrádek, Czech Republic

A balanced immune system relies on regulatory T cells (Tregs) to prevent excessive immune responses. While thymus-derived Tregs are well known, a unique subset of microbiota-specific, peripherally induced RORγt⁺ Tregs has emerged as a critical regulator of intestinal immunity and the crosstalk with microbial communities. Our preliminary data reveal that RORγt⁺ Tregs originate in mesenteric lymph nodes during early-life microbiota colonization and, remarkably, can persist long-term as a clonally stable population even in the absence of microbial antigens. Moreover, once established, these RORγt⁺ Tregs suppress the induction of newly arising counterparts. This suggests that these cells function as an immunological “memory,” safeguarding beneficial primordial microbiota and offering a novel paradigm for gut immune homeostasis. Furthermore, we have identified a previously unrecognized surface marker, Sca-1, which enables precise tracking and characterization of this memory subset. Together, our findings offer a novel perspective on RORγt⁺ Treg developmental dynamics and suggest that their role in the crosstalk between the immune system and microbiota is more important for shaping microbial composition than previously expected.

Gut Microbiome and Personality and Social Decision-Making

Martyna Malcher, Natalia Wichrowska, Wiktoria Manowska, Karolina Reysowska, Julia Kwoczek & Jurand Sobiecki

The Fahrenheit Union of Universities in Gdańsk, Poland

Emerging research into the gut–brain axis consistently demonstrates that the gut microbiome plays a key role in shaping human cognition, emotion, and behavior. Drawing on recent studies that link microbiome composition to personality traits and social decision-making, our presentation offers an integrative overview of these developments while presenting our novel survey data on gut health habits. Previous investigations have revealed that variations in microbial diversity and specific bacterial taxa are associated with social traits, including sociability and fairness sensitivity, as well as with overall personality dimensions such as neuroticism and extraversion. Complementarily, experimental work has indicated that targeted dietary interventions using pre- and probiotic supplements can modulate gut microbiota, subsequently influencing socio-affective behaviors such as altruistic punishment in economic decision-making tasks. In our presentation, we build on these interdisciplinary insights by not only reviewing the current literature but also incorporating primary survey data. Our survey assessed a range of habits related to gut microbiome health, including dietary patterns, consumption of fermented foods and probiotic supplements, and self-reported gut health symptoms. By merging literature on gut microbiome influences on personality and social decision-making with empirical data on everyday gut health habits, our presentation proposes an integrative framework for understanding how nutrition and microbial ecology together contribute to mental well-being and social behavior. This holistic approach not only underscores the bidirectional nature of the gut–brain dialogue but also highlights potential avenues for personalized, behavior-based interventions aimed at enhancing both gut and mental health.

Herring roe oil exerts anti-psoriatic and immunomodulatory effects on the IL-17/23 signaling axis in immune and skin cells and promotes SPM biosynthesis

Jennifer Mildenberger¹, T. Ringheim-Bakka³, A. Saliani⁴, Nina Therese Solberg², Tone-Kari K. Østbye², Vibeke Høst², Federico Petrucelli³, M. Busygina³, Runhild Gammelsæter³, J. Dalli⁴, Sissel B. Rønning², Mona E. Pedersen²

1 Møreforsking AS, Borgundveien 340, 6009 Ålesund, Norway
2 Nofima AS, Osloveien 1, 1433 Ås, Norway
3 Arctic Bioscience AS, Industrivegen 42, 6155 Ørsta, Norway
4 William Harvey Research Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, Charterhouse Square, London. UK. EC1M 6BQ.

Immune-mediated inflammatory disease (IMID) is a major public health issue which can affect a number of organs with a profound impact on life quality. Psoriasis is a chronic IMID of the skin which presents with both local and systemic inflammation. Several systemic treatment options are available for moderate to severe psoriasis, while low-risk oral treatments for milder forms are limited. Herring roe oil (HRO) has previously shown beneficial effects on the clinical presentation of psoriasis, however, cell-specific effects remained unknown. We here present results which suggest that HRO exerts effects on different cell types on major psoriasis-driving aspects, reducing the secretion of IL-23 and IL-17 from macrophages and T-cells, respectively, as well as interfering with IL-17-induced signaling and proliferation of keratinocytes in co-culture with fibroblasts. We have also explored the influence of HRO on the biosynthesis of lipid mediators and specialized pro-resolving mediators (SPMs). We observed broad SPM biosynthesis associated with a shift towards a protective and possibly reparative macrophage phenotype as well as promotion of biosynthesis of pro-resolving lipid mediators in the skin cell co-culture, thus demonstrating a possible mechanism for resolution of inflammation in the skin niche using HRO.

TLR9 Deficiency Modulates Microbiota and Tumor Development in a Colitis-Associated Cancer Model Under Different Dietary Regimens

Eliska Pivrncova, Marketa Tenglerova, Stepan Coufal, Miloslav Kverka, Klara Kostovcikova

Institute of Microbiology of the CAS, v. v. i. Videnska 1083, 142 00 Prague 4 – Krc

Toll-like receptor 9 (TLR9) is an innate immune sensor recognizing microbial DNA, and its absence alters host-microbe interactions. In the context of colitis-associated colorectal cancer (CAC), TLR9 expression may modulate inflammatory responses and tumorigenesis. This study investigates how TLR9 deficiency affects tumor development and gut microbiota composition under a standard control diet (CD) or a high-protein diet (HPD).
CAC was induced in wild-type (WT) and TLR9 KO mice using an AOM-DSS protocol, with mice maintained on either CD or HPD. Microbiota analysis was conducted by 16S rRNA Illumina MiSeq instrument followed by microbiome bioinformatics performed by Qiime 2.
TLR9 KO mice fed with HPD developed significantly more macroscopic tumors compared to WT and TLR9 KO mice fed with CD. Furthermore, TLR9 KO mice on CD showed significantly lower production of IFNγ, TNFα, IL-4 and IL-13 in mLN, significantly lower production of IL-4 and IL-13 in spleen and significantly lower production of IL-1β, IL-17 and S100A8 in the colon.
Microbiota analysis revealed significant clustering by disease status and diet (PERMANOVA p=0.001). Alpha diversity was significantly higher in healthy controls than in CAC samples, except for shannon index in TLR9 KO mice on CD, where the values after CAS induction increased. Before CAC induction, TLR9 KO mice on CD showed significantly lower abundances of Parasutterella, Bifidobacterium, Akkermansia, Gastranaerophilus, and HTT002, and higher abundance of Rikenellaceae_RC9_gut_group and Enterococcus compared to HPD (p<0.01). After induction, Alloprevotella and Rikenellaceae_RC9_gut_group decreased on CD, while significant increase was observed in Escherichia/Shigella in CD and HPD-fed TLR9 KO mice (p<0.01).
Our findings suggest that components of HPD interact with host receptors (potentially other TLRs) and, via microbiota shifts, trigger immune pathways that override the absence of TLR9, promoting inflammation and tumor progression.
Supported by Czech Academy of Sciences (Lumina Quaeruntum Program, grant number: LQ200202105).

Mucosal microbiota alterations in primary sclerosis cholangitis persist after liver transplantation and are associated with clinical features independently from geography

Petra Polakovičova ¹, Monika Cahova ¹, Marie Heczkova ¹, Kristian Holm ⁴, Mojmir Hlavaty ², Alena Bohdanecka ¹, Pavel Drastich ², Filip Tichanek ³, Johannes R. Hov ^4,#, Lukas Bajer^2,#

1 Institute for Clinical and Experimental Medicine, Center for Experimental Medicine, Prague
2 Institute for Clinical and Experimental Medicine, Department of Hepatogastroenterology, Prague
3 Institute for Clinical and Experimental Medicine, Department of Data Science, Prague
4 Research Institute of Internal Medicine, Oslo University Hospital Rikshospitalet, Norway

Background and Aim: PSC-associated alteration of gut microbiota has already been described, but the effect of geographic origin has not been addressed yet. We aimed to explore the disease-specific mucosal microbial pattern independent of regional conditions with respect to liver transplantation (LTx), gut inflammation (IBD), and PSC recurrence (rPSC).
Methods: The 16S ribosomal RNA gene (V3–V4) from ileocolonic biopsies was sequenced on 167 PSC (pre_LTx), 105 liver transplanted (post_LTx, 38 of which developed recurrence), and 96 healthy (HC) controls from Norway and the Czech Republic.
Results: Alpha diversity was lower in all PSC groups than in HC. Machine learning models distinguished HC well from pre_LTx (avg_AUC ileum 0.95; colon 0.92) and post_LTx patients (avg_AUC ileum 0.9; colon 0.94), while the separation between pre_LTx and post_LTx (avg_AUC ileum 0.8; colon 0.77) or rPSC vs non-rPSC (avg_AUC colon 0.81) was less precise. The models did not discriminate between PSC patients with and without IBD (avg_AUC ileum 0.64; colon 0.55) or between rPSC and non-rPSC in the ileum (avg_AUC 0.58). The models' performance on merged and region-specific datasets was comparable. The shared region-independent PSC microbiota was dominated by Enterococcus, Pseudomonas, Veillonella, Klebsiella, and Streptococcus, while some common commensals were underrepresented. Microbial Dysbiosis Index negatively correlated with alpha diversity and serum albumin, while a positive correlation was observed with markers of cholestatic disease (ALP, GGT) and liver fibrosis (APRI). rPSC seems to be associated with the dysregulation of a specific subset of colonic microbes.
Conclusions: Our findings revealed a distinct mucosal microbiota composition associated with PSC that partly persists after LTx. It is correlated with the severity of PSC liver injury but not with markers of intestinal inflammation. The altered colon microbiota might contribute to the PSC recurrence.

Non-colonizing Lactococci expressing IL-23 inhibitors modulate experimental colitis in mice

Milan Raska,¹, Leona Raskova Kafkova ¹, Petr Kosztyu ¹, Kristyna Sloupenska ¹, Katerina Zachova ¹, Milan Kuchař ², Natalya Panova ², Jozef Skarda ^3,4, Stephen Walimbwa ¹, Ales Berlec ^5,6, Petr Maly ²

1 Department of Immunology, Faculty of Medicine and Dentistry, Palacky University Olomouc, Hnevotinska 3, 779 00, Olomouc, Czech Republic
2 Laboratory of Ligand Engineering, Institute of Biotechnology of the Czech Academy of Sciences, BIOCEV Research Center, Prumyslova 595, 252 50, Vestec, Czech Republic
3 Department of Pathology, University Hospital Ostrava and Faculty of Medicine, University of Ostrava, 17. Listopadu 1790/5, 70852, Ostrava, Czech Republic
4 Institute of Clinical and Molecular Pathology, Faculty of Medicine and Dentistry, Palacky University Olomouc, Hnevotinska 3, 779 00, Olomouc, Czech Republic
6 Department of Biotechnology, Jožef Stefan Institute, Jamova 39, SI-1000, Ljubljana, Slovenia
7 Faculty of Pharmacy, University of Ljubljana, Askerceva 7, SI-1000, Ljubljana, Slovenia

Background. Interleukin 23 is a crucially involved in intestinal autoimmune disorders. Inhibition of IL-23 signaling has been validated in inflammatory bowel disease (IBD) as a therapeutic target. Here, we tested small binding proteins of non-immunoglobulin origin, inhibiting the IL-23 cytokine (subunit p19) and IL-23 receptor (subunit IL-23R).
Methods. Recombinant Lactococcus bacteria producing IL-23 or IL-23R binders/inhibitors were generated and engineered for oral delivery. In dextran sulfate sodium (DSS) and trinitrobenzene sulfonic acid (TNBS)-induced colitis models, protectivity of IL-23 and IL-23R binders expressing Lactococci was tested. Colon histology, cell infiltration, local and systemic inflammatory cytokines were analyzed.
Results. Statistical analyses of DSS colitis showed reductions in disease activity index, colon shortening, mucosal inflammatory cell infiltration when selected IL-23 binder was orally delivered by L. lactis bacteria. The protective effect of IL-23 binder expressing L. lactis was even more pronounced in TNBS colitis.
Interpretation. Oral administration of non-colonizing bacteria L. lactis expressing IL-23p19 inhibitor is a promising strategy for developing novel non-invasive IBD-modifying therapy.

Immune Response to Microbial Transglutaminase and Cross-Reactivity to Tissue Transglutaminase in a Murine Model

Maliha Rizwan, Michal Kraus, Veronika Motuzova, Janaina L S Donadio, Stepan Coufal, Miloslav Kverka

Laboratory of Cellular and Molecular Immunology, Institute of Microbiology of the Czech Academy of Sciences, Prague, Czech Republic

While microbial transglutaminase (mTG) is widely used in the food industry due to its ability to modify proteins to improve food quality, tissue transglutaminase (tTG) is a known autoantigen in celiac disease (CD). Despite their structural differences, both catalyze the modification of gluten. Here we aim to study the differences in immune response to them mTG and tTG and even whether we can induce cross-reactivity from mTG to tTG.
We immunized C57BL/6 mice subcutaneously with mTG from Streptoverticillium mobaranese with CFA/IFA. One month after immunization, we analyzed the antibody response to mTG and tTG from serum samples by ELISA and T cell response to these antigens from cells isolated from mesenteric lymph nodes by multicolor flow cytometry. We also cultured excised Payer’s patches from parenterally immunized mice to analyze changes in cytokine production.
We detected a high anti-mTG IgG response and also anti-tTG IgM in mouse sera of immunized mice. Immunization increased proportions of CD4^⁺CD44^⁺, CD4^⁺CD69^⁺, CD4^⁺CD122^⁺, and CD4^⁺ CD25^⁺, T cells population. Interestingly parenteral immunization increased IL-10 in Payer’s patches of the immunized mice.
Subcutaneous immunization with mTG induced an antibody response to tTG, triggered a stronger regulatory response in the inductive site of mucosal immunity, and brought T cells to a more mature state.

Gut Microbiota from Anorexia Nervosa Donors Exacerbates Disease Progression in a Mouse Model

Radka Roubalová¹, Petra Procházková¹, Janet Ježková^1,2, Kristýna Coufalová^1,2, Kateřina Zadáková^1,2, Hana Papežová³

1 Laboratory of Cellular and Molecular Immunology, Institute of Microbiology of the Czech Academy of Sciences, Prague, Czechia
2 First Faculty of Medicine, Charles University, Prague, Czechia
3 Department of Psychiatry, First Faculty of Medicine, Charles University and General University Hospital in Prague, Prague, Czechia

Patients suffering from the eating disorder anorexia nervosa (AN) are characterized by very low body weight, an intense fear of weight gain, a distorted body image, and often exhibit hyperactivity. Previously, we observed the gut microbiome dysbiosis in these patients with a reduced abundance of short-chain fatty acid (SCFA)-producing bacterial taxa. Correspondingly, the levels of dominant SCFAs acetate, propionate, and butyrate were reduced in their stool samples. Additionally, the levels of various neurotransmitters (dopamine, serotonin, GABA) were significantly lower in the stool of patients with AN than in healthy controls.
To investigate the role of the microbiome in appetite regulation, we used the activity-based anorexia (ABA) model - a rodent model that integrates two key features of AN: food restriction and hyperactivity. We transplanted fecal microbiota from healthy controls and from patients with either acute or chronic AN into mice. Mice receiving microbiota from AN patients exhibited reduced expression of the orexigenic (hunger-signaling) peptides neuropeptide Y (NPY) and agouti-related peptide (AgRP), which are produced by GABAergic neurons in the hypothalamus. Furthermore, mice transplanted with microbiota from acute AN patients showed increased expression of certain GABA receptors that inhibit the satiety pathway. This may serve as a compensatory mechanism for reduced GABA levels in the brain, which could result in insufficient inhibition of the satiety pathway. Accordingly, we observed reduced food intake in mice transplanted with the microbiota from patients with acute AN compared to mice with microbiota from chronic patients and healthy controls. This mirrors the nature of the disease, where patients in an acute stage of illness usually restrict their food, while chronic patients suffer to a greater extent from purgative behavior, with episodes of overeating followed by compensatory behavior (vomiting, laxative abuse).

Potential substitutes for wheat gluten in a gluten–free diet – Chlorella, oats and potatoes may induce IgE–sensitization in celiac patients

Iva Hoffmanová¹, Věra Hábová², Adéla Sánchez Szczepanková^2,3, Helena Tlaskalová–Hogenová², Daniel Sánchez²

1 Department of Internal Medicine, Second Faculty of Medicine, Charles University and University Hospital in Motol, Prague, Czech Republic,

2 Laboratory of Cellular and Molecular Immunology, Institute of Microbiology of the Czech Academy of Sciences, Prague, Czech Republic

3 First Faculty of Medicine, Charles University, Prague, Czech Republic

The expanding incidence of celiac disease (CLD) requires an increase in the quantity and quality of gluten–free foods, which remain the only lifelong treatment for patients with CLD, and a gluten-free diet (GFD) is also used in the treatment of non-celiac gluten sensitivity and wheat allergy. The selection of new gluten–free foodstuffs is complicated by the increasing incidence of food allergies (FA) and their coincidence with CLD. We hypothesized that IgE–sensitization of CLD patients to wheat foodstuff alternatives might occur. For this reason, we tested IgE antibodies (Ab) against Chlorella sorokiniana, oats, and potatoes (nutritionally and technologically suitable for replacing wheat in a GFD and improving its quality) in patients with CLD, patients with FA, and healthy controls (88 individuals) using Western blot. IgE Ab against Chlorella proteins were found in 10 out of 22 patients with active CLD and 7/22 CLD patients on a GFD; IgE Ab reactivity against potatoes and oats was low in patients with active CLD (4/22 and 2/22, respectively) and similar to controls. In patients with FA, IgE Ab against oats were found in 20 out of 22, IgE Ab against Chlorella in 17/ 22, and IgE Ab against potatoes in 13/22. The occurrence of IgE Ab against food proteins is associated with a significant risk of IgE–mediated food allergies and anaphylaxis. This fact should be considered when selecting crops for new, improved gluten–free foods and as a potential reason for incomplete remission in CLD patients on a GFD.

The high levels of beta-defensin 2 reflect the severity of psoriasis and normalises after treatment with IL-17 inhibitors

Tereza Ludvikova ¹, Stepan Coufal ¹, Tomas Thon ¹, Zaneta Slavickova ¹, Michaela Novakova ², Jan Hugo ³, Miloslav Kverka ¹, Helena Tlaskalova-Hogenova ¹, Petr Bohac ², Zuzana Jiraskova Zakostelska ¹, Zuzana Reiss ¹, Filip Rob ²

1 Laboratory of Cellular and Molecular Immunology, Institute of Microbiology of the Czech Academy of Sciences, Czech Republic,
2 Department of Dermatovenerology, Second Faculty of Medicine, Charles University, Bulovka University Hospital, Prague, Czech Republic,
3 Department of Dermatovenereology, Third Faculty of Medicine, Charles University and University Hospital Kralovske Vinohrady, Prague, Czech Republic

Inhibitors of interleukin-17 (IL-17) are in use with high efficacy for treatment of psoriasis. High BMI may influence not only disease severity but also response outcome. The aim of this study was to analyze longitudinal changes in serum levels of chemerin, a pro-inflammatory adipokine, and beta defensin-2 (hBD-2), anti-microbial molecule previously described as a reliable marker of disease activity.
We collected serum samples from 13 healthy controls (HC) and 48 patients before, 1 and 6 months after anti-IL-17 treatment. The severity of psoriasis was assessed using Psoriasis Area and Severity Index (PASI). Levels of chemerin and hBD-2 were quantified by ELISA.
The PASI score decreased significantly after 1 (p<0.0001) and 6 (p=0.002) months, demonstrating the efficacy of treatment. Compared to HC, psoriasis patients have higher levels of hBD-2 (p<0.0001) prior the treatment. During treatment, the level of hBD-2 decreased significantly during the first month from a median value of 6596 pg/ml (IQR 1991.0-13888.0) to 62 pg/ml (IQR 8.5-632.6, p<0.0001) and remained stable after six months of treatment (median value 80 pg/ml, IQR 23.5- 534.8). We found no changes in chemerin serum levels between psoriasis patients and HC, as well chemerin levels during treatment were similar.
Patients with psoriasis have higher serum level of hBD-2, main decrease was within first month and normalizes during treatment. Our observation confirms association of hBD-2 with psoriasis severity and treatment outcome. We did not find any significant relation between hBD-2 and chemerin serum level during IL-17 treatment. In next step we will analyze also the changes in the skin and gut microbes during the IL-17 treatment.
This study was supported by Ministry of Health of the Czech Republic grant no. NU22J-05-00056 and grant no. NW24-06-00509.

Synbiotic Intervention for Better Relaxation and Cognitive Performance

Jurand Sobiecki, Wiktoria Manowska, Oliwia Kosecka, Zofia Kujawińska & Marianna Szczepaniak

Institute of Psychology, University of Gdańsk, Poland

Recent advances in gut–brain axis research highlight the gut microbiome as a key factor influencing mental well-being, emotional regulation, and social functioning. This proposed interdisciplinary project builds on previous initiatives conducted within the Biological Basis of Well-Being Centre initiative, extending the scope of prior SEA-EU Alliance (Stress Relief Initiative) and Fahrenheit Universities (Gutt Microbiome and Psychosocial Functioning)-funded projects. The planned study investigates the effects of a 7-week synbiotic intervention—combining selected probiotic strains with prebiotics—on cognitive performance, relaxation, body composition, and self-reported well-being. Using a placebo-controlled design, the study integrates neurophysiological, cognitive, and physical (body composition analysis) measurements. Relaxation is assessed during massage chair sessions, while cognitive engagement is tested through tasks requiring sustained attention and rapid decision-making. The project aims to determine whether daily synbiotic supplementation enhances neurocognitive functioning and physiological recovery. This integrative approach captures multidimensional outcomes across neural, cognitive, and somatic domains, offering novel insights into the psychophysiological benefits of gut microbiota modulation. In the long term, this research seeks to inform the development of scalable, low-cost, and evidence-based interventions aimed at enhancing mental and physical health. Findings from this study could guide future public health strategies, academic well-being programs, and therapeutic approaches addressing stress, burnout, and lifestyle-related cognitive decline in both clinical and non-clinical populations.

Pathways Between Gut Microbiome and Well-Being: Systematic Review and Meta-Analysis on Inflammation and Depression

Martyna Szyperska, Iwona Ulenberg, Wiktoria Stickel, Martyna Malcher & Jurand Sobiecki

The Fahrenheit Union of Universities in Gdańsk, Poland

The gut microbiome is emerging as a pivotal factor in human health and behavior, with growing evidence implicating its role in modulating inflammatory responses that may contribute to depressive symptomatology. However, a comprehensive and systematic synthesis of the literature exploring the association between inflammatory markers and depression has yet to be undertaken. This presentation introduces our systematic review and meta-analysis—designed following PRISMA guidelines—to broadly examine the relationship between key inflammatory markers and depressive symptoms across diverse populations and study designs. Our analysis aggregates evidence from research that has investigated cytokines such as interleukin-6, tumor necrosis factor-alpha, and interleukin-1β in relation to mood disturbances. By incorporating data from studies with varying methodological frameworks, we aimed to evaluate not only the strength and consistency of the inflammation–depression link but also to probe the potential directionality and underlying mechanisms of this association. This approach enables us to address critical questions regarding whether systemic inflammation acts as a precipitating factor for depression, a secondary consequence, or part of a bidirectional interplay. The presentation will outline our methodology, including study selection criteria, data extraction procedures, and statistical techniques to manage heterogeneity among studies. We will discuss potential confounding factors and the importance of standardized biomarker assessment protocols that could enhance future research in this field. Finally, implications for integrative mental health interventions will be highlighted, considering how targeting inflammatory pathways and optimizing gut microbiome health might contribute to novel therapeutic strategies for depression. This overview invites dialogue and interdisciplinary collaboration as we seek to refine our approach and ultimately provide a more robust understanding of the psychoneuroimmune interplay underpinning depressive disorders.

The fate of hyaluronan administered into the intestinal loop of germ-free mice.

Romana Šínová¹, Matěj Šimek¹, Martin Schwarzer², Dagmar Šrůtková², Tomáš Hudcovic², Kristina Nešporová¹, Vladimír Velebný¹

1 Contipro a.s., Dolní Dobrouč 401, 56102, Dolní Dobrouč, Czech Republic
2 Institute of Microbiology of the Czech Academy of Sciences, Nový Hrádek, Czech Republic

The beneficial effects of hyaluronan (HA) as a dietary supplement have been shown in several studies; however, the exact biological mechanism remains unclear. HA is cleaved by the gut microbiome into unsaturated oligosaccharides (∆oHA), which become bioavailable and penetrate the intestinal epithelium and central circulation. The Mw of HA shifted from 1600 kDa to approximately 150–600 kDa in the stomach and small intestine (1). We investigated the intestinal absorption of HA of defined Mw, with a particular focus on its uptake by goblet cells, M cells within Peyer’s patches, and Paneth cells in the small intestine, as well as its potential translocation into the systemic circulation.
Material & Methods: Different Mw of 13C-HA and biotin-HA were injected into the intestinal loop with Peyer's patches of germ-free mice. The concentration of 13C-HA in the plasma and organs was determined by LC-MS. The localization of biotin-labeled HA in the small intestine and its colocalization with the goblet cells, Paneth cells, and M-cells were visualized using histofluorescence and confocal microscopy.
Results: The absorption of biotin-labeled ∆oHA and MMW HA by intestinal epithelium was confirmed. ∆oHA exhibits a diffuse distribution throughout the cross-section of the small intestine, with limited colocalization observed in Paneth cells. In contrast, MMW HA shows distinct colocalization with goblet cells and M cells in the Peyer's patches. 13C-HA (∆oHA, MMW, HMW) can translocate into the plasma, mesenteric nodes, spleen, liver, and kidney.
Discussion: Our data suggest that HA of various Mw may penetrate through the small intestine barrier, potentially mediated by M-cells and goblet cells with (GAPs) Goblet cell-associated antigen passages.
Reference: Šimek M. et al., Molecular weight and gut microbiota determine the bioavailability of orally administered hyaluronic acid. Carbohydr Polym. 2023 Aug 1;313:120880. doi: 10.1016/j.carbpol.2023.120880.

T-cell profiles and serum biomarkers differ in ameliorating and deteriorating atopic dermatitis in paediatric patients with food allergy within the first two years of life

Tomas Thon¹ and Eliska Kopelentova², Dagmar Srutkova³, Stepan Coufal¹, Miloslav Kverka¹, Stepanka Capkova⁴, Jana Cadova⁴, Anna Sediva², Helena Tlaskalova-Hogenova¹, Zuzana Jiraskova Zakostelska¹ and Andrea Polouckova²

1 Laboratory of Cellular and Molecular Immunology, Institute of Microbiology of the Czech Academy of Sciences, Prague, Czech Republic.
2 Department of Immunology, 2nd Faculty of Medicine, Charles University and Motol University Hospital, Prague, Czech Republic.
3 Laboratory of Gnotobiology, Institute of Microbiology of the Czech Academy of Sciences, Novy Hradek, Czech Republic.
4 Department of Paediatric Dermatology, Motol University Hospital, Prague, Czech Republic

Introduction: Atopic dermatitis (AD) is a chronic inflammatory skin disorder frequently associated with food allergy (FA). There is a growing need to define disease endotypes for more targeted therapeutic strategies due to high heterogeneity of AD and FA.
Here, we perform an analysis of serum markers and T cell populations to identify the differences in immune responses of patients with AD and FA who exhibited amelioration or deterioration of AD.
Methods: We collected serum and PBMCs from 107 children with AD and FA within the first year of live and 1 year after. AD severity was assessed using clinical score. Serum markers related to epithelial damage (E-FABP, I-FABP, L-FABP), immune regulation (CCL17, CCL11, IL-18), and microbial translocation (ASCA IgA, ASCA IgG, LBP) were measured by ELISA. T cell subsets were characterized by flow cytometry.
Results: We found that ameliorating patients showed reduced levels of E-FABP, I-FABP, IL-18, and LBP after 1 year. Independent of disease progression, all AD patients with FA exhibited decreased levels of L-FABP and CCL17, and increased levels of ASCA IgA, ASCA IgG, and CCL11 after 1 year.
T cell analysis showed a reduction in gut-homing Tregs and cytotoxic T cells in ameliorating patients at the second sampling, with no changes in deteriorating patients. Skin-homing Tregs did not change in any of the groups, while skin-homing cytotoxic T cells increased only in deteriorating patients.
Conclusion: We have described the unique signatures associated with disease improvement in children with AD. The result could serve as part of the criteria for distinguishing disease endotypes.
This study was supported by the Ministry of Health of the Czech Republic grant no. NU20-05-00038; by the Academy of Sciences of the Czech Republic (LQ200202105) and by the Ministry of Education, Youth and Sports of the Czech Republic (grant number CZ.02.01.01/00/22_008/0004597).

Immunostimulatory Potential of Staphylococcus aureus-Derived Vesicles in the Context of Infective Endocarditis

Kristyna Turkova^1,2,³, Veronika Mlcekova^1,2, Miriam Sandanusova^1,2, Hana Obrucova^4,5, Tomas Freiberger^4,5, Julia Orlovska ^1,2, Lukas Kubala ^1,2,³, Gabriela Ambrozova²

1 Department of Experimental Biology, Faculty of Science, Masaryk University, Brno, Czech Republic
2 Institute of Biophysics, Academy of Sciences of the Czech Republic, Brno, Czech Republic
3 International Clinical Research Center, St. Anne´s University Hospital, Brno, Czech Republic
4 Department of Clinical Immunology and Allergology, Faculty of Medicine, Masaryk University Brno, Czech Republic
5 Centre of Cardiovascular and Transplantation Surgery, Brno, Czech Republic

Infective endocarditis (IE) is a severe inflammatory disease of the heart's inner lining, primarily caused by gram-positive bacteria such as Streptococcus, Staphylococcus, and Enterococcus, accounting for 80–90% of cases. Recent research has highlighted the role of bacterial extracellular vesicles (bEVs) as potential contributors to bacterial virulence and immune modulation.

This study focuses on bEVs derived from a clinical strain of Staphylococcus aureus 3.496, characterized by whole genome sequencing using Oxford Nanopore Technologies. bEVs were isolated through a series of centrifugation, filtration, ultracentrifugation, and sucrose cushion purification steps. Comprehensive characterization was conducted using nanoparticle tracking analysis (NTA), cryo-electron microscopy (Cryo-EM), FTIR spectroscopy, Western blotting, and protein quantification. Functional assays revealed that stimulation of RAW264.7 macrophages with purified bEVs significantly increased the production of proinflammatory mediators, including TNFα, IL-6, nitric oxide, and inducible nitric oxide synthase (iNOS). The vesicles, approximately 80 nm in diameter with a lipid bilayer structure, triggered a pronounced inflammatory response, suggesting their role in host immune activation. These findings support the involvement of Staphylococcus aureus-derived bEVs in the pathogenesis of IE, with ongoing proteomic analyses aimed at identifying associated virulence factors within the vesicular cargo.

The work was supported by project National Institute for Research of Metabolic and Cardiovascular Diseases (Programme EXCELES, Project No. LX22NPO5104) - Funded by the European Union – Next Generation EU.

Lactiplantibacillus plantarum WJL promotes the growth of postnatal milk-fed germ-free piglets in a strain-dependent manner

Barbora Valášková ¹, Dagmar Šrůtková ¹, Tereza Švábová ¹, Tereza Novotná ¹, Anna Jelínková ¹, Gautam Umesh Kumar¹, Kateřina Štěpánová ¹, Sudhanshu Shekhar ¹, Marek Šinkora ¹, Schwarzer Martin ¹

1 Laboratory of Gnotobiology, IMIC CAS, Nový Hrádek, Czech Republic

Introduction
Our group has previously shown that the specific strain Lactoplantibacillus plantarum (Lp) WJL can promote the growth of malnourished Drosophila larvae and juvenile gnotobiotic and conventional mice in a strain-specific manner (1, 2). Due to its anatomical, physiological and genetic similarity to humans, the pig is often used as a translational animal model in biomedical research. We therefore investigated the ability of LpWJL to promote postnatal growth of gnotobiotic piglets.
Materials and methods
Germ-free (GF) minipigs (Animal Research Institute, Kostelec nad Orlicí, Czech Republic) were obtained by hysterectomy on gestation day 112. Piglets were reared in fiberglass isolators with a heated floor and were bottle-fed autoclaved cow’s milk-based diet (Mlékárna Hlinsko, Czech Republic) 6–7 times a day. Food intake was recorded. On day 4 of life, piglets were colonized with either LpWJL or strain LpNIZO2877, and the degree of colonization in feces was checked regularly. Body weight and length were measured daily until sacrifice on day 15. Serum levels of insulin-like growth factor 1 (IGF-1) were measured by ELISA.
Results
Preliminary data show that both LpWJL and LpNIZO2877 successfully colonized the milk-fed piglets. Compared to GF- and LpNIZO2877-colonized animals, piglets colonized with LpWJL showed improved growth and significantly higher serum IGF-1 levels, which is consistent with our previous results in the Drosophila and mouse models.
This work was supported by the Czech Science Foundation grant number 21-19640M.
References:
(1) Schwarzer M et al: Lactobacillus plantarum strain maintains growth of infant mice during chronic undernutrition. Science 2016, doi: 10.1126/science.aad8588
(2) Schwarzer M et al: Microbe-mediated intestinal NOD2 stimulation improves linear growth of undernourished infant mice. Science 2023, doi: 10.1126/science.ade9767

Composition of the skin microbiota and immunoregulatory responses distinguish IgE and non-IgE cow’s milk allergy in patients with atopic dermatitis

Tomas Thon¹, Eliska Kopelentova², Dagmar Srutkova³, Stepan Coufal¹, Jakub Kreisinger⁴, Filip Rob⁵, Zuzana Reiss¹, Miloslav Kverka¹, Stepanka Capkova⁶, Lucie Bulantova⁷, Vojtech Bystry⁷, Martin Schwarzer³, Anna Sediva², Helena Tlaskalova-Hogenova¹, Zuzana Jiraskova Zakostelska^1* and Andrea Polouckova²

1 Laboratory of Cellular and Molecular Immunology, Institute of Microbiology of the Czech Academy of Sciences, Prague, Czech Republic.
2 Department of Immunology, 2nd Faculty of Medicine, Charles University and Motol University Hospital, Prague, Czech Republic.
3 Laboratory of Gnotobiology, Institute of Microbiology of the Czech Academy of Sciences, Novy Hradek, Czech Republic.
4 Laboratory of Animal Evolutionary Biology, Faculty of Science, Department of Zoology, Charles University, Prague, Czech Republic.
5 Department of Dermatovenerology, Second Faculty of Medicine, Charles University, Prague, Czech Republic.
6 Department of Paediatric Dermatology, Motol University Hospital, Prague, Czech Republic 7CEITEC-Central European Institute of Technology, Masaryk University, Brno, Czech Republic.

The increasing incidence of infants with early-onset atopic dermatitis (AD) associated with food allergy (FA) is a serious problem worldwide. A characterisation of AD and FA endotypes has become essential for the development of personalised treatments, as each endotype may respond differently to immunomodulatory therapies.Therefore, we investigated markers associated with changes in the microbiota and immune responses within the gut-skin axis of cow's milk allergy (CMA) patients with AD to characterise the course of the diseases and distinguish between IgE and non-IgE CMA endotypes. We report that the skin microbiota of patients with IgE CMA differs greatly from healthy controls (HC) and from patients with non-IgE CMA, despite similar AD severity. Far less pronounced than in the composition of the skin microbiota, we observed changes in composition of bacteria and fungi in gut of both allergy groups compared to HC. Further, E-FABP, a marker of epithelial barrier integrity, was elevated in patients with IgE CMA compared to non-IgE CMA patients. Regarding the immune response to bacteria, we found a significant increase in CD14 levels in patients with non-IgE CMA compared to IgE CMA patients. Additionally, patients with non-IgE CMA have more T-regulatory lymphocytes in peripheral blood mononuclear cells that migrate into the intestine than patients with IgE CMA. There were no significant differences between the groups in the frequency of T-regulatory lymphocytes migrating into the skin. Myeloid dendritic cells in non-IgE CMA patients showed increased CLEC7A+CD209+ expression after zymosan stimulation, indicating an altered fungal response. These findings provide insights into the complex interplay between the damaged epithelial barrier, gut and skin microbiome, and immune responses in AD patients with different types of FA. This study was supported by the Ministry of Health of the Czech Republic grant no. NU20-05-00038; Youth and Sports of the Czech Republic (grant number CZ.02.01.01/00/22_008/0004597).