Mucosal fungi promote gut barrier function and social behavior via Type 17 immunity.

Fungal communities (the mycobiota) are an integral part of the gut microbiota, and the disruption of their integrity contributes to local and gut-distal pathologies. Yet, the mechanisms by which intestinal fungi promote homeostasis remain unclear. We characterized the mycobiota biogeography along the gastrointestinal tract and identified a subset of fungi associated with the intestinal mucosa of mice and humans. Mucosa-associated fungi (MAF) reinforced intestinal epithelial function and protected mice against intestinal injury and bacterial infection. Notably, intestinal colonization with a defined consortium of MAF promoted social behavior in mice. The gut-local effects on barrier function were dependent on IL-22 production by CD4+ T helper cells, whereas the effects on social behavior were mediated through IL-17R-dependent signaling in neurons. Thus, the spatial organization of the gut mycobiota is associated with host-protective immunity and epithelial barrier function and might be a driver of the neuroimmune modulation of mouse behavior through complementary Type 17 immune mechanisms.

Human gut mycobiota tune immunity via CARD9-dependent induction of anti-fungal IgG antibodies.

Antibodies mediate natural and vaccine-induced immunity against viral and bacterial pathogens, whereas fungi represent a widespread kingdom of pathogenic species for which neither vaccine nor neutralizing antibody therapies are clinically available. Here, using a multi-kingdom antibody profiling (multiKAP) approach, we explore the human antibody repertoires against gut commensal fungi (mycobiota). We identify species preferentially targeted by systemic antibodies in humans, with Candida albicans being the major inducer of antifungal immunoglobulin G (IgG). Fungal colonization of the gut induces germinal center (GC)-dependent B cell expansion in extraintestinal lymphoid tissues and generates systemic antibodies that confer protection against disseminated C. albicans or C. auris infection. Antifungal IgG production depends on the innate immunity regulator CARD9 and CARD9+CX3CR1+ macrophages. In individuals with invasive candidiasis, loss-of-function mutations in CARD9 are associated with impaired antifungal IgG responses. These results reveal an important role of gut commensal fungi in shaping the human antibody repertoire through CARD9-dependent induction of host-protective antifungal IgG.

Gut Mycobiota in Immunity and Inflammatory Disease.

The mammalian intestine is colonized by a wealth of microorganisms-including bacteria, viruses, protozoa, and fungi-that are all integrated into a functional trans-kingdom community. Characterization of the composition of the fungal community-the mycobiota-has advanced further than the much-needed mechanistic studies. Recent findings have revealed roles for the gut mycobiota in the regulation of host immunity and in the development and progression of human diseases of inflammatory origin. We review these findings here while placing them in the context of the current understanding of the pathways and cellular networks that induce local and systemic immune responses to fungi in the gastrointestinal tract. We discuss gaps in knowledge and argue for the importance of considering bacteria-fungal interactions as we aim to define the roles of mycobiota in immune homeostasis and immune-associated pathologies.

Immune regulation by fungal strain diversity in inflammatory bowel disease.

The fungal microbiota (mycobiota) is an integral part of the complex multikingdom microbial community colonizing the mammalian gastrointestinal tract and has an important role in immune regulation1-6. Although aberrant changes in the mycobiota have been linked to several diseases, including inflammatory bowel disease3-9, it is currently unknown whether fungal species captured by deep sequencing represent living organisms and whether specific fungi have functional consequences for disease development in affected individuals. Here we developed a translational platform for the functional analysis of the mycobiome at the fungal-strain- and patient-specific level. Combining high-resolution mycobiota sequencing, fungal culturomics and genomics, a CRISPR-Cas9-based fungal strain editing system, in vitro functional immunoreactivity assays and in vivo models, this platform enables the examination of host-fungal crosstalk in the human gut. We discovered a rich genetic diversity of opportunistic Candida albicans strains that dominate the colonic mucosa of patients with inflammatory bowel disease. Among these human-gut-derived isolates, strains with high immune-cell-damaging capacity (HD strains) reflect the disease features of individual patients with ulcerative colitis and aggravated intestinal inflammation in vivo through IL-1ß-dependent mechanisms. Niche-specific inflammatory immunity and interleukin-17A-producing T helper cell (TH17 cell) antifungal responses by HD strains in the gut were dependent on the C. albicans-secreted peptide toxin candidalysin during the transition from a benign commensal to a pathobiont state. These findings reveal the strain-specific nature of host-fungal interactions in the human gut and highlight new diagnostic and therapeutic targets for diseases of inflammatory origin.

Sensing Microbial Viability through Bacterial RNA Augments T Follicular Helper Cell and Antibody Responses.

Live vaccines historically afford superior protection, yet the cellular and molecular mechanisms mediating protective immunity remain unclear. Here we found that vaccination of mice with live, but not dead, Gram-negative bacteria heightened follicular T helper cell (Tfh) differentiation, germinal center formation, and protective antibody production through the signaling adaptor TRIF. Complementing the dead vaccine with an innate signature of bacterial viability, bacterial RNA, recapitulated these responses. The interferon (IFN) and inflammasome pathways downstream of TRIF orchestrated Tfh responses extrinsically to B cells and classical dendritic cells. Instead, CX3CR1+CCR2- monocytes instructed Tfh differentiation through interleukin-1ß (IL-1ß), a tightly regulated cytokine secreted upon TRIF-dependent IFN licensing of the inflammasome. Hierarchical production of IFN-ß and IL-1ß dictated Tfh differentiation and elicited the augmented humoral responses characteristic of live vaccines. These findings identify bacterial RNA, an innate signature of microbial viability, as a trigger for Tfh differentiation and suggest new approaches toward vaccine formulations for coordinating augmented Tfh and B cell responses.

Rapid antigen diversification through mitotic recombination in the human malaria parasite Plasmodium falciparum.

Malaria parasites possess the remarkable ability to maintain chronic infections that fail to elicit a protective immune response, characteristics that have stymied vaccine development and cause people living in endemic regions to remain at risk of malaria despite previous exposure to the disease. These traits stem from the tremendous antigenic diversity displayed by parasites circulating in the field. For Plasmodium falciparum, the most virulent of the human malaria parasites, this diversity is exemplified by the variant gene family called var, which encodes the major surface antigen displayed on infected red blood cells (RBCs). This gene family exhibits virtually limitless diversity when var gene repertoires from different parasite isolates are compared. Previous studies indicated that this remarkable genome plasticity results from extensive ectopic recombination between var genes during mitotic replication; however, the molecular mechanisms that direct this process to antigen-encoding loci while the rest of the genome remains relatively stable were not determined. Using targeted DNA double-strand breaks (DSBs) and long-read whole-genome sequencing, we show that a single break within an antigen-encoding region of the genome can result in a cascade of recombination events leading to the generation of multiple chimeric var genes, a process that can greatly accelerate the generation of diversity within this family. We also found that recombinations did not occur randomly, but rather high-probability, specific recombination products were observed repeatedly. These results provide a molecular basis for previously described structured rearrangements that drive diversification of this highly polymorphic gene family.

From Birth and Throughout Life: Fungal Microbiota in Nutrition and Metabolic Health.

The human gastrointestinal tract is home to a vibrant, diverse ecosystem of prokaryotic and eukaryotic microorganisms. The gut fungi (mycobiota) have recently risen to prominence due to their ability to modulate host immunity. Colonization of the gut occurs through a combination of vertical transmission from the maternal mycobiota and environmental and dietary exposure. Data from human and animal studies demonstrate that nutrition strongly affects the mycobiota composition and that changes in the fungal communities can aggravate metabolic diseases. The mechanisms pertaining to the mycobiota's influence on host health, pathology, and resident gastrointestinal communities through intrakingdom, transkingdom, and immune cross talk are beginning to come into focus, setting the stage for a new chapter in microbiota-host interactions. Herein, we examine the inception, maturation, and dietary modulation of gastrointestinal and nutritional fungal communities and inspect their impact on metabolic diseases in humans.

Profound mycobiome differences between segregated mouse colonies do not influence Th17 responses to a newly introduced gut fungal commensal.

Gut mycobiota dysbiosis can negatively impact the outcome of several diseases of inflammatory origin, suggesting a role of the mycobiota in influencing the host immunity. However, it is unknown whether the gut mycobiota composition can create an immune environment that would influence the immune response to a newly introduced intestinal fungus. Using ITS1 deep sequencing, we evaluated the mycobiome structure of C57BL/6J mice acquired from Jackson (JAX) or bred in a controlled environment at a dedicated room in our own mouse facility (WCM-CE) for several generations. We found that C57BL/6J mice from these segregated mouse colonies harbor dramatically different mycobiota. To assess whether the mycobiota make up can influence immune responses to colonization with a fungus foreign to the murine GI tract, we colonized JAX and WCM-CE mice with the human commensal C. albicans and measured Th17 responses in the gut. We found that independent of mycobiota composition, mice produced strong Th17 responses to gastrointestinal C. albicans colonization. Our data suggest that different mouse colonies can carry dramatically different mycobiota. Nevertheless, strong Th17 responses to a newly introduced opportunistic commensal fungus are potently induced independent of the mycobiota background in this experimental setting.

Macrophage interactions with fungi and bacteria in inflammatory bowel disease.

PURPOSE OF REVIEW: In this review, we discuss recent advances into delineating the dual role of intestinal phagocytes in health and during intestinal disease. We further discuss the key role of gut-resident macrophages in recognition of bacterial and fungal microbiota in the gut. RECENT FINDINGS: Inflammatory bowel disease (IBD) commonly manifests with pathologic changes in the composition of gut bacterial and fungal microbiota. Intestinal macrophages are key regulators of the balance between tolerogenic immunity and inflammation. Recent studies have highlighted the role of resident intestinal macrophages in the control of commensal fungi and bacteria in the steady state and during dysbiosis. The dual role of these cells in maintaining intestinal homeostasis and responding to microbiota dysbiosis during inflammation is being increasingly studied. SUMMARY: It is becoming increasingly clear that an aberrant proinflammatory response to microbiota by infiltrating monocytes plays a role in the development of intestinal inflammation. Intestinal mononuclear phagocytes with characteristics of macrophages play an important role in limiting fungal and bacterial overgrowth under these conditions, but can be influenced by the inflammatory environment to further propel inflammation. Better understanding of the interaction of intestinal macrophages with host microbiota including commensal fungi and bacteria, provides an opportunity for the development of more targeted therapies for IBD.

Deficiency of Protein Tyrosine Phosphatase Non-Receptor Type 2 in Intestinal Epithelial Cells Has No Appreciable Impact on Dextran Sulphate Sodium Colitis Severity But Promotes Wound Healing.

BACKGROUND/AIMS: The protein tyrosine phosphatase non-receptor type 2 (PTPN2) is known to mediate susceptibility to inflammatory bowel diseases. Cell culture experiments suggest that PTPN2 influences barrier function, autophagy and secretion of pro-inflammatory cytokines. PTPN2 knockout mice die a few weeks after birth due to systemic inflammation, emphasizing the importance of this phosphatase in inflammatory processes. The aim of this study was to investigate the role of PTPN2 in colon epithelial cells by performing dextran sulphate sodium (DSS)-induced colitis in PTPN2xVilCre mice. METHODS: Acute colitis was induced by administering 2.5 or 2% DSS for 7 days and chronic colitis by 4 cycles of treatment using 1% DSS. Body weight of mice was measured regularly and colonoscopy was done at the end of the experiments. Mice were sacrificed afterwards and colon specimens were obtained for H&E staining. For analysis of wound healing, mechanical wounds were introduced during endoscopy and wound closure assessed by daily colonoscopy. RESULTS: Although colonoscopy and weight development suggested changes in colitis severity, the lack of any influence of PTPN2 deficiency on histological scoring for inflammation severity after acute or chronic DSS colitis indicates that colitis severity is not influenced by epithelial-specific loss of PTPN2. Chronic colitis induced the development of aberrant crypt foci more frequently in PTPN2xVilCre mice compared to their wild type littermates. On the other hand, loss of PTPN2-induced enhanced epithelial cell proliferation and promoted wound closure. CONCLUSIONS: Loss of PTPN2 in intestinal epithelial cells (IECs) has no significant influence on inflammation in DSS colitis. Obviously, loss of PTPN2 in IECs can be compensated in vivo, thereby suppressing a phenotype. This lack of a colitis-phenotype might be due to enhanced epithelial cell proliferation and subsequent increased wound-healing capacity of the epithelial layer.

Oral administration of dextran sodium sulphate induces a caecum-localized colitis in rabbits.

Trichuris suis ova (TSO) have shown promising results in the treatment of inflammatory bowel disease (IBD) but the mechanisms which underlies this therapeutic effect cannot be studied in mice and rats as T. suis fails to colonize the rodent intestine, whilst hatching in humans and rabbits. As a suitable rabbit IBD model is currently not available, we developed a rabbit colitis model by administration of dextran sodium sulphate (DSS). White Himalayan rabbits (n = 12) received 0.1% DSS in the daily water supply for five days. Clinical symptoms were monitored daily, and rabbits were sacrificed at different time points. A genomewide expression analysis was performed with RNA isolated from caecal lamina propria mononuclear cells (LPMC) and intestinal epithelial cells (IEC). The disease activity index of DSS rabbits increased up to 2.1 ± 0.4 (n = 6) at day 10 (controls <0.5). DSS induced a caecum-localized pathology with crypt architectural distortion, stunted villous surface and inflammatory infiltrate in the lamina propria. The histopathology score reached a peak of 14.2 ± 4.9 (n = 4) at day 10 (controls 7.7 ± 0.9, n = 5). Expression profiling revealed an enrichment of IBD-related genes in both LPMC and IEC. Innate inflammatory response, Th17 signalling and chemotaxis were among the pathways affected significantly. We describe a reproducible and reliable rabbit model of DSS colitis. Localization of the inflammation in the caecum and its similarities to IBD make this model particularly suitable to study TSO therapy in vivo.

Beyond the gut.

Mycobiota modulate immunity and behavior.

Mycobiota-induced IgA antibodies regulate fungal commensalism in the gut and are dysregulated in Crohn's disease.

Secretory immunoglobulin A (sIgA) plays an important role in gut barrier protection by shaping the resident microbiota community, restricting the growth of bacterial pathogens and enhancing host protective immunity via immunological exclusion. Here, we found that a portion of the microbiota-driven sIgA response is induced by and directed towards intestinal fungi. Analysis of the human gut mycobiota bound by sIgA revealed a preference for hyphae, a fungal morphotype associated with virulence. Candida albicans was a potent inducer of IgA class-switch recombination among plasma cells, via an interaction dependent on intestinal phagocytes and hyphal programming. Characterization of sIgA affinity and polyreactivity showed that hyphae-associated virulence factors were bound by these antibodies and that sIgA influenced C. albicans morphotypes in the murine gut. Furthermore, an increase in granular hyphal morphologies in patients with Crohn's disease compared with healthy controls correlated with a decrease in antifungal sIgA antibody titre with affinity to two hyphae-associated virulence factors. Thus, in addition to its importance in gut bacterial regulation, sIgA targets the uniquely fungal phenomenon of hyphal formation. Our findings indicate that antifungal sIgA produced in the gut can play a role in regulating intestinal fungal commensalism by coating fungal morphotypes linked to virulence, thereby providing a protective mechanism that might be dysregulated in patients with Crohn's disease.

Fungal Trans-kingdom Dynamics Linked to Responsiveness to Fecal Microbiota Transplantation (FMT) Therapy in Ulcerative Colitis.

Fecal microbiota transplantation (FMT) targeting gut microbiota has recently been successfully applied to ulcerative colitis. However, only a subset of patients responds to FMT, and there is a pressing need for biomarkers of responsiveness. Fungi (the mycobiota) represent a highly immunologically reactive component of the gut microbiota. We analyzed samples from a large randomized controlled trial of FMT for ulcerative colitis (UC). High Candida abundance pre-FMT was associated with a clinical response, whereas decreased Candida abundance post-FMT was indicative of ameliorated disease severity. High pre-FMT Candida was associated with increased bacterial diversity post-FMT, and the presence of genera was linked to FMT responsiveness. Although we detected elevated anti-Candida antibodies in placebo recipients, this increase was abrogated in FMT recipients. Our data suggest that FMT might reduce Candida to contain pro-inflammatory immunity during intestinal disease and highlight the utility of mycobiota-focused approaches to identify FMT responders prior to therapy initiation.

Modulation of the fungal mycobiome is regulated by the chitin-binding receptor FIBCD1.

Host-microbiota interactions are critical in regulating mammalian health and disease. In addition to bacteria, parasites, and viruses, beneficial communities of fungi (the mycobiome) are important modulators of immune- and tissue-homeostasis. Chitin is a major component of the fungal cell wall, and fibrinogen C containing domain 1 (FIBCD1) is a chitin-binding protein; however, the role of this molecule in influencing host-mycobiome interactions in vivo has never been examined. Here, we identify direct binding of FIBCD1 to intestinal-derived fungi and demonstrate that epithelial-specific expression of FIBCD1 results in significantly reduced fungal colonization and amelioration of fungal-driven intestinal inflammation. Collectively, these results identify FIBCD1 as a previously unrecognized microbial pattern recognition receptor through which intestinal epithelial cells can recognize and control fungal colonization, limit fungal dysbiosis, and dampen intestinal inflammation.

Laboratory mice born to wild mice have natural microbiota and model human immune responses.

Laboratory mouse studies are paramount for understanding basic biological phenomena but also have limitations. These include conflicting results caused by divergent microbiota and limited translational research value. To address both shortcomings, we transferred C57BL/6 embryos into wild mice, creating "wildlings." These mice have a natural microbiota and pathogens at all body sites and the tractable genetics of C57BL/6 mice. The bacterial microbiome, mycobiome, and virome of wildlings affect the immune landscape of multiple organs. Their gut microbiota outcompete laboratory microbiota and demonstrate resilience to environmental challenges. Wildlings, but not conventional laboratory mice, phenocopied human immune responses in two preclinical studies. A combined natural microbiota- and pathogen-based model may enhance the reproducibility of biomedical studies and increase the bench-to-bedside safety and success of immunological studies.

Response to Fungal Dysbiosis by Gut-Resident CX3CR1+ Mononuclear Phagocytes Aggravates Allergic Airway Disease.

Sensing of the gut microbiota, including fungi, regulates mucosal immunity. Whether fungal sensing in the gut can influence immunity at other body sites is unknown. Here we show that fluconazole-induced gut fungal dysbiosis has persistent effects on allergic airway disease in a house dust mite challenge model. Mice with a defined community of bacteria, but lacking intestinal fungi were not susceptible to fluconazole-induced dysbiosis, while colonization with a fungal mixture recapitulated the detrimental effects. Gut-resident mononuclear phagocytes (MNPs) expressing the fractalkine receptor CX3CR1 were essential for the effect of gut fungal dysbiosis on peripheral immunity. Depletion of CX3CR1+ MNPs or selective inhibition of Syk signaling downstream of fungal sensing in these cells ameliorated lung allergy. These results indicate that disruption of intestinal fungal communities can have persistent effects on peripheral immunity and aggravate disease severity through fungal sensing by gut-resident CX3CR1+ MNPs.

CX3CR1+ mononuclear phagocytes control immunity to intestinal fungi.

Intestinal fungi are an important component of the microbiota, and recent studies have unveiled their potential in modulating host immune homeostasis and inflammatory disease. Nonetheless, the mechanisms governing immunity to gut fungal communities (mycobiota) remain unknown. We identified CX3CR1+ mononuclear phagocytes (MNPs) as being essential for the initiation of innate and adaptive immune responses to intestinal fungi. CX3CR1+ MNPs express antifungal receptors and activate antifungal responses in a Syk-dependent manner. Genetic ablation of CX3CR1+ MNPs in mice led to changes in gut fungal communities and to severe colitis that was rescued by antifungal treatment. In Crohn's disease patients, a missense mutation in the gene encoding CX3CR1 was identified and found to be associated with impaired antifungal responses. These results unravel a role of CX3CR1+ MNPs in mediating interactions between intestinal mycobiota and host immunity at steady state and during inflammatory disease.

The Proton-activated Receptor GPR4 Modulates Intestinal Inflammation.

BACKGROUND AND AIMS: During active inflammation, intraluminal intestinal pH is decreased in patients with inflammatory bowel disease [IBD]. Acidic pH may play a role in IBD pathophysiology. Recently, proton-sensing G-protein coupled receptors were identified, including GPR4, OGR1 [GPR68], and TDAG8 [GPR65]. We investigated whether GPR4 is involved in intestinal inflammation. METHODS: The role of GPR4 was assessed in murine colitis models by chronic dextran sulphate sodium [DSS] administration and by cross-breeding into an IL-10 deficient background for development of spontaneous colitis. Colitis severity was assessed by body weight, colonoscopy, colon length, histological score, cytokine mRNA expression, and myeloperoxidase [MPO] activity. In the spontaneous Il-10-/- colitis model, the incidence of rectal prolapse and characteristics of lamina propria leukocytes [LPLs] were analysed. RESULTS: Gpr4-/- mice showed reduced body weight loss and histology score after induction of chronic DSS colitis. In Gpr4-/-/Il-10-/- double knock-outs, the onset and progression of rectal prolapse were significantly delayed and mitigated compared with Gpr4+/+/Il-10-/- mice. Double knock-out mice showed lower histology scores, MPO activity, CD4+ T helper cell infiltration, IFN-γ, iNOS, MCP-1 [CCL2], CXCL1, and CXCL2 expression compared with controls. In colon, GPR4 mRNA was detected in endothelial cells, some smooth muscle cells, and some macrophages. CONCLUSIONS: Absence of GPR4 ameliorates colitis in IBD animal models, indicating an important regulatory role in mucosal inflammation, thus providing a new link between tissue pH and the immune system. Therapeutic inhibition of GPR4 may be beneficial for the treatment of IBD.