Microbiota-derived inosine programs protective CD8+ T cell responses against influenza in newborns.

The immunological defects causing susceptibility to severe viral respiratory infections due to early-life dysbiosis remain ill-defined. Here, we show that influenza virus susceptibility in dysbiotic infant mice is caused by CD8+ T cell hyporesponsiveness and diminished persistence as tissue-resident memory cells. We describe a previously unknown role for nuclear factor interleukin 3 (NFIL3) in repression of memory differentiation of CD8+ T cells in dysbiotic mice involving epigenetic regulation of T cell factor 1 (TCF 1) expression. Pulmonary CD8+ T cells from dysbiotic human infants share these transcriptional signatures and functional phenotypes. Mechanistically, intestinal inosine was reduced in dysbiotic human infants and newborn mice, and inosine replacement reversed epigenetic dysregulation of Tcf7 and increased memory differentiation and responsiveness of pulmonary CD8+ T cells. Our data unveils new developmental layers controlling immune cell activation and identifies microbial metabolites that may be used therapeutically in the future to protect at-risk newborns.

Epithelial regulation of microbiota-immune cell dynamics.

The mammalian gastrointestinal tract hosts a diverse community of trillions of microorganisms, collectively termed the microbiota, which play a fundamental role in regulating tissue physiology and immunity. Recent studies have sought to dissect the cellular and molecular mechanisms mediating communication between the microbiota and host immune system. Epithelial cells line the intestine and form an initial barrier separating the microbiota from underlying immune cells, and disruption of epithelial function has been associated with various conditions ranging from infection to inflammatory bowel diseases and cancer. From several studies, it is now clear that epithelial cells integrate signals from commensal microbes. Importantly, these non-hematopoietic cells also direct regulatory mechanisms that instruct the recruitment and function of microbiota-sensitive immune cells. In this review, we discuss the central role that has emerged for epithelial cells in orchestrating intestinal immunity and highlight epithelial pathways through which the microbiota can calibrate tissue-intrinsic immune responses.

Microbiota-derived butyrate restricts tuft cell differentiation via histone deacetylase 3 to modulate intestinal type 2 immunity.

Tuft cells in mucosal tissues are key regulators of type 2 immunity. Here, we examined the impact of the microbiota on tuft cell biology in the intestine. Succinate induction of tuft cells and type 2 innate lymphoid cells was elevated with loss of gut microbiota. Colonization with butyrate-producing bacteria or treatment with butyrate suppressed this effect and reduced intestinal histone deacetylase activity. Epithelial-intrinsic deletion of the epigenetic-modifying enzyme histone deacetylase 3 (HDAC3) inhibited tuft cell expansion in vivo and impaired type 2 immune responses during helminth infection. Butyrate restricted stem cell differentiation into tuft cells, and inhibition of HDAC3 in adult mice and human intestinal organoids blocked tuft cell expansion. Collectively, these data define a HDAC3 mechanism in stem cells for tuft cell differentiation that is dampened by a commensal metabolite, revealing a pathway whereby the microbiota calibrate intestinal type 2 immunity.

Kruppel-like factor 2+ CD4 T cells avert microbiota-induced intestinal inflammation.

Intestinal colonization by antigenically foreign microbes necessitates expanded peripheral immune tolerance. Here we show commensal microbiota prime expansion of CD4 T cells unified by the Kruppel-like factor 2 (KLF2) transcriptional regulator and an essential role for KLF2+ CD4 cells in averting microbiota-driven intestinal inflammation. CD4 cells with commensal specificity in secondary lymphoid organs and intestinal tissues are enriched for KLF2 expression, and distinct from FOXP3+ regulatory T cells or other differentiation lineages. Mice with conditional KLF2 deficiency in T cells develop spontaneous rectal prolapse and intestinal inflammation, phenotypes overturned by eliminating microbiota or reconstituting with donor KLF2+ cells. Activated KLF2+ cells selectively produce IL-10, and eliminating IL-10 overrides their suppressive function in vitro and protection against intestinal inflammation in vivo. Together with reduced KLF2+ CD4 cell accumulation in Crohn's disease, a necessity for the KLF2+ subpopulation of T regulatory type 1 (Tr1) cells in sustaining commensal tolerance is demonstrated.

Microbiota-derived butyrate dampens linaclotide stimulation of the guanylate cyclase C pathway in patient-derived colonoids.

BACKGROUND & AIMS: Disorders of gut-brain interaction (DGBI) are complex conditions that result in decreased quality of life and a significant cost burden. Linaclotide, a guanylin cyclase C (GCC) receptor agonist, is approved as a DGBI treatment. However, its efficacy has been limited and variable across DGBI patients. Microbiota and metabolomic alterations are noted in DGBI patients, provoking the hypothesis that the microbiota may impact the GCC response to current therapeutics. METHODS: Human-derived intestinal organoids were grown from pediatric DGBI, non-IBD colon biopsies (colonoids). Colonoids were treated with 250 nM linaclotide and assayed for cGMP to develop a model of GCC activity. Butyrate was administered to human colonoids overnight at a concentration of 1 mM. Colonoid lysates were analyzed for cGMP levels by ELISA. For the swelling assay, colonoids were photographed pre- and post-treatment and volume was measured using ImageJ. Principal coordinate analyses (PCoA) were performed on the Bray-Curtis dissimilarity and Jaccard distance to assess differences in the community composition of short-chain fatty acid (SCFA) producing microbial species in the intestinal microbiota from pediatric patients with IBS and healthy control samples. KEY RESULTS: Linaclotide treatment induced a significant increase in [cGMP] and swelling of patient-derived colonoids, demonstrating a human in vitro model of linaclotide-induced GCC activation. Shotgun sequencing analysis of pediatric IBS patients and healthy controls showed differences in the composition of commensal SCFA-producing bacteria. Butyrate exposure significantly dampened linaclotide-induced cGMP levels and swelling in patient-derived colonoids. CONCLUSIONS & INFERENCES: Patient-derived colonoids demonstrate that microbiota-derived butyrate can dampen human colonic responses to linaclotide. This study supports incorporation of microbiota and metabolomic assessment to improve precision medicine for DGBI patients.

IEC-intrinsic IL-1R signaling holds dual roles in regulating intestinal homeostasis and inflammation.

Intestinal epithelial cells (IECs) constitute a critical first line of defense against microbes. While IECs are known to respond to various microbial signals, the precise upstream cues regulating diverse IEC responses are not clear. Here, we discover a dual role for IEC-intrinsic interleukin-1 receptor (IL-1R) signaling in regulating intestinal homeostasis and inflammation. Absence of IL-1R in epithelial cells abrogates a homeostatic antimicrobial program including production of antimicrobial peptides (AMPs). Mice deficient for IEC-intrinsic IL-1R are unable to clear Citrobacter rodentium (C. rodentium) but are protected from DSS-induced colitis. Mechanistically, IL-1R signaling enhances IL-22R-induced signal transducer and activator of transcription 3 (STAT3) phosphorylation in IECs leading to elevated production of AMPs. IL-1R signaling in IECs also directly induces expression of chemokines as well as genes involved in the production of reactive oxygen species. Our findings establish a protective role for IEC-intrinsic IL-1R signaling in combating infections but a detrimental role during colitis induced by epithelial damage.

Intestinal epithelial HDAC3 and MHC class II coordinate microbiota-specific immunity.

Aberrant immune responses to resident microbes promote inflammatory bowel disease and other chronic inflammatory conditions. However, how microbiota-specific immunity is controlled in mucosal tissues remains poorly understood. Here, we found that mice lacking epithelial expression of microbiota-sensitive histone deacetylase 3 (HDAC3) exhibited increased accumulation of commensal-specific CD4+ T cells in the intestine, provoking the hypothesis that epithelial HDAC3 may instruct local microbiota-specific immunity. Consistent with this, microbiota-specific CD4+ T cells and epithelial HDAC3 expression were concurrently induced following early-life microbiota colonization. Further, epithelium-intrinsic ablation of HDAC3 decreased commensal-specific Tregs, increased commensal-specific Th17 cells, and promoted T cell-driven colitis. Mechanistically, HDAC3 was essential for NF-κB-dependent regulation of epithelial MHC class II (MHCII). Epithelium-intrinsic MHCII dampened local accumulation of commensal-specific Th17 cells in adult mice and protected against microbiota-triggered inflammation. Remarkably, HDAC3 enabled the microbiota to induce MHCII expression on epithelial cells and limit the number of commensal-specific T cells in the intestine. Collectively, these data reveal a central role for an epithelial histone deacetylase in directing the dynamic balance of tissue-intrinsic CD4+ T cell subsets that recognize commensal microbes and control inflammation.

Metagenomic analysis of the fecal microbiome in colorectal cancer patients compared to healthy controls as a function of age.

BACKGROUND AND AIMS: Colorectal cancer (CRC) incidence is increasing in young patients without a clear etiology. Emerging data have implicated the fecal microbiome in CRC carcinogenesis. However, its impact on young onset CRC is poorly defined. METHODS: We performed a meta-analysis of fecal metagenomics sequencing data from n = 692 patients with CRC and n = 602 healthy controls from eleven studies to evaluate features of the fecal metagenome associated with CRC. We hypothesized that known carcinogenic virulence factors (colibactin, fadA) and species abundance may be differentially enriched in young CRC patients relative to older CRC patients and controls. RESULTS: Summary odds ratios (OR) for CRC were increased with the presence of colibactin (OR 1.92 95% CI 1.08-3.38), fadA (OR 4.57 95% CI 1.63-12.85), and F. nucleatum (OR 6.93 95% CI 3.01-15.96) in meta-analysis models adjusted for age, gender, and body mass index. The OR for CRC for the presence of E.coli was 2.02 (0.92-4.45). An increase in the prevalence of Fusobacterium nucleatum (OR = 1.40 [1.18; 1.65]) and Escherichia coli (OR = 1.14 [1.02; 1.28]) per 10-year increase in age was observed in models including samples from both CRC and healthy controls. Species relative abundance was differentially enriched in young CRC patients for five species-Intestinimonas butyriciproducens, Holdemania filiformis, Firimicutues bacterium CAG 83, Bilophilia wadsworthia, and Alistipes putredinis. CONCLUSION: In this study, we observed strong associations with CRC status for colibactin, fadA, and Fusobacterium nucleatum with CRC relative to controls. In addition, we identified several microbial species differentially enriched in young colorectal cancer patients. Studies targeting the young CRC patients are warranted to elucidate underlying preclinical mechanisms.

Good intentions gone wrong: The B cell block to epithelial repair.

Cellular dynamics that influence mucosal healing are not well understood. In this issue of Immunity, Frede, Czarnewski, Monasterio et al. find that B cells accumulate in the colon following intestinal injury. These B cells impair epithelial repair by hindering local stromal-epithelial interactions.

Dietary phytate primes epithelial antibacterial immunity in the intestine.

Although diet has long been associated with susceptibility to infection, the dietary components that regulate host defense remain poorly understood. Here, we demonstrate that consuming rice bran decreases susceptibility to intestinal infection with Citrobacter rodentium, a murine pathogen that is similar to enteropathogenic E. coli infection in humans. Rice bran naturally contains high levels of the substance phytate. Interestingly, phytate supplementation also protected against intestinal infection, and enzymatic metabolism of phytate by commensal bacteria was necessary for phytate-induced host defense. Mechanistically, phytate consumption induced mammalian intestinal epithelial expression of STAT3-regulated antimicrobial pathways and increased phosphorylated STAT3, suggesting that dietary phytate promotes innate defense through epithelial STAT3 activation. Further, phytate regulation of epithelial STAT3 was mediated by the microbiota-sensitive enzyme histone deacetylase 3 (HDAC3). Collectively, these data demonstrate that metabolism of dietary phytate by microbiota decreases intestinal infection and suggests that consuming bran and other phytate-enriched foods may represent an effective dietary strategy for priming host immunity.

Neonatal microbiota-epithelial interactions that impact infection.

Despite modern therapeutic developments and prophylactic use of antibiotics during birth or in the first few months of life, enteric infections continue to be a major cause of neonatal mortality and morbidity globally. The neonatal period is characterized by initial intestinal colonization with microbiota and concurrent immune system development. It is also a sensitive window during which perturbations to the environment or host can significantly impact colonization by commensal microbes. Extensive research has demonstrated that these early life alterations to the microbiota can lead to enhanced susceptibility to enteric infections and increased systemic dissemination in newborns. Various contributing factors continue to pose challenges in prevention and control of neonatal enteric infections. These include alterations in the gut microbiota composition, impaired immune response, and effects of maternal factors. In addition, there remains limited understanding for how commensal microbes impact host-pathogen interactions in newborns. In this review, we discuss the recent recognition of initial microbiota-epithelial interactions that occur in neonates and can regulate susceptibility to intestinal infection. These studies suggest the development of neonatal prophylactic or therapeutic regimens that include boosting epithelial defense through microbiota-directed interventions.

Histone Deacetylase Inhibition by Gut Microbe-Generated Short-Chain Fatty Acids Entrains Intestinal Epithelial Circadian Rhythms.

BACKGROUND & AIMS: The circadian clock orchestrates ∼24-hour oscillations of gastrointestinal epithelial structure and function that drive diurnal rhythms in gut microbiota. Here, we use experimental and computational approaches in intestinal organoids to reveal reciprocal effects of gut microbial metabolites on epithelial timekeeping by an epigenetic mechanism. METHODS: We cultured enteroids in media supplemented with sterile supernatants from the altered Schaedler Flora (ASF), a defined murine microbiota. Circadian oscillations of bioluminescent PER2 and Bmal1 were measured in the presence or absence of individual ASF supernatants. Separately, we applied machine learning to ASF metabolomics to identify phase-shifting metabolites. RESULTS: Sterile filtrates from 3 of 7 ASF species (ASF360 Lactobacillus intestinalis, ASF361 Ligilactobacillus murinus, and ASF502 Clostridium species) induced minimal alterations in circadian rhythms, whereas filtrates from 4 ASF species (ASF356 Clostridium species, ASF492 Eubacterium plexicaudatum, ASF500 Pseudoflavonifactor species, and ASF519 Parabacteroides goldsteinii) induced profound, concentration-dependent phase shifts. Random forest classification identified short-chain fatty acid (SCFA) (butyrate, propionate, acetate, and isovalerate) production as a discriminating feature of ASF "shifters." Experiments with SCFAs confirmed machine learning predictions, with a median phase shift of 6.2 hours in murine enteroids. Pharmacologic or botanical histone deacetylase (HDAC) inhibitors yielded similar findings. Further, mithramycin A, an inhibitor of HDAC inhibition, reduced SCFA-induced phase shifts by 20% (P < .05) and conditional knockout of HDAC3 in enteroids abrogated butyrate effects on Per2 expression. Key findings were reproducible in human Bmal1-luciferase enteroids, colonoids, and Per2-luciferase Caco-2 cells. CONCLUSIONS: Gut microbe-generated SCFAs entrain intestinal epithelial circadian rhythms by an HDACi-dependent mechanism, with critical implications for understanding microbial and circadian network regulation of intestinal epithelial homeostasis.

Epigenetic regulation by gut microbiota.

The gastrointestinal tract is continuously exposed to trillions of commensal microbes, collectively termed the microbiota, which are environmental stimuli that can direct health and disease within the host. In addition to well-established bacterial sensing pathways, microbial signals are also integrated through epigenetic modifications that calibrate the transcriptional program of host cells without altering the underlying genetic code. Microbiota-sensitive epigenetic changes include modifications to the DNA or histones, as well as regulation of non-coding RNAs. While microbiota-sensitive epigenetic mechanisms have been described in both local intestinal cells and as well in peripheral tissues, further research is required to fully decipher the complex relationship between the host and microbiota. This Review highlights current understandings of epigenetic regulation by gut microbiota and important implications of these findings in guiding therapeutic approaches to prevent or combat diseases driven by impaired microbiota-host interactions.

Commensal segmented filamentous bacteria-derived retinoic acid primes host defense to intestinal infection.

Interactions between the microbiota and mammalian host are essential for defense against infection, but the microbial-derived cues that mediate this relationship remain unclear. Here, we find that intestinal epithelial cell (IEC)-associated commensal bacteria, segmented filamentous bacteria (SFB), promote early protection against the pathogen Citrobacter rodentium, independent of CD4+ T cells. SFB induced histone modifications in IECs at sites enriched for retinoic acid receptor motifs, suggesting that SFB may enhance defense through retinoic acid (RA). Consistent with this, inhibiting RA signaling suppressed SFB-induced protection. Intestinal RA levels were elevated in SFB mice, despite the inhibition of mammalian RA production, indicating that SFB directly modulate RA. Interestingly, RA was produced by intestinal bacteria, and the loss of bacterial-intrinsic aldehyde dehydrogenase activity decreased the RA levels and increased infection. These data reveal RA as an unexpected microbiota-derived metabolite that primes innate defense and suggests that pre- and probiotic approaches to elevate RA could prevent or combat infections.

Inflammation-Associated Microbiota Composition Across Domestic Animals.

Domestic animals represent important resources for understanding shared mechanisms underlying complex natural diseases that arise due to both genetic and environmental factors. Intestinal inflammation, particularly inflammatory bowel disease (IBD), is a significant health challenge in humans and domestic animals. While the etiology of IBD is multifactorial, imbalance of symbiotic gut microbiota has been hypothesized to play a central role in disease pathophysiology. Advances in genomic sequencing and analytical pipelines have enabled researchers to decipher the composition of the intestinal microbiota during health and in the context of naturally occurring diseases. This review compiles microbiome genomic data across domestic species and highlights a common occurrence of gut microbiome dysbiosis during idiopathic intestinal inflammation in multiple species, including dogs, cats, horses, cows, and pigs. Current microbiome data obtained from animals with intestinal inflammation are mostly limited to taxonomical analyses in association with broad clinical phenotype. In general, a pathogen or pathosymbiont were not detected. Rather, functional potential of the altered microbiota has been suggested to be one of the key etiologic factors. Among the domestic species studied, canine analyses are currently the most advanced with incorporation of functional profiling of microbiota. Canine IBD parallels features of the disease in humans, thus canines represent a strong natural model for human IBD. While deeper analyses of metagenomic data, coupled with host molecular analyses are needed, comparative studies across domestic species can reveal shared microbial alterations and regulatory mechanisms that will improve our understanding of intestinal inflammation in both animals and humans.

Epithelial sensing of microbiota-derived signals.

The gastrointestinal tract harbors trillions of microbial species, collectively termed the microbiota, which establish a symbiotic relationship with the host. Decades of research have emphasized the necessity of microbial signals in the development, maturation, and function of host physiology. However, changes in the composition or containment of the microbiota have been linked to the development of several chronic inflammatory diseases, including inflammatory bowel diseases. Intestinal epithelial cells (IECs) are in constant contact with the microbiota and are critical for maintaining intestinal homeostasis. Signals from the microbiota are directly sensed by IECs and influence intestinal health by calibrating immune cell responses and fortifying intestinal barrier function. IECs detect commensal microbes through engagement of common pattern recognition receptors or by sensing the production of microbial-derived metabolites. Deficiencies in these microbial-detecting pathways in IECs leads to impaired epithelial barrier function and altered intestinal homeostasis. This Review aims to highlight the pathways by which IECs sense microbiota-derived signals and the necessity of these detection pathways in maintaining epithelial barrier integrity.

IL-10-producing Tfh cells accumulate with age and link inflammation with age-related immune suppression.

Aging results in profound immune dysfunction, resulting in the decline of vaccine responsiveness previously attributed to irreversible defects in the immune system. In addition to increased interleukin-6 (IL-6), we found aged mice exhibit increased systemic IL-10 that requires forkhead box P3-negative (FoxP3-), but not FoxP3+, CD4+T cells. Most IL-10-producing cells manifested a T follicular helper (Tfh) phenotype and required the Tfh cytokines IL-6 and IL-21 for their accrual, so we refer to them as Tfh10 cells. IL-21 was also required to maintain normal serum levels of IL-6 and IL-10. Notably, antigen-specific Tfh10 cells arose after immunization of aged mice, and neutralization of IL-10 receptor signaling significantly restored Tfh-dependent antibody responses, whereas depletion of FoxP3+ regulatory and follicular regulatory cells did not. Thus, these data demonstrate that immune suppression with age is reversible and implicate Tfh10 cells as an intriguing link between "inflammaging" and impaired immune responses with age.

Microbiota-derived metabolite promotes HDAC3 activity in the gut.

The coevolution of mammalian hosts and their beneficial commensal microbes has led to development of symbiotic host-microbiota relationships1. Epigenetic machinery permits mammalian cells to integrate environmental signals2; however, how these pathways are fine-tuned by diverse cues from commensal bacteria is not well understood. Here we reveal a highly selective pathway through which microbiota-derived inositol phosphate regulates histone deacetylase 3 (HDAC3) activity in the intestine. Despite the abundant presence of HDAC inhibitors such as butyrate in the intestine, we found that HDAC3 activity was sharply increased in intestinal epithelial cells of microbiota-replete mice compared with germ-free mice. This divergence was reconciled by the finding that commensal bacteria, including Escherichia coli, stimulated HDAC activity through metabolism of phytate and production of inositol-1,4,5-trisphosphate (InsP3). Both intestinal exposure to InsP3 and phytate ingestion promoted recovery following intestinal damage. Of note, InsP3 also induced growth of intestinal organoids derived from human tissue, stimulated HDAC3-dependent proliferation and countered butyrate inhibition of colonic growth. Collectively, these results show that InsP3 is a microbiota-derived metabolite that activates a mammalian histone deacetylase to promote epithelial repair. Thus, HDAC3 represents a convergent epigenetic sensor of distinct metabolites that calibrates host responses to diverse microbial signals.