Genetic manipulation of gut microbes enables single-gene interrogation in a complex microbiome.

Hundreds of microbiota genes are associated with host biology/disease. Unraveling the causal contribution of a microbiota gene to host biology remains difficult because many are encoded by nonmodel gut commensals and not genetically targetable. A general approach to identify their gene transfer methodology and build their gene manipulation tools would enable mechanistic dissections of their impact on host physiology. We developed a pipeline that identifies the gene transfer methods for multiple nonmodel microbes spanning five phyla, and we demonstrated the utility of their genetic tools by modulating microbiome-derived short-chain fatty acids and bile acids in vitro and in the host. In a proof-of-principle study, by deleting a commensal gene for bile acid synthesis in a complex microbiome, we discovered an intriguing role of this gene in regulating colon inflammation. This technology will enable genetically engineering the nonmodel gut microbiome and facilitate mechanistic dissection of microbiota-host interactions.

Dysregulation of ILC3s unleashes progression and immunotherapy resistance in colon cancer.

Group 3 innate lymphoid cells (ILC3s) regulate immunity and inflammation, yet their role in cancer remains elusive. Here, we identify that colorectal cancer (CRC) manifests with altered ILC3s that are characterized by reduced frequencies, increased plasticity, and an imbalance with T cells. We evaluated the consequences of these changes in mice and determined that a dialog between ILC3s and T cells via major histocompatibility complex class II (MHCII) is necessary to support colonization with microbiota that subsequently induce type-1 immunity in the intestine and tumor microenvironment. As a result, mice lacking ILC3-specific MHCII develop invasive CRC and resistance to anti-PD-1 immunotherapy. Finally, humans with dysregulated intestinal ILC3s harbor microbiota that fail to induce type-1 immunity and immunotherapy responsiveness when transferred to mice. Collectively, these data define a protective role for ILC3s in cancer and indicate that their inherent disruption in CRC drives dysfunctional adaptive immunity, tumor progression, and immunotherapy resistance.

Single-Cell Analysis of Crohn's Disease Lesions Identifies a Pathogenic Cellular Module Associated with Resistance to Anti-TNF Therapy.

Clinical benefits of cytokine blockade in ileal Crohn's disease (iCD) are limited to a subset of patients. Here, we applied single-cell technologies to iCD lesions to address whether cellular heterogeneity contributes to treatment resistance. We found that a subset of patients expressed a unique cellular module in inflamed tissues that consisted of IgG plasma cells, inflammatory mononuclear phagocytes, activated T cells, and stromal cells, which we named the GIMATS module. Analysis of ligand-receptor interaction pairs identified a distinct network connectivity that likely drives the GIMATS module. Strikingly, the GIMATS module was also present in a subset of patients in four independent iCD cohorts (n = 441), and its presence at diagnosis correlated with failure to achieve durable corticosteroid-free remission upon anti-TNF therapy. These results emphasize the limitations of current diagnostic assays and the potential for single-cell mapping tools to identify novel biomarkers of treatment response and tailored therapeutic opportunities.

Ketone Body Signaling Mediates Intestinal Stem Cell Homeostasis and Adaptation to Diet.

Little is known about how metabolites couple tissue-specific stem cell function with physiology. Here we show that, in the mammalian small intestine, the expression of Hmgcs2 (3-hydroxy-3-methylglutaryl-CoA synthetase 2), the gene encoding the rate-limiting enzyme in the production of ketone bodies, including beta-hydroxybutyrate (ßOHB), distinguishes self-renewing Lgr5+ stem cells (ISCs) from differentiated cell types. Hmgcs2 loss depletes ßOHB levels in Lgr5+ ISCs and skews their differentiation toward secretory cell fates, which can be rescued by exogenous ßOHB and class I histone deacetylase (HDAC) inhibitor treatment. Mechanistically, ßOHB acts by inhibiting HDACs to reinforce Notch signaling, instructing ISC self-renewal and lineage decisions. Notably, although a high-fat ketogenic diet elevates ISC function and post-injury regeneration through ßOHB-mediated Notch signaling, a glucose-supplemented diet has the opposite effects. These findings reveal how control of ßOHB-activated signaling in ISCs by diet helps to fine-tune stem cell adaptation in homeostasis and injury.

Intestinal CXCR6+ ILC3s migrate to the kidney and exacerbate renal fibrosis via IL-23 receptor signaling enhanced by PD-1 expression.

Group 3 innate lymphoid cells (ILC3s) regulate inflammation and tissue repair at mucosal sites, but whether these functions pertain to other tissues-like the kidneys-remains unclear. Here, we observed that renal fibrosis in humans was associated with increased ILC3s in the kidneys and blood. In mice, we showed that CXCR6+ ILC3s rapidly migrated from the intestinal mucosa and accumulated in the kidney via CXCL16 released from the injured tubules. Within the fibrotic kidney, ILC3s increased the expression of programmed cell death-1 (PD-1) and subsequent IL-17A production to directly activate myofibroblasts and fibrotic niche formation. ILC3 expression of PD-1 inhibited IL-23R endocytosis and consequently amplified the JAK2/STAT3/RORγt/IL-17A pathway that was essential for the pro-fibrogenic effect of ILC3s. Thus, we reveal a hitherto unrecognized migration pathway of ILC3s from the intestine to the kidney and the PD-1-dependent function of ILC3s in promoting renal fibrosis.

Intestinal Dysmotility Syndromes following Systemic Infection by Flaviviruses.

Although chronic gastrointestinal dysmotility syndromes are a common worldwide health problem, underlying causes for these disorders are poorly understood. We show that flavivirus infection of enteric neurons leads to acute neuronal injury and cell death, inflammation, bowel dilation, and slowing of intestinal transit in mice. Flavivirus-primed CD8+ T cells promote these phenotypes, as their absence diminished enteric neuron injury and intestinal transit delays, and their adoptive transfer reestablished dysmotility after flavivirus infection. Remarkably, mice surviving acute flavivirus infection developed chronic gastrointestinal dysmotility that was exacerbated by immunization with an unrelated alphavirus vaccine or exposure to a non-infectious inflammatory stimulus. This model of chronic post-infectious gastrointestinal dysmotility in mice suggests that viral infections with tropism for enteric neurons and the ensuing immune response might contribute to the development of bowel motility disorders in humans. These results suggest an opportunity for unique approaches to diagnosis and therapy of gastrointestinal dysmotility syndromes.

Dietary and Microbial Oxazoles Induce Intestinal Inflammation by Modulating Aryl Hydrocarbon Receptor Responses.

Genome-wide association studies have identified risk loci associated with the development of inflammatory bowel disease, while epidemiological studies have emphasized that pathogenesis likely involves host interactions with environmental elements whose source and structure need to be defined. Here, we identify a class of compounds derived from dietary, microbial, and industrial sources that are characterized by the presence of a five-membered oxazole ring and induce CD1d-dependent intestinal inflammation. We observe that minimal oxazole structures modulate natural killer T cell-dependent inflammation by regulating lipid antigen presentation by CD1d on intestinal epithelial cells (IECs). CD1d-restricted production of interleukin 10 by IECs is limited through activity of the aryl hydrocarbon receptor (AhR) pathway in response to oxazole induction of tryptophan metabolites. As such, the depletion of the AhR in the intestinal epithelium abrogates oxazole-induced inflammation. In summary, we identify environmentally derived oxazoles as triggers of CD1d-dependent intestinal inflammatory responses that occur via activation of the AhR in the intestinal epithelium.

Ambient oxygen levels regulate intestinal dysbiosis and GVHD severity after allogeneic stem cell transplantation.

The severity of T cell-mediated gastrointestinal (GI) diseases such as graft-versus-host disease (GVHD) and inflammatory bowel diseases correlates with a decrease in the diversity of the host gut microbiome composition characterized by loss of obligate anaerobic commensals. The mechanisms underpinning these changes in the microbial structure remain unknown. Here, we show in multiple specific pathogen-free (SPF), gnotobiotic, and germ-free murine models of GI GVHD that the initiation of the intestinal damage by the pathogenic T cells altered ambient oxygen levels in the GI tract and caused dysbiosis. The change in oxygen levels contributed to the severity of intestinal pathology in a host intestinal HIF-1α- and a microbiome-dependent manner. Regulation of intestinal ambient oxygen levels with oral iron chelation mitigated dysbiosis and reduced the severity of the GI GVHD. Thus, targeting ambient intestinal oxygen levels may represent a novel, non-immunosuppressive strategy to mitigate T cell-driven intestinal diseases.

IL-17RA-signaling in Lgr5+ intestinal stem cells induces expression of transcription factor ATOH1 to promote secretory cell lineage commitment.

The Th17 cell-lineage-defining cytokine IL-17A contributes to host defense and inflammatory disease by coordinating multicellular immune responses. The IL-17 receptor (IL-17RA) is expressed by diverse intestinal cell types, and therapies targeting IL-17A induce adverse intestinal events, suggesting additional tissue-specific functions. Here, we used multiple conditional deletion models to identify a role for IL-17A in secretory epithelial cell differentiation in the gut. Paneth, tuft, goblet, and enteroendocrine cell numbers were dependent on IL-17A-mediated induction of the transcription factor ATOH1 in Lgr5+ intestinal epithelial stem cells. Although dispensable at steady state, IL-17RA signaling in ATOH1+ cells was required to regenerate secretory cells following injury. Finally, IL-17A stimulation of human-derived intestinal organoids that were locked into a cystic immature state induced ATOH1 expression and rescued secretory cell differentiation. Our data suggest that the cross talk between immune cells and stem cells regulates secretory cell lineage commitment and the integrity of the mucosa.

CTLA-4-expressing ILC3s restrain interleukin-23-mediated inflammation.

Interleukin (IL-)23 is a major mediator and therapeutic target in chronic inflammatory diseases that also elicits tissue protection in the intestine at homeostasis or following acute infection1-4. However, the mechanisms that shape these beneficial versus pathological outcomes remain poorly understood. To address this gap in knowledge, we performed single-cell RNA sequencing on all IL-23 receptor-expressing cells in the intestine and their acute response to IL-23, revealing a dominance of T cells and group 3 innate lymphoid cells (ILC3s). Unexpectedly, we identified potent upregulation of the immunoregulatory checkpoint molecule cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) on ILC3s. This pathway was activated by gut microbes and IL-23 in a FOXO1- and STAT3-dependent manner. Mice lacking CTLA-4 on ILC3s exhibited reduced regulatory T cells, elevated inflammatory T cells and more-severe intestinal inflammation. IL-23 induction of CTLA-4+ ILC3s was necessary and sufficient to reduce co-stimulatory molecules and increase PD-L1 bioavailability on intestinal myeloid cells. Finally, human ILC3s upregulated CTLA-4 in response to IL-23 or gut inflammation and correlated with immunoregulation in inflammatory bowel disease. These results reveal ILC3-intrinsic CTLA-4 as an essential checkpoint that restrains the pathological outcomes of IL-23, suggesting that disruption of these lymphocytes, which occurs in inflammatory bowel disease5-7, contributes to chronic inflammation.

Development of antifibrotic therapy for stricturing Crohn's disease: lessons from randomized trials in other fibrotic diseases.

Intestinal fibrosis is considered an inevitable complication of Crohn's disease (CD) that results in symptoms of obstruction and stricture formation. Endoscopic or surgical treatment is required to treat the majority of patients. Progress in the management of stricturing CD is hampered by the lack of effective antifibrotic therapy; however, this situation is likely to change because of recent advances in other fibrotic diseases of the lung, liver, and skin. In this review, we summarize data from randomized controlled trials (RCTs) of antifibrotic therapies in these conditions. Multiple compounds have been tested for antifibrotic effects in other organs. According to their mechanisms, they were categorized into growth factor modulators, inflammation modulators, 5-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors, intracellular enzymes and kinases, renin-angiotensin system (RAS) modulators, and others. From our review of the results from the clinical trials and discussion of their implications in the gastrointestinal tract, we have identified several molecular candidates that could serve as potential therapies for intestinal fibrosis in CD.

IFN-λ suppresses intestinal inflammation by non-translational regulation of neutrophil function.

Interferon-λ (IFN-λ) is a central regulator of mucosal immunity; however, its signaling specificity relative to that of type I interferons is poorly defined. IFN-λ can induce antiviral interferon-stimulated genes (ISGs) in epithelia, while the effect of IFN-λ in non-epithelial cells remains unclear. Here we report that neutrophils responded to IFN-λ. We found that in addition to inducing ISG transcription, IFN-λ (but not IFN-ß) specifically activated a translation-independent signaling pathway that diminished the production of reactive oxygen species and degranulation in neutrophils. In mice, IFN-λ was elicited by enteric viruses and acted on neutrophils to decrease oxidative stress and intestinal damage. Thus, IFN-λ acted as a unique immunomodulatory agent by modifying transcriptional and non-translational neutrophil responses, which might permit a controlled development of the inflammatory process.

Active eosinophils regulate host defence and immune responses in colitis.

In the past decade, single-cell transcriptomics has helped to uncover new cell types and states and led to the construction of a cellular compendium of health and disease. Despite this progress, some difficult-to-sequence cells remain absent from tissue atlases. Eosinophils-elusive granulocytes that are implicated in a plethora of human pathologies1-5-are among these uncharted cell types. The heterogeneity of eosinophils and the gene programs that underpin their pleiotropic functions remain poorly understood. Here we provide a comprehensive single-cell transcriptomic profiling of mouse eosinophils. We identify an active and a basal population of intestinal eosinophils, which differ in their transcriptome, surface proteome and spatial localization. By means of a genome-wide CRISPR inhibition screen and functional assays, we reveal a mechanism by which interleukin-33 (IL-33) and interferon-γ (IFNγ) induce the accumulation of active eosinophils in the inflamed colon. Active eosinophils are endowed with bactericidal and T cell regulatory activity, and express the co-stimulatory molecules CD80 and PD-L1. Notably, active eosinophils are enriched in the lamina propria of a small cohort of patients with inflammatory bowel disease, and are closely associated with CD4+ T cells. Our findings provide insights into the biology of eosinophils and highlight the crucial contribution of this cell type to intestinal homeostasis, immune regulation and host defence. Furthermore, we lay a framework for the characterization of eosinophils in human gastrointestinal diseases.

Cytokine Networks in the Pathophysiology of Inflammatory Bowel Disease.

Inflammatory bowel disease (IBD) is a chronic inflammatory disease of the gastrointestinal tract. Cytokine-targeted therapies have transformed the treatment of IBD, providing control of symptoms and longer relapse-free periods. However, many patients fail to respond, highlighting the need for therapies tailored to the underlying cell and molecular disease drivers. Here we discuss the progression of IBD from the perspective of remodeling of cytokine networks. We place well-established and under-studied cytokine modules in the context of cellular interactions, their dynamic regulation in early and late stages of disease (i.e., fibrosis), and their current and potential use in the clinic. Examining how particular cytokine networks drive distinct features and phases of IBD will shed light on the etiology of IBD and provide a basis for more effective treatments.

ZBTB46 defines and regulates ILC3s that protect the intestine.

RORγt is a lineage-specifying transcription factor that is expressed by immune cells that are enriched in the gastrointestinal tract and promote immunity, inflammation and tissue homeostasis1-15. However, fundamental questions remain with regard to the cellular heterogeneity among these cell types, the mechanisms that control protective versus inflammatory properties and their functional redundancy. Here we define all RORγt+ immune cells in the intestine at single-cell resolution and identify a subset of group 3 innate lymphoid cells (ILC3s) that expresses ZBTB46, a transcription factor specifying conventional dendritic cells16-20. ZBTB46 is robustly expressed by CCR6+ lymphoid-tissue-inducer-like ILC3s that are developmentally and phenotypically distinct from conventional dendritic cells, and its expression is imprinted by RORγt, fine-tuned by microbiota-derived signals and increased by pro-inflammatory cytokines. ZBTB46 restrains the inflammatory properties of ILC3s, including the OX40L-dependent expansion of T helper 17 cells and the exacerbated intestinal inflammation that occurs after enteric infection. Finally, ZBTB46+ ILC3s are a major source of IL-22, and selective depletion of this population renders mice susceptible to enteric infection and associated intestinal inflammation. These results show that ZBTB46 is a transcription factor that is shared between conventional dendritic cells and ILC3s, and identify a cell-intrinsic function for ZBTB46 in restraining the pro-inflammatory properties of ILC3s and a non-redundant role for ZBTB46+ ILC3s in orchestrating intestinal health.

ADAR1 prevents autoinflammation by suppressing spontaneous ZBP1 activation.

The RNA-editing enzyme adenosine deaminase acting on RNA 1 (ADAR1) limits the accumulation of endogenous immunostimulatory double-stranded RNA (dsRNA)1. In humans, reduced ADAR1 activity causes the severe inflammatory disease Aicardi-Goutières syndrome (AGS)2. In mice, complete loss of ADAR1 activity is embryonically lethal3-6, and mutations similar to those found in patients with AGS cause autoinflammation7-12. Mechanistically, adenosine-to-inosine (A-to-I) base modification of endogenous dsRNA by ADAR1 prevents chronic overactivation of the dsRNA sensors MDA5 and PKR3,7-10,13,14. Here we show that ADAR1 also inhibits the spontaneous activation of the left-handed Z-nucleic acid sensor ZBP1. Activation of ZBP1 elicits caspase-8-dependent apoptosis and MLKL-mediated necroptosis of ADAR1-deficient cells. ZBP1 contributes to the embryonic lethality of Adar-knockout mice, and it drives early mortality and intestinal cell death in mice deficient in the expression of both ADAR and MAVS. The Z-nucleic-acid-binding Zα domain of ADAR1 is necessary to prevent ZBP1-mediated intestinal cell death and skin inflammation. The Zα domain of ADAR1 promotes A-to-I editing of endogenous Alu elements to prevent dsRNA formation through the pairing of inverted Alu repeats, which can otherwise induce ZBP1 activation. This shows that recognition of Alu duplex RNA by ZBP1 may contribute to the pathological features of AGS that result from the loss of ADAR1 function.

Altered TMPRSS2 usage by SARS-CoV-2 Omicron impacts infectivity and fusogenicity.

The SARS-CoV-2 Omicron BA.1 variant emerged in 20211 and has multiple mutations in its spike protein2. Here we show that the spike protein of Omicron has a higher affinity for ACE2 compared with Delta, and a marked change in its antigenicity increases Omicron's evasion of therapeutic monoclonal and vaccine-elicited polyclonal neutralizing antibodies after two doses. mRNA vaccination as a third vaccine dose rescues and broadens neutralization. Importantly, the antiviral drugs remdesivir and molnupiravir retain efficacy against Omicron BA.1. Replication was similar for Omicron and Delta virus isolates in human nasal epithelial cultures. However, in lung cells and gut cells, Omicron demonstrated lower replication. Omicron spike protein was less efficiently cleaved compared with Delta. The differences in replication were mapped to the entry efficiency of the virus on the basis of spike-pseudotyped virus assays. The defect in entry of Omicron pseudotyped virus to specific cell types effectively correlated with higher cellular RNA expression of TMPRSS2, and deletion of TMPRSS2 affected Delta entry to a greater extent than Omicron. Furthermore, drug inhibitors targeting specific entry pathways3 demonstrated that the Omicron spike inefficiently uses the cellular protease TMPRSS2, which promotes cell entry through plasma membrane fusion, with greater dependency on cell entry through the endocytic pathway. Consistent with suboptimal S1/S2 cleavage and inability to use TMPRSS2, syncytium formation by the Omicron spike was substantially impaired compared with the Delta spike. The less efficient spike cleavage of Omicron at S1/S2 is associated with a shift in cellular tropism away from TMPRSS2-expressing cells, with implications for altered pathogenesis.

Inulin fibre promotes microbiota-derived bile acids and type 2 inflammation.

Dietary fibres can exert beneficial anti-inflammatory effects through microbially fermented short-chain fatty acid metabolites^1,2, although the immunoregulatory roles of most fibre diets and their microbiota-derived metabolites remain poorly defined. Here, using microbial sequencing and untargeted metabolomics, we show that a diet of inulin fibre alters the composition of the mouse microbiota and the levels of microbiota-derived metabolites, notably bile acids. This metabolomic shift is associated with type 2 inflammation in the intestine and lungs, characterized by IL-33 production, activation of group 2 innate lymphoid cells and eosinophilia. Delivery of cholic acid mimics inulin-induced type 2 inflammation, whereas deletion of the bile acid receptor farnesoid X receptor diminishes the effects of inulin. The effects of inulin are microbiota dependent and were reproduced in mice colonized with human-derived microbiota. Furthermore, genetic deletion of a bile-acid-metabolizing enzyme in one bacterial species abolishes the ability of inulin to trigger type 2 inflammation. Finally, we demonstrate that inulin enhances allergen- and helminth-induced type 2 inflammation. Taken together, these data reveal that dietary inulin fibre triggers microbiota-derived cholic acid and type 2 inflammation at barrier surfaces with implications for understanding the pathophysiology of allergic inflammation, tissue protection and host defence.

Identification of environmental factors that promote intestinal inflammation.

Genome-wide association studies have identified risk loci linked to inflammatory bowel disease (IBD)1-a complex chronic inflammatory disorder of the gastrointestinal tract. The increasing prevalence of IBD in industrialized countries and the augmented disease risk observed in migrants who move into areas of higher disease prevalence suggest that environmental factors are also important determinants of IBD susceptibility and severity2. However, the identification of environmental factors relevant to IBD and the mechanisms by which they influence disease has been hampered by the lack of platforms for their systematic investigation. Here we describe an integrated systems approach, combining publicly available databases, zebrafish chemical screens, machine learning and mouse preclinical models to identify environmental factors that control intestinal inflammation. This approach established that the herbicide propyzamide increases inflammation in the small and large intestine. Moreover, we show that an AHR-NF-κB-C/EBPß signalling axis operates in T cells and dendritic cells to promote intestinal inflammation, and is targeted by propyzamide. In conclusion, we developed a pipeline for the identification of environmental factors and mechanisms of pathogenesis in IBD and, potentially, other inflammatory diseases.

Regulation of intestinal immunity and tissue repair by enteric glia.

Tissue maintenance and repair depend on the integrated activity of multiple cell types1. Whereas the contributions of epithelial2,3, immune4,5 and stromal cells6,7 in intestinal tissue integrity are well understood, the role of intrinsic neuroglia networks remains largely unknown. Here we uncover important roles of enteric glial cells (EGCs) in intestinal homeostasis, immunity and tissue repair. We demonstrate that infection of mice with Heligmosomoides polygyrus leads to enteric gliosis and the upregulation of an interferon gamma (IFNγ) gene signature. IFNγ-dependent gene modules were also induced in EGCs from patients with inflammatory bowel disease8. Single-cell transcriptomics analysis of the tunica muscularis showed that glia-specific abrogation of IFNγ signalling leads to tissue-wide activation of pro-inflammatory transcriptional programs. Furthermore, disruption of the IFNγ-EGC signalling axis enhanced the inflammatory and granulomatous response of the tunica muscularis to helminths. Mechanistically, we show that the upregulation of Cxcl10 is an early immediate response of EGCs to IFNγ signalling and provide evidence that this chemokine and the downstream amplification of IFNγ signalling in the tunica muscularis are required for a measured inflammatory response to helminths and resolution of the granulomatous pathology. Our study demonstrates that IFNγ signalling in enteric glia is central to intestinal homeostasis and reveals critical roles of the IFNγ-EGC-CXCL10 axis in immune response and tissue repair after infectious challenge.