Charting the cellular biogeography in colitis reveals fibroblast trajectories and coordinated spatial remodeling.

Gut inflammation involves contributions from immune and non-immune cells, whose interactions are shaped by the spatial organization of the healthy gut and its remodeling during inflammation. The crosstalk between fibroblasts and immune cells is an important axis in this process, but our understanding has been challenged by incomplete cell-type definition and biogeography. To address this challenge, we used multiplexed error-robust fluorescence in situ hybridization (MERFISH) to profile the expression of 940 genes in 1.35 million cells imaged across the onset and recovery from a mouse colitis model. We identified diverse cell populations, charted their spatial organization, and revealed their polarization or recruitment in inflammation. We found a staged progression of inflammation-associated tissue neighborhoods defined, in part, by multiple inflammation-associated fibroblasts, with unique expression profiles, spatial localization, cell-cell interactions, and healthy fibroblast origins. Similar signatures in ulcerative colitis suggest conserved human processes. Broadly, we provide a framework for understanding inflammation-induced remodeling in the gut and other tissues.

Nociceptor neurons direct goblet cells via a CGRP-RAMP1 axis to drive mucus production and gut barrier protection.

Neuroepithelial crosstalk is critical for gut physiology. However, the mechanisms by which sensory neurons communicate with epithelial cells to mediate gut barrier protection at homeostasis and during inflammation are not well understood. Here, we find that Nav1.8+CGRP+ nociceptor neurons are juxtaposed with and signal to intestinal goblet cells to drive mucus secretion and gut protection. Nociceptor ablation led to decreased mucus thickness and dysbiosis, while chemogenetic nociceptor activation or capsaicin treatment induced mucus growth. Mouse and human goblet cells expressed Ramp1, receptor for the neuropeptide CGRP. Nociceptors signal via the CGRP-Ramp1 pathway to induce rapid goblet cell emptying and mucus secretion. Notably, commensal microbes activated nociceptors to control homeostatic CGRP release. In the absence of nociceptors or epithelial Ramp1, mice showed increased epithelial stress and susceptibility to colitis. Conversely, CGRP administration protected nociceptor-ablated mice against colitis. Our findings demonstrate a neuron-goblet cell axis that orchestrates gut mucosal barrier protection.

Nucleophosmin 1 promotes mucosal immunity by supporting mitochondrial oxidative phosphorylation and ILC3 activity.

Nucleophosmin 1 (NPM1) is commonly mutated in myelodysplastic syndrome (MDS) and acute myeloid leukemia. Concurrent inflammatory bowel diseases (IBD) and MDS are common, indicating a close relationship between IBD and MDS. Here we examined the function of NPM1 in IBD and colitis-associated colorectal cancer (CAC). NPM1 expression was reduced in patients with IBD. Npm1+/- mice were more susceptible to acute colitis and experimentally induced CAC than littermate controls. Npm1 deficiency impaired the function of interleukin-22 (IL-22)-producing group three innate lymphoid cells (ILC3s). Mice lacking Npm1 in ILC3s exhibited decreased IL-22 production and accelerated development of colitis. NPM1 was important for mitochondrial biogenesis and metabolism by oxidative phosphorylation in ILC3s. Further experiments revealed that NPM1 cooperates with p65 to promote mitochondrial transcription factor A (TFAM) transcription in ILC3s. Overexpression of Npm1 in mice enhanced ILC3 function and reduced the severity of dextran sulfate sodium-induced colitis. Thus, our findings indicate that NPM1 in ILC3s protects against IBD by regulating mitochondrial metabolism through a p65-TFAM axis.

Somatic Evolution in Non-neoplastic IBD-Affected Colon.

Inflammatory bowel disease (IBD) is a chronic inflammatory disease associated with increased risk of gastrointestinal cancers. We whole-genome sequenced 446 colonic crypts from 46 IBD patients and compared these to 412 crypts from 41 non-IBD controls from our previous publication on the mutation landscape of the normal colon. The average mutation rate of affected colonic epithelial cells is 2.4-fold that of healthy colon, and this increase is mostly driven by acceleration of mutational processes ubiquitously observed in normal colon. In contrast to the normal colon, where clonal expansions outside the confines of the crypt are rare, we observed widespread millimeter-scale clonal expansions. We discovered non-synonymous mutations in ARID1A, FBXW7, PIGR, ZC3H12A, and genes in the interleukin 17 and Toll-like receptor pathways, under positive selection in IBD. These results suggest distinct selection mechanisms in the colitis-affected colon and that somatic mutations potentially play a causal role in IBD pathogenesis.

RNA Sensing by Gut Piezo1 Is Essential for Systemic Serotonin Synthesis.

Gastrointestinal enterochromaffin cells regulate bone and gut homeostasis via serotonin (5-hydroxytryptamine [5-HT]) production. A recent report suggested that gut microbes regulate 5-HT levels; however, the precise underlying molecular mechanisms are unexplored. Here, we reveal that the cation channel Piezo1 in the gut acts as a sensor of single-stranded RNA (ssRNA) governing 5-HT production. Intestinal epithelium-specific deletion of mouse Piezo1 profoundly disturbed gut peristalsis, impeded experimental colitis, and suppressed serum 5-HT levels. Because of systemic 5-HT deficiency, conditional knockout of Piezo1 increased bone formation. Notably, fecal ssRNA was identified as a natural Piezo1 ligand, and ssRNA-stimulated 5-HT synthesis from the gut was evoked in a MyD88/TRIF-independent manner. Colonic infusion of RNase A suppressed gut motility and increased bone mass. These findings suggest gut ssRNA as a master determinant of systemic 5-HT levels, indicating the ssRNA-Piezo1 axis as a potential prophylactic target for treatment of bone and gut disorders.

Molecular Pathways of Colon Inflammation Induced by Cancer Immunotherapy.

Checkpoint blockade with antibodies specific for the PD-1 and CTLA-4 inhibitory receptors can induce durable responses in a wide range of human cancers. However, the immunological mechanisms responsible for severe inflammatory side effects remain poorly understood. Here we report a comprehensive single-cell analysis of immune cell populations in colitis, a common and severe side effect of checkpoint blockade. We observed a striking accumulation of CD8 T cells with highly cytotoxic and proliferative states and no evidence of regulatory T cell depletion. T cell receptor (TCR) sequence analysis demonstrated that a substantial fraction of colitis-associated CD8 T cells originated from tissue-resident populations, explaining the frequently early onset of colitis symptoms following treatment initiation. Our analysis also identified cytokines, chemokines, and surface receptors that could serve as therapeutic targets for colitis and potentially other inflammatory side effects of checkpoint blockade.

pH sensing controls tissue inflammation by modulating cellular metabolism and endo-lysosomal function of immune cells.

Extracellular acidification occurs in inflamed tissue and the tumor microenvironment; however, a systematic study on how pH sensing contributes to tissue homeostasis is lacking. In the present study, we examine cell type-specific roles of the pH sensor G protein-coupled receptor 65 (GPR65) and its inflammatory disease-associated Ile231Leu-coding variant in inflammation control. GPR65 Ile231Leu knock-in mice are highly susceptible to both bacterial infection-induced and T cell-driven colitis. Mechanistically, GPR65 Ile231Leu elicits a cytokine imbalance through impaired helper type 17 T cell (TH17 cell) and TH22 cell differentiation and interleukin (IL)-22 production in association with altered cellular metabolism controlled through the cAMP-CREB-DGAT1 axis. In dendritic cells, GPR65 Ile231Leu elevates IL-12 and IL-23 release at acidic pH and alters endo-lysosomal fusion and degradation capacity, resulting in enhanced antigen presentation. The present study highlights GPR65 Ile231Leu as a multistep risk factor in intestinal inflammation and illuminates a mechanism by which pH sensing controls inflammatory circuits and tissue homeostasis.

Cooperative Metabolic Adaptations in the Host Can Favor Asymptomatic Infection and Select for Attenuated Virulence in an Enteric Pathogen.

Pathogen virulence exists on a continuum. The strategies that drive symptomatic or asymptomatic infections remain largely unknown. We took advantage of the concept of lethal dose 50 (LD50) to ask which component of individual non-genetic variation between hosts defines whether they survive or succumb to infection. Using the enteric pathogen Citrobacter, we found no difference in pathogen burdens between healthy and symptomatic populations. Iron metabolism-related genes were induced in asymptomatic hosts compared to symptomatic or naive mice. Dietary iron conferred complete protection without influencing pathogen burdens, even at 1000× the lethal dose of Citrobacter. Dietary iron induced insulin resistance, increasing glucose levels in the intestine that were necessary and sufficient to suppress pathogen virulence. A short course of dietary iron drove the selection of attenuated Citrobacter strains that can transmit and asymptomatically colonize naive hosts, demonstrating that environmental factors and cooperative metabolic strategies can drive conversion of pathogens toward commensalism.

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.

Hobit- and Blimp-1-driven CD4+ tissue-resident memory T cells control chronic intestinal inflammation.

Although tissue-resident memory T cells (TRM cells) have been shown to regulate host protection in infectious disorders, their function in inflammatory bowel disease (IBD) remains to be investigated. Here we characterized TRM cells in human IBD and in experimental models of intestinal inflammation. Pro-inflammatory TRM cells accumulated in the mucosa of patients with IBD, and the presence of CD4+CD69+CD103+ TRM cells was predictive of the development of flares. In vivo, functional impairment of TRM cells in mice with double knockout of the TRM-cell-associated transcription factors Hobit and Blimp-1 attenuated disease in several models of colitis, due to impaired cross-talk between the adaptive and innate immune system. Finally, depletion of TRM cells led to a suppression of colitis activity. Together, our data demonstrate a central role for TRM cells in the pathogenesis of chronic intestinal inflammation and suggest that these cells could be targets for future therapeutic approaches in IBD.

IL-17 metabolically reprograms activated fibroblastic reticular cells for proliferation and survival.

Lymph-node (LN) stromal cell populations expand during the inflammation that accompanies T cell activation. Interleukin-17 (IL-17)-producing helper T cells (TH17 cells) promote inflammation through the induction of cytokines and chemokines in peripheral tissues. We demonstrate a critical requirement for IL-17 in the proliferation of LN and splenic stromal cells, particularly fibroblastic reticular cells (FRCs), during experimental autoimmune encephalomyelitis and colitis. Without signaling via the IL-17 receptor, activated FRCs underwent cell cycle arrest and apoptosis, accompanied by signs of nutrient stress in vivo. IL-17 signaling in FRCs was not required for the development of TH17 cells, but failed FRC proliferation impaired germinal center formation and antigen-specific antibody production. Induction of the transcriptional co-activator IκBζ via IL-17 signaling mediated increased glucose uptake and expression of the gene Cpt1a, encoding CPT1A, a rate-limiting enzyme of mitochondrial fatty acid oxidation. Hence, IL-17 produced by locally differentiating TH17 cells is an important driver of the activation of inflamed LN stromal cells, through metabolic reprogramming required to support proliferation and survival.

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.

Biased allosteric activation of ketone body receptor HCAR2 suppresses inflammation.

Hydroxycarboxylic acid receptor 2 (HCAR2), modulated by endogenous ketone body ß-hydroxybutyrate and exogenous niacin, is a promising therapeutic target for inflammation-related diseases. HCAR2 mediates distinct pathophysiological events by activating Gi/o protein or ß-arrestin effectors. Here, we characterize compound 9n as a Gi-biased allosteric modulator (BAM) of HCAR2 and exhibit anti-inflammatory efficacy in RAW264.7 macrophages via a specific HCAR2-Gi pathway. Furthermore, four structures of HCAR2-Gi complex bound to orthosteric agonists (niacin or monomethyl fumarate), compound 9n, and niacin together with compound 9n simultaneously reveal a common orthosteric site and a unique allosteric site. Combined with functional studies, we decipher the action framework of biased allosteric modulation of compound 9n on the orthosteric site. Moreover, co-administration of compound 9n with orthosteric agonists could enhance anti-inflammatory effects in the mouse model of colitis. Together, our study provides insight to understand the molecular pharmacology of the BAM and facilitates exploring the therapeutic potential of the BAM with orthosteric drugs.

Cognate recognition of microbial antigens defines constricted CD4+ T cell receptor repertoires in the inflamed colon.

Key aspects of intestinal T cells, including their antigen specificity and their selection by the microbiota and other intestinal antigens, as well as the contribution of individual T cell clones to regulatory and effector functions, remain unresolved. Here we tracked adoptively transferred T cell populations to specify the interrelation of T cell receptor repertoire and the gut antigenic environment. We show that dominant TCRα clonotypes were shared between interferon-γ- and interleukin-17-producing but not regulatory Foxp3+ T cells. Identical TCRα clonotypes accumulated in the colon of different individuals, whereas antibiotics or defined colonization correlated with the expansion of distinct expanded T cell clonotypes. Our results demonstrate key aspects of intestinal CD4+ T cell activation and suggest that few microbial species exert a dominant effect on the intestinal T cell repertoire during colitis. We speculate that dominant proinflammatory T cell clones might provide a therapeutic target in human inflammatory bowel disease.

Non-canonical NF-κB signaling limits the tolerogenic ß-catenin-Raldh2 axis in gut dendritic cells to exacerbate intestinal pathologies.

Dendritic cell (DC) dysfunction is known to exacerbate intestinal pathologies, but the mechanisms compromising DC-mediated immune regulation in this context remain unclear. Here, we show that intestinal dendritic cells from a mouse model of experimental colitis exhibit significant levels of noncanonical NF-κB signaling, which activates the RelB:p52 heterodimer. Genetic inactivation of this pathway in DCs alleviates intestinal pathologies in mice suffering from colitis. Deficiency of RelB:p52 diminishes transcription of Axin1, a critical component of the ß-catenin destruction complex, reinforcing ß-catenin-dependent expression of Raldh2, which imparts tolerogenic DC attributes by promoting retinoic acid synthesis. DC-specific impairment of noncanonical NF-κB signaling leads to increased colonic numbers of Tregs and IgA+ B cells, which promote luminal IgA production and foster eubiosis. Experimentally introduced ß-catenin haploinsufficiency in DCs with deficient noncanonical NF-κB signaling moderates Raldh2 activity, reinstating colitogenic sensitivity in mice. Finally, inflammatory bowel-disease patients also display a deleterious noncanonical NF-κB signaling signature in intestinal DCs. In sum, we establish how noncanonical NF-κB signaling in dendritic cells can subvert retinoic acid synthesis to fuel intestinal inflammation.

Oxysterol Sensing through the Receptor GPR183 Promotes the Lymphoid-Tissue-Inducing Function of Innate Lymphoid Cells and Colonic Inflammation.

Group 3 innate lymphoid cells (ILC3s) sense environmental signals and are critical for tissue integrity in the intestine. Yet, which signals are sensed and what receptors control ILC3 function remain poorly understood. Here, we show that ILC3s with a lymphoid-tissue-inducer (LTi) phenotype expressed G-protein-coupled receptor 183 (GPR183) and migrated to its oxysterol ligand 7α,25-hydroxycholesterol (7α,25-OHC). In mice lacking Gpr183 or 7α,25-OHC, ILC3s failed to localize to cryptopatches (CPs) and isolated lymphoid follicles (ILFs). Gpr183 deficiency in ILC3s caused a defect in CP and ILF formation in the colon, but not in the small intestine. Localized oxysterol production by fibroblastic stromal cells provided an essential signal for colonic lymphoid tissue development, and inflammation-induced increased oxysterol production caused colitis through GPR183-mediated cell recruitment. Our findings show that GPR183 promotes lymphoid organ development and indicate that oxysterol-GPR183-dependent positioning within tissues controls ILC3 activity and intestinal homeostasis.

Microbiota-Induced TNF-like Ligand 1A Drives Group 3 Innate Lymphoid Cell-Mediated Barrier Protection and Intestinal T Cell Activation during Colitis.

Inflammatory bowel disease (IBD) results from a dysregulated interaction between the microbiota and a genetically susceptible host. Genetic studies have linked TNFSF15 polymorphisms and its protein TNF-like ligand 1A (TL1A) with IBD, but the functional role of TL1A is not known. Here, we found that adherent IBD-associated microbiota induced TL1A release from CX3CR1+ mononuclear phagocytes (MNPs). Using cell-specific genetic deletion models, we identified an essential role for CX3CR1+MNP-derived TL1A in driving group 3 innate lymphoid cell (ILC3) production of interleukin-22 and mucosal healing during acute colitis. In contrast to this protective role in acute colitis, TL1A-dependent expression of co-stimulatory molecule OX40L in MHCII+ ILC3s during colitis led to co-stimulation of antigen-specific T cells that was required for chronic T cell colitis. These results identify a role for ILC3s in activating intestinal T cells and reveal a central role for TL1A in promoting ILC3 barrier immunity during colitis.

C. difficile exploits a host metabolite produced during toxin-mediated disease.

Several enteric pathogens can gain specific metabolic advantages over other members of the microbiota by inducing host pathology and inflammation. The pathogen Clostridium difficile is responsible for a toxin-mediated colitis that causes 450,000 infections and 15,000 deaths in the United States each year1; however, the molecular mechanisms by which C. difficile benefits from this pathology remain unclear. To understand how the metabolism of C. difficile adapts to the inflammatory conditions that its toxins induce, here we use RNA sequencing to define, in a mouse model, the metabolic states of wild-type C. difficile and of an isogenic mutant that lacks toxins. By combining bacterial and mouse genetics, we demonstrate that C. difficile uses sorbitol derived from both diet and host. Host-derived sorbitol is produced by the enzyme aldose reductase, which is expressed by diverse immune cells and is upregulated during inflammation-including during toxin-mediated disease induced by C. difficile. This work highlights a mechanism by which C. difficile can use a host-derived nutrient that is generated during toxin-induced disease by an enzyme that has not previously been associated with infection.

The Crohn's Disease Risk Factor IRGM Limits NLRP3 Inflammasome Activation by Impeding Its Assembly and by Mediating Its Selective Autophagy.

Several large-scale genome-wide association studies genetically linked IRGM to Crohn's disease and other inflammatory disorders in which the IRGM appears to have a protective function. However, the mechanism by which IRGM accomplishes this anti-inflammatory role remains unclear. Here, we reveal that IRGM/Irgm1 is a negative regulator of the NLRP3 inflammasome activation. We show that IRGM expression, which is increased by PAMPs, DAMPs, and microbes, can suppress the pro-inflammatory responses provoked by the same stimuli. IRGM/Irgm1 negatively regulates IL-1ß maturation by suppressing the activation of the NLRP3 inflammasome. Mechanistically, we show that IRGM interacts with NLRP3 and ASC and hinders inflammasome assembly by blocking their oligomerization. Further, IRGM mediates selective autophagic degradation of NLRP3 and ASC. By suppressing inflammasome activation, IRGM/Irgm1 protects from pyroptosis and gut inflammation in a Crohn's disease experimental mouse model. This study for the first time identifies the mechanism by which IRGM is protective against inflammatory disorders.

Intestinal Lactobacillus murinus-derived small RNAs target porcine polyamine metabolism.

Gut microbiota plays a vital role in host metabolism; however, the influence of gut microbes on polyamine metabolism is unknown. Here, we found germ-free models possess elevated polyamine levels in the colon. Mechanistically, intestinal Lactobacillus murinus-derived small RNAs in extracellular vesicles down-regulate host polyamine metabolism by targeting the expression of enzymes in polyamine metabolism. In addition, Lactobacillus murinus delays recovery of dextran sodium sulfate-induced colitis by reducing polyamine levels in mice. Notably, a decline in the abundance of small RNAs was observed in the colon of mice with colorectal cancer (CRC) and human CRC specimens, accompanied by elevated polyamine levels. Collectively, our study identifies a specific underlying mechanism used by intestinal microbiota to modulate host polyamine metabolism, which provides potential intervention for the treatment of polyamine-associated diseases.