The enteric nervous system relays psychological stress to intestinal inflammation.

Mental health profoundly impacts inflammatory responses in the body. This is particularly apparent in inflammatory bowel disease (IBD), in which psychological stress is associated with exacerbated disease flares. Here, we discover a critical role for the enteric nervous system (ENS) in mediating the aggravating effect of chronic stress on intestinal inflammation. We find that chronically elevated levels of glucocorticoids drive the generation of an inflammatory subset of enteric glia that promotes monocyte- and TNF-mediated inflammation via CSF1. Additionally, glucocorticoids cause transcriptional immaturity in enteric neurons, acetylcholine deficiency, and dysmotility via TGF-ß2. We verify the connection between the psychological state, intestinal inflammation, and dysmotility in three cohorts of IBD patients. Together, these findings offer a mechanistic explanation for the impact of the brain on peripheral inflammation, define the ENS as a relay between psychological stress and gut inflammation, and suggest that stress management could serve as a valuable component of IBD care.

Serotonin reduction in post-acute sequelae of viral infection.

Post-acute sequelae of COVID-19 (PASC, "Long COVID") pose a significant global health challenge. The pathophysiology is unknown, and no effective treatments have been found to date. Several hypotheses have been formulated to explain the etiology of PASC, including viral persistence, chronic inflammation, hypercoagulability, and autonomic dysfunction. Here, we propose a mechanism that links all four hypotheses in a single pathway and provides actionable insights for therapeutic interventions. We find that PASC are associated with serotonin reduction. Viral infection and type I interferon-driven inflammation reduce serotonin through three mechanisms: diminished intestinal absorption of the serotonin precursor tryptophan; platelet hyperactivation and thrombocytopenia, which impacts serotonin storage; and enhanced MAO-mediated serotonin turnover. Peripheral serotonin reduction, in turn, impedes the activity of the vagus nerve and thereby impairs hippocampal responses and memory. These findings provide a possible explanation for neurocognitive symptoms associated with viral persistence in Long COVID, which may extend to other post-viral syndromes.

Multiscale 3D genome organization underlies ILC2 ontogenesis and allergic airway inflammation.

Innate lymphoid cells (ILCs) are well-characterized immune cells that play key roles in host defense and tissue homeostasis. Yet, how the three-dimensional (3D) genome organization underlies the development and functions of ILCs is unknown. Herein, we carried out an integrative analysis of the 3D genome structure, chromatin accessibility and gene expression in mature ILCs. Our results revealed that the local 3D configuration of the genome is rewired specifically at loci associated with ILC biology to promote their development and functional differentiation. Importantly, we demonstrated that the ontogenesis of ILC2s and the progression of allergic airway inflammation are determined by a unique local 3D configuration of the region containing the ILC-lineage-defining factor Id2, which is characterized by multiple interactions between the Id2 promoter and distal regulatory elements bound by the transcription factors GATA-3 and RORα, unveiling the mechanism whereby the Id2 expression is specifically controlled in group 2 ILCs.

Quantitative control of Ets1 dosage by a multi-enhancer hub promotes Th1 cell differentiation and protects from allergic inflammation.

Multi-enhancer hubs are spatial clusters of enhancers present across numerous developmental programs. Here, we studied the functional relevance of these three-dimensional structures in T cell biology. Mathematical modeling identified a highly connected multi-enhancer hub at the Ets1 locus, comprising a noncoding regulatory element that was a hotspot for sequence variation associated with allergic disease in humans. Deletion of this regulatory element in mice revealed that the multi-enhancer connectivity was dispensable for T cell development but required for CD4+ T helper 1 (Th1) differentiation. These mice were protected from Th1-mediated colitis but exhibited overt allergic responses. Mechanistically, the multi-enhancer hub controlled the dosage of Ets1 that was required for CTCF recruitment and assembly of Th1-specific genome topology. Our findings establish a paradigm wherein multi-enhancer hubs control cellular competence to respond to an inductive cue through quantitative control of gene dosage and provide insight into how sequence variation within noncoding elements at the Ets1 locus predisposes individuals to allergic responses.

Protein kinase R and the integrated stress response drive immunopathology caused by mutations in the RNA deaminase ADAR1.

The RNA deaminase ADAR1 is an essential negative regulator of the RNA sensor MDA5, and loss of ADAR1 function triggers inappropriate activation of MDA5 by self-RNAs. Mutations in ADAR, the gene that encodes ADAR1, cause human immune diseases, including Aicardi-Goutières syndrome (AGS). However, the mechanisms of MDA5-dependent disease pathogenesis in vivo remain unknown. Here we generated mice with a single amino acid change in ADAR1 that models the most common human ADAR AGS mutation. These Adar mutant mice developed lethal disease that required MDA5, the RIG-I-like receptor LGP2, type I interferons, and the eIF2α kinase PKR. A small-molecule inhibitor of the integrated stress response (ISR) that acts downstream of eIF2α phosphorylation prevented immunopathology and rescued the mice from mortality. These findings place PKR and the ISR as central components of immunopathology in vivo and identify therapeutic targets for treatment of human diseases associated with the ADAR1-MDA5 axis.

The lncRNA ALPHA specifically targets chikungunya virus to control infection.

Arthropod-borne viruses, including the alphavirus chikungunya virus (CHIKV), cause acute disease in millions of people and utilize potent mechanisms to antagonize and circumvent innate immune pathways including the type I interferon (IFN) pathway. In response, hosts have evolved antiviral counterdefense strategies that remain incompletely understood. Recent studies have found that long noncoding RNAs (lncRNAs) regulate classical innate immune pathways; how lncRNAs contribute to additional antiviral counterdefenses remains unclear. Using high-throughput genetic screening, we identified a cytoplasmic antiviral lncRNA that we named antiviral lncRNA prohibiting human alphaviruses (ALPHA), which is transcriptionally induced by alphaviruses and functions independently of IFN to inhibit the replication of CHIKV and its closest relative, O'nyong'nyong virus (ONNV), but not other viruses. Furthermore, we showed that ALPHA interacts with CHIKV genomic RNA and restrains viral RNA replication. Together, our findings reveal that ALPHA and potentially other lncRNAs can mediate non-canonical antiviral immune responses against specific viruses.

Genetic Variation in Type 1 Diabetes Reconfigures the 3D Chromatin Organization of T Cells and Alters Gene Expression.

Genetics is a major determinant of susceptibility to autoimmune disorders. Here, we examined whether genome organization provides resilience or susceptibility to sequence variations, and how this would contribute to the molecular etiology of an autoimmune disease. We generated high-resolution maps of linear and 3D genome organization in thymocytes of NOD mice, a model of type 1 diabetes (T1D), and the diabetes-resistant C57BL/6 mice. Multi-enhancer interactions formed at genomic regions harboring genes with prominent roles in T cell development in both strains. However, diabetes risk-conferring loci coalesced enhancers and promoters in NOD, but not C57BL/6 thymocytes. 3D genome mapping of NODxC57BL/6 F1 thymocytes revealed that genomic misfolding in NOD mice is mediated in cis. Moreover, immune cells infiltrating the pancreas of humans with T1D exhibited increased expression of genes located on misfolded loci in mice. Thus, genetic variation leads to altered 3D chromatin architecture and associated changes in gene expression that may underlie autoimmune pathology.

NLRP6 inflammasome orchestrates the colonic host-microbial interface by regulating goblet cell mucus secretion.

Mucus production by goblet cells of the large intestine serves as a crucial antimicrobial protective mechanism at the interface between the eukaryotic and prokaryotic cells of the mammalian intestinal ecosystem. However, the regulatory pathways involved in goblet cell-induced mucus secretion remain largely unknown. Here, we demonstrate that the NLRP6 inflammasome, a recently described regulator of colonic microbiota composition and biogeographical distribution, is a critical orchestrator of goblet cell mucin granule exocytosis. NLRP6 deficiency leads to defective autophagy in goblet cells and abrogated mucus secretion into the large intestinal lumen. Consequently, NLRP6 inflammasome-deficient mice are unable to clear enteric pathogens from the mucosal surface, rendering them highly susceptible to persistent infection. This study identifies an innate immune regulatory pathway governing goblet cell mucus secretion, linking nonhematopoietic inflammasome signaling to autophagy and highlighting the goblet cell as a critical innate immune player in the control of intestinal host-microbial mutualism. PAPERCLIP:

Microbiota keep the intestinal clock ticking.

Daily metabolic patterns are regulated in a circadian manner often via the hypothalamic axis. Mukherji et al. now report a surprising role for commensal bacteria in the circadian regulation of glucocorticoid production by intestinal epithelial cells.

Group 1 Innate Lymphoid Cell Lineage Identity Is Determined by a cis-Regulatory Element Marked by a Long Non-coding RNA.

Commitment to the innate lymphoid cell (ILC) lineage is determined by Id2, a transcriptional regulator that antagonizes T and B cell-specific gene expression programs. Yet how Id2 expression is regulated in each ILC subset remains poorly understood. We identified a cis-regulatory element demarcated by a long non-coding RNA (lncRNA) that controls the function and lineage identity of group 1 ILCs, while being dispensable for early ILC development and homeostasis of ILC2s and ILC3s. The locus encoding this lncRNA, which we termed Rroid, directly interacted with the promoter of its neighboring gene, Id2, in group 1 ILCs. Moreover, the Rroid locus, but not the lncRNA itself, controlled the identity and function of ILC1s by promoting chromatin accessibility and deposition of STAT5 at the promoter of Id2 in response to interleukin (IL)-15. Thus, non-coding elements responsive to extracellular cues unique to each ILC subset represent a key regulatory layer for controlling the identity and function of ILCs.

NLRP6 inflammasome regulates colonic microbial ecology and risk for colitis.

Inflammasomes are multiprotein complexes that function as sensors of endogenous or exogenous damage-associated molecular patterns. Here, we show that deficiency of NLRP6 in mouse colonic epithelial cells results in reduced IL-18 levels and altered fecal microbiota characterized by expanded representation of the bacterial phyla Bacteroidetes (Prevotellaceae) and TM7. NLRP6 inflammasome-deficient mice were characterized by spontaneous intestinal hyperplasia, inflammatory cell recruitment, and exacerbation of chemical colitis induced by exposure to dextran sodium sulfate (DSS). Cross-fostering and cohousing experiments revealed that the colitogenic activity of this microbiota is transferable to neonatal or adult wild-type mice, leading to exacerbation of DSS colitis via induction of the cytokine, CCL5. Antibiotic treatment and electron microscopy studies further supported the role of Prevotellaceae as a key representative of this microbiota-associated phenotype. Altogether, perturbations in this inflammasome pathway, including NLRP6, ASC, caspase-1, and IL-18, may constitute a predisposing or initiating event in some cases of human IBD.

Nmes1 is a novel regulator of mucosal response influencing intestinal healing potential.

The initiation of tissue remodeling following damage is a critical step in preventing the development of immune-mediated diseases. Several factors contribute to mucosal healing, leading to innovative therapeutic approaches for managing intestinal disorders. However, uncovering alternative targets and gaining mechanistic insights are imperative to enhance therapy efficacy and broaden its applicability across different intestinal diseases. Here we demonstrate that Nmes1, encoding for Normal Mucosa of Esophagus-Specific gene 1, also known as Aa467197, is a novel regulator of mucosal healing. Nmes1 influences the macrophage response to the tissue remodeling cytokine IL-4 in vitro. In addition, using two murine models of intestinal damage, each characterized by a type 2-dominated environment with contrasting functions, the ablation of Nmes1 results in decreased intestinal regeneration during the recovery phase of colitis, while enhancing parasitic egg clearance and reducing fibrosis during the advanced stages of Schistosoma mansoni infection. These outcomes are associated with alterations in CX3CR1+ macrophages, cells known for their wound-healing potential in the inflamed colon, hence promising candidates for cell therapies. All in all, our data indicate Nmes1 as a novel contributor to mucosal healing, setting the basis for further investigation into its potential as a new target for the treatment of colon-associated inflammation.

Ontogeny and heterogeneity of innate lymphoid cells and the noncoding genome.

Since their discovery a decade ago, it has become evident that innate lymphoid cells (ILCs) play critical roles in protective immune responses against intracellular and extracellular pathogens but are also central regulators of epithelial barrier integrity and tissue homeostasis. ILCs populate almost every tissue in mammalian organisms; therefore, not surprisingly, dysregulation of their functions contributes to the development and progression of multiple inflammatory and metabolic diseases. Our knowledge of the transcriptional programs governing the development, differentiation, and functions of the different groups of ILCs has increased dramatically in the last ten years. However, with the advent of new technologies, an unprecedented level of heterogeneity, plasticity, and developmental complexity has started to be revealed. In this review, we highlight recent advances in our understanding of ILC development and their biological functions. In particular, we aim to emphasize how our increasing knowledge of the chromatin landscape and the noncoding genome of these innate lymphocytes is allowing us to better understand their development and functions in different contexts during homeostasis and inflammation. Moreover, we propose that the design of more refined genetic tools to study tissue-specific ILCs and their functions can be accomplished by leveraging our understanding of how specific noncoding elements of the genome regulate gene expression in ILCs.

Inflammasomes: far beyond inflammation.

Nearly a decade ago, the concept of inflammasomes was introduced. Since then, the biochemical characterization of the inflammasomes has led to a richer understanding of innate immune responses in the context of infection and sterile inflammation. This has provided the rationale for successful clinical therapies for a spectrum of hereditary periodic fever syndromes and potentially for some metabolic pathologies.

A new perspective on mesenchymal-immune interactions in adipose tissue.

The mammalian immune system has crucial homeostatic functions in different adipose depots. However, white adipose tissue (WAT) inflammation is a hallmark of obesity and can contribute to type 2 diabetes mellitus (T2DM). Recently, mesenchymal cells were identified as highly heterogenous populations displaying specialized immune functions in immune cell migration, activation, survival, and overall lymphoid tissue organization in several tissues. How they regulate the inflammatory milieu within different adipose depots remains unknown. Using recently published single-cell RNA-sequencing (scRNAseq) data sets, we analyze cytokine and chemokine expression of mouse WAT mesenchymal cell subpopulations to highlight potential immunological heterogeneity and specialization, hypothesizing on their immunological functions. This new perspective on immune-mesenchymal cell interactions in adipose tissue may promote studies that heighten our understanding of immune cell processes within WAT during health and obesity. We hope that these studies redefine our knowledge of the roles of mesenchymal cells in regulating adipose tissue inflammation and physiology.

Increased activity of IRE1 improves the clinical presentation of EAE.

Activation of the endoplasmic reticulum (ER) stress sensor inositol-requiring enzyme-1α (IRE1α) contributes to neuronal development and is known to induce neuronal remodeling in vitro and in vivo. On the contrary, excessive IRE1 activity is often detrimental and may contribute to neurodegeneration. To determine the consequences of increased activation of IRE1α, we used a mouse model expressing a C148S variant of IRE1α with increased and sustained activation. Surprisingly, the mutation did not affect the differentiation of highly secretory antibody-producing cells but exhibited a beneficial effect in a mouse model of experimental autoimmune encephalomyelitis (EAE). Although mechanical allodynia was unaffected, significant improvement in motor function was found in IRE1C148S mice with EAE relative to wild type (WT) mice. Coincident with this improvement, there was reduced microgliosis in the spinal cord of IRE1C148S mice, with reduced expression of proinflammatory cytokine genes. This was accompanied by reduced axonal degeneration and enhanced 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) levels, suggesting improved myelin integrity. Interestingly, while the IRE1C148S mutation is expressed in all cells, the reduction in proinflammatory cytokines and in the microglial activation marker ionized calcium-binding adapter molecule (IBA1), along with preservation of phagocytic gene expression, all point to microglia as the cell type contributing to the clinical improvement in IRE1C148S animals. Our data suggest that sustained increase in IRE1α activity can be beneficial in vivo, and that this protection is cell type and context dependent. Considering the overwhelming but conflicting evidence for the role of ER stress in neurological diseases, a better understanding of the function of ER stress sensors in physiological contexts is clearly needed.

RIPK1 activates distinct gasdermins in macrophages and neutrophils upon pathogen blockade of innate immune signaling.

Injection of effector proteins to block host innate immune signaling is a common strategy used by many pathogenic organisms to establish an infection. For example, pathogenic Yersinia species inject the acetyltransferase YopJ into target cells to inhibit NF-κB and MAPK signaling. To counteract this, detection of YopJ activity in myeloid cells promotes the assembly of a RIPK1-caspase-8 death-inducing platform that confers antibacterial defense. While recent studies revealed that caspase-8 cleaves the pore-forming protein gasdermin D to trigger pyroptosis in macrophages, whether RIPK1 activates additional substrates downstream of caspase-8 to promote host defense is unclear. Here, we report that the related gasdermin family member gasdermin E (GSDME) is activated upon detection of YopJ activity in a RIPK1 kinase-dependent manner. Specifically, GSDME promotes neutrophil pyroptosis and IL-1ß release, which is critical for anti-Yersinia defense. During in vivo infection, IL-1ß neutralization increases bacterial burden in wild-type but not Gsdme-deficient mice. Thus, our study establishes GSDME as an important mediator that counteracts pathogen blockade of innate immune signaling.

The intestinal parasite Cryptosporidium is controlled by an enterocyte intrinsic inflammasome that depends on NLRP6.

The apicomplexan parasite Cryptosporidium infects the intestinal epithelium. While infection is widespread around the world, children in resource-poor settings suffer a disproportionate disease burden. Cryptosporidiosis is a leading cause of diarrheal disease, responsible for mortality and stunted growth in children. CD4 T cells are required to resolve this infection, but powerful innate mechanisms control the parasite prior to the onset of adaptive immunity. Here, we use the natural mouse pathogen Cryptosporidium tyzzeri to demonstrate that the inflammasome plays a critical role in initiating this early response. Mice lacking core inflammasome components, including caspase-1 and apoptosis-associated speck-like protein, show increased parasite burden and caspase 1 deletion solely in enterocytes phenocopies whole-body knockout (KO). This response was fully functional in germfree mice and sufficient to control Cryptosporidium infection. Inflammasome activation leads to the release of IL-18, and mice that lack IL-18 are more susceptible to infection. Treatment of infected caspase 1 KO mice with recombinant IL-18 is remarkably efficient in rescuing parasite control. Notably, NOD-like receptor family pyrin domain containing 6 (NLRP6) was the only NLR required for innate parasite control. Taken together, these data support a model of innate recognition of Cryptosporidium infection through an NLRP6-dependent and enterocyte-intrinsic inflammasome that leads to the release of IL-18 required for parasite control.

An in vivo screen of noncoding loci reveals that Daedalus is a gatekeeper of an Ikaros-dependent checkpoint during haematopoiesis.

Haematopoiesis relies on tightly controlled gene expression patterns as development proceeds through a series of progenitors. While the regulation of hematopoietic development has been well studied, the role of noncoding elements in this critical process is a developing field. In particular, the discovery of new regulators of lymphopoiesis could have important implications for our understanding of the adaptive immune system and disease. Here we elucidate how a noncoding element is capable of regulating a broadly expressed transcription factor, Ikaros, in a lymphoid lineage-specific manner, such that it imbues Ikaros with the ability to specify the lymphoid lineage over alternate fates. Deletion of the Daedalus locus, which is proximal to Ikaros, led to a severe reduction in early lymphoid progenitors, exerting control over the earliest fate decisions during lymphoid lineage commitment. Daedalus locus deletion led to alterations in Ikaros isoform expression and a significant reduction in Ikaros protein. The Daedalus locus may function through direct DNA interaction as Hi-C analysis demonstrated an interaction between the two loci. Finally, we identify an Ikaros-regulated erythroid-lymphoid checkpoint that is governed by Daedalus in a lymphoid-lineage-specific manner. Daedalus appears to act as a gatekeeper of Ikaros's broad lineage-specifying functions, selectively stabilizing Ikaros activity in the lymphoid lineage and permitting diversion to the erythroid fate in its absence. These findings represent a key illustration of how a transcription factor with broad lineage expression must work in concert with noncoding elements to orchestrate hematopoietic lineage commitment.

Genetic targeting of Card19 is linked to disrupted NINJ1 expression, impaired cell lysis, and increased susceptibility to Yersinia infection.

Cell death plays a critical role in inflammatory responses. During pyroptosis, inflammatory caspases cleave Gasdermin D (GSDMD) to release an N-terminal fragment that generates plasma membrane pores that mediate cell lysis and IL-1 cytokine release. Terminal cell lysis and IL-1ß release following caspase activation can be uncoupled in certain cell types or in response to particular stimuli, a state termed hyperactivation. However, the factors and mechanisms that regulate terminal cell lysis downstream of GSDMD cleavage remain poorly understood. In the course of studies to define regulation of pyroptosis during Yersinia infection, we identified a line of Card19-deficient mice (Card19lxcn) whose macrophages were protected from cell lysis and showed reduced apoptosis and pyroptosis, yet had wild-type levels of caspase activation, IL-1 secretion, and GSDMD cleavage. Unexpectedly, CARD19, a mitochondrial CARD-containing protein, was not directly responsible for this, as an independently-generated CRISPR/Cas9 Card19 knockout mouse line (Card19Null) showed no defect in macrophage cell lysis. Notably, Card19 is located on chromosome 13, immediately adjacent to Ninj1, which was recently found to regulate cell lysis downstream of GSDMD activation. RNA-seq and western blotting revealed that Card19lxcn BMDMs have significantly reduced NINJ1 expression, and reconstitution of Ninj1 in Card19lxcn immortalized BMDMs restored their ability to undergo cell lysis in response to caspase-dependent cell death stimuli. Card19lxcn mice exhibited increased susceptibility to Yersinia infection, whereas independently-generated Card19Null mice did not, demonstrating that cell lysis itself plays a key role in protection against bacterial infection, and that the increased infection susceptibility of Card19lxcn mice is attributable to loss of NINJ1. Our findings identify genetic targeting of Card19 being responsible for off-target effects on the adjacent gene Ninj1, disrupting the ability of macrophages to undergo plasma membrane rupture downstream of gasdermin cleavage and impacting host survival and bacterial control during Yersinia infection.