Gasdermin D licenses MHCII induction to maintain food tolerance in small intestine.

The intestinal epithelial cells (IECs) constitute the primary barrier between host cells and numerous foreign antigens; it is unclear how IECs induce the protective immunity against pathogens while maintaining the immune tolerance to food. Here, we found IECs accumulate a less recognized 13-kD N-terminal fragment of GSDMD that is cleaved by caspase-3/7 in response to dietary antigens. Unlike the 30-kD GSDMD cleavage fragment that executes pyroptosis, the IEC-accumulated GSDMD cleavage fragment translocates to the nucleus and induces the transcription of CIITA and MHCII molecules, which in turn induces the Tr1 cells in upper small intestine. Mice treated with a caspase-3/7 inhibitor, mice with GSDMD mutation resistant to caspase-3/7 cleavage, mice with MHCII deficiency in IECs, and mice with Tr1 deficiency all displayed a disrupted food tolerance phenotype. Our study supports that differential cleavage of GSDMD can be understood as a regulatory hub controlling immunity versus tolerance in the small intestine.

Humanized mouse liver reveals endothelial control of essential hepatic metabolic functions.

Hepatocytes, the major metabolic hub of the body, execute functions that are human-specific, altered in human disease, and currently thought to be regulated through endocrine and cell-autonomous mechanisms. Here, we show that key metabolic functions of human hepatocytes are controlled by non-parenchymal cells (NPCs) in their microenvironment. We developed mice bearing human hepatic tissue composed of human hepatocytes and NPCs, including human immune, endothelial, and stellate cells. Humanized livers reproduce human liver architecture, perform vital human-specific metabolic/homeostatic processes, and model human pathologies, including fibrosis and non-alcoholic fatty liver disease (NAFLD). Leveraging species mismatch and lipidomics, we demonstrate that human NPCs control metabolic functions of human hepatocytes in a paracrine manner. Mechanistically, we uncover a species-specific interaction whereby WNT2 secreted by sinusoidal endothelial cells controls cholesterol uptake and bile acid conjugation in hepatocytes through receptor FZD5. These results reveal the essential microenvironmental regulation of hepatic metabolism and its human-specific aspects.

Phase separation drives RNA virus-induced activation of the NLRP6 inflammasome.

NLRP6 is important in host defense by inducing functional outcomes including inflammasome activation and interferon production. Here, we show that NLRP6 undergoes liquid-liquid phase separation (LLPS) upon interaction with double-stranded RNA (dsRNA) in vitro and in cells, and an intrinsically disordered poly-lysine sequence (K350-354) of NLRP6 is important for multivalent interactions, phase separation, and inflammasome activation. Nlrp6-deficient or Nlrp6K350-354A mutant mice show reduced inflammasome activation upon mouse hepatitis virus or rotavirus infection, and in steady state stimulated by intestinal microbiota, implicating NLRP6 LLPS in anti-microbial immunity. Recruitment of ASC via helical assembly solidifies NLRP6 condensates, and ASC further recruits and activates caspase-1. Lipoteichoic acid, a known NLRP6 ligand, also promotes NLRP6 LLPS, and DHX15, a helicase in NLRP6-induced interferon signaling, co-forms condensates with NLRP6 and dsRNA. Thus, LLPS of NLRP6 is a common response to ligand stimulation, which serves to direct NLRP6 to distinct functional outcomes depending on the cellular context.

Epithelial Nlrp10 inflammasome mediates protection against intestinal autoinflammation.

Unlike other nucleotide oligomerization domain-like receptors, Nlrp10 lacks a canonical leucine-rich repeat domain, suggesting that it is incapable of signal sensing and inflammasome formation. Here we show that mouse Nlrp10 is expressed in distal colonic intestinal epithelial cells (IECs) and modulated by the intestinal microbiome. In vitro, Nlrp10 forms an Apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC)-dependent, m-3M3FBS-activated, polyinosinic:polycytidylic acid-modulated inflammasome driving interleukin-1ß and interleukin-18 secretion. In vivo, Nlrp10 signaling is dispensable during steady state but becomes functional during autoinflammation in antagonizing mucosal damage. Importantly, whole-body or conditional IEC Nlrp10 depletion leads to reduced IEC caspase-1 activation, coupled with enhanced susceptibility to dextran sodium sulfate-induced colitis, mediated by altered inflammatory and healing programs. Collectively, understanding Nlrp10 inflammasome-dependent and independent activity, regulation and possible human relevance might facilitate the development of new innate immune anti-inflammatory interventions.

Human hemato-lymphoid system mice: current use and future potential for medicine.

To directly study complex human hemato-lymphoid system physiology and respective system-associated diseases in vivo, human-to-mouse xenotransplantation models for human blood and blood-forming cells and organs have been developed over the past three decades. We here review the fundamental requirements and the remarkable progress made over the past few years in improving these systems, the current major achievements reached by use of these models, and the future challenges to more closely model and study human health and disease and to achieve predictive preclinical testing of both prevention measures and potential new therapies.

Enteric Nervous System-Derived IL-18 Orchestrates Mucosal Barrier Immunity.

Mucosal barrier immunity is essential for the maintenance of the commensal microflora and combating invasive bacterial infection. Although immune and epithelial cells are thought to be the canonical orchestrators of this complex equilibrium, here, we show that the enteric nervous system (ENS) plays an essential and non-redundant role in governing the antimicrobial protein (AMP) response. Using confocal microscopy and single-molecule fluorescence in situ mRNA hybridization (smFISH) studies, we observed that intestinal neurons produce the pleiotropic cytokine IL-18. Strikingly, deletion of IL-18 from the enteric neurons alone, but not immune or epithelial cells, rendered mice susceptible to invasive Salmonella typhimurium (S.t.) infection. Mechanistically, unbiased RNA sequencing and single-cell sequencing revealed that enteric neuronal IL-18 is specifically required for homeostatic goblet cell AMP production. Together, we show that neuron-derived IL-18 signaling controls tissue-wide intestinal immunity and has profound consequences on the mucosal barrier and invasive bacterial killing.

tRNA-m1A modification promotes T cell expansion via efficient MYC protein synthesis.

Naive T cells undergo radical changes during the transition from dormant to hyperactive states upon activation, which necessitates de novo protein production via transcription and translation. However, the mechanism whereby T cells globally promote translation remains largely unknown. Here, we show that on exit from quiescence, T cells upregulate transfer RNA (tRNA) m1A58 'writer' proteins TRMT61A and TRMT6, which confer m1A58 RNA modification on a specific subset of early expressed tRNAs. These m1A-modified early tRNAs enhance translation efficiency, enabling rapid and necessary synthesis of MYC and of a specific group of key functional proteins. The MYC protein then guides the exit of naive T cells from a quiescent state into a proliferative state and promotes rapid T cell expansion after activation. Conditional deletion of the Trmt61a gene in mouse CD4+ T cells causes MYC protein deficiency and cell cycle arrest, disrupts T cell expansion upon cognate antigen stimulation and alleviates colitis in a mouse adoptive transfer colitis model. Our study elucidates for the first time, to our knowledge, the in vivo physiological roles of tRNA-m1A58 modification in T cell-mediated pathogenesis and reveals a new mechanism of tRNA-m1A58-controlled T cell homeostasis and signal-dependent translational control of specific key proteins.

Effector TH17 Cells Give Rise to Long-Lived TRM Cells that Are Essential for an Immediate Response against Bacterial Infection.

Adaptive immunity provides life-long protection by generating central and effector memory T cells and the most recently described tissue resident memory T (TRM) cells. However, the cellular origin of CD4 TRM cells and their contribution to host defense remain elusive. Using IL-17A tracking-fate mouse models, we found that a significant fraction of lung CD4 TRM cells derive from IL-17A-producing effector (TH17) cells following immunization with heat-killed Klebsiella pneumonia (Kp). These exTH17 TRM cells are maintained in the lung by IL-7, produced by lymphatic endothelial cells. During a memory response, neither antibodies, γδ T cells, nor circulatory T cells are sufficient for the rapid host defense required to eliminate Kp. Conversely, using parabiosis and depletion studies, we demonstrated that exTH17 TRM cells play an important role in bacterial clearance. Thus, we delineate the origin and function of airway CD4 TRM cells during bacterial infection, offering novel strategies for targeted vaccine design.

Circulating NK cells establish tissue residency upon acute infection of skin and mediate accelerated effector responses to secondary infection.

Natural killer (NK) cells are present in the circulation and can also be found residing in tissues, and these populations exhibit distinct developmental requirements and are thought to differ in terms of ontogeny. Here, we investigate whether circulating conventional NK (cNK) cells can develop into long-lived tissue-resident NK (trNK) cells following acute infections. We found that viral and bacterial infections of the skin triggered the recruitment of cNK cells and their differentiation into Tcf1hiCD69hi trNK cells that share transcriptional similarity with CD56brightTCF1hi NK cells in human tissues. Skin trNK cells arose from interferon (IFN)-γ-producing effector cells and required restricted expression of the transcriptional regulator Blimp1 to optimize Tcf1-dependent trNK cell formation. Upon secondary infection, trNK cells rapidly gained effector function and mediated an accelerated NK cell response. Thus, cNK cells redistribute and permanently position at sites of previous infection via a mechanism promoting tissue residency that is distinct from Hobit-dependent developmental paths of NK cells and ILC1 seeding tissues during ontogeny.

A metabolic switch orchestrated by IL-18 and the cyclic dinucleotide cGAMP programs intestinal tolerance.

Tissues are exposed to diverse inflammatory challenges that shape future inflammatory responses. While cellular metabolism regulates immune function, how metabolism programs and stabilizes immune states within tissues and tunes susceptibility to inflammation is poorly understood. Here, we describe an innate immune metabolic switch that programs long-term intestinal tolerance. Intestinal interleukin-18 (IL-18) stimulation elicited tolerogenic macrophages by preventing their proinflammatory glycolytic polarization via metabolic reprogramming to fatty acid oxidation (FAO). FAO reprogramming was triggered by IL-18 activation of SLC12A3 (NCC), leading to sodium influx, release of mitochondrial DNA, and activation of stimulator of interferon genes (STING). FAO was maintained in macrophages by a bistable switch that encoded memory of IL-18 stimulation and by intercellular positive feedback that sustained the production of macrophage-derived 2'3'-cyclic GMP-AMP (cGAMP) and epithelial-derived IL-18. Thus, a tissue-reinforced metabolic switch encodes durable immune tolerance in the gut and may enable reconstructing compromised immune tolerance in chronic inflammation.

Circuit Design Features of a Stable Two-Cell System.

Cell communication within tissues is mediated by multiple paracrine signals including growth factors, which control cell survival and proliferation. Cells and the growth factors they produce and receive constitute a circuit with specific properties that ensure homeostasis. Here, we used computational and experimental approaches to characterize the features of cell circuits based on growth factor exchange between macrophages and fibroblasts, two cell types found in most mammalian tissues. We found that the macrophage-fibroblast cell circuit is stable and robust to perturbations. Analytical screening of all possible two-cell circuit topologies revealed the circuit features sufficient for stability, including environmental constraint and negative-feedback regulation. Moreover, we found that cell-cell contact is essential for the stability of the macrophage-fibroblast circuit. These findings illustrate principles of cell circuit design and provide a quantitative perspective on cell interactions.

Deubiquitination of NLRP6 inflammasome by Cyld critically regulates intestinal inflammation.

The inflammasome NLRP6 plays a crucial role in regulating inflammation and host defense against microorganisms in the intestine. However, the molecular mechanisms by which NLRP6 function is inhibited to prevent excessive inflammation remain unclear. Here, we demonstrate that the deubiquitinase Cyld prevents excessive interleukin 18 (IL-18) production in the colonic mucosa by deubiquitinating NLRP6. We show that deubiquitination inhibited the NLRP6-ASC inflammasome complex and regulated the maturation of IL-18. Cyld deficiency in mice resulted in elevated levels of active IL-18 and severe colonic inflammation following Citrobacter rodentium infection. Further, in patients with ulcerative colitis, the concentration of active IL-18 was inversely correlated with CYLD expression. Thus, we have identified a novel regulatory mechanism that inhibits the NLRP6-IL-18 pathway in intestinal inflammation.

T cell-derived interleukin-22 drives the expression of CD155 by cancer cells to suppress NK cell function and promote metastasis.

Although T cells can exert potent anti-tumor immunity, a subset of T helper (Th) cells producing interleukin-22 (IL-22) in breast and lung tumors is linked to dismal patient outcome. Here, we examined the mechanisms whereby these T cells contribute to disease. In murine models of lung and breast cancer, constitutional and T cell-specific deletion of Il22 reduced metastases without affecting primary tumor growth. Deletion of the IL-22 receptor on cancer cells decreases metastasis to a degree similar to that seen in IL-22-deficient mice. IL-22 induced high expression of CD155, which bound to the activating receptor CD226 on NK cells. Excessive activation led to decreased amounts of CD226 and functionally impaired NK cells, which elevated the metastatic burden. IL-22 signaling was also associated with CD155 expression in human datasets and with poor patient outcomes. Taken together, our findings reveal an immunosuppressive circuit activated by T cell-derived IL-22 that promotes lung metastasis.

The Stromal Intervention: Regulation of Immunity and Inflammation at the Epithelial-Mesenchymal Barrier.

The immune system safeguards organ integrity by employing a balancing act of inflammatory and immunosuppressive mechanisms designed to neutralize foreign invaders and resolve injury. Maintaining or restoring a state of immune homeostasis is particularly challenging at barrier sites where constant exposure to immunogenic environmental agents may induce destructive inflammation. Recent studies underscore the role of epithelial and mesenchymal barrier cells in regulating immune cell function and local homeostatic and inflammatory responses. Here, we highlight immunoregulatory circuits engaging epithelial and mesenchymal cells in the intestine, airways, and skin and discuss how immune communications with hematopoietic cells and the microbiota orchestrate local immune homeostasis and inflammation.

Publisher Correction: Subsets of ILC3-ILC1-like cells generate a diversity spectrum of innate lymphoid cells in human mucosal tissues.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Subsets of ILC3-ILC1-like cells generate a diversity spectrum of innate lymphoid cells in human mucosal tissues.

Innate lymphoid cells (ILCs) are tissue-resident lymphocytes categorized on the basis of their core regulatory programs and the expression of signature cytokines. Human ILC3s that produce the cytokine interleukin-22 convert into ILC1-like cells that produce interferon-γ in vitro, but whether this conversion occurs in vivo remains unclear. In the present study we found that ILC3s and ILC1s in human tonsils represented the ends of a spectrum that included additional discrete subsets. RNA velocity analysis identified an intermediate ILC3-ILC1 cluster, which had strong directionality toward ILC1s. In humanized mice, the acquisition of ILC1 features by ILC3s showed tissue dependency. Chromatin studies indicated that the transcription factors Aiolos and T-bet cooperated to repress regulatory elements active in ILC3s. A transitional ILC3-ILC1 population was also detected in the human intestine. We conclude that ILC3s undergo conversion into ILC1-like cells in human tissues in vivo, and that tissue factors and Aiolos were required for this process.

Epithelial IL-18 Equilibrium Controls Barrier Function in Colitis.

The intestinal mucosal barrier controlling the resident microbiome is dependent on a protective mucus layer generated by goblet cells, impairment of which is a hallmark of the inflammatory bowel disease, ulcerative colitis. Here, we show that IL-18 is critical in driving the pathologic breakdown of barrier integrity in a model of colitis. Deletion of Il18 or its receptor Il18r1 in intestinal epithelial cells (Δ/EC) conferred protection from colitis and mucosal damage in mice. In contrast, deletion of the IL-18 negative regulator Il18bp resulted in severe colitis associated with loss of mature goblet cells. Colitis and goblet cell loss were rescued in Il18bp(-/-);Il18r(Δ/EC) mice, demonstrating that colitis severity is controlled at the level of IL-18 signaling in intestinal epithelial cells. IL-18 inhibited goblet cell maturation by regulating the transcriptional program instructing goblet cell development. These results inform on the mechanism of goblet cell dysfunction that underlies the pathology of ulcerative colitis.

Microbiota-Modulated Metabolites Shape the Intestinal Microenvironment by Regulating NLRP6 Inflammasome Signaling.

Host-microbiome co-evolution drives homeostasis and disease susceptibility, yet regulatory principles governing the integrated intestinal host-commensal microenvironment remain obscure. While inflammasome signaling participates in these interactions, its activators and microbiome-modulating mechanisms are unknown. Here, we demonstrate that the microbiota-associated metabolites taurine, histamine, and spermine shape the host-microbiome interface by co-modulating NLRP6 inflammasome signaling, epithelial IL-18 secretion, and downstream anti-microbial peptide (AMP) profiles. Distortion of this balanced AMP landscape by inflammasome deficiency drives dysbiosis development. Upon fecal transfer, colitis-inducing microbiota hijacks this microenvironment-orchestrating machinery through metabolite-mediated inflammasome suppression, leading to distorted AMP balance favoring its preferential colonization. Restoration of the metabolite-inflammasome-AMP axis reinstates a normal microbiota and ameliorates colitis. Together, we identify microbial modulators of the NLRP6 inflammasome and highlight mechanisms by which microbiome-host interactions cooperatively drive microbial community stability through metabolite-mediated innate immune modulation. Therefore, targeted "postbiotic" metabolomic intervention may restore a normal microenvironment as treatment or prevention of dysbiosis-driven diseases.

IL-10 constrains sphingolipid metabolism to limit inflammation.

Interleukin-10 (IL-10) is a key anti-inflammatory cytokine that can limit immune cell activation and cytokine production in innate immune cell types1. Loss of IL-10 signalling results in life-threatening inflammatory bowel disease in humans and mice-however, the exact mechanism by which IL-10 signalling subdues inflammation remains unclear2-5. Here we find that increased saturated very long chain (VLC) ceramides are critical for the heightened inflammatory gene expression that is a hallmark of IL-10 deficiency. Accordingly, genetic deletion of ceramide synthase 2 (encoded by Cers2), the enzyme responsible for VLC ceramide production, limited the exacerbated inflammatory gene expression programme associated with IL-10 deficiency both in vitro and in vivo. The accumulation of saturated VLC ceramides was regulated by a decrease in metabolic flux through the de novo mono-unsaturated fatty acid synthesis pathway. Restoring mono-unsaturated fatty acid availability to cells deficient in IL-10 signalling limited saturated VLC ceramide production and the associated inflammation. Mechanistically, we find that persistent inflammation mediated by VLC ceramides is largely dependent on sustained activity of REL, an immuno-modulatory transcription factor. Together, these data indicate that an IL-10-driven fatty acid desaturation programme rewires VLC ceramide accumulation and aberrant activation of REL. These studies support the idea that fatty acid homeostasis in innate immune cells serves as a key regulatory node to control pathologic inflammation and suggests that 'metabolic correction' of VLC homeostasis could be an important strategy to normalize dysregulated inflammation caused by the absence of IL-10.