Modulating microbiome-immune axis in the deployment-related chronic diseases of Veterans: report of an expert meeting.

The present report summarizes the United States Department of Veterans Affairs (VA) field-based meeting titled "Modulating microbiome-immune axis in the deployment-related chronic diseases of Veterans." Our Veteran patient population experiences a high incidence of service-related chronic physical and mental health problems, such as infection, irritable bowel syndrome (IBS), inflammatory bowel disease (IBD), various forms of hematological and non-hematological malignancies, neurologic conditions, end-stage organ failure, requiring transplantation, and posttraumatic stress disorder (PTSD). We report the views of a group of scientists who focus on the current state of scientific knowledge elucidating the mechanisms underlying the aforementioned disorders, novel therapeutic targets, and development of new approaches for clinical intervention. In conclusion, we dovetailed on four research areas of interest: 1) microbiome interaction with immune cells after hematopoietic cell and/or solid organ transplantation, graft-versus-host disease (GVHD) and graft rejection, 2) intestinal inflammation and its modification in IBD and cancer, 3) microbiome-neuron-immunity interplay in mental and physical health, and 4) microbiome-micronutrient-immune interactions during homeostasis and infectious diseases. At this VA field-based meeting, we proposed to explore a multi-disciplinary, multi-institutional, collaborative strategy to initiate a roadmap, specifically focusing on host microbiome-immune interactions among those with service-related chronic diseases to potentially identify novel and translatable therapeutic targets.

ScRNA-seq defines dynamic T-cell subsets in longitudinal colon and peripheral blood samples in immune checkpoint inhibitor-induced colitis.

Immune checkpoint inhibitors (ICIs) are increasingly being used to manage multiple tumor types. Unfortunately, immune-related adverse events affect up to 60% of recipients, often leading to treatment discontinuation in settings where few alternative cancer therapies may be available. Checkpoint inhibitor induced colitis (ICI-colitis) is a common toxicity for which the underlying mechanisms are poorly defined. To better understand the changing colon-specific and peripheral immune environments over the course of progression and treatment of colitis, we collected blood and colon tissue from a patient with Merkel cell carcinoma who developed colitis on treatment with pembrolizumab. We performed single-cell RNA sequencing and T-cell receptor sequencing on samples collected before, during and after pembrolizumab and after various interventions to mitigate toxicity. We report T-cells populations defined by cytotoxicity, memory, and proliferation markers at various stages of colitis. We show preferential depletion of CD8+ T cells with biologic therapy and nominate both circulating and colon-resident T-cell subsets as potential drivers of inflammation and response to immune suppression. Our findings highlight the need for further exploration of the colon immune environment and rationalize future studies evaluating biologics for ICI-colitis, including in the context of ICI re-challenge.

IL-12 and IL-23 pathway inhibition in inflammatory bowel disease.

Interleukin-12 (IL-12) and interleukin-23 (IL-23), which belong to the IL-12 family of cytokines, have a key role in intestinal homeostasis and inflammation and are implicated in the pathogenesis of inflammatory bowel disease. Upon their secretion by antigen-presenting cells, they exert both pro-inflammatory and anti-inflammatory receptor-mediated effects. An increased understanding of these biological effects, particularly the pro-inflammatory effects mediated by IL-12 and IL-23, has led to the development of monoclonal antibodies that target a subunit common to IL-12 and IL-23 (p40; targeted by ustekinumab and briakinumab), or the IL-23-specific subunit (p19; targeted by risankizumab, guselkumab, brazikumab and mirikizumab). This Review provides a summary of the biology of the IL-12 family cytokines IL-12 and IL-23, discusses the role of these cytokines in intestinal homeostasis and inflammation, and highlights IL-12- and IL-23-directed drug development for the treatment of Crohn's disease and ulcerative colitis.

Pattern Recognition Receptor Signaling and Cytokine Networks in Microbial Defenses and Regulation of Intestinal Barriers: Implications for Inflammatory Bowel Disease.

Inflammatory bowel disease is characterized by defects in epithelial function and dysregulated inflammatory signaling by lamina propria mononuclear cells including macrophages and dendritic cells in response to microbiota. In this review, we focus on the role of pattern recognition receptors in the inflammatory response as well as epithelial barrier regulation. We explore cytokine networks that increase inflammation, regulate paracellular permeability, cause epithelial damage, up-regulate epithelial proliferation, and trigger restitutive processes. We focus on studies using patient samples as well as speculate on pathways that can be targeted to more holistically treat patients with inflammatory bowel disease.

Ubiquitination of ATF6 by disease-associated RNF186 promotes the innate receptor-induced unfolded protein response.

Properly balancing microbial responses by the innate immune system through pattern recognition receptors (PRRs) is critical for intestinal immune homeostasis. Ring finger protein 186 (RNF186) genetic variants are associated with inflammatory bowel disease (IBD). However, functions for the E3 ubiquitin ligase RNF186 are incompletely defined. We found that upon stimulation of the PRR nucleotide-binding oligomerization domain containing 2 (NOD2) in human macrophages, RNF186 localized to the ER, formed a complex with ER stress sensors, ubiquitinated the ER stress sensor activating transcription factor 6 (ATF6), and promoted the unfolded protein response (UPR). These events, in turn, led to downstream signaling, cytokine secretion, and antimicrobial pathway induction. Importantly, RNF186-mediated ubiquitination of K152 on ATF6 was required for these outcomes, highlighting a key role for ATF6 ubiquitination in PRR-initiated functions. Human macrophages transfected with the rare RNF186-A64T IBD risk variant and macrophages from common rs6426833 RNF186 IBD risk carriers demonstrated reduced NOD2-induced outcomes, which were restored by rescuing UPR signaling. Mice deficient in RNF186 or ATF6 demonstrated a reduced UPR in colonic tissues, increased weight loss, and less effective clearance of bacteria with dextran sodium sulfate-induced injury and upon oral challenge with Salmonella Typhimurium. Therefore, we identified that RNF186 was required for PRR-induced, UPR-associated signaling leading to key macrophage functions; defined that RNF186-mediated ubiquitination of ATF6 was essential for these functions; and elucidated how RNF186 IBD risk variants modulated these outcomes.

The Tight Junction Protein ZO-1 Is Dispensable for Barrier Function but Critical for Effective Mucosal Repair.

BACKGROUNDS & AIMS: Increased permeability is implicated in the pathogenesis of intestinal disease. In vitro and in vivo studies have linked down-regulation of the scaffolding protein ZO-1, encoded by the TJP1 gene, to increased tight junction permeability. This has not, however, been tested in vivo. Here, we assessed the contributions of ZO-1 to in vivo epithelial barrier function and mucosal homeostasis. METHODS: Public Gene Expression Omnibus data sets and biopsy specimens from patients with inflammatory bowel disease (IBD) and healthy control individuals were analyzed. Tjp1f/f;vil-CreTg mice with intestinal epithelial-specific ZO-1 knockout (ZO-1KO.IEC) mice and Tjp1f/f mice littermates without Cre expression were studied using chemical and immune-mediated models of disease as well as colonic stem cell cultures. RESULTS: ZO-1 transcript and protein expression were reduced in biopsy specimens from patients with IBD. Despite mildly increased intestinal permeability, ZO-1KO.IEC mice were healthy and did not develop spontaneous disease. ZO-1KO.IEC mice were, however, hypersensitive to mucosal insults and displayed defective repair. Furthermore, ZO-1-deficient colonic epithelia failed to up-regulate proliferation in response to damage in vivo or Wnt signaling in vitro. ZO-1 was associated with centrioles in interphase cells and mitotic spindle poles during division. In the absence of ZO-1, mitotic spindles failed to correctly orient, resulting in mitotic catastrophe and abortive proliferation. ZO-1 is, therefore, critical for up-regulation of epithelial proliferation and successful completion of mitosis. CONCLUSIONS: ZO-1 makes critical, tight junction-independent contributions to Wnt signaling and mitotic spindle orientation. As a result, ZO-1 is essential for mucosal repair. We speculate that ZO-1 down-regulation may be one cause of ineffective mucosal healing in patients with IBD.

The E3 ubiquitin ligase RNF186 and RNF186 risk variants regulate innate receptor-induced outcomes.

Balancing microbial-induced cytokines and microbial clearance is critical at mucosal sites such as the intestine. How the inflammatory bowel disease (IBD)-associated gene RNF186 regulates this balance is unclear. We found that macrophages from IBD-risk rs6426833 carriers in the RNF186 region showed reduced cytokines to stimulation through multiple pattern recognition receptors (PRRs). Upon stimulation of PRRs, the E3-ubiquitin ligase RNF186 promoted ubiquitination of signaling complex molecules shared across PRRs and those unique to select PRRs. Furthermore, RNF186 was required for PRR-initiated signaling complex assembly and downstream signaling. RNF186, along with its intact E3-ubiquitin ligase activity, was required for optimal PRR-induced antimicrobial reactive oxygen species, reactive nitrogen species, and autophagy pathways and intracellular bacterial clearance in human macrophages and for bacterial clearance in intestinal myeloid cells. Cells transfected with the rare RNF186-A64T IBD-risk variant and macrophages from common rs6426833 RNF186 IBD-risk carriers demonstrated a reduction in these RNF186-dependent outcomes. These studies identify mechanisms through which RNF186 regulates innate immunity and show that RNF186 IBD-risk variants demonstrate a loss of function in PRR-initiated outcomes.

TNFSF15 Promotes Antimicrobial Pathways in Human Macrophages and These Are Modulated by TNFSF15 Disease-Risk Variants.

BACKGROUND & AIMS: TNFSF15 genetic variants leading to increased TNF superfamily member 15 (TNFSF15) expression confer risk for inflammatory bowel disease (IBD), and TNFSF15 is being explored as a therapeutic target in IBD patients. Although the focus for TNFSF15-mediated inflammatory outcomes has been predominantly on its action on T cells, TNFSF15 also promotes inflammatory outcomes in human macrophages. Given the critical role for macrophages in bacterial clearance, we hypothesized that TNFSF15 promotes antimicrobial pathways in human macrophages and that macrophages from TNFSF15 IBD risk carriers with higher TNFSF15 expression have an advantage in these antimicrobial outcomes. METHODS: We analyzed protein expression, signaling, bacterial uptake, and intracellular bacterial clearance in human monocyte-derived macrophages through flow cytometry, enzyme-linked immunosorbent assay, and gentamicin protection. RESULTS: Autocrine/paracrine TNFSF15 interactions with death receptor 3 (DR3) were required for optimal levels of pattern-recognition-receptor (PRR)-induced bacterial clearance in human macrophages. TNFSF15 induced pyruvate dehydrogenase kinase 1-dependent bacterial uptake and promoted intracellular bacterial clearance through reactive oxygen species, nitric oxide synthase 2, and autophagy up-regulation. The TNFSF15-initiated TNF receptor-associated factor 2/receptor-interacting protein kinase 1/RIP3 pathway was required for mitogen-activated protein kinase and nuclear factor-κB activation, and, in turn, induction of each of the antimicrobial pathways; the TNFSF15-initiated Fas-associated protein with death domain/mucosa-associated lymphoid tissue lymphoma translocation protein 1/caspase-8 pathway played a less prominent role in antimicrobial functions, despite its key role in TNFSF15-induced cytokine secretion. Complementation of signaling pathways or antimicrobial pathways restored bacterial uptake and clearance in PRR-stimulated macrophages where TNFSF15:DR3 interactions were inhibited. Monocyte-derived macrophages from high TNFSF15-expressing rs6478108 TT IBD risk carriers in the TNFSF15 region showed increased levels of the identified antimicrobial pathways. CONCLUSIONS: We identify that autocrine/paracrine TNFSF15 is required for optimal PRR-enhanced antimicrobial pathways in macrophages, define mechanisms regulating TNFSF15-dependent bacterial clearance, and determine how the TNFSF15 IBD risk genotype modulates these outcomes.

T Cell-Intrinsic IRF5 Regulates T Cell Signaling, Migration, and Differentiation and Promotes Intestinal Inflammation.

IRF5 polymorphisms are associated with multiple immune-mediated diseases, including ulcerative colitis. IRF5 contributions are attributed to its role in myeloid lineages. How T cell-intrinsic IRF5 contributes to inflammatory outcomes is not well understood. We identify a previously undefined key role for T cell-intrinsic IRF5. In mice, IRF5 in CD4+ T cells promotes Th1- and Th17-associated cytokines and decreases Th2-associated cytokines. IRF5 is required for the optimal assembly of the TCR-initiated signaling complex and downstream signaling at early times, and at later times binds to promoters of Th1- and Th17-associated transcription factors and cytokines. IRF5 also regulates chemokine receptor-initiated signaling and, in turn, T cell migration. In vivo, IRF5 in CD4+ T cells enhances the severity of experimental colitis. Importantly, human CD4+ T cells from high IRF5-expressing disease-risk genetic carriers demonstrate increased chemokine-induced migration and Th1/Th17 cytokines and reduced Th2-associated and anti-inflammatory cytokines. These data demonstrate key roles for T cell-intrinsic IRF5 in inflammatory outcomes.

Myeloid Cell-Intrinsic IRF5 Promotes T Cell Responses through Multiple Distinct Checkpoints In Vivo, and IRF5 Immune-Mediated Disease Risk Variants Modulate These Myeloid Cell Functions.

Common IRF5 genetic risk variants associated with multiple immune-mediated diseases are a major determinant of interindividual variability in pattern-recognition receptor (PRR)-induced cytokines in myeloid cells. However, how myeloid cell-intrinsic IRF5 regulates the multiple distinct checkpoints mediating T cell outcomes in vivo and IRF5-dependent mechanisms contributing to these distinct checkpoints are not well defined. Using an in vivo Ag-specific adoptive T cell transfer approach into Irf5-/- mice, we found that T cell-extrinsic IRF5 regulated T cell outcomes at multiple critical checkpoints, including chemokine-mediated T cell trafficking into lymph nodes and PDK1-dependent soluble Ag uptake, costimulatory molecule upregulation, and secretion of Th1 (IL-12)- and Th17 (IL-23, IL-1ß, and IL-6)-conditioning cytokines by myeloid cells, which then cross-regulated Th2 and regulatory T cells. IRF5 was required for PRR-induced MAPK and NF-κB activation, which, in turn, regulated these key outcomes in myeloid cells. Importantly, mice with IRF5 deleted from myeloid cells demonstrated T cell outcomes similar to those observed in Irf5-/- mice. Complementation of IL-12 and IL-23 was able to restore T cell differentiation both in vitro and in vivo in the context of myeloid cell-deficient IRF5. Finally, human monocyte-derived dendritic cells from IRF5 disease-associated genetic risk carriers leading to increased IRF5 expression demonstrated increased Ag uptake and increased PRR-induced costimulatory molecule expression and chemokine and cytokine secretion compared with monocyte-derived dendritic cells from low-expressing IRF5 genetic variant carriers. These data establish that myeloid cell-intrinsic IRF5 regulates multiple distinct checkpoints in T cell activation and differentiation and that these are modulated by IRF5 disease risk variants.

Myeloid Cell Expression of LACC1 Is Required for Bacterial Clearance and Control of Intestinal Inflammation.

BACKGROUND & AIMS: Loss-of-function variants in the laccase domain containing 1 (LACC1) gene are associated with immune-mediated diseases, including inflammatory bowel disease. It is not clear how LACC1 balances defenses against intestinal bacteria vs intestinal inflammation or what cells are responsible for this balance in humans or mice. METHODS: Lacc1-/- mice and mice with myeloid-specific disruption of Lacc1 (Lacc1Δmye) were given oral Salmonella Typhimurium or dextran sodium sulfate. CD45RBhiCD4+T cells were transferred to Lacc1-/-Rag2-/- mice to induce colitis. Organs were collected and analyzed by histology and protein expression. Bone marrow-derived macrophages and dendritic cells, lamina propria macrophages, and mesenteric lymph node dendritic cells were examined. We performed assays to measure intestinal permeability, cell subsets, bacterial uptake and clearance, reactive oxygen species, nitrite production, autophagy, signaling, messenger RNA, and cytokine levels. RESULTS: Lacc1-/- mice developed more severe T-cell transfer colitis than wild-type mice and had an increased burden of bacteria in intestinal lymphoid organs, which expressed lower levels of T helper (Th) 1 and Th17 cytokines and higher levels of Th2 cytokines. Intestinal lymphoid organs from mice with deletion of LACC1 had an increased burden of bacteria after oral administration of S Typhimurium and after administration of dextran sodium sulfate compared with wild-type mice. In macrophages, expression of LACC1 was required for toll-like receptor-induced uptake of bacteria, which required PDK1, and of mitogen-activated protein kinase (MAPK)- and nuclear factor κB-dependent induction of reactive oxygen species, reactive nitrogen species, and autophagy. Expression of LACC1 by dendritic cells was required for increasing expression of Th1 and Th17 cytokines and reducing expression of Th2 cytokines upon coculture with CD4+ T cells. Mice with LACC1-deficient myeloid cells had an increased burden of bacteria and altered T-cell cytokines in intestinal lymphoid organs, similar to Lacc1-/- mice. Complementation of cytokines produced by myeloid cells to cocultures of LACC1-deficient myeloid cells and wild-type CD4+ T cells restored T-cell cytokine regulation. When S Typhimurium-infected Lacc1Δmye mice were injected with these myeloid cell-derived cytokines, intestinal tissues increased production of Th1 and Th17 cytokines, and bacteria were reduced. CONCLUSIONS: Disruption of Lacc1 in mice increases the burden of bacteria in intestinal lymphoid organs and intestinal inflammation after induction of chronic colitis. LACC1 expression by myeloid cells in mice is required to clear bacteria and to regulate adaptive T-cell responses against microbes.

Disease Risk-Associated Genetic Variants in STAT1 and STAT4 Function in a Complementary Manner to Increase Pattern-Recognition Receptor-Induced Outcomes in Human Macrophages.

STAT proteins can regulate both pro- and anti-inflammatory cytokine signaling. Therefore, identifying consequences of modulating expression of a given STAT is ultimately critical for determining its potential as a therapeutic target and for defining the mechanisms through which immune-mediated disease variants in STAT genes contribute to disease pathogenesis. Genetic variants in the STAT1/STAT4 region are associated with multiple immune-mediated diseases, including inflammatory bowel disease (IBD). These diseases are characterized by dysregulated cytokine secretion in response to pattern-recognition receptor (PRR) stimulation. We found that the common IBD-associated rs1517352 C risk allele increased both STAT1 and STAT4 expression in human monocyte-derived macrophages (MDMs). We therefore hypothesized that the STAT1/STAT4 variant might regulate PRR-initiated responses in a complementary and cooperative manner because of the important role of autocrine/paracrine cytokines in modulating PRR-initiated signaling. STAT1 and STAT4 were required for PRR- and live bacterial-induced secretion of multiple cytokines. These outcomes were particularly dependent on PRR-initiated autocrine/paracrine IL-12-induced STAT4 activation to generate IFN-γ, with autocrine IFN-γ then signaling through STAT1. STAT1 and STAT4 also promoted bacterial-induced cytokines in intestinal myeloid cells and PRR-enhanced antimicrobial pathways in MDMs. Importantly, MDMs from rs1517352 C IBD risk allele carriers demonstrated increased TLR4-, IFN-γ- and IL-12-induced STAT1 and STAT4 phosphorylation and cytokine secretion and increased TLR4-enhanced antimicrobial pathways. Taken together, STAT1 and STAT4 expression is coregulated by a shared genetic region, and STAT1 /STAT4-immune disease-associated variants modulate IFN-γ- and IL-12-associated outcomes, and in turn, PRR-induced outcomes, highlighting that these genes cooperate to regulate pathways relevant to disease pathogenesis.

IL23 Promotes Antimicrobial Pathways in Human Macrophages, Which Are Reduced With the IBD-Protective IL23R R381Q Variant.

BACKGROUND & AIMS: Interleukin (IL)23 is a major contributor to inflammatory bowel disease (IBD) pathogenesis and is being pursued as a therapeutic target, both through targeting IL23 alone or in combination with IL12. Unexpected trial outcomes highlight the importance of understanding the cell types through which IL23 regulates immune responses, and how IL23 and IL12 compare in these responses. Macrophages are key players in IBD, and IL23 recently was found to promote inflammatory outcomes in human macrophages. This raises the possibility that IL23 may be required for additional essential macrophage functions, in particular microbial clearance, such that either blocking the IL23 pathway or the IL23R-R381Q IBD-protective variant may reduce macrophage-mediated microbial clearance. METHODS: We analyzed protein expression, signaling, bacterial uptake, and intracellular bacterial clearance in human monocyte-derived macrophages through Western blot, flow cytometry, and gentamicin protection. RESULTS: Autocrine/paracrine IL23 was critical for optimal levels of pattern-recognition-receptor (PRR)-induced intracellular bacterial clearance in human macrophages. Mechanisms regulated by IL23 included induction of pyruvate dehydrogenase kinase 1-dependent bacterial uptake, and up-regulation of reactive oxygen species through nicotinamide adenine dinucleotide phosphate oxidase members, nitric oxide synthase 2, and autophagy through ATG5 and ATG16L1. Complementing these pathways in IL23R-deficient macrophages restored PRR-induced bacterial uptake and clearance. Janus kinase 2, TYK2, and STAT3 were required for IL23-induced mechanisms. IL23 and IL12 induced antimicrobial pathways to similar levels in human macrophages. Relative to IL23R-R381, transfected IL23R-Q381, or monocyte-derived macrophages from IL23R-Q381 carriers showed reduced bacterial uptake and clearance. CONCLUSIONS: We identify that autocrine/paracrine IL23 is required for optimal PRR-enhanced macrophage bacterial uptake and intracellular bacterial clearance, define mechanisms regulating IL23R-induced bacterial clearance, and determine how the IBD-protective IL23R-R381Q variant modulates these processes.

IL23 induces IL23R recycling and amplifies innate receptor-induced signalling and cytokines in human macrophages, and the IBD-protective IL23R R381Q variant modulates these outcomes.

OBJECTIVE: The interleukin (IL)23 pathway contributes to IBD pathogenesis and is being actively studied as a therapeutic target in patients with IBD. Unexpected outcomes in these therapeutic trials have highlighted the importance of understanding the cell types and mechanisms through which IL23 regulates immune outcomes. How IL23 regulates macrophage outcomes and the consequences of the IL23R R381Q IBD-protective variant on macrophages are not well defined; macrophages are key players in IBD pathogenesis and inflammation. DESIGN: We analysed protein and RNA expression, signalling and localisation in human monocyte-derived macrophages (MDMs) through western blot, ELISA, real-time PCR, flow cytometry, immunoprecipitation and microscopy. RESULTS: IL23R was critical for optimal levels of pattern-recognition receptor (PRR)-induced signalling and cytokines in human MDMs. In contrast to the coreceptor IL12Rß1, IL23 induced dynamic IL23R cell surface regulation and this required clathrin and dynamin-mediated endocytosis and endocytic recycling-dependent pathways; these pathways were essential for IL23R-mediated outcomes. The IBD-protective IL23R R381Q variant showed distinct outcomes. Relative to IL23R R381, HeLa cells expressing IL23R Q381 showed decreased IL23R recycling and reduced assembly of IL23R Q381 with Janus kinase/signal transducer and activator of transcription pathway members. In MDMs from IL23R Q381 carriers, IL23R accumulated in late endosomes and lysosomes on IL23 treatment and cells demonstrated decreased IL23R- and PRR-induced signalling and cytokines relative to IL23R R381 MDMs. CONCLUSION: Macrophage-mediated inflammatory pathways are key contributors to IBD pathogenesis, and we identify an autocrine/paracrine IL23 requirement in PRR-initiated human macrophage outcomes and in human intestinal myeloid cells, establish that IL23R undergoes ligand-induced recycling, define mechanisms regulating IL23R-induced signalling and determine how the IBD-protective IL23R R381Q variant modulates these processes.

LACC1 Required for NOD2-Induced, ER Stress-Mediated Innate Immune Outcomes in Human Macrophages and LACC1 Risk Variants Modulate These Outcomes.

LACC1 genetic variants are associated with multiple immune-mediated diseases. However, laccase domain containing-1 (LACC1) functions are incompletely defined. We find that upon stimulation of the pattern-recognition receptor (PRR) NOD2, LACC1 localizes to the endoplasmic reticulum (ER) and forms a complex with ER-stress sensors. All three ER-stress branches, PERK, IRE1α, and ATF6, are required for NOD2-induced signaling, cytokines, and antimicrobial pathways in human macrophages. LACC1, and its localization to the ER, is required for these outcomes. Relative to wild-type (WT) LACC1, transfection of the common Val254 and rare Arg284 immune-mediated disease-risk LACC1 variants into HeLa cells and macrophages, as well as macrophages from LACC1 Val254 carriers, shows reduced NOD2-induced ER stress-associated outcomes; these downstream outcomes are restored by rescuing ER stress. Therefore, we identify ER stress to be essential in PRR-induced outcomes in macrophages, define a critical role for LACC1 in these ER stress-dependent events, and elucidate how LACC1 disease-risk variants mediate these outcomes.

STAT3 and STAT5 Signaling Thresholds Determine Distinct Regulation for Innate Receptor-Induced Inflammatory Cytokines, and STAT3/STAT5 Disease Variants Modulate These Outcomes.

Genetic variants in the STAT3/STAT5A/STAT5B region are associated with immune-mediated diseases, including inflammatory bowel disease (IBD). However, how STAT3 and STAT5 regulate the critical balance between pro- and anti-inflammatory cytokines and how common disease-associated genetic variants (e.g., rs12942547) in the region modulate this balance are incompletely understood. We found that upon pattern-recognition receptor (PRR) stimulation of human monocyte-derived macrophages (MDMs), decreasing STAT3, STAT5a, and STAT5b expression led to a progressive decrease in anti-inflammatory cytokines, whereas proinflammatory cytokines initially decreased but then increased when STAT3 or STAT5 expression fell below a critical threshold. Mechanisms regulating STAT3- and STAT5-dependent inflammatory cytokine outcomes included negative feedback from autocrine/paracrine IL-10, TGF-ß, IL-4, IL-13, IL-22, and TSLP secretion and SOCS1/SOCS2/SOCS3 induction. MDMs from rs12942547 AA disease-risk carriers demonstrated increased STAT3, STAT5a, and STAT5b expression and increased PRR-induced STAT3 and STAT5 phosphorylation relative to GG MDMs. Both pro- and anti-inflammatory cytokine secretion was decreased in MDMs from GG carriers, as STAT3, STAT5a, and STAT5b expression was above the threshold for reciprocal regulation of these cytokines. Taken together, we identify that the threshold of STAT3, STAT5a, and STAT5b expression determines if PRR-induced proinflammatory cytokines are increased or decreased, define mechanisms for this reciprocal regulation, and elucidate consequences for disease variants in the STAT3/STAT5A/STAT5B region, indicating that considering signaling thresholds and targeting specific cell types might be beneficial when evaluating therapeutic interventions in this pathway.

Correction to: Reducing IRF5 expression attenuates colitis in mice, but impairs the clearance of intestinal pathogens.

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

Reducing IRF5 expression attenuates colitis in mice, but impairs the clearance of intestinal pathogens.

IRF5 genetic variants leading to decreased IRF5 expression reduce risk for ulcerative colitis. However, how IRF5 regulates intestinal inflammation and contributes to the balance between defenses against intestinal pathogens and inflammation in vivo, and the cells mediating this balance, are not known. We found that deleting IRF5 in mice led to reduced intestinal inflammation in the T cell transfer colitis model, with reduced Th1 and Th17, and increased Th2 cytokines. However, with orally-administered invasive S. Typhimurium, IRF5-/- mice demonstrated an increased bacterial burden in the context of reduced Th1 and Th17 cytokines. IRF5 in macrophages was required for PDK1-dependent phagocytosis and for NFκB-dependent pathways mediating intracellular bacterial clearance. Despite reduced bacterial clearance pathways, in IRF5-/- mice exposed to high levels of resident intestinal bacteria after DSS-induced injury, the lower levels of inflammatory cytokines were associated with reduced intestinal permeability, and in turn, reduced bacterial translocation and intestinal inflammation. Consistent with the myeloid cell-intrinsic roles for IRF5 in vitro, mice with IRF5 deleted from myeloid cells demonstrated outcomes similar to those observed in IRF5-/- mice. While these data suggest that inhibition of IRF5 may be therapeutic in colitis, this needs to be balanced with the identified IRF5 role in protecting against intestinal pathogens.

Challenges in IBD Research: Precision Medicine.

Precision medicine is part of five focus areas of the Challenges in IBD research document, which also includes preclinical human IBD mechanisms, environmental triggers, novel technologies, and pragmatic clinical research. The Challenges in IBD Research document provides a comprehensive overview of current gaps in inflammatory bowel diseases (IBD) research and delivers actionable approaches to address them. It is the result of a multidisciplinary input from scientists, clinicians, patients, and funders, and represents a valuable resource for patient centric research prioritization. In particular, the precision medicine section is focused on highlighting the main gap areas that must be addressed to get closer to treatments tailored to the biological and clinical characteristics of each patient, which is the aim of precision medicine. The main gaps were identified in: 1) understanding and predicting the natural history of IBD: disease susceptibility, activity, and behavior; 2) predicting disease course and treatment response; and 3) optimizing current and developing new molecular technologies. Suggested approaches to bridge these gaps include prospective longitudinal cohort studies to identify and validate precision biomarkers for prognostication of disease course, and prediction and monitoring of treatment response. To achieve this, harmonization across studies is key as well as development of standardized methods and infrastructure. The implementation of state-of-the-art molecular technologies, systems biology and machine learning approaches for multi-omics and clinical data integration and analysis will be also fundamental. Finally, randomized biomarker-stratified trials will be critical to evaluate the clinical utility of validated signatures and biomarkers in improving patient outcomes and cost-effective care.

Twist1 and Twist2 Induce Human Macrophage Memory upon Chronic Innate Receptor Treatment by HDAC-Mediated Deacetylation of Cytokine Promoters.

Intestinal tissues are continuously exposed to microbial products that stimulate pattern-recognition receptors (PRRs). Ongoing PRR stimulation can confer epigenetic changes in macrophages, which can then regulate subsequent immune outcomes and adaptation to the local environment. Mechanisms leading to these changes are incompletely understood. We found that short-term stimulation of the PRR NOD2 in primary human monocyte-derived macrophages resulted in increased H3 and H4 acetylation of cytokine promoters, consistent with the increased cytokine secretion observed. However, with prolonged NOD2 stimulation, both the acetylation and cytokine secretion were dramatically decreased. Chronic NOD2 stimulation upregulated the transcription factors Twist1 and Twist2, which bound to the promoters of the histone deacetylases HDAC1 and HDAC3 and induced HDAC1 and HDAC3 expression. HDAC1 and HDAC3 then mediated histone deacetylation at cytokine promoters and, in turn, cytokine downregulation under these conditions. Similar regulation was observed upon chronic stimulation of multiple PRRs. Consistent with the chronic microbial exposure in the intestinal environment, TWIST1, TWIST2, HDAC1, and HDAC3 were upregulated in human intestinal relative to peripheral macrophages. Importantly, complementing HDAC1 and HDAC3 in Twist1/Twist2-deficient monocyte-derived macrophages restored the reduced histone acetylation on cytokine promoters and the decreased cytokine secretion with chronic NOD2 stimulation. Taken together, we identify mechanisms wherein Twist1 and Twist2 promote chromatin modifications, resulting in macrophage instruction and adaptation to conditions in the intestinal microenvironment.