The dendritic cell lineage: ontogeny and function of dendritic cells and their subsets in the steady state and the inflamed setting.

Dendritic cells (DCs) form a remarkable cellular network that shapes adaptive immune responses according to peripheral cues. After four decades of research, we now know that DCs arise from a hematopoietic lineage distinct from other leukocytes, establishing the DC system as a unique hematopoietic branch. Recent work has also established that tissue DCs consist of developmentally and functionally distinct subsets that differentially regulate T lymphocyte function. This review discusses major advances in our understanding of the regulation of DC lineage commitment, differentiation, diversification, and function in situ.

The cis-Regulatory Atlas of the Mouse Immune System.

A complete chart of cis-regulatory elements and their dynamic activity is necessary to understand the transcriptional basis of differentiation and function of an organ system. We generated matched epigenome and transcriptome measurements in 86 primary cell types that span the mouse immune system and its differentiation cascades. This breadth of data enable variance components analysis that suggests that genes fall into two distinct classes, controlled by either enhancer- or promoter-driven logic, and multiple regression that connects genes to the enhancers that regulate them. Relating transcription factor (TF) expression to the genome-wide accessibility of their binding motifs classifies them as predominantly openers or closers of local chromatin accessibility, pinpointing specific cis-regulatory elements where binding of given TFs is likely functionally relevant, validated by chromatin immunoprecipitation sequencing (ChIP-seq). Overall, this cis-regulatory atlas provides a trove of information on transcriptional regulation through immune differentiation and a foundational scaffold to define key regulatory events throughout the immunological genome.

Recirculating Intestinal IgA-Producing Cells Regulate Neuroinflammation via IL-10.

Plasma cells (PC) are found in the CNS of multiple sclerosis (MS) patients, yet their source and role in MS remains unclear. We find that some PC in the CNS of mice with experimental autoimmune encephalomyelitis (EAE) originate in the gut and produce immunoglobulin A (IgA). Moreover, we show that IgA+ PC are dramatically reduced in the gut during EAE, and likewise, a reduction in IgA-bound fecal bacteria is seen in MS patients during disease relapse. Removal of plasmablast (PB) plus PC resulted in exacerbated EAE that was normalized by the introduction of gut-derived IgA+ PC. Furthermore, mice with an over-abundance of IgA+ PB and/or PC were specifically resistant to the effector stage of EAE, and expression of interleukin (IL)-10 by PB plus PC was necessary and sufficient to confer resistance. Our data show that IgA+ PB and/or PC mobilized from the gut play an unexpected role in suppressing neuroinflammation.

Host-Protozoan Interactions Protect from Mucosal Infections through Activation of the Inflammasome.

While conventional pathogenic protists have been extensively studied, there is an underappreciated constitutive protist microbiota that is an integral part of the vertebrate microbiome. The impact of these species on the host and their potential contributions to mucosal immune homeostasis remain poorly studied. Here, we show that the protozoan Tritrichomonas musculis activates the host epithelial inflammasome to induce IL-18 release. Epithelial-derived IL-18 promotes dendritic cell-driven Th1 and Th17 immunity and confers dramatic protection from mucosal bacterial infections. Along with its role as a "protistic" antibiotic, colonization with T. musculis exacerbates the development of T-cell-driven colitis and sporadic colorectal tumors. Our findings demonstrate a novel mutualistic host-protozoan interaction that increases mucosal host defenses at the cost of an increased risk of inflammatory disease.

Crosstalk between muscularis macrophages and enteric neurons regulates gastrointestinal motility.

Intestinal peristalsis is a dynamic physiologic process influenced by dietary and microbial changes. It is tightly regulated by complex cellular interactions; however, our understanding of these controls is incomplete. A distinct population of macrophages is distributed in the intestinal muscularis externa. We demonstrate that, in the steady state, muscularis macrophages regulate peristaltic activity of the colon. They change the pattern of smooth muscle contractions by secreting bone morphogenetic protein 2 (BMP2), which activates BMP receptor (BMPR) expressed by enteric neurons. Enteric neurons, in turn, secrete colony stimulatory factor 1 (CSF1), a growth factor required for macrophage development. Finally, stimuli from microbial commensals regulate BMP2 expression by macrophages and CSF1 expression by enteric neurons. Our findings identify a plastic, microbiota-driven crosstalk between muscularis macrophages and enteric neurons that controls gastrointestinal motility. PAPERFLICK:

Microbiotas from Humans with Inflammatory Bowel Disease Alter the Balance of Gut Th17 and RORγt+ Regulatory T Cells and Exacerbate Colitis in Mice.

Microbiota are thought to influence the development and progression of inflammatory bowel disease (IBD), but determining generalizable effects of microbiota on IBD etiology requires larger-scale functional analyses. We colonized germ-free mice with intestinal microbiotas from 30 healthy and IBD donors and determined the homeostatic intestinal T cell response to each microbiota. Compared to microbiotas from healthy donors, transfer of IBD microbiotas into germ-free mice increased numbers of intestinal Th17 cells and Th2 cells and decreased numbers of RORγt+ Treg cells. Colonization with IBD microbiotas exacerbated disease in a model where colitis is induced upon transfer of naive T cells into Rag1-/- mice. The proportions of Th17 and RORγt+ Treg cells induced by each microbiota were predictive of human disease status and accounted for disease severity in the Rag1-/- colitis model. Thus, an impact on intestinal Th17 and RORγt+ Treg cell compartments emerges as a unifying feature of IBD microbiotas, suggesting a general mechanism for microbial contribution to IBD pathogenesis.

Innate lymphoid cells integrate stromal and immunological signals to enhance antibody production by splenic marginal zone B cells.

Innate lymphoid cells (ILCs) regulate stromal cells, epithelial cells and cells of the immune system, but their effect on B cells remains unclear. Here we identified RORγt(+) ILCs near the marginal zone (MZ), a splenic compartment that contains innate-like B cells highly responsive to circulating T cell-independent (TI) antigens. Splenic ILCs established bidirectional crosstalk with MAdCAM-1(+) marginal reticular cells by providing tumor-necrosis factor (TNF) and lymphotoxin, and they stimulated MZ B cells via B cell-activation factor (BAFF), the ligand of the costimulatory receptor CD40 (CD40L) and the Notch ligand Delta-like 1 (DLL1). Splenic ILCs further helped MZ B cells and their plasma-cell progeny by coopting neutrophils through release of the cytokine GM-CSF. Consequently, depletion of ILCs impaired both pre- and post-immune TI antibody responses. Thus, ILCs integrate stromal and myeloid signals to orchestrate innate-like antibody production at the interface between the immune system and circulatory system.

The Preprint Club: A blueprint for community-based peer review: A blueprint for community-based peer review.

Cross-institutional journal clubs focused on preprints are a new approach to community-based peer review and allow ERCs to gain experience.

NLRP1B and NLRP3 Control the Host Response following Colonization with the Commensal Protist Tritrichomonas musculis.

Commensal intestinal protozoa, unlike their pathogenic relatives, are neglected members of the mammalian microbiome. These microbes have a significant impact on the host's intestinal immune homeostasis, typically by elevating anti-microbial host defense. Tritrichomonas musculis, a protozoan gut commensal, strengthens the intestinal host defense against enteric Salmonella infections through Asc- and Il1r1-dependent Th1 and Th17 cell activation. However, the underlying inflammasomes mediating this effect remain unknown. In this study, we report that colonization with T. musculis results in an increase in luminal extracellular ATP that is followed by increased caspase activity, higher cell death, elevated levels of IL-1ß, and increased numbers of IL-18 receptor-expressing Th1 and Th17 cells in the colon. Mice deficient in either Nlrp1b or Nlrp3 failed to display these protozoan-driven immune changes and lost resistance to enteric Salmonella infections even in the presence of T. musculis These findings demonstrate that T. musculis-mediated host protection requires sensors of extracellular and intracellular ATP to confer resistance to enteric Salmonella infections.

Vasoactive intestinal peptide promotes host defense against enteric pathogens by modulating the recruitment of group 3 innate lymphoid cells.

Group 3 innate lymphoid cells (ILC3s) control the formation of intestinal lymphoid tissues and play key roles in intestinal defense. They express neuropeptide vasoactive intestinal peptide (VIP) receptor 2 (VPAC2), through which VIP modulates their function, but whether VIP exerts other effects on ILC3 remains unclear. We show that VIP promotes ILC3 recruitment to the intestine through VPAC1 independent of the microbiota or adaptive immunity. VIP is also required for postnatal formation of lymphoid tissues as well as the maintenance of local populations of retinoic acid (RA)-producing dendritic cells, with RA up-regulating gut-homing receptor CCR9 expression by ILC3s. Correspondingly, mice deficient in VIP or VPAC1 suffer a paucity of intestinal ILC3s along with impaired production of the cytokine IL-22, rendering them highly susceptible to the enteric pathogen Citrobacter rodentium This heightened susceptibility to C. rodentium infection was ameliorated by RA supplementation, adoptive transfer of ILC3s, or by recombinant IL-22. Thus, VIP regulates the recruitment of intestinal ILC3s and formation of postnatal intestinal lymphoid tissues, offering protection against enteric pathogens.

Neutralizing Anti-Granulocyte Macrophage-Colony Stimulating Factor Autoantibodies Recognize Post-Translational Glycosylations on Granulocyte Macrophage-Colony Stimulating Factor Years Before Diagnosis and Predict Complicated Crohn's Disease.

BACKGROUND & AIMS: Anti-granulocyte macrophage-colony stimulating factor autoantibodies (aGMAbs) are detected in patients with ileal Crohn's disease (CD). Their induction and mode of action during or before disease are not well understood. We aimed to investigate the underlying mechanisms associated with aGMAb induction, from functional orientation to recognized epitopes, for their impact on intestinal immune homeostasis and use as a predictive biomarker for complicated CD. METHODS: We characterized using enzyme-linked immunosorbent assay naturally occurring aGMAbs in longitudinal serum samples from patients archived before the diagnosis of CD (n = 220) as well as from 400 healthy individuals (matched controls) as part of the US Defense Medical Surveillance System. We used biochemical, cellular, and transcriptional analysis to uncover a mechanism that governs the impaired immune balance in CD mucosa after diagnosis. RESULTS: Neutralizing aGMAbs were found to be specific for post-translational glycosylation on granulocyte macrophage-colony stimulating factor (GM-CSF), detectable years before diagnosis, and associated with complicated CD at presentation. Glycosylation of GM-CSF was altered in patients with CD, and aGMAb affected myeloid homeostasis and promoted group 1 innate lymphoid cells. Perturbations in immune homeostasis preceded the diagnosis in the serum of patients with CD presenting with aGMAb and were detectable in the noninflamed CD mucosa. CONCLUSIONS: Anti-GMAbs predict the diagnosis of complicated CD long before the diagnosis of disease, recognize uniquely glycosylated epitopes, and impair myeloid cell and innate lymphoid cell balance associated with altered intestinal immune homeostasis.

Tissue-resident macrophages self-maintain locally throughout adult life with minimal contribution from circulating monocytes.

Despite accumulating evidence suggesting local self-maintenance of tissue macrophages in the steady state, the dogma remains that tissue macrophages derive from monocytes. Using parabiosis and fate-mapping approaches, we confirmed that monocytes do not show significant contribution to tissue macrophages in the steady state. Similarly, we found that after depletion of lung macrophages, the majority of repopulation occurred by stochastic cellular proliferation in situ in a macrophage colony-stimulating factor (M-Csf)- and granulocyte macrophage (GM)-CSF-dependent manner but independently of interleukin-4. We also found that after bone marrow transplantation, host macrophages retained the capacity to expand when the development of donor macrophages was compromised. Expansion of host macrophages was functional and prevented the development of alveolar proteinosis in mice transplanted with GM-Csf-receptor-deficient progenitors. Collectively, these results indicate that tissue-resident macrophages and circulating monocytes should be classified as mononuclear phagocyte lineages that are independently maintained in the steady state.

RORgammat and commensal microflora are required for the differentiation of mucosal interleukin 22-producing NKp46+ cells.

The mucosal immune system of the intestine is separated from a vast array of microbes by a single layer of epithelial cells. Cues from the commensal microflora are needed to maintain epithelial homeostasis, but the molecular and cellular identities of these cues are unclear. Here we provide evidence that signals from the commensal microflora contribute to the differentiation of a lymphocyte population coexpressing stimulatory natural killer cell receptors and the transcription factor RORgammat that produced interleukin 22 (IL-22). The emergence of these IL-22-producing RORgammathiNKp46+NK1.1(int) cells depended on RORgammat expression, which indicated that these cells may have been derived from lymphoid tissue-inducer cells. IL-22 released by these cells promoted the production of antimicrobial molecules important in the maintenance of mucosal homeostasis.

GM-CSF controls nonlymphoid tissue dendritic cell homeostasis but is dispensable for the differentiation of inflammatory dendritic cells.

GM-CSF (Csf-2) is a critical cytokine for the in vitro generation of dendritic cells (DCs) and is thought to control the development of inflammatory DCs and resident CD103(+) DCs in some tissues. Here we showed that in contrast to the current understanding, Csf-2 receptor acts in the steady state to promote the survival and homeostasis of nonlymphoid tissue-resident CD103(+) and CD11b(+) DCs. Absence of Csf-2 receptor on lung DCs abrogated the induction of CD8(+) T cell immunity after immunization with particulate antigens. In contrast, Csf-2 receptor was dispensable for the differentiation and innate function of inflammatory DCs during acute injuries. Instead, inflammatory DCs required Csf-1 receptor for their development. Thus, Csf-2 is important in vaccine-induced CD8(+) T cell immunity through the regulation of nonlymphoid tissue DC homeostasis rather than control of inflammatory DCs in vivo.

Neutrophil ageing is regulated by the microbiome.

Blood polymorphonuclear neutrophils provide immune protection against pathogens, but may also promote tissue injury in inflammatory diseases. Although neutrophils are generally considered to be a relatively homogeneous population, evidence for heterogeneity is emerging. Under steady-state conditions, neutrophil heterogeneity may arise from ageing and replenishment by newly released neutrophils from the bone marrow. Aged neutrophils upregulate CXCR4, a receptor allowing their clearance in the bone marrow, with feedback inhibition of neutrophil production via the IL-17/G-CSF axis, and rhythmic modulation of the haematopoietic stem-cell niche. The aged subset also expresses low levels of L-selectin. Previous studies have suggested that in vitro-aged neutrophils exhibit impaired migration and reduced pro-inflammatory properties. Here, using in vivo ageing analyses in mice, we show that neutrophil pro-inflammatory activity correlates positively with their ageing whilst in circulation. Aged neutrophils represent an overly active subset exhibiting enhanced αMß2 integrin activation and neutrophil extracellular trap formation under inflammatory conditions. Neutrophil ageing is driven by the microbiota via Toll-like receptor and myeloid differentiation factor 88-mediated signalling pathways. Depletion of the microbiota significantly reduces the number of circulating aged neutrophils and dramatically improves the pathogenesis and inflammation-related organ damage in models of sickle-cell disease or endotoxin-induced septic shock. These results identify a role for the microbiota in regulating a disease-promoting neutrophil subset.

Interactions Between Diet and the Intestinal Microbiota Alter Intestinal Permeability and Colitis Severity in Mice.

BACKGROUND & AIMS: It is not clear how the complex interactions between diet and the intestinal microbiota affect development of mucosal inflammation or inflammatory bowel disease. We investigated interactions between dietary ingredients, nutrients, and the microbiota in specific pathogen-free (SPF) and germ-free (GF) mice given more than 40 unique diets; we quantified individual and synergistic effects of dietary macronutrients and the microbiota on intestinal health and development of colitis. METHODS: C56BL/6J SPF and GF mice were placed on custom diets containing different concentrations and sources of protein, fat, digestible carbohydrates, and indigestible carbohydrates (fiber). After 1 week, SPF and GF mice were given dextran sulfate sodium (DSS) to induce colitis. Disease severity was determined based on the percent weight change from baseline, and modeled as a function of the concentration of each macronutrient in the diet. In unchallenged mice, we measured intestinal permeability by feeding mice labeled dextran and measuring levels in blood. Feces were collected and microbiota were analyzed by 16S rDNA sequencing. We collected colons from mice and performed transcriptome analyses. RESULTS: Fecal microbiota varied with diet; the concentration of protein and fiber had the strongest effect on colitis development. Among 9 fiber sources tested, psyllium, pectin, and cellulose fiber reduced the severity of colitis in SPF mice, whereas methylcellulose increased severity. Increasing dietary protein increased the density of the fecal microbiota and the severity of colitis in SPF mice, but not in GF mice or mice given antibiotics. Psyllium fiber reduced the severity of colitis through microbiota-dependent and microbiota-independent mechanisms. Combinatorial perturbations to dietary casein protein and psyllium fiber in parallel accounted for most variation in gut microbial density and intestinal permeability in unchallenged mice, as well as the severity of DSS-induced colitis; changes in 1 ingredient could be offset by changes in another. CONCLUSIONS: In an analysis of the effects of different dietary components and the gut microbiota on mice with and without DSS-induced colitis, we found complex mixtures of nutrients affect intestinal permeability, gut microbial density, and development of intestinal inflammation.

Interleukin-1ß-induced IRAK1 ubiquitination is required for TH-GM-CSF cell differentiation in T cell-mediated inflammation.

Accumulating evidence suggests granulocyte macrophage-colony stimulating factor (GM-CSF) can function as an inflammatory mediator, but whether GM-CSF-producing CD4+ T cells (TH-GM-CSF) are a distinct T helper cell subset is lacking. Herein we demonstrate that interleukin (IL)-1ß exclusively drives differentiation of naïve CD4+ T cells into TH-GM-CSF cells via inducing ubiquitination of IL-1 receptor-associated kinase 1 (IRAK1) and subsequent activation of the transcription factor NF-kappaB (NF-κB), independent of RAR-related orphan receptor gamma (RORγt) required for TH17 differentiation. In vivo, TH-GM-CSF cells are present in murine Citrobacter Rodentium infections and mediate colitis following adoptive transfer of CD4+ T cells into Rag1-/- mice via GM-CSF-induced macrophage activation. The TH-GM-CSF cell phenotype is stable and distinct from the TH17 genetic program, but IL-1ß can convert pre-formed TH17 cells into TH-GM-CSF cells, thereby accounting for previously reported associations between IL-17 and GM-CSF. Together, our results newly identify IL-1ß/NF-κB-dependent TH-GM-CSF cells as a unique T helper cell subset and highlight the importance of CD4+ T cell-derived GM-CSF induced macrophage activation as a previously undescribed T cell effector mechanism.