The small and large intestine contain related mesenchymal subsets that derive from embryonic Gli1+ precursors.

The intestinal lamina propria contains a diverse network of fibroblasts that provide key support functions to cells within their local environment. Despite this, our understanding of the diversity, location and ontogeny of fibroblasts within and along the length of the intestine remains incomplete. Here we show that the small and large intestinal lamina propria contain similar fibroblast subsets that locate in specific anatomical niches. Nevertheless, we find that the transcriptional profile of similar fibroblast subsets differs markedly between the small intestine and colon suggesting region specific functions. We perform in vivo transplantation and lineage-tracing experiments to demonstrate that adult intestinal fibroblast subsets, smooth muscle cells and pericytes derive from Gli1-expressing precursors present in embryonic day 12.5 intestine. Trajectory analysis of single cell RNA-seq datasets of E12.5 and adult mesenchymal cells suggest that adult smooth muscle cells and fibroblasts derive from distinct embryonic intermediates and that adult fibroblast subsets develop in a linear trajectory from CD81+ fibroblasts. Finally, we provide evidence that colonic subepithelial PDGFRαhi fibroblasts comprise several functionally distinct populations that originate from an Fgfr2-expressing fibroblast intermediate. Our results provide insights into intestinal stromal cell diversity, location, function, and ontogeny, with implications for intestinal development and homeostasis.

cDC1-derived IL-27 regulates small intestinal CD4+ T cell homeostasis in mice.

The small intestinal lamina propria contains large numbers of IFNγ-producing T helper (Th1) cells that play important roles in intestinal homeostasis and host defense, but the mechanisms underlying their development remain poorly understood. Here, we demonstrate that Th1 cells accumulate in the SI-LP after weaning and are maintained there long term. While both Th17 and Th1 cell accumulation in the SI-LP was microbiota dependent, Th1 cell accumulation uniquely required IL-27 and MHCII expression by cDC1. This reflected a requirement for IL-27 signaling in the priming of Th1 cells rather than for their maintenance once in the mucosa. cDC1-derived IL-27 was essential for maintaining the Th1-Th17 balance within the SI-LP, and in its absence, remaining Th1 cells expressed enhanced levels of Th17 signature genes. In conclusion, we identify cDC1-derived IL-27 as a key regulator of SI-LP Th1-Th17 cell homeostasis.

IRF8 deficiency induces the transcriptional, functional, and epigenetic reprogramming of cDC1 into the cDC2 lineage.

Conventional dendritic cells (cDCs) consist of two major functionally and phenotypically distinct subsets, cDC1 and cDC2, whose development is dependent on distinct sets of transcription factors. Interferon regulatory factor 8 (IRF8) is required at multiple stages of cDC1 development, but its role in committed cDC1 remains unclear. Here, we used Xcr1-cre to delete Irf8 in committed cDC1 and demonstrate that Irf8 is required for maintaining the identity of cDC1. In the absence of Irf8, committed cDC1 acquired the transcriptional, functional, and chromatin accessibility properties of cDC2. This conversion was independent of Irf4 and was associated with the decreased accessibility of putative IRF8, Batf3, and composite AP-1-IRF (AICE)-binding elements, together with increased accessibility of cDC2-associated transcription-factor-binding elements. Thus, IRF8 expression by committed cDC1 is required for preventing their conversion into cDC2-like cells.

The porcine large intestine contains developmentally distinct submucosal lymphoid clusters and mucosal isolated lymphoid follicles.

Gut-associated lymphoid tissues (GALT) serve as key priming sites for intestinal adaptive immune responses. Most of our understanding of GALT function and development arises from studies in mice. However, the diversity, structure and cellular composition of GALT differs markedly between mammalian species and the developmental window in which distinct GALT structures develop in large mammals remains poorly understood. Given the importance of pigs as models of human disease, as well as their role in livestock production, we adapted a recently developed protocol for the isolation of human GALT to assess the diversity, development and immune composition of large intestinal GALT in neonatal and adult pigs. We demonstrate that the large intestine of adult pigs contains two major GALT types; multifollicular submucosal GALT that we term submucosal lymphoid clusters (SLC) which develop prenatally, and as yet undescribed mucosal isolated lymphoid follicles (M-ILF), which arise after birth. Using confocal laser microscopy and flow cytometry, we additionally assess the microanatomy and lymphocyte composition of SLC and M-ILF, compare them to jejunal Peyer's patches (PP), and describe the maturation of these structures. Collectively, our results provide a deeper understanding of the diversity and development of GALT within the porcine large intestine.

Identification and characterization of murine glycoprotein 2-expressing intestinal dendritic cells.

The intestinal lamina propria (LP) contains distinct subsets of classical dendritic cells (cDC), each playing key non-redundant roles in intestinal immune homeostasis. Here, we show that glycoprotein 2 (GP2), a GPI-anchored protein and receptor for bacterial type-I fimbriae, is selectively expressed by CD103+CD11b+ cDC in the murine small intestine (SI). GP2 expression was induced on CD103+CD11b+ cDC within the SI-LP and was regulated by IRF4, TGFßR1- and retinoic acid signalling. Mice selectively lacking Gp2 on CD103+CD11b+ cDC (huLang-Cre.gp2fl/fl mice) had normal numbers and proportions of innate and adaptive immune cells in the SI-LP suggesting that GP2 expression by CD103+CD11b+ cDC is not required for intestinal immune homoeostasis.

Intestinal cDC1 drive cross-tolerance to epithelial-derived antigen via induction of FoxP3+CD8+ Tregs.

Although CD8+ T cell tolerance to tissue-specific antigen (TSA) is essential for host homeostasis, the mechanisms underlying peripheral cross-tolerance and whether they may differ between tissue sites remain to be fully elucidated. Here, we demonstrate that peripheral cross-tolerance to intestinal epithelial cell (IEC)-derived antigen involves the generation and suppressive function of FoxP3+CD8+ T cells. FoxP3+CD8+ Treg generation was dependent on intestinal cDC1, whose absence led to a break of tolerance and epithelial destruction. Mechanistically, intestinal cDC1-derived PD-L1, TGFß, and retinoic acid contributed to the generation of gut-tropic CCR9+CD103+FoxP3+CD8+ Tregs Last, CD103-deficient CD8+ T cells lacked tolerogenic activity in vivo, indicating a role for CD103 in FoxP3+CD8+ Treg function. Our results describe a role for FoxP3+CD8+ Tregs in cross-tolerance in the intestine for which development requires intestinal cDC1.

Human gut-associated lymphoid tissues (GALT); diversity, structure, and function.

Gut-associated lymphoid tissues (GALT) are the key antigen sampling and adaptive immune inductive sites within the intestinal wall. Human GALT includes the multi-follicular Peyer's patches of the ileum, the vermiform appendix, and the numerous isolated lymphoid follicles (ILF) which are distributed along the length of the intestine. Our current understanding of GALT diversity and function derives primarily from studies in mice, and the relevance of many of these findings to human GALT remains unclear. Here we review our current understanding of human GALT diversity, structure, and composition as well as their potential for regulating intestinal immune responses during homeostasis and inflammatory bowel disease (IBD). Finally, we outline some key remaining questions regarding human GALT, the answers to which will advance our understanding of intestinal immune responses and provide potential opportunities to improve the treatment of intestinal diseases.

Identification, isolation and analysis of human gut-associated lymphoid tissues.

Gut-associated lymphoid tissues (GALTs) comprise key intestinal immune inductive sites, including the Peyer's patches of the small intestine and different types of isolated lymphoid follicle (ILF) found along the length of the gut. Our understanding of human GALT is limited due to a lack of protocols for their isolation. Here we describe a technique that, uniquely among intestinal cell isolation protocols, allows identification and isolation of all human GALT, as well as GALT-free intestinal lamina propria (LP). The technique involves the mechanical separation of intestinal mucosa from the submucosa, allowing the identification and isolation of submucosal ILF (SM-ILF), LP-embedded mucosal ILF (M-ILF) and LP free of contaminating lymphoid tissue. Individual SM-ILF, M-ILF and Peyer's patch follicles can be subsequently digested for downstream cellular and molecular characterization. The technique, which takes 4-10 h, will be useful for researchers interested in intestinal immune development and function in health and disease.

Complete Topological Mapping of a Cellular Protein Interactome Reveals Bow-Tie Motifs as Ubiquitous Connectors of Protein Complexes.

The network topology of a protein interactome is shaped by the function of each protein, making it a resource of functional knowledge in tissues and in single cells. Today, this resource is underused, as complete network topology characterization has proved difficult for large protein interactomes. We apply a matrix visualization and decoding approach to a physical protein interactome of a dendritic cell, thereby characterizing its topology with no prior assumptions of structure. We discover 294 proteins, each forming topological motifs called "bow-ties" that tie together the majority of observed protein complexes. The central proteins of these bow-ties have unique network properties, display multifunctional capabilities, are enriched for essential proteins, and are widely expressed in other cells and tissues. Collectively, the bow-tie motifs are a pervasive and previously unnoted topological trend in cellular interactomes. As such, these results provide fundamental knowledge on how intracellular protein connectivity is organized and operates.

Immune Profiling of Human Gut-Associated Lymphoid Tissue Identifies a Role for Isolated Lymphoid Follicles in Priming of Region-Specific Immunity.

The intestine contains some of the most diverse and complex immune compartments in the body. Here we describe a method for isolating human gut-associated lymphoid tissues (GALTs) that allows unprecedented profiling of the adaptive immune system in submucosal and mucosal isolated lymphoid follicles (SM-ILFs and M-ILFs, respectively) as well as in GALT-free intestinal lamina propria (LP). SM-ILF and M-ILF showed distinct patterns of distribution along the length of the intestine, were linked to the systemic circulation through MAdCAM-1+ high endothelial venules and efferent lymphatics, and had immune profiles consistent with immune-inductive sites. IgA sequencing analysis indicated that human ILFs are sites where intestinal adaptive immune responses are initiated in an anatomically restricted manner. Our findings position ILFs as key inductive hubs for regional immunity in the human intestine, and the methods presented will allow future assessment of these compartments in health and disease.

Deletion of IRF4 in Dendritic Cells Leads to Delayed Onset of T Cell-Dependent Colitis.

Classical dendritic cells (cDC) can be classified into two major subsets: Irf8-dependent cDC1 and Irf4-expressing cDC2. Although these subsets play distinct roles in intestinal immune homeostasis, their functions in T cell-driven colitis remain unknown. To assess the role of IRF4 expression in cDC2 in T cell-driven colitis, CD11c-Cre.Irf4 fl/fl and Irf4 fl/fl mice were backcrossed onto a Rag-1 -/- background and used as recipients of CD45RBhiCD4+ T cells. Colitis score and innate immune cell influx were reduced in Cre+ mice 4 wk posttransfer, and these changes were associated with reduced CD4+ T cell counts in both the mesenteric lymph nodes and colon. By 7 wk, colitis score and colon CD4+ T cell numbers were similar in Cre+ and Cre- mice despite a selective reduction in Th17 cells in the colon of Cre+ mice and a continued reduction in CD4+ T cell numbers in mesenteric lymph nodes. Cotransfer of CD25+CD45RBlo CD4+ T cells prevented CD45RBhiCD4+ T cell-driven colitis in both Cre+ and Cre- recipients, demonstrating that IRF4 expression by cDC is not required for CD4+ regulatory T cell-mediated control of colitis. Collectively these results suggest a role for IRF4 expression in cDC2 in the generation of colitogenic CD4+ T cells, which becomes redundant as colitis progresses.

Guidelines for the use of flow cytometry and cell sorting in immunological studies (second edition).

These guidelines are a consensus work of a considerable number of members of the immunology and flow cytometry community. They provide the theory and key practical aspects of flow cytometry enabling immunologists to avoid the common errors that often undermine immunological data. Notably, there are comprehensive sections of all major immune cell types with helpful Tables detailing phenotypes in murine and human cells. The latest flow cytometry techniques and applications are also described, featuring examples of the data that can be generated and, importantly, how the data can be analysed. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid, all written and peer-reviewed by leading experts in the field, making this an essential research companion.

SMAC mimetics promote NIK-dependent inhibition of CD4+ TH17 cell differentiation.

Second mitochondria-derived activator of caspase (SMAC) mimetics (SMs) are selective antagonists of the inhibitor of apoptosis proteins (IAPs), which activate noncanonical NF-κB signaling and promote tumor cell death. Through gene expression analysis, we found that treatment of CD4+ T cells with SMs during T helper 17 (TH17) cell differentiation disrupted the balance between two antagonistic transcription factor modules. Moreover, proteomics analysis revealed that SMs altered the abundance of proteins associated with cell cycle, mitochondrial activity, and the balance between canonical and noncanonical NF-κB signaling. Whereas SMs inhibited interleukin-17 (IL-17) production and ameliorated TH17 cell-driven inflammation, they stimulated IL-22 secretion. Mechanistically, SM-mediated activation of NF-κB-inducing kinase (NIK) and the transcription factors RelB and p52 directly suppressed Il17a expression and IL-17A protein production, as well as the expression of a number of other immune genes. Induction of IL-22 production correlated with the NIK-dependent reduction in cMAF protein abundance and the enhanced activity of the aryl hydrocarbon receptor. Last, SMs also increased IL-9 and IL-13 production and, under competing conditions, favored the differentiation of naïve CD4+ T cells into TH2 cells rather than TH17 cells. These results demonstrate that SMs shape the gene expression and protein profiles of TH17 cells and inhibit TH17 cell-driven autoimmunity.

Retinoic Acid Signaling in Thymic Epithelial Cells Regulates Thymopoiesis.

Despite the essential role of thymic epithelial cells (TEC) in T cell development, the signals regulating TEC differentiation and homeostasis remain incompletely understood. In this study, we show a key in vivo role for the vitamin A metabolite, retinoic acid (RA), in TEC homeostasis. In the absence of RA signaling in TEC, cortical TEC (cTEC) and CD80loMHC class IIlo medullary TEC displayed subset-specific alterations in gene expression, which in cTEC included genes involved in epithelial proliferation, development, and differentiation. Mice whose TEC were unable to respond to RA showed increased cTEC proliferation, an accumulation of stem cell Ag-1hi cTEC, and, in early life, a decrease in medullary TEC numbers. These alterations resulted in reduced thymic cellularity in early life, a reduction in CD4 single-positive and CD8 single-positive numbers in both young and adult mice, and enhanced peripheral CD8+ T cell survival upon TCR stimulation. Collectively, our results identify RA as a regulator of TEC homeostasis that is essential for TEC function and normal thymopoiesis.

Irf4-dependent CD103+CD11b+ dendritic cells and the intestinal microbiome regulate monocyte and macrophage activation and intestinal peristalsis in postoperative ileus.

OBJECTIVE: Postoperative ileus (POI), the most frequent complication after intestinal surgery, depends on dendritic cells (DCs) and macrophages. Here, we have investigated the mechanism that activates these cells and the contribution of the intestinal microbiota for POI induction. DESIGN: POI was induced by manipulating the intestine of mice, which selectively lack DCs, monocytes or macrophages. The disease severity in the small and large intestine was analysed by determining the distribution of orally applied fluorescein isothiocyanate-dextran and by measuring the excretion time of a retrogradely inserted glass ball. The impact of the microbiota on intestinal peristalsis was evaluated after oral antibiotic treatment. RESULTS: We found that Cd11c-Cre+ Irf4flox/flox mice lack CD103+CD11b+ DCs, a DC subset unique to the intestine whose function is poorly understood. Their absence in the intestinal muscularis reduced pathogenic inducible nitric oxide synthase (iNOS) production by monocytes and macrophages and ameliorated POI. Pathogenic iNOS was produced in the jejunum by resident Ly6C- macrophages and infiltrating chemokine receptor 2-dependent Ly6C+ monocytes, but in the colon only by the latter demonstrating differential tolerance mechanisms along the intestinal tract. Consistently, depletion of both cell subsets reduced small intestinal POI, whereas the depletion of Ly6C+ monocytes alone was sufficient to prevent large intestinal POI. The differential role of monocytes and macrophages in small and large intestinal POI suggested a potential role of the intestinal microbiota. Indeed, antibiotic treatment reduced iNOS levels and ameliorated POI. CONCLUSIONS: Our findings reveal that CD103+CD11b+ DCs and the intestinal microbiome are a prerequisite for the activation of intestinal monocytes and macrophages and for dysregulating intestinal motility in POI.

Different populations of CD11b+ dendritic cells drive Th2 responses in the small intestine and colon.

T-helper 2 (Th2) cell responses defend against parasites. Although dendritic cells (DCs) are vital for the induction of T-cell responses, the DC subpopulations that induce Th2 cells in the intestine are unidentified. Here we show that intestinal Th2 responses against Trichuris muris worms and Schistosoma mansoni eggs do not develop in mice with IRF-4-deficient DCs (IRF-4f/f CD11c-cre). Adoptive transfer of conventional DCs, in particular CD11b-expressing DCs from the intestine, is sufficient to prime S. mansoni-specific Th2 responses. Surprisingly, transferred IRF-4-deficient DCs also effectively prime S. mansoni-specific Th2 responses. Egg antigens do not induce the expression of IRF-4-related genes. Instead, IRF-4f/f CD11c-cre mice have fewer CD11b+ migrating DCs and fewer DCs carrying parasite antigens to the lymph nodes. Furthermore, CD11b+CD103+ DCs induce Th2 responses in the small intestine, whereas CD11b+CD103- DCs perform this role in the colon, revealing a specific functional heterogeneity among intestinal DCs in inducing Th2 responses.

Regionalized Development and Maintenance of the Intestinal Adaptive Immune Landscape.

The intestinal immune system has the daunting task of protecting us from pathogenic insults while limiting inflammatory responses against the resident commensal microbiota and providing tolerance to food antigens. This role is particularly impressive when one considers the vast mucosal surface and changing landscape that the intestinal immune system must monitor. In this review, we highlight regional differences in the development and composition of the adaptive immune landscape of the intestine and the impact of local intrinsic and environmental factors that shape this process. To conclude, we review the evidence for a critical window of opportunity for early-life exposures that affect immune development and alter disease susceptibility later in life.

Eosinophils are key regulators of perivascular adipose tissue and vascular functionality.

Obesity impairs the relaxant capacity of adipose tissue surrounding the vasculature (PVAT) and has been implicated in resultant obesity-related hypertension and impaired glucose intolerance. Resident immune cells are thought to regulate adipocyte activity. We investigated the role of eosinophils in mediating normal PVAT function. Healthy PVAT elicits an anti-contractile effect, which was lost in mice deficient in eosinophils, mimicking the obese phenotype, and was restored upon eosinophil reconstitution. Ex vivo studies demonstrated that the loss of PVAT function was due to reduced bioavailability of adiponectin and adipocyte-derived nitric oxide, which was restored after eosinophil reconstitution. Mechanistic studies demonstrated that adiponectin and nitric oxide are released after activation of adipocyte-expressed ß3 adrenoceptors by catecholamines, and identified eosinophils as a novel source of these mediators. We conclude that adipose tissue eosinophils play a key role in the regulation of normal PVAT anti-contractile function.

IRF8 Transcription Factor Controls Survival and Function of Terminally Differentiated Conventional and Plasmacytoid Dendritic Cells, Respectively.

Interferon regulatory factor-8 (IRF8) has been proposed to be essential for development of monocytes, plasmacytoid dendritic cells (pDCs) and type 1 conventional dendritic cells (cDC1s) and remains highly expressed in differentiated DCs. Transcription factors that are required to maintain the identity of terminally differentiated cells are designated "terminal selectors." Using BM chimeras, conditional Irf8(fl/fl) mice and various promotors to target Cre recombinase to different stages of monocyte and DC development, we have identified IRF8 as a terminal selector of the cDC1 lineage controlling survival. In monocytes, IRF8 was necessary during early but not late development. Complete or late deletion of IRF8 had no effect on pDC development or survival but altered their phenotype and gene-expression profile leading to increased T cell stimulatory function but decreased type 1 interferon production. Thus, IRF8 differentially controls the survival and function of terminally differentiated monocytes, cDC1s, and pDCs.

Long-term persistence of human donor alveolar macrophages in lung transplant recipients.

BACKGROUND: Alveolar macrophages (AMFs) are critical regulators of lung function, and may participate in graft rejection following lung transplantation. Recent studies in experimental animals suggest that most AMFs are self-maintaining cells of embryonic origin, but knowledge about the ontogeny and life span of human AMFs is scarce. METHODS: To follow the origin and longevity of AMFs in patients with lung transplantation for more than 100 weeks, we obtained transbronchial biopsies from 10 gender-mismatched patients with lung transplantation. These were subjected to combined in situ hybridisation for X/Y chromosomes and immunofluorescence staining for macrophage markers. Moreover, development of AMFs in humanised mice reconstituted with CD34+ umbilical cord-derived cells was assessed. RESULTS: The number of donor-derived AMFs was unchanged during the 2 year post-transplantation period. A fraction of the AMFs proliferated locally, demonstrating that at least a subset of human AMFs have the capacity to self-renew. Lungs of humanised mice were found to abundantly contain populations of human AMFs expressing markers compatible with a monocyte origin. Moreover, in patients with lung transplantation we found that recipient monocytes seeded the alveoli early after transplantation, and showed subsequent phenotypical changes consistent with differentiation into proliferating mature AMFs. This resulted in a stable mixed chimerism between donor and recipient AMFs throughout the 2-year period. CONCLUSIONS: The finding that human AMFs are maintained in the lung parenchyma for several years indicates that pulmonary macrophage transplantation can be a feasible therapeutic option for patients with diseases caused by dysfunctional AMFs. Moreover, in a lung transplantation setting, long-term persistence of donor AMFs may be important for the development of chronic graft rejection.