Intra- and Inter-cellular Rewiring of the Human Colon during Ulcerative Colitis.

Genome-wide association studies (GWAS) have revealed risk alleles for ulcerative colitis (UC). To understand their cell type specificities and pathways of action, we generate an atlas of 366,650 cells from the colon mucosa of 18 UC patients and 12 healthy individuals, revealing 51 epithelial, stromal, and immune cell subsets, including BEST4+ enterocytes, microfold-like cells, and IL13RA2+IL11+ inflammatory fibroblasts, which we associate with resistance to anti-TNF treatment. Inflammatory fibroblasts, inflammatory monocytes, microfold-like cells, and T cells that co-express CD8 and IL-17 expand with disease, forming intercellular interaction hubs. Many UC risk genes are cell type specific and co-regulated within relatively few gene modules, suggesting convergence onto limited sets of cell types and pathways. Using this observation, we nominate and infer functions for specific risk genes across GWAS loci. Our work provides a framework for interrogating complex human diseases and mapping risk variants to cell types and pathways.

C1orf106 is a colitis risk gene that regulates stability of epithelial adherens junctions.

Polymorphisms in C1orf106 are associated with increased risk of inflammatory bowel disease (IBD). However, the function of C1orf106 and the consequences of disease-associated polymorphisms are unknown. Here we demonstrate that C1orf106 regulates adherens junction stability by regulating the degradation of cytohesin-1, a guanine nucleotide exchange factor that controls activation of ARF6. By limiting cytohesin-1-dependent ARF6 activation, C1orf106 stabilizes adherens junctions. Consistent with this model, C1orf106-/- mice exhibit defects in the intestinal epithelial cell barrier, a phenotype observed in IBD patients that confers increased susceptibility to intestinal pathogens. Furthermore, the IBD risk variant increases C1orf106 ubiquitination and turnover with consequent functional impairments. These findings delineate a mechanism by which a genetic polymorphism fine-tunes intestinal epithelial barrier integrity and elucidate a fundamental mechanism of cellular junctional control.

Three Unique Interstitial Macrophages in the Murine Lung at Steady State.

The current paradigm in macrophage biology is that some tissues mainly contain macrophages from embryonic origin, such as microglia in the brain, whereas other tissues contain postnatal-derived macrophages, such as the gut. However, in the lung and in other organs, such as the skin, there are both embryonic and postnatal-derived macrophages. In this study, we demonstrate in the steady-state lung that the mononuclear phagocyte system is comprised of three newly identified interstitial macrophages (IMs), alveolar macrophages, dendritic cells, and few extravascular monocytes. We focused on similarities and differences between the three IM subtypes, specifically, their phenotype, location, transcriptional signature, phagocytic capacity, turnover, and lack of survival dependency on fractalkine receptor, CX3CR1. Pulmonary IMs were located in the bronchial interstitium but not the alveolar interstitium. At the transcriptional level, all three IMs displayed a macrophage signature and phenotype. All IMs expressed MER proto-oncogene, tyrosine kinase, CD64, CD11b, and CX3CR1, and were further distinguished by differences in cell surface protein expression of CD206, Lyve-1, CD11c, CCR2, and MHC class II, along with the absence of Ly6C, Ly6G, and Siglec F. Most intriguingly, in addition to the lung, similar phenotypic populations of IMs were observed in other nonlymphoid organs, perhaps highlighting conserved functions throughout the body. These findings promote future research to track four distinct pulmonary macrophages and decipher the division of labor that exists between them.

TMEM258 Is a Component of the Oligosaccharyltransferase Complex Controlling ER Stress and Intestinal Inflammation.

Significant insights into disease pathogenesis have been gleaned from population-level genetic studies; however, many loci associated with complex genetic disease contain numerous genes, and phenotypic associations cannot be assigned unequivocally. In particular, a gene-dense locus on chromosome 11 (61.5-61.65 Mb) has been associated with inflammatory bowel disease, rheumatoid arthritis, and coronary artery disease. Here, we identify TMEM258 within this locus as a central regulator of intestinal inflammation. Strikingly, Tmem258 haploinsufficient mice exhibit severe intestinal inflammation in a model of colitis. At the mechanistic level, we demonstrate that TMEM258 is a required component of the oligosaccharyltransferase complex and is essential for N-linked protein glycosylation. Consequently, homozygous deficiency of Tmem258 in colonic organoids results in unresolved endoplasmic reticulum (ER) stress culminating in apoptosis. Collectively, our results demonstrate that TMEM258 is a central mediator of ER quality control and intestinal homeostasis.

A protein-truncating R179X variant in RNF186 confers protection against ulcerative colitis.

Protein-truncating variants protective against human disease provide in vivo validation of therapeutic targets. Here we used targeted sequencing to conduct a search for protein-truncating variants conferring protection against inflammatory bowel disease exploiting knowledge of common variants associated with the same disease. Through replication genotyping and imputation we found that a predicted protein-truncating variant (rs36095412, p.R179X, genotyped in 11,148 ulcerative colitis patients and 295,446 controls, MAF=up to 0.78%) in RNF186, a single-exon ring finger E3 ligase with strong colonic expression, protects against ulcerative colitis (overall P=6.89 × 10(-7), odds ratio=0.30). We further demonstrate that the truncated protein exhibits reduced expression and altered subcellular localization, suggesting the protective mechanism may reside in the loss of an interaction or function via mislocalization and/or loss of an essential transmembrane domain.

Flow Cytometric Analysis of Mononuclear Phagocytes in Nondiseased Human Lung and Lung-Draining Lymph Nodes.

RATIONALE: The pulmonary mononuclear phagocyte system is a critical host defense mechanism composed of macrophages, monocytes, monocyte-derived cells, and dendritic cells. However, our current characterization of these cells is limited because it is derived largely from animal studies and analysis of human mononuclear phagocytes from blood and small tissue resections around tumors. OBJECTIVES: Phenotypic and morphologic characterization of mononuclear phagocytes that potentially access inhaled antigens in human lungs. METHODS: We acquired and analyzed pulmonary mononuclear phagocytes from fully intact nondiseased human lungs (including the major blood vessels and draining lymph nodes) obtained en bloc from 72 individual donors. Differential labeling of hematopoietic cells via intrabronchial and intravenous administration of antibodies within the same lobe was used to identify extravascular tissue-resident mononuclear phagocytes and exclude cells within the vascular lumen. Multiparameter flow cytometry was used to identify mononuclear phagocyte populations among cells labeled by each route of antibody delivery. MEASUREMENTS AND MAIN RESULTS: We performed a phenotypic analysis of pulmonary mononuclear phagocytes isolated from whole nondiseased human lungs and lung-draining lymph nodes. Five pulmonary mononuclear phagocytes were observed, including macrophages, monocyte-derived cells, and dendritic cells that were phenotypically distinct from cell populations found in blood. CONCLUSIONS: Different mononuclear phagocytes, particularly dendritic cells, were labeled by intravascular and intrabronchial antibody delivery, countering the notion that tissue and blood mononuclear phagocytes are equivalent systems. Phenotypic descriptions of the mononuclear phagocytes in nondiseased lungs provide a precedent for comparative studies in diseased lungs and potential targets for therapeutics.

Transcriptome analysis highlights the conserved difference between embryonic and postnatal-derived alveolar macrophages.

Alveolar macrophages (AMs) reside on the luminal surfaces of the airways and alveoli where they maintain host defense and promote alveolar homeostasis by ingesting inhaled particulates and regulating inflammatory responses. Recent studies have demonstrated that AMs populate the lungs during embryogenesis and self-renew throughout life with minimal replacement by circulating monocytes, except under extreme conditions of depletion or radiation injury. Here we demonstrate that on a global scale, environment appears to dictate AM development and function. Indeed, transcriptome analysis of embryonic host-derived and postnatal donor-derived AMs coexisting within the same mouse demonstrated >98% correlation and overall functional analyses were similar. However, we also identified several genes whose expression was dictated by origin rather than environment. The most differentially expressed gene not altered by environment was Marco, a gene recently demonstrated to have enhancer activity in embryonic-derived but not postnatal-derived tissue macrophages. Overall, we show that under homeostatic conditions, the environment largely dictates the programming and function of AMs, whereas the expression of a small number of genes remains linked to the origin of the cell.

Cutting Edge: Roles for Batf3-Dependent APCs in the Rejection of Minor Histocompatibility Antigen-Mismatched Grafts.

In transplantation, a major obstacle for graft acceptance in MHC-matched individuals is the mismatch of minor histocompatibility Ags. Minor histocompatibility Ags are peptides derived from polymorphic proteins that can be presented by APCs on MHC molecules. The APC subtype uniquely responsible for the rejection of minor Ag-mismatched grafts has not yet been identified. In this study, we examined graft rejection in three mouse models: 1) mismatch of male-specific minor Ags, 2) mismatch of minor Ags distinct from male-specific minor Ags, and 3) skin transplant. This study demonstrates that in the absence of pathogen-associated molecular patterns, Batf3-dependent dendritic cells elicit the rejection of cells and grafts expressing mismatched minor Ags. The implication of our findings in clinical transplantation may be significant, as minor Ag reactivity has been implicated in the pathogenesis of multiple allograft tissues.

MHC-restricted phosphopeptides from insulin receptor substrate-2 and CDC25b offer broad-based immunotherapeutic agents for cancer.

Cancer cells display novel phosphopeptides in association with MHC class I and II molecules. In this study, we evaluated two HLA-A2-restricted phosphopeptides derived from the insulin receptor substrate (IRS)-2 and the cell-cycle regulator CDC25b. These proteins are both broadly expressed in multiple malignancies and linked to cancer cell survival. Two phosphopeptides, termed pIRS-21097-1105 and pCDC25b38-46, served as targets of strong and specific CD8 T-cell memory responses in normal human donors. We cloned T-cell receptor (TCR) cDNAs from murine CD8 T-cell lines specific for either pIRS-21097-1105 or pCDC25b38-46. Expression of these TCRs in human CD8 T cells imparted high-avidity phosphopeptide-specific recognition and cytotoxic and cytokine-secreting effector activities. Using these cells, we found that endogenously processed pIRS-21097-1105 was presented on HLA-A2(+) melanomas and breast, ovarian, and colorectal carcinomas. Presentation was correlated with the level of the Ser(1100)-phosphorylated IRS-2 protein in metastatic melanoma tissues. The highest expression of this protein was evident on dividing malignant cells. Presentation of endogenously processed pCDC25b38-46 was narrower, but still evident on HLA-A2(+) melanoma, breast carcinoma, and lymphoblastoid cells. Notably, pIRS-21097-1105-specific and pCDC25b38-46-specific TCR-expressing human CD8 T cells markedly slowed tumor outgrowth in vivo. Our results define two new antigens that may be developed as immunotherapeutic agents for a broad range of HLA-A2(+) cancers.

Dendritic cell subsets require cis-activation for cytotoxic CD8 T-cell induction.

Dendritic cells (DCs) are required for the induction of cytotoxic T cells (CTL). In most tissues, including the lung, the resident DCs fall into two types expressing the integrin markers CD103 and CD11b. The current supposition is that DC function is predetermined by lineage, designating the CD103(+) DC as the major cross-presenting DC able to induce CTL. Here we show that Poly I:C (TLR3 agonist) or R848 (TLR7 agonist) do not activate all endogenous DCs. CD11b(+) DCs can orchestrate a CTL response in vivo in the presence of a TLR7 agonist but not a TLR3 agonist, whereas CD103(+) DCs require ligation of TLR3 for this purpose. This selectivity does not extend to antigen cross-presentation for T-cell proliferation but is required for induction of cytotoxicity. Thus, we demonstrate that the ability of DCs to induce functional CTLs is specific to the nature of the pathogen-associated molecular pattern (PAMP) encountered by endogenous DC.

Circulating but not immobilized N-deglycosylated von Willebrand factor increases platelet adhesion under flow conditions.

The role of von Willebrand factor (VWF) as a shear stress activated platelet adhesive has been related to a coiled-elongated shape conformation. The forces dominating this transition have been suggested to be controlled by the proteins polymeric architecture. However, the fact that 20% of VWF molecular weight originates from glycan moieties has so far been neglected in these calculations. In this study, we present a systematic experimental investigation on the role of N-glycosylation for VWF mediated platelet adhesion under flow. A microfluidic flow chamber with a stenotic compartment that allows one to mimic various physiological flow conditions was designed for the efficient analysis of the adhesion spectrum. Surprisingly, we found an increase in platelet adhesion with elevated shear rate, both qualitatively and quantitatively fully conserved when N-deglycosylated VWF (N-deg-VWF) instead of VWF was immobilized in the microfluidic channel. This has been demonstrated consistently over four orders of magnitude in shear rate. In contrast, when N-deg-VWF was added to the supernatant, an increase in adhesion rate by a factor of two was detected compared to the addition of wild-type VWF. It appears that once immobilized, the role of glycans is at least modified if not-as found here for the case of adhesion-negated. These findings strengthen the physical impact of the circulating polymer on shear dependent platelet adhesion events. At present, there is no theoretical explanation for an increase in platelet adhesion to VWF in the absence of its N-glycans. However, our data indicate that the effective solubility of the protein and hence its shape or conformation may be altered by the degree of glycosylation and is therefore a good candidate for modifying the forces required to uncoil this biopolymer.

Pulmonary dendritic cell development and antigen acquisition.

Pulmonary dendritic cells (DCs) constantly sample the tissue and traffic inhaled antigens to the lung-draining lymph node where they normally orchestrate an appropriate immune response. The dynamic ability of these professional antigen-presenting cells to promote tolerance or immunity has been intensively studied by several groups, including ours. Distinct DC subsets in both lymphoid and non-lymphoid tissues have been described based on their surface molecule expression and location. Current efforts to unravel DC development and function are providing insight into the various roles each subset offers the immune system. Elucidating DC functions, particularly in the lung, may then allow use of the inherent ability of these cells for enhanced vaccine strategies and therapeutics for pulmonary infections and diseases.

CD103+ pulmonary dendritic cells preferentially acquire and present apoptotic cell-associated antigen.

Cells undergoing programmed cell death (apoptosis) are removed in situ by macrophages and dendritic cells (DCs) through a specialized form of phagocytosis (efferocytosis). In the lung, there are two primary DC subsets with the potential to migrate to the local lymph nodes (LNs) and initiate adaptive immune responses. In this study, we show that only CD103(+) DCs were able to acquire and transport apoptotic cells to the draining LNs and cross present apoptotic cell-associated antigen to CD8 T cells. In contrast, both the CD11b(hi) and the CD103(+) DCs were able to ingest and traffic latex beads or soluble antigen. CD103(+) DCs selectively exhibited high expression of TLR3, and ligation of this receptor led to enhanced in vivo cytotoxic T cell responses to apoptotic cell-associated antigen. The selective role for CD103(+) DCs was confirmed in Batf3(-/-) mice, which lack this DC subtype. Our findings suggest that CD103(+) DCs are the DC subset in the lung that captures and presents apoptotic cell-associated antigen under homeostatic and inflammatory conditions and raise the possibility for more focused immunological targeting to CD8 T cell responses.