The Ubiquitin Binding Protein TAX1BP1 Mediates Autophagasome Induction and the Metabolic Transition of Activated T Cells.

During immune responses, naive T cells transition from small quiescent cells to rapidly cycling cells. We have found that T cells lacking TAX1BP1 exhibit delays in growth of cell size and cell cycling. TAX1BP1-deficient T cells exited G0 but stalled in S phase, due to both bioenergetic and biosynthetic defects. These defects were due to deficiencies in mTOR complex formation and activation. These mTOR defects in turn resulted from defective autophagy induction. TAX1BP1 binding of LC3 and GABARAP via its LC3-interacting region (LIR), but not its ubiquitin-binding domain, supported T cell proliferation. Supplementation of TAX1BP1-deficient T cells with metabolically active L-cysteine rescued mTOR activation and proliferation but not autophagy. These studies reveal that TAX1BP1 drives a specialized form of autophagy, providing critical amino acids that activate mTOR and enable the metabolic transition of activated T cells.

A20 upregulation during treated HIV disease is associated with intestinal epithelial cell recovery and function.

TRIAL REGISTRATION: ClinicalTrials.gov Clinical Trial NCT00594880.

Effect of I-scan Electronic Chromoendoscopy on Detection of Adenomas During Colonoscopy.

BACKGROUND & AIMS: I-scan is an electronic chromoendoscopy technology that improves resolution of epithelial and mucosal surfaces and vessels. We performed a randomized controlled trial to compare detection of adenomas by i-scan vs standard high-definition white-light (HDWL) colonoscopy. METHODS: From February 1 through December 31, 2017, 740 outpatients (50-75 years old) undergoing screening and surveillance for colorectal neoplasia were randomly assigned to groups that received colonoscopies with i-scan 1 (surface and contrast enhancement) or HDWL. When lesions and polyps were detected, endoscopists could switch between i-scan 1 and HDWL imaging to confirm their finding; polyps were collected and analyzed by histology. The primary outcome was adenoma detection rate (ADR, proportion of subjects with at least 1 adenoma of any size); secondary outcomes included detection of sessile serrated polyps and neoplasias, along with location, size, and morphology of polyps. We performed intent to treat and per-protocol analyses (on 357 patients evaluated by i-scan and 358 evaluated by HDWL colonoscopy) to assess the primary and secondary outcomes. RESULTS: There were no differences in baseline characteristics between the groups. In the intent to treat analysis, the ADR was significantly higher in the i-scan 1 group (47.2%) than in the HDWL colonoscopy group (37.7%) (P = .01). In the per-protocol analysis, the ADR in the i-scan 1 group (47.6%) was also significantly higher than in the HDWL group (37.2%) (P = .005), but this effect was not consistent among all endoscopists. There was no difference between groups in detection of sessile serrated polyps. However, the rate of neoplasia detection was significantly higher in the i-scan 1 group (56.4%) than in the than the HDWL group (46.1%) (P = .005). In secondary analyses, the increase in ADR was associated with improved detection of diminutive flat adenomas in the right colon. CONCLUSION: In a prospective randomized trial, higher proportions of patients with adenomas were identified in a group that underwent colonoscopy with i-scan 1 than in a group evaluated by HDWL colonoscopy. This effect was mainly due to improved detection of diminutive, flat right sided adenomas. I-scan 1 technology may benefit some endoscopists. ClinicalTrials.gov no: NCT02811419.

Tetramers reveal IL-17-secreting CD4+ T cells that are specific for U1-70 in lupus and mixed connective tissue disease.

Antigen-specific CD4(+) T cells are implicated in the autoimmune disease systemic lupus erythematosus (SLE), but little is known about the peptide antigens that they recognize and their precise function in disease. We generated a series of MHC class II tetramers of I-E(k)-containing peptides from the spliceosomal protein U1-70 that specifically stain distinct CD4(+) T-cell populations in MRL/lpr mice. The T-cell populations recognize an epitope differing only by the presence or absence of a single phosphate residue at position serine(140). The frequency of CD4(+) T cells specific for U1-70(131-150):I-E(k) (without phosphorylation) correlates with disease severity and anti-U1-70 autoantibody production. These T cells also express RORγt and produce IL-17A. Furthermore, the U1-70-specific CD4(+) T cells that produce IL-17A are detected in a subset of patients with SLE and are significantly increased in patients with mixed connective tissue disease. These studies provide tools for studying antigen-specific CD4(+) T cells in lupus, and demonstrate an antigen-specific source of IL-17A in autoimmune disease.

The U1-snRNP complex: structural properties relating to autoimmune pathogenesis in rheumatic diseases.

The U1 small nuclear ribonucleoprotein particle (snRNP) is a target of autoreactive B cells and T cells in several rheumatic diseases including systemic lupus erythematosus (SLE) and mixed connective tissue disease (MCTD). We propose that inherent structural properties of this autoantigen complex, including common RNA-binding motifs, B and T-cell epitopes, and a unique stimulatory RNA molecule, underlie its susceptibility as a target of the autoimmune response. Immune mechanisms that may contribute to overall U1-snRNP immunogenicity include epitope spreading through B and T-cell interactions, apoptosis-induced modifications, and toll-like receptor (TLR) activation through stimulation by U1-snRNA. We conclude that understanding the interactions between U1-snRNP and the immune system will provide insights into why certain patients develop anti-U1-snRNP autoimmunity, and more importantly how to effectively target therapies against this autoimmune response.

Dysregulation of CD4+ and CD8+ resident memory T, myeloid, and stromal cells in steroid-experienced, checkpoint inhibitor colitis.

BACKGROUND: Colitis caused by checkpoint inhibitors (CPI) is frequent and is treated with empiric steroids, but CPI colitis mechanisms in steroid-experienced or refractory disease are unclear. METHODS: Using colon biopsies and blood from predominantly steroid-experienced CPI colitis patients, we performed multiplexed single-cell transcriptomics and proteomics to nominate contributing populations. RESULTS: CPI colitis biopsies showed enrichment of CD4+resident memory (RM) T cells in addition to CD8+ RM and cytotoxic CD8+ T cells. Matching T cell receptor (TCR) clonotypes suggested that both RMs are progenitors that yield cytotoxic effectors. Activated, CD38+ HLA-DR+ CD4+ RM and cytotoxic CD8+ T cells were enriched in steroid-experienced and a validation data set of steroid-naïve CPI colitis, underscoring their pathogenic potential across steroid exposure. Distinct from ulcerative colitis, CPI colitis exhibited perturbed stromal metabolism (NAD+, tryptophan) impacting epithelial survival and inflammation. Endothelial cells in CPI colitis after anti-TNF and anti-cytotoxic T-lymphocyte-associated antigen 4 (anti-CTLA-4) upregulated the integrin α4ß7 ligand molecular vascular addressin cell adhesion molecule 1 (MAdCAM-1), which may preferentially respond to vedolizumab (anti-α4ß7). CONCLUSIONS: These findings nominate CD4+ RM and MAdCAM-1+ endothelial cells for targeting in specific subsets of CPI colitis patients.

Single-cell and spatial multi-omics highlight effects of anti-integrin therapy across cellular compartments in ulcerative colitis.

Ulcerative colitis (UC) is driven by immune and stromal subsets, culminating in epithelial injury. Vedolizumab (VDZ) is an anti-integrin antibody that is effective for treating UC. VDZ is known to inhibit lymphocyte trafficking to the intestine, but its broader effects on other cell subsets are less defined. To identify the inflammatory cells that contribute to colitis and are affected by VDZ, we performed single-cell transcriptomic and proteomic analyses of peripheral blood and colonic biopsies in healthy controls and patients with UC on VDZ or other therapies. Here we show that VDZ treatment is associated with alterations in circulating and tissue mononuclear phagocyte (MNP) subsets, along with modest shifts in lymphocytes. Spatial multi-omics of formalin-fixed biopsies demonstrates trends towards increased abundance and proximity of MNP and fibroblast subsets in active colitis. Spatial transcriptomics of archived specimens pre-treatment identifies epithelial-, MNP-, and fibroblast-enriched genes related to VDZ responsiveness, highlighting important roles for these subsets in UC.

Single-cell and spatial multi-omics highlight effects of anti-integrin therapy across cellular compartments in ulcerative colitis.

Ulcerative colitis (UC) is driven by immune and stromal subsets, culminating in epithelial injury. Vedolizumab (VDZ) is an anti-integrin antibody that is effective for treating UC. VDZ is known to inhibit lymphocyte trafficking to the intestine, but its broader effects on other cell subsets are less defined. To identify the inflammatory cells that contribute to colitis and are affected by VDZ, we perform single-cell transcriptomic and proteomic analyses of peripheral blood and colonic biopsies in healthy controls and patients with UC on VDZ or other therapies. Here we show that VDZ treatment is associated with alterations in circulating and tissue mononuclear phagocyte (MNP) subsets, along with modest shifts in lymphocytes. Spatial multi-omics of formalin-fixed biopsies demonstrates trends towards increased abundance and proximity of MNP and fibroblast subsets in active colitis. Spatial transcriptomics of archived specimens pre-treatment identifies epithelial-, MNP-, and fibroblast-enriched genes related to VDZ responsiveness, highlighting important roles for these subsets in UC.

A20 Restricts NOS2 Expression and Intestinal Tumorigenesis in a Mouse Model of Colitis-Associated Cancer.

BACKGROUND AND AIMS: Colon cancer can occur sporadically or in the setting of chronic inflammation, such as in patients with inflammatory bowel disease. We previously showed that A20, a critical negative regulator of tumor necrosis factor signal transduction, could regulate sporadic colon cancer development. In this report, we investigate whether A20 also acts as a tumor suppressor in a model of colitis-associated cancer. METHODS: Colitis and colitis-associated tumors were induced in wild-type and A20 intestinal epithelial cell-specific knockout (A20dIEC) mice using dextran sodium sulfate and azoxymethane. Clinicopathologic markers of inflammation were assessed in conjunction with colonic tumor burden. Gene expression analyses and immunohistochemistry were performed on colonic tissue and intestinal enteroids. Nitric oxide (NO) production and activity were assessed in whole colonic lysates and mouse embryonic fibroblasts. RESULTS: A20dIEC mice develop larger tumors after treatment with dextran sodium sulfate and azoxymethane than wild-type mice. In addition to elevated markers of inflammation, A20dIEC mice have significantly enhanced expression of inducible nitric oxide synthase (iNOS), a well-known driver of neoplasia. Enhanced iNOS expression is associated with the formation of reactive nitrogen species and DNA damage. Loss of A20 also enhances NO-dependent cell death directly. CONCLUSION: Mechanistically, we propose that A20 normally restricts tumor necrosis factor-induced nuclear factor kappa B-dependent production of iNOS in intestinal epithelial cells, thereby protecting against colitis-associated tumorigenesis. We also propose that A20 plays a direct role in regulating NO-dependent cell death.

Vedolizumab Antagonizes MAdCAM-1-Dependent Human Placental Cytotrophoblast Adhesion and Invasion In Vitro.

BACKGROUND: Anti-α4ß7 (Vedolizumab) treats inflammatory bowel disease (IBD) by blocking the interaction between integrin α4ß7 on leukocytes and mucosal addressin cell-adhesion molecule-1 (MAdCAM-1) on the gut endothelium. Women with IBD often require continuing biologic therapy during pregnancy to avoid disease flare. To date, there have been no reports of an increase in adverse events with Vedolizumab use during pregnancy. Notably, integrins play a major role in human placental development during pregnancy. It is unknown whether Vedolizumab disrupts placental cell (cytotrophoblast) invasion and/or adhesion by blocking interactions with MAdCAM-1. We therefore investigated human placental expression of MAdCAM-1, the role of MAdCAM-1/α4ß7 interactions in cytotrophoblast invasion/adhesion in vitro, and whether Vedolizumab administration in vivo alters the placental structure. METHODS: Histological sections of placentas from normal pregnancies were evaluated for MAdCAM-1 expression by immunofluorescence. The impacts of Vedolizumab or anti-integrin ß7 on human cytotrophoblast invasion and adhesion were assessed. Histology results from term placentas of 2 patients with IBD receiving Vedolizumab were compared to those of untreated healthy controls. RESULTS: Placental MAdCAM-1 expression was predominantly associated with invading extravillous cytotrophoblasts at the maternal-fetal interface. Treatment of isolated primary cytotrophoblasts with Vedolizumab or anti-integrin ß7 significantly reduced Matrigel invasion, adherence to a MAdCAM-1-coated substrate, and interactions with HuT-78 cells. Placentas from 2 Vedolizumab-treated patients with IBD exhibited pronounced pathologic features as compared to healthy control specimens. CONCLUSIONS: This study revealed a previously unrecognized role for α4ß7 and MAdCAM-1 in human placentation. More clinical and histological data from Vedolizumab-treated pregnant patients will be necessary to determine whether this medication poses any risk to the mother and fetus.

Intestinal inflammation alters the antigen-specific immune response to a skin commensal.

Resident microbes in skin and gut predominantly impact local immune cell function during homeostasis. However, colitis-associated neutrophilic skin disorders suggest possible breakdown of this compartmentalization with disease. Using a model wherein neonatal skin colonization by Staphylococcus epidermidis facilitates generation of commensal-specific tolerance and CD4+ regulatory T cells (Tregs), we ask whether this response is perturbed by gut inflammation. Chemically induced colitis is accompanied by intestinal expansion of S. epidermidis and reduces gut-draining lymph node (dLN) commensal-specific Tregs. It also results in reduced commensal-specific Tregs in skin and skin-dLNs and increased skin neutrophils. Increased CD4+ circulation between gut and skin dLN suggests that the altered cutaneous response is initiated in the colon, and resistance to colitis-induced effects in Cd4creIl1r1fl/fl mice implicate interleukin (IL)-1 in mediating the altered commensal-specific response. These findings provide mechanistic insight into observed connections between inflammatory skin and intestinal diseases.

Microbial signals, MyD88, and lymphotoxin drive TNF-independent intestinal epithelial tissue damage.

Anti-TNF antibodies are effective for treating patients with inflammatory bowel disease (IBD), but many patients fail to respond to anti-TNF therapy, highlighting the importance of TNF-independent disease. We previously demonstrated that acute deletion of 2 IBD susceptibility genes, A20 (Tnfaip3) and Abin-1 (Tnip1), in intestinal epithelial cells (IECs) sensitized mice to both TNF-dependent and TNF-independent death. Here we show that TNF-independent IEC death after A20 and Abin-1 deletion was rescued by germ-free derivation or deletion of MyD88, while deletion of Trif provided only partial protection. Combined deletion of Ripk3 and Casp8, which inhibits both apoptotic and necroptotic death, completely protected against death after acute deletion of A20 and Abin-1 in IECs. A20- and Abin-1-deficient IECs were sensitized to TNF-independent, TNFR1-mediated death in response to lymphotoxin α (LTα) homotrimers. Blockade of LTα in vivo reduced weight loss and improved survival when combined with partial deletion of MyD88. Biopsies of inflamed colon mucosa from patients with IBD exhibited increased LTA and IL1B expression, including a subset of patients with active colitis on anti-TNF therapy. These data show that microbial signals, MyD88, and LTα all contribute to TNF-independent intestinal injury.

Naive CD4 t cell proliferation is controlled by mammalian target of rapamycin regulation of GRAIL expression.

In this study, we demonstrate that the E3 ubiquitin ligase gene related to anergy in lymphocytes (GRAIL) is expressed in quiescent naive mouse and human CD4 T cells and has a functional role in inhibiting naive T cell proliferation. Following TCR engagement, CD28 costimulation results in the expression of IL-2 whose signaling through its receptor activates the Akt-mammalian target of rapamycin (mTOR) pathway. Activation of mTOR allows selective mRNA translation, including the epistatic regulator of GRAIL, Otubain-1 (Otub1), whose expression results in the degradation of GRAIL and allows T cell proliferation. The activation of mTOR appears to be the critical component of IL-2R signaling regulating GRAIL expression. CTLA4-Ig treatment blocks CD28 costimulation and resultant IL-2 expression, whereas rapamycin and anti-IL-2 treatment block mTOR activation downstream of IL-2R signaling. Thus, all three of these biotherapeutics inhibit mTOR-dependent translation of mRNA transcripts, resulting in blockade of Otub1 expression, maintenance of GRAIL, and inhibition of CD4 T cell proliferation. These observations provide a mechanistic pathway sequentially linking CD28 costimulation, IL-2R signaling, and mTOR activation as important requirements for naive CD4 T cell proliferation through the regulation of Otub1 and GRAIL expression. Our findings also extend the role of GRAIL beyond anergy induction and maintenance, suggesting that endogenous GRAIL regulates general cell cycle and proliferation of primary naive CD4 T cells.

Differential regulation of chemokines by IL-17 in colonic epithelial cells.

The IL-23/IL-17 pathway plays an important role in chronic inflammatory diseases, including inflammatory bowel disease. In inflammatory bowel disease, intestinal epithelial cells are an important source of chemokines that recruit inflammatory cells. We examined the effect of IL-17 on chemokine expression of HT-29 colonic epithelial cells. IL-17 strongly repressed TNF-alpha-stimulated expression of CXCL10, CXCL11, and CCL5, but synergized with TNF-alpha for induction of CXCL8, CXCL1, and CCL20 mRNAs. For CXCL10, IL-17 strongly inhibited promoter activity but had no effect on mRNA stability. In contrast, for CXCL8, IL-17 slightly decreased promoter activity but stabilized its normally unstable mRNA, leading to a net increase in steady-state mRNA abundance. IL-17 synergized with TNF-alpha in transactivating the epidermal growth factor receptor (EGFR) and in activating ERK and p38 MAPK. The p38 and ERK pathway inhibitors SB203580 and U0126 reversed the repressive effect of IL-17 on CXCL10 mRNA abundance and promoter activity and also reversed the inductive effect of IL-17 on CXCL8 mRNA, indicating that MAPK signaling mediates both the transcriptional repression of CXCL10 and the stabilization of CXCL8 mRNA by IL-17. The EGFR kinase inhibitor AG1478 partially reversed the effects of IL-17 on CXCL8 and CXCL10 mRNA, demonstrating a role for EGFR in downstream IL-17 signaling. The overall results indicate a positive effect of IL-17 on chemokines that recruit neutrophils (CXCL8 and CXCL1), and Th17 cells (CCL20). In contrast, IL-17 represses expression of CXCL10, CXCL11, and CCR5, three chemokines that selectively recruit Th1 but not other effector T cells.

A20 in dendritic cells restrains intestinal anti-bacterial peptide expression and preserves commensal homeostasis.

Microbial dysbiosis commonly occurs in patients with inflammatory bowel diseases (IBD). Exogenous causes of dysbiosis such as antibiotics and diet are well described, but host derived causes are understudied. A20 is a potent regulator of signals triggered by microbial pattern molecules, and A20 regulates susceptibility to intestinal inflammation in mice and in humans. We now report that mice lacking A20 expression in dendritic cells, A20FL/FL CD11c-Cre mice (or A20dDC mice), spontaneously develop colitogenic intestinal dysbiosis that is evident upon weaning and precedes the onset of colitis. Intestines from A20dDC mice express increased amounts of Reg3ß and Reg3γ, but not Ang4. A20 deficient DCs promote gut microbiota perturbation in the absence of adaptive lymphocytes. Moreover, A20 deficient DCs directly induce expression of Reg3ß and Reg3γ but not Ang 4 in normal intestinal epithelial cell enteroid cultures in the absence of other cell types. These findings reveal a pathophysiological pathway in which defective expression of an IBD susceptibility gene in DCs drives aberrant expression of anti-bacterial peptides and luminal dysbiosis that in turn confers host susceptibility to intestinal inflammation.

Correction: Anti-TNF and thiopurine therapy in pregnant IBD patients does not significantly alter a panel of B-cell and T-cell subsets in 1-year-old infants.

The original version of this article contained an error in Fig. 2, in which part of the text in the legend was omitted. This has now been corrected in the PDF and HTML versions of the paper.Furthermore, the figure legends were missing for the Supplementary figure files. The HTML has now been updated to include a corrected version of the Supplementary Information.