A lymphocyte chemoaffinity axis for lung, non-intestinal mucosae and CNS.

Tissue-selective chemoattractants direct lymphocytes to epithelial surfaces to establish local immune environments, regulate immune responses to food antigens and commensal organisms, and protect from pathogens. Homeostatic chemoattractants for small intestines, colon, and skin are known1 2, but chemotropic mechanisms selective for respiratory tract and other non-intestinal mucosal tissues (NIMT) remain poorly understood. Here we leveraged diverse omics datasets to identify GPR25 as a lymphocyte receptor for CXCL17, a chemoattractant cytokine whose expression by epithelial cells of airways, upper gastrointestinal and squamous mucosae unifies the NIMT and distinguishes them from intestinal mucosae. Single-cell transcriptomic analyses show that GPR25 is induced on innate lymphocytes prior to emigration to the periphery, and is imprinted in secondary lymphoid tissues on activated B and T cells responding to immune challenge. GPR25 characterizes B and T tissue resident memory and regulatory T lymphocytes in NIMT and lungs in humans and mediates lymphocyte homing to barrier epithelia of the airways, oral cavity, stomach, biliary and genitourinary tracts in mouse models. GPR25 is also expressed by T cells in cerebrospinal fluid and CXCL17 by neurons, suggesting a role in CNS immune regulation. We reveal widespread imprinting of GPR25 on regulatory T cells, suggesting a mechanistic link to population genetic evidence that GPR25 is protective in autoimmunity3,4. Our results define a GPR25-CXCL17 chemoaffinity axis with the potential to integrate immunity and tolerance at non-intestinal mucosae and the CNS.

Chemerin triggers migration of a CD8 T cell subset with natural killer cell functions.

The recruitment of cells with effector functions into the tumor microenvironment holds potential for delaying cancer progression. We show that subsets of human CD28-effector CD8 T cells, CCR7- CD45RO+ effector memory, and CCR7- CD45RO- effector memory RA phenotypes, express the chemerin receptor CMKLR1 and bind chemerin via the receptor. CMKLR1-expressing human CD8 effector memory T cells present gene, protein, and cytotoxic features of NK cells. Active chemerin promotes chemotaxis of CMKLR1-expressing CD8 effector memory cells and triggers activation of the α4ß1 integrin. In an experimental prostate tumor mouse model, chemerin expression is downregulated in the tumor microenvironment, which is associated with few tumor-infiltrating CD8+ T cells, while forced overexpression of chemerin by mouse prostate cancer cells leads to an accumulation of intra-tumor CD8+ T cells. Furthermore, α4 integrin blockade abrogated the chemerin-dependent recruitment of CD8+ T effector memory cells into implanted prostate tumors in vivo. The results identify a role for chemerin:CMKLR1 in defining a specialized NK-like CD8 T cell, and suggest the use of chemerin-dependent modalities to target effector CMKLR1-expressing T cells to the tumor microenvironment for immunotherapeutic purposes.

Novel CMKLR1 Inhibitors for Application in Demyelinating Disease.

Small molecules that disrupt leukocyte trafficking have proven effective in treating patients with multiple sclerosis (MS). We previously reported that chemerin receptor chemokine-like receptor 1 (CMKLR1) is required for maximal clinical and histological experimental autoimmune encephalomyelitis (EAE); and identified CMKLR1 small molecule antagonist 2-(α-naphthoyl) ethyltrimethylammonium iodide (α-NETA) that significantly suppressed disease onset in vivo. Here we directly compared α-NETA versus FDA-approved MS drug Tecfidera for clinical efficacy in EAE; characterized key safety/toxicity parameters for α-NETA; identified structure-activity relationships among α-NETA domains and CMKLR1 inhibition; and evaluated improved α-NETA analogs for in vivo efficacy. α-NETA proved safe and superior to Tecfidera in suppressing clinical EAE. In addition, we discovered structurally differentiated α-NETA analogs (primarily ortho- or para-methoxy substitutions) with significantly improved target potency in vitro and improved efficacy in vivo. These findings suggest that α-NETA-based CMKLR1 inhibitors may prove safe and effective in treating demyelinating diseases and potentially other autoimmune disorders.

DGAT1 inhibits retinol-dependent regulatory T cell formation and mediates autoimmune encephalomyelitis.

The balance of effector versus regulatory T cells (Tregs) controls inflammation in numerous settings, including multiple sclerosis (MS). Here we show that memory phenotype CD4+ T cells infiltrating the central nervous system during experimental autoimmune encephalomyelitis (EAE), a widely studied animal model of MS, expressed high levels of mRNA for Dgat1 encoding diacylglycerol-O-acyltransferase-1 (DGAT1), an enzyme that catalyzes triglyceride synthesis and retinyl ester formation. DGAT1 inhibition or deficiency attenuated EAE, with associated enhanced Treg frequency; and encephalitogenic, DGAT1-/- in vitro-polarized Th17 cells were poor inducers of EAE in adoptive recipients. DGAT1 acyltransferase activity sequesters retinol in ester form, preventing synthesis of retinoic acid, a cofactor for Treg generation. In cultures with T cell-depleted lymphoid tissues, retinol enhanced Treg induction from DGAT1-/- but not from WT T cells. The WT Treg induction defect was reversed by DGAT1 inhibition. These results demonstrate that DGAT1 suppresses retinol-dependent Treg formation and suggest its potential as a therapeutic target for autoimmune inflammation.

The expression and regulation of chemerin in the epidermis.

Chemerin is a protein ligand for the G protein-coupled receptor CMKLR1 and also binds to two atypical heptahelical receptors, CCRL2 and GPR1. Chemerin is a leukocyte attractant, adipokine, and antimicrobial protein. Although chemerin was initially identified as a highly expressed gene in healthy skin keratinocytes that was downregulated during psoriasis, the regulation of chemerin and its receptors in the skin by specific cytokines and microbial factors remains unexplored. Here we show that chemerin, CMKLR1, CCRL2 and GPR1 are expressed in human and mouse epidermis, suggesting that this tissue may be both a source and target for chemerin mediated effects. In human skin cultures, chemerin is significantly downregulated by IL-17 and IL-22, key cytokines implicated in psoriasis, whereas it is upregulated by acute phase cytokines oncostatin M and IL-1ß. Moreover, we show that human keratinocytes in vitro and mouse skin in vivo respond to specific microbial signals to regulate expression levels of chemerin and its receptors. Furthermore, in a cutaneous infection model, chemerin is required for maximal bactericidal effects in vivo. Together, our findings reveal previously uncharacterized regulators of chemerin expression in skin and identify a physiologic role for chemerin in skin barrier defense against microbial pathogens.

Transcriptional programs of lymphoid tissue capillary and high endothelium reveal control mechanisms for lymphocyte homing.

Lymphocytes are recruited from blood by high-endothelial venules (HEVs). We performed transcriptomic analyses and identified molecular signatures that distinguish HEVs from capillary endothelium and that define tissue-specific HEV specialization. Capillaries expressed gene programs for vascular development. HEV-expressed genes showed enrichment for genes encoding molecules involved in immunological defense and lymphocyte migration. We identify capillary and HEV markers and candidate mechanisms for regulated recruitment of lymphocytes, including a lymph node HEV-selective transmembrane mucin; transcriptional control of functionally specialized carbohydrate ligands for lymphocyte L-selectin; HEV expression of molecules for transendothelial migration; and metabolic programs for lipid mediators of lymphocyte motility and chemotaxis. We also elucidate a carbohydrate-recognition pathway that targets B cells to intestinal lymphoid tissues, defining CD22 as a lectin-homing receptor for mucosal HEVs.

Robust expansion of dendritic cells in vivo by hydrodynamic FLT3L-FC gene transfer.

Due to low numbers of endogenous dendritic cells (DCs) in vivo, exogenous DC-poietin Fms-like tyrosine kinase 3-ligand (FLT3L) is routinely used to generate DC for subsequent studies. We engineered a novel FLT3L-FC DNA construct that, when combined with hydrodynamic gene transfer (HDT), induced robust DC expansion in mice. DC generated in vivo by FLT3L-FC HDT produced cytokines in response to stimulation by an array of TLR agonists and promoted T cell proliferation. The FLT3L-FC protein produced in vivo spontaneously homodimerized to enable effective FLT signaling and the FC-domain enhanced its plasma half-life, providing an improved reagent and method to boost DC numbers.

Canonical and noncanonical Wnt proteins program dendritic cell responses for tolerance.

Ag-presenting dendritic cells (DCs) interpret environmental signals to orchestrate local and systemic immune responses. They govern the balance between tolerance and inflammation at epithelial surfaces, where the immune system must provide robust pathogen responses while maintaining tolerance to commensal flora and food Ags. The Wnt family of secreted proteins, which control epithelial and hematopoietic development and homeostasis, is emerging as an important regulator of inflammation. In this study, we show that canonical and noncanonical Wnts directly stimulate murine DC production of anti-inflammatory cytokines. Wnt3A triggers canonical ß-catenin signaling and preferentially induces DC TGF-ß and VEGF production, whereas Wnt5A induces IL-10 through alternative pathways. The Wnts also alter DC responses to microbe- or pathogen-associated molecular patterns, inhibiting proinflammatory cytokine induction in response to TLR ligands and promoting DC generation of Foxp3(+) regulatory T cells. Moreover, although both Wnts suppress proinflammatory responses to bacterial endotoxin and to TLR1/2, TLR7, and TLR9 ligands, Wnt5A, but not Wnt3A, inhibits IL-6 production in response to the viral mimic, polyinosinic:polycytidylic acid. Thus, Wnt family members directly and differentially regulate DC functions, an ability that may contribute to the balance between tolerance and inflammation at epithelial sites of exposure to microbes and environmental Ags.

Activation of p38 mitogen-activated protein kinase by norepinephrine in T-lineage cells.

The catecholamine norepinephrine (NE) stimulates T lymphocytes through a beta-adrenergic receptor (ßAR)/adenylyl cyclase (AC)/cyclic AMP (cAMP)/protein kinase A (PKA) pathway, leading to altered cell responsiveness and apoptosis. p38 Mitogen-activated protein kinase (MAPK), a major intracellular signalling mediator for cellular and environmental stressors, is involved in the production of immune modulators and in the regulation of T-cell development, survival and death. In these studies we investigated the relationship among NE signalling, p38 MAPK activity and T-cell death. We showed that NE stimulation of BALB/c mouse thymocytes and S49 thymoma cells selectively increases the dual phosphorylation and activity of p38α MAPK. p38 MAPK activation involves the ßAR, Gs protein, AC, cAMP and PKA, as determined through the use of a ßAR antagonist, activators of AC and cAMP, and S49 clonal mutants deficient in Gs and PKA. Dual phosphorylation of p38 MAPK is also dependent on its own catalytic activity. Inhibition of p38 MAPK activity revealed its involvement in cAMP-mediated activating transcription factor-2 (ATF-2) phosphorylation, Fas ligand messenger RNA (mRNA) up-regulation, and cell death. These results identify a mechanism through which NE stimulation of the ßAR/Gs/PKA pathway activates p38 MAPK, which can be potentiated by autophosphorylation, and leads to changes in T-cell dynamics, in part through the regulation of Fas ligand mRNA expression.

Thy-1 mRNA destabilization by norepinephrine a 3' UTR cAMP responsive decay element and involves RNA binding proteins.

Thy-1 is a cell surface protein important in immunologic and neurologic processes, including T cell activation and proliferation, and neuronal outgrowth. In murine thymocytes, Thy-1 is downregulated in response to norepinephrine (NE) through posttranscriptional destabilization of its mRNA mediated by ßAR/AC/cAMP/PKA signaling. In this study we investigated factors involved in NE/cAMP-mediated Thy-1 mRNA destabilization in S49 thymoma cells, and identified a region containing two copies of the AUUUA regulatory element (ARE), a motif commonly associated with mRNA decay, in the Thy-1 mRNA 3' UTR. Insertion of the Thy-1 ARE region into a reporter gene, resulted in cAMP induced destabilization of the reporter gene mRNA. RNA-protein binding studies revealed multiple Thy-1 ARE binding proteins, including AUF1, HuR, and TIAR. RNA silencing of HuR enhanced cAMP-mediated downregulation of Thy-1 mRNA, in contrast, silencing AUF1 had no effect. Immunoblotting revealed multiple proteins phosphorylated by PKA as a result of NE or cAMP signaling. These results reveal that the machinery of NE/cAMP modulation of Thy-1 mRNA decay involves a cAMP responsive ARE in its 3' UTR and multiple site specific ARE binding proteins. These findings add to our knowledge of Thy-1 mRNA regulation and provide insight into the regulation of ARE containing mRNAs, which impacts stress-related immunosuppression.