Bim regulates the survival and suppressive capability of CD8+ FOXP3+ regulatory T cells during murine GVHD.

CD8+ Foxp3+ T cells (Tregs) are a potent regulatory population whose functional and ontological similarities to CD4+ Fox3+ T cells have not been well delineated. Using an experimental model of graft-versus-host disease (GVHD), we observed that CD8+ Tregs were significantly less potent than CD4+ Tregs for the suppression of GVHD. To define the mechanistic basis for this observation, we examined the T-cell repertoire and the transcriptional profile of in vivo-derived CD4+ and CD8+ Tregs that emerged early during this disease. Polyclonal and alloantigen-induced CD8+ Tregs had repertoire diversity that was similar to that of conventional CD8+ T cells, indicating that a restricted repertoire was not the proximate cause of decreased suppression. Transcriptional profiling revealed that CD8+ Tregs possessed a canonical Treg transcriptional signature that was similar to that observed in CD4+ Tregs, yet distinct from conventional CD8+ T cells. Pathway analysis, however, demonstrated that CD8+ Tregs had differential gene expression in pathways involved in cell death and survival. This was further confirmed by detailed mRNA sequence analysis and protein expression studies, which demonstrated that CD8+ Tregs had increased expression of Bim and reduced expression of Mcl-1. Transplantation with CD8+ Foxp3+ Bim-/- Tregs resulted in prolonged Treg survival and reduced GVHD lethality compared with wild-type CD8+ Tregs, providing functional confirmation that increased expression of Bim was responsible for reduced in vivo efficacy. Thus, Bim regulates the survival and suppressive capability of CD8+ Tregs, which may have implications for their use in regulatory T-cell therapy.

Loss of FFAR2 promotes colon cancer by epigenetic dysregulation of inflammation suppressors.

Free fatty acid receptor 2 (FFAR2, also named GPR43), is activated by short-chain fatty acids (SCFAs), such as butyrate, that are produced when gut bacteria ferment dietary fiber. FFAR2 has been suggested to regulate colonic inflammation, which is a major risk factor for the development of colon cancer and is also linked to epigenetic dysregulation in colon carcinogenesis. The current study assessed whether FFAR2, acting as an epigenetic regulator, protects against colon carcinogenesis. To mimic the mild inflammation that promotes human colon cancer, we treated mice with dextran sodium sulfate (DSS) overnight, which avoids excessive inflammation but induces mild inflammation that promotes colon carcinogenesis in the ApcMin/+ and the azoxymethane (AOM)-treated mice. Our results showed that FFAR2 deficiency promotes the development of colon adenoma in the ApcMin/+ /DSS mice and the progression of adenoma to adenocarcinoma in the AOM/DSS mice. FFAR2's downstream cAMP-PKA-CREB pathway was enhanced, leading to overexpression of histone deacetylases (HDACs) in the FFAR2-deficient mice. ChIP-qPCR analysis revealed differential binding of H3K27me3 and H3K4me3 histone marks onto the promoter regions of inflammation suppressors (e.g., sfrp1, dkk3, socs1), resulting in decreased expression of these genes in the FFAR2-deficient mice. Also, more neutrophils infiltrated into tumors and colon lamina propria of the FFAR2-deficient mice. Depletion of neutrophils blocked the progression of colon tumors. In addition, FFAR2 is required for butyrate to suppress HDAC expression and hypermethylation of inflammation suppressors. Therefore, our results suggest that FFAR2 is an epigenetic tumor suppressor that acts at multiple stages of colon carcinogenesis.

Loss of free fatty acid receptor 2 enhances colonic adenoma development and reduces the chemopreventive effects of black raspberries in ApcMin/+ mice.

We previously showed that black raspberries (BRBs) have beneficial effects in human colorectal cancer and a mouse model of colorectal cancer (ApcMin/+). The current study investigated the role of free fatty acid receptor 2 (FFAR2) in colon carcinogenesis and whether the FFAR2 signaling pathway contributes to BRB-mediated chemoprevention in mice. FFAR2 (also named GPR43) is a member of the G-protein-coupled receptor family that is expressed in leukocytes and colon. ApcMin/+ and ApcMin/+-FFAR2-/- mice were given a control diet or the control diet supplemented with 5% BRBs for 8 weeks. FFAR2 deficiency promoted colonic polyp development, with 100% incidence and increased polyp number and size. The ApcMin/+ mice developed colonic tubular adenoma, whereas the ApcMin/+-FFAR2-/- mice developed colonic tubular adenoma with high-grade dysplasia. FFAR2 deficiency also enhanced the cAMP-PKA-CREB-HDAC pathway, downstream of FFAR2 signaling, and increased activation of the Wnt pathway, and raised the percentage of GR-1+ neutrophils in colonic lamina propria (LP) and increased infiltration of GR-1+ neutrophils into colonic polyps. BRBs suppressed colonic polyp development and inhibited the cAMP-PKA-CREB-HDAC and Wnt pathways in the ApcMin/+ mice but not the ApcMin/+-FFAR2-/- mice. They also increased the percentage of GR-1+ neutrophils and cytokine secretion in colonic LP and decreased the infiltration of GR-1+ neutrophils and IL-1ß expression in colon polyps of ApcMin/+ mice but not ApcMin/+-FFAR2-/- mice. These results suggest that loss of FFAR2 drives colon tumorigenesis and that BRBs require functional FFAR2 to be chemopreventive. BRBs have the potential to modulate the host immune system, thereby enhancing the antitumor immune microenvironment.

A colitogenic memory CD4+ T cell population mediates gastrointestinal graft-versus-host disease.

Damage to the gastrointestinal tract is a major cause of morbidity and mortality in graft-versus-host disease (GVHD) and is attributable to T cell-mediated inflammation. In this work, we identified a unique CD4+ T cell population that constitutively expresses the ß2 integrin CD11c and displays a biased central memory phenotype and memory T cell transcriptional profile, innate-like properties, and increased expression of the gut-homing molecules α4ß7 and CCR9. Using several complementary murine GVHD models, we determined that adoptive transfer and early accumulation of ß2 integrin-expressing CD4+ T cells in the gastrointestinal tract initiated Th1-mediated proinflammatory cytokine production, augmented pathological damage in the colon, and increased mortality. The pathogenic effect of this CD4+ T cell population critically depended on coexpression of the IL-23 receptor, which was required for maximal inflammatory effects. Non-Foxp3-expressing CD4+ T cells produced IL-10, which regulated colonic inflammation and attenuated lethality in the absence of functional CD4+Foxp3+ T cells. Thus, the coordinate expression of CD11c and the IL-23 receptor defines an IL-10-regulated, colitogenic memory CD4+ T cell subset that is poised to initiate inflammation when there is loss of tolerance and breakdown of mucosal barriers.

Blockade of interleukin-27 signaling reduces GVHD in mice by augmenting Treg reconstitution and stabilizing Foxp3 expression.

Reestablishment of competent regulatory pathways has emerged as a strategy to reduce the severity of graft-versus-host disease (GVHD), and recalibrate the effector and regulatory arms of the immune system. However, clinically feasible, cost-effective strategies that do not require extensive ex vivo cellular manipulation have remained elusive. In the current study, we demonstrate that inhibition of the interleukin-27p28 (IL-27p28) signaling pathway through antibody blockade or genetic ablation prevented lethal GVHD in multiple murine transplant models. Moreover, protection from GVHD was attributable to augmented global reconstitution of CD4+ natural regulatory T cells (nTregs), CD4+ induced Tregs (iTregs), and CD8+ iTregs, and was more potent than temporally concordant blockade of IL-6 signaling. Inhibition of IL-27p28 also enhanced the suppressive capacity of adoptively transferred CD4+ nTregs by increasing the stability of Foxp3 expression. Notably, blockade of IL-27p28 signaling reduced T-cell-derived-IL-10 production in conventional T cells; however, there was no corresponding effect in CD4+ or CD8+ Tregs, indicating that IL-27 inhibition had differential effects on IL-10 production and preserved a mechanistic pathway by which Tregs are known to suppress GVHD. Targeting of IL-27 therefore represents a novel strategy for the in vivo expansion of Tregs and subsequent prevention of GVHD without the requirement for ex vivo cellular manipulation, and provides additional support for the critical proinflammatory role that members of the IL-6 and IL-12 cytokine families play in GVHD biology.

E-cadherin is critical for collective sheet migration and is regulated by the chemokine CXCL12 protein during restitution.

Chemokines and other immune mediators enhance epithelial barrier repair. The intestinal barrier is established by highly regulated cell-cell contacts between epithelial cells. The goal of these studies was to define the role for the chemokine CXCL12 in regulating E-cadherin during collective sheet migration during epithelial restitution. Mechanisms regulating E-cadherin were investigated using Caco2(BBE) and IEC-6 model epithelia. Genetic knockdown confirmed a critical role for E-cadherin in in vitro restitution and in vivo wound repair. During restitution, both CXCL12 and TGF-ß1 tightened the monolayer by decreasing the paracellular space between migrating epithelial cells. However, CXCL12 differed from TGF-ß1 by stimulating the significant increase in E-cadherin membrane localization during restitution. Chemokine-stimulated relocalization of E-cadherin was paralleled by an increase in barrier integrity of polarized epithelium during restitution. CXCL12 activation of its cognate receptor CXCR4 stimulated E-cadherin localization and monolayer tightening through Rho-associated protein kinase activation and F-actin reorganization. These data demonstrate a key role for E-cadherin in intestinal epithelial restitution.

Chemokine stimulation promotes enterocyte migration through laminin-specific integrins.

Intestinal homeostasis is regulated in part by the single cell layer of the mucosal epithelium. This physical barrier is a prominent part of the innate immune system and possesses an intrinsic ability to heal damage and limit infection. The restitutive epithelial migration phase of healing requires dynamic integrin adhesion to the extracellular matrix. Previously, we have shown that the homeostatic chemokine CXCL12 utilizes intracellular calcium to increase enterocyte migration on laminin. The aim of these studies was to investigate integrin specificity and, in turn, functional responses elicited by CXCL12 stimulation. Analysis of cellular adhesion and spreading revealed CXCL12 preferentially activated laminin-specific integrins compared with collagen IV-binding integrins. Laminin-specific cell adhesion and spreading elicited by CXCL12 was dependent on intracellular calcium. CXCL12 increased activated ß1-integrins on the surface of epithelial cells compared with untreated cells. RT-PCR confirmed expression of the laminin-binding integrins-α3ß1, -α6ß1, and -α6ß4. Interestingly, shRNA-mediated depletion of laminin-specific α3- or α6-integrin subunits revealed differential functions. α3-Integrin knockdown reduced basal as well as inducible restitution. Depletion of α6-integrin specifically abolished CXCL12-stimulated, but not TGF-ß1 or basal, migration. Depletion with either shα3-integrin or shα6-integrin prevented CXCL12-evoked cell spreading. Our data indicate that CXCL12 stimulates the inside-out activation of laminin-specific integrins to promote cell migratory functions. Together, our findings support the notion that extracellular mediators within the gastrointestinal mucosa coordinate cell-matrix interactions during epithelial restitution.

Calcium mobilization triggered by the chemokine CXCL12 regulates migration in wounded intestinal epithelial monolayers.

Restitution of intestinal epithelial barrier damage involves the coordinated remodeling of focal adhesions in actively migrating enterocytes. Defining the extracellular mediators and the intracellular signaling pathways regulating those dynamic processes is a key step in developing restitution-targeted therapies. Previously we have determined that activation of the chemokine receptor CXCR4 by the cognate ligand CXCL12 enhances intestinal epithelial restitution through reorganization of the actin cytoskeleton. The aim of these studies was to investigate the role of calcium effectors in CXCL12-mediated restitution. CXCL12 stimulated release of intracellular calcium in a dose-dependent manner. Inhibition of intracellular calcium flux impaired CXCL12-mediated migration of IEC-6 and CaCo2 cells. Pharmacological blockade and specific shRNA depletion of the phospholipase-C (PLCbeta3) isoform attenuated CXCL12-enhanced migration, linking receptor activation with intracellular calcium flux. Immunoblot analyses demonstrated CXCL12 activated the calcium-regulated focal adhesion protein proline-rich tyrosine kinase-2 (Pyk2) and the effector proteins paxillin and p130(Cas). Interruption of Pyk2 signaling potently blocked CXCL12-induced wound closure. CXCL12-stimulated epithelial cell migration was enhanced on laminin and abrogated by intracellular calcium chelation. These results suggest CXCL12 regulates restitution through calcium-activated Pyk2 localized to active focal adhesions. Calcium signaling pathways may therefore provide a novel avenue for enhancing barrier repair.