The role of the CCL25-CCR9 axis in beta-cell function: potential for therapeutic intervention in type 2 diabetes.

BACKGROUND AND PURPOSE: Chemokines are known to play essential roles mediating immunity and inflammation in many physiological and pathophysiological processes, with reports linking their action to the development of obesity, insulin resistance and type 2 diabetes (T2D). Given our findings of highly upregulated mRNA expression of the chemokine receptor CCR9 in islets from obese human donors, we have determined the effects of CCR9 activation by CCL25 on islet function and viability. BASIC PROCEDURES: RT-qPCR was used to measure expression of 384 GPCR mRNAs in human islets from organ donors with normal and elevated BMI. mRNA encoding CCR9, a receptor that was highly upregulated in islets from obese donors, was also quantified in islets from lean and high-fat diet (HFD) mice. The effects of CCR9 activation by exogenous CCL25 in human and mouse islets and its inhibition by the CCR9 antagonist vercirnon on insulin secretion, apoptosis and cAMP accumulation were examined using standard techniques. MAIN FINDINGS: The qPCR analysis showed altered expression of several GPCRs in islets isolated from lean and obese donors. CCR9 displayed over 90-fold upregulation in islets from obese individuals, and it was also significantly upregulated in islets from obese mice. In isolated human and mouse islets exogenous CCL25 inhibited glucose-induced insulin secretion in a concentration-dependent manner, enhanced cytokine-induced apoptosis and significantly reduced forskolin-induced elevation in cAMP levels. These detrimental effects of CCL25 in islets were blocked by vercirnon, which had no effect on its own. PRINCIPAL CONCLUSIONS: We have shown that CCL25 acts via the Gαi-coupled receptor CCR9 to impair beta-cell function by inhibiting insulin secretion and promoting cytokine-induced apoptosis. Upregulation of CCR9 in islets in obesity, possibly secondary to accumulation of passenger immune cells, may predispose to metabolic dysfunction and our data suggest that CCL25 downregulation or CCR9 inhibition could be explored to treat T2D.

Toll-Like Receptor 4 Promotes Th17 Lymphocyte Infiltration Via CCL25/CCR9 in Pathogenesis of Experimental Autoimmune Encephalomyelitis.

Toll-like receptor 4 (TLR4) is a key component in innate immunity and has been linked to central nervous system (CNS) inflammation diseases, such as multiple sclerosis (MS), an inflammatory disorder induced by autoreactive Th17 cells. In our study, we found that TLR4 deficient (TLR4-/-) mice were inadequate to induce experimental autoimmune encephalomyelitis (EAE), characterized by low clinic score and weight loss, alleviative demyelinating, as well as decreased inflammatory cell infiltration in the spinal cord. In the lesion area of EAE mice, loss of TLR4 down-regulated the secretion of inflammatory cytokines and chemokine CCL25. Furthermore, the expression of CCR9 was decreased and chemotactic migration was attenuated in TLR4-/- Th17 cells. Our results demonstrate that TLR4 may mediate Th17 infiltration through CCL25/CCR9 signal during pathogenesis of EAE. Graphical Abstract Immunofluorescent staining of RORγt (green) and CCR9 (red) in spinal cords. TLR4 deficiency down-regulates CCR9 expression in infiltrating lymphocytes.

A Subset of CCL25-Induced Gut-Homing T Cells Affects Intestinal Immunity to Infection and Cancer.

Protective immunity relies upon differentiation of T cells into the appropriate subtype required to clear infections and efficient effector T cell localization to antigen-rich tissue. Recent studies have highlighted the role played by subpopulations of tissue-resident memory (TRM) T lymphocytes in the protection from invading pathogens. The intestinal mucosa and associated lymphoid tissue are densely populated by a variety of resident lymphocyte populations, including αß and γδ CD8+ intraepithelial T lymphocytes (IELs) and CD4+ T cells. While the development of intestinal γδ CD8+ IELs has been extensively investigated, the origin and function of intestinal CD4+ T cells have not been clarified. We report that CCR9 signals delivered during naïve T cell priming promote the differentiation of a population of α4ß7+ IFN-γ-producing memory CD4+ T cells, which displays a TRM molecular signature, preferentially localizes to the gastrointestinal (GI) tract and associated lymphoid tissue and cannot be mobilized by remote antigenic challenge. We further show that this population shapes the immune microenvironment of GI tissue, thus affecting effector immunity in infection and cancer.

CCL25 chemokine-guided stem cell attraction: an assessment of possible benefits and risks.

Due to its chemoattraction potential on mesenchymal stromal cells of the CCL25/CCR9 axis, local application of CCL25 to severely damaged tissues may be a promising approach for regenerative therapies. Analysis of the given data revealed that CCL25/CCR9 signaling has a crucial role in regulation of an adult immune homeostasis. CCR9 expression variations resulted in dysfunctional immune response in colitis, rheumatoid arthritis and endometriosis. Regarding oncology, different neoplastic tissues exploit CCL25-dependent CCR9 signaling for either local proliferation or migration processes. The CCR9 pathway likely can trigger crosstalk between the Akt and NOTCH pathway and thus participate in the regulation of the neoplastic behavior. In conclusion, the designated application-tissue requires precise molecular analysis of possible CCR9 expression due to its proto-oncogenic characteristics.

Atypical chemokine receptor ACKR2 controls branching morphogenesis in the developing mammary gland.

Macrophages are important regulators of branching morphogenesis during development and postnatally in the mammary gland. Regulation of macrophage dynamics during these processes can therefore have a profound impact on development. We demonstrate here that the developing mammary gland expresses high levels of inflammatory CC-chemokines, which are essential in vivo regulators of macrophage migration. We further demonstrate that the atypical chemokine receptor ACKR2, which scavenges inflammatory CC-chemokines, is differentially expressed during mammary gland development. We have previously shown that ACKR2 regulates macrophage dynamics during lymphatic vessel development. Here, we extend these observations to reveal a novel role for ACKR2 in regulating the postnatal development of the mammary gland. Specifically, we show that Ackr2-/- mice display precocious mammary gland development. This is associated with increased macrophage recruitment to the developing gland and increased density of the ductal epithelial network. These data demonstrate that ACKR2 is an important regulator of branching morphogenesis in diverse biological contexts and provide the first evidence of a role for chemokines and their receptors in postnatal development processes.

Imiquimod-induced CCR9 Ameliorates murine TNBS Colitis.

AIMS: To investigate whether Imiquimod (IMQ) as TLR7 ligand protects mice from colonic inflammation and the mechanisms underlying in such immunoregulatory conditions.　METHODS: Murine colitis was induced to Balb/c mice by administration of trinitrobenzene sulfonic acid (TNBS) with or without daily intraperitoneal administration of IMQ.　Colitis was evaluated by body weight decreases and by histological score.　Also colonic mRNA expression was measured by RT-PCR.　To confirm the induction of Regulatory T cells (Tregs) by type-1 IFN from pDCs, we generated mouse bone marrow-derived pDCs and co-cultured these with CD4+ T cells isolated from mouse spleen with or without IMQ stimulation.　Cytokine production in the culture supernatant was measured by ELISA and the number of Tregs were analyzed by flow cytometry.　Spleen and mesenteric lymph nodes (MLN) from IMQ-treated mice were collected, and mRNA expressions of cytokine were measured by RT-PCR and cytokine productions were measured by ELISA.　Tregs and chemokine expressions were analyzed in colon of TNBS-induced colitis mouse by immunohistochemistry.　RESULTS: Administration of IMQ significantly suppressed colonic inflammation of TNBS-induced colitis.　In the colons of IMQ-treated mice, mRNA expression of TNF-α was decreased, and strong expressions of IL-6, IFN-ß and TGF-ß were detected.　IL-10 and TGF-ß productions were increased in the supernatant of co-cultured cells stimulated with IMQ, although we were unable to detect Treg differentiaton in IMQ-stimulated co-cultured cells.　In MLN of IMQ-treated mice, strong expressions of TLR7, IFN-ß, TGF-ß and Foxp3 mRNA were detected.　IL-10 production from MLN cells was also increased in the IMQ-treated group.　Finally, Tregs in the inflamed colon and CCR9 in MLN of IMQ-treated mice were detected.　CONCLUSION: These results suggest that IMQ protects mice from TNBS colitis through induction of CCR9, which regulates accumulation of Tregs in the inflamed colon.

Role of inflammatory macrophages and CCR9/CCL25 chemokine axis in the pathogenesis of liver injury as a therapeutic target.

It is widely known that a variety of immune cells in the liver contribute to the pathogenesis of liver diseases. Recently, roles of chemokines/chemokines receptors axes regarding the migration of immune competent cells to the inflamed liver have been reported as possible therapeutic targets. We here showed that CCR9+ CD11b+ macrophages play an important role during the course of Concanavalin A-induced murine acute liver injury as well as human acute hepatitis via the production of inflammatory cytokines and the Th1 induction. Further analysis using liver-shielded radiation and bone marrow (BM) transplantation model mice revealed that these CCR9+ CD11b+ macrophages are originated from BM-derived monocytes, but not liver resident macrophages (Kupffer cells). Furthermore, these CD11b+ inflammatory macrophages in contact with hepatic stellate cells contribute to the pathogenesis of murine experimental liver fibrosis via CCR9/CCL25 axis. Collectively, these results with further verification in human samples clarify the pathogenic role of CCR9/CCL25 axis as therapeutic target of a variety of liver diseases.

Unexpected Regulatory Role of CCR9 in Regulatory T Cell Development.

T cells reactive to microbiota regulate the pathogenesis of inflammatory bowel disease (IBD). As T cell trafficking to intestines is regulated through interactions between highly specific chemokine-chemokine receptors, efforts have been made to develop intestine-specific immunosuppression based on blocking these key processes. CCR9, a gut-trophic chemokine receptor expressed by lymphocytes and dendritic cells, has been implicated in the regulation of IBD through mediating recruitment of T cells to inflamed sites. However, the role of CCR9 in inducing and sustaining inflammation in the context of IBD is poorly understood. In this study, we demonstrate that CCR9 deficiency in effector T cells and Tregs does not affect the development of colitis in a microbiota antigen-specific, T cell-mediated model. However, Treg cells express higher levels of CCR9 compared to those in effector T cells. Interestingly, CCR9 inhibits Treg cell development, in that CCR9-/- mice demonstrate a high level of Foxp3+ Tregs, and ligation of CCR9 by its ligand CCL25 inhibits Treg cell differentiation in vitro. Collectively, our data indicate that in addition to acting as a gut-homing molecule, CCR9 signaling shapes immune responses by inhibiting Treg cell development.

CCR9-mediated signaling through ß-catenin and identification of a novel CCR9 antagonist.

Elevated levels of chemokine receptor CCR9 expression in solid tumors may contribute to poor patient prognosis. In this study, we characterized a novel CCR9-mediated pathway that promotes pancreatic cancer cell invasion and drug resistance, indicating that CCR9 may play a critical role in cancer progression through activation of ß-catenin. We noted that the CCL25/CCR9 axis in pancreatic cancer cells induced the activation of ß-catenin, which enhanced cell proliferation, invasion, and drug resistance. CCR9-mediated activation of ß-catenin and the resulting downstream effects were effectively inhibited by blockade of the PI3K/AKT pathway, but not by antagonism of Wnt. Importantly, we discovered that CCR9/CCL25 increased the lethal dose of gemcitabine, suggesting decreased efficacy of anti-cancer drugs with CCR9 signaling. Through in silico computational modeling, we identified candidate CCR9 antagonists and tested their effects on CCR9/ß-catenin regulation of cell signaling and drug sensitivity. When combined with gemcitabine, it resulted in synergistic cytotoxicity. Our results show that CCR9/ß-catenin signaling enhances pancreatic cancer invasiveness and chemoresistance, and may be a highly novel therapeutic target.

The atypical chemokine receptor CCRL1 shapes functional CCL21 gradients in lymph nodes.

Afferent lymph-borne dendritic cells essentially rely on the chemokine receptor CCR7 for their transition from the subcapsular lymph node sinus into the parenchyma, a migratory step driven by putative gradients of CCR7 ligands. We found that lymph node fringes indeed contained physiological gradients of the chemokine CCL21, which depended on the expression of CCRL1, the atypical receptor for the CCR7 ligands CCL19 and CCL21. Lymphatic endothelial cells lining the ceiling of the subcapsular sinus, but not those lining the floor, expressed CCRL1, which scavenged chemokines from the sinus lumen. This created chemokine gradients across the sinus floor and enabled the emigration of dendritic cells. In vitro live imaging revealed that spatially confined expression of CCRL1 was necessary and sufficient for the creation of functional chemokine gradients.

A myriad of functions and complex regulation of the CCR7/CCL19/CCL21 chemokine axis in the adaptive immune system.

The chemokine receptor CCR7 and its ligands CCL19 and CCL21 control a diverse array of migratory events in adaptive immune function. Most prominently, CCR7 promotes homing of T cells and DCs to T cell areas of lymphoid tissues where T cell priming occurs. However, CCR7 and its ligands also contribute to a multitude of adaptive immune functions including thymocyte development, secondary lymphoid organogenesis, high affinity antibody responses, regulatory and memory T cell function, and lymphocyte egress from tissues. In this survey, we summarise the role of CCR7 in adaptive immunity and describe recent progress in understanding how this axis is regulated. In particular we highlight CCX-CKR, which scavenges both CCR7 ligands, and discuss its emerging significance in the immune system.

C-C motif chemokine receptor 9 positive macrophages activate hepatic stellate cells and promote liver fibrosis in mice.

UNLABELLED: Chemokine receptors mediate migration of immune cells into the liver, thereby promoting liver inflammation. C-C motif chemokine receptor (CCR) 9(+) macrophages are crucial in the pathogenesis of acute liver inflammation, but the role and underlying mechanisms of this macrophage subset in chronic liver injury and subsequent liver fibrosis are not fully understood. We confirmed that tumor necrosis factor alpha (TNF-α)-producing CCR9(+) macrophages accumulated during the initiation of carbon tetrachloride (CCl4 )-induced liver injury, and CCR9 deficiency attenuated the degree of liver damage. Accumulation of CCR9(+) macrophages persisted prominently during the process of liver fibrosis induced by repetitive CCl4 or thioacetamide (TAA)/leptin administration. Increased CCR9 expression was also found on activated hepatic stellate cells (HSCs). Importantly, experimental liver fibrosis was significantly ameliorated in CCR9(-/-) mice compared with wild-type (WT) mice, assessed by α-smooth muscle actin (α-SMA) immunostain, Sirius red staining, and messenger RNA (mRNA) expression levels of α-SMA, collagen 1α1, transforming growth factor (TGF)-ß1, and tissue inhibitor of metalloproteinase (TIMP)-1. Accumulated CD11b(+) macrophages in CCl4 -treated WT mice showed marked increases in TNF, NO synthase-2, and TGF-ß1 mRNA expression compared with CCR9(-/-) mice, implying proinflammatory and profibrogenic properties. Hepatic CD11b(+) macrophages from CCl4 -treated WT mice (i.e., CCR9(+) macrophages), but not CD8(+) T lymphocytes or non-CD11b(+) cells, significantly activated HSCs in vitro compared with those from CCR9(-/-) mice. TNF-α or TGF-ß1 antagonism attenuated CCR9(+) macrophage-induced HSC activation. Furthermore, C-C motif chemokine ligand (CCL) 25 mediated migration and, to a lesser extent, activation of HSCs in vitro. CONCLUSION: Accumulated CD11b(+) macrophages are critical for activating HSCs through the CCR9/CCL25 axis and therefore promote liver fibrosis. CCR9 antagonism might be a novel therapeutic target for liver fibrosis.

CCR9+ macrophages are required for eradication of peritoneal bacterial infections and prevention of polymicrobial sepsis.

Sepsis is a systemic inflammatory response to infection associated with multiple organ dysfunction syndrome and a high mortality rate. In septic shock induced by severe peritonitis, early response of peritoneal macrophages against infected microbes is vital in preventing the spread of infection. We found that the mucosal homing receptor CCR9, is induced in peritoneal macrophages in response to inflammatory stimulation. We used a cecal ligation and puncture (CLP) model of sepsis to determine the role of CCR9 with respect to peritoneal macrophages, and controlling peritoneal infection and systemic inflammation. CCR9(-/-) mice showed aggravated septic shock with higher mortality rates compared with wild-type (WT) mice. Six hours after CLP, CCR9(-/-) mice demonstrated a greater inflammatory response. This was associated with higher production of inflammatory cytokines, such as IL-6, TNF and IP-10 in peritoneal lavage compared with WT mice. Although the numbers of peritoneal bacteria were elevated in CCR9(-/-) mice subjected to CLP compared with WT mice, this was normalized in CCR9(-/-) mice subjected to CLP through the adoptive transfer of WT peritoneal macrophages. We conclude that CCR9 is required for recruitment of peritoneal macrophages in the steady state to control systemic sepsis during early phases of peritoneal infection.

Expression, regulation, and function of atypical chemerin receptor CCRL2 on endothelial cells.

Chemokine (CC motif) receptor-like 2 (CCRL2) binds leukocyte chemoattractant chemerin and can regulate local levels of the attractant, but does not itself support cell migration. In this study, we show that CCRL2 and VCAM-1 are upregulated on cultured human and mouse vascular endothelial cells (EC) and cell lines by proinflammatory stimuli. CCRL2 induction is dependent on NF-κB and JAK/STAT signaling pathways, and activated endothelial cells specifically bind chemerin. In vivo, CCRL2 is constitutively expressed at high levels by lung endothelial cells and at lower levels by liver endothelium; and liver but not lung EC respond to systemic LPS injection by further upregulation of the receptor. Plasma levels of total chemerin are elevated in CCRL2(-/-) mice and are significantly enhanced after systemic LPS treatment in CCRL2(-/-) mice compared with wild-type mice. Following acute LPS-induced pulmonary inflammation in vivo, chemokine-like receptor 1 (CMKLR1)(+) NK cell recruitment to the airways is significantly impaired in CCRL2(-/-) mice compared with wild-type mice. In vitro, chemerin binding to CCRL2 on endothelial cells triggers robust adhesion of CMKLR1(+) lymphoid cells through an α(4)ß(1) integrin/VCAM-1-dependent mechanism. In conclusion, CCRL2 is expressed by EC in a tissue- and activation-dependent fashion, regulates circulating chemerin levels and its bioactivity, and enhances chemerin- and CMKLR1-dependent lymphocyte/EC adhesion in vitro and recruitment to inflamed airways in vivo. Its expression and/or induction on EC by proinflammatory stimuli provide a novel and specific mechanism for the local enrichment of chemerin at inflammatory sites, regulating the recruitment of CMKLR1(+) cells.

Thymus-expressed chemokine promotes survival of PC12 cells via PI3K pathway.

We reported previously that CCR9 was neuroprotective in the mouse hippocampal neurons. This study was aimed to investigate if thymus-expressed chemokine (TECK)/CCL25 could promote survival of PC12 cells though its receptor CCR9. pEGFP-N1/CCR9 recombinant was constructed and transfected into PC12 cells. Along with this, 50 nM NGF was used to induce PC12 cells to differentiate into sympathetic-like neurons. We show here that under serum-free conditions and within a concentration range (50-200 nM), TECK rescued pEGFP-N1/CCR9 transfected PC12 cells from undergoing apoptosis in serum-free medium; however, it did not exert a similar effect on the cells in the control. On the other hand, the PC12 cells succumbed to a higher concentration of TECK (≥ 300 nM). Bim expression was up-regulated in PC12 cells cultured in serum-free medium in the absence of factors or with anti-TECK+TECK; however, it was not up-regulated in TECK-treated PC12 cells. p-Akt was detected at 15 min which lasted for at least 60 min when PC12 cells were cultured in serum-free medium with TECK. Additionally, it was shown that such an effect was effectively blocked by PI3K inhibitor, Wortmannin. These data suggest that TECK promotes survival of serum-deprived PC12 cells through its receptor, CCR9, most likely via the PI3K/Akt signaling pathway.

CCL25 mediates migration, invasion and matrix metalloproteinase expression by breast cancer cells in a CCR9-dependent fashion.

Breast cancer (BrCa) is one of the most frequently diagnosed cancers and the second leading cause of cancer-related deaths in North American women. Most deaths are caused by metastasis, and BrCa is characterized by a distinct metastatic pattern involving lymph nodes, bone marrow, lung, liver and brain. Migration of metastatic cells share many similarities with leukocyte trafficking, which are regulated by chemokines and their receptors. The current study evaluates the expression and functional role of CCR9, and its only known ligand, CCL25, in BrCa cell migration and invasion. Quantitative immunohistochemical analysis showed that both moderately and poorly differentiated BrCa tissue expressed significantly more (P<0.0001) CCR9 compared to non-neoplastic breast tissue. Interestingly, poorly differentiated BrCa tissue expressed significantly more (P<0.0001) CCR9 compared to moderately differentiated BrCa tissue. Similarly, CCR9 was highly expressed by the aggressive breast cancer cell line (MDA-MD-231) compared to the less aggressive MCF-7. Migration as well as invasion assays were used to evaluate the functional interaction between CCR9 and CCL25 in BrCa cell lines (MDA-MB-231 and MCF-7). Neutralizing CCR9-CCL25 interactions significantly impaired the migration and invasion of BrCa cells. Furthermore, CCL25 enhanced the expression of MMP-1, -9, -11 and -13 active proteins by BrCa cells in a CCR9-dependent fashion. These studies show CCR9 is functionally and significantly expressed by BrCa (poorly > moderately differentiated) tissue and cells as well as that CCL25 activation of this receptor promotes breast tumor cell migration, invasion and MMP expression, which are key components of BrCa metastasis.

GSK-1605786, a selective small-molecule antagonist of the CCR9 chemokine receptor for the treatment of Crohn's disease.

GSK-1605786 (CCX-282; Traficet-EN), a selective antagonist of the CC chemokine receptor (CCR9), is being developed by GlaxoSmithKline plc under license from ChemoCentryx Inc for the potential treatment of inflammatory bowel disease, including Crohn's disease and celiac disease. CCR9 is a tissue-specific lymphocyte trafficking molecule that selectively attracts both B- and T-cells to the small gut. Inhibition of CCR9 by GSK-1605786 may inhibit B- and T-cell entry to the small gut and ameliorate inflammation while leaving immune function at other anatomical sites unaffected. GSK-1605786 was assessed as a treatment for moderate-to-severe Crohn's disease in the phase II/III PROTECT-1 trial and as a treatment for celiac disease in a phase II trial. Data suggest that GSK-1605786 is efficacious in patients with Crohn's disease with the advantage of being orally bioavailable.

Novel mechanisms for the control of renin synthesis and release.

Renin is the key regulated step in the enzymatic cascade that leads to angiotensin generation and the control of blood pressure and fluid/electrolyte homeostasis. In the adult unstressed animal, renin is synthesized and released by renal juxtaglomerular cells. However, when homeostasis is threatened, the number of cells that express and release renin increases and extends beyond the juxtaglomerular area; the result is an increase in circulating renin and the reestablishment of homeostasis. The increase in the number of renin cells, a process termed recruitment, is achieved by dedifferentiation and re-expression of renin in cells derived from the renin lineage. The mechanisms that regulate the related processes of reacquisition of the renin phenotype, renin synthesis, and renin release are beginning to be understood. Numerous studies point to cAMP as a central common factor for the regulation of renin phenotype. In addition, we are seeing the emergence of gap junctions and microRNAs as new and promising avenues for a more complete understanding of the complex regulation of the renin cell.

CCR7 and CCR9 together recruit hematopoietic progenitors to the adult thymus.

T lymphopoiesis requires settling of the thymus by bone marrow-derived precursors throughout adult life. Progenitor entry into the thymus is selective, but the molecular basis of this selectivity is incompletely understood. The chemokine receptor CCR9 has been demonstrated to be important in this process. However, progenitors lacking CCR9 can still enter the thymus, suggesting a role for additional molecules. Here we report that the chemokine receptor CCR7 is also required for efficient thymic settling. CCR7 is selectively expressed on bone marrow progenitors previously shown to have the capacity to settle the thymus, and CCR7(-/-) progenitors are defective in settling the thymus. We further demonstrate that CCR7 sustains thymic settling in the absence of CCR9. Mice deficient for both CCR7 and CCR9 have severe reductions in the number of early thymic progenitors, and in competitive assays CCR7(-/-)CCR9(-/-) double knockout progenitors are almost completely restricted from thymic settling. However, these mice possess near-normal thymic cellularity. Compensatory expansion of intrathymic populations can account for at least a part of this recovery. Together our results illustrate the critical role of chemokine receptor signaling in thymic settling and help to clarify the cellular identity of the physiologic thymic settling progenitors.