GPR15-C10ORF99 functional pairing initiates colonic Treg homing in amniotes.

Regulatory T lymphocyte (Treg) homing reactions mediated by G protein-coupled receptor (GPCR)-ligand interactions play a central role in maintaining intestinal immune homeostasis by restraining inappropriate immune responses in the gastrointestinal tract. However, the origin of Treg homing to the colon remains mysterious. Here, we report that the C10ORF99 peptide (also known as CPR15L and AP57), a cognate ligand of GPR15 that controls Treg homing to the colon, originates from a duplication of the flanking CDHR1 gene and is functionally paired with GPR15 in amniotes. Evolutionary analysis and experimental data indicate that the GPR15-C10ORF99 pair is functionally conserved to mediate colonic Treg homing in amniotes and their expression patterns are positively correlated with herbivore diet in the colon. With the first herbivorous diet in early amniotes, a new biological process (herbivorous diet short-chain fatty acid-C10ORF99/GPR15-induced Treg homing colon immune homeostasis) emerged, and we propose an evolutionary model whereby GPR15-C10ORF99 functional pairing has initiated the first colonic Treg homing reaction in amniotes. Our findings also highlight that GPCR-ligand pairing leads to physiological adaptation during vertebrate evolution.

Natural cystatin C fragments inhibit GPR15-mediated HIV and SIV infection without interfering with GPR15L signaling.

GPR15 is a G protein-coupled receptor (GPCR) proposed to play a role in mucosal immunity that also serves as a major entry cofactor for HIV-2 and simian immunodeficiency virus (SIV). To discover novel endogenous GPR15 ligands, we screened a hemofiltrate (HF)-derived peptide library for inhibitors of GPR15-mediated SIV infection. Our approach identified a C-terminal fragment of cystatin C (CysC95-146) that specifically inhibits GPR15-dependent HIV-1, HIV-2, and SIV infection. In contrast, GPR15L, the chemokine ligand of GPR15, failed to inhibit virus infection. We found that cystatin C fragments preventing GPR15-mediated viral entry do not interfere with GPR15L signaling and are generated by proteases activated at sites of inflammation. The antiretroviral activity of CysC95-146 was confirmed in primary CD4+ T cells and is conserved in simian hosts of SIV infection. Thus, we identified a potent endogenous inhibitor of GPR15-mediated HIV and SIV infection that does not interfere with the physiological function of this GPCR.

Enforced gut homing of murine regulatory T cells reduces early graft-versus-host disease severity.

Damage to the gastrointestinal tract following allogeneic hematopoietic stem cell transplantation is a significant contributor to the severity and perpetuation of graft-versus-host disease. In preclinical models and clinical trials, we showed that infusing high numbers of regulatory T cells reduces graft-versus-host disease incidence. Despite no change in in vitro suppressive function, transfer of ex vivo expanded regulatory T cells transduced to overexpress G protein-coupled receptor 15 or C-C motif chemokine receptor 9, specific homing receptors for colon or small intestine, respectively, lessened graft-versus-host disease severity in mice. Increased regulatory T cell frequency and retention within the gastrointestinal tissues of mice that received gut homing T cells correlated with lower inflammation and gut damage early post-transplant, decreased graft-versus-host disease severity, and prolonged survival compared with those receiving control transduced regulatory T cells. These data provide evidence that enforced targeting of ex vivo expanded regulatory T cells to the gastrointestinal tract diminishes gut injury and is associated with decreased graft-versus-host disease severity.

Tyrosine sulfation and O-glycosylation of chemoattractant receptor GPR15 differentially regulate interaction with GPR15L.

GPR15 is a G-protein-coupled receptor (GPCR) that directs lymphocyte homing to the colon and skin. Recent studies have identified a chemokine-like protein GPR15L (also known as C10orf99) as a functional ligand of GPR15. In this study, we examined the structural elements that regulate the GPR15-GPR15L interaction with primary focus on post-translational modifications (PTMs) of receptor N-terminus and on the C-terminus of the ligand. Our findings reveal that the GPR15 receptor is sulfated on the N-terminal tyrosine residue(s) and disruption of tyrosine sulfation inhibits binding of GPR15L. In contrast, the disruption of O-glycosylation on the N-terminal threonine or serine residues, or the removal of α2,3-linked sialic acids from O-glycans, enhances the GPR15L binding. Thus, GPR15 represents a unique chemoattractant receptor in which different N-terminal PTMs regulate its ligand binding in a contrasting manner. We further demonstrate that, unlike canonical chemokines, GPR15L activity critically requires its extreme C-terminal residue and that its hydrophobicity may be a key attribute that facilitates an optimal interaction with the receptor. Our results reveal novel insights into chemoattractant receptor-ligand interaction and provide a valid footing for potential intervention targeting the GPR15-GPR15L axis.

The G protein-coupled receptor 15 (GPR15) regulates cutaneous immunology by maintaining dendritic epidermal T cells and regulating the skin microbiome.

The G protein-coupled receptor 15 (GPR15) regulates homing of different T-cell populations into the gut, thus, preserving tissue homeostasis. Its potential role in the preservation of homeostasis on other body interfaces, including the skin, is less well understood. We addressed the impact of GPR15 on cutaneous T-cell populations and the skin microbiome under steady-state conditions. Genetic deficiency in GPR15 substantially altered the composition of skin-resident T-cell populations. Precisely, dendritic epidermal T cells were almost absent in the epidermis of Gpr15-/- mice. The niche of dendritic epidermal T cells in the epidermis was, instead, populated by αß TCR+ T cells. These changes were associated with shifts in the skin microbiota in Gpr15-/- mice. Collectively, our results uncover a role of GPR15 in the regulation of the cutaneous immune system and, thus, highlight the receptor as important general regulator of tissue homeostasis of exterior body interfaces.

The Role of GPR15 Function in Blood and Vasculature.

Since the first prominent description of the orphan G protein-coupled receptor 15 (GPR15) on lymphocytes as a co-receptor for the human immunodeficiency virus (HIV) type 1 and 2 and the first report about the GPR15-triggered cytoprotective effect on vascular endothelial cells by recombinant human thrombomodulin, several decades passed before the GPR15 has been recently deorphanized. Because of new findings on GPR15, this review will summarize the consequences of GPR15 signaling considering the variety of GPR15-expressing cell types and of GPR15 ligands, with a focus on blood and vasculature.

GPR15+ T cells are Th17 like, increased in smokers and associated with multiple sclerosis.

Smoking is a risk factor for the development and progression of multiple sclerosis (MS); however, the pathogenic effects of smoking are poorly understood. We studied the smoking-associated chemokine receptor-like molecule GPR15 in relation to relapsing-remitting MS (RRMS). Using microarray analyses and qPCR we found elevated GPR15 in blood cells from smokers, and increased GPR15 expression in RRMS. By flow cytometry we detected increased frequencies of GPR15 expressing T and B cells in smokers, but no difference between patients with RRMS and healthy controls. However, after cell culture with the autoantigens myelin basic protein (MBP) and myelin oligodendrocyte glycoprotein, frequencies of MBP-reactive and non-proliferating GPR15+CD4+ T cells were increased in patients with RRMS compared with healthy controls. GPR15+CD4+ T cells produced IL-17 and were enriched in the cerebrospinal fluid (CSF). Furthermore, in the CSF of patients with RRMS, GPR15+ T cells were associated with CCR6+CXCR3+/CCR6-CXCR3+ phenotypes and correlated positively with concentrations of the newly identified GPR15-ligand (GPR15L), myelin degradation and disability. In conclusion, we have identified a proinflammatory cell type linking smoking with pathogenic immune cell functions in RRMS.

Specific induction of the unique GPR15 expression in heterogeneous blood lymphocytes by tobacco smoking.

Purpose: In the peripheral blood, it has been shown that smoking is, to date, the only specific condition leading to an increase in GPR15+ T cells. We, therefore, aimed to characterize GPR15-expressing blood T cells in more detail. Materials and Methods: The whole transcriptome by RNAseq as a proxy for protein expression was analyzed in GPR15+ and GPR15- T cells. A deep immuno-phenotyping was conducted for the identification of T cell subtypes. Results: The expression of GPR15 seemed to be unique, not concomitantly accompanied with the expression of another protein. According to different T cell subtypes, there is no single cell type prominently represented in GPR15+ T cells. The individually different proportions of GPR15+ cells among each GPR15-expressing T cell subtypes in blood were strongly associated with chronic smoking. Indeed, the frequency of GPR15+ T cell subtypes can be effectively used as a highly convincing biomarker for tobacco smoking. Conclusions: While the chronic smoking-induced enrichment of GPR15+ T cells in blood might indicate a systemic inflammation, by the widespread presence in different T cell subtypes, GPR15 could feature a general impact on maintaining the systemic homeostasis to putatively prevent harm from smoking.

An Integrated Pan-Cancer Analysis and Structure-Based Virtual Screening of GPR15.

G protein-coupled receptor 15 (GPR15, also known as BOB) is an extensively studied orphan G protein-coupled receptors (GPCRs) involving human immunodeficiency virus (HIV) infection, colonic inflammation, and smoking-related diseases. Recently, GPR15 was deorphanized and its corresponding natural ligand demonstrated an ability to inhibit cancer cell growth. However, no study reported the potential role of GPR15 in a pan-cancer manner. Using large-scale publicly available data from the Cancer Genome Atlas (TCGA) and the Genotype-Tissue Expression (GTEx) databases, we found that GPR15 expression is significantly lower in colon adenocarcinoma (COAD) and rectal adenocarcinoma (READ) than in normal tissues. Among 33 cancer types, GPR15 expression was significantly positively correlated with the prognoses of COAD, neck squamous carcinoma (HNSC), and lung adenocarcinoma (LUAD) and significantly negatively correlated with stomach adenocarcinoma (STAD). This study also revealed that commonly upregulated gene sets in the high GPR15 expression group (stratified via median) of COAD, HNSC, LUAD, and STAD are enriched in immune systems, indicating that GPR15 might be considered as a potential target for cancer immunotherapy. Furthermore, we modelled the 3D structure of GPR15 and conducted structure-based virtual screening. The top eight hit compounds were screened and then subjected to molecular dynamics (MD) simulation for stability analysis. Our study provides novel insights into the role of GPR15 in a pan-cancer manner and discovered a potential hit compound for GPR15 antagonists.

Tobacco-smoking induced GPR15-expressing T cells in blood do not indicate pulmonary damage.

BACKGROUND: Recently, it was shown that chronic tobacco smoking evokes specific cellular and molecular changes in white blood cells by an excess of G protein-coupled receptor 15 (GPR15)-expressing T cells as well as a hypomethylation at DNA CpG site cg05575921 in granulocytes. In the present study, we aimed to clarify the general usefulness of these two biomarkers as putative signs of non-cancerous change in homeostasis of the lungs. METHODS: In a clinical cohort consisting of 42 patients with chronic obstructive pulmonary disease (COPD), interstitial lung disease (ILD) and pneumonia and a control cohort of 123 volunteers, the content of GPR15-expressing blood cells as well as the degree of methylation at cg05575921 were analysed by flow-cytometry and pyrosequencing, respectively. Smoking behaviour was estimated by questionnaire and cotinine level in plasma. RESULTS: Never-smoking patients could be distinguished from former and current smokers by both the proportion of GPR15-expressing T cells as well as cg05575921 methylation in granulocytes, with 100% and 97% specificity and 100% sensitivity, respectively. However, both parameters were not affected by lung diseases. The degrees of both parameters were not changed neither in non-smoking nor smoking patients, compared to appropriate control cohorts of volunteers. CONCLUSIONS: The degree of GPR15-expressing cells among T cells as well as the methylation at cg05575921 in granulocytes in blood are both rather signs of tobacco-smoking induced systemic inflammation because they don't indicate specifically non-cancerous pathological changes in the lungs.

Orphan chemoattractant receptor GPR15 mediates dendritic epidermal T-cell recruitment to the skin.

Homing of murine dendritic epidermal T cells (DETCs) from the thymus to the skin is regulated by specific trafficking receptors during late embryogenesis. Once in the epidermis, Vγ3δ1 TCR DETCs are maintained through self-renewal and participate in wound healing. GPR15 is an orphan G protein-linked chemoattractant receptor involved in the recruitment of regulatory T cells to the colon. Here we show that GPR15 is highly expressed on fetal thymic DETC precursors and on recently recruited DETCs, and mediates the earliest seeding of the epidermis, which occurs at the time of establishment of skin barrier function. DETCs in GPR15(-/-) mice remain low at birth, but later participation of CCR10 and CCR4 in DETC homing allows DETCs to reach near normal levels in adult skin. Our findings establish a role for GPR15 in skin lymphocyte homing and suggest that it may contribute to lymphocyte subset targeting to diverse epithelial sites.

Orphan receptor GPR15/BOB is up-regulated in rheumatoid arthritis.

Chemokine receptors on leukocytes mediate the recruitment and accumulation of these cells within affected joints in chronic inflammatory diseases such as rheumatoid arthritis (RA). Identification of involved receptors offers potential for development of therapeutic interventions. The objective of this study was to investigate the expression of orphan receptor GPR15/BOB in the synovium of RA and non-RA patients and in peripheral blood of RA patients and healthy donors. GPR15/BOB protein and messenger RNA expression were examined in RA and non-RA synovium by immunofluorescence and reverse-transcription polymerase chain reaction (RT-PCR) respectively. GPR15/BOB expression on peripheral blood leukocytes was analysed by flow cytometry and GPR15/BOB messenger RNA was examined in peripheral blood monocytes by RT-PCR. GPR15/BOB protein was observed in CD68+ and CD14+ macrophages in synovia, with greater expression in RA synovia. GPR15/BOB protein was expressed in all patient synovia whereas in non-RA synovia expression was low or absent. Similarly GPR15/BOB messenger RNA was detected in all RA and a minority of non-RA synovia. GPR15/BOB protein was expressed on peripheral blood leukocytes from RA and healthy individuals with increased expression by monocytes and neutrophils in RA. GPR15/BOB messenger RNA expression was confirmed in peripheral blood monocytes. In conclusion GPR15/BOB is expressed by macrophages in synovial tissue and on monocytes and neutrophils in peripheral blood, and expression is up-regulated in RA patients compared to non-RA controls. This orphan receptor on monocytes/macrophages and neutrophils may play a role in RA pathophysiology.

GPR15LG regulates psoriasis-like inflammation by down-regulating inflammatory factors on keratinocytes.

Psoriasis is a common chronic inflammatory skin disease characterized by aberrant proliferation of keratinocytes and infiltration of immune cells. We previously found that GPR15LG protein is highly expressed in psoriasis lesional skin and it positively regulates psoriatic keratinocyte proliferation. Our data also showed that GPR15LG could regulate the activity of NF-κB pathway, which is associated with psoriatic inflammation. In the present study, we demonstrated that Gpr15lg (ortholog of GPR15LG) knockdown attenuated the severity of imiquimod (IMQ)-induced psoriasis-like inflammation in mice. Such an effect was achieved by down-regulating the expression of inflammatory cytokines interleukin (IL)-1α, IL-1ß, tumor necrosis factor (TNF)-α and S100A7. Consistently, GPR15LG knockdown in vitro significantly downgraded the expression of inflammatory factors in the cellular model of psoriasis. These results suggested that GPR15LG could be involved in the development of psoriasis by regulating inflammation.

Blocking GPR15 Counteracts Integrin-dependent T Cell Gut Homing in Vivo.

BACKGROUND AND AIMS: The G protein coupled receptor GPR15 is expressed on and functionally important for T cells homing to the large intestine. However, the precise mechanisms by which GPR15 controls gut homing have been unclear. Thus, we aimed to elucidate these mechanisms as well as to explore the potential of targeting GPR15 for interfering with T cell recruitment to the colon in inflammatory bowel disease [IBD]. METHODS: We used dynamic adhesion and transmigration assays, as well as a humanised in vivo model of intestinal cell trafficking, to study GPR15-dependent effects on gut homing. Moreover, we analysed GPR15 and integrin expression in patients with and without IBD, cross-sectionally and longitudinally. RESULTS: GPR15 controlled T cell adhesion to MAdCAM-1 and VCAM-1 upstream of α4ß7 and α4ß1 integrin, respectively. Consistently, high co-expression of these integrins with GPR15 was found on T cells from patients with IBD, and GPR15 also promoted T cell recruitment to the colon in humanised mice. Anti-GPR15 antibodies effectively blocked T cell gut homing in vitro and in vivo. In vitro data, as well as observations in a cohort of patients treated with vedolizumab, suggest that this might be more effective than inhibiting α4ß7. CONCLUSIONS: GPR15 seems to have a broad, but organ-selective, impact on T cell trafficking and is therefore a promising target for future therapy of IBD. Further studies are needed.

GPR15 in colon cancer development and anti-tumor immune responses.

Introduction: The chemoattractant receptor, G protein-coupled receptor 15 (GPR15), promotes colon homing of T cells in health and colitis. GPR15 function in colon cancer is largely unexplored, motivating our current studies. Methods: In human study, immune cells were isolated from tumor tissues and healthy surgical tumor margins (STM), and their proportions as well as expression of GPR15 was analyzed by flow cytometry. In mouse studies, colon cancer was induced in GPR15-deficient (KO) and GPR15-suficient (Het) mice using azoxymethane (AOM) and dextran sulfate sodium (DSS) solution in drinking water. Serial endoscopy was performed in mice to monitor and visualize the distal region of colon. Mice were euthanized 10 weeks after the initial DSS administration, and the colon length and the number of polyps were recorded. Next, we identified the effects of GPR15L on established tumors in the MC38-colorectal cancer (CRC) mouse model. Immune cells were isolated from the mice colons or tumors and assessed by flow cytometry. Results: Our analysis of human CRC tissue revealed a significant reduction in GPR15+ immune cell frequencies in tumors compared to 'tumor-free' surgical margins. Similarly, our data analysis using The Cancer Genome Atlas (TCGA) indicated that lower GPR15 expression is associated with poor survival in human colon cancer. In the AOM/DSS colitis-associated colon cancer model, we observed increased colonic polyps and lower survival in Gpr15 +-KO compared to Gpr15-Het mice. Analysis of immune cell infiltrates in the colonic polyps showed significantly decreased CD8+ T cells and increased IL-17+ CD4+ and IL-17+ CD8+ T cells in Gpr15-KO than in Het mice. Consistent with a protective role of GPR15, administration of GPR15L to established tumors in the MC38-CRC model increased CD45+ cell infiltration, enhanced TNFa expression on CD4+ and CD8+ T cells at the tumor site and dramatically reduced tumor burden. Discussion: Our findings highlight an important, unidentified role of the GPR15-GPR15L axis in promoting a tumor-suppressive immune microenvironment and unveils a novel, colon-specific therapeutic target for CRC.

Polyphenol and glucosinolate-derived AhR modulators regulate GPR15 expression on human CD4+ T cells.

Diets high in fruit and vegetables are perceived to be beneficial for intestinal homeostasis, in health as well as in the context of inflammatory bowel diseases (IBDs). Recent breakthroughs in the field of immunology have highlighted the importance of the ligand-activated transcription factor aryl hydrocarbon receptor (AhR) as a critical regulator of mucosal immunity, including the intestinal trafficking of CD4+ helper T cells, an immune cell subset implicated in a wide range of homeostatic and pathogenic processes. Specifically, the AhR has been shown to directly regulate the expression of the chemoattractant receptor G Protein-Coupled Receptor 15 (GPR15) on CD4+ T cells. GPR15 is an important gut homing marker whose expression on CD4+ T cells in the peripheral circulation is elevated in patients suffering from ulcerative colitis, raising the possibility that, in this setting, the beneficial effect of a diet rich in fruits and vegetables may be mediated through the modulation of GPR15 expression. To address this, we screened physiologically-relevant polyphenol and glucosinolate metabolites for their ability to affect both AhR activity and GPR15 expression. Our complementary approach and associated findings suggest that polyphenol and glucosinolate metabolites can regulate GPR15 expression on human CD4+ T cells in an AhR-dependent manner.

Endocytic proteins mediating GPR15 receptor internalization provide insight into the underlying mechanisms.

GPR15 is a G protein-coupled receptor involved in immune disorders such as human immunodeficiency virus-induced enteropathy, multiple sclerosis, and colitis. Yet, the important endocytosis mechanism of GPR15 remained unclear. This study determined the participation of endocytic machinery proteins, including Gα proteins, G protein-coupled receptor kinases (GRKs), protein kinase C, arrestins, clathrin, caveolin, and dynamin in GPR15 internalization. The results demonstrate that GPR15 internalization is moderately dependent on GRKs and clathrin, and highly dependent on caveolin and dynamin. Moreover, a bystander arrestin recruitment assay showed that GPR15 recruits arrestin-3 to the cell membrane upon agonist stimulation, although GPR15 internalizes in an arrestin-independent manner. Overall, our study provides novel insights into ß-arrestin recruitment and receptor internalization mechanisms for the recently deorphanized GPR15.

Structure-activity relationship of GPR15L peptide analogues and investigation of their interaction with the GPR15 receptor.

The 57-mer full-length GPR15L(25-81) peptide has been identified as the principal endogenous agonist of the G protein-coupled receptor GPR15. Its main activity resides in the C-terminal 11-mer GPR15L(71-81), which has full efficacy but ~40-fold lower potency than the full-length peptide. Here, we systematically investigated the structure-activity relationship of GPR15L(71-81) by truncations/extensions, alanine-scanning, and N- and C-terminal capping. The synthesized peptide analogues were tested at GPR15 stably expressed in HEK293A cells using a homogenous time-resolved Förster resonance energy transfer-based Gi cAMP functional assay. We show that the C-terminal α carboxyl group and the residues Leu78 , Pro75 , Val74 , and Trp72 are critical for receptor interaction and contribute significantly to the peptide potency. Furthermore, we tested the ability of GPR15L(71-81), C-terminally amidated GPR15L(71-81), and GPR15L(25-81) to activate the three GPR15 receptor mutants in a bioluminescence resonance energy transfer-based G protein activation assay. The results demonstrate that the Lys192 and Glu272 residues in GPR15 are important for the potency of the GPR15L peptide. Overall, our study identifies critical residues in the peptide and receptor sequences for future drug design.

Emerging roles of a chemoattractant receptor GPR15 and ligands in pathophysiology.

Chemokine receptors play a central role in the maintenance of immune homeostasis and development of inflammation by directing leukocyte migration to tissues. GPR15 is a G protein-coupled receptor (GPCR) that was initially known as a co-receptor for human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV), with structural similarity to other members of the chemoattractant receptor family. Since the discovery of its novel function as a colon-homing receptor of T cells in mice a decade ago, GPR15 has been rapidly gaining attention for its involvement in a variety of inflammatory and immune disorders. The recent identification of its natural ligand C10orf99, a chemokine-like polypeptide strongly expressed in gastrointestinal tissues, has established that GPR15-C10orf99 is a novel signaling axis that controls intestinal homeostasis and inflammation through the migration of immune cells. In addition, it has been demonstrated that C10orf99-independent functions of GPR15 and GPR15-independent activities of C10orf99 also play significant roles in the pathophysiology. Therefore, GPR15 and its ligands are potential therapeutic targets. To provide a basis for the future development of GPR15- or GPR15 ligand-targeted therapeutics, we have summarized the latest advances in the role of GPR15 and its ligands in human diseases as well as the molecular mechanisms that regulate GPR15 expression and functions.

Delineation of the GPR15 receptor-mediated Gα protein signalling profile in recombinant mammalian cells.

The GPR15 receptor is a G protein-coupled receptor (GPCR), which is activated by an endogenous peptide GPR15L(25-81) and a C-terminal peptide fragment GPR15L(71-81). GPR15 signals through the Gi/o pathway to decrease intracellular cyclic adenosine 3',5'-monophosphate (cAMP). However, the activation profiles of the GPR15 receptor within Gi/o subtypes have not been examined. Moreover, whether the receptor can also couple to Gs , Gq/11 and G12/13 is unclear. Here, GPR15L(25-81) and GPR15L(71-81) are used as pharmacological tool compounds to delineate the GPR15 receptor-mediated Gα protein signalling using a G protein activation assay and second messenger assay conducted on living cells. The results show that the GPR15 receptor preferentially couples to Gi/o rather than other pathways in both assays. Within the Gi/o family, the GPR15 receptor activates all the subtypes (Gi1 , Gi2 , Gi3 , GoA , GoB and Gz ). The Emax and activation rates of Gi1, Gi2 , Gi3, GoA and GoB are similar, whilst the Emax of Gz is smaller and the activation rate is significantly slower. The potencies of both peptides toward each Gi/o subtype have been determined. Furthermore, the GPR15 receptor signals through Gi/o to inhibit cAMP accumulation, which could be blocked by the application of the Gi/o inhibitor pertussis toxin.