Chemokine Receptor CCR5 Research Articles

MAIT cells in chronic HBV infection‐related liver fibrosis: Friends or foes?

MAIT cells in chronic HBV infection‐related liver fibrosis: Friends or foes?

Inflammasome is a central player in B cell development and homing.

Whereas the role of the nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain-containing protein (NLRP) 3 pathway in innate immunity has been extensively studied, little attention has been paid to its contribution to adaptive immunity. Studies in animal models and human subjects have shown the contribution of NLRP3 to the T cell compartment, and its role in B lymphocyte functions has been proposed. Here, we report that ablation of nlrp3 in mice led to altered B cell development in the bone marrow, and distorted expression of B cell subsets that play innate-like functions, that is, marginal zone B cells in the spleen and B-1a cells in the peritoneal cavity. Mechanistically, in the absence of NLRP3 expression, the transcription factor IRF4, previously found to interact with NLRP3 in the nucleus of lymphocytes, was up-regulated. NLRP3 ablation reduced the expression of the chemokine receptors CXCR4 and CCR7 in an IRF4-dependent manner, indicating that the presence of NLRP3 is critical for optimal expression of chemokine receptors on B cells. We conclude that activation of the NLRP3 inflammasome plays a role in B cell development, homing, and retention in lymphoid organs.

CCL20/CCR6 chemokine signaling is not essential for pathogenesis in an experimental autoimmune encephalomyelitis mouse model of multiple sclerosis

Multiple sclerosis is an autoimmune disease in which the immune system attacks the nerve myelin sheath. The balance between pathogenic Th17 cells and regulatory Treg cells, both of which express the chemokine receptor CCR6 is critical for determining disease activity. It has been postulated that CCL20, the cognate ligand of CCR6, produced by the blood-brain barrier attracts these immune cells to the central nervous system (CNS). However, the pathological phenotypes of the experimental model of multiple sclerosis in CCR6-knockout (KO) mice are inconclusive, while this has not been addressed in CCL20-KO mice.To address this, we generated CCL20-KO and CCR6-KO mice using the CRISPR/Cas9 system. Clinical phenotypes of experimental autoimmune encephalomyelitis (EAE) in the chronic phase were slightly exacerbated in both mutant mice relative to those in wild-type (WT) mice. Inflammatory cell infiltration and demyelination in the CNS were similar in the KO and WT mice. CNS CD4+ T cell counts were the same for mutant and WT mice. The mutant and WT mice did not differ significantly in the proportions of Th17 and Treg cells in the CNS, or in IL-17 and TGF-β mRNA expression in the CNS.These findings suggest that CCL20/CCR6-mediated cell migration is not necessarily required for the onset of EAE, and may be compensated for by other chemokine signals.

Multifaceted Defects in Monocytes in Different Phases of Chronic Hepatitis B Virus Infection: Lack of Restoration after Antiviral Therapy.

Monocytes play an important role in the control of microbial infection, but monocyte biology during chronic hepatitis B virus (HBV) infection (CHI) remains inadequately studied. We investigated the frequency, phenotype, and functions of monocyte subsets in different phases of CHI, namely, immune tolerance (IT), hepatitis B early antigen (HBeAg)-positive/HBeAg-negative chronic hepatitis B (EP-/EN-CHB, respectively), and inactive carrier (IC), identified factors responsible for their functional alterations, and determined the impact of antiviral therapy on these cells. Flow cytometric analysis indicated that HLA-DR+ CD14++ CD16- classical monocytes were significantly reduced while HLA-DR+ CD14++ CD16+ intermediate and HLA-DR+ CD14+ CD16++ nonclassical monocytes were expanded in IT and EP-/EN-CHB compared with those in IC and healthy controls (HC). In comparison to IC/HC, monocytes in IT and CHB exhibited diminished expression of Toll-like receptor 2 (TLR-2)/TLR-4/TLR-9 and cytokines interleukin-12 (IL-12)/tumor necrosis factor alpha (TNF-α)/IL-6 but produced higher levels of IL-10/transforming growth factor β (TGF-β). Further, monocytes in CHB/IT showed impaired phagocytosis and oxidative response relative to those in IC/HC. In vitro assays indicated that high titers of hepatitis B surface antigen (HBsAg) present in IT/CHB and of IL-4 in CHB triggered the functional defects in monocytes via induction of β-catenin. Additionally, monocyte-derived M1 macrophages of CHB/IT produced fewer proinflammatory and more anti-inflammatory cytokines than those of IC/HC, while in CHB/IT, the monocytes skewed the differentiation of CD4+ T cells more toward regulatory T cells and a Th2 phenotype. Moreover, monocytes in CHB and IT overexpressed chemokine receptor CCR2, which coincided with increased intrahepatic accumulation of β-catenin+ CD14+ cells. One year of tenofovir therapy failed to normalize monocyte functions or reduce serum HBsAg/IL-4 levels. Taken together, monocytes are functionally perturbed mostly in IT and EP-/EN-CHB phases. Targeting intramonocytic β-catenin or reducing HBsAg/IL-4 levels might restore monocyte function and facilitate viral clearance. IMPORTANCE Chronic HBV infection (CHI) is a major cause of end-stage liver disease for which pharmacological treatments currently available are inadequate. Chronically HBV-infected patients fail to mount an efficient immune response to the virus, impeding viral clearance and recovery from hepatitis. Monocytes represent a central part of innate immunity, but a comprehensive understanding on monocyte involvement in CHI is still lacking. We here report a multitude of defects in monocytes in chronically HBV-infected patients that include alteration in subset distribution, Toll-like receptor expression, cytokine production, phagocytic activity, oxidative response, migratory ability, polarization of monocyte-derived macrophages, and monocyte-T-cell interaction. We demonstrated that high levels of hepatitis B virus surface antigen and IL-4 potentiate these defects in monocytes via β-catenin induction while therapy with the nucleotide analog tenofovir fails to restore monocyte function. Our findings add to the continuing effort to devise new immunotherapeutic strategies that could reverse the immune defects in CHI.

Proteomic and Transcriptomic Profiling Reveals Insights into Mechanisms of Leukemic Immune Escape

Despite the high rates of complete remissions achievable by modern frontline therapies, relapse of acute lymphoblastic leukemia (ALL) remains common and is associated with poor long-term survival. Relapse occurs due to the outgrowth of cells from the residual leukemia niche. These are able to evade populations of leukemia-specific memory/effector CD8+ and CD4+ T-cells that are robustly detectable in most patients. This implies a need for better understanding of basic mechanisms underlying relapse and leukemic immune evasion. The presence of phenotypically exhausted PD1+/- TIM3+ CD4+ T-cells in diagnostic bone marrow biopsy samples has been associated with inferior clinical outcomes in clinical studies of ALL patients. This suggests that CD4+ T-cell dysfunction facilitates immune escape by residual leukemic cells. To examine this, we used single-cell RNAseq/CITESEq to study CD4+ T-cells in diagnostic bone marrow biopsy samples and a murine model of BCR-ABL-rearranged ALL. In both settings, we discovered that FOXP3-PD1+ TIM3+ CD4+ T-cells are primarily composed of a unique population capable of helper and cytotoxic functions ("T-helper cytotoxic" or THCTX cells). THCTX cells expressed the effector cytokines TNF and IFNγ, but also the suppressive cytokine IL-10. In advanced leukemia, THCTX cells maintained IFNγ expression but lost the capacity for TNF synthesis, suggestive of a unique state of exhaustion. Administration of an anti-PDL1 antibody in combination with the BCR-ABL inhibitor nilotinib markedly improved long-term survival from 10% to 70% in preclinical models. Treatment benefit from dual therapy was dependent on the presence of CD4+ T-cells and was associated with the clonal expansion of leukemia-specific THCTX cells. Expanded THCTX clones had relatively preserved expression of the helper molecule CD40L as well as of ligands for the CCR5 chemokine receptor, which plays a key role in recruitment of memory/effector T-cells. Congruent with these observations, anti-PDL1 checkpoint blockade was also associated with higher frequencies of Granzyme+ CD8+ T-cells. T-cell exhaustion occurs due to chronic TCR stimulation occurring in the presence of concerted microenvironmental cues from surrounding cell types. To understand how the topography of the leukemia niche contributes to the observed exhaustion of CD4+ T-cells, we performed spatial proteomic profiling of mouse splenic tissue during leukemia development. Leukemic cells express GFP and were recognized with an anti-GFP antibody. Early leukemic infiltration led to pronounced disruption of the immune structures of the spleen and the adoption of novel cell neighborhoods (Figure 1). Infiltration was marked by loss of boundaries between the red and white pulp, and dissolution of the marginal and mantle zones. CD4+ and CD8+ T-cells were diminished in frequency, infrequently observed in proximity to leukemic cells, and expressed exhaustion markers, suggestive of an immune desert phenotype. Overall, these findings support a model in which ALL progression occurs due to the accrued dysfunction of CD4+ THCTX cells marked by a loss of helper and chemotactic functions. This is coincident with profound disruption and reorganization of the lymphoid environment into T-cell poor zones. These findings provide mechanistic insights into the high relapse risk observed among patients with CD4+ T-cell phenotypic exhaustion. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Effect of azithromycin on migration of peripheral blood NK cells from patients with chronic obstructive pulmonary disease

Currently, no drugs have been identified that could slow progression of chronic obstructive pulmonary disease (COPD), or have a significant impact on patient mortality. Therefore, research continues aimed at studying the mechanisms of COPD development and searching for drugs that affect its molecular pathogenesis. The aim of our work was to determine the ability of azithromycin combined with corticosteroids to affect the migration of peripheral blood NK cells from the COPD patients. In the present study, we have measured expression of chemokine receptors CCR5, CCR6, CCR7, CXCR3, CXCR4, CXCR6 on the surface of peripheral blood NK cells (CD3- CD56+) by means of flow cytometry in 54 smoking patients with COPD, 21 healthy smokers, and 20 healthy non-smokers. Moreover, the effect of azithromycin (10 µg/mL) and budesonide (10 nM) on the migration of NK cells from COPD patients (n = 8) towards CCL5 (10 nM) and CXCL10 (10 nM) was determined. We found that the percentage of NK cells expressing CXCR3 and CCR5 chemokine receptors was increased in smoking patients with COPD compared with healthy smokers and healthy non-smokers. However, the proportion of these NK cell subsets did not differ between healthy smokers and healthy non-smokers. There were no significant differences in the percentage of NK cells expressing CXCR4, CXCR6, CCR6, CCR7 chemokine receptors between the three groups of subjects. Addition of budesonide to the cell suspensions decreased the migration of blood NK cells towards CCL5 and CXCL10. Azithromycin was also shown to suppress the migration of blood NK cells towards these chemokines. The combination of azithromycin and budesonide was more potent at inhibiting NK cell chemotaxis towards CCL5 and CXCL10 than any of these drugs added alone. Our results demonstrate a change in the chemokine receptor profile of NK cells in COPD patients and indicate the advantages of the combined use of corticosteroids and azithromycin for COPD treatment.

IMMU-21. GLIOMA-DERIVED FACTORS RECRUIT AND INDUCE AN IMMUNE SUPPRESSIVE PHENOTYPE IN BONE MARROW-DERIVED CCR2+ MYELOID CELLS

Abstract INTRODUCTION Infiltrating immune-suppressive myeloid cells represent a tumor supportive population that contributes to immune checkpoint inhibitor resistance and poor survival in Glioblastoma (GBM) patients. We have previously characterized monocytic-myeloid derived suppressor cells (M-MDSCs) based on their dual expression of chemokine receptors CCR2 and CX3CR1(CCR2+/CX3CR1+). Genetic and pharmacologic targeting of CCR2, in combination with PD-1 blockade, reduced the percentage of M-MDSCs in the glioma-microenvironment and slowed the progression of KR158 and 005GSC murine gliomas. Additional studies are needed to investigate the chemokines responsible for the tumor recruitment of CCR2+/CX3CR1+ cells and the impact of glioma derived factors on their immune suppressive phenotype. OBJECTIVE Evaluate the effect of glioma derived factors on the migration and suppression of bone marrow CCR2+/CX3CR1+ myeloid cells. METHODS A transwell migration assay was utilized to determine the migratory ability of CCR2+/CX3CR1+ cells to KR158B conditioned in the presence of CCL2 and CCL7 neutralizing antibodies. Ly6G-/GR1+ cells were isolated from bone marrow cultured with KR158B conditioned media and co-cultured with freshly isolated T-cells to examine their immune-suppressive phenotype. RESULTS KR158B gliomas differentially upregulate cytokines including CCL2, IL6, G-CSF, GM-CSF as compared to healthy naive brains. KR158B conditioned media increased the percentage of bone marrow-derived CCR2+/CX3CR1+ cells that are CD11b+, Ly6Chi, and Ly6G-. Bone marrow-derived CCR2+/CX3CR1+ cells expanded in KR158B condition media suppress both CD4+ and CD8+ T cell proliferation. Bone marrow-derived CCR2+/CX3CR1+ cells migrate to recombinant CCL2 and CCL7 as well as KR158B glioma conditioned media. Migration to conditioned media is completely inhibited by the combination of CCL2 and CCL7 neutralizing antibodies. High CCL2 and CCL7 are associated with poor prognosis in human GBM. CONCLUSION Glioma-derived CCL2 and CCL7 mediate migration of CCR2+ myeloid cells into the tumor microenvironment in a redundant manner. Additional glioma-derived factors induce CCR2+/CX3CR1+ myeloid cells to a CD4/8+ T cell suppressive state.

Overexpression of the Chemokine Receptor CCR7 In Patients with Hepatocellular Carcinoma: Correlation with Disseminated Circulating Tumor Cells.

Overexpression of the Chemokine Receptor CCR7 In Patients with Hepatocellular Carcinoma: Correlation with Disseminated Circulating Tumor Cells.

Myeloid- and hepatocyte-specific deletion of group VIA calcium-independent phospholipase A2 leads to dichotomous opposing phenotypes during MCD diet-induced NASH

Polymorphisms of phospholipase A2VIA (iPLA2β or PLA2G6) are associated with body weights and blood C-reactive protein. The role of iPLA2β/PLA2G6 in non-alcoholic steatohepatitis (NASH) is still elusive because female iPla2β-null mice showed attenuated hepatic steatosis but exacerbated hepatic fibrosis after feeding with methionine- and choline-deficient diet (MCDD). Herein, female mice with myeloid- (MPla2g6−/−) and hepatocyte- (LPla2g6−/−) specific PLA2G6 deletion were generated and phenotyped after MCDD feeding. Without any effects on hepatic steatosis, MCDD-fed MPla2g6−/− mice showed further exaggeration of liver inflammation and fibrosis as well as elevation of plasma TNFα, CCL2, and circulating monocytes. Bone-marrow-derived macrophages (BMDMs) from MPla2g6−/− mice displayed upregulation of PPARγ and CEBPα proteins, and elevated release of IL6 and CXCL1 under LPS stimulation. LPS-stimulated BMDMs from MCDD-fed MPla2g6−/− mice showed suppressed expression of M1 Tnfa and Il6, but marked upregulation of M2 Arg1, Chil3, IL10, and IL13 as well as chemokine receptors Ccr2 and Ccr5. This in vitro shift was associated with exaggeration of hepatic M1/M2 cytokines, chemokines/chemokine receptors, and fibrosis genes. Contrarily, MCDD-fed LPla2g6−/− mice showed a complete protection which was associated with upregulation of Ppara/PPARα and attenuated expression of Pparg/PPARγ, fatty-acid uptake, triglyceride synthesis, and de novo lipogenesis genes. Interestingly, LPla2g6−/− mice fed with chow or MCDD displayed an attenuation of blood monocytes and elevation of anti-inflammatory lipoxin A4 in plasma and liver. Thus, PLA2G6 inactivation specifically in myeloid cells and hepatocytes led to opposing phenotypes in female mice undergoing NASH. Hepatocyte-specific PLA2G6 inhibitors may be further developed for treatment of this disease.

Inflammatory Chemokine Receptors Support Inflammatory Macrophage and Dendritic Cell Maturation.

Dendritic cells form clusters in vivo, but the mechanism behind this has not been determined. In this article, we demonstrate that monocytes from mice deficient in the chemokine receptors CCR1, CCR2, CCR3, and CCR5 display reduced clustering in vitro, which is associated with impaired dendritic cell and macrophage differentiation. We further show that the differentiating cells themselves produce ligands for these receptors that function, in a redundant manner, to regulate cell clustering. Deletion of, or pharmacological blockade of, more than one of these receptors is required to impair clustering and differentiation. Our data show that chemokines and their receptors support clustering by increasing expression of, and activating, cell-surface integrins, which are associated with cell-cell interactions and, in the context of monocyte differentiation, with reduced expression of Foxp1, a known transcriptional suppressor of monocyte differentiation. Our data therefore provide a mechanism whereby chemokines and their receptors typically found in inflammatory environments can interact to promote murine monocyte differentiation to macrophages and dendritic cells.

Activation of the Chemokine Receptor CCR1 and Preferential Recruitment of Gαi Suppress RSV Replication: Implications for Developing Novel Respiratory Syncytial Virus Treatment Strategies.

Respiratory syncytial virus (RSV) is a major pathogen that can cause acute respiratory infectious diseases of the upper and lower respiratory tract, especially in children, elderly individuals, and immunocompromised people. Generally, following viral infection, respiratory epithelial cells secrete cytokines and chemokines to recruit immune cells and initiate innate and/or adaptive immune responses. However, whether chemokines affect viral replication in nonimmune cells is rarely clear. In this study, we detected that chemokine CCL5 was highly expressed, while expression of its receptor, CCR1, was downregulated in respiratory epithelial cells after RSV infection. When we overexpressed CCR1 on respiratory epithelial cells in vivo or in vitro, viral load was significantly suppressed, which can be restored by the neutralizing antibody for CCR1. Interestingly, the antiviral effect of CCR1 was not related to type I interferon (IFN-I), apoptosis induction, or viral adhesion or entry inhibition. In contrast, it was related to the preferential recruitment and activation of the adaptor Gαi, which promoted inositol 1,4,5-triphosphate receptor type 3 (ITPR3) expression, leading to inhibited STAT3 phosphorylation; explicitly, phosphorylated STAT3 (p-STAT3) was verified to be among the important factors regulating the activity of HSP90, which has been previously reported to be a chaperone of RSV RNA polymerase. In summary, we are the first to reveal that CCR1 on the surface of nonimmune cells regulates RSV replication through a previously unknown mechanism that does not involve IFN-I induction. IMPORTANCE Our results revealed a novel mechanism by which RSV escapes innate immunity. That is, although it induces high CCL5 expression, RSV might attenuate the binding of CCL5 by downregulating the expression of CCR1 in respiratory epithelial cells to weaken the inhibitory effect of CCR1 on HSP90 activity and thereby facilitate RSV replication in nonimmune cells. This study provides a new target for the development of co-antiviral inhibitors against other components of the host and co-molecular chaperone/HSP90 and provides a scientific basis for the search for effective broad-spectrum antiviral drugs.

Tetrabromobisphenol A effects on differentiating mouse embryonic stem cells reveals unexpected impact on immune system.

Tetrabromobisphenol A (TBBPA) is a potent flame retardant used in numerous appliances and a major pollutant in households and ecosystems. In vertebrates, it was shown to affect neurodevelopment, the hypothalamic-pituitary-gonadal axis and thyroid signaling, but its toxicity and modes of actions are still a matter of debate. The molecular phenotype resulting from exposure to TBBPA is only poorly described, especially at the level of transcriptome reprogramming, which further limits our understanding of its molecular toxicity. In this work, we combined functional genomics and system biology to provide a system-wide description of the transcriptomic alterations induced by TBBPA acting on differentiating mESCs, and provide potential new toxicity markers. We found that TBBPA-induced transcriptome reprogramming affect a large collection of genes loosely connected within the network of biological pathways, indicating widespread interferences on biological processes. We also found two hotspots of action: at the level of neuronal differentiation markers, and surprisingly, at the level of immune system functions, which has been largely overlooked until now. This effect is particularly strong, as terminal differentiation markers of both myeloid and lymphoid lineages are strongly reduced: the membrane T cell receptor (Cd79a, Cd79b), interleukin seven receptor (Il7r), macrophages cytokine receptor (Csf1r), monocyte chemokine receptor (Ccr2). Also, the high affinity IgE receptor (Fcer1g), a key mediator of allergic reactions, is strongly induced. Thus, the molecular imbalance induce by TBBPA may be stronger than initially realized.

CCR7-mediated T follicular helper cell differentiation is associated with the pathogenesis and immune microenvironment of spinal cord injury-induced immune deficiency syndrome.

Spinal cord injury-induced immune deficiency syndrome (SCI-IDS) is a disorder characterized by systemic immunosuppression secondary to SCI that dramatically increases the likelihood of infection and is difficult to treat. T follicular helper (Tfh) cells regulated by chemokine receptor CCR7 are associated with SCI-IDS after acute SCI. The present study explored the roles of CCR7 in SCI-IDS occurrence and immune microenvironment composition. Gene expression profile data of peripheral blood leukocytes from SCI and non-SCI subjects were collected from the Gene Expression Omnibus database. According to differential gene expression analysis, a protein-protein interaction (PPI) network, and risk model construction, the CCR7 expression level was prominently related to acute SCI and CCR7 expression was significantly downregulated after acute SCI. Next, we constructed a clinical prediction model and used it to identify patients with acute SCI. Using Gene Ontology (GO) analysis and gene set enrichment analysis (GSEA), we discovered that immune-related biological processes, such as T cell receptor signaling pathway, were suppressed, whereas chemokine-related signaling pathways were activated after acute SCI. Immune infiltration analysis performed using single sample GSEA and CIBERSORT suggested that Tfh cell function was significantly correlated with the CCR7 expression levels and was considerably reduced after acute SCI. Acute SCI was divided into two subtypes, and we integrated multiple classifiers to analyze and elucidate the immunomodulatory relationships in both subtypes jointly. The results suggested that CCR7 suppresses the immunodeficiency phenotype by activating the chemokine signaling pathway in Tfh cells. In conclusion, CCR7 exhibits potential as a diagnostic marker for acute SCI.

CCR3 blockage elicits polyploidization associated with the signatures of epithelial-mesenchymal transition in carcinoma cell lines.

Malignant features such as the acquisition of metastatic ability, stemness of cells, and therapeutic resistance of cancer cells are associated with epithelial-mesenchymal transition (EMT) accompanied by changes in motility and morphology. Recent reports implicated that the formation of polyploid giant cancer cells (PGCCs) in human malignancy correlated with the EMT processes. Chemokines are often involved in the regulation of cancer cell migration into tissues, and various types of human cancers exhibit enhanced expression of chemokine receptors, which could augment intrinsic potentials such as invasive activity, proliferating ability, and survival capacity in cancer cells. Nevertheless, the contribution of CCR3 in malignant cancer cells is controversial because it is a well-known primal receptor for the migration of eosinophils, one of the cells of the innate immune system. Here, we explored the blockage of chemokine receptor CCR3 in carcinoma cell lines and found that inhibition of CCR3 induced the formation of polyploid giant cells and stabilization of β-catenin via the PI3K/Akt/GSK-3β signaling pathway, which are processes associated with EMT. As a result of CCR3 inhibition, converted cells acquired enhanced mobile and proliferation abilities. In summary, these data indicate that modulation of the CCR3/PI3K/Akt/GSK-3β signaling pathway regulates polyploidization associated with the EMT processes.

A Functional Network Driven by MicroRNA-125a Regulates Monocyte Trafficking in Acute Inflammation.

During the onset of acute inflammation, rapid trafficking of leukocytes is essential to mount appropriate immune responses towards an inflammatory insult. Monocytes are especially indispensable for counteracting the inflammatory stimulus, neutralising the noxa and reconstituting tissue homeostasis. Thus, monocyte trafficking to the inflammatory sites needs to be precisely orchestrated. In this study, we identify a regulatory network driven by miR-125a that affects monocyte adhesion and chemotaxis by the direct targeting of two adhesion molecules, i.e., junction adhesion molecule A (JAM-A), junction adhesion molecule-like (JAM-L) and the chemotaxis-mediating chemokine receptor CCR2. By investigating monocytes isolated from patients undergoing cardiac surgery, we found that acute yet sterile inflammation reduces miR-125a levels, concomitantly enhancing the expression of JAM-A, JAM-L and CCR2. In contrast, TLR-4-specific stimulation with the pathogen-associated molecular pattern (PAMP) LPS, usually present within the perivascular inflamed area, resulted in dramatically induced levels of miR-125a with concomitant repression of JAM-A, JAM-L and CCR2 as early as 3.5 h. Our study identifies miR-125a as an important regulator of monocyte trafficking and shows that the phenotype of human monocytes is strongly influenced by this miRNA, depending on the type of inflammatory stimulus.

A RORγt+ cell instructs gut microbiota-specific Treg cell differentiation.

The mutualistic relationship of gut-resident microbiota and the host immune system promotes homeostasis that ensures maintenance of the microbial community and of a largely non-aggressive immune cell compartment1,2. The consequences of disturbing this balance include proximal inflammatory conditions, such as Crohn's disease, and systemic illnesses. This equilibrium is achieved in part through the induction of both effector and suppressor arms of the adaptive immune system. Helicobacter species induce T regulatory (Treg) and T follicular helper (TFH) cells under homeostatic conditions, but induce inflammatory T helper 17 (TH17) cells when induced Treg (iTreg) cells are compromised3,4. How Helicobacter and other gut bacteria direct T cells to adopt distinct functions remains poorly understood. Here we investigated the cells and molecular components required for iTreg cell differentiation. We found that antigen presentation by cells expressing RORγt, rather than by classical dendritic cells, was required and sufficient for induction of Treg cells. These RORγt+ cells-probably type 3 innate lymphoid cells and/or Janus cells5-require the antigen-presentation machinery, the chemokine receptor CCR7 and the TGFβ activator αv integrin. In the absence of any of these factors, there was expansion of pathogenic TH17 cells instead of iTreg cells, induced by CCR7-independent antigen-presenting cells. Thus, intestinal commensal microbes and their products target multiple antigen-presenting cells with pre-determined features suited to directing appropriate T cell differentiation programmes, rather than a common antigen-presenting cell that they endow with appropriate functions.

In vitro-induced Foxp3+ CD8+ regulatory T cells suppress allergic IgE response in the gut.

Several subsets of CD8+ T cells are known to have a suppressive function in different tissues and diseases in mice and humans. Due to the lack of a consensus on the phenotype of regulatory CD8+ T cells and very low frequency in the body, its clinical use as adoptive cellular therapy has not advanced much. In the present work, using DNA methyltransferase inhibitor 5-Aza-2'-deoxycytidine (Aza), we efficiently and stably differentiated naïve CD8+ T cells (CD8+ CD25- CD44- cells) into the CD8+ Foxp3+ regulatory CD8+ T cells (CD8 Tregs). We also generated OVA peptide257-264 -specific CD8+ Foxp3+ Tregs. Compared with activated CD8 T cells, Aza plus TGF-β-induced CD8+ Foxp3+ Tregs showed significantly increased surface expression of CD39, CD73, CD122, CD62L, and CD103, and secreted TGF-βand suppressed the proliferation of effector CD4+ T cells. Interestingly, CD8+ Foxp3+ Tregs exhibited low expression of perforin and granzyme required for cytotoxic function. Analysis of chemokine receptors showed that TGF-β+Aza induced CD8+ Foxp3+ Tregs expressed gut-tropic chemokine receptors CCR6 and CCR9, and chemokine receptors CCR7 and CXCR3 required for mobilization into the spleen, lymph nodes, and gut-associated lymphoid tissues. Adoptive transfer of induced CD8+ Foxp3+ Tregs restored cholera toxin-induced breakdown of oral tolerance to OVA by regulating OVA-specific IgE and IgG1. Altogether, we showed an efficient method to generate antigen-specific CD8+ Foxp3+ Tregs, and the adoptive transfer of these cells induces oral tolerance by suppressing allergic response and maintaining intestinal homeostasis.

IL-10 Overexpression After BCG Vaccination Does Not Impair Control of Mycobacterium tuberculosis Infection.

Control of tuberculosis depends on the rapid expression of protective CD4+ T-cell responses in the Mycobacterium tuberculosis (Mtb)-infected lungs. We have recently shown that the immunomodulatory cytokine IL-10 acts intrinsically in CD4+ T cells and impairs their parenchymal migratory capacity, thereby preventing control of Mtb infection. Herein, we show that IL-10 overexpression does not impact the protection conferred by the established memory CD4+ T-cell response, as BCG-vaccinated mice overexpressing IL-10 only during Mtb infection display an accelerated, BCG-induced, Ag85b-specific CD4+ T-cell response and control Mtb infection. However, IL-10 inhibits the migration of recently activated ESAT-6-specific CD4+ T cells into the lung parenchyma and impairs the development of ectopic lymphoid structures associated with reduced expression of the chemokine receptors CXCR5 and CCR7. Together, our data support a role for BCG vaccination in preventing the immunosuppressive effects of IL-10 in the fast progression of Mtb infection and may provide valuable insights on the mechanisms contributing to the variable efficacy of BCG vaccination.

822 RPT193, a CCR4 inhibitor, improves the inflammatory skin transcriptomic profile in patients with atopic dermatitis

The chemokine receptor CCR4 mediates CCL17- and CCL22-driven chemotaxis of Th2 cells into the skin of patients with atopic dermatitis (AD). RPT193 is an oral CCR4 inhibitor that has been developed to treat AD and other allergic inflammatory diseases. The safety and efficacy of RPT193 as monotherapy for moderate-to-severe AD was previously described in a placebo-controlled, double-blinded Phase 1 study. Here, we present the skin and peripheral biomarker analysis from this Phase 1 trial. Following 28 days of 400 mg daily RPT193 treatment, RNA-seq and/or RT-PCR demonstrated significant downregulation of general inflammation- (MMP12; p<0.01), innate immunity- (IL8; p<0.01), T-cell/T-cell activation- (IL2, CCL19, CCR7, ICOS; p<0.05), Th1- (CCL2; p<0.05), Th2- (CCL22, CCR4; p<0.05), and Th17/Th22-related markers (IL22, CCL20, CCR6, S100A8, S100A9, S100A12, PI3/Elafin; p<0.05) compared to baseline in RPT193-treated, but not placebo-treated, subjects.

Potent CCR3 Receptor Antagonist, SB328437, Suppresses Colonic Eosinophil Chemotaxis and Inflammation in the Winnie Murine Model of Spontaneous Chronic Colitis.

Eosinophils and their regulatory molecules have been associated with chronic intestinal inflammation and gastrointestinal dysfunctions; eosinophil accumulation in the gut is prominent in inflammatory bowel disease (IBD). The chemokine receptor CCR3 plays a pivotal role in local and systemic recruitment and activation of eosinophils. In this study, we targeted CCR3-ligand interactions with a potent CCR3 receptor antagonist, SB328437, to alleviate eosinophil-associated immunological responses in the Winnie model of spontaneous chronic colitis. Winnie and C57BL/6 mice were treated with SB328437 or vehicle. Clinical and histopathological parameters of chronic colitis were assessed. Flow cytometry was performed to discern changes in colonic, splenic, circulatory, and bone marrow-derived leukocytes. Changes to the serum levels of eosinophil-associated chemokines and cytokines were measured using BioPlex. Inhibition of CCR3 receptors with SB328437 attenuated disease activity and gross morphological damage to the inflamed intestines and reduced eosinophils and their regulatory molecules in the inflamed colon and circulation. SB328437 had no effect on eosinophils and their progenitor cells in the spleen and bone marrow. This study demonstrates that targeting eosinophils via the CCR3 axis has anti-inflammatory effects in the inflamed intestine, and also contributes to understanding the role of eosinophils as potential end-point targets for IBD treatment.