CCR5 Ligands Research Articles

A CCR4 antagonist attenuates atopic dermatitis-like skin inflammation by inhibiting the recruitment and expansion of Th2 cells and Th17 cells.

CCR4 is a major trafficking receptor for T-helper (Th) 2 cells and Th17 cells and is considered as a potential therapeutic target for atopic dermatitis (AD). The CCR4 ligands CCL17 and CCL22 have been reported to be upregulated in the skin lesions of AD patients. Of note, thymic stromal lymphopoietin (TSLP), a master regulator of the Th2 immune response, promotes the expression of CCL17 and CCL22 in AD skin lesions. Here, we investigated the role of CCR4 in an AD mouse model induced by MC903, a TSLP inducer. Topical application of MC903 to ear skin increased the expression of not only TSLP but also CCL17, CCL22, the Th2 cytokine IL-4, and the Th17 cytokine IL-17A. Consistently, MC903 induced AD-like skin lesions as shown by increased epidermal thickness; increased infiltration of eosinophils, mast cells, type 2 innate lymphoid cells, Th2 cells, and Th17 cells; and elevated serum levels of total IgE. We also found increased expansion of Th2 cells and Th17 cells in the regional lymph nodes (LNs) of AD mice. Compound 22, a CCR4 inhibitor, ameliorated AD-like skin lesions with reduction of Th2 cells and Th17 cells in the skin lesions and regional LNs. We further confirmed that compound 22 diminished the expansion of Th2 cells and Th17 cells in the coculture of CD11c+ dendritic cells (DCs) and CD4+ T cells derived from the regional LNs of AD mice. Collectively, CCR4 antagonists may exhibit anti-allergic effects by inhibiting both the recruitment and expansion of Th2 cells and Th17 cells in AD.

CCR4 and CCR7 differentially regulate thymocyte localization with distinct outcomes for central tolerance.

Central tolerance ensures autoreactive T cells are eliminated or diverted to the regulatory T cell lineage, thus preventing autoimmunity. To undergo central tolerance, thymocytes must enter the medulla to test their T-cell receptors (TCRs) for autoreactivity against the diverse self-antigens displayed by antigen-presenting cells (APCs). While CCR7 is known to promote thymocyte medullary entry and negative selection, our previous studies implicate CCR4 in these processes, raising the question of whether CCR4 and CCR7 play distinct or redundant roles in central tolerance. Here, synchronized positive selection assays, two-photon time-lapse microscopy, and quantification of TCR-signaled apoptotic thymocytes, demonstrate that CCR4 and CCR7 promote medullary accumulation and central tolerance of distinct post-positive selection thymocyte subsets in mice. CCR4 is upregulated within hours of positive selection signaling and promotes medullary entry and clonal deletion of immature post-positive selection thymocytes. In contrast, CCR7 is expressed several days later and is required for medullary localization and negative selection of mature thymocytes. In addition, CCR4 and CCR7 differentially enforce self-tolerance, with CCR4 enforcing tolerance to self-antigens presented by activated APCs, which express CCR4 ligands. Our findings show that CCR7 expression is not synonymous with medullary localization and support a revised model of central tolerance in which CCR4 and CCR7 promote early and late stages of negative selection, respectively, via interactions with distinct APC subsets.

Chemokine CCL19 promotes type 2 helper T cell differentiation and allergic airway inflammation

Abstract Allergic asthma is largely driven by the actions of allergen-specific CD4 +type 2 T helper (Th2) cells. Following allergen inhalation, these cells are induced by the interaction of naïve CD4 +T cells with antigen-bearing dendritic cells (DCs) in lung-draining lymph nodes (LNs). The molecular mechanisms underlying the induction of T helper cells have been well studied and include the migration of allergen-bearing DCs from the lung to regional LNs in a manner dependent on the chemokine receptor, CCR7. Previous studies have demonstrated that CCL21, a CCR7 ligand, is required for the accumulation of naïve T cells and activated DCs in the LNs, but the function of another ligand, CCL19, has been unknown. In the present study, we investigated the role of CCL19 in allergic airway inflammation using CCL19-deficient mice. In a mouse model of asthma, eosinophils and lymphocytes were significantly reduced in airways of CCL19-deficient mice compared with those of wildtype (WT) animals, and production of IL-4 and IL-13 in the lung was significantly diminished, suggesting that CCL19 is required for type 2 immune responses. Co-cultures of naïve WT CD4 +T cells with CCL19-deficient DCs produced less type 2 cytokines than did T cells co-cultured with WT DCs. Further, fibroblastic reticular cells (FRCs) from CCL19-deficient mouse LNs produced less Th2 cytokine production compared with WT FRCs. Addition of recombinant CCL19 to the culture of naïve CD4 +T cells increased phosphorylation of STAT5 and gene expression of Il2, Il2ra, Il2rb, Il2rg, Sta5a, Stat5b and Stat6. Together, our findings suggest that CCL19 produced by DCs and FRCs promotes Th2 differentiation through the activation of STAT5-signaling, and that this promotes allergic airway inflammation. This work was supported by Intramural Research Program of NIEHS, NIH (ZIA ES102025-09).

CCR4 and CCR7 differentially regulate thymocyte localization with distinct outcomes for central tolerance

Abstract Central tolerance ensures autoreactive thymocytes are either eliminated via clonal deletion or are diverted to the regulatory T-cell lineage, thus minimizing T cell autoimmunity. To undergo central tolerance, thymocytes must enter the medulla to test their TCRs for autoreactivity against the diverse self-antigens displayed by medullary antigen presenting cells (APCs). While CCR7 is known to promote thymocyte medullary entry and negative selection against some self-antigens, our previous studies implicate CCR4 in these processes, raising the question of whether CCR4 and CCR7 play distinct or redundant roles in central tolerance. Here, we use synchronized positive selection assays and 2-photon timelapse microscopy to demonstrate that CCR4 and CCR7 are expressed sequentially after positive selection and promote medullary accumulation and central tolerance of distinct post-positive selection thymocyte subsets. CCR4 is upregulated within hours of positive selection signaling and promotes medullary entry and clonal deletion of immature post-positive selection thymocytes. In contrast, CCR7 is expressed several days later and promotes medullary entry and negative selection of mature thymocytes. Unlike CCR7 ligands, which are expressed by medullary thymic epithelial cells, CCR4 ligands are expressed by hematopoietic APCs, and we find that CCR4 enforces thymocyte tolerance to self-antigens presented by activated thymic APCs. Our findings demonstrate that CCR7 expression is neither necessary nor sufficient for thymocyte medullary entry and support a revised model of central tolerance in which CCR4 and CCR7 promote early and late stages of central tolerance, respectively, via interactions with distinct APC subsets. Supported by a grant from the NIH/NIAID (R01AI104870)

Extravasation of pathogenic human type 17 Th cells requires both endothelial cell-bound and non-cell bound chemokines

Abstract Extravasation of pro-inflammatory T cells is critical for their effectiveness in host defense and for mediating tissue damage in immune-mediated disease. These cells co-express multiple chemokine receptors and understanding the activities of individual receptors in extravasation is essential for exploiting the chemokine system for therapeutic benefit – either to block or enhance the trafficking of these cells into sites of inflammation. CCR6 is expressed on all human Th17 cells, and we found that CCR6 +CCR2 +cells are enriched both in cells that express a pathogenic Th17 signature, including the ability to produce GM-CSF and IFNγ, and are unusually efficient at transendothelial migration (TEM). Ligands for CCR6, CCR5 and CXCR3 were secreted by and bound to endothelial cells activated with TNFα and IFNγ, and these receptors could all mediate firm arrest of CCR6 +CCR2 +cells under flow - but could not mediate TEM. TEM required CCR2, whose ligands, CCL2, CCL7 and CCL8, were all secreted by activated endothelial cells but failed to bind to endothelial cell surfaces. Chemokines were secreted from the basal as well as the apical sides of endothelial cells. Pre-treating CCR6 +CCR2 +cells with CCL2 or transducing endothelial cells to express a surface-bound CCL2-CXCL9 chimera led to both CCR2-mediated arrest and, importantly, inhibition of TEM. Taken together, our data suggest that while arrest requires endothelial surface-bound chemokine, TEM requires a transendothelial gradient maintained by the rapid dilution of non-bound luminal chemokine by flow. Consequently, mediating arrest versus TEM are not inherent, but nonetheless mutually exclusive activities of receptors that depend, respectively, on binding vs. non-binding chemokines. NIH

Studies in humans and mice reveal a critical role for CCR2 in trafficking of pDCs during infection with SARS-CoV-2

Abstract Although GWAS have implicated chemokine receptors in affecting disease severity, the role of chemokines in COVID-19 pathogenesis has not been defined. To bridge this gap, we surveyed expression of 14 chemokine receptors on blood leukocytes from 19 hospitalized COVID-19 patients vs. healthy controls. Among many differences between patients and controls, we found that CCR2, which we have shown mediates lymphocyte transendothelial migration, was down on patient MAIT cells and pDCs, whose numbers in blood are depressed in COVID-19. If a chemokine receptor mediates extravasation, lower levels of the receptor on blood cells could result from efficient, and thereby preferential migration of leukocytes expressing higher levels of the receptor into infected tissue. In support of this mechanism, we found enrichment of CCR2 highcells among MAIT cells migrating to the CCR2 ligand, CCL2, in vitro. To test if CCR2 mediates leukocyte extravasation in COVID-19 we infected transgenic K18-hACE2 Ccr2 reporter mice that were Ccr2 +/RFP(Ccr2 sufficient) or Ccr2 RFP/RFP(Ccr2 deficient) with SARS-CoV-2 intranasally and analyzed pDCs. Blood pDCs were RFP +and their numbers were decreased in Ccr2 RFP/RFPmice at baseline. Over seven days after infection with SARS-CoV-2 (LD 50), pDCs increased and then decreased in the blood, while increasingly progressively in lungs in both Ccr2 +/RFPand Ccr2 RFP/RFPmice. However, absolute numbers of lung pDCs and ratios of lung/blood pDCs were markedly diminished, as was survival, in Ccr2 RFP/RFPmice. We propose that increased mortality in Ccr2-deficient mice may be due in part to inadequate numbers of lung pDCs, leading to local deficiencies in type 1 and III interferons, which are critical for defense against SARS-CoV-2.

Abstract LB202: Obesity-trained C1q+ macrophages compromiseT cell anti-tumor immunity

Abstract Adipose tissue in the mammary gland undergoes dramatic remodeling during obesity and is involved in numerous metabolic diseases. However, how obesity-driven remodeling in adipose tissue regulates anti-tumor immunity remains unclear. Here we provide the detailed cellular atlases of mouse mammary fat pad and tumor at single-cell resolution in lean and obese mice. By leveraging scRNA-seq, we found that high-fat feeding causes a marked increase of macrophage in adipose tissue. Interestingly, a subset of macrophages expressing high levels of C1q complex genes is accumulated in obese subjects. We found that glucocorticoid may as a key regulator of C1q+ macrophage identity. Synthetic glucocorticoid, Dexamethasone, triggers C1q expression in macrophages. We found that C1q+ macrophage derived C1q complex could suppress CD8+ T cell response, thus fueling breast cancer progression. In mouse TNBC tumor, we uncovered a specific inflammatory responsive tumor subset in obese tumors with high CCR2 ligand expression. Obesity-induced TNFα exposure triggers of breast cancer cells reprogramming and CCR2 ligand production. Consequently, CCR2 ligands promote macrophage into tumor. Moreover, depletion of macrophages by anti-CSF1R suppresses tumor growth in obese mice. Our findings define a novel fundamental mechanism of C1q+ macrophage involved in anti-tumor response in obesity. Citation Format: Tao Zhang, Shimeng Liu, Myles Brown. Obesity-trained C1q+ macrophages compromiseT cell anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB202.

Abstract 2946: Depleting hCCR8 mAb Therapy #1: Characterization of a broad collection of anti-hCCR8 mAbs

Abstract Background: Tumor-infiltrating Tregs (TITR) constitute a sub-family of immuno-suppressive cells abundantly found in multiple solid cancers. They play a critical role in tumor progression and their specific depletion has been recently proposed as a novel therapeutic strategy to fight cancers. Based on the transcriptional analysis of tumor infiltrating immune cells, the G-protein coupled receptor CCR8 was found to be expressed on murine and human TITR, but not on proinflammatory effector T cells. The CCR8 receptor therefore represents a unique TITR target for the development of novel depletive antibody-based therapeutics. Methods: We have generated a broad collection of monoclonal antibodies (mAb) directed against the human CCR8 receptor. This collection was widely characterized using different sources of recombinant and native hCCR8 positive cell subsets. Moreover, their binding and activities in several functional assays were assessed with different approaches (BRET, Flow Cytometry, including the inhibition of recruitment of different G-proteins to the CCR8 receptor). A comprehensive molecular and pharmacological characterization of each mAb of our proprietary library was generated. mAb binding against different peptides mimicking different states of post-translational modifications of the N-terminus of CCR8 was also monitored. Finally, using two surrogate antibodies directed against the mouse receptor, we tested the role of CCR8 antagonism and Treg depletion in multiple syngeneic models. Results: A comprehensive characterization of Domain Therapeutics’ mAb library revealed distinct mAb cellular reactivity profiles, different epitope recognition patterns (based on binding to different peptides with different post-translational modifications), subnanomolar antagonist activity of downstream signaling, and insurmountable property with regards to the CCR8 ligand CCL1. Moreover, we demonstrated that treatment with a depleting anti-mCCR8 mAb translated into robust anti-tumor activity in multiple syngeneic mouse models. Conclusion: Due to its high and relatively specific expression on tumor-infiltrating Tregs, CCR8 represents an attractive novel target to derive novel immunotherapies. At Domain Therapeutics, a mAb library of several dozen antibodies with distinct and differentiated binding and pharmacological activity profile was successfully discovered and patent-protected. This collection constitutes a unique source of future clinical candidates for the development of a best-in-class well-differentiated anti-hCCR8 depleting antibody for the treatment of cancers. Citation Format: Claudine Vermot-Desroches, Iseulys Richert, Malaury Schappler, Alice Gentil Dit Maurin, Megane Jeannelle, Solene Rose, Luc Baron, Quentin Ruet, Camille Dietsch, Pauline Urquia, Safia Ayachi, Orphee Blanchard, Maria Dolores Garcia Fernandez, Christel Franchet, Nathalie Lenne, Stephan Schann. Depleting hCCR8 mAb Therapy #1: Characterization of a broad collection of anti-hCCR8 mAbs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2946.

CCL5/CCR5-mediated peripheral inflammation exacerbates blood‒brain barrier disruption after intracerebral hemorrhage in mice

BackgroundOwing to metabolic disequilibrium and immune suppression, intracerebral hemorrhage (ICH) patients are prone to infections; according to a recent global analysis of stroke cases, approximately 10 million new-onset ICH patients had experienced concurrent infection. However, the intrinsic mechanisms underlying the effects of infection related peripheral inflammation after ICH remain unclear.MethodsLipopolysaccharide (LPS) was intraperitoneally injected into ICH model mice to induce peripheral inflammation. Neurobehavioral deficits, blood‒brain barrier (BBB) disruption, and the expression of CCR5, JAK2, STAT3, and MMP9 were evaluated after treatment with recombinant CCL5 (rCCL5) (a CCR5 ligand), maraviroc (MVC) (an FDA-approved selective CCR5 antagonist), or JAK2 CRISPR plasmids.ResultsOur study revealed that severe peripheral inflammation increased CCL5/CCR5 axis activation in multiple inflammatory cell types, including microglia, astrocytes, and monocytes, and aggravated BBB disruption and neurobehavioral dysfunction after ICH, possibly in part through the JAK2/STAT3 signaling pathway.ConclusionsCCR5 might be a potential target for the clinical treatment of infection-induced exacerbation of BBB disruption following ICH.

The Chemokine Receptor CCR1 Mediates Microglia Stimulated Glioma Invasion

Glioblastoma multiforme (GBM) is the most aggressive form of adult brain tumor which is highly resistant to conventional treatment and therapy. Glioma cells are highly motile resulting in infiltrative tumors with poorly defined borders. Another hallmark of GBM is a high degree of tumor macrophage/microglia infiltration. The level of these tumor-associated macrophages/microglia (TAMs) correlates with higher malignancy and poorer prognosis. We previously demonstrated that inhibition of TAM infiltration into glioma tumors with the CSF-1R antagonist pexidartinib (PLX3397) can inhibit glioma cell invasion in-vitro and in-vivo. In this study, we demonstrate an important role for the chemokine receptor CCR1 in mediating microglia/TAM stimulated glioma invasion. Using two structurally distinct CCR1 antagonists, including a novel inhibitor "MG-1-5", we were able to block microglial activated GL261 glioma cell invasion in a dose dependent manner. Interestingly, treatment of a murine microglia cell line with glioma conditioned media resulted in a strong induction of CCR1 gene and protein expression. This induction was attenuated by inhibition of CSF-1R. In addition, glioma conditioned media treatment of microglia resulted in a rapid upregulation of gene expression of several CCR1 ligands including CCL3, CCL5, CCL6 and CCL9. These data support the existence of tumor stimulated autocrine loop within TAMs which ultimately mediates tumor cell invasion.

Met-Signaling Controls Dendritic Cell Migration in Skin by Regulating Podosome Formation and Function

Signaling through the HGF receptor/Met in skin-resident Langerhans cells (LCs) and dermal dendritic cells (DCs) is essential for their emigration toward draining lymph nodes upon inflammation-induced activation. In this study, we addressed the role of Met signaling in distinct steps of LC/dermal DC emigration from the skin by employing a conditionally Met-deficient mouse model (Metflox/flox). We found that Met deficiency severely impaired podosome formation in DCs and concomitantly decreased the proteolytic degradation of gelatin. Accordingly, Met-deficient LCs failed to efficiently cross the extracellular matrix-rich basement membrane between the epidermis and the dermis. We further observed that HGF-dependent Met activation reduced the adhesion of bone marrow-derived LCs to various extracellular matrix factors and enhanced the motility of DCs in three-dimensional collagen matrices, which was not the case for Met-deficient LCs/DCs. We found no impact of Met signaling on the integrin-independent amoeboid migration of DCs in response to the CCR7 ligand CCL19. Collectively, our data show that the Met-signaling pathway regulates the migratory properties of DC in HGF-dependent and HGF-independent manners.

Chemokine Receptors—Structure-Based Virtual Screening Assisted by Machine Learning

Chemokines modulate the immune response by regulating the migration of immune cells. They are also known to participate in such processes as cell-cell adhesion, allograft rejection, and angiogenesis. Chemokines interact with two different subfamilies of G protein-coupled receptors: conventional chemokine receptors and atypical chemokine receptors. Here, we focused on the former one which has been linked to many inflammatory diseases, including: multiple sclerosis, asthma, nephritis, and rheumatoid arthritis. Available crystal and cryo-EM structures and homology models of six chemokine receptors (CCR1 to CCR6) were described and tested in terms of their usefulness in structure-based drug design. As a result of structure-based virtual screening for CCR2 and CCR3, several new active compounds were proposed. Known inhibitors of CCR1 to CCR6, acquired from ChEMBL, were used as training sets for two machine learning algorithms in ligand-based drug design. Performance of LightGBM was compared with a sequential Keras/TensorFlow model of neural network for these diverse datasets. A combination of structure-based virtual screening with machine learning allowed to propose several active ligands for CCR2 and CCR3 with two distinct compounds predicted as CCR3 actives by all three tested methods: Glide, Keras/TensorFlow NN, and LightGBM. In addition, the performance of these three methods in the prediction of the CCR2/CCR3 receptor subtype selectivity was assessed.

IFNα and 5-Aza-2'-deoxycytidine combined with a dendritic-cell targeting DNA vaccine alter tumor immune cell infiltration in the B16F10 melanoma model.

DNA vaccines containing a fusion of the gene encoding chemokine MIP-3α (CCL20), the ligand for CCR6 on immature dendritic cells (DCs), to melanoma-associated antigen genes have enhanced anti-tumor immunity and efficacy compared to those lacking the chemokine gene. Previous work has shown that type-I interferon (IFNα or IFN) and 5-Aza-2'-deoxycytidine (5Aza) significantly enhance the therapeutic benefit of DNA vaccines as measured by reduced tumor burden and improved mouse survival. Here, we explored mouse intratumoral immune correlates underlying the therapeutic benefit of this combination regimen (vaccine, IFN, and 5Aza) as compared to vaccine alone and IFN and 5Aza without vaccine, focusing on chemokine mRNA expression by qRT-PCR and inflammatory cellular infiltration into the tumor microenvironment (TME) by flow cytometry and immunohistochemistry (IHC). The combination group significantly upregulated intratumoral mRNA expression of key immune infiltration chemokines XCL1 and CXCL10. Flow cytometric analyses of tumor suspensions exhibited greater tumor infiltration of CD8+ DCs, CCR7+ DCs, and NK cells in the combination group, as well as reduced levels of myeloid-derived suppressor cells (MDSCs) in vaccinated groups. The mice receiving combination therapy also had greater proportions of effector/memory T-cells (Tem), in addition to showing an enhanced infiltration of Tem and central memory CD8+ T-cells, (Tcm). Tem and Tcm populations both correlated with smaller tumor size. Immunohistochemical analysis of tumors confirmed that CD8+ cells were more abundant overall and especially in the tumor parenchyma with combination therapy. Efficient targeting of antigen to immature DCs with a chemokine-fusion vaccine offers a potential alternative approach to classic and dendritic cell-based vaccines. Combining this approach with IFNα and 5Aza treatments significantly improved vaccine efficacy. This treatment creates an environment of increased inflammatory chemokines that facilitates the trafficking of CD8+ DCs, NK cells, and CD8+ T-cells, especially memory cells, while reducing the number of MDSCs. Importantly, in the combination group, CD8+ cells were more able to penetrate the tumor mass in addition to being more numerous. Further analysis of the pathways engaged by our combination therapy is expected to provide additional insights into melanoma pathogenesis and facilitate the development of novel treatment strategies.

LncRNA15691 promotes T-ALL infiltration by upregulating CCR9 via increased MATR3 stability.

Our previous studies demonstrated that CCR9 plays an important role in several aspects of T-cell acute lymphoblastic leukemia progression and that CCR9 is a potential therapeutic target. However, the underlying mechanism that regulates CCR9 expression remains incompletely understood. In this study, bioinformatics analysis and validation in clinical samples revealed the lncRNA15691 to be positively correlated with CCR9 mRNA expression and significantly upregulated in T-cell acute lymphoblastic leukemia samples and CCR9high T-cell acute lymphoblastic leukemia cell lines. LncRNA15691, a previously uncharacterized lncRNA, was found to be located in both the cytoplasm and the nucleus via fluorescence in situ hybridization assay. In addition, lncRNA15691 upregulated the expression of CCR9 and was involved in T-cell acute lymphoblastic leukemia cell invasion. In vivo experiments showed that lncRNA15691 promoted leukemia cell homing/infiltration into the bone marrow, blood, and spleen, whereas the CCR9 ligand, CCL25, augmented the extramedullary infiltration of CCR9low leukemia cells overexpressing lncRNA15691 into blood, spleen, and liver. Subsequently, RNA protein pull-down assays, coupled with liquid chromatography-tandem mass spectrometry, were used to uncover potential lncRNA15691-interacting proteins, which were then validated by RNA immunoprecipitation. These mechanistic studies revealed that lncRNA15691 upregulated CCR9 expression via directly binding to and stabilizing MATR3 by inhibiting its nuclear degradation mediated by PKA. Collectively, our study revealed a novel mechanism of regulating CCR9 expression and implicated lncRNA15691 as a potential novel biomarker for T-cell acute lymphoblastic leukemia infiltration.

Glucocorticoids Impair the 7α-Hydroxycholesterol-Enhanced Innate Immune Response.

Glucocorticoids suppress the vascular inflammation that occurs under hypercholesterolemia, as demonstrated in an animal model fed a high-cholesterol diet. However, the molecular mechanisms underlying these beneficial effects remain poorly understood. Because cholesterol is oxidized to form cholesterol oxides (oxysterols) that are capable of inducing inflammation, we investigated whether glucocorticoids affect the immune responses evoked by 7α-hydroxycholesterol (7αOHChol). The treatment of human THP-1 monocytic cells with dexamethasone (Dex) and prednisolone (Pdn) downregulated the expression of pattern recognition receptors (PRRs), such as TLR6 and CD14, and diminished 7αOHChol-enhanced response to FSL-1, a TLR2/6 ligand, and lipopolysaccharide, which interacts with CD14 to initiate immune responses, as determined by the reduced secretion of IL-23 and CCL2, respectively. Glucocorticoids weakened the 7αOHChol-induced production of CCL2 and CCR5 ligands, which was accompanied by decreased migration of monocytic cells and CCR5-expressing Jurkat T cells. Treatment with Dex or Pdn also reduced the phosphorylation of the Akt-1 Src, ERK1/2, and p65 subunits. These results indicate that both Dex and Pdn impair the expression of PRRs and their downstream products, chemokine production, and phosphorylation of signaling molecules. Collectively, glucocorticoids suppress the innate immune response and activation of monocytic cells to an inflammatory phenotype enhanced or induced by 7αOHChol, which may contribute to the anti-inflammatory effects in hypercholesterolemic conditions.

Pharmacological Evidence of the Important Roles of CCR1 and CCR3 and Their Endogenous Ligands CCL2/7/8 in Hypersensitivity Based on a Murine Model of Neuropathic Pain.

Neuropathic pain treatment remains a challenging issue because the therapies currently used in the clinic are not sufficiently effective. Moreover, the mechanism of neuropathy is still not entirely understood; however, much evidence indicates that chemokines are important factors in the initial and late phases of neuropathic pain. To date, the roles of CCR1, CCR3 and their endogenous ligands have not been extensively studied; therefore, they have become the subject of our research. In the present comprehensive behavioral and biochemical study, we detected significant time-dependent and long-lasting increases in the mRNA levels of CCR1 and/or CCR3 ligands, such as CCL2/3/4/5/6/7/8/9, in the murine spinal cord after chronic constriction injury of the sciatic nerve, and these increases were accompanied by changes in the levels of microglial/macrophage, astrocyte and neutrophil cell markers. ELISA results suggested that endogenous ligands of CCR1 and CCR3 are involved in the development (CCL2/3/5/7/8/9) and persistence (CCL2/7/8) of neuropathic pain. Moreover, intrathecal injection of CCL2/3/5/7/8/9 confirmed their possible strong influence on mechanical and thermal hypersensitivity development. Importantly, inhibition of CCL2/7/8 production and CCR1 and CCR3 blockade by selective/dual antagonists effectively reduced neuropathic pain-like behavior. The obtained data suggest that CCL2/7/8/CCR1 and CCL7/8/CCR3 signaling are important in the modulation of neuropathic pain in mice and that these chemokines and their receptors may be interesting targets for future investigations.

Cutting Edge: CCR9 Promotes CD8+ T Cell Recruitment to the Brain during Congenital Cytomegalovirus Infection.

CD8+ T lymphocytes infiltrate the brain during congenital CMV infection and promote viral clearance. However, the mechanisms by which CD8+ T cells are recruited to the brain remain unclear. Using a mouse model of congenital CMV, we found a gut-homing chemokine receptor (CCR9) was preferentially expressed in CD8+ T cells localized in the brain postinfection. In the absence of CCR9 or CCL25 (CCR9's ligand) expression, CD8+ T cells failed to migrate to key sites of infection in the brain and protect the host from severe forms of disease. Interestingly, we found that expression of CCR9 on CD8+ T cells was also responsible for spatial temporal positioning of T cells in the brain. Collectively, our data demonstrate that the CMV-infected brain uses a similar mechanism for CD8+ T cell homing as the small intestine.

CCL17 Promotes Colitis-Associated Tumorigenesis Dependent on the Microbiota.

Colorectal cancer is one of the most common cancers and a major cause of mortality. Proinflammatory and antitumor immune responses play critical roles in colitis-associated colon cancer. CCL17, a chemokine of the C-C family and ligand for CCR4, is expressed by intestinal dendritic cells in the steady state and is upregulated during colitis in mouse models and inflammatory bowel disease patients. In this study, we investigated the expression pattern and functional relevance of CCL17 for colitis-associated colon tumor development using CCL17-enhanced GFP-knockin mice. CCL17 was highly expressed by dendritic cells but also upregulated in macrophages and intermediary monocytes in colon tumors induced by exposure to azoxymethane and dextran sodium sulfate. Despite a similar degree of inflammation in the colon, CCL17-deficient mice developed fewer tumors than did CCL17-competent mice. This protective effect was abrogated by cohousing, indicating a dependency on the microbiota. Changes in microbiota diversity and composition were detected in separately housed CCL17-deficient mice, and these mice were more susceptible to azoxymethane-induced early apoptosis in the colon affecting tumor initiation. Immune cell infiltration in colitis-induced colon tumors was not affected by the lack of CCL17. Taken together, our results indicate that CCL17 promotes colitis-associated tumorigenesis by influencing the composition of the intestinal microbiome and reducing apoptosis during tumor initiation.

Abstract 11914: VSIG4+ Resident Tissue Macrophages Govern Cardiac Remodeling and Function After Acute Myocardial Infarction in Mice

Introduction. Resident tissue macrophages (RTM) are essential cellular hubs regulating tissue homeostasis beyond their classical immune surveillance functions. Of note, after acute myocardial infarction (AMI), cardiac RTM have been shown to inhibit fibrosis, promote angiogenesis, and foster the anti-inflammatory response to injury by interacting with other immune cell types, such as monocytes and T lymphocytes, to drive them towards pro-healing phenotypes. Hypothesis. Using Single Cell RNA Sequencing, we identified a new subpopulation of cardiac RTM characterized by the expression of VSIG4 (B7 family-related protein V-set and Ig domain-containing 4). In this work, we hypothesized that VSIG4+ RTM display protective function in the infarcted heart. Method and results. Visg4 deficient mice showed adverse cardiac remodeling and worsened cardiac function at day 14 and 28 after the onset of ischemia when compared to their wild-type littermates. Echocardiography-based transthoracic injection of FACS-sorted cardiac VISG4+ macrophages into infarcted hearts 2 weeks post-AMI, improved cardiac function in both WT and Vsig4 -/- mice, underlying the protective role of VSIG4+ RTM in this pathological setting. Conversely, injection of Vsig4 -/- macrophages impaired cardiac function in WT and Vsig4 -/- animals. VSIG4 deficiency is associated with a higher number of neutrophils, inflammatory monocytes and macrophages but also with a reduction in the amount of CCR5+ regulatory T lymphocytes in the cardiac tissue without any changes in blood, bone marrow and spleen. In cultured peritoneal macrophages, a prototypical example of RTM, IL4 and IL13 stimulation improved VSIG4 mRNA and protein levels through IL4 receptor alpha and IL13 receptor alpha1 activation. IL4 and IL13 treatment also increased secretion of CCL3 and CCL4, two chemokine ligands of CCR5, these effects were blunted in VSIG4 deficient macrophages. Conclusion. VSIG4+ RTM are key regulators of cardiac remodeling after AMI likely through their ability to hamper inflammation and recruit protective regulatory T cells in infarcted heart.

Cytosolic DNA sensor activation inhibits HIV infection of macrophages.

Cytosolic recognition of microbial DNA in macrophages results in the activation of the interferon (IFN)-dependent antiviral innate immunity. Here, we examined whether activating DNA sensors in peripheral blood monocyte-derived macrophages (MDMs) can inhibithuman immunodeficiency virus(HIV). We observed that the stimulation of MDMs with poly(dA:dT) or poly(dG:dC) (synthetic ligands for the DNA sensors) inhibited HIV infection and replication. MDMs treated with poly(dA:dT) or poly(dG:dC) expressed higher levels of both type I and type III IFNs than untreated cells. Activation of the DNA sensors in MDMs also induced the expression of the multiple intracellular anti-HIV factors, including IFN-stimulated genes (ISGs: ISG15, ISG56, Viperin, OAS2, GBP5, MxB, and Tetherin) and the HIV restriction microRNAs (miR-29c, miR-138, miR-146a, miR-155, miR-198, and miR-223). In addition, the DNA sensor activation of MDM upregulated the expression of the CC chemokines (RANTES, MIP-1α, MIP-1β), the ligands for HIV entry coreceptor CCR5. These observations indicate that the cytosolic DNA sensors have a protective role in the macrophage intracellular immunity against HIV and that targeting the DNA sensors has therapeutic potential for immune activation-based anti-HIV treatment.