Recruitment Of CCR2 Research Articles

Abstract Mo015: Myeloid Specific PD-L1 Deletion Promotes Cardiac Dysfunction And Myocardial Inflammation

Background: Immunotherapy, particularly PD-1/PD-L1 blockers, is effective in cancer treatment but can cause rare, but potentially fatal heart issues. Despite the significant impact of these adverse events, there is limited research into the underlying mechanisms of immune checkpoint inhibitor (ICI)-mediated cardiotoxicity. PD-L1, which is abundantly expressed in cardiac tissue, particularly exhibits high expression levels over myeloid cells. However, the precise role of myeloid-specific PD-L1 signaling in mediating autoimmune cardiac disease pathology remains unclear. Methods and Results: To study the role of myeloid cell-specific PD-L1 (myeloid-PD-L1) in cardiac pathophysiology, we generated myeloid-specific PD-L1 KO mice, using Lyz2tm1(Cre) Ifo systems. As expected, PD-L1 expression was significantly down-regulated in Mφs from myeloid-PD-L1 KOs. The cardiac function was followed by echocardiography. Controls and myeloid-PD-L1-KO hearts had comparable ventricular function at 1 month of age. Interestingly, as early as 2 months of age, myeloid-PD-L1-KOs displayed marked cardiac dysfunction as reflected by reduced LVEF, LVFS, and HF markers. Immune profiling was conducted whether chronic inflammation contributes to cardiac dysfunction and remodeling at a later age as observed at 2 and 3 months of age. Interestingly, excessive systemic inflammation and substantial immune infiltration (innate and adoptive), activation, and a pro-inflammatory cytokine storm were detected in the KO hearts. Furthermore, myeloid-PD-L1 KO hearts showed increased recruitment of pro-inflammatory CCR2+ monocytes and macrophages. Conclusion: Our data with myeloid-PD-L1 KOs suggest that deleting PD-L1 in myeloid lineage promotes myocardial and systemic inflammation through innate and adaptive immune cell activation and recruitment of CCR2+ MΦs. Subsequently, this leads to impaired cardiac pathophysiology. Thus, these translational studies will directly evaluate the potential of targeting myeloid-PD-L1 signaling to alleviate cardiac damage.

Interstitial macrophage phenotypes in Schistosoma-induced pulmonary hypertension.

Schistosomiasis is a common cause of pulmonary hypertension (PH) worldwide. Type 2 inflammation contributes to the development of Schistosoma-induced PH. Specifically, interstitial macrophages (IMs) derived from monocytes play a pivotal role by producing thrombospondin-1 (TSP-1), which in turn activates TGF-β, thereby driving the pathology of PH. Resident and recruited IM subpopulations have recently been identified. We hypothesized that in Schistosoma-PH, one IM subpopulation expresses monocyte recruitment factors, whereas recruited monocytes become a separate IM subpopulation that expresses TSP-1. Mice were intraperitoneally sensitized and then intravenously challenged with S. mansoni eggs. Flow cytometry on lungs and blood was performed on wildtype and reporter mice to identify IM subpopulations and protein expression. Single-cell RNA sequencing (scRNAseq) was performed on flow-sorted IMs from unexposed and at day 1, 3 and 7 following Schistosoma exposure to complement flow cytometry based IM characterization and identify gene expression. Flow cytometry and scRNAseq both identified 3 IM subpopulations, characterized by CCR2, MHCII, and FOLR2 expression. Following Schistosoma exposure, the CCR2+ IM subpopulation expanded, suggestive of circulating monocyte recruitment. Schistosoma exposure caused increased monocyte-recruitment ligand CCL2 expression in the resident FOLR2+ IM subpopulation. In contrast, the vascular pathology-driving protein TSP-1 was greatest in the CCR2+ IM subpopulation. Schistosoma-induced PH involves crosstalk between IM subpopulations, with increased expression of monocyte recruitment ligands by resident FOLR2+ IMs, and the recruitment of CCR2+ IMs which express TSP-1 that activates TGF-β and causes PH.

Abstract P3089: Selective Activation Of Cardiac Macrophages To Study Injury And Repair

Objective: Pathophysiological inflammatory responses play an important role during ischemia-mediated heart injury and remodeling, where monocytes and macrophages appear to be critically involved. Cardiac macrophages are generally classified as a recruited, monocyte-derived subset that is Ccr2 + and a tissue resident subset that is typically Ccr2 - . Recent studies have shown that these two subsets have divergent functions with Ccr2 - macrophages being pro-regenerative while Ccr2 + macrophages are pro-inflammatory. Here we selectively manipulated the content and/or activation of these two macrophage subsets in the heart. Methods and Results: To study the function of Ccr2 + cardiac macrophages in the heart, we have overexpressed Ccl-2 (Mcp-1) ligand specifically in cardiomyocytes by using adeno-associated virus serotype-9 (AAV9) mediated gene transfer. Postnatal day 4 (p4) Ccr2 RFP/+ pups were injected with either AAV9-Ccl-2 or AAV9-control. At two months of age, we observed increased Ccl-2 transcripts and protein expression in the heart as measured by qRT-PCR and ELISA, respectively. By quantifying immune cells using flow cytometry, we observed increased infiltration of Ccr2 + macrophages (CD45 + CD11b + CD64 + ) in the hearts of AAV9-Ccl2 injected mice compared to controls. However, echocardiography analysis revealed no change in cardiac function in adult Ccl-2 overexpressed mice. Moreover, the recruitment of Ccr2 + macrophages into the myocardium did not cause fibroblast expansion or activation. Conclusion: Our results show that cardiomyocyte-specific overexpression of Ccl-2 induces profound infiltration of Ccr2 + macrophages into the myocardium but this did not alter cardiac function or cause fibrosis at two or six months of age.

Immunomodulatory effect of first-in-human PeriCord cardiac bioimplant: preliminary data of the PERISCOPE clinical trial

Abstract Background The PERISCOPE is a first-in-human, phase I, double blind, one centre clinical trial to test the safety of the PeriCord, an advanced therapy medicinal product (PEI18–140), for the treatment of patients with infarcted myocardial tissue. The PeriCord is a GMP-complying allogeneic engineered tissue graft consisting on a decellularised pericardial matrix colonised with umbilical cord Wharton's jelly mesenchymal stromal cells. The PeriCord implantation has shown to promote damaged tissue revascularisation, reduce infarct size, adverse remodelling and fibrosis, and ultimately improve cardiac function preclinically. Purpose To assess the safety and monitor the monocyte and cytokine response after PeriCord implantation in patients with infarcted myocardial tissue. Methods The PERISCOPE clinical trial has been approved by AEMPS and the Local Ethics Committee of our institution (Eudra-CT 2018–001964–49). Twelve patients with transmural myocardial infarction (>50% by MRI) who were candidates for surgical revascularisation were included: two roll-in and 10 randomised into a Control group (n=5; CABG) or Treatment group (n=5; n=7 in total; CABG and PeriCord implantation over the transmural myocardial scar). In conjunction with clinical follow-up, whole blood was sampled at baseline and 3-, 6-, and 9-days post-surgery for monocyte populations analysis by flow cytometry and plasma cytokine and monocyte chemoattractant chemokine levels by multiplex ELISA. Results The target patient enrolment has been met and no adverse effects related to PeriCord implantation were observed so far. Recruitment of CCR2+ activated monocytes in peripheral blood peaked on day 3 post-surgery in both groups (increase in the %CCR2+ monocytes of 6.1±5.7% in Control, p=0.38; and 6.5±1.8% in PeriCord Treated patients, p=0.0004), concomitant with a surge in the monocyte chemoattractant CCL2 plasma levels. At the same time, there was a decrease in the number of “non-classical” monocytes regardless of treatment (decrease in CD14-/CD16+ monocytes of 16.7±11.1% in Control, p<0.01; and 24.0±15.0% in PeriCord Treated patients, p<0.001) and a marked reduction in CX3CR1 expression in monocytes of PeriCord treated patients (decrease of 53.2±20.6% in mean fluorescence intensity at day 3, p<0.01), while plasma CX3CL1 levels were undetectable. Both populations recovered on day 9 only in treated patients (p<0.05). Conclusions Implantation of the PeriCord in myocardial infarction patients did not promote adverse reactions and modulated circulating monocyte subsets that have been associated with post-infarction myocardial tissue healing (CCR2 and CX3CR1). These results will be analysed for correlation with therapeutic benefits by the end of patient follow-up. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Instituto Carlos III, Generalitat de Catalunya

Circulating monocytes associated with anti-PD-1 resistance in human biliary cancer induce Tcell paralysis.

Suppressive myeloid cells can contribute to immunotherapy resistance, but their role in response to checkpoint inhibition (CPI) in anti-PD-1 refractory cancers, such as biliary tract cancer (BTC), remains elusive. We use multiplexed single-cell transcriptomic and epitope sequencing to profile greater than 200,000 peripheral blood mononuclear cells from advanced BTC patients (n= 9) and matched healthy donors (n= 8). Following anti-PD-1 treatment, CD14+ monocytes expressing high levels of immunosuppressive cytokines and chemotactic molecules (CD14CTX) increase in the circulation of patients with BTC tumors that are CPI resistant. CD14CTX can directly suppress CD4+ Tcells and induce SOCS3 expression in CD4+ Tcells, rendering them functionally unresponsive. The CD14CTX gene signature associates with worse survival in patients with BTC as well as in other anti-PD-1 refractory cancers. These results demonstrate that monocytes arising after anti-PD-1 treatment can induce Tcell paralysis as a distinct mode of tumor-mediated immunosuppression leading to CPI resistance.

The Null Hypothesis of IFN-γ and Monocyte Function in Leishmaniasis

The phagosomal pathogen Leishmania appears unaffected by deliberate changes in the early Th1/Th2 balance. In this issue, Carneiro etal. explain these paradoxical results by showing that manipulations affecting IFN-γ-mediated phagocyte activation are counteracted by effects on IFN-γ-dependent recruitment of CCR2+ monocytes permissive to parasite growth.

Abstract A98: Cancer-associated fibroblasts promote immunosuppression by inducing NOX2-expressing monocytic MDSCs in lung squamous cell carcinoma

Abstract Cancer-associated fibroblasts (CAFs) are activated fibroblasts that constitute the stromal component in the tumor microenvironment (TME). Although CAFs have been shown to promote tumor growth and mediate resistance to chemotherapy, their roles and potential mechanisms by which they may contribute to immune suppression in lung squamous cell carcinoma (LSCC) remain largely unexplored. Here, we identified a positive correlation between CAF and monocytic myeloid cell abundances in 501 primary LSCCs by mining the TCGA dataset. We further validated this finding in an independent cohort using imaging mass cytometry and found a significant spatial interaction between CAFs and monocytic myeloid cells in the TME. To delineate the interplay between CAFs and monocytic myeloid cells, we used chemotaxis assays to show that LSCC patient-derived CAFs promoted recruitment of CCR2+ monocytes via CCL2, which could be reversed by CCR2 inhibition. Using a three-dimensional culture system, we found that CAFs polarized monocytes to adopt a myeloid-derived suppressor cell (MDSC) phenotype characterized by robust suppression of autologous CD8+ T-cell proliferation and IFNγ production. To elucidate additional immunosuppressive mechanisms of CAF-educated MDSCs, we performed proteomic profiling and identified the NADPH oxidase 2 (NOX2) complex to be highly enriched in MDSCs. Pharmacologic inhibition of NOX2 activity in CAF-induced MDSCs restored CD8+ T-cell proliferation. Taken together, our study highlights a pivotal role of CAFs in regulating monocyte recruitment and differentiation and demonstrates that NOX2 inhibition abrogates the CAF-MDSC axis, illuminating a potential therapeutic path to reversing the CAF-mediated immunosuppressive microenvironment. Citation Format: Handan Xiang, Carlo Ramil, Josephine Hai, Chunsheng Zhang, Huijun Wang, Amanda A. Watkins, Roshi Afshar, Peter Georgiev, Xuelei Song, Dongyu Sun, Andrey Loboda, Yanlin Jia, Lily Y Moy, An Chi, Philip E. Brandish. Cancer-associated fibroblasts promote immunosuppression by inducing NOX2-expressing monocytic MDSCs in lung squamous cell carcinoma [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A98.

Treatment with Cestode Parasite Antigens Results in Recruitment of CCR2 + Myeloid Cells, the Adoptive Transfer of Which Ameliorates Colitis

Awareness of the immunological underpinnings of host-parasite interactions may reveal immune signaling pathways that could be used to treat inflammatory disease in humans. Previously we showed that infection with the rat tapeworm, Hymenolepis diminuta, used as a model helminth, or systemic delivery of worm antigen (HdAg) significantly reduced the severity of dinitrobenzene sulfonic acid (DNBS)-induced colitis in mice. Extending these analyses, intraperitoneal injection of HdAg dose-dependently suppressed dextran sodium sulfate (DSS)-induced colitis, and this was paralleled by reduced gamma interferon (IFN-γ), interleukin-17 (IL-17), and tumor necrosis factor alpha (TNF-α) production and increased IL-10 production from mitogen-activated splenocytes. Treatment with HdAg resulted in a CCR2-dependent recruitment of CDllb+ F4/80+ Ly6Chi Gr-1lo monocyte-like cells into the peritoneum 24 h later that were predominantly programmed death ligand 1 (PD-L1) positive and CXCR2 negative. In vitro assays indicated that these cells were unable to suppress T cell proliferation but enhanced IL-10 and IL-4 production from activated T cells. Adoptive transfer of the HdAg-recruited monocytic cells into naive mice blocked DSS-induced colitis. These findings add to the variety of means by which treatment with parasitic helminth-derived antigens can ameliorate concomitant disease. A precise understanding of the mechanism(s) of action of HdAg and other helminth-derived antigens (and a parallel consideration of putative side effects) may lead to the development of novel therapies for human idiopathic disorders such as inflammatory bowel disease.

IL-4 signaling drives a unique arginase+/IL-1β+ microglia phenotype and recruits macrophages to the inflammatory CNS: consequences of age-related deficits in IL-4Rα after traumatic spinal cord injury.

Alternative activation of microglia/macrophages (M2a) by interleukin (IL)-4 is purported to support intrinsic growth and repair processes after CNS injury. Nonetheless, alternative activation of microglia is poorly understood in vivo, particularly in the context of inflammation, injury, and aging. Here, we show that aged mice (18-19 months) had reduced functional recovery after spinal cord injury (SCI) associated with impaired induction of IL-4 receptor α (IL-4Rα) on microglia. The failure to successfully promote an IL-4/IL-4Rα response in aged mice resulted in attenuated arginase (M2a associated), IL-1β, and chemokine ligand 2 (CCL2) expression, and diminished recruitment of IL-4Rα(+) macrophages to the injured spinal cord. Furthermore, the link between reduced IL-4Rα expression and reduced arginase, IL-1β, and CCL2 expression was confirmed using adult IL-4Rα knock-out (IL-4Rα(KO)) mice. To better understand IL-4Rα-mediated regulation of active microglia, a series of studies was completed in mice that were peripherally injected with lipopolysaccharide and later provided IL-4 by intracerebroventricular infusion. These immune-based studies demonstrate that inflammatory-induced IL-4Rα upregulation on microglia was required for the induction of arginase by IL-4. In addition, IL-4-mediated reprogramming of active microglia enhanced neurite growth ex vivo and increased inflammatory gene expression (i.e., IL-1β and CCL2) and the corresponding recruitment of CCR2(+)/IL-4Rα(+)/arginase(+) myeloid cells in vivo. IL-4 reprogrammed active microglia to a unique and previously unreported phenotype (arginase(+)/IL-1β(+)) that augmented neurite growth and enhanced recruitment of peripheral IL-4Rα(+) myeloid cells to the CNS. Moreover, this key signaling cascade was impaired with age corresponding with reduced functional recovery after SCI.

CCR2 Deficiency Promotes Exacerbated Chronic Erosive Neutrophil-Dominated Chikungunya Virus Arthritis

Chikungunya virus (CHIKV) is a member of a globally distributed group of arthritogenic alphaviruses that cause weeks to months of debilitating polyarthritis/arthralgia, which is often poorly managed with current treatments. Arthritic disease is usually characterized by high levels of the chemokine CCL2 and a prodigious monocyte/macrophage infiltrate. Several inhibitors of CCL2 and its receptor CCR2 are in development and may find application for treatment of certain inflammatory conditions, including autoimmune and viral arthritides. Here we used CCR2(-/-) mice to determine the effect of CCR2 deficiency on CHIKV infection and arthritis. Although there were no significant changes in viral load or RNA persistence and only marginal changes in antiviral immunity, arthritic disease was substantially increased and prolonged in CCR2(-/-) mice compared to wild-type mice. The monocyte/macrophage infiltrate was replaced in CCR2(-/-) mice by a severe neutrophil (followed by an eosinophil) infiltrate and was associated with changes in the expression levels of multiple inflammatory mediators (including CXCL1, CXCL2, granulocyte colony-stimulating factor [G-CSF], interleukin-1β [IL-1β], and IL-10). The loss of anti-inflammatory macrophages and their activities (e.g., efferocytosis) was also implicated in exacerbated inflammation. Clear evidence of cartilage damage was also seen in CHIKV-infected CCR2(-/-) mice, a feature not normally associated with alphaviral arthritides. Although recruitment of CCR2(+) monocytes/macrophages can contribute to inflammation, it also appears to be critical for preventing excessive pathology and resolving inflammation following alphavirus infection. Caution might thus be warranted when considering therapeutic targeting of CCR2/CCL2 for the treatment of alphaviral arthritides. Here we describe the first analysis of viral arthritis in mice deficient for the chemokine receptor CCR2. CCR2 is thought to be central to the monocyte/macrophage-dominated inflammatory arthritic infiltrates seen after infection with arthritogenic alphaviruses such as chikungunya virus. Surprisingly, the viral arthritis caused by chikungunya virus in CCR2-deficient mice was more severe, prolonged, and erosive and was neutrophil dominated, with viral replication and persistence not being significantly affected. Monocytes/macrophages recruited by CCL2 thus also appear to be important for both preventing even worse pathology mediated by neutrophils and promoting resolution of inflammation. Caution might thus be warranted when considering the use of therapeutic agents that target CCR2/CCL2 or inflammatory monocytes/macrophages for the treatment of alphaviral (and perhaps other viral) arthritides. Individuals with diminished CCR2 responses (due to drug treatment or other reasons) may also be at risk of exacerbated arthritic disease following alphaviral infection.

Endothelial CCR2 Signaling Induced by Colon Carcinoma Cells Enables Extravasation via the JAK2-Stat5 and p38MAPK Pathway

Increased expression of the chemokine CCL2 in tumor cells correlates with enhanced metastasis, poor prognosis, and recruitment of CCR2(+)Ly6C(hi) monocytes. However, the mechanisms driving tumor cell extravasation through the endothelium remain elusive. Here, we describe CCL2 upregulation in metastatic UICC stage IV colon carcinomas and demonstrate that tumor cell-derived CCL2 activates the CCR2(+) endothelium to increase vascular permeability invivo. CCR2 deficiency prevents colon carcinoma extravasation and metastasis. Of note, CCR2 expression on radio-resistant cells or endothelial CCR2 expression restores extravasation and metastasis in Ccr2(-/-) mice. Reduction of CCR2 expression on myeloid cells decreases but does not prevent metastasis. CCL2-induced vascular permeability and metastasis is dependent on JAK2-Stat5 and p38MAPK signaling. Our study identifies potential targets for treating CCL2-dependent metastasis.

Stressor-enhanced colitis is abrogated in mice deficient in CCL2 (61.6)

Abstract Exposing mice to a social stressor called social disruption (SDR) has been reported to increase CD11b+Ly-6ChiCCR2+ inflammatory monocytes in the circulation and spleen. This study was designed to test the hypothesis that stressor-induced exacerbation of pathogen-induced colitis is dependent upon CCL2-mediated recruitment of inflammatory monocytes. Mice deficient in CCL2 (i.e., CCL2-/-), a chemokine involved in the recruitment of CCR2+ monocytes, and wild type C57BL/6 mice were challenged with the colonic pathogen Citrobacter rodentium during exposure to SDR. Exposing mice to SDR, which consists of intermale aggression for 2 hrs a night on 6 consecutive nights, during challenge with C.rodentium significantly increased pathogen-induced colitis. This increase was marked by an enhanced accumulation of F4/80+ colonic monocytes/macrophages, increased pathogen burden and pathogen-induced colonic histopathology, and increased inflammatory cytokine and chemokine gene expression in comparison to C. rodentium-challenged non-stressed control. These stressor effects were dependent upon CCL2, since exposing CCL2-/- mice to SDR did not lead to an increased accumulation of colonic monocytes/macrophages in the colon and did not significantly increase gene expression or pathogen-induced histopathology. Together, these data illustrate the deleterious effects of stressor-enhanced recruitment of inflammatory monocytes during bacterial challenge.