CCR2-KO Mice Research Articles

Evidence of Alveolar Macrophage Metabolic Shift Following Stereotactic Body Radiation Therapy -Induced Lung Fibrosis in Mice

Radiation-induced pneumopathy is the main dose-limiting factor in cases of chest radiation therapy. Macrophage infiltration is frequently observed in irradiated lung tissues and may participate in lung damage development. Radiation-induced lung fibrosis can be reproduced in rodent models using whole thorax irradiation but suffers from limits concerning the role played by unexposed lung volumes in damage development. Here, we used an accurate stereotactic body radiation therapy preclinical model irradiating 4% of the mouse lung. Tissue damage development and macrophage populations were followed by histology, flow cytometry, and single-cell RNA sequencing. Wild-type and CCR2 KO mice, in which monocyte recruitment is abrogated, were exposed to single doses of radiation, inducing progressive (60 Gy) or rapid (80 Gy) lung fibrosis. Numerous clusters of macrophages were observed around the injured area, during progressive as well as rapid fibrosis. The results indicate that probably CCR2-independent recruitment and/or in situ proliferation may be responsible for macrophage invasion. Alveolar macrophages experience a metabolic shift from fatty acid metabolism to cholesterol biosynthesis, directing them through a possible profibrotic phenotype. Depicted data revealed that the origin and phenotype of macrophages present in the injured area may differ from what has been previously described in preclinical models exposing large lung volumes, representing a potentially interesting trail in the deciphering of radiation-induced lung damage processes. Our study brings new possible clues to the understanding of macrophage implications in radiation-induced lung damage, representing an interesting area for exploration in future studies.

CCR2+ monocytes/macrophages drive steroid hormone imbalance-related prostatic fibrosis

Benign Prostatic Hyperplasia (BPH) is a complex condition leading to Lower Urinary Tract Symptoms in aging men, characterized by cellular proliferation, smooth muscle dysfunction, inflammation, and fibrosis. While BPH is known to involve heightened macrophage infiltration, the specific contribution of infiltrating monocytes/macrophages to the disease mechanism remains uncertain. This research explores the impact of reducing circulating monocytes and subsequently limiting their tissue infiltration by using Ccr2 knockout (Ccr2-KO) mice. Ccr2-KO and wild type mice were implanted with testosterone and estradiol (T + E2, 25 mg + 2.5 mg) pellets. Urinary function was assessed via weekly void spot assays over 12 weeks, and prostatic macrophage levels were visualized and quantified in tissue sections using an F4/80 antibody. Additionally, Ki-67 staining was used to evaluate cell proliferation, and picrosirius red staining to assess collagen accumulation. Increased voiding frequency which developed in T + E2 mice, was significantly ameliorated in Ccr2-KO mice, however, both Ccr2-KO and wild type (WT) mice showed increased bladder weights after three month, representing a hypertrophic response to bladder outlet obstruction. T + E2 substantially increased the density of macrophages in WT but not Ccr2-KO mouse prostate. Proliferation rate, as indicated by Ki-67 positivity, was elevated in the vental and anterior prostate lobes but was only marginally reduced in Ccr2-KO mice. Most importantly, a significant prostatic collagen accumulation was observed in WT mice that was markedly reduced by Ccr2 deficiency post T + E2 treatment. The absence of Ccr2 mitigates urinary dysfunction and alters prostatic macrophage levels and collagen accumulation in steroid hormone imbalance. These findings suggest a crucial role for monocyte infiltration, giving rise to macrophages or other cell derivatives, to drive fibrosis.

Ccr2-dependent monocytes exacerbate intestinal inflammation and modulate gut serotonergic signaling following traumatic brain injury.

Traumatic brain injury (TBI) leads to acute gastrointestinal dysfunction and mucosal damage, resulting in feeding intolerance. C-C motif chemokine receptor 2 (Ccr2 + ) monocytes are crucial immune cells that regulate the gut's inflammatory response via the brain-gut axis. Using Ccr2 ko mice, we investigated the intricate interplay between these cells to better elucidate the role of systemic inflammation after TBI. A murine-controlled cortical impact model was used, and results were analyzed on postinjury days 1 and 3. The experimental groups included (1) sham C57Bl/6 wild type (WT), (2) TBI WT, (3) sham Ccr2 ko , and (4) TBI Ccr2 ko . Mice were euthanized on postinjury days 1 and 3 to harvest the ileum and study intestinal dysfunction and serotonergic signaling using a combination of quantitative real-time polymerase chain reaction, immunohistochemistry, fluorescein isothiocyanate-dextran motility assays, and flow cytometry. Student's t test and one-way analysis of variance were used for statistical analysis, with significance achieved when p < 0.05. Traumatic brain injury resulted in severe dysfunction and dysmotility of the small intestine in WT mice as established by significant upregulation of inflammatory cytokines iNOS , Lcn2 , TNFα , and IL1β and the innate immunity receptor toll-like receptor 4 ( Tlr4 ). This was accompanied by disruption of genes related to serotonin synthesis and degradation. Notably, Ccr2 ko mice subjected to TBI showed substantial improvements in intestinal pathology. Traumatic brain injury Ccr2 ko groups demonstrated reduced expression of inflammatory mediators ( iNOS , Lcn2 , IL1β , and Tlr4 ) and improvement in serotonin synthesis genes, including tryptophan hydroxylase 1 ( Tph1 ) and dopa decarboxylase ( Ddc ). Our study reveals a critical role for Ccr2 + monocytes in modulating intestinal homeostasis after TBI. Ccr2 + monocytes aggravate intestinal inflammation and alter gut-derived serotonergic signaling. Therefore, targeting Ccr2 + monocyte-dependent responses could provide a better understanding of TBI-induced gut inflammation. Further studies are required to elucidate the impact of these changes on brain neuroinflammation and cognitive outcomes.

Immune Responses in Oral Papillomavirus Clearance in the MmuPV1 Mouse Model.

Human papillomavirus (HPV)-induced oropharyngeal cancer now exceeds HPV-induced cervical cancer, with a noticeable sex bias. Although it is well established that women have a more proficient immune system, it remains unclear whether immune control of oral papillomavirus infections differs between sexes. In the current study, we use genetically modified mice to target CCR2 and Stat1 pathways, with the aim of investigating the role of both innate and adaptive immune responses in clearing oral papillomavirus, using our established papillomavirus (MmuPV1) infection model. Persistent oral MmuPV1 infection was detected in Rag1ko mice with T and B cell deficiencies. Meanwhile, other tested mice were susceptible to MmuPV1 infections but were able to clear the virus. We found sex differences in key myeloid cells, including macrophages, neutrophils, and dendritic cells in the infected tongues of wild type and Stat1ko mice but these differences were not observed in CCR2ko mice. Intriguingly, we also observed a sex difference in anti-MmuPV1 E4 antibody levels, especially for two IgG isotypes: IgG2b and IgG3. However, we found comparable numbers of interferon-gamma-producing CD8 T cells stimulated by E6 and E7 in both sexes. These findings suggest that males and females may use different components of innate and adaptive immune responses to control papillomavirus infections in the MmuPV1 mouse model. The observed sex difference in immune responses, especially in myeloid cells including dendritic cell (DC) subsets, may have potential diagnostic and prognostic values for HPV-associated oropharyngeal cancer.

Development of Novel Protein-Drug Conjugates for the Treatment of Chronic Myelomonocytic Leukemia

Introduction: Chronic myelomonocytic leukemia (CMML) is a myeloid stem cell neoplasm with few effective therapies and poor prognosis. We have developed CCL2-drug conjugates that specifically bind, internalize and eliminate cells of the disease clone via inhibition of the key metabolic enzyme NAMPT. CCL2 is a chemokine which binds CCR2, a receptor expressed predominantly on the classical monocytes canonically expanded in CMML, while CMML stem and progenitors display ~100-fold higher CCR2 expression compared with healthy equivalents ( Ferrall-Fairbanks et al, Blood Cancer Discov 2022 3(6):536-553). CCL2-NAMPTi conjugates thus represent an attractive and novel strategy, co-opting both lineage specificity and therapeutic window to deliver payload drugs for efficient and targeted cell killing. Methods: Four NAMPTi warheads were made in adaptable seven-step synthetic route and covalently conjugated to human CCL2 though TGase. We screened the range of CCL2-NAMPTi conjugates on CCR2-expressing cell lines (THP-1; MV411) for their ability to reduce NAD/NADH and induce cytotoxicity. The lead CCL2-NAMPTi conjugate was identified based on its ability to reduce NAD/NADH levels, induce cell death, and show impermeability when using the unconjugated warhead alone. Plasma stability studies confirmed conjugate stability in mouse and human plasma with no release of unconjugated active warhead. The lead compound was then tested on CD14+ cells isolated from CMML patient peripheral blood (PB); examining NAD/NADH levels, cell viability and induction of caspase 3/8. To better understand its specificity and target cell population, we conducted in vivo studies using fluorescently labelled CCL2 in wild type and CCR2 KO mice. Therapeutic effectiveness of the lead conjugate was evaluated by means of NAD/NADH reduction in THP1 subcutaneous models. Results: All CCL2-NAMPTi tested induced cell death in THP-1 cells (IC50 1.1-7.2 nM) following 72 hours incubation. The lead compound, MM015, effectively reduced NAD/NADH levels in CCR2+ cell lines (THP-1; MV411) 24-48 hours following treatment (IC 50 0.1-0.8 nM), with a corresponding decrease in cell viability at 72 and 96 hours (IC 50 1.1- 2.1 nM). No effect was observed on CCR2- cell lines (Jurkat; PC9). Specificity of the lead conjugate was confirmed by measuring NAD/NADH levels and cytotoxicity in presence of 100-fold excess of unconjugated CCL2. Efficacy studies on primary CMML PB samples confirmed that MM015 exposure specifically reduced NAD/NADH levels (n=6, IC 50 0.5-15 nM) and cell viability (n=6, IC 50 1.9-250 nM), and induced caspase 3/8 activation. Flow cytometry analysis of CMML PB confirmed CCR2 expression was largely restricted to monocytes and precursors, as compared with other immune cell subsets (n=6). Accordingly, efficacy measures in treated whole PB were exclusively seen in the CD14+ population, sparing lymphoid and other lineage cells, demonstrating ability of CCL2-NAMPTi to selectively target the CMML disease clone. In vivo experiments using fluorescently labelled CCL2 confirmed selective uptake of intravenously administered human CCL2 by CCR2+ cells within mouse blood, bone marrow and in THP-1 subcutaneous tumour cells. No uptake of fluorescently labelled CCL2 was observed in CCR2 KO mice, further confirming CCR2 specific uptake. Additionally, measurement of human CCL2 in CCR2 KO mouse peripheral blood by ELISA confirmed CCR2 dependent CCL2 uptake, 1 hour following intravenous administration. In vivo efficacy studies showed consistent reduction of NAD/NADH levels in xenografted tumor cells, and monocytic cells in the murine BM compartment, 24 hours following IV injection of MM015. Conclusion: We have developed a novel drug delivery strategy utilizing CCL2-NAMPTi conjugates to selectively target and eliminate key CMML cell populations. CCL2-NAMPTi can effectively reduce NAD levels and induce subsequent cell death in CCR2-expressing cell lines, primary CMML cells and in vivo models. This innovative approach holds therapeutic promise to address this major unmet clinical need.

Temporal changes in glucose metabolism reflect polarization in resident and monocyte-derived macrophages after myocardial infarction.

Metabolic reprogramming from glycolysis to the mitochondrial tricarboxylic acid (TCA) cycle and oxidative phosphorylation may mediate macrophage polarization from the pro-inflammatory M1 to the anti-inflammatory M2 phenotype. We hypothesized that changes in cardiac macrophage glucose metabolism would reflect polarization status after myocardial infarction (MI), ranging from the early inflammatory phase to the later wound healing phase. MI was induced by permanent ligation of the left coronary artery in adult male C57BL/6J mice for 1 (D1), 3 (D3), or 7 (D7) days. Infarct macrophages were subjected to metabolic flux analysis or gene expression analysis. Monocyte versus resident cardiac macrophage metabolism was assessed using mice lacking the Ccr2 gene (CCR2 KO). By flow cytometry and RT-PCR, D1 macrophages exhibited an M1 phenotype while D7 macrophages exhibited an M2 phenotype. Macrophage glycolysis (extracellular acidification rate) was increased at D1 and D3, returning to basal levels at D7. Glucose oxidation (oxygen consumption rate) was decreased at D3, returning to basal levels at D7. At D1, glycolytic genes were elevated (Gapdh, Ldha, Pkm2), while TCA cycle genes were elevated at D3 (Idh1 and Idh2) and D7 (Pdha1, Idh1/2, Sdha/b). Surprisingly, Slc2a1 and Hk1/2 were increased at D7, as well as pentose phosphate pathway (PPP) genes (G6pdx, G6pd2, Pgd, Rpia, Taldo1), indicating increased PPP activity. Macrophages from CCR2 KO mice showed decreased glycolysis and increased glucose oxidation at D3, and decreases in Ldha and Pkm2 expression. Administration of dichloroacetate, a pyruvate dehydrogenase kinase inhibitor, robustly decreased pyruvate dehydrogenase phosphorylation in the non-infarcted remote zone, but did not affect macrophage phenotype or metabolism in the infarct zone. Our results indicate that changes in glucose metabolism and the PPP underlie macrophage polarization following MI, and that metabolic reprogramming is a key feature of monocyte-derived but not resident macrophages.

Cardiac macrophages regulate isoproterenol-induced Takotsubo-like cardiomyopathy.

Takotsubo syndrome (TTS) is an acute, stress-induced cardiomyopathy that occurs predominantly in women after extreme physical and/or emotional stress. To date, our understanding of the molecular basis for TTS remains unknown and, consequently, specific therapies are lacking. Myocardial infiltration of monocytes and macrophages in TTS has been documented in clinical studies. However, the functional importance of these findings remains poorly understood. Here, we show that a single high dose of isoproterenol (ISO) in mice induced a TTS-like cardiomyopathy phenotype characterized by female predominance, severe cardiac dysfunction, and robust myocardial infiltration of macrophages. Single-cell RNA-Seq studies of myocardial immune cells revealed that TTS-like cardiomyopathy is associated with complex activation of innate and adaptive immune cells in the heart, and macrophages were identified as the dominant immune cells. Global macrophage depletion (via clodronate liposome administration) or blockade of macrophage infiltration (via a CCR2 antagonist or in CCR2-KO mice) resulted in recovery of cardiac dysfunction in ISO-challenged mice. In addition, damping myeloid cell activation by HIF1α deficiency or exposure to the immunomodulatory agent bortezomib ameliorated ISO-induced cardiac dysfunction. Collectively, our findings identify macrophages as a critical regulator of TTS pathogenesis that can be targeted for therapeutic gain.

CX3CR1 But Not CCR2 Expression Is Required for the Development of Autoimmune Peripheral Neuropathy in Mice.

One hallmark of Guillain-Barre syndrome (GBS), a prototypic autoimmune peripheral neuropathy (APN) is infiltration of leukocytes (macrophages and T cells) into peripheral nerves, where chemokines and their receptors play major roles. In this study, we aimed to understand the potential contribution of chemokine receptors CCR2 and CX3CR1 in APN by using a well-established mouse model, B7.2 transgenic (L31) mice, which possesses a predisposed inflammatory background. We crossbred respectively CCR2KO and CX3CR1KO mice with L31 mice. The disease was initiated by partial ligation on one of the sciatic nerves. APN pathology and neurological function were evaluated on the other non-ligated sciatic nerve/limb. Our results revealed that L31/CX3CR1KO but not L31/CCR2KO mice were resistant to APN. CX3CR1 is needed for maintaining circulating monocyte and CD8+ T cell survival. While migration of a significant number of activated CD8+ T cells to peripheral nerves is essential in autoimmune response in nerve, recruitment of monocytes into PNS seems optional. Disease onset is independent of CCR2 mediated blood-derived macrophage recruitment, which can be replaced by compensatory proliferation of resident macrophages in peripheral nerve. CX3CR1 could also contribute to APN via its critical involvement in maintaining nerve macrophage phagocytic ability. We conclude that blockade of CX3CR1 signaling may represent an interesting anti-inflammatory strategy to improve therapeutic management for GBS patients.

The CCR2/MCP-1 Chemokine Pathway and Lung Adenocarcinoma.

Simple SummaryMyeloid-derived suppressor cells (MDSCs) are present at the sites of many solid tumors and dampen host immune responses. We studied how these MDSCs accumulate and how this process might be interrupted using lung cancer cell lines growing in immunocompetent mice. We found that tumor cells release a chemokine, MCP-1, that attracts MDSCs as a result of their expression of the MCP-1 receptor, CCR2. We showed that mice lacking CCR2, or the use of a small-molecule inhibitor of CCR2, prevented MDSC recruitment to the tumors and derepressed host T cell responses, allowing immune activation and inhibition of tumor growth. These data suggest that CCR2 inhibitors may be a new tool for cancer immunotherapy.Host anti-tumor immunity can be hindered by various mechanisms present within the tumor microenvironment, including the actions of myeloid-derived suppressor cells (MDSCs). We investigated the role of the CCR2/MCP-1 pathway in MDSC-associated tumor progression in murine lung cancer models. Phenotypic profiling revealed maximal expression of CCR2 by tumor-resident MDSCs, and MCP-1 by transplanted TC1 tumor cells, respectively. Use of CCR2-knockout (CCR2-KO) mice showed dependence of tumor growth on CCR2 signaling. Tumors in CCR2-KO mice had fewer CCR2low MDSCs, CD4 T cells and Tregs than WT mice, and increased infiltration by CD8 T cells producing IFN-γ and granzyme-B. Effects were MDSC specific, since WT and CCR2-KO conventional T (Tcon) cells had comparable proliferation and production of inflammatory cytokines, and suppressive functions of WT and CCR2-KO Foxp3+ Treg cells were also similar. We used a thioglycolate-induced peritonitis model to demonstrate a role for CCR2/MCP-1 in trafficking of CCR2+ cells to an inflammatory site, and showed the ability of a CCR2 antagonist to inhibit such trafficking. Use of this CCR2 antagonist promoted anti-tumor immunity and limited tumor growth. In summary, tumor cells are the prime source of MCP-1 that promotes MDSC recruitment, and our genetic and pharmacologic data demonstrate that CCR2 targeting may be an important component of cancer immunotherapy.

CCL2/CCR2 signaling protects against bladder cancer growth in a T cell dependent manner

Abstract The chemokine CCL2 (C-C motif ligand 2) is an important signaling axis underlying recruitment of pro-tumorigenic myeloid cells and is associated with worse outcomes in several tumor models. Currently, anti-CCL2 antibodies are being tested in cancer trials for solid tumors. To test CCL2 signaling in bladder carcinogenesis, wild type (WT) mice and mice deficient in CCL2 (CCL2KO) were given the urothelial carcinogen N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN). Surprisingly, tumor incidence was higher in CCL2KO mice demonstrating an unanticipated protective role for CCL2 signaling in bladder cancer (BC). We then orthotopically challenged WT, CCL2KO, and CCR2KO mice (lacking the major CCL2 receptor) with MB49 BC and confirmed the protective effect of CCL2/CCR2. WT mice had significantly more intratumoral T cells compared to CCL2KO mice suggesting that CCL2 is involved in recruiting T cells to the bladder. Further, depletion of T cells abolished this protective effect of CCL2. Adoptive transfer of CCR2+ T cells into CCR2KO mice restored protection against MB49. CCR2+ T cells were also more activated, functional and tumor specific compared to their CCR2− counterparts. We found that anti-CCL2 promotes BC growth highlighting a concern for use of anti-CCL2 in BC. Moreover, intravesical recombinant CCL2 (rCCL2) either alone or in combination with intravesical gemcitabine reduced bladder tumor and improved survival of mice with MB49 BC. We further developed a slow-release encapsulated nanoparticle formulation of rCCL2 which successfully treated MB49, C3H mice with MBT-2 BC and reduced urine hematuria in our first ever humanized model of orthotopic BC. Our results are rapidly translatable and identify a novel treatment strategy in BC.

Critical roles of astrocytic-CCL2-dependent monocyte infiltration in a DJ-1 knockout mouse model of delayed brain repair.

Monocyte-derived macrophages play a role in the repair of the injured brain. We previously reported that a deficiency of the Parkinson's disease (PD)-associated gene DJ-1 delays repair of brain injury produced by stereotaxic injection of ATP, a component of damage-associated molecular patterns. Here, we show that a DJ-1 deficiency attenuates monocyte infiltration into the damaged brain owing to a decrease in C-C motif chemokine ligand 2 (CCL2) expression in astrocytes. Like DJ-1-knockout (KO) mice, CCL2 receptor (CCR2)-KO mice showed defects in monocyte infiltration and delayed recovery of brain injury, as determined by 9.4 T magnetic resonance imaging analysis and immunostaining for tyrosine hydroxylase and glial fibrillary acid protein. Notably, transcriptome analyses showed that genes related to regeneration and synapse formation were similarly downregulated in injured brains of DJ-1-KO and CCR2-KO mice compared with the injured wild-type brain. These results indicate that defective astrogliosis in DJ-1-KO mice is associated with decreased CCL2 expression and attenuated monocyte infiltration, resulting in delayed repair of brain injury. Thus, delayed repair of brain injury could contribute to the development of PD. MAIN POINTS: A DJ-1 deficiency attenuates infiltration of monocytes owing to a decrease in CCL2 expression in astrocytes, which in turn led to delay in repair of brain injury.

43 Activation of CCL2/CCR2 Signaling Axis Is Responsible for Spinal Cord Inflammation and Loss of Muscle Mass in Mice After Burn Injury

Abstract Introduction Burn-induced systemic inflammation (BISI) can induce inflammatory responses in peripheral nervous system and also cause neuroinflammation in the central nervous system, leading to morbidity and mortality. The mechanisms of neuromotor impairment involving CCL2/CCR2 signaling axis are largely unknown. We hypothesized that BISI causes neuroinflammation in the spinal cord, and this inflammatory response can lead to motor neuronal damage, disintegration of nerve termini and eventually muscle wasting. Methods Cytokines/chemokines were estimated by Cytokine Antibody Array. Nerve termini and muscle fibers in abdominal muscle, and motor neuronal apoptosis in the spinal cord were analyzed by fluorescent microscopy and immunochemical analyses. Loss of muscle mass was assessed by wet weight of the muscles compared with control in mice after BISI. Results Our data showed that CCL2 was significantly increased in the serum (&amp;gt;2.4 fold) at day 1 and 3 after BISI in vivo. Iin the extracellular medium (&amp;gt;2.0 fold) of organotypic spinal cord explant after exposure to exogenous histones ex vivo at day1. Neuroinflammation in the spinal cord after BISI caused motor neuronal apoptosis, nerve termini disintegration, and loss of muscle mass of soleus (63.4 + 5.1%), tibialis (72.3 + 2.7%) and gastrocnemius (70.2 + 1.1%) compared to sham burn injured wild-type (WT) or YFP-neurons expressing transgenic mice. In contrast, GTS-21 reduced neuroinflammation and significantly prevented loss of muscle mass of soleus (86.8 + 0.2%), tibialis (85.4 + 4.5%) and gastrocnemius (89.0 + 4.0%). Depletion of macrophages by PBS-liposomes in burned mice showed transmigration of CCR2-containing monocytes-derived macrophages (CCR2-MDMΦ) in the ventral horn of the spinal cord, and led to the loss of all three muscles (soleus, 62.4 + 2.6%; tibialis, 86.3 + 3.5; gastrocnemius, 81.4 + 1.7%). Nevertheless, depletion of macrophages by Clophosomes in burned mice inhibited the transmigration of CCR2-MDMΦ, and protected muscle mass (soleus, 96.6 + 3.9%; tibialis, 101 + 3.9%; gastrocnemius, 98.3 + 1.2%) significantly. Subsequently, we demonstrated that the knocking out of CCR2 gene in mice protected motor neuronal apoptosis and inhibited loss of muscle mass of soleus, tibialis and gastrocnemius from 63.4 + 5.1%, 72.3 + 2.7% and 70.2 + 1.1% in WT mice to 82.1 + 4.7%, 85.4 + 4.5% and 95.8 + 3.2% in CCR2KO mice, respectively, after BISI. Conclusions Collectively, these results suggest activated macrophages creep to the spinal cord through CCL2/CCR2 signaling axis after BISI to cause neuroinflammation, leading to motor neuronal damage, nerve disintegration and muscle wasting. Applicability of Research to Practice These findings will be useful to understand the pathology occurring in burned patients and to develop the therapeutics measures to protect them from BISI.

P6303Developmental origin of cardiac macrophages in steady state and myocardial infarction

Abstract Background Macrophages are the most abundant immune cells in the myocardial tissue in steady state. The sterile inflammation caused by myocardial infarction triggers a massive immune reaction, which leads to a profound influx of neutrophils and monocytes. In the postacute phase of infarction macrophages play an essential role in reparative processes. Recently, it has become clear that macrophages in the heart have a dual developmental origin from embryonic and bone marrow (BM) hematopoiesis. In this study, we sought to investigate the contribution of embryonic derived macrophages to the cardiac macrophage pool in steady state as well as the acute and chronic phase after ischemia/reperfusion injury. Methods/Results To address the origin of macrophages in steady state we used different models of lineage tracing to determine the developmental origin of cardiac macrophages. Using FLT3-Cre mice and radiation-independent CD45.1/.2 bone marrow chimera, we found that the resident macrophage population in the heart is mainly independent of definitive hematopoiesis (approximately 70–80% of cardiac macrophages). The BM-dependent population on the other hand is replenished by blood-derived monocytes. Further we used the radiation-independent CD45.1/.2 bone marrow chimera to characterize the origin of macrophages at different time points after I/R-injury. In the acute phase after myocardial infarction we observed a profound influx of BM-derived macrophages in the infarct region and also in the remote area. 30 days after I/R-injury the composition of the resident macrophage pool was mainly comprised of BM-independent macrophages, similar to steady state conditions. To address the role of BM-derived macrophages we used CCR2-ko mice, which have low numbers of inflammatory monocytes in peripheral blood. CCR2-ko mice showed reduced macrophage numbers in the acute phase after myocardial infarction. Using positron emission tomography we investigated the influence of CCR2-deficiency on cardiac function after I/R-injury. In comparison to WT mice, CCR2-ko mice showed a significantly increased infarct size. Cardiac remodeling, determined by end-diastolic volume, on the other hand was improved in CCR2-ko mice. The ejection fraction was similar in both groups. Conclusion The cardiac macrophage pool is mainly comprised of BM-independent macrophages. In response to I/R-injury monocyte-derived macrophages transiently enter the myocardium but do not persist in significant numbers over time. The influx of BM-derived macrophages after I/R-injury was reduced using CCR2-ko mice, which led to improved cardiac remodeling. Our findings are of potential importance for understanding the cardiac immune response and for the therapeutic targeting of macrophages in inflammatory conditions. Acknowledgement/Funding German Society of Cardiology, German Centre for Cardiovascular Research, LMU Excellence, SFB 914

Abstract 094: Adaptive Cardiac Remodeling to Chronic Pressure Overload Requires the Expression of Placental Growth Factor in the Spleen to Recruit Reparative Macrophages to the Left Ventricle

Cardiac remodeling is the process established by the left ventricle (LV) to respond to different challenging stimuli. Although immunity has been clearly shown to participate to LV remodeling in myocardial infarction, how immune response participates to hypertensive chronic stress is poorly understood. We found that Placental Growth Factor (PlGF), belonging to the VEGF family, is necessary for adaptive remodeling to pressure overload and modulates the recruitment of innate immune cells in the LV. Here we asked where PlGF does exert its effects: i) directly in the myocardium or ii) by modulating immune organs? As TAC induced PlGF both in cardiac tissue and in the spleen, we devised a strategy to assess the contribution of these two tissues. We generated chimeric mice by spleen transplantation between WT and KO mice, obtaining: 1) WT mice with PlGF KO spleen and 2) PlGF KO mice with WT spleen (EF% 78 ± 0,7), finding that only mice with PlGF expressed in the spleen (EF% 68 ± 1,5) established adaptive remodeling to pressure overload, as assessed by echocardiography (p&lt;0.01). Since in the absence of PlGF we observed a consistent reduction of LV macrophages, we characterized the subsets of innate immune cells by flow cytometry. While WT mice subjected to TAC showed early LV recruitment of non resident CD11b + CD64 + Timd4 - CCR2 low CX 3 CR1 hi macrophages, PlGF KO mice failed to show this response. To investigate the potential relevance of the spleen as reservoir of myeloid cells with adaptive/reparative functions for LV, we induced TAC in splenectomized mice, finding that they didn’t develop adaptive remodeling, exhibiting early HFrEF. To investigate the role of reparative macrophages, that are mostly characterized by the expression of the CX 3 CR1 receptor, and of pro-inflammatory monocytes that express high levels and accumulate through CCR2, we also subjected CCR2 KO and CX 3 CR1 KO mice to TAC. While CCR2 KO behaved similarly to WT mice, CX 3 CR1 KO were unable to establish adaptive remodeling and early developed HFrEF similarly to mice deprived of the spleen and to PlGF KO mice. Overall our results indicate that pressure overload induces PlGF in the spleen as an immunomodulator capable to deploy reparative macrophages that sustain adaptive remodeling of the LV.

Abstract 1965: Inhibiting the CCR2/MCP-1 chemokine pathway blocks MDSC recruitment and promotes anti-tumor immunity

Abstract Host anti-tumor immunity, including the actions of cytotoxic T cells, plays a key role in curtailing the growth of “hot” tumors but can be hindered by multiple mechanisms. One such mechanism involves the local recruitment and expansion of myeloid derived suppressor cells (MDSCs) within the tumor microenvironment. We investigated the role of the CCR2/MCP-1 axis in MDSC-associated tumor progression using the TC1 lung tumor cell line implanted into syngeneic C57BL/6 mice. Phenotypic profiling of TC1 tumors revealed maximal expression of CCR2 by tumor resident MDSCs (p&amp;lt;0.01 vs. neutrophils, tumor-associated macrophages/TAM or T cells), and MCP-1 by transplanted tumor cells (p&amp;lt;0.01 vs. all types of leukocytes), respectively. Additionally, utilization of CCR2 knockout (CCR2KO) mice showed the dependence of progression of TC1 tumor on CCR2 signaling (p&amp;lt;0.01). Tumors in CCR2KO mice had fewer CCR2lowMDSCs (p&amp;lt;0.01), CD4 T cells (p&amp;lt;0.01), and Foxp3+ Treg cells (p&amp;lt;0.05). Conversely, CD8 T cell infiltration was significantly augmented in tumors from CCR2KO mice (p&amp;lt;0.05), and these CD8 T cells were capable of making higher levels of IFN-γ (p&amp;lt;0.01) and granzyme-B (p&amp;lt;0.05) upon restimulation than CD8 T cells from WT tumor-bearing mice. These effects were tumor-specific and not the result of genetic ablation of CCR2 on T cells, since the adoptive transfer of WT or CCR2KO cells into B2D6/F1 mice showed that CCR2KO cells had comparable activation, proliferation, inflammatory cytokine production, and Treg-mediated suppressive function as WT cells (all p&amp;gt;0.05). We next used a thioglycolate-induced peritonitis model of inflammation to validate the role of CCR2 and MCP-1 in trafficking of CCR2+ cells to the site of inflammation. We showed the ability of a CCR2 antagonist to inhibit trafficking of CCR2+ cells to the site of inflammation, in a dose-dependent manner, with a maximal effect at a dose of 10 mg/kg (p&amp;lt;0.05). We then tested the compound at this inhibitory concentration for its ability to impair CCR2+ cell recruitment to MCP-1 expressing TC1 tumors. Use of the CCR2 antagonist blocked growth TC1 tumors (p&amp;lt;0.001) and markedly suppressed intratumoral MDSC numbers (p&amp;lt;0.01), while boosting the numbers of cytotoxic CD8 T cells (p&amp;lt;0.005) and intratumoral expression of IFN-γ, TNF-α, granzyme-B and perforin (all p&amp;lt;0.01). In summary, fully complementary genetic and pharmacologic data indicate that CCR2 targeting may be an important new component of immuno-oncology based therapies. Note: This abstract was not presented at the meeting. Citation Format: Payal Mittal, Liqing Wang, Tatiana Akimova, Craig A. Leach, Jose C. Clemente, Mathew Sender, Yao Chen, Brandon Turenen, Wayne W. Hancock. Inhibiting the CCR2/MCP-1 chemokine pathway blocks MDSC recruitment and promotes anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1965.

Chemokine receptors CCR2 and CX3CR1 regulate viral encephalitis-induced hippocampal damage but not seizures

Viral encephalitis is a major risk factor for the development of seizures, epilepsy, and hippocampal damage with associated cognitive impairment, markedly reducing quality of life in survivors. The mechanisms underlying seizures and hippocampal neurodegeneration developing during and after viral encephalitis are only incompletely understood, hampering the development of preventive treatments. Recent findings suggest that brain invasion of blood-born monocytes may be critically involved in both seizures and brain damage in response to encephalitis, whereas the relative role of microglia, the brain's resident immune cells, in these processes is not clear. CCR2 and CX3CR1 are two chemokine receptors that regulate the responses of myeloid cells, such as monocytes and microglia, during inflammation. We used Ccr2-KO and Cx3cr1-KO mice to understand the role of these receptors in viral encephalitis-associated seizures and neurodegeneration, using the Theiler's virus model of encephalitis in C57BL/6 mice. Our results show that CCR2 as well as CX3CR1 plays a key role in the accumulation of myeloid cells in the CNS and activation of hippocampal myeloid cells upon infection. Furthermore, by using Cx3cr1-creER+/-tdTomatoSt/Wt reporter mice, we show that, with regard to CD45 and CD11b expression, some microglia become indistinguishable from monocytes during CNS infection. Interestingly, the lack of CCR2 or CX3CR1 receptors was associated with almost complete prevention of hippocampal damage but did not prevent seizure development after viral CNS infection. These data are compatible with the hypothesis that CNS inflammatory mechanism(s) other than the infiltrating myeloid cells trigger the development of seizures during viral encephalitis.

CCR2 Contributes to F4/80+ Cells Migration Along Intramembranous Bone Healing in Maxilla, but Its Deficiency Does Not Critically Affect the Healing Outcome.

Bone healing depends of a transient inflammatory response, involving selective migration of leukocytes under the control of chemokine system. CCR2 has been regarded as an essential receptor for macrophage recruitment to inflammation and healing sites, but its role in the intramembranous bone healing on craniofacial region remains unknown. Therefore, we investigated the role of CCR2 on F4/80+ cells migration and its consequences to the intramembranous healing outcome. C57BL/6 wild-type (WT) and CCR2KO mice were subjected to upper right incisor extraction, followed by micro-computed tomography, histological, immunological, and molecular analysis along experimental periods. CCR2 was associated with F4/80+ cells influx to the intramembranous bone healing in WT mice, and CCR2+ cells presented a kinetics similar to F4/80+ and CCR5+ cells. By contrast, F4/80+ and CCR5+ cells were significantly reduced in CCR2KO mice. The absence of CCR2 did not cause major microscopic changes in healing parameters, while molecular analysis demonstrated differential genes expression of several molecules between CCR2KO and WT mice. The mRNA expression of TGFB1, RUNX2, and mesenchymal stem cells markers (CXCL12, CD106, OCT4, NANOG, and CD146) was decreased in CCR2KO mice, while IL6, CXCR1, RANKL, and ECM markers (MMP1, 2, 9, and Col1a2) were significantly increased in different periods. Finally, immunofluorescence and FACS revealed that F4/80+ cells are positive for both CCR2 and CCR5, suggesting that CCR5 may account for the remaining migration of the F4/80+ cells in CCR2KO mice. In summary, these results indicate that CCR2+ cells play a primary role in F4/80+ cells migration along healing in intramembranous bones, but its deficiency does not critically impact healing outcome.

PO-112 The senescence associated secretory phenotype (SASP)-factor CcL2 fosters vascular dysfunction and endothelial cell loss in radiation-induced lung disease

Introduction Blood vessels are critical targets of the radiation response. Acute vascular damage and dysfunction upon irradiation as well as delayed endothelial cell loss are particularly prominent in the radiation response of normal tissues. However, the mechanisms underlying radiation-induced adverse effects in the vascular compartment remain elusive and no causative radioprotective treatment is available to date. Material and methods Here we investigated the therapeutic potential of Ccl2 signalling inhibition to specifically protect endothelial cells (EC) from injury of radiation-induced acute and late toxicity in lungs after whole thorax irradiation (WTI) using a mouse model of radiation-induced pneumopathy as well as ex vivo cultured human lung tissue. Results and discussions RT-induced vascular dysfunction and associated adverse effects can be efficiently antagonised by inhibition of Ccl2 signalling using either the selective Ccl2 inhibitor bindarit (BIN) or mice deficient for the main Ccl2 receptor CCR2 (KO). BIN-treatment efficiently counteracted the RT-induced expression of Ccl2, normalised endothelial cell (EC) morphology and vascular function and limited lung inflammation and metastasis early after irradiation (acute effects). A similar protection of the vascular compartment was detected by loss of Ccl2 signalling in lungs of CCR2-KO mice. Long-term Ccl2 signalling inhibition also significantly limited EC loss and accompanied fibrosis progression as adverse late effect. With respect to the human situation, we further confirmed that Ccl2 secreted by RT-induced senescent epithelial cells resulted in the activation of normally quiescent but DNA-damaged EC finally leading to EC loss in ex vivo cultured human normal lung tissue. Abrogation of certain aspects of the secretome of irradiated resident lung cells, in particular signalling inhibition of the senescence associated secretory phenotype (SASP)-factor Ccl2 secreted predominantly by RT-induced senescent epithelial cells resulted in protection of the endothelial compartment. Conclusion Radioprotection of the normal tissue via Ccl2 signalling inhibition without simultaneous protection or preferable radiosensitization of tumour tissue might improve local tumour control and survival, because higher doses of radiation could be used.

Cell-intrinsic and spatially divergent tumor programmed death ligand 1 (PD-L1) signals modify local and systemic anti-tumor immunity through novel chemokine effects

Abstract PD-L1, expressed on many tumors, inhibits anti-tumor T cells by programmed death (PD)-1 engagement. Tumor PD-L1 predicts anti-PD-L1 treatment outcomes through incompletely understood mechanisms. We used naturally PD-L1+ murine B16 melanoma (ctrl) and made PD-L1KO B16 by CRISPR/Cas9. PD-L1KO B16 grew faster when ctrl B16 was on the opposite flank (trans). By contrast, ctrl B16 grew slower when trans to PD-L1KO B16. Anti-PD-L1 slowed ctrl but not PD-L1KO B16 tumors as expected. However, when PD-L1KO was trans to ctrl αPD-L1 now also slowed PD-L1KO growth. Anti-PD-L1 increased CD3+ T cell infiltration into ctrl and PD-L1KO tumors similarly in vivo, but increased natural killer (NK) cell numbers and functions (e.g., CD107a, IFN-g) in PD-L1KO tumors &amp;gt; ctrl. CD4+ and CD8+ T cells chemotaxed to PD-L1KO slightly &amp;gt; ctrl, but NK cells migrated 2-fold more to PD-L1KO vs ctrl. PD-L1KO tumors produced more CCL2 than ctrl in vitro and in vivo, and this CCL2 induced CCR2+ NK chemotaxis in vitro. Both NK cells and T cells were required for optimal trans responses to anti-PD-L1 treatment in vivo. In wild type mice, anti-CCL2 reduced growth of trans PD-L1KO tumors &amp;gt; ctrl, and reduced anti-PD-L1 efficacy against PD-L1KO&amp;gt; ctrl tumors. In CCR2KO mice, anti-PD-L1 efficacy was retained against trans ctrl B16, but abolished against PD-L1KO trans tumors. PD-L1 also regulated other chemokines recruiting receptor+ cells, with similar effects seen in MB49 bladder cancer and 4T1 breast cancer cells. Thus, tumor PD-L1 alters immune infiltrates and anti-PD-L1 efficacy through novel chemokine effects. Our models help understand anti-PD-L1 (and likely anti-PD-1) responses based on tumor PD-L1 expression and can define ways to improve immunotherapy for PD-L1 null tumors.

Regulators of mycobacterial granuloma formation – CCL2 and VEGF-A

Abstract Granulomas are macrophage dominated lesions and are a central feature of mycobacterial infections. These cell aggregates are dynamically changing structures as the life span of the granuloma recruited effector cells is relatively short and the cells have to be replaced. CCL2 and VEGF-A are required for granuloma maintenance as blocking the action of these chemokines results in reduction of granuloma size and number after BCG or Mtb infections. We seek to elucidate the relevance of these two cytokines in the timeline of mycobacterial infections and their relative contribution to granuloma formation. CD11c+ cell immigration is impaired, costimulatory molecule expression is lower and granulomas are smaller while the bacterial burden is higher in the liver when CCR2KO mice are infected i.p. with M. bovis BCG. Similarly, in animals that are selectively deficient in macrophage VEGF-A production the granulomas are smaller. Granuloma cells produce VEGF-A. Caseating granulomas, like the ones induced in C3HeB/FeJ (Kramnik) mice are hypoxic and hypoxia is a strong inducer of VEGF-A production. Sarcoid lesions induced by BCG or Mtb in B6 mice are not hypoxic but we show that ATP released from dead cells induces VEGF-A in a subpopulation of macrophages in the granulomas. Additionally, VEGF production proved to be dependent on granuloma size: bigger granulomas produce larger quantities of VEGF-A while smaller lesions produce less or none. This finding suggests that unlike other chemokines such as CCL2, which is described to be involved in both granuloma initiation and maintenance, VEGF-A may support an increased lesion size in the late acute phase of infection. Regulating cellular recruitment may provide new therapies for granulomatous diseases.