Inhibition Of CCL2 Research Articles

Mechanism Exploration of Astaxanthin in the Treatment of Adriamycin-induced Cardiotoxicity Based on Network Pharmacology and Experimental Validation.

Astaxanthin (AXT), a natural antioxidant recognized for its therapeutic potential in cancer and cardiovascular diseases, holds promise in mitigating adriamycin-induced cardiotoxicity (AIC). Nevertheless, the underlying mechanisms of AXT in AIC mitigation remain to be elucidated. Consequently, this study endeavors to elucidate the mechanism of AXT against AIC, employing an integrated approach. Network pharmacology, molecular docking, and molecular dynamics simulations were harnessed to explore the molecular mechanism underlying AXT's action against AIC. Furthermore, the in-vitro AIC model was established with the H9c2 cell to generate transcriptome data for validation. A total of 533 putative AXT targets and 1478 AIC-related genes were initially screened by database retrieval and bioinformatics analysis. A total of 248 potential targets of AXT against AIC and several signaling pathways were identified by network pharmacology and enrichment analysis. Two core genes (CCL2 and NOS3) and the AGE-RAGE signaling pathway in diabetic complications were further highlighted by transcriptome validation based on the AIC in-vitro model. Additionally, molecular docking and dynamics analyses supported the robust binding affinity of AXT with the core targets. The study suggested that AXT might ameliorate AIC through the inhibition of CCL2 and NOS3 as well as AGE-RAGE signaling, which provide a theoretical basis for the development of a strategy against AIC.

Ephrin A1 Stimulates CCL2 Secretion to Facilitate Pre-metastatic Niche Formation and Promote Gastric Cancer Liver Metastasis.

The liver is a primary target for distal metastasis of gastric cancer (GC). The hepatic pre-metastatic niche (PMN) facilitates crucial communications between primary tumor and liver, thereby playing an essential role in hepatic metastasis. Identification of the molecular mechanisms driving PMN formation in GC could facilitate development of strategies to prevent and treat liver metastasis. Here, we uncovered a role for ephrin A1 (EFNA1) signaling in development of the PMN. EFNA1 overexpression in GC cells significantly increased CCL2 secretion through the Hippo-YAP pathway. Secreted CCL2 activated hepatic stellate cells (HStCs) within the hepatic PMN via the WNT/β-catenin pathway. Inhibition of CCL2 significantly suppressed HStC activation and reduced liver metastasis triggered by EFNA1 signaling in GC cells. Moreover, high CCL2 expression correlated with poor survival in GC patients. Overall, these findings reveal that EFNA1 signaling in GC cells upregulates CCL2, which activates HStCs to engender establishment of a hepatic pre-metastatic niche that supports liver metastasis.

Choroid plexus CCL2‒CCR2 signaling orchestrates macrophage recruitment and cerebrospinal fluid hypersecretion in hydrocephalus

The choroid plexus (ChP) serves as the principal origin of cerebrospinal fluid (CSF). CSF hypersecretion due to ChP inflammation has emerged as an important pathogenesis of hydrocephalus recently. Nevertheless, the precise mechanisms of ChP inflammation and the ensuing CSF hypersecretion in hydrocephalus remain ill-defined. In the present study, we elucidate the critical role of macrophages in the pathogenesis of ChP inflammation. Specifically, we identify the chemokine CCL2, released by ChP epithelial cells, recruits CCR2+ monocytes to the ChP thereby inciting hydrocephalus pathogenesis. The accumulated ChP macrophages increase the inflammation in ChP epithelial cells through TNF-α/TNFR1/NF-κB signaling cascade, thereby leading to CSF hypersecretion. Strikingly, augmentation of ChP‒CCL2 using an adeno-associated viral approach (AAV) exacerbates macrophage recruitment, activation, and ventriculomegaly in rat PHH models. Systemic application of Bindarit, a specific CCL2 inhibitor, significantly inhibits ChP macrophage infiltration and activation and reduces CSF secretion rate. Furthermore, the administration of CCR2 antagonist (INCB 3284) reduces ChP macrophage accumulation and ventriculomegaly. This study not only unveils the ChP CCL2‒CCR2 signaling in the pathophysiology of hydrocephalus but also unveils Bindarit as a promising therapeutic choice for the management of posthemorrhagic hydrocephalus.

STING signaling activation modulates macrophage polarization via CCL2 in radiation-induced lung injury

BackgroundRadiation-induced lung injury (RILI) is a prevalent complication of thoracic radiotherapy in cancer patients. A comprehensive understanding of the underlying mechanisms of RILI is essential for the development of effective prevention and treatment strategies.MethodsTo investigate RILI, we utilized a mouse model that received 12.5 Gy whole-thoracic irradiation. The evaluation of RILI was performed using a combination of quantitative real-time polymerase chain reaction (qRT-PCR), enzyme-linked immunosorbent assay (ELISA), histology, western blot, immunohistochemistry, RNA sequencing, and flow cytometry. Additionally, we established a co-culture system consisting of macrophages, lung epithelial cells, and fibroblasts for in vitro studies. In this system, lung epithelial cells were irradiated with a dose of 4 Gy, and we employed STING knockout macrophages. Translational examinations were conducted to explore the relationship between STING expression in pre-radiotherapy lung tissues, dynamic changes in circulating CCL2, and the development of RILI.ResultsOur findings revealed significant activation of the cGAS-STING pathway and M1 polarization of macrophages in the lungs of irradiated mice. In vitro studies demonstrated that the deficiency of cGAS-STING signaling led to impaired macrophage polarization and RILI. Through RNA sequencing, cytokine profiling, and rescue experiments using a CCL2 inhibitor called Bindarit, we identified the involvement of CCL2 in the regulation of macrophage polarization and the development of RILI. Moreover, translational investigations using patient samples collected before and after thoracic radiotherapy provided additional evidence supporting the association between cGAS-STING signaling activity, CCL2 upregulation, and the development of radiation pneumonitis.ConclusionsThe cGAS-STING signaling pathway plays a crucial role in regulating the recruitment and polarization of macrophages, partly through CCL2, during the pathogenesis of RILI.

Twist1 in podocytes ameliorates podocyte injury and proteinuria by limiting CCL2-dependent macrophage infiltration.

The transcription factor Twist1 regulates several processes that could impact kidney disease progression, including epithelial cell differentiation and inflammatory cytokine induction. Podocytes are specialized epithelia that exhibit features of immune cells and could therefore mediate unique effects of Twist1 on glomerular disease. To study Twist1 functions in podocytes during proteinuric kidney disease, we employed a conditional mutant mouse in which Twist1 was selectively ablated in podocytes (Twist1-PKO). Deletion of Twist1 in podocytes augmented proteinuria, podocyte injury, and foot process effacement in glomerular injury models. Twist1 in podocytes constrained renal accumulation of monocytes/macrophages and glomerular expression of CCL2 and the macrophage cytokine TNF-α after injury. Deletion of TNF-α selectively from podocytes had no impact on the progression of proteinuric nephropathy. By contrast, the inhibition of CCL2 abrogated the exaggeration in proteinuria and podocyte injury accruing from podocyte Twist1 deletion. Collectively, Twist1 in podocytes mitigated urine albumin excretion and podocyte injury in proteinuric kidney diseases by limiting CCL2 induction that drove monocyte/macrophage infiltration into injured glomeruli. Myeloid cells, rather than podocytes, further promoted podocyte injury and glomerular disease by secreting TNF-α. These data highlight the capacity of Twist1 in the podocyte to mitigate glomerular injury by curtailing the local myeloid immune response.

STEM-30. NECROPTOTIC ASTROCYTES CONTRIBUTE TO MAINTAINING STEMNESS OF DISSEMINATED MEDULLOBLASTOMA THROUGH CCL2 SECRETION

Abstract Medulloblastoma (MB) with metastases at diagnosis and recurrence correlates with poor prognosis. Unfortunately, the molecular mechanism underlying metastases growth has received less attention than primary therapy-naïve MB. Though astrocytes have been frequently detected in brain tumors, their roles in regulating the stemness properties of MB stem-like cells (MBSCs) in disseminated lesions remain elusive. Effects of tumor-associated astrocytes (TAA)-secreted CCL2 on MBSCs self-renewal was determined by immunostaining analysis. Necroptosis of TAA was examined by measuring necrosome activity. Alterations in Notch signaling were examined after inhibition of CCL2. Progression of MBSCs-derived tumors was evaluated after pharmaceutical blockage of necroptosis. TAA, as the essential components of disseminated tumor, produced high level of CCL2 to shape inflammation microenvironment, which stimulated the enrichment of MBSCs in disseminated MB. In particular, CCL2 played a pivotal role in maintaining stem-like properties via JAK2/STAT3 mediated activation of Notch signaling. Loss of CCL2/CCR2 function repressed JAK2/STAT3-Notch pathway and impaired MBSCs proliferation, leading to a dramatic reduction of stemness, tumorigenicity and metastasizing capability. Furthermore, necroptosis-induced CCL2 release depended on RIP1/RIP3/MLKL activation in TAA, which promoted the oncogenic phenotype. Blockade of necroptosis resulted in CCL2 deprivation and compromised MBSCs self-proliferation, indicating MBSCs outsourced CCL2 from necroptotic TAA. Finally, CCL2 was upregulated in high-risk stages of MB, further supporting its value as a prognostic indicator. These findings highlighted the critical role of CCL2/CCR2 in Notch signaling activation in MBSCs, and revealed a necroptosis-associated glial cytokine microenvironment driving stemness maintenance in disseminations.

IL1B triggers inflammatory cytokine production in bovine oviduct epithelial cells and induces neutrophil accumulation via CCL2.

The oviduct is essential for reproduction. We previously showed that oviduct epithelial cells (OECs) isolated from aged cows expressed higher levels of inflammatory cytokines, including interleukin (IL) 1A and IL1B. In addition, aging is associated with tissue dysfunction and cellular senescence via a senescence-associated secretory phenotype (SASP) and immune cell accumulation. We investigated whether IL1A or IL1B causes SASP production, cellular senescence, and inflammatory responses in bovine OECs. The OECs were isolated from bovine oviducts from young (mean 50.3months) and aged cows (mean 157.0months) and cultured. Treatment with IL1A or IL1B induced SASP production (IL8, IL6, TNFA, and CCL2) and mRNA expression of cell adhesion molecules in bovine OECs, but both IL1s did not induce cellular senescence in OECs and migration of polymorphonuclear neutrophils (PMNs). Cultured medium of OECs treated with IL1s, especially IL1B, dramatically induced PMN migration. Treatment with the CCL2 inhibitor, but not IL8 or its receptor CXCR2 inhibitors, significantly reduced immune cell migration in IL1B-treated OEC-cultured medium. Treatment with IL1B increased PMN adhesion to OECs, resulting in further SASP production in OECs due to a PMN-OEC interaction. We suggest that senescence-associated IL1s cause SASP production in bovine OECs and CCL2 induced by IL1B is essential for the migration of immune cells to OECs. Specifically, IL1B regulates PMN migration and adhesion to bovine OECs, and PMNs accelerate inflammatory cytokine production from bovine OECs via a direct interaction. These phenomena may contribute to chronic oviductal inflammation, resulting in subfertility.

Elevated CCL2 causes Leydig cell malfunction in metabolic syndrome.

Metabolic syndrome (MetS), which is associated with chronic inflammation, predisposes males to hypogonadism and subfertility. The underlying mechanism of these pathologies remains poorly understood. Homozygous leptin-resistant obese db/db mice are characterized by small testes, low testicular testosterone, and a reduced number of Leydig cells. Here we report that IL-1β, CCL2 (also known as MCP-1), and corticosterone concentrations were increased in the testes of db/db mice relative to those in WT controls. Cultured murine and human Leydig cells responded to cytokine stress with increased CCL2 release and apoptotic signals. Chemical inhibition of CCL2 rescued Leydig cell function in vitro and in db/db mice. Consistently, we found that Ccl2-deficient mice fed with a high-energy diet were protected from testicular dysfunction compared with similarly fed WT mice. Finally, a cohort of infertile men with a history of MetS showed that reduction of CCL2 plasma levels could be achieved by weight loss and was clearly associated with recovery from hypogonadism. Taken together, we conclude that CCL2-mediated chronic inflammation is, to a large extent, responsible for the subfertility in MetS by causing damage to Leydig cells.

Cortistatin protects against inflammatory airway diseases through curbing CCL2 and antagonizing NF-κB signaling pathway

Asthma is a chronic inflammatory disease affecting millions of people around the world, yet much remains unknown about its underlying mechanisms. Cortistatin (CST) is a neuropeptide which is reported to be a potential endogenous anti-inflammatory factor in several conditions. To testify the potential involvement of CST in airway inflammatory reaction, an ovalbumin (OVA)-induced mice model was established in wild-type (WT) as well as CST-knockout (Cort-/-) mice. Thereafter, lung tissue or cell samples were gathered in each group, and histological analysis as well as cell counting assay indicated that Cort-/- mice exhibited exaggeration of asthma compared with WT control group. Moreover, mRNA sequencing assay revealed that CCL2 was a potential target of CST in asthma, and administration of CCL2 inhibitor alleviated airway inflammation of asthma in Cort-/- mice. Moreover, NF-κB signaling pathway might be closely associated with the protective function of CST in asthma, as enhanced activation of NF-κB signaling pathway was observed in OVA-induced asthma model of Cort-/- mice, and SN50, an inhibitor of NF-κB signaling pathway, antagonized asthma development in Cort-/- mice. In summary, CST might represent as a promising target for the treatment of asthma through interacting with CCL2 and NF-κB signaling pathway.

Inhibition of CCL2 by bindarit alleviates diabetes-associated periodontitis by suppressing inflammatory monocyte infiltration and altering macrophage properties

Diabetes-associated periodontitis (DP) aggravates diabetic complications and increases mortality from diabetes. DP is caused by diabetes-enhanced host immune-inflammatory responses to bacterial insult. In this study, we found that persistently elevated CCL2 levels in combination with proinflammatory monocyte infiltration of periodontal tissues were closely related to DP. Moreover, inhibition of CCL2 by oral administration of bindarit reduced alveolar bone loss and increased periodontal epithelial thickness by suppressing periodontal inflammation. Furthermore, bindarit suppressed the infiltration of proinflammatory monocytes and altered the inflammatory properties of macrophages in the diabetic periodontium. This finding provides a basis for the development of an effective therapeutic approach for treating DP.

MiR-511-3p protects against cockroach allergen-induced lung inflammation by antagonizing CCL2.

miR-511-3p, encoded by CD206/Mrc1, was demonstrated to reduce allergic inflammation and promote alternative (M2) macrophage polarization. Here, we sought to elucidate the fundamental mechanism by which miR-511-3p attenuates allergic inflammation and promotes macrophage polarization. Compared with WT mice, the allergen-challenged Mrc1-/- mice showed increased airway hyperresponsiveness (AHR) and inflammation. However, this increased AHR and inflammation were significantly attenuated when these mice were pretransduced with adeno-associated virus-miR-511-3p (AAV-miR-511-3p). Gene expression profiling of macrophages identified Ccl2 as one of the major genes that was highly expressed in M2 macrophages but antagonized by miR-511-3p. The interaction between miR-511-3p and Ccl2 was confirmed by in silico analysis and mRNA-miR pulldown assay. Further evidence for the inhibition of Ccl2 by miR-511-3p was given by reduced levels of Ccl2 in supernatants of miR-511-3p-transduced macrophages and in bronchoalveolar lavage fluids of AAV-miR-511-3p-infected Mrc1-/- mice. Mechanistically, we demonstrated that Ccl2 promotes M1 macrophage polarization by activating RhoA signaling through Ccr2. The interaction between Ccr2 and RhoA was also supported by coimmunoprecipitation assay. Importantly, inhibition of RhoA signaling suppressed cockroach allergen-induced AHR and lung inflammation. These findings suggest a potentially novel mechanism by which miR-511-3p regulates allergic inflammation and macrophage polarization by targeting Ccl2 and its downstream Ccr2/RhoA axis.

Increased Antiangiogenic Effect by Blocking CCL2-dependent Macrophages in a Rodent Glioblastoma Model: Correlation Study with Dynamic Susceptibility Contrast Perfusion MRI

When glioblastoma multiforme (GBM) is treated with anti-vascular endothelial growth factor (VEGF) agents, it commonly exhibits tumor progression due to the development of resistance, which results in a dismal survival rate. GBM tumors contain a large number of monocytes/macrophages, which have been shown to be resistant to the effects of bevacizumab. It has been reported that tumor-associated macrophages (TAMs) promote resistance to bevacizumab treatment. Therefore, it is important to target TAMs in the GBM microenvironment. TAMs, which depend on chemokine ligand 2 (CCL2) for differentiation and survival, induce the expression of proangiogenic factors such as VEGF. Dynamic susceptibility contrast (DSC)-MR imaging is an advanced technique that provides information on tumor blood volume and can potentially predict the response to several treatments, including anti-angiogenic agents such as bevacizumab, in human GBM. In this study, we used a CCL2 inhibitor, mNOX-E36, to suppress the recruitment of TAMs in a CCL2-expressing rat GBM model and investigated the effect of combination therapy with bevacizumab using DSC-MR imaging. We demonstrated that the inhibition of CCL2 blocked macrophage recruitment and angiogenesis, which resulted in decreased tumor volume and blood volume in CCL2-expressing GBM in a rat model. Our results provide direct evidence that CCL2 expression can increase the resistance to bevacizumab, which can be assessed noninvasively with the DSC-MR imaging technique. This study shows that the suppression of CCL2 can play an important role in increasing the efficacy of anti-angiogenic treatment in GBM by inhibiting the recruitment of CCL2-dependent macrophages.

Intervention of Inflammatory Monocyte Activity Limits Dermal Fibrosis

Monocytes and monocyte-derived cells are important players in the initiation, progression, and resolution of inflammatory skin reactions. As inflammation is a prerequisite for fibrosis development, we focused on the role of monocytes in cutaneous fibrosis, the clinical hallmark of patients suffering from systemic sclerosis. Investigating the function of monocytes in reactive oxygen species-induced dermal fibrosis, we observed that early monocyte depletion partially reduced disease severity. Low numbers of inflammatory Ly6Chigh monocytes, as well as inhibition of CCR2 and CCL2 in wild type animals by a specific L-RNA aptamer, mitigated disease parameters, indicating a pivotal role for CCR2+ inflammatory monocytes and the CCR2/CCL2 axis in fibrosis development. Of note, mice lacking splenic reservoirs failed to recruit monocytes to the skin and developed less fibrosis. Furthermore, enforced monocyte conversion into noninflammatory, patrolling Ly6Clow monocytes by a nuclear receptor Nur77-agonist also resulted in significantly impaired cutaneous inflammation and dermal fibrosis. Most evident, pronounced monocyte conversion in interferon stimulated gene 12-deficient mice with pronounced nuclear Nur77 signaling completely protected from dermal fibrosis. Our study shows that inflammatory monocytes that are recruited from splenic reservoirs play a key role in the development of skin fibrosis and can be therapeutically challenged by forced conversion via the Nur77/interferon stimulated gene 12 axis.

Crosstalk between cancer cells and tumor associated macrophages is required for mesenchymal circulating tumor cell-mediated colorectal cancer metastasis

BackgroundTumor-associated macrophages (TAMs) are major components of tumor microenvironment that frequently associated with tumor metastasis in human cancers. Circulating tumor cell (CTC), originating from primary tumor sites, is considered to be the precursors of tumor metastasis. However, the regulatory mechanism of TAMs in CTC-mediated tumor metastasis still remains unclear.MethodsImmunohistochemical staining was used to detect the macrophages infiltration (CD68 and CD163), epithelial–mesenchymal transition (EMT) markers (E-cadherin and Vimentin) expression in serial sections of human colorectal cancer (CRC) specimens. Then, the correlations between macrophages infiltration and clinicopathologic features, mesenchymal CTC ratio, and patients’ prognosis were analyzed. A co-culture assay in vitro was used to evaluate the role of TAMs on CRC EMT, migration and invasion, and ELISA, luciferase reporter assay and CHIP were performed to uncover the underlying mechanism. Furthermore, an in vivo model was carried out to confirm the effect of TAMs on mesenchymal CTC-mediated metastasis.ResultsClinically, CD163+ TAMs infiltrated in invasive front was associated with EMT, mesenchymal CTC ratio, and poor prognosis in patients with CRC. CRC–conditioned macrophages regulated EMT program to enhance CRC cells migration and invasion by secreting IL6. TAMs-derived IL6 activated the JAK2/STAT3 pathway, and activated STAT3 transcriptionally inhibited the tumor suppressor miR-506-3p in CRC cells. miR-506-3p, a key miRNA regulating FoxQ1, was downregulated in CRC cells, resulting in increased FoxQ1 expression, which in turn led to the production of CCL2 that promoted macrophage recruitment. Inhibition of CCL2 or IL6 broke this loop and reduced macrophage migration and mesenchymal CTC-mediated metastasis, respectively.ConclusionsOur data indicates that TAMs induce EMT program to enhance CRC migration, invasion, and CTC-mediated metastasis by regulating the JAK2/STAT3/miR-506-3p/FoxQ1 axis, which in turn leads to the production of CCL2 that promote macrophage recruitment, revealing a new cross-talk between immune cells and tumor cells in CRC microenvironment.

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.

Inhibition of Radiation-Induced Ccl2 Signaling Protects Lungs from Vascular Dysfunction and Endothelial Cell Loss.

Aims: Radiation-induced normal tissue toxicity often precludes the application of curative radiation doses. Here we investigated the therapeutic potential of chemokine C-C motif ligand 2 (Ccl2) signaling inhibition to protect normal lung tissue from radiotherapy (RT)-induced injury. Results: RT-induced vascular dysfunction and associated adverse effects can be efficiently antagonized by inhibition of Ccl2 signaling 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, normalized 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 signaling in lungs of CCR2-KO mice. Long-term Ccl2 signaling 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. Innovation: Abrogation of certain aspects of the secretome of irradiated resident lung cells, in particular signaling inhibition of the senescence-associated secretory phenotype-factor Ccl2 secreted predominantly by RT-induced senescent epithelial cells, resulted in protection of the endothelial compartment. Conclusions: Radioprotection of the normal tissue via Ccl2 signaling inhibition without simultaneous protection or preferable radiosensitization of tumor tissue might improve local tumor control and survival, because higher doses of radiation could be used.

Abstract 3908: The mechanism of premetastatic niche formation through FBXW7/NOTCH/CCL2 pathway and clinical trial using CCL2 inhibitor “SK-818” for breast cancer patients

Abstract Background Metastasis is a major cause of death in cancer patients, & elucidation of the mechanisms is expected to provide a basis for the development of new cancer treatment. Premetastatic niche whereby tumors prepare defined organs for metastasized cells is considered to be crucial for the development of metastasis, which consists of host cells such as monocytes, macrophages, & other inflammatory cells. However, the mechanism of how tumor cells communicate with host cells to develop a premetastatic niche remains unclear. FBXW7 is the F-box protein component of an SCF-type ubiquitin ligase, in which it functions as a receptor responsible for substrate recognition followed by the degradation of the substrate. Some part of the substrates of FBXW7 regulates the recruitment of monocytes, macrophages, & other inflammatory cells. Therefore, we hypothesized that expression of FBXW7 in the host cells is a key determinant of the formation of premetastatic niches. Materials & Methods To investigate the role of FBXW7 in the formation of premetastatic niche, we developed bone marrow-specific FBXW7-deficient mice. 1. We transferred B16F10 melanoma cells or E0771 mouse breast cancer cells into the tail vein or mammary fat pad of the mice & the wild type mice to evaluate the metastatic potential to lung. 2. We examined the serum concentrations of various cytokines or chemokines in the mice before & after E0771 cells transplantation using cytokine array. 3. To examine whether the enhanced metastasis apparent in FBXW7-deficient mice is dependent on the CCL2/CCR2 pathway, we treated FBXW7-deficient mice with SK-818, a CCR2 antagonist. 4. We measured the expression of FBXW7 mRNA in peripheral blood of breast cancer patients by qRT-PCR & examined the relationship between FBXW7 mRNA expression & prognosis. Results 1. The extent of lung metastasis of B16F10 or E0771 cell was markedly enhanced in FBXW7-deficient mice. 2. FBXW7-deficient mice exhibited increased serum levels of CCL2. Moreover, accumulation of NOTCH & consequent transcriptional activation of CCL2 was observed in bone marrow-derived stromal cells of FBXW7-deficient mice. 3. Administration of SK-818 blocked the enhancement of lung metastasis in FBXW7-deficient mice. 4. In human breast cancer patients, low FBXW7 mRNA expression in peripheral blood showed high concentration of CCL2 in serum & poor prognosis. Conclusions We found that the FBXW7/NOTCH/CCL2 pathway play a key role in the regulation of cancer metastasis through the formation of premetastatic niches, & that SK-818 inhibit cancer metastasis in mice. SK-818 is currently administered clinically for the treatment of patients with hepatitis B virus infection in Japan. Based on these findings, we have been conducting Phase I clinical trial to assess the safety of SK-818 for breast cancer patients. Citation Format: Miwa Noda, Takaaki Masuda, Naoki Hayashi, Takahiro Kogawa, Shinji Ohno, Keiichi Nakayama, Yohsuke Kuroda, Hidetoshi Eguchi, Shuhei Ito, Masafumi Inomata, Koshi Mimori. The mechanism of premetastatic niche formation through FBXW7/NOTCH/CCL2 pathway and clinical trial using CCL2 inhibitor “SK-818” for breast cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3908. doi:10.1158/1538-7445.AM2017-3908

Molecular mechanisms promoting progression of autoimmune myocarditis to dilated cardiomyopathy.

Abstract Myocarditis is an inflammatory disease of the myocardium, described by infiltration of immune cells and cardiomyocyte necrosis. It is a major cause of sudden death in young adults. Patients often progress to a more severe form of the disease termed dilated cardiomyopathy (DCM). It is characterized by extensive fibrosis leading to impaired cardiac function. Immunosuppressive treatments with corticosteroids (CSs) have not been effective in preventing the disease. We hypothesized that macrophage migration inhibitory factor (MIF), being the only known pro-inflammatory cytokine induced by CSs may play a role in resistance to CSs. Also, MIF counter-regulates CS-mediated immunosuppression. Using the experimental autoimmune myocarditis (EAM) animal model, we observed that MIF−/− mice treated with Dexamethasone (Dex) were highly resistant to EAM and progression to DCM. Furthermore, from a translational approach, treatment using small molecule inhibitors of MIF combined with Dex recapitulated this phenotype in wild type mice. We observed that treated mice showed decreased expression of key chemokines and extracellular matrix molecules compared with controls, implicating these molecules in disease progression. Currently, we are investigating these molecules by selectively inhibiting them in order to understand their contribution to disease pathogenesis. We observed that inhibition of CCL2 after, although not prior to establishment of EAM attenuated progression to DCM. Thereby, suggesting that CCL2 is predominantly involved during the fibrotic phase aiding in progression as opposed to the induction phase of the disease. Besides a novel approach to prevent DCM, our studies may provide new insights into the mechanisms driving DCM.

Low-Intensity Training and the C5a Complement Antagonist NOX-D21 Rescue the mdx Phenotype through Modulation of Inflammation

Inflammatory events occurring in dystrophic muscles contribute to the progression of Duchenne muscular dystrophy (DMD). Low-intensity training (LIT) attenuates the phenotype of mdx mice, an animal model for DMD. Therefore, we postulated that LIT could have anti-inflammatory properties. We assessed levels of inflammatory cytokines and infiltrated immune cells in gastrocnemius muscle of mdx mice after LIT. We detected high levels of complement component C5a, chemokine ligand (CCL) 2, CD68+ monocytes/macrophages, and proinflammatory M1 macrophages in muscles of mdx mice. LIT decreased CCL2 levels, increased CD68+ cell numbers, and shifted the macrophage population to the regenerative M2 type. We investigated whether inhibition of C5a or CCL2 with L-aptamers could mimic the effects of LIT. Although no effect of CCL2 inhibition was detected, treatment with the C5a inhibitor, NOX-D21, rescued the phenotype of nonexercised mdx mice, but not of exercised ones. In both cases, the level of CD68+ cells increased and macrophage populations leaned toward the inflammatory M1 type. In muscles of nonexercised treated mice, the level of IL-1 receptor antagonist increased, damage decreased, and fibers were switched toward the glycolytic fast type; in muscles of exercised mice, fibers were switched to the oxidative slow type. These results reveal the effects of LIT on the inflammatory status of mdx mice and suggest that NOX-D21 could be an anti-inflammatory drug for DMD.

C-C Motif Chemokine 5 Attenuates Angiotensin II–Dependent Kidney Injury by Limiting Renal Macrophage Infiltration

Inappropriate activation of the renin angiotensin system (RAS) is a key contributor to the pathogenesis of essential hypertension. During RAS activation, infiltration of immune cells into the kidney exacerbates hypertension and renal injury. However, the mechanisms underpinning the accumulation of mononuclear cells in the kidney after RAS stimulation remain unclear. C-C motif chemokine 5 (CCL5) drives recruitment of macrophages and T lymphocytes into injured tissues, and we have found that RAS activation induces CCL5 expression in the kidney during the pathogenesis of hypertension and renal fibrosis. We therefore evaluated the contribution of CCL5 to renal damage and fibrosis in hypertensive and normotensive models of RAS stimulation. Surprisingly, during angiotensin II-induced hypertension, CCL5-deficient (knockout, KO) mice exhibited markedly augmented kidney damage, macrophage infiltration, and expression of proinflammatory macrophage cytokines compared with wild-type controls. When subjected to the normotensive unilateral ureteral obstruction model of endogenous RAS activation, CCL5 KO mice similarly developed more severe renal fibrosis and greater accumulation of macrophages in the kidney, congruent with enhanced renal expression of the macrophage chemokine CCL2. In turn, pharmacologic inhibition of CCL2 abrogated the differences between CCL5 KO and wild-type mice in kidney fibrosis and macrophage infiltration after unilateral ureteral obstruction. These data indicate that CCL5 paradoxically limits macrophage accumulation in the injured kidney during RAS activation by constraining the proinflammatory actions of CCL2.