Inhibition of CCL7 improves endothelial dysfunction and vasculopathy in mouse models of diabetes mellitus.

Diabetic vascular disease is a major complication of diabetes mellitus (DM). Chemokine C-C motif ligand 7 (CCL7) attracts macrophages and monocytes, amplifying inflammatory processes in the vasculature. We hypothesized a causal role for CCL7 in diabetic vasculopathy. CCL7 concentrations were higher in the plasma of patients with type 2 DM, as well as in supernatants from their endothelial progenitor cells (EPCs). High-glucose stimulation increased the secretion of CCL7 from human dermal microvascular endothelial cells (HDMECs) through the c-Fos and c-Jun signaling pathways. CCL7 inhibition using knockdown or neutralization antibody treatment reversed the high glucose-induced impaired tube formation and migration abilities of EPCs, human aortic endothelial cells, human coronary artery endothelial cells, and HDMECs. Administration of recombinant human CCL7 protein impaired tube formation and migration abilities by down-regulating the AKT-endothelial nitric oxide synthase and AKT/nuclear factor erythroid 2-related factor 2/heme oxygenase-1/vascular endothelial growth factor/stromal cell-derived factor-1 pathways and by up-regulating ERK/phosphorylated p65/interleukin-1ß/interleukin-6/tumor necrosis factor-α pathways through CC chemokine receptor 3 in endothelial cells. Ccl7 knockout in streptozotocin-treated mice showed improved neovasculogenesis in ischemic limbs and accelerated wound repair, with increased circulating EPCs and capillary density. CCL7 antibody treatment in db/db mice and high-fat diet-induced hyperglycemia mice showed improved neovasculogenesis in ischemic limbs and wound areas, accompanied by up-regulation of angiogenic proteins and down-regulation of inflammatory proteins. Endothelial cell-specific Ccl7-knockout mice showed ameliorated diabetic vasculopathy in streptozotocin-induced DM. This study highlights the potential of CCL7 as a therapeutic target for diabetic vasculopathy.

Muscle cell-derived Ccl8 is a negative regulator of skeletal muscle regeneration.

Skeletal muscles undergo robust regeneration upon injury, and infiltrating immune cells play a major role in not only clearing damaged tissues but also regulating the myogenic process through secreted cytokines. Chemokine C-C motif ligand 8 (Ccl8), along with Ccl2 and Ccl7, has been reported to mediate inflammatory responses to suppress muscle regeneration. Ccl8 is also expressed by muscle cells, but a role of the muscle cell-derived Ccl8 in myogenesis has not been reported. In this study, we found that knockdown of Ccl8, but not Ccl2 or Ccl7, led to increased differentiation of C2C12 myoblasts. Analysis of existing single-cell transcriptomic datasets revealed that both immune cells and muscle stem cells (MuSCs) in regenerating muscles express Ccl8, with the expression by MuSCs at a much lower level, and that the temporal patterns of Ccl8 expression were different in MuSCs and macrophages. To probe a function of muscle cell-derived Ccl8 in vivo, we utilized a mouse system in which Cas9 was expressed in Pax7+ myogenic progenitor cells (MPCs) and Ccl8 gene editing was induced by AAV9-delivered sgRNA. Depletion of Ccl8 in Pax7+ MPCs resulted in accelerated muscle regeneration after barium chloride-induced injury in both young and middle-aged mice, and intramuscular administration of a recombinant Ccl8 reversed the phenotype. Accelerated regeneration was also observed when Ccl8 was depleted in Myf5+ or MyoD+ MPCs by similar approaches. Our results suggest that muscle cell-derived Ccl8 plays a unique role in regulating the initiation of myogenic differentiation during injury-induced muscle regeneration.

P21 facilitates macrophage chemotaxis by promoting CCL7 in the lung epithelial cell lines treated with radiation and bleomycin.

BACKGROUND: Interstitial lung diseases (ILDs) can be induced and even exacerbated by radiotherapy in thoracic cancer patients. The roles of immune responses underlying the development of these severe lung injuries are still obscure and need to be investigated. METHODS: A severe lung damage murine model was established by delivering 16 Gy X-rays to the chest of mice that had been pre-treated with bleomycin (BLM) and thus hold ILDs. Bioinformatic analyses were performed on the GEO datasets of radiation-induced lung injury (RILI) and BLM-induced pulmonary fibrosis (BIPF), and RNA-sequencing data of the severely damaged lung tissues. The screened differentially expressed genes (DEGs) were verified in lung epithelial cell lines by qRT-PCR assay. The injured lung tissue pathology was analyzed with H&E and Masson's staining, and immunohistochemistry staining. The macrophage chemotaxis and activity promoted by the stressed epithelial cells were determined by using a cell co-culture system. The expressions of p21 in MLE-12 and Beas-2B cells were detected by qRT-PCR, western blot, and immunofluorescence. The concentration of CCL7 in cell supernatant was measured by ELISA assay. In some experiments, Beas-2B cells were transfected with p21-siRNA or CCL7-siRNA before irradiation and/or BLM treatment. RESULTS: After the treatment of irradiation and/or BLM, the inflammatory and immune responses, chemokine-mediated signaling pathways were steadily activated in the severely injured lung, and p21 was screened out by the bioinformatic analysis and further verified to be upregulated in both mouse and human lung epithelial cell lines. The expression of P21 was positively correlated with macrophage infiltration in the injured lung tissues. Co-culturing with stressed Beas-2B cells or its conditioned medium containing CCL7 protein, U937 macrophages were actively polarized to M1-phase and their migration ability was obviously increased along with the damage degree of Beas-2B cells. Furthermore, knockdown p21 reduced CCL7 expression in Beas-2B cells and then decreased the chemotaxis of co-cultured macrophages. CONCLUSIONS: P21 promoted CCL7 release from the severely injured lung epithelial cell lines and contributed to the macrophage chemotaxis in vitro, which provides new insights for better understanding the inflammatory responses in lung injury.

Staphylococcus aureus Infection Induces the Production of the Neutrophil Chemoattractants CXCL1, CXCL2, CXCL3, CXCL5, CCL3, and CCL7 by Murine Osteoblasts.

Staphylococcus aureus is the principal causative agent of osteomyelitis, a serious bacterial infection of bone that is associated with progressive inflammatory damage. Bone-forming osteoblasts have increasingly been recognized to play an important role in the initiation and progression of detrimental inflammation at sites of infection and have been demonstrated to release an array of inflammatory mediators and factors that promote osteoclastogenesis and leukocyte recruitment following bacterial challenge. In the present study, we describe elevated bone tissue levels of the potent neutrophil-attracting chemokines CXCL1, CXCL2, CXCL3, CXCL5, CCL3, and CCL7 in a murine model of posttraumatic staphylococcal osteomyelitis. RNA sequencing (RNA-Seq) gene ontology analysis of isolated primary murine osteoblasts showed enrichment in differentially expressed genes involved in cell migration and chemokine receptor binding and chemokine activity following S. aureus infection, and a rapid increase in the expression of mRNA encoding CXCL1, CXCL2, CXCL3, CXCL5, CCL3, and CCL7, in these cells. Importantly, we have confirmed that such upregulated gene expression results in protein production with the demonstration that S. aureus challenge elicits the rapid and robust release of these chemokines by osteoblasts and does so in a bacterial dose-dependent manner. Furthermore, we have confirmed the ability of soluble osteoblast-derived chemokines to elicit the migration of a neutrophil-like cell line. As such, these studies demonstrate the robust production of CXCL1, CXCL2, CXCL3, CXCL5, CCL3, and CCL7 by osteoblasts in response to S. aureus infection, and the release of such neutrophil-attracting chemokines provides an additional mechanism by which osteoblasts could drive the inflammatory bone loss associated with staphylococcal osteomyelitis.

Serum CCL7 Is a Novel Prognostic Biomarker of Metastatic Colorectal Cancer.

BACKGROUND/AIM: Colorectal cancer is the third most common cancer globally, and the poor prognosis of patients with metastatic colorectal cancer (mCRC) warrants urgent attention. We previously obtained 10 candidate serum biomarkers for mCRC. Our aim with this study was to determine the prognostic performance of the pre-treatment serum C-C motif chemokine ligand 7 (CCL7) concentration in patients with mCRC. PATIENTS AND METHODS: Protein concentrations of CCL7 were examined using ELISA and immunohistochemistry for serum (n=110) and surgical specimens (n=85), respectively, of patients with mCRC. The relationship between protein concentration and prognosis was examined using Cox regression analysis, receiver operator characteristic curve analysis and the Kaplan-Meier method. RESULTS: The overall survival (OS) of patients with high concentrations of serum CCL7 was significantly poorer than that of patients with low concentrations. Patients with a high CCL7 concentration in the stroma had significantly poorer outcomes than those with a low concentration. The concentrations of carcinoembryonic antigen (CEA) and carbohydrate antigen 19-9 were significantly higher in the high-CCL7 group, compared to those in the low-CCL7 group. Univariate and multivariate analysis revealed that serum CCL7 concentration was a significant prognostic factor for mCRC. The combination of serum CCL and CEA concentrations was also useful in this regard (area under the curve=0.71). CONCLUSION: The combined pre-treatment serum levels of CCL7 and CEA are useful prognostic biomarkers for mCRC.

KLF15 suppresses tumor growth and metastasis in Triple-Negative Breast Cancer by downregulating CCL2 and CCL7.

Kruppel like factor 15 (KLF15), a transcriptional factor belonging to the Kruppel-like factor (KLF) family of genes, has recently been reported as a tumor suppressor gene in breast cancer. However, the specific mechanisms by which KLF15 inhibits BrCa have not been elucidated. Here we investigated the role and mechanism of KLF15 in triple-negative breast cancer (TNBC). KLF15 expression and methylation were detected by RT-qPCR, RT-PCR and methylation-specific PCR in breast cancer cell lines and tissues. The effects of KLF15 on TNBC cell functions were examined via various cellular function assays. The specific anti-tumor mechanisms of KLF15 were further investigated by RNA sequence, RT-qPCR, Western blotting, luciferase assay, ChIP, and bioinformatics analysis. As the results showed that KLF15 is significantly downregulated in breast cancer cell lines and tissues, which promoter methylation of KLF15 partially contributes to. Exogenous expression of KLF15 induced apoptosis and G2/M phase cell cycle arrest, suppressed cell proliferation, metastasis and in vivo tumorigenesis of TNBC cells. Mechanism studies revealed that KLF15 targeted and downregulated C-C motif chemokine ligand 2 (CCL2) and CCL7. Moreover, transcriptome and metabolome analysis revealed that KLF15 is involved in key anti-tumor regulatory and metabolic pathways in TNBC. In conclusion, KLF15 suppresses cell growth and metastasis in TNBC by downregulating CCL2 and CCL7. KLF15 may be a prognostic biomarker in TNBC.

Aberrant hyperexpression of the RNA binding protein FMRP in tumors mediates immune evasion.

Many human cancers manifest the capability to circumvent attack by the adaptive immune system. In this work, we identified a component of immune evasion that involves frequent up-regulation of fragile X mental retardation protein (FMRP) in solid tumors. FMRP represses immune attack, as revealed by cancer cells engineered to lack its expression. FMRP-deficient tumors were infiltrated by activated T cells that impaired tumor growth and enhanced survival in mice. Mechanistically, FMRP's immunosuppression was multifactorial, involving repression of the chemoattractant C-C motif chemokine ligand 7 (CCL7) concomitant with up-regulation of three immunomodulators-interleukin-33 (IL-33), tumor-secreted protein S (PROS1), and extracellular vesicles. Gene signatures associate FMRP's cancer network with poor prognosis and response to therapy in cancer patients. Collectively, FMRP is implicated as a regulator that orchestrates a multifaceted barrier to antitumor immune responses.

The role of CCL2, CCL7, ICAM-1, and VCAM-1 in interaction of endothelial cells and natural killer cells.

Natural killer (NK) cell-based therapy has been studied for the treatment of patients with cancers, but the inadequate infiltration of NK cells into solid tumors remains a big challenge to its clinical application. In this study, we examined the interaction between NK cells and endothelial cells, which might play a major role in NK cell homing to solid tumors. We found that endothelial cells were activated by TNF-α and IL-1ß, which were produced by tumor-associated CD11b+ cells, which included F4/80+ macrophages. TNF-α-treated endothelial cells increased NK cell migration by producing CCL2 and CCL7, which was proved by transwell and imaging assays. TNF-α-treated endothelial cells adhered well to NK cells, which was due to a TNF-α-induced increase in ICAM-1 and VCAM-1 expression on endothelial cells. Imaging data confirmed that TNF-α-treated endothelial cells transfected with ICAM-1 or VCAM-1 siRNAs did not establish stable contacts with NK cells. Taken together, our data suggest that CCL2, CCL7, ICAM-1, and VCAM-1 expressed by endothelial cells will be potential targets to guide adequate interaction with NK cells, which is a crucial step for NK cell homing to the tumor microenvironment.

LINC01094/SPI1/CCL7 Axis Promotes Macrophage Accumulation in Lung Adenocarcinoma and Tumor Cell Dissemination.

Objective: Infiltration of tumor-associated macrophages is closely linked to the malignant development of human cancers. This research studies the function of C-C motif chemokine ligand 7 (CCL7) in the macrophage accumulation in lung adenocarcinoma (LUAD) and the underpinning mechanism. Methods: The expression profile of CCL7 in LUAD and its correlations with patient's prognosis and macrophage infiltration were predicted via bioinformatics systems. Artificial up- or downregulation of CCL7 was induced in LUAD cells to explore its function in the mobility, EMT of cancer cells, and migration of M2 macrophages. Cancer cells were implanted in NOD/SCID mice to induce xenograft tumors. The CCL7-related transcription factors or factors were predicted by bioinformatic tools, and the molecular interactions were confirmed by immunoprecipitation or luciferase assays. Results: CCL7 was highly expressed in LUAD and linked to increased TAM infiltration. Knockdown of CCL7 suppressed the chemotaxis and M2 skewing of macrophages, and it blocked the EMT and mobility of LUAD cells. CCL7 downregulation also suppressed macrophage infiltration in xenograft tumors in mice. Spi-1 proto-oncogene (SPI1) was confirmed as an upstream factor activating CCL7 transcription, and LINC01094 was found to bind to SPI1 to promote its nuclear translocation. Upregulation of SPI1 restored the chemotactic migration and M2 polarization of macrophages in LUAD cells. Conclusion: This paper reveals that LINC01094 binds to SPI1 to promote its nuclear translocation, which further activates CCL7 transcription by binding to its promoter, leading to M2 macrophage accumulation and dissemination of tumor cells.

CCL7 playing a dominant role in recruiting early OCPs to facilitate osteolysis at metastatic site of colorectal cancer.

BACKGROUND: Chemoattractant is critical to recruitment of osteoclast precursors and stimulates tumor bone metastasis. However, the role of chemoattractant in bone metastasis of colorectal cancer (CRC) is still unclear. METHODS: Histochemistry analysis and TRAP staining were utilized to detect the bone resorption and activation of osteoclasts (OCs) after administration of CCL7 neutralizing antibody or CCR1 siRNA. qRT-PCR analysis and ELISA assay were performed to detect the mRNA level and protein level of chemoattractant. BrdU assay and Tunel assay were used to detect the proliferation and apoptosis of osteoclast precursors (OCPs). The migration of OCPs was detected by Transwell assay. Western blots assay was performed to examine the protein levels of pathways regulating the expression of CCL7 or CCR1. RESULTS: OCPs-derived CCL7 was significantly upregulated in bone marrow after bone metastasis of CRC. Blockage of CCL7 efficiently prevented bone resorption. Administration of CCL7 promoted the migration of OCPs. Lactate promoted the expression of CCL7 through JNK pathway. In addition, CCR1 was the most important receptor of CCL7. CONCLUSION: Our study indicates the essential role of CCL7-CCR1 signaling for recruitment of OCPs in early bone metastasis of CRC. Targeting CCL7 or CCR1 could restore the bone volume, which could be a potential therapeutical target. Video Abstract.

Bindarit Reduces Bone Loss in Ovariectomized Mice by Inhibiting CCL2 and CCL7 Expression via the NF-κB Signaling Pathway.

OBJECTIVE: To investigate the changes in proinflammatory cytokines and chemokines, namely, C-C motif ligand (CCL) 2 and CCL7, in postmenopausal osteoporosis (PMOP) and to develop a new drug, bindarit (Bnd), for PMOP in an ovariectomized (OVX) mouse model. METHODS: Bone marrow macrophages (BMMs) from the femurs of five women with PMOP and five premenopausal women without osteoporosis were detected by RNA sequencing. BMMs from mice were differentiated into osteoclasts and treated with a synthetic inhibitor of CCL2 and CCL7, Bnd, or 17 beta estradiol (E2 ). Mouse BMMs were differentiated into osteoclasts with or without Bnd for 7 days and analyzed by RNA sequencing. Osteoblasts of mice were induced to undergo osteoblastogenesis and treated with Bnd. OVX mice were treated with E2 or Bnd after surgery. The protein and mRNA expression of CCL2 and CCL7 was detected using immunostaining and qPCR, respectively, in OVX and aged mice and in cells cultured in vitro. Osteoclast formation was detected using a tartrate-resistant acid phosphatase (TRAP) assay in vitro and in vivo. Alkaline phosphatase (ALP), runt-related transcription factor 2 (Runx2) and osteocalcin (OCN) were detected using immunostaining to evaluate osteogenesis. Microcomputed tomography was conducted to analyze trabecular bone parameters, the structure model index, bone mineral density and other variables. Nuclear factor-κB (NF-κB) signaling pathway-related protein phosphorylation of IKKα/ß (p-IKKα/ß) and p-NFκB p65 was examined using western blotting. RESULTS: CCL2, CCL7 and their receptor of C-C chemokine receptor-2 (CCR2), and the NF-κB signaling pathway, were significantly increased in women with PMOP. CCL2 and CCL7 protein and mRNA expression was increased in OVX mice and aged female mice, but the increases were attenuated by E2 and Bnd. E2 and Bnd effectively inhibited osteoclastogenesis and the protein expression of CCL2 and CCL7 both in vitro and in vivo and reduced bone loss in OVX mice. Bnd did not affect the mineralization of osteoblasts directly in vitro but reduced bone turnover in vivo. p-IKKα/ß and p-NFκB p65 levels were increased in BMMs of mice after differentiation into osteoclasts but were significantly decreased by Bnd. CONCLUSION: The proinflammatory cytokines and chemokines CCL2, CCL7 and CCR2 were correlated with PMOP. Bnd attenuated the increases in CCL2 and CCL7 levels to affect osteoporosis in OVX mice via the NFκB signaling pathway. Thus, Bnd may be useful as a new therapeutic for the prevention of PMOP.

Immunosuppressive tumor microenvironment in uterine serous carcinoma via CCL7 signal with myeloid-derived suppressor cells.

Serous carcinoma of the uterus (USC) is a pathological subtype of high-grade endometrial cancers, with no effective treatment for advanced cases. Since such refractory tumors frequently harbor antitumor immune tolerance, many immunotherapies have been investigated for various malignant tumors using immuno-competent animal models mimicking their local immunities. In this study, we established an orthotopic mouse model of high-grade endometrial cancer and evaluated the local tumor immunity to explore the efficacy of immunotherapies against USC. A multivariate analysis of 62 human USC cases revealed that the tumor-infiltrating cell status, few CD8+ cells and abundant myeloid-derived suppressor cells (MDSCs), was an independent prognostic factor (P < 0.005). A murine endometrial cancer cell (mECC) was obtained from C57BL/6 mice via endometrium-specific deletion of Pten and Tp53, and another high-grade cell (HPmECC) was established by further overexpressing Myc in mECCs. HPmECCs exhibited higher capacities of migration and anchorage-independent proliferation than mECCs (P < 0.01, P < 0.0001), and when both types of cells were inoculated into the uterus of C57BL/6 mice, the prognosis of mice bearing HPmECC-derived tumors was significantly poorer (P < 0.001). Histopathological analysis of HPmECC orthotopic tumors showed serous carcinoma-like features with prominent tumor infiltration of MDSCs (P < 0.05), and anti-Gr-1 antibody treatment significantly prolonged the prognosis of HPmECC-derived tumor-bearing mice (P < 0.05). High CCL7 expression was observed in human USC and HPmECC, and MDSCs migration was promoted in a CCL7 concentration-dependent manner. These results indicate that antitumor immunity is suppressed in USC due to increased number of tumor-infiltrating MDSCs via CCL signal.

CCL7 and TGF-ß secreted by MSCs play opposite roles in regulating CRC metastasis in a KLF5/CXCL5-dependent manner.

CXCL5 is overexpressed in colorectal cancer (CRC) and promotes distant metastasis and angiogenesis of tumors; however, the underlying mechanism that mediates CXCL5 overexpression in CRC remains unclear. Here, we successfully extracted and identified primary mesenchymal stromal cells (MSCs) and verified the promoting effects of tumor-associated MSCs on CRC proliferation and metastasis in vivo and in vitro. We found that MSCs not only promoted the expression of CXCL5 by secreting CCL7 but also secreted TGF-ß to inhibit this process. After secretion, CCL7/CCR1 activated downstream CBP/P300 to acetylate KLF5 to promote CXCL5 transcription, while TGF-ß reversed the effect of KLF5 on transcription activation by regulating SMAD4. Taken together, our results indicate that MSCs in the tumor microenvironment promoted the progression and metastasis of CRC and regulated the expression of CXCL5 in CRC cells by secreting CCL7 and TGF-ß. KLF5 is the key site of these processes and plays a dual role in CXCL5 regulation. MSCs and their secreted factors may serve as potential therapeutic targets in the tumor environment.

Oxidative stress induces inflammation of lens cells and triggers immune surveillance of ocular tissues.

Recent reports have challenged the notion that the lens is immune-privileged. However, these studies have not fully identified the molecular mechanism(s) that promote immune surveillance of the lens. Using a mouse model of targeted glutathione (GSH) deficiency in ocular surface tissues, we have investigated the role of oxidative stress in upregulating cytokine expression and promoting immune surveillance of the eye. RNA-sequencing of lenses from postnatal day (P) 1-aged Gclcf/f;Le-CreTg/- (KO) and Gclcf/f;Le-Cre-/- control (CON) mice revealed upregulation of many cytokines (e.g., CCL4, GDF15, CSF1) and immune response genes in the lenses of KO mice. The eyes of KO mice had a greater number of cells in the aqueous and vitreous humors at P1, P20 and P50 than age-matched CON and Gclcw/w;Le-CreTg/- (CRE) mice. Histological analyses revealed the presence of innate immune cells (i.e., macrophages, leukocytes) in ocular structures of the KO mice. At P20, the expression of cytokines and ROS content was higher in the lenses of KO mice than in those from age-matched CRE and CON mice, suggesting that oxidative stress may induce cytokine expression. In vitro administration of the oxidant, hydrogen peroxide, and the depletion of GSH (using buthionine sulfoximine (BSO)) in 21EM15 lens epithelial cells induced cytokine expression, an effect that was prevented by co-treatment of the cells with N-acetyl-l-cysteine (NAC), a antioxidant. The in vivo and ex vivo induction of cytokine expression by oxidative stress was associated with the expression of markers of epithelial-to-mesenchymal transition (EMT), α-SMA, in lens cells. Given that EMT of lens epithelial cells causes posterior capsule opacification (PCO), we propose that oxidative stress induces cytokine expression, EMT and the development of PCO in a positive feedback loop. Collectively these data indicate that oxidative stress induces inflammation of lens cells which promotes immune surveillance of ocular structures.

Glioma-derived CCL2 and CCL7 mediate migration of immune suppressive CCR2+/CX3CR1+ M-MDSCs into the tumor microenvironment in a redundant manner.

Glioblastoma (GBM) is the most common and malignant primary brain tumor, resulting in poor survival despite aggressive therapies. GBM is characterized in part by a highly heterogeneous and immunosuppressive tumor microenvironment (TME) made up predominantly of infiltrating peripheral immune cells. One significant immune cell type that contributes to glioma immune evasion is a population of immunosuppressive, hematopoietic cells, termed myeloid-derived suppressor cells (MDSCs). Previous studies suggest that a potent subset of myeloid cells, expressing monocytic (M)-MDSC markers, distinguished by dual expression of chemokine receptors CCR2 and CX3CR1, utilize CCR2 to infiltrate into the TME. This study evaluated the T cell suppressive function and migratory properties of CCR2+/CX3CR1+ MDSCs. Bone marrow-derived CCR2+/CX3CR1+ cells adopt an immune suppressive cell phenotype when cultured with glioma-derived factors. Recombinant and glioma-derived CCL2 and CCL7 induce the migration of CCR2+/CX3CR1+ MDSCs with similar efficacy. KR158B-CCL2 and -CCL7 knockdown murine gliomas contain equivalent percentages of CCR2+/CX3CR1+ MDSCs compared to KR158B gliomas. Combined neutralization of CCL2 and CCL7 completely blocks CCR2-expressing cell migration to KR158B cell conditioned media. CCR2+/CX3CR1+ cells are also reduced within KR158B gliomas upon combination targeting of CCL2 and CCL7. High levels of CCL2 and CCL7 are also associated with negative prognostic outcomes in GBM patients. These data provide a more comprehensive understanding of the function of CCR2+/CX3CR1+ MDSCs and the role of CCL2 and CCL7 in the recruitment of these immune suppressive cells and further support the significance of targeting this chemokine axis in GBM.

Distal Lung Microenvironment Triggers Release of Mediators Recognized as Potential Systemic Biomarkers for Idiopathic Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic lung disease with an unmet need of biomarkers that can aid in the diagnostic and prognostic assessment of the disease and response to treatment. In this two-part explorative proteomic study, we demonstrate how proteins associated with tissue remodeling, inflammation and chemotaxis such as MMP7, CXCL13 and CCL19 are released in response to aberrant extracellular matrix (ECM) in IPF lung. We used a novel ex vivo model where decellularized lung tissue from IPF patients and healthy donors were repopulated with healthy fibroblasts to monitor locally released mediators. Results were validated in longitudinally collected serum samples from 38 IPF patients and from 77 healthy controls. We demonstrate how proteins elevated in the ex vivo model (e.g., MMP7), and other serum proteins found elevated in IPF patients such as HGF, VEGFA, MCP-3, IL-6 and TNFRSF12A, are associated with disease severity and progression and their response to antifibrotic treatment. Our study supports the model's applicability in studying mechanisms involved in IPF and provides additional evidence for both established and potentially new biomarkers in IPF.

Sex-dependent acrolein sensitivity in mice is associated with differential lung cell, protein, and transcript changes.

Acrolein is a reactive inhalation hazard. Acrolein's initial interaction, which in itself can be function-altering, is followed by time-dependent cascade of complex cellular and pulmonary responses that dictate the severity of the injury. To investigate the pathophysiological progression of sex-dependent acrolein-induced acute lung injury, C57BL/6J mice were exposed for 30 min to sublethal, but toxic, and lethal acrolein. Male mice were more sensitive than female mice. Acrolein of 50 ppm was sublethal to female but lethal to male mice, and 75 ppm was lethal to female mice. Lethal and sublethal acrolein exposure decreased bronchoalveolar lavage (BAL) total cell number at 3 h after exposure. The cell number decrease was followed by progressive total cell and neutrophil number and protein increases. The BAL total cell number in female mice exposed to a sublethal, but not lethal dose, returned to control levels at 16 h. In contrast, BAL protein content and neutrophil number were higher in mice exposed to lethal compared to sublethal acrolein. RNASeq pathway analysis identified greater increased lung neutrophil, glutathione metabolism, oxidative stress responses, and CCL7 (aka MCP-3), CXCL10 (aka IP-10), and IL6 transcripts in males than females, whereas IL10 increased more in female than male mice. Thus, the IL6:IL10 ratio, an indicator of disease severity, was greater in males than females. Further, H3.3 histone B (H3F3B) and pro-platelet basic protein (PPBP aka CXCL7), transcripts increased in acrolein exposed mouse BAL and plasma at 3 h, while H3F3B protein that is associated with neutrophil extracellular traps formation increased at 12 h. These results suggest that H3F3B and PPBP transcripts increase may contribute to extracellular H3F3B and PPBP proteins increase.

Upregulation of CCL7, CCL20, CXCL2, IL-1ß, IL-6 and MMP-9 in Skin Samples of PCB Exposed Individuals-A Preliminary Study.

Polychlorinated biphenyls (PCBs) are well known immunotoxic and carcinogenic compounds. Although cutaneous symptoms are the hallmark of exposure to these compounds, exact pathophysiologic mechanisms are not well understood. We took skin biopsies from moderately high PCB exposed workers (n = 25) after an informed consent and investigated the expression of immunological markers such as CCL-7, CCL-20, CXCL2, IL-1ß and IL-6, as well as the matrix metalloproteinase MMP-9, EPGN and NRF2 by RT-qPCR, and compared expression levels with plasma PCB levels. Statistical analyses showed a significant correlation between CCL-20, CXCL2, IL-6, IL-1ß, CCL-7 and MMP-9 and PCB serum levels. EPGN and NRF2 were not correlated to PCB levels in the blood. We found a significant correlation of genes involved in autoimmune, auto-inflammatory and carcinogenesis in skin samples of PCB exposed individuals with elevated plasma PCB levels. Confirmation of these findings needs to be performed in bigger study groups and larger gen-sets, including multiple housekeeping genes. Further study needs to be performed to see whether a chronical exposure to these and similar compounds can cause higher incidence of malignancies and inflammatory disease.

Redox-sensitive activation of CCL7 by BRG1 in hepatocytes during liver injury.

Liver injuries induced by various stimuli share in common an acute inflammatory response, in which circulating macrophages home to the liver parenchyma to participate in the regulation of repair, regeneration, and fibrosis. In the present study we investigated the role of hepatocyte-derived C-C motif ligand 7 (CCL7) in macrophage migration during liver injury focusing on its transcriptional regulation. We report that CCL7 expression was up-regulated in the liver by lipopolysaccharide (LPS) injection (acute liver injury) or methionine-and-choline-deficient (MCD) diet feeding (chronic liver injury) paralleling increased macrophage infiltration. CCL7 expression was also inducible in hepatocytes, but not in hepatic stellate cells or in Kupffer cells, by LPS treatment or exposure to palmitate in vitro. Hepatocyte-specific deletion of Brahma-related gene 1 (BRG1), a chromatin remodeling protein, resulted in a concomitant loss of CCL7 induction and macrophage infiltration in the murine livers. Of interest, BRG1-induced CCL7 transcription and macrophage migration was completely blocked by the antioxidant N-acetylcystine. Further analyses revealed that BRG1 interacted with activator protein 1 (AP-1) to regulate CCL7 transcription in hepatocytes in a redox-sensitive manner mediated in part by casein kinase 2 (CK2)-catalyzed phosphorylation of BRG1. Importantly, a positive correlation between BRG1/CCL7 expression and macrophage infiltration was identified in human liver biopsy specimens. In conclusion, our data unveil a novel role for BRG1 as a redox-sensitive activator of CCL7 transcription.