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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-1β/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.

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., (© 2024 The Author(s). The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

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.

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Takacs, Kreiger, Luo, Tian, Garcia, Deleyrolle, Mitchell and Harrison.)

Background: Chemokine-mediated neuroinflammation plays an important role in the pathogenesis of neuropathic pain. The chemokine CC motif ligand 7 (CCL7) and its receptor CCR2 have been reported to contribute to neuropathic pain via astrocyte-microglial interaction in the spinal cord. Whether CCL7 in the trigeminal ganglion (TG) involves in trigeminal neuropathic pain and the involved mechanism remain largely unknown., Methods: The partial infraorbital nerve transection (pIONT) was used to induce trigeminal neuropathic pain in mice. The expression of Ccl7 , Ccr1 , Ccr2 , and Ccr3 was examined by real-time quantitative polymerase chain reaction. The distribution of CCL7, CCR2, and CCR3 was detected by immunofluorescence double-staining. The activation of extracellular signal-regulated kinase (ERK) was examined by Western blot and immunofluorescence. The effect of CCL7 on neuronal excitability was tested by whole-cell patch clamp recording. The effect of selective antagonists for CCR1, CCR2, and CCR3 on pain hypersensitivity was checked by behavioral testing., Results: Ccl7 was persistently increased in neurons of TG after pIONT, and specific inhibition of CCL7 in the TG effectively relieved pIONT-induced orofacial mechanical allodynia. Intra-TG injection of recombinant CCL7 induced mechanical allodynia and increased the phosphorylation of ERK in the TG. Incubation of CCL7 with TG neurons also dose-dependently enhanced the neuronal excitability. Furthermore, pIONT increased the expression of CCL7 receptors Ccr1 , Ccr2 , and Ccr3 . The intra-TG injection of the specific antagonist of CCR2 or CCR3 but not of CCR1 alleviated pIONT-induced orofacial mechanical allodynia and reduced ERK activation. Immunostaining showed that CCR2 and CCR3 are expressed in TG neurons, and CCL7-induced hyperexcitability of TG neurons was decreased by antagonists of CCR2 or CCR3., Conclusion: CCL7 activates ERK in TG neurons via CCR2 and CCR3 to enhance neuronal excitability, which contributes to the maintenance of trigeminal neuropathic pain. CCL7-CCR2/CCR3-ERK pathway may be potential targets for treating trigeminal neuropathic pain.

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-1β, 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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

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.

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., (© 2022. The Author(s).)

Chemokines are key components in the pathology of chronic diseases. Chemokine CC motif ligand 7 (CCL7) is believed to be associated with cardiovascular disease, diabetes mellitus, and kidney disease. CCL7 may play a role in inflammatory events by attracting macrophages and monocytes to further amplify inflammatory processes and contribute to disease progression. However, CCL7-specific pathological signaling pathways need to be further confirmed in these chronic diseases. Given the multiple redundancy system among chemokines and their receptors, further experimental and clinical studies are needed to clarify whether direct CCL7 inhibition mechanisms could be a promising therapeutic approach to attenuating the development of cardiovascular disease, diabetes mellitus, and kidney disease., (© 2022. The Author(s).)

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., Competing Interests: The authors report no conflict of interest., (Copyright © 2022 Zhuo Wu et al.)

Microglial activation in the spinal cord contributes to the development of inflammatory pain. Monocyte chemotactic protein 3 (MCP3) can induce microglial activation, resulting in increased pain sensitivity; however, the underlying mechanism remains poorly understood. 3,5-dicaffeoylquinic acid (3,5-DCQA) has shown protective effects against inflammation-related diseases, but the effect of 3,5-DCQA on microglial activation and inflammatory pain is not evaluated. This study aimed to investigate the effects of 3,5-DCQA on microglial activation-induced inflammatory pain. Furthermore, the underlying mechanism inhibited by 3,5-DCQA via MCP3 suppression was studied. To induce microglial activation, LPS was treated in BV2 microglial cells. The LPS-induced microglial activation and pro-inflammatory cytokines production were significantly reduced by 3,5-DCQA treatment in BV2 cells. Moreover, 3,5-DCQA suppressed LPS-induced MCP3 expression, resulting in reduced phosphorylation of JAK2/STAT3. Interestingly, the suppressed JAK2/STAT3 signaling enhanced autophagy induction in BV2 cells. The increased autophagy by 3,5-DCQA and knockout of MCP3 inhibited LPS-induced inflammatory response in BV2 cells. To establish the inflammatory pain, CFA was injected into the right paw of mice. The CFA-induced pain hypersensitivity and foot swelling were attenuated by the oral administration of 3,5-DCQA. Moreover, CFA-induced microglial activation was reduced and the autophagy markers were recovered in the spinal cord of 3,5-DCQA-administered mice. Similar results were observed in cultured primary microglia. Our findings indicate that 3,5-DCQA attenuates inflammation-mediated pain hypersensitivity by enhancing autophagy through inhibition of MCP3-induced JAK2/STAT3 signaling. Therefore, 3,5-DCQA could be a potential therapeutic agent for alleviating inflammatory pain., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

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., (© 2022. The Author(s).)

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 Gclc f/f ;Le-Cre Tg/- (KO) and Gclc f/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 Gclc w/w ;Le-Cre Tg/- (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., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Cellular senescence is closely related to tissue aging including bone. Bone homeostasis is maintained by the tight balance between bone-forming osteoblasts and bone-resorbing osteoclasts, but it undergoes deregulation with age, causing age-associated osteoporosis, a main cause of which is osteoblast dysfunction. Oxidative stress caused by the accumulation of reactive oxygen species (ROS) in bone tissues with aging can accelerate osteoblast senescence and dysfunction. However, the regulatory mechanism that controls the ROS-induced senescence of osteoblasts is poorly understood. Here, we identified Peptidyl arginine deiminase 2 (PADI2), a post-translational modifying enzyme, as a regulator of ROS-accelerated senescence of osteoblasts via RNA-sequencing and further functional validations. PADI2 downregulation by treatment with H 2 O 2 or its siRNA promoted cellular senescence and suppressed osteoblast differentiation. CCL2, 5, and 7 known as the elements of the senescence-associated secretory phenotype (SASP) which is a secretome including proinflammatory cytokines and chemokines emitted by senescent cells and a representative feature of senescence, were upregulated by H 2 O 2 treatment or Padi2 knockdown. Furthermore, blocking these SASP factors with neutralizing antibodies or siRNAs alleviated the senescence and dysfunction of osteoblasts induced by H 2 O 2 treatment or Padi2 knockdown. The elevated production of these SASP factors was mediated by the activation of NFκB signaling pathway. The inhibition of NFκB using the pharmacological inhibitor or siRNA effectively relieved H 2 O 2 treatment- or Padi2 knockdown-induced senescence and osteoblast dysfunction. Together, our study for the first time uncover the role of PADI2 in ROS-accelerated cellular senescence of osteoblasts and provide new mechanistic and therapeutic insights into excessive ROS-promoted cellular senescence and aging-related bone diseases., (© 2022. The Author(s).)

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.

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., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Microglia are immune cells of the central nervous system that mediate neuroinflammation. It is widely known that microglia-mediated inflammation in the brain contribute to the widespread tissue damage and neurological deficits in traumatic brain injury (TBI). However, the mechanisms responsible for this inflammatory response remain elusive. Here, we investigated the role of astrocyte-derived chemokine (C-C motif) ligand 7 (CCL7) in microglial-controlled inflammation following TBI. Our results demonstrated that astrocyte-derived CCL7 induced microglial activation and the release of proinflammatory mediators in the cortex and serum of rats that underwent experimental TBI. Furthermore, CCL7 knockout improved microglia-controlled inflammation, brain morphology and neurological dysfunction following TBI. In vitro, CCL7-siRNA attenuated the LPS-induced expression of pro-inflammatory markers in the co-culture of microglia and astrocytes. Collectively, our findings uncover an important role for astrocyte-derived CCL7 in promoting microglia-mediated inflammation after TBI and suggests CCL7 could serve as a potential therapeutic strategy for attenuating TBI by inhibiting microglial activation., (Copyright © 2021 Elsevier B.V. All rights reserved.)

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., (© 2021 The Authors. Physiological Reports published by Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society.)

Chemokine C-C motif ligand 7 (CCL7), a member of CC chemokine subfamily, plays pivotal roles in numerous inflammatory diseases. Hyper-activation of inflammation is an important characteristic of abdominal aortic aneurysm (AAA). Therefore, in the present study, we aimed to determine the effect of CCL7 on AAA formation. CCL7 abundance in aortic tissue and macrophage infiltration were both increased in angiotensin II (Ang II)-induced AAA mice. Ex vivo, CCL7 promoted macrophage polarization towards M1 phenotype. This effect was reversed by the blockage of CCR1, a receptor of CCL7. CCL7 up-regulated JAK2/STAT1 protein level in macrophage, and CCL7-induced M1 activation was suppressed by JAK2/STAT1 pathway inhibition. To verify the effect of CCL7 on AAA in vivo, either CCL7-neutralizing antibody (CCL7-nAb) or vehicles were intraperitoneally injected 24 hours prior to Ang II infusion and subsequently every three days for 4 weeks. CCL7-nAb administration significantly attenuated Ang II-induced luminal and external dilation as well as pathological remodelling. Immunostaining showed that CCL7-nAb administration significantly decreased aneurysmal macrophage infiltration. In conclusion, CCL7 contributed to Ang II-induced AAA by promoting M1 phenotype of macrophage through CCR1/JAK2/STAT1 signalling pathway., (© 2021 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Background: Food proteins differ in their allergenic potential. Currently, there is no predictive and validated bio-assay to evaluate the allergenicity of novel food proteins. The objective of this study was to investigate the potential of a human peripheral blood mononuclear cell (PBMC) gene expression assay to identify biomarkers to predict the allergenicity of legume proteins., Results: PBMCs from healthy donors were exposed to weakly and strongly allergenic legume proteins (2S albumins, and 7S and 11S globulins from white bean, soybean, peanut, pea and lupine) in three experiments. Possible biomarkers for allergenicity were investigated by exposing PBMCs to a protein pair of weakly (white bean) and strongly allergenic (soybean) 7S globulins in a pilot experiment. Gene expression was measured by RNA-sequencing and differentially expressed genes were selected as biomarkers. 153 genes were identified as having significantly different expression levels to the 7S globulin of white bean compared to soybean. Inclusion of multiple protein pairs from 2S albumins (lupine and peanut) and 7S globulins (white bean and soybean) in a larger study, led to the selection of CCL2, CCL7, and RASD2 as biomarkers to distinguish weakly from strongly allergenic proteins. The relevance of these three biomarkers was confirmed by qPCR when PBMCs were exposed to a larger panel of weakly and strongly allergenic legume proteins (2S albumins, and 7S and 11S globulins from white bean, soybean, peanut, pea and lupine)., Conclusions: The PBMC gene expression assay can potentially distinguish weakly from strongly allergenic legume proteins within a protein family, though it will be challenging to develop a generic method for all protein families from plant and animal sources. Graded responses within a protein family might be of more value in allergenicity prediction instead of a yes or no classification.

Bone metastasis remains the most frequent and the deadliest complication of prostate cancer (PCa). Mechanisms leading to the homing of tumor cells to bone remain poorly characterized. Role of chemokines in providing navigational cues to migrating cancer cells bearing specific receptors is well established. Bone is an adipocyte-rich organ since 50 to 70% of the adult bone marrow (BM) volume comprise bone marrow adipocytes (BM-Ads), which are likely to produce chemokines within the bone microenvironment. Using in vitro migration assays, we demonstrated that soluble factors released by human primary BM-Ads are able to support the directed migration of PCa cells in a CCR3-dependent manner. In addition, we showed that CCL7, a chemokine previously involved in the CCR3-dependent migration of PCa cells outside of the prostate gland, is released by human BM-Ads. These effects are amplified by obesity and ageing, two clinical conditions known to promote aggressive and metastatic PCa. In human tumors, we found an enrichment of CCR3 in bone metastasis vs. primary tumors at mRNA levels using Oncomine microarray database. In addition, immunohistochemistry experiments demonstrated overexpression of CCR3 in bone versus visceral metastases. These results underline the potential importance of BM-Ads in the bone metastatic process and imply a CCR3/CCL7 axis whose pharmacological interest needs to be evaluated.

The balance between the responsiveness of the intestinal immune system and the gut environment is fundamental for the maintenance of intestinal homeostasis, which is required for an adequate recognition of entering antigens. The disruption of this homeostasis by exaggerated immune response to harmless antigens can lead to the development of intestinal disorders such as inflammatory bowel disease. Stromal cells are sessile non-hematopoietic cells that build the backbone of the lymph node, an important site for the immune response induction, but also contribute to immune response and tolerance induction. However, the knowledge about the role of stromal cells in the regulation of inflammatory responses is still limited. Therefore, in this study we analyzed the influence of stromal cells on the development of chronic intestinal inflammation. Here, we show that intestinal inflammation alters the immune activation of the mesenteric lymph node-derived stromal cells. Podoplanin + and CD21/35 + stromal cells showed increased expression of MHC class II molecules, but CD106 expression on CD21/35 + cells was reduced. Stromal cells secreted cytokines and chemokines such as CCL7 and CXCL16 influenced the gut-homing phenotype and proliferation of CD4 + and CD8 + T cells. Furthermore, stromal cells of peripheral lymph nodes transplanted into the mesentery attenuated colitis severity in B6- Il10 -/- mice. The reduced colitis severity in these mice was associated with increased expression of IL4 and distinct activation pattern of stromal cells derived from transplanted peripheral lymph nodes. Altogether, our results demonstrate that lymph node stromal cells impact development of chronic colitis via T cell induction. Moreover, lymph node stromal cells from different draining area due to neonatally imprinted processes distinctly regulate the induction of immune responses., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Basic, Peppermüller, Bolsega, Bleich, Bornemann, Bode and Buettner.)

Streptococcus mutans is a gram-positive bacterium that causes tooth decay. The exopolyssacharides, mostly glucans synthesized by the bacterium are responsible for establishing pathogenic bio-films associated with dental caries disease. The regulatory immune and inflammatory reactions implicated by the synthesized glucans are still not clearly understood. In this study, a water-soluble exopolyssacharide (WSP) was extracted from culture of Str. mutans. The structural properties of WSP, [α-(1 → 3, 1 → 6)-D-glucan] were confirmed using Fourier-transform infrared spectroscopy and 13 C-nuclear magnetic resonance spectroscopy. Furthermore, the effects of WSP on the global gene expression of the macrophage-like RAW 264.7 cells were analyzed using mRNA-seq analysis. Using Gene Ontology analysis, we compiled a total of 24,421 genes that were upregulated or downregulated by more than 5.0-fold and 0.3-fold, respectively. Most of the transcripts were grouped under immune response and inflammation-related gene categories. Among the 802 immunity-related genes analyzed, chemokine ligand 7 (Ccl7), interleukin-1β (IL-1β), interleukin-1α (IL-1α) and interleukin-6 (IL-6) were upregulated after WSP exposure. In addition, among a total of 344 genes related to inflammation, Ccl7, IL-1α and IL-6 were upregulated. These results suggest that [α-(1 → 3, 1 → 6)-D-glucan] from Str. mutans produces activates macrophages and may contribute to the immune and inflammatory response to periodontal disease., Competing Interests: Declaration of competing interest The authors declare no conflict of interests., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Traumatic brain injury (TBI) is the leading cause of death in the global population. Disturbed inflammatory processes after TBI exacerbate secondary brain injury and contribute to unfavorable outcomes. Multiple inflammatory events that accompany brain trauma, such as glial activation, chemokine release, or the initiation of the complement system cascade, have been identified as potential targets for TBI treatment. However, the participation of chemokines in the complement activation remains unknown. Our studies sought to determine the changes in the expression of the molecules involved in the CCL2/CCL7/CCL12/CCR2 pathway in the injured brain and the effect of CCL2, CCL7, and CCL12 (10, 100, and 500 ng/mL) on the classic and lectin complement pathways and inflammatory factors in microglial cell cultures. Brain injury in mice was modeled by controlled cortical impact (CCI). Our findings indicate a time-dependent upregulation of CCL2, CCL7, and CCL12 at the mRNA and protein levels within the cortex, striatum, and/or thalamus beginning 24 h after the trauma. The analysis of the expression of the receptor of the tested chemokines, CCR2, revealed its substantial upregulation within the injured brain areas mainly on the mRNA level. Using primary cortical microglial cell cultures, we observed a substantial increase in the expression of CCL2, CCL7, and CCL12 after 24 h of LPS (100 ng/mL) treatment. CCL2 stimulation of microglia increased the level of IL-1β mRNA but did not influence the expression of IL-18, IL-6, and IL-10. Moreover, CCL2 significantly increased the expression of Iba1, a marker of microglia activation. CCL2 and CCL12 upregulated the expression of C1qa but did not influence the expression of C1ra and C1s1 (classical pathway); moreover, CCL2 increased ficolin A expression and reduced collectin 11 expression (lectin pathway). Additionally, we observed the downregulation of pentraxin 3, a modulator of the complement cascade, after CCL2 and CCL12 treatment. We did not detect the expression of ficolin B, Mbl1, and Mbl2 in microglial cells. Our data identify CCL2 as a modulator of the classical and lectin complement pathways suggesting that CCL2 may be a promising target for pharmacological intervention after brain injury. Moreover, our study provides evidence that CCL2 and two other CCR2 ligands may play a role in the development of changes in TBI., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The efficacy of checkpoint immunotherapy to non-small cell lung cancer (NSCLC) largely depends on the tumor microenvironment (TME). Here, we demonstrate that CCL7 facilitates anti-PD-1 therapy for the Kras LSL-G12D/+ Tp53 fl/fl (KP) and the Kras LSL-G12D/+ Lkb1 fl/fl (KL) NSCLC mouse models by recruiting conventional DC 1 (cDC1) into the TME to promote T cell expansion. CCL7 exhibits high expression in NSCLC tumor tissues and is positively correlated with the infiltration of cDC1 in the TME and the overall survival of NSCLC patients. CCL7 deficiency impairs the infiltration of cDC1 in the TME and the subsequent expansion of CD8 + and CD4 + T cells in bronchial draining lymph nodes and TME, thereby promoting tumor development in the KP mouse model. Administration of CCL7 into lungs alone or in combination with anti-PD-1 significantly inhibits tumor development and prolongs the survival of KP and KL mice. These findings suggest that CCL7 potentially serves as a biomarker and adjuvant for checkpoint immunotherapy of NSCLC.

Muscle regeneration depends on a robust albeit transient inflammatory response. Persistent inflammation is a feature of age-related regenerative deficits, yet the underlying mechanisms are poorly understood. Here, we find inflammatory-related CC-chemokine-receptor 2 (Ccr2) expression in non-hematopoietic myogenic progenitors (MPs) during regeneration. After injury, the expression of Ccr2 in MPs corresponds to the levels of its ligands, the chemokines Ccl2, 7, and 8. We find stimulation of Ccr2-activity inhibits MP fusion and contribution to myofibers. This occurs in association with increases in MAPKp38δ/γ signaling, MyoD phosphorylation, and repression of the terminal myogenic commitment factor Myogenin. High levels of Ccr2-chemokines are a feature of regenerating aged muscle. Correspondingly, deletion of Ccr2 in MPs is necessary for proper fusion into regenerating aged muscle. Finally, opportune Ccr2 inhibition after injury enhances aged regeneration and functional recovery. These results demonstrate that inflammatory-induced activation of Ccr2 signaling in myogenic cells contributes to aged muscle regenerative decline.

The therapeutic strategies for advanced gastric cancer (GC) remain unsatisfying and limited. Therefore, it is still imperative to fully elucidate the mechanisms underlying GC metastasis. Here, we report a novel role of SRY-box transcription factor 18 (SOX18), a member of the SOX family, in promoting GC metastasis. The elevated expression of SOX18 was positively correlated with distant metastasis, higher AJCC stage, and poor prognosis in human GC. SOX18 expression was an independent and significant risk factor for the recurrence and survival in GC patients. Up-regulation of SOX18 promoted GC invasion and metastasis, whereas down-regulation of SOX18 decreased GC invasion and metastasis. Melanoma cell adhesion molecule (MCAM) and C-C motif chemokine ligand 7 (CCL7) are direct transcriptional targets of SOX18. Knockdown of MCAM and CCL7 significantly decreased SOX18-mediated GC invasion and metastasis, while the stable overexpression of MCAM and CCL7 reversed the decrease in cell invasion and metastasis that was induced by the inhibition of SOX18. A mechanistic investigation indicated that the upregulation of SOX18 that was mediated by the CCL7-CCR1 pathway relied on the ERK/ELK1 pathway. SOX18 knockdown significantly reduced CCL7-enhanced GC invasion and metastasis. Furthermore, BX471, a specific CCR1 inhibitor, significantly reduced the SOX18-mediated GC invasion and metastasis. In human GC tissues, SOX18 expression was positively correlated with CCL7 and MCAM expression, and patients with positive coexpression of SOX18/CCL7 or SOX18/MCAM had the worst prognosis. In conclusion, we defined a CCL7-CCR1-SOX18 positive feedback loop that played a pivotal role in GC metastasis, and targeting this pathway may be a promising therapeutic option for the clinical management of GC.

Liver metastases remain a major cause of death from gastrointestinal tract cancers and other malignancies, such as breast and lung carcinomas. Understanding the underlying biology is essential for the design of effective therapies. We previously identified the chemokine CCL7 and its receptor CCR3 as critical mediators of invasion and metastasis in lung and colon carcinoma cells. Here we show that the CCL7/CCR3 axis regulates a late stage in invadopodia genesis namely, the targeting of MMP-9 to the invadopodia complex, thereby promoting invadopodia maturation and collagen degradation. We show that this process could be blocked by overexpression of a dominant negative RhoA in highly invasive cells, while a constitutively active RhoA upregulated invadopodia maturation in CCL7-silenced and poorly invasive and metastatic cells and also enhanced their metastatic potential in vivo, collectively, implicating RhoA activation in signaling downstream of CCL7. Blockade of the ERK or PI3K pathways by chemical inhibitors also inhibited invadopodia formation, but affected the initiation stage of invadopodia genesis. Our data implicate CCL7/CCR3 signaling in invadopodia maturation and suggest that chemokine signaling acts in concert with extracellular matrix-initiated signals to promote invasion and liver metastasis., Competing Interests: Declaration of competing interest The authors of this manuscript have no conflict of interest to declare., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Located within red pulp cords, splenic red pulp macrophages (RPMs) are constantly exposed to the blood flow, clearing senescent red blood cells (RBCs) and recycling iron from hemoglobin. Here, we studied the mechanisms underlying RPM homeostasis, focusing on the involvement of stromal cells as these cells perform anchoring and nurturing macrophage niche functions in lymph nodes and liver. Microscopy revealed that RPMs are embedded in a reticular meshwork of red pulp fibroblasts characterized by the expression of the transcription factor Wilms' Tumor 1 (WT1) and colony stimulating factor 1 (CSF1). Conditional deletion of Csf1 in WT1 + red pulp fibroblasts, but not white pulp fibroblasts, drastically altered the RPM network without altering circulating CSF1 levels. Upon RPM depletion, red pulp fibroblasts transiently produced the monocyte chemoattractants CCL2 and CCL7, thereby contributing to the replenishment of the RPM network. Thus, red pulp fibroblasts anchor and nurture RPM, a function likely conserved in humans., Competing Interests: Declaration of Interests The authors declare no competing interests, (Copyright © 2020 Elsevier Inc. All rights reserved.)

Type 1 reactions (T1R) an inflammatory condition, of local skin patches in 30-40% leprosy patients during the course of MDT. IL-17A and IL-17F play an important role in regulating skin inflammation through neutrophils. In the present study, we have analyzed 18 of each T1R and Non-reactions (NR) patients through flow cytometry and qPCR. Interestingly we found that, CD3 + CD4 + gated IL-17A + IL-17F + cells were significantly high in T1R in both MLSA stimulated PBMCs and skin lesions as compared to NR leprosy patients. Hierarchical clustering analysis of gene expression showed that CXCL6, CXCL5, CCL20, CCL7, MMP13 and IL-17RB expression were significantly associated with IL-17A and IL-17F expression (Spearman r 2  = 0.77 to 0.98), neutrophils and monocyte markers respectively. In this study, the inflammation noted in lesions of T1R is a different phenotype of Th17 which produce double positive IL-17A + IL17F + and also contributes IL-17 producing neutrophils and thus would be useful for monitoring, diagnosis and treatment response before reactions episodes., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Microglia, the tissue-resident macrophages in the central nervous system, are important for the initiation and perpetuation of neuroinflammation. Peroxisome proliferator-activated receptor gamma (PPARγ) is a ligand-inducible transcription factor and plays an important role in fatty acid metabolism. Our previous study found that 1-O-alkyl glycerophosphate (AGP), a naturally occurring ether analog of lysophosphatidic acid, is a high-affinity, partial agonist of PPARγ. In this study, we investigated the role of AGP in microglial activation and illustrated the underlying molecular mechanism. We found that AGP treatment increased the production of intracellular reactive oxygen species and induced PPARγ activation in microglial cells. Interestingly, AGP also up-regulated the expression levels of the cluster of differentiation 36 (CD36) scavenger receptor, a high-affinity receptor for oxidized low-density lipoproteins. The findings suggest that AGP induces PPARγ activation, enhances CD36 expression and increases the production of intracellular reactive oxygen species (ROS) in microglial cells., (Copyright © 2019. Published by Elsevier Inc.)

Acetaminophen (APAP) overdose-induced hepatotoxicity is the leading cause of drug-induced liver injury worldwide. The related injury pathogenesis is mainly focused on the liver. Here, the authors report that gut barrier disruption may also be involved in APAP hepatotoxicity. APAP administration led to gut leakiness and colonic epithelial chemokine (C-C motif) ligand 7 (CCL7) up-regulation. Intestinal epithelial cell (IEC)-specific CCL7 transgenic mice (CCL7 tgIEC mice) showed markedly increased myosin light chain kinase phosphorylation, and elevated gut permeability and bacterial translocation into the liver compared to wild-type mice. Global transcriptome analysis revealed that the expression of hepatic proinflammatory genes was enhanced in CCL7 tgIEC mice compared with wild-type animals. Moreover, CCL7 overexpression in intestinal epithelial cells significantly augmented APAP-induced acute liver injury. These data provide new evidence that dysfunction of CCL7-mediated gut barrier integrity may be an important contributor to APAP-induced hepatotoxicity., (Copyright © 2020 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

The aim of the study was to clarify the distinctive features of stem cells for effective cell-based therapy strategies in regenerative medicine. The expression levels of cytokines secreted from stem cells from exfoliated deciduous teeth (SHED), dental pulp stem cells (DPSCs), and bone marrow derived mesenchymal stem cells (BMMSCs) were examined to identify the details of their characteristics. A total of 174 cytokines were analyzed using cytokine antibody array, and their expression levels were confirmed by an enzyme-linked immunosorbent assay. These results indicated that 11 cytokines that were related to tissue regeneration, including growth factors, chemokines, and inflammatory cytokines, were identical in SHED, DPSCs, and BMMSCs. The comparative analyses between SHED and BMMSCs revealed that hepatocyte growth factor (HGF), matrix metalloproteinase-3, and stromal cell derived factor 1 (SDF-1) were expressed 6.7-, 2.5-, and 2.1-fold higher, respectively, in SHEDs. HGF was also expressed 3.4-fold higher in DPSCs than BMMSCs. Monocyte chemoattractant protein-1, and-3 were expressed more strongly in BMMSCs. SHED contained significantly higher SDF-1 levels than DPSCs. The distinct cytokine secretion indicated that they had different character besides basic MSC features. This knowledge of diagnostic cytokines analysis secreted from SHED, DPSCs, and BMMSCs extends our understanding, and can provide a novel therapeutic paradigm shift for functional cell-based therapy.

Atherosclerotic plaque development depends on chronic inflammation of the arterial wall. A dysbiotic gut microbiota can cause low-grade inflammation, and microbiota composition was linked to cardiovascular disease risk. However, the role of this environmental factor in atherothrombosis remains undefined. To analyze the impact of gut microbiota on atherothrombosis, we rederived low-density lipoprotein receptor-deficient ( Ldlr -/- ) mice as germfree (GF) and kept these mice for 16 weeks on an atherogenic high-fat Western diet (HFD) under GF isolator conditions and under conventionally raised specific-pathogen-free conditions (CONV-R). In spite of reduced diversity of the cecal gut microbiome, caused by atherogenic HFD, GF Ldlr -/- mice and CONV-R Ldlr -/- mice exhibited atherosclerotic lesions of comparable sizes in the common carotid artery. In contrast to HFD-fed mice, showing no difference in total cholesterol levels, CONV-R Ldlr -/- mice fed control diet (CD) had significantly reduced total plasma cholesterol, very-low-density lipoprotein (VLDL), and LDL levels compared with GF Ldlr -/- mice. Myeloid cell counts in blood as well as leukocyte adhesion to the vessel wall at the common carotid artery of GF Ldlr -/- mice on HFD were diminished compared to CONV-R Ldlr -/- controls. Plasma cytokine profiling revealed reduced levels of the proinflammatory chemokines CCL7 and CXCL1 in GF Ldlr -/- mice, whereas the T-cell-related interleukin 9 (IL-9) and IL-27 were elevated. In the atherothrombosis model of ultrasound-induced rupture of the common carotid artery plaque, thrombus area was significantly reduced in GF Ldlr -/- mice relative to CONV-R Ldlr -/- mice. Ex vivo , this atherothrombotic phenotype was explained by decreased adhesion-dependent platelet activation and thrombus growth of HFD-fed GF Ldlr -/- mice on type III collagen. IMPORTANCE Our results demonstrate a functional role for the commensal microbiota in atherothrombosis. In a ferric chloride injury model of the carotid artery, GF C57BL/6J mice had increased occlusion times compared to colonized controls. Interestingly, in late atherosclerosis, HFD-fed GF Ldlr -/- mice had reduced plaque rupture-induced thrombus growth in the carotid artery and diminished ex vivo thrombus formation under arterial flow conditions., (Copyright © 2019 Kiouptsi et al.)

Background: Remifentanil-induced hyperalgesia (RIH) may be a potential component for acute confusional state and chronic pain after surgery, but specific mechanism remains elusive. Chemokines and inflammatory mediators associated neuroinflammation is involved in central nociceptive sensitization. The contribution of AMPA receptor to excitatory synaptic transmission in pathologic pain has been indicated. This study investigated whether chemokine CCL7 modulated spinal phosphorylation of AMPA receptor via interleukin (IL)-18 in a rat model of RIH., Methods: Mechanical allodynia and thermal hyperalgesia after remifentanil infusion were verified by assessment of paw withdrawal threshold and paw withdrawal latency, respectively. A neutralizing antibody against CCL7, IL-18 antagonist and recombinant CCL7 were employed to identify the mechanisms. Levels of CCL7 and its receptors, expressions of IL-18 and its receptor IL-18R, and the phosphorylation of GluA1 in the spinal cord dorsal horn were evaluated., Results: We reported that acute exposure to remifentanil caused and maintained behavioral RIH with up-regulation of expression in CCL7/CCR2 and IL-18/IL-18R and the phosphorylation of GluA1 in the dorsal horn. Spinal inhibition of CCL7/CCR2 impaired RIH, spinal expression of IL-18/IL-18R and the phosphorylation of GluA1. Central blockage of IL-18/IL-18R reduced spinal phosphorylation of GluA1 and behavioral RIH. Also, intrathecal injection of exogenous CCL7 facilitated acute pain behaviors and spinal phosphorylation of GluA1 in naïve mice, which was suppressed by co-administration of IL-18 antagonist., Conclusion: Our current findings demonstrate that spinal CCL7/CCR2 modulation of GluA1-containing AMPA receptor activation via IL-18 and IL-18R over-expression is an important pathogenesis of remifentanil-induced hyperalgesia in rats., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The process of vascular remodeling is associated with increased hypoxia. However, the contribution of hypoxia-inducible factor 1α (HIF1α), the key transcription factor mediating cellular hypoxic responses, to vascular remodeling is established, but not completely understood. In the angiotensin II (Ang II)-induced vascular remodeling model, HIF1α was increased and activated in vascular smooth muscle cells (VSMCs). Selective genetic disruption of Hif1a in VSMCs markedly ameliorated Ang II-induced vascular remodeling, as revealed by decreased blood pressure, aortic thickness, collagen deposition, inflammation, and aortic stiffness. VSMC Hif1a deficiency also specifically suppressed Ang II-induced infiltration of CD45 + CD11b + F4/80 + CD206 - M1 macrophages into the vessel. Mechanistically, HIF1α deficiency in VSMCs dramatically suppressed the expression of CCL7, a chemokine critical for macrophage recruitment. Bioinformatic analysis and chromatin immunoprecipitation assays revealed three functional hypoxia-response elements in the Ccl7 promoter, indicating that Ccl7 is a direct HIF1α target gene. Blocking CCL7 with antibody in vivo alleviated Ang II-induced hypertension and vascular remodeling, coincident with decreased macrophage infiltration. This study provides direct evidence that HIF1α activation in VSMCs exacerbates Ang II-induced macrophage infiltration and resultant vascular remodeling via its target gene Ccl7, and thus may serve as a potential therapeutic target for remodeling-related vascular disease.

Background: Syndecans are heparan sulfate proteoglycans that occur in membrane-bound or soluble forms. Syndecan-3, the least well-characterised of the syndecan family, is highly expressed on synovial endothelial cells in rheumatoid arthritis patients. Here, it binds pro-inflammatory chemokines with evidence for a role in chemokine presentation and leukocyte trafficking into the joint, promoting the inflammatory response. In this study, we explored the role of soluble syndecan-3 as a binder of chemokines and as an anti-inflammatory and therapeutic molecule., Methods: A human monocytic cell line and CD14+ PBMCs were utilised in both Boyden chamber and trans-endothelial migration assays. Soluble syndecan-3 was tested in antigen-induced and collagen-induced in vivo arthritis models in mice. ELISA and isothermal fluorescence titration assays assessed the binding affinities. Syndecan-3 expression was identified by flow cytometry and PCR, and levels of shedding by ELISA., Results: Using in vitro and in vivo models, soluble syndecan-3 inhibited leukocyte migration in vitro in response to CCL7 and its administration in murine models of rheumatoid arthritis reduced histological disease severity. Using isothermal fluorescence titration, the binding affinity of soluble syndecan-3 to inflammatory chemokines CCL2, CCL7 and CXCL8 was determined, revealing little difference, with K d s in the low nM range. TNFα increased cell surface expression and shedding of syndecan-3 from cultured human endothelial cells. Furthermore, soluble syndecan-3 occurred naturally in the sera of patients with rheumatoid arthritis and periodontitis, and its levels correlated with syndecan-1., Conclusions: This study shows that the addition of soluble syndecan-3 may represent an alternative therapeutic approach in inflammatory disease.

The complex neuroimmunological interactions mediated by chemokines are suggested to be responsible for the development of neuropathic pain. The lack of knowledge regarding the detailed pathomechanism of neuropathy is one reason for the lack of optimally efficient therapies. Recently, several lines of evidence indicated that expression of CCR2 is increased in spinal cord neurons and microglial cells after peripheral nerve injury. It was previously shown that administration of CCR2 antagonists induces analgesic effects; however, the role of CCR2 ligands in neuropathic pain still needs to be explained. Thus, the goal of our studies was to investigate the roles of CCL2, CCL7, and CCL12 in neuropathic pain development and opioid effectiveness. The experiments were conducted on primary glial cell cultures and two groups of mice: naive and neuropathic. We used chronic constriction injury (CCI) of the sciatic nerve as a neuropathic pain model. Mice intrathecally received chemokines (CCL2, CCL7, CCL12) at a dose of 10, 100 or 500 ng, neutralizing antibodies (anti-CCL2, anti-CCL7) at a dose of 1, 4 or 8 μg, and opioids (morphine, buprenorphine) at a dose of 1 μg. The pain-related behaviors were assessed using the von Frey and cold plate tests. The biochemical analysis of mRNA expression of glial markers, CCL2, CCL7 and CCL12 was performed using quantitative reverse transcriptase real-time PCR. We demonstrated that CCI of the sciatic nerve elevated spinal expression of CCL2, CCL7 and CCL12 in mice, in parallel with microglia and astroglial activation markers. Moreover, intrathecal injection of CCL2 and CCL7 induced pain-related behavior in naive mice in a dose-dependent manner. Surprisingly, intrathecal injection of CCL12 did not influence nociceptive transmission in naive or neuropathic mice. Additionally, we showed for the first time that intrathecal injection of CCL2 and CCL7 neutralizing antibodies not only attenuated CCI-induced pain-related behaviors in mice but also augmented the analgesia induced by morphine and buprenorphine. In vitro studies suggest that both microglia and astrocytes are an important cellular sources of the examined chemokines. Our results revealed the crucial roles of CCL2 and CCL7, but not CCL12, in neuropathic pain development and indicated that pharmacological modulation of these factors may serve as a potential therapeutic target for new (co)analgesics., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Resident and inflammatory mononuclear phagocytes (MPh) with functional plasticity in the intestine are critically involved in the pathology of Inflammatory Bowel Diseases (IBD), in which the mechanism remains incompletely understood. In the present study, we found that increased expression of E3 ligase FBXW7 in the inflamed intestine was significantly correlated to IBD severity in both human diseases and mice model. Myeloid-Fbxw7 deficiency protected mice from dextran sodium sulfate (DSS) and 2,6,4-trinitrobenzene sulfonic acid (TNBS) induced colitis. Fbxw7 deficiency resulted in decreased production of chemokines CCL2 and CCL7 by colonic CX3CR1hi resident macrophages and reduced accumulation of CX3CR1int pro-inflammatory MPh in colitis colon tissue. Mice received AAV-shFbxw7 administration showed significantly improved survival rate and alleviated colitis. Mechanisms screening demonstrated that FBXW7 suppresses H3K27me3 modification and promotes Ccl2 and Ccl7 expression via degradation of histone-lysine N-methyltransferase EZH2 in macrophages. Taken together, our results indicate that FBXW7 degrades EZH2 and increases Ccl2/Ccl7 in CX3CR1hi macrophages, which promotes the recruiting CX3CR1int pro-inflammatory MPh into local colon tissues with colitis. Targeting FBXW7 might represent a potential therapeutic approach for intestine inflammation intervention.

Whereas prior studies have demonstrated an important immunomodulatory role for the neuronal cholinergic system in the heart, the role of the non-neuronal cholinergic system is not well understood. To address the immunomodulatory role of the non-neuronal cholinergic system in the heart we used a previously validated diphtheria toxin (DT)-induced cardiomyocyte ablation model (Rosa26-DTMlc2v-Cre mice). DT-injected Rosa26-DTMlc2v-Cre mice were treated with diluent or Pyridostigmine Bromide (PYR), a reversible cholinesterase inhibitor. PYR treatment resulted in increased survival and decreased numbers of MHC-IIlowCCR2+ macrophages in DT-injected Rosa26-DTMlc2v-Cre mice compared to diluent treated Rosa26-DTMlc2v-Cre mice. Importantly, the expression of CCL2/7 mRNA and protein was reduced in the hearts of PYR-treated mice. Backcrossing Rosa26-DTMlc2v-Cre mice with a transgenic mouse line (Chat-ChR2) that constitutively overexpresses the vesicular acetylcholine transporter (VAChT) resulted in decreased expression of Ccl2/7 mRNA and decreased numbers of CD68+ cells in DT-injured Rosa26-DTMlc2v-Cre/Chat-ChR2 mouse hearts, consistent with the pharmacologic studies with PYR. In vitro studies with cultures of LPS-stimulated peritoneal macrophages revealed a concentration-dependent reduction in CCL2 secretion following stimulation with ACh, nicotine and muscarine. Viewed together, these findings reveal a previously unappreciated immunomodulatory role for the non-neuronal cholinergic system in regulating homeostatic responses in the heart following tissue injury.

During disease, cells experience various stresses that manifest as an accumulation of misfolded proteins and eventually lead to cell death. To combat this stress, cells activate a pathway called unfolded protein response that functions to maintain endoplasmic reticulum (ER) homeostasis and determines cell fate. We recently reported a hitherto unknown mechanism of regulating ER stress via a novel post-translational modification called Fic-mediatedadenylylation/AMPylation. Specifically, we showed that the human Fic (filamentation induced by cAMP) protein, HYPE/FicD, catalyzes the addition of an adenosine monophosphate (AMP) to the ER chaperone, BiP, to alter the cell's unfolded protein response-mediated response to misfolded proteins. Here, we report that we have now identified a second target for HYPE-alpha-synuclein (αSyn), a presynaptic protein involved in Parkinson's disease. Aggregated αSyn has been shown to induce ER stress and elicit neurotoxicity in Parkinson's disease models. We show that HYPE adenylylates αSyn and reduces phenotypes associated with αSyn aggregation invitro, suggesting a possible mechanism by which cells cope with αSyn toxicity., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Leukocyte migration, a hallmark of the inflammatory response, is stimulated by the interactions between chemokines, which are expressed in injured or infected tissues, and chemokine receptors, which are G protein-coupled receptors (GPCRs) expressed in the leukocyte plasma membrane. One mechanism for the regulation of chemokine receptor signaling is biased agonism, the ability of different chemokine ligands to preferentially activate different intracellular signaling pathways via the same receptor. To identify features of chemokines that give rise to biased agonism, we studied the activation of the receptor CCR1 by the chemokines CCL7, CCL8, and CCL15(Δ26). We found that, compared to CCL15(Δ26), CCL7 and CCL8 exhibited biased agonism towards cAMP inhibition and away from β-Arrestin 2 recruitment. Moreover, N-terminal substitution of the CCL15(Δ26) N-terminus with that of CCL7 resulted in a chimera with similar biased agonism to CCL7. Similarly, N-terminal truncation of CCL15(Δ26) also resulted in signaling bias between cAMP inhibition and β-Arrestin 2 recruitment signals. These results show that the interactions of the chemokine N-terminal region with the receptor transmembrane region play a key role in selecting receptor conformations coupled to specific signaling pathways., Competing Interests: The authors declare no conflict of interest.

In the present study, we sought to elucidate the mechanisms by which monocytes migrate into the pleural space in the presence of anaphylatoxins in tuberculous pleural effusion (TPE). Monocytes in both pleural effusion and blood were counted, and their phenotypic characteristics were analyzed. Activation of the complement system was detected in TPE. The effects of Mpt64 and anaphylatoxins on the production of chemokines in pleural mesothelial cells (PMCs) were measured. The chemoattractant activity of chemokines produced by PMCs for monocytes was observed. Levels of CD14 + CD16 + monocytes were significantly higher in TPE than in blood. Three pathways of the complement system were activated in TPE. C3a-C3aR1, C5a-C5aR1, CCL2-CCR2, CCL7-CCR2, and CX3CL1-CX3CR1 were coexpressed in PMCs and monocytes isolated from TPE. Moreover, we initially found that Mpt64 stimulated the expression of C3a and C5a in PMCs. C3a and C5a not only induced CCL2, CCL7, and CX3CL1 expression in PMCs but also stimulated production of IL-1β, IL-17, and IL-27 in monocytes. C3a and C5a stimulated PMCs to secrete CCL2, CCL7, and CX3CL1, which recruited CD14 + CD16 + monocytes to the pleural cavity. As a result, the infiltration of CD14 + CD16 + monocytes engaged in the pathogenesis of TPE by excessive production of inflammatory cytokines.

We describe a method, termed cryoAPEX, which couples chemical fixation and high-pressure freezing of cells with peroxidase tagging (APEX) to allow precise localization of membrane proteins in the context of a well-preserved subcellular membrane architecture. Further, cryoAPEX is compatible with electron tomography. As an example, we apply cryoAPEX to obtain a high-resolution three-dimensional contextual map of the human FIC (filamentation induced by cAMP) protein, HYPE (also known as FICD). HYPE is a single-pass membrane protein that localizes to the endoplasmic reticulum (ER) lumen and regulates the unfolded protein response. Alternate cellular locations for HYPE have been suggested. CryoAPEX analysis shows that, under normal and/or resting conditions, HYPE localizes robustly within the subdomains of the ER and is not detected in the secretory pathway or other organelles. CryoAPEX is broadly applicable for assessing both lumenal and cytosol-facing membrane proteins., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

FIC proteins regulate molecular processes from bacteria to humans by catalyzing post-translational modifications (PTM), the most frequent being the addition of AMP or AMPylation. In many AMPylating FIC proteins, a structurally conserved glutamate represses AMPylation and, in mammalian FICD, also supports deAMPylation of BiP/GRP78, a key chaperone of the unfolded protein response. Currently, a direct signal regulating these FIC proteins has not been identified. Here, we use X-ray crystallography and in vitro PTM assays to address this question. We discover that Enterococcus faecalis FIC (EfFIC) catalyzes both AMPylation and deAMPylation and that the glutamate implements a multi-position metal switch whereby Mg 2+ and Ca 2+ control AMPylation and deAMPylation differentially without a conformational change. Remarkably, Ca 2+ concentration also tunes deAMPylation of BiP by human FICD. Our results suggest that the conserved glutamate is a signature of AMPylation/deAMPylation FIC bifunctionality and identify metal ions as diffusible signals that regulate such FIC proteins directly.

The chemokine CCL7 (MCP3) is known to promote the recruitment of many innate immune cell types including monocytes and neutrophils to sites of bacterial and viral infection and eosinophils and basophils to sites of allergic inflammation. CCL7 upregulation has been associated with many inflammatory settings including infection, cardiovascular disease, and the tumor microenvironment. CCL7's pleotropic effects are due in part to its ability to bind numerous chemokine receptors, namely CCR1, CCR2, CCR3, CCR5, and CCR10. CCL7-blockade or CCL7-deficiency is often marked by decreased inflammation and poor pathogen control. In the context of Leishmania major infection, CCL7 is specifically upregulated in the skin one-2 weeks after infection but its role in L. major control is unclear. To determine CCL7's impact on the response to L. major we infected WT and CCL7 -/- C57BL/6 mice. L. major infection of CCL7-deficient mice led to an unexpected increase in inflammation in the infected skin 2 weeks post-infection. A broad increase in immune cell subsets was observed but was dominated by enhanced neutrophilic infiltration. Increased neutrophil recruitment was associated with an enhanced IL-17 gene profile in the infected skin. CCL7 was shown to directly antagonize neutrophil migration in vitro and CCL7 add-back in vivo specifically reduced neutrophil influx into the infected skin revealing an unexpected role for CCL7 in limiting neutrophil recruitment during L. major infection. Enhanced neutrophilic infiltration in CCL7-deficient mice changed the balance of L. major infected host cells with an increase in the ratio of infected neutrophils over monocytes/macrophages. To determine the consequence of CCL7 deficiency on L. major control we analyzed parasite load cutaneously at the site of infection and viscerally in the draining LN and spleen. The CCL7 -/- mice supported robust cutaneous parasite control similar to their WT C57BL/6 counterparts. In contrast, CCL7-deficiency led to greater parasite dissemination and poor parasite control in the spleen. Our studies reveal a novel role for CCL7 in negatively regulating cutaneous inflammation, specifically neutrophils, early during L. major infection. We propose that CCL7-mediated dampening of the early immune response in the skin may limit the ability of the parasite to disseminate without compromising cutaneous control.

Curcumin has been reported to have anti-fibrotic effect. However, the anti-fibrotic mechanism of curcumin for liver fibrosis remains obscure. In the presenting study, we aimed to investigate whether curcumin reduce chemokines secretion by inhibiting kupffer cells (KCs) activation to decrease Ly6C hi monocyte infiltration in the treatment of liver fibrosis. Liver fibrosis was induced by intraperitoneal carbon tetrachloride (CCl 4 )-injection in mice. Mice in curcumin group received curcumin treatment by gavage. Pretreatment with curcumin significantly protected mice from liver inflammation and fibrosis. Compared to CCl 4 group, mice in the curcumin group showed significantly less intrahepatic infiltration of Ly6C hi monocytes, but no difference of other leucocyte subtypes. Moreover, curcumin significantly reduced Ly6C hi monocytes associated pro-inflammatory and pro-fibrogenic cytokines, which was in line with the decreased numbers of intrahepatic Ly6C hi monocytes. Further study found that curcumin is able to decrease KCs activation and monocyte chemokines, which explains why curcumin can reduce Ly6C hi monocytes infiltration during liver fibrosis. In vitro, we discovered that curcumin prevents the polarization of macrophages toward M1 and reduces monocyte chemokines secretion, which is involved with ERK1/2 and p38 pathways. Taken together, for the first time, we verified that curcumin can reduce chemokines secretion by inhibiting KCs activation to decrease Ly6C hi monocyte infiltration in the treatment of liver fibrosis. These results suggested that curcumin may be considered a promising candidate in the prevention and treatment of liver fibrosis., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

Background: Bone marrow mesenchymal stem cells (BMSCs) and acupuncture are known to mitigate tissue damage. This study aimed to investigate the therapeutic effects of combined electroacupuncture (EA) stimulation and BMSC injection in a rat model of anal sphincter injury-induced faecal incontinence (FI)., Methods: 60 Sprague-Dawley rats were randomly divided into five groups: sham-operated control, FI, FI+EA, FI+BMSC, and FI+BMSC+EA. The anorectal tissues were collected on days 1, 3, 7 and 14. Repair of the injured anal sphincter was compared using haematoxylin and eosin (HE) and immunocytochemiscal analyses with sarcomeric α actinin. The expression of stromal cell derived factor-1 (SDF-1) and monocyte chemoattractant protein-3 (MCP-3) was detected by quantitative reverse transcription PCR to evaluate the effects of EA on the homing of BMSCs., Results: The therapeutic effect of combined EA+BMSCs on damaged tissue was the strongest among all the groups as indicated by HE and immunohistochemical staining. The expression of SDF-1 and MCP-3 was significantly increased by combined EA and BMSC treatment when compared with the other groups (P=0.01 to P<0.05), suggesting promotive effects of EA on the homing of BMSCs., Conclusion: The combination of EA and BMSC transplantation effectively repaired the impaired anal sphincters. The underlying mechanism might be associated with apparent promotive effects of EA on the homing of BMSCs. Our study provides a theoretical basis for the development of a non-surgical treatment method for FI secondary to muscle impairment., Competing Interests: Competing interests: None declared., (© Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.)

Liver metastases remain a major cause of death from gastrointestinal tract cancers as well as from other malignancies such as breast and lung carcinomas and melanoma. Understanding the underlying biology is essential for the design of effective targeted therapies. We previously reported that collagen IV α1/α2 overexpression in non-metastatic lung carcinoma (M27 colIV ) cells increased their metastatic ability, specifically to the liver and documented high collagen IV levels in surgical resections of liver metastases from diverse tumor types. Here, we aimed to elucidate the functional relevance of collagen IV to metastatic outgrowth in the liver. Gene expression profiling revealed in M27 colIV cells significant increases in the expression of chemokines CCL5 (5.7-fold) and CCL7 (2.6-fold) relative to wild-type cells, and this was validated by qPCR and western blotting. Similarly, in human colon carcinoma KM12C and KM12SM cells with divergent liver-colonizing potentials, CCL7 and CCL5 production correlated with type IV collagen expression and the metastatic phenotype. CCL7 silencing by short hairpin RNA (shRNA) reduced experimental liver metastasis in both cell types, whereas CCL5 silencing reduced metastasis of M27 colIV cells, implicating these cytokines in metastatic expansion in the liver. Subsequent functional analyses implicated both MEK/ERK and PI3K signaling upstream of CCL7 upregulation and identified CCL7 (but not CCL5) as a critical migration/invasion factor, acting via the chemokine receptor CCR3. Chemokine CCL5 was identified as a regulator of the T-cell immune response in the liver. Loss of CCL7 in KM12SM cells was also associated with altered E-cadherin and reduced vimentin and Snail expression, implicating it in epithelial-to-mesenchymal transition in these cells. Moreover, in clinical specimens of colon cancer liver metastases analyzed by immunohistochemistry, CCL5 and CCL7 levels paralleled those of collagen IV. The results identify the chemokines CCL5 and CCL7 as type IV collagen-regulated genes that promote liver metastasis by distinct and complementary mechanisms.