Integrating machine learning algorithms and single-cell analysis to identify gut microbiota-related macrophage biomarkers in atherosclerotic plaques.

Objective: The relationship between macrophages and the gut microbiota in patients with atherosclerosis remains poorly defined, and effective biological markers are lacking. This study aims to elucidate the interplay between gut microbial communities and macrophages, and to identify biomarkers associated with the destabilization of atherosclerotic plaques. The goal is to enhance our understanding of the underlying molecular pathways and to pave new avenues for diagnostic approaches and therapeutic strategies in the disease. Methods: This study employed Weighted Gene Co-expression Network Analysis (WGCNA) and differential expression analysis on atherosclerosis datasets to identify macrophage-associated genes and quantify the correlation between these genes and gut microbiota gene sets. The Random Forest algorithm was utilized to pinpoint PLEK, IRF8, BTK, CCR1, and CD68 as gut microbiota-related macrophage genes, and a nomogram was constructed. Based on the top five genes, a Non-negative Matrix Factorization (NMF) algorithm was applied to construct gut microbiota-related macrophage clusters and analyze their potential biological alterations. Subsequent single-cell analyses were conducted to observe the expression patterns of the top five genes and the interactions between immune cells. Finally, the expression profiles of key molecules were validated using clinical samples from atherosclerosis patients. Results: Utilizing the Random Forest algorithm, we ultimately identified PLEK, IRF8, CD68, CCR1, and BTK as gut microbiota-associated macrophage genes that are upregulated in atherosclerotic plaques. A nomogram based on the expression of these five genes was constructed for use as an auxiliary tool in clinical diagnosis. Single-cell analysis confirmed the specific expression of gut microbiota-associated macrophage genes in macrophages. Clinical samples substantiated the high expression of PLEK in unstable atherosclerotic plaques. Conclusion: Gut microbiota-associated macrophage genes (PLEK, IRF8, CD68, CCR1, and BTK) may be implicated in the pathogenesis of atherosclerotic plaques and could serve as diagnostic markers to aid patients with atherosclerosis.

Role of CCR1/5/7 in hepatocellular carcinoma: a study on prognostic evaluation, molecular subtyping, and association with immune infiltration.

This study aims to assess the prognostic value of the C-C motif chemokine receptor (CCR) gene family in hepatocellular carcinoma (HCC) and its relationship with immune infiltration and molecular subtypes of HCC. The evaluation of the GSE14520 dataset and TCGA database confirmed the prognostic significance of CCR. Building upon the correlation between CCR1, CCR5, and CCR7 and favorable prognosis, we further validated the prognostic importance of CCR1, CCR5, and CCR7 in ICGC database and an independent cohort from Guangxi autonomous region. Then, we constructed a risk prognosis model. Additionally, we observed significant positive correlations between CCR1, CCR5, and CCR7 and the infiltration of B cells, T cells, and macrophages in HCC. Subsequently, we conducted CCK assays, Transwell assays, and colony formation assays to evaluate the molecular biological functions of CCR1, CCR5, and CCR7. These experiments further confirmed that upregulation of CCR1, CCR5, and CCR7 can individually inhibit the proliferation, migration, and stemness of HCC cells. By analyzing the relationship between expression levels and tumor mutation frequency, we discovered that patients with high CCR1 expression were more likely to be classified as non-proliferative HCC. Similar conclusions were observed for CCR5 and CCR7. The association of CCR1, CCR5, and CCR7 with the molecular subtypes of HCC suggests that they may serve as intermediary molecules linking immune status and molecular subtypes in HCC. In summary, CCR1, CCR5, and CCR7 have the potential to serve as prognostic biomarkers for HCC and regulate HCC progression by influencing immune cell infiltration.

Expression of the chemokine receptor CCR1 decreases sensitivity to bortezomib in multiple myeloma cell lines.

BACKGROUND: The proteasome inhibitor bortezomib is one of the primary therapies used for the haematological malignancy multiple myeloma (MM). However, intrinsic or acquired resistance to bortezomib, via mechanisms that are not fully elucidated, is a barrier to successful treatment in many patients. Our previous studies have shown that elevated expression of the chemokine receptor CCR1 in MM plasma cells in newly diagnosed MM patients is associated with poor prognosis. Here, we hypothesised that the poor prognosis conferred by CCR1 expression is, in part, due to a CCR1-mediated decrease in MM plasma cell sensitivity to bortezomib. METHODS: In order to investigate the role of CCR1 in MM cells, CCR1 was knocked out in human myeloma cell lines OPM2 and U266 using CRISPR-Cas9. Additionally, CCR1 was overexpressed in the mouse MM cell line 5TGM1. The effect of bortezomib on CCR1 knockout or CCR1-overexpressing cells was then assessed by WST-1 assay, with or without CCL3 siRNA knockdown or addition of recombinant human CCL3. NSG mice were inoculated intratibially with OPM2-CCR1KO cells and were treated with 0.7 mg/kg bortezomib or vehicle twice per week for 3 weeks and GFP+ tumour cells in the bone marrow were quantitated by flow cytometry. The effect of CCR1 overexpression or knockout on unfolded protein response pathways was assessed using qPCR for ATF4, HSPA5, XBP1, ERN1 and CHOP and Western blot for IRE1α and p-Jnk. RESULTS: Using CCR1 overexpression or CRIPSR-Cas9-mediated CCR1 knockout in MM cell lines, we found that CCR1 expression significantly decreases sensitivity to bortezomib in vitro, independent of the CCR1 ligand CCL3. In addition, CCR1 knockout rendered the human MM cell line OPM2 more sensitive to bortezomib in an intratibial MM model in NSG mice in vivo. Moreover, CCR1 expression negatively regulated the expression of the unfolded protein response receptor IRE1 and downstream target gene XBP1, suggesting this pathway may be responsible for the decreased bortezomib sensitivity of CCR1-expressing cells. CONCLUSIONS: Taken together, these studies suggest that CCR1 expression may be associated with decreased response to bortezomib in MM cell lines.

CFL1 restores the migratory capacity of bone marrow mesenchymal stem cells in primary Sjögren's syndrome by regulating CCR1 expression.

BACKGROUND: Primary Sjögren's syndrome (pSS) is a chronic systemic autoimmune disease. There is no relevant research on whether the migratory ability of bone marrow mesenchymal stem cells (BM-MSC) is impaired in patients with pSS (pSS-BMMSC). METHODS: Trajectories and velocities of BM-MSC were analyzed. Transwell migration assay and wound healing assay were used to investigate the migratory capacity of BM-MSC. The proliferative capacity of BM-MSC was evaluated by EDU and CCK8 assay. RNA-seq analysis was then performed to identify the underlying mechanism of lentivirus-mediated cofilin-1 overexpression BM-MSC (BMMSCCFL1). The therapeutic efficacy of BMMSCCFL1 was evaluated in NOD mice. RESULTS: The migratory capacity of pSS-BMMSC was significantly reduced compared to normal volunteers (HC-BMMSC). The expression of the motility-related gene CFL1 was decreased in pSS-BMMSC. Lentivirus-mediated CFL1 overexpression of pSS-BMMSC promoted the migration capacity of pSS-BMMSC. Furthermore, RNA-seq revealed that CCR1 was the downstream target gene of CFL1. To further elucidate the mechanism of CFL1 in regulating BM-MSC migration and proliferation via the CCL5/CCR1 axis, we performed a rescue experiment using BX431 (a CCR1-specific inhibitor) to inhibit CCR1. The results showed that CCR1 inhibitors suppressed the migration and proliferation capacity of MSC induced by CFL1. CONCLUSION: The pSS-BMMSC leads to impaired migration and proliferation, and overexpression of CFL1 can rescue the functional deficiency and alleviate disease symptoms in NOD mice. Mechanically, CFL1 can regulate the expression level of the downstream CCL5/CCR1 axis to enhance the migration and proliferation of BM-MSC.

Inhibition of CCR1 attenuates neuroinflammation via the JAK2/STAT3 signaling pathway after subarachnoid hemorrhage.

BACKGROUND AND PURPOSE: Neuroinflammation is an important mechanism underlying brain injury caused by subarachnoid hemorrhage (SAH). C-C chemokine receptor type 1 (CCR1)-mediated inflammation is involved in the pathology of many central nervous system diseases. Herein, we investigated whether inhibition of CCR1 alleviated neuroinflammation after experimental SAH and aimed to elucidate the mechanisms of its potential protective effects. METHODS: To analyze SAH transcriptome data R studio was used, and a mouse model of SAH was established using endovascular perforations. In this model, the selective CCR1 antagonist Met-RANTES (Met-R) and the CCR1 agonist recombinant CCL5 (rCCL5) were administered 1 h after SAH induction. To investigate the possible downstream mechanisms of CCR1, the JAK2 inhibitor AG490 and the JAK2 activator coumermycin A1 (C-A1) were administered 1 h after SAH induction. Furthermore, post-SAH evaluation, including SAH grading, neurological function tests, Western blot, the terminal deoxynucleotidyl transferase dUTP nick end labeling assay, and Fluoro-Jade B and fluorescent immunohistochemical staining were performed. Cerebrospinal fluid (CSF) samples were detected by ELISA. RESULTS: CCL5 and CCR1 expression levels increased significantly following SAH. Met-R significantly improved neurological deficits in mice, decreased apoptosis and degeneration of ipsilateral cerebral cortex neurons, reduced infiltrating neutrophils, and promoted microglial activation after SAH induction. Furthermore, Met-R inhibited the expression of p-JAK2, p-STAT3, interleukin-1ß, and tumor necrosis factor-α. However, the protective effects of Met-R were abolished by C-A1 treatment. Furthermore, rCCL5 injection aggravated neurological dysfunction and increased the expression of p-JAK2, p-STAT3, interleukin-1ß, and tumor necrosis factor-α in SAH mice, all of which were reversed by the administration of AG490. Finally, the levels of CCL5 and CCR1 were elevate in the CSF of SAH patient and high level of CCL5 and CCR1 levels were associated with poor outcome. CONCLUSION: The present results suggested that inhibition of CCR1 attenuates neuroinflammation after SAH via the JAK2/STAT3 signaling pathway, which may provide a new target for the treatment of SAH.

The Chemokine Receptor CCR1 Mediates Microglia Stimulated Glioma Invasion.

Glioblastoma multiforme (GBM) is the most aggressive form of adult brain tumor which is highly resistant to conventional treatment and therapy. Glioma cells are highly motile resulting in infiltrative tumors with poorly defined borders. Another hallmark of GBM is a high degree of tumor macrophage/microglia infiltration. The level of these tumor-associated macrophages/microglia (TAMs) correlates with higher malignancy and poorer prognosis. We previously demonstrated that inhibition of TAM infiltration into glioma tumors with the CSF-1R antagonist pexidartinib (PLX3397) can inhibit glioma cell invasion in-vitro and in-vivo. In this study, we demonstrate an important role for the chemokine receptor CCR1 in mediating microglia/TAM stimulated glioma invasion. Using two structurally distinct CCR1 antagonists, including a novel inhibitor "MG-1-5", we were able to block microglial activated GL261 glioma cell invasion in a dose dependent manner. Interestingly, treatment of a murine microglia cell line with glioma conditioned media resulted in a strong induction of CCR1 gene and protein expression. This induction was attenuated by inhibition of CSF-1R. In addition, glioma conditioned media treatment of microglia resulted in a rapid upregulation of gene expression of several CCR1 ligands including CCL3, CCL5, CCL6 and CCL9. These data support the existence of tumor stimulated autocrine loop within TAMs which ultimately mediates tumor cell invasion.

Dysfunction of CCR1+ decidual macrophages is a potential risk factor in the occurrence of unexplained recurrent pregnancy loss.

Recurrent pregnancy loss (RPL) puzzles 1-3% of women of childbearing age worldwide. Immunological factors account for more than 60% of cases of unexplained RPL (URPL); however, the underlying mechanism remains unclear. Here, using single-cell sequencing data and functional experiments with clinical samples, we identified a distinct population of CCR1+ decidual macrophages (dMφ) that were preferentially enriched in the decidua from normal early pregnancies but were substantially decreased in patients with URPL. Specific gene signatures endowed CCR1+ dMφ with immunosuppressive and migration-regulatory properties, which were attenuated in URPL. Additionally, CCR1+ dMφ promoted epithelial-to-mesenchymal transition (EMT) to promote trophoblast migration and invasion by activating the ERK1/2 signaling pathway. Decidual stromal cell (DSC)-derived CCL8 was the key regulator of CCR1+ dMφ as CCL8 recruited peripheral CCR1+ monocytes, induced a CCR1+ dMφ-like phenotype, and reinforced the CCR1+ dMφ-exerted modulation of trophoblasts. In patients with URPL, CCL8 expression in DSCs was decreased and trophoblast EMT was defective. Our findings revealed that CCR1+ dMφ play an important role in immune tolerance and trophoblast functions at the maternal-fetal interface. Additionally, decreased quantity and dysregulated function of CCR1+ dMφ result in URPL. In conclusion, we provide insights into the crosstalk between CCR1+ dMφ, trophoblasts, and DSCs at the maternal-fetal interface and macrophage-targeted interventions of URPL.

Evolving polarisation of infiltrating and alveolar macrophages in the lung during metastatic progression of melanoma suggests CCR1 as a therapeutic target.

Metastatic tumour progression is facilitated by tumour associated macrophages (TAMs) that enforce pro-tumour mechanisms and suppress immunity. In pulmonary metastases, it is unclear whether TAMs comprise tissue resident or infiltrating, recruited macrophages; and the different expression patterns of these TAMs are not well established. Using the mouse melanoma B16F10 model of experimental pulmonary metastasis, we show that infiltrating macrophages (IM) change their gene expression from an early pro-inflammatory to a later tumour promoting profile as the lesions grow. In contrast, resident alveolar macrophages (AM) maintain expression of crucial pro-inflammatory/anti-tumour genes with time. During metastatic growth, the pool of macrophages, which initially contains mainly alveolar macrophages, increasingly consists of infiltrating macrophages potentially facilitating metastasis progression. Blocking chemokine receptor mediated macrophage infiltration in the lung revealed a prominent role for CCR2 in Ly6C+ pro-inflammatory monocyte/macrophage recruitment during metastasis progression, while inhibition of CCR2 signalling led to increased metastatic colony burden. CCR1 blockade, in contrast, suppressed late phase pro-tumour MR+Ly6C- monocyte/macrophage infiltration accompanied by expansion of the alveolar macrophage compartment and accumulation of NK cells, leading to reduced metastatic burden. These data indicate that IM has greater plasticity and higher phenotypic responsiveness to tumour challenge than AM. A considerable difference is also confirmed between CCR1 and CCR2 with regard to the recruited IM subsets, with CCR1 presenting a potential therapeutic target in pulmonary metastasis from melanoma.

CCR1 and CCR5 mediate cancer-induced myelopoiesis and differentiation of myeloid cells in the tumor.

BACKGROUND: Cancer-induced 'emergency' myelopoiesis plays a key role in tumor progression by inducing the accumulation of myeloid cells with a suppressive phenotype peripherally and in the tumor. Chemokine receptors (CCRs) and, in particular, CCR1, CCR2, CCR5, and CCR7 are emerging as key regulators of myeloid cell trafficking and function but their precise role has not been completely clarified yet because of the signal redundancy, integration, and promiscuity of chemokines and of the expression of these CCRs on other leukocyte subsets. METHODS: We used the 4PD nanoparticle for the in vivo targeted silencing of CCR1, CCR2, CCR5, and/or CCR7 in the myeloid cells of tumor bearing mice to evaluate the effect of treatments on tumor growth, myeloid cell trafficking and polarization. We used flow and image cytometry and functional assays to monitor changes in the tumor microenvironment and depletion experiments and immune deficient mice to determine the role of Ly6G+cells during tumor progression. We further evaluated in vitro the impact of chemokine receptor inhibition and tumor derived factors on myeloid cell differentiation from mouse and human hematopoietic stem and precursors cells (HSPCs) using flow cytometry, transcriptome analysis, cytokines beads arrays, functional assays, and mice deficient for CCR1 or CCR5. RESULTS: 4PD-mediated in vivo silencing of CCR1 and CCR5 on myeloid cells and myeloid precursors was necessary and sufficient to inhibit tumor progression. Functional studies indicated that this antitumor effect was not mediated by alteration of myeloid cell chemotaxes but rather by the repolarization of polymorphonuclear myeloid-derived suppressor cells (MDSCs) into tumoricidal neutrophils. Transcriptome functional and cytokine analysis indicated that tumor derived factors induced CCL3 and CCL4 in HSPCs that, through the autocrine engagement of CCR1 and CCR5, induced HSPCs differentiation in MDSCs. These finding were confirmed across mice with different genetic backgrounds and using HSPCs from umbilical cord blood and peripheral blood of patients with cancer. CONCLUSIONS: Our data support the notion that CCR1 and CCR5 and their ligands are a master immunological hub activated by several tumor derived factors. Activation of this pathway is necessary for the differentiation of MDSCs and protumoral macrophages.

Suppression of C-C chemokine receptor 1 is a key regulation for colon cancer chemoprevention in AOM/DSS mice by fucoxanthin.

Fucoxanthin (Fx) has shown potential cancer chemopreventive functions in a carcinogenic murine azoxymethane/dextran sodium sulfate (AOM/DSS) model. However, the molecular mechanisms based on transcriptome profiles in vivo remain poorly understood. We investigated Fx-dependent alterations of the transcriptome with cancer-associated proteins in colorectal mucosal tissue obtained from AOM/DSS mice with or without Fx treatment. Fx administration (50 mg/kg body weight for 14 weeks) significantly prevented the onset of colorectal adenocarcinoma in AOM/DSS mice. A transcriptome analysis revealed that 11 signals, including adhesion, cell cycle, chemokine receptor, interleukin, MAPK, PI3K/AKT, p53, RAS, STAT, TGF-ß, and Wnt were remarkably altered by Fx administration. In particular, chemokine (C-C motif) receptor 1 (Ccr1), which is contained in a gene set related to cytokine-cytokine receptor interactions, was the only significantly down-regulated gene after Fx administration for both 7 and 14 weeks. CCR1, AKT, Cyclin D1, and Smad2 were found to play central roles in the 11 signals shown above. Fx administration significantly down-regulated CCR1 (0.3- and 0.5-fold in mucosal crypts and lamina propria, respectively), pAKT(Ser473) (0.2-fold in mucosal crypts), Cyclin D1 (0.4-fold in mucosal crypts), and pSmad2(Ser465/467) (0.7-fold in mucosal crypts) compared with proteins in these tissues of control mice after Fx administration for 14 weeks. Our findings suggested that Fx exerts a chemopreventive effect in AOM/DSS mice through attenuation of CCR1 expression along with 11 cancer-associated signals.

Diet-induced obesity accelerates oral carcinogenesis by recruitment and functional enhancement of myeloid-derived suppressor cells.

Although obesity has been associated with an increased risk and aggressiveness of many types of carcinoma, whether it promotes squamous cell carcinoma remains unclear. To reveal the role of obesity in oral squamous cell carcinoma (OSCC) initiation and development, we used 4NQO-induced OSCC model mice to examine the impact of dietary obesity on carcinogenesis. The results showed that high-fat diet (HFD)-induced obesity significantly promoted the incidence of OSCC and altered the local immune microenvironment with the expansion of CD11b+Gr1+ myeloid-derived suppressor cells (MDSCs). The underlying mechanism that induced an immunosuppressive local microenvironment in obesity was the recruitment of MDSCs through the CCL9/CCR1 axis and enhancement of MDSC immunosuppressive function via intracellular fatty acid uptake. Furthermore, clinical samples verified the increase in infiltrated CD33+ (a marker of human MDSCs) cells in obese OSCC patients, and data from the TCGA dataset confirmed that CD33 expression was positively correlated with local adipocytes in OSCC. Survival analysis showed that enrichment of adipocytes and high expression of CD33 were associated with poor prognosis in OSCC patients. Strikingly, depletion of MDSCs significantly ameliorated HFD-promoted carcinogenesis in 4NQO-induced model mice. These findings indicate that obesity is also an important risk factor for OSCC, and cancer immunotherapy, especially targeting MDSCs, may exhibit greater antitumor efficacy in obese patients.

CCL7 contributes to angiotensin II-induced abdominal aortic aneurysm by promoting macrophage infiltration and pro-inflammatory phenotype.

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.

CCL23 suppresses liver cancer progression through the CCR1/AKT/ESR1 feedback loop.

With the ability to activate certain signaling pathways, chemokines and their receptors may facilitate tumor progression at key steps, including proliferation, immunomodulation, and metastasis. Nevertheless, their prognostic value and regulatory mechanism warrant thorough studies in liver cancer. Here, by screening the expression profiles of all known chemokines in independent liver cancer cohorts, we found that CCL23 was frequently downregulated at mRNA and protein levels in liver cancer. Decreased CCL23 correlated with shortened patient survival, enrichment of signatures related to cancer stem cell property, and metastatic potential. In addition to serving as a tumor suppressor through recruiting CD8+ T cell infiltration in liver cancer, CCL23 could repress cancer cell proliferation, stemness, and mobility. Mechanistically, the expression of CCL23 was transcriptionally regulated by ESR1. On the other hand, CCL23 could suppress the activation of AKT signaling and thus promote the expression of ESR1, forming a feedback loop in liver cancer cells. Collectively, these findings reveal that loss of CCL23 drives liver cancer progression by coordinating immune evasion and metastasis initiation. Targeting the ESR1/CCL23/CCR1/AKT regulatory axis could be an effective therapeutic strategy.

Stromal CCL5 Promotes Breast Cancer Progression by Interacting with CCR3 in Tumor Cells.

Chemokines secreted from stromal cells have important roles for interactions with carcinoma cells and regulating tumor progression. C-C motif chemokine ligand (CCL) 5 is expressed in various types of stromal cells and associated with tumor progression, interacting with C-C chemokine receptor (CCR) 1, 3 and 5 expressed in tumor cells. However, the expression on CCL5 and its receptors have so far not been well-examined in human breast carcinoma tissues. We therefore immunolocalized CCL5, as well as CCR1, 3 and 5, in 111 human breast carcinoma tissues and correlated them with clinicopathological characteristics. Stromal CCL5 immunoreactivity was significantly correlated with the aggressive phenotype of breast carcinomas. Importantly, this tendency was observed especially in the CCR3-positive group. Furthermore, the risk of recurrence was significantly higher in the patients with breast carcinomas positive for CCL5 and CCR3 but negative for CCR1 and CCR5, as compared with other patients. In summary, the CCL5-CCR3 axis might contribute to a worse prognosis in breast cancer patients, and these findings will contribute to a better understanding of the significance of the CCL5/CCRs axis in breast carcinoma microenvironment.

Loganin alleviates macrophage infiltration and activation by inhibiting the MCP-1/CCR2 axis in diabetic nephropathy.

BACKGROUND/AIMS: The theory of inflammation is one of the important theories in the pathogenesis of diabetic nephropathy (DN). We herein aimed to explore whether loganin affected macrophage infiltration and activation upon diabetic nephropathy (DN) by a spontaneous DN mice and a co-culture system of glomerular mesangial cells (GMCs) and macrophage cells (RAW264.7) which was induced by advanced glycation end products (AGEs). METHODS AND KEY FINDINGS: Loganin showed remarkable capacity on protecting renal from damage by mitigating diabetic symptoms, improving the histomorphology of the kidney, decreasing the expression of extracellular matrix such as FN, COL-IV and TGF-ß, reversing the production of IL-12 and IL-10 and decreasing the number of infiltrating macrophages in the kidney. Moreover, loganin showed markedly effects by suppressing iNOS and CD16/32 expressions (M1 markers), increasing Arg-1 and CD206 expressions (M2 markers), which were the phenotypic transformation of macrophage. These effects may be attributed to the inhibition of the receptor for AGEs (RAGE) /monocyte chemotactic protein-1 (MCP-1)/CC chemokine receptor 2 (CCR2) signaling pathway, with significantly down-regulated expressions of RAGE, MCP-1 and CCR2 by loganin. Loganin further decreased MCP-1 secretion when RAGE was silenced, which means other target was involved in regulating the MCP-1 expression. While loganin combinated with the inhibitor of CCR2 exerted stronger anti-inhibition effects of iNOS expression, suggesting that CCR2 was the target of loganin in regulating the activation of macrophages. SIGNIFICANCE: Loganin could ameliorate DN kidney damage by inhibiting macrophage infiltration and activation via the MCP-1/CCR2 signaling pathway in DN.

Prevention of CaCl2-induced aortic inflammation and subsequent aneurysm formation by the CCL3-CCR5 axis.

Inflammatory mediators such as cytokines and chemokines are crucially involved in the development of abdominal aortic aneurysm (AAA). Here we report that CaCl2 application into abdominal aorta induces AAA with intra-aortic infiltration of macrophages as well as enhanced expression of chemokine (C-C motif) ligand 3 (CCL3) and MMP-9. Moreover, infiltrating macrophages express C-C chemokine receptor 5 (CCR5, a specific receptor for CCL3) and MMP-9. Both Ccl3-/- mice and Ccr5-/- but not Ccr1-/- mice exhibit exaggerated CaCl2-inducced AAA with augmented macrophage infiltration and MMP-9 expression. Similar observations are also obtained on an angiotensin II-induced AAA model. Immunoneutralization of CCL3 mimics the phenotypes observed in CaCl2-treated Ccl3-/- mice. On the contrary, CCL3 treatment attenuates CaCl2-induced AAA in both wild-type and Ccl3-/- mice. Consistently, we find that the CCL3-CCR5 axis suppresses PMA-induced enhancement of MMP-9 expression in macrophages. Thus, CCL3 can be effective to prevent the development of CaCl2-induced AAA by suppressing MMP-9 expression.

Multiple myeloma hinders erythropoiesis and causes anaemia owing to high levels of CCL3 in the bone marrow microenvironment.

Anaemia is the most common complication of myeloma and is associated with worse clinical outcomes. Although marrow replacement with myeloma cells is widely considered a mechanistic rationale for anaemia, the exact process has not been fully understood. Our large cohort of 1363 myeloma patients had more than 50% of patients with moderate or severe anaemia at the time of diagnosis. Anaemia positively correlated with myeloma cell infiltration in the bone marrow (BM) and worse patient outcomes. The quantity and erythroid differentiation of HSPCs were affected by myeloma cell infiltration in the BM. The master regulators of erythropoiesis, GATA1 and KLF1, were obviously downregulated in myeloma HSPCs. However, the gene encoding the chemokine CCL3 showed significantly upregulated expression. Elevated CCL3 in the BM plasma of myeloma further inhibited the erythropoiesis of HSPCs via activation of CCL3/CCR1/p38 signalling and suppressed GATA1 expression. Treatment with a CCR1 antagonist effectively recovered GATA1 expression and rescued erythropoiesis in HSPCs. Myeloma cell infiltration causes elevated expression of CCL3 in BM, which suppresses the erythropoiesis of HSPCs and results in anaemia by downregulating the level of GATA1 in HSPCs. Thus, our study indicates that targeting CCL3 would be a potential strategy against anaemia and improve the survival of myeloma patients.

Endothelial cell­derived CCL15 mediates the transmigration of fibrocytes through the CCL15­CCR1 axis in vitro.

Wound healing is a complex physiological process in which fibrocytes serve a vital role. However, the mechanism underlying the recruitment of fibrocytes during wound healing remains largely unknown. The present study aimed to investigate whether endothelial cells are involved in the recruitment of fibrocytes in wound healing. To mimic the in vivo angiogenic process, a co­culture system consisting of endothelial cells and fibrocytes was achieved using a permeable Transwell co­culture system. The expression of chemokines produced by endothelial cells with or without co­culture was then measured using a gene chip. Based on the dataset from chip analysis, chemokine ligand 15 (CCL15) produced by endothelial cells was identified, which likely serves a regulatory role in mediating the transmigration of fibrocytes. Overexpression of CCL15 in endothelial cells or chemokine receptor 1 (CCR1) in fibrocytes promoted the transmigration of fibrocytes, whilst silencing the expression of CCL15 in endothelial cells or that of CCR1 in fibrocytes attenuated the transmigration of fibrocytes. Results from the present study suggested that the CCL15­CCR1 axis between endothelial cells and fibrocytes serves a vital role in mediating the recruitment of fibrocytes during wound healing.

Omental macrophages secrete chemokine ligands that promote ovarian cancer colonization of the omentum via CCR1.

The omentum is the most common site of ovarian cancer metastasis. Immune cell clusters called milky spots are found throughout the omentum. It is however unknown if these immune cells contribute to ovarian cancer metastasis. Here we report that omental macrophages promote the migration and colonization of ovarian cancer cells to the omentum through the secretion of chemokine ligands that interact with chemokine receptor 1 (CCR1). We found that depletion of macrophages reduces ovarian cancer colonization of the omentum. RNA-sequencing of macrophages isolated from mouse omentum and mesenteric adipose tissue revealed a specific enrichment of chemokine ligand CCL6 in omental macrophages. CCL6 and the human homolog CCL23 were both necessary and sufficient to promote ovarian cancer migration by activating ERK1/2 and PI3K pathways. Importantly, inhibition of CCR1 reduced ovarian cancer colonization. These findings demonstrate a critical mechanism of omental macrophage induced colonization by ovarian cancer cells via CCR1 signaling.

KSHV enhances mesenchymal stem cell homing and promotes KS-like pathogenesis.

Kaposi's sarcoma (KS) tends to occur in injured or inflamed sites of the body, which is described as the "Koebner phenomenon". KS is also unique in its extraordinary angio-hyperplastic inflammatory phenotype. Recently, evidence has accrued indicating that KS may derive from KSHV-infected mesenchymal stem cells (MSCs), which possess enhanced migration and homing ability. Inspired by these findings, we hypothesized that KS may arise from KSHV-infected MSCs that chemotactically migrate to preexisting inflammatory or injured sites. Here we report that KSHV infection of human MSCs significantly up-regulated expression of several chemokine receptors and enhanced cell migration ability in vitro. Furthermore, using a wound mouse model, we demonstrated that KSHV infection dramatically promotes MSCs migrating and settling in the wound sites. In addition, two mice in the KSHV-infected group showed purpura and tumors with KS-like features. Taken together, KSHV-enhanced MSC migration ability and inflammatory microenvironment play crucial roles in KS development.