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The serine/threonine/tyrosine kinase 1 (STYK1) is a receptor protein-tyrosine kinase (RPTK)-like molecule that is detected in several human organs. STYK1 plays an important role in promoting tumorigenesis and metastasis in various cancers. By analyzing the expression of RTKs in immune cells in the database of 2013 Immunological Genome Project, we found that STYK1 was principally expressed in NK cells. In order to investigate the function of STYK1, we used CRISPR/Cas9 technology to generate STYK1-deleted mice, we found STYK1 deletion mice have normal number, development, and function of NK cells in spleen and bone marrow in tumor-free resting state. To examine the tumor surveillance of STYK1 in vivo, we utilized a variety of tumor models, including NK cell-specific target cell (ß2M and RMA-S) clearance experiments in vivo, subcutaneous and intravenous injection of B16F10 melanoma model, and the spontaneous breast cancer model MMTV-PyMT. Surprisingly, we discovered that deletion of the oncogenic STYK1 promoted the four-model tumor progression, and we observed a reduction of NK cell accumulation in the tumor tissues of STYK1 deletion mice compared to WT mice. In order to study the mechanism of STYK1 in NK, RNA sequence of STYK1−/− and WT NK have unveiled a disparity in the signaling pathways linked to migration and adhesion in STYK1−/− NK cells. Further analysis of chemokine receptors associated with NK cell migration revealed that STYK1-deficient NK cells exhibited a significant reduction in CCR2 expression. The STYK1 expression was negatively associated with tumor progression in glioma patients. Overall, our study found the expression of STYK1 in NK cell mediates NK cell anti-tumor response through regulating CCR2 and infiltrating into tumor tissue. [ABSTRACT FROM AUTHOR]

The focus of nanoparticles in vivo trafficking has been mostly on their tissue-level biodistribution and clearance. Recent progress in the nanomedicine field suggests that the targeting of nanoparticles to immune cells can be used to modulate the immune response and enhance therapeutic delivery to the diseased tissue. In the presence of tumor lesions, monocytic-myeloid-derived suppressor cells (M-MDSCs) expand significantly in the bone marrow, egress into peripheral blood, and traffic to the solid tumor, where they help maintain an immuno-suppressive tumor microenvironment. In this study, we investigated the interaction between PAMAM dendrimers and M-MDSCs in two murine models of glioblastoma, by examining the cell-level biodistribution kinetics of the systemically injected dendrimers. We found that M-MDSCs in the tumor and lymphoid organs can efficiently endocytose hydroxyl dendrimers. Interestingly, the trafficking of M-MDSCs from the bone marrow to the tumor contributed to the deposition of hydroxyl dendrimers in the tumor. M-MDSCs showed different capacities of endocytosing dendrimers of different functionalities in vivo. This differential uptake was mediated by the unique serum proteins associated with each dendrimer surface functionality. The results of this study set up the framework for developing dendrimer-based immunotherapy to target M-MDSCs for cancer treatment. [Display omitted] • Monocytic MDSC (M-MDSC) in the tumor show high capacity of taking up dendrimers. • The recruitment of M-MDSCs contribute to the dendrimer accumulation in the tumor. • Dendrimer surface chemistry affects their interactions with M-MDSCs. • Dendrimer protein corona mediates the interactions between dendrimers and M-MDSCs. [ABSTRACT FROM AUTHOR]

Psychosocial stress may alter cortisol and/or affect the normal functioning of the immune system. Curcuminoids can promote beneficial effects in neuropsychiatric diseases. We evaluated whether curcumin supplementation for 15 consecutive days (1800 mg/day) would decrease systemic MCP-1, sCD14, and TNF alpha levels in patients with moderate anxiety (n = 81). A total number of 81 subjects were enrolled in this study, divided into the following groups according to their Hamilton scores: a control group including patients without anxiety who were not taking curcumin (Cont, n = 22) and an anxiety group including patients with moderate anxiety (Anx, n = 22). The curcumin-treated patients experienced moderate anxiety, and they take curcumin for 15 consecutive days (Anx-Cur (after), n = 15, 1800 mg/day). An evaluation of 128 patients was conducted, which allowed for their assignment to the study groups according to their scores on Hamilton scale II. The cortisol levels were quantified in salivary samples through ELISA (ng/mL), and malonaldehyde (MDA) levels were measured in plasma via the TBARS assay as an index of lipoperoxidation. Several systemic proinflammatory cytokines (pg/mL: MCP-1, TNF alpha, IL-1 beta) and mediators were quantified through ELISA (pg/mL), including systemic sCD14 levels as a marker of monocyte activation. A two-way bifactorial ANOVA was conducted to evaluate the contributions of the anxiety factor (Anx) and/or curcumin factor (Cur) in all the tested markers, including interactions between both factors. High systemic MCP-1 and elevated sCD14 levels were observed in patients with moderate anxiety, which were reduced with curcumin supplementation. In addition, curcumin prevented cortisol overexpression and decreased MDA levels as an antioxidant response in these patients. Collectively, curcumin presented anti-chemotactic effects by reducing systemic MCP-1 levels in anxiety. Curcumin decreased systemic MCP-1 as well as sCD14 levels in patients with moderate anxiety. [ABSTRACT FROM AUTHOR]

Cardiovascular disease remains one of the leading causes of death globally. Recent advancements in sequencing technologies have led to the identification of a unique population of macrophages within the heart, termed cardiac resident macrophages (CRMs), which exhibit self‐renewal capabilities and play crucial roles in regulating cardiac homeostasis, inflammation, as well as injury and repair processes. This literature review aims to elucidate the origin and phenotypic characteristics of CRMs, comprehensively outline their contributions to cardiac homeostasis and further summarize their functional roles and molecular mechanisms implicated in the onset and progression of cardiovascular diseases. These insights are poised to pave the way for novel therapeutic strategies centred on targeted interventions based on the distinctive properties of resident macrophages. [ABSTRACT FROM AUTHOR]

Chemokine ligands and their receptors have acquired less attention than pro- and anti-inflammatory cytokines in schizophrenia (SCZ). Thus, we aimed to examine the impact of functional polymorphisms of the chemokine genes CXCL10, CXCL16, CXCR4, and CCR2 in the development of SCZ. Using PCR–RFLP, we analyzed the selected polymorphisms in a Tunisian cohort composed of 200 patients with SCZ and 200 healthy controls. Our preliminary data suggest that the minor allele A of CXCL10 rs4256246 is significantly associated with likelihood of SCZ (PAdjusted = 0.00002) and more precisely to paranoid patients with late-onset SCZ (PAdjusted = 0.0007). However, the mutated allele T of CXCR4 rs2228014 showed a significant protective impact against SCZ (PAdjusted = 0.000007) and especially to male sex (PAdjusted = 0.000003). This effect persists among the undifferentiated patients with early-onset SCZ (PAdjusted = 0.002). Following the stratified analyses, CCR2 rs1799864 and CXCL16 rs2277680 were significantly correlated with the clinical symptoms among disorganized patients. As regards haplotype analysis, we noted that GATG haplotype was associated with protection against SCZ (PAdjusted = 0.0087) but the AGCG haplotype was correlated with susceptibility to this disease (PAdjusted = 0.014). Our preliminary results suggested that CXCL10 rs4256246 enhanced susceptibility to SCZ, while CXCR4 rs2228014 seemed to be protective factor. Furthermore, we identified a substantial correlation between CCR2 rs1799864 and CXCL16 rs2277680 with the clinical signs of the disorder. To validate these results and clarify the functional significance of the targeted polymorphisms in SCZ, more independent research is needed. [ABSTRACT FROM AUTHOR]

Simple Summary: Prostate cancer is the second most common cancer among males and is a significant cause of morbidity and mortality. The chemokine C-C motif ligand 2 (CCL2) and its receptor C-C motif chemokine receptor 2 (CCR2) are expressed in prostate cancer but their prognostic value is unclear. This study investigated CCL2 and CCR2 as prognostic biomarkers in locally advanced prostate cancer using the large TROG 03.04 RADAR clinical trial cohort. The results demonstrate that despite being expressed in prostate tumours, CCR2 and CCL2 have no prognostic value in the disease. This study investigated the prognostic value of the chemokine C-C motif ligand 2 (CCL2) and its receptor C-C motif chemokine receptor 2 (CCR2) expression in locally advanced prostate cancer treated with radiotherapy and androgen deprivation using the 10-year outcome data from the TROG 03.04 RADAR clinical trial. CCL2 and CCR2 protein expression in prostate cancer biopsies at the time of diagnosis were quantified by immunohistochemistry and digital quantification. CCR2 protein expression was detected in prostate cancer cells and was associated with prostate-specific antigen serum concentration (p = 0.045). However, neither CCL2 nor CCR2 tissue expression could predict prostate cancer progression, or other clinicopathological parameters including perineural invasion and patient outcome. In serum samples, CCL2 concentration at the time of diagnosis, as assayed by enzyme-linked immunosorbent assay, was significantly higher in patients with prostate cancer compared with benign prostatic hyperplasia (median difference 0.22 ng/mL, 95% CI, 0.17–0.30) (p < 0.0001) and normal controls (median difference 0.13 ng/mL, 95% CI, 0.13–0.17) (p < 0.0001). However, circulating CCL2 was not statistically significant as a predictor of disease progression and patient outcome. In conclusion, this study shows that although CCL2 and CCR2 are expressed in prostate cancer, with an increased level of CCL2 in the serum, neither CCL2 nor CCR2 expression has a clinical prognostic value in locally advanced prostate cancer. [ABSTRACT FROM AUTHOR]

CC chemokine receptor 2 (CCR2) has been linked to many inflammatory and immune diseases, making it a relevant drug target. Yet, all CCR2 antagonists developed so far have failed in clinical trials; thus, novel strategies are needed to target this receptor. Targeted protein degradation represents a novel approach to inhibit protein function by hijacking the cellular degradation machinery, such as the proteasome, to degrade the protein of interest. Here, we aimed to determine the amenability of CCR2 to chemically induced degradation by using a CCR2 fusion protein containing a HaloTag7 and HiBiT tag (CCR2-HaloTag-HiBiT). After characterization of the CCR2 construct, we used luminescence-based assays and immunofluorescence to quantify CCR2 levels, as well as a label-free, phenotypic assay to investigate the functional effect of CCR2 degradation. Treatment with HaloPROTAC3, which selectively degrades HaloTag fusion proteins, led to concentration- and time-dependent degradation of CCR2-HaloTag-HiBiT. HaloPROTAC3 induced degradation via the proteasome, as degradation was fully blocked with proteasomal inhibitors. Finally, functional assays showed that degradation of CCR2-HaloTag-HiBiT leads to a reduced functional response after agonist stimulation. Overall, our results indicate that CCR2 is amenable to targeted degradation, paving the way for the future development of CCR2 chemical degraders. [ABSTRACT FROM AUTHOR]

Background and Aim: Cenicriviroc (CVC) is a CCR2/CCR5 antagonist that has been shown to be effective in the treatment of inflammatory and fibrotic diseases. Our study evaluated its efficacy in colitis. Methods: Mouse models of DSS‐induced acute and chronic colitis were established. The efficacy of CVC in colitis was assessed by disease activity index (DAI) scores, histological assessment of inflammation and fibrosis, and expression assays of key molecules. In in vitro experiments, HT29 cell line was exposed to TNFα to study inflammatory signaling in intestinal epithelial cells. CCD‐18Co colonic myofibroblasts and human primary colonic fibroblasts were activated by TGFβ1 to mimic fibroblast activation. Results: In HT29 cells, CVC significantly reduced mRNA expression of CCL5 (P < 0.01) but had no effect on CCL2. Furthermore, CVC reduced downstream CX3CL1 (P < 0.01) and TNFα (P < 0.05) expression, thereby inhibiting inflammatory progression. In acute colitis mice, CVC significantly reduced DAI scores and serum TNFα levels (P < 0.05) and attenuated colonic inflammation as shown by HE staining. Meanwhile, CVC had no adverse effects on the liver, heart, and kidney of mice. On the other hand, in cellular models of chronic colitis, CVC decreased the expression of fibrosis markers, including FN, CTGF, α‐SMA, and MMP9, and inhibited TGFβ1‐induced fibrotic activation (P < 0.01). In addition, CVC attenuated colonic fibrosis in chronic colitis mice. Moreover, CVC significantly promoted autophagy, which contributed to its regulation of inflammation. Conclusions: CVC significantly inhibited inflammation through CCL5/CCR5 signaling without damaging vital organs and suppressed fibrotic activation in chronic colitis, suggesting its great potential to relieve colonic inflammation and fibrosis. [ABSTRACT FROM AUTHOR]

Abstract Background Since the 1990s, evidence has accumulated that macrophages promote peripheral nerve regeneration and are required for enhancing regeneration in the conditioning lesion (CL) response. After a sciatic nerve injury, macrophages accumulate in the injury site, the nerve distal to that site, and the axotomized dorsal root ganglia (DRGs). In the peripheral nervous system, as in other tissues, the macrophage response is derived from both resident macrophages and recruited monocyte-derived macrophages (MDMs). Unresolved questions are: at which sites do macrophages enhance nerve regeneration, and is a particular population needed. Methods Ccr2 knock-out (KO) and Ccr2 gfp/gfp knock-in/KO mice were used to prevent MDM recruitment. Using these strains in a sciatic CL paradigm, we examined the necessity of MDMs and residents for CL-enhanced regeneration in vivo and characterized injury-induced nerve inflammation. CL paradigm variants, including the addition of pharmacological macrophage depletion methods, tested the role of various macrophage populations in initiating or sustaining the CL response. In vivo regeneration, measured from bilateral proximal test lesions (TLs) after 2 d, and macrophages were quantified by immunofluorescent staining. Results Peripheral CL-enhanced regeneration was equivalent between crush and transection CLs and was sustained for 28 days in both Ccr2 KO and WT mice despite MDM depletion. Similarly, the central CL response measured in dorsal roots was unchanged in Ccr2 KO mice. Macrophages at both the TL and CL, but not between them, stained for the pro-regenerative marker, arginase 1. TL macrophages were primarily CCR2-dependent MDMs and nearly absent in Ccr2 KO and Ccr2 gfp/gfp KO mice. However, there were only slightly fewer Arg1+ macrophages in CCR2 null CLs than controls due to resident macrophage compensation. Zymosan injection into an intact WT sciatic nerve recruited Arg1+ macrophages but did not enhance regeneration. Finally, clodronate injection into Ccr2 gfp KO CLs dramatically reduced CL macrophages. Combined with the Ccr2 gfp KO background, depleting MDMs and TL macrophages, and a transection CL, physically removing the distal nerve environment, nearly all macrophages in the nerve were removed, yet CL-enhanced regeneration was not impaired. Conclusions Macrophages in the sciatic nerve are neither necessary nor sufficient to produce a CL response.

Osteoarthritis (OA) is characterized by a complex interplay of molecular signals orchestrated by the CCL2/CCR2 axis. The pathogenesis of OA has been revealed to be influenced by a multifaceted effect of CCL2/CCR2 signaling on inflammation, cartilage degradation, and joint homeostasis. The CCL2/CCR2 axis promotes immune cell recruitment and tips the balance toward degeneration by influencing chondrocyte behavior. Insights into these intricate pathways will offer novel therapeutic approaches, paving the way for targeted interventions that may redefine OA management in the future. This review article explores the molecular symphony through the lens of the CCL2/CCR2 axis, providing a harmonious blend of current knowledge and future directions on OA treatment. Furthermore, in this study, through a meticulous review of recent research, the key players and molecular mechanisms that amplify the catabolic cascade within the joint microenvironment are identified, and therapeutic approaches to targeting the CCL2/CCR axis are discussed. [ABSTRACT FROM AUTHOR]

BACKGROUND: Viral infections can cause acute respiratory distress syndrome (ARDS), systemic inflammation, and secondary cardiovascular complications. Lung macrophage subsets change during ARDS, but the role of heart macrophages in cardiac injury during viral ARDS remains unknown. Here we investigate how immune signals typical for viral ARDS affect cardiac macrophage subsets, cardiovascular health, and systemic inflammation. METHODS: We assessed cardiac macrophage subsets using immunofluorescence histology of autopsy specimens from 21 patients with COVID-19 with SARS-CoV-2--associated ARDS and 33 patients who died from other causes. In mice, we compared cardiac immune cell dynamics after SARS-CoV-2 infection with ARDS induced by intratracheal instillation of Tolllike receptor ligands and an ACE2 (angiotensin-converting enzyme 2) inhibitor. RESULTS: In humans, SARS-CoV-2 increased total cardiac macrophage counts and led to a higher proportion of CCR2+ (C-C chemokine receptor type 2 positive) macrophages. In mice, SARS-CoV-2 and virus-free lung injury triggered profound remodeling of cardiac resident macrophages, recapitulating the clinical expansion of CCR2+ macrophages. Treating mice exposed to virus-like ARDS with a tumor necrosis factor α--neutralizing antibody reduced cardiac monocytes and inflammatory MHCIIlo CCR2+ macrophages while also preserving cardiac function. Virus-like ARDS elevated mortality in mice with pre-existing heart failure. CONCLUSIONS: Our data suggest that viral ARDS promotes cardiac inflammation by expanding the CCR2+ macrophage subset, and the associated cardiac phenotypes in mice can be elicited by activating the host immune system even without viral presence in the heart. [ABSTRACT FROM AUTHOR]

STUDY QUESTION Does the chemokine/chemokine receptor axis, involved in immune cell trafficking, contribute to the pathology of testicular inflammation and how does activin A modulate this network? SUMMARY ANSWER Testicular chemokines and their receptors (especially those essential for trafficking of monocytes) are elevated in orchitis, and activin A modulates the expression of the chemokine/chemokine receptor network to promote monocyte/macrophage and T cell infiltration into the testes, causing extensive tissue damage. WHAT IS KNOWN ALREADY The levels of CC motif chemokine receptor (CCR)2 and its ligand CC motif chemokine ligand (CCL)2 are increased in experimental autoimmune orchitis (EAO) compared with healthy testes, and mice deficient in CCR2 are protected from EAO-induced tissue damage. Activin A induces CCR2 expression in macrophages, promoting their migration. Moreover, there is a positive correlation between testicular activin A concentration and the severity of autoimmune orchitis. Inhibition of activin A activity by overexpression of follistatin (FST) reduces EAO-induced testicular damage. STUDY DESIGN, SIZE, DURATION EAO was induced in 10–12-week-old male C57BL/6J (wild-type; WT) and B6.129P2-Ccr2tm1Mae/tm1Mae (Ccr2−/−) mice (n = 6). Adjuvant (n = 6) and untreated (n = 6) age-matched control mice were also included. Testes were collected at 50 days after the first immunization with testicular homogenate in complete Freund's adjuvant. In another experimental setup, WT mice were injected with a non-replicative recombinant adeno-associated viral vector carrying a FST315-expressing gene cassette (rAAV-FST315; n = 7–9) or an empty control vector (n = 5) 30 days prior to EAO induction. Appropriate adjuvant (n = 4–5) and untreated (n = 4–6) controls were also examined. Furthermore, human testicular biopsies exhibiting focal leukocytic infiltration and impaired spermatogenesis (n = 17) were investigated. Biopsies showing intact spermatogenesis were included as controls (n = 9). Bone-marrow-derived macrophages (BMDMs) generated from WT mice were treated with activin A (50 ng/ml) for 6 days. Activin-A-treated or untreated BMDMs were then co-cultured with purified mouse splenic T cells for two days to assess chemokine and cytokine production. PARTICIPANTS/MATERIALS, SETTING, METHODS Quantitative real-time PCR (qRT-PCR) was used to analyze the expression of chemokines in total testicular RNA collected from mice. Immunofluorescence staining was used to detect activin A, F4/80, and CD3 expression in mouse testes. The expression of chemokine/chemokine-receptor-encoding genes was examined in human testicular biopsies by qRT-PCR. Correlations between chemokine expression levels and either the immune cell infiltration density or the mean spermatogenesis score were analyzed. Immunofluorescence staining was used to evaluate the expression of CD68 and CCR2 in human testicular biopsies. RNA isolated from murine BMDMs was used to characterize these cells in terms of their chemokine/chemokine receptor expression levels. Conditioned media from co-cultures of BMDMs and T cells were collected to determine chemokine levels and the production of pro-inflammatory cytokines tumor necrosis factor (TNF) and interferon (IFN)-γ by T cells. MAIN RESULTS AND THE ROLE OF CHANCE Induction of EAO in the testes of WT mice increased the expression of chemokine receptors such as Ccr1 (P  < 0.001), Ccr2 (P  < 0.0001), Ccr3 (P  < 0.0001), Ccr5 (P  < 0.0001), CXC motif chemokine receptor (Cxcr)3 (P  < 0.01), and CX3C motif chemokine receptor (Cx3cr)1 (P  < 0.001), as well as that of most of their ligands. Ccr2 deficiency reversed some of the changes associated with EAO by reducing the expression of Ccr1 (P  < 0.0001), Ccr3 (P  < 0.0001), Ccr5 (P  < 0.01), Cxcr3 (P  < 0.001), and Cx3cr1 (P  < 0.0001). Importantly, the biopsies showing impaired spermatogenesis and concomitant focal leukocytic infiltration exhibited higher expression of CCL2 (P  < 0.01), CCR1 (P  < 0.05), CCR2 (P  < 0.001), and CCR5 (P  < 0.001) than control biopsies with no signs of inflammation and intact spermatogenesis. The gene expression of CCR2 and its ligand CCL2 correlated positively with the immune cell infiltration density (P  < 0.05) and negatively with the mean spermatogenesis score (P  < 0.001). Moreover, CD68+ macrophages expressing CCR2 were present in human testes with leukocytic infiltration with evidence of tubular damage. Treatment of BMDMs, as surrogates for testicular macrophages, with activin A increased their expression of Ccr1 , Ccr2 , and Ccr5 while reducing their expression of Ccl2 , Ccl3 , Ccl4 , Ccl6 , Ccl7 Ccl8 , and Ccl12. These findings were validated in vivo , by showing that inhibiting activin A activity by overexpressing FST in EAO mice decreased the expression of Ccr2 (P  < 0.05) and Ccr5 (P  < 0.001) in the testes. Interestingly, co-culturing activin-A-treated BMDMs and T cells reduced the levels of CCL2 (P  < 0.05), CCL3/4 (P  < 0.01), and CCL12 (P  < 0.05) in the medium and attenuated the production of TNF (P  < 0.05) by T cells. The majority of cells secreting activin A in EAO testes were identified as macrophages. LARGE SCALE DATA N/A. LIMITATIONS, REASONS FOR CAUTION BMDMs were used as surrogates for testicular macrophages. Hence, results obtained from the in vitro experiments might not be fully representative of the situation in the testes in vivo. Moreover, since total RNA was extracted from the testicular tissue to examine chemokine expression, the contributions of individual cell types as producers of specific chemokines may have been overlooked. WIDER IMPLICATIONS OF THE FINDINGS Our data indicate that macrophages are implicated in the development and progression of testicular inflammation by expressing CCR2 and activin A, which ultimately remodel the chemokine/chemokine receptor network and recruit other immune cells to the site of inflammation. Consequently, inhibition of CCR2 or activin A could serve as a potential therapeutic strategy for reducing testicular inflammation. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by the International Research Training Group in 'Molecular pathogenesis on male reproductive disorders', a collaboration between Justus Liebig University (Giessen) and Monash University (Melbourne) (GRK1871/1-2) funded by the Deutsche Forschungsgemeinschaft and Monash University, a National Health and Medical Research Council of Australia Ideas Grant (1184867), and the Victorian Government's Operational Infrastructure Support Programme. The authors declare no competing financial interests. [ABSTRACT FROM AUTHOR]

Background: Since the 1990s, evidence has accumulated that macrophages promote peripheral nerve regeneration and are required for enhancing regeneration in the conditioning lesion (CL) response. After a sciatic nerve injury, macrophages accumulate in the injury site, the nerve distal to that site, and the axotomized dorsal root ganglia (DRGs). In the peripheral nervous system, as in other tissues, the macrophage response is derived from both resident macrophages and recruited monocyte-derived macrophages (MDMs). Unresolved questions are: at which sites do macrophages enhance nerve regeneration, and is a particular population needed. Methods: Ccr2 knock-out (KO) and Ccr2gfp/gfp knock-in/KO mice were used to prevent MDM recruitment. Using these strains in a sciatic CL paradigm, we examined the necessity of MDMs and residents for CL-enhanced regeneration in vivo and characterized injury-induced nerve inflammation. CL paradigm variants, including the addition of pharmacological macrophage depletion methods, tested the role of various macrophage populations in initiating or sustaining the CL response. In vivo regeneration, measured from bilateral proximal test lesions (TLs) after 2 d, and macrophages were quantified by immunofluorescent staining. Results: Peripheral CL-enhanced regeneration was equivalent between crush and transection CLs and was sustained for 28 days in both Ccr2 KO and WT mice despite MDM depletion. Similarly, the central CL response measured in dorsal roots was unchanged in Ccr2 KO mice. Macrophages at both the TL and CL, but not between them, stained for the pro-regenerative marker, arginase 1. TL macrophages were primarily CCR2-dependent MDMs and nearly absent in Ccr2 KO and Ccr2gfp/gfp KO mice. However, there were only slightly fewer Arg1+ macrophages in CCR2 null CLs than controls due to resident macrophage compensation. Zymosan injection into an intact WT sciatic nerve recruited Arg1+ macrophages but did not enhance regeneration. Finally, clodronate injection into Ccr2gfp KO CLs dramatically reduced CL macrophages. Combined with the Ccr2gfp KO background, depleting MDMs and TL macrophages, and a transection CL, physically removing the distal nerve environment, nearly all macrophages in the nerve were removed, yet CL-enhanced regeneration was not impaired. Conclusions: Macrophages in the sciatic nerve are neither necessary nor sufficient to produce a CL response. [ABSTRACT FROM AUTHOR]

Investigating the role of podocytes in proteinuric disease is imperative to address the increasing global burden of chronic kidney disease (CKD). Studies strongly implicate increased levels of monocyte chemoattractant protein-1 (MCP-1/CCL2) in proteinuric CKD. Since podocytes express the receptor for MCP-1 (i.e., CCR2), we hypothesized that podocyte-specific MCP-1 production in response to stimuli could activate its receptor in an autocrine manner, leading to further podocyte injury. To test this hypothesis, we generated podocyte-specific MCP-1 knockout mice (Podo-Mcp-1fl/fl) and exposed them to proteinuric injury induced by either angiotensin II (Ang II; 1.5 mg/kg/d, osmotic minipump) or Adriamycin (Adr; 18 mg/kg, intravenous bolus). At baseline, there were no between-group differences in body weight, histology, albuminuria, and podocyte markers. After 28 days, there were no between-group differences in survival, change in body weight, albuminuria, kidney function, glomerular injury, and tubulointerstitial fibrosis. The lack of protection in the knockout mice suggests that podocyte-specific MCP-1 production is not a major contributor to either Ang II- or Adr-induced glomerular disease, implicating that another cell type is the source of pathogenic MCP-1 production in CKD. [ABSTRACT FROM AUTHOR]

Background and PurposeThe risk of skin injuries in space is increasing with longer space missions and a growing astronaut population. This highlights the importance of understanding the adverse effects of weightlessness on wound healing. The objective of this research was to examine the therapeutic potential of Low-Level Light Therapy (LLLT) on skin healing processes under simulated microgravity (SMG) conditions and uncover the underlying molecular mechanisms, thus providing innovative solutions and a sound theoretical basis for space skin injuries.MethodsHindlimb unloading (HU) mice models were used to simulate weightlessness conditions, with or without a complete management of LLLT for 14 days. A systematic testing consisting of HE, Masson and immunohistochemical staining was performed against the standardized mouse tissue specimens. In vitro assessment of cellular biological functions under SMG conditions was carried out in the rotation system of culture (RSOC) using HaCaT and NIH3T3 cell-lines.ResultsUnder SMG conditions, LLLT significantly reduced skin wound area in HU mice, especially on Days 10 (p < 0.001), accompanied by increased collagen deposition and elevated levels of Ki67 and CD31. Moreover, LLLT showed impressive anti-inflammatory effects represented by the reduced in pro-inflammatory markers including LY6G, F4/80 and CD86, as well as the decreased levels of IL-1β, IL-6 and TNF-α. Conversely, an elevation in the anti-inflammatory marker CD206 was observed. By employing bioinformatics technology, we further found the PI3K/AKT signaling was prominent in the KEGG pathway analysis and CCR2 acted as a hub gene in the interaction network. Therefore, we demonstrated that LLLT could enhance the phosphorylation of PI3K/AKT and reduce CCR2 expression under SMG conditions, while CCR2 knockdown promoted the phosphorylation of PI3K/AKT, suggesting an important role of CCR2/PI3K/AKT signal axis in LLLT-accelerated wound healing under SMG conditions.ConclusionLLLT induced activation of the PI3K/AKT signaling pathway through suppression of CCR2 expression, which significantly enhanced skin wound healing under SMG conditions.s.

Introduction: Indole-3-carbinol (I3C) is found in cruciferous vegetables and used as a dietary supplement. It is known to act as a ligand for aryl hydrocarbon receptor (AhR). In the current study, we investigated the role of AhR and the ability of I3C to attenuate LPS-induced Acute Respiratory Distress Syndrome (ARDS). Methods: To that end, we induced ARDS in wild-type C57BL/6 mice, Ccr2gfp/gfp KI/KO mice (mice deficient in the CCR2 receptor), and LyZcreAhRfl/fl mice (mice deficient in the AhR on myeloid linage cells). Additionally, mice were treated with I3C (65 mg/kg) or vehicle to investigate its efficacy to treat ARDS. Results: I3C decreased the neutrophils expressing CXCR2, a receptor associated with neutrophil recruitment in the lungs. In addition, LPS-exposed mice treated with I3C revealed downregulation of CCR2+ monocytes in the lungs and lowered CCL2 (MCP-1) protein levels in serum and bronchoalveolar lavage fluid. Loss of CCR2 on monocytes blocked the recruitment of CXCR2+ neutrophils and decreased the total number of immune cells in the lungs during ARDS. In addition, loss of the AhR on myeloid linage cells ablated I3Cmediated attenuation of CXCR2+ neutrophils and CCR2+ monocytes in the lungs from ARDS animals. Interestingly, scRNASeq showed that in macrophage/monocyte cell clusters of LPS-exposed mice, I3C reduced the expression of CXCL2 and CXCL3, which bind to CXCR2 and are involved in neutrophil recruitment to the disease site. Discussion: These findings suggest that CCR2+ monocytes are involved in the migration and recruitment of CXCR2+ neutrophils during ARDS, and the AhR ligand, I3C, can suppress ARDS through the regulation of immune cell trafficking. [ABSTRACT FROM AUTHOR]

Obesity and metabolic diseases, such as insulin resistance, type 2 diabetes, nonalcoholic fatty liver disease, and cardiovascular ailments, represent formidable global health challenges, bearing considerable implications for both morbidity and mortality rates. It has become increasingly evident that chronic, low‐grade inflammation plays a pivotal role in the genesis and advancement of these conditions. The involvement of C‐C chemokine ligand 2 (CCL2) and its corresponding receptor, C‐C chemokine receptor 2 (CCR2), has been extensively documented in numerous inflammatory maladies. Recent evidence indicates that the CCL2/CCR2 pathway extends beyond immune cell recruitment and inflammation, exerting a notable influence on the genesis and progression of metabolic syndrome. The present review seeks to furnish a comprehensive exposition of the CCL2‐CCR2 signaling axis within the context of obesity and metabolic disorders, elucidating its molecular mechanisms, functional roles, and therapeutic implications. [ABSTRACT FROM AUTHOR]

Background: The chemokine ligand CCL2 and its cognate receptor CCR2 have been implicated in the pathogenesis of a wide variety of diseases. Hence, the inhibition of the CCL2/CCR2 signaling pathway has been of great attention in recent studies. Among suggested medications, statins known as HMG-COA reductase inhibitors with their pleiotropic effects are widely under investigation. Method: A comprehensive literature search on Scopus and PubMed databases was conducted using the keywords 'CCL2', 'CCR2', 'monocyte chemoattractant protein-1', 'HMG-COA reductase inhibitor', and 'statin'. Both experimental and clinical studies measuring CCL2/CCR2 expressions following statin therapy were identified excluding the ones focused on cardiovascular diseases. Results: Herein, we summarized the effects of statins on CCL2 and CCR2 expression in various pathologic conditions including immune-mediated diseases, nephropathies, diabetes, rheumatic diseases, neuroinflammation, inflammatory bowel diseases, gynecologic diseases, and cancers. Conclusion: For the most part, statins play an inhibitory role on the CCL2–CCR2 axis which implies their potential to be further developed as therapeutic options in non-cardiovascular diseases either alone or in combination with other conventional treatments. However, the existing literature mostly focused on experimental models and is therefore inadequate to reach a conclusion. [ABSTRACT FROM AUTHOR]

Osteoarthritis (OA) is characterized by a complex interplay of molecular signals orchestrated by the CCL2/CCR2 axis. The pathogenesis of OA has been revealed to be influenced by a multifaceted effect of CCL2/CCR2 signaling on inflammation, cartilage degradation, and joint homeostasis. The CCL2/CCR2 axis promotes immune cell recruitment and tips the balance toward degeneration by influencing chondrocyte behavior. Insights into these intricate pathways will offer novel therapeutic approaches, paving the way for targeted interventions that may redefine OA management in the future. This review article explores the molecular symphony through the lens of the CCL2/CCR2 axis, providing a harmonious blend of current knowledge and future directions on OA treatment. Furthermore, in this study, through a meticulous review of recent research, the key players and molecular mechanisms that amplify the catabolic cascade within the joint microenvironment are identified, and therapeutic approaches to targeting the CCL2/CCR axis are discussed.

CC chemokine receptor 2 (CCR2) has been linked to many inflammatory and immune diseases, making it a relevant drug target. Yet, all CCR2 antagonists developed so far have failed in clinical trials; thus, novel strategies are needed to target this receptor. Targeted protein degradation represents a novel approach to inhibit protein function by hijacking the cellular degradation machinery, such as the proteasome, to degrade the protein of interest. Here, we aimed to determine the amenability of CCR2 to chemically induced degradation by using a CCR2 fusion protein containing a HaloTag7 and HiBiT tag (CCR2-HaloTag-HiBiT). After characterization of the CCR2 construct, we used luminescence-based assays and immunofluorescence to quantify CCR2 levels, as well as a label-free, phenotypic assay to investigate the functional effect of CCR2 degradation. Treatment with HaloPROTAC3, which selectively degrades HaloTag fusion proteins, led to concentration- and time-dependent degradation of CCR2-HaloTag-HiBiT. HaloPROTAC3 induced degradation via the proteasome, as degradation was fully blocked with proteasomal inhibitors. Finally, functional assays showed that degradation of CCR2-HaloTag-HiBiT leads to a reduced functional response after agonist stimulation. Overall, our results indicate that CCR2 is amenable to targeted degradation, paving the way for the future development of CCR2 chemical degraders.

Back pain and radiculopathy caused by disc herniation are major health issues worldwide. While macrophages are key players in disc herniation induced inflammation, their roles and origins in disease progression remain unclear. We aim to study the roles of monocytes and derivatives in a mouse model of disc herniation. Using a CCR2-CreER; R26R-EGFP (Ai6) transgenic mouse strain, we fate-mapped C-C chemokine receptor type 2 (CCR2) expressing monocytes and derivatives at disc herniation sites, and employed a CCR2RFP/RFP mouse strain and a CCR2-specific antagonist to study the effects of CCR2+ monocytes on local inflammatory responses, pain level, and disc degeneration by immunostaining, flow cytometry, and histology. CCR2+ monocytes (GFP+) increased at the sites of disc hernia over postoperative day 4, 6, and 9 in CCR2-CreER; Ai6 mice. F4/80+ cells increased, and meanwhile, CD11b+ cells trended downward. Co-localization analysis revealed that both GFP+CD11b+ and GFP+F4/80+ constituted the majority of CD11b+ and F4/80+ cells at disc hernia sites. Fluorescence activated cell sorter purified GFP+ cells exhibited higher cytokine expressions than GFP- cells. Inhibition of CCR2 signaling reduced infiltration of monocytes and macrophages, alleviated pain, maintained disc height, and reduced osteoclast activity in adjacent cortical bone for up to 1 month. Our findings suggest that circulating CCR2+ monocytes play important roles in initiating and promoting the local inflammatory responses, pain sensitization, and degenerative changes after disc herniation, and thus may serve as therapeutic targets for disc herniation induced back and leg pain. [Display omitted] [ABSTRACT FROM AUTHOR]

Monocytes play a significant role in the pathogenesis of most inflammatory diseases, including autoimmune diseases. Herein, different subpopulations of monocytes often play differential, partially antagonistic roles, in the regulation of tissue populations. Pemphigoid diseases constitute a group of autoimmune blistering skin diseases featuring a marked infiltration of the dermis with immune cells, including monocytes. The monocyte subsets infiltrating the skin, however, have largely remained elusive. Monocyte adhesion and recruitment into the inflamed tissues are regulated by chemokine receptors, most prominently by CCR2 and CX3CR1. To delineate the involvement of monocyte populations in autoimmune blistering skin diseases, we spatiotemporally monitored the dynamic spectrum of monocyte populations that infiltrate the inflamed skin using multiphoton intravital imaging and reporter mice for chemokine receptors. Experimental epidermolysis bullosa acquisita (EBA) was induced by injection of anti-murine type VII collagen (amCOLVII) IgG into the Csf1rEGFP-reporter mice, where circulating myeloid cells, such as monocytes and neutrophils, express an EGFP. EGFP+ cells, including neutrophils and monocytes, were present in the skin, immediately after the deposition of the amCOLVII antibody at the dermal-epidermal junction. To investigate the recruitment and involvement of different monocyte-derived cell populations in the disease course further, EBA was induced in CCR2RFP/+-reporter and CX3CR1GFP/+-reporter mice. A comparable distribution of red fluorescent protein (RFP)+ or green fluorescent protein (GFP)+ was found in both diseased mice and their respective controls over time, indicating the similar recruitment of monocytes into the skin following the binding of autoantibodies. Experiments were extended to the CCR2RFP/RFP-deficient and CX3CR1GFP/GFP-deficient mice to determine whether monocyte recruitment and disease severity are compromised in the absence of the receptor. A comparable pattern was seen in the recruitment of monocytes into the skin in both reporter and deficient mice. However, in contrast to similar disease severity between CX3CR1-deficient and reporter mice, CCR2-deficient mice developed significantly less disease than CCR2-reporter mice, as indicated by the percentage of affected area of ears. Collectively, our observations indicate that while CCR2 and CX3CR1 receptors are not involved in the recruitment of monocytes into the skin, CCR2 deficiency is associated with improved disease outcomes in experimental EBA in mice. [ABSTRACT FROM AUTHOR]

Objective: Approximately 80% of people with Coronavirus disease 2019 (COVID-19) are asymptomatic, and only a small proportion of cases show serious consequences leading to hospitalization. The interplay between chemokines and their receptors can affect the severity of several infectious diseases, such as severe acute respiratory syndrome and Middle East Respiratory Syndrome. The interplay of monocyte chemoattractant protein-1 (MCP-1) with its receptor C-C motif chemokine receptor 2 (CCR2) may affect the pathogenesis of COVID-19 by functioning in the dispatch of lymphocytes and monocytes/macrophages to the infection site. Materials and Methods: The serum MCP-1 and CCR2 concentrations were measured using the enzyme-linked immunosorbent assay (ELISA) in 49 asymptomatic, 50 severe, and 57 critical COVID-19 cases. Results: Serum MCP-1 levels were considerably higher in critical cases than in cases in the other two groups, suggesting an increased risk for disease severity (p = 0.008; p = 0.01, respectively). Serum CCR2 levels were significantly higher in asymptomatic cases than in critical cases suggesting a protective role against disease severity (p = 0.001). Conclusion: MCP-1 and CCR2 may be candidate biomarkers for the prediction of disease severity. Therefore, by measuring serum levels of MCP-1 and CCR2 early, the disease course can be predicted, and necessary precautions can be taken before the disease becomes severe. [ABSTRACT FROM AUTHOR]

Tryptophan and its derivatives perform a variety of biological functions; however, the role and specific mechanism of many tryptophan derivatives in intestinal inflammation remain largely unclear. Here, we identified that an Escherichia coli strain (Ec-TMU) isolated from the feces of tinidazole-treated individuals, and indole-3-lactic acid (ILA) in its supernatant, decreased the susceptibility of mice to dextran sulfate sodium-induced colitis. Ec-TMU and ILA contribute to the relief of colitis by inhibiting the production of epithelial CCL2/7, thereby reducing the accumulation of inflammatory macrophages in vitro and in vivo. Mechanistically, ILA downregulates glycolysis, NF-κB, and HIF signaling pathways via the aryl hydrocarbon receptor, resulting in decreased CCL2/7 production in epithelial cells. Clinical evidence suggests that the fecal ILA level is negatively correlated with the progression indicator of inflammatory bowel diseases. These results demonstrate that ILA has the potential to regulate intestinal homeostasis by modulating epithelium-macrophage interactions. [ABSTRACT FROM AUTHOR]

IntroductionIndole-3-carbinol (I3C) is found in cruciferous vegetables and used as a dietary supplement. It is known to act as a ligand for aryl hydrocarbon receptor (AhR). In the current study, we investigated the role of AhR and the ability of I3C to attenuate LPS-induced Acute Respiratory Distress Syndrome (ARDS).MethodsTo that end, we induced ARDS in wild-type C57BL/6 mice, Ccr2gfp/gfp KI/KO mice (mice deficient in the CCR2 receptor), and LyZcreAhRfl/fl mice (mice deficient in the AhR on myeloid linage cells). Additionally, mice were treated with I3C (65 mg/kg) or vehicle to investigate its efficacy to treat ARDS.ResultsI3C decreased the neutrophils expressing CXCR2, a receptor associated with neutrophil recruitment in the lungs. In addition, LPS-exposed mice treated with I3C revealed downregulation of CCR2+ monocytes in the lungs and lowered CCL2 (MCP-1) protein levels in serum and bronchoalveolar lavage fluid. Loss of CCR2 on monocytes blocked the recruitment of CXCR2+ neutrophils and decreased the total number of immune cells in the lungs during ARDS. In addition, loss of the AhR on myeloid linage cells ablated I3C-mediated attenuation of CXCR2+ neutrophils and CCR2+ monocytes in the lungs from ARDS animals. Interestingly, scRNASeq showed that in macrophage/monocyte cell clusters of LPS-exposed mice, I3C reduced the expression of CXCL2 and CXCL3, which bind to CXCR2 and are involved in neutrophil recruitment to the disease site.DiscussionThese findings suggest that CCR2+ monocytes are involved in the migration and recruitment of CXCR2+ neutrophils during ARDS, and the AhR ligand, I3C, can suppress ARDS through the regulation of immune cell trafficking.

Notch-RBP-J signaling plays an essential role in the maintenance of myeloid homeostasis. However, its role in monocyte cell fate decisions is not fully understood. Here, we showed that conditional deletion of transcription factor RBP-J in myeloid cells resulted in marked accumulation of blood Ly6Clo monocytes that highly expressed chemokine receptor CCR2. Bone marrow transplantation and parabiosis experiments revealed a cell-intrinsic requirement of RBP-J for controlling blood Ly6CloCCR2hi monocytes. RBP-J-deficient Ly6Clo monocytes exhibited enhanced capacity competing with wildtype counterparts in blood circulation. In accordance with alterations of circulating monocytes, RBP-J deficiency led to markedly increased population of lung tissues with Ly6Clo monocytes and CD16.2+ interstitial macrophages. Furthermore, RBP-J deficiency-associated phenotypes could be genetically corrected by further deleting Ccr2 in myeloid cells. These results demonstrate that RBP-J functions as a crucial regulator of blood Ly6Clo monocytes and thus derived lung-resident myeloid populations, at least in part through regulation of CCR2.

Investigating the role of podocytes in proteinuric disease is imperative to address the increasing global burden of chronic kidney disease (CKD). Studies strongly implicate increased levels of monocyte chemoattractant protein-1 (MCP-1/CCL2) in proteinuric CKD. Since podocytes express the receptor for MCP-1 (i.e., CCR2), we hypothesized that podocyte-specific MCP-1 production in response to stimuli could activate its receptor in an autocrine manner, leading to further podocyte injury. To test this hypothesis, we generated podocyte-specific MCP-1 knockout mice (Podo-Mcp-1fl/fl) and exposed them to proteinuric injury induced by either angiotensin II (Ang II; 1.5 mg/kg/d, osmotic minipump) or Adriamycin (Adr; 18 mg/kg, intravenous bolus). At baseline, there were no between-group differences in body weight, histology, albuminuria, and podocyte markers. After 28 days, there were no between-group differences in survival, change in body weight, albuminuria, kidney function, glomerular injury, and tubulointerstitial fibrosis. The lack of protection in the knockout mice suggests that podocyte-specific MCP-1 production is not a major contributor to either Ang II- or Adr-induced glomerular disease, implicating that another cell type is the source of pathogenic MCP-1 production in CKD.

Monocytes play a significant role in the pathogenesis of most inflammatory diseases, including autoimmune diseases. Herein, different subpopulations of monocytes often play differential, partially antagonistic roles, in the regulation of tissue populations. Pemphigoid diseases constitute a group of autoimmune blistering skin diseases featuring a marked infiltration of the dermis with immune cells, including monocytes. The monocyte subsets infiltrating the skin, however, have largely remained elusive. Monocyte adhesion and recruitment into the inflamed tissues are regulated by chemokine receptors, most prominently by CCR2 and CX3CR1. To delineate the involvement of monocyte populations in autoimmune blistering skin diseases, we spatiotemporally monitored the dynamic spectrum of monocyte populations that infiltrate the inflamed skin using multiphoton intravital imaging and reporter mice for chemokine receptors. Experimental epidermolysis bullosa acquisita (EBA) was induced by injection of anti-murine type VII collagen (amCOLVII) IgG into the Csf1rEGFP-reporter mice, where circulating myeloid cells, such as monocytes and neutrophils, express an EGFP. EGFP+ cells, including neutrophils and monocytes, were present in the skin, immediately after the deposition of the amCOLVII antibody at the dermal-epidermal junction. To investigate the recruitment and involvement of different monocyte-derived cell populations in the disease course further, EBA was induced in CCR2RFP/+-reporter and CX3CR1GFP/+-reporter mice. A comparable distribution of red fluorescent protein (RFP)+ or green fluorescent protein (GFP)+ was found in both diseased mice and their respective controls over time, indicating the similar recruitment of monocytes into the skin following the binding of autoantibodies. Experiments were extended to the CCR2RFP/RFP-deficient and CX3CR1GFP/GFP-deficient mice to determine whether monocyte recruitment and disease severity are compromised in the absence of the receptor. A comparable pattern was seen in the recruitment of monocytes into the skin in both reporter and deficient mice. However, in contrast to similar disease severity between CX3CR1-deficient and reporter mice, CCR2-deficient mice developed significantly less disease than CCR2-reporter mice, as indicated by the percentage of affected area of ears. Collectively, our observations indicate that while CCR2 and CX3CR1 receptors are not involved in the recruitment of monocytes into the skin, CCR2 deficiency is associated with improved disease outcomes in experimental EBA in mice.

Migraine, especially chronic migraine, is highly debilitating and still lacks effective treatment. The persistent headache arises from activation and sensitization of primary afferent neurons in the trigeminovascular pathway, but the underlying mechanisms remain incompletely understood. Animal studies indicate that signalling through chemokine C-C motif ligand 2 (CCL2) and C-C motif chemokine receptor 2 (CCR2) mediates the development of chronic pain after tissue or nerve injury. Some migraine patients had elevated CCL2 levels in CSF or cranial periosteum. However, whether the CCL2-CCR2 signalling pathway contributes to chronic migraine is not clear. Here, we modelled chronic headache with repeated administration of nitroglycerin (NTG, a reliable migraine trigger in migraineurs) and found that both Ccl2 and Ccr2 mRNA were upregulated in dura and trigeminal ganglion (TG) tissues that are implicated in migraine pathophysiology. In Ccl2 and Ccr2 global knockout mice, repeated NTG administration did not evoke acute or persistent facial skin hypersensitivity as in wild-type mice. Intraperitoneal injection of CCL2 neutralizing antibodies inhibited chronic headache-related behaviours induced by repeated NTG administration and repetitive restraint stress, suggesting that the peripheral CCL2-CCR2 signalling mediates headache chronification. We found that CCL2 was mainly expressed in TG neurons and cells associated with dura blood vessels, whereas CCR2 was expressed in subsets of macrophages and T cells in TG and dura but not in TG neurons under both control and disease states. Deletion of Ccr2 gene in primary afferent neurons did not alter NTG-induced sensitization, but eliminating CCR2 expression in either T cells or myeloid cells abolished NTG-induced behaviours, indicating that both CCL2-CCR2 signalling in T cells and macrophages are required to establish chronic headache-related sensitization. At cellular level, repeated NTG administration increased the number of TG neurons that responded to calcitonin-gene-related peptide (CGRP) and pituitary adenylate cyclase activating polypeptide (PACAP) as well as the production of CGRP in wild-type but not Ccr2 global knockout mice. Lastly, co-administration of CCL2 and CGRP neutralizing antibodies was more effective in reversing NTG-induced behaviours than individual antibodies. Taken together, these results suggest that migraine triggers activate CCL2-CCR2 signalling in macrophages and T cells. This consequently enhances both CGRP and PACAP signalling in TG neurons, ultimately leading to persistent neuronal sensitization underlying chronic headache. Our work not only identifies the peripheral CCL2 and CCR2 as potential targets for chronic migraine therapy, but also provides proof-of-concept that inhibition of both peripheral CGRP and CCL2-CCR2 signalling is more effective than targeting either pathway alone. [ABSTRACT FROM AUTHOR]

Homeostasis of the host immune system is regulated by white blood cells with a variety of cell surface receptors for cytokines. Chemotactic cytokines (chemokines) activate their receptors to evoke the chemotaxis of immune cells in homeostatic migrations or inflammatory conditions towards inflamed tissue or pathogens. Dysregulation of the immune system leading to disorders such as allergies, autoimmune diseases, or cancer requires efficient, fast-acting drugs to minimize the long-term effects of chronic inflammation. Here, we performed structure-based virtual screening (SBVS) assisted by the Keras/TensorFlow neural network (NN) to find novel compound scaffolds acting on three chemokine receptors: CCR2, CCR3, and one CXC receptor, CXCR3. Keras/TensorFlow NN was used here not as a typically used binary classifier but as an efficient multi-class classifier that can discard not only inactive compounds but also low- or medium-activity compounds. Several compounds proposed by SBVS and NN were tested in 100 ns all-atom molecular dynamics simulations to confirm their binding affinity. To improve the basic binding affinity of the compounds, new chemical modifications were proposed. The modified compounds were compared with known antagonists of these three chemokine receptors. Known CXCR3 compounds were among the top predicted compounds; thus, the benefits of using Keras/TensorFlow in drug discovery have been shown in addition to structure-based approaches. Furthermore, we showed that Keras/TensorFlow NN can accurately predict the receptor subtype selectivity of compounds, for which SBVS often fails. We cross-tested chemokine receptor datasets retrieved from ChEMBL and curated datasets for cannabinoid receptors. The NN model trained on the cannabinoid receptor datasets retrieved from ChEMBL was the most accurate in the receptor subtype selectivity prediction. Among NN models trained on the chemokine receptor datasets, the CXCR3 model showed the highest accuracy in differentiating the receptor subtype for a given compound dataset. [ABSTRACT FROM AUTHOR]

γδT cells are important innate immune cells that are involved in the occurrence and development of autoimmune diseases such as systemic lupus erythematosus (SLE). Lupus nephritis (LN) is a serious complication of SLE, characterized by the accumulation of immune cells (including γδT cells) in the target organs to participate in the disease process. Therefore, clarifying how γδT cells chemotactically migrate to target organs may be a key to developing therapeutic methods against LN. Enzyme‐linked immunosorbent assay (ELISA) was used to detect serum levels of chemokines in LN patients and healthy controls. Real‐time polymerase chain reaction (RT‐PCR) and flow cytometry were used to measure the expression of chemokine receptors on the surface of γδT cells. The chemotactic migration ability of γδT cells was detected by Transwell assay. Signalling pathway activation of γδT cells was detected by Automated Capillary Electrophoresis Immunoassay and flow cytometry. The serum levels of chemokines, including monocyte chemoattractant protein‐1 (MCP‐1) in LN patients, were significantly increased. CCR2, the receptor of MCP‐1, was also highly expressed on the surface of peripheral γδT cells in LN patients. In addition, the exogenous addition of MCP‐1 can enhance chemotactic migration of γδT cells in LN patients. MCP‐1 could activate STAT3 signalling in LN patients' peripheral γδT cells. γδT cells might participate in the pathogenesis of LN through MCP‐1/CCR2 axis. This finding provides new opportunities for developing treatment methods against LN by targeting MCP‐1/CCR2 axis. [ABSTRACT FROM AUTHOR]

Monocytes in hepatitis C virus (HCV) infection play a critical role in chronic liver inflammation and fibrosis. We studied circulating monocytes and monocyte receptors in patients with HCV infection who were naive to treatment and those who received direct acting antiviral therapy and achieved sustained virological response. CD64+ CCR2+ (M1-like) and CD206+ CD163+ CX3CR1+ (M2-like) monocyte numbers and receptor expression were evaluated by flow cytometry. Higher expression of the monocyte chemokine receptor CCR2 predicted the severity of liver fibrosis, independent of successful treatment and viral clearance (R2 = 0.235, p = 0.002), whereas monocyte CX3CR1 expression was lower in both treated and untreated patients compared with controls (p = 0.011). The expression of the scavenger receptor CD163 was lower in patients with successful treatment (p = 0.005), supporting its role as a marker of treatment response. CD64+ CCR2+ (M1-like) and CD206+ CD163+ CX3CR1+ (M2-like) monocyte numbers were not altered with fibrosis progression or treatment response. Our findings reflect the diverse functions of monocytes in liver inflammation, fibrosis, and therapy. However, HCV clearance did not lead to complete monocyte reconstitution. Targeting monocytes and their chemokine receptors bears therapeutic potential to reduce liver fibrosis and improve disease outcome. [ABSTRACT FROM AUTHOR]

Obesity rates continue to rise, and obese individuals are at higher risk for multiple types of cancer, including breast cancer. Obese mammary fat is a site of chronic, macrophage‐driven inflammation, which enhances fibrosis within adipose tissue. Elevated fibrosis within the mammary gland may contribute to risk for obesity‐associated breast cancer. To understand how inflammation due to obesity enhanced fibrosis within mammary tissue, we utilized a high‐fat diet model of obesity and elimination of CCR2 signaling in mice to identify changes in immune cell populations and their impact on fibrosis. We observed that obesity increased a population of CD11b+ cells with the ability to form myofibroblast‐like colonies in vitro. This population of CD11b+ cells is consistent with fibrocytes, which have been identified in wound healing and chronic inflammatory diseases but have not been examined in obesity. In CCR2‐null mice, which have limited ability to recruit myeloid lineage cells into obese adipose tissue, we observed reduced mammary fibrosis and diminished fibrocyte colony formation in vitro. Transplantation of myeloid progenitor cells, which are the cells of origin for fibrocytes, into the mammary glands of obese CCR2‐null mice resulted in significantly increased myofibroblast formation. Gene expression analyses of the myeloid progenitor cell population from obese mice demonstrated enrichment for genes associated with collagen biosynthesis and extracellular matrix remodeling. Together these results show that obesity enhances recruitment of fibrocytes to promote obesity‐induced fibrosis in the mammary gland. [ABSTRACT FROM AUTHOR]

Background: Persistent liver damage contributes to the development of liver fibrosis, marked by an accumulation of extracellular matrix. Macrophages play a pivotal role in this process, with the CCL2-CCR2 and CX3CR1-CX3CL1 axes serving as key regulators of macrophage recruitment, liver infiltration, and differentiation. In this study, utilizing a rat model of carbon tetrachloride (CCL4)-induced liver fibrosis, we aimed to investigate the impact of imatinib and bone marrow-derived mesenchymal stem cells (BM-MSCs) on the expression of these axis. Methods: Sixteen Sprague-Dawley rats were divided into four groups: healthy, liver fibrosis, imatinibrecipient, and BM-MSC-recipient. Treatment effects were evaluated using histopathology and Sirus-red staining. Quantitative real-time PCR was employed to analyze changes in the expression of the genes CCL2, CCR2, CX3CL1, and CX3CR1. Results: Histopathological assessments revealed the efficacy of imatinib and BM-MSCs in mitigating liver fibrosis. Our findings demonstrated a significant reduction in CCL2 and CCR2 expression in both imatinib and BM-MSCs treatment groups compared to the liver fibrosis group. Conversely, the gene expression of CX3CL1 and CX3CR1 increased in both therapeutic groups compared to the liver fibrosis groups. Conclusion: Conclusion: The notable decrease in CCL2-CCR2 genes in both therapeutic groups suggests that BM-MSCs and imatinib may contribute to a decline in inflammatory macrophages within the liver. The lower CCL2-CCR2 expression in imatinib-recipient rats indicates better efficacy in modulating the recruitment of inflammatory macrophages. The elevated expression of CX3CL1 in BM-MSC-recipient rats suggests a greater impact on the polarization of LY6Chigh (inflammatory) to LY6Clow (anti-inflammatory) macrophages, warranting further investigation. [ABSTRACT FROM AUTHOR]

Atherosclerosis is driven by a diverse range of cellular and molecular processes. In the present study, we sought to better understand how statins mitigate proatherogenic inflammation. 48 male New Zealand rabbits were divided into eight groups, each including 6 animals. The control groups received normal chow for 90 and 120 days. Three groups underwent a hypercholesterolemic diet (HCD) for 30, 60, and 90 days. Another three groups underwent HCD for 3 months, followed by normal chow for one month, with or without rosuvastatin or fluvastatin. The cytokine and chemokine expressions were assessed in the samples of thoracic and abdominal aorta. Rosuvastatin significantly reduced MYD88, CCL4, CCL20, CCR2, TNF-α, IFN-β, IL-1b, IL-2, IL-4, IL-8, and IL-10, both in the thoracic and abdominal aorta. Fluvastatin also downregulated MYD88, CCR2, IFN-β, IFN-γ, IL-1b, IL-2, IL-4, and IL-10 in both aortic segments. Rosuvastatin curtailed the expression of CCL4, IFN-β, IL-2, IL-4, and IL-10 more effectively than fluvastatin in both types of tissue. MYD88, TNF-α, IL-1b, and IL-8 showed a stronger downregulation with rosuvastatin compared to fluvastatin only in the thoracic aorta. The CCL20 and CCR2 levels reduced more extensively with rosuvastatin treatment only in abdominal aortic tissue. In conclusion, statin therapy can halt proatherogenic inflammation in hyperlipidemic animals. Rosuvastatin may be more effective in downregulating MYD88 in atherosclerotic thoracic aortas. [ABSTRACT FROM AUTHOR]

C-C motif ligand 2 (CCL2), also known as monocytic chemotactic protein 1 (MCP-1), is an integral chemotactic factor which recruits macrophages for the immune response. Together with its receptors (e.g., CCR2, ACKR1, and ACKR2), they exert noticeable influences on various diseases of different systems. At the maternal-fetal interface, CCL2 is detected to be expressed in trophoblasts, decidual tissue, the myometrium, and others. Meanwhile, existing reports have determined a series of physiological regulators of CCL2, which functions in maintaining normal recruitment of immunocytes, tissue remodeling, and angiogenesis. However, abnormal levels of CCL2 have also been reported to be associated with adverse pregnancy outcomes such as spontaneous abortion, preeclampsia and preterm labor. In this review, we concentrate on CCL2 expression at the maternal-fetal interface, as well as its precise regulatory mechanisms and classic signaling pathways, to reveal the multidimensional aspects of CCL2 in pregnancy. [ABSTRACT FROM AUTHOR]

Kidney macrophages are comprised of both monocyte-derived and tissue resident populations; however, the heterogeneity of kidney macrophages and factors that regulate their heterogeneity are poorly understood. Herein, we performed single cell RNA sequencing (scRNAseq), fate mapping, and parabiosis to define the cellular heterogeneity of kidney macrophages in healthy mice. Our data indicate that healthy mouse kidneys contain four major subsets of monocytes and two major subsets of kidney resident macrophages (KRM) including a population with enriched Ccr2 expression, suggesting monocyte origin. Surprisingly, fate mapping data using the newly developed Ms4a3Cre Rosa Stopf/f TdT model indicate that less than 50% of Ccr2+ KRM are derived from Ly6chi monocytes. Instead, we find that Ccr2 expression in KRM reflects their spatial distribution as this cell population is almost exclusively found in the kidney cortex. We also identified Cx3cr1 as a gene that governs cortex specific accumulation of Ccr2+ KRM and show that loss of Ccr2+ KRM reduces the severity of cystic kidney disease in a mouse model where cysts are mainly localized to the kidney cortex. Collectively, our data indicate that Cx3cr1 regulates KRM heterogeneity and niche-specific disease progression. [ABSTRACT FROM AUTHOR]

We examined the effect of chronic restraint stress and the counteractive effects of daily exercise on the molecular basis of the brain-bone marrow (BM) interactions, by especially focusing on the paraventricular nucleus (PVN) of the hypothalamus. Male Wistar rats were assigned into control, restraint stress, and stress + daily spontaneous exercise (SE) groups. BM and hypothalamic gene expression profiles were examined through the undertaking of RT-PCR and microarrays, respectively. The inflammatory blood cell population was investigated through flow cytometry. Through the use of immunohistochemistry, we examined the presence of BM-derived C-C chemokine receptor type 2 (CCR2)-expressing microglial cells in the rat PVN. The gene expression levels of BM inflammatory factors such as those of interleukin 1 beta and CCR2, and the inflammatory blood cell population were found to be significantly higher in both restrained groups compared with control group. Interestingly, chronic restraint stress alone activated the recruitment of BM-derived CCR2-expressing microglial cells into the PVN, whereas daily spontaneous exercise prevented it. A notable finding was that restraint stress upregulated relative gene expression of hypothalamic matrix metalloproteinase 3 (MMP3), which increases the permeability of the blood-brain barrier (BBB), and that exercise managed to normalize it. Moreover, relative expression of some hypothalamic genes directly involved in the facilitation of cell migration was downregulated by daily exercise. Our findings suggest that daily spontaneous exercise can reduce the numbers of BM-derived CCR2-expressing microglial cells into the PVN through the prevention of stress-induced changes in the hypothalamic gene expression. NEW & NOTEWORTHY Chronic restraint stress can upregulate MMP3 gene expression in the rat hypothalamus, whereas daily spontaneous exercise can prevent this stress-induced effect. Stress-induced BM-derived inflammatory cell recruitment into the rat PVN can be prevented by daily spontaneous exercise. Stress-induced increase of hypothalamic MMP3 gene expression may be responsible for BBB injury, thereby allowing for BM-derived inflammatory cells to be recruited and to accumulate in the rat PVN, and to be subsequently involved in the onset of stress-induced hypertension. [ABSTRACT FROM AUTHOR]

Abstract Background Neuroinflammation and neuronal apoptosis are closely associated with a poor prognosis in patients with subarachnoid hemorrhage (SAH). We investigated the role of C–C motif chemokine receptor 2 (CCR2) in SAH. Methods Pre-processed RNA-seq transcriptome datasets GSE167110 and GSE79416 from the Gene Expression Omnibus (GEO) database were screened for genes differentially expressed between mice with SAH and control mice, using bioinformatics analysis. The endovascular perforation model was performed to establish SAH. RS504393 (a CCR2 antagonist) and LY294002 (PI3K inhibitor) were administered to explore the mechanism of neuroinflammation after SAH. SAH grading, neurological scoring, brain water content and blood–brain barrier (BBB) permeability determination, enzyme-linked immunosorbent assay (ELISA), western blotting, and immunofluorescence were performed. An in vitro model of SAH was induced in H22 cells by hemin treatment. The protective mechanism of CCR2 inhibition was studied by adding RS504393 and LY294002. Clinical cerebrospinal fluid (CST) samples were detected by ELISA. Results Expression of CCR2 was upregulated in both datasets and was identified as a hub gene. CCR2 expression was significantly upregulated in the cytoplasm of neurons after SAH, both in vitro and in vivo. RS significantly reduced the brain water content and blood–brain barrier permeability, alleviated neuroinflammation, and reduced neuronal apoptosis after SAH. Additionally, the protective effects of CCR2 inhibition were abolished by LY treatment. Finally, the levels of CCR2, inflammatory factors, and apoptotic factors were elevated in the CSF of patients with SAH. CCR2 levels were associated with patient outcomes at the 6-month follow-up. Conclusion CCR2 expression was upregulated in both in vitro and in vivo SAH models. Additionally, inhibition of CCR2, at least partly through the PI3K/AKT pathway, alleviated neuroinflammation and neuronal apoptosis in vivo and in vitro. CCR2 levels in the CSF have a moderate diagnostic value for 6-month outcome prediction in patients with SAH.

Background: Familial Mediterranean Fever (FMF) is an autoinflammatory disease and may express as various clinical findings. Chemokines are crucial elements of the inflammatory process. MCP-1 and its’ receptor CCR2 are the main chemokines for monocytes/macrophages that may play critical roles in FMF. Thus, it was aimed to investigate the MCP-1 (A-2518G) and CCR2 (G190A) polymorphisms and MCP-1 expression level, which may affect MEFV gene function. Material and Method: Patients with FMF were identified according to the Tel-Hashomer criteria. DNA and RNA were isolated from the obtained blood samples. Genotyping analysis was performed by PCR-RFLP technique. In addition, expression analyzes were performed by Real-time PCR method. The obtained results were evaluated statistically. Results: A total of 229 individuals (125 male and 104 female) were included in the study. While 120 individuals had FMF clinic, and 107 individuals did not have. The remaining two individuals had suspicious clinical status. In addition, while 75 individuals were homozygous mutants, 77 individuals were heterozygous mutants, and 77 individuals did not carry mutation in the MEFV gene. No significant relationship was found in between both FMF clinic and MEFV genotypes, and MCP-1 (A-2518G) and CCR2 (G190A) genotypes. In the expression analysis, MCP-1 expression increased in patients with FMF clinic compared to those without. In addition, MCP-1 expression was increased in the heterozygous MEFV group compared to those without mutation, moreover, the expression level was highest in homozygous MEFV group. In addition, according to the MCP-1 (A-2518G) genotyping, MCP-1 expression elevated in the homozygous as well as the heterozygous groups, compared to the Wild type group. Conclusion: MCP-1 expression is increased in FMF disease, which may explain the clinical differences between FMF patients. MEFV mutations may exacerbate inflammation by increasing MCP-1 transcription. MCP-1 expression is increased in patients with MCP-1(A-2518G) mutations, which aggravates FMF clinic. MCP-1 expression may be assessed as a marker in suspicious cases. Keywords: Familial Mediterranean Fever, MCP-1, CCR2, expression

Abstract Acute graft‐versus‐host disease (aGvHD) remains a major threat to a successful outcome after allogeneic hematopoietic stem cell transplantation (HSCT). Although antibody‐based targeting of the CD40/CD40 ligand costimulatory pathway can prevent aGvHD, side effects hampered their clinical application, prompting a need for other ways to interfere with this important dendritic T‐cell costimulatory pathway. Here, we used small interfering RNA (siRNA) complexed with β‐glucan allowing the binding and uptake of the siRNA/β‐glucan complex (siCD40/schizophyllan [SPG]; chemical modifications called NJA‐312, NJA‐302, and NJA‐515) into Dectin1+ cells, which recognize this pathogen‐associated molecular pattern receptor. aGvHD was induced by the transplantation of splenocytes and bone marrow cells from C57BL/6J into CBF1 mice. Splenic dendritic cells retained Dectin1 expression after HSCT but showed lower expression after irradiation. The administration of siCD40/SPG, NJA‐312, and NJA‐302 ameliorated aGvHD‐mediated lethality and tissue damage of spleen and liver, but not skin. Multiple NJA‐312high injections prevented aGvHD but resulted in early weight loss in allogeneic HSCT mice. In addition, NJA‐312 treatment caused delayed initial donor T and B‐cell recovery but resulted in stable chimerism in surviving mice. Mechanistically, NJA‐312 reduced organ damage by suppressing CCR2+, F4/80+, and IL17A‐expressing cell accumulation in spleen, liver, and thymus but not the skin of mice with aGvHD. Our work demonstrates that siRNA targeting of CD40 delivered via the PAMP‐recognizing lectin Dectin1 changes the immunological niche, suppresses organ‐specific murine aGvHD, and induces immune tolerance after organ transplantation. Our work charts future directions for therapeutic interventions to modulate tissue‐specific immune reactions using Pathogen‐associated molecular pattern (PAMP) molecules like 1,3‐β‐glucan for cell delivery of siRNA.

Although excessive immune responses by Th17 cells, a helper T cell subset, are implicated in the pathogenesis of inflammatory bowel disease (IBD), the mechanism by which its localization in an inflamed colon is regulated remains unclear. Chemokines and their receptors are involved in the pathogenesis of IBD, however, the relative significance of each receptor on Th17 cells remains unknown. We generated C–C motif chemokine receptor 2 (CCR2) knockout (KO) and CCR6 KO mice in the syngeneic background using the CRISPR/Cas9 system and found that the phenotypes of experimental colitis worsened in both mutant mice. Surprisingly, the phenotype of colitis in CCR2/CCR6‐double knockout (CCR2/6 DKO) mice was opposite to that of the single‐deficient mice, with significantly milder experimental colitis (p <.05). The same was true for the symptoms in CCR6 KO mice, but not in wild type mice treated with a CCR2 inhibitor, propagermanium. Colonic CCR2+CCR6+ Th17 cells produced a potentially pathogenic cytokine GM‐CSF whose levels in the gut were significantly reduced in CCR2/6 DKO mice (p <.05). These results suggest that GM‐CSF‐producing CCR2+CCR6+ Th17 cells are pathogenic and are attracted to the inflamed colon by either CCR2 or CCR6 gradient, which subsequently exacerbates experimental colitis in mice. [ABSTRACT FROM AUTHOR]

Status epilepticus (SE) evokes leukocyte infiltration in the frontoparietal cortex (FPC) without the blood-brain barrier disruption. Monocyte chemotactic protein-1 (MCP-1) and macrophage inflammatory protein-2 (MIP-2) regulate leukocyte recruitments into the brain parenchyma. Epigallocatechin-3-gallate (EGCG) is an antioxidant and a ligand for non-integrin 67-kDa laminin receptor (67LR). However, it is unknown whether EGCG and/or 67LR affect SE-induced leukocyte infiltrations in the FPC. In the present study, SE infiltrated myeloperoxidase (MPO)-positive neutrophils, as well as cluster of differentiation 68 (CD68)-positive monocytes in the FPC are investigated. Following SE, MCP-1 was upregulated in microglia, which was abrogated by EGCG treatment. The C–C motif chemokine receptor 2 (CCR2, MCP-1 receptor) and MIP-2 expressions were increased in astrocytes, which were attenuated by MCP-1 neutralization and EGCG treatment. SE reduced 67LR expression in astrocytes, but not endothelial cells. Under physiological conditions, 67LR neutralization did not lead to MCP-1 induction in microglia. However, it induced MIP-2 expression and extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation in astrocytes and leukocyte infiltration in the FPC. Co-treatment of EGCG or U0126 (an ERK1/2 inhibitor) attenuated these events induced by 67LR neutralization. These findings indicate that the EGCG may ameliorate leukocyte infiltration in the FPC by inhibiting microglial MCP-1 induction independent of 67LR, as well as 67LR-ERK1/2-MIP-2 signaling pathway in astrocytes. [ABSTRACT FROM AUTHOR]

Background: The study aimed to identify core biomarkers related to diagnosis and immune microenvironment regulation and explore the immune molecular mechanism of diabetic nephropathy (DN) through bioinformatics analysis. Methods: GSE30529, GSE99325, and GSE104954 were merged with removing batch effects, and different expression genes (DEGs) were screened at a criterion |log2FC| >0.5 and adjusted P <0.05. KEGG, GO, and GSEA analyses were performed. Hub genes were screened by conducting PPI networks and calculating node genes using five algorithms with CytoHubba, followed by LASSO and ROC analysis to accurately identify diagnostic biomarkers. In addition, two different GEO datasets, GSE175759 and GSE47184, and an experiment cohort with 30 controls and 40 DN patients detected by IHC, were used to validate the biomarkers. Moreover, ssGSEA was performed to analyze the immune microenvironment in DN. Wilcoxon test and LASSO regression were used to determine the core immune signatures. The correlation between biomarkers and crucial immune signatures was calculated by Spearman analysis. Finally, cMap was used to explore potential drugs treating renal tubule injury in DN patients. Results: A total of 509 DEGs, including 338 upregulated and 171 downregulated genes, were screened out. "chemokine signaling pathway" and "cell adhesion molecules" were enriched in both GSEA and KEGG analysis. CCR2, CX3CR1, and SELP, especially for the combination model of the three genes, were identified as core biomarkers with high diagnostic capabilities with striking AUC, sensitivity, and specificity in both merged and validated datasets and IHC validation. Immune infiltration analysis showed a notable infiltration advantage for APC co-stimulation, CD8+ T cells, checkpoint, cytolytic activity, macrophages, MHC class I, and parainflammation in the DN group. In addition, the correlation analysis showed that CCR2, CX3CR1, and SELP were strongly and positively correlated with checkpoint, cytolytic activity, macrophages, MHC class I, and parainflammation in the DN group. Finally, dilazep was screened out as an underlying compound for DN analyzed by CMap. Conclusions: CCR2, CX3CR1, and SELP are underlying diagnostic biomarkers for DN, especially in their combination. APC co-stimulation, CD8+ T cells, checkpoint, cytolytic activity, macrophages, MHC class I, and parainflammation may participate in the occurrence and development of DN. At last, dilazep may be a promising drug for treating DN. [ABSTRACT FROM AUTHOR]

Myocardial injury is a serious complication of sepsis associated with high morbidity and mortality. Our previous work has confirmed that silibinin (SIL) alleviates septic myocardial injury, but the specific molecular mechanism has not been fully elucidated. This study aimed to identify its potential targets through network pharmacology combined with experimental verification. Firstly, a total of 29 overlapping genes between sepsis and SIL targets were obtained from RNA-seq analysis and the known databases. Subsequently, KEGG and GO analysis showed that these genes were enriched in immune response and cytokine-cytokine receptor interaction pathways. Notably, CCR2 was identified as an important candidate hub by protein-protein interaction analysis and molecular docking approach. In vivo experiments showed that SIL treatment significantly improved survival rate and cardiac function in septic mice, accompanied by decreased CCR2 expression. Moreover, in vitro experiments obtained the similar results. Especially, CCR2 siRNA attenuated inflammation response. In conclusion, this study systematically elucidated the key target of SIL in the treatment of septic myocardial injury. These findings provide valuable insights into the targets of sepsis and offer new avenues for exploring drug effect systematically. [Display omitted] • This study provides a rich resource of disease-drug interaction targets and the complex pharmacological network was constructed. • CCR2 was identified as an important candidate hub. • Experiments validation showed that SIL treatment protected septic myocardial injury, accompanied by decreased CCR2 expression. • CCR2-governed signaling plays an important role in the etiology of septic myocardial injury and cardioprotection of SIL. [ABSTRACT FROM AUTHOR]

Aims/hypothesis: Accumulating evidence suggests that leucocytes play a critical role in diabetes-induced vascular lesions and other abnormalities that characterise the early stages of diabetic retinopathy. However, the role of monocytes has yet to be fully investigated; therefore, we used Ccr2−/− mice to study the role of CCR2+ inflammatory monocytes in the pathogenesis of diabetes-induced degeneration of retinal capillaries. Methods: Experimental diabetes was induced in wild-type and Ccr2−/− mice using streptozotocin. After 2 months, superoxide levels, expression of inflammatory genes, leucostasis, leucocyte- and monocyte-mediated cytotoxicity against retinal endothelial cell death, retinal thickness and visual function were evaluated. Retinal capillary degeneration was determined after 8 months of diabetes. Flow cytometry of peripheral blood for differential expression of CCR2 in monocytes was assessed. Results: In nondiabetic mice, CCR2 was highly expressed on monocytes, and Ccr2−/− mice lack CCR2+ monocytes in the peripheral blood. Diabetes-induced retinal superoxide, expression of proinflammatory genes Inos and Icam1, leucostasis and leucocyte-mediated cytotoxicity against retinal endothelial cells were inhibited in diabetic Ccr2-deficient mice and in chimeric mice lacking Ccr2 only from myeloid cells. In order to focus on monocytes, these cells were immuno-isolated after 2 months of diabetes, and they significantly increased monocyte-mediated endothelial cell cytotoxicity ex vivo. Monocytes from Ccr2-deficient mice caused significantly less endothelial cell death. The diabetes-induced retinal capillary degeneration was inhibited in Ccr2−/− mice and in chimeric mice lacking Ccr2 only from myeloid cells. Conclusions/interpretation: CCR2+ inflammatory monocytes contribute to the pathogenesis of early lesions of diabetic retinopathy. [ABSTRACT FROM AUTHOR]

Natural killer (NK) cells are a subset of lymphocytes that offer great potential for cancer immunotherapy due to their natural anti-tumor activity and the possibility to safely transplant cells from healthy donors to patients in a clinical setting. However, the efficacy of cell-based immunotherapies using both T and NK cells is often limited by a poor infiltration of immune cells into solid tumors. Importantly, regulatory immune cell subsets are frequently recruited to tumor sites. In this study, we overexpressed two chemokine receptors, CCR4 and CCR2B, that are naturally found on T regulatory cells and tumor-resident monocytes, respectively, on NK cells. Using the NK cell line NK-92 as well as primary NK cells from peripheral blood, we show that genetically engineered NK cells can be efficiently redirected using chemokine receptors from different immune cell lineages and migrate towards chemokines such as CCL22 or CCL2, without impairing the natural effector functions. This approach has the potential to enhance the therapeutic effect of immunotherapies in solid tumors by directing genetically engineered donor NK cells to tumor sites. As a future therapeutic option, the natural anti-tumor activity of NK cells at the tumor sites can be increased by co-expression of chemokine receptors with chimeric antigen receptors (CAR) or T cell receptors (TCR) on NK cells can be performed in the future. [ABSTRACT FROM AUTHOR]

Chemokines modulate the immune response by regulating the migration of immune cells. They are also known to participate in such processes as cell–cell adhesion, allograft rejection, and angiogenesis. Chemokines interact with two different subfamilies of G protein-coupled receptors: conventional chemokine receptors and atypical chemokine receptors. Here, we focused on the former one which has been linked to many inflammatory diseases, including: multiple sclerosis, asthma, nephritis, and rheumatoid arthritis. Available crystal and cryo-EM structures and homology models of six chemokine receptors (CCR1 to CCR6) were described and tested in terms of their usefulness in structure-based drug design. As a result of structure-based virtual screening for CCR2 and CCR3, several new active compounds were proposed. Known inhibitors of CCR1 to CCR6, acquired from ChEMBL, were used as training sets for two machine learning algorithms in ligand-based drug design. Performance of LightGBM was compared with a sequential Keras/TensorFlow model of neural network for these diverse datasets. A combination of structure-based virtual screening with machine learning allowed to propose several active ligands for CCR2 and CCR3 with two distinct compounds predicted as CCR3 actives by all three tested methods: Glide, Keras/TensorFlow NN, and LightGBM. In addition, the performance of these three methods in the prediction of the CCR2/CCR3 receptor subtype selectivity was assessed. [ABSTRACT FROM AUTHOR]

Chronic levels of inflammation lead to autoimmune diseases such as rheumatoid arthritis and atherosclerosis. A key molecular mediator responsible for the progression of these diseases is Chemokine C–C motif ligand 2 (CCL2), a homodimerized cytokine that dissociates into monomeric form and binds to the CCR2 receptor. CCL2, also known as monocyte chemoattractant protein‐1 (MCP‐1), attracts monocytes to migrate to areas of injury and mature into macrophages, leading to positive feedback inflammation with further release of proinflammatory molecules such as IL‐1β and TNF‐α. Sequestering CCL2 to prevent its binding to CCR2 may prevent this inflammatory activity. Prior work adapted an α‐helical CCL2‐binding peptide (WKNFQTI) from murine CCR2 through extracellular loop analysis. Here, higher‐affinity peptide binders are computationally designed through homology modeling and energy calculations, yielding an 11‐amino acid peptide with high binding affinity. In addition, Rosetta mutations improves binding affinity in silico with blockage of the CCL2 dimerization site. Future work in analyzing binding kinetics and in vivo inflammation abrogation will confirm the accuracy of computational modeling techniques in de novo rational design of CCL2 cytokine binders. [ABSTRACT FROM AUTHOR]