Regulating Macrophage Infiltration Research Articles

The analgesic effects of Yu-Xue-Bi tablet (YXB) on mice with inflammatory pain by regulating LXA4-FPR2-TRPA1 pathway

BackgroundOxylipins including lipoxin A4 (LXA4) facilitate the resolution of inflammation and possess analgesic properties by inhibiting macrophage infiltration and transient receptor potential (TRP) protein expression. Yu-Xue-Bi Tablet (YXB) is a traditional Chinese patent medicine used to relieve inflammatory pain. Our previous research has shown that the analgesic effect of YXB is related to inhibiting peripheral inflammation and regulating macrophage infiltration, but the mechanism is not yet clear. The purpose of this study is to explore the mechanisms of YXB on mice models with Complete Freund’s Adjuvant (CFA)-induced inflammatory pain from the perspective at the resolution of inflammation.MethodsMechanical allodynia thresholds and heat hypersensitivity were measured using the Von Frey test and the hot plate test respectively. The open field test and the tail suspension test were employed to measure anxiety and depressive behaviors respectively. The expression of CD68+ and the proportion of F4/80+CD11b+ cells were measured by immunofluorescence staining and flow cytometry. The expression of transient receptor potential ankyrin 1(TRPA1) was measured by immunofluorescence staining and western blotting. Oxylipins omics analysis provided quantitative data on oxylipins in the paws, and enzyme linked immunosorbent assay (ELISA) was used to measure the levels of LXA4 there. Immunofluorescence staining was used to perform the expression of Leukotriene A4 hydroxylase (LTA4H) in the paws of mice. The impact of injecting the formyl peptide receptor 2(FPR2) antagonist WRW4 and the TRPA1 agonist AITC into the left paws was observed, focusing on the expression of mechanical allodynia thresholds, the expression of CD68+, TRPA1 in the paws, and Calcitonin gene-related peptide (CGRP) in the L5 spinal dorsal horn.ResultsYXB elevated mechanical allodynia thresholds, alleviated heat hypersensitivity and anxiety and depressive behaviors in CFA mice. It significantly reduced the number of CD68+ and proportion of F4/80+CD11b+ within the paws, thereby decreasing macrophage infiltration. Additionally, it diminished the expression of TRPA1 in the paws and TRPV1 in the DRG, leading to an inhibition of peripheral sensitization. Through quantitative analysis, it was found that YXB could modulate DHA-derived oxylipins and LXA4. ELISA results indicated that YXB elevated the levels of LXA4 and inhibited the expression of LAT4H in the paws. Furthermore, the pro-resolution and analgesic effects of YXB were hindered after administration of the FPR2 antagonist. Compared with the AITC group, YXB showed no significant improvement in anti-inflammatory and analgesic effects.ConclusionsYXB can regulate the oxylipins of paws in CFA mice to promote the resolution of inflammation. The LXA4-FPR2-TRPA1 pathway is a key mechanism for the resolution of inflammation and analgesic effects.

Cenicriviroc Suppresses and Reverses Steatohepatitis by Regulating Macrophage Infiltration and M2 Polarization in Mice.

The inhibition of hepatic macrophage and Kupfer cell recruitment and activation is a potential strategy for treating insulin resistance and nonalcoholic steatohepatitis (NASH). Cenicriviroc (CVC), a dual C-C chemokine receptor 2 (CCR2) and CCR5 antagonist, has shown antifibrotic activity in murine models of NASH and has been evaluated in clinical trials on patients with NASH. This study investigated the effects of CVC on macrophage infiltration and polarization in a lipotoxic model of NASH. C57BL/6 mice were fed a high-cholesterol, high-fat (CL) diet or a CL diet containing 0.015% CVC (CL + CVC) for 12 weeks. Macrophage recruitment and activation were assayed by immunohistochemistry and flow cytometry. CVC supplementation attenuated excessive hepatic lipid accumulation and peroxidation and alleviated glucose intolerance and hyperinsulinemia in the mice that were fed the CL diet. Flow cytometry analysis revealed that compared with the CL group, mice fed the CL + CVC diet had fewer M1-like macrophages, more M2-like macrophages, and fewer T cell counts, indicating that CVC caused an M2-dominant shift of macrophages in the liver. Similarly, CVC decreased lipopolysaccharide-stimulated M1-like macrophage activation, whereas it increased interleukin-4-induced M2-type macrophage polarization in vitro. In addition, CVC attenuated hepatic fibrosis by repressing hepatic stellate cell activation. Lastly, CVC reversed insulin resistance as well as steatosis, inflammation, and fibrosis of the liver in mice with pre-existing NASH. In conclusion, CVC prevented and reversed hepatic steatosis, insulin resistance, inflammation, and fibrogenesis in the liver of NASH mice via M2 macrophage polarization.

CXCL1-CXCR2 axis mediates inflammatory response after sciatic nerve injury by regulating macrophage infiltration

Macrophages play a crucial role in the inflammatory response following sciatic nerve injury. Studies have demonstrated that C-X-C motif chemokine (CXCL) 1 recruit macrophages by binding to C-X-C chemokine receptor (CXCR) 2 and participates in the inflammatory response of various diseases. Based on these findings, we aimed to explore the role of the CXCL1-CXCR2 axis in the repair process after peripheral nerve injury. Initially, we simulated sciatic nerve injury and observed an increased expression of CXCL1 and CXCR2 in the nerves of the injury group. Both in vivo and in vitro experiments confirmed that the heightened CXCL1 expression occurs in Schwann cells and is secreted, while the elevated CXCR2 is expressed by recruited macrophages. In addition, in vitro experiments demonstrated that the binding of CXCL1 to CXCR2 can activate the NLRP3 inflammasome and promote the production of interleukin-1 beta (IL-1β) in macrophages. However, after mice were subjected to sciatic nerve injury, the number of macrophages and the expression of inflammatory factors in the sciatic nerve were reduced following treatment with the CXCR2 inhibitor SB225002. Simultaneously, we evaluated the sciatic nerve function index, the expression of p75 neurotrophic factor receptor (p75NTR), and myelin proteins, and all of these results were improved with the use of SB225002. Thus, our results suggest that after sciatic nerve injury, the CXCL1-CXCR2 axis mediates the inflammatory response by promoting the recruitment and activation of macrophages, which is detrimental to the repair of the injured nerves. In contrast, treatment with SB225002 promotes the repair of injured sciatic nerves.

Zhoushi Qi Ling decoction inhibits the progression of castration-resistant prostate cancer in vivo by regulating macrophage infiltration via IL6-STAT3 signaling

Background and aimProstate cancer is a leading malignant tumor in men, associated with a high rate of mortality. Androgen deprivation therapy is commonly used to treat prostate cancer, which contributes to the progression of castration-resistant prostate cancer (CRPC). The current therapy has a low survival rate in patients with CRPC. Our study aims to develop a novel effective approach for CRPC treatment and improve survival benefits. Experimental procedureCRPC cell line PC-3-Luc expressing luciferase and the CRPC cell line PC-3-IL6-Luc stably overexpressing IL-6 were used to establish the xenograft tumor mouse model. The tumor was monitored weekly using Bioluminescence imaging. Infiltrated macrophages were quantified by fluorescence-activated cell sorting using flow cytometry. IL6 mRNA level was determined using quantitative real-time PCR. The protein levels of total STAT3 and phosphorylated STAT3 were determined using Western blot. Results and conclusionZhoushi Qi Ling decoction (ZQD) treatment significantly reduced PC3 the xenograft tumor progression and the number of infiltrated macrophages when compared with saline treatment. IL6 mRNA level was remarkedly suppressed by ZQD treatment. Notably, the protein level of phosphorylated STAT3 was significantly decreased in PC3 the xenograft tumor treated with ZQD compared to saline treatment. Our findings demonstrated that ZQD treatment significantly reduced the progression of prostate cancer, evidenced by the reduced population of infiltrated macrophages and the inhibition of the IL6/STAT3 pathway.

CD11b mediates hypertensive cardiac remodeling by regulating macrophage infiltration and polarization

IntroductionLeukocyte infiltration is an early event during cardiac remodeling frequently leading to heart failure (HF). Integrins mediate leukocyte infiltration during inflammation. However, the importance of specific integrins in hypertensive cardiac remodeling is still unclear. ObjectivesTo elucidate the significance of CD11b in hypertensive cardiac remodeling. MethodsAngiotensin (Ang II) or deoxycorticosterone acetate (DOCA)-salt was used to induce cardiac remodeling in mice of gene knockout (KO), bone marrow (BM) chimera, and the CD11b neutralizing antibody or agonist leukadherin-1 (LA1) treatment. ResultsOur microarray data showed that integrin subunits Itgam (CD11b) and Itgb2 (CD18) were the most highly upregulated in Ang II-infused hearts. CD11b expression and CD11b/CD18+ myelomonocytes were also time-dependently increased. KO or pharmacological blockade of CD11b greatly attenuated cardiac remodeling and macrophage infiltration and M1 polarization induced by Ang II or DOCA-salt. This protection was verified in wild-type mice transplanted with CD11b-deficient BM cells. Conversely, administration of CD11b agonist LA1 showed the opposite effects. Further, CD11b KO reduced Ang II-induced macrophage adhesion and M1 polarization, leading to reduction of cardiomyocyte enlargement and fibroblast differentiation in vitro. The numbers of CD14+CD11b+CD18+ monocytes and CD15+CD11b+CD18+ granulocytes were obviously higher in HF patients than in normal controls. ConclusionOur data demonstrate an important role of CD11b+ myeloid cells in hypertensive cardiac remodeling, and suggest that HF may benefit from targeting CD11b.

Pb Induces MCP-1 in the Choroid Plexus.

Simple SummaryOur data in this study show that oral Pb exposure can induce MCP-1 expression along with monocyte and macrophage infiltration in the choroid plexus. Both NF-κB and p38 MAPK pathways modulate Pb-induced MCP-1 expression in CP epithelial cells.Lead (Pb) is an environmental element that has been implicated in the development of dementia and Alzheimer’s disease (AD). Additionally, innate immune activation contributes to AD pathophysiology. However, the mechanisms involved remain poorly understood. The choroid plexus (CP) is not only the site of cerebrospinal fluid (CSF) production, but also an important location for communication between the circulation and the CSF. In this study, we investigated the involvement of the CP during Pb exposure by evaluating the expression of the monocyte chemoattractant protein-1 (MCP-1). MCP-1 is highly expressed in the CP compared to other CNS tissues. MCP-1 regulates macrophage infiltration and is upregulated in AD brains. Our study revealed that Pb exposure stimulated MCP-1 expression, along with a significantly increased macrophage infiltration into the CP. By using cultured Z310 rat CP cells, Pb exposure stimulated MCP-1 expression in a dose-related fashion and markedly activated both NF-κB and p38 MAP kinase. Interestingly, both SB 203580, a p38 inhibitor, and BAY 11-7082, an NF-κB p65 inhibitor, significantly blocked Pb-induced MCP-1 expression. However, SB203580 did not directly inhibit NF-κB p65 phosphorylation. In conclusion, Pb exposure stimulates MCP-1 expression via the p38 and NF-κB p65 pathways along with macrophage infiltration into the CP.

Fos regulates macrophage infiltration against surrounding tissue resistance by a cortical actin-based mechanism in Drosophila.

The infiltration of immune cells into tissues underlies the establishment of tissue-resident macrophages and responses to infections and tumors. Yet the mechanisms immune cells utilize to negotiate tissue barriers in living organisms are not well understood, and a role for cortical actin has not been examined. Here, we find that the tissue invasion of Drosophila macrophages, also known as plasmatocytes or hemocytes, utilizes enhanced cortical F-actin levels stimulated by the Drosophila member of the fos proto oncogene transcription factor family (Dfos, Kayak). RNA sequencing analysis and live imaging show that Dfos enhances F-actin levels around the entire macrophage surface by increasing mRNA levels of the membrane spanning molecular scaffold tetraspanin TM4SF, and the actin cross-linking filamin Cheerio, which are themselves required for invasion. Both the filamin and the tetraspanin enhance the cortical activity of Rho1 and the formin Diaphanous and thus the assembly of cortical actin, which is a critical function since expressing a dominant active form of Diaphanous can rescue the Dfos macrophage invasion defect. In vivo imaging shows that Dfos enhances the efficiency of the initial phases of macrophage tissue entry. Genetic evidence argues that this Dfos-induced program in macrophages counteracts the constraint produced by the tension of surrounding tissues and buffers the properties of the macrophage nucleus from affecting tissue entry. We thus identify strengthening the cortical actin cytoskeleton through Dfos as a key process allowing efficient forward movement of an immune cell into surrounding tissues.

Gasdermin D Is a Novel Prognostic Biomarker and Relates to TMZ Response in Glioblastoma.

Simple SummaryGSMD family were crucial regulators of pyroptosis. We used WB, IHC and bioinformatics to explore expression and potential role of GSDMs family in the progression of GBM. We found that only GSDMD expression was upregulated in glioma compared with nontumor brain tissues both in the public datasets and in-house cohort. High GSDMD expression was significantly associated with WHO grade IV, IDH 1/2 wild-type, mesenchymal subtypes and shorter overall survival.Moreover, GSDMD expression increased with after treating with TMZ in a time-dependent manner. We conculded that GSDMD was a novel prognostic biomarker, as well as TMZ-treatment response marker in glioma.The gasdermin (GSDM) family act as executioners during pyroptosis. However, its expression and biological role in glioma remain to be determined. This study carried out gene expression from six public datasets. Westerns blots and immunohistochemistry (IHC) staining were employed to examine GSDM expression in glioma in an in-house cohort. Kaplan–Meier and Cox regression analyses were performed to evaluate the prognostic role of GSDMs in glioma. Association between gene expression and immune infiltration was assessed by IHC and immunofluorescence (IF) staining of tissue sections. TMZ-induced pyroptosis was assessed by observation of morphological changes, WB and ELISA detection. Only GSDMD expression was upregulated in glioma compared with nontumor brain tissues both in the public datasets and in-house cohort. High GSDMD expression was significantly associated with WHO grade IV, IDH 1/2 wild-type and mesenchymal subtypes. Besides, high GSDMD expression was associated with shorter overall survival and could be used as an independent risk factor for poor outcomes in LGG and GBM. GO enrichment analysis and IHC validation revealed that GSDMD expression might participate in regulating macrophage infiltration and polarization. TMZ treatment induced the pyroptosis in GBM cells and GSDMD expression increased with after treating with TMZ in a time-dependent manner. Moreover, knocking down GSDMD obviously decreased IL-1β expression and reduced TMZ-induced pyroptosis in in vitro. GSDMD was a novel prognostic biomarker, as well as TMZ-treatment response marker in glioma.

Interleukin-7 aggravates myocardial ischaemia/reperfusion injury by regulating macrophage infiltration and polarization.

Interleukin (IL)‐7 is known to enhance the macrophages cytotoxic activity and that macrophages play a pivotal role in the development and progression of myocardial ischaemia/reperfusion (I/R) injury. However, the effects of IL‐7 on macrophages infiltration and polarization in myocardial I/R injury are currently unclear. This study aimed to evaluate the effects of the IL‐7 expression on myocardial I/R injury and their relationship with macrophages. The data showed that IL‐7 expression in mouse heart tissue increases following I/R injury and that IL‐7 knockout or anti‐IL‐7 antibody treatment significantly improve I/R injury, including reduction in myocardial infarction area, a serum troponin T level decreases and an improvement in cardiac function. On the other hand, recombinant IL‐7 (rIL‐7) supplementation induces opposite effects and the anti‐IL‐7 antibody significantly reduces the cardiomyocyte apoptosis and macrophage infiltration. rIL‐7 cannot directly cause apoptosis, but it can induce cardiomyocyte apoptosis through macrophages, in addition to increase the macrophages migration in vitro. Anti‐IL‐7 antibody affects the cytokine production in T helper (Th) 1 and Th2 cells and also promotes the macrophages differentiation to M2 macrophages. However, anti‐IL‐7 antibody does not reduce the M1 macrophage number, and it only increases the ratio of M2/M1 macrophages in mice heart tissues after I/R injury. Taking together, these data reveal that IL‐7 plays an intensifying role in myocardial I/R injury by promoting cardiomyocyte apoptosis through the regulation of macrophage infiltration and polarization.

Recombinant sulfated CCR2 peptide trap reduces retinal degeneration in mice

Chemokine receptors are generally sulfated at tyrosine residues of the N-terminal region. Tyrosine sulfation of the C–C chemokine receptor type 2 (CCR2) enhances its interaction with the chemokine ligand CCL2. Here, we generated a recombinant sulfated CCR2 peptide trap (mCCR2-S2) and investigated its effects on retinal degeneration in mice. Treatment with mCCR2-S2 reduced choroidal neovascularization (CNV) in a laser-induced CNV mouse model. In NaIO3-injected mice, treatment with mCCR2-S2 increased the outer nuclear layer thickness and rhodopsin expression in the retinas compared to that in mice treated with mCCR2-wild-type or glutathione S-transferase controls. Furthermore, glial fibrillary acidic protein (GFAP) expression and macrophage infiltration were decreased in mCCR2-S2-treated retinas. Recombinant mCCR2-S2 suppressed CNV development and retinal degeneration, possibly by regulating macrophage infiltration. Thus, the sulfated form of the CCR2 peptide trap may be a useful tool for treating patients with retinal degeneration, such as those with age-related macular degeneration and intraocular inflammatory disorders.

FOXE1-Dependent Regulation of Macrophage Chemotaxis by Thyroid Cells In Vitro and In Vivo.

Forkhead box E1 (FOXE1) is a lineage-restricted transcription factor involved in thyroid cancer susceptibility. Cancer-associated polymorphisms map in regulatory regions, thus affecting the extent of gene expression. We have recently shown that genetic reduction of FOXE1 dosage modifies multiple thyroid cancer phenotypes. To identify relevant effectors playing roles in thyroid cancer development, here we analyse FOXE1-induced transcriptional alterations in thyroid cells that do not express endogenous FOXE1. Expression of FOXE1 elicits cell migration, while transcriptome analysis reveals that several immune cells-related categories are highly enriched in differentially expressed genes, including several upregulated chemokines involved in macrophage recruitment. Accordingly, FOXE1-expressing cells induce chemotaxis of co-cultured monocytes. We then asked if FOXE1 was able to regulate macrophage infiltration in thyroid cancers in vivo by using a mouse model of cancer, either wild type or with only one functional FOXE1 allele. Expression of the same set of chemokines directly correlates with FOXE1 dosage, and pro-tumourigenic M2 macrophage infiltration is decreased in tumours with reduced FOXE1. These data establish a novel link between FOXE1 and macrophages recruitment in the thyroid cancer microenvironment, highlighting an unsuspected function of this gene in the crosstalk between neoplastic and immune cells that shape tumour development and progression.

Specialized Pro-Resolving Mediators as Potential Regulators of Inflammatory Macrophage Responses in COVID-19.

The recent outbreak of SARS-CoV2 has emerged as one of the biggest pandemics of our century, with outrageous health, social and economic consequences globally. Macrophages may lay in the center of COVID-19 pathogenesis and lethality and treatment of the macrophage-induced cytokine storm has emerged as essential. Specialized pro-resolving mediators (SPMs) hold strong therapeutic potentials in the management of COVID-19 as they can regulate macrophage infiltration and cytokine production but also promote a pro-resolving macrophage phenotype. In this review, we discuss the homeostatic functions of SPMs acting directly on macrophages on various levels, towards the resolution of inflammation. Moreover, we address the molecular events that link the lipid mediators with COVID-19 severity and discuss the clinical potentials of SPMs in COVID-19 immunotherapeutics.

H2S alleviates renal injury and fibrosis in response to unilateral ureteral obstruction by regulating macrophage infiltration via inhibition of NLRP3 signaling

Renal fibrosis is a key pathological feature in chronic kidney diseases (CKDs). Dysregulation of hydrogen sulfide (H2S) homeostasis is implicated in the pathogenesis of CKDs. Here, C57/BL6 mice were allocated to Sham and unilateral ureteral obstruction (UUO) groups, which were treated with NaHS or NLRP3 inflammasome inhibitor 16673-34-0 for 3–14 days. UUO mice displayed downregulation of H2S production and increased macrophage infiltration in obstructed kidneys. H2S donor NaHS treatment attenuated renal damage and fibrosis and inhibited M1 and M2 macrophage infiltration. NLPR3 inflammasome was activated and levels of phosphorylated nuclear factor κB (NF-κB) p65 subunit, phosphorylated signal transducer and activator of transcription 6 (STAT6) and interleukin (IL)-4 protein were increased in the kidneys after UUO. NLRP3 inhibitor inactivated NF-κB and IL-4/STAT6 signaling, suppressed M1 and M2 macrophage infiltration and attenuated renal damage and fibrosis in UUO mice. NaHS treatment also suppressed NLRP3, NF-κB and IL-4/STAT6 activation in the obstructed kidneys. In conclusion, the therapeutic effects of H2S on UUO-induced renal injury and fibrosis are at least in part by inhibition of M1 and M2 macrophage infiltration. H2S suppresses NLRP3 activation and subsequently inactivates NF-κB and IL-4/STAT6 signaling, which may contribute to the anti-inflammatory and anti-fibrotic effects of H2S.

Bone marrow stromal cells transplantation promotes the resolution and recanalization of deep vein thrombosis in rabbits through regulating macrophage infiltration and angiogenesis.

This study aims to validate whether bone marrow stromal cells (BMSCs) transplantation could promote the resolution and recanalization of deep vein thrombosis (DVT) and to explore the underlying mechanism. The right hind femoral vein was embolized to establish the DVT rabbit model. BMSCs from New Zealand white rabbits were isolated and identified, and then injected into DVT rabbits. After that, the extent of angiogenesis was determined by the amount of capillaries that were positive for antibody against vWF. Macrophage infiltration was measured by immunohistochemistry with F4/80 antibody. M1 or M2 macrophages were identified as F4/80 + CD11c + or F4/80 + CD206 + cells by using flow cytometry analysis, respectively. BMSCs were successfully isolated and identified. BMSCs transplantation promotes macrophage infiltration and angiogenesis in DVT rabbits. BMSCs transplantation causes M1/M2 polarization, altered cytokine production and increased monocyte chemotactic protein 1 (MCP-1) protein expression in DVT rabbits. However, injection of MCP-1 protein not only reversed the effects of BMSCs transplantation on macrophage infiltration and angiogenesis, but also reversed the effects of BMSCs transplantation on M1/M2 polarization and cytokine production in DVT rabbits. BMSCs transplantation promotes the resolution and recanalization of DVT in rabbits through regulating macrophage infiltration and angiogenesis, the underlying mechanism is associated with MCP-1 expression.

Microvascular endothelial cells engulf myelin debris and promote macrophage recruitment and fibrosis after neural injury.

The clearance of damaged myelin sheaths is critical to ensure functional recovery from neural injury. Here we show a previously unidentified role for microvessels and their lining endothelial cells in engulfing myelin debris in spinal cord injury (SCI) and experimental autoimmune encephalomyelitis (EAE). We demonstrate that IgG opsonization of myelin debris is required for its effective engulfment by endothelial cells and that the autophagy-lysosome pathway is crucial for degradation of engulfed myelin debris. We further show that endothelial cells exert critical functions beyond myelin clearance to promote progression of demyelination disorders by regulating macrophage infiltration, pathologic angiogenesis and fibrosis in both SCI and EAE. Unexpectedly, myelin debris engulfment induces endothelial-to-mesenchymal transition, a process that confers upon endothelial cells the ability to stimulate the endothelial-derived production of fibrotic components. Overall, our study demonstrates that the processing of myelin debris through the autophagy-lysosome pathway promotes inflammation and angiogenesis and may contribute to fibrotic scar formation.

TIPE1 suppresses osteosarcoma tumor growth by regulating macrophage infiltration.

Osteosarcoma is the most common primary malignancy of the bone, and macrophages play a promotional role during osteosarcoma development and progression. TIPE1 is known to function as a tumor suppressor in diverse cancers by inducing cell arrest and apoptosis. However, the biological function of TIPE1 in osteosarcoma is still unclear. The purpose of this study was to investigate the expression and function of TIPE1 in osteosarcoma. In the present study, TIPE1 expression in osteosarcoma cancer cells was determined by qPCR and western blotting. A subcutaneous tumor model was established to investigate the potential anti-tumor activity of TIPE1 in osteosarcoma. Further, flow cytometry, western blotting, immunofluorescence staining, and ELISA were performed to clarify the underlying mechanism by which TIPE1 regulates growth of osteosarcoma. Our results suggest that TIPE1 is downregulated in osteosarcoma cancer cells, and ectopic expression TIPE1 significantly inhibited osteosarcoma tumor growth in vivo. Furthermore, TIPE1 inhibits the infiltration of macrophages in osteosarcoma tumor by suppressing MCP-1 expression in osteosarcoma cells. Further in vivo study revealed that inhibition of MCP-1/CCR2 axis by Bindarit blocked the inhibitory effect of TIPE1 on osteosarcoma growth. Collectively, our results demonstrate the anti-tumor role of TIPE1 in osteosarcoma and reveal a novel therapy target for osteosarcoma.

Expression of soluble epoxide hydrolase in renal tubular epithelial cells regulates macrophage infiltration and polarization in IgA nephropathy.

Tubulointerstitial inflammatory cell infiltration and activation contribute to kidney inflammation and fibrosis. Epoxyeicosatrienoic acids (EETs), which are rapidly metabolized to dihydroxyeicosatrienoic acids by the soluble epoxide hydrolase (sEH), have multiple biological functions, including vasodilation, anti-inflammatory action, and others. Inhibition of sEH has been demonstrated to attenuate inflammation in many renal disease models. However, the relationship between sEH expression and macrophage polarization in the kidney remains unknown. In this study, we investigated the relationships between the level of sEH and clinical and pathological parameters in IgA nephropathy. The level of sEH expression positively correlated with proteinuria and infiltration of macrophages. sEH-positive tubules were found to be surrounded by macrophages. Furthermore, we found that incubation of immortalized human proximal tubular HK-2 cells with total urinary protein and overexpression of sEH promoted inflammatory factor production, which was associated with M1 polarization. We also exposed RAW264.7 mouse leukemic monocytes/macrophages to different HK-2 cell culture media conditioned by incubation with various substances affecting sEH amount or activity. We found that the upregulation of sEH promoted M1 polarization. However, pharmacological inhibition of sEH and supplementation with EETs reversed the conditioning effects of urinary proteins by inhibiting M1 polarization through the NF-κB pathway and stimulating M2 polarization through the phosphatidylinositol 3-kinase pathway. These data suggest that inhibition of sEH could be a new strategy to prevent the progression of inflammation and to attenuate renal tubulointerstitial fibrosis.

1447 Vitamin D attenuates acute skin inflammation following nitrogen mustard exposure by targeting M1 macrophages

Cutaneous exposure to alkylating agents such as nitrogen mustard (NM) causes severe blistering with systemic complications long after the exposure. We have previously shown that a single dose of vitamin D (VD) is sufficient to rescue NM-exposed mice from pancytopenia and death and that early intervention with VD is of critical importance. However, the effect of VD on cellular infiltration and local milieu in the skin is unknown. Here we propose that VD promotes skin recovery by preventing dermal infiltration including pro-inflammatory macrophages to maintain a cellular distribution that accelerates skin repair to restore immune equilibrium. Dermal derived cells isolated from skin of NM-exposed mice were analyzed by flow cytometry for total cell counts and macrophage characterization. Mice treated with VD strongly inhibited cellular infiltration to the inflamed skin on day 2 post exposure (p=0.01) especially F4/80+ macrophages (p=0.02). Decreased infiltration was accompanied by a concomitant decrease in tissue pro-inflammatory cytokine, inducible nitric oxide synthase. On day 5 post NM, VD expanded the number of cells populating the skin suggesting that these cells were conducive to skin repair (p=0.01). To assess the distribution of pro-inflammatory M1 and anti-inflammatory M2 macrophages modulated by VD, F4/80 macrophages were stained for Ly6C and CD206 respectively. The findings demonstrate that compared to NM-exposed mice, VD intervention restrained the expansion of F4/80+Ly6C+ M1 macrophages in the inflamed skin on day 6 (p=0.04). Furthermore, a clear opposing trend is observed between M1 and M2 macrophage populations in the skin of VD-treated mice on day 6 (p=0.005), that is absent in NM-only mice, that sets the foundation for accelerated skin recovery. Taken together our results define a critical role for vitamin D in regulating macrophage infiltration and its phenotype to diminish local inflammation and accelerate skin repair.

Activating transcription factor 3 regulates chemokine expression in contracting C2C12 myotubes and in mouse skeletal muscle after eccentric exercise

Activating transcription factor (ATF) 3 regulates chemokine expression in various cell types and tissues. Herein, we studied this regulation in contracting muscle cells in vitro, and in skeletal muscle after muscle-damaging exercise in vivo. C2C12 myotubes with normal or low ATF3 levels (atf3_siRNA) were electrically stimulated (EPS). Also, ATF3-knockout (ATF3-KO) and control mice ran downhill until exhaustion, and muscles were analyzed post-exercise. EPS increased ATF3 levels in myotubes (P < 0.01). Chemokine C-C motif ligand (ccl) 2 mRNA increased post-EPS, but atf3_siRNA attenuated the response (P < 0.05). Atf3_siRNA up-regulated ccl6 basal mRNA, and down-regulated ccl9 and chemokine C-X-C motif ligand (cxcl) 1 basal mRNAs. Post-exercise, ATF3-KO mice showed exacerbated mRNA levels of ccl6 and ccl9 in soleus (P < 0.05), and similar trends were observed for ccl2 and interleukin (il) 1β (P < 0.09). In quadriceps, il6 mRNA level increased only in ATF3-KO (P < 0.05), and cxcl1 mRNA showed a similar trend (P = 0.082). Cluster of differentiation-68 (cd68) mRNA, a macrophage marker, increased in quadriceps and soleus independently of genotype (P < 0.001). Our data demonstrate that ATF3 regulates chemokine expression in muscle cells in vitro and skeletal muscle in vivo, but the regulation differs in each model. Cells other than myofibers may thus participate in the response observed in skeletal muscle. Our results also indicate that ATF3-independent mechanisms would regulate macrophage infiltration upon muscle-damaging exercise. The implications of chemokine regulation in skeletal muscle remain to be determined.

Long-term spironolactone treatment reduces coronary TRPC expression, vasoconstriction, and atherosclerosis in metabolic syndrome pigs

Coronary transient receptor potential canonical (TRPC) channel expression is elevated in metabolic syndrome (MetS). However, differential contribution of TRPCs to coronary pathology in MetS is not fully elucidated. We investigated the roles of TRPC1 and TRPC6 isoforms in coronary arteries of MetS pigs and determined whether long-term treatment with a mineralocorticoid receptor inhibitor, spironolactone, attenuates coronary TRPC expression and associated dysfunctions. MetS coronary arteries exhibited significant atherosclerosis, endothelial dysfunction, and increased histamine-induced contractions. Immunohistochemical studies revealed that TRPC6 immunostaining was significantly greater in the medial layer of MetS pig coronary arteries compared to that in Lean pigs, whereas little TRPC6 immunostaining was found in atheromas. Conversely, TRPC1 immunostaining was weak in the medial layer but strong in MetS atheromas, where it was predominantly localized to macrophages. Spironolactone treatment significantly decreased coronary TRPC expression and dysfunctions in MetS pigs. In vivo targeted delivery of the dominant-negative (DN)-TRPC6 cDNA to the coronary wall reduced histamine-induced calcium transients in the MetS coronary artery medial layer, implying a role for TRPC6 in mediating calcium influx in MetS coronary smooth muscles. Monocyte adhesion was increased in Lean pig coronary arteries cultured in the presence of aldosterone; and spironolactone antagonized this effect, suggesting that coronary mineralocorticoid receptor activation may regulate macrophage infiltration. TRPC1 expression in atheroma macrophages was associated with advanced atherosclerosis, whereas medial TRPC6 upregulation correlated with increased histamine-induced calcium transients and coronary contractility. We propose that long-term spironolactone treatment may be a therapeutic strategy to decrease TRPC expression and coronary pathology associated with MetS.