Polarization Of Peritoneal Macrophages Research Articles

A Graphene Composite Film Based Wearable Far-Infrared Therapy Apparatus (GRAFT) for Effective Prevention of Postoperative Peritoneal Adhesion.

Postoperative peritoneal adhesion (PPA) is the most frequent complication after abdominal surgery. Current anti-adhesion strategies largely rely on the use of physical separating barriers creating an interface blocking peritoneal adhesion, which cannot reduce inflammation and suffers from limited anti-adhesion efficacy with unwanted side effects. Here, by exploiting the alternative activated macrophages to alleviate inflammation in adhesion development, a flexible graphene-composite-film (F-GCF) generating far-infrared (FIR) irradiation that effectively modulates the macrophage phenotype toward the anti-inflammatory M2 type, resulting in reduced PPA formation, is designed. The anti-adhesion effect of the FIR generated by F-GCF is determined in the rat abdominal wall abrasion-cecum defect models, which exhibit reduced incidence and area of PPA by 67.0% and 92.1% after FIR treatment without skin damage, significantly superior to the clinically used chitosan hydrogel. Notably, within peritoneal macrophages, FIR reduces inflammation reaction and promotes tissue plasminogen activator (t-PA) level via the polarization of peritoneal macrophages through upregulating Nr4a2 expression. To facilitate clinical use, a wirelessly controlled, wearable, F-GCF-based FIR therapy apparatus (GRAFT) is further developed and its remarkable anti-adhesion ability in the porcine PPA model is revealed. Collectively, the physical, biochemical, and in vivo preclinical data provide compelling evidence demonstrating the clinical-translational value of FIR in PPA prevention.

Effects of Increased Mitochondrial DNA Mutation on the Macrophage Response to Listeria Monocytogenes

Mitochondria are important for cellular function, and as cells divide, their mitochondria also divide by replicating their DNA. The integrity of mitochondria DNA (mtDNA) replication, carried out by Polymerase G (PolG), is critical for the maintenance of mitochondria and their functions. In this study, mice carrying a mutant PolG, PolGD257A, were used to determine the effect of increased mtDNA mutations on the macrophage population and polarization in response to bacterial and cytokine challenge. It was hypothesized that increased mtDNA mutations will inhibit pathogen clearance by macrophages. To test this hypothesis, the PolGD257A mice were used, along with Listeria monocytogenes (LM) as a model of bacterial infection. Three days post LM infection, the bacterial load and the macrophage population was determined in the spleen and liver of PolGD257A and WT mice. No statistical difference was observed in the bacterial load in the liver or spleen, or in the macrophage population in the spleen of the PolGD257A and WT mice. However, the PolGD257A/D257A mice were associated with a higher percentage of macrophages in the liver during LM infection. Polarization of peritoneal macrophages into classically activated (M1) and alternatively activated (M2) macrophages was also studied in vitro. In a single experiment, increased mtDNA mutations in PolGD257A mice seemed to elicit increased M1 and decreased M2 macrophage polarization. Replication of the experiment is warranted to confirm these results. These experimental findings could lead to a better understanding of the role of the mitochondria and macrophages in infectious disease.

Exposure duration of ambient fine particulate matter determines the polarization of macrophages.

Ambient fine particulate matter (FPM) promotes airway inflammation and aggravates respiratory and cardiovascular diseases. Macrophage polarization plays an essential role in FPM-induced inflammation and tissue repair. The balance of pro-inflammatory M1-type and anti-inflammatory M2-type macrophages determines the fate of tissues and is involved in the pathogenesis of various FPM-induced diseases. The mechanism of macrophage polarization induced by FPM is still not fully understood. Here, we explored the effect of ambient FPM exposure duration on the polarization of peritoneal macrophages. Mice were exposed to concentrated ambient FPM for different duration. Markers of M1-type macrophage and M2-type macrophage in peritoneal macrophages were detected. We found that macrophage polarization was affected by FPM both in vitro and in vivo. Acute FPM stimulation in vitro and short-term concentrated ambient FPM exposure in vivo promoted the expression of NLRP3 and NOS2 and inhibited the expression of ARG1 and CD206. With the extension of concentrated ambient FPM exposure time, ARG1 was gradually up-regulated, and NLRP3 was gradually down-regulated. These results indicate that FPM exposure duration interferes with macrophage polarization. This may provide new insight into the treatment of patients exposed to FPM.

Depression Exacerbates Dextran Sulfate Sodium-Induced Colitis via IRF5-Mediated Macrophage Polarization.

Patients with inflammatory bowel disease (IBD) and concurrent depression are predisposed to severer disease activity and a worse prognosis. Macrophage polarization toward the M1 phenotype may contribute to the exacerbation of IBD with comorbid depression. Moreover, interferon regulatory factor 5 (IRF5) is involved in the pathogenesis of IBD. The aim of this study was to explore the role of IRF5 in macrophage polarization in the impact of depression upon colitis. Depressive-like behavior was induced by repeated forced swim stress. Colon length, disease activity index (DAI), colon morphology, histology, ultrastructure of epithelial barrier, lamina propria macrophage polarization, and expression of IRF5 were compared between DSS colitis rats with and without depressive-like behavior. IRF5 shRNA was constructed to affect the rat peritoneal macrophages polarization in vitro. After IRF5 shRNA lentivirus was introduced into colon by enema, the colitis severity, lamina propria macrophage polarization, and TNF-α, IL-1β, and IL-10 of colon tissues were measured. The study found severer colonic inflammation in depressed versus non-depressed DSS-colitis rats. Depressed DSS-colitis rats exhibited smaller subepithelial macrophages size and reduced intracellular granule diversity compared with nondepressed DSS-colitis rats. Increased polarization toward the M1 phenotype, elevated expression of IRF5, and co-expression of IRF5 with CD86 were found in depressed versus nondepressed DSS-colitis rats. Lentivirus-mediated shRNA interference with IRF5 expression switched rat peritoneal macrophage polarization from the M1 to the M2 phenotype, downregulated TNF-α, IL-1β expression to a greater extent in depressed versus nondepressed colitis rats. IRF5-mediated macrophage polarization may likely underlie the deterioration of DSS-induced colitis caused by depression.

Mesenchymal stem cells regulate M1 polarization of peritoneal macrophages through the CARD9-NF-κB signaling pathway in severe acute pancreatitis.

Macrophages release large numbers of proinflammatory cytokines that trigger inflammatory cascade reactions, which promote the rapid development of severe acute pancreatitis (SAP) from local to systemic inflammation. The ability of mesenchymal stem cells (MSCs) to suppress inflammation is related to inhibition of M1 polarization of macrophages. Our previous studies revealed that caspase recruitment domain protein 9 (CARD9) was involved in SAP inflammation and activation of the CARD9-NF-κB signaling pathway plays an important proinflammatory role in SAP. At present, there is no effective treatment to control the inflammatory response in SAP. Therefore, the aim of the present study was to determine whether MSCs regulate the polarization of macrophages through the CARD9-NF-κB signaling pathway in SAP. Short hairpin RNA interference technology and coculture in vitro were used to assess the activation status of the CARD9-NF-κB signal pathway in macrophages. Furthermore, flow cytometry was used to determine the polarization state of macrophages. The results showed MSCs inhibited CARD9 expression in vivo and in vitro (P < .05), alleviated inflammation induced by proinflammatory cytokines, and inhibited the phosphorylation of NF-κB in macrophages both in vivo and in vitro. Meanwhile, MSCs downregulated the CARD9-NF-κB signal pathway and inhibited M1 polarization of macrophages. In conclusion, MSCs regulate M1 polarization of peritoneal macrophages through the CARD9-NF-κB signaling pathway in SAP and transplantation of MSCs presents an effective treatment option for SAP.

Secretome of adipose-derived mesenchymal stem cells establish anti-inflammatory milieu by developing M2b/c macrophage

Abstract Mesenchymal stem cells (MSCs) express immunomodulatory and anti-inflammatory effect toward immune system with several mechanisms such as secreting bioactive molecules. Distinct mechanisms of their immune actions, however, are not fully understood. As one of the important cells in immune system, macrophages play a vital role in innate immunity with two functional phenotypes, classically activated (M1) and alternatively activated (M2) macrophages. In this study, immunomodulatory ability of adipose-derived MSCs (AD-MSCs) secretome toward peritoneal macrophage was studied. Secretome was concentrated from human AD-MSCs using 3K TFF membrane. Murine peritoneal macrophages were cultured for 24 hours with secretome of AD-MSCs and 4 hours with LPS. Macrophages were analyzed by flow cytometry, ELISA, and real-time PCR. LC-MS/MS proteomics analysis of secretome was performed and acquired protein sets were analyzed by DAVID Bioinformatics. Secretome of AD-MSCs increased cell viability of peritoneal macrophages. Secretome treated peritoneal macrophages resulted in reduced M1 markers such as CD86, iNOS, and TNFα and increased M2 markers such as CD206, Arginase-1, and IL-10. Also, peritoneal macrophage expressed upregulation of mRNA level of LIGHT and MerTK, M2b and M2c markers, respectively. 83 proteins were identified in secretome according to the proteomics analysis. Several proteins among them were selected as candidates after confirmation of inhibiting the effect of secretome with neutralizing antibodies. In conclusion, secretome of AD-MSCs induced polarization of peritoneal macrophages from M1 macrophages to M2 macrophages and made them create anti-inflammatory milieu by several key secreted proteins. Supported by the National Research Foundation of Korea(NRF) grant funded by the Korea government(MSIT) (2021R1F1A1049630) and the Ministry of Health and Welfare, Republic of Korea (HI14C1324)

The glycolysis inhibitor 2-deoxyglucose ameliorates adjuvant-induced arthritis by regulating macrophage polarization in an AMPK-dependent manner

Macrophages are highly plastic cells critical for the development of rheumatoid arthritis (RA). Macrophages exhibit a high degree of pro-inflammatory plasticity in RA, accompanied by a metabolic reprogramming from oxidative phosphorylation (OXPHOS) to glycolysis. 2-deoxyglucose (2-DG), a glycolysis inhibitor, has previously been shown to exhibit anti-inflammatory and anti-arthritic properties. However, the specific mechanisms of inflammatory modulation by 2-DG remain unclear. This study used 2-DG to treat rats with adjuvant arthritis (AA) and investigated its specific anti-arthritic mechanisms in the murine-derived macrophage cell line RAW264.7 in vitro. 2-DG reduced the arthritis index as well as alleviated cellular infiltration, synovial hyperplasia, and bone erosion in AA rats. Moreover, 2-DG treatment modulated peritoneal macrophage polarization, increasing levels of the arginase1 (Arg1) and decreasing expression of the inducible nitric oxide synthase (iNOS). 2-DG activated AMP-activated protein kinase (AMPK) via phosphorylation and reduced activation of the nuclear factor κB (NF-κB) in peritoneal macrophages of AA rats. In vitro, we verified that 2-DG promoted macrophage transition from M1 to M2-type by upregulating the expression of p-AMPKα and suppressing NF-κB activation in LPS-stimulated RAW264.7 cells. LPS-induced macrophages exhibited a metabolic shift from glycolysis to OXPHOS following 2-DG treatment, as observed by reduced extracellular acidification rate (ECAR), lactate export, glucose consumption, as well as an elevated oxygen consumption rate (OCR) and intracellular ATP concentration. Importantly, changes in polarization and metabolism in response to 2-DG were dampened after AMPKα knockdown. These findings indicate that the anti-arthritic 2-DG effect is mediated by a modulation of macrophage polarization in an AMPK-dependent manner.

PRMT4 inhibitor TP-064 inhibits the pro-inflammatory macrophage lipopolysaccharide response in vitro and ex vivo and induces peritonitis-associated neutrophilia in vivo

Previous in vitro studies have shown that protein arginine N-methyltransferase 4 (PRMT4) is a co-activator for an array of cellular activities, including NF-κB-regulated pro-inflammatory responses. Here we investigated the effect of PRMT4 inhibitor TP-064 treatment on macrophage inflammation in vitro and in vivo.Exposure of RAW 264.7 monocyte/macrophages to TP-064 was associated with a significant decrease in the production of pro-inflammatory cytokines upon a lipopolysaccharide challenge. Similarly, thioglycollate-elicited peritoneal cells isolated from wildtype mice treated with TP-064 showed lowered mRNA expression levels and cytokine production of pro-inflammatory mediators interleukin (IL)-1β, IL-6, IL-12p40, and tumor necrosis factor-α in response to lipopolysaccharide exposure. However, TP-064-treated mice exhibited an ongoing pro-inflammatory peritonitis after 5 days of thioglycollate exposure, as evident from a shift in the peritoneal macrophage polarization state from an anti-inflammatory LY6ClowCD206hi to a pro-inflammatory LY6ChiCD206low phenotype. In addition, TP-064-treated mice accumulated (activated) neutrophils within the peritoneum as well as in the blood (7-fold higher; P < 0.001) and major organs such as kidney and liver, without apparent tissue toxicity. TP-064 treatment downregulated hepatic mRNA expression levels of the PRMT4 target genes glucose-6-phosphatase catalytic subunit (−50%, P < 0.05) and the cyclin-dependent kinases 2 (−50%, P < 0.05) and 4 (−30%, P < 0.05), suggesting a direct transcriptional effect of PRMT4 also in hepatocytes.In conclusion, we have shown that the PRMT4 inhibitor TP-064 induces peritonitis-associated neutrophilia in vivo and inhibits the pro-inflammatory macrophage lipopolysaccharide response in vitro and ex vivo. Our findings suggest that TP-064 can possibly be applied as therapy in NF-κB-based inflammatory diseases.

Absence of TNF Leads to Alternative Activation in Peritoneal Macrophages in Experimental Listeria Monocytogenes Infection

ABSTRACT Macrophages are crucial effectors of innate immunity against the pathogenic bacterium Listeria monocytogenes. The pro-inflammatory cytokine tumour necrosis factor-α (TNF) has been shown to be crucial for resistance to L. monocytogenes and mice deficient in TNF signalling succumb quickly after infection. However, the mechanisms underlying TNF-mediated defence against L. monocytogenes infection have not been completely elucidated. Here, we demonstrate that TNF concurrently functions to support a pro-inflammatory M1 phenotype while actively blocking macrophage polarization to the M2 phenotype. Compared to WT mice, peritoneal macrophages in TNF-deficient mice inoculated with L. monocytogenes respond with M2 polarization by upregulating Arg1. Consistently, TNF blockade in vitro resulted in M2 polarization in peritoneal macrophages during L. monocytogenes infection. Additionally, TNF promotes the transition from M2 to M1 polarization in peritoneal macrophages. Further investigation of peritoneal macrophage polarization suggested the NF-κB pathway is involved in the TNF-dependent M2 to M1 shift. Conversely, treatment of peritoneal macrophage with a PPARγ agonist blunted the expression of M1 genes induced by TNF and reduced NF-κB signalling pathway activation. Competing signalling mechanisms therefore play an essential role in the ability of peritoneal macrophage to resolve L. monocytogenes infections with TNF playing an essential role in driving M1 polarization. Abbreviations: LPM: large peritoneal macrophage; SPM: small peritoneal macrophage; LLO: listeriolysin O; iNOS: inducible nitric oxide synthase; DCs: dendritic cells

Gadolinium Chloride Inhibits the Production of Liver Interleukin-27 and Mitigates Liver Injury in the CLP Mouse Model.

Background Liver macrophages play an important regulatory role in the inflammatory response of liver injury after severe infection. Interleukin- (IL-) 27 is an inflammatory cytokine that plays an important role in diseases caused by bacterial infection. However, the relationship between IL-27 and liver macrophages in liver injury after severe infection is not yet clear. Methods A cecal ligation puncture (CLP) model was established in wild-type (WT) and IL-27 receptor- (WSX-1-) deficient (IL-27r−/−) mice, and recombinant IL-27 and gadolinium chloride (GdCl3) were injected into WT mice in the designated groups. The serum and liver IL-27, IL-6, tumor necrosis factor alpha (TNF-α), and IL-1β expression levels were evaluated by ELISA, quantitative PCR, or Western blotting; serum ALT and AST were detected by detection kits; and the severity of liver damage was evaluated by hematoxylin and eosin staining and the TUNEL assay of the liver tissue from the different groups. Liver macrophage polarization was evaluated by immunofluorescence. In addition, the polarization of peritoneal macrophage was evaluated by flow cytometry. Results The serum and liver IL-27 expression levels were elevated in WT mice after CLP-induced severe infection, which were consistent with the changes in HE scores in the liver tissue. The levels of serum ALT, AST, liver IL-6, TNF-α, and IL-1β mRNA and liver pathological injury scores were further increased when pretreated with recombinant IL-27 in WT mice, but these levels were decreased in IL-27r−/− mice after CLP-induced severe infection compared to WT mice. In WT mice pretreated with GdCl3, liver pathological scores, serum ALT and AST, TUNEL-positive cell proportion from liver tissues, liver IL-27 expression, and the liver macrophages M1 polarization proportion decreased after CLP; however, the serum IL-27, IL-6, TNF-α, and IL-1β levels and the pathological lung and kidney scores were not significantly changed. When supplemented with exogenous IL-27, the liver pathological scores, serum ALT, AST, TUNEL-positive cell proportion of liver tissues, liver IL-27 expression, and the liver macrophage M1 polarization proportion increased. The in vitro, IL-27 expression increased in peritoneal macrophages when stimulated with LPS. Recombinant IL-27 together with LPS promoted the elevations in IL-6, TNF-α, and IL-1β levels in supernatant and the M1 polarization of peritoneal macrophages. Conclusion IL-27 is an important cytokine in the inflammatory response to liver injury after severe infection. The reduction of liver injury by gadolinium chloride in severe infection mice models may relate to the inhibition of liver IL-27 production. These changes may be mainly related to the decrease of liver macrophages M1 polarization. IL-27 may have a positive feedback on these macrophages.

Oxamate, an inhibitor of lactate dehydrogenase, can stimulate M2 polarization of peritoneal macrophages in mice with Lewis lung carcinoma.

Inhibition of aerobic glycolysis of cancer cells is considered a promising therapeutic strategy for the treatment of neoplasms. Some inhibitors of energy metabolism can affect not only tumor cells but also the functional polarization of tumor-associated macrophages, which may either enhance the antitumor effect of such agents or impair their antitumor efficacy. To investigate the effect of oxamate, a lactate dehydrogenase (LDH) inhibitor, on the polarization of peritoneal macrophages (PMP) in both intact mice and mice with transplanted Lewis lung carcinoma (LLC). The low-metastatic LLC variant, LLC/R9, was transplanted to female C57Bl/6mice. Sodium oxamate was used as the test agent at concentrations of 0.02, 0.2, and 2mg/ml. Macrophage polarization in tumor-bearing mice was estimated on day 23after tumor transplantation by assessing nitric oxide (NO) production and arginase activity as functional indices of PMPs polarization. Oxamate can affect the functional polarization of PMPs in both intact mice and animals with transplanted LLC/R9. Oxamate in all studied concentrations changed the markers of PMPs polarization in intact mice (decreasing NO levels and activating arginase activity) that indicated the stimulation of M2polarization. In tumor-bearing animals, stimulation of M2polarization is observed at low concentrations of oxamate (0.02mg/ml), but its high concentrations (2.0mg/ml) causes M1polarization, which is characterized by three-fold increase in the level of NO and a decrease in the level of arginase activity. Oxamate, an inhibitor of LDH, can stimulate M2polarization of peritoneal macrophages of mice bearing LLC in a dose-dependent manner.

Cannabinoid type 2 receptor manipulates skeletal muscle regeneration partly by regulating macrophage M1/M2 polarization in IR injury in mice

AimsThe beneficial effects of cannabinoid type 2 receptor (CB2R) activation have been verified in various tissue repair processes. Our recent study revealed CB2R activation promotes myogenesis partly through Nrf2 signaling in a mouse skeletal muscle ischemia-reperfusion (IR) injury model. Other relevant mechanisms need to be further elucidated. Macrophages orchestrate tissue regeneration mainly by changing their phenotype and function. The aim of this study was to investigate the role of CB2R in IR-induced skeletal muscle regeneration, focusing on its impact on macrophage polarization and the consequences on myogenesis. Main methodsThe effects of CB2R on skeletal muscle regeneration, and the macrophage infiltration and M1/M2 polarization were tested with the IR injury model in wild type (WT) and CB2R knockout (CB2R-KO) mice. The effect of CB2R on peritoneal macrophage polarization, and its impact on the myoblasts differentiation was evaluated by co-culture experiments in vitro. Key findingsThe present study revealed the myofiber regeneration was hindered in the CB2R-KO mice. The infiltration of M1 macrophages and relevant markers' protein expression were enhanced in the CB2R-KO mice, while that of M2 macrophages was decreased compared with the WT mice. The in vitro studies further demonstrated that the absence of CB2R promoted M1 polarization while inhibited M2 polarization. The promoted M1 polarization and retarded M2 polarization in CB2R-KO macrophages hindered myoblasts differentiation. SignificanceOverall, these results suggested CB2R plays a beneficial effect on skeletal muscle regeneration partly by regulating macrophage M1/M2 polarization after IR injury in mice.

High glucose contributes to the polarization of peritoneal macrophages to the M2 phenotype invivo and invitro.

Glucose is the primary osmotic medium used in most peritoneal dialysis (PD) solutions, and long-term exposure to high glucose is a major contributor to peritoneal fibrosis. Our previous study revealed that M2 macrophages participate in the development of PD-related fibrosis in a rat model. In the present study, the effects of high glucose on peritoneal macrophage polarization in vivo and in vitro were further evaluated. Continuous ambulatory PD (CAPD) patients with an overnight dwell of 1.5 or 2.5% glucose dialysate were recruited for this study. Overnight effluent samples from patients with CAPD (2,000 ml) were centrifuged to collect cells from the peritoneal cavity. J774A.1 cells (murine macrophages from ascites) were cultured in different concentrations of glucose. Macrophage phenotype markers were detected by flow cytometry. The levels of cytokines in PD effluent and the supernatant of murine macrophages were detected by enzyme-linked immunosorbent assays. The activity of arginase was determined by quantitative colorimetric analysis. In total, 107 CAPD subjects (92 patients using 1.5% glucose dialysate and 15 patients using 2.5% glucose dialysate) were recruited. The percentage of M1 macrophages (CD14- and CCr7-positive cells) in the 1.5 and 2.5% glucose dialysate groups was 23.0±13.3 and 24.9±12.0%, respectively. The difference was not significant (P>0.05). The percentage of M2 macrophages (CD14- and CD206-positive cells) in the 1.5% glucose dialysate group (36.2±11.4%) was significantly decreased compared to the 2.5% glucose dialysate group (43.2±7.4%) (P<0.05). Murine macrophages were cultured in a high-glucose in vitro environment, and the percentage of M1 macrophages in 138.8 mmol/l glucose medium significantly increased over time. The percentage of M2 macrophages increased in a glucose concentration-dependent and time-dependent manner. Arginase 1 in murine macrophages and the level of transforming growth factor β1 in the supernatant increased in a glucose concentration-dependent manner. In conclusion, high glucose contributed to the polarization of peritoneal macrophages to the M2 phenotype, which may play an important role in the pathogenesis of PD-related fibrosis.

MSC-secreted TGF-\u03b2 regulates lipopolysaccharide-stimulated macrophage M2-like polarization via the Akt/FoxO1 pathway

BackgroundAn uncontrolled inflammatory response is a critical pathophysiological feature of sepsis. Mesenchymal stem cells (MSCs) induce macrophage phenotype polarization and reduce inflammation in sepsis. MSC-secreted transforming growth factor beta (TGF-β) participated in the immune modulatory function of MSCs. However, the underlying mechanism of MSC-secreted TGF-β was not fully elucidated in regulation macrophage M2-like polarization.MethodsThe paracrine effects of MSCs on macrophage polarization were studied using a co-culture protocol with LPS-stimulated RAW264.7 cells/mouse peritoneal macrophages and MSCs. The effect of TGF-β in the co-culture system was blocked by the TGF-β receptor inhibitor. To determine the role of MSC-secreted TGF-β, we used recombinant TGF-β to culture with LPS-stimulated RAW264.7 cells. In addition, we employed antibody microarray analysis to determine the mechanisms of MSC secreted TGF-β on LPS-stimulated RAW264.7 cell/mouse peritoneal macrophage M2-like polarization. Furthermore, we used an Akt inhibitor and a FoxO1 inhibitor to inhibit the Akt/FoxO1 pathway. The nuclear translocation of FoxO1 was detected by Western blot.ResultsMSCs induced LPS-stimulated RAW264.7 cell/mouse peritoneal macrophage polarization towards the M2-like phenotype and significantly reduced pro-inflammatory cytokine levels via paracrine, which was inhibited by TGF-β receptor inhibitor. Furthermore, we found that MSC-secreted TGF-β enhanced the macrophage phagocytic ability. The antibody microarray analysis and Western blot verified that TGF-β treatment activated the Akt/FoxO1 pathway in LPS-stimulated macrophages, TGF-β-induced FoxO1 nuclear translocation and obviously expressed in the cytoplasm, the effects of TGF-β regulatory effects on LPS-stimulated macrophage were inhibited by pre-treatment with Akt inhibitor and FoxO1 inhibitor.ConclusionsTGF-β secreted by MSCs could skew LPS-stimulated macrophage polarization towards the M2-like phenotype, reduce inflammatory reactions, and improve the phagocytic ability via the Akt/FoxO1 pathway, providing potential therapeutic strategies for sepsis.

Dendritic Cell Activating Receptor 1 (DCAR1) Associates With FcεRIγ and Is Expressed by Myeloid Cell Subsets in the Rat.

Dendritic cell activating receptor-1 (DCAR1) is a cell-surface receptor encoded by the Antigen Presenting Lectin-like gene Complex (APLEC). We generated a mouse monoclonal antibody against rat DCAR1, and used this to characterize receptor expression and function. Rat DCAR1 was expressed on minor subsets of myeloid cells in lymphoid tissue, but was uniformly expressed at a high level by eosinophils, and at a low level by neutrophils. It was expressed by eosinophils in the peritoneal cavity and the lamina propria of the gut, and by subsets of macrophages or dendritic cells at these sites. Polarization of peritoneal macrophages showed that DCAR1 expression was absent on M1 macrophages, and increased on M2 macrophages. DCAR1 could be expressed as a homodimer and its associated with the activating adaptor protein FcεRIγ. This association allowed efficient phagocytosis of antibody-coated beads. Additionally, cross-linking of DCAR1 on the surface of rat eosinophils lead to production of reactive oxygen species. These data show that DCAR1 is an activating receptor. Its expression on M2 macrophages and eosinophils suggests that it may play a role in the immune response to parasites.

Stabilized Interleukin-4-Loaded Poly(lactic-co-glycolic) Acid Films Shift Proinflammatory Macrophages toward a Regenerative Phenotype in Vitro.

Macrophages are immune cells involved in wound healing and tissue regeneration; however, the sustained presence of proinflammatory macrophages in wound sites impairs healing. In this study, we shifted peritoneal macrophage polarization away from a proinflammatory (M1) phenotype through exposure to stabilized interleukin-4 (IL-4) in poly(lactic-co-glycolic acid) films in combination with topographical guidance from electrospun poly-L-lactic acid fibers. To our knowledge, this was the first study to stabilize IL-4 with bovine serum albumin (BSA) within a biomaterial. When IL-4 was coloaded with BSA for stabilization, we saw increased IL-4 bioactivity compared to no added stabilization, trehalose stabilization, or murine serum albumin stabilization. We observed increased elongation of peritoneal macrophages, increased RNA expression of anti-inflammatory marker arginase-1, increased ratio of interleukin-10/interleukin- 12 p40 RNA, and decreased protein expression of proinflammatory markers (interleukin-12 p40 and RANTES) compared to controls. Taken together, these results suggest the macrophages were less proinflammatory and were a more pro-resolving phenotype. When stabilized with BSA, IL-4-loaded films effectively shift macrophage polarization state and are thus promising scaffolds to reduce inflammation within in vivo injury models.

[Effects of histone methyltransferase inhibitor on the polarization of peritoneal macrophages in septic mice].

To investigate the effect of histone methyltransferase (EZH2) inhibitor on the polarization of peritoneal macrophages in septic mice. Thirty-six healthy male C57BL/6J mice were divided into three groups by random number table method (n = 12): sham operated group (Sham group), sepsis model group (CLP group) and EZH2 inhibitor treatment group (CLP+3-DZNeP group). Sepsis animal model was established by cecum ligation and puncture (CLP); Sham group was challenged only by cecum traction without ligation. 3-Deazaneplanocin A (3-DZNeP) 1 mg/kg was intraperitoneal injected 24 hours before and 1 hour after CLP in CLP+3-DZNeP group. Eight mice in each group were sacrificed at 24 hours after surgery. The levels of proinflammatory cytokines interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in peritoneal lavatory fluid were detected by high throughput liquid protein chip. The expression levels of inducible nitrogenase (iNOS) and macrophage mannose receptor (CD206) were analyzed by flow cytometry. Mouse peritoneal macrophages were isolated and purified by adherent method, the protein expressions of EZH2, peroxisome proliferator-activated receptorγ (PPARγ) were detected by Western Blot. The remaining 4 mice were sacrificed at 48 hours after surgery, the histopathological changes of lung and kidney tissue were evaluated by hematoxylin-eosin (HE) staining. Compared with Sham group, the infiltration of inflammatory cells in lung and kidney of the CLP group, the levels of IL-6 and TNF-α in peritoneal lavatory fluid were significant increased [IL-6 (ng/L): 7 794.75±405.56 vs. 78.63±74.09, TNF-α (ng/L): 147.25±25.19 vs. 18.20±5.03, both P < 0.01], the percentage of M1 type macrophages was significantly increased [iNOS+ F4/80+: (13.18±8.80)% vs. (1.57±0.77)%, P < 0.05], and the protein expression of EZH2 was significantly increased (EZH2/GAPDH: 0.84±0.11 vs. 0.11±0.03, P < 0.01), while the protein expression of PPARγ was significantly decreased (PPARγ/GAPDH: 0.09±0.01 vs. 0.27±0.09, P < 0.01). Compared with CLP group, the histopathological changes of lung and kidney in CLP+3-DZNeP group were significantly alleviated, the levels of IL-6 and TNF-α in peritoneal lavatory fluid were significantly decreased [IL-6 (ng/L): 4 207.10±876.60 vs. 7 794.75±405.56, TNF-α (ng/L): 63.00±25.37 vs. 147.25±25.19, both P < 0.01 ], the percentage of M1 type macrophages was significantly decreased [iNOS+ F4/80+: (3.64±0.89)% vs. (13.18±8.80)%, P < 0.05], while the percentage of M2 type macrophages was significantly increased [CD206+ F4/80+: (17.68±5.63)% vs. (7.60±3.17)%, P < 0.01], the protein expression of EZH2 was significantly decreased (EZH2/GAPDH: 0.53±0.09 vs. 0.84±0.11, P < 0.05), and the protein expression of PPARγ was significantly increased (PPARγ/GAPDH: 0.39±0.14 vs. 0.09±0.01, P < 0.05). Sepsis induces high expression of EZH2 in peritoneal macrophages, and may induce polarization of M1 type macrophages by inhibiting the expression of PPARγ protein. EZH2 inhibitor 3-DZNeP can lessen the inflammatory cytokines release by inhibiting the M1 type macrophages polarization.

Abdominal paracentesis drainage ameliorates severe acute pancreatitis in rats by regulating the polarization of peritoneal macrophages.

AIMTo investigate the role of peritoneal macrophage (PM) polarization in the therapeutic effect of abdominal paracentesis drainage (APD) on severe acute pancreatitis (SAP).METHODSSAP was induced by 5% Na-taurocholate retrograde injection in Sprague-Dawley rats. APD was performed by inserting a drainage tube with a vacuum ball into the lower right abdomen of the rats immediately after the induction of SAP. To verify the effect of APD on macrophages, PMs were isolated and cultured in an environment, with the peritoneal inflammatory environment simulated by the addition of peritoneal lavage in complete RPMI 1640 medium. Hematoxylin and eosin staining was performed. The levels of pancreatitis biomarkers amylase and lipase as well as the levels of inflammatory mediators in the blood and peritoneal lavage were determined. The polarization phenotypes of the PMs were identified by detecting the marker expression of M1/M2 macrophages via flow cytometry, qPCR and immunohistochemical staining. The protein expression in macrophages that had infiltrated the pancreas was determined by Western blot.RESULTSAPD treatment significantly reduced the histopathological scores and levels of amylase, lipase, tumor necrosis factor-α and interleukin (IL)-1β, indicating that APD ameliorates the severity of SAP. Importantly, we found that APD treatment polarized PMs towards the M2 phenotype, as evidenced by the reduced number of M1 macrophages and the reduced levels of pro-inflammatory mediators, such as IL-1β and L-selectin, as well as the increased number of M2 macrophages and increased levels of anti-inflammatory mediators, such as IL-4 and IL-10. Furthermore, in an in vitro study wherein peritoneal lavage from the APD group was added to the cultured PMs to simulate the peritoneal inflammatory environment, PMs also exhibited a dominant M2 phenotype, resulting in a significantly lower level of inflammation. Finally, APD treatment increased the proportion of M2 macrophages and upregulated the expression of the anti-inflammatory protein Arg-1 in the pancreas of SAP model rats.CONCLUSIONThese findings suggest that APD treatment exerts anti-inflammatory effects by regulating the M2 polarization of PMs, providing novel insights into the mechanism underlying its therapeutic effect.

[Effects of allogeneic bone marrow mesenchymal stem cells on polarization of peritoneal macrophages in rats with sepsis].

Objective: To explore the effects of allogeneic bone marrow mesenchymal stem cells (BMSCs) on polarization of peritoneal macrophages isolated from rats with sepsis induced by endotoxin/lipopolysaccharide (LPS). Methods: (1) BMSCs were isolated, cultured and purified from 5 SD rats with whole bone marrow adherent method. The third passage of cells were collected for morphologic observation, detection of expressions of stem cell surface markers CD29, CD44, CD45, and CD90 with flow cytometer, and identification of osteogenic and adipogenic differentiation. (2) Another 45 SD rats were divided into sham injury group (SI, n=5), LPS control group (LC, n=20), and BMSCs-treated group (BT, n=20) according to the random number table. Rats in groups LC and BT were injected with LPS (5 mg/kg) via tail vein to induce sepsis; rats in group SI were injected with the same amount of normal saline to simulate the damage. At post injury hour (PIH) 1, rats in group BT were given 1 mL BMSCs (2×10(6)/mL) via tail vein injection; rats in another two groups were injected with equal volume of phosphate buffer saline. Five rats in group SI at PIH 24 and in groups LC and BT at PIH 6, 12, 24, and 48 were sacrificed to harvest lung tissue for pathological observation with HE staining. In addition, rats in group SI at PIH 24 and in groups LC and BT at PIH 24 and 48 were simultaneously performed with intraperitoneal injection of low-glucose DMEM. Then peritoneal fluid was harvested to culture peritoneal macrophages. Flow cytometer was used to assess the positive expression of cell makers of macrophages including CD68 (making gate), CD11c, and CD206 in group SI at PIH 24 and in groups LC and BT at PIH 24 and 48. Data were processed with one-way analysis of variance and LSD test. Results: (1) The third passage of cells showed uniform fiber-like shape similar to fibroblasts. These cells showed positive expressions of CD29, CD44, CD90 and weak positive expression of CD45. They were able to differentiate into osteoblasts and adipocytes. These cells were identified as BMSCs. (2) At PIH 24, the structure of pulmonary alveoli of rats in group SI was clear and complete with no congestion or inflammatory cell infiltration. At PIH 6, the structure of pulmonary alveoli of rats in groups LC and BT was clear with a small amount of inflammatory cell infiltration, slight congestion and pulmonary interstitial thickening. At PIH 12, the inflammatory responses in lung tissue of rats in group LC were more severe than those in group BT with a large amount of inflammatory cell infiltration, serious congestion, and obvious pulmonary interstitial thickening. The pathological results of rats in group BT at PIH 12 was consistent with the results at PIH 6. At PIH 24, the pathological results of rats in groups LC and BT were similar to the results at PIH 12. At PIH 48, the structure of pulmonary alveoli tissue of rats in group LC was still severely disrupted, with a large number of inflammatory cell infiltration and congestion in lung tissue, but pulmonary interstitial thickening was slightly alleviated than before. The condition of rats in group BT nearly recovered to that in group SI. (3) At PIH 24, the positive expression rate of CD11c in peritoneal macrophages of rats in group LC [(83±10)%] was close to that in group BT [(87±7)%, P>0.05], and they were both significantly higher than the rate in group SI [(55±12)%, with P values below 0.01]. The positive expression rate of CD11c in peritoneal macrophages of rats in group LC [(59±11)%] at PIH 48 was close to that in group SI at PIH 24 (P>0.05), and they were both significantly higher than the rate in group BT [(20±11)%] at PIH 48 (with P values below 0.01). At PIH 24, the positive expression percentages of CD206 in peritoneal macrophages of rats were similar among the three groups (with P values above 0.05). The positive expression percentage of CD206 in peritoneal macrophages of rats in group SI at PIH 24 was close to that in group BT at PIH 48 (P>0.05), and they were both significantly lower than the percentage in group LC at PIH 48 (with P values below 0.01). Conclusions: BMSCs can reduce the pathological inflammatory responses in the lung of rats with sepsis and inhibit peritoneal macrophages from polarizing into M1 phenotype, whereas they can not promote macrophages to polarize into M2 phenotype.

M2 polarization of murine peritoneal macrophages induces regulatory cytokine production and suppresses T-cell proliferation.

Bone-marrow-derived macrophages are divided into two phenotypically and functionally distinct subsets, M1 and M2 macrophages. Recently, it was shown that adoptive transfer of M2-polarized peritoneal macrophages reduced the severity of experimental colitis in mice. However, it is still unclear whether peritoneal macrophages possess the same ability to be polarized to cells with functionally different phenotypes and cytokine production patterns as bone-marrow-derived macrophages. To address this question, we examined the ability of peritoneal macrophages to be polarized to the M1 and M2 phenotypes and determined the specific cytokine profiles of cells with each phenotype. We showed that peritoneal macrophages, as well as bone-marrow-derived macrophages, were differentiated into M1 and M2 phenotypes following stimulation with interferon-γ (IFN-γ) and interleukin-4 (IL-4)/IL-13, respectively. Following invitro stimulation with lipopolysaccharide, M2-polarized peritoneal macrophages predominantly expressed T helper type 2 (Th2) cytokines and regulatory cytokines, including IL-4, IL-13, transforming growth factor-β and IL-10, whereas M1-polarized peritoneal macrophages expressed negligible amounts of Th1 and pro-inflammatory cytokines. ELISA showed that M2-polarized peritoneal macrophages produced significantly more IL-10 than M1-polarized peritoneal macrophages. Notably, M2-polarized peritoneal macrophages contributed more to the suppression of T-cell proliferation than did M1-polarized peritoneal macrophages. The mRNA expression of Th2 cytokines, including IL-4 and IL-13, increased in T-cells co-cultured with M2-polarized macrophages. Hence, our findings showed that M2 polarization of peritoneal macrophages induced regulatory cytokine production and suppressed T-cell proliferation invitro, and that resident peritoneal macrophages could be used as a new adoptive transfer therapy for autoimmune/inflammatory diseases after polarization to the regulatory phenotype exvivo.