Neglected immunoregulation: M2 polarization of macrophages triggered by low-dose irradiation plays an important role in bone regeneration.

Current studies have found that low-dose irradiation (IR) can promote bone regeneration. However, mechanism studies of IR-triggered bone regeneration mainly focus on the effects of osteoblasts, neglecting the role of the surrounding immune microenvironment. Here in this study, in vitro proliferation experiments showed that low-dose IR ≤2 Gy could promote the proliferation of bone marrow mesenchymal stem cells (BMSCs), and qRT-PCR assay showed that low-dose IR ≤2 Gy could exert the M2 polarization of Raw264.7 cells, while IR >2 Gy inhibited BMSC proliferation and triggered M1 polarization in Raw264.7 cells. The ALP and mineralized nodules staining showed that low-dose IR ≤2 Gy not only promoted osteoblast mineralization through IR-triggered osteoblast proliferation but also through M2 polarization of Raw264.7 cells, while high-dose IR >2 Gy had the opposite effect. The co-incubation of BMSC with low-dose IR irradiated Raw264.7 cell supernatants increased the mRNA expression of BMP-2 and Osx. The rat cranial defects model revealed that low-dose IR ≤2 Gy gradually promoted bone regeneration, while high-dose IR >2 Gy inhibited bone regeneration. Detection of macrophage polarity in peripheral blood samples showed that low-dose IR ≤2 Gy increased the expression of CD206 and CD163, but decreased the expression of CD86 and CD80 in macrophages, which indicated M2 polarization of macrophages in vivo, while high-dose IR had the opposite effect. Our finding innovatively revealed that low-dose IR ≤2 Gy promotes bone regeneration not only by directly promoting the proliferation of osteoblasts but also by triggering M2 polarization of macrophages, which provided a new perspective for immune mechanism study in the treatment of bone defects with low-dose IR.

The expression of matrix metalloproteinase-12 in the peritoneum of rats with continuous peritoneal dialysis.

BACKGROUND: Peritoneal dialysis (PD) is an important alternative treatment for end-stage renal disease. Continuous exposure to non-physiological fluids during PD is associated with pathological responses, such as sustained microinflammation, leading to tissue fibrosis and angiogenesis. However, the effect of PD fluid on submesothelial cells has not yet been investigated in detail. METHODS: We investigated the association between macrophages and the expression of matrix metalloproteinase-12 (MMP-12), an elastin proteinase secreted by macrophages, in the peritoneal tissue of rats undergoing continuous PD. RESULTS: Morphological data revealed that the submesothelial layer of the peritoneum in PD model rats was markedly thickened, with fibrosis and angiogenesis. In the fibrillization area, elastin was disorganized and fragmented, and macrophages accumulated, which tended to have M2 characteristics. The expression of MMP-12 was enhanced by continuous exposure to PD fluid, suggesting that MMP-12 expression may be involved in PD fluid-induced peritoneal damage. CONCLUSIONS: The results of this study may lead to a better understanding of the mechanisms underlying fibrosis in PD.

Macrophage-Specific Coxsackievirus and Adenovirus Receptor Deletion Enhances Macrophage M1 Polarity in CVB3-Induced Myocarditis.

The coxsackievirus and adenovirus receptor (CAR) is very well known as an epithelial tight junction and cardiac intercalated disc protein; it mediates attachment and infection via the coxsackievirus B3 (CVB3) and type 5 adenovirus. Macrophages play important roles in early immunity during viral infections. However, the role of CAR in macrophages is not well studied in relation to CVB3 infection. In this study, the function of CAR was observed in the Raw264.7 mouse macrophage cell line. CAR expression was stimulated by treatment with lipopolysaccharide (LPS) and tumor necrosis factor-α (TNF-α). In thioglycollate-induced peritonitis, the peritoneal macrophage was activated and CAR expression was increased. The macrophage-specific CAR conditional knockout mice (KO) were generated from lysozyme Cre mice. The expression of inflammatory cytokine (IL-1ß and TNF-α) was attenuated in the KO mice's peritoneal macrophage after LPS treatment. In addition, the virus was not replicated in CAR-deleted macrophages. The organ virus replication was not significantly different in both wild-type (WT) and KO mice at days three and seven post-infection (p.i). However, the inflammatory M1 polarity genes (IL-1ß, IL-6, TNF-α and MCP-1) were significantly increased, with increased rates of myocarditis in the heart of KO mice compared to those of WT mice. In contrast, type1 interferon (IFN-α and ß) was significantly decreased in the heart of KO mice. Serum chemokine CXCL-11 was increased in the KO mice at day three p.i. compared to the WT mice. The attenuation of IFN-α and ß in macrophage CAR deletion induced higher levels of CXCL-11 and more increased CD4 and CD8 T cells in KO mice hearts compared to those of WT mice at day seven p.i. These results demonstrate that macrophage-specific CAR deletion increased the macrophage M1 polarity and myocarditis in CVB3 infection. In addition, chemokine CXCL-11 expression was increased, and stimulated CD4 and CD8 T cell activity. Macrophage CAR may be important for the regulation of innate-immunity-induced local inflammation in CVB3 infection.

The Apolipoprotein A-I Mimetic L-4F Attenuates Monocyte Activation and Adverse Cardiac Remodeling after Myocardial Infarction.

Excessive inflammation after myocardial infarction (MI) can promote infarct expansion and adverse left ventricular (LV) remodeling. L-4F, a mimetic peptide of apolipoprotein A-I (apoA-I), exhibits anti-inflammatory and anti-atherogenic properties; however, whether L-4F imparts beneficial effects after myocardial infarction (MI) is unknown. Here we demonstrate that L-4F suppresses the expansion of blood, splenic, and myocardial pro-inflammatory monocytes and macrophages in a mouse model of reperfused MI. Changes in immune cell profiles were accompanied by alleviation of post-MI LV remodeling and dysfunction. In vitro, L-4F also inhibited pro-inflammatory and glycolytic gene expression in macrophages. In summary, L-4F treatment prevents prolonged and excessive inflammation after MI, in part through modulation of pro-inflammatory monocytes and macrophages, and improves post-MI LV remodeling. These data suggest that L-4F could be a used as a therapeutic adjunct in humans with MI to limit inflammation and alleviate the progression to heart failure.

Naoxintong attenuates Ischaemia/reperfusion Injury through inhibiting NLRP3 inflammasome activation.

Naoxintong (NXT) is a Chinese Materia Medica standardized product extracted from 16 various kinds of Chinese traditional herbal medicines including Salvia miltiorrhiza, Angelica sinensis, Astragali Radix. Naoxintong is clinically effective in treating ischaemia heart disease. Nucleotide-binding oligomerization domain-Like Receptor with a Pyrin domain 3 (NLRP3) inflammasome has been critically involved in myocardial ischaemia/reperfusion (I/R) injury. Here, we have been suggested that NXT might attenuate myocardial I/R injury via suppression of NLRP3 inflammasome activation. Male C57BL6 mice were subjected to myocardial I/R injury via 45 min. coronary ligation and release for the indicated times. Naoxintong (0.7 g/kg/day) and PBS were orally administrated for 2 weeks before surgery. Cardiac function assessed by echocardiography was significantly improved in the NXT group compared to PBS group at day 2 after myocardial I/R. NLRP3 inflammasome activation is crucially involved in the initial inflammatory response after myocardial I/R injury, leading to cleaved caspase-1, mature interleukin (IL)-1ß production, accompanying by macrophage and neutrophil infiltration. The cardioprotective effect of NXT was associated with a diminished NLRP3 inflammasome activation, decreased pro-inflammatory macrophage (M1 macrophages) and neutrophil infiltration after myocardial I/R injury. In addition, serum levels of IL-1ß, indicators of NLRP3 inflammasome activation, were also significantly suppressed in the NXT treated group after I/R injury. Naoxintong exerts cardioprotive effects at least partly by suppression of NLRP3 inflammasome activation in this I/R injury model.

Secreted ectodomain of sialic acid-binding Ig-like lectin-9 and monocyte chemoattractant protein-1 promote recovery after rat spinal cord injury by altering macrophage polarity.

Engrafted mesenchymal stem cells from human deciduous dental pulp (SHEDs) support recovery from neural insults via paracrine mechanisms that are poorly understood. Here we show that the conditioned serum-free medium (CM) from SHEDs, administered intrathecally into rat injured spinal cord during the acute postinjury period, caused remarkable functional recovery. The ability of SHED-CM to induce recovery was associated with an immunoregulatory activity that induced anti-inflammatory M2-like macrophages. Secretome analysis of the SHED-CM revealed a previously unrecognized set of inducers for anti-inflammatory M2-like macrophages: monocyte chemoattractant protein-1 (MCP-1) and the secreted ectodomain of sialic acid-binding Ig-like lectin-9 (ED-Siglec-9). Depleting MCP-1 and ED-Siglec-9 from the SHED-CM prominently reduced its ability to induce M2-like macrophages and to promote functional recovery after spinal cord injury (SCI). The combination of MCP-1 and ED-Siglec-9 synergistically promoted the M2-like differentiation of bone marrow-derived macrophages in vitro, and this effect was abolished by a selective antagonist for CC chemokine receptor 2 (CCR2) or by the genetic knock-out of CCR2. Furthermore, MCP-1 and ED-Siglec-9 administration into the injured spinal cord induced M2-like macrophages and led to a marked recovery of hindlimb locomotor function after SCI. The inhibition of this M2 induction through the inactivation of CCR2 function abolished the therapeutic effects of both SHED-CM and MCP-1/ED-Siglec-9. Macrophages activated by MCP-1 and ED-Siglec-9 extended neurite and suppressed apoptosis of primary cerebellar granule neurons against the neurotoxic effects of chondroitin sulfate proteoglycans. Our data suggest that the unique combination of MCP-1 and ED-Siglec-9 repairs the SCI through anti-inflammatory M2-like macrophage induction.

Macrophage phenotypic subtypes diametrically regulate epithelial-mesenchymal plasticity in breast cancer cells.

BACKGROUND: Metastatic progression of breast cancer involves phenotypic plasticity of the carcinoma cells moving between epithelial and mesenchymal behaviors. During metastatic seeding and dormancy, even highly aggressive carcinoma cells take on an E-cadherin-positive epithelial phenotype that is absent from the emergent, lethal metastatic outgrowths. These phenotypes are linked to the metastatic microenvironment, though the specific cells and induction signals are still to be deciphered. Recent evidence suggests that macrophages impact tumor progression, and may alter the balance between cancer cell EMT and MErT in the metastatic microenvironment. METHODS: Here we explore the role of M1/M2 macrophages in epithelial-mesenchymal plasticity of breast cancer cells by coculturing epithelial and mesenchymal cells lines with macrophages. RESULTS: We found that after polarizing the THP-1 human monocyte cell line, the M1 and M2-types were stable and maintained when co-cultured with breast cancer cells. Surprisingly, M2 macrophages may conferred a growth advantage to the epithelial MCF-7 cells, with these cells being driven to a partial mesenchymal phenotypic as indicated by spindle morphology. Notably, E-cadherin protein expression is significantly decreased in MCF-7 cells co-cultured with M2 macrophages. M0 and M1 macrophages had no effect on the MCF-7 epithelial phenotype. However, the M1 macrophages impacted the highly aggressive mesenchymal-like MDA-MB-231 breast cancer cells to take on a quiescent, epithelial phenotype with re-expression of E-cadherin. The M2 macrophages if anything exacerbated the mesenchymal phenotype of the MDA-MB-231 cells. CONCLUSION: Our findings demonstrate M2 macrophages might impart outgrowth and M1 macrophages may contribute to dormancy behaviors in metastatic breast cancer cells. Thus EMT and MErT are regulated by selected macrophage phenotype in the liver metastatic microenvironment. These results indicate macrophage could be a potential therapeutic target for limiting death due to malignant metastases in breast cancer.

Counter-regulation of the ligand-receptor pair Leda-1/Pianp and Pilrα during the LPS-mediated immune response of murine macrophages.

Liver endothelial differentiation-associated protein-1 (Leda-1/Pianp) is a type-I-transmembrane protein that is able to bind and activate immune inhibitory receptor Pilrα. Here we show that Leda-1/Pianp is strain-specifically expressed in lymphoid organs and macrophages of Th2-prone BALB/c mice but not of Th1-prone C57BL/6J mice. LPS stimulation of BALB/c bone marrow-derived macrophages (BMM) and macrophage-like Raw 264.7 cells conversely regulated Leda-1/Pianp and Pilrα expression. Pilrα induction was caused by LPS-mediated transcriptional modulation and increased mRNA expression. On the other hand, the LPS-mediated decline of Leda-1/Pianp expression was the result of proteolytic degradation by matrix metalloproteinases. In summary, these findings demonstrate that counter-regulation of the ligand-receptor pair Leda-1/Pianp and Pilrα is part of the complex innate immune response of macrophages and its genetically determined strain-specific modulation.

Enhanced wound regeneration by PGS/PLA fiber dressing containing platelet-rich plasma: an in vitro study.

Novel wound dressings with therapeutic effects are being continually designed to improve the wound healing process. In this study, the structural, chemical, physical, and biological properties of an electrospun poly glycerol sebacate/poly lactide acid/platelet-rich plasma (PGS/PLA-PRP) nanofibers were evaluated to determine its impacts on in vitro wound healing. Results revealed desirable cell viability in the Fibroblast (L929) and macrophage (RAW-264.7) cell lines as well as human umbilical vein endothelial cells (HUVEC). Cell migration was evident in the scratch assay (L929 cell line) so that it promoted scratch contraction to accelerate in vitro wound healing. Moreover, addition of PRP to the fiber structure led to enhanced collagen deposition (~ 2 times) in comparison with PGS/PLA scaffolds. While by addition PRP to PGS/PLA fibers not only decreased the expression levels of pro-inflammatory cytokines (IL-6 and TNF-α) in RAW-264.7 cells but also led to significantly increased levels of cytokine (IL-10) and the growth factor (TGF-ß), which are related to the anti-inflammatory phase (M2 phenotype). Finally, PGS/PLA-PRP was found to induce a significant level of angiogenesis by forming branching points, loops, and tubes. Based on the results obtained, the PGS/PLA-PRP dressing developed might be a promising evolution in skin tissue engineering ensuring improved wound healing and tissue regeneration.

The clinical impact of macrophage polarity after Kasai portoenterostomy in biliary atresia.

Introduction: Biliary atresia (BA) is a cholestatic hepatopathy caused by fibrosing destruction of intrahepatic and extrahepatic bile ducts, and its etiology has not been clearly revealed. In BA, liver fibrosis progression is often observed even after Kasai portoenterostomy (KPE), and more than half of cases require liver transplantation in their lifetime in Japan. Macrophages play an important role in liver fibrosis progression and are classically divided into proinflammatory (M1) and fibrotic macrophages (M2), whose phenotypic transformation is called "macrophage polarity." The polarity has been reported to reflect the tissue microenvironment. In this study, we examined the relationship between macrophage polarity and the post-KPE clinical course. Materials and methods: Thirty BA patients who underwent KPE in our institution from 2000 to 2020 were recruited. Multiple immunostainings for CD68, CD163, CK19, and α-SMA were carried out on liver biopsy specimens obtained at KPE. ROC curves were calculated based on each clinical event, and the correlation with the clinical data was analyzed. Results and discussion: The M2 ratio, defined as the proportion of M2 macrophages (CD163-positive cells), was correlated inversely with the occurrence of postoperative cholangitis (AUC: 0.7602). The patients were classified into M2 high (n = 19) and non-high (n = 11) groups based on an M2 ratio value obtained from the Youden index ( = 0.918). As a result, pathological evaluations (Metavir score, αSMA area fraction, and CK19 area fraction) were not significantly different between these groups. In mild liver fibrosis cases (Metavir score = 0-2), the M2 non-high group had a significantly lower native liver survival rate than the high group (p = 0.02). Moreover, 4 out of 8 cases in the M2 non-high group underwent early liver transplantation within 2 years after KPE. Conclusions: Non-M2 macrophages, including M1 macrophages, may be correlated with postoperative cholangitis, and the M2 non-high group in mild liver fibrosis cases had a significantly lower native liver survival rate than the high group, requiring early liver transplantation in this study. Preventing advanced liver fibrosis is a key factor in improving native liver survival for BA patients, and liver macrophages may play important roles in liver homeostasis and the promotion of inflammation and fibrosis.

Temporal dynamics of cardiac immune cell accumulation following acute myocardial infarction.

Acute myocardial infarction (MI) causes sterile inflammation, which is characterized by recruitment and activation of innate and adaptive immune system cells. Here we delineate the temporal dynamics of immune cell accumulation following MI by flow cytometry. Neutrophils increased immediately to a peak at 3 days post-MI. Macrophages were numerically the predominant cells infiltrating the infarcted myocardium, increasing in number over the first week post-MI. Macrophages are functionally heterogeneous, whereby the first responders exhibit high expression levels of proinflammatory mediators, while the late responders express high levels of the anti-inflammatory cytokine IL-10; these macrophages can be classified into M1 and M2 macrophages, respectively, based on surface-marker expression. M1 macrophages dominated at 1-3 days post-MI, whereas M2 macrophages represented the predominant macrophage subset after 5 days. The M2 macrophages expressed high levels of reparative genes in addition to proinflammatory genes to the same levels as in M1 macrophages. The predominant subset of dendritic cells (DCs) was myeloid DC, which peaked in number on day 7. Th1 and regulatory T cells were the predominant subsets of CD4(+) T cells, whereas Th2 and Th17 cells were minor populations. CD8(+) T cells, γδT cells, B cells, natural killer (NK) cells and NKT cells peaked on day 7 post-MI. Timely reperfusion reduced the total number of leukocytes accumulated in the post-MI period, shifting the peak of innate immune response towards earlier and blunting the wave of adaptive immune response. In conclusion, these results provide important knowledge necessary for developing successful immunomodulatory therapies.

M1 to M2 induction in macrophages using a retinoic acid-releasing mesenchymal stem cell scaffold.

BACKGROUND: Modulation of macrophage polarization is required for effective tissue repair and regenerative therapies. Therapeutic modulation of macrophages from an inflammatory M1 to a fibrotic M2 phenotype could help in diseases, such as chronic wounds, which are stalled in a prolonged and heightened inflammatory stage within the wound healing process. OBJECTIVE: This study evaluates the efficiency of a pullulan/gelatin nanofiber scaffold loaded with retinoic acid (RA) and adipose-derived mesenchymal stem cells (ASCs) to modulate M1 to M2 anti-inflammatory transition. METHODS: Scaffolds were fabricated by electrospinning, and crosslinked using ethylene glycol diglycidyl ether (EGDE). Exposure of RA and/or ASCs to cultured macrophages have been shown to promote M1 to M2 transition. Pullulan was chosen as a scaffold material due to its ability to quench reactive oxygen species, key signaling molecules that play an important role in the progression of inflammation, as well as for its excellent mechanical properties. Gelatin was chosen as an additional scaffold component due to the presence of cell-binding motifs and its biocompatibility. Scaffold compositions examined were 75:25 and 50:50, pullulan:gelatin. The scaffolds were crosslinked in 1:70 and 1:50 EGDE:EtOH. The scaffold composition was determined via FTIR. For the present study, the 75:25 pullulan:gelatin crosslinked with 1:70 EGDE:EtOH, forming nanofibers 328 ± 47.9 nm (mean ± SD) in diameter, was chosen as the scaffold composition due to its lower degradation and release rate, which allows a sustained delivery of RA. RESULTS: The scaffold composition degraded to approximately 80% after 14 days, with approximately 38% of the drug released after 7 days. THP-1 monocytic cells were induced into a M1 macrophage phenotype through stimulation with lipopolysaccharide (LPS) and gamma interferon (IFN-γ). These M1 macrophages were the exposed to scaffolds loaded with RA and ASCs, to induce differentiation to an M2 phenotype. CONCLUSION: Gene expression quantitation by qPCR showed a reduction of M1 biomarkers, tumor necrosis factor alpha (TNFα) and interleukin 1ß (IL1ß), and an increase of M2 biomarker CCL22 after 2 days of exposure, suggesting successful M1 to M2 transition.

Developmental and homeostatic signaling transmitted by the G-protein coupled receptor FPR2.

Formyl peptide receptor 2 (FPR2) and its mouse counterpart Fpr2 are the members of the G protein-coupled receptor (GPCR) family. FPR2 is the only member of the FPRs that interacts with ligands from different sources. FPR2 is expressed in myeloid cells as well as epithelial cells, endothelial cells, neurons, and hepatocytes. During the past years, some unusual properties of FPR2 have attracted intense attention because FPR2 appears to possess dual functions by activating or inhibiting intracellular signal pathways based on the nature, concentration of the ligands, and the temporal and spatial settings of the microenvironment in vivo, the cell types it interacts with. Therefore, FPR2 controls an abundant array of developmental and homeostatic signaling cascades, in addition to its "classical" capacity to mediate the migration of hematopoietic and non-hematopoietic cells including malignant cells. In this review, we summarize recent development in FPR2 research, particularly in its role in diseases, therefore helping to establish FPR2 as a potential target for therapeutic intervention.

A20 affects macrophage polarization through the NLRP3 inflammasome signaling pathway and promotes breast cancer progression.

Breast cancer is the most common malignant tumor in females, and the majority of patients succumb to metastasis. The present study aimed to investigate the association between tumor necrosis factor alpha-induced protein 3 (A20), NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) and tumor-associated macrophage polarization, and their effects on the proliferation and metastasis of breast cancer cells. The expression of A20 in breast cancer cells was analyzed by reverse transcription-quantitative PCR (RT-qPCR) and western blotting. RT-qPCR and western blotting were also used to confirm the transfection efficiency. The viability, clone formation, migration, invasion and angiogenesis of transfected breast cancer cells were detected by Cell Counting Kit-8, colony formation, wound healing, Transwell and tube formation assays, respectively. Activated macrophages, namely M1 and M2 type macrophages, were observed by double staining immunofluorescence. The levels of M1 and M2 macrophage markers were analyzed by qPCR. The expression of angiogenesis-related proteins and NLRP3 inflammasome activation-associated proteins was detected by western blotting. The results revealed that A20 was highly expressed in breast cancer cells. Interference with A20 inhibited the proliferation, invasion, migration and angiogenesis of breast cancer cells, and inhibited the M2-like polarization of macrophages. Interference with A20 promoted the activation of the NLRP3 inflammasome. The NLRP3 inhibitor MCC950 alleviated the effect of interference with A20 to promote macrophage proliferation and recruitment, as well as M2-like polarization. In conclusion, interference with A20 inhibited macrophage proliferation and M2-like polarization through the NLRP3 inflammasome signaling pathway to inhibit breast cancer progression.

Codelivery of HBx-siRNA and Plasmid Encoding IL-12 for Inhibition of Hepatitis B Virus and Reactivation of Antiviral Immunity.

Chronic hepatitis B is a critical cause of many serious liver diseases such as hepatocellular carcinoma (HCC). The main challenges in hepatitis B treatment include the rebound of hepatitis B virus (HBV)-related antigen levels after drug withdrawal and the immunosuppression caused by the virus. Herein, we demonstrate that the HBV-related antigen can be effectively inhibited and antiviral immunity can be successfully reactivated through codelivery of the small interfering RNA (siRNA) targeting HBV X protein (HBx) and the plasmid encoding interleukin 12 (pIL-12) to hepatocytes and immune cells. After being treated by the siRNA/pIL-12 codelivery system, HBx mRNA and hepatitis B surface antigen (HBsAg) are dramatically reduced in HepG2.215 cells. More importantly, the downregulated CD47 and programmed death ligand 1 (PD-L1) and the upregulated interferon-ß promoter stimulator-1 (IPS-1), retinoic acid-inducible gene-1 (RIG-1), CD80, and human leukocyte antigen-1 (HLA-1) in treated HepG2.215 cells indicate that the immunosuppression is reversed by the codelivery system. Furthermore, the codelivery system results in inhibition of extracellular regulated protein kinases (ERK) and phosphoinositide-3-kinase (PI3K)/protein kinase B (Akt) pathways, as well as downregulation of B-cell lymphoma-2 (Bcl-2) and upregulation of p53, implying its potential in preventing the progression of HBV-induced HCC. In addition, J774A.1 macrophages treated by the codelivery system were successfully differentiated into the M1 phenotype and expressed enhanced cytokines with anti-hepatitis B effects such as interleukin 6 (IL-6) and tumor necrosis factor-α (TNF-α). Therefore, we believe that codelivery of siRNA and pIL-12 can effectively inhibit hepatitis B virus, reverse virus-induced immunosuppression, reactivate antiviral immunity, and hinder the progression of HBV-induced hepatocellular carcinoma. This investigation provides a promising approach for the synergistic treatment of HBV infection.

Liposome-mediated small RNA delivery to convert the macrophage polarity: A novel therapeutic approach to treat inflammatory uterine disease.

Macrophages are present in all tissues for maintaining tissue homeostasis, and macrophage polarization plays a vital role in alleviating inflammation. Therefore, specific delivery of polarization modulators to macrophages in situ is critical for treating inflammatory diseases. We demonstrate that a size-controlled miRNA-encapsulated macrophage-targeting liposomes (miR/MT-Lip) specifically targets macrophages to promote M1-to-M2 polarization conversion, alleviating inflammation without cytotoxicity. miR/MT-Lip, approximately 1.2 µm, showed excellent internalization through phagocytosis and/or macropinocytosis in macrophages. miR-10a/MT-Lip, but not scramble miR-Fluorescein amidite (FAM)/MT-Lip as control, effectively converted the polarization of lipopolysaccharide (LPS)-induced M1 macrophages to M2 in vitro. When miR-10a/MT-Lip was intravenously delivered to mice insulted with LPS for inflammation, the proportion of M2 macrophages was significantly increased without disturbing the population of other immune cells. Furthermore, scramble miR-FAM/MT-Lip was mainly detected in macrophages, but not other immune cells. When our miR/MT-Lip was administered to mice with Asherman's syndrome that suffer from infertility because of sterile uterine inflammation, macrophage-specific targeting of miR-10a/MT-Lip facilitated M1-to-M2 conversion for angiogenesis in the impaired uterus, resulting in restoration of healthy uterine conditions. The results indicate that our MT-Lip encapsulating small RNAs has excellent potential to treat various inflammatory disorders by fine-tuning macrophage polarization in vivo without any side effects.

Endothelial Dysfunction in Diabetes.

Diabetes is a worldwide health issue closely associated with cardiovascular events. Given the pandemic of obesity, the identification of the basic underpinnings of vascular disease is strongly needed. Emerging evidence has suggested that endothelial dysfunction is a critical step in the progression of atherosclerosis. However, how diabetes affects the endothelium is poorly understood. Experimental and clinical studies have illuminated the tight link between insulin resistance and endothelial dysfunction. In addition, macrophage polarization from M2 towards M1 contributes to the process of endothelial damage. The possibility that novel classes of anti-hyperglycemic agents exert beneficial effects on the endothelial function and macrophage polarization has been raised. In this review, we discuss the current status of knowledge regarding the pathological significance of insulin signaling in endothelium. Finally, we summarize recent therapeutic strategies against endothelial dysfunction with an emphasis on macrophage polarity.

Macrophage repolarization using emu oil-based electrospun nanofibers: possible application in regenerative medicine.

In the regenerative medicine therapies, the availability of engineered scaffolds that modulate inflammatory states is highly required. The aim of this study was to evaluate the efficiency of electrospun nanofibrous scaffolds containing natural substances with anti-inflammatory properties such as Emu oil (EO) to control inflammation and re-polarization of macrophages toward M2 anti-inflammatory phonotype. For this purpose, bead free and smooth EO-blended PCL/PEG electrospun nanofibrous mats were successfully fabricated and characterized using FE-SEM, FTIR, and Universal Testing Machine. GC/MS findings of pure EO revealed the fatty acids composition. MTT results showed that macrophage viability on EO-PCL/PEG nanofibres was higher than on PCL/PEG nanofibres and control (p ≤ .05). Additionally, the presence of EO into nanofibres was found to influence on macrophage morphologies, using FE-SEM. qPCR results showed a reduction in iNOS-2 and an increase in Arg-1 levels of macrophages seeded on EO-PCL/PEG nanofibres, indicating the successfully polarization of the macrophages to M2 phenotype. The change in macrophage phenotype on EO-based nanofibres could suppress the inflammation in LPS/IFN-γ stimulated macrophages as evidenced by a major reduction in pro-inflammatory cytokine levels TNF-α, IL-1ß, and IL-6. Conclusively, the results demonstrated that EO-based nanofibres efficiently modulated RAW264.7 macrophage polarity toward an anti-inflammatory M2 phenotype.

Macrophage repolarization using CD44-targeting hyaluronic acid-polylactide nanoparticles containing curcumin.

The aim of this study was to evaluate the efficiency of using a natural substance, curcumin, encapsulated in CD44-targeting hyaluronate-polylactide (HA-PLA) nanoparticles (NPs) for the modulation of macrophage polarity from the pro-inflammatory M1 to anti-inflammatory M2 phenotype. For this purpose, the characterization of the NPs was monitored using 1HNMR, FTIR, DLS and FE-SEM. The effects of curcumin-encapsulated HA-PLA NPs on the viability of LPS/IFN-γ stimulated peritoneal macrophages were determined using MTT assay. The cellular uptake of free curcumin and nano-formulated curcumin was assessed using confocal microscopy. Also, the expression levels of iNOS-2 (M1 marker), Arg-1 (M2 marker) and also pro-inflammatory cytokines were measured by real-time PCR. Data showed that the nano-formulated curcumin with spherical shape, an average diameter of 102.5 nm and high cellular uptake was significantly less toxic to peritoneal macrophages. Furthermore, the nano-formulated curcumin effectively indicated a reduction in iNOS-2 and an increase in Arg-1 levels than free curcumin. The change in macrophage phenotype by curcumin-encapsulated HA-PLA NPs could suppress the inflammation in LPS/IFN-γ stimulated macrophages as evidenced by a major reduction in pro-inflammatory cytokines. Conclusively, the results suggested that the curcumin formulation with CD44-targeting HA-PLA NPs might be a promising platform for the treatment of inflammatory diseases.

Persistent expression of neutrophil gelatinase-associated lipocalin and M2 macrophage markers and chronic fibrosis after acute kidney injury.

Recent epidemiologic studies revealed a correlation between acute kidney injury (AKI) episodes and the progression to chronic kidney disease (CKD). Although the severity and duration of the initial insult likely predict the development of CKD, information regarding tissue markers predictive of early development of renal fibrosis is limited. We investigated key markers in fibrotic kidney in rats and mice. Seven- to eight-week-old male Sprague-Dawley rats underwent bilateral ischemia-reperfusion injury (IRI). Kidney tissues were collected to determine the markers correlated with the severity of kidney fibrosis. In a separate set, a specific chemokine (C-C motif) receptor 2 (CCR2) inhibitor, RS-102895, was administered to 9-week-old male C57BL/6J mice that underwent unilateral IRI (9.2 mg/kg/day in drinking water for 17 days) to investigate whether blockade of the monocyte chemotactic protein-1 (MCP-1) signaling was sufficient to prevent fibrosis. Among candidate tissue markers, neutrophil gelatinase-associated lipocalin (NGAL) and MCP-1 mRNA expressions were correlated with kidney fibrosis. Studies on macrophage polarity showed that mRNA expression of M2, but not M1 macrophage markers, were correlated with acute-phase serum creatinine and fibrosis. Pharmacological blockade of the MCP-1-CCR2 signaling downregulated CCR2, which was insufficient to improve fibrosis in mouse unilateral IRI model, suggesting that additional, redundant pathways contribute to fibrosis. These findings suggested that tissue NGAL expression and M2 macrophage markers are promising markers that show severity of kidney fibrosis. Mechanistic involvement of these markers in CKD pathogenesis warrant additional investigation.