Expression Of M2 Macrophage Markers Research Articles

Water extract of ginseng and astragalus regulates macrophage polarization and synergistically enhances DDP's anticancer effect

Ethnopharmacological relevanceIn traditional Chinese medicine, supplementing Qi and strengthening body resistance are an important principle of anticancer treatment. Panax ginseng C.A.Mey. (ginseng) and Astragalus membranaceus Bunge (astragalus) are the representative herbs for this therapeutic principle. Aim of the studyThis study aims to explore the effect of the water extract of ginseng and astragalus (WEGA) on regulating macrophage polarization and mediating anticancer in the tumor microenvironment. Materials and methodsA549 cells were cultured in tumor-associated macrophage (TAM) supernatant with various concentrations of WEGA (0, 5, 10, 20 mg/mL). A549 cell proliferation was determined through methyl thiazole tetrazolium (MTT) assay and real-time cell analysis (RTCA), respectively. In vivo experiments were performed with a Lewis lung cancer (LLC) xenograft mouse model. Forty-eight mice were divided into six groups and treated with saline, WEGA, or cis-diamine dichloro platinum (DDP) with dosage of WEGA (0, 30, 60, 120 mg/kg body weight/day). The different groups were administered with drugs via oral or intraperitoneal injection once a day for 21 consecutive days. Tumor inhibition rate, spleen index, thymus index, cytokine, protein, and mRNA expression levels were detected in mice. ResultsIn a co-culture system, WEGA remarkably inhibited A549 cell proliferation, promoted the expression of M1 macrophage markers and inhibited M2 TAMs markers. Therefore, WEGA affected the biological behavior of cancer cells by regulating the expression of some markers relevant to macrophage polarization. In addition, the group of WEGA and DDP chemotherapy effectively inhibited the transplanted tumor growth in mice and improved weight loss and immunosuppressive with the cisplatin inducing. ConclusionsThis study provides mechanistic insights into the anticancer effect of WEGA through the regulation of macrophage polarization and highlights that WEGA could be a novel option for integrative cancer therapies.

Leishmania infection activates host mTOR for its survival by M2macrophage polarization.

Mammalian target of rapamycin (mTOR) is a central regulator of growth and immunity of host cells. It's involvement in cancer and tuberculosis is well documented but least explored in Leishmania donovani invasion of host cells. Therefore, in the present study, we aimed to investigate the role of mTOR in M2 macrophage polarization for Leishmania survival. We observed that Leishmania infection activated host mTOR pathway characterized by phosphorylation of mTOR, 70S6K and 4-EBP1. Inhibition of mTOR resulted in decreased parasite load and percent infectivity. Moreover, Leishmania infection triggered cell proliferation as was evidenced by increased expression of cyclin A and p-RPS6. mTOR activation during Leishmania infection resulted in reduced expression of M1 macrophage markers (eg, ROS, NO, iNOS, NOX-1, IL-12, IL-1β and TNF-α), and increased expression of M2 macrophage markers (eg, arginase-1, IL-10, TGF-β, CD206 and CD163). Furthermore, we observed that in case of Leishmania infection, mTOR inhibition increased the translocation of NF-κB to nucleus and deactivation of STAT-3. Eventually, we observed that inhibition of M2 macrophage polarization reduced Leishmania survival inside macrophages. Therefore, our findings suggest that mTOR plays a crucial role in regulation of M2 macrophage polarization and direct the innate immune homeostasis towards parasite survival inside host.

Angiopoietin-like 8 Improves Insulin Resistance and Attenuates Adipose Tissue Inflammation in Diet-Induced Obese Mice

Angiopoietin-like 8 (ANGPTL8) is closely linked to obesity-associated metabolic diseases and insulin resistance. The aim of the current study was to investigate the ability of ANGPTL8 to reverse insulin resistance in obese mice. The administration of ANGPTL8 reduced weight gain and improved glucose tolerance in mice with diet-induced obesity. In addition, ANGPTL8 administration modified macrophage infiltration, reduced monocyte chemoattractant protein-1 (MCP-1) and interleukin-1β(IL-1β) levels, and increased adiponectin gene expression in inguinal white adipose tissue (iWAT). Moreover, the exposure of a cultured peritoneal macrophage line to ANGPTL8 reduced the mRNA expression of M1 macrophage markers (TNF-α and IL-1β) upon stimulation with lipopolysaccharides in a dose-dependent manner. By contrast, when incubated with IL-4, exposure of macrophages to ANGPTL8 increased the mRNA expression of M2 macrophage markers (Arg1 and Chi3l3) in a dose-dependent manner. Collectively, the results of the present study demonstrated that treatment with ANGPTL8 can attenuate adipose tissue inflammation through regulation of macrophage polarization, and thus, it could be useful for improving insulin resistance.

TanshinoneIIA Alleviates Inflammatory Response and Directs Macrophage Polarization in Lipopolysaccharide-Stimulated RAW264.7 Cells.

TanshinoneIIA (TanIIA) has been demonstrated to possess numerous biological effects. However, the specific effect of TanIIA on macrophage polarization has not been reported. In this study, it was revealed that TanIIA might play a pivotal role in macrophage polarization. As our results indicated, cell morphology was changed in RAW264.7 cells which were treated with LPS or LPS/TanIIA (0.1μM, 1μM, 10μM). Subsequently, pro-inflammatory cytokine TNF-α and anti-inflammatory cytokine IL-10 were measured by ELISA kits. Furthermore, TanIIA enhanced the expression of M2 macrophage markers (Arg1 and FIZZ1) and decreased the expression of markers associated with M1 macrophage polarization (iNOS and IL-1β). Increased expression of CD206 was also detected by flow cytometry in TanIIA-treated groups. Mechanistically, it was revealed that TanIIA modulated macrophage polarization by ameliorating mitochondrial function and regulating TLR4-HMGB1/CEBP-β pathway. In addition, increased expression of miR-155 was observed in RAW264.7 cells incubated with LPS and were effectively inhibited by TanIIA. Taken together, it was suggested that TanIIA inhibits inflammatory response and promotes macrophage polarization toward an M2 phenotype, which provides new evidence for the anti-inflammation activity of TanIIA.

Exosomal MALAT1 derived from oxidized low-density lipoprotein-treated endothelial cells promotes M2 macrophage polarization.

Oxidized low-density lipoprotein (oxLDL)-induced injury and apoptosis of endothelial cells are important initial events in numerous cardiovascular diseases. Following activation by oxLDL, monocytes adhere to endothelial cells, migrate into the subendothelial spaces and then undergo differentiation into macrophages, which subsequently induces the formation of atherosclerotic lesions. However, the mechanisms underlying the activation of macrophage differentiation by oxLDL-treated endothelial cells remain unclear. In the present study, it was demonstrated that exosomal metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) was increased in oxLDL-treated human umbilical vein endothelial cells. When co-cultured with monocytes, exosomes extracted from oxLDL-treated HUVECs were endocytosed. Furthermore, exosomes derived from oxLDL-treated endothelial cells were revealed to promote M2 macrophage polarization, as reverse transcription-quantitative polymerase chain reaction, western blotting and ELISA analyses demonstrated increases in the expression of M2 macrophage markers, including macrophage mannose receptor 1 (also termed CD206), arginase-1 and interleukin (IL)-10, and decreases in the expression of the M1 macrophage marker, IL-12. Furthermore, the suppression of MALAT1 expression in monocytes was demonstrated to reverse exosome-mediated M2 macrophage polarization. In conclusion, the results of the present study revealed a novel mechanism underlying the onset of atherogenesis associated with endothelial cells and macrophages: Exosomal MALAT1 derived from oxLDL-treated endothelial cells promoted M2 macrophage polarization. This result may provide a novel scientific basis for the understanding of atherosclerosis progression.

Oral Administration of α-Asarone Promotes Functional Recovery in Rats With Spinal Cord Injury.

α-asarone, a bioactive compound found in Acorus plant species, has been shown to exhibit neuroprotective, anti-oxidative, anti-inflammatory, and cognitive-enhancing effects. However, the effects of α-asarone on spinal cord injury (SCI) have not yet been elucidated. The present study investigated the effects of α-asarone on the mRNA of pro-inflammatory cytokines, macrophage polarization toward an anti-inflammatory M2 phenotype, and angiogenesis in rats with compressive SCI. α-Asarone was orally administered (10 mg/kg) once per day for 14 days following moderate static compression SCI. Compared to controls, α-asarone treatment significantly improved locomotor score, prevented neuroinflammation, and facilitated angiogenesis in the spinal cord at 14 days after SCI. Furthermore, α-asarone significantly reduced the TNF-α, IL-1β, IL-6, monocyte chemoattractant protein 1 (MCP-1), macrophage inflammatory protein 2 (MIP-2), and inducible nitric oxide synthase (iNOS) levels but increased the IL-4, IL-10, and arginase 1 levels at 24 h after SCI. At 7 and 14 days after SCI, immunohistochemistry showed reduced reactive gliosis and neuroinflammation and an increased expression of M2 macrophage markers and angiogenesis. The results suggest that the inhibition of pro-inflammatory cytokines, macrophage polarization toward an anti-inflammatory M2 phenotype, and angiogenesis by α-asarone may be some of the mechanisms underlying the α-asarone-mediated neuroprotective effects on an injured spinal cord.

Expression of M2 macrophage markers YKL-39 and CCL18 in breast cancer is associated with the effect of neoadjuvant chemotherapy.

High activity of enzyme TOP2a in tumor cells is known to be associated with sensitivity to anthracycline chemotherapy, but 20% of such patients do not show clinical response. Tumor microenvironment, including tumor-associated macrophages (TAM), is an essential factor defining the efficiency of chemotherapy. In the present study, we analyzed the expression of M2 macrophage markers, YKL-39 and CCL18, in tumors of breast cancer patients received anthracycline-based NAC. Patients were divided into two groups according to the level of doxorubicin sensitivity marker TOP2a: DOX-Sense and DOX-Res groups. Expression levels of TOR2a, CD68, YKL-39 and CCL18 genes were analyzed by qPCR, the amplification of TOR2a gene locus was assessed by the microarray assay. Clinical and pathological responses to neoadjuvant chemotherapy were assessed. We found that the average level of TOP2a expression in patients of DOX-Sense group was almost 10 times higher than in patients of DOX-Res group, and the expression of CD68 was 3 times higher in the DOX-Sense group compared to DOX-Res group. We demonstrated that expression levels of M2-derived cytokines but not the amount of TAM is indicative for clinical and pathological chemotherapy efficacy in breast cancer patients. Out of 8 patients from DOX-Sense group who did not respond to neoadjuvant chemotherapy (NAC), 7 patients had M2+ macrophage phenotype (YKL-39+CCL18- or YKL-39-CCL18+) and only one patient had M2- macrophage phenotype (YKL-39-CCL18-). In DOX-Res group, out of 14 patients who clinically responded to NAC 9 patients had M2- phenotype and only 5 patients had M2+ macrophage phenotype. Among pathological non-responders in DOX-Sense group, 19 (82%) patients had M2+ tumor phenotype and only 4 (18%) patients had M2- phenotype. In DOX-Res group, all 5 patients who pathologically responded to NAC had M2 phenotype (YKL-39-CCL18-). Unlike the clinical response to NAC, the differences in the frequency of M2+ and M2- phenotypes between pathologically responding and non-responding patients within DOX-Sense and DOX-Res groups were statistically significant. Thus, we showed that in patients with breast cancer who received anthracycline-containing NAC the absence of clinical response is associated with the presence of M2+ macrophage phenotype (YKL-39-CCL18 + or YKL-39 + CCL18-) based on TOP2a overexpression data.

Vein wrapping promotes M2 macrophage polarization in a rat chronic constriction injury model.

Although the therapeutic potential of vein wrapping (VW) for recurrent compressive neuropathy has been widely reported, the mechanisms underlying this technique have not been characterized. M2 macrophages induced by interleukin-4 (IL-4) or interleukin-10 (IL-10) have an anti-inflammatory function and play an important role in peripheral nerve repair. To evaluate whether VW promotes M2 polarization, we divided chronic constriction injury (CCI) rats into untreated and VW (CCI + VW)-treated groups. Pain withdrawal thresholds in both groups were evaluated using von Frey filaments. Expression of the anti-inflammatory cytokines IL-4 and IL-10 in vein and nerve were quantified using real time polymerase chain reaction (RT-PCR), and expression of the anti-inflammatory M2 macrophage markers CD206 and arginase-1 (Arg1) after VW was assessed by RT-PCR and immunohistochemistry. To evaluate the effect of exogenous IL-4 or IL-10 on M2 macrophage-marker expression, CD11b-positive macrophages isolated from sciatic nerve were stimulated with recombinant IL-4 and IL-10. VW significantly increased the pain withdrawal threshold. IL-4 and IL-10 mRNA expression was higher in veins than in the sciatic nerve. VW significantly increased CD206 and Arg1 mRNA expression compared to the CCI group. The number of CD206- and Arg1-immunoreactive cells in nerve bundles was twofold higher in the CCI + VW than CCI group. Application of exogenous IL-4 doubled CD206 and Arg1 mRNA expression in CD11b-positive macrophages. These results show that vein-derived IL-4 potentiates the benefit of VW through the activation of M2 macrophages in the sciatic nerve. Our results may help to optimize current procedures for treating recurrent compressive neuropathy. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.

Abstract P1-05-03: JNK signaling regulates tumor cell–tumor-associated macrophage cross-talk in triple-negative breast cancer

Abstract Despite advances in our understanding of the molecular mechanisms underlying the aggressiveness of triple-negative breast cancer (TNBC), the contribution of tumor-associated macrophages (TAMs) to TNBC pathogenesis has not been therapeutically exploited. TAMs are the most abundant cell types in the tumor microenvironment (TME) and the key contributor to tumor progression and invasion. We have found that c-Jun NH2-terminal kinase (JNK), a member of the MAPK family and a major regulator of inflammation, contributes to TNBC tumorigenesis by promoting the cancer stem-like cell phenotype. However, whether the JNK pathway regulates TAMs and their cross-talk with tumor cells in TNBC remains unknown. Here, we tested the hypothesis that JNK signaling contributes to TNBC aggressiveness by promoting the tumor cell–TAM cross-talk that facilitates TNBC cell invasiveness. We found that, among 80 patients with primary inflammatory breast cancer (IBC), TNBC tumors (n=18) had 2-fold more TAMs than non-TNBC tumors (n=62, P=0.05) and that high TAM counts were correlated with shorter disease-free survival of patients with IBC (P=0.05). Both JNK1 and c-Jun were highly activated in TAMs, and JNK-IN-8, a pan-inhibitor of JNK, suppressed c-Jun activation. JNK-IN-8 also increased expression of M1 macrophage markers (CD80 and HLA-DR) but reduced expression of TAM markers (CD163 and CD206), suggesting that JNK suppresses M1 macrophage differentiation but promotes TAM differentiation. Co-culture with TAMs significantly enhanced migration and invasion of HCC70 and MDA-MB-468 human and 4T1 murine TNBC cells. Similarly, an enhancement in TNBC cells migration and invasion was observed following culture with TAM-conditioned medium, suggesting that TAMs enhance TNBC cellular activities through paracrine signaling. In addition, inhibition of JNK signaling in TNBC cells or in TAMs by JNK-IN-8 significantly suppressed TAM-promoted enhancement of TNBC cell migration and invasion. These studies strongly suggest that JNK regulates M1/TAM differentiation and TNBC cell–TAM cross-talk. Furthermore, cytokine/chemokine profiling analysis showed that, of the identified molecules, MCP-1 (secreted by TAMs) and VEGF (secreted by TNBC cells) had the highest expression levels and that their expression was dramatically reduced following JNK-IN-8 treatment. Stimulation with recombinant VEGF increased proliferation of MDA-MB-468 cells, and stimulation with recombinant MCP-1 enhanced migration of the cells. These findings suggest that VEGF and MCP-1 are involved in JNK-mediated TNBC cell–TAM cross-talk. Together, our results suggest that JNK signaling regulates tumor cell–TAM cross-talk through MCP-1– and/or VEGF-mediated paracrine signaling and that JNK is an important therapeutic target in TNBC. Further animal studies using JNK-knockout TNBC cells co-injected with TAMs are needed to confirm our in vitro findings. Citation Format: Xie X, Otsuka S, Chu K, Lu AY, Tripathy D, Dalby KN, Hittelman WN, Van Laere S, Bartholomeusz C, Ueno NT. JNK signaling regulates tumor cell–tumor-associated macrophage cross-talk in triple-negative breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P1-05-03.

Differential response to caloric restriction of retroperitoneal, epididymal, and subcutaneous adipose tissue depots in rats

The beneficial actions of caloric restriction (CR) are partially mediated by metabolic remodeling of white adipose tissue (WAT). Recently, we showed that CR enhances de novo fatty acid (FA) biosynthesis and mitochondrial biogenesis, particularly in WAT. Here, to better understand the response of WAT to CR, we compare the effects of CR on three WAT depots in rats: retroperitoneal (rWAT), epididymal (eWAT) and subcutaneous (sWAT). Computed tomography and histological analysis showed that CR reduced the volume and average size of rWAT adipocytes. In all WAT depots, CR markedly upregulated the expression of proteins involved in FA biosynthesis in fed rats. In visceral WAT (rWAT and eWAT), hormone-sensitive lipase (lipolytic form) phosphorylation was increased by CR under fed conditions, and decreased by CR under fasted conditions. Conversely, in sWAT, hormone-sensitive lipase phosphorylation was increased by CR under fasted conditions. CR enhanced the effect of feeding on AKT activity in sWAT (indicative of a positive effect on insulin sensitivity) but not in rWAT or eWAT. These data suggest that CR improves lipid metabolism in an insulin signaling-dependent manner in sWAT only. The effects of CR on adipokine (adiponectin and leptin) expression were also different among rWAT, eWAT and sWAT, and CR reduced the gene expression of M2 macrophage markers in rWAT and sWAT, but not in eWAT. We conclude that CR differentially affects the characteristics of WAT depots in rats, including adipocyte size, lipid metabolism, insulin signaling, adipocytokine profile and macrophage infiltration.

Heme oxygenase-1 induction by methylene blue protects RAW264.7 cells from hydrogen peroxide-induced injury

Although methylene blue (MB) has showed strong antioxidant effect, its effect related with heme oxygenase-1 (HO-1) is still unclear. Thus, we investigated the effects of MB on HO-1 protein content and enzyme activity, and its protective effect against hydrogen peroxide (H2O2)-induced oxidative damage in RAW264.7 macrophage. The cell viability and the release of lactate dehydrogenase of RAW264.7 were determined. The mitochondrial functions were valuated through these indexes: content of adenosine triphosphate, superoxide dismutase, concentration of reactive oxygen species and mitochondrial membrane potential. Meanwhile, high content screening tested generation of ROS, MMP and intracellular concentration of calcium ion. qRT-PCR valuated macrophage phenotype markers expression. Lastly, flow cytometry and caspase-3 detection analyzed RAW264.7 apoptosis. Our data showed that (1) Both pretreatment and posttreatment of MB increased HO-1 protein content and enzyme activity; (2) MB rescued cells from H2O2-induced mitochondrial dysfunction; (3) High content screening revealed that MB alleviated the changes including generation of reactive oxygen species, mitochondrial membrane potential and intracellular concentration of calcium ion in H2O2 exposed RAW264.7; (4) MB attenuated H2O2-induced apoptosis; (5) MB pretreatment decreased the expression of M1 macrophage markers (Tnf and Nos2) while increasing the expression of M2 macrophage markers (Mrc1 and Il10); (6) The beneficial effect of MB was abolished by zinc protoporphyrin IX (HO-1 activity inhibitor) or HO-1 siRNA. In summary, MB protects RAW264.7 cells from H2O2-induced injury through up-regulation HO-1.

Preconditioning of murine mesenchymal stem cells synergistically enhanced immunomodulation and osteogenesis

BackgroundMesenchymal stem cells (MSCs) are capable of immunomodulation and tissue regeneration, highlighting their potential translational application for treating inflammatory bone disorders. MSC-mediated immunomodulation is regulated by proinflammatory cytokines and pathogen-associated molecular patterns such as lipopolysaccharide (LPS). Previous studies showed that MSCs exposed to interferon gamma (IFN-γ) and the proinflammatory cytokine tumor necrosis factor alpha (TNF-α) synergistically suppressed T-cell activation.MethodsIn the current study, we developed a novel preconditioning strategy for MSCs using LPS plus TNF-α to optimize the immunomodulating ability of MSCs on macrophage polarization.ResultsPreconditioned MSCs enhanced anti-inflammatory M2 macrophage marker expression (Arginase 1 and CD206) and decreased inflammatory M1 macrophage marker (TNF-α/IL-1Ra) expression using an in-vitro coculture model. Immunomodulation of MSCs on macrophages was significantly increased compared to the combination of IFN-γ plus TNF-α or single treatment controls. Increased osteogenic differentiation including alkaline phosphate activity and matrix mineralization was only observed in the LPS plus TNF-α preconditioned MSCs. Mechanistic studies showed that increased prostaglandin E2 (PGE2) production was associated with enhanced Arginase 1 expression. Selective cyclooxygenase-2 inhibition by Celecoxib decreased PGE2 production and Arginase 1 expression in cocultured macrophages.ConclusionsThe novel preconditioned MSCs have increased immunomodulation and bone regeneration potential and could be applied to the treatment of inflammatory bone disorders including periprosthetic osteolysis, fracture healing/nonunions, and osteonecrosis.

Amelogenin induces M2 macrophage polarisation via PGE2/cAMP signalling pathway

ObjectivesAmelogenin, the major component of the enamel matrix derivative (EMD), has been suggested as a bioactive candidate for periodontal regeneration. Apart from producing a regenerative effect on periodontal tissues, amelogenin has also been reported to have an anti-inflammatory effect. However, the precise molecular mechanisms underlying these effects remain unclear. In the present study, we examined the immunomodulatory effects of amelogenin on macrophages. DesignHuman phorbol 12-myristate 13-acetate (PMA)-differentiated U937 macrophages and CD14+ peripheral blood-derived monocytes (PBMC)-derived macrophages were stimulated with recombinant amelogenin (rM180). After performing a detailed microarray analysis, the effects of rM180 on macrophage phenotype and signal transduction pathways were evaluated by real-time polymerase chain reaction, enzyme-linked immunosorbent assay, confocal microscopy and flow cytometry. ResultsThe microarray analysis demonstrated that rM180 increased the expression of anti-inflammatory genes in lipopolysaccharide (LPS)-challenged macrophages after 24h, while it temporarily up-regulated inflammatory responses at 4h. rM180 significantly enhanced the expression of M2 macrophage markers (CD163 and CD206). rM180-induced M2 macrophage polarisation was associated with morphological changes as well as vascular endothelial growth factor (VEGF) production. rM180 enhanced prostaglandin E2 (PGE2) expression, and the activation of the cAMP/cAMP-responsive element binding (CREB) signaling pathway was involved in amelogenin-induced M2 macrophage polarisation. Blocking of PGE2 signaling by indomethacin specifically abrogated rM180 with or without LPS-induced M2 shift in PBMC-derived macrophages. ConclusionAmelogenin could reprogram macrophages into the anti-inflammatory M2 phenotype. It could therefore contribute to the early resolution of inflammation in periodontal lesions and provide a suitable environment for remodeling-periodontal tissues.

TSG-6 Secreted by Human Adipose Tissue-derived Mesenchymal Stem Cells Ameliorates DSS-induced colitis by Inducing M2 Macrophage Polarization in Mice

Previous studies have revealed that mesenchymal stem cells (MSCs) alleviate inflammatory bowel disease (IBD) by modulating inflammatory cytokines in the inflamed intestine. However, the mechanisms underlying these effects are not completely understood. We sought to investigate the therapeutic effects of human adipose tissue-derived (hAT)-MSCs in an IBD mouse model and to explore the mechanisms of the regulation of inflammation. Dextran sulfate sodium-induced colitis mice were infused with hAT-MSCs intraperitoneally and colon tissues were collected on day 10. hAT-MSCs were shown to induce the expression of M2 macrophage markers and to regulate the expression of pro- and anti-inflammatory cytokines in the colon. Quantitative real time-PCR analyses demonstrated that less than 20 hAT-MSCs, 0.001% of all intraperitoneally injected hAT-MSCs, were detected in the inflamed colon. To investigate the effects of hAT-MSC-secreted factors in vitro, transwell co-culture system was used, demonstrating that tumour necrosis factor-α-induced gene/protein 6 (TSG-6) released by hAT-MSCs induces M2 macrophages. In vivo, hAT-MSCs transfected with TSG-6 small interfering RNA, administered intraperitoneally, were not able to induce M2 macrophage phenotype switch in the inflamed colon and had no significant effects on IBD severity. In conclusion, hAT-MSC-produced TSG-6 can ameliorate IBD by inducing M2 macrophage switch in mice.

Kinetics of Corneal Antigen Presenting Cells in Experimental Dry Eye Disease

ObjectiveTo evaluate dry eye disease (DED)-induced alterations in subsets of corneal antigen presenting cells (APCs) in a mouse model of experimental DED.Methods and AnalysisSeven to 8-week-old female C57BL/6 mice were...

M2-like macrophage polarization in high lactic acid-producing head and neck cancer.

Reprogramming of glucose metabolism in tumor cells is referred to as the Warburg effect and results in increased lactic acid secretion into the tumor microenvironment. We have previously shown that lactic acid has important roles as a pro‐inflammatory and immunosuppressive mediator and promotes tumor progression. In this study, we examined the relationship between the lactic acid concentration and expression of LDHA and GLUT1, which are related to the Warburg effect, in human head and neck squamous cell carcinoma (HNSCC). Tumors expressing lower levels of LDHA and GLUT1 had a higher concentration of lactic acid than those with higher LDHA and GLUT1 expression. Lactic acid also suppressed the expression of LDHA and GLUT1 in vitro. We previously reported that lactic acid enhances expression of an M2 macrophage marker, ARG1, in murine macrophages. Therefore, we investigated the relationship between the lactic acid concentration and polarization of M2 macrophages in HNSCC by measuring the expression of M2 macrophage markers, CSF1R and CD163, normalized using a pan‐macrophage marker, CD68. Tumors with lower levels of CD68 showed a higher concentration of lactic acid, whereas those with higher levels of CSF1R showed a significantly higher concentration of lactic acid. A similar tendency was observed for CD163. These results suggest that tumor‐secreted lactic acid is linked to the reduction of macrophages in tumors and promotes induction of M2‐like macrophage polarization in human HNSCC.

Role of BDNF in Macrophage Migration and M2 Phenotype in Chondrosarcoma Microenvironment

Chondrosarcoma is the second most common malignancy of bone after myeloma and osteosarcoma. Brain derived neurotrophic factor (BDNF) is a small-molecule protein from neurotrophin family of growth factors that is associated with disease status and outcomes of chondrosarcoma. Tumor-associated macrophages are M2-polarized macrophages that infiltrate tumor microenvironment and promote tumorigenesis. However, the role between BDNF and macrophages in chondrosarcoma microenvironment remains largely unknown. Here this study revealed that conditioned medium (CM) from BDNF-treated chondrosarcoma cells dramatically enhanced macrophage migration. BDNF also promotes the adhesion between macrophages and chondrosarcoma cells. Furthermore, knockdown of BDNF in chondrosarcoma cells diminished the expression of M2 macrophage markers. The above results illustrate that BDNF promotes macrophage migration and M2 phenotype in chondrosarcoma microenvironment.

Nanoparticle-Delivered IRF5 siRNA Facilitates M1 to M2 Transition, Reduces Demyelination and Neurofilament Loss, and Promotes Functional Recovery After Spinal Cord Injury in Mice.

Macrophage activation and persistent inflammation contribute to the pathogenesis of spinal cord injury (SCI), and different phenotypes of macrophages play diverse roles in the pathological process of SCI. After SCI, there is an acute phase of alternatively activated (M2) macrophage infiltration, followed by a long-lasting phase of classically activated (M1) macrophage accumulation in the wound. The long-lasting predominance of M1 macrophages may derail healing and compromise organ functions. Based on the previous findings that the transcription factor interferon regulatory factor 5 (IRF5) up-regulates genes associated with M1 macrophages, we attempted to examine the effect of silencing IRF5 on SCI progression. IRF5 expression was assessed with Western blotting or immunohistochemistry. Macrophage phenotypes were measured with flow cytometry or immunohistochemistry. M1- or M2-related cytokines were measured with a Luminex assay kit. IRF5 siRNA was delivered into the macrophages infiltrated into the wound of SCI mice through lipidoid nanoparticle. Locomotor functions were measured with Basso Mouse Scale (BMS) scoring. Myelination was assessed with luxol fast blue staining. Myelin binding protein, neurofilaments, synaptic markers, and cytokines in the wound area were measured with Western blotting. The Mann-Whitney U test was used for statistical analyses. After SCI, significant elevation of IRF5 was evident on day 1, peaked on day 7, and gradually decreased thereafter. Similar dynamic change in the expression of CD86, a typical M1 marker, was observed. In contrast, there was a transient increase in the expression of CD206, a typical M2 marker, which peaked 6h after SCI, and returned to baseline within 1day. Macrophages isolated from the epicenter at day 3 after SCI were predominantly M1 phenotype, and a siRNA-mediated knockdown of IRF5 resulted in a reduced expression of M1 macrophage markers and increased expression of M2 macrophage markers. Nanoparticle-mediated delivery of IRF5 siRNA to SCI mouse model resulted in a dramatic decrease in the number of M1 macrophages and a significant increase in the number of M2 macrophages in the wound. This was associated with a robust inflammation resolution, attenuation of demyelination and neurofilament loss, and significant improvement of locomotor function (p < 0.05). IRF5 may serve as a therapeutic target to promote post-SCI recovery.

SOCS3 is a modulator of human macrophage phagocytosis.

Suppressor of cytokine signaling (SOCS) proteins are recognized as key feedback inhibitors modulating the inflammatory activities of macrophages, but comparatively little is known about whether and how they affect phagocytosis. Here, we evaluated the role of SOCS3 in driving the inflammatory phenotype and phagocytic uptake of apoptotic cells by human macrophages and the signaling pathways that are necessary for efficient phagocytosis. In M1-activated human monocyte-derived macrophages, SOCS3 silencing, using short interfering RNA technology, resulted in a decreased expression of proinflammatory markers and an increased expression of M2 macrophage markers. Strikingly, we demonstrated for the first time that SOCS3 knockdown significantly enhances the phagocytic capacity of M1 macrophages for carboxylate-modified beads and apoptotic neutrophils. With the use of live-cell video microscopy, we showed that SOCS3 knockdown radically affects the temporal dynamics of particle engulfment, enabling more rapid uptake of a second target and delaying postengulfment processing, as evidenced by deferred acquisition of phagosome maturation markers. SOCS3 knockdown impacts on phagocytosis through increased PI3K and Ras-related C3 botulinum toxin substrate 1 (Rac1) activity, pathways essential for engulfment and clearance of apoptotic cells. Enhanced phagocytosis in SOCS3-silenced cells was reversed by pharmacological PI3K inhibition. Furthermore, we revealed that actin polymerization, downstream of PI3K/Rac1 activation, was significantly altered in SOCS3-silenced cells, providing a mechanism for their greater phagocytic activity. The findings support a new model, whereby SOCS3 not only plays an important role in driving macrophage inflammatory responses but modulates key signaling pathways organizing the actin cytoskeleton to regulate the efficiency of phagocytic processes.

The Rhizome Mixture of Anemarrhena asphodeloides and Coptis chinensis Attenuates Mesalazine-Resistant Colitis in Mice.

We investigated the effect of DWac on the gut microbiota composition in mice with 2,3,6-trinitrobenzenesulfonic acid- (TNBS-) induced colitis. Treatment with DWac restored TNBS-disturbed gut microbiota composition and attenuated TNBS-induced colitis. Moreover, we examined the effect of DWac in mice with mesalazine-resistant colitis (MRC). Intrarectal injection of TNBS in MRC mice caused severe colitis, as well as colon shortening, edema, and increased myeloperoxidase activity. Treatment with mesalazine (30 mg/kg) did not attenuate TNBS-induced colitis in MRC mice, whereas treatment with DWac (30 mg/kg) significantly attenuated TNBS-induced colitis. Moreover, treatment with the mixture of mesalazine (15 mg/kg) and DWac (15 mg/kg) additively attenuated colitis in MRC mice. Treatment with DWac and its mixture with mesalazine inhibited TNBS-induced activation of NF-κB and expression of M1 macrophage markers but increased TNBS-suppressed expression of M2 macrophage markers. Furthermore, these inhibited TNBS-induced T-bet, RORγt, TNF-α, and IL-17 expression but increased TNBS-suppressed Foxp3 and IL-10 expression. However, Th2 cell differentiation and GATA3 and IL-5 expression were not affected. These findings suggest that DWac can ameliorate MRC by increasing the polarization of M2 macrophage and correcting the disturbance of gut microbiota and Th1/Th17/Treg, as well as additively attenuating MRC along with mesalazine.