Percentage Of M2 Macrophages Research Articles

Protective Role of Matrix Metalloproteinase-2 in Allergic Bronchial Asthma.

Inflammation, reversible obstruction, and hyperresponsiveness of the airways are characteristic findings of bronchial asthma. Several evidence has demonstrated the involvement of matrix metalloproteinase-2 in allergic airway inflammation. Matrix metalloproteinase-2 may promote aberrant tissue remodeling in late stages of allergic airway inflammation. However, whether matrix metalloproteinase-2 is detrimental or protective in early stages of allergic airway inflammation remains unclear. To evaluate this here we compared the severity of allergic bronchial asthma between mice overexpressing human matrix metalloproteinase-2 and wild type mice. After sensitization and challenge with an allergen, mice overexpressing the human matrix metalloproteinase-2 showed a significant reduction in airway hyperresponsiveness and in the expression of Th2 cytokines and IgE compared to their wild type counterparts. An inhibitor of matrix metalloproteinases abolished this beneficial effect of human matrix metalloproteinase-2 overexpression. Allergen-sensitized and challenged human matrix metalloproteinase-2 transgenic mice had enhanced percentage of M1 macrophages with increased expression of inducible nitric oxide synthase and STAT1 activation in the lungs compared to their wild type counterparts. There was no difference in the percentage of regulatory T cells between mouse groups. The results of this study showed that matrix metalloproteinase-2 is protective in allergic bronchial asthma by promoting polarization of macrophages to M1 phenotype.

Efficacy of a new oncolytic adenovirus armed with IL-13 against oral carcinoma models.

PurposeThe efficacy of traditional therapies for oral carcinoma (OC) is limited. Oncolytic adenovirus, a novel strategy of cancer therapy, shows potential use in OC treatment. However, its clinical application is limited by pre-existing neutralizing antibodies. Thus, this study aimed to examine the efficacy of a new modified adenovirus against OC in vitro and in vivo.Materials and methodsA multiple modified adenovirus (MMAD) armed with IL-13 (MMAD-IL-13) was constructed, and its effect on Cal-27 cells was examined. The potency of MMAD-IL-13 was examined in vitro and in vivo. For in vitro experiment, CCK-8 kit was used to determine the IC50 of MMAD-IL-3 in OC cell lines. For in vivo experiment, Cal-27 xenograft models were used to determine the antitumor effect of MMAD-IL-13. Apoptosis was measured in Cal-27 cells by Western blotting assay. Immunity response was detected in Cal-27 xenograft models 7 days after intratumoral injection with MMAD-IL-13. The potency of MMAD and MMAD-IL-13 was compared in Cal-27 peripheral blood mononuclear cells (PBMCs) models.ResultsMMAD-IL-13 was successfully constructed; the harvested virus could be replicated and they overexpressed human IL-13 in Cal-27 cells. Compared with MMAD, MMAD-IL-13 showed enhanced antitumor effect in vitro by inducing apoptosis and reducing percentage of M2 macrophages in tumor environment in vivo. MMAD-IL-13 also showed potent antitumor effect in Cal-27, SCC-4, and Tca8113 cells in vitro and in Cal-27 xenograft models in vivo. However, MMAD-IL13 did not harm normal human oral epithelial cells in vitro and exhibited no effect on body weight in Cal-27 xenograft models. In Cal-27 PBMC models, MMAD-IL-13 showed stronger antitumor effect than MMAD.ConclusionA new oncolytic adenovirus carrying the human IL-13 gene was constructed. This virus effectively led to remission of tumor development and death of OC cells in vivo and in vitro, showing its potential as a clinical cancer therapy.

Treatment with 3,4-dihydroxyphenylethyl alcohol glycoside ameliorates sepsis-induced ALI in mice by reducing inflammation and regulating M1 polarization

The bioactive phenylethanoid 3,4-dihydroxyphenylethyl alcohol glycoside (DAG) is a component isolated from Sargentodoxa cuneata. The effects of DAG on acute lung injury (ALI) are largely unknown. Here, the effects of DAG on sepsis-induced ALI were investigated, and the related mechanisms were explored. Male C57BL/6 mice were used to establish a sepsis-induced ALI model. Levels of inflammatory cytokines were determined using real-time quantitative reverse transcription PCRs (qRT-PCR) and enzyme-linked immunosorbent assays (ELISAs). Pathological changes in the lung tissues were evaluated using haematoxylin and eosin (HE) staining. Mouse survival was quantified, and macrophage polarization was analyzed using flow cytometry. Our results showed that, in septic mice, pretreatment with DAG significantly improved survival, reduced histological damage in the lung, and suppressed the inflammatory response by inhibiting the activation of the NF-κB, STAT3, and p38 MAPK signaling pathways. Moreover, DAG treatment reduced the percentage of M1 macrophages in the bronchoalveolar lavage fluid (BALF) and spleen. In addition, DAG treatment decreased the production of pro-inflammatory cytokines and suppressed the activation of the NF-κB, STAT3, and p38 MAPK signaling pathways in LPS-induced MH-S cells. DAG treatment also reduced the relative abundances of M1 macrophages and M1 macrophage markers by suppressing the activation of the Notch1 signaling pathway. Thus, our results provided new insights for the development of drugs to treat ALI.

Effects of dietary glutamine supplementation on immune cell polarization and muscle regeneration in diabetic mice with limb ischemia.

Diabetes is a chronic inflammatory disorder resulting in endothelial dysfunction which contributes to peripheral arterial disease and limb ischemia. Leukocytes play critical roles in vascular and tissue remodelling after ischemia. This study investigated the effects of dietary glutamine (GLN) supplementation on immune cell polarization in diabetic mice subjected to limb ischemia. Diabetes was induced by an intraperitoneal injection of streptozotocin for 5 consecutive days in C57BL/6J mice. Diabetic mice were fed the AIN-93 diet or an AIN-93 diet in which a part of the casein was replaced by GLN. After 3 weeks of the dietary intervention, mice were subjected to unilateral femoral artery ligation to induce limb ischemia. GLN supplementation enhanced the proportion of anti-inflammatory monocytes and regulatory T cells in the blood. Expression of C-C motif chemokine receptor 5 by activated CD4+ T cells was promoted and prolonged in the GLN-supplemented group. GLN downregulated the percentage of M1 macrophages in muscle tissues which was correlated with lower levels of C-C motif chemokine ligand 2 in plasma. The muscle M1/M2 ratio was also reduced in the GLN group. Gene expression of interleukin-6 was suppressed by GLN supplementation, while expression levels of the peroxisome proliferator-activated receptor γ and myogenic differentiation 1 genes were elevated in post-ischemic muscles. Histological findings also indicated that muscle regeneration was accelerated in the GLN group. GLN supplementation in diabetic mice may exert more-balanced polarization of CD4+ T cells, monocytes, and macrophages, thus attenuating inflammatory responses and contributing to muscle regeneration after limb ischemia.

Sex Differences in Human Monocyte/Macrophages in Response to IL‐4

BackgroundAllergic asthma is a common chronic inflammatory disease of the lungs that affects 10% of the population in developed countries. Asthma shows a sex difference with more boys than girls diagnosed before puberty. In adulthood, women are twice as likely to have asthma than men. Women can also have increased disease severity and be less responsive to corticosteroid treatment. Sex hormones therefore may be important in asthma. Asthmatics have more and a greater percentage of M2 macrophages in their lungs than healthy individuals. Mouse studies have also demonstrated that M2 macrophages contribute to worse allergic lung inflammation. Furthermore, estrogen (E2) stimulates the production of M2 macrophage‐related cytokines that promote lung remodeling. How sex steroids affect monocyte‐macrophages to enhance M2 polarization is still unknown.Aim of the studyTo determine the molecular basis of sex differences and E2/androgen modulation of IL‐4‐induced M2 macrophage differentiation.ResultsOur preliminary data showed that both human monocytes and monocyte derived‐macrophages (MDM) from asthmatic donors upregulated M2 genes (CCL22, CCL17, TGM2, and MMP12) than cells from healthy donors, on IL‐4 stimulation. Moreover, expression of CCL17, TGM2, and CD200R was higher in MDM from asthmatic donors than in monocytes. IL‐4‐induced CCL17 and MMP12 shown a trend to greater expression in asthmatic female than male MDM. To explain the differences in IL‐4 responsiveness, we measured IL‐4 receptors (IL‐4R) on monocytes from men and women. Expression of γC, part of the type‐I IL‐4R complex, was greater on the surface of monocytes from healthy female compared to male donors. Conversely, IL‐13Rα2, an inhibitory receptor for IL‐13, was higher on male monocytes. To determine how responsive macrophages may be to E2 stimulation, we quantified estrogen receptor (ER) expression in MDM from healthy and allergic donors. MDM from allergic donors expressed more mRNA for ERα and less ERβ (linked to inhibition of E2 responses) and AR compared with MDM from a healthy donor. These data match the increased expression of ERα we measured in alveolar macrophages from female mice compared to male mice during allergic lung inflammation. Expression of CD206 greater on IL‐4‐stimulated cells in the presence of E2, mirroring our findings of increased M2 gene expression in mouse macrophages treated with E2 in vitro and in ovariectomized female mice implanted with E2 in vivo.ConclusionsOur preliminary results suggest that MDM from asthmatic donors are more sensitive to IL‐4 with enhanced M2 polarization. Sex differences in IL‐4R expression suggest increased IL‐4/‐13 responsiveness in female compared to male monocytes. ERα expression is higher in allergic MDMs and likely promotes enhanced M2 phenotype in the presence of E2. E2 promoted M2 polarization in human and mouse macrophages. Understanding the differences in monocyte‐macrophage responses between women and men and the effect of sex steroids on asthma can lead to developing better therapies for asthma according to the sex of the patient.Support or Funding InformationNIH grant R01 HL124477This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Differences in immune tumor microenvironment between primary and metastatic castration-resistant prostate cancer.

58 Background: Androgen deprivation therapy (ADT)-induced immunosuppression has been described in animal models as contributing to castration-resistant prostate cancer (CRPC) development, but the clinical relevance of these observations has not been established. In this study, we compared the immune tumor microenvironment (iTME) of primary prostate cancer (PPC) versus metastatic CRPC (mCRPC). Methods: Genomic and clinically annotated data were obtained of PPC and mCRPC from the TCGA provisional and SU2C/PCF databases. Patients without RNA-seq data were excluded. 22 iTME cell subsets were estimated using CIBERSORT LM22 algorithm. Relative cell proportions and gene expression in log-transformed FPKM were compared using Wilcoxon tests. Results: 499 PPC and 118 mCRPC were studied. Compared to PPC, mCRPC had higher percentage of M2 macrophages (24.6% vs. 10.2%, p<0.0001). High M2 in mCRPC was associated with 2.5-, 6- and 19.5-fold increases in IL-10, VEGFA and ARG1 mRNA levels, respectively (p<0.0001). T- cell exhaustion markers, LAG3 and CD160, were upregulated in mCRPC by 43.2% and 266.7% (p<0.0001). Although CD8 T-cells were higher in mCRPC, the CD8/M2 ratio was lower (1.1 vs. 1.5, p=0.002). Prior use of abiraterone or enzalutamide in the mCRPC cohort (n=54) did not alter iTME cell fractions compared to AR antagonist naïve mCRPC. The detailed iTME comparisons were listed in Table. Conclusions: Our data indicate the development of tumor-favoring iTME in the course of progression to mCRPC while on ADT. The iTME is characterized by increased M2 macrophage infiltration that may provide a novel and important therapeutic target. [Table: see text]

Tumoral NOX4 recruits M2 tumor-associated macrophages via ROS/PI3K signaling-dependent various cytokine production to promote NSCLC growth

M2-type tumor-associated macrophages (TAMs) infiltration contributes to cancer malignant progression. However, the mechanisms for controlling recruitment and M2 polarization of macrophages by cancer cells are largely unclear. NADPH oxidase 4 (NOX4) is abundantly expressed in non-small cell lung cancer (NSCLC) and mediates cancer progression. NOXs are in close relation with cancer-related inflammation, nevertheless, whether tumoral NOXs influence microenvironmental macrophages remains undentified. This study found that there was a close association between NOX4 expression and macrophage chemotaxis in patients with NSCLC analyzed using TCGA RNA-sequencing data. NOX4 in NSCLC cells (A549 and Calu-1 cell lines) efficiently enhanced murine peritoneal macrophage migration and induces M2 polarization. Immunohistochemical analysis of clinical specimens confirmed the positive correlation of NOX4 and CD68 or CD206. The mechanical study revealed that tumoral NOX4-induced reactive oxygen species (ROS) stimulated various cytokine production, including CCL7, IL8, CSF-1 and VEGF-C, via PI3K/Akt signaling-dependent manner. Blockade of the function of these cytokines reversed NOX4 effect on macrophages. Specifically, the results showed that tumoral NOX4-educated M2 macrophages exhibited elevated JNK activity, expressed and released HB-EGF, thus facilitating NSCLC proliferation in vitro. Pretreatment of macrophages with JNK inhibitor blocked tumoral NOX4-induced HB-EGF production in M2 macrophages. Finally, in a xenograft mouse model, overexpression of NOX4 in A549 cells enhanced the tumor growth. Elimination of ROS by NAC or inhibition of NOX4 activity by GKT137831 suppressed tumor growth accompanied by reduction in macrophage infiltration and the percentage of M2 macrophages. In conclusion, our study indicates that tumoral NOX4 recruits M2 TAMs via ROS/PI3K signaling-dependent various cytokine production, thus contributing NSCLC cell growth.

Electroacupuncture at ST-36 ameliorates DSS-induced acute colitis via regulating macrophage polarization induced by suppressing NLRP3/IL-1β and promoting Nrf2/HO-1

BackgroundElectroacupuncture (EA) at ST-36 can attenuate acute experimental colitis, but the mechanisms are unclear. We investigated the role of macrophages in the anti-inflammatory effects of EA and its molecular mechanisms. MethodsMale C57BL/6 mice were randomized into five groups: normal control, dextran sulfate sodium (DSS)-induced acute colitis (DSS), DSS with sham EA (SEA), DSS with high-frequency EA (HEA) and DSS with low-frequency EA (LEA). Body weight, colon length, DAI score and histological score were evaluated during colitis progression. Serum and colonic levels of pro- and anti-inflammatory cytokines were detected with ELISA, cytometric beads array, RT-PCR and western blotting analysis. Colonic macrophage subsets were determined using flow cytometry. Magnetic-activated cell sorting was applied to isolate colonic macrophages, and molecular mechanisms were explored with western blotting, RT-PCR and immunofluorescence. Results(1) Compared with the DSS group, HEA and LEA attenuated body weight loss and decreased DAI and histological scores. (2) Serum levels and colonic protein and mRNA levels of IL-1β, TNFα, IL-6, IL-12 and IL17 were markedly decreased with HEA and LEA. IL-10 level was increased with HEA. (3) M1 macrophage percentage increased, while M2 macrophage percentage decreased in the DSS group; HEA and LEA reversed these proportions. (4) NLRP3/IL-1β protein and mRNA levels in isolated macrophages decreased with HEA and LEA compared with the DSS treatment group; (5) HEA increased Nrf2/HO-1 levels compared with levels in DSS mice. ConclusionThe anti-inflammatory effects of EA on DSS-induced acute colitis may rely on regulating macrophage polarization, NLRP3/IL-1β suppression and Nrf2/HO-1 promotion.

Abstract 17163: Nox4 Deletion Attenuates Age-Associated Vascular Inflammation and Atherosclerosis Burden in Hyperlipidemic Mice by Modulating Macrophage Phenotype

Introduction: Increased vascular reactive oxygen species (ROS) levels and chronic inflammation are hallmarks of vascular aging. We previously reported that expression of NOX4 NADPH oxidase increases with age in mouse and human arteries and correlates with inflammatory cytokine secretion and atherosclerotic lesion progression. Hypothesis: To determine whether NOX4 promotes atherosclerosis in aging, we assessed vascular inflammation and atherosclerosis burden in Nox4 -/- / Apoe -/- and Apoe -/- mice. Results: Aortic atherosclerotic lesion area was not different between 5-month old (young) Nox4 -/- / Apoe -/- and Apoe -/- mice fed a Western diet for 3 months. Lesion area increased significantly in 16-month old (aged) compared with young mice but was 51% lower in Nox 4 -/- / Apoe -/- versus Apoe -/- mice (n=12, P <0.0001). Immunofluorescence analysis of aorta transverse sections showed 30% lower ROS levels and significantly reduced expression of inflammatory cytokines - CCL2 and IL6 - in atherosclerotic lesions of aged Nox4 -/- / Apoe -/- versus Apoe -/- mice. Flow cytometry analysis of atherosclerotic aortas showed significantly higher number of CD11b + macrophages in the lesions of aged Apoe -/- versus Nox4 -/- /Apoe -/- mice. However, the number of M1 pro-inflammatory macrophages (CD38 + ) was not different, whereas percentage of M2 macrophages (Egr2 + ) was significantly higher in aged Nox4 -/- Apoe -/- versus Apoe -/- mice (46% vs. 33%, respectively). In addition, Nox4 deletion increased the ratio of M2/M1 macrophages in the atherosclerotic aortas from aged Apoe -/- mice (0.56 and 0.43 in Nox 4 -/- / Apoe -/- and Apoe -/- , respectively). Macrophages isolated from Nox4 -/- mice showed lower number of M1 cells after IFNγ+LPS treatment and higher number of M2 cells after IL4 treatment versus macrophages from the wild-type. Furthermore, wild-type macrophages showed significantly higher secreted IL1β levels after IFNγ+LPS treatment versus macrophages from Nox4 -/- mice. Conclusions: These data suggest that increased vascular NOX4 levels in aged Apoe -/- mice promote a proinflammatory environment, enhancing plaque macrophage accumulation and skewing polarization to M1 phenotype resulting in inflammasome activation and atherosclerotic lesion expansion.

M2 macrophages are closely associated with accelerated clavicle fracture healing in patients with traumatic brain injury: a retrospective cohort study

BackgroundMounting evidence indicate patients with traumatic brain injury (TBI) have an accelerated fracture healing. The healing process of bone fractures is greatly dependent on infiltrated macrophages. The macrophages are categorized into M1 or M2 phenotypes with different functions. This study is aimed to address the potential role of subtypes of macrophages in the process of fracture healing in patients with TBI.MethodsTwenty-five cases of clavicle fracture alone (CF group) and 22 cases of clavicle fracture concomitant with TBI (CFT group) were retrospectively analyzed in this study. Callus tissues were harvested during operations. The expressions of COX-2, CD206, and CD68 were measured with immunohistochemistry.ResultsThe percentages of M2 macrophages in total macrophages increased after bone fracture in both groups, while the percentages of M1-type macrophages are decreased. Interestingly, the increased percentages of M2 macrophages are significantly higher in CFT group than in CF group. Compared to CF group, the fracture callus volume was much larger (21.9 vs 8.5 cm3) and the fracture healing time was much shorter (82.2 vs 127.0 days) in CFT group. The percentage of M2 macrophages was negatively correlated with fracture healing time in patients (r = − 0.575, p < 0.01).ConclusionsThe findings suggest that the percentages of M2 macrophages in callus tissues increased dramatically during the repairing stage in both CF and CFT group. Percentages of M2 macrophages are associated with accelerated fracture healing in patients with TBI. M2 macrophage polarization during the stage of bone regeneration may play a vital role in promoting bone fracture healing.

Astragaloside IV inhibits lung cancer progression and metastasis by modulating macrophage polarization through AMPK signaling

BackgroundAccumulating evidence suggests that M2-polarized tumor-associated macrophages (TAMs) play an important role in cancer progression and metastasis, making M2 polarization of TAMs an ever more appealing target for therapeutic intervention. Astragaloside IV (AS-IV), a saponin component isolated from Astragali radix, has been reported to inhibit the invasion and metastasis of lung cancer, but its effects on TAMs during lung cancer progression have not been investigated.MethodsHuman THP-1 monocytes were induced to differentiate into M2 macrophages through treatments with IL-4, IL-13, and phorbol myristate acetate (PMA). We used the lung cancer cell lines A549 and H1299 cultured in conditioned medium from M2 macrophages (M2-CM) to investigate the effects of AS-IV on tumor growth, invasion, migration, and angiogenesis of lung cancer cells. Macrophage subset distribution, M1 and M2 macrophage-associated markers, and mRNA expression were analyzed by flow cytometry and quantitative PCR. The activation of adenosine monophosphate-activated protein kinase (AMPK) signaling pathways that mediate M2-CM–promoted tumor migration was detected using western blotting.ResultsHere we found that AS-IV significantly inhibited IL-13 and IL-4–induced M2 polarization of macrophages, as illustrated by reduced expression of CD206 and M2-associated genes, and that AS-IV suppressed the M2-CM–induced invasion, migration, and angiogenesis of A549 and H1299 cells. In vivo experiments demonstrated that AS-IV greatly inhibited tumor growth and reduced the number of metastases of Lewis lung cancer. The percentage of M2 macrophages was decreased in tumor tissue after AS-IV treatment. Furthermore, AS-IV inhibited AMPKα activation in M2 macrophages, and silencing of AMPKα partially abrogated the inhibitory effect of AS-IV.ConclusionsAS-IV reduced the growth, invasion, migration, and angiogenesis of lung cancer by blocking the M2 polarization of macrophages partially through the AMPK signaling pathway, which appears to play an important role in AS-IV’s ability to inhibit the metastasis of lung cancer.

PO-287 ’Annex’in A1 to the breast tumour microenvironment by aiding in macrophage polarisation

IntroductionTumor-associated macrophages (TAMs) choreograph various aspects of the tumour microenvironment. Macrophages exhibit cellular plasticity and can be polarised to M1 or M2 subtypes in the presence of different microenvironment ’signals’. Annexin A1 (ANXA1), an anti-inflammatory protein is highly expressed in the metastatic breast cancer. Many studies have found that the TAMs exhibit a higher pro-tumorigenic and anti-inflammatory, M2 phenotype.Material and methodsGene Expression Omnibus (GEO) and ArrayExpress were used to assess the association between TAMs and breast cancer in the patients. Percentage of M2 macrophages was evaluated using flow cytometry in the breast tumours from the MMTV mice and the mammary fat pad of the control mice. Macrophage education by breast cancer was assessed by ex vivo differentiation of bone-marrow derived macrophages (BMDMs) in the presence or absence of breast cancer conditioned media by flow cytometry, ELISA, and mRNA expression.Results and discussionsClinically, we found that the M2 TAMs were highly enriched in the claudin-low breast cancer subtype and was strongly associated with ANXA1 gene expression. In the MMTV mouse model, M2 TAMs were higher in the breast tumours as compared to the mammary tissues along with higher expression of ANXA1 and other M2 markers. Additionally, BMDMs were skewed to a more M2 TAM-like phenotype upon co-culture with the breast cancer cells, with enhanced migratory and invasive properties and phagocytic potential, which was reduced in the ANXAI-deficient BMDMs. In another in vivo mouse model employed in our study, we found that the pro-tumorigenic M2 macrophages were significantly reduced in the breast tumours isolated from the ANXA1-deficient mice as compared to the wild type, when 4 T1 had been injected orthotopically in them.ConclusionCollectively, our data describe a novel regulatory role of Annexin A1 in promoting macrophage polarisation to a more M2 phenotype in the breast tumour microenvironment both ex vivo and in vivo, thus presenting itself and its associated receptor as a potential drug target.Furthermore, our secretome data has also revealed some interesting results and Affymetrix microarray profiling and RNA sequencing studies are underway to explore the unique signature molecules and the signalling mechanism involved in governing this dynamic process.

Berberine inhibits macrophage M1 polarization via AKT1/SOCS1/NF-κB signaling pathway to protect against DSS-induced colitis

Berberine has been reported to have protective effects in colitis treatment. However, the detailed mechanisms remain unclear. Herein, we demonstrated that berberine could protect against dextran sulfate sodium (DSS)-induced colitis in mice by regulating macrophage polarization. In the colitis mouse model, berberine ameliorated DSS-induced colon shortening and colon tissue injury. Moreover, berberine-treated mice showed significant reduction in the disease activity index (DAI), pro-inflammatory cytokines expression and macrophages infiltration compared with the DSS-treated mice. Notably, berberine significantly reduced the percentage of M1 macrophages. In vitro analysis also confirmed the inhibitory effects of berberine on macrophages M1 polarization in RAW267.4 cells. Further investigation showed that berberine promoted AKT1 expression in mRNA and protein level. Silence of AKT1 abolished the inhibitory effect of berberine on macrophages M1 polarization. The berberine-induced AKT1 expression promoted suppressers of cytokine signaling (SOCS1) activation, which inhibited nuclear factor-kappa B (NF-κB) phosphorylation. In addition, we also found that berberine activated AKT1/SOCS1 signaling pathway but inhibited p65 phosphorylation in macrophages in vivo. Therefore, we concluded that berberine played a regulatory role in macrophages M1 polarization in DSS-induced colitis via AKT1/SOCS1/NF-κB signaling pathway. This unexpected property of berberine may provide a potential explanation for its protective effects in colitis treatment.

A phase I study to evaluate the safety of trigriluzole (BHV-4157) in combination with PD-1 blocking antibodies.

TPS80 Background: The Metabotropic Glutamate Receptor 1 (GRM1) is expressed in 60-100% of human melanomas, breast cancers, and other solid tumors. Riluzole, an FDA-approved drug for ALS, inhibits GRM1 signal transduction. A phase 0 trial in melanoma patients demonstrated that riluzole suppressed signaling through the MAPK and PI3K/AKT pathways but no objective responses were seen in a phase 2 study.The clinical use of riluzole outside of ALS has been limited by: (i) poor oral bioavailability, (ii) extensive first-pass hepatic metabolism, (iii) high variability in PK parameters, (iv) food-related decrease in bioavailability, and (v) dose-related hepatotoxicity. Trigriluzole (BHV-4157) is a third generation prodrug of riluzole with improved PK/PD properties. In our MASS20 in vivo murine model of melanoma, GRM1 signal transduction appears to suppress tumor immunity through up-regulation of M-CSF and CCL2 expression with a subsequent increase in the percentage of M2 macrophages in the tumor microenvironment. In an immunocompetent mouse model, treatment with trigriluzole and anti-PD-1 antibody was more effective than either drug alone. Methods: Subjects with advanced or refractory solid cancers or lymphoma will be treated with increasing doses of trigriluzole in this phase 1b study. The dosing cohorts of trigriluzole will be using a semi-Bayesian modified toxicity probability interval dose escalation procedure. Trigriluzole monotherapy will be given for a 14-day lead in period and then patients will receive trigriluzole in combination with nivolumab 240mg IV every 2 weeks. After the maximum tolerated dose (MTD) of trigriluzole is identified, it will be checked in a cohort of 6 patients in combination with pembrolizumab. Total sample size will be 12 – 27 subjects. PD-1-directed therapy prior to entry is allowed. Blood samples and optional biopsies will be collected for correlative analysis. PBMCs and fresh tumor tissue will be grafted into NSG mice to create a patient-derived autologous double-humanized murine model for each participant. Clinical trial information: NCT03229278.

The effects of ethanolic extract of Phaleria macrocarpa (Scheff.) Boerl leaf on macrophage phagocytic activity in diabetic rat model

Diabetic patients suffer inflammation and immune deficiency as a consequence of the decrease in macrophage phagocytic activity, thus making them vulnerable to infection. The ability of Ethanolic Extract of Phaleria macrocarpa Leaf (EEPML) to increase macrophage phagocytic activity has also a potential in the diabetic case. EEPML also has anti-inflammatory effect. In this study the EEPML potential to increase peritoneal macrophage phagocytic activity and change M1 and M2 macrophage percentage in diabetic rat model is investigated. This was a quasi experimental study with post test only control group design. Fourty five male Sprague Dawley rats within the age of 8 weeks were classified into normal control group, diabetic control group with solvent, diabetic with 7mg/200g, 14mg/200g, and 28mg/200g of EEPML peroral administration, once a day. The diabetic rat model was made with streptozotocin and nicotinamide injection. The rats were terminated in 3 rd , 14 th and 25 th day of extract administration. Peritoneal fluid was isolated then cultured for macrophage phagocytic activity assay with latex beads. M1 and M2 macrophage percentage was analyzed using flow cytometry with anti CD40 and CD206 antibody. Result of statistical analysis show that active macrophage and phagocytic index mean of EEPML rat groups on day 3, 14 and 25 was significantly higher than the control group. The mean of M1 macrophage percentage of EEPML rat groups was significantly higher than control on day 3 and 14, and lower on day 25, while mean of M2 macrophage percentage didn’t show any significant difference within groups. Conclusion of this study is administration of EEPML increases peritoneal macrophage phagocytic activity on day 3, 14 and 25. This is also increases M1 macrophage percentage on day 14, decrease M1 macrophage percentage on day 25, and doesn’t change peritoneal M2 macrophage percentage in diabetic rat model.

Interleukin-4 mediates the analgesia produced by low-intensity exercise in mice with neuropathic pain.

Peripheral nerve injury (PNI) activates the immune system, resulting in increased proinflammatory cytokines at the site of injury and in the spinal cord dorsal horn. Exercise modulates the immune system promoting an anti-inflammatory phenotype of macrophages in uninjured muscle, and increases in anti-inflammatory cytokines can promote healing and analgesia. We proposed that PNI will decrease, and treadmill exercise will increase, release of anti-inflammatory cytokines at the site of injury and in the spinal cord. We show that 2 weeks of treadmill exercise improves neuropathic pain behaviors in mice: mechanical hyperalgesia, escape and avoidance behavior, and spontaneous locomotor activity. Peripheral nerve injury reduced anti-inflammatory cytokines (interleukin-4 [IL-4], IL-1ra, and IL-5) at the site of nerve injury and in the spinal dorsal horn, whereas exercise restored IL-4, IL-1ra, and IL-5 concentrations to preinjury levels. IL4 mice and mice treated with IL-4 antibody did not develop analgesia to treadmill exercise. Using immunohistochemical staining of the sciatic nerve, treadmill exercise increased the percentage of M2 macrophages (secretes anti-inflammatory cytokines) and decreased M1 macrophages (secretes proinflammatory cytokines) when compared with sedentary mice. The increased M2 and decreased M1 macrophages in exercised mice did not occur in IL-4 mice. In the spinal cord, PNI increased glial cell activation, brain-derived neurotrophic factor and β-nerve growth factor levels, and decreased IL-4 and IL-1ra levels, whereas treadmill exercise suppressed glial cells activation (Glial Fibrillary Acidic Protein and Iba1 immunoreactivity), reduced brain-derived neurotrophic factor and β-nerve growth factor, and increased IL-4, IL-1ra, and IL-5 concentrations. Our results suggest that IL-4 mediates the analgesia produced by low-intensity exercise by modulating peripheral and central neuroimmune responses in mice with neuropathic pain.

Nerve-specific, xenogeneic extracellular matrix hydrogel promotes recovery following peripheral nerve injury.

Peripheral nerve possesses the inherent ability to regrow and recover following injury. However, nerve regeneration is often slow and incomplete due to limitations associated with the local microenvironment during the repair process. Manipulation of the local microenvironment at the site of nerve repair, therefore, represents a significant opportunity for improvement in downstream outcomes. Macrophages and Schwann cells play a key role in the orchestration of early events after peripheral nerve injury. We describe the production, characterization, and use of an injectable, peripheral nerve-specific extracellular matrix-based hydrogel (PNSECM) for promoting modulation of the local macrophage and Schwann cell responses at the site of nerve repair in a rodent model of sciatic nerve injury. We show that PNSECM hydrogels largely maintain the matrix structure associated with normal native peripheral nerve tissue. PNSECM hydrogels were also found to promote increased macrophage invasion, higher percentages of M2 macrophages and enhanced Schwann cell migration when used as a lumen filler in a rodent model of nerve gap repair using an inert nerve guidance conduit. These results suggest that an injectable PNSECM hydrogel can provide a supportive, bioactive scaffold which promotes repair of peripheral nerve in vivo. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 450-459, 2018.

Role of Bone Marrow Mononuclear Cell Seeding for Nanofiber Vascular Grafts.

Electrospinning is a promising technology that provides biodegradable nanofiber scaffolds for cardiovascular tissue engineering. However, success with these materials has been limited, and the optimal combination of scaffold parameters for a tissue-engineered vascular graft (TEVG) remains elusive. The purpose of the present study is to evaluate the effect of bone marrow mononuclear cell (BM-MNC) seeding in electrospun scaffolds to support the rational design of optimized TEVGs. Nanofiber scaffolds were fabricated from co-electrospinning a solution of polyglycolic acid and a solution of poly(ι-lactide-co-ɛ-caprolactone) and characterized with scanning electron microscopy. Platelet activation and cell seeding efficiency were assessed by ATP secretion and DNA assays, respectively. Cell-free and BM-MNC seeded scaffolds were implanted in C57BL/6 mice (n = 15/group) as infrarenal inferior vena cava (IVC) interposition conduits. Animals were followed with serial ultrasonography for 6 months, after which grafts were harvested for evaluation of patency and neotissue formation by histology and immunohistochemistry (n = 10/group) and PCR (n = 5/group) analyses. BM-MNC seeding of electrospun scaffolds prevented stenosis compared with unseeded scaffolds (seeded: 9/10 patent vs. unseeded: 1/10 patent, p = 0.0003). Seeded vascular grafts demonstrated concentric laminated smooth muscle cells, a confluent endothelial monolayer, and a collagen-rich extracellular matrix. Platelet-derived ATP, a marker of platelet activation, was significantly reduced after incubating thrombin-activated platelets in the presence of seeded scaffolds compared with unseeded scaffolds (p < 0.0001). In addition, reduced macrophage infiltration and a higher M2 macrophage percentage were observed in seeded grafts. The beneficial effects of BM-MNC seeding apply to electrospun TEVG scaffolds by attenuating stenosis through the regulation of platelet activation and inflammatory macrophage function, leading to well-organized neotissue formation. BM-MNC seeding is a valuable technique that can be used in the rational design of optimal TEVG scaffolds.

Regulatory effect of bone marrow mesenchymal stem cells on polarization of macrophages

Objective: To examine the regulatory effect of bone marrow mesenchymal stem cells (BM-MSCs) on the polarization of bone marrow-derived macrophages, and to provide a theoretical support for the application of mesenchymal stem cells in the treatment of liver fibrosis. Methods: MSCs and macrophages were first isolated from the bone marrow of mice. Macrophages were polarized to M1 macrophages with lipopolysaccharide (LPS) and interferon-γ (IFN-γ), and to M2 macrophages with interleukin-4 (IL-4). The macrophages were then co-cultured with BM-MSCs in a Transwell for 24 h, and changes in the percentages of M1 and M2 macrophages were examined using flow cytometry. The mRNA levels of the M1 macrophage-associated cytokines, tumor necrosis factor-α (TNF-α) and interleukin-23a (IL-23a), and M2 macrophage-associated molecules, arginase-1 (Arg-1) and CD163, were measured by real-time quantitative PCR. The two samples were compared using the t test, and P < 0.05 was considered as statistically significant. Results: Flow cytometry showed that the percentage of M1 macrophages was significantly lower in the (macrophage + LPS + IFN-γ + BM-MSC) co-culture group than in the (macrophage + LPS + IFN-γ) group (62.5% ± 4.6% vs 86.6% ± 6.9%, t = 5.034, P = 0.0073). In addition, the relative mRNA expression of TNF-α and IL-23a was also significantly reduced in the co-culture group compared with those in the macrophage control group as measured by RT-qPCR (t = 11.57 and 10.57, respectively, P < 0.05). Compared with that in the macrophage control group, the percentage of M2 macrophages in the (macrophage+BM-MSC) co-culture group was significantly increased (89.5% ± 5.8% vs 70.1% ± 6.3%, t = 3.924, P = 0.0172), along with significantly elevated relative mRNA expression of Arg1 (14.35±1.05 vs 1.00±0.03, t = 21.96, P < 0.05) and CD163 (3.04 ± 0.27 vs 1.00 ± 0.03, t = 13.14, P < 0.05). Conclusion: BM-MSCs can inhibit LPS + IFN-γ-induced polarization to M1 macrophages and promote polarization to M2 macrophages through the release of paracrine factors.

Protectin DX increases survival in a mouse model of sepsis by ameliorating inflammation and modulating macrophage phenotype

Recently, a serial of studies have demonstrated that lipid mediators derived from Omega-3 fatty acid docosahexaenoic acid have pro-resolving or anti-inflammatory effects in many inflammatory diseases. Here, we sought to evaluate whether Protectin DX (PDX, an isomer of Protecin D1), a newly identified lipid mediator, could protect mice against sepsis and explore the underling mechanism. Animal model of sepsis was established by cecum ligation and puncture (CLP). We found that PDX increased overall survival rate within eight days and attenuated multiple organ injury in septic mice. In addition, PDX reduced pro-inflammatory cytokines and bacterial load 24 h after CLP. Moreover, PDX promoted phagocytosis of peritoneal macrophages and increased the percentage of M2 macrophages in peritoneum of septic mice. In vitro, M2 macrophage markers (Arg1 and Ym1) and its transcriptional regulator (peroxisome proliferator-activated receptor-γ, PPAR-γ) were upregulated in Raw264.7 macrophages challenged with PDX. GW9662 (a PPAR-γ inhibitor) and PPAR-γ siRNA abrogated the induction of Arg1 and Ym1 by PDX in Raw264.7 cells. Taken together, our results suggest that PDX is able to promote M2 polarization, enhance phagocytosis activity of macrophage and accelerate resolution of inflammation, finally leading to increased survival rate of septic mice.