Levels Of M2 Macrophages Research Articles

Peroxisome proliferator-activated receptor-γ-mediated polarization of macrophages in Neospora caninum infection

BackgroundNeospora caninum is an apicomplexan parasite closely related Toxoplasma gondii, which causes neurological disease and abortion in multiple animal species. Macrophage polarization plays an important role in host immune responses to parasites infection, such as Toxoplasma gondii, Leishmania, Trypanosoma cruzi. However, the dynamics of macrophage polarization, as well as the possible mechanism that regulate macrophage polarization, during N. caninum infection remains unclear. MethodsThe M1 and M2-phenotypic markers of peritoneal macrophages from mice infected with tachyzoites of Nc-1 were analyzed by flow cytometry (FCM) analysis. Then J774A.1 cells were respectively treated with GW9662 and RGZ, and stimulated by tachyzoites of Nc-1. M1 and M2-phenotypic markers were determined by FCM and ELISA. And the activations of PPAR-γ and NF-κB were determined by Western blotting. ResultsIn this study, our data showed that macrophages were preferentially differentiated into the M1 type during the acute stage of N. caninum infection, while the level of M2 macrophages significantly increased during the chronic stage of infection. In vitro study, compared with the GW9662 group and RGZ group, N. caninum can promote M2-polarized phenotype through up-regulate the activity of PPAR-γ and inhibting NF-κB activation. ConclusionIn conclusion, this study demonstrated that macrophages are plastic since M1 differentiated macrophages can express M2 markers with N. caninum infection through up-regulating the activity of PPAR-γ and inhibting NF-κB activation and may be providing new insights for the prevention and treatment of N. caninum infection.

GRM1 to induce M-CSF and CCL2 expression to lead to an upregulation of M2 macrophages in melanoma.

122 Background: GRM1 transgenic mice in an immune competent background (C57BL6) develope melanoma with 100% penetrance (TG-3 mice). Ectopic expression of GRM1 in immortalized mouse melanocytes (MelanA) produce cells (MASS20) that are tumorigenic in both SCID and immune competent C57BL6 mice. We have examined the downstream events of GRM1 signaling that allow transformed cells to evade immune destruction. Methods: Cytokine antibody arrays and Western blot were used to examine differences in cytokine expression between MASS20 cells and parental controls. Next, we cultured C57BL6 bone marrow cells in MASS20 conditioned medium for 24 and 48 hours and determined resultant M2 macrophage levels. Finally, we used oral weekly liposome clodronate (LC) treatment to deplete macrophages in murine models to determine the effect on tumor growth. Results: MASS20 cells produce higher levels of M-CSF and CCL2 as compared with control cells. These two cytokines recruit and induce the polarization of tumor associated macrophages (TAM) to increase the M2 fraction resulting in accelerated tumor progression. Culturing C57BL6 bone marrow cells in MASS20 conditioned medium increased expression of the M2 macrophage marker arginase1 at both time points. Weekly oral LC treatment to C57BL6 mice greatly inhibited the growth of MASS20 tumors as compared to growth in control animals. Single-cell suspensions of spleen and tumor cells from these animals were subjected to flow cytometry and showed that LC treatment depleted macrophages by 90% in the spleen, and increased the percentage of cd8+T and cd335+ NK cells in tumors from treated mice as compared to controls. Finally, we treated TG-3 transgenic mice with weekly oral LC and found that treated animals still developed tumors, but progression was greatly slowed, and survival greatly prolonged, compared to untreated animals. Within 1 week of stopping LC in the treated animals there was rapid growth of the tumors. These findings suggest a link between GRM1-signal transduction and TAM-induced suppression of tumor-immunity in melanoma. Conclusions: We conclude that GRM1-induced suppression of tumor immunity occurs through induction of M2 macrophages in the tumor microenvironment.

Abstract A17: Differential gene expression and tumor mutanome analysis reveal significantly enriched pathways associated with higher tumor burden of M1 and M2 macrophages

Abstract Many attributes of the tumor microenvironment, such as the level of CD8 T-cells in the tumor, higher levels of pro-inflammatory cytokine network dominated by interferon signaling, antigen processing and presentation correlate with superior efficacy of checkpoint control inhibitors. Tumors lacking CD8 T-cells are less responsive to checkpoint control blockade, and therefore other therapeutic modalities for treating these tumors need to be explored. In this study, we set out to identify a set of core pathways associated with the absence or presence of specific immune cell types in tumors. These core pathways can be modulated to alter the immune profile of these unresponsive tumors and sensitize them to checkpoint control blockade. To this end, we created a robust bioinformatic solution OncoPeptTUME, to systematically investigate the immune landscape of tumors from RNA-seq data, using a set of proprietary immune cell type-specific gene expression signatures. Using a scoring method derived from Single Cell Gene Set Enrichment Analysis (ssGSEA), we quantitated the relative abundance of different immune cell types present in 9345 tumors across 33 cancers available in the TCGA dataset. We first validated our approach by selecting high and low CD8 T-cell containing tumors across many different cancers. Differential gene expression analysis between these two sets of tumors identified upregulated genes in PD-1 signaling, IFN-α, β and γ pathways, TCR signaling, antigen processing and presentation as previously reported in multiple studies. Similarly, tumors with high infiltration of myeloid derived suppressor cells (MDSCs) showed high level expression of a large number of inhibitory receptors associated with innate immune cells, demonstrating potential mechanism of immunesuppression in these tumors. Few studies, however, have investigated tumor intrinsic and extrinsic factors that favor infiltration of macrophages and induce them to differentiate into M1 and M2 functional states. The M1 and M2 macrophages regulate the inflammatory state of the tumor microenvironment by producing cytokines, chemokines and growth factors thereby making them susceptible or resistant to immune-mediated elimination. We applied M1 and M2-specific gene expression signatures on CD8 T-cell depleted tumors and identified 236 tumors having high or low M1 or M2 macrophages. Differential gene expression analysis reveal that tumors containing high M1 macrophages have significant upregulation of IFN-α/β signaling, tryptophan catabolism, IL1-β processing and CASP1 inflammosome activity. By contrast, tumors with low M1 infiltrated macrophages have upregulation of FGFR1c-Klotho pathway genes. Since differentiation of macrophages into M1 or M2 occurs in the tissue microenvironment, FGFR1 signaling may regulate macrophage phenotype rather than their migration into the tumor. In support of this hypothesis, we detected higher level of M2 macrophages in tumors that are depleted of M1 macrophages. We also analyzed genetic alterations in tumor cells that favor higher levels of M2 macrophages by examining non-synonymous somatic mutations in the coding sequences of genes. We observed loss- of-function mutations in p53 gene across many different cancers (breast, glioblastoma, stomach adenocarcinoma and lung squamous cell carcinoma) showing higher burden of M2 macrophages compared to the M1 type. Our analysis demonstrate that combining expression signatures with tumor mutanome analysis can provide a powerful tool to assess the tumor microenvironment and identify pathways that promote, or exclude infiltration/differentiation of specific immune cells. Citation Format: Nitin Mandloi, Ashwini Patil, Rekha Sathian, Aparna Mohan, Malini Manoharan, Ravi Gupta, Hiranjith Govindamangalam, Amit Chaudhuri. Differential gene expression and tumor mutanome analysis reveal significantly enriched pathways associated with higher tumor burden of M1 and M2 macrophages. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr A17.

Activation of myeloid and endothelial cells by CD40L gene therapy supports T-cell expansion and migration into the tumor microenvironment

CD40 is an interesting target in cancer immunotherapy due to its ability to stimulate T-helper 1 immunity via maturation of dendritic cells and to drive M2 to M1 macrophage differentiation. Pancreatic cancer has a high M2 content that has shown responsive to anti-CD40 agonist therapy and CD40 may thus be a suitable target for immune activation in these patients. In this study, a novel oncolytic adenovirus armed with a trimerized membrane-bound extracellular CD40L (TMZ-CD40L) was evaluated as a treatment of pancreatic cancer. Further, the CD40L mechanisms of action were elucidated in cancer models. The results demonstrated that the virus transferring TMZ-CD40L had oncolytic capacity in pancreatic cancer cells and could control tumor progression. TMZ-CD40L was a potent stimulator of human myeloid cells and T-cell responses. Further, CD40L-mediated stimulation increased tumor-infiltrating T cells in vivo, which may be due to a direct activation of endothelial cells to upregulate receptors for lymphocyte attachment and transmigration. In conclusion, CD40L-mediated gene therapy is an interesting concept for the treatment of tumors with high levels of M2 macrophages, such as pancreatic cancer, and an oncolytic virus as carrier of CD40L may further boost tumor killing and immune activation.

Mesenchymal Stem Cell-Educated Macrophages Ameliorate LPS-Induced Systemic Response.

Both bone marrow and adipose-derived mesenchymal stem cells (ASCs) have immunomodulatory effects. The goal of this study was to determine whether ASCs-educated macrophages could directly ameliorate LPS-induced systemic response in a mouse model. Mouse peritoneal macrophages were cocultured with ASCs in a Transwell system for 2 days to educate macrophages. Mice were divided into 5 groups: control, LPS, LPS + ASCs, LPS + untreated macrophages, and LPS + educated macrophages. Educated macrophages decreased lung inflammation, weight loss, pulmonary edema, and inflammatory cytokine response. In vitro, ASCs increased expression of M2 macrophages independent of direct cell-to-cell contact when macrophages were treated with LPS or serum from patients with acute respiratory distress syndrome (ARDS). When macrophages were cultured with serum from ARDS patients who were treated with ASCs or placebo in our previous clinical trial, there was no difference in M2 macrophage levels before and after ASCs treatment indicating a suboptimal response to the treatment protocol. ASCs also reduced the levels of LPS-induced proinflammatory cytokines in vitro which were mimicked by IL-10 and blocked by antibodies for IL-10 and IL-10 receptor supporting the notion that educated macrophages exert their anti-inflammatory effects via IL-10-dependent mechanisms.

Progression of inflammation during immunodeficient mouse skeletal muscle regeneration.

The skeletal muscle injury triggers the inflammatory response which is crucial for damaged muscle fiber degradation and satellite cell activation. Immunodeficient mice are often used as a model to study the myogenic potential of transplanted human stem cells. Therefore, it is crucial to elucidate whether such model truly reflects processes occurring under physiological conditions. To answer this question we compared skeletal muscle regeneration of BALB/c, i.e. animals producing all types of inflammatory cells, and SCID mice. Results of our study documented that initial stages of muscles regeneration in both strains of mice were comparable. However, lower number of mononucleated cells was noticed in regenerating SCID mouse muscles. Significant differences in the number of CD14-/CD45+ and CD14+/CD45+ cells between BALB/c and SCID muscles were also observed. In addition, we found important differences in M1 and M2 macrophage levels of BALB/c and SCID mouse muscles identified by CD68 and CD163 markers. Thus, our data show that differences in inflammatory response during muscle regeneration, were not translated into significant modifications in muscle regeneration.

Interleukin-10 conjugated electrospun polycaprolactone (PCL) nanofibre scaffolds for promoting alternatively activated (M2) macrophages around the peripheral nerve in vivo

Macrophages play a key role in tissue regeneration following peripheral nerve injury by preparing the surrounding parenchyma for regeneration, however, they can be damaging if the response is excessive. Interleukin 10 (IL-10) is a cytokine that promotes macrophages toward an anti-inflammatory/wound healing state (M2 phenotype). The bioactive half-life of IL-10 is dependent on the cellular microenvironment and ranges from minutes to hours in vivo. Our objective was to extend the in vivo bioavailability and bioactivity of IL-10 by attaching the protein onto nanofibrous scaffolds and demonstrating increased expression levels of M2 macrophages when placed around healthy intact peripheral nerves. IL-10 was adsorbed and covalently bound to electrospun poly(ε-caprolactone) (PCL) nanofibrous scaffolds. In vivo bioavailability and bioactivity of IL-10 was confirmed by wrapping IL-10 conjugated nanofibres around the sciatic nerves of Wistar rats and quantifying M2 macrophages immunohistochemically double labelled with ED1 and either arginase 1 or CD206. IL-10 remained immobilised to PCL scaffolds for more than 120days when stored in phosphate buffered saline at room temperature and for up to 14days when implanted around the sciatic nerve. IL-10 conjugated nanofibres successfully induced macrophage polarisation towards the M2 activated state within the scaffold material as well as the adjacent tissue surrounding the nerve. PCL biofunctionalised nanofibres are useful for manipulating the cellular microenvironment. Materials such as these could potentially lead to new therapeutic strategies for nervous tissue injuries as well as provide novel investigative tools for biological research.

The promotion of functional urinary bladder regeneration using anti-inflammatory nanofibers

Current attempts at tissue regeneration utilizing synthetic and decellularized biologic-based materials have typically been met in part by innate immune responses in the form of a robust inflammatory reaction at the site of implantation or grafting. This can ultimately lead to tissue fibrosis with direct negative impact on tissue growth, development, and function. In order to temper the innate inflammatory response, anti-inflammatory signals were incorporated through display on self-assembling peptide nanofibers to promote tissue healing and subsequent graft compliance throughout the regenerative process. Utilizing an established urinary bladder augmentation model, the highly pro-inflammatory biologic scaffold (decellularized small intestinal submucosa) was treated with anti-inflammatory peptide amphiphiles (AIF-PAs) or control peptide amphiphiles and used for augmentation. Significant regenerative advantages of the AIF-PAs were observed including potent angiogenic responses, limited tissue collagen accumulation, and the modulation of macrophage and neutrophil responses in regenerated bladder tissue. Upon further characterization, a reduction in the levels of M2 macrophages was observed, but not in M1 macrophages in control groups, while treatment groups exhibited decreased levels of M1 macrophages and stabilized levels of M2 macrophages. Pro-inflammatory cytokine production was decreased while anti-inflammatory cytokines were up-regulated in treatment groups. This resulted in far fewer incidences of tissue granuloma and bladder stone formation. Finally, functional urinary bladder testing revealed greater bladder compliance and similar capacities in groups treated with AIF-PAs. Data demonstrate that AIF-PAs can alleviate galvanic innate immune responses and provide a highly conducive regenerative milieu that may be applicable in a variety of clinical settings.

Abstract 3846: Pleiotrophin in the tumor microenvironment induces expression of VEGFR2 on macrophages and promotes an anti-inflammatory phenotype

Abstract Macrophages are an abundant immune cell in the tumor microenvironment that contribute to tumor growth and metastasis. Macrophage recruitment into tumors is mediated by multiple cytokines, including vascular endothelial growth factor-A (VEGF). We are interested in investigating the effects of VEGF inhibition on macrophages and other stromal cells within the tumor. We demonstrated recently that macrophages (systemic and tumor associated) in tumor-bearing animals express VEGFR2 in contrast to macrophages from non tumor-bearing animals. Furthermore, we found that selective inhibition of VEGFR2 inhibits macrophage infiltration into tumors in vivo. These results demonstrate that when VEGFR2 is expressed on macrophages it is the dominant receptor driving VEGF-induced migration in vitro and in vivo. Additionally, we found that the cytokine pleiotrophin (PTN) is sufficient to induce VEGFR2 expression on macrophages. PTN is expressed by many human breast cancer cells (e.g., MDA-MB-231) and the majority of human primary breast tumors. However, the connection between PTN and macrophage phenotype is still unexplored. Macrophages can be divided into two functional sub-types: M1 or classically activated macrophages and M2 or alternatively activated macrophages. M1 macrophages are powerful effector cells that produce pro-inflammatory cytokines and are capable of destroying tumor cells. In contrast, M2 macrophages inhibit the inflammatory response and promote tumor growth and angiogenesis. We employed the MMTV-PyMT transgenic, spontaneous breast cancer model to characterize PTN expression, immune cell infiltration, and immune cell function during the course of tumor progression. We found that PTN levels correlate with VEGFR2+ macrophage and M2 macrophage levels, which all peak during the transition from hyperplasia to early malignancy. Whereas, MDSC and Treg levels as well as levels of the anti-inflammatory cytokines IL-10 and IL-1β peak during the early carcinoma stage. We identified that PTN not only induces VEGFR2 expression by macrophages but also promotes an M2 phenotype in vitro. In addition, we have evidence that VEGF can modulate the level of PTN expression through VEGFR1. These results suggest a mechanism through which anti-VEGF therapy modulates PTN and M2 macrophage levels in vivo. Based on these observations, we hypothesize that PTN is a critical cytokine involved in breast tumor progression through promotion of a VEGFR2+, M2 macrophage phenotype in the tumor microenvironment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3846.