M2 Macrophage Population Research Articles

Abstract 1043: CSF-1R inhibitor prevented pre-metastatic lung niches in metastatic mammary tumor

Abstract Exosomes are small vesicle cellular products originating from the endocytic pathway abundantly secreted by tumor cells. These exosomes have the ability to alter their immediate microenvironment (ME) through cell-cell interaction by fusion with plasma membrane and subsequent endocytosis or release of their cargo. Exosomes are critical modulators of pre-metastatic niche creation by increasing the recruitment of inflammatory cells. In breast cancer, tumor-derived exosomes recruit myeloid derived suppressor cells in the creation of an immunosuppressive pro-tumorigenic lung niches. Colony stimulating factor 1-receptor (CSF1R), which is a key regulator of myeloid cell proliferation, survival, and differentiation can be blocked by a selective inhibitor GW2580. Overexpression of CSF-1 has been implicated in the creation of increased number of metastatic niches in numerous cancers. In our study, we have demonstrated that the exosomes secreted under hypoxic conditions can initiate early pre-metastatic niche creation in lungs in a metastatic breast cancer model compared to normoxia-secreted exosomes. Exosomes were injected intravenously into Balb/c female mice three days after the implantation of 4T1 breast tumor and continued for a week with injections on alternated days. The animals were pre-treated with GW2580 the day before tumor implantation and continued for a week concomitantly with exosomes on alternate days. Lungs, bone marrow, spleen and brain tissues were collected and analyzed by flow cytometer to detect myeloid and angiogenic cells populations. GW2580 was able to prevent myeloid cell infiltration in lungs and bone marrow. Further, we observed significant increase in anti-tumorigenic M1-macrophage population in the lungs of exosome treated animals pre-treated with GW2580. However, these findings were not observed in the bone marrows of the same group. Vasculogenic leukocyte and angiogenic myeloid cell populations were significantly decreased in the lung of exosome treated animals pre-treated with GW2580. A similar decrease in these populations of cells was not seen in the lungs of animals pre-treated with GW2580 only. These surprising results have led us to hypothesize that GW2580 treatment can prevent the effects of exosomes, which causes infiltration of myeloid cells in the lung to create metastatic niche. These observations indicate a role of CSF1R inhibitor in preventing the distant metastatic niche formation. Citation Format: Thaiz F. Borin, Kartik Angara, Mohammad Rashid, Adarsh Shankar, Asm Iskander, Roxan Ara, Meenu Jain, Bhagelu R. Achyut, Ali S. Arbab. CSF-1R inhibitor prevented pre-metastatic lung niches in metastatic mammary tumor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1043. doi:10.1158/1538-7445.AM2017-1043

Anti-PD1 treatment to induce M1 polarization of tumor infiltrating macrophages in a 3D ex vivo system of lung cancer patients.

e23090 Background: Nivolumab and pembrolizumab treatment targeting the PD1/PD-L1 axis has demonstrated increased survival benefit in subpopulations of patients with advanced non-small cell lung cancer (NSCLC). The impact of these therapeutics on tumor immune microenvironment is not fully understood. Classical M1 macrophages are critical components involved in the inflammatory response and antitumor immunity. In this study, we evaluated the effect of PD1/PD-L1 blockade on M1 polarization of tumor-associated macrophages (TAMs) and activation of cytotoxic T-cells in a 3D ex vivo system of NSCLC. Methods: Fresh tumor tissues obtained from consented patients with NSCLC at the time of surgical resection were utilized in a 3D ex vivo tumor miscrosphere assay. 3D tumor microspheres were treated with nivolumab or pembrolizumab at 10 mg/ml for 36 hours within an intact tumor microenvironment. Flow cytometry analysis was performed to evaluate treatment-mediated TAM polarization, activation of T-cells and changes in CD4 and CD8 subpopulations. A multiplex human cytokine assay was used to simultaneously analyze the differential secretion of cytokines. Additionally, a NanoString platform containing probes to quantitate 770 immune function genes was used to determine potential positive or negative associations between expression of immune function genes and TIL activation by ex vivo treatment. Results: Both nivolumab and pembrolizumab treatment increased population of M1 macrophages (CD68+, CD80+, CD163-) and simultaneous release of MIP1b, IFN-ɣ, TNF-a, and GM-CSF cytokines as well as expression of genes related to the M1 phenotype that was accompanied by activation of CD8 cells assessed by Ki67 and CD107a expression and increased expression of genes involved in the IFNg pathway. Conclusions: Our studies showed that anti PD1/PD-L1 treatment leads to M1 macrophage polarization and T-cell activation in subgroups of NSCLC patients emphasizing the importance of comprehensive analysis of tumor immune microenvironment for a better understanding of the mechanism of action of immuno-oncology drugs that may help developing rationale combination treatments in NSCLC.

Blunting age-associated chronic inflammation preserves hematopoiesis and immunity and reduces leukemogenesis

Abstract Aging is the single most important prognostic factor for the development of many cancers. While mutational accumulation may increase the risk of cancer in aged individuals, declining immunity due to chronic inflammation is also a likely contributing factor. We have demonstrated that reducing aging-associated chronic inflammation abrogates fitness declines in B-progenitor cells, and significantly reduces leukemogenesis in aged mice (Henry et al., JCI, 2015). In addition to preserving the function of B-progenitor cells, subsequent studies have revealed that reducing chronic inflammation in aged mice augments immune responses. Specifically, reducing inflammation in aged mice (≥22 months) results in a two-fold reduction in the number of splenic M2 macrophage and T-regulatory cell populations, as well as, a three-fold reduction in the surface expression of the inhibitory protein PD-L1 on innate immune cells. Reducing chronic inflammation in aged mice also results in a three-fold increase in the percentage of interferon-gamma producing CD4+ and CD8+ T-cell lymphocytes when stimulated. These phenotypes were observed in aged, anti-inflammatory transgenic mice (alpha-1-anti-trypsin and interleukin-37) and in geriatric mice (≥27 months) treated with recombinant interleukin-37. Overall, these findings suggest that reducing chronic inflammation in aged populations can rejuvenate hematopoiesis and immunity, while also creating a less permissive environment for leukemogenesis.

Bosutinib Therapy Ameliorates Lung Inflammation and Fibrosis in Experimental Silicosis.

Silicosis is an occupational lung disease for which no effective therapy exists. We hypothesized that bosutinib, a tyrosine kinase inhibitor, might ameliorate inflammatory responses, attenuate pulmonary fibrosis, and thus improve lung function in experimental silicosis. For this purpose, we investigated the potential efficacy of bosutinib in the treatment of experimental silicosis induced in C57BL/6 mice by intratracheal administration of silica particles. After 15 days, once disease was established, animals were randomly assigned to receive DMSO or bosutinib (1 mg/kg/dose in 0.1 mL 1% DMSO) by oral gavage, twice daily for 14 days. On day 30, lung mechanics and morphometry, total and differential cell count in alveolar septa and granuloma, levels of interleukin (IL)-1β, tumor necrosis factor (TNF)-α, interferon (IFN)-γ, IL-4, transforming growth factor (TGF)-β, and vascular endothelial growth factor in lung homogenate, M1 and M2 macrophages, total leukocytes, and T cells in BALF, lymph nodes, and thymus, and collagen fiber content in alveolar septa and granuloma were analyzed. In a separate in vitro experiment, RAW264.7 macrophages were exposed to silica particles in the presence or absence of bosutinib. After 24 h, gene expressions of arginase-1, IL-10, IL-12, inducible nitric oxide synthase (iNOS), metalloproteinase (MMP)-9, tissue inhibitor of metalloproteinase (TIMP)-1, and caspase-3 were evaluated. In vivo, in silicotic animals, bosutinib, compared to DMSO, decreased: (1) fraction area of collapsed alveoli, (2) size and number of granulomas, and mononuclear cell granuloma infiltration; (3) IL-1β, TNF-α, IFN-γ, and TGF-β levels in lung homogenates, (4) collagen fiber content in lung parenchyma, and (5) viscoelastic pressure and static lung elastance. Bosutinib also reduced M1 cell counts while increasing M2 macrophage population in both lung parenchyma and granulomas. Total leukocyte, regulatory T, CD4+, and CD8+ cell counts in the lung-draining lymph nodes also decreased with bosutinib therapy without affecting thymus cellularity. In vitro, bosutinib led to a decrease in IL-12 and iNOS and increase in IL-10, arginase-1, MMP-9, and TIMP-1. In conclusion, in the current model of silicosis, bosutinib therapy yielded beneficial effects on lung inflammation and remodeling, therefore resulting in lung mechanics improvement. Bosutinib may hold promise for silicosis; however, further studies are required.

Macropinocytosis of Nab-paclitaxel Drives Macrophage Activation in Pancreatic Cancer.

Pancreatic cancer is a devastating disease that is largely refractory to currently available treatment strategies. Therapeutic resistance is partially attributed to the dense stromal reaction of pancreatic ductal adenocarcinoma tumors that includes a pervasive infiltration of immunosuppressive (M2) macrophages. Nab-paclitaxel (trade name Abraxane) is a nanoparticle albumin-bound formulation of paclitaxel that, in combination with gemcitabine, is currently the first-line treatment for pancreatic cancer. Here, we show that macrophages internalized nab-paclitaxel via macropinocytosis. The macropinocytic uptake of nab-paclitaxel induced macrophage immunostimulatory (M1) cytokine expression and synergized with IFNγ to promote inducible nitric oxide synthase expression in a TLR4-dependent manner. Nab-paclitaxel was internalized by tumor-associated macrophages in vivo, and therapeutic doses of nab-paclitaxel alone, and in combination with gemcitabine, increased the MHCII+CD80+CD86+ M1 macrophage population. These data revealed an unanticipated role for nab-paclitaxel in macrophage activation and rationalized its potential use to target immune evasion in pancreatic cancer. Cancer Immunol Res; 5(3); 182-90. ©2017 AACR.

Primary human monocytes differentiate into M2 macrophages and involve Notch-1 pathway.

The current study investigates whether inhibiting the Notch-1 signaling pathway in primary human monocytes enhances M2 macrophage differentiation. We generated a primary human monocyte cell culture model to understand the effect of the Notch-1 signaling pathway. Monocytes were treated with Notch-1 inhibitors DAPT or siRNA. Our data show that there was a significant increase in the M1 macrophage population demonstrated by iNOS marker in the primary human monocytes treated with apoptotic-conditioned medium (ACM). Next, the levels of pro-inflammatory cytokines IL-6 and MCP-1, as well as TNF-α, increased in ACM media (p < 0.05). Furthermore, M1 macrophages and pro-inflammatory cytokines were reduced following DAPT or siRNA treatment. Comparatively, there was a significant increase in M2 macrophages, as demonstrated by an increase in CD206 and arginase-1 positive cells treated with DAPT or siRNA (p < 0.05). Furthermore, a significant increase in the associated anti-inflammatory cytokines IL-10 and IL-1RA was also observed with respect to control groups (p < 0.05). We conclude that blocking the Notch-1 pathway with DAPT or siRNA attenuates pro-inflammatory cytokines, enhances M2 macrophage differentiation, and increases anti-inflammatory cytokines in primary human monocytes. As a result, Notch-1 pathway inhibition has potential therapeutic applications of inflammatory disease.

Attenuation of obesity-induced inflammation in mice orally administered with salmon cartilage proteoglycan, a prophylactic agent

Obesity is associated with chronic inflammation of adipose tissue and causes development of type 2 diabetes. M1 macrophage population was increased in adipose tissue of obese mouse. M1 macrophages induce insulin resistance through the secretion of proinflammatory cytokines. Our previous studies demonstrated that salmon cartilage proteoglycan (PG) suppresses excess inflammation in various mouse inflammatory diseases. In this study, we examined the effect of PG on type 2 diabetes using high-fat-diet (HFD) induced obese mouse model. Oral PG administration enhanced the population of small adipocytes (area less than 1000 μm2) without body and tissue weight gain. In addition, PG administration suppressed mRNA expression of TNF-α, IL-6 and CXCL2 in adipose tissue. The proportion of M1 macrophages was decreased by PG administration. In addition, PG administration suppressed hyperglycemia after intraperitoneal glucose injection. Fasted serum insulin level was decreased in PG-administered mice. Moreover, insulin-stimulated phosphorylation of Akt was enhanced in the liver and gastrocnemius skeletal muscle of PG-administered mice. These data suggested that PG administration improves hyperglycemia and insulin sensitivity in obese mice by modulation of M1 macrophages which secrete proinflammatory cytokines in adipose tissue and activation of Akt in liver and skeletal muscle.

Abstract B75: Targeting the immune-microenvironment with combined inhibition of MEK and STAT3 in a mouse model of pancreatic cancer

Abstract Introduction: Activating KRAS mutations are commonly found in PDAC and lead to constitutive downstream activation of MEK, which results in uncontrolled proliferation. We have previously shown that MEK inhibition results in activation of STAT3 signaling which confers drug resistance and continued cancer cell growth while combined STAT3 and MEK inhibition overcomes this resistance. Since STAT3 is a critical mediator of cytokine signaling and MEK is a mediator of cytokine production, we sought to determine the effects of MEK and STAT3 inhibition on the immune tumor microenvironment (TME). Tumor infiltrating immune/inflammatory cells, such as regulatory T cells (Tregs), myeloid –derived suppressor cells (MDSCs) and macrophages support tumor growth and contribute to therapeutic resistance. We hypothesized that combined MEK and STAT3 inhibition down regulates the suppressive immune infiltrates and promotes an anti-tumor microenvironment. Experimental procedure: To understand the effect of MEK and/or STAT3 inhibition of PDAC cells, three dimensional spheroid cultures of PDAC cells (MiaPaCa-2, Panc-1, BxPC3) were prepared. Spheroid cultures were treated with inhibitors to MEK (AZD6244) and/or STAT3 (AZD1480) for 10 days. At the end of treatment, spheroids were quantified for size and metabolic activity. To determine in vivo effects, Ptf1a Cre/+ ; LSL-KrasG12D; Tgfbr2fl/fl (PKT) mice were treated with either vehicle, MEK inhibition, STAT3 inhibition, or the combination for 2 weeks. Post-treated pancreatic tissue was extracted, weighed, and examined for pancreatic integrity using immuno-histological and enzymatic analyses. Alternately, the pancreas and spleen were extracted from the mice, cells were isolated from the tissue, and subsequently labeled with antigens for macrophages (CD45, F4/80, CD86, CD80, CD206, CD204), myeloid cells (CD45, CD11b, Ly6g, Ly6c), and T cells (CD45, CD3e, CD4, CD8, CD25, FoxP3) before assessing population percentages by flow cytometric analysis. Results: Treatment with MEK inhibition resulted in slightly reduced spheroid size and metabolic activity; however, combined MEK/STAT3 treatment led to a significant decrease in spheroid size as well as metabolic activity. In PKT mice, treatment with combined inhibitors for MEK and STAT3 resulted in the enhanced suppression of tumor formation compared to either agent alone. Histological analysis of combined MEK/STAT3 significantly inhibited tumor size, maintained a higher percentage of pancreatic integrity, displaying increased percentage of normal acini, reduced CK-19 staining, reduced collagen deposition and minimal alcian blue stain. Analysis of the tumor immune infiltrates revealed a significant reduction in the immunosuppressive/tumor promoting myeloid derived suppressor cell (MDSC) population (CD45+CD11b+Ly6g+Ly6c+) and regulatory T cell population (CD45+CD3e+CD4+CD25+FoxP3+) in the pancreas of mice treated with combined MEK/STAT3 inhibition compared to control mice or mice treated with single agents. Alternately, combined MEK/STAT3 inhibition promoted an increased neutrophil population (7AAD-CD11b+Ly6c+Ly6g-), but a decreased inflammatory M1 macrophage population (CD45+F4/80+CD80+CD86+). Conclusions: Combined MEK/STAT3 inhibition downregulates the tumor promoting immune infiltrates resulting in dramatically reduced tumor burden and enhanced normal pancreatic tissue in a highly aggressive mouse model of pancreatic cancer. Citation Format: Casey Roberts, Michael N. VanSaun, Purushottam Lamichhane, Fanuel Messaggio, Krisztina Kovacs, Supriya Srinivasan, Xizi Dai, Jennifer Barretta, Nagaraj Nagathihalli, Nipun B. Merchant.{Authors}. Targeting the immune-microenvironment with combined inhibition of MEK and STAT3 in a mouse model of pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr B75.

A role of lamina propria ILC1 in the impaired host antibacterial resistance of chronic alcohol consuming mice

Abstract Bacterial translocation and subsequent sepsis influenced by ILC1 in lamina propria were studied in chronic alcohol consuming mice (CAC mice) exposed to Enterococcus faecalis oral infection. CAC mice were prepared by an oral gavage of 0.6 ml/day of 20% EtOH for 12 weeks. Control mice received water in the same fashion. All mice were decontaminated and orally infected with 106 CFU/mouse of E. faecalis. The numbers of bacteria in mesenteric lymph nodes (MLNs) and liver of these mice were measured by the colony counting method. Intestines were removed from control and CAC mice, and digested with 100 U/ml of collagenase. Mononuclear cells were isolated from the cell suspension by Percoll density gradient centrifugation. Lineage negative (Lin−) and positive (Lin+) cells were purified magnetically from the mononuclear cells, and analyzed for the population of M1 macrophages (Mϕ) and innate lymphoid cells (ILCs) by flow cytometry. In the results, enterococcal translocation and subsequent sepsis were severely shown in CAC mice, as compared with those of control mice. The number of IFNγ-producing ILC1 was not increased in lamina propria of CAC mice orally infected with E. faecalis, while the large number of ILC1 was isolated from lamina propria of control mice infected with the pathogen. Also, an adequate number of M1Mϕ was isolated from lamina propria of control mice orally infected with E. faecalis, whereas M1Mϕ were not isolated significantly from lamina propria of CAC mice infected with the pathogen. Quiescent Mϕ switch to M1Mϕ in response to IFNγ stimulation. These results suggest that lamina propria ILC1 play an important role in the increased susceptibility of CAC mice to E. faecalis translocation and subsequent sepsis.

Cichorium intybus Linn. Extract Prevents Type 2 Diabetes Through Inhibition of NLRP3 Inflammasome Activation.

This study provides the scientific basis for the inhibitory effect of the aerial parts of Cichorium intybus Linn. (C. intybus) on the activation of the NLRP3 inflammasome in vitro and on high-fat diet (HFD)-induced type-2 diabetes (T2D). Lipopolysaccharide (LPS)-primed bone marrow-derived macrophages were used to study the effects methanolic extract of C. intybus leaf (CI) on inflammasome activation. An insulin resistance model (mice fed a HFD) was used to study the in vivo effect of CI on T2D. CI attenuated interleukin-1β (IL-1β) secretion by inhibiting the activation of the NLRP3 inflammasome in mouse bone marrow macrophages. The CI treatment attenuated the intracellular movement of NLRP3 in Triton X-100 insoluble fraction, without affecting the expression of other NLRP3 inflammasome-related proteins. Attenuated IL-1β secretion may improve glucose metabolism in the HFD-fed insulin resistance mouse model. CI also attenuated the infiltration of M1 macrophages and increased the M2 macrophage population in white adipose tissue. Collectively, our data showed that CI inhibits IL-1β secretion through attenuation of NLRP3 inflammasome activation, leading to an antidiabetic effect by improving glucose metabolism and inhibiting metainflammation.

A reappraisal of macrophage polarization in glioblastoma: Histopathological and immunohistochemical findings and review of the literature

The survival rate in glioblastoma multiforme patients has scarcely improved in the last decades; however, many new therapeutic strategies have been theorized or developed for these neoplasias. Recently, the inverse correlation observed between patient prognosis and tumor-associated macrophages (TAMs) density in solid tumors has encouraged the development of anti-tumor strategies aiming to target TAMs. As expected, TAMs polarization is influenced by both macrophage localization and tumor microenvironment signals, resulting in a more complex scenario than the simple M1/M2 activation status. Macrophage polarization in glioblastoma has not yet been fully elucidated, and most results have been obtained in experimental non-human settings, with some apparent contradiction. The authors performed a histopathological and immunohistochemical study of 37 cases of glioblastoma in order to characterize the M1 and M2 macrophage populations within TAMs. A high prevalence of CD163+ M2-polarized macrophages was detected in this cohort, whereas iNOS+ macrophages were rarely found. The down-regulation of CD68 expression in microglia/macrophage infiltrating glioblastomas is also reported for the first time. Such a finding is associated with a specific location of TAMs within the lesion, as confirmed by the fact that CD68 staining was lower than CD163, mainly in perivascular areas.The authors discuss the recent literature about the global scenario of macrophage plasticity and polarization in glioblastoma, and suggest some pivotal points for therapeutic applications.

BMP-7 Treatment Increases M2 Macrophage Differentiation and Reduces Inflammation and Plaque Formation in Apo E-/- Mice.

Inflammation plays a fundamental role in the inception and development of atherosclerosis (ATH). Mechanisms of inflammation include the infiltration of monocytes into the injured area and subsequent differentiation into either pro-inflammatory M1 macrophages or anti-inflammatory M2 macrophages. We have previously published data suggesting bone morphogenetic protein-7 (BMP-7) enhances M2 macrophage differentiation and anti-inflammatory cytokine secretion in vitro. In this regard, we hypothesized BMP-7 would inhibit plaque formation in an animal model of ATH through monocytic plasticity mediation. ATH was generated in male and female Apo E-/- mice via partial left carotid artery (PLCA) ligation and mice were divided into 3 groups: Sham, PLCA, and PLCA+BMP-7 (200ug/kg; i.v.). Our data suggest that BMP-7 inhibits plaque formation and increases arterial systolic velocity. Furthermore, we report inhibition of monocyte infiltration and a decrease in associated pro-inflammatory cytokines (MCP-1, TNF-α, and IL-6) in the PLCA+BMP-7 mice. In contrast, our data suggest a significant (p<0.05) increase in M2 macrophage populations with consequential enhanced anti-inflammatory cytokine (IL-1RA, IL-10, and Arginase 1) expression following BMP-7 treatment. We have also observed that mechanisms promoting monocyte into M2 macrophage differentiation by BMP-7 involve the upregulation and activation of the BMP-7 receptor (BMP-7RII). In conclusion, we report that BMP-7 has the potential to mediate cellular plasticity and mitigate the inflammatory immune response, which results in decreased plaque formation and improved blood velocity.

Synthesis and evaluation of multivalent M2pep peptides for targeting alternatively activated M2 macrophages

The tumor microenvironment in the majority of cancers is known to favor polarization of tumor-associated macrophages (TAMs) to alternatively activated M2 phenotype, promoting disease progression and reducing patient survival. Effective therapy targeting this M2 macrophage population is thus a promising adjuvant to approved cancer therapies. One of the challenges in targeting M2-like TAMs is a lack of high affinity targeting ligand with good selectivity over anti-tumor M1-like TAMs. We have previously identified an M2 macrophage-targeting peptide (M2pep) that binds preferentially to murine M2 macrophages and M2-like TAMs. A fusion peptide of M2pep with pro-apoptotic peptide KLA (M2pepKLA) was further used to reduce TAM population in vivo but high concentrations and frequent dosing were required due to low binding affinity of M2pep for M2 macrophage. The goal of this study was to develop more potent TAM depletion constructs by increasing the valency of both the M2pep targeting and KLA drug domains. Divalent and tetravalent displays of M2pep ([M2pep]2–Biotin and [M2pep]4–Biotin) were synthesized and evaluated for improvement in binding avidity to the murine macrophages. High avidity and selective binding of [M2pep]2–Biotin to M2 macrophages were achieved with at least 10-fold lower concentration than required for monovalent M2pep activity. Increasing M2pep valency to four, however, resulted in a reduction in both binding activity and selectivity. Surprisingly, both divalent and tetravalent M2pep, without conjugation of any cytotoxic drug cargo, exhibited M2 macrophage-selective toxicity not observed in monovalent M2pep treatment. We next synthesized divalent M2pep with monovalent and divalent KLA ([M2pep]2–[KLA] and [M2pep]2–[KLA]2) to evaluate its enhanced potency compared to M2pepKLA. While both constructs were significantly more toxic than M2pepKLA to primary, bone marrow-derived M2 macrophage, desired selectivity was retained only with [M2pep]2–[KLA]. Finally, we evaluated all multivalent M2pep and M2pepKLA analogs using a syngeneic CT-26 tumor cell suspension. In this setting, [M2pep]4–Biotin and [M2pep]2–[KLA]2 exhibited selective toxicity to both M2-like TAMs and malignant cells but not to M1-like TAMs. Therefore, these constructs are promising anti-cancer constructs with dual-modality mechanisms: malignant cell killing and TAM-based immunomodulation.

The re-polarisation of M2 and M1 macrophages and its role on cancer outcomes

The anti-tumour and pro-tumour roles of Th1/Th2 immune cells and M1/M2 macrophages have been documented by numerous experimental studies. However, it is still unknown how these immune cells interact with each other to control tumour dynamics. Here, we use a mathematical model for the interactions between mouse melanoma cells, Th2/Th1 cells and M2/M1 macrophages, to investigate the unknown role of the re-polarisation between M1 and M2 macrophages on tumour growth. The results show that tumour growth is associated with a type-II immune response described by large numbers of Th2 and M2 cells. Moreover, we show that (i) the ratio k of the transition rates k12 (for the re-polarisation M1→M2) and k21 (for the re-polarisation M2→M1) is important in reducing tumour population, and (ii) the particular values of these transition rates control the delay in tumour growth and the final tumour size. We also perform a sensitivity analysis to investigate the effect of various model parameters on changes in the tumour cell population, and confirm that the ratio k alone and the ratio of M2 and M1 macrophage populations at earlier times (e.g., day 7) cannot always predict the final tumour size.

Characterization of M1/M2 Tumour-Associated Macrophages (TAMs) and Th1/Th2 Cytokine Profiles in Patients with NSCLC

Lung cancer is one of the most commonly reported cancers, and is known to be associated with a poor prognosis. The function of tumour-associated macrophages (TAMs) in lung cancer patients is multifaceted and the literature shows conflicting roles. (I) To analyze the Th1 and Th2 cytokine levels that contribute to the differentiation of M1 and M2 macrophage populations in the serum of patients with NSCLC versus non-cancer controls; and (II) To characterize the M1 and M2 macrophage populations within TAMs in different subtypes of NSCLC compared to non-tumour tissue. The Th1 and Th2 cytokine levels were analyzed in serum using the Bio-Plex assay. In addition, TAMs subsets from non-tumour and tumour tissues were analyzed using immunohistochemistry (IHC). The level of IL-1β, IL-4, IL-6 and IL-8 was found to be increased in the serum of patients with large cell carcinoma but not in other NSCLC subtypes compared to non-cancer controls. In addition, the expression of CD68 and M2 marker CD163 was found to be increased (P ≤ 0.0001) in all NSCLC subtypes compared to non-tumour tissues. In contrast, the expression of iNOS (M1 marker) was decreased in the tumour tissue of patients with adenocarcinoma (P ≤ 0.01) and squamous carcinoma (P ≤ 0.05) but not in large cell carcinoma compared to non-tumour tissue. The results of this study indicate that NSCLC might have the ability to alter phenotype within the lung tumour areas in the local environment (TAMs) but not in the bloodstream in the systemic environment (serum) except for large cell carcinoma.

Abstract 2348: Expression of CECR1 by activated M2-type macrophages in glioma

Abstract Objectives Glial neoplasms harbor macrophages which are mainly of the M2 type and secrete Th2 cytokines. These M2 macrophages are not only involved in immune suppression but also serve functions in angiogenesis. Recent studies have shown that the adenosine deaminase CECR1 is involved in the maintenance of vascular homeostasis and M2-macrophage differentiation. Here we aim to identify the expressional levels of CECR1 in M1 and M2 macrophage populations in gliomas and investigate the role of these cells in glioma development. Methods To identify the relation between CECR1 expression and the macrophage subtypes, tissue samples of human glioma and normal brain controls were used for Real Time PCR and immunostaining. For in vitro functional studies, we generated M1 and M2-like macrophages by differentiating CD14+ monocytes isolated from human peripheral blood in GM-CSF and M-CSF medium. With and without stimulation of GBM conditioned medium, CECR1 level was measured by RT-PCR and western blot. CECR1 protein was added to the macrophage culture medium and the expression level of the M1 marker CD80 and the M2 marker CD163 were measured by flow cytometry and confocal microscopy. Results The macrophages were predominantly present in perivascular spaces, but also in the tumor tissue and neuropil of the normal brain. At the mRNA level the expression of CECR1 was positively correlated with the M2 markers CD204 and IL-10. Immunohistochemical investigation revealed that CECR1 is localized in microglia and macrophages in low-grade gliomas and control brain and it overlaps and co-localizes with the M2-markers CD204 and CD163, but not the M1-marker CD80. In vitro experiments showed that CECR1 expression is higher in the M-CSF-induced M2-macrophages than in their GM-CSF-induced M1 counterparts. Under the stimulation of GBM conditioned medium, increased CECR1 is detected in both M1 and M2-like macrophages. In addition, exogenous CECR1 greatly affects both M-CSF and GM-CSF induced macrophages’ response by up-regulating CD163 in M1-like macrophages and increasing CD163 positive cells in M2-like macrophages. As a result, CECR1 skews macrophage differentiation towards the M2 phenotype. Conclusion This study is the first to show that the M2-macrophage, not the M1-macrophage in glioma, is the main source of CECR1. In addition, CECR1 is shown to be a potent regulator of M2-macrophages. High levels of CECR1 is able to skew GM-CSF driven M1 macrophage differentiation towards an M2 macrophage phenotype. We propose that CECR1 expression by macrophages promotes M2-macrophage differentiation under the influence of an adenosine-rich glioma microenvironment to support tumor growth and tumor angiogenesis. The findings may well direct the search for new anti-angiogenic target molecules for the treatment of glioma. Citation Format: Changbin Zhu, Marcel M. van der Weiden, Adrea Scchetti, Thierry P.P. van den Bosch, Ihsan Chrifi, Maarten M. Brandt, Dana A.M. Mustafa, Caroline Cheng, Johan M. Kros. Expression of CECR1 by activated M2-type macrophages in glioma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2348. doi:10.1158/1538-7445.AM2015-2348

TNF-a mediated inflammatory macrophage polarization contributes to the pathogenesis of steroid-induced osteonecrosis in mice.

The phenotypic polarization of macrophages are involved in steroid-induced osteonecrosis (ON). This study tried to investigate the detrimental and beneficial roles of M1/M2 macrophages associated with TNF-a in ON. Mice ON model was induced by the injection of methylprednisolone. After that, flow cytometry technique, immunohistochemistry, immunofluorescence, ELISA, and RT-PCR methods were used to investigate the expression pattern of macrophages and the expression of inflammatory cytokines. During the progression of ON, massive chronic inflammatory cells infiltrated into the necrotic zone, represented by the infiltration of macrophages. In the early stage of ON, there was high TNF-a activity; and a large population of M1 macrophages infiltrated into the necrotic zone. On the contrary, the expression of TNF-a gradually decreased; simultaneously, a larger M2 cell population presented in the necrotic zone in the late stage of ON. The increased M2 macrophages could be beneficial for resolving inflammation and promoting tissue repair, confirmed by the histologic findings of appositional new bone formation around the necrotic bone. Thus, it showed that TNF-a-mediated alteration of M1/M2 macrophage polarization contributed to the pathogenesis of steroid-induced osteonecrosis. M1-polarized macrophages appeared to be disruptive in the early stage of ON, while M2-polarized macrophages played an important role in the late stage during the pathogenesis of ON.

M1-/M2-macrophages contribute to the development of GST-P-positive preneoplastic lesions in chemically-induced rat cirrhosis

Glutathione S-transferase placental form (GST-P) expression in hepatocyte foci is regarded as a preneoplastic change in rats. We aimed to reveal the contribution of polarized macrophages in development of GST-P-positive pseudolobules (PLs) in chemically-induced rat cirrhosis. F344 rats were injected with thioacetamide (100mg/kg BW, twice a week, intraperitoneally). Macrophage immunophenotypes and expression of M1-/M2-related factors were analyzed by immunohistochemistry, real-time RT-PCR and laser microdissection. GST-P-positive foci/clusters were clearly observed at post-first injection week 15. GST-P-positive PLs were distinguishable at weeks 20-32. Microarray analysis revealed upregulation of preneoplastic genes in GST-P-positive PLs at week 32. M1 (CD68+, Iba1+)-and M2 (CD163+, CD204+, Gal-3+)-macrophages were greater in number in the GST-P-positive PLs, whereas MHC class II-positive (M1) macrophage number was fewer in the GST-P-positive PLs. Expression of both M1 (IFN-γ, IL-1β, TNF-α, Iba1)- and M2 (IL-4, TGF-β1, IL-10)-related factors were higher in GST-P-positive PLs. Our results showed that both M1- and M2-macrophage populations contribute to the development of hepatic preneoplastic lesions. MHC class II-positive macrophages may be related to anti-tumor progression, since their kinetics showed reverse pattern to other macrophage phenotypes.

The recruitment of macrophages to the pre-ovulatory ovary (CAM4P.146)

Abstract Ovulation requires the chemotaxis of leukocytes from the spleen to the ovary. Hormonal signals that are released during the peri-ovulatory period initiate the release of specific leukocyte populations that infiltrate the ovary and facilitate the release of oocytes. We identified a specific population of monocyte that infiltrates the ovary during a specific period of time, as well as M1 and M2-like macrophage populations. Before ovulation occurs, a Ly-6hi monocyte is likely promoting inflammation and follicular rupture. The Ly-6c high monocyte infiltration will start to diminish as they differentiate into M1 macrophages to further promote inflammation. These studies will utilize a super-ovulation protocol to initiate ovulation in immature balb/c mice. Ovaries will be collected at several times post hCG injection and analyzed by multi-color flow cytometry. Oviducts will be collected from mice 20 hours after hCG administration and cumulous oocyte complexes will be collected, counted and averaged to determine ovulation rate. Studies should establish that without these subsets of monocytes and macrophages, the rate of ovulation is reduced. In this regard, in the ovary, the Ly-6c high monocytes may function as a source of M1 macrophages to initiate the inflammatory process.

Krüppel-like factor 4 is a transcriptional regulator of M1/M2 macrophage polarization in alcoholic liver disease.

Macrophages play an important role in inflammation and liver injury. In ALD, activated macrophages, including M1 (proinflammatory) and M2 (anti-inflammatory) macrophages, are present in the liver. As KLF4 has been described as a regulator of macrophage polarization, we investigated its role in ALD. Chronic alcohol feeding in C57Bl/6 mice led to increased expression of M1 (TNF-α, MCP1, and IL-1β) and M2 (Arg1, Mrc1, and IL-10) genes and the frequency of CD206+CD163+ M2 macrophages in the liver. KLF4 mRNA and protein levels were increased in the livers of EtFed compared with PF mice. In macrophages, in vivo and in vitro, EtOH increased KLF4 levels, transcriptional activity, and expression of M1 and M2 genes. KLF4 knockdown and overexpression experiments demonstrated alcohol-dependent and -independent functions of KLF4 in regulating M1 and M2 markers. KLF4 siRNA treatment, alone and in synergy with alcohol, increased the levels of M1 markers. In contrast, KLF4 overexpression increased the levels of M2 and decreased M1 markers, and this was enhanced further by alcohol. KLF4 was regulated by alcohol and its metabolites. KLF4 mRNA and activity were increased in the presence of 4-MP, an inhibitor of ADH, and CYP2E1. However, inhibition of acetaldehyde breakdown attenuated KLF4 induction and promoted M1 polarization. We conclude that KLF4 regulates M1 and M2 markers in ALD. EtOH promotes KLF4 and M2 phenotype, whereas acetaldehyde attenuates KLF4 and promotes M1 macrophage, which may explain the increased presence of M1 and M2 macrophage populations in ALD.