M2 Gene Expression Research Articles

Microparticles Incorporating Dual Apoptotic Factors to Inhibit Inflammatory Effects in Macrophages

New approaches to treat autoimmune diseases are needed, and we can be inspired by mechanisms in immune tolerance to guide the design of these approaches. Efferocytosis, the process of phagocyte-mediated apoptotic cell (AC) disposal, represents a potent tolerogenic mechanism that we could draw inspiration from to restore immune tolerance to specific autoantigens. ACs engage multiple avenues of the immune response to redirect aberrant immune responses. Two such avenues are: phosphatidylserine on the outer leaflet of the cell and engaging the aryl hydrocarbon receptor (AhR) pathway. We incorporated these two avenues into one acetalated dextran (Ace-DEX) microparticle (MP) for evaluation in vitro. First phosphatidylserine (PS) was incorporated into Ace-DEX MPs and evaluated for cellular association and mediators of cell tolerance including IL-10 production and M2 associated gene expression when particles were cultured with peritoneal macrophages (PMacs). Further PS Ace-DEX MPs were evaluated as an agent to suppress LPS stimulated PMacs. Then, AhR agonist 2-(1′H-indole-3′-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE) was incorporated into Ace-DEX MPs and expression of M2 and IL-10 genes was evaluated in PMacs. Further the ITE and PS Ace-DEX MPs (PS/ITE MPs) were evaluated for suppression of T cell priming and Th1 polarization. Our results indicate that the PS/ITE-MPs stimulated anti-inflammatory cytokine expression and suppressed inflammation following LPS stimulation of PMacs. Moreover, PS/ITE MPs induced the anti-inflammatory enzyme IDO1 and suppressed macrophage-mediated T cell priming and Th1 polarization. These findings suggest that PS and ITE-loaded Ace-DEX MPs could be a promising therapeutic tool for suppressing inflammation.

Unmasking the dynamics of Mycoplasma gallisepticum: deciphering HD11 macrophage polarization for innovative infection control strategies

Mycoplasma gallisepticum (MG) is a highly contagious avian respiratory pathogen characterized by rapid spread, widespread distribution, and long-term persistence of infection. Previous studies have shown that chicken macrophage HD11 cells play a critical role in the replication and immunomodulation of MG. Macrophages are multifunctional immunomodulatory cells that polarize into different functions and morphologies in response to exogenous stimuli. However, the effect of MG infection on HD11 polarization is not well understood. In this study, we observed a time-dependent increase in both the expression of the MG-related virulence protein pMGA1.2 and the copy number of MG upon MG infection. Polarization studies revealed an upregulation of M1-type marker genes in MG-infected HD11 cells, suggesting that MG mainly induces HD11 macrophages towards M1-type polarization. Furthermore, MG activated the inflammatory vesicle NLRP3 signaling pathway, and NLRP3 inhibitors affected the expression of M1 and M2 marker genes, indicating the crucial regulatory role of the NLRP3 signaling pathway in MG-induced polarization of HD11 macrophages. Our findings reveal a novel mechanism of MG infection, namely the polarization of MG-infected HD11 macrophages. This discovery suggests that altering the macrophage phenotype to inhibit MG infection may be an effective control strategy. These findings provide new perspectives on the pathogenic mechanism and control measures of MG.

Directionally non-rotating electric field therapy delivered through implanted electrodes as a glioblastoma treatment platform: A proof-of-principle study.

While directionally rotating tumor-treating fields (TTF) therapy has garnered considerable clinical interest in recent years, there has been comparatively less focus on directionally non-rotating electric field therapy (dnEFT). We explored dnEFT generated through customized electrodes as a glioblastoma therapy in in vitro and in vivo preclinical models. The effects of dnEFT on tumor apoptosis and microglia/macrophages in the tumor microenvironment were tested using flow-cytometric and qPCR assays. In vitro, dnEFT generated using a clinical-grade spinal cord stimulator showed antineoplastic activity against independent glioblastoma cell lines. In support of the results obtained using the clinical-grade electrode, dnEFT delivered through a customized, 2-electrode array induced glioblastoma apoptosis. To characterize this effect in vivo, a custom-designed 4-electrode array was fabricated such that tumor cells can be implanted into murine cerebrum through a center channel equidistant from the electrodes. After implantation with this array and luciferase-expressing murine GL261 glioblastoma cells, mice were randomized to dnEFT or placebo. Relative to placebo-treated mice, dnEFT reduced tumor growth (measured by bioluminescence) and prolonged survival (median survival gain of 6.5 days). Analysis of brain sections following dnEFT showed a notable increase in the accumulation of peritumoral macrophage/microglia with increased expression of M1 genes (IFNγ, TNFα, and IL-6) and decreased expression of M2 genes (CD206, Arg, and IL-10) relative to placebo-treated tumors. Our results suggest therapeutic potential in glioblastoma for dnEFT delivered through implanted electrodes, supporting the development of a proof-of-principle clinical trial using commercially available deep brain stimulator electrodes.

Single-cell characterization of macrophages in uveal melanoma uncovers transcriptionally heterogeneous subsets conferring poor prognosis and aggressive behavior

Uveal melanoma (UM) is the most frequent primary intraocular malignancy with high metastatic potential and poor prognosis. Macrophages represent one of the most abundant infiltrating immune cells with diverse functions in cancers. However, the cellular heterogeneity and functional diversity of macrophages in UM remain largely unexplored. In this study, we analyzed 63,264 single-cell transcriptomes from 11 UM patients and identified four transcriptionally distinct macrophage subsets (termed MΦ-C1 to MΦ-C4). Among them, we found that MΦ-C4 exhibited relatively low expression of both M1 and M2 signature genes, loss of inflammatory pathways and antigen presentation, instead demonstrating enhanced signaling for proliferation, mitochondrial functions and metabolism. We quantified the infiltration abundance of MΦ-C4 from single-cell and bulk transcriptomes across five cohorts and found that increased MΦ-C4 infiltration was relevant to aggressive behaviors and may serve as an independent prognostic indicator for poor outcomes. We propose a novel subtyping scheme based on macrophages by integrating the transcriptional signatures of MΦ-C4 and machine learning to stratify patients into MΦ-C4-enriched or MΦ-C4-depleted subtypes. These two subtypes showed significantly different clinical outcomes and were validated through bulk RNA sequencing and immunofluorescence assays in both public multicenter cohorts and our in-house cohort. Following further translational investigation, our findings highlight a potential therapeutic strategy of targeting macrophage subsets to control metastatic disease and consistently improve the outcome of patients with UM.

Heparin Affinity-Based IL-4 Delivery to Modulate Macrophage Phenotype and Endothelial Cell Activity In Vitro.

Macrophages play a pivotal role in wound healing and tissue regeneration, as they are rapidly recruited to the site of injury or implanted foreign material. Depending on their interaction with the material, macrophages can develop different phenotypes, with the M1 pro-inflammatory and M2 pro-regenerative phenotypes being highly involved in tissue regeneration. M2 macrophages mitigate inflammation and promote tissue regeneration and extracellular matrix remodeling. In this study, we engineered a gelatin-heparin-methacrylate (GelMA-HepMA) hydrogel that gradually releases interleukin-4 (IL-4), a cytokine that modulates macrophages to adopt the M2 phenotype. Methacrylation of heparin improved the retention of both heparin and IL-4 within the hydrogel. The GelMA-HepMA hydrogel and IL-4 synergistically downregulated M1 gene expression and upregulated M2 gene expression in macrophages within 48 h of in vitro cell culture. However, the M2-like macrophage phenotype induced by the GelMA-HepMA-IL-4 hydrogel did not necessarily further improve endothelial cell proliferation and migration in vitro.

756 Effects of Bioengineered Allogeneic Cellular Constructs on Macrophage Phenotype in the Context of Burns

Abstract Introduction The persistent inflammatory and stress responses generate in burn patients have a strong effect on the immune system. Macrophages that are activated in response to burn injuries polarize towards an immunosuppressive phenotype commonly known as M2b. Furthermore, due to sepsis, the chronic exposure to the pro-inflammatory environment also leads to an exhausted macrophage phenotype. Together these effects compromise macrophage behavior that influences the healing outcome. In clinical trials, bioengineered allogeneic cellular constructs (BACCs) have shown potential in promoting healing in patients with deep partial thickness burns. However, the mechanism(s) behind these successful effects are not totally understood yet. For this reason, the aim of this study is to investigate how the crosstalk with the BACC influence different burn-related macrophage phenotype. Methods Primary human macrophages (N=4 healthy donors) were co-cultured with the BACC or collagen controls. Macrophages were isolated over 6 days of co-culture and their RNA was extracted for gene expression analysis, and conditioned media was collected to analyze the secretion of proteins. To assess phenotype changes, we performed gene set analysis using single sample gene set enrichment. To investigate changes in the phenotype of macrophages relevant to burn wounds macrophages were polarized to the immune suppressive M2b phenotype or LPS-exhausted phenotypes before co-culturing them with the BACC. Undifferentiated macrophages (M0) were used as controls. Results Gene expression analysis revealed a shift in undifferentiated macrophage phenotype towards an M2 phenotype by day 3 and 6 in co-culture as demonstrated by an increase in the expression of multiple M2 genes, including MRC1 and CCL18. Secretory analysis revealed a decrease in the secretory of inflammatory proteins, like interleukin 6. Conclusions Collectively these results demonstrate that BACCs promote the transition of macrophage phenotype towards a pro-reparative M2 phenotype which may contribute to the construct’s pro-healing capabilities. Applicability of Research to Practice This study provides insight into the mechanism by which BACCs promote healing in the burn wound environment.

Rabies virus uniquely reprograms the transcriptome of human monocyte-derived macrophages.

Macrophages are amongst the first immune cells that encounter rabies virus (RABV) at virus entry sites. Activation of macrophages is essential for the onset of a potent immune response, but insights into the effects of RABV on macrophage activation are scarce. In this study we performed high-throughput sequencing on RNA extracted from macrophages that were exposed to RABV for 48 hours, and compared their transcriptional profiles to that of non-polarized macrophages (M0), and macrophages polarized towards the canonical M1, M2a and M2c phenotypes. Our analysis revealed that RABV-stimulated macrophages show high expression of several M1, M2a and M2c signature genes. Apart from their partial resemblance to these phenotypes, unbiased clustering analysis revealed that RABV induces a unique and distinct polarization program. Closer examination revealed that RABV induced multiple pathways related to the interferon- and antiviral response, which were not induced under other classical polarization strategies. Surprisingly, our data show that RABV induces an activated rather than a fully suppressed macrophage phenotype, triggering virus-induced activation and polarization. This includes multiple genes with known antiviral (e.g. APOBEC3A, IFIT/OAS/TRIM genes), which may play a role in anti-RABV immunity.

Myeloid Phenotypes in Tracheostomy-Associated Granulation Tissue.

Tracheostomy-associated granulation tissue is a common, recurrent problem occurring secondary to chronic mucosal irritation. Although granulation tissue is composed of predominantly innate immune cells, the phenotype of monocytes and macrophages in tracheostomy-associated granulation tissue is unknown. This study aims to define the myeloid cell population in granulation tissue secondary to tracheostomy. Granulation tissue biopsies were obtained from 8 patients with tracheostomy secondary to laryngotracheal stenosis. Cell type analysis was performed by flow cytometry and gene expression was measured by quantitative real-time polymerase chain reaction. These methods and immunohistochemistry were used to define the monocyte/macrophage population in granulation tissue and were compared to tracheal autopsy control specimens. Flow cytometry demonstrated macrophages (CD45+CD11b+) and monocytes (CD45+FSClow SSClow ) represent 23.2 ± 6% of the granulation tissue cell population. The M2 phenotype (CD206) is present in 77 ± 11% of the macrophage population and increased compared to the M1 phenotype (p=0.012). Classical monocytes (CD45+CD14high CD16low ) were increased in granulation tissue compared to controls (61.2 ± 7% and 30 ± 8.5%, p=0.038). Eighty-five percent of macrophages expressed pro-inflammatory S100A8/A9 and 36 ± 4% of macrophages co-localized CD169, associated with tissue-resident macrophages. M2 gene expression (Arg1/CD206) was increased in granulation tissue (3.7 ± 0.4, p=0.035 and 3.5 ± 0.5, p=0.047) whereas M1 gene expression (CD80/CD86) was similar to controls (p=0.64, p=0.3). Immunohistochemistry of granulation tissue demonstrated increased cells co-localizing CD11b and CD206. M2 macrophages are the dominant macrophage phenotype in tracheostomy-associated granulation tissue. The role of this cell type in promoting ongoing inflammation warrants future investigation to identify potential treatments for granulation tissue secondary to tracheostomy. 3 Laryngoscope, 133:2346-2356, 2023.

Abstract 10174: Intravenous Muse Cell Transplantation Enhances Angiogenesis and Reduces Muscle Fibrosis in a Mouse Hindlimb Ischemia Model

Introduction: Multilineage-differentiating stress enduring (Muse) cells, a unique subpopulation of mesenchymal stem cells (MSCs), have been proposed as a promising cell source for regenerative medicine. However, the therapeutic potential of the Muse cells in ischemic limb disease remains unclear. In this study, we aimed to investigate therapeutic potential of Muse cell transplantation for the treatment of severe limb ischemia. Methods and Results: Muse cells isolated from human MSCs, non-Muse MSCs, or mouse bone marrow-derived mononuclear cells (BM-MNCs) were administered intravenously or intramuscularly to a mouse model of hindlimb ischemia (HLI). When the same number of cells were administered, blood flow recovery of the ischemic limb was significantly enhanced in the mice administered with Muse cells over 2 weeks compared to the others. Intravenous Muse cell administration resulted in comparable blood flow recovery to intramuscular Muse cell administration. Immunohistochemistry analysis revealed that intravenous Muse cell administration increased microvascular density and ameliorated interstitial fibrosis in the ischemic limb. We detected mCherry-labeled Muse cells expressing CD31 and human genomic DNA in the ischemic limb 1 week after the intravenous Muse cell administration. Muse cells produced higher VEGF compared to non-Muse MSCs, and Muse cell administration increased tissue VEGF level and expression of angiogenesis-related genes in the ischemic limb. Moreover, we found that Muse cell administration increased M2 macrophage and gene expression ratio of anti-inflammatory genes to proinflammatory gene, iNOS, in the ischemic limb. Lastly, we administered Muse cell intravenously at day 1 and 3, or high-dose BM-MNCs intramuscularly at day1 after the HLI surgery and found that repetitive Muse cell administration resulted in significantly enhanced blood flow recovery in the ischemic limb compared to high-dose BM-MNCs administration. Conclusions: Using an animal model, we demonstrated that intravenous Muse cell transplantation exerts robust therapeutic effects on limb ischemia injury. Therefore, Muse cell-based therapy may provide a novel therapeutic approach for patients with severe limb ischemia.

Evaluation of Forkhead BOX M1 (FOXM1) gene expression in colorectal cancer.

Forkhead Box M1 (FOXM1)-a key cell cycle regulator is a member of the Forkhead transcription factor family. It plays a key role in embryogenesis and cell proliferation and has been strongly linked to various solid tumors. We sought to understand the regulation of FOXM1 in colorectal cancer (CRC), as well as if and to what extent other clinicopathological characteristics are associated with FOXM1. The investigation comprised 98 CRC samples and normal tissues (controls). All colon cancer patients had a colonoscopy and targeted biopsy. All rectal cancer patients had a CT and MRI. Real-time PCR, Immunohistochemistry, and Western blotting were used to evaluate FOXM1 expression, and the findings were analyzed using SPSS (v.26). FOXM1 mRNA and protein expression were substantially upregulated in tumor tissues, with the majority of these proteins localized in nucleo-cytoplasm. Elevated protein levels of FOXM1 were strongly correlated with lower education level, larger tumor size, lymph node status, lymphovascular invasion (LVI), perineural invasion (PNI), lymph node metastasis (LNM), tumor invasion depth (subserosal and serosal invasion), late stage (III and IV), localization (nucleo-cytoplasmic), intensity (strong) and recurrence. Based on survival analysis, FOXM1 overexpression and nucleo-cytoplasmic localization were associated with shorter disease-free survival while stage and PNI were linked to poorer overall and disease-free survival. According to the results of the Cox regression analysis, stage and PNI were significant predictors of prognosis in CRC patients. FOXM1 expression was elevated in CRC and was linked to reduced disease-free survival. These findings support prior reports and hence FOXM1 can be an important prognostic marker for CRC and a promising therapeutic target. Additionally, we found a link between poor disease-free survival and FOXM1's nucleo-cytoplasmic localization. However, since the sample size of this study was small, further research is needed to validate our findings.

Abstract 1059: Paricalcitol, hydroxychloroquine and gemcitabine promote antitumor effects and modulate immune profile in pancreatic ductal adenocarcinoma

Abstract Background Pancreatic ductal adenocarcinoma (PDAC) has low (<5%) five-year survival. We hypothesized combined Vitamin D3 (Paricalcitol; Par) and hydroxychloroquine (HCQ) impacts stroma, anti-tumor immunity, and autophagy, thereby sensitizing PDAC to gemcitabine (Gem). Methods For subcutaneous efficacy, 5-6-week-old female nude mice (n=5/group) were injected with KPC-Luc cells and randomized to: 1) vehicle (PBS), 2) Gem plus HCQ, 3) Gem plus Par, and 4) Gem, HCQ, and Par once tumors were palpable. Gem and Par (twice weekly) were administered via intraperitoneal injection. HCQ was given orally (daily). For orthotropic efficacy and single cell analysis, female C57BL/6J mice (n=6/group) were injected with 2×105 KPC-Luc cells into the pancreas tail. On day 5, animals were randomized to 1) PBS and 2) combined Gem/HCQ/Par as above. Tumors were analyzed via bioluminescent imaging. After 15 days, animals were euthanized, tumors were weighed and dissociated into single cells. High viability samples were used to generate scRNA-Seq libraries using 10X genomics droplet approach. Results Gem, HCQ, and Par significantly (p<0.001) reduced tumor growth/weight versus controls in both models. Single cell transcriptome profiles of 11,188 cells (controls) and 7,624 cells (Gem/HCQ/Par) were obtained. Split UMAP plots of samples were generated with cell clusters annotated from established gene markers. Eight clusters expressing PDAC-associated genes were designated as PDAC cells. The tumor microenvironment (TME) showed dramatic post-treatment changes with significant decreases in PDAC cells (p=0.007) and increases in B cells, monocytes/macrophages (p=0.0006, 0.03 respectively), and increased T/NK cells in treated versus control mice. Supervised analysis among T cells showed more activation (Cd44, Tnfrsf4/9) and exhaustion (Ctla4, Lag3, Pdcd1) associated gene expression post-treatment, indicating robust T cell response. ssGSEA analysis showed significant (p<.001) activation of Th1/Th2/Th17 pathways. Monocyte/macrophages clusters analysis revealed greater expression of M1 genes and M2 genes in treated and control samples, respectively. Modified EMT was observed in stromal cells with enhanced MHC1 expression. Conclusion HCQ and Par enhance the efficacy of Gem in two mice PDAC models. Single cell transcriptome analyses revealed the combination treatment promoted immune shifts in the PDAC TME. Effects of Par and HCQ and standard chemotherapy drugs, Gem and nab-paclitaxel are being studied in an ongoing clinical trial (NCT04524702). Citation Format: Purnachandra Nagaraju Ganji, Beena Elizabeth Thomas, Maggie Phillips, Cameron Herting, Amanda Ruggieri, Jessica Millian Keilson, Michael Kim Turgeon, Sisira Saraswatula, William Pilcher, Gregory B. Lesinski, Manoj Bhasin, Bassel F. El-Rayes. Paricalcitol, hydroxychloroquine and gemcitabine promote antitumor effects and modulate immune profile in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1059.

Paternal stress alters synaptic density and expression of GAP-43, GRIN1, M1 and SYP genes in the hippocampus and cortex of offspring of stress-induced male rats

Adverse experiences during pregnancy have a negative impact on the neuronal structure and behavior of offspring, but the effects of a father's life events on the outcome of progeny are scarce. The present study is intended to investigate whether paternal stress affects the offspring brain structure, especially those regions concerned with learning and formation of memory, namely the hippocampus (HC) and prefrontal cortex (PFC), and also the expression of certain genes linked to learning and memory in the offspring. Induced stress to male rats by five stressors, one per day followed by allowing them to mate with the normal, unstressed female. Synaptophysin immunoreactivity was assessed in the tissue sections of the HC and PFC as well as expression of genes concerned with learning and memory was evaluated by RT-PCR in the progeny of stress-received males. The progeny of stressed rats had reduced antisynaptophysin immunoreactivity in the HC and PFC. The synaptic density in HC was less in the A-S (Offspring of male rats who received stress during adulthood) and PA-S (offspring of male rats who received stress during both adolescence and adulthood) than in P-S (offspring of male rats who received stress during adolescence) and C-C (offspring of control) groups. Similar results were observed even in the PFC. The results of post hoc tests proved that the HC and PFC of the progeny of stress-exposed rats exhibited considerably less synaptic density than control (P<0.05), and the levels of expression of GAP-43, GRIN1, M1, and SYP genes in HC and PFC were down-regulated. This study concludes that paternal adverse experiences can affect the offspring's synaptic plasticity and also the genes, which can regulate learning and formation of memory.

Corilagin Ameliorates Con A-Induced Hepatic Injury by Restricting M1 Macrophage Polarization.

Immune-mediated hepatic injury plays a key role in the initiation and pathogenesis of diverse liver diseases. However, treatment choice for immune-mediated hepatic injury remains limited. Corilagin, a natural ellagitannin extracted from various traditional Chinese medicines, has been demonstrated to exhibit multiple pharmacological activities, such as anti-inflammatory, anti-tumor, and hepatoprotective properties. The present study aimed to investigate the effects of corilagin on immune-mediated hepatic injury using a murine model of concanavalin A (Con A)-induced hepatitis, which is well-characterized to study acute immune-mediated hepatitis. Herein, mice were administered corilagin (25 mg/kg) intraperitoneally twice at 12 h intervals, and 1 h later, the mice were challenged with Con A (20 mg/kg body weight); serum and liver samples were collected after 12 h. The results showed that corilagin significantly increased the survival of mice and reduced serum alanine transaminase (ALT) and aspartate aminotransferase (AST) levels. In addition, corilagin markedly improved histopathological damage, hepatocyte apoptosis, and oxidative stress in the liver. The activation of M1 macrophages in the hepatic mononuclear cells was also significantly reduced compared with that in the control group. The expression of M1 macrophage-associated proinflammatory cytokines and genes, including interleukin (IL)-6, IL-12, and inducible nitric oxide synthase (iNOS), was also decreased after corilagin treatment. Finally, the results demonstrated that corilagin regulated macrophage polarization by modulating the mitogen-activated protein kinases (MAPK), nuclear factor (NF)-κB, and interferon regulatory factor (IRF) signaling pathways. Thus, the findings indicate that corilagin protects mice from Con A-induced immune-mediated hepatic injury by limiting M1 macrophage activation via the MAPK, NF-κB, and IRF signaling pathways, suggesting corilagin as a possible treatment choice for immune-mediated hepatic injury.

Macrophage Polarization Related to Crystal Phases of Calcium Phosphate Biomaterials.

Calcium phosphate (CaP) materials influence macrophage polarization during bone healing. However, the effect of the crystal phase of CaP materials on the immune response of bone remains unclear. In this study, the effect of the crystal phases of CaP materials on the regulation of macrophage polarization was investigated. Human THP-1 cells and mouse RAW 264 cells were cultured with octacalcium phosphate (OCP) and its hydrolyzed form Ca-deficient hydroxyapatite to assess the expression of pro-inflammatory M1 and anti-inflammatory M2 macrophage-related genes. OCP inhibited the excessive inflammatory response and switched macrophages to the anti-inflammatory M2 phenotype, which promoted the expression of the interleukin 10 (IL10) gene. In contrast, HL stimulated an excessive inflammatory response by promoting the expression of pro-inflammatory M1 macrophage-related genes. To observe changes in the microenvironment induced by OCP and HL, inorganic phosphate (Pi) and calcium ion (Ca2+) concentrations and pH value in the medium were measured. The expression of the pro-inflammatory M1 macrophage-related genes (tumor necrosis factor alpha (TNFα) and interlukin 1beta (IL1β)) was closely related to the increase in ion concentration caused by the increase in the CaP dose. Together, these results suggest that the microenvironment caused by the crystal phase of CaP materials may be involved in the immune-regulation capacity of CaP materials.

Re-visit the Concept of M1 versus M2 Phenotypes of BV2 Microglia and Test Their Effects on Stroke Outcome in Mice

Abstract Backgrounds: Whether there are distinctive macrophage functional phenotypes of M1 versus M2 has been debated. We re-examined them by studying M1/M2 gene and protein expressions in cultured BV2 microglial cells and their effects on stroke outcomes in vivo. Methods: BV2 microglia cells were cultured and polarized with lipopolysaccharide (LPS) and interleukin-4 (IL-4) to produce M (LPS) and M (IL-4) phenotypes, which were originally defined as M1 and M2 phenotypes, respectively. Typical M1 and M2 gene or protein expression patterns were analyzed in M (LPS) and M (IL-4) phenotypes and their distinctive effects on stroke outcomes were compared. Results: M (LPS) and M (IL-4) had distinctive morphologies. M (IL-4) had significantly higher gene expressions of the typical M2 markers and other anti-inflammatory genes, while M (LPS) had higher gene expression of typical M1 markers and other pro-inflammatory genes. Nevertheless, M2 gene expressions were also enhanced in M (LPS), and M1 gene expressions were increased in M (IL-4), although with relatively lower levels. Adoptive transfer of M (IL-4) reduced infarction and improved neurological scores, while M (LPS) macrophages generated the opposite effect. Fluorescence activated cell sorting (FACS) and confocal studies suggest that M (IL-4) inhibited, while M (LPS) promoted the infiltration of monocyte-derived macrophages and iNOS-positive cells. Conclusions: M (LPS) and M (IL-4) from cultured BV2 cells indeed are distinctive functional phenotypes, but it is inaccurate to simply classify them into M1 and M2 phenotypes based on a few typical gene and protein markers.

The microenvironment of DLBCL is characterized by noncanonical macrophages recruited by tumor-derived CCL5

AbstractTissue invasion by tumor cells induces a host inflammatory response that variably impacts tumorigenesis. This has been well documented for tumor-associated macrophages (TAMs) that could play a pro/M2- or an anti/M1-tumoral function. TAMs frequently infiltrate diffuse large B-cell lymphoma (DLBCL), an aggressive neoplasm arising from germinal center–experienced B cells. However, the pathway leading to the presence of TAMs in DLBCL remains unknown, and their impact is unclear. Here, we show that some DLBCL tumor cells expressed the chemokine CCL5, enabling the differential recruitment of blood monocytes through their expression of CCR1 and CCR5. CCL5 expression by DLBCL was not related to molecular subtypes, and healthy tonsillar B cells did not produce this chemokine, implying a posttransformation event. A single-cell analysis revealed that most DLBCL TAMs had a noncanonical gene signature with the concomitant expression of M1 and M2 genes. The presence of noncanonical TAMs may explain the lack of impact of macrophages on DLBCL development reported in some survival studies.

Effects of Macrophage Colony-Stimulating Factor Isoforms on Leukemia Progression in Mouse Acute Myeloid Leukemia Model

Oncogenic events occur by a variety of mechanisms, including point mutations, in-frame deletions or insertions, epigenetic modifications or even surrounding changes. As an important growth factor, M-CSF plays different roles in complicated pathological context and there are complex heterogeneities between M-CSF different splicing isoforms (sM-CSF and mM-CSF). The role of M-CSF and the difference between the two isoforms in hematological malignancies, which are significantly different from solid tumors, is poorly understood. In this study, we studied the different effects of mM-CSF and sM-CSF on both intrinsic factor-leukemia cells and surrounding linker-macrophage in acute myeloid leukemia mice model.The high level expression of M-CSF was found in myeloid leukemia patients from bloodspot database. We established MLL-AF9 induced AML mouse model with high level expression of sM-CSF or mM-CSF. We found mM-CSF could delay the progression of leukemia. Subsequently, we investigated the effect of the two isoforms on both leukemia cells and macrophages. More proliferative cells were detected in leukemia cells with high level M-CSF isoforms. In vitro colony assay experiments showed sM-CSF could promote the self-renew of leukemia cells. Morphological and flow cytometry analysis indicated M-CSF isoforms had different potential in promoting leukemia cell differentiation. Moreover, myeloid differentiation associated transcription factor-Cebpe and Runx1 might account for differentiation promoting effect by mM-CSF. As for macrophages, M-CSF isoforms could increase monocytes and macrophages in leukemic microenvironment by both growth-promoting and recruitment effects. Additionally, mM-CSF promoted the expression of both M1 and M2-associated genes. Meanwhile mM-CSF promoted the specific phagocytosis potential of macrophages to leukemia cells. Furthermore, the gene expression profiles of LAMs (leukemia associated macrophages) revealed mM-CSF did make macrophages a special phenotype with enhanced oxidative phosphorylation, metabolism and phagocytosis pathway. Moreover, phagocytosis of LAMs educated by mM-CSF may be mediated by oxidative phosphorylation.Our results suggested that the diverse effects of M-CSF two isoforms in leukemic microenvironment and mM-CSF delayed the leukemia course by affecting both leukemia cell differentiation and macrophage frequencies along with specific phagocytosis to leukemia cells. The work reveals that both the intrinsic and surrounding factors participate in tumor progression and the complex of carcinogenesis and the interaction between tumor cell itself and microenvironment linkers should be further studied.This work was supported by Grants 81370634 and 81570153 from the National Natural Science Foundation of China (NSFC); program 2016-I2M-2-006 from CAMS Initiative for Innovative Medicine; grant 17JCZDJC35000 from the Tianjin Science and Technology Committee. DisclosuresNo relevant conflicts of interest to declare.

1199-P: The Oncogene MDM2 Induces M1 Macrophage Polarization and Adipose Tissue Inflammation in Obesity

Obesity is characterized by chronic low-grade inflammation that contributes to the pathogenesis of type 2 diabetes. Adipose tissue macrophages predominantly switch to a pro-inflammatory phenotype known as M1 during obesity. The accumulation of pro-inflammatory M macrophages in obese adipose tissue results in the secretion of a cluster of pro-inflammatory cytokines that instigate adipose tissue inflammation and insulin resistance. Here we discovered that expression of oncoprotein murine double minute 2 (MDM2) was elevated in obese white adipose tissues. Also, MDM2 protein expression was drastically induced in macrophages activated with lipopolysaccharide (LPS) and interferon-γ (IFNγ). Mice with myeloid cells-specific MDM2 haploinsufficiency (Mye-MDM2 KO mice) were generated and subjected to high-fat-diet (HFD) feeding. After 16 weeks of HFD feeding, obesity-induced glucose intolerance and insulin resistance were alleviated in Mye-MDM2 KO mice with reference to their wild type (WT) controls. Systemic inflammation induced by LPS injection was also reduced in Mye-MDM2 KO mice. In vitro, expression of M1 genes induced by LPS and IFNγ were inhibited in bone marrow-derived macrophages with haplodeficiency of MDM2. Similarly, pharmacological inhibition of MDM2 with the small molecule inhibitor Nutlin-3a attenuates pro-inflammatory cytokines secretion by macrophages. Mechanistically, haplodeletion of MDM2 led to decreased expression of glycolytic genes in BMDM during M1 activation. Taken together, we demonstrated a novel role of MDM2 in regulating glycolysis and M1 macrophage activation in addition to its role as a negative regulator of p53 tumour suppressor protein, suggesting it as a potential therapeutic target for treating type 2 diabetes. Disclosure K. Cheng: None. Funding Hong Kong General Research Fund (17100717)

Macrophage LAMTOR1 Deficiency Prevents Dietary Obesity and Insulin Resistance Through Inflammation-Induced Energy Expenditure.

Here, we studied the metabolic function of LAMTOR1 from macrophages using LAMTOR1 macrophage-specific knockout (MKO) mice. LAMTOR1 MKO mice showed resistance to high-fat diet (HFD)-induced obesity, lipid steatosis, and glucose metabolic disorders, with elevated levels of pro-inflammatory cytokines. The energy expenditure, oxygen consumption, and CO2 production increased significantly in HFD-fed MKO vs. wild-type (WT) mice. HE and immunohistochemistry staining showed a remarkable CD68+ Kupffer cell accumulation in the liver. Additionally, flow cytometry revealed that the proportion of macrophages and monocytes increased significantly in the liver of MKO mice. Of note, these macrophages were probably derived from the bone marrow since the proportion of CD11b+ cells as well as the proliferative activity was also increased in the context of femoral bone marrow cells. In addition, the Kupffer cells of both WT and KO mice were double-positive for the M1 (CD86) and M2 (CD206) markers. However, the expression of both M1 and M2 macrophage-related genes was increased in the liver of HFD-fed KO mice. Murine primary hepatocytes and Kupffer cells were further isolated and incubated with oleic acid for 24 h. The glucose output of primary hepatocytes from MKO mice was not affected. However, decreased lipid tolerance was observed in LAMTOR1-deficient Kupffer cells. Overall, our results suggest that LAMTOR1 deficiency in macrophages prevents obesity and metabolic disorders via the accumulation of Kupffer cells in the liver and the consequent hyper-inflammation and increased energy expenditure. Therefore, our results provide a new perspective for macrophage-derived LAMTOR1 in the context of systemic metabolism.

Absence of TNF Leads to Alternative Activation in Peritoneal Macrophages in Experimental Listeria Monocytogenes Infection

ABSTRACT Macrophages are crucial effectors of innate immunity against the pathogenic bacterium Listeria monocytogenes. The pro-inflammatory cytokine tumour necrosis factor-α (TNF) has been shown to be crucial for resistance to L. monocytogenes and mice deficient in TNF signalling succumb quickly after infection. However, the mechanisms underlying TNF-mediated defence against L. monocytogenes infection have not been completely elucidated. Here, we demonstrate that TNF concurrently functions to support a pro-inflammatory M1 phenotype while actively blocking macrophage polarization to the M2 phenotype. Compared to WT mice, peritoneal macrophages in TNF-deficient mice inoculated with L. monocytogenes respond with M2 polarization by upregulating Arg1. Consistently, TNF blockade in vitro resulted in M2 polarization in peritoneal macrophages during L. monocytogenes infection. Additionally, TNF promotes the transition from M2 to M1 polarization in peritoneal macrophages. Further investigation of peritoneal macrophage polarization suggested the NF-κB pathway is involved in the TNF-dependent M2 to M1 shift. Conversely, treatment of peritoneal macrophage with a PPARγ agonist blunted the expression of M1 genes induced by TNF and reduced NF-κB signalling pathway activation. Competing signalling mechanisms therefore play an essential role in the ability of peritoneal macrophage to resolve L. monocytogenes infections with TNF playing an essential role in driving M1 polarization. Abbreviations: LPM: large peritoneal macrophage; SPM: small peritoneal macrophage; LLO: listeriolysin O; iNOS: inducible nitric oxide synthase; DCs: dendritic cells