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TLR-3 Stimulation Skews M2 Macrophages to M1 Through IFN-?? Signaling and Restricts Tumor Progression.

ABSTRACT: During tumor progression, macrophages shift their protective M1-phenotype to pro-tumorigenic M2-subtype. Therefore, conversion of M2 to M1 phenotype may be a potential therapeutic intervention. TLRs are important pathogen recognition receptors expressed by cells of the immune system. Recently, a crucial role of TLR-3 has been suggested in cancer. Consequently, in the current study, we defined the role of TLR-3 in the reversion of M2-macrophages to M1. We analyzed the role of TLR-3 stimulation for skewing M2-macrophages to M1 at mRNA and protein level through qRT-PCR, flow cytometry, western blotting, and ELISA. The effectiveness of TLR-3L stimulation to revert M2-macrophages to M1 was evaluated in the murine tumor model. To determine the role of IFN-?? signaling in vitro and in vivo, we used Ifnar1^-/- macrophages and anti-IFN-?? antibodies, respectively. We observed upregulation of M1-specific markers MHC-II and costimulatory molecules like CD86, CD80, and CD40 on M2-macrophages upon TLR-3 stimulation. In contrast, reduced expression of M2-indicators CD206, Tim-3, and pro-inflammatory cytokines was noticed. The administration of TLR-3L in the murine tumor reverted the M2-macrophages to M1-phenotype and regressed the tumor growth. The mechanism deciphered for macrophage reversion and controlling the tumor growth is dependent on IFN-?? signaling pathway. The results indicate that the signaling through TLR-3 is important in protection against tumors by skewing M2-macrophages to protective M1-subtype.

SUBMITTER: Vidyarthi A

PROVIDER: S-EPMC6060442 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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TLR-3 Stimulation Skews M2 Macrophages to M1 Through IFN-αβ Signaling and Restricts Tumor Progression.

Vidyarthi Aurobind A Khan Nargis N Agnihotri Tapan T Negi Shikha S Das Deepjyoti K DK Aqdas Mohammad M Chatterjee Deepyan D Colegio Oscar R OR Tewari Manoj K MK Agrewala Javed N JN

Frontiers in immunology 20180719

During tumor progression, macrophages shift their protective M1-phenotype to pro-tumorigenic M2-subtype. Therefore, conversion of M2 to M1 phenotype may be a potential therapeutic intervention. TLRs are important pathogen recognition receptors expressed by cells of the immune system. Recently, a crucial role of TLR-3 has been suggested in cancer. Consequently, in the current study, we defined the role of TLR-3 in the reversion of M2-macrophages to M1. We analyzed the role of TLR-3 stimulation fo ...[more]

PMID: 30072995

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Project description:BackgroundToll-like receptors (TLRs) and macrophages play an important role in rheumatoid arthritis (RA). Currently, it is not clear whether inflammatory M1 or anti-inflammatory M2 predominate among the resident macrophages in the synovium. In the present study, we set out to investigate the impact of TLR stimulation on monocyte-derived M1 and M2 macrophage function and phenotype by mimicking the exposure to abundant TLR agonists as occurs in the context of RA. The response of macrophage subsets to TLR2 and TLR4 activation was evaluated on cluster of differentiation (CD) marker profile; cytokine secretion; gene expression; and NF-?B, interferon regulatory factors 3 and 7 (IRF3/7), and mitogen-activated protein kinase (MAPK) activation.MethodsHuman monocytes were isolated from peripheral blood of healthy individuals and patients with RA and differentiated into M1-like and M2-like macrophages by granulocyte-macrophage colony-stimulating factor (GM-CSF) and macrophage colony-stimulating factor (M-CSF), respectively. Cells were either (1) stimulated with TLR ligands Pam3 or lipopolysaccharide (LPS) or (2) classically activated via interferon (IFN)-?/LPS. Cytokine production was measured by enzyme-linked immunosorbent assay, and gene expression was measured by qPCR. Cells were stained for CD markers and analyzed by fluorescence-activated cell sorting. NF-?B, IRF3/7, and MAPKs were detected by Western blotting.ResultsMonocyte-derived macrophages of healthy donors (HD) or patients with RA displayed comparable subset-specific phenotypes upon exposure to TLR agonists. CD14 and CD163 marker expression on M2 macrophages did not change upon TLR2 and TLR4 engagement. By contrast, M2 gene markers HMOX1, FOLR2, and SLC40A1 were decreased. Importantly, M2 macrophages derived from HD or patients with RA showed both a decreased ratio of interleukin (IL)-10/IL-6 and IL-10/IL-8 upon stimulation with TLR2 ligand Pam3 compared with TLR4 ligand LPS. Gene expression of TLR2 was increased, whereas TLR4 expression was decreased, by TLR ligand stimulation. MAPKs p38, extracellular signal-regulated kinase 1/2, and c-Jun N-terminal kinase were activated more strongly in M2 than in M1 macrophages by Pam3 or LPS.ConclusionsWe show that the anti-inflammatory activity of M2 macrophages is reduced in the presence of abundant TLR2 ligands without significant changes in cell surface markers. Thus, the classical M1/M2 paradigm based on cellular markers does not apply to macrophage functions in inflammatory conditions such as RA.

Project description:Rationale: Breast cancer (BrCa) is the most common cancer worldwide, and the 5-year relative survival rate has declined in patients diagnosed at stage IV. Advanced BrCa is considered as incurable, which still lack effective treatment strategies. Identifying and characterizing new tumor suppression genes is important to establish effective prognostic biomarkers or therapeutic targets for late-stage BrCa. Methods: RNA-seq was applied in BrCa tissues and normal breast tissues. Through analyzing differentially expressed genes, DRD2 was selected for further analysis. And expression and promoter methylation status of DRD2 were also determined. DRD2 functions were analyzed by various cell biology assays in vitro. Subcutaneous tumor model was used to explore DRD2 effects in vivo. A co-cultivated system was constructed to investigate interactions of DRD2 and macrophages in vitro. WB, IHC, IF, TUNEL, qRT-PCR, Co-IP, Antibody Array, and Mass Spectrum analysis were further applied to determine the detailed mechanism. Results: In BrCa, DRD2 was found to be downregulated due to promoter methylation. Higher expression of DRD2 positively correlated with longer survival times especially in HER2-positive patients. DRD2 also promoted BrCa cells sensitivity to Paclitaxel. Ectopic expression of DRD2 significantly inhibited BrCa tumorigenesis. DRD2 also induced apoptosis as well as necroptosis in vitro and in vivo. DRD2 restricted NF-κB signaling pathway activation through interacting with β-arrestin2, DDX5 and eEF1A2. Interestingly, DRD2 also regulated microenvironment as it facilitated M1 polarization of macrophages, and triggered GSDME-executed pyroptosis. Conclusion: Collectively, this study novelly manifests the role of DRD2 in suppressing BrCa tumorigenesis, predicting prognosis and treatment response. And this study further reveals the critical role of DRD2 in educating M1 macrophages, restricting NF-κB signaling pathway and triggering different processes of programmed cell death in BrCa. Taking together, those findings represent a predictive and therapeutic target for BrCa.

Dataset Information

TLR-3 Stimulation Skews M2 Macrophages to M1 Through IFN-?? Signaling and Restricts Tumor Progression.

Publications

TLR-3 Stimulation Skews M2 Macrophages to M1 Through IFN-αβ Signaling and Restricts Tumor Progression.

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