M2 Phenotype Polarization Research Articles

Advanced glycation end products enhance M1 macrophage polarization by activating the MAPK pathway

Backgroundβ-cell dysfunction is one of the core pathogenetic mechanisms of type 2 diabetes mellitus (T2DM). However, there are currently no effective therapeutic strategies to preserve β-cell mass and function. The role of islet macrophage phenotype reprogramming in β-cell dysfunction has attracted great attention. Given that advanced glycation end products (AGEs) are major pathogenic factors in T2DM, we investigated the effect of AGEs on macrophage activation and their role in β-cell dysfunction. MethodsWe examined cytokine secretion, M1 and M2 macrophage-associated marker expression and MAPK phosphorylation levels in AGEs-stimulated macrophages. MIN6 cells were cocultured with AGEs-pretreated macrophages to study the effect of AGEs-induced macrophage activation on β-cell dysfunction. ResultsWe found that AGEs treatment significantly enhanced macrophage secretion of proinflammatory cytokines. The expression of M1 macrophage markers, such as iNOS and the surface marker CD11c, was significantly upregulated, whereas the expression of M2 macrophage markers, such as Arg1 and CD206, was reciprocally downregulated upon AGEs stimulation. AGEs treatment predominantly activated the MAPK pathway, and the inhibition of the MAPK pathway partially attenuated the AGEs-induced polarization of macrophages. In addition, coculture with AGEs-pretreated macrophages significantly inhibited the expression of molecules involved in β-cell function and was accompanied by the impairment of glucose-stimulated insulin secretion (GSIS) in MIN6 cells. ConclusionAGEs enhance the expression of proinflammatory molecules by activating the MAPK pathway. Moreover, these data imply that AGEs induce macrophage M1 phenotype polarization but restrain M2 polarization, which might contribute to β-cell dysfunction in the pathogenesis of T2DM.

The role of resveratrol as a natural modulator in glia activation in experimental models of stroke.

Stroke is one of the most important causes of death and disability in modern and developing societies. In a stroke, both the glial cells and neurons develop apoptosis due to decreased cellular access to glucose and oxygen. Resveratrol (3, 5, 4'-trihydroxy-trans-stilbene) as a herbal compound shows neuroprotective and glioprotective effects. This article reviews how resveratrol can alleviate symptoms after stroke to help neurons to survive by modulating some signaling pathways in glia. Various databases such as ISI Web of Knowledge, Scopus, Medline, PubMed, and Google Scholar, were searched from 2000 to February 2020 to gather the required articles using appropriate keywords. Resveratrol enhances anti-inflammatory and decreases inflammatory cytokines by affecting the signaling pathways in microglia such as AMP-activated protein kinase (5' adenosine monophosphate-activated protein kinase, AMPK), SIRT1 (sirtuin 1) and SOCS1 (suppressor of cytokine signaling 1). Furthermore, through miR-155 overexpressing in microglia, resveratrol promotes M2 phenotype polarization. Resveratrol also increases AMPK and inhibits GSK-3β (glycogen synthase kinase 3 beta) activity in astrocytes, which release energy, makes ATP available to neurons and reduces reactive oxygen species (ROS). Besides, resveratrol increases oligodendrocyte survival, which can lead to maintaining post-stroke brain homeostasis. These results suggest that resveratrol can be considered a novel therapeutic agent for the reduction of stroke symptoms that can not only affect neuronal function but also play an important role in reducing neurotoxicity by altering glial activity and signaling.

Araloside C attenuates atherosclerosis by modulating macrophage polarization via Sirt1-mediated autophagy.

Atherosclerosis-related cardiovascular disease is still the predominant cause of death worldwide. Araloside C (AsC), a natural saponin, exerts extensive anti-inflammatory properties. In this study, we explored the protective effects and mechanism of AsC on macrophage polarization in atherosclerosis in vivo and in vitro. Using a high-fat diet (HFD)-fed ApoE-/- mouse model and RAW264.7 macrophages exposed to ox-LDL, AsC was evaluated for its effects on polarization and autophagy. AsC significantly reduced the plaque area in atherosclerotic mice and lipid accumulation in ox-LDL-treated macrophages, promoted M2 phenotype macrophage polarization, increased the number of autophagosomes and modulated the expression of autophagy-related proteins. Moreover, the autophagy inhibitor 3-methyladenine and BECN1 siRNA obviously abolished the antiatherosclerotic and M2 macrophage polarization effects of AsC. Mechanistically, AsC targeted Sirt1and increased its expression, and this increase in expression was associated with increased autophagy and M2 phenotype polarization. In contrast, the effects of AsC were markedly blocked by EX527 and Sirt1 siRNA. Altogether, AsC attenuates foam cell formation and lessens atherosclerosis by modulating macrophage polarization via Sirt1-mediated autophagy.

M2 Phenotype Macrophages Colocalize with Schwann Cells in Human Dental Pulp

Macrophages are immune cells with high plasticity that perform many functions related to tissue injury and repair. They are generally categorized as 2 functional phenotypes: M1 (proinflammatory) and M2 (anti-inflammatory and prohealing). To investigate the role of macrophages in human dental pulp, we examined the localization and distributional alterations of macrophages in healthy dental pulp as well as during the reparative process of pulp capping with mineral trioxide aggregate (MTA) and in cariously inflamed pulp of adult human teeth. We also quantified the populations of M1/M2 macrophages in healthy dental pulp by flow cytometric analysis. CD68+CD86+ cells (M1 phenotype) and CD68+CD163+ cells (M2 phenotype) were 2.11% ± 0.50% and 44.99% ± 2.22%, respectively, of 2.96% ± 0.41% CD68+ cells (pan-macrophages) in whole healthy dental pulp. Interestingly, M2 phenotype macrophages were associated with Schwann cells in healthy pulp, during mineralized bridge formation, and in pulp with carious infections in vivo. Furthermore, the M2 macrophages associated with Schwann cells expressed brain-derived neurotrophic factor (BDNF) under all in vivo conditions. Moreover, we found that plasma cells expressed BDNF. Coculture of Schwann cells isolated from human dental pulp and human monocytic cell line THP-1 showed that Schwann cells induced M2 phenotypic polarization of THP-1 cell-derived macrophages. The THP-1 macrophages that maintained contact with Schwann cells were stimulated, leading to elongation of their cell shape and expression of M2 phenotype marker CD163 in cocultures. In summary, we revealed the spatiotemporal localization of macrophages and potent induction of the M2 phenotype by Schwann cells in human dental pulp. M2 macrophages protect neural elements, whereas M1 cells promote neuronal destruction. Therefore, suppressing the neurodestructive M1 phenotype and maintaining the neuroprotective M2 phenotype of macrophages by Schwann cells may be critical for development of effective treatment strategies to maintain the viability of highly innervated dental pulp.

Targeted Delivery of Chlorogenic Acid to Effectively Promote the Polarization of TAMs for the Treatment of Glioblastoma

Tumor-associated macrophages (TAMs) generally display an immunosuppressive M2 phenotype and promote tumor progression and metastasis, suggesting their potential value as a target in cancer immunotherapy. Chlorogenic acid (CHA) has been identified as a potent immunomodulator that promotes the polarization of TAMs from an M2 to an M1 phenotype. However, rapid clearance in vivo and low tumor accumulation have compromised the immunotherapeutic efficacy of CHA in clinical trials. In this study, we developed mannosylated liposomes for targeted delivery of CHA to TAMs. The immunoregulatory effects of CHA along with the overall antitumor efficacy of CHA-encapsulated mannosylated liposomes were investigated through in vitro and in vivo experiments. The prepared CHA-encapsulated mannosylated liposomes exhibited an ideal particle size, favorable stability, and preferential accumulation in tumors via the mannose receptor-mediated TAMs-targeting effects. Further, CHA-encapsulated mannosylated liposomes inhibited G422 glioma tumor growth by efficiently promoting the polarization of the pro-tumorigenic M2 phenotype to the anti-tumorigenic M1 phenotype. Overall, these findings indicate that CHA-encapsulated mannosylated liposomes have great potential to enhance the immunotherapeutic efficacy of CHA by inducing a shift from the M2 to the M1 phenotype.

Targeted ferritin nanoparticle encapsulating CpG oligodeoxynucleotides induces tumor-associated macrophage M2 phenotype polarization into M1 phenotype and inhibits tumor growth.

Tumor-associated macrophages (TAM) are primarily of the M2 type that facilitates tumor growth, metastasis, and immunosuppression. Therefore, repolarizing the TAMs to the pro-inflammatory M1 type is a promising therapeutic strategy against cancer. Toll-like receptor (TLR) agonists like CpG oligodeoxynucleotides (CpG ODNs) can induce anti-tumor macrophages, however, their applications in vivo are limited by the lack of effective delivery approaches. Naked CpG ODNs fail to penetrate cell membranes and are easily cleared by nucleases, which can potentially trigger an inflammatory response in serum by systemic administration. Nanoparticles can deliver TLR agonists to the target TAMs following systemic administration and selectively accumulate in tumors and macrophages, and eventually trigger TLR signaling and M1 polarization. In this study, we developed a nanoparticle vector for the targeted delivery of CpG ODNs to M2 type TAMs by encapsulating the CpG ODNs inside human ferritin heavy chain (rHF) nanocages surface modified with a murine M2 macrophage-targeting peptide M2pep. These M2pep-rHF-CpG nanoparticles repolarized M2 TAMs to the M1 type and inhibited tumor growth in 4T1 tumor-bearing mice after intravenous injection. Furthermore, M2pep-rHF-CpG also reversed the phenotype of cultured human macrophages in vitro. Interestingly, the empty M2pep-rHF nanoparticles lacking CpG ODNs also exhibited anti-tumor ability. Taken together, M2pep-rHF nanoparticles offer a novel anti-cancer therapeutic strategy via targeted delivery of CpG ODNs to M2 type TAMs, and M2pep-rHF-CpG or M2pep-rHF nanoparticles may become promising medicines for tumor immunotherapy.

Long-lived tumor-associated macrophages in glioma.

BackgroundThe tumor microenvironment plays a major tumor-supportive role in glioma. In particular, tumor-associated macrophages (TAMs), which can make up to one-third of the tumor mass, actively support tumor growth, invasion, and angiogenesis. Predominantly alternatively activated (M2-polarized) TAMs are found in late-stage glioma in both human and mouse tumors, as well as in relapse samples from patients. However, whether tumor-educated M2 TAMs can actively contribute to the emergence and growth of relapse is currently debated.MethodsTo investigate whether tumor-educated stromal cells remaining in the brain after surgical removal of the primary tumor can be long-lived and retain their tumor-supporting function, we developed a transplantation mouse model and performed lineage-tracing.ResultsWe discovered that macrophages can survive transplantation and stay present in the tumor much longer than previously suggested, while sustaining an M2-polarized protumorigenic phenotype. Transplanted tumors showed a more aggressive growth and faster polarization of the TAMs toward an M2 phenotype compared with primary tumors, a process dependent on the presence of few cotransplanted macrophages.ConclusionsOverall, we propose a new way for tumor-educated TAMs to contribute to glioma aggressiveness by long survival and stable protumorigenic features. These properties could have a relapse-supporting effect.

Rebamipide-loaded chitosan nanoparticles accelerate prostatic wound healing by inhibiting M1 macrophage-mediated inflammation via the NF-κB signaling pathway.

A large proportion of benign prostatic hyperplasia (BPH) patients suffer from lower urinary tract symptoms after surgery due to the presence of prostatic urothelium wounds. Rebamipide (RBM) exerts wound healing promotion and anti-inflammatory effects on various tissues, including the urothelium. However, intravesical administration of RBM is hindered due to its low solubility and resulting unsustainable drug concentrations in the bladder. In this study, RBM-loaded chitosan nanoparticles (RBM/CTS NPs) were prepared using the ionic cross-linking method. Physicochemical characteristics and the wound healing promotion effect, as well as in vitro influence on macrophages were evaluated. The results show that RBM/CTS NPs are spherical with uniform size distribution, while slower and sustained in vitro release of RBM is presented. In vivo, faster wound healing and improved re-epithelialization progress were observed after treatment with RBM/CTS NPs in a model of thulium laser resection of the prostate (TmLRP). The degree of local inflammatory response decreased, as confirmed by decreasing numbers of pro-inflammatory M1 phenotype macrophages and levels of IL-1β, IL-6, IL-12 and TNF-α in the urine of canines. We also found that RBM/CTS NPs suppress macrophage M1 polarization induced by lipopolysaccharide and interferon-γ and inhibit the activation of the NF-κB signaling pathway. Therefore, as a novel therapeutic strategy, intravesical administration of RBM/CTS NPs can effectively avoid drug intolerance and drug wastage, accelerating the postoperative wound repairing of the prostatic urethra by suppressing macrophage M1 phenotype polarization.

Enhanced osteogenic differentiation of BMSCs and M2-phenotype polarization of macrophages on a titanium surface modified with graphene oxide for potential implant applications

Osseointegration at the bone–implant interface is a complex biological process that is triggered by the immune-inflammatory response and mediated by various cell types such as, bone-forming cells and immune cells, especially macrophages. The polarization of macrophages to inflammatory/regenerative (M1/M2) phenotypes, as well as the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) at the bone–implant interface, significantly affects implant osseointegration and even causes implant failure. Graphene oxide (GO) is a promising candidate for performing implant surface functionalization to modulate the interactions between implants and cells. Herein, we explored the effects of a GO coating on the osteogenic differentiation of BMSCs and the polarization of macrophages to enhance the application of GO surface modification in improving bone–implant integration. In the present study, a large particle sandblasting and acid etching (SLA) surface that is commonly used in clinical practice was selected as the control group, and GO was deposited on the SLA surface by the ultrasonic atomization spraying technique. The surface characteristics of these two groups, including the surface morphology, roughness, wettability, protein adsorption capacity and cell compatibility, were assessed. Then, the effects of GO surface modification on the osteogenic differentiation of BMSCs and the polarization of macrophages were evaluated. The results showed that the GO coating was successfully fabricated on the titanium substrates, which endowed the SLA surface with improved hydrophilicity and protein adsorption capacity and reduced roughness. Compared with the SLA surface, the GO-modified surface significantly enhanced the osteogenic differentiation of BMSCs and the M2-phenotype polarization of macrophages in vitro. This dual-regulatory role is of great significance in achieving rapid osseointegration as well as resolving the poor osseointegration associated with macrophage-related inflammation.

The immunomodulatory role of irisin on osteogenesis via AMPK-mediated macrophage polarization

Bone healing is thought to be closely related to macrophages. Irisin, a cleaved hormone-like myokine, is well known to participate in immunoregulation and regulates bone metabolism. However, whether irisin could influence osteogenesis by affecting macrophage polarization is remain unknown. Here, the present study aims to investigate the potential immunomodulatory role of irisin on macrophages polarization and its subsequent impact on osteogenesis. We demonstrated that irisin increased cell viability without toxic effect in both Raw264.7 macrophages and MC3T3-E1 cells. Furthermore, irisin treatment polarized M0 and M1 macrophages towards M2 phenotype, with increased expression of CD206-APC, ARG-1 and TGF-β1, and decreased expression of CD86-PE and TNF-α. In addition, the direct co-cultured test of Raw264.7 macrophages and pre-osteoblastic MC3T3-E1 cells showed that irisin-treated M0 and M1 macrophages promoted osteogenesis with obvious formation of mineralized particles. Interestingly, irisin exposure robustly activated AMPK-α signaling, as manifested by increased expression of phosphorylated AMPK-α. Knockdown of AMPK-α by siRNA significantly suppressed the phosphorylation of AMPK-α, abrogated irisin-induced polarization of M2 phenotype, and inhibited the osteogenic ability of Raw264.7 macrophages. Taken together, our findings showed that irisin-induced M2 polarization enhanced osteogenesis in osteoblasts, and this effect might be associated with activation of AMPK.

Bladder cancer cell‑secreted exosomal miR‑21 activates the PI3K/AKT pathway in macrophages to promote cancer progression.

Tumour-associated macrophages (TAMs) compose a major component of the tumour microenvironment and form in this microenvironment prior to cancer metastasis. However, the detailed mechanisms of TAM remodelling in the context of bladder cancer have not been clearly defined. The present study collected exosomes from the conditioned medium of human bladder T24 cancer cells. The effects of macrophages treated with exosomes derived from T24 cells on bladder cancer cell migration and invasion were analysed by Transwell assays. The expression levels of endogenous and exosomal microRNA-21 (miR-21) were examined by reverse transcription-quantitative PCR, while the expression level of the target protein was analysed by western blot analysis. Luciferase reporter plasmids and mutants were used to confirm direct targeting. The effects of miR-21 on bladder cancer cell migration and invasion were analysed by Transwell and Matrigel assays following miR-21 transfection. It was identified that exosomes derived from bladder cancer cells polarized THP-1 cell-derived macrophages into the M2 phenotype, and TAM-mediated pro-migratory and pro-invasive activity was determined. Moreover, it was found that miR-21 was highly expressed in exosomes derived from bladder cancer cells as well as in macrophages treated with exosomes. In addition, macrophages transfected with miR-21 exhibited M2 polarization and promoted T24 cell migratory and invasive ability. Mechanistically, exosomal miR-21 derived from bladder cancer cells inhibited phosphatase and tensin homolog activation of the PI3K/AKT signalling pathway in macrophages and enhanced STAT3 expression to promote M2 phenotypic polarization. The present results suggest that exosomal miR-21 can promote cancer progression by polarizing TAMs.

Curdlan (Alcaligenes faecalis) (1→3)-β-d-Glucan Oligosaccharides Drive M1 Phenotype Polarization in Murine Bone Marrow-Derived Macrophages via Activation of MAPKs and NF-κB Pathways.

Functional oligosaccharides, particularly curdlan (1→3)-β-d-glucan oligosaccharides (GOS), play important roles in modulating host immune responses. However, the molecular mechanisms underlying the immunostimulatory effects of GOS on macrophage polarization are not clear. In this work, GOS (5–1000 µg/mL) were non-toxic to bone marrow-derived macrophages (BMDMs) with improved pinocytic and bactericidal capacities. Incubation with GOS (100 µg/mL) induced M1 phenotype polarization of BMDMs as evidenced by increased CD11c+/CD86+ (10.1%) and M1 gene expression of inducible nitric oxide synthase, interleukin (IL)-1β, and chemokine C-C-motif ligand 2. Accordingly, the secretion of cytokines IL-1β, IL-6, monocyte chemotactic protein-1, and tumor necrosis factor-α, as well as the nitrite release of BMDMs were increased by GOS (100 µg/mL). Expression of mitogen-activated protein kinases (MAPKs) of phosphorylated (p)-c-Jun amino-terminal kinase, p-extracellular signal regulated kinase, and p-p38 in BMDMs were increased by GOS, as well as the p-Stat1. Moreover, nuclear factor-kappa B (NF-κB) p-p65 expression in BMDMs was promoted by GOS while it suppressed IκBα expression. Receptor blocking with anti-CR3 (CD11b/CD18) and anti-toll-like receptor (TLR) 2 antibodies diminished GOS induced M1 phenotype polarization with reduced mRNA expression of M1 genes, decreased cytokine and nitrite releases, and suppressed signaling pathway activation. Thus, CR3 (CD11b/CD18) and TLR2 mediated activation of MAPKs and NF-κB pathways are responsible for GOS induced polarization of BMDMs.

XIAOPI Formula Inhibits Breast Cancer Stem Cells via Suppressing Tumor-Associated Macrophages/C-X-C Motif Chemokine Ligand 1 Pathway.

Macrophages are the most abundant stromal cells associated with the host immune system in multiple malignancies including breast cancer. With proven clinical efficacy and no noticeable adverse effects, XIAOPI formula (XPS) has been approved for breast hyperplasia treatment by the State Food and Drug Administration of China (SFDA) in 2018. The existing knowledge about the anti-breast cancer activities and mechanisms of XPS has been very limited. The present study aimed to investigate whether XPS could exert an anti-breast cancer effect by regulating tumor-associated macrophages (TAMs) in tumor microenvironment. Herein, breast cancer cells and TAMs were co-cultured using the transwell co-culture system to simulate the coexistence of them. XPS could significantly inhibit the proliferation, colony formation, breast cancer stem cells (CSCs) subpopulation, mammosphere formation abilities as well as stemness-related genes expression in both human and mouse breast cancer cells in the co-culture system. Additionally, XPS could suppress M2 phenotype polarization as well as C-X-C motif chemokine ligand 1 (CXCL1) expression and secretion of TAMs. Notably, further mechanistic explorations verified TAMs/CXCL1 as the critical target of XPS in inhibiting breast CSCs self-renewal in the co-culture system as the exogenous CXCL1 administration could abrogate the inhibitory effect of XPS on breast CSCs self-renewal. More importantly, XPS significantly inhibited mammary tumor growth, breast CSCs subpopulation, and TAMs/CXCL1 activity in mouse 4T1-Luc xenografts in vivo without any detectable side effects. Taken together, this study not only uncovers the immunomodulatory mechanism of XPS in treating breast cancer but also sheds novel insights into TAMs/CXCL1 as a potential molecular target for breast CSCs elimination.

Lachnum polysaccharide suppresses S180 sarcoma by boosting anti-tumor immune responses and skewing tumor-associated macrophages toward M1 phenotype

Therapeutic strategies that targeting tumor-associated macrophages (TAMs) reprogramming play a crucial role in ameliorating the immunosuppressive tumor microenvironment and boosting anti-tumor immune responses. In this study, we demonstrated that Lachnum polysaccharide (LEP) could work as an immunomodulator to reset TAMs from pro-tumor M2 to anti-tumor M1 phenotype. Mechanistically, LEP promoted Th1 polarization and the secretion of IFN-γ, which played a key role in M1 phenotype polarization. In parallel, LEP might directly activate M1 macrophages via TLR4 mediated NF-κB signaling pathway. Moreover, LEP also resulted in the accumulation of anti-tumor immune cells and decreased the infiltration of immunosuppressive cells such as myeloid-derived suppressor cells (MDSCs) and Treg cells, thereby potentiating anti-tumor immunity. In summary, these results revealed a novel mechanism of the anti-tumor effect of LEP and provided a potential new avenue targeting TAMs and cancer immunotherapy.

Novel H2S-Releasing hydrogel for wound repair via in situ polarization of M2 macrophages

Hydrogen sulfide (H2S), as a gaseous messenger, exhibits potential therapeutic effects in biological and clinical applications. Herein, an in situ forming biomimetic hyaluronic acid (HA) hydrogel was used as a matrix to dope a pH-controllable H2S donor, JK1, to form a novel HA-JK1 hybrid system. This HA-JK1 hydrogel was designed as an ideal delivery scaffold for JK1 with pH-dependent prolonged H2S releasing profile. In vitro study suggested that JK1 could induce the polarization of M2 phenotype indicating a higher pro-healing efficiency of macrophages. The in vivo studies on dermal wounds showed that the HA-JK1 hybrid hydrogel significantly accelerated the wound regeneration process through enhanced re-epithelialization, collagen deposition, angiogenesis and cell proliferation. Furthermore, the in vivo results also demonstrated a higher level of M2 polarization in HA-JK1 treated group with reduced inflammation and improved wound remodeling effects, which was consistent with the in vitro results. These observations could be considered as a key to the efficient wound treatment. Therefore, we suggest that HA-JK1 can be used as a novel wound dressing material toward cutaneous wound model in vivo. This system should significantly enhance wound regeneration through the release of H2S that induces the expression of M2 macrophage phenotype.

Pulmonary fibroblasts-secreted CXCL10 polarizes alveolar macrophages under pro-inflammatory stimuli

BackgroundDuring acute lung injury, lung fibroblasts produce chemokines that assist the activation and migration of resident macrophages. The interactions between pulmonary fibroblasts and alveolar macrophages demonstrate the early event in the recruitment of immune cells, and the production of chemokines appear to be central mediators of the initiation and progression of inflammatory responses. In this study, the aim was to investigate the signaling pathway leading to CXCL10 secretion and the effects of CXCL10 released by activated fibroblasts on regulating macrophage polarization in a pro-inflammatory microenvironment. MethodsThe expression of chemokines CCL2, CCL5, CXCL10, and CXCL12, and the phosphorylation of signaling molecules STAT3, FAK, GSK3αβ and PKCδ were investigated by real time-PCR, ELISA, or Western blot on TNFα- or IL-1β-activated MRC-5 pulmonary fibroblasts. By collecting conditioned medium from TNFα-activated fibroblasts, the expression of iNOS and arginase I on MH-S alveolar macrophages were examined by real-time PCR. Surface markers CD86 and CD206 expressions on alveolar macrophages were also evaluated by flow cytometry. ResultsWe found that CXCL10 production was significantly elevated on MRC-5 fibroblasts under TNFα- or IL-1β treatment. In addition, we revealed that TNFα and IL-1β initiated phosphorylation of STAT3, FAK, GSK3αβ and PKCδ signaling cascade, leading to the elevation of CXCL10 expression. Moreover, conditioned medium collected from TNFα-activated MRC-5 fibroblasts increased iNOS and CD86 expressions and decreased arginase I and CD206 expressions on MH-S alveolar macrophages, and neutralization of CXCL10 abolished these observed phenomena. ConclusionThese results suggest that CXCL10 is crucial in activated fibroblasts-promoted M1 phenotype polarization of alveolar macrophages. In this regard, targeting fibroblasts-released CXCL10 may be promising as anti-inflammatory therapy against acute lung injury.

Abstract 2800: Oral cancer cell-derived extracellular vesicles can modulate an immunosuppressive microenvironment through M2 phenotype polarization

Abstract Oral squamous cell carcinoma (OSCC) is the most prevalent malignant tumor among head and neck cancers, with 300,000 new cases and 145,000 deaths around the world per year, with less than 50% survival rate of advanced cases after five years of diagnosis. The molecular mechanisms involved in immunosurveillance and oral cancer development are poorly understood and, neither molecular markers nor immunotherapy response predictors are available. However, recent findings in molecular biology and immunology of cancer cell have shown that tumor-stroma communication promotes intense suppressive signaling in the microenvironment, which may contribute to failure of therapy. Tumor-stroma communication is not an unidirectional process, driven only by cancer cells. Other resident cells, like cancer-associated fibroblasts (CAFs), immune cells as macrophages and lymphocytes, endothelial cells and tumor stem cells, which are in an intense cross-talk by cell-cell contact, secreted factors and other components of the extracellular matrix (ECM), can alter and be altered by the microenvironment. In this context, the cancer-derived extracellular vesicles (EVs) acquire a significant pathological role in cell-cell communication by changing the phenotype of the recipient cells, promoting tumor growth and metastasis. Here, we explored the role of three different source of EVs, originated from normal gingival keratinocyte (HMK) and from less (SCC9) and high (HSC3) aggressive OSCC cell lines in the modulation of monocyte-derived macrophage M1 or M2 phenotypes, and their ability to contribute to the suppressive phenotype in the tumor microenvironment. CD163 expression, a marker of M2 macrophages, was significantly increased in CD14+ monocytes stimulated by HSC3-EVs, in comparison with non-stimulated monocytes, HMK- and SCC9-EVs-stimulated cells. In addition, CD64+/CD86+/CD80+ M1 macrophage subset, differentiated by GM-CSF/IFN-gamma, showed a decreased expression of the T-cell co-stimulatory molecule, CD86 after stimulus with HSC3-EVs. Furthermore, the mass spectrometry analysis of HSC3-EV protein contents identified a set of up- and down-regulated candidate proteins that may be involved in immunoregulation and suppressive pathways. Since the M2 macrophage phenotype is associated with increased angiogenesis, immunological tolerance and development of neoplastic cells, the M1 subset, the classically activated macrophages, may recognize and eliminate cancer cells. Thus, our results indicate that EVs from aggressive cells HSC3 may contribute to immunosuppressive tumor microenvironment in oral cancer by deviating macrophages into a tumor-promoting instead of a tumor-controlling phenotype. Citation Format: Ana Karina Oliveira, Thiago Andrade Patente, Rodrigo Nalio Ramos, Ariane Busso Lopes, Romênia Ramos Domingues, Mariane Tami Amano, Eliana Blini Marengo, Jose Alexandre Barbuto, Adriana Franco Squina. Oral cancer cell-derived extracellular vesicles can modulate an immunosuppressive microenvironment through M2 phenotype polarization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2800.

Abstract 121: S100A9 inhibitor Tasquinimod: A novel strategy to inhibit small cell lung cancer progression and metastasis

Abstract Small cell lung cancer (SCLC) treatment is a major clinical challenge at present as it is highly refractory to available drugs. The MDSCs/macrophages are known to help SCLC develop resistant to available therapies. S100A9 (Migration inhibitory factor-related protein 14 (MRP14) is an EF-hand calcium-binding protein that has been involved in cell migration, invasion, proliferation, and tumor metastasis in various type of cancers, however not much is known about its role in SCLC. In this study, we found that S100A9 protein is highly up-regulated in various types of pulmonary neuroendocrine carcinomas (NEC) patient tissues compared to normal using tissue microarrays. We also observed that SCLC patients with higher S100A9 expression have significantly increased numbers of macrophage in the stroma. We have also shown that pre-treatment of the cells with S100A9 inhibitor (Tasquinimod) suppressed in-vitro cell migration, invasion, and colony formation. In addition, we analyzed the efficacy of S100A9 inhibitor against SCLC using in vivo mouse models. S100A9 inhibitor significantly reduces tumor growth and metastasis in SCLC in xenograft mouse models. We further observed that S100A9 inhibitor suppressed myeloid-derived suppressor cells (MDSC) populations and TAMs of the M2-polarized phenotype in SCLC. Moreover, we found myeloid cells sequestered from tumors of treated mice expressed were MI type as they showed higher levels of inducible nitric oxide synthase (iNos), and lower levels of arginase-1. Molecular analysis revealed that Tasquinimod decreases expression of IL6, IL10, and TGF-β1 in the cancer cells which helps inhibit macrophage activation to TAMs. Reduced proliferation and vascularization were observed in the tumors obtained from animals treated with S100A9 inhibitor. We also observed S100A9 inhibitor suppressed osteolytic bone formation in ex-vivo resorption assay. Overall, our studies, for the first time, show that Tasquinimod that targets S100A9 signaling could be used as a novel therapeutic strategy against SCLC. Citation Format: Salha E. Sassi, Dinesh Ahirwar, Ramesh K. Ganju. S100A9 inhibitor Tasquinimod: A novel strategy to inhibit small cell lung cancer progression and metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 121.

Exosomal miRNA-19b-3p of tubular epithelial cells promotes M1 macrophage activation in kidney injury.

Tubulointerstitial inflammation is a common characteristic of acute and chronic kidney injury. However, the mechanism by which the initial injury of tubular epithelial cells (TECs) drives interstitial inflammation remains unclear. This paper aims to explore the role of exosomal miRNAs derived from TECs in the development of tubulointerstitial inflammation. Global microRNA(miRNA) expression profiling of renal exosomes was examined in a LPS induced acute kidney injury (AKI) mouse model and miR-19b-3p was identified as the miRNA that was most notably increased in TEC-derived exosomes compared to controls. Similar results were also found in an adriamycin (ADR) induced chronic proteinuric kidney disease model in which exosomal miR-19b-3p was markedly released. Interestingly, once released, TEC-derived exosomal miR-19b-3p was internalized by macrophages, leading to M1 phenotype polarization through targeting NF-κB/SOCS-1. A dual-luciferase reporter assay confirmed that SOCS-1 was the direct target of miR-19b-3p. Importantly, the pathogenic role of exosomal miR-19b-3p in initiating renal inflammation was revealed by the ability of adoptively transferred of purified TEC-derived exosomes to cause tubulointerstitial inflammation in mice, which was reversed by inhibition of miR-19b-3p. Clinically, high levels of miR-19b-3p were found in urinary exosomes and were correlated with the severity of tubulointerstitial inflammation in patients with diabetic nephropathy. Thus, our studies demonstrated that exosomal miR-19b-3p mediated the communication between injured TECs and macrophages, leading to M1 macrophage activation. The exosome/miR-19b-3p/SOCS1 axis played a critical pathologic role in tubulointerstitial inflammation, representing a new therapeutic target for kidney disease.

Pyruvate Dehydrogenase Kinase Is a Metabolic Checkpoint for Polarization of Macrophages to the M1 Phenotype.

Metabolic reprogramming during macrophage polarization supports the effector functions of these cells in health and disease. Here, we demonstrate that pyruvate dehydrogenase kinase (PDK), which inhibits the pyruvate dehydrogenase-mediated conversion of cytosolic pyruvate to mitochondrial acetyl-CoA, functions as a metabolic checkpoint in M1 macrophages. Polarization was not prevented by PDK2 or PDK4 deletion but was fully prevented by the combined deletion of PDK2 and PDK4; this lack of polarization was correlated with improved mitochondrial respiration and rewiring of metabolic breaks that are characterized by increased glycolytic intermediates and reduced metabolites in the TCA cycle. Genetic deletion or pharmacological inhibition of PDK2/4 prevents polarization of macrophages to the M1 phenotype in response to inflammatory stimuli (lipopolysaccharide plus IFN-γ). Transplantation of PDK2/4-deficient bone marrow into irradiated wild-type mice to produce mice with PDK2/4-deficient myeloid cells prevented M1 polarization, reduced obesity-associated insulin resistance, and ameliorated adipose tissue inflammation. A novel, pharmacological PDK inhibitor, KPLH1130, improved high-fat diet-induced insulin resistance; this was correlated with a reduction in the levels of pro-inflammatory markers and improved mitochondrial function. These studies identify PDK2/4 as a metabolic checkpoint for M1 phenotype polarization of macrophages, which could potentially be exploited as a novel therapeutic target for obesity-associated metabolic disorders and other inflammatory conditions.