Gene Expression Of M2 Markers Research Articles

PARP14 Contributes to the Development of the Tumor-Associated Macrophage Phenotype.

Cancers reprogram macrophages (MΦs) to a tumor-growth-promoting TAM (tumor-associated MΦ) phenotype that is similar to the anti-inflammatory M2 phenotype. Poly(ADP-ribose) polymerase (PARP) enzymes regulate various aspects of MΦ biology, but their role in the development of TAM phenotype has not yet been investigated. Here, we show that the multispectral PARP inhibitor (PARPi) PJ34 and the PARP14 specific inhibitor MCD113 suppress the expression of M2 marker genes in IL-4-polarized primary murine MΦs, in THP-1 monocytic human MΦs, and in primary human monocyte-derived MΦs. MΦs isolated from PARP14 knockout mice showed a limited ability to differentiate to M2 cells. In a murine model of TAM polarization (4T1 breast carcinoma cell supernatant transfer to primary MΦs) and in a human TAM model (spheroids formed from JIMT-1 breast carcinoma cells and THP-1-MΦs), both PARPis and the PARP14 KO phenotype caused weaker TAM polarization. Increased JIMT-1 cell apoptosis in co-culture spheroids treated with PARPis suggested reduced functional TAM reprogramming. Protein profiling arrays identified lipocalin-2, macrophage migration inhibitory factor, and plasminogen activator inhibitor-1 as potential (ADP-ribosyl)ation-dependent mediators of TAM differentiation. Our data suggest that PARP14 inhibition might be a viable anticancer strategy with a potential to boost anticancer immune responses by reprogramming TAMs.

Etomidate inhibits tumor growth of glioblastoma by regulating M1 macrophage polarization.

Glioblastoma (GBM) is a common primary central nervous system tumor. Although the multimodal integrated treatment for GBM has made great progress in recent years, the overall survival time of GBM is still short. Thus, novel treatments for GBM are worth further investigation and exploration. This study aimed to investigate the effects of etomidate on GBM tumor growth and the underlying mechanism. A xenograft tumor model was established and treated with etomidate to assess tumor growth. Immunohistochemistry (IHC) assay evaluated the positive rate of Ki67 cells in tumor tissues. Cell counting kit (CCK)-8 and EdU assays accessed the cell viability and proliferation. Immunofluorescence (IF) staining detected the distribution of macrophage markers in tumor tissues. The percentages of M1- and M2-like macrophages in tumor-associated macrophages (TAMs) and co-culture system (macrophages and GBM cells) were detected using flow cytometry. Macrophage polarization-related genes were measured using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Etomidate treatment inhibited the tumor growth, and increased the CD86+ cells but decreased the CD206+ cells in TAMs. The gene expression of M1 markers was increased in TAMs of etomidate-treated mice, whereas that of M2 markers was decreased. Moreover, etomidate treatment increased the number of CD86+ M1-like macrophages co-cultured with tumor cells but decreased that of CD206+ M2-like macrophages, with the upregulation of M1 markers and downregulation of M2 markers. Etomidate inhibited GBM tumor growth by promoting M1 macrophage polarization, suggesting a new insight into the clinical treatment of GBM.

Pulsatilla Saponins Inhibit Experimental Lung Metastasis of Melanoma via Targeting STAT6-Mediated M2 Macrophages Polarization.

Pulsatilla saponins (PS) extracts from Pulsatilla chinensis (Bge.) Regel, are a commonly used traditional Chinese medicine. In the previous study, we found Pulsatilla saponins displayed anti-tumor activity without side effects such as bone marrow suppression. However, the mechanism of the anti-tumor effect was not illustrated well. Since M2-like tumor-associated macrophages (TAMs) that required activation of the signal transducer and activator of transcription 6 (STAT6) for polarization are the important immune cells in the tumor microenvironment and play a key role in tumor progress and metastasis, this study aimed to confirm whether Pulsatilla saponins could inhibit the development and metastasis of tumors by inhibiting the polarization of M2 macrophages. We investigated the relevance of M2 macrophage polarization and the anti-tumor effects of Pulsatilla saponins in vitro and in vivo. In vitro, Pulsatilla saponins could decrease the mRNA level of M2 marker genes Arg1, Fizz1, Ym1, and CD206, and the down-regulation effect of phosphorylated STAT6 induced by IL-4; moreover, the conditioned medium (CM) from bone marrow-derived macrophages (BMDM) treated with Pulsatilla saponins could inhibit the proliferation and migration of B16-F0 cells. In vivo, Pulsatilla saponins could reduce the number of lung metastasis loci, down-regulate the expression of M2 marker genes, and suppress the expression of phosphorylated STAT6 in tumor tissues. Furthermore, we used AS1517499 (AS), a STAT6 inhibitor, to verify the role of PS on M2 macrophage polarization both in vitro and in vivo. We found that Pulsatilla saponins failed to further inhibit STAT6 activation; the mRNA level of Arg1, Fizz1, Ym1, and CD206; and the proliferation and migration of B16-F0 cells after AS1517499 intervention in vitro. Similar results were obtained in vivo. These results illustrated that Pulsatilla saponins could effectively suppress tumor progress by inhibiting the polarization of M2 macrophages via the STAT6 signaling pathway; this revealed a novel mechanism for its anti-tumor activity.

The Role of Interferon Regulatory Factor 1 in Regulating Microglial Activation and Retinal Inflammation.

Microglia are resident immune cells in the central nervous system (CNS). Microglial activation plays a prominent role in neuroinflammation and CNS diseases. However, the underlying mechanisms of microglial activation are not well understood. Here, we report that the transcription factor interferon regulatory factor 1 (IRF1) plays critical roles in microglial activation and retinal inflammation by regulating pro- and anti-inflammatory gene expression. IRF1 expression was upregulated in activated retinal microglia compared to those at the steady state. IRF1 knockout (KO) in BV2 microglia cells (BV2ΔIRF1) created by CRISPR/Cas9 genome-editing technique causes decreased microglia proliferation, migration, and phagocytosis. IRF1-KO decreased pro-inflammatory M1 marker gene expression induced by lipopolysaccharides (LPS), such as IL-6, COX-2, and CCL5, but increased anti-inflammatory M2 marker gene expression by IL-4/13, such as Arg-1, CD206, and TGF-β. Compared to the wild-type cells, microglial-conditioned media (MCM) of activated BV2ΔIRF1 cell cultures reduced toxicity or death to several retinal cells, including mouse cone photoreceptor-like 661 W cells, rat retinal neuron precursor R28 cells, and human ARPE-19 cells. IRF1 knockdown by siRNA alleviated microglial activation and retinal inflammation induced by LPS in mice. Together, the findings suggest that IRF1 plays a vital role in regulating microglial activation and retinal inflammation and, therefore, may be targeted for treating inflammatory and degenerative retinal diseases.

CTLA4-Ig treatment induces M1\u2013M2 shift in cultured monocyte-derived macrophages from healthy subjects and rheumatoid arthritis patients

BackgroundIn rheumatoid arthritis (RA), macrophages play an important role in modulating the immunoinflammatory response through their polarisation into “classically” (M1) or “alternatively activated” (M2) phenotypes. In RA, CTLA4-Ig (abatacept) reduces the inflammatory activity of macrophages by interacting with the costimulatory molecule CD86. The study aimed to investigate the efficacy of CTLA4-Ig treatment to induce an M2 phenotype both in M1-polarised monocyte-derived macrophages (MDMs) obtained from healthy subjects (HS) and in cultured MDMs obtained from active RA patients.MethodsCultured MDMs were obtained from peripheral blood mononuclear cells of 7 active RA patients and from 10 HS after stimulation with phorbol myristate acetate (5 ng/mL) for 24 h. HS-MDMs were then stimulated with lipopolysaccharide (LPS, 1 mg/mL) for 4 h to induce M1-MDMs. M1-MDMs and RA-MDMs were treated with CTLA4-Ig (100 μM and 500 μM) for 3, 12, 24, and 48 h. The gene expression of CD80, CD86, and TLR4 (M1 markers); CD163, CD204, and CD206 (surface M2 markers); and MerTK (functional M2 marker) was evaluated by qRT-PCR. The protein synthesis of surface M2 markers was investigated by Western blotting. The statistical analysis was performed by the Wilcoxon t-test.ResultsIn LPS-induced HS-M1-MDMs, CTLA4-Ig 100 μM and 500 μM significantly downregulated the gene expression of M1 markers (3 h p<0.01 for all molecules; 12 h p<0.05 for TLR4 and CD86) and significantly upregulated that of M2 markers, primarily after 12 h of treatment (CD163: p < 0.01 and p < 0.05; CD206: p < 0.05 and p < 0.01; CD204: p < 0.05 by 100 mg/mL). Moreover, in these cells, CTLA4-Ig 500 μM increased the protein synthesis of surface M2 markers (p < 0.05). Similarly, in RA-MDMs, the CTLA4-Ig treatment significantly downregulated the gene expression of M1 markers at both concentrations primarily after 12 h (p < 0.05). Furthermore, both concentrations of CTLA4-Ig significantly upregulated the gene expression of CD206 (after 3 h of treatment; p < 0.05), CD163, and MerTK (after 12 h of treatment, p < 0.05), whereas CD204 gene expression was significantly upregulated by the high concentration of CTLA4-Ig (p < 0.05). The protein synthesis of all surface markers was increased primarily by CTLA4-Ig 500 μM, significantly for CD204 and CD206 after 24 h of treatment (p < 0.05).ConclusionsCTLA4-Ig treatment seems to induce the in vitro shift from M1 to M2 macrophages, of both HS-M1-MDMs and RA-MDMs, as observed by the significant downregulation exerted on selected M1 markers and the upregulation of selected M2 markers suggesting an additional mechanism for its modulation of the RA inflammatory process.

Sulforaphane attenuates dextran sodium sulphate induced intestinal inflammation via IL-10/STAT3 signaling mediated macrophage phenotype switching

Innate immunity, particularly macrophages, is critical for intestinal homeostasis. Sulforaphane, a dietary isothiocyanate from cruciferous vegetables, has been reported to protect against intestinal inflammation. However, the role of macrophages in sulforaphane mediated intestinal inflammation and the underlying molecular mechanisms have not been studied yet. In this study, sulforaphane effectively attenuated dextran sodium sulphate (DSS) induced intestinal inflammation in murine model. Of note, sulforaphane skewed the switching from classically (M1) to alternatively (M2) activated phenotype both in intestinal and bone marrow-derived macrophages (BMDMs). The expression levels of M1 associated maker genes induced by DSS or lipopolysaccharide (LPS) plus interferon gamma-γ (IFN-γ) were suppressed by sulforaphane while M2 marker gene expression levels were improved. This resulted in alteration of inflammatory mediators, particularly interleukin-10 (IL-10), both in colon tissues and culture medium of BMDMs. Subsequently, IL-10 was found to mediate the sulforaphane induced M2 phenotype switching of BMDMs through the activation of STAT3 signaling. This was confirmed by immunofluorescence analysis with increased number of p-STAT3-positive cells in the colon sections. Moreover, anti-IL-10 neutralizing antibody significantly interfered M2 phenotyping of BMDMs induced by sulforaphane with reduced STAT3 phosphorylation. Findings here introduced a potential utilization of sulforaphane for intestinal inflammation treatment with macrophages as the therapeutic targets.

Micro-Vibration Stimulation Promotes M2 Polarization of Macrophages Beneficial for Osteogenesis Via Activating FAK/P38MAPK and Suppressing Ca &lt;sup&gt;2+&lt;/sup&gt;/ERK/ p65NF-κB Signaling Pathways

The emergence of osteoimmunology has demonstrated that bone system is closely related to immune system. However, most studies have investigated the interaction between immune cells and bone-related cells under a static cell culture, the effects of mechanical stimuli on the inflammatory responses of macrophages remain elusive. In this work, micro-vibration stimulation (MVS) was introduced into regulating macrophage polarization and functional status. The results found that MVS with 40 Hz in frequency and 0.3 g in magnitude could significantly raise the fraction of CD206-positive M2 population, up-regulate the expression of M2 marker genes, and enhance the secretion of M2 marker proteins in RAW 264.7 macrophages. It suggested that calcium ion and focal adhesion played an important role in transmitting MVS signal. Further inhibitor studies and western blotting analyses indicated that MVS-induced M2 polarization might be attributed to the activation of p38MAPK signaling and suppression of the ERK1/2 and p65NF-κB signaling. Moreover, MVS-mediated macrophage secretion enhanced osteoblastic differentiation of MC3T3-E1 pre-osteoblasts via Wnt/β-catenin and ERK1/2 signaling in a paracrine way. These findings highlight a critical role of MVS in inducing M2 polarization of macrophage to create a pro-osteogenic environment, which offers a drug-free modulatory strategy to regulate immune response and promote osteogenesis.

Comparative study of the effects of ziram and disulfiram on human monocyte-derived macrophage functions and polarization: involvement of zinc.

Ziram, a zinc dithiocarbamate is widely used worldwide as a fungicide in agriculture. In order to investigate ziram-induced changes in macrophage functions and polarization, human monocytes-derived macrophages in culture were treated with ziram at 0.01-10μmol.L-1 for 4-24h. To characterize zinc involvement in these changes, we also determined the effects of disulfiram alone (dithiocarbamate without zinc) or in co-incubation with ZnSO4. We have shown that ziram and disulfiram at 0.01μmol.L-1 increased zymosan phagocytosis. In contrast, ziram at 10μmol.L-1 completely inhibited this phagocytic process, the oxidative burst triggered by zymosan and the production of TNF-α, IL-1β, IL-6, and CCL2 triggered by LPS. Disulfiram had the same effects on these macrophages functions only when combined with zinc (10μmol.L-1). In contrast, at 10μmol.L-1 ziram and zinc associated-disulfiram induced expression of several antioxidants genes HMOX1, SOD2, and catalase, which could suggest the induction of oxidative stress. This oxidative stress could be involved in the increase in late apoptosis induced by ziram (10μmol.L-1) and zinc associated-disulfiram. Concerning gene expression profiles of membrane markers of macrophage polarization, ziram at 10μmol.L-1 had two opposite effects. It inhibited the gene expression of M2 markers (CD36, CD163) in the same way as the disulfiram-zinc co-treatment. Conversely, ziram induced gene expression of other M2 markers CD209, CD11b, and CD16 in the same way as treatment with zinc alone. Disulfiram-zinc association had no significant effects on these markers. These results taken together show that ziram via zinc modulates macrophages to M2-like anti-inflammatory phenotype which is often associated with various diseases.

CD68+ tumor-associated myeloid cells as the target of adenosine-induced gene products and predictor of response to adenosine blockade with ciforadenant (cifo) in renal cell cancer (RCC).

5025 Background: Adenosine in the tumor microenvironment (TME) is immunosuppressive and may play a role in resistance to immunotherapy. We described an adenosine induced gene expression signature (AS, Fong, Cancer Disc 2020) that correlates with response to therapy with cifo, an adenosine A2A receptor antagonist, as monotherapy or in combination with atezolizumab in refractory RCC. These genes express chemokines that signal through CCR2 and CXCR2 to recruit myeloid cells including immunosuppressive tumor associated-M2 macrophages, which are thought to mediate resistance to anti-PD(L)1 treatment. We now identify tumor infiltrating CD68+ myeloid cells as the effector cell for adenosine mediated immunosuppression. Methods: 82 RCC pts have been treated in an ongoing Phase 1/1b trial evaluating cifo (100mg po bid) monotherapy or combination with atezolizumab (840mg IV q 2 weeks). Tumor biopsies, obtained at screening and on therapy, are available for analysis in 32 pts to date. RNA expression was measured in tumors using Nanostring. Immunohistochemistry (IHC) for CD68 was performed on biopsies with CD68+ tumors defined as &gt; 4% tumor area containing CD68+ cells. Results: Pt characteristics are median age 63; median prior therapies 3, with 72% failing prior anti-PD(L)1. Gene expression of M2 markers consisting of CD68 (p = 0.0008) and CD163 (p = 0.03) was higher in baseline samples from AS+ compared to AS- pts. By IHC, 10 pts had CD68+ cells infiltrating the tumor; 9 of 10 AS+. Tumor regression was observed in 6 of 10 CD68+ pts (N = 3 monotherapy and 3 combination) including 4 partial responses (PR, RECIST). No PRs and 2 minor responses were seen in 22 pts who were CD68- (p &lt; 0.005). Median time to progression was not reached for CD68+ vs 2 mo for CD68-. Paired biopsies showed a significant reduction in infiltrating CD68+ cells (p = 0.03) with treatment including 2 of 2 evaluable PRs. Conclusions: Adenosine immunosuppression is mediated by M2 macrophages, which can be reversed by cifo. Enumerating tumor infiltrating CD68+ cells may be a valuable biomarker for identifying pts that will respond to adenosine blockade. Clinical trial information: NCT02655822 .

Downregulation of HDAC8 expression decreases CD163 levels and promotes the apoptosis of macrophages by activating the ERK signaling pathway in recurrent spontaneous miscarriage

Recurrent spontaneous miscarriage (RSM) is a systemic disorder that has been defined as two or more pregnancies lost before the 20th week of gestation. Although the impaired function of macrophages at the maternal-fetal interface has been reported to be associated with RSM, the underlying mechanisms have not been fully elucidated. Here, we revealed that HDAC8 plays a critical role in RSM. Our results show that the mRNA and protein expression of HDAC8 was decreased in decidual macrophages from RSM patients. Moreover, the knockdown of HDAC8 resulted in a significant decrease in CD163 expression and an increase in apoptosis in dTHP-1 macrophages. Mechanistically, the ERK signaling pathway was activated in HDAC8-knockdown macrophages. When HDAC8-knockdown cells were pretreated with the ERK inhibitor U0126, expression levels of CD163, activated caspases 3, 7 and 9, and the apoptosis rate, were rescued. Taken together, our current results suggest that HDAC8 plays an important role in macrophage activation and apoptosis and may contribute to maintaining normal pregnancy by increasing the expression of M2 marker genes and inhibiting the apoptosis of macrophages at the maternal-fetal interface.

GPETAFLR, a peptide from Lupinus angustifolius L. prevents inflammation in microglial cells and confers neuroprotection in brain

ABSTRACT Objectives: Neuroinflammation is a complex inflammatory process in the central nervous system (CNS) where microglia may play a critical role. GPETAFLR is a peptide isolated from Lupinus angustifolius L. protein hydrolysates with functional activity in mononuclear phagocytes. However, it is unknown whether GPETAFLR has neuroprotective effects. Methods: We analysed the potential anti-neuroinflammatory activity of GPETAFLR by using two different models of neuroinflammation: BV-2 microglial cells and mice with high-fat diet (HFD)-induced obesity. Results: GPETAFLR hampered LPS-induced upregulation of pro-inflammatory and M1 marker genes in BV-2 cells. This effect was accompanied by an unchanged expression of anti-inflammatory IL-10 gene and by an increased expression of M2 marker genes. GPETAFLR also increased the transcriptional activity of M2 marker genes, while the microglia population remained unchanged in number and M1/M2 status in brain of mice with high-fat diet (HFD)-induced obesity. Furthermore, GPETAFLR counteracted HFD-induced downregulation of IL-10 and upregulation of pro-inflammatory markers in the mouse brain, both at gene and protein levels. Discussion: This is the first report describing that a peptide from plant origin robustly restrained the pro-inflammatory activation of microglial cells in cultures and in brain. Our data suggest that GPETAFLR might be instrumental in maintaining CNS homeostasis by inhibiting neuroinflammation.

Asiaticoside loading into polylactic-co-glycolic acid electrospun nanofibers attenuates host inflammatory response and promotes M2 macrophage polarization.

Polylactic-co-glycolic acid (PLGA) is one of the most promising synthetic materials for tissue engineering due to its excellent biocompatibility, good mechanical properties, and tunable biodegradation time. However, the accumulation of PLGA degradative products could cause significant host inflammatory response, a microenvironment favoring tissue fibrosis that is mainly mediated by M1 subtype macrophage. Drug loading is an emerging technology to modify electrospun nanofibers, and asiaticoside (AS) was demonstrated as an anti-inflammatory drug. This study investigated the potential effect of AS incorporating into PLGA electrospun nanofibers on modulating host inflammatory response. The results showed that AS co-electrospun with PLGA nanofibers could significantly reduce the infiltration of inflammatory cells at the implantation site as opposed to the site of regular PLGA nanofibers. In particular, immunohistochemistry demonstrated decreased M1 macrophage infiltration whereas increased M2 macrophage infiltration in the implantation site of AS-PLGA nanofibers when compared to the PLGA implantation site. In vitro study also revealed that culture of human fibroblasts on PLGA nanofibers resulted in significantly enhanced gene expression of inflammatory cytokines when compared to non-seeded fibroblasts, but these genes were significantly downregulated when seeded on AS-PLGA. Furthermore, culture of macrophage on AS-PLGA led to upregulated M2 marker gene expression and downregulated M1 marker gene expression. Collectively, these results indicate that, AS might be an ideal drug for loading into electrospun polymer nanofibers and thus favoring for tissue regeneration via mediating macrophage polarization.

Effects of stress and cortisol on the polarization of carp macrophages

In teleost fish, myelopoiesis is maintained both in the head (HK) and trunk kidney (TK), but only the HK holds the endocrine cells that produce the stress hormone cortisol. We now compared the effects of prolonged restraint stress (in vivo) and cortisol (in vitro) on the polarization of HK and TK-derived carp macrophages.Monocytes/macrophages from both sources were treated in vitro with cortisol, lipopolysaccharide or with both factors combined. In vivo, fish were challenged by a prolonged restraint stress. Gene expression of several markers typical for classical M1 and alternative M2 macrophage polarization, as well as glucocorticoid receptors, were measured.Cells from both sources did not differ in the constitutive gene expression of glucocorticoid receptors, whereas they significantly differed in their response to cortisol and stress. In the LPS-stimulated HK monocytes/macrophages, cortisol in vitro counteracted the action of LPS while the effects of cortisol on the activity of TK monocytes/macrophages were less explicit.In vivo, restraint stress up-regulated gene expression of M2 markers in freshly isolated HK monocytes/macrophages, while at the same time it did not affect TK monocytes/macrophages. Moreover, LPS-stimulated HK monocytes/macrophages from stressed animals showed only minor differences in the gene expression of M1 and M2 markers, compared to LPS-treated monocytes/macrophages from control fish. In contrast, stress-induced changes in TK-derived LPS-treated cells were more pronounced. However, these changes did not clearly indicate whether in TK monocytes/macrophages stress will stimulate classical or alternative polarization.Altogether, our results imply that cortisol in vitro and stress in vivo direct HK, but not TK, monocytes/macrophages to the path of alternative polarization. These findings reveal that like in mammals, also in fish the glucocorticoids form important stimulators of alternative macrophage polarization.

188-OR: Partial Depletion of CD206 M2-Like Macrophages Enhances Preadipocyte Differentiation via Reduction of Wnt Signaling

Several pieces of evidence indicate that Wnt/β-catenin pathway may regulate the mesenchymal stem cells fate proliferation in vivo. The Wnt signaling is also known to inhibit preadipocyte differentiation into adipocyte. Adipose tissue macrophages play important roles in the maintenance of tissue homeostasis by regulating insulin sensitivity via their anti-inflammatory actions. However, how CD206 M2-like macrophages linked to Wnt signaling in maintaining the preadipocyte differentiation, remains elusive. In the present study, we aimed to investigate the physiological role of CD206 linked to Wnt signaling in preadipocyte differentiation. We show that C1q, which can mediate Wnt signaling, is abundantly expressed in CD206 M2-like macrophages in epididymal adipose tissue. Then, we examined whether CD206 M2-macrophages depletion activate or inhibit Wnt signaling and preadipocyte differentiation. Here, we show that the expression of M2 marker genes was reduced with a reduced expression of Wnt signaling genes in adipose tissue of CD206-reduced mice. Western blot analysis confirmed reduced protein level of β-catenin in CD206-reduced mice that can contribute to regulate the preadipocyte/progenitor’s activity. We presumed that the reduction of C1q-induced Wnt signaling promoted adipogenesis. We also found that depletion of CD206 M2-like macrophages resulted in the generation of the smaller adipocyte, upregulated expression of metabolically favorable genes and enhanced insulin sensitivity in both chow and high-fat diet-fed CD206-reduced mice. Taken together, we show that partial depletion of CD206 M2-like macrophages inhibits Wnt/β-catenin pathway activity in mice and increases preadipocyte differentiation, thus ameliorating insulin resistance in lean and obese mice. Disclosure A. Nawaz: Research Support; Spouse/Partner; Astellas Pharma Inc., Astellas Pharma Inc., AstraZeneca, Daiichi Sankyo Company, Limited, Eli Lilly and Company. Research Support; Self; Japan Society for the Promotion of Science. Research Support; Spouse/Partner; Merck Sharp &amp; Dohme Corp., Mitsubishi Tanabe Pharma Corporation, Novartis Pharmaceuticals Corporation, Novo Nordisk A/S, Ono Pharmaceutical Co., Ltd., Pfizer Inc., Sanofi, Shionogi &amp; Co., Ltd., Takeda Pharmaceutical Company Limited. T. Nakagawa: None. K. Yagi: None. S. Fujisaka: None. K. Tobe: Advisory Panel; Self; Novo Nordisk A/S. Board Member; Self; Japan Diabetes Society. Research Support; Self; Bristol-Myers Squibb Company. K. Okabe: None. T. Kado: None. J. Liu: None. M. Bilal: None. Funding Japan Society for the Promotion of Science

Astragalus Polysaccharide RAP Induces Macrophage Phenotype Polarization to M1 via the Notch Signaling Pathway.

Macrophages occur in polarized phenotypes, whose characteristics determine the role they play in tumor growth. The M1 phenotype macrophages promote tumoricidal responses and suppress tumor growth. Our previous study showed that a polysaccharide isolated from Radix Astragali, named RAP, was itself non-cytotoxic but induced RAW264.7 cells’ cytotoxicity against cancer cells. The current study was undertaken to determine its mechanism. Series studies was conducted to show that RAP is able to induce much higher gene expression of M1 markers, including iNOS, IL-6, TNF-a, and CXCL10, compared with the control group. When RAP-induced BMDMs were transplanted together with 4T1 tumor cells in BALB/c mice, both tumor volume and tumor weight decreased. Further studies indicated that RAP induces the Notch signaling pathway in RAW264.7 cells. The function of Notch signaling in macrophage polarization was confirmed by using γ-secretase inhibitor. These results suggested that Astragalus polysaccharide RAP induces macrophage’s polarization to M1 phenotype via the Notch signaling pathway.

Effect of Propofol on the Production of Inflammatory Cytokines by Human Polarized Macrophages.

Macrophages are key immune system cells involved in inflammatory processes. Classically activated (M1) macrophages are characterized by strong antimicrobicidal properties, whereas alternatively activated (M2) macrophages are involved in wound healing. Severe inflammation can induce postoperative complications during the perioperative period. Invasive surgical procedures induce polarization to M1 macrophages and associated complications. As perioperative management, it is an important strategy to regulate polarization and functions of macrophages during inflammatory processes. Although propofol has been found to exhibit anti-inflammatory activities in monocytes and macrophages, it is unclear whether propofol regulates the functions of M1 and M2 macrophages during inflammatory processes. This study therefore investigated the effects of propofol on human macrophage polarization. During M1 polarization, propofol suppressed the production of IL-6 and IL-1β but did not affect TNF-α production. In contrast, propofol did not affect the gene expression of M2 markers, such as IL-10, TGF-β, and CD206, during M2 polarization. Propofol was similar to the GABAA agonist muscimol in inducing nuclear translocation of nuclear factor-E2-related factor 2 (Nrf2) and inhibiting IL-6 and IL-1β, but not TNF-α, production. Knockdown of Nrf2 using siRNA significantly reduced the effect of propofol on IL-6 and IL-1β production. These results suggest that propofol prevents inflammatory responses during polarization of human M1 macrophages by suppressing the expression of IL-6 and IL-1β through the GABAA receptor and the Nrf2-mediated signal transduction pathway.

Role of Host GPR120 in Mediating Dietary Omega-3 Fatty Acid Inhibition of Prostate Cancer.

GPR120, a G protein-coupled receptor for long-chain polyunsaturated fatty acids (FAs), mediates the anti-inflammatory effects of omega-3 (ω-3) FAs. We investigated whether host or tumor GPR120 plays a role in the anti-prostate cancer effects of ω-3 FAs. MycCap prostate cancer allografts were grown in immunocompetent wild-type (WT) and GPR120 knockout (KO) mice fed ω-3 (fish oil) or ω-6 (corn oil) diets. Immune cell infiltration was quantified by flow cytometry, and gene expression of immune cell markers in isolated tumor-associated macrophages (TAMs) was quantified by quantitative real-time polymerase chain reaction. Archived tissue from a fish oil intervention trial was used to correlate gene expression of GPR120 with cell cycle progression (CCP) genes and Ki67 index (n = 11-15 per group). All statistical tests were two-sided. In WT mice (n = 7 per group), dietary ω-3 FAs decreased MycCap allograft tumor growth (mean [SD] final tumor volume ω-6 = 491 [437] mm3 vs ω-3 = 127 [77] mm3, P = .04), whereas in global GPR120KO mice (n = 7 per group) ω-3 FAs had no anticancer effects. Dietary ω-3 FAs inhibited GPR120KO-MycCaP allografts grown in WT mice (n = 8 per group; mean [SD] final tumor volume ω-6 = 776 [767] mm3 vs ω-3 = 36 [34] mm3, P = .02). Omega-3 FA treatment decreased the number of M2-like TAMs in tumor tissue and gene expression of M2 markers in isolated TAMs compared with ω-6 controls in WT (n = 7 per group) but not in GPR120KO mice (n = 7 per group). In human tissue, higher expression of stromal GPR120 correlated with greater reduction in expression of CCP genes in men with prostate cancer on a high-ω-3 diet (r = -.57, P = .04). Host GPR120 plays a central role in the anti-prostate cancer effects of dietary ω-3 FAs. Future studies are required to determine if the anticancer effects of ω-3 FAs are mediated through inhibition of M2-like macrophages and if host GPR120 status predicts anticancer effects of dietary ω-3 FAs in men with prostate cancer.

Reduced inflammatory response by incorporating magnesium into porous TiO2 coating on titanium substrate.

The implant materials with proper anti-inflammatory and osteogenic properties may be promising for orthopedic applications. The inflammatory response induced by biomaterials has been regarded as one of the critical factors in determining in vivo fate of implants. Therefore, a novel bone biomaterial should have inflammation regulatory effects instead of being completely bio-inert. In the present work, the inflammation regulatory effects of exogenous magnesium (Mg) ions were investigated. Under the stimulation of lipopolysaccharide (LPS), macrophages exposed to Mg2+ exhibited down-regulated gene expressions of M1 markers (CD86, CD11c and inducible nitric oxide synthase (iNOS)) and pro-inflammatory cytokines (tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and IL-1β), up-regulated gene expression of M2 marker CD163 and decreased TNF-α release, indicating that Mg2+ could switch macrophages from M1 to M2 phenotype. Thereafter, micro-arc oxidation (MAO) technique was employed to fabricate Mg-containing ceramic coatings on titanium substrates. Macrophages grown on Mg-containing surface were switched from M1 to M2 phenotype with the stimulation of LPS, evidenced by suppressed gene expressions of M1 markers (CD86, CD11c and iNOS) and pro-inflammatory cytokines (TNF-α and IL-1β), promoted gene expression of M2 marker CD163 and decreased TNF-α release. Moreover, gene expressions of bone morphogenetic protein-2 (BMP-2), BMP-6 and vascular endothelial growth factor (VEGF) were up-regulated on Mg incorporated MAO surface without LPS stimulation. Together, Mg could be used as an anti-inflammatory agent for suppressing inflammation and mediating osteogenesis. The integration of Mg in biomaterials could endow bone biomaterials with anti-inflammatory property.

Gefitinib inhibits M2-like polarization of tumor-associated macrophages in Lewis lung cancer by targeting the STAT6 signaling pathway.

M2-like polarized tumor-associated macrophages (TAMs) play a pivotal role in promoting cancer cell growth, invasion, metastasis and angiogenesis. The identification of M2-like TAMs during tumor progression is an attractive approach for cancer therapy. In this study, we investigated the relevance of macrophage polarization and the antitumor effect of gefitinib in Lewis Lung cancer (LLC) in vitro and in vivo. Gefitinib at a concentration below 2.5 μmol/L did not cause significant growth inhibition on LLC and RAW 264.7 cell lines and bone marrow-derived macrophage (BMDMs). However, a small concentration of gefitinib (0.62 μmol/L) significantly inhibited IL-13-induced M2-like polarization of macrophages, evidenced by the decreased expression of the M2 surface markers CD206 and CD163, down-regulation of specific M2-marker genes (Mrc1, Ym1, Fizz1, Arg1, IL-10 and CCL2) as well as inhibition of M2-like macrophage-mediated invasion and migration of LLC cells. In RAW 264.7 cells, gefitinib inhibits IL-13-induced phosphorylation of STAT6, which was a crucial signaling pathway in macrophage M2-like polarization. In LLC mice metastasis model, oral administration of gefitinib (75 mg·kg-1·d-1, for 21 d) significantly reduced the number of lung metastasis nodules, down-regulated the expression of M2 marker genes and the percentages CD206+ and CD68+ macrophages in tumor tissues. These results demonstrated that gefitinib effectively inhibits M2-like polarization both in vitro and in vivo, revealing a novel potential mechanism for the chemopreventative effect of gefitinib.

Abstract 142: IL-10 Regulates Inflammation to Improve LV Physiology After Myocardial Infarction by Stimulating M2 Macrophage Polarization and Fibroblast Activation

Inflammation resolution is important for scar formation following myocardial infarction (MI) and requires the coordinated actions of macrophages and fibroblasts. In this study, we hypothesized that exogenous interleukin-10 (IL-10), an anti-inflammatory cytokine, promotes post-MI repair through actions on these cardiac cell types. To test this hypothesis, C57BL/6J mice (male, 3-6 months old, n=24/group) were treated with saline or IL-10 (50 μg/kg/day) by osmotic mini-pump infusion starting at day 1 post-MI and sacrificed at day 7 post-MI. IL-10 infusion doubled plasma IL-10 concentrations by day 7 post-MI. Despite similar infarct areas and mortality rates, IL-10 treatment significantly decreased LV dilation (1.6-fold for end-systolic volume and 1.4-fold for end-diastolic volume) and improved ejection fraction 1.8-fold (both p&lt;0.05). IL-10 treatment attenuated inflammation at day 7 post-MI, evidenced by decreased numbers of Mac-3+ macrophages in the infarct (p&lt;0.05). LV macrophages isolated from day 7 post-MI mice treated with IL-10 showed significantly elevated gene expression of M2 markers (Arg1, Ym1 and TGF-β1; all p&lt;0.05). We further performed RNA-seq analysis on post-MI cardiac macrophages and identified 410 significantly different genes (155 increased, 225 decreased by IL-10 treatment). By functional network analysis grouping, the majority of genes (133 out of 410) were part of the cellular assembly and repair functional group. Of these, Hyaluronidase 3 (Hyal 3) showed the largest fold change between groups. IL-10 treatment decreased Hyal3 by 28%, which reduced hyaluronan degradation and limited collagen deposition (all p&lt;0.05). In addition, ex vivo IL-10 treatment increased fibroblast activation (proliferation, migration, and collagen production), an effect that was both directly and indirectly influenced by macrophage M2 polarization. Combined, our results indicate that in vivo infusion of IL-10 post-MI improves the LV microenvironment to dampen inflammation and facilitate cardiac wound healing.