M1-related Cytokines Research Articles

Macrophage polarization in human gingival tissue in response to periodontal disease.

Although accumulating evidence indicates that macrophages are central players in the destructive and reparative phases of periodontal disease, their polarization states at different stages of periodontal inflammation remain unclear. We collected gingival biopsies from patients with chronic periodontitis (P group), gingivitis (G group), or periodontally healthy individuals (H group). Polarized macrophages were identified through immunofluorescence. M1- and M2-related cytokines were detected by immunohistochemistry. Compared with the H group, the P group had more M1 cells (higher M1/M2 ratio) and significantly higher TNF-α, IFN-γ, IL-6, and IL-12 levels. Although the G group also exhibited higher TNF-α and IL-12 levels than the H group, they had similar M1/M2 ratios. The M1/M2 ratio and IFN-γ and IL-6 levels were significantly higher in the P than the G group. Among M2-related cytokines, IL-4 levels were significantly higher in the G than the H group. The M1/M2 ratio was positively correlated with clinical probing depth (PD), and both were positively correlated with IFN-γ and IL-6. PD was negatively correlated with IL-4. Macrophage polarization in gingival tissue may be responsible for the development and progression of inflammation-induced tissue destruction, and modulating macrophage function may be a potential strategy for periodontal disease management.

Axl Involved in Mineral Trioxide Aggregate Induces Macrophage Polarization

IntroductionIn this study, we examined the effect of mineral trioxide aggregate (MTA) on macrophage polarization and the potential involvement of Axl/nuclear factor kappa B (NF-κB) signaling in mediating the effect of MTA. MethodsThe human monocyte cell line THP-1 was cultured with MTA solution for 1, 2, or 3 days, and the population change of M2 macrophages was analyzed by flow cytometry. Expression of M2 cytokines was examined by enzyme-linked immunosorbent assay. Phagocytosis and angiogenesis-induction ability were also assayed. The involvement of Axl/NF-κB signaling in MTA-treated cells was examined by analyzing phosphorylation status of Axl, Akt, IKKα/β, and IκBα. Specific inhibitors for Axl/Akt/NF-κB signaling were added to MTA-treated THP-1 cells, and their cytokine expression change was examined. ResultsFlow cytometry analysis showed that MTA treatment increased CD206＋ cells in a time-dependent way. After MTA treatment, the expression of M2-related cytokines was up-regulated. MTA also enhanced phagocytic ability and the ability of THP-1 cells to induce angiogenesis. Treatment of MTA led to activate Axl/Akt/NF-kB signal axis by phosphorylation of Axl, Akt, IKKα/β, IκBα, and p65. In addition, MTA-induced interleukin 10, transforming growth factor beta, and vascular endothelial growth factor expression was suppressed as specific inhibitors were added. ConclusionsOur findings indicate that MTA is able to induce macrophage polarization toward the M2 phenotype, with up-regulation of interleukin 10, transforming growth factor beta, and vascular endothelial growth factor, and that Axl/Akt/NF-κB signaling participates in this process. These results provide the cellular and molecular basis of MTA's anti-inflammatory action in clinical applications.

Anti-inflammatory signaling by mammary tumor cells mediates prometastatic macrophage polarization in an innovative intraductal mouse model for triple-negative breast cancer

BackgroundMurine breast cancer models relying on intraductal tumor cell inoculations are attractive because they allow the study of breast cancer from early ductal carcinoma in situ to metastasis. Using a fully immunocompetent 4T1-based intraductal model for triple-negative breast cancer (TNBC) we aimed to investigate the immunological responses that guide such intraductal tumor progression, focusing on the prominent role of macrophages.MethodsIntraductal inoculations were performed in lactating female mice with luciferase-expressing 4T1 mammary tumor cells either with or without additional RAW264.7 macrophages, mimicking basal versus increased macrophage-tumor cell interactions in the ductal environment. Imaging of 4T1-derived luminescence was used to monitor primary tumor growth and metastases. Tumor proliferation, hypoxia, disruption of the ductal architecture and tumor immune populations were determined immunohistochemically. M1- (pro-inflammatory) and M2-related (anti-inflammatory) cytokine levels were determined by Luminex assays and ELISA to investigate the activation state of the macrophage inoculum. Levels of the metastatic proteins matrix metalloproteinase 9 (MMP-9) and vascular endothelial growth factor (VEGF) as well as of the immune-related disease biomarkers chitinase 3-like 1 (CHI3L1) and lipocalin 2 (LCN2) were measured by ELISA to evaluate disease progression at the protein level.ResultsMice intraductally co-injected with macrophages showed severe splenomegaly with faster ductal breakthrough of tumor cells and increased metastases in axillary lymph nodes and lungs. These mice showed higher M1-related cytokines in the early disease stages (at 1 to 3 weeks post-inoculation) due to the pro-inflammatory nature of RAW264.7 macrophages with increased Ly6G-positive neutrophils and decreased anti-inflammatory macrophages in the tumor microenvironment. However, upon metastasis (at 5 weeks post-inoculation), a prominent increase in M2-related cytokine levels was detected and established a tumor microenvironment with similar immune populations and cytokine responses as in mice which received only 4T1 tumor cells. The observed tumor-associated immune responses and the increased metastasis were associated with significantly induced local and systemic levels of MMP-9, VEGF, CHI3L1 and LCN2.ConclusionsThe current experimental study with an innovative immunocompetent intraductal model for TNBC pinpoints towards a metastasis-supporting M1 to M2 macrophage polarization in the mammary ducts mediated by 4T1-derived signaling. We propose to explore this process as immunotherapeutic target.

MiR-98 modulates macrophage polarization and suppresses the effects of tumor-associated macrophages on promoting invasion and epithelial\u2013mesenchymal transition of hepatocellular carcinoma

BackgroundTumor-associated macrophages (TAMs) are generally recognized as a promoter of tumor progression. miR-98 has been shown to suppress the proliferation, migration, invasion and epithelial–mesenchymal transition (EMT) of hepatocellular carcinoma (HCC) cells. Here, we aim to investigate the role of miR-98-mediated macrophage polarization in HCC progression.MethodsHuman blood monocytes were isolated from healthy male donors and incubated with culture medium collected from HepG2 cells for 7 days. The phenotype of the macrophages was detected. The protein expression was detected by Western blot. Levels of cytokines secreted in culture medium were measured using the specific enzyme-linked immunosorbent assay kits. To explore the role of miR-98 in HCC-conditioned TAMs, HCC cells HepG2 and SMMC7721 were cultured with conditioned medium from HCC-conditioned TAMs that had been transfected with miR-98 mimic/inhibitor. Cell proliferation, migration and invasion assays were performed.ResultsHCC-conditioned TAMs possessed M2-like phenotype, including increased protein expression of CD163 and TNF-αlow, IL-1βlow, TGF-βhigh and IL-10high phenotype. HCC-conditioned TAMs also promoted proliferation, migration, invasion and EMT of HepG2 and SMMC7721 cells. Furthermore, miR-98 modulated macrophage polarization from M2 to M1 in HCC-conditioned TAMs, as evidenced by the alteration of M1- or M2-related cytokines. Moreover, miR-98 mimic significantly suppressed the HCC-conditioned TAMs-mediated promotion of cell migration, invasion and EMT in HepG2 and SMMC7721 cells compared with negative control, whereas miR-98 inhibitor exerted reversed effects.ConclusionsmiR-98 may play a vital role in regulating macrophage polarization, thereby suppressing the TAMs-mediated promotion of invasion and EMT in HCC.

The Flavonoid Quercetin Ameliorates Liver Inflammation and Fibrosis by Regulating Hepatic Macrophages Activation and Polarization in Mice

At present, there are no effective antifibrotic drugs for patients with chronic liver disease; hence, the development of antifibrotic therapies is urgently needed. Here, we performed an experimental and translational study to investigate the potential and underlying mechanism of quercetin treatment in liver fibrosis, mainly focusing on the impact of quercetin on macrophages activation and polarization. BALB/c mice were induced liver fibrosis by carbon tetrachloride (CCl4) for 8 weeks and concomitantly treated with quercetin (50 mg/kg) or vehicle by daily gavage. Liver inflammation, fibrosis, and hepatic stellate cells (HSCs) activation were examined. Moreover, massive macrophages accumulation, M1 macrophages and their related markers, such as tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, and monocyte chemotactic protein-1 (MCP-1) in livers were analyzed. In vitro, we used Raw 264.7 cells to examine the effect of quercetin on M1-polarized macrophages activation. Our results showed that quercetin dramatically ameliorated liver inflammation, fibrosis, and inhibited HSCs activation. These results were attributed to the reductive recruitment of macrophages (F4/80+ and CD68+) into the liver in quercetin-treated fibrotic mice confirmed by immunostaining and expression levels of marker molecules. Importantly, quercetin strongly inhibited M1 polarization and M1-related inflammatory cytokines in fibrotic livers when compared with vehicle-treated mice. In vitro, studies further revealed that quercetin efficiently inhibited macrophages activation and M1 polarization, as well as decreased the mRNA expression of M1 macrophage markers such as TNF-α, IL-1β, IL-6, and nitric oxide synthase 2. Mechanistically, the inhibition of M1 macrophages by quercetin was associated with the decreased levels of Notch1 expression on macrophages both in vivo and in vitro. Taken together, our data indicated that quercetin attenuated CCl4-induced liver inflammation and fibrosis in mice through inhibiting macrophages infiltration and modulating M1 macrophages polarization via targeting Notch1 pathway. Hence, quercetin holds promise as potential therapeutic agent for human fibrotic liver disease.

Targeting the NF-κb-Dependent HIF-1β Pathway Reprograms Macrophage Polarization Induced By Oxidized LDL

Introduction: Plasticity is one of the hallmarks of macrophages, an essential component of innate and adaptive immunity. In response to various stimuli, macrophages can differentiate or polarize to either pro-inflammatory M1 or anti-inflammatory M2 phenotype, which determines whether inflammation is initiated and promoted or terminated and resolved. Of note, the phenotype of polarized M1-M2 macrophages may be reversed in certain circumstances, providing an opportunity to treat inflammatory disorders (e.g., atherosclerosis) and inflammation-related diseases (e.g., cancer) by targeting macrophage polarization. Emerging evidence supports that transcriptional regulation play an important role in polarization and function of macrophages via reprogramming expression of pro- versus anti-inflammatory genes. However, despite well-established cross-talk between two major transcriptional factors, NF-κB and hypoxia-inducible factor (HIF), it remains unclear whether NF-κB interacts with HIFs (particularly HIF-1β, a regulatory subunit of the active HIF complex) in reprogramming macrophages. Here, we investigated the mechanism underlying macrophage activation induced by oxidized low density lipoprotein (oxLDL), a central event of uncontrolled inflammation in atherosclerosis.Materials and Methods: The murine macrophage cell line RAW264.7 and human THP-1 cell line-derived macrophages were employed. After exposed to oxLDL, cells were analyzed by qPCR, Western blot, flow cytometry (Cytometric Bead Array, CBA), and ELISA analyses to monitor expression of M1 and M2 markers and related cytokines, as well as activation of the NF-κB and HIF pathways. The shRNA approach was used to knock down expression of target genes for functional evaluation. The findings from in vitro experiments involving cell lines were then validated in primary samples obtained from healthy donors (n = 10) and patients with coronary heart disease (n = 22) and stroke (n = 11).Results: Exposure to oxLDL triggered M1 polarization of murine and human macrophages, characterized by expression of iNOS and robust production of M1 pro-inflammatory cytokines (e.g., TNF-α, MCP-1, IL-1β, IL-6) but not M2 anti-inflammatory cytokines (e.g., IL-10, TGF-β). In contrast, protein level of the M2 marker Arg1 was clearly decreased after treated with oxLDL. Notably, exposure of macrophages to oxLDL resulted in markedly increased expression of HIF-1α and -1β, in association with activation of both canonical and non-canonical NF-κB pathways. Functionally, whereas inhibition of NF-κB activation by the IKK inhibitor parthenolide almost completely prevented M1 polarization and promoted M2 polarization, knockdown of HIF-1β by shRNA also largely reversed macrophage polarization from M1 to M2 after exposed to oxLDL. These results were confirmed in human macrophages differentiated by PMA from primary peripheral blood monocytes obtained from patients with coronary heart disease or ischemic stroke, and normal donors. These events were accompanied by a clear reversal of oxLDL-induced morphological changes of macrophages. Mechanistically, inhibition of NF-κB activation dramatically diminished expression of HIF-1α and -1β induced by oxLDL. However, while shRNA knockdown of HIF-1β sharply blocked HIF-1α expression in macrophages exposed to oxLDL, it failed to impair activation of NF-κB. These findings indicate that oxLDL-induced HIF-1β expression is dependent on NF-κB activation, which in turn activates the HIF pathway via HIF-1α up-regulation probably by stabilizing HIF-1α protein.Conclusion: HIF-1β (encoded by ARNT) is identified for the first time as a novel target that reprograms M1-M2 polarization of macrophages, at least after exposure to oxLDL, a risk factor of atherosclerosis. HIF-1β is further demonstrated to act as downstream of NF-κB to induce activation of the HIF pathway. Importantly, these findings suggest that HIF-1β might serve as a therapeutic target for the treatment of inflammatory disorders such as atherosclerosis, and probably immune diseases and cancer as well. DisclosuresNo relevant conflicts of interest to declare.

Echinacea purpurea Extract Polarizes M1 Macrophages in Murine Bone Marrow-Derived Macrophages Through the Activation of JNK.

Echinacea purpurea is an indigenous North American purple cone flower used by North Americans for treatment of various infectious diseases and wounds. This study investigated the effect of polysaccharide enriched extract of Echinacea purpurea (EE) on the polarization of macrophages. The results showed that 100 µg/mL of EE could markedly activate the macrophage by increasing the expression of CD80, CD86, and MHCII molecules. Meanwhile, EE upregulated the markers of classically activated macrophages (M1) such as CCR7 and the production of IL-1β, IL-6, IL-12p70, TNF-αand NO. The functional tests showed that EE enhanced the phagocytic and intracellular bactericidal activity of macrophage against ST. Furthermore, we demonstrated that JNK are required for EE-induced NO and M1-related cytokines production. Together, these results demonstrated that EE can polarize macrophages towards M1 phenotype, which is dependent on the JNK signaling pathways. J. Cell. Biochem. 118: 2664-2671, 2017. © 2017 Wiley Periodicals, Inc.

Metformin-treated cancer cells modulate macrophage polarization through AMPK-NF-κB signaling.

Accumulating evidence is indicating metformin to possess the potential ability in preventing tumor development and suppressing cancer growth. However, the exact mechanism of its antitumorigenic effects is still not clear. We found that metformin suppressed the ability of cancer to skew macrophage toward M2 phenotype. Metformin treated cancer cells increased macrophage expression of M1-related cytokines IL-12 and TNF-α and attenuated M2-related cytokines IL-8, IL-10, and TGF-β expression. Furthermore, metformin treated cancer cells displayed inhibited secretion of IL-4, IL-10 and IL-13; cytokines important for inducing M2 macrophages. Conversely, M1 inducing cytokine IFN-γ was upper-regulated in cancer cells. Additionally, through increasing AMPK and p65 phosphorylation, metformin treatment activated AMPK-NF-κB signaling of cancer cells that participate in regulating M1 and M2 inducing cytokines expression. Moreover, Compound C, an AMPK inhibitor, significantly increased IL-4, IL-10, and IL-13 expression while BAY-117082, an NF-κB inhibitor, decreased expression. In metformin-treated tumor tissue, the percentage of M2-like macrophages decreased while M1-like macrophages increased. These findings suggest that metformin activates cancer AMPK-NF-κB signaling, a pathway involved in regulating M1/M2 expression and inducing genes for macrophage polarization to anti-tumor phenotype.

Iron Reduces M1 Macrophage Polarization in RAW264.7 Macrophages Associated with Inhibition of STAT1.

Iron metabolism in inflammation has been mostly characterized in macrophages exposed to pathogens or inflammatory conditions. The aim of this study is to investigate the cross-regulatory interactions between M1 macrophage polarization and iron metabolism. Firstly, we characterized the transcription of genes related to iron homeostasis in M1 RAW264.7 macrophages stimulated by IFN-γ. The molecular signature of M1 macrophages showed high levels of iron storage (ferritin), a low level of iron export (ferroportin), and changes of iron regulators (hepcidin and transferrin receptors), which favour iron sequestration in the reticuloendothelial system and are benefit for inflammatory disorders. Then, we evaluated the effect of iron on M1 macrophage polarization. Iron significantly reduced mRNA levels of IL-6, IL-1β, TNF-α, and iNOS produced by IFN-γ-polarized M1 macrophages. Immunofluorescence analysis showed that iron also reduced iNOS production. However, iron did not compromise but enhanced the ability of M1-polarized macrophages to phagocytose FITC-dextran. Moreover, we demonstrated that STAT1 inhibition was required for reduction of iNOS and M1-related cytokines production by the present of iron. Together, these findings indicated that iron decreased polarization of M1 macrophages and inhibited the production of the proinflammatory cytokines. The results expanded our knowledge about the role of iron in macrophage polarization.

Pinocembrin protects hemorrhagic brain primarily by inhibiting toll-like receptor 4 and reducing M1 phenotype microglia

Neuroinflammation is a major contributor to intracerebral hemorrhage (ICH) progression, but no drug is currently available to reduce this response and protect against ICH-induced injury. Recently, the natural product pinocembrin has been shown to ameliorate neuroinflammation and is undergoing a phase II clinical trial for ischemic stroke treatment. In this study, we examined the efficacy of pinocembrin in an ICH model, and further examined its effect on microglial activation and polarization. In vivo, pinocembrin dose-dependently reduced lesion volume by ∼47.5% and reduced neurologic deficits of mice at 72h after collagenase-induced ICH. The optimal dose of pinocembrin (5mg/kg) suppressed microglial activation as evidenced by decreases in CD68-positive microglia and reduced proinflammatory cytokines tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6. Pinocembrin also reduced the number of classically activated M1-like microglia without affecting M2-like microglia in the perilesional region. Additionally, pinocembrin decreased the expression of toll-like receptor (TLR)4 and its downstream target proteins TRIF and MyD88. The protection by pinocembrin was lost in microglia-depleted mice and in TLR4lps-del mice, and pinocembrin failed to decrease the number of M1-like microglia in TLR4lps-del mice. In lipopolysaccharide-stimulated BV-2 cells or primary microglia, pinocembrin decreased M1-related cytokines and markers (IL-1β, IL-6, TNF-α, and iNOS), NF-κB activation, and TLR4 expression, but it did not interfere with TLR4/MyD88 and TLR4/TRIF interactions or affect microglial phagocytosis of red blood cells. Inhibition of the TLR4 signaling pathway and reduction in M1-like microglial polarization might be the major mechanism by which pinocembrin protects hemorrhagic brain. With anti-inflammatory properties, pinocembrin could be a promising new drug candidate for treating ICH and other acute brain injuries.

Nanoparticle-Delivered IRF5 siRNA Facilitates M1 to M2 Transition, Reduces Demyelination and Neurofilament Loss, and Promotes Functional Recovery After Spinal Cord Injury in Mice.

Macrophage activation and persistent inflammation contribute to the pathogenesis of spinal cord injury (SCI), and different phenotypes of macrophages play diverse roles in the pathological process of SCI. After SCI, there is an acute phase of alternatively activated (M2) macrophage infiltration, followed by a long-lasting phase of classically activated (M1) macrophage accumulation in the wound. The long-lasting predominance of M1 macrophages may derail healing and compromise organ functions. Based on the previous findings that the transcription factor interferon regulatory factor 5 (IRF5) up-regulates genes associated with M1 macrophages, we attempted to examine the effect of silencing IRF5 on SCI progression. IRF5 expression was assessed with Western blotting or immunohistochemistry. Macrophage phenotypes were measured with flow cytometry or immunohistochemistry. M1- or M2-related cytokines were measured with a Luminex assay kit. IRF5 siRNA was delivered into the macrophages infiltrated into the wound of SCI mice through lipidoid nanoparticle. Locomotor functions were measured with Basso Mouse Scale (BMS) scoring. Myelination was assessed with luxol fast blue staining. Myelin binding protein, neurofilaments, synaptic markers, and cytokines in the wound area were measured with Western blotting. The Mann-Whitney U test was used for statistical analyses. After SCI, significant elevation of IRF5 was evident on day 1, peaked on day 7, and gradually decreased thereafter. Similar dynamic change in the expression of CD86, a typical M1 marker, was observed. In contrast, there was a transient increase in the expression of CD206, a typical M2 marker, which peaked 6h after SCI, and returned to baseline within 1day. Macrophages isolated from the epicenter at day 3 after SCI were predominantly M1 phenotype, and a siRNA-mediated knockdown of IRF5 resulted in a reduced expression of M1 macrophage markers and increased expression of M2 macrophage markers. Nanoparticle-mediated delivery of IRF5 siRNA to SCI mouse model resulted in a dramatic decrease in the number of M1 macrophages and a significant increase in the number of M2 macrophages in the wound. This was associated with a robust inflammation resolution, attenuation of demyelination and neurofilament loss, and significant improvement of locomotor function (p < 0.05). IRF5 may serve as a therapeutic target to promote post-SCI recovery.

Circulating cell-free DNA indicates M1/M2 responses during septic peritonitis

Circulating cell-free DNA (cfDNA) has been widely suggested as clinical indicator in diseases, including sepsis. It was thought that the cfDNA was coming from the cell lysis, necrosis and apoptosis caused by tissue damages during sepsis. M1 or M2 macrophage-type responses kill or repair in vivo, which is highly relevant with the tissue damages in sepsis. The correlation between cfDNA and M1/M2 responses during sepsis was never investigated. Here, we used bacteria injection induced septic peritonitis mouse model in both M1-dominant C57bl/6 and M2-dominant Balb/c mouse strains. We found that M2-dominant Balb/c mice showed better prognosis of septic peritonitis than C57bl/6 mice, which is corresponded with lower level of cfDNA in septic Balb/c mice compared to septic C57bl/6 mice. By assessing the M1 and M2 related cytokines in both septic Balb/c and C57bl/6 mice, we found out that Balb/c mice has lower tumor necrosis factor α (TNFα) and higher interleukin 10 (IL-10) productions than C57bl/6 mice during septic peritonitis. Especially, when monitoring the monocyte subtypes in peripheral blood of these septic mice, we found out that C57bl/6 showed higher inflammatory (Ly6Chigh) monocyte (corresponding to M1 macrophage) proportion than Balb/c mice. Interestingly, we find out that cfDNA is highly correlated with the ratio of Ly6Chigh monocytes versus Ly6Clow monocytes, which represents M1/M2 (killing/healing) responses. Our study suggested that the cfDNA is a good indicator for evaluating M1/M2 responses in septic peritonitis.

Effects of montelukast on M2-related cytokine and chemokine in M2 macrophages

Asthma is a chronic airway inflammatory disease mediated by T-helper (Th)2 cells. Montelukast (trade name Singulair) is a cysteinyl leukotriene receptor antagonist used for asthma treatment. Mirroring Th1-Th2 polarization, two distinct states of macrophages have been recognized: the classically activated (M1) macrophages and the alternatively activated (M2) macrophages. M2 polarization is known to be a response to the Th2 cytokines; however, the effects of montelukast on M2 macrophages have not been well characterized. The aim of the present study was to investigate the effects of montelukast on the expression of cytokines and chemokines in M2-like macrophages, and to explore possible intracellular signaling pathways. The human monocytic leukemia cell line THP-1 and human monocytes from healthy donors were cultured with interleukin-4 for M2 polarization, and then the cells were pretreated with or without montelukast before lipopolysaccharide (LPS) stimulation. Supernatants were collected to determine interleukin-10, I-309/CCL1, and MDC/CCL22 levels by enzyme-linked immunosorbent assay. Intracellular signaling was investigated using nuclear factor (NF)-κB inhibitors, mitogen-activated protein kinase (MAPK) inhibitors, and western blot analysis. LPS-induced interleukin-10 and I-309/CCL1 expression was significantly suppressed by montelukast in THP-1-derived and human monocyte-derived M2 macrophages after LPS stimulation. MDC/CCL22 expression was only significantly suppressed by montelukast in THP-1-derived M2 macrophages after 48hours of incubation. In western blot analysis, montelukast was able to suppress LPS-induced MAPK-phospho-p38 and NF-κB-phospho-p65 expression. Montelukast suppressed LPS-induced M2-related cytokines and chemokines in alternatively activated macrophages, and the effects might be mediated through the MAPK-p38 and NF-κB-p65 pathways.

IL-6 influences the balance between M1 and M2 macrophages in a mouse model of irritant contact dermatitis

Abstract Irritant contact dermatitis (ICD) is an acute inflammatory response that is ranked the 2nd most prevalent occupational injury associated with workman’s compensation. Interleukin-6 (IL-6), a pro-inflammatory cytokine produced in the skin, is closely associated with healing and decreased levels are associated with more severe ICD. The phenotypic nature of the macrophage infiltrate during skin inflammation can greatly influence damage and healing during ICD. The pro-inflammatory, classically activated (M1) macrophage produce cytokines such as IL-6, IL-1, and TNFα, while alternatively activated (M2) macrophages produce IL-10 and TGFβ, and are thought to be associated with tissue repair. IL-6 may play a regulatory role in mediating the M1 versus M2 phenotypic change in skin. To examine the role of IL-6 in ICD, IL-6KO and C57 mice were exposed to benzoalkonium chloride (BKC) for 7 days. IL-6KO mice displayed decreased expression of CD206 (M2) as compared to CD86 (M1) and the ration of CD86:CD206 was significantly increased in IL-6KO mice compared to control. Multiplex protein analysis showed expression of multiple M1-related cytokines such IFN-g, CCL3, CCL4, CCL5, and CCL7 significantly modulated in IL6-KO BKC treated mice compared to C57. Additionally, M2 related cytokine/chemokine receptors IL-4R and CCR1 were significantly dysregulated in IL-6KO BKC treated mice. IL-6 treatment of RAW cells enhanced the expression of CD206 in manner similar to IL-4. Overall, these results further confirm a role for IL-6 in macrophage polarization and could eventually result in the development of therapies for dermatitis based on this cytokines activity.

Increased M1 Macrophages Infiltration Is Associated with Thrombogenesis in Rheumatic Mitral Stenosis Patients with Atrial Fibrillation.

Atrial fibrillation (AF) is the most common arrhythmia. In patients with AF, the role of macrophage subsets in thrombogenesis is unclear. In the present study, we analyzed the role of M1 and M2 macrophages and related cytokines in thrombogenesis of AF. Immunohistochemistry, Western blot, and TUNEL assay were used to detect M1/M2 macrophage infiltration, the expression pattern of IL-1β and inflammasome components, and apoptosis of cardiomyocytes in 71 specimens obtained from the left atrial appendage of patients with rheumatic mitral stenosis (MS) with or without thrombosis. We demonstrated that proinflammatory M1 macrophages were predominant in the atrium of MS patients with AF and thrombus. NLRP3 inflammasomes and IL-1β, which are primarily functional in macrophages, were activated in those patients. We also showed that increased cell death was associated with thrombogenesis in MS patients. These data indicate that infiltration of M1 macrophages and over-activation of NLRP3 inflammasomes may play a role in progressive atrial inflammation and thrombogenesis in rheumatic mitral stenosis patients with AF.

The MSHA strain of Pseudomonas aeruginosa (PA-MSHA) inhibits gastric carcinoma progression by inducing M1 macrophage polarization.

Macrophages play crucial roles in promoting tumor development and progression. In the present study, we found that the mannose-sensitive hemagglutination pilus strain of Pseudomonas aeruginosa (PA-MSHA) was efficient in inducing M1 macrophage polarization. PA-MSHA treatment increases expression of M1-related cytokines and promotes activation of murine peritoneal macrophages (MPM). Interestingly, PA-MSHA inhibits cell proliferation and migration and induces the apoptosis of gastric carcinoma cells. These effects of PA-MSHA on M1 polarization were associated with activation of NF-κB expression. Thus, inducing polarization of M1 by PA-MSHA may be one potential strategy for inhibiting gastric carcinoma progression in mice.

Bifidobacterium pseudocatenulatum CECT7765 induces an M2 anti-inflammatory transition in macrophages from patients with cirrhosis

Patients with cirrhosis show recurrent access of bacterial products into the bloodstream inducing a multi-altered immunological status leading to relevant complications. We aimed at evaluating Bifidobacterium pseudocatenulatum CECT7765 effect on the host's macrophage function. Patients with cirrhosis and ascites were included. Granulocyte-macrophage colony-stimulating factor (GM-CSF) monocyte-derived and ascitic fluid (AF) macrophages were cultured with M-CSF, lipopolysaccharide (LPS) and/or the bifidobacterial strain. Pellets and supernatants were evaluated for gene expression of M1 and M2-related genes and cytokine secretion. Cell surface expression molecules were evaluated by flow cytometry. Kupffer cells from bile duct ligated and CCl4 rats were also evaluated. Experiments were run on GM-CSF blood-derived and AF macrophages from 10 patients with cirrhosis and 10 healthy donors. Different macrophage morphology was observed by optical microscopy in cells stimulated with bifidobacteria vs. LPS. M2-like expression of CD206, CD163 and CD16 was significantly increased in macrophages after stimulation with the bifidobacterial strain vs. LPS. B. pseudocatenulatum CECT7765 was able to significantly change the cytokine secretion pattern of blood-derived and AF macrophages and Kupffer cells from bile duct ligated and CCl4 cirrhotic rats compared to that induced by LPS. B. pseudocatenulatum CECT7765 was also effective in inducing a phenotype transition and a functional change from an M1- to an M2-related gene expression and cytokine secretion pattern in AF macrophages even after LPS-pretreatment. B. pseudocatenulatum CECT7765 did not reduce AF macrophage bacterial killing capacity. B. pseudocatenulatum CECT7765 induces a morphologic, phenotypic and functional transition towards an anti-inflammatory profile in GM-CSF monocyte-derived and AF macrophages from patients with cirrhosis that may help in controlling sustained inflammation in decompensated cirrhosis.

Abstract 473: Functional polarization of macrophage triggered by tumor-conditioned medium

Abstract Purpose: Macrophages are important components of leukocyte infiltrates in various tumor stroma. Macrophages that infiltrate into tumor tissues are called tumor-associated macrophages (TAMs). TAMs show anti-tumorigenesis(M1) or pro-tumorigenesis (M2) functions depending on the cytokine milieu of the tumor microenvironment. TAMs perform some key homeostatic functions that allow tumor maintenance and growth. However, the signals involved in communication between tumor cells and macrophages are poorly defined. The aim of this study is to explore the macrophage polarization treated with the tumor-conditioned medium. Methods: Ana-1 macrophages, human THP-1 monocytes, and bone-marrow-derived macrophages (BMDMs) from C57BL/6J mice were incubated with HCT116-conditioned medium, respectively. Then we detected M1/M2 specific molecular markers by flow cytometry, Western blot and Real-time PCR. Flow cytometry was used to quantitatively assess the change of macrophage populations in macrophages treated with conditioned medium or normal medium. CD68(human) and CD11b(human), F4/80(mouse) and CD11b(mouse) were used as total-macrophage markers, CD206 as a marker of M2 macrophage. Secretory cytokine profile of the polarized macrophages was measured by cytokine array. Then, HCT116 cells were treated with conditioned medium of M2-like macrophage and applied to cell growth and colony formation assay. Results: By Western blot, we found that HCT116-conditioned medium could induce BMDMs and Ana-1 cells to overexpress arginase1(Arg1), but Ana-1 cells to express low inducible nitric oxide synthase(iNOS). Flow cytometry showed high expression level of CD68(human) and CD11b(human), F4/80(mouse) and CD11b(mouse), CD206 in HCT116-conditioned medium induced macrophages. Additionally, mRNA level of Arg1 was higher and M2-related cytokines IL-10,IL13 were also significantly upregulated, while mRNA level of iNOS was lower and M1-related cytokines IL-12,IFN-γwere downregulated in HCT116-conditioned medium induced macrophages. Conditioned medium of M2-like macrophages promoted the HCT116 cell growth and proliferation. Conclusions: HCT116 can secrete the soluble factors into conditioned medium, which would induce TAM polarization. Subsequently, conditioned medium of M2-like macrophages can promote tumor cells growth. Citation Format: Weina Zhang. Functional polarization of macrophage triggered by tumor-conditioned medium. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 473. doi:10.1158/1538-7445.AM2015-473

CD163+ tumor-associated macrophage is a prognostic biomarker and is associated with therapeutic effect on malignant pleural effusion of lung cancer patients.

CD163+ tumor-associated macrophages (TAMs) play an important role in the progression of cancer. However, the significance of CD163+ TAMs in malignant pleural effusion (MPE) is still unclear. The aim of this study is to evaluate the prognostic value of CD163+ TAMs in MPE, and the regulatory effect of an immune adjuvant (pseudomonas aeruginosa - mannose-sensitive hemagglutinin, PA-MSHA, which is used for MPE treatment in clinic) on CD163+ TAMs in MPE. Here, we found that the percentage of CD163+ TAMs in MPE was significantly higher than that in non-malignant pleural effusion (P<0.001). More importantly, CD163+ TAMs in MPE patients were an independent prognostic factor for progression-free survival. M2-related cytokines were highly expressed in MPE-derived CD163+ TAMs than in MPE-derived CD163- macrophages (P<0.05). CD163+ TAMs frequency in MPE patients was obviously reduced after PA-MSHA treatment in clinic (P<0.05). After treatment with PA-MSHA, M2 macrophages were re-educated to M1 macrophages in vitro. TLR4 blocking antibody inhibited M2 macrophages polarization to M1 macrophages induced by PA-MSHA. These findings highlight that accumulation of CD163+ TAMs in MPE caused by lung cancer is closely correlated with poor prognosis. CD163+ TAMs are associated with therapeutic effect in MPE. PA-MSHA re-educates CD163+ TAMs to M1 macrophages through TLR4-mediated pathway in MPE.

Glycyrrhizic Acid Promotes M1 Macrophage Polarization in Murine Bone Marrow-Derived Macrophages Associated with the Activation of JNK and NF-κB.

The roots and rhizomes of Glycyrrhiza species (licorice) have been widely used as natural sweeteners and herbal medicines. The aim of this study is to investigate the effect of glycyrrhizic acid (GA) from licorice on macrophage polarization. Both phenotypic and functional activities of murine bone marrow-derived macrophages (BMDMs) treated by GA were assessed. Our results showed that GA obviously increased the cell surface expression of CD80, CD86, and MHCII molecules. Meanwhile, GA upregulated the expression of CCR7 and the production of TNF-α, IL-12, IL-6, and NO (the markers of classically activated (M1) macrophages), whereas it downregulated the expression of MR, Ym1, and Arg1 (the markers of alternatively activated (M2) macrophage). The functional tests showed that GA dramatically enhanced the uptake of FITC-dextran and E. coli K88 by BMDMs and decreased the intracellular survival of E. coli K88 and S. typhimurium. Moreover, we demonstrated that JNK and NF-κB activation are required for GA-induced NO and M1-related cytokines production, while ERK1/2 pathway exhibits a regulatory effect via induction of IL-10. Together, these findings indicated that GA promoted polarization of M1 macrophages and enhanced its phagocytosis and bactericidal capacity. The results expanded our knowledge about the role of GA in macrophage polarization.