CD73 Activity is Dispensable for the Polarization of M2 Macrophages

The ectoenzyme CD73 catalyzes the hydrolysis of AMP, and is one of the most important producers of extracellular adenosine. On regulatory T cells, CD73 is necessary for immunosuppressive functions, and on Th17 cells CD73-generated adenosine exerts anti-inflammatory effects. However, the expression and function of CD73 in pro-inflammatory M1 and in immunosuppressive M2 macrophages is largely unknown. Here we show that CD73 expression and enzyme activity were induced in in vitro polarized pro-inflammatory human M(LPS+TNF) monocytes/macrophages, while CD73 was absent from immunosuppressive M(IL-4+M-CSF)-polarized macrophages. Inhibition of CD73 activity with the inhibitor AMPCP did not affect the polarization of human monocytes. In mice, CD73 was present on resident peritoneal macrophages. In striking contrast, elicited peritoneal macrophages remained CD73 negative regardless of their polarization towards either a pro-inflammatory M(LPS) or anti-inflammatory M(IL-4c) direction. Finally, the ability of peritoneal macrophages to polarize to pro- and anti-inflammatory cells was perfectly normal in CD73-deficient mice in vivo. These data indicate that, in contrast to other major leukocyte subpopulations, CD73 activity on macrophages does not play a major role in their polarization and that in mice host CD73 on any cell type is not required in vivo for peritoneal macrophage polarization towards either a pro- or an anti-inflammatory direction.     

Anti-Inflammatory Activity of S. Marianum and N. Sativa Extracts on Macrophages

Background:Nigella sativa (N. sativa) and Silybum marianum (S. marianum) are used to regulate macrophage polarization in lipopolysaccharide-induced RAW 264.7 cells and thioglycollate-elicited peritoneal inflammation.Methods:Cytotoxicity assays and acute toxicity tests were performed to investigate the safe dose and toxicity of the prepared extracts. Also, nitric oxide production was determined by Griess assay on RAW264.7 and peritoneal macrophage supernatants. After RNA extraction from macrophages, real-time PCR was performed to measure the relative gene expression of tumor necrosis factor (TNF)-α, interleukin (IL)-6, transforming growth factor (TGF)-β, and IL-10. Finally, regulatory T cells (Treg cells) were counted by flow cytometry.Results:S. marianum methanolic extract (SME), N. sativa ethanolic extract (NEE), and their mixture (SME+NEE) decreased NO levels significantly in RAW264.7 and peritoneal murine macrophages. N. sativa ethanolic extract significantly increased IL-10 gene expression and significantly decreased IL-6 and TNF-α expression in RAW264.7 cells. In mixture-treated peritoneal macrophages, IL-10 and TGF-β expression were significantly increased, while IL-6 and TNF-α were significantly decreased. Also, the percentage of Treg cells was significantly greater in the mixture-treated cells than in controls.Conclusion:These results suggest that an SME and NEE mixture has anti-inflammatory and immunomodulatory activities and may be useful in the treatment of diseases of immunopathologic origin characterized by macrophage hyperactivation.Key Words: Cytokine, Inflammation, Nigella sativa, Nitric oxide, Silybum marianum     

Gingipain of <Emphasis Type="Italic">Porphyromonas gingivalis</Emphasis> manipulates M1 macrophage polarization through C5a pathway

Gingipains secreted by Porphyromonas gingivalis (P. gingivalis, Pg) play an important role in maintaining macrophage infiltrating. And, this study is to evaluate effects of gingipain on M1 macrophage polarization after exposure to Porphyromonas gingivalis (P. gingivalis, Pg) and if these effects are through complement component 5a (C5a) pathway. Mouse RAW264.7 macrophages were exposed to gingipain extracts, Escherichia coli lipopolysaccharides (Ec-LPS), Pg-LPS with or without the C5aR antagonist: PMX-53 for 24 h. Then, gene expressions and protein of IL-12, IL-23, iNOS, IL-10, TNF-α, IL-1β, and IL-6 were determined by qRT-PCR and ELISA assays. Surface markers CD86 for M1 and CD206 for M2 were also evaluated by flow cytometry. The results show that gingipain extracts alone increased expressions of IL-12, IL-23, iNOS, TNF-α, IL-1β, and IL-6, but not IL-10. Gingipain extracts plus Ec-LPS decreased expressions of IL-12, IL-23, iNOS, TNF-α, IL-1β, and IL-6 in which Ec-LPS induced increase. For gingipain extracts plus Pg-LPS-treated RAW264.7, macrophages, gingipain extracts enhanced expressions of IL-12 and IL-23 in which Pg-LPS induced increase, but not iNOS and IL-10 while gingipain extracts decreased expressions of TNF-α, IL-1β, and IL-6 in which Pg-LPS induced increase. Interestingly, PMX-53 increased expressions of IL-12, IL-23, and iNOS when RAW264.7 macrophages were treated with gingipain extracts plus Ec-LPS or Pg-LPS and PMX-53, while PMX-53 decreased expressions of TNF-α, IL-1β, and IL-6. Changes of CD86-positive macrophages were consistent with cytokine changes. Our data indicate that gingipain is a critical regulator, more like a promoter to manipulate M1 macrophage polarization in order to benefit P. gingivalis infection through the C5a pathway.     

Intimal lining layer macrophages but not synovial sublining macrophages display an IL-10 polarized-like phenotype in chronic synovitis

Introduction

Synovial tissue macrophages play a key role in chronic inflammatory arthritis, but the contribution of different macrophage subsets in this process remains largely unknown. The main in vitro polarized macrophage subsets are classically (M1) and alternatively (M2) activated macrophages, the latter comprising interleukin (IL)-4 and IL-10 polarized cells. Here, we aimed to evaluate the polarization status of synovial macrophages in spondyloarthritis (SpA) and rheumatoid arthritis (RA).

Methods

Expression of polarization markers on synovial macrophages, peripheral blood monocytes, and in vitro polarized monocyte-derived macrophages from SpA versus RA patients was assessed by immunohistochemistry and flow cytometry, respectively. The polarization status of the intimal lining layer and the synovial sublining macrophages was assessed by double immunofluorescence staining.

Results

The expression of the IL-10 polarization marker cluster of differentiation 163 (CD163) was increased in SpA compared with RA intimal lining layer, but no differences were found in other M1 and M2 markers between the diseases. Furthermore, no significant phenotypic differences in monocytes and in vitro polarized monocyte-derived macrophages were seen between SpA, RA, and healthy controls, indicating that the differential CD163 expression does not reflect a preferential M2 polarization in SpA. More detailed analysis of intimal lining layer macrophages revealed a strong co-expression of the IL-10 polarization markers CD163 and cluster of differentiation 32 (CD32) but not any of the other markers in both SpA and RA. In contrast, synovial sublining macrophages had a more heterogeneous phenotype, with a majority of cells co-expressing M1 and M2 markers.

Conclusions

The intimal lining layer but not synovial sublining macrophages display an IL-10 polarized-like phenotype, with increased CD163 expression in SpA versus RA synovitis. These differences in the distribution of the polarized macrophage subset may contribute to the outcome of chronic synovitis.     

Overexpression of IL-10 in C2D Macrophages Promotes a Macrophage Phenotypic Switch in Adipose Tissue Environments

Adipose tissue macrophages are a heterogeneous collection of classically activated (M1) and alternatively activated (M2) macrophages. Interleukin 10 (IL-10) is an anti-inflammatory cytokine, secreted by a variety of cell types including M2 macrophages. We generated a macrophage cell line stably overexpressing IL-10 (C2D-IL10) and analyzed the C2D-IL10 cells for several macrophage markers after exposure to adipocytes compared to C2D cells transfected with an empty vector (C2D-vector). C2D-IL10 macrophage cells expressed more CD206 when co-cultured with adipocytes than C2D-vector cells; while the co-cultured cell mixture also expressed higher levels of Il4, Il10, Il1β and Tnf. Since regular C2D cells traffic to adipose tissue after adoptive transfer, we explored the impact of constitutive IL-10 expression on C2D-IL10 macrophages in adipose tissue in vivo. Adipose tissue-isolated C2D-IL10 cells increased the percentage of CD206⁺, CD301⁺, CD11c⁻CD206⁺ (M2) and CD11c⁺CD206⁺ (M1b) on their cell surface, compared to isolated C2D-vector cells. These data suggest that the expression of IL-10 remains stable, alters the C2D-IL10 macrophage cell surface phenotype and may play a role in regulating macrophage interactions with the adipose tissue.     

Induction of Murine Macrophage M2 Polarization by Cigarette Smoke Extract via the JAK2/STAT3 Pathway

Cigarette smoking is a major pathogenic factor in lung cancer. Macrophages play an important role in host defense and adaptive immunity. These cells display diverse phenotypes for performing different functions. M2 type macrophages usually exhibit immunosuppressive and tumor-promoting characteristics. Although macrophage polarization toward the M2 phenotype has been observed in the lungs of cigarette smokers, the molecular basis of the process remains unclear. In this study, we evaluated the possible mechanisms for the polarization of mouse macrophages that are induced by cigarette smoking (CS) or cigarette smoke extract (CSE). The results showed that exposure to CSE suppressed the production of reactive oxygen species (ROS) and nitric oxide (NO) and down-regulated the phagocytic ability of Ana-1 cells. The CD163 expressions on the surface of macrophages from different sources were significantly increased in in vivo and in vitro studies. The M1 macrophage cytokines TNF-α, IL-12p40 and enzyme iNOS decreased in the culture supernatant, and their mRNA levels decreased depending on the time and concentration of CSE. In contrast, the M2 phenotype macrophage cytokines IL-10, IL-6, TGF-β1 and TGF-β2 were up-regulated. Moreover, phosphorylation of JAK2 and STAT3 was observed after the Ana-1 cells were treated with CSE. In addition, pretreating the Ana-1 cells with the STAT3 phosphorylation inhibitor WP1066 inhibited the CSE-induced CD163 expression, increased the mRNA level of IL-10 and significantly decreased the mRNA level of IL-12. In conclusion, we demonstrated that the M2 polarization of macrophages induced by CS could be mediated through JAK2/STAT3 pathway activation.     

Chlamydia psittaci plasmid-encoded CPSIT_P7 induces macrophage polarization to enhance the antibacterial response through TLR4-mediated MAPK and NF-κB pathways

Although the protective effects of Chlamydia psittaci plasmid-encoded protein CPSIT_P7 as vaccine antigens to against chlamydial infection have been confirmed in our previous study, the function and mechanism of CPSIT_P7 inducing innate immunity in the antibacterial response remain unknown. Here, we found that plasmid protein CPSIT_P7 could induce M1 macrophage polarization upregulating the genes of the surface molecule CD86, proinflammatory cytokines (TNF-α, IL-6, and IL-1β), and antibacterial effector NO synthase 2 (iNOS). During M1 macrophage polarization, macrophages acquire phagocytic and microbicidal competence, which promotes the host antibacterial response. As we observed that CPSIT_P7-induced M1 macrophages could partially reduce the infected mice pulmonary Chlamydia psittaci load. Furthermore, CPSIT_P7 induced M1 macrophage polarization through the TLR4-mediated MAPK and NF-κB pathways. Collectively, our results highlight the effect of CPSIT_P7 on macrophage polarization and provide new insights into new prevention and treatment strategies for chlamydial infection.     

Abietic acid attenuates sepsis-induced lung injury by inhibiting nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway to inhibit M1 macrophage polarization

Lung injury is one of the leading causes of death in sepsis. Abietic acid (AA) has demonstrated anti-inflammatory and bacteriostatic properties. Herein, we established a mouse model of sepsis by cecal ligation and puncture, and intraperitoneally injected AA to treat. Lung injury was assessed by H&E staining and the inflammation in bronchoalveolar lavage fluid (BALF) were assessed by counting the number of inflammatory cells and detecting the content of inflammatory factors. Meanwhile, we also designed to study the effect of AA on lipopolysaccharide (LPS)-induced inflammatory response and macrophage marker gene expression in RAW264.7 cells in vitro. In this study, we found that AA inhibited LPS-induced secretion of inflammatory mediators (IL-1β, TNF-α, IL-6 and MIP-2), and decreased the expression of M1 macrophage e markers (CD16 and iNOS) and p-p65 protein, while increased the expression of M2 macrophage markers (CD206 and Arg-1) in RAW264.7 cells in vitro. In vivo, the therapy of AA not only rescued septic animals, but also attenuated lung injury in sepsis mice. Moreover, AA decreased the number of total cells, neutrophils and macrophages, the conceration of total protein, and the levels of inflammatory mediators in BALF of sepsis mice. Further, we found that AA inhibited M1 macrophage polarization and blocked nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway in BALF of sepsis mice. In conclusion, Abietic acid attenuates sepsis-induced lung injury, and its mechanism may be related to reducing inflammation by inhibiting NF-κB signaling to inhibit M1 macrophage polarization.     

IL-10 is required for polarization of macrophages to M2-like phenotype by mycobacterial DnaK (heat shock protein 70)

Macrophages are key cells in the innate immune system. They phagocytose pathogens and cellular debris, promote inflammation, and have important roles in tumor immunity. Depending on the microenvironment, macrophages can polarize to M1 (inflammatory) or M2 (anti-inflammatory) phenotypes. Extracellular DnaK (the bacterial ortholog of the mammalian Hsp70) from Mycobacterium tuberculosis (Mtb) was described to exert immune modulatory roles in an IL-10 dependent manner. We have previously observed that endotoxin-free DnaK can polarize macrophages to an M2-like phenotype. However, the mechanisms that underlie this polarization need to be further investigated. IL-10 has been described to promote macrophage polarization, so we investigated the involvement of this cytokine in macrophages stimulated with extracellular DnaK. IL-10 was required to induce the expression of M2 markers - Ym1 and Fizz, when macrophages were treated with DnaK. Blockade of IL-10R also impaired DnaK induced polarization, demonstrating the requirement of the IL-10/IL-10R signaling pathway in this polarization. DnaK was able to induce TGF-β mRNA in treated macrophages in an IL-10 dependent manner. However, protein TGF-β could not be detected in culture supernatants. Finally, using an in vivo allogeneic melanoma model, we observed that DnaK-treated macrophages can promote tumor growth in an IL-10-dependent manner. Our results indicate that the IL-10/IL-10R axis is required for DnaK-induced M2-like polarization in murine macrophages.     

Extracellular Mycobacterial DnaK Polarizes Macrophages to the M2-Like Phenotype

Macrophages are myeloid cells that play an essential role in inflammation and host defense, regulating immune responses and maintaining tissue homeostasis. Depending on the microenvironment, macrophages can polarize to two distinct phenotypes. The M1 phenotype is activated by IFN-γ and bacterial products, and displays an inflammatory profile, while M2 macrophages are activated by IL-4 and tend to be anti-inflammatory or immunosupressive. It was observed that DnaK from Mycobacterium tuberculosis has immunosuppressive properties, inducing a tolerogenic phenotype in dendritic cells and MDSCs, contributing to graft acceptance and tumor growth. However, its role in macrophage polarization remains to be elucidated. We asked whether DnaK was able to modulate macrophage phenotype. Murine macrophages, derived from bone marrow, or from the peritoneum, were incubated with DnaK and their phenotype compared to M1 or M2 polarized macrophages. Treatment with DnaK leads macrophages to present higher arginase I activity, IL-10 production and FIZZ1 and Ym1 expression. Furthermore, DnaK increased surface levels of CD206. Importantly, DnaK-treated macrophages were able to promote tumor growth in an allogeneic melanoma model. Our results suggest that DnaK polarizes macrophages to the M2-like phenotype and could constitute a virulence factor and is an important immunomodulator of macrophage responses.     

Smoking status and anti-inflammatory macrophages in bronchoalveolar lavage and induced sputum in COPD

Background

Macrophages have been implicated in the pathogenesis of COPD. M1 and M2 macrophages constitute subpopulations displaying pro- and anti-inflammatory properties. We hypothesized that smoking cessation affects macrophage heterogeneity in the lung of patients with COPD. Our aim was to study macrophage heterogeneity using the M2-marker CD163 and selected pro- and anti-inflammatory mediators in bronchoalveolar lavage (BAL) fluid and induced sputum from current smokers and ex-smokers with COPD.

Methods

114 COPD patients (72 current smokers; 42 ex-smokers, median smoking cessation 3.5 years) were studied cross-sectionally and underwent sputum induction (M/F 99/15, age 62 ± 8 [mean ± SD] years, 42 (31-55) [median (range)] packyears, post-bronchodilator FEV₁ 63 ± 9% predicted, no steroids past 6 months). BAL was collected from 71 patients. CD163⁺ macrophages were quantified in BAL and sputum cytospins. Pro- and anti-inflammatory mediators were measured in BAL and sputum supernatants.

Results

Ex-smokers with COPD had a higher percentage, but lower number of CD163⁺ macrophages in BAL than current smokers (83.5% and 68.0%, p = 0.04; 5.6 and 20.1 ×10⁴/ml, p = 0.001 respectively). The percentage CD163⁺ M2 macrophages was higher in BAL compared to sputum (74.0% and 30.3%, p < 0.001). BAL M-CSF levels were higher in smokers than ex-smokers (571 pg/ml and 150 pg/ml, p = 0.001) and correlated with the number of CD163⁺ BAL macrophages (Rs = 0.38, p = 0.003). No significant differences were found between smokers and ex-smokers in the levels of pro-inflammatory (IL-6 and IL-8), and anti-inflammatory (elafin, and Secretory Leukocyte Protease Inhibitor [SLPI]) mediators in BAL and sputum.

Conclusions

Our data suggest that smoking cessation partially changes the macrophage polarization in vivo in the periphery of the lung towards an anti-inflammatory phenotype, which is not accompanied by a decrease in inflammatory parameters.     

Does iron overload in metabolic syndrome affect macrophage profile? A case control study

AimsDysmetabolic iron overload syndrome (DIOS) is common but the clinical relevance of iron overload is not understood. Macrophages are central cells in iron homeostasis and inflammation. We hypothesized that iron overload in DIOS could affect the phenotype of monocytes and impair macrophage gene expression.MethodsThis study compared 20 subjects with DIOS to 20 subjects with metabolic syndrome (MetS) without iron overload, and 20 healthy controls. Monocytes were phenotyped by Fluorescence-Activated Cell Sorting (FACS) and differentiated into anti-inflammatory M2 macrophages in the presence of IL-4. The expression of 38 genes related to inflammation, iron metabolism and M2 phenotype was assessed by real-time PCR.ResultsFACS showed no difference between monocytes across the three groups. The macrophagic response to IL-4-driven differentiation was altered in four of the five genes of M2 phenotype (MRC1, F13A1, ABCA1, TGM2 but not FABP4), in DIOS vs Mets and controls demonstrating an impaired M2 polarization. The expression profile of inflammatory genes was not different in DIOS vs MetS. Several genes of iron metabolism presented a higher expression in DIOS vs MetS: SCL11A2 (a free iron transporter, +76 %, p = 0.04), SOD1 (an antioxidant enzyme, +27 %, p = 0.02), and TFRC (the receptor 1 of transferrin, +59 %, p = 0.003).ConclusionsIn DIOS, macrophage polarization toward the M2 alternative phenotype is impaired but not associated with a pro-inflammatory profile. The up regulation of transferrin receptor 1 (TFRC) in DIOS macrophages suggests an adaptive role that may limit iron toxicity in DIOS.     

Direct effects of airborne PM2.5 exposure on macrophage polarizations

BackgroundExposure of atmospheric particulate matter with an aerodynamic diameter less than 2.5 μm (PM_2.5) is epidemiologically associated with illnesses. Potential effects of air pollutants on innate immunity have raised concerns. As the first defense line, macrophages are able to induce inflammatory response. However, whether PM_2.5 exposure affects macrophage polarizations remains unclear.MethodsWe used freshly isolated macrophages as a model system to demonstrate effects of PM_2.5 on macrophage polarizations. The expressions of cytokines and key molecular markers were detected by real-time PCR, and flow cytometry. The specific inhibitors and gene deletion technologies were used to address the molecular mechanisms.ResultsPM_2.5 increased the expression of pro-inflammatory cytokines granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-6 (IL-6), interleukin-1β (IL-1β), tumor necrosis factor alpha (TNFα). PM_2.5 also enhanced the lipopolysaccharide (LPS)-induced M1 polarization even though there was no evidence in the change of cell viability. However, PM_2.5 significantly decreased the number of mitochondria in a dose dependent manner. Pre-treatment with NAC, a scavenger of reactive oxygen species (ROS), prevented the increase of ROS and rescued the PM_2.5-impacted M1 but not M2 response. However, mTOR deletion partially rescued the effects of PM_2.5 to reduce M2 polarization.ConclusionsPM_2.5 exposure significantly enhanced inflammatory M1 polarization through ROS pathway, whereas PM_2.5 exposure inhibited anti-inflammatory M2 polarization through mTOR-dependent pathway.General significanceThe present studies suggested that short-term exposure of PM_2.5 acts on the balance of inflammatory M1 and anti-inflammatory M2 macrophage polarizations, which may be involved in air pollution-induced immune disorders and diseases. This article is part of a Special Issue entitled Air Pollution, edited by Wenjun Ding, Andrew J. Ghio and Weidong Wu.     

Docosahexaenoic acid attenuates macrophage-induced inflammation and improves insulin sensitivity in adipocytes-specific differential effects between LC n-3 PUFA

ObjectiveAdipose tissue inflammation with immune cell recruitment plays a key role in obesity-induced insulin resistance (IR). Long-chain (LC) n-3 polyunsaturated fatty acids (PUFA) eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have anti-inflammatory potential; however, their individual effects on adipose IR are ill defined. We hypothesized that EPA and DHA may differentially affect macrophage-induced IR in adipocytes.MethodsJ774.2 macrophages pretreated with EPA or DHA (50 μM for 5 days) were stimulated with lipopolysaccharide (LPS, 100 ng/ml for 30 min–48 h). Cytokine secretion profiles and activation status of macrophages were assessed by enzyme-linked immunosorbent assay and flow cytometry. Pretreated macrophages were seeded onto transwell inserts and placed over 3T3-L1 adipocytes for 24–72 h; effects on adipocyte–macrophage cytokine cross-talk and insulin-stimulated ³H-glucose transport into adipocytes were monitored.ResultsDHA had more potent anti-inflammatory effects relative to EPA, with marked attenuation of LPS-induced nuclear factor (NF)κB activation and tumor necrosis factor (TNF)α secretion in macrophages. DHA specifically enhanced anti-inflammatory interleukin (IL)-10 secretion and reduced the expression of proinflammatory M1 (F4/80⁺/CD11⁺) macrophages. Co-culture of DHA-enriched macrophages with adipocytes attenuated IL-6 and TNFα secretion while enhancing IL-10 secretion. Conditioned media (CM) from DHA-enriched macrophages attenuated adipocyte NFκB activation. Adipocytes co-cultured with DHA-enriched macrophages maintained insulin sensitivity with enhanced insulin-stimulated ³H-glucose transport, GLUT4 translocation and preservation of insulin-receptor substrate-1 expression compared to co-culture with untreated macrophages. We confirmed that IL-10 expressed by DHA-enriched macrophages attenuates the CM-induced proinflammatory IR phenotype in adipocytes.ConclusionsWe demonstrate an attenuated proinflammatory phenotype of DHA-pretreated macrophages, which when co-cultured with adipocytes partially preserved insulin sensitivity.     

In Vitro Comparison of the Effects of Probiotic,Commensal and Pathogenic Strains on Macrophage Polarization

Macrophages are important with respect to both innate and adaptive immune responses and are known to differentiate into pro-inflammatory M1- or anti-inflammatory M2-phenotypes following activation. In order to study how different bacteria affect macrophage polarization, we exposed murine RAW 264.7 macrophages to sixteen different strains representing probiotic strains, pathogens, commensals and strains of food origin. Increased inducible nitric oxide synthase (iNOS) or arginase-1 gene expression indicates M1 or M2 polarization, respectively, and was quantified by qRT-PCR. Strains of Escherichia and Salmonella elevated iNOS expression more so than strains of Enterococcus, Lactobacillus and Lactococcus, indicating that Gram-negative strains are more potent M1 inducers. However, strain-specific responses were observed. For instance, Escherichia coli Nissle 1917 was a poor inducer of iNOS gene expression compared to the other E. coli strains, while Enterococcus faecalis Symbioflor-1 was more potent in this respect compared to all the eleven Gram-positive strains tested. Macrophage polarization was further characterized by quantifying secreted pro- and anti-inflammatory cytokines. Exposure to the pathogen E. coli 042 produced a cytokine profile indicating M1 differentiation, which is in accordance with the PCR data. However, exposure to most strains resulted in either high or low secretion levels of all cytokines tested, rather than a clear M1 or M2 profile. In general, the Gram-negative strains induced high levels of cytokine secretion compared to the Gram-positive strains. Interestingly, strains of human origin had a higher impact on macrophages compared to strains of food origin.     

Effective-components combination improves airway remodeling in COPD rats by suppressing M2 macrophage polarization via the inhibition of mTORC2 activity

BackgroundIn chronic obstructive pulmonary disease (COPD), M2 macrophages release multiple tissue repair-related factors, leading to airway remodeling, a significant pathological characteristic. Meanwhile, effective-components combination (ECC), derived from Bufei Yishen formula (BYF), is an effective treatment for COPD.PurposeTo determine the potential mechanisms of ECC in airway remodeling in COPD by suppressing M2 macrophage polarization.MethodsWe established a rat COPD Model using exposure to cigarette smoke and bacterial infection to investigate the efficacy of ECC. We also treated macrophages with IL-4 for 12 h to explore the in vivo effect of ECC on M2 macrophage polarization and mTORC2 signals.ResultsThe disease severity of COPD rats could be alleviated by ECC treatment, which improved pulmonary function and alleviated pathological injuries in lung tissue and the inflammatory cytokine levels. Meanwhile, ECC could ameliorate airway remodeling by reducing collagen deposition, hindering airway mucus hypersecretion and smooth muscle cell proliferation, and reducing the number of M2 macrophages in the lung tissues of COPD rats. Furthermore, with IL-4-induced macrophages, we found that ECC could suppress M2 macrophage polarization by decreasing the levels of M2 macrophage markers. Finally, we discovered that ECC inhibited mTORC2 activity by examining p-mTOR²⁴⁸¹ and its downstream protein p-Akt⁴⁷³.ConclusionsECC exerts beneficial effects on airway remodeling in COPD rats, likely by suppressing M2 macrophage polarization via the inhibition of mTORC2 activity.     

Macrophage Polarization Reflects T Cell Composition of Tumor Microenvironment in Pediatric Classical Hodgkin Lymphoma and Has Impact on Survival

Macrophages have been implicated in the pathogenesis of classical Hodgkin lymphoma (cHL) and have been suggested to have a negative impact on outcome. Most studies addressing the role of macrophages in cHL have relied on identification of macrophages by generic macrophage antigens, e.g., CD68. We have therefore conducted an in situ analysis of macrophage polarization in a series of 100 pediatric cHL (pcHL) cases using double staining immunohistochemistry, combining CD68 or CD163 with pSTAT1 (M1-like) or CMAF (M2-like). M1- or M2-polarised microenvironment was defined by an excess of one population over the other (>1.5). Expression of STAT1 and LYZ genes was also evaluated by RT-qPCR. Patients <14 years and EBV+ cases displayed higher numbers of CD68+pSTAT1+ cells than older children and EBV- cases, respectively (P=0.01 and P=0.02). A cytotoxic tumor microenvironment, defined by a CD8+/FOXP3+ ratio >1.5 was associated with higher numbers of CD68+pSTAT1+ (P=0.025) and CD163+pSTAT1+ macrophages (P<0.0005). Levels of STAT1 and LYZ expression were associated with the numbers of CD68+pSTAT1+ macrophages. EBV+ cHL cases disclosed a predominant M1 polarized microenvironment similar to Th1 mediated inflammatory disorders, while EBV- cHL showed a predominant M2 polarized microenvironment closer to Th2 mediated inflammatory diseases. Better overall-survival (OS) was observed in cases with higher numbers of CD163+pSTAT1+ macrophages (P=0.02) while larger numbers of CD163+CMAF+ macrophages were associated with worse progression-free survival (PFS) (P=0.02). Predominant M1-like polarization as disclosed by CD163+pSTAT1+/CD163+CMAF+ ratio > 1.5 was associated with better OS (P= 0.037). In conclusion, macrophage polarization in pcHL correlates with prevalent local T cell response and may be influenced by the EBV-status of neoplastic cells. Besides, M1-like and M2-like macrophages displayed differential effects on outcome in pcHL.     

Maternal hypercholesterolemia exacerbates atherosclerosis lesions in female offspring through potentiating macrophage polarization toward an inflammatory M1 phenotype

Maternal hypercholesterolemia induces early onset of cardiovascular diseases in offspring; however, its underlying mechanism remains poorly understood. We hypothesized that maternal hypercholesterolemia increases offspring susceptibility to atherosclerosis in adulthood through developmental modifications of macrophages. Female apolipoprotein E (ApoE)-deficient mice were fed a Western-type diet (WD) or a control diet (CD) prior to and throughout gestation and lactation. The offspring were all fed a WD after weaning. Sixteen-week-old female offspring of WD-fed dams showed a significant increase in atherosclerotic lesions of the aorta and aortic root compared with those of CD-fed dams. This effect was associated with increased macrophage accumulation within lesions, macrophage inflammation and an increase in circulating Ly6C^high monocyte and F4/80 macrophage counts. We further evidenced that in utero WD exposure promoted macrophage polarization toward the M1 phenotype by elevating M1 markers (Cd86, Inos/Nos2) without affecting M2 markers (Cd206, Arg1). Proinflammatory cytokine synthesis was also enhanced in response to LPS. Finally, maternal WD intake strongly inhibited the macrophage expression of Pparg and Lxra, which was associated with aberrant DNA methylation of Lxra promoter. Our findings demonstrate that maternal hypercholesterolemia exacerbates atherosclerosis, in part by altering the epigenetic state of the macrophage genome of the offspring, imprinting gene expression, and changing macrophage polarization, which ultimately contributes to plaque macrophage burden.     

Proanthocyanidins from the bark of Metasequoia glyptostroboides ameliorate allergic contact dermatitis through directly inhibiting T cells activation and Th1/Th17 responses

ObjectiveThe leaves and bark of Metasequoia glyptostroboides are used as anti-microbic, analgesic and anti-inflammatory drug for dermatic diseases in Chinese folk medicine. However, the pharmacological effects and material basis responsible for the therapeutic use of this herb have not yet been well studied. The objectives of this study were to evaluate the anti-inflammatory effects of the proanthocyanidin fraction from the bark of M. glyptostroboides (MGEB) and to elucidate its immunological mechanisms.Materials and methodsThe anti-inflammatory activity of MGEB was evaluated using 2,4-dinitrofluorobenzene (DNFB)-induced allergic contact dermatitis (ACD) in mice. Its potential mechanisms were further investigated by determining its effects on Con A-induced T cell activation and Th1/Th17 responses in vitro.ResultsBoth intraperitoneal injection and oral administration of MGEB significantly reduced the ear swelling in DNFB-induced ACD mice. MGEB inhibited Con A-induced proliferation and the expression levels of cell surface molecules CD69 and CD25 of T cells in vitro. MGEB also significantly decreased the production of Th1/Th17 specific cytokines (IL-2, IFN-γ and IL-17) and down-regulated their mRNA expression levels in activated T-cells.ConclusionsMGEB could ameliorate ACD, at least in part, through directly inhibiting T cells activation and Th1/Th17 responses.