Protein Expression In Macrophages Research Articles

Assessing the role of phosphorylated S6 ribosomal protein in the pathological diagnosis of pulmonary antibody-mediated rejection

BackgroundPulmonary antibody-mediated rejection is still a challenging diagnosis as C4d has poor sensitivity. Previous studies have indicated that the phosphorylated S6 ribosomal protein, a component of the mammalian target of rapamycin (mTOR) pathway, is correlated with de novo donor-specific antibodies in lung transplantation. The objective of this study was to evaluate the phosphorylation of S6 ribosomal protein as a surrogate for AMR diagnosis in lung transplant patients. MethodsThis multicentre retrospective study analyzed transbronchial biopsies from 216 lung transplanted patients, 114 with antibody-mediated rejection and 102 without (19 with acute cellular rejection, 17 with ischemia/reperfusion injury, 18 with infection, and 48 without post-transplant complications). Immunohistochemistry was used to quantify phosphorylated S6 ribosomal protein expression in macrophages, endothelium, epithelium, and inter-pathologist agreement was assessed. ResultsMedian phosphorylated S6 ribosomal protein expression values were higher in antibody-mediated rejection cases than in controls for all cell components, with the highest sensitivity in macrophages (0.9) and the highest specificity in endothelial expression (0.8). The difference was mainly significant in macrophages compared to other post-lung transplantation complications. Inter-pathologist agreement was moderate for macrophages and endothelium, with higher agreement when phosphorylated S6 ribosomal protein expression was dichotomized into positive/negative. The inclusion of phosphorylated S6 ribosomal protein in the diagnostic algorithm could have increased antibody-mediated rejection certainty levels by 25%. ConclusionsThe study supports the role of the mTOR pathway in antibody-mediated rejection-related graft injury and suggests that tissue phosphorylation of S6 ribosomal protein could be a useful surrogate for a more accurate pathological diagnosis of lung antibody-mediated rejection.

Azilsartan inhibits inflammation-triggered bone resorption and osteoclastogenesis in vivo via suppression of TNF-α expression in macrophages.

Hypertension is a major risk factor for cardiovascular disease (CVD) and is associated with increased bone loss due to excessive activity of the local renin-angiotensin system (RAS). Angiotensinogen/Angiotensin (ANG) II/Angiotensin II type 1 receptor (AT1R) axis is considered as the core axis regulating RAS activity. Azilsartan is an FDA-approved selective AT1R antagonist that is used to treat hypertension. This study aimed to determine whether azilsartan affects formation of osteoclast, resorption of bone, and the expression of cytokines linked with osteoclastogenesis during lipopolysaccharide (LPS)-triggered inflammation in vivo. In vivo, following a 5-day supracalvarial injection of LPS or tumor necrosis factor-alpha (TNF-α) with or without azilsartan, the proportion of bone resorption and the number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells, which are identified as osteoclasts on mice calvariae were counted. The mRNA expression levels of TRAP, cathepsin K, receptor activator of NF-κB ligand (RANKL), and TNF-α were also evaluated. In vitro, the effect of azilsartan (0, 0.01, 0.1, 1, and 10 μM) on RANKL and TNF-α-triggered osteoclastogenesis were investigated. Also, whether azilsartan restrains LPS-triggered TNF-α mRNA and protein expression in macrophages and RANKL expression in osteoblasts were assessed. Furthermore, western blotting for analysis of mitogen-activated protein kinases (MAPKs) signaling was conducted. Azilsartan-treated calvariae exhibited significantly lower bone resorption and osteoclastogenesis than those treated with LPS alone. In vivo, LPS with azilsartan administration resulted in lower levels of receptor activator of RANKL and TNF-α mRNA expression than LPS administration alone. Nevertheless, azilsartan did not show inhibitory effect on RANKL- and TNF-α-triggered osteoclastogenesis in vitro. Compared to macrophages treated with LPS, TNF-α mRNA and protein levels were lower in macrophages treated by LPS with azilsartan. In contrast, RANKL mRNA and protein expression levels in osteoblasts were the same in cells co-treated with azilsartan and LPS and those exposed to LPS only. Furthermore, azilsartan suppressed LPS-triggered MAPKs signaling pathway in macrophages. After 5-day supracalvarial injection, there is no difference between TNF-α injection group and TNF-α with azilsartan injection group. These findings imply that azilsartan prevents LPS-triggered TNF-α production in macrophages, which in turn prevents LPS-Triggered osteoclast formation and bone resorption in vivo.

Mycobacterium tuberculosis induces immune evasion in macrophages through RNA-binding protein tristetraprolin

Abstract Immune evasion of Mycobacterium tuberculosis (Mtb) facilitates intracellular bacterial growth. The mechanisms of immune evasion, however, are still not fully understood. In this study, we reveal that tristetraprolin (TTP), one of the best characterized RNA-binding proteins controlling stability of the target mRNAs, mediates innate immune evasion of mycobacteria. We found that TTP knockout mice displayed reduced bacterial burden in the early stage after Mtb aerosol challenge. Macrophages deficient in TTP also showed an inhibition in mycobacterial growth. Live mycobacteria induced TTP protein expression in macrophages, which was blocked by mTOR inhibitor rapamycin. Rapamycin promoted TTP protein degradation. Proteasome inhibitor MG-132 recovered the shortened TTP protein half-life induced by rapamycin. Ubiquitination mediated TTP protein degradation triggered by rapamycin in infected macrophages. TTP induction suppressed the expression of iNOS/TNF-α/IL-12/IL-23, and weakened the bactericidal effects in infected macrophages, whereas blocking mTOR pathway with rapamycin enhanced the bactericidal effects of infected macrophages through TTP inhibition. Overall, this study reveals a novel role for RNA-binding protein TTP in Mtb immune evasion mechanisms and provides a potential target for host-directed therapy against TB. R21AI171734 & R21AI151936

Knee Alignment Correction by High Tibial Osteotomy Reduces Symptoms and Synovial Inflammation in Knee Osteoarthritis Accompanied by Macrophage Phenotypic Change From M1 to M2.

This study aimed to determine whether alignment correction by high tibial osteotomy (HTO) can change the biologic intraarticular microenvironment of osteoarthritic (OA) knees. Synovial tissue (ST) and fluid (SF) were collected from the affected knees of 31 OA patients during initial HTO and plate removal surgeries. Changes in gene expression in ST were investigated by microarray and real-time polymerase chain reaction (PCR). ST specimens were also evaluated histologically using synovitis scores and immunofluorescence staining to determine macrophage polarity. Cytokines and chemokines in SF were analyzed by enzyme-linked immunosorbent assays. The mechanism of macrophage polarization was investigated in human peripheral blood mononuclear cell-derived macrophages and fibroblast-like synoviocytes (FLS) stimulated with cartilage fragments. We also evaluated Spearman correlations between Knee Injury and Osteoarthritis Outcome scores (KOOS) and macrophage-related gene expression. The microarray results indicated down-regulated inflammatory genes and pathways. Real-time PCR determined that genes expressing proinflammatory IL1B and IL6 were down-regulated and M2 macrophage-related IL1RA, IL10, CCL18, and CD206 were up-regulated. Histologic findings revealed attenuated synovitis scores and a shift from M1 to M2 macrophages. Interleukin-1β (IL-1β) concentrations in SF decreased after HTO. Cartilage fragments were responsible for M1 macrophage polarization and proinflammatory gene and protein expression in macrophages, whereas cartilage fragments up-regulated only IL-6 protein in FLS. Postoperative KOOS positively correlated with the expression of the M2-related genes CCL18 and CD206. Correction of lower limb alignment with HTO attenuated synovial inflammation and changed macrophage polarization from M1 to M2, suggesting an improved intraarticular environment in knee OA.

Branched-chain amino acids promotes the repair of exercise-induced muscle damage via enhancing macrophage polarization.

The repair of exercise-induced muscle damage (EIMD) is closely related with inflammation. Branched-chain amino acids (BCAAs), as a nutritional supplement, promote EIMD repair; however, the underlying mechanism remains unclear. In vivo, Sprague-Dawley rats were subjected to Armstrong's eccentric exercise (a 120-min downhill run with a slope of -16° and a speed of 16mmin-1) to induce EIMD and BCAA supplement was administered by oral gavage. Protein expression of macrophages (CD68 and CD163) and myogenic regulatory factors (MYOD and MYOG) in gastrocnemius was analyzed. Inflammatory cytokines and creatine kinase (CK) levels in serum was also measured. In vitro, peritoneal macrophages from mice were incubated with lipopolysaccharide (LPS) or IL-4 with or without BCAAs in culture medium. For co-culture experiment, C2C12 cells were cultured with the conditioned medium from macrophages prestimulated with LPS or IL-4 in the presence or absence of BCAAs. The current study indicated BCAA supplementation enhanced the M1/M2 polarization of macrophages in skeletal muscle during EIMD repair, and BCAAs promoted M1 polarization through enhancing mTORC1-HIF1α-glycolysis pathway, and promoted M2 polarization independently of mTORC1. In addition, BCAA-promoted M1 macrophages further stimulated the proliferation of muscle satellite cells, whereas BCAA-promoted M2 macrophages stimulated their differentiation. Together, these results show macrophages mediate the BCAAs' beneficial impacts on EIMD repair via stimulating the proliferation and differentiation of muscle satellite cells, shedding light on the critical role of inflammation in EIMD repair and the potential nutritional strategies to ameliorate muscle damage.

LC3-Associated Phagocytosis Is Impaired in Bone Marrow Macrophages during Ageing Leading to Defective Processing of Apoptotic Cells and Suppression of Sting Activation

Introduction Macrophage phagocytic function declines with ageing, with potential deleterious consequences on inflammation, immune response, maintenance of tissue health, and cancer. Paradoxically, we and others have found that with increasing age, macrophage number increases within the bone marrow (BM) microenvironment, supporting the hypothesis that the ageing macrophages are functionally impaired. Phagocytosis by mononuclear cells occurs through either an LC3-dependent or -independent mechanism. LC3-associated phagocytosis (LAP) is physiologically triggered by pathogens and cellular debris via interaction with phagocyte surface receptors, including T cell membrane protein 4 (TIM-4) and FcR. Here, we investigate whether age-related macrophage functional impairment is associated with LC3. Results BM was isolated from young (8-12 weeks) and aged (18-24 months) female C57Bl/6 mice and macrophages were analysed using flow cytometry and isolated via FACS. We show increased BM macrophage numbers in old BM (2 fold), but no difference in total phagocytic potential as measured by uptake of E.coli bioparticles or apoptotic debris, when compared to young. Furthermore, using immunofluorescent microscopy to visualise macrophage phagocytosis, we observed that the phagosomes generated in BM macrophages from aged animals did not recruit LC3, whereas in young animals LC3 was recruited. Consequently, macrophages from older animals accumulated large amounts of E.coli bioparticles and apoptotic bodies when compared to young macrophages. This suggests defective processing of the E.coli bioparticles by the phagosome once the phagosome has formed. To understand if this is due to lack of LC3 protein, we next compared gene expression and protein levels of LC3 in young and old animals. There was no loss in LC3 RNA or protein expression in macrophages from old and young. As we have recently reported that BM macrophage LAP mediates the stimulator of interferon genes (STING) pathway, we examined STING activation in old and young BM macrophages by measuring Gbp2, Irf7, and Ifit3 gene expression. Results showed a significant reduction in STING mediated gene expression in BM macrophages from aged mice compared to young. Summary These data suggest the decline in macrophage phagocytic function associated with age occurs because of a failure of LC3-mediated phagosome clearance. Furthermore, we hypothesise that the observed increase in BM macrophage numbers in older animals is, at least in part, a compensatory mechanism for the defective individual macrophage phagocytic processing seen, helping preserve overall BM phagocytic function.

Abstract 15494: Characterization of Adiponectin Kinetics and Adiponectin R2 Pathway in Thp-1 Macrophages and Foam Cells

Background: Adiponectin (APN) is an adipokine most abundantly secreted by adipocytes and possesses vasculoprotective properties. We and others have previously showed that APN receptor 2 (adipoR2) pathway is impaired in foam cells when compared to macrophages. Nevertheless, the functional role of adipoR2 pathway in foam cells has not been fully investigated. We hypothesize that APN kinetics involving AdipoR2 and its subsequent downstream signaling peroxisome proliferator-activated receptor-α (PPAR-α) expression are altered in foam cells when compared to macrophages. Methods: We used THP-1 human macrophage-derived foam cells loaded with oxidized LDL (60μg/ml) and 3H-cholesterol (2μCi/ml) versus control macrophages. APN isomers and PPAR-α protein expression were detected by gradient SDS-PAGE, and Western blotting, respectively. Media cell cultures mixtures were analysed under protease inhibitor by a size-exclusion centrifugal filter molecular weight cut-off 10 kDa. Results: PPAR-α activation significantly increased in APN-treated macrophages (24h) when compared with APN-treated foam cells (24h) with a maximum fold difference of 1.90±1.21, p=0.002 at 10μg APN/mL (PPAR-α: β actin ratio, spectral count; n=10 repeats for each). As a result, in foam cells, APN stimulated PPAR-α with a lower Km molar efficiency (0.06 ± 0.13 μM) and lower velocity Vmax 0.67±0.08 /h-as compared to macrophages (0.023±0.02 μM and 1.37±0.11 /h respectively, p=0.01 for both). In macrophages, APN had smaller dissociation constant Kd (0.037±0.56 μM) than in foam cells (0.10±1.30 μM). APN increased significantly PPAR-α activity rate over time reaching a maximum of 2h, with 1.19±0.02 PPAR-α protein expression in macrophages vs 0.85±0.057 in foam cells. Additionally, the APN dimer/monomer ratio increased over 24h when compared to foam cells, reaching a maximum at 2h in macrophages. Strong correlations were noted between PPARα and APN dimers: in macrophages (r=0.85; p=0.006) and in foam cells (r=0.68, p=0.05). Conclusion: APN stimulates PPARα activity in macrophages, but significantly less in foam cells (weaker kinetic affinity process), suggesting that APN interaction and binding with adipoR2 and subsequent downstream PPARα expression are affected in foam cells.

Protective Effects of Polydatin from Grapes and Reynoutria japonica Houtt. on Damaged Macrophages Treated with Acetaminophen

The unregulated use of acetaminophen (APAP), an antipyretic and analgesic drug, harms hepatocytes and kidney cells, leading to liver failure and acute kidney injury. Herein, we investigate whether APAP damages macrophages in the immune system by observing its effects on macrophage proliferation and apoptosis. Using proteomics, we analyzed the effects of APAP on macrophage protein expression profiles and evaluated whether polydatin, the active ingredient in grapes and wine, can repair the damaged cells. The results showed that APAP alters the morphology and physiological processes of macrophages, inhibits macrophage proliferation, and promotes apoptosis. We observed 528 differentially expressed proteins when 500 µg/mL APAP was administered to the cells. These proteins are involved in biological processes including cell division, apoptosis, and acute phase response. Overall, our findings demonstrate that APAP harms the immune system by damaging macrophages and that polydatin can repair this damage.

Induction of ALOX5 during Polarization of M1 Macrophages by Multi‐walled Carbon Nanotubes

Fibrogenic carbon nanotubes (CNTs) induce the polarization of M1 and M2 macrophages in mouse lungs. Polarization of the macrophages differentiates the production of proinflammatory and pro‐resolving lipid mediators (LMs) to mediate acute inflammation and its resolution in a time‐dependent manner. In the present study, we examined the molecular mechanism by which multi‐walled CNTs (MWCNTs) induce M1 polarization in vitro, with focus on induction of arachidonate 5‐lipoxygenase (ALOX5), which is required to produce proinflammatory LMs, such as leukotriene B4 (LTB4). Treatment of J774A.1 macrophages with MWCNTs at a concentration of 2.5 µg/mL increased the expression of ALOX5 mRNA by 2.0‐fold at 1‐day and 2.5‐fold at 3‐day post‐exposure. At a concentration of 10 µg/mL, MWCNTs increased the ALOX5 mRNA expression by 3.5‐fold at 1‐day and 5.0‐fold at 3‐day post‐exposure. Immunoblotting shows that MWCNTs significantly increased the ALOX5 protein expression in macrophages. Luciferase reporter assays with the mouse ALOX5 promoter of 2 kilobase upstream of translation start codon demonstrate that the ALOX5 promoter activity increased more than 5‐fold over background. This activity was significantly elevated following 5’‐deletion analyses of ALOX5 promoter and further enhanced by MWCNT treatment, implicating a transcriptional mechanism in the induction of ALOX5. MWCNTs preferentially induced the expression of CD68, a cell surface marker of M1, by 2.5‐fold and induction was persistently high to 3‐day post‐exposure. Moreover, both the expression and activity of the inducible nitric oxide synthase, an intracellular marker of M1 macrophages, were increased by MWCNTs, indicating induction of M1 polarization. Consistent with this notion, MWCNTs increased the capacity of the macrophages to produce proinflammatory cytokines, such as tumor necrosis factor‐α (TNF‐α) and interleukin‐1β (IL‐1β), and proinflammatory LMs, such as LTB4 and prostaglandin E2 (PGE2), as measured by enzyme‐linked immunosorbent assays. Taken together, these results support that persistent exposure to MWCNTs polarizes macrophages to M1 cells with induction of ALOX5 and elevated production of proinflammatory LMs to boost the acute inflammatory response to fibrogenic nanoparticles.

Long Non-coding RNA GAS5/miR-520-3p/SOCS3 Axis Regulates Inflammatory Response in Lipopolysaccharide-Induced Macrophages.

Sepsis is mainly caused by infection, and inflammation plays a vital role in the progression of sepsis. Increasing evidence shows the regulatory mechanism of long non-coding RNA growth arrest-specific 5 (GAS5) in inflammatory response. However, the potential role of GAS5 in sepsis was not really clear. Here, we set to investigate the role and mechanism of GAS5 in the inflammatory response of lipopolysaccharide (LPS)-induced macrophages in vitro. Levels of GAS5, microRNA-520-3p, suppressor of cytokine signaling 3 (SOCS3) and inflammatory cytokines tumor necrosis factor-α (TNF-α), and interleukin (IL)-6 and IL-1β were determined by quantitative real-time polymerase chain reaction (qRT-PCR). Nitric oxide (NO) release was measured through flow cytometry. The levels of TNF-α, IL-6, and IL-1β were also detected using ELISA. Dual-luciferase reporter and RNA pull-down assays were performed to clarify the relationship between miR-520-3p and GAS5 or SOCS3. Western blot was carried out to assess SOCS3 protein expression in macrophages. GAS5 level was remarkably decreased in sepsis serum and it was inversely related to the severity of sepsis. Upregulation of GAS5 repressed inflammatory response in LPS-induced macrophages, and the inhibitory effect was returned by miR-520-3p mimics. Moreover, miR-520-3p inhibitor downregulated the levels of inflammatory factors of TNF-α, IL-6, and IL-1β, as well as suppressed NO release. Mechanically, GAS5 functioned as a sponge of miR-520-3p and miR-520-3p directly targeted SOCS3. GAS5 regulated inflammatory response by the miR-520-3p/SOCS3 axis in LPS-induced macrophages, which furnished a novel therapeutic idea in clinical treatment of inflammation-induced sepsis.

BHLHE40 promotes macrophage pro-inflammatory gene expression and functions.

Macrophages are the principal innate immune cells that populate all major organs and provide the first line of cellular defense against infections and/or injuries. The immediate and early-responding macrophages must mount a robust pro-inflammatory response to protect the host by eliminating deleterious agents. The effective pro-inflammatory macrophage response requires the activation of complex transcriptional programs that modulate the dynamic regulation of inflammatory and metabolic gene expression. Therefore, transcription factors that govern pro-inflammatory and metabolic gene expression play an essential role in shaping the macrophage inflammatory response. Herein, we identify the basic helix-loop-helix family member e40 (BHLHE40), as a critical transcription factor that promotes broad pro-inflammatory and glycolytic gene expression by elevating HIF1α levels in macrophages. Our in vivo studies revealed that myeloid-BHLHE40 deficiency significantly attenuates macrophage and neutrophil recruitment to the site of inflammation. Our integrated transcriptomics and gene set enrichment analysis (GSEA) studies show that BHLHE40 deficiency broadly curtails inflammatory signaling pathways, hypoxia response, and glycolytic gene expression in macrophages. Utilizing complementary gain- and loss-of-function studies, our analyses uncovered that BHLHE40 promotes LPS-induced HIF1α mRNA and protein expression in macrophages. More importantly, forced overexpression of oxygen stable form of HIF1α completely reversed attenuated pro-inflammatory and glycolytic gene expression in BHLHE40-deficient macrophages. Collectively, these results demonstrate that BHLHE40 promotes macrophage pro-inflammatory gene expression and functions by elevating HIF1α expression in macrophages.

Role of Macrophages in Cytotoxicity, Reactive Oxygen Species Production and DNA Damage in 1,2-Dichloropropane-Exposed Human Cholangiocytes In Vitro.

1,2-Dichloropropane (1,2-DCP), a synthetic chlorinated organic compound, was extensively used in the past in offset color proof-printing. In 2014, the International Agency for Research on Cancer (IARC) reclassified 1,2-DCP from its initial Group 3 to Group 1. Prior to the reclassification, cholangiocarcinoma was diagnosed in a group of workers exposed to 1,2 -DCP in an offset color proof-printing company in Japan. In comparison with other forms of cholangiocarcinoma, 1,2-DCP-induced cholangiocarcinoma was of early onset and accompanied by extensive pre-cancerous lesions in large bile ducts. However, the mechanism of 1,2-DCP-induced cholangiocarcinoma is poorly understood. Inflammatory cell proliferation was observed in various sites of the bile duct in the noncancerous hepatic tissues of the 1,2-DCP-induced cholangiocarcinoma. The aim of this study was to enhance our understanding of the mechanism of 1,2-DCP-related cholangiocarcinogenesis. We applied an in vitro system to investigate the effects of 1,2-DCP, using MMNK-1 cholangiocytes cultured alone or with THP-1 macrophages. The cultured cells were exposed to 1,2-DCP at 0, 0.1, 0.2, 0.4, and 0.8 mM for 24 h, and then assessed for cell proliferation, cell cytotoxicity, DNA damage, and ROS production. Exposure to 1,2-DCP increased proliferation of MMNK-1 cholangiocytes cultured alone, but not those cultured with macrophages. 1,2-DCP also increased LDH cytotoxicity, DNA damage, and ROS production in MMNK-1 cholangiocytes co-cultured with macrophages but not those cultured alone. 1,2-DCP increased TNFα and IL-1β protein expression in macrophages. The results highlight the role of macrophages in enhancing the effects of 1,2-DCP on cytotoxicity, ROS production, and DNA damage in cholangiocytes.

Culture of Mycobacterium smegmatis in Different Carbon Sources to Induce In Vitro Cholesterol Consumption Leads to Alterations in the Host Cells after Infection: A Macrophage Proteomics Analysis.

During tuberculosis, Mycobacterium uses host macrophage cholesterol as a carbon and energy source. To mimic these conditions, Mycobacterium smegmatis can be cultured in minimal medium (MM) to induce cholesterol consumption in vitro. During cultivation, M. smegmatis consumes MM cholesterol and changes the accumulation of cell wall compounds, such as PIMs, LM, and LAM, which plays an important role in its pathogenicity. These changes lead to cell surface hydrophobicity modifications and H2O2 susceptibility. Furthermore, when M. smegmatis infects J774A.1 macrophages, it induces granuloma-like structure formation. The present study aims to assess macrophage molecular disturbances caused by M. smegmatis after cholesterol consumption, using proteomics analyses. Proteins that showed changes in expression levels were analyzed in silico using OmicsBox and String analysis to investigate the canonical pathways and functional networks involved in infection. Our results demonstrate that, after cholesterol consumption, M. smegmatis can induce deregulation of protein expression in macrophages. Many of these proteins are related to cytoskeleton remodeling, immune response, the ubiquitination pathway, mRNA processing, and immunometabolism. The identification of these proteins sheds light on the biochemical pathways involved in the mechanisms of action of mycobacteria infection, and may suggest novel protein targets for the development of new and improved treatments.

Cannabidiol-Driven Alterations to Inflammatory Protein Landscape of Lipopolysaccharide-Activated Macrophages In Vitro May Be Mediated by Autophagy and Oxidative Stress.

Background: The nonpsychotropic phytocannabinoid cannabidiol (CBD) presents itself as a potentially safe and effective anti-inflammatory treatment relative to clinical standards. In this present study, we compare the capacity of CBD to the corticosteroid dexamethasone (Dex) in altering the secreted protein landscape of activated macrophages and speculate upon the mechanism underpinning these alterations. Materials and Methods: Human THP-1 monocytes were differentiated into macrophages (THP-1 derived macrophages [tMACs]), activated with lipopolysaccharide (LPS), and then treated with 5, 10, 25, 50, or 100 μM CBD or 10 μM Dex for 24 h. Following treatment, cytotoxicity of CBD and protein expression levels from culture supernatants and from whole cell lysates were assessed for secreted and intracellular proteins, respectively. Results: High concentration (50 and 100 μM) CBD treatments exhibit a cytotoxic effect on LPS-activated tMACs following the 24-h treatment. Relative to the LPS-activated and untreated control (M[LPS]), both 25 μM CBD and 10 μM Dex reduced expression of pro-inflammatory markers-tumor necrosis factor alpha, interleukin 1 beta, and regulated on activation, normal T cell expressed and secreted (RANTES)-as well as the pleiotropic marker interleukin-6 (IL-6). A similar trend was observed for anti-inflammatory markers interleukin-10 and vascular endothelial growth factor (VEGF). Dex further reduced secreted levels of monocyte chemoattractant protein-1 in addition to suppressing IL-6 and VEGF beyond treatments with CBD. The anti-inflammatory capacity of 25 μM CBD was concurrent with reduction in levels of phosphorylated mammalian target of rapamycin Ser 2448, endothelial nitric oxide synthase, and induction of cyclooxygenase 2 relative to M(LPS). This could suggest that the observed effects on macrophage immune profile may be conferred through inhibition of mammalian target of rapamycin complex 1 and ensuing induction of autophagy. Conclusion: Cumulatively, these data demonstrate cytotoxicity of high concentration CBD treatment. The data reported herein largely agree with other literature demonstrating the anti-inflammatory effects of CBD. However, there is discrepancy within literature surrounding efficacious concentrations and effects of CBD on specific secreted proteins. These data expand upon previous work investigating the effects of CBD on inflammatory protein expression in macrophages, as well as provide insight into the mechanism by which these effects are conferred.

Calcitriol in the Presence of Conditioned Media from Metastatic Breast Cancer Cells Enhances Ex Vivo Polarization of M2 Alternative Murine Bone Marrow-Derived Macrophages.

Simple SummaryIn this study, we stimulated bone marrow-derived macrophages to M0, M1, and M2 subtypes, with or without calcitriol, or with or without 4T1 (metastatic), 67NR (non-metastatic), and Eph4-Ev (normal) cell culture supernatants (CMs) to test their effect on polarization. We showed that calcitriol increased the expression of Cd206 and Spp1 mRNA and CD36, CCL2, and arginase levels for M2 macrophages and decreased Cd80 and Spp1 mRNA and IL-1, IL-6, OPN, and iNOS for M1 macrophages. 4T1 CM influenced the expression of the studied genes and proteins to a greater extent than 67NR and Eph4; the strongest effect was noted for M2 macrophages. We show that calcitriol and 4T1 CM enhance the polarization of M2 macrophages and M2 macrophages differentiated with calcitriol-stimulated migration of 4T1 and 67NR cells. We indicate that the immunosuppressive properties of calcitriol may unfavorably affect the tumor microenvironment, and supplementation with vitamin D in oncological patients may not always bring benefits.In this study, we differentiated murine bone marrow-derived macrophages (BMDMs) into M0, M1, and M2 in the presence or absence of calcitriol. Real-time PCR analysis of gene expression, FACS analysis of surface markers, and chemokine/cytokine production assays were performed. In addition, the effect of the conditioned media (CM) from murine breast cancer 4T1 (metastatic) and 67NR (non-metastatic) and Eph4-Ev (normal) cells with and without calcitriol on the polarization of M1/M2 cells was determined. We found that calcitriol enhanced the differentiation of M2 macrophages, which was manifested by increased expression of Cd206 and Spp1 mRNA and CD36, Arg, and CCL2 in M2 BMDMs and by decreased expression of Cd80 and Spp1 mRNA and IL-1, IL-6, OPN, and iNOS in M1 BMDMs. 4T1 CM showed a higher effect on the gene and protein expression in macrophages than 67NR and Eph4-Ev, with the greatest effect observed on M2 macrophages which increased their differentiation and properties characteristic of alternative macrophages. Moreover, M2 macrophages differentiated with calcitriol-stimulated migration of 4T1 and 67NR cells through fibronectin and collagen type IV, respectively. Overall, our results indicated that vitamin D supplementation may not always be beneficial, especially in relation to cancers causing excessive, pathological activation of the immune system.

Hypercapnia Suppresses Macrophage Antiviral Activity and Increases Mortality of Influenza A Infection via Akt1.

Hypercapnia (HC), elevation of the partial pressure of CO2 in blood and tissues, is a risk factor for mortality in patients with severe acute and chronic lung diseases. We previously showed that HC inhibits multiple macrophage and neutrophil antimicrobial functions and increases the mortality of bacterial pneumonia in mice. In this study, we show that normoxic HC increases viral replication, lung injury, and mortality in mice infected with influenza A virus (IAV). Elevated CO2 increased IAV replication and inhibited antiviral gene and protein expression in macrophages in vivo and in vitro. HC potentiated IAV-induced activation of Akt, whereas specific pharmacologic inhibition or short hairpin RNA knockdown of Akt1 in alveolar macrophages blocked HC's effects on IAV growth and the macrophage antiviral response. Our findings suggest that targeting Akt1 or the downstream pathways through which elevated CO2 signals could enhance macrophage antiviral host defense and improve clinical outcomes in hypercapnic patients with advanced lung disease.

Anti-inflammation Effects of Sinomenine on Macrophages through Suppressing Activated TLR4/NF-κB Signaling Pathway.

Sinomenine (SN) has been used in the clinical treatment of systemic lupus erythematosus and rheumatoid arthritis for many years. Studies showed that SN held protective effects such as anti-inflammation, scavenging free radicals and suppressing immune response in many autoimmune diseases. The purpose of the present study is to explore the mechanism of anti-inflammation of SN on lipopolysaccharide (LPS)-induced macrophages activation and investigate whether the TLR4/NF-κB signaling pathway participated in. Macrophages isolated from mouse peritoneal cavity were stimulated by 1 µg/mL LPS for 24 h. And then the cells were treated with various concentrations of SN, TLR4 inhibitor respectively for additional 48 h. Drug toxicity was detected by MTT assay and Transwell experiment was used to assess chemotaxis. Furthermore, TLR4 and MyD88 mRNA levels were detected by real-time PCR. Western blotting was used to examine TLR4, MyD88 and phosphorylated IκB protein expression in macrophages. Immunofluorescence assay was applied to observe p65 NF-κB protein expression in macrophage nucleus. We extracted macrophages with high purity and activity from the abdominal cavity of mice. SN remarkably inhibited the chemotaxis and secretion function of LPS-stimulated macrophages. It also down-regulated both the protein levels of inflammatory cytokines (TNF-α, IL-1β and IL-6) and the RNA and protein levels of the key factors (TLR4, MyD88, P-IκB) in TLR4 pathway. The expression of p65 NF-κB protein in nuclei was down-regulated, which was correlated with a similar decrease in P-IκB protein level. In conclusion, SN can inhibit the LPS induced immune responses in macrophages by blocking the activated TLR4/NF-κB signaling pathway. These results may provide a therapeutic approach to regulate inflammatory responses.

Inhibitory Effects of Gynura procumbens Ethanolic Extract on Nitric Oxide Production and Inducible Nitric Oxide Synthase (iNOS) Protein Expression in Macrophages

Nitric oxide (NO) overproduction by inducible nitric oxide synthase (iNOS) may be associated with acute and chronic inflammations. Macrophages as important cells in the innate immune system are able to be stimulated and can lead to iNOS activation and excessive NO production. Gynura procumbens is a medicinal plant traditionally used in treating various ailments including inflammation but the mechanism of anti-inflammatory activity of this plant is still elusive. This study was carried out to investigate the anti-inflammatory therapeutic effects of Gynura procumbens ethanolic extract on NO production and iNOS protein expression in RAW 264.7 macrophages stimulated with lipopolysaccharide (LPS). Cell viability of RAW 264.7 macrophages treated with Gynura procumbens ethanolic extract was determined by MTT assay. NO production was determined by Griess assay following Gynura procumbens ethanolic extract treatment alone or in combination with LPS stimulation. Protein expression of iNOS was determined by western blot. RAW 264.7 macrophages viability of more than 90% was observed after 24 h treatment with Gynura procumbens ethanolic extract concentration range of 3.9 μg/mL to 500 μg/mL. Significant inhibition of NO production level has been identified in LPS-stimulated RAW 264.7 cells pre-treated with 250 μg/mL Gynura procumbens ethanolic extract (p<0.05) while all selected concentrations of Gynura procumbens ethanolic extract showed no significant alteration of NO production in the absence of LPS stimulation. Pre-treatment of 250 μg/mL Gynura procumbens ethanolic extract also demonstrated significant suppression of iNOS protein expression in LPS-stimulated RAW 264.7 cells (p<0.05). In conclusion, this study demonstrates that Gynura procumbens ethanolic extract exhibits anti-inflammatory potential through inhibition of NO production and iNOS protein expression in LPS-stimulated macrophages, suggesting that this plant could be further researched for its beneficial use in inflammatory disorders.

Protective effects and mechanisms of omega-3 polyunsaturated fatty acid on intestinal injury and macrophage polarization in peritoneal dialysis rats.

ABSTRACTAimThis study was conducted to investigate the chronic injury of peritoneal glucose injection on the peritoneum and intestine and the protective effects of omega‐3 polyunsaturated fatty acid (ω‐3PUFA) during peritoneal dialysis (PD).MethodsPeritoneal dialysis animal models were established by intraperitoneal injection of 4.25% glucose for 28 days. Protein expression in ileum and peritoneum was measured by immunofloresence and immunohistochemistry. Protein expression in macrophages was measured by Western blot. Fibrosis was analyzed by Masson staining.ResultsPeritoneal dialysis significantly increased the structural injury and decreased junction‐related protein ZO‐1 and occludin expression in ileum, the expression of proteins relating to the activation of M2 (Erg2, IRF4), but not M1 (CD38, IRF5) macrophages. PD significantly increased the expression of TGF‐β1, VEGF and ALK5 protein in peritoneal tissues. PD significantly increased fibrosis (Masson staining) and the expression of fibroblast marker α‐SMA in peritoneal tissues. Injection of macrophage clean reagent and ω‐3PUFA significantly inhibited M2 activation, and decreased Masson staining, α‐SMA, TGF‐β1, VEGF and ALK5 protein expression in peritoneal tissues in PD treated rats. ω‐3PUFA injection significantly decreased PD‐induced injury in ileum and normalized the expression of ZO‐1 and occludin in the ileum of PD rats.ConclusionOmega‐3 fatty acids can provide a protective role on PD‐induced peritoneal fibrosis and injury of the intestine.

Proteomic Analysis of Human Macrophage Polarization Under a Low Oxygen Environment.

Macrophages are innate immune cells involved in a number of physiological functions ranging from responses to infectious pathogens to tissue homeostasis. The various functions of these cells are related to their activation states, which is also called polarization. The precise molecular description of these various polarizations is a priority in the field of macrophage biology. It is currently acknowledged that a multidimensional approach is necessary to describe how polarization is controlled by environmental signals. In this report, we describe a protocol designed to obtain the proteomic signature of various polarizations in human macrophages. This protocol is based on a label-free quantification of macrophage protein expression obtained from in-gel fractionated and Lys C/trypsin-digested cellular lysis content. We also provide a protocol based on in-solution digestion and isoelectric focusing fractionation to use as an alternative. Because oxygen concentration is a relevant environmental parameter in tissues, we use this protocol to explore how atmospheric composition or a low oxygen environment affects the classification of macrophage polarization.