CCL2 and Interleukin-6 Promote Survival of Human CD11b+ Peripheral Blood Mononuclear Cells and Induce M2-type Macrophage Polarization

CCL2 and interleukin (IL)-6 are among the most prevalent cytokines in the tumor microenvironment, with expression generally correlating with tumor progression and metastasis. CCL2 and IL-6 induced expression of each other in CD11b⁺ cells isolated from human peripheral blood. It was demonstrated that both cytokines induce up-regulation of the antiapoptotic proteins cFLIP_L (cellular caspase-8 (FLICE)-like inhibitory protein), Bcl-2, and Bcl-X_L and inhibit the cleavage of caspase-8 and subsequent activation of the caspase-cascade, thus protecting cells from apoptosis under serum deprivation stress. Furthermore, both cytokines induced hyperactivation of autophagy in these cells. Upon CCL2 or IL-6 stimulation, CD11b⁺ cells demonstrated a significant increase in the mannose receptor (CD206) and the CD14⁺/CD206⁺ double-positive cells, suggesting a polarization of macrophages toward the CD206⁺ M2-type phenotype. Caspase-8 inhibitors mimicked the cytokine-induced up-regulation of autophagy and M2 polarization. Furthermore, E64D and leupeptin, which are able to function as inhibitors of autophagic degradation, reversed the effect of caspase-8 inhibitors in the M2-macrophage polarization, indicating a role of autophagy in this mechanism. Additionally, in patients with advanced castrate-resistant prostate cancer, metastatic lesions exhibited an increased CD14⁺/CD206⁺ double-positive cell population compared with normal tissues. Altogether, these findings suggest a role for CCL2 and IL-6 in the survival of myeloid monocytes recruited to the tumor microenvironment and their differentiation toward tumor-promoting M2-type macrophages via inhibition of caspase-8 cleavage and enhanced autophagy.     

Autophagy protects PC12 cells against deoxynivalenol toxicity via the Class III PI3K/beclin 1/Bcl-2 pathway

Deoxynivalenol (DON) is a major mycotoxin from the trichothecene family of mycotoxins produced by Fusarium fungi. It can cause a variety of adverse effects on human and farm animal health. Here, we determined the effect of DON on the Class III phosphatidylinositol 3-kinase (PIK3C3)/beclin 1/B cell lymphoma-2 (Bcl-2) pathway in PC12 cells and the relationship between autophagy and apoptosis. The effects of DON were evaluated based on the apoptosis ratio; the typical indicators of autophagy, including cellular morphology, acridine orange- and monodansylcadaverine-labeled vacuoles, green fluorescent protein–microtubule associated protein 1 light chain 3 (LC3) localization, and LC3 immunofluorescence; and the expression of key autophagy-related genes and proteins, that is, PIK3C3, beclin 1, Bcl-2, LC3, and p62. The relationship between autophagy and apoptosis was analyzed by western blot analysis and flow cytometry. DON-induced PC12 cell morphological changes and autophagy significantly. PIK3C3, beclin 1, and LC3 increased in tandem with the DON concentration used; Bcl-2 and p62 expression decreased as DON concentrations increased. Moreover, the PIK3C3/beclin 1/Bcl-2 signaling pathway played a role in DON-induced autophagy. Our findings suggest that DON can induce autophagy by activating the PIK3C3/beclin 1/Bcl-2 signaling pathway and that autophagy may play a positive role in reducing DON-induced apoptosis.     

miR-34a-5p was involved in chronic intermittent hypoxia-induced autophagy of human coronary artery endothelial cells via Bcl-2/beclin 1 signal transduction pathway

Autophagy refers to the genetically regulated process to regulate the survival and death of cells, which is conserved in evolution. Typically, autophagy exerts a vital part under physiopathological conditions. Whether autophagy can be resulted from chronic intermittent hypoxia (CIH), a prominent characteristic of obstructive sleep apnea-hypopnea syndrome (OSAHS), remains to be investigated. Furthermore, microRNAs (miRNAs) can serve as the regulating factors in a variety of benign and malignant diseases; nonetheless, it remains to be fully illustrated about the way by which miRNAs modulate autophagy. According to our results, for human coronary artery endothelial cells (HCAECs), CIH increased the expression of autophagy-associated proteins, which depended on the concentration and time; besides, it could promote autophagic vacuole (AV) formation. In addition, CIH could activate beclin 1, which was dependent on dose and time. In HCAECs, microRNA-34a-5p (miR-34a-5p) was overexpressed after exposed to CIH, and its target protein B-cell lymphoma 2 (Bcl-2) was downregulated. Moreover, inhibiting miR-34a-5p increased Bcl-2 and p62 expression, while downregulating beclin 1, Vps34, Atg5, and LC3 levels, implying the role of miR-34a-5p in CIH-induced autophagy. Moreover, exogenous upregulation of Bcl-2 could block miR-34a-5p influence on CIH-induced autophagy through suppressing beclin 1 expression. Additionally, beclin 1 could enhance the autophagy induced by CIH. In conclusion, overexpression of miR-34a-5p activated beclin 1 through Bcl-2 inhibition in CIH and participated in CIH-induced autophagy.     

Mechanistic determinants of MBNL activity

Muscleblind-like (MBNL) proteins are critical RNA processing factors in development. MBNL activity is disrupted in the neuromuscular disease myotonic dystrophy type 1 (DM1), due to the instability of a non-coding microsatellite in the DMPK gene and the expression of CUG expansion (CUG^exp) RNAs. Pathogenic interactions between MBNL and CUG^exp RNA lead to the formation of nuclear complexes termed foci and prevent MBNL function in pre-mRNA processing. The existence of multiple MBNL genes, as well as multiple protein isoforms, raises the question of whether different MBNL proteins possess unique or redundant functions. To address this question, we coexpressed three MBNL paralogs in cells at equivalent levels and characterized both specific and redundant roles of these proteins in alternative splicing and RNA foci dynamics. When coexpressed in the same cells, MBNL1, MBNL2 and MBNL3 bind the same RNA motifs with different affinities. While MBNL1 demonstrated the highest splicing activity, MBNL3 showed the lowest. When forming RNA foci, MBNL1 is the most mobile paralog, while MBNL3 is rather static and the most densely packed on CUG^exp RNA. Therefore, our results demonstrate that MBNL paralogs and gene-specific isoforms possess inherent functional differences, an outcome that could be enlisted to improve therapeutic strategies for DM1.     

Tissue transglutaminase cross-links beclin 1 and regulates autophagy in MPP-treated human SH-SY5Y cells

Tissue transglutaminase (tTG) is a cross-linking enzyme involved in protein aggregation during Parkinson’s disease (PD) pathogenesis. Autophagy is inhibited by tTG activation via a mechanism in which cross-linking of beclin 1, an autophagy initiator at the level of the endoplasmic reticulum (ER), has been implicated. We reported increased tTG protein levels and activity at the ER in both PD brain and in a PD-mimicking cell system. Here we characterized the interaction between tTG and beclin 1 at the ER membrane and the role of tTG in reduced autophagy in an in vitro model of PD, using differentiated SH-SY5Y neurons treated with the PD-mimic MPP⁺. We found that under PD-mimicking conditions, beclin 1 and tTG partially colocalized at the ER, beclin 1 levels increased at the ER, and tTG readily cross-linked beclin 1 which was prevented by enzymatic blockade of tTG. Under these conditions, accumulation of beclin 1 at the ER was enhanced by inhibition of tTG activity. In line with these observations and the role of beclin 1 in autophagy, levels of the autophagy marker protein LC3II in MPP⁺-treated cells, were significantly increased by inhibition of tTG activity. Our data provide first evidence for a role of tTG-mediated regulation of beclin 1 and autophagy in MPP⁺-treated human SH-SY5Y cells.     

M2-polarized tumor-associated macrophages promote epithelial-mesenchymal transition via activation of the AKT3/PRAS40 signaling pathway in intrahepatic cholangiocarcinoma

Tumor-associated macrophages (TAMs) have been considered as a major component of the tumor microenvironment. However, the crosstalk between M2-polarized tumor-associated macrophages (M2-TAMs) and intrahepatic cholangiocarcinoma (ICC) remains undetermined. In the present study, we aimed to clarify the role of M2-TAMs in ICC and the underlying mechanism. The in vitro assay demonstrated M2-TAMs promoted epithelial-mesenchymal transition (EMT) of ICC cells, resulting in enhanced cell invasion and metastasis ability. Moreover, M2-TAMs modulated the microenvironment of ICC by increasing the secretion of cytokines (GM-CSF, tumor necrosis factor-α [TNF-α], ICAM-1, interleukin-6 [IL-6], etc) and chemokines (CCL1, CCL3, etc). In addition, p-AKT (Ser473) and p-PRAS40 (Thr246) were upregulated in ICC cells when cocultured with M2-TAMs or treated with M2-TAMs secreted core cytokines (GM-CSF, TNF-α, ICAM-1, and IL-6). Consistently, AKT3 silencing (but not AKT1 silencing and AKT2 silencing) markedly inhibited phosphorylation of AKT and PRAS40 of ICC cells and inhibited the EMT process when cocultured with M2-TAMs. Taken together, the current data indicated that M2-TAMs promoted ICC cells EMT, partially through increasing secretion of cytokines and chemokines, thus modulating the microenvironment and activating the AKT3/PRAS40 signaling pathway.     

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.     

LncRNA GIHCG promoted the proliferation and migration of renal cell carcinoma through regulating miR-499a-5p/XIAP axis

BackgroundOur previous study demonstrated that lncRNA GIHCG is upregulated in renal cell carcinoma (RCC) and that knockdown of lncRNA GIHCG suppresses the proliferation and migration of RCC cells. However, the mechanism of lncRNA GIHCG in RCC needs further exploration.MethodsThe proliferation, cell cycle, migration, and apoptosis of RCC cells were tested using CCK-8, flow cytometry, wound healing and Annexin-V/-FITC/PI flow cytometry assays, respectively. Dual-luciferase reporter and RNA pull-down or RNA immunoprecipitation assays (RIPs) were performed to analyze the interactions among lncRNA GIHCG, miR-499a-5p and XIAP. A tumour xenograft study was conducted to verify the function of lncRNA GIHCG in RCC development in vivo.ResultsKnockdown of lncRNA GIHCG inhibited cell proliferation and migration and induced G0/G1 arrest while promoting apoptosis. Overexpression of lncRNA GIHCG led to the opposite results. LncRNA GIHCG sponged miR-499a-5p and downregulated its expression in RCC cells. MiR-499a-5p overexpression suppressed RCC cell growth. MiR-499a-5p targeted XIAP and inhibited its expression. LncRNA GIHCG knockdown reduced the growth of tumour xenografts in vivo and the expression of XIAP while increasing miR-499a-5p levels.ConclusionLncRNA GIHCG accelerated the development of RCC by targeting miR-499a-5p and increasing XIAP levels.     

Autophagy induced by 2-deoxy-D-glucose suppresses intracellular multiplication of Legionella pneumophila in A/J mouse macrophages

Legionella pneumophila Philadelphia-1 (Lp-1) can grow intracellularly in A/J mouse peritoneal macrophages (A/J Mφ). We previously reported that 2-deoxy-D-glucose (2dG), when added in macrophage culture media, inhibited the intracellular multiplication of Lp-1 in A/J Mφ. We found that 1mM of 2dG caused LC3-II-conversion that reflects an induction of autophagy and that 1 and 10mM of 2dG induced apoptosis associated with caspase-4 activation. We therefore investigated whether 2dG-induced autophagy or apoptosis suppresses the replication of Lp-1 in 2dG-treated A/J Mφ. When autophagy-related 5 (Atg5) was knocked down by RNA interference, the Atg5-siRNA-transfected cells revealed an enhanced replication of Lp-1 in A/J Mφ compared with the nontargetting siRNA-transfected cells. However, caspase-4 inhibitor did not affect the 2dG-induced inhibition of intracellular multiplication of Lp-1 in A/J Mφ. These findings suggested that autophagy, not apoptosis, suppressed the intracellular growth of Lp-1 in A/J Mφ when 1 or 10 mM of 2dG were added to the culture media.     

HCC cells with high levels of Bcl-2 are resistant to ABT-737 via activation of the ROS–JNK–autophagy pathway

The Bcl-2 inhibitor ABT-737 has shown promising antitumor efficacy in vivo and in vitro. However, some reports have demonstrated that HCC cells are resistant to ABT-737, and the corresponding molecular mechanisms of this resistance are not well known. In this study, we found that HCC cells with high levels of Bcl-2 were markedly resistant to ABT-737 compared to HCC cells with low levels of Bcl-2. In HCC cells with high levels of Bcl-2 (such as HepG2 cells), ABT-737 induced protective autophagy via the sequential triggering of reactive oxygen species (ROS) accumulation, short-term activation of JNK, enhanced phosphorylation of Bcl-2, and dissociation of Beclin 1 from the Bcl-2/Beclin 1 complex. Moreover, autophagy suppressed the overactivation of the ROS–JNK pathway and protected against apoptosis. In HCC cells with low levels of Bcl-2 (i.e., Huh7 cells), ABT-737 induced apoptosis via the sequential stimulation of ROS, sustained activation of JNK, enhanced translocation of Bax from the cytosol to the mitochondria, and release of cytochrome c. In sum, this study indicated that the activation of the ROS–JNK–autophagy pathway may be an important mechanism by which HCC cells with high levels of Bcl-2 are resistant to ABT-737.     

The regulatory peptide pidotimod facilitates M2 macrophage polarization and its function

Pidotimod is a synthetic dipeptide with biological and immunological activity in innate immune responses. It has been reported that pidotimod could promote functional maturation of dendritic cells, but little is known about the regulation of macrophages. Recent studies have demonstrated that M1 or M2 polarized macrophages are of great importance for responses to microorganism infection or host mediators. The aim of this study was to determine the effectiveness of pidotimod on mouse bone marrow-derived macrophage polarization and its function. The results showed that pidotimod had no influence on M1-polarized macrophage. While interestingly, a significant increase of M2 marker gene expression (Arg1, Fizz1, Ym1, MR) was observed (p < 0.01) in IL-4-induced M2 macrophage treated with pidotimod. In addition, cell surface expression of mannose receptor was dramatically enhanced using fluorescence activated cell sorter (FACS) analysis. Furthermore, the function of M2 macrophage was also determinated. The results showed that the supernatant of pidotimod-treated M2 macrophage could increase the migration (p < 0.05) and enhance the wound closure rate (p < 0.05) of MLE-12 cells. Collectively, it could be concluded that pidotimod significantly facilitated IL-4-induced M2 macrophage polarization and improves its function.