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TLR2-dependent selective autophagy regulates NF-κB lysosomal degradation in hepatoma-derived M2 macrophage differentiation.

ABSTRACT: Autophagy is a lysosomal pathway for cellular homeostasis control. Both non-selective bulk autophagy and selective autophagy of specific proteins or organelles have been found. Selective autophagy prevents cells from pathogen invasion and stress damage, but its role in regulating transcriptional factors is not clear. Using a macrophage cell differentiation model, the role of autophagy in nuclear factor-κB (NF-κB) regulation is investigated. The bone marrow-derived macrophages (BMDMs) will differentiate into a M2-like phenotype in the presence of hepatoma tumor cell condition medium (CM). The TLR2 signaling drives this M2 polarization and causes NF-κB p65 degradation via lysosome-dependent pathway. The CM-induced ubiquitinated- NF-κB p65 forms aggresome-like structures (ALS) in the cytoplasm of cultured and hepatoma-associated M2 macrophages. This NF-κB p65-contained ALS is recognized by p62/SQSTM1 and degraded by selective autophagy. Treatment with the lysosomal inhibitor bafilomycin A1 or the knockdown of Atg5 can prevent CM-induced NK-κB p65 degradation and induce M2 macrophages to produce a high level of pro-inflammatory cytokines. Furthermore, TLR2 signal induces sustained phosphorylation of extracellular signal-regulated kinase 1/2 to facilitate this autophagy-dependent NF-κB regulation. Our finding provides a novel pathway of NF-κB regulation by p62/SQSTM1-mediated selective autophagy.

SUBMITTER: Chang CP

PROVIDER: S-EPMC3569990 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The polymorphic membrane protein D (PmpD) is a highly conserved outer membrane protein which plays an important role in pathogenesis during Chlamydia psittaci infection. In this study, we evaluated the ability of the N-terminus of PmpD (PmpD-N) to modulate the functions of chicken macrophages and the signaling pathway(s) involved in PmpD-N-induced Toll-like receptors (TLRs), as well as interleukin (IL)-6 and IL-10 cytokine secretions. Thus, HD11 macrophages were treated with exogenous and intracellular PmpD-N of C. psittaci. The chlamydial growth was evaluated by enumeration of chlamydial loads in the infected macrophages. The phagocytic function of macrophages following PmpD-N treatment was detected by fluorescein-labeled Escherichia coli (E. coli). The concentration of nitric oxide (NO) secreted by HD11 macrophages was measured by the amount of NO2- in the culture supernatant using the Griess method. The cytokine secretions were assessed using multiplex cytokine ELISA kits. Expression levels of TLRs, myeloid differentiation factor 88 (MyD88), and nuclear factor kappa B (NF-κB) were analyzed by a Western blotting assay, as well as a luciferase assay, while NF-κB p65 nuclear translocation was assessed by confocal microscopy. The nuclear translocation of the transcription factor NF-κB was confirmed by evaluating its ability to combine with the corresponding promoter using the electrophoretic mobility shift assay (EMSA). After treatment with exogenous or endogenous PmpD-N, chlamydial loads and phagocytic functions were reduced significantly compared with those of the plasmid vector group, while NO secretions were reduced significantly compared with those of the lipopolysaccharide (LPS) treatment. Stimulation of HD11 cells with PmpD-N provoked the secretion of the Th2 cytokines, IL-6, and IL-10 and upregulated the expression of TLR2, TLR4, MyD88, and NF-κB. Furthermore, inhibition of TLR2, MyD88, and NF-κB in HD11 cells significantly decreased IL-6 and IL-10 cytokine levels, while NO production and phagocytosis increased significantly, strongly suggesting their involvement in PmpD-N-induced Th2 cytokine secretion and macrophage dysfunction. Our data indicate that C. psittaci PmpD-N inhibited macrophage functions by activating the Th2 immune response and the TLR2/MyD88/NF-κB signaling pathway.

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TLR2-dependent selective autophagy regulates NF-κB lysosomal degradation in hepatoma-derived M2 macrophage differentiation.

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