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Inflammation-induced inhibition of chaperone-mediated autophagy maintains the immunosuppressive function of murine mesenchymal stromal cells.

ABSTRACT: Macroautophagy has been implicated in modulating the therapeutic function of mesenchymal stromal cells (MSCs). However, the biological function of chaperone-mediated autophagy (CMA) in MSCs remains elusive. Here, we found that CMA was inhibited in MSCs in response to the proinflammatory cytokines interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α). In addition, suppression of CMA by knocking down the CMA-related lysosomal receptor lysosomal-associated membrane protein 2 (LAMP-2A) in MSCs significantly enhanced the immunosuppressive effect of MSCs on T cell proliferation, and as expected, LAMP-2A overexpression in MSCs exerted the opposite effect on T cell proliferation. This effect of CMA on the immunosuppressive function of MSCs was attributed to its negative regulation of the expression of chemokine C-X-C motif ligand 10 (CXCL10), which recruits inflammatory cells, especially T cells, to MSCs, and inducible nitric oxide synthase (iNOS), which leads to the subsequent inhibition of T cell proliferation via nitric oxide (NO). Mechanistically, CMA inhibition dramatically promoted IFN-γ plus TNF-α-induced activation of NF-κB and STAT1, leading to the enhanced expression of CXCL10 and iNOS in MSCs. Furthermore, we found that IFN-γ plus TNF-α-induced AKT activation contributed to CMA inhibition in MSCs. More interestingly, CMA-deficient MSCs exhibited improved therapeutic efficacy in inflammatory liver injury. Taken together, our findings established CMA inhibition as a critical contributor to the immunosuppressive function of MSCs induced by inflammatory cytokines and highlighted a previously unknown function of CMA.

SUBMITTER: Zhang J

PROVIDER: S-EPMC8167126 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:RationaleThe NLRP3 (NLR [NOD-like receptor] family, pyrin domain containing 3) inflammasome is an important driver of atherosclerosis. Our previous study shows that chaperone-mediated autophagy (CMA), one of the main lysosomal degradative process, has a regulatory role in lipid metabolism of macrophages. However, whether the NLRP3 inflammasome is regulated by CMA, and the role of CMA in atherosclerosis remains unclear.ObjectiveTo determine the role of CMA in the regulation of NLRP3 inflammasome and atherosclerosis.Methods and resultsThe expression of CMA marker, LAMP-2A (lysosome-associated membrane protein type 2A), was first analyzed in ApoE-/- mouse aortas and human coronary atherosclerotic plaques, and a significant downregulation of LAMP-2A in advanced atherosclerosis in both mice and humans was observed. To selectively block CMA, we generated macrophage-specific conditional LAMP-2A knockout mouse strains in C57BL/6 mice and ApoE-/- mice. Deletion of macrophage LAMP-2A accelerated atherosclerotic lesion formation in the aortic root and the whole aorta in ApoE-/- mice. Mechanistically, LAMP-2A deficiency promoted NLRP3 inflammasome activation and subsequent release of mature IL (interleukin)-1β in macrophages and atherosclerotic plaques. Furthermore, gain-of-function studies verified that restoration of LAMP-2A levels in LAMP-2A-deficient macrophages greatly attenuated NLRP3 inflammasome activation. Importantly, we identified the NLRP3 protein as a CMA substrate and demonstrated that LAMP-2A deficiency did not affect the NLRP3 mRNA levels but hindered degradation of the NLRP3 protein through CMA pathway.ConclusionsCMA function becomes impaired during the progression of atherosclerosis, which increases NLRP3 inflammasome activation and secretion of IL-1β, promoting vascular inflammation and atherosclerosis progression. Our study unveils a new mechanism by which NLRP3 inflammasome is regulated in macrophages and atherosclerosis, thus providing a new insight into the role of autophagy-lysosomal pathway in atherosclerosis. Pharmacological activation of CMA may provide a novel therapeutic strategy for atherosclerosis and other NLRP3 inflammasome/IL-1β-driven diseases.

Project description:Mesenchymal stromal cells (MSCs) have received attention as promising therapeutic agents for the treatment of various diseases. However, poor post-transplantation viability is a major hurdle in MSC-based therapy, despite encouraging results in many inflammatory disorders. Recently, three dimensional (3D)-cultured MSCs (MSC3D) were shown to have higher cell survival and enhanced anti-inflammatory effects, although the underlying mechanisms have not yet been elucidated. In this study, we investigated the molecular mechanisms by which MSC3D gain the potential for enhanced cell viability. Herein, we found that macroautophagy/autophagy was highly induced and ROS production was suppressed in MSC3D as compared to 2D-cultured MSCs (MSC2D). Interestingly, inhibition of autophagy induction caused decreased cell viability and increased apoptotic activity in MSC3D. Furthermore, modulation of ROS production was closely related to the survival and apoptosis of MSC3D. We also observed that HMOX1 (heme oxygenase 1) was significantly up-regulated in MSC3D. In addition, gene silencing of HMOX1 caused upregulation of ROS production and suppression of the genes related to autophagy. Moreover, inhibition of HIF1A (hypoxia inducible factor 1 subunit alpha) caused suppression of HMOX1 expression in MSC3D, indicating that the HIF1A-HMOX1 axis plays a crucial role in the modulation of ROS production and autophagy induction in MSC3D. Finally, the critical role of autophagy induction on improved therapeutic effects of MSC3D was further verified in dextran sulfate sodium (DSS)-induced murine colitis. Taken together, these results indicated that autophagy activation and modulation of ROS production mediated via the HIF1A-HMOX1 axis play pivotal roles in enhancing the viability of MSC3D.Abbreviations: 3D: three dimensional; 3MA: 3 methlyadenine; AMPK: AMP-activated protein kinase; Baf A1: bafilomycin A1; CFSE: carboxyfluorescein succinimidyl ester; CoCl2: cobalt chloride; CoPP: cobalt protoporphyrin; DSS: dextran sulfate sodium; ECM: extracellular matrix; FOXO3/FOXO3A: forkhead box O3; HIF1A: hypoxia inducible factor 1 subunit alpha; HMOX1/HO-1: heme oxygenase 1; HSCs: hematopoietic stem cells; IL1A/IL-1α: interleukin 1 alpha; IL1B/IL-1β: interleukin 1 beta; IL8: interleukin 8; KEAP1: kelch like ECH associated protein 1; LAMP1: lysosomal associated membrane protein 1; LAMP2: lysosomal associated membrane protein 2; MSC2D: 2D-cultured MSCs; MSC3D: 3D-cultured MSCs; MSCs: mesenchymal stromal cells; NFE2L2/NRF2: nuclear factor, erythroid 2 like 2; PGE2: prostaglandin E2; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; PINK1: PTEN induced kinase 1; ROS: reactive oxygen species; siRNA: small interfering RNA; SIRT1: sirtuin 1; SOD2: superoxide dismutase 2; SQSTM1/p62: sequestosome 1; TGFB/TGF-β: transforming growth factor beta.

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Inflammation-induced inhibition of chaperone-mediated autophagy maintains the immunosuppressive function of murine mesenchymal stromal cells.

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