The Role Of Chaperone-mediated Autophagy Of Macrophages And Its Molecularmechanism In The Development Of NASH

BackgroundThe prevalence of non-alcoholic fatty liver disease（NAFLD）is increasing worldwide,including non-alcoholic fatty liver,non-alcoholic steatohepatitis（NASH）,non-alcoholic fatty liver fibrosis,liver cirrhosis and liver cancer.However,except for lifestyle intervention,there is no specific treatment approved for improving NAFLD/NASH.Therefore,it is of great clinical significance to explore the pathogenesis of NAFLD/NASH and to seek promising therapeutic targets.As one of the important types of autophagy,chaperone-mediated autophagy（CMA）is the only autophagy pathway that selectively degrades soluble proteins in lysosomes,and is involved in aging,tumors,glucose and lipid metabolism,and inflammatory responses.Lysosome-associated membrane protein 2A（LAMP2A）,as the rate-limiting molecule of CMA,recognizes the substrate protein carried by hsc70,and transfers the substrate protein to the lysosome for degradation with the assistance of lysosomal resident partners.Preliminary study of our group has shown that the expression level of hepatic LAMP2A in NAFLD patients is down-regulated with the severity of the disease,and deficient CMA in hepatocytes inhibited the degradation of lipid droplet-coated protein plin5,aggravating the progression of the disease.Recent studies have shown that macrophages,as an important component of the innate immune system,also play an important role in NAFLD/NASH.Liver embryo-derived Kupffer cells can trigger inflammation in the early stage of disease and secrete chemokines to promote the recruitment of monocytes to the liver.Monocyte derived macrophages（MDM）secrete a large number of pro-inflammatory cytokines to amplify the inflammatory responses of the liver,promoting disease progression.Studies have shown that CMA affects the phenotype of macrophages and is involved in disease progression.Deficient CMA in tumor-associated macrophages leads to increased secretion of pro-inflammatory factors and promotes tumor invasion;in the progression of atherosclerosis,the function of CMA in macrophages becomes impaired,promoting NLRP3inflammasome activation.However,the role of CMA of macrophages in NAFLD/NASH remains unclear.AimWe aimed to study the role of CMA of macrophages in the development of NASH and to find a promising NASH therapeutic target.MethodsWild-type mice were fed a high-fat-fructose-cholesterol diet（HFFC）or a Methionine choline deficient diet（MCD）to induce the murine NASH model.Western-Blot,RT-q PCR and FACS technology were employed to detect LAMP2A expression of liver macrophages in NASH.BMDMs and RAW264.7 cell lines were treated with palmitic acid（PA）to evaluate CMA function of macrophages.To further explore the role of CMA in NASH,we established myeloid macrophage CMA-deficient mice,and then administrated HFFC diet or MCD diet to induce the murine NASH model,and evaluated the effect of deficient CMA of macrophages on NASH-related pathologies such as liver injury,insulin resistance,steatosis,inflammation,and fibrosis in mice.We screened the molecules that regulated NASH progression by mass spectrometry,and we clarified the mechanism of CMA in regulating the molecule by co-immunoprecipitation and immunofluorescence expriments.Results1.HE staining results showed that increased hepatocyte ballooning and inflammation in the liver of the wild-type mice which were fed with MCD or HFFC diets;and oil red staining results showed that the liver of those mice increased lipid droplets,indicating that we successfully establish a diet-induced murine NASH model.2.Western-Blot and RT-q PCR were used to examine the expression of LAMP2A in the liver macrophages of NASH mice,and the results showed that both the protein and m RNA levels of LAMP2A of macrophages were significantly down-regulated;Flow cytometry results also showed that the expression of LAMP2A of macrophages was down-regulated,and the macrophages in the liver were mainly MDM.After PA stimulated BMDMs and RAW264.7 in vitro,Western-Blot and RT-q PCR results showed that the protein and m RNA expression of LAMP2A was down-regulated in a time-dependent manner.It indicated that the CMA function of liver macrophages in NASH mice was impaired.3.Myeloid macrophage CMA-deficient mice(Lyz2-Cre⁺-LAMP2A^fl/fl,LAMP2A^ΔMΦ)were constructed by Cre-loxp technology.Agarose gel electrophoresis was used to identify mouse genotype,Western-Blot and RT-q PCR results showed that the protein and m RNA expression of LAMP2A was significantly down-regulated.These results showed that Myeloid macrophage CMA-deficient mice were successfully constructed.4.In the HFFC diet-induced NASH model,compared with the control group,the body weight of the LAMP2A^ΔMΦmice increased more significantly from the 8th week;IPITT and IPGTT results showed that the ability of the LAMP2A^ΔMΦmice in regulating blood glucose decreased compared with the control group;Serum biochemical results showed that the ALT level was elevated in LAMP2A^ΔMΦmice.The results of HE and Oil Red staining showed that hepatocyte ballooning,lipid droplets and inflammation were increased in LAMP2A^ΔMΦmice,suggesting that deficient CMA exacerbated HFFC diet-induced NASH-related phenotypes.5.In the MCD diet-induced NASH model,serum biochemical results showed that the levels of ALT and AST in LAMP2A^ΔMΦmice were increased;HE and oil red staining results showed that LAMP2A^ΔMΦmice hepatocyte ballooning,lipid droplets,and inflammation were increased;Masson staining results showed increased collagen formation in LAMP2A^ΔMΦmice;Immunohistochemical staining results showed increasedα-SMA expression in LAMP2A^ΔMΦmice;Western-Blot and RT-q PCR results showed that the expression of liver fibrosis-related molecules,such asα-SMA and Collagen I,were significantly increased in LAMP2A^ΔMΦmice,indicating that decicient CMA aggravated MCD diet-induced NASH-related phenotypes.6.RT-q PCR results in MCD and HFFC diet-induced NASH models showed that the m RNA levels of hepatic proinflammatory cytokines,such as IL-6,IL-1β,and TNFα,were increased in LAMP2A^ΔMΦmice.Immunohistochemical results showed that the number of F4/80⁺macrophages were increased in LAMP2A^ΔMΦmice;Flow cytometry results showed that the number of liver macrophages and pro-inflammatory macrophages（CD86⁺CD206^-MΦ）in LAMP2A^ΔMΦmice were increased.7.Isolating the murine bone marrow derived macrophages,and LPS+IFN-γwas used to induce M1 macrophages and IL-4 to induce M2 macrophages,respectively.Western-Blot and RT-q PCR results showed that the increased expression of M1-type classical molecule i NOS,CD86 and IL-6 and the decreased expression of M2-type classical molecule Arg1 in macrophages derived from LAMP2A^ΔMΦmice.8.A knockdown of LAMP2A in RAW264.7 cell line,and LPS+IFN-γwas used to induce M1 macrophages and IL-4 to induce M2 macrophages,respectively.Western-Blot and RT-q PCR results showed that with the decline of LAMP2A,the expression of M1-type classical molecules i NOS,CD86 and IL-6 were increased,and the expression of M2-type classical molecule Arg1 was decreased.9.Using LPS+PA to stimulate primary bone marrow-derived macrophages from LAMP2A^ΔMΦand LAMP2A^fl/flmice in vitro,proteomics results showed that the expression of Nup85,which regulates macrophage recruitment,was up-regulated,and database analysis showed that the Nup85 protein contains a KFERQ-like motif.It is suggested that Nup85 may be a substrate of CMA.10.RT-q PCR results showed that the m RNA of Nup85 did not change significantly after LAMP2A knockout;Western-Blot results showed that the protein expression of Nup85 was up-regulated after LAMP2A knockout;Western-Blot results showed that the expression of Nup85 was up-regulated in LAMP2A^fl/fl mice’s BMDMs treated with inhibitors,and the expression of Nup85 was not up-regulated in LAMP2A^ΔMΦmice’s BMDMs.Co-immunoprecipitation results showed that Nup85 interacted with CMA elements,LAMP2A and hsc70;immunofluorescence staining results showed that Nup85 and LAMP2A co-localized.And inhibition of Nup85 attenuated macrophage recruitment by CMA deficiency.ConclusionsLAMP2A-mediated CMA function of hepatic macrophages was impaired in NASH mice,resulting in increased Nup85 expression,which plays an important role in regulating the recruitment of macrophages.The upregulation of Nup85 increased the infiltration of macrophages in liver,promoting the progression of NASH.