Macrophage SCAP Contributes To Metaflammation And Lean Nafld By Activating STING-NF-κB Signaling Pathway

Objective:Nonalcoholic fatty liver disease（NAFLD）is commonly considered to be associated with obesity,but not all obese patients develop NAFLD;instead,there are increasing reports of a large proportion of NAFLD patients with normal or even lower body mass index（BMI）,often referred to as"lean NAFLD".Lean NAFLD is defined as a’metabolically unhealthy normal weight’NAFLD,which is characterized by a decrease in subcutaneous fat and ectopic fat deposition in the liver.Although lean NAFLD has normal body weight,hepatic lipid deposition,insulin resistance and metabolic abnormalities are still present.Thus,the"location"and"type"of obesity have a more significant impact on an individual’s metabolic health than the total mass of fat.The pathogenesis of lean NAFLD appears complex and is still poorly understood,therefore it is of great value to clarify the pathogenesis of lean NAFLD and to find effective therapeutic targets.Sterol regulatory element binding protein（SREBP）cleavage-activating protein（SCAP）is a cholesterol sensor that regulates signal transduction for intracellular cholesterol homeostasis.Recent studies have shown that Dysregulation of SCAP could affect the development of metabolic disease.Increased expression of SCAP and its conformational changes in inflammatory states lead to an imbalance in cholesterol homeostasis.At the meantime,SCAP promotes the activation of inflammatory signaling pathways to aggravate the progression of metabolic diseases.The role of macrophage SCAP in the development of lean NAFLD is unclear.Therefore,we focused on the role of macrophage SCAP in the paigen diet（PD）-induced lean NAFLD model to provide new therapeutic targets for the prevention and treatment of lean NAFLD.Methods:（1）Firstly,we induced a lean NAFLD model by a12-week-PD-feeding and recording the weight change during that period.GTT and ITT tests were performed and lipids were measured after 12weeks of PD-feeding.The vital organ tissues were weighed after sacrificing to screen out the organs with significant differences.HE staining of liver sections was performed to observe hepatocyte steatosis and lobular inflammatory infiltration;Oil Red O staining,intrahepatic TC and TG assays were performed to observe lipid accumulation in the liver;HE staining of epididymal white adipose tissue（e WAT）was performed to observe adipocyte size;F4/80 immunohistochemistry and inflammatory factor assays（m RNA,protein blotting,ELISA）were performed on e WAT and liver tissues to observe macrophage infiltration and the degree of tissue inflammation;m RNA levels of lipid metabolism genes in e WAT and liver tissue were performed to observe lipid metabolism disorders.（2）We further observed the expression of macrophage SCAP using the detection method of immunofluorescence and flow cytometry in the e WAT and liver tissues of PD（NCD）-fed mice.Macrophage SCAP-specific knockout mice(Lys M-Cre SCAP^fl/fl)were generated by breeding Lys M-Cre mice with SCAP^fl/fl mice.Cre-negative SCAP^fl/fl littermates were used as controls.Genotypes were identified by PCR,and the extent and specificity of SCAP knockdown were identified by q RT-PCR by extracting primary bone marrow-derived macrophages（BMDMs）.The selected mice were fed with NCD and PD for 12 weeks.HE staining,F4/80 immunohistochemistry staining,inflammatory factor m RNA of e WAT and liver tissue,and lipid accumulation in the liver were observed of NCD-fed mice.GTT and ITT experiments were performed on 12-week-PD-fed mice,and blood lipids were detected.HE staining,Oil Red O staining,F4/80immunohistochemistry,inflammatory factor（m RNA,Western blot,ELISA）,and lipid metabolism gene m RNA levels were analyzed on e WAT and liver tissue to observe the phenotype of macrophage SCAP knockout mice.An overexpression（knockdown）macrophage SCAP/adipocyte（hepatocyte）co-culture model was constructed in vitro.Adipocytes（hepatocytes）were stained with Oil Red O to observe lipid accumulation alterations,and lipid metabolism genes and inflammatory factor m RNA levels were analyzed to verify in vivo findings.（3）The total protein,cytosol and nuclear proteins of liver tissue were extracted for Western blot analyzed of P-P65 and P65 changes in12-week-NCD（PD）-fed mice;The expression of IKKα,IKKβ,IKKα/β,IκBα,P-IκBα,STING,TBK1,P-TBK1 in liver was detected by western blotting to verify the changes of STING-NF-κB signaling pathway;Immunofluorescence staining was used to detect the co-localization and expression of F4/80 and P65 in e WAT and liver tissue;STING immunohistochemical staining and STING-F4/80 immunofluorescence colocalization analyzed in e WAT and liver tissues.Overexpression or knockdown of SCAP in RAW264.7 macrophages and m RNA expression of inflammatory factors was detected after treatment with LPS;Western blot analyzed of total protein,cytosol and nuclear protein of P-P65 and P65;Western blot analyzed of STING-NF-κB signaling pathway;P65 nuclear localization was detected by immunofluorescence.The co-localization of STING and SCAP in liver tissue and RAW264.7 cells was detected by immunofluorescence,and the interaction of SCAP with STING and TBK1was detected by co-immunoprecipitation.The colocalization of STING/Golgi,TBK1/Golgi was analyzed in SCAP knockdown cells by immunofluorescence;The co-localization of STING/Golgi and TBK1/Golgi was detected by immunofluorescence under the conditions of overexpressing SCAP and overexpressing SCAP treated with25-hydroxycholesterol,and the changes of P65,P-TBK1,STING and P-P65 were detected by western blotting.（4）We induced steatohepatitis and liver fibrosis in SCAP^ΔMφand SCAP^fl/flmice by 16-week-PD-feeding.HE staining,Sirius red staining,α-SMA immunohistochemical staining,and F4/80 immunohistochemical staining were performed on mouse liver tissue to observe changes in fibrosis and macrophage infiltration.The expressions of fibrosis genes（Col1a1,Col4a4,TGF-β,α-SMA）and proteins（Col1a1,α-SMA）were examined;the m RNA and protein expressions of inflammatory factors were examined.The expressions of P-P65,P-TBK1 and STING were examined by western blotting.Resluts:（1）After fed either NCD or PD for 12 weeks,compared with the NCD control group,the body weight of the mice was clear decreased in the PD-fed group,which was accompanied by marked manifestations of metabolic syndrome,hyperlipidemia,impaired glucose tolerance and insulin sensitivity.PD-fed mice displayed increased liver weight with significant reduction in e WAT weight.HE staining showed that adipocytes were smaller in e WAT,hepatocyte steatosis and lobular inflammatory infiltration were more clear in the PD-fed mice than in NCD-fed mice.Oil Red O staining showed obvious intracellular hepatocyte accumulation of neutral lipids in the PD-fed mice.Macrophages significantly infiltrated in e WAT and liver tissues of the PD-fed mice,The expression of inflammatory factors were increased in both tissues of PD-fed mice.e WAT and liver tissues exhibited different phenotypes of lipid metabolism disorders under PD-induced inflammation state:the adipose tissue was characterized by increased lipolysis,however,the liver was characterized by increased synthesis and lipid uptake.（2）Macrophage SCAP expression was abnormally increased in e WAT and liver tissues of PD-fed mice.We generated macrophage/monocyte-specific SCAP knockout mice Lys M-Cre+SCAP^fl/fl(SCAP^△Mφ).There were no difference in e WAT and liver tissue phenotype of SCAP^△Mφmice and SCAP^fl/fl mice,under NCD-feeding condition.Metabolism-related indicators were significantly ameliorated in SCAP^△Mφmice of 12 weeks PD-fed;The weight of e WAT increased,the weight of liver decreased,the infiltration of macrophages in the tissue and the inflammatory response were alleviated,and the lipid metabolism disorder was improved.Co-culture experiments in vitro confirm the effects of macrophage SCAP on hepatocyte（adipocyte）inflammation and lipid metabolism.（3）Macrophage SCAP deletion alleviates the inflammatory response in the e WAT and liver tissue of PD-fed mice via inhibition of STING-NF-κB signaling.SCAP promotes inflammatory response through STING-NF-κB signaling activation in vitro.SCAP recruits STING and TBK1 to the Golgi and activates NF-κB signaling.（4）Macrophage SCAP deletion ameliorates the degree of liver fibrosis through inhibiting STING-NF-κB signaling activation in 16-week-PD-fed mice.Conclusions:Our findings suggest that SCAP acts as a novel regulator of the macrophage inflammatory response and the pathogenesis of lean NAFLD by activating the STING-NF-κB signaling pathway.Inhibition of macrophage SCAP may represent a new therapeutic strategy for the treatment of lean NAFLD.