The Role Of Otulin In Negatively Regulating The LUBAC-NF-κB Pathway In The Pathogenesis Of Systemic Lupus Erythematosus

Background:Systemic lupus erythematosus(SLE)is a chronic autoimmune disease characterized by multiple production of autoantibodies,activation of complement and immune deposition of complex.The prevalence of SLE in our country is about 70 per 100,000 and is increasing year by year.The treatment of SLE mainly includes non-steroidal anti-inflammatory drugs,glucocorticoids,hydroxychloroquine and immunosuppressive agents.Although these drugs can significantly improve the prognosis of patients with SLE,they also bring side effects such as bone marrow suppression,eye disease,and femoral head necrosis.Therefore,the research on the mechanism of SLE and the development of targeted drugs have important clinical significance.Ubiquitin(Ub)is a reversible covalent modification of protein after translation,and it can participate in multiple signaling pathways in cells.Linear ubiquitination is a special type of ubiquitination modification that plays a critical role in innate and adaptive immunity.LUBAC is the only known E3 ligase capable of generating linear ubiquitination chains.It consists of HOIP(HOIL1-interacting protein),HOIL-1L(heme-oxidazid IRP2 ubiquitin ligase)and SHARPIN(Shank associated RHdomain interacting protein).OTULIN(OTU domain DUB with Linear linkage specificity)is a deubiquitination enzyme which can specifically removes linear ubiquitination chains.Researchers have found that the PIM structure of OTULIN is combined with the PUB of HOIP through the N-terminal.OTULIN participates in a variety of cell functions such as apoptosis,necroptosis,signaling transduction,innate and adaptive immunity through regulating LUBAC.However,no studies have been conducted on OTULIN/LUBAC in patients with SLE.This study intends to explore the following issues through in vivo and vitro research1.The expression of OTULIN and HOIP in PBMCs(peripheral blood mononuclear cells)of patients with SLE was detected to analyze the correlation between the expression of OTULIN/HOIP and the disease activity related indicators of SLE.2.To detect the expression of OTULIN and HOIP in THP-1 cells treated with serum from SLE patients in vitro.3.To investigate the possible mechanism of OTULIN-LUBAC-NF-κB in the occurrence of SLE.Objective1 To detect the expression and effect of OTULIN and HOIP in peripheral blood mononuclear cells of SLE patients.1.1 MethodThis study includes 71 patients with SLE,and 15 healthy volunteers of the same age and sex were selected as controls.SLE diagnoses are in line with the SLE classification criteria established by 1997 ACR,2012 SLICC or 2019 EULAR/ACR.OTULIN positive cells in CD19+B cells,CD4+T cells,CD8+T cells,and CD 14+monocytes were detected by flow cytometry.The expression of OTULIN and HOIP in PBMCs of SLE patients were detected by Western blot,immunofluorescence and other methods.71 patients with SLE were grouped by SLEDAI score,and the Correlation between the expression of OTULIN/HOIP and SLE disease activity indicators were analysed(white blood cells,platelets,HB(hemoglobin),anti-dsDNA antibodies,complement C3,C4,ESR,CRP,immunoglobulin IgG,IgA,IgM,24-hour proteinurine)1.2 Results1.2.1.Compared with healthy controls,HOIP protein in each group of SLE patients increased(p<0.05);HOIP protein in stable group have no significant difference with mild activity group(p>0.05),HOIP protein in medium/high activity group were significantly higher than that in stable and mild activity group(p<0.05);Compared with healthy controls,OTULIN protein in SLE patients in medium/high activity group were significantly increased(p<0.05).There were no significant difference in OTULIN protein in mild activity group compared with the stable activity group(P>0.05).OTULIN and HOIP protein in PBMCs ofSLE patients were positively correlated with the SLEDAI score.OTULIN protein were significantly positively correlated with HOIP.OTULIN protein in PBMCs of SLE patients were negatively correlated with HB,complement C3 and C4,and positively correlated with proteinuria(p<0.05).1.2.2 Flow cytometry showed that OTULIN were expressed in CD 19+B cells,CD4+T cells,CD8+T cells and CD14+ in PBMCs of SLE patients,and were maily expressed in CD 14+monocytes.1.2.3 The results of immunofluorescence showed that both OTULIN and HOIP were expressed in PBMCs of SLE patients,and were co-localized in the cytoplasm.ConclusionThe HOIP and OTULIN protein in PBMCs of SLE patients were increased,and were positively correlated with the SLEDAI score.OTULIN protein in PBMCs of SLE patients were negatively correlated with HB,complement C3,C4,and were positively correlated with proteinuria,suggesting that OTULIN/HOIP might be one of the indicators of SLE disease activity.Futhermore,the expression of OTULIN protein is positively correlated with HOIP.Immunofluorescence showed that OTULIN and HOIP are co-localized in cytoplasm,suggesting that OTULIN might be combined with HOIP and play a certain protective effect on immune inflammation in SLE.Objective 2 To investigate the possible machanism of OTULIN-LUBAC-NF-κB pathway in THP-1 stimulated with serum of SLE patients in vitro.2.1 MethodTaking THP-1 cells as the research object,divided into the following groups:NC(negative control)group(THP-1 cells without any treatment),OTULIN overexpression group,OTULIN knockdown group,OTULIN knockdown plus HOIP inhibitor intervention group and vector or nonsense chain intervention group.The serum of healthy people were used as control.1×106 cells were added to each well of a 6-well cell culture plate,and cells were collected after 24 hours of stimulation.Quantitative RT-PCR and Western Blot were used to detect the expression of HOIP and OTULIN at gene and protein levels.The level of IL-1β in the cell culture supernatant were detected by ELISA,and the activation of NF-κB signaling pathway(the expression levels of p-IKBa and p-p65)were detected.2.2 Results2.2.1 Compared with the THP-1 cells cultured with serum of healthy controls,OTULIN and HOIP mRNA and protein levels in THP-1 cells stimulated with serum of lupus patients were increased significantly(p<0.05).2.2.2 Compared with simple lupus serum stimulated group,OTULIN protein in THP-1 cells increased after the intervention of OTULIN overexpression lentivirus(p<0.05),accompanied by a decrease of HOIP protein(p<0.05),OTULIN protein were significantly down-regulated(p<0.05)in OTULIN knockdown group,and HOIP were significantly upregulated(p<0.05).However,OTULIN and HOIP protein in THP-1 cells transfected with empty vector(CON386)or nonsense chain(CON387)viruses have no significantly different from those in non-intervention group.2.2.3 Compared with HC(health control)serum stimulated group,p-IKBa and p-p65 protein significantly increased after lupus serum stimulation,as well as the increase of IL1β(p<0.05).Compared with simple lupus serum intervention group,p-IKBa and p-p65 in OTULIN overexpressed THP-1 cells significantly down-regulated,companied with the decreased of IL-1β(p<0.05).On the contrary,compared with simple lupus serum intervention group,p-IKBα and p-p65 and IL-1β in THP-1 cells increased in OTULIN knockdown THP-1 cells(p<0.05).On the basis of above experiments,p-IKBα and p-p65 protein and IL-1β down-regulated in OTULIN knockdown combined with the intervention of HOIP inhibitors(p<0.05).2.3 ConclusionAfter intervention with lupus serum,OTULIN and HOIP mRNA and protein upregulated in THP-1 cells.Overexpression of OTULIN in THP-1 cells could be able to downregulate HOIP,and significantly inhibited the activation of NF-κB signaling pathway and IL-1β release stimulated by lupus serum;OTULIN knockdown in THP-1 cells showed increased of HOIP,and resulted in more severe activation of NF-κB pathway as well as the release of IL-1β after lupus serum stimulation,HOIP inhibitor could reverse the changes above.These results suggested that OTULIN maight negatively regulate the LUBAC-NFκB pathway to inhibit immune inflammation.