Research On The Role Of Alarmin IL-33in The Pathogenesis Of Lupus Nephritis

Background and ObjectivesSystemic lupus erythematosus (SLE) is a specific autoimmune disease thatinvolves in genetics, hormones, environment, infection and other factors. It could becharacterized by multi-system organ involvement and lots of autoantibodies in serum.The kidney is the easiest target organ involvement and lupus nephritis(LN) is the mostimportant clinical manifestation of SLE.LN greatly impacts on the prognosis andrenal failure is a major cause of death in SLE patients. The pathogenesis of LN is notabsolute clear.Diseases modifying antirheumatic drugs (DMARDS) are still the maintreatment of LN currently. Although the emergence of biologics brings more treatmentoptions, but the results are still unsatisfactory.So,for deeper research on thepathogenesis of LN and development of new drugs remains to be done.Innate immune system abnormalities occur in the early onset of SLE, even beforeclinical manifestations, and throughout the course of the disease. Pathogen associatedmolecular patterns(PAMPs) and damage associated molecular patterns(DAMPs) arethe ways to recognize exogenous and endogenous danger signals by innate immunesystem. They can be expressed on cells of the innate immune pattern recognitionreceptors (PRRs) identified, then initiate an immune response.’Alarmins’ are a group of endogenous proteins or molecules that releasing fromcells during cellular demise to alert the host innate immune system.Alarmins bind the specific receptors expressing on the immune cells and then act the nuclear factorNF-KB axis, recruit monocyte/macrophages, neutrophils and special T cells, therebyinitiate innate and specific immune response which involve SLE, rheumatoid arthritis(RA), atherosclerosis, hepatitis and cancer disease process. Different alarmins have asimilar pattern of immune activation.Interleukin-33(IL-33) is the newest partner of alarmins. Its specific receptor isST2. ST2is known to exist in a transmembrane form (ST2L) and also alternativelyspliced to produce a secreted soluble form (sST2). IL-33stimulates target cells bybinding to ST2, thereby activating nuclear factor (NF)-kB and mitogen-activatedprotein kinase pathways, releasing lots of inflammatory factors and inducing tissuedamage.sST2treatment can attenuate inflammatory tissue damage from the researchof RA, acute kidney injury induced by drugs, asthma and etc in vivo.The clinical researches have found that serum of IL-33or sST2significantlyincreased in the SLE patients. The level of IL-33or sST2is positively correlated withSLE disease activity index (SLEDAI).The therapy with anti-IL-33protein canattenuate severity of the LN in the animal model of SLE. The above results suggestthat IL-33/ST2pathway may participate in the kidney target SLE immune activation,and antagonized the activated pathway may become a new approach for the treatmentof LN. But the mechanism about how the IL-33/ST2pathway involving in lupusnephritis inflammation injury is still unclear. Our study aimed to clarify thesequestions: Whether IL-33derived from the kidney of LN.Which cells may release theIL-33, and whether its level is associated with the severity of kidney damage. Furthermore, we will study which immune cells express ST2receptor in peripheral blood,and how IL-33and immune cells interact to injure the kidney.Material and Methods:1. Grouping Matched group:8weeks old SPF level female C57BL/6j mice;LN modelgroup:8weeks old SPF level female MRL/1pr mice; Five for each group. Twogroups were observed in general and tested the level of urinary protein. All of the mice sacrificed in18weeks,testing the level of serum ds-DNA,BUN and Scr andobserving on renal lesion in renal tissue of HE, PASM, MASSON staining.2. Real time PCR examed the renal tissue IL-33mRNA expression level in2groupsof mice. Western blot examed the renal tissue IL-33protein expression level in2groups of mice and then analyzed the correlation between IL-33expression levelsand urinary protein, serum ds-DNA.3. Immunohistochemistry expressed IL-33in renal tissue of position, further more,double immunofluorescence localized IL-33in derived cells.4. ELISA tested serum levels of IL-33, sST2in the LN patients with or without flare.5. Flow cytometry Detected the expression levels of surface ST2receptors onperipheral blood CD4+T cells and CD14+mononuclear cells in SLE patients andthe healthy control.Results:1. The general situation and urinary protein levels in miceTwo groups of mice were survived before sacrificing. C57group is a goodmental state, good appetite, activity, fur shiny, no hair, no joint swelling of limbs.MRL/lpr group decreased appetite, lazy in different degree edema, color of hair lacksluster, but no obvious epilation. Some mice had mild to moderate swelling of kneejoint. The testing of urine protein began form14weeks old every two weeks. AllMRL/lpr mice detected in deferent degree urine protein but did not increase overall.The urine protein of C57mice always was negative.2. The level of serum BUN, Scr and ds-DNA in miceMRL/lpr mice serum BUN level is10.04±1.79mmol/L,while C57mice’s is7.42±1.00mmol/L.There is no statistical significance between two groups(p=0.300).MRL/lpr mice serum Scr level is25.60±7.30ummol/L,while C57mice’s is25.75±8.54ummol/L.There is also no statistical significance between twogroups(p=0.878).The serum ds-DNA is positive in all MRL/lpr mice(from2+to4+),but negative in C57mice. There is statistical significance between two groups (p=0.003)3. General and special pathology staining in miceHE staining: Renal glomerular structure clearly displays and the ball can beseen in the vascular loop in C57mice. Disordered glomerular hypertrophy and largelymphocytes infiltrate in renal interstitium in MRL/lpr mice. PASM staining:MRL/lpr mice’s Glomerular basement membrane is thick, endothelial and epithelialcells hyperplasia, while the basement membrane in C57mice is normal. Massonstaining: lots of eosinophilic immune complex deposit in glomerular and protein tubes in the renal tubules in MRL/lpr mice. There is neither eosinophilic immune complexnor protein tubes in the kidney of C57mice.4. IL-33expression in the mice kidney tissueMRL/lpr group increase the expression of IL-33mRNA by an average of2.73±1.34times comparing with C57group, a significant difference between two groups(p=0.045). It indicates that MRL/lpr mice has up-regulated the expression of IL-33ingene levels. By western blot, MRL/lpr group also significantly increased theexpression of IL-33protein (OD value0.558±0.094vs.0.034±0.002).The expressionof C57is little, a significant difference between two groups (p=0.0005). It indicatesthat MRL/lpr mice have up-regulated the expression of IL-33in protein levels.5. Correlation analysis between IL-33and urine protein/serum ds-DNA in renal tissueof MRL/lpr miceThe spearman correlation coefficient indicates that a significant positivecorrelation between the changes of MRL/lpr mice in renal tissue of IL-33gray ratioand semi quantitative urine protein (r2=0.72, p=0.037).Although there is nosignificant relation between il-33and Semi quantitative ds-DNA level (r2=0.386,p=0.215), but positive correlation trend between them.6. The expression of IL-33in renal tissue cells of miceBy immunochemistry, IL-33expresses in cells of the glomerular basementmembrane zone, but rare in renal interstitial in MRL/lpr. C57significantly reduces theexpression of IL-33.Further, double Immunofluorescence shows that the expression ofIL-33by Glomerular endothelial cells. 7. The expression of ST2receptor in Peripheral blood cells of LN and healthy controlST2receptor is up-regulates on the surface of CD4+T cells and CD14+mononuclear cells in Peripheral blood of LN patients, compared with the healthycontrol. There is significant different between them.Conclusions:1. MRL/lpr renal tissue up-regulates IL-33expression. The level positively relates tourinary protein level which could reflect the degree of kidney damage.2. IL-33comes from Glomerular endothelial cells in the renal tissue when the tissuesuffers injury.3. LN patients up-regulate ST2receptor on the surface of CD4+T cells and CD14+mononuclear cells in Peripheral blood. It may indicate that IL-33inducesinflammatory injury by Mediating peripheral blood mononuclear cells and CD4+Tcells infiltrating into the renal tissue.