Association Study Of Serum IRF4Level And Single Nucleotide Polymorphisms In IRF4with Systemic Lupus Erythematosus

Background Systemic lupus erythematosus （SLE） is a complex autoimmune diseaserelevant to immunological defects caused by abnormal immune regulation andexcessive production of autoantibodies. At present though the pathogenesis of SLE isnot clarified, it is confirmed to be intimately connected with many kinds of factors,including genetic factors, environmental factors, sex hormones, the immunity and so on.Among the wide variety of immunological aberrations associated with SLE, in recentyears a group of transcription factors IRF family have received much attention.IFN-regulatory factors （IRFs） form a group of transcription factors that have thepotential to contribute to the pathogenesis of systemic lupus. The members involved weknown are IRF1, IRF3, IRF5and IRF7. IRF1can trigger proinflammatory cytokineexpression in tubular epithelial cells and immune cells of the kidney after ischemia.IRF5is required for immune cell maturation and for TLR signaling, both contributing toluPus. IRF3, IRF7could induce the typeⅠinterferon production upon TLR7signalingpathways that plays a decisive role in SLE pathogenesis. While IRF4shows a variety ofregulating function in adaptive immunity, as IRF4participates in B/T cells and plasmacells mature, the conversion of immune globulin，the ability of regulatory T cellsinhibiting Th2response and Th17T cells induced. The researchers also found that theSPI1of Ets family can regulate down CD68exPression on the macrophages and itspromoter activity by interaction with IRF4. In fact, SPI1has been confirmed relationwith SLE pathogenesis, and CD68has an increasing expression level in SLE patients.Since IRF4is found to be necessary for the maturation of T cells, B cells, and plasmacells, for the ability of regulatory T （Treg） cells to inhibit T helper （Th）2cells, and forthe differentiation of Th17cells, which are crucial in the pathogenesis of SLE, the rolesof IRF4in the initiation and progression of SLE can not be neglected. To the best of our knowledge, there was no study that researched the association between IRF4and SLEin Chinese Han population. So our study aims to determinate the correlation betweenIRF4and SLE from serum and gene polymorphism, providing a new train of thought toexplore the pathogenesis of SLE.Part I Association of IRF4and SLE in serum levelObjective To explore the correlation of interferon regulatory factor4and systemiclupus erythematosus （SLE） from the serum level, looking for new therapeutic targetsand improving the life quality of SLE patients.Methods Enzyme-linked immunosorbent experiment （ELISA） was performed todeterminate IRF4expression level in serum in a case-control study with90SLE patientsand healthy controls respectively and related statistical analysis were done combinedwith the questionnaire data.Results No statistically significant differences of the serum IRF4expression level werefound in SLE patients and controls, lupus nephritis group and no lupus nephritis group,active disease and inactive disease group （P>0.05respectively）. In SLE patients, theserum IRF4exPression level and disease activity index score was not found obviouscorrelation. The level of serum IRF4at low comPlement （C3↓） Positive grouP andnegative group was found statistical difference （Z=3.141, P=0.002）, other laboratoryindex positive group and negative group did not find significant difference.Conclusion In this experiment we found no correlation between IRF4and SLE in theserum level, however, it could deepen the understanding of SLE and the resultsprovided a new train of thought in the research of SLE pathogenesis. Part II Association of IRF4Single Nucleotide Polymorphism with SLEObjective Our purpose is to evaluate whether the IRF4gene Single NucleotidePolymorphism （SNP rs872071） is associated with susceptibility to SLE in a ChineseHan population.Methods A case-control study was performed with a total of663patients with SLE（588females,75males; mean age35.17±11.39years, range from10to76years） wererecruited from Anhui Provincial Hospital and the First Affiliated Hospital of AnhuiMedical University. The diagnosis of SLE was fullfilled the American College ofRheumatology1997criteria for SLE. Disease activity was assessed by the SLE DiseaseActivity Index （SLEDAI）. Demographic data and clinical data were collected fromhospital records or by questionnaire and reviewed by experienced physicians. Renalinvolvement of SLE was defined according to the ACR criteria, i.e. any one of thefollowing:（i） persistent proteinuria≥0.5g/day,（ii） the presence of active cellular castsor （iii） biopsy evidence of lupus nephritis （LN）. A total of658healthy blood donors（580females,78males; mean age34.36±11.99years, range from19to81years） wereincluded as controls, all of whom were without any rheumatologic conditions. EDTAanti-coagulated venous blood samples were collected from all participants. GenomicDNA was extracted from peripheral blood lymphocytes by standard procedures usingFlexi Gene DNA kits （Qiagen）. All subjects were Chinese Han population and gavetheir written consent to participate. A tagger SNP rs872071of IRF4with a minor allelefrequency （MAF） bigger than0.05, was genotyped by TaqMan SNP assay usingAssay-on-Demand probes and primers （Applied Biosystems, Foster City, CA, USA;catalogue numbers C_<sub><sub>8770093₁0） and ABI Prism7300Real-time fluorescencequantitative PCR （Applied Biosystems, Foster City, CA, USA）. Reaction conditionswere initial denaturation at95℃for10minutes followed by50cycles of denaturationat95℃for15s, and annealing/extension at60℃for1min. Results （1） IRF4gene polymorphism（rs872071）had no significant differencebetween SLE patients and healthy controls（A/G vs. G/G P=0.543, OR=0.872,95%CI=0.562-1.355, G vs. A P=0.512, OR=1.058,95%CI=0.893-1.254, A/A+A/G vs. G/GP=0.475, OR=0.857,95%CI=0.562-1.308）.（2） The allele frequencies （A/G） genotypeby Systemic Lupus Erythematosus Disease Activity Index （SLEDAI） score analysis inSLE patients no strong correlation was found（G vs. A P=0.326, OR=0.832,95%CI0.576-1.202, A/A+A/G vs. G/G P=0.466, OR=1.356,95%CI=0.597-3.080）.（3）Similarly in a subgroup analysis by clinical manifestation of lupus nephritis, nosignificant differences were found between non-LN group and LN group （G/G vs. A/Gvs. A/A χ²=0.611, P=0.631, G vs. A χ²=0.411, P=0.521）. Conclusion This findingsuggests that IRF4gene polymorphism is not associated with SLE in Chinese Hanpopulation, but further studies are needed to establish the role of IRF4in SLE withlarger sample sized, and in different ethnic populations.