Explore The Role For Activation Of Basophils Mediated By Toll Like Receptor 2 In Systemic Lupus Erythematosus

Background and Objective:Systemic lupus erythematosus（SLE）is one of rheumatic diseases with high morbidity,mortality and disability rate.It can result in the multisystem damage,such as the blood system,the respiratory system,the digestive system,the nervous system and the kidney system.Besides,it is characterized by the presence of a variety of autoantibodies in the serum.The typical antibodies include antinuclear antibodies,ds-DNA antibodies,Smith antibodies and other autoantibodies.The main causes of death include renal failure,infection,and central nervous system disorders^[1].However,its pathogenesis is still not clearnow.Immune disorders are the core of its morbidity and it cannot be cured now.Up to now,no matter the hormone,immunosuppressant or the biologics in recent years targeting at various cytokines and inflammatory factors,the treatments have great individual difference on effect and high recurrence rate.Therefore,the exploration of new targets for treatment remains the focus of the study of SLE.In addition,the dysfunction of T and B lymphocyte plays an important role in the pathogenesis of SLE.There has been a long-term dispute on whether the SLE is caused by the high activity of B cells or the abnormality of function of T help cells.Now,we believe that both of them are causes.SLE is not only a B lymphocyte disease but also a T lymphocyte disease.SLE is characterized by a large number of autoantibodies in the body.More than 95%patients have the antibodies in the body,which indicates the abnormal activity of B cells.The significant increase of immunoglobulin in serum can be observed in the vitro culture of B cells.In the 70s,people believed that the low or deficiency T suppressor cell was the main cause of lupus.With the further study of cytokine and CD4T help cells,they found that T cells,especially the T help cells,was also significant in the pathogenesis of lupus as the B cells.CD4T help cells can be divided into Th1 and Th2.The role of Th1/Th2 response disturbance in the pathogenesis of lupus has gradually aroused the attention of researchers in recent years.We all know that there are various cytokines in SLE patients and it is associated with the severity of SLE.The cytokine expression in the body is different while it is dominated by different Th1 or Th2 cells.For example,if it is dominated by the Th1 immune response,the cytokine in body would mainly include IFN-γ,IL-2,TNF.If it is dominated by theTh2 immune response,the expression of IL-4,IL-5,IL-6,IL-10,IL-13 would increase^[2].The series of cytokines secreted by T cells can affect the function of B cells.Th2 cells can assist in the proliferation of B cells by secreting IL-4.Under normal circumstances,T help cells can express little CD40L,but the increase of the expression of CD40L of T help cells will be observed in active lupus patients.Besides,the duration of CD40L expression has increased after activating T cells in vitro.CD40L can bind to CD40 on the surface of B cells,promoting the proliferation and activation of B cells and participating in the pathogenesis of SLE.Basophils have been known for more than 140 years.Under normal circumstances,peripheral blood leukocyte has few basophils（<0.5%）.It is one of the major effector cells that mediate hypersensitivity^[3].With the rapid development of monoclonal antibody technology and high purity cell classification technology in recent years,we can purify and culture basophils now.Therefore,we have the better understanding on its phenotype and function and find many new biological effects of basophils.In addition to the classic hypersensitivity mediated by IgE,basophils also play an important role in immune response and immune regulation^[4-5].It is involved in the autoimmune diseases by secreting various cytokines and inflammatory factors after activation.Basophils can generate the Th2 type cytokine after activating,such as IL-4 and IL-13,mediating the differentiation of Th0 into Th2 cells.It inhibits the Th1immune response and affects the balance of Th1/Th2 by enhancing the Th2 immune response^[6].Basophils affect the function of B cells by changing the balance of Th1and Th2 and participate in the pathogenesis of various autoimmune diseases.Activation is the key step for basophils to play the biological function of immune regulation.All the time,the activation methods of basophils known by us is the classical IgE mediated activation mechanism,but other activation mechanisms are found now.The activation mechanisms of basophils mediated by the toll like receptor（TLR）is gradually discovered by researchers^[7].Basophils significantly express the TLR2receptor.Up to now,the study of TLR2 mainly focuses on its natural immune function in the infection of bacteria,viruses,fungi and other pathogens.TLR2 has significant antibacterial and antiviral effects.Most studies focus on the prevention and treatment of sepsis and septic shock,but only few studies focus on the immune system disease.Thus,few researches have studied that whether TLR2 can regulate the release of cytokines and inflammatory factors,affect the balance of Th1/Th2 and participate in the pathogenesis of autoimmune diseases by mediating basophil activation.Thus,the study firstly tests the number and activation of the peripheral blood basophils of SLE patients,determine whether the number of basophils in the peripheral blood of SLE patients changes and evaluate the correlation between basophils and disease activity.It takes healthy volunteers and RA patients as the control group,test the number and activation of basophil in peripheral blood of RA patients and healthy controls,and compare tha twith the number of basophils in peripheral blood of patients with SLE.Meanwhile,we also analyze the relationship between the number of basophil in peripheral blood in the normal control group and RA patients and the correlation between the activation of basophil in peripheral blood of RA patients and the indexes of disease activity（DAS28,CCP,CRP and others）and discuss the difference between the number of basophils in the two diseases,exploring new simple and effective methods for monitoring the condition of patients with SLE.Then,it takes human basophil systemKU-812 as the research objects and determines the role of basophils by activating basophils mediated by TLR2 in the serum of patientsin SLE,activating intracellular conduction pathway and promote the release of inflammatory factors and cytokine for elucidating the possible mechanism of basophil in the pathogenesis of SLE,provide a new theoretical basis for the application of basophil as a therapeutic target of SLE.Methods:（1）Collection of clinical data and experimental specimens:30 patients with SLE in the initial treatment phase hospitalized in the rheumatology department of Shengjing Hospital from October 2015 to September 2017 are selected.All selected cases conform to the SLE classification standardrecommended by ACR2012.The personal histories of all patients are recorded in detail.Patient with severe other systemic diseases,other immune diseases and infection would be excluded,the disease courses of which are shorter than 1 year.The control group includes 30 healthy volunteers and 27 rheumatoid arthritis patients in the initial treatment phase with the matched gender and age.All selected RA patients conform to the RA classification standardrecommended by ACR2009.The personal histories of all patients are recorded in detail.Patient with severe other systemic diseases,other immune diseases and infection would be excluded,the disease courses of which are shorter than 1 year.2mL EDTA anticoagulant peripheral blood of SLE patients,RA patients and healthy volunteers who conform to the selected conditions will be collected.The laboratory and general clinical data collection and the SLEDAI scoring had been carried out on SLE patients.The laboratory and general clinical data collection and the DAS28scoring had been carried out on RA patients.（2）Test the number and activation of peripheral blood basophil in patients with SLE,patients with RA and healthy volunteers through flow cytometry.Analyze the number and activation change of peripheral blood basophil in patients with SLE,patients with RA and healthy volunteers.Analyze correlation between the number and activation of peripheral blood basophil in patients SLE and the indexes of disease activity of SLE,such as SLEDAI score,serum complement C3 and C4,anti-ds-DNA antibody,CRP,ESR,urine protein and immunoglobulin.Carry out the statistical analysisof the number change and activationof peripheral blood basophil in patientswith RA and the indexes of disease activity of RA,such as DAS28 score,CCP,CRP and ESR.（3）We also performed in vitro experiments to induce the differentiation and maturation of human KU-812 basophils.The basophils were then stimulated using the serum from healthy volunteers,serum from SLE patients and serum from SLE patients plus TLR2 agonist or antagonist.The supernatants from KU-812 cell cultures were collected and the cells were collected.（4）The concentration of interleukin-4（IL-4）、IFN-γand tumor necrosis factor-α（TNF-α）secreted by the cells were measured using ELISA kits.The activation of TLR-2 antibody and nuclear factor kB（Nuclear factor-kappa B,NF-kB）was detected by Western blot（Western blot,WB）.Result:The absolute count of basophil in peripheral blood of SLE patients is significantly lower than that in the control group and the RA patients（SLE vs Control P﹤0.001;SLE vs RA P=0.018）.Compared with the control group and the RA patients,the activation of basophil in peripheral blood of SLE patients has significantly increased（SLE vs Control P=0.001;SLE vs RA P=0.047）.The activation status of basophil has significant statistical difference among the SLE group with different disease activity（P﹤0.001,P=0.001,P=0.031）.With the increase of SLEDAI score,the activation of basophil in peripheral blood has obviously increased.The activation of basophil in peripheral blood of SLE patients is negatively correlated with the serum complement（C3 P=0.004,r=﹣0.512 and C4P=0.010,r=﹣0.460）and positively correlated with the ds-DNA（P<0.001,r=0.648）and the immunoglobulin（ImmunoglobulinG P=0.039,r=0.378;ImmunoglobulinA P=0.008,r=0.477;ImmunoglobulinM P=0.002,r=0.549）.There is no significant correlation between the activation of basophil in peripheral blood of SLE patients and24h urine protein（P=0.584）,ESR（P=0.488）,CRP（P=0.843）.Compared with the control group,the absolute count of basophil in peripheral blood of RA patients is also statistically significant（RA vs Control P=0.039）.The activation is significantly higher than that in the control group（RA vs Control P=0.037）,and the activation status of basophil has significant statistical difference among the RA group with different disease activity（P=0.011,P=0.037,P=0.002）.With the increase of DAS28 score,the activation of basophil in peripheral blood has obviously increased.The activation of basophil in peripheral blood of RA patients is positively correlated with the CCP（P<0.001,r=0.831）and the CRP（P=0.001,r=0.588）,but there is no significant correlation between the activation of basophil in peripheral blood of RA patients and ESR（P=0.885,r=0.029）.While stimulating basophils in SLE patients with TLR2agonist,it is found that the expressions of TLR2 and NF-kB are higher than that in the control group.TLR2activate the intracellular NF-kB pathway,significantly increases the TNF-αand IL-4 and reduces the IFN-γin the serum of SLE patients by mediating the activation of basophils in patients with SLE.Compared with the ratio of IFN-γ/IL-4 in the serum of healthy volunteer（P=0.0195）,it indicates that IL-4 is increased in patients with SLE and Th2 immune response is dominant.The increasing level of IL-4 and TNF-αin SLE serumis irrelevant to the disease activity（P>0.05）.TLR2 affects the cytokine secretion by mediating the activationof basophils and adjusts the balance between Th1 and Th2 for participating in the pathogenesis of SLE.Conclusion:1、Compared with the control group and RA patients,the number of basophils in peripheral blood is significantly decreased and the activation is obviously increased in patients with SLE.They are related to the disease activity of the SLE,which indicates basophils may therefore be useful as an early monitoring index of SLE activity.Compared with the control group,the number of basophils in peripheral blood is decreased and the activation is obviously increased in patients with RA.They are related to the disease activity of the RA,which indicates that the peripheral blood basophil of RA patients is also significant on reflecting the disease activity of RA.2、TLR2 activate the intracellular NF-kB pathway and promote the secretion of inflammatory factor TNF-αby mediating the activation of basophil in the serum of patients with SLE.Meanwhile,activated basophils will promote the secretion of Th2type cytokine IL-4,inhibit the secretion of Th1 type cytokine IFN-γ,influence the balance between Th1 and Th2,enhance the humoral immune responseand participate in the pathogenesis of SLE.3、Because the TLR2 agonist PGN in the study comes from Gram-positive bacterium,Staphylococcus aureus.PGN is a major surface component of cell wall.Speculated that pathogenic microorganism infection,such as bacteria and viruses,may play a role in SLE through the basophil activation mediated by the TLR2.With the further study on basophils and TLR2receptors,it is expected to provide the new method for the treatment of SLE by taking TLR2 as a target to block the activation of basophil.