The Role Of Liver X Receptors In The Pathogenesis Of Behcet’s Disease And Vogt-koyanagi-harada Syndrome

BackgroundUveitis is a kind of inflammatory disease involved uvea, vitreous body,retina and retinal vessels which is also called intraocular inflammation. Itcan be classified into various types according to the different incipientpositions and involved tissues. There are infectious, traumatic andautoimmune uveitis in terms of etiology; anterior, intermediate, posterioruveitis and panuveitis according to the position of inflammation; alsoFuchs syndrome, Behcet’s disease and Vogt-Koyanagi-Harada syndrome interms of relevance with systemic disease. Behcet’s disease is a disease withmulti-organs and systems involved which is characterized with uveitis,polymorphic skin lesion, oral ulcer and genital ulcer.Vogt-Koyanagi-Harada syndrome is also an inflammatory disease withmulti-organs and systems involved which is characterized with choroiditis,chorioretinitis, papillitis, neuroretinitis and granulomatous panuveitis, oftenaccompanied by systemic manifestations such as changes of skin and hair,auditory dysfunction and meningeal irritation sign. Both of the two uveitis entities which are characterized by wide range of involved tissues, frequentoccurrence, difficult to cure and bad pognosis belong to the types mostdifficult to deal with and most common in our China. The etiology andpathogenesis are both unclear of the two diseases, although various theoriesexist including bacteria infection, virus infection and autoimmunity withthe last kind most accepted now. Liver X receptor is a kind of nuclearreceptor. Refer to the functions of liver X receptor, the focus was on lipidmetabolism at earlier times and was transferred to modulation ofinflammation and immunity recently. The ligands of Liver X receptorsinclude natural oxysteroids and syntherized agonist such as T0901317andGW3965. Thus the following studies were designed:1. The relationshipbetween expression level of liver X receptors and Behcet’s disease;2. Therelationship between expression level of liver X receptors andVogt-Koyanagi-Harada disease;3. The role of liver X receptors in thefunction of CD4+T cells. We could find out whether liver X receptor plays arole in the pathogenesis of Behcet’s disease and Vogt-Koyanagi-Haradasyndrome and provide a novel approach to deal with these uveitis entitieshard to cure and easy to recur. Part Ⅰ Therelationship between expression level of liver Xreceptors and Behcet’s diseasePurposeThe expression of Liver X Receptor in Behcet’s disease patients andnormal controls as well as the relationship with disease development wereinvestigated.MethodsPeripheral blood mononuclear cells of BD patients (including patientswith active and inactive disease) and healthy controls were isolated fromfreshly drawn blood by Ficoll-Hypaque density gradient centrifugation.Total RNA of the PBMCs were used to obtain cDNA according to themanufacturer’s instructions. Real-time quantitative PCR was performedusing the cDNA to detect the expression of LXR in PBMCs. Magneticbeads were used to separate CD4+T cells according to the manufacturer’sprotocol. To determine the expression of LXR in patients and normalcontrols, freshly isolated CD4+T cells were used and analyzed by indirectflow cytometry.ResultsmRNA expression of LXRβ in PBMCs and protein expression of LXRβin purified CD4+T cells were both significantly lower in patients withactive BD than in healthy controls. LXRβ expression was not altered in BDpatients with inactive disease both in PBMCs and CD4+T cells. no differences could be detected for mRNA expression in PBMCs and proteinexpression in CD4+T cells of LXRα between the three groups tested.ConclusionsIn this study, we found that the mRNA expression of LXRβ in PBMCsfrom uveitis patients with BD was lower as compared to healthy controls.LXRβ protein expression was also lower in purified CD4+T cells from BDpatients. Expression of the LXRα isoform both at the level of PBMCs andCD4+T cells in BD patients was not altered. The fact that LXR expressionwas only decreased in our uveitis patients with active ocular inflammationand not during a quiescent episode supports the role for LXR in thedevelopment of these diseases.PartⅡ The relationship between expression level of liver Xreceptors and Vogt-Koyanagi-Harada diseasePurposeThe Liver X receptor is becoming an important regulator ofinflammatory and immune responses. The expression of Liver X Receptorin Vogt-Koyanagi-Harada disease patients and normal controls as well asthe relationship with disease development were investigated.Methods Peripheral blood mononuclear cells of VKH patients (including patientswith active and inactive disease) and healthy controls were isolated fromfreshly drawn blood by Ficoll-Hypaque density gradient centrifugation.Magnetic beads were used to separate CD4+T cells. Real-time quantitativePCR and flow cytometry were used to evaluate the expression of LXR.ResultsAs for VKH patients, mRNA expression of LXRβ in PBMCs from activedisease patients was significantly decreased compared with normal controls.LXRβ mRNA expression in PBMCs from inactive VKH patients was notdifferent from healthy controls. LXRβ protein expression in CD4+T cellswas not different between the healthy control, inactive VKH and activeVKH groups. On the other hand, no differences could be detected formRNA expression in PBMCs and protein expression in CD4+T cells ofLXRα between the various groups tested.ConclusionsIn this study, we found that the mRNA expression of LXRβ in PBMCsfrom uveitis patients with BD or VKH was lower as compared to healthycontrols. LXRβ protein expression was also lower in purified CD4+T cellsfrom BD patients but not in VKH patients. The reason for the differencemay be due to the fact that a lower level of LXRβ in the PBMCs of VKH patients existed mainly in other types of cells but not in CD4+T cells.PartⅢ The role of liver X receptors in the function ofCD4+T cellsPurposeThe exact role of LXR in immune regulation is still not completelyknown. We investigated whether LXR might have an impact on CD4+Thelper cells and therefore play a role in the pathogenesis of BD and VKHdisease.MethodsPeripheral blood mononuclear cells of patients (including BD and VKHpatients) and healthy controls were isolated from freshly drawn blood byFicoll-Hypaque density gradient centrifugation. Magnetic beads were usedto separate CD4+T cells. Effects of LXR on CD4+T cells were detected byflow cytometry and Elisa. Flow cytometry and RT-PCR were performed tostudy the mechanisms involved in the effect of an LXR agonist GW3965on CD4+T cells.ResultsThe frequency of Th17and Th1cells along with the relevant cytokines IL17, IFNγ and corresponding transcriptional factors RORC, T-bet were alldecreased following LXR activation by agonist GW3965. LXR activationinhibited proliferation but had no influence on apoptosis of CD4+T cells.LXR controlled the expression of inflammatory cytokines through an effecton Nfκb phosphorylation. LXR activation was accompanied by anincreased mRNA expression of apolipoprotein-e.ConclusionsOur findings provide an evidence for a role of LXR in the pathogenesisof intraocular inflammation caused by BD and VKH disease and suggestthat LXR activation can be a potential modulator of aberrant immuneresponses by inhibiting Th1and Th17cell responses.