P-糖蛋白单克隆抗体改善MRL/lpr狼疮鼠肾脏病变的研究

目的 探讨P-糖蛋白(P-glyeoprotein,P-gp)单克隆抗体(UIC2)在改善MRL/lpr狼疮鼠肾脏病变中的作用.方法 24只14周龄雌性MRl/lpr狼疮鼠分为3组:P-gp单克隆抗体1次治疗组(G1)、P-gp单克隆抗体3次治疗组(G2)、对照组(G0).观察体重;采用考马斯亮蓝法检测24 h尿蛋白定量;采用酶联免疫吸附法试验(enzyme linked immunosorbent assay,ELISA)检测抗双链DNA(dsDNA)抗体水平;采用酶法检测血清肌酐水平;采用苏木素-伊红染色、免疫荧光和电镜观察肾脏病理改变.结果 ①22周时G1组和G2组体重高于G0组(P<0.05).② 24 h尿蛋白定量22周时Gl组[(1.9±1.1)mg]和G2组[(1.4±0.9)mg]低于G0组[(3.1±1.9)mg](P<0.05);26周时G1组[(2.4±1.4)mg]和G2组[(1.8±1.1)mg]低于G0组[(5.3±2.2)mg](P<0.05).③26周时血清肌酐G1组[(7.0±2.9)μmol/L]和G2组[(6.1±2.5)μmol/L]低于G0组[(12.7±1.3)umol/L](P<0.05).④19周时,抗dsDNA抗体滴度G1组[(43±19)×102 U/mL]和G2组[(45±32)×102 U/mL]低于G0组[(87±39)×102 U/mL](P<0.05);26周时G2组[(35±11)×102 U/mL]低于G0组[(59±35)×102 U/mL].⑤肾小球新月体形成率G1组(0.11±0.05)和G2组(0.09土0.01)低于G0组(0.23±0.07),G2组较Gl组减低(P均<0.05).结论 P-gp单克隆抗体可改善MRl/lpr狼疮鼠肾脏病变的进展,减少尿蛋白、降低血清肌酐,对狼疮肾炎有显著疗效.     

Selective silencing of DNA-specific B lymphocytes delays lupus activity in MRL/lpr mice

The pathological DNA-specific B lymphocytes in lupus are logical targets for a selected therapeutic intervention. We have hypothesized that it should be possible to suppress selectively the activity of these B cells in lupus mice by administering to them an artificial molecule that cross-links their surface immunoglobulins with the inhibitory FcgammaIIb surface receptors. A hybrid molecule was constructed by coupling the DNA-mimicking DWEYSVWLSN peptide to a monoclonal anti-mouse FcgammaRIIb antibody. This chimeric antibody was added to cultured spleen cells from sick MRL/lpr mice, immunized with diphtheria toxoid, resulting in reduction of the numbers of anti-DNA but not of anti-diphtheria IgG antibody-producing cells. Intravenous infusions with the DNA-peptide antibody chimera to 7-wk-old animals prevented the appearance of IgG anti-DNA antibodies and of albuminuria in the next 2 months. The administration of the DNA-peptide chimeric antibody to 18 wk-old mice with full-blown disease resulted in the maintenance of a flat level of IgG anti-DNA antibodies and in delay of the aggravation of the lupus glomerulonephritis. The use of chimeric antibodies targeting inhibitory B lymphocyte receptors represents a novel approach for the selective suppression of autoreactive disease-associated B cells in autoimmune diseases.     

Lack of association of Vβ8+ T cells with lupus-like syndrome in MRL-lpr/lpr mice

To evaluate the role of Vβ8⁺ T cells in the development of lupus-like autoimmune syndrome in MRL-lpr/lpr mice, we treated them with the F23.1 anti-Vβ8 monoclonal antibody (mAb) from birth to 4 months of age. Here we report that almost complete depletion of Vβ8⁺ T cells by the F23.1 mAb treatment neither inhibited nor delayed the development of hypergammaglobulinemia, autoantibody production and autoimmune glomerulonephritis in MRL-lpr/lpr mice. In addition, the F23.1 mAb treatment did not prevent the development of lymphadenopathy and the generation of a CD4^?CD8^? double-negative T cell subset, characteristically accumulating in lpr lymph nodes. Our results strongly argue against the idea that the Vβ8⁺ T cells play a critical role in the development of lupus-like autoimmune syndrome in MRL-lpr/lpr mice.     

Reduced concentrations of the B cell cytokine interleukin 38 are associated with cardiovascular disease risk in overweight subjects

The IL-1 family member IL-38 (IL1F10) suppresses inflammatory and autoimmune conditions. Here, we report that plasma concentrations of IL-38 in 288 healthy Europeans correlate positively with circulating memory B cells and plasmablasts. IL-38 correlated negatively with age (p = 0.02) and was stable in 48 subjects for 1 year. In comparison with primary keratinocytes, IL1F10 expression in CD19⁺ B cells from PBMC was lower, whereas cell-associated IL-38 expression was comparable. In vitro, IL-38 is released from CD19⁺ B cells after stimulation with rituximab. Intravenous LPS in humans failed to induce circulating IL-38, compared to 100-fold induction of IL-6 and IL-1 receptor antagonist. In a cohort of 296 subjects with body mass index > 27 at high risk for cardiovascular disease, IL-38 plasma concentrations were significantly lower than in healthy subjects (p < 0.0001), and lowest in those with metabolic syndrome (p < 0.05). IL-38 also correlated inversely with high sensitivity C-reactive protein (p < 0.01), IL-6, IL-1Ra, and leptin (p < 0.05). We conclude that a relative deficiency of the B cell product IL-38 is associated with increased systemic inflammation in aging, cardiovascular and metabolic disease, and is consistent with IL-38 as an anti-inflammatory cytokine.     

白细胞介素10信号转导机制对C57BL/6和MRL/lpr~-小鼠腹腔巨噬细胞功能及活性影响的比较

目的：体外比较白细胞介素10（IL-10）信号转导机制对C57BL/6和MRL/lpr-小鼠腹腔巨噬细胞功能及活性的影响。方法：分别分离C57BL/6和MRL/lpr-小鼠腹腔巨噬细胞进行培养；用不同浓度IL-10（0.01、0.1、1和10ng/ml）进行刺激,用MTT法测定比较两种品系小鼠腹腔巨噬细胞的增殖反应能力；Real time PCR分别测定其产生前炎性细胞因子IL-1α、M-CSF、TNF-α的量；用100ng/mlIL-10分别刺激两种品系小鼠腹腔巨噬细胞10分钟,免疫印迹法比较两种细胞中JAK1、TYK2、STAT3及磷酸化水平。结果：C57BL/6小鼠巨噬细胞数及其产生的前炎性细胞因子量随IL-10刺激浓度的增加而递减；MRL/lpr-小鼠巨噬细胞数及其前炎性细胞因子IL-1α、M-CSF的量随IL-10刺激浓度的增加而递增,但巨噬细胞产生TNF-α的量随IL-10刺激浓度的增加而递减；MRL/lpr-小鼠细胞内信号转导分子的磷酸化水平低于C57BL/6。结论：MRL/lpr-小鼠的细胞免疫和炎症反应不能被IL-10抑制,可能是由于其信号转导过程中的缺陷所致。     

新西兰小鼠CD8+T细胞数量异常的易感基因定位

目的 :定位自发性SLE模型 (NZB×NZW)F1小鼠CD8 T细胞数量异常的遗传易感基因。方法 :建立 (NZB×NZW )F1×NZW回交小鼠模型 ,采用覆盖小鼠 1 9条常染色体的多态性微卫星遗传标记及数量性状位点分析进行基因定位。结果 :CD8 T细胞数量异常的易感基因与小鼠第 1条染色体尾端 92 3cM处的微卫星遗传标记D1Mit36肯定连锁 (Lods值 >3) ,且回交小鼠该遗传标记B W型组的外周血淋巴细胞中CD8 T细胞百分比明显低于W W型组 (P <0 0 0 1 )。结论 :(NZB×NZW )F1小鼠外周血CD8 T细胞数量异常的候选易感基因位于第 1条染色体尾端 92 3cM附近 ,来源于NZB小鼠。     

Lack of T cells in Act1-deficient mice results in elevated IgM-specific autoantibodies but reduced lupus-like disease

Act1 is a negative regulator of B‐cell activation factor of the TNF family (BAFF) and CD40L‐induced signaling. BALB/C mice lacking Act1 develop systemic autoimmunity resembling systemic lupus erythematosus (SLE) and Sjögren's syndrome (SjS). SLE and SjS are characterized by anti‐nuclear IgG autoantibody (ANA‐IgG) production and inflammation of peripheral tissues. As autoantibody production can occur in a T‐cell dependent or T‐cell independent manner, we investigated the role of T‐cell help during Act1‐mediated autoimmunity. Act1‐deficiency was bred onto C57Bl/6 (B6.Act1^?/?) mice and B6.TCRβ^?/?TCRδ^?/?Act1^?/? (TKO) mice were generated. While TCRβ/δ‐sufficient B6.Act1^?/? mice developed splenomegaly and lymphadenopathy, hypergammaglobulinemia, elevated levels of ANA‐IgG, and kidney pathology, TKO mice failed to develop any such signs of disease. Neither B6.Act1^?/? nor TKO mice developed SjS‐like disease, suggesting that epigenetic interactions on the BALB/C background are responsible for this phenotype in BALB/C.Act1^?/? mice. Interestingly, BAFF‐driven transitional B‐cell abnormalities, previously reported in BALB/C.Act1^?/? mice, were intact in B6.Act1^?/? mice and largely independent of T cells. In conclusion, T cells are necessary for the development of SLE‐like disease in B6.Act1^?/? mice, but not BAFF‐driven transitional B‐cell differentiation.     

Immunomodulation of autoimmunity in MRL/lpr mice with syngeneic bone marrow transplantation (SBMT).

MRL-lpr/lpr mice spontaneously develop a severe autoimmune syndrome, characterized by massive generalized lymphadenopathy, arthritis, arteritis, dermatitis and immune complex-mediated glomerulonephritis. Bone marrow transplantation (BMT) from MHC-matched systemic lupus erythematosus (SLE)-resistant donors to susceptible recipients has proved effective in correcting autoimmune manifestations in autoimmune-prone mice. We investigated the effect of syngeneic BMT from MRL/lpr (donor) to immunocompromised MRL/lpr (recipient), after purging the bone marrow inoculum with MoAbs against mature T cells (anti-Thy 1.2). All the untreated mice developed lymphadenopathy and by the age of 36 weeks five of the eight were dead; in contrast, all the mice which underwent syngeneic BMT following acute immunosuppression with total body irradiation (900 cGy) (TBI) remained disease-free. In an additional experiment, it was found that conditioning with cyclophosphamide (CY) before BMT was more effective than TBI in inhibiting delayed-onset autoimmune manifestations (mean survival 350 days in the CY group and 305 days in the TBI group, versus 197 days in untreated controls). Under both immunosuppressive regimens T cell-depleted bone marrow grafts produced far better results than did unmanipulated BMT. Following syngeneic BMT the incidence of proteinuria and the level of serum anti-DNA (dd) antibodies were significantly reduced, compared with that of the age-matched untreated controls. CY was more effective than TBI in reducing the anti-DNA titres. Likewise, T depletion of bone marrow inocula before BMT induced a more drastic drop in autoantibodies, following both CY and TBI conditioning protocols. After syngeneic BMT (either CY or TBI) no signs of lymphadenopathy were observed even at an advanced age. Upon histopathological examination, the BMT-treated mice displayed normal glomeruli with occasional minimal signs of glomerulonephritis. Syngeneic T cell-depleted BMT following acute cytoreduction of anti-self immune lymphocytes may represent a new therapeutic approach for drug-resistant autoimmune diseases.     

Therapeutic efficacy of anti-CD19 CAR-T cells in a mouse model of systemic lupus erythematosus

Dysregulated B-cell activation plays pivotal roles in systemic lupus erythematosus (SLE), which makes B-cell depletion a potential strategy for SLE treatment. The clinical success of anti-CD19 CAR-T cells in treating B-cell malignancies has attracted the attention of researchers. In this study, we aimed to investigate the feasibility of applying anti-CD19 CAR-T cell therapy to SLE treatment in a mouse disease model. We constructed murine anti-CD19 CARs with either CD28 or 4-1BB as the intracellular costimulatory motif and evaluated the therapeutic function of the corresponding CAR-T cells by infusing them into MRL-lpr mice. Furthermore, anti-CD19 CAR-T cells were transferred to MRL-lpr mice before the onset of disease to determine their role in SLE prevention. According to our observations, compared with antibody treatment, the adoptive transfer of our anti-CD19 CAR-T cells showed a more sustained B-cell-depletion effect in MRL-lpr mice. The transfer of syngeneic anti-CD19 CAR-T cells not only prevented disease pathogenesis before the onset of disease symptoms but also displayed therapeutic benefits at a later stage after disease progression. We also tried to optimize the treatment strategy and found that compared with CAR-T cells with the CD28 costimulatory motif, CAR-T cells with the 4-1BB costimulatory motif showed better therapeutic efficiency without cell enrichment. Taken together, these results show that anti-CD19 CAR-T cell therapy was effective in the prevention and treatment of a murine model of SLE, indicating its potential for clinical use in patients.     

KN-93, an inhibitor of calcium/calmodulin-dependent protein kinase IV,promotes generation and function of Foxp3+ regulatory T cells in MRL/lpr mice

Abstract

Objective: Foxp3⁺ regulatory T cells (T_reg) are pivotal for the maintenance of peripheral tolerance and prevent development of autoimmune diseases. We have reported that calcium/calmodulin-dependent protein kinase IV (CaMK4) deficient MRL/lpr mice display less disease activity by promoting IL-2 production and increasing the activity of T_reg cells. To further define the mechanism of CaMK4 on T_reg cells in systemic lupus erythematosus (SLE), we used the Foxp3-GFP reporter mice and treated them with KN-93, an inhibitor of CaMK4. Methods: We generated MRL/lpr Foxp3-GFP mice to record T_reg cells; stimulated naïve CD4⁺ T cells from MRL/lpr Foxp3-GFP mice under T_reg polarizing conditions in the absence or presence of KN-93; evaluated the number of GFP positive cells in lymphoid organs and examined skin and kidney pathology at 16 weeks of age. We also examined the infiltration of cells and recruitment of T_reg cells in the kidney. Results: We show that culture of MRL/lpr Foxp3-GFP T cells in the presence of KN-93 promotes T_reg differentiation in a dose-dependent manner. Treatment of MRL/lpr Foxp3-GFP mice with KN-93 results in a significant induction of T_reg cells in the spleen, peripheral lymph nodes and peripheral blood and this is accompanied by decreased skin and kidney damage. Notably, KN-93 clearly diminishes the accumulation of inflammatory cells along with reciprocally increased T_reg cells in target organ. Conclusion: Our results indicate that KN-93 treatment enhances the generation of T_reg cells in vitro and in vivo highlighting its potential therapeutic use for the treatment of human autoimmune diseases.     

IL-10RA基因多态性及其与系统性红斑狼疮关系的研究

目的:确定SLE模型小鼠IL-10RA基因变异及其与SLE表现型是否存在关联。方法:用微卫星遗传标记及数量性状位点(QTL)分析方法确定SLE模型小鼠B/W F1的SLE易感基因精确染色体定位并选取候选易感基因,对候选易感基因进行测序分析,选取有基因序列异常的候选易感基因进行PCR-SSCP分析,确定候选易感基因碱基序列变异位点与抗染色质抗体、抗DNA抗体,抗组蛋白抗体及蛋白尿等SLE表现型的相关关系。结果:QTL分析结果表明B/W F1×NZB小鼠抗染色质抗体易感基因与NZW型IL-10RA基因紧密连锁;测序分析发现IL-10RA基因编码区有18处碱基变异,其中7处碱基变异将导致编码氨基酸的变异;抗染色质抗体、抗DNA抗体,抗组蛋白抗体及蛋白尿等SLE表现型与NZW型IL-10RA基因密切相关。另一种SLE模型小鼠MRL的IL-10RA基因存在相同变异。结论:NZW小鼠IL-10RA基因编码区碱基序列存在变异,B/W F1×NZB小鼠SLE表现型与NZW小鼠第9染色体IL-10RA编码区碱基变异相关,提示IL-10RA可能是SLE模型小鼠的一个SLE易感基因。     

In vivo activation of Treg cells with a CD28 superagonist prevents and ameliorates chronic destructive arthritis in mice

Although regulatory T (Treg) cells are necessary to prevent autoimmune diseases, including arthritis, whether Treg cells can ameliorate established inflammatory disease is controversial. Using the glucose‐6‐phosphate isomerase (G6PI)‐induced arthritis model in mice, we aimed to determine the therapeutic efficacy of increasing Treg cell number and function during chronic destructive arthritis. Chronic destructive arthritis was induced by transient depletion of Treg cells prior to immunization with G6PI. At different time points after disease induction, mice were treated with a CD28 superagonistic antibody (CD28SA). CD28SA treatment during the induction phase of arthritis ameliorated the acute signs of arthritis and completely prevented the development of chronic destructive arthritis. CD28SA treatment of mice with fully developed arthritis induced a significant reduction in clinical and histological signs of arthritis. When given during the chronic destructive phase of arthritis, 56 days after disease induction, CD28SA treatment resulted in a modest reduction of clinical signs of arthritis and a reduction in histopathological signs of joint inflammation. Our data show that increasing the number and activation of Treg cells by a CD28SA is therapeutically effective in experimental arthritis.     

Dissecting genetic control of autoimmunity in NOD congenic mice

My lab investigates genetic control of autoimmune disease and autoimmune phenotypes using a series of nonobese diabetic (NOD) congenic mice. NOD congenic mice have regions from B6/B10 introgressed onto the NOD genetic background, which reduces the severity/incidence of autoimmune diabetes. We have demonstrated, however, that while diabetes is reduced, other autoimmune phenotypes and diseases arise in NOD congenic mice. Mapping the genomic regions responsible for these phenotypes has produced novel insights into genetic control of autoimmunity. This review will illustrate some of the genetically controlled phenotypes we have investigated, which shed light upon autoimmune features relevant to human type 1 diabetes, systemic lupus erythematosus, and primary biliary cirrhosis.     

Indole-3-carbinol improves survival in lupus-prone mice by inducing tandem B- and T-cell differentiation blockades

Indole-3-carbinol (I3C), derived from cruciferous vegetables, alters estrogen metabolism. Since lupus is estrogen dependent, we reasoned that I3C might be effective in SLE. I3C significantly thwarted disease progression and prolonged survival in (NZB × NZW) F1 mice. Immunofluorescent and serologic analyses in treated animals indicated a transient blockade in B-cell maturation with increased immature B cells, decreased mature B cells, and a significant reduction of certain autoantibodies. Subsequently, a delay in T-cell maturation occurred in the treated group, manifested by significantly increased naive T cells, decreased mature and memory T cells, and decreased CD4:CD8 T-cell ratios. T cells from the I3C cohort, stimulated in vitro with various mitogens, exhibited enhanced responsiveness. Con A-stimulated T cells from I3C-treated mice produced Th1 cytokines, whereas those from control animals produced Th2 cytokines. Our studies suggest immunological mechanisms by which I3C ameliorates SLE in mice and provide a rationale for its use as an adjunctive therapy for human lupus.     

IL-17 producing CD4 T cells mediate accelerated ischemia/reperfusion-induced injury in autoimmunity-prone mice

Elements of the innate and adaptive immune response have been implicated in the development of tissue damage after ischemic reperfusion (I/R). Here we demonstrate that T cells infiltrate the intestine of C57BL/6 mice subjected to intestinal I/R during the first hour of reperfusion. The intensity of the T cell infiltration was higher in B6.MRL/lpr mice subjected to intestinal I/R and reflected more severe tissue damage than that observed in control mice. Depletion of T cells limited I/R damage in B6.MRL/lpr mice, whereas repletion of B6.MRL/lpr lymph node-derived T cells into the I/R-resistant Rag-1^−/− mouse reconstituted tissue injury. The tissue-infiltrating T cells were found to produce IL-17. Finally, IL-23 deficient mice, which are known not to produce IL-17, displayed significantly less intestinal damage when subjected to I/R. Our data assign T cells a major role in intestinal I/R damage by virtue of producing the pro-inflammatory cytokine IL-17.     

CTLA-4Ig对B6.MRL-Faslpr/J狼疮小鼠脾脏Th17和Treg细胞表达的影响与其对小鼠狼疮样病征干预作用的相关性研究

目的:初步探讨B7阻断剂CD28与细胞毒T淋巴细胞相关抗原4免疫球蛋白(CTLA-4Ig)对B6.MRL-Faslpr/J狼疮小鼠脾脏Th17和调节性T细胞(Treg细胞)表达的影响与其对小鼠狼疮样病征干预作用之间的相关性。方法:将4个月龄大小雌性B6.MRL-Faslpr/J狼疮小鼠16只,随机分为观察组(Ⅰ组)和对照组(Ⅱ组),分别静脉注射CTLA-4Ig及等量PBS,检测小鼠干预前后24 h尿蛋白、ANA抗体、ds-DNA抗体及干预结束2周后血清IL-17A、脾脏中Th17细胞和Treg细胞百分比。结果:末次干预2周后Ⅰ组的24 h尿蛋白、血清ANA及ds-DNA较Ⅱ组下降均有统计学意义(均P0.05)。末次干预2周后Ⅰ组血清中IL-17A、脾脏Th17细胞比例均较Ⅱ组低,而脾脏的Treg细胞占CD4+T淋巴细胞的比例高于Ⅱ组,差异均有统计学意义(均P0.05)。结论:CTLA4-Ig具有减轻B6.MRL-Faslpr/J狼疮鼠狼疮样病征的作用;上调Treg细胞、下调Th17细胞可能是CTLA-4Ig减轻B6.MRL-Faslpr/J狼疮鼠狼疮样病征的重要机制之一。