Therapeutic Effects Of IL-35 On NOD/Ltj Mice With Spontaneous Sjogren’s Syndrome And Related Mechanisms

Objective: Primary Sjogren’s Syndrome(p SS)is a chronic autoimmune disease that usually affects the exocrine glands,including the lips and lacrimal glands,causing patients to complain of dry mouth and dry eyes and often becoming the first symptom of medical treatment.The pathological features lie in the presence of organ-specific or non-specific autoantibodies in blood circulation,and the exocrine glands will be damaged by lymphocytes.Without timely and reasonable treatment,multiple organs may be damaged and develop into systemic chronic immune diseases.The pathological sections of p SS patients will observe lymphocyte infiltration,gland duct damage,and acinar atrophy in the interstitium of salivary glands,and the lymphocytes are mainly CD4+T cells,the pathogenesis of which is not clear at present.It is widely believed by scholars at home and abroad that the proportion of immune cells dominated by CD4+T cells is unbalanced,resulting in the production of a variety of pro-inflammatory cytokines,which are involved in the pathogenesis of p SS as an important pathogenic factor.In recent years,more and more attention has been paid to the role of T cell subpopulation imbalance and the pathogenic role of various cytokines secreted in p SS and other autoimmune diseases.T helper cell 17(Th17),as a new CD4+T cell subpopulation,plays an important role in the pathogenesis of p SS by secreting various cytokines such as interleukin-17(IL-17),interleukin-6(IL-6),and tumor necrosis factor α(TNF-α)to induce an inflammatory response.Participate in the pathogenesis of p SS.Regulatory T cells(Tregs)are inhibitory T cells,which inhibit the activation and proliferation of effector T cells to maintain peripheral immune tolerance and play an important inhibitory role in a variety of autoimmune diseases.Th17 and Treg balance each other to maintain a steady-state,but changes in the local cytokine microenvironment in the body can easily induce juvenile T cells to differentiate into Th17 cells or Treg cells in different directions,and the ratio is easily unbalanced,thus leading to differentiation and proliferation of pathological cells and triggering autoimmune diseases.A variety of autoimmune diseases are associated with the altered Th17/Treg ratio,and studies have shown that the imbalance of Th17/Treg also plays an important role in the pathogenesis of Sjogren’s syndrome(SS).Some clinical and animal tests in recent years suggest that after the improvement of the ratio imbalance of Th17/Treg,the common clinical symptoms of dry mouth and eyes in SS patients can be alleviated,and histopathology shows that the inflammatory response of exocrine glands such as the submandibular gland can be alleviated.Interleukin 35(IL-35)is a novel cytokine that promotes Treg cell proliferation and inhibits the proliferation and function of CD4 effector T cells,including Th1 cells and Th17 cells,in animal models of Rheumatoid arthritis(RA).Currently,there are few studies on THE role of IL-35 in Sjogren’s syndrome.We treated NOD/Ltj mice in the SS animal model with IL-35 and analyzed the pathological changes of excretory glands in NOD/Ltj mice after IL-35 treatment,as well as the effects on Th17 and Treg subsets.To explore whether IL-35 can play a role in SS treatment by regulating Th17/Treg balance.In addition,several studies have shown that JAK/STAT signaling pathway is involved in the pathogenesis of SS,and the signal path can be a lot of types I and type II cytokine activation,IL-2,IL-6 is one of the important types I cytokine,in an inflammatory response,apoptosis,stress,cell cycle and movement,growth and so on the many kinds of pathological and physiological process play an important role.IL-35 may regulate the JAK/STAT signaling pathway in RA,cardiovascular disease,Systemic Lupus erythematosus(SLE),acute kidney injury(AKI),and other diseases.However,the lip gland biopsy specimens of SS patients indicated an abnormal JAK/STAT signaling pathway.Since JAK/STAT signaling pathway is also an important upstream signaling pathway involved in Th17 and Treg differentiation,in summary,we speculated that an abnormal JAK/STAT signaling pathway may also exist in NOD/Ltj mice as SS animal model,and IL-35 may also regulate this signaling pathway.Thus,Th17/Treg balance can be adjusted to play a therapeutic role in p SS.At present,there is no relevant research at home and abroad,so we carried out a series of studies on this,trying to find new targets for the treatment of p SS.Non-obese diabetic mice(NOD/Ltj non-obese diabetic mice,NOD/Ltj for short)are animal models of spontaneous SS.At the age of 10 weeks,the focal infiltration of lymphocytes in salivary glands is obvious,and the salivary gland secretion function begins to decline,and the disease gradually worsens with the increase of the age of mice.Loss of salivary gland secretory function at 20 weeks of age;A variety of specific autoantibodies of SS patients exist in their serum,and these disease characteristics are similar to human SS,which has become a typical animal model for studying spontaneous Sjogren’s syndrome in recent years.NOD/Ltj mice were also used as experimental research objects in this study.Materials and methods:Part I: A total of 10 SPF 9-week-old ICR mice and 40 NOD/Ltj mice of9-week-old SS animal models were selected as research objects.ICR mice and NOD/Ltj mice were divided into two groups: C was the blank control group(ICR mice);M was the model control group(NOD/Ltj mice).Hydroxychloroquine(HCQ)+M was used as the control group(NOD/Ltj mice + hydroxychloroquine solution 0.5m L/day).IL-35 I+M was low-dose IL-35 intervention group I(NOD/Ltj mice +IL-353 μg/day).IL-35 II+M was IL-35 intervention group II high-dose group(NOD/Ltj mice +IL-35 6 μg/day).There were 10 mice in each group.After 1 week of adaptive feeding,10-week-old mice were given the drug for 6 weeks after onset,and the mice were killed after the end of an administration.During the experiment,the general condition and biological behavior,body weight,water intake,and saliva flow of the mice were monitored.The weight of the submandibular gland and spleen was measured.Submandibular gland index and spleen index were calculated.HE staining method was used to evaluate and analyze the histopathological changes of mouse submandibular gland and pancreas,and to explore the pathological effects of IL-35 treatment on immune damage of exocrine gland in NOD/Ltj mice.Cytokines closely related to Th17 and Treg cell subsets in peripheral blood of mice in each group were detected by enzyme-linked immunosorbent assay(ELISA): Il-17(an important downstream cytokine secreted by Th17),IL-6(an important cytokine promoting the differentiation of Th17),interleukin 10(IL-10,an important downstream cytokine secreted by Tregs),and IL-2(an important cytokine promoting the differentiation of Treg)in peripheral blood were analyzed after IL-35 treatment.The proportion of Th17 and Treg subgroups in the submandibular gland and peripheral blood of mice were detected by multiple immunofluorescences and flow cytometry(FCM),respectively,and the effects of IL-35 treatment on the proportion of Th17/Treg in submandibular gland tissue and peripheral blood were analyzed.Quantitative reverse transcription-polymerase chain reaction(RT-q PCR)was used to detect the expression of JAK/STAT pathway-related genes in the submandibular gland: Il-6 /JAK2/STAT3 pathway genes related to Th17 differentiation and development(IL-6,IL-6r,JAK2,STAT3),IL-2/JAK3/STAT5 pathway genes related to Treg differentiation and development(IL-2,IL-2rα,IL-2rβ,JAK3,STAT5),To explore whether the corresponding signaling pathway genes are involved in the pathogenesis of NOD/Ltj mice and the effect of IL-35 on the expression of corresponding signaling pathway genes.Western Blotting was used to detect the expression of JAK/STAT pathway-related proteins in the submandibular gland: Th17 differentiation and development related IL-6/JAK2/STAT3 pathway proteins(IL-6,IL-6r,JAK2,P-JAK2,STAT3,P-STAT3),IL-2/JAK3/STAT5 pathway proteins related to differentiation and development of Treg(IL-2,IL-2rα,IL-2rβ,JAK3,P-JAK3,STAT5,P-STAT5),to explore whether the corresponding JAK/STAT signaling pathway is involved in pathogenesis in NOD/Ltj mice.And the effect of IL-35 on the corresponding signaling pathway.Part II: There were 10 ICR mice and 40 NOD/Ltj model mice.The spleen CD4+T cells in each group were sorted by magnetic beads,and the sorted CD4+T cells were divided into 5 groups: C is ICR blank control group,M is NOD/Ltj model control group,(IL-35 I,IL-35 II,and IL-35 III)+M is NOD/Ltj mouse spleen CD4+T cell culture with 25 ng/m L,50 ng/m L and 100 ng/m L IL-35,respectively.The freshly isolated and extracted CD4+T cells were suspended in a T cell culture medium and co-cultured with anti-CD3,anti-CD28,and different concentrations of IL-35.The following tests were performed at 24,48,and 72 hours respectively: The specific transcription factor of Th17 cells,Retinoic acid associated lone nucleus receptor transcription factor(RORγt)and Treg cells,fork-head family transcription factor(Foxp3)were detected by RT-q PCR.The proportion of Th17 cells and Treg cells was detected by FCM.The relationship between IL-35 and the regulation of the Th17/Treg ratio was analyzed at the cellular level in vitro,and the optimal concentration and duration of IL-35 regulating the Th17/Treg ratio were found.Part III:(1)Ten ICR mice and 80 NOD/Ltj mice were selected for magnetic beads sorting of spleen CD4+T cells(same as in Part II).The selected CD4+T cells were cultured in vitro and divided into six groups: C was ICR negative control group;M was NOD/Ltj model control group;IL-35 +M group was added with IL-35 100 ng/m L;IL-6 +M group was added with IL-6 100 ng/m L;IL-6 +JSI-124+M group was added with IL-6 100 ng/m L,JSI-124(0.5 μmol/L);IL-6 +IL-35+M group was added with IL-6 100 ng/m L and IL-35 100 ng/m L.After 72 h co-culture,RORγt m RNA level was detected by RT-q PCR.The proportion of Th17 cells was detected by FCM.Western Blotting was used to detect the expression of JAK2/STAT3 signaling pathway proteins.To explore whether the regulation of IL-35 on Th17 is realized through the IL-6-mediated /JAK2/STAT3 signaling pathway,and to verify this by applying the specific blocker JSI-124 of this signaling pathway.(2)The magnetic beads sorting of mice spleen CD4+T cells was the same as the above method.The sorted CD4+T cells were cultured in vitro and divided into six groups: group C,M and IL-35+M were the same as(1).IL-2 +M group was added with IL-2 100 ng/m L;IL-2 +AG490+M add IL-2 100 ng/m L,AG490(50 μmol/L);IL-2 +IL-35+M group was added with IL-2 100 ng/m L and IL-35 100 ng/m L.After72 h co-culture,Foxp3 m RNA level was detected by RT-q PCR.The proportion of Treg cells was detected by FCM.Western Blotting was used to detect the expression of JAK3/STAT5 signaling pathway proteins involved in Treg differentiation.To explore whether the regulation of IL-35 on Treg balance is achieved through the IL-2-mediated JAK3/STAT5 signaling pathway,and to verify this by applying AG490,a specific blocker of this signaling pathway.Results:1.NOD/Ltj mice showed a gradual decrease in body weight,a gradual increase in water intake,and a significant decrease in saliva volume.IL-35 treatment group could significantly improve the above symptoms in a dose-dependent manner.In NOD/Ltj mice,the submandibular gland index was significantly decreased and the spleen index was significantly increased.IL-35 treatment group could significantly increase the submandibular gland index and decrease the spleen index in a dose-related manner.Pathology showed that lymphocyte infiltration in submandibular gland tissue was obvious in NOD/Ltj mice,and the IL-35 treatment group could significantly reduce lymphocyte infiltration in the submandibular gland in a dose-dependent manner.Multiple immunofluorescence results showed that the proportion of Th17 cells in the submandibular gland of NOD/Ltj mice increased,while the proportion of Treg cells decreased.IL-35 treatment group could down-regulate the proportion of Th17 cells and up-regulate the proportion of Treg cells in a dose-dependent manner.ELISA showed that the levels of cytokines IL-17 and IL-6 in peripheral blood of NOD/Ltj mice were increased,while the levels of IL-10 and IL-2 were decreased.The IL-35 treatment group decreased IL-17 and IL-6levels and increased IL-10 and IL-2 levels in a dose-dependent manner.The proportion of Th17 and Treg cells in peripheral blood detected by the FCM method showed that the proportion of Th17 cells in peripheral blood of NOD/Ltj mice increased while the proportion of Treg cells decreased.IL-35 treatment group could down-regulate the proportion of Th17 cells and up-regulate the proportion of Treg cells in a dose-related manner.RT-q PCR results showed that the expression of IL-6/JAK2/STAT3 pathway-related genes(IL-6,IL-6r,JAK2,and STAT3)related to Th17 cell differentiation in NOD/Ltj mice was significantly up-regulated,and the expression of these genes was significantly down-regulated in the IL-35 treatment group in a dose-dependent manner.The expression of IL-2/JAK3/STAT5pathway-related genes(IL-2,IL-2rα,IL-2rβ,JAK3,and STAT5)related to Treg cell differentiation was significantly down-regulated,and the expression of these genes was significantly up-regulated in the IL-35 treatment group in a dose-dependent manner.Western Blotting results showed that: The expression of IL-6/JAK2/STAT3 pathway proteins(IL-6,IL-6r,P-JAK2,P-STAT3)related to Th17 cell differentiation was significantly up-regulated in NOD/Ltj mice submandibular gland,and the expression of these proteins was significantly down-regulated in the IL-35 treatment group in a dose-dependent manner.The expressions of IL-2/JAK3/STAT5 pathway proteins(IL-2,IL-2rα,IL-2rβ,P-JAK3,and P-STAT5)related to Treg cell differentiation were significantly down-regulated in NOD/Ltj mice submandibular gland,and the expression of these proteins was significantly up-regulated in the IL-35 treatment group in a dose-dependent manner.2.CD4+T cells from the spleen of NOD/Ltj mice were cultured with different concentrations of IL-35 for 24 h,48 h,and 72 h,and it was found that RORγt m RNA expression level and Th17 cell proportion were down-regulated when IL-35 concentration was 100ng/m L at 72 h.The up-regulation of Foxp3 m RNA expression and proportion of Treg cells were the most significant(P < 0.01).3.(1)When the spleen CD4+ cells of NOD/Ltj mice were co-cultured for 72 h,IL-6 activated JAK2/STAT3 pathway,up-regulated RORγt expression,and increased the proportion of Th17 cells.After the addition of JAK2/STAT3 pathway blocker JSI-124,the above effects of IL-6 were significantly reduced.These results suggested that IL-6 up-regulated RORγt expression and Th17 cell proportion through JAK2/STAT3 pathway.After co-culture with CD4+ cells from the spleen of IL-6 and NOD/Ltj mice,IL-35 significantly inhibited the activation of the JAK2/STAT3 pathway,down-regulated the expression of RORγt m RNA,and reduced the proportion of Th17 cells,suggesting that IL-35 could inhibit the expression of RORγt by inhibiting the IL-6/JAK2/STAT3 pathway.Inhibit the differentiation and proliferation of Th17 cells and down-regulate the proportion of Th17 cells.(2)When the spleen CD4+ cells of NOD/Ltj mice were co-cultured for 72 h,IL-2 could activate JAK3/STAT5 pathway,up-regulate Foxp3 m RNA expression,and increase the proportion of Treg cells.After the addition of JAK3/STAT5 pathway blocker AG490,the above effects of IL-2 were significantly reduced.It was suggested that IL-2 up-regulated Foxp3 expression and increased the proportion of Treg cells through JAK3/STAT5 pathway.After co-culture with CD4+ cells from the spleen of IL-2 and NOD/Ltj mice,IL-35 can significantly activate JAK3/STAT5 pathway,up-regulate Foxp3 m RNA expression and increase the proportion of Treg cells,suggesting that IL-35 can promote Foxp3 expression by enhancing IL-2/JAK3/STAT5 pathway.It promoted the proliferation of Treg cells and up-regulated the proportion of Treg cells.In conclusion:1.IL-35 can effectively improve the immune-inflammatory damage of the exocrine gland in NOD/Ltj mice and regulate the balance of Treg/Th17 cells.2.IL-35 regulates Treg/Th17 cell balance in a time-and dose-dependent manner.3.IL-35 regulation of Treg/Th17 cell balance may be achieved through the following mechanisms: inhibition of IL-6/JAK2/STAT3 pathway down-regulates RORγt expression,thus inhibiting Th17 differentiation and proliferation;Enhanced IL-2/JAK3/STAT5 pathway up-regulated Foxp3 expression,thus promoting differentiation and proliferation of Treg;It provides a new idea of diagnosis and treatment for SS.