â… .Interleukin-17 Modulates The Ability Of Diabetogenic T Cells To Mediate Type 1 Diabetes In NOD Mouse â…¡.Chronic Ethanol Feeding Decrease PBR And StAR Of Leydig Cells In Rat

PerfaceInterleukin-17(IL-17),also called IL-17A,is mainly expressed in activated CD4+ T cells.The gene location for IL-17 is 6p12 and is a homodimetric glycoprotein consisting of 155 amino acids of 35kDa molecular weight.The IL-17 receptor(IL-17R)is a typeâ… transmembrane protein,and its mRNA is distributed ubiquitously in various tissues,and its engagement activates both transcription factor NF-KB and kinase Jnk pathway.Now,it is known that IL-17 is a preinflammatory factor secreted by IL-17 effector CD4 cells(TH-17 cells)which differentiated from naive CD4 T cells.IL-23 is a key trigger for inducing the development of IL-17 T cells from naive CD4 T cells.At present,it has been proved that the expression of IL-17 has closely linked to a growing list of autoimmune and inflammatory diseases such as rheumatoid arthritis, lupus,asthma and allograft rejection.IL-17 produced from TH-17 cells mainly concerned to inflammation reaction in tissues by stimulation of inflammation factors and cytokines.Approximately 60 genes were upregulated by IL-17,many encoding proinflammatory molecules.IL-17 also increase TNF regulating the expression concerned to inflammatory genes.So,the published data have indicated that IL-17 is a proinflammatory mediator on multiple cells types in vitro and involved in specific inflammation processes leading to the mobilization of granulocytes.IL-17 is one of important pathways through which CD4 acts in inflammation in tissue.Type 1 Diabetes is a slow progressive autoimmune disease resulting in complete destruction of the insulin-production beta(β)cells in the pancreatic islets. As the pathogenic process and histopathologieal stages in nonbese diabetes(NOD) closely parallels of insulin-dependent diabetes meUitus(IDDM)in human,NOD mice are prone to develop diabetes spontaneously.The cellular infiltration into the pancreas that leads to insulitis and beta-cell destruction can begin as early as 3 to 4 weeks of age in the NOD mouse and up to 12 weeks of age,the animal may be overtly diabetic. The BDC2.5 CD4 T cells originally were isolated from islet-infiltrating leucocytes in NOD islets and have the ability to recognize specifically islet cells antigens and kill them.So,this kind of cells is called diabetogenic T cells.In 1993,Katz et al generated a strain of mice carrying the rearranged TCRα(Vcα)andβ(Vβ4)genes from a diabetogenic T cells isolated from a NOD mouse, which is called BDC2.5/NOD TCR transgenie mouse(also called BDC2.5/NOD). Compared to NOD,BDC2.5 T(BDC T)cells are increased in BDC2.5/NOD mice in the periphery,and approximately 95%of CD4 T and CD8 T cells in BDC2.5 mice displayed Vβ4.The process of disease development is similar to NOD.Based on these works,Katz et al reported that the transgene crossed from BDC2.5/NOD mice onto NOD.scid background,and produced a new strain, BDC2.5/NOD.scid with sudc distinguished characters as:(1)Only had CD4 T cells in this model and specifically express BDC2.5 TCR on the CD4 T cells.Other T cells subset and B cells had been deleted.(2)Hasten the development of insulitis and diabetes.BDC2.5/NOD.scid mice develop insulitis just from 14-16 days of age and become severe insulitis and diabetes by 22-30 days of age.All BDC2.5/NOD.scid mice died from diabetic complications on or before 33 days of age.The BDC2.5/NOD.scid mice had dramatically compressed the time of the development of diabetes and allowed us to determine by direct in situ cellular infiltration and apoptotic change ofβcells.Katz's experiments suggest that the expression of IL-17 has a closely relationship with the development of insulitis and pre-diabetes.They observed the high level of plasma IL-17 as early as 10 days of life in BDC2.5/NOD.scid mice, concurrent with the onset of severe insulitis and per-diabetes.Whereas,at the time of the diabetes onset(21 days),the level of IL-17 plummet dramatically.Conversely,the levels of IL-17 tends to vary minimally in BDC/NOD and NOD mice.Moreover, treatment of NOD.scid recipient mice of activated BDC2.5 T cells with anti-IL-17 antibody did not prevent the onset of diabetes significantly.On the other hand,the study of IL-17 knockout mice proposed that IL-17 is critical for proliferation of CD4+ T cells.Therefore,whether the adjustment of IL-17 expression in BDC T cells themselves would affect the transfer diabetes from BDCT cells and the development of diabetes?To understand the influence of IL-17 on Type 1 diabetes,we have constructed MSCV-IRES-Thy1.1(MIT)vector with IL-17 and pMND BANSHEE Thy1.1-U6 (pMND BANSHEE)vector with siRNA IL-17(small interfering RNA of IL-17, abbreviated to silL-17 here)and cloned to produce the target viruses for transduction BDC2.5 T cells.We hope to observe the endogenous regulation of IL-17 to the BDC2.5 T cells in the development of Type 1 diabetes animal models.Objectives(1)To explore and establish a stable expression system for IL-17 and si-17 in BDC T cells.(2)To test the inhibition of si-17 to IL-17 in BDC T cells.(3)To transfer the IL-17 transgenic BDC T cells to NOD.scid mice and identify the effect on inducing diabetes and suppressing as the expression of si-17 in BDC T in vivo.(4)To study the mechanism of IL-17 in the development of type 1 diabetes.Methods1.The construction of retrovirus vector with IL-17 and siIL-17:IL-17 gene is linked to MSCV-IRES-Thy1.1 retrovirus vector by gene recombination with thy1.1 as marker of target gene.The MIT with anti-apoptosis gene of Bcl₂ and empty vector of MIT are as positive controls,and the blank control of Mock is as negative control.Both U6 promotor and Thy1.1 marker were inserted into the cloning site of pMND-BANSHEE to construct the integrated retrovirus vector for our experiment and the siIL-17 gene was inserted between the cloning sites to construct siIL-17 retrovirus vector.2.Production of virus by Phoenix cells:The phoenix cells were cultured and passaged to keep logarithmic growth.The target DNA was transfected into phoenix cells with aid of calcium-phosphate.After culture for 24hs,the virus supernatant was harvested.The phoenix cells were identified by Thyl.1 antibody and flow cytometry before and after transfection.3.Activation of BDC T cells and virus transfection:The Spleen cells from BDC/NOD mice were stimulated by mouse CD3 and CD28 antibodies for about 24hs.BDC T cells marked CD4 & V[34 antibodies were checked by flow cytometry and the activation percentage of cells were identified by marked CD69 & CD62L antibodies.4.The infection of BDC T cells and the expression of IL-17BDC T cells were infected with target virus.After incubation,the infection percentage of T cells marked Thyl.1 & Thyl.2 antibodies was determined by flow cytometry and sorted them.The expression of IL-17 in the double positive cells was assayed by ELISA.5.Transfer infected BDC T cells to NOD.scid mice:A large number of infected BDC T cells were produced and Thy1.1/Thy1.2 T cells were sorted by MACS Separation Columns.Each of NOD.scid mouse was injected the infected BDC T cells of and injection of BDC T cells as well as PBS injected as negative controls.The mice blood sugar was assayed.6.The plasma tumor necrosis factor -alpha(TNF-α),interferon-gamma(IFN-γ), interleukin-6(IL-6)and IL-4 were assay by ELISA and pancreas from NOD.scid were stained with hematoxylin and eosin(H&E).The spleen cells and PLN cells have been checked by Flow cytometry.Results1.The recombinant retrovirus are the virus DNA with the target genes of IL- 17 or siIL-17 respectively.The results from sequence and digestion are the same as the expected fragments of them.2.Transfected phoenix cells by retrovirus with target genes: Identified the phoenix cells by specific antibodies and flow cytometry,They show Thy1.1 negative before transfection and the transfected percentage increase significantly in MIT,Bcl₂-MIT,IL-17 vector and siIL-17 vector of(96.1±4.2)%, (93.2±2.2)%,(93.5±3.2)%and(89.5±4.7)%respectively in the post-transfection, compared with Mock control(0.4±0.1)%.3.Activate BDC T cells:Marked the spleen cells of pre- and post-activated with CD4/CD69 & CD4/CD62L antibodies.Compared to pre-activated,the percentage of post-activated BDC T cells increased from(3.4±1.5)%and(8.6±2.8)%to(33.7±6.9)%and (25.5±5.3)%,respectively.The results showed that the T cells were optimal for infection.4.Infect BDC T cells by retrovirus with target genes:Marked the infected BDC T cells with Thy1.1/Thy1.2 antibodies and flow,the percentage of infected BDC T cells in Mock(blank control),MIT(empty vector),Bcl₂ (positive control),IL-17 and silL-17 were(0.6±0.3)%,(7.2±2.4)%,(6.8±2.6)%, (6.4±2.4)%and(4.6±1.8)%,respectively.5.Identified the sorted transgenic BDC T cells:Marked the sorted cells by Thy1.1/Thy1.2/BDC2.5 TCR antibodies and flow cytometry,the results suggested that:â‘ the sorted cells marked by Thy1.1/Thyl.2/BDC2.5 TCR antibodies were BDC2.5 T cells.â‘¡Compared to pre-sorted,the percentage of Thy1.1/Thy1.2 double positive IL-17 cells increased from(5.1±2.4)%to(97.2±1.2)%,and from(4.6±1.6)%to(83.2±6.3)%in double positive siIL-17 cells.6.IL-17 expression in transgenic BDC T cells in vivo:IL-17 levels in IL-17 transgenic BDC T cells increase significantly,and even more after activation.Conversely,the expression of IL-17 is inhibited in siIL-17 BDC T cells and IL-17 levels decrease.7.Determined the blood sugar of NOD.scid mice and histopathology:The blood sugar from tails in NOD.scid mice are assayed at 4pm.By the end of observation(36 days),All of the NOD.scid mice(6 mice)injected IL-17 BDC T cells have diabetes and there is no diabetes in the NOD.scid mice of siIL-17 BDC T cells injected and negative control as well as BDC T cells groups(Thy1.1-/Thy1.2+)to observe until the end of observation.Pancreases H&E stained showed that there were distinct severe insulitis and destruction of islets in NOD.scid mice injected IL-17 BDC T cells,and peri- or moderate insulitis in NOD.scid mice whether injected siIL-17 BDC T cells or BDC T cells,are normal in negative control group.8.The plasma levels of TNF-α,IFN-γ,IL-6 and IL-4:The results show that the plasma levels of IL-17,TNF-α,IFN-γand IL-6 in IL-17 mice are higher than those of in silL-17 mice or in control(P＜0.01).Whereas, there are no difference in the plasma cytokines levels between the silL-17 mice and control(P＞0.05).The plasma level of IL-4 in the IL-17 mice has not been detected and are very low in the siIL-17 mice and control.9.Check the spleen cells and PLN cells by Flow cytometry:In the diabetes mice of injected IL-17 BDC T cells,the recruitment and proliferation of BDC T cells in spleen and PLN increase markably and the percentage of dendritic cells rise.Conclusions1.The BDC T cells with high expression IL-17 can be obtained by infecting them with IL-17 retrovirus in vitro.The expression of siIL-17 in BDC T cells can efficiently suppress the expression of IL-17,namely BDC T cells of low expression.2.Packaging system of phoenix cells is a safety and efficient "packaging machine" for retrovirus.3.Transfer BDC T cells with high IL-17 expression can induce and speed up the development of diabetes in NOD.scid mice,and BDC T cells with si-17 expression can inhibit the development of diabetes in NOD.scid mice by suppression of IL-17 expression in BDC T cells.4.IL-17 can activate the host DC and BDC T cells and result in the infiltration of lymphocytes to islets,and BDC T cells can destroy the isletsβcells by release TNF-α,IFN-γ,IL-6 as well as inhabitation of IL-4 cytokines.5.Dendritic cells(DC)in spleen and PLN may induce the activation and proliferation of BDC T cells with IL-17 high-expression,and moreover damage ofβcells in islets. PerfaeeThe ethanol has been becomes broadly addicted substance for abuses at present. Excessive ethanol consumption may be among the causes of the various disadvantageous influence to the male reproductive system.Now,people believe that ethanol consumption is one of important factor which the rate of male infertility incidents rise year by year.Numerous epidemiological data and experiments confirmed that,excessive ethanol consumption can obviously reduce serum testosterone level.Ethanol could reportedly affect the activity of HPA axis,which is believed as the possible mechanism of ethanol.Moreover,ethanol can act to the Leydig cells directly and inhibit the production of testosterone.Recently,it was raised that PBR and StAR are functionally linked to leydig cells testosterone synthesis.Both are located on the outer membrane in mitochondria and plays a key role in the transport of cholesterol(substrate)from outer to inner membrane ofmitochondria.,known as the rate-limiting step in the steroidogenic stage of testosterone synthesis.Studies show that PBR was present exclusively in the interstitial Leydig cells and immuno- histochemical localization of StAR in leydig cells stained moderately to intensely.Series of experiments shown that targeted disruption of the PBR gene in R2C Leydig cells arrested cholesterol transport into mitochondria and steroid formation;transfection of the PBR-disrupted cells with a PBR cDNA rescued steroidogenesis.Whereas,StAR knockout mice exhibited multiple hormonal abnormalities.Therefore,both PBR and StAR play an very important role in steroidogenesis.Document shown that ethanol can affected PBR expression the kidney and increased binding of PBR with its ligand.Ethanol administration to rats for 30 days resulted in a significant decrease in the density of PBR in the olfactory bulb,and the reason may be concerned with neurotoxicity from ethanol.Similarly,experiments showed that ethanol rapidly induces the increased StAR expression and translocation in rat adrenal gland,and this StAR protein expression paralleled increases in plasma pregnenolone,progesterone and corticosterone levels.The experiments from Herman showed that the levels of StAR and of PBR were significantly decreased within 10 minutes of drug administration intragastrically,and by 48 hours,STAR,PBR levels had increased above control values.Whereas,Calvo reported the binding of ligand to PBR was increased in the kidneys of rats treated with ethanol for 12 weeks,but not to ethanol increased.So,above researches suggested that the trend of ethanol impacting on PBR and StAR be not clear.Based on above studies,it is necessary to deeply understand the relationship between ethanol and PBR & STAR,and mechanism of inhibition to testosterone. Therefore,we focus on the objective and methods as follow.Objectives1.To observe the change of testes tissue by Hematoxylin and Eosin staining.2.To understand the inhibition of ethanol to testosterone by assaying the rat serum testosterone level.3.To determine the StAR mRNA expression levels by extraction of total RNA from testes tissues.Furthermore,to investigate the influence of ethanol on PBR and StAR protein western-blotting.4.To determine the location and expression of PBR and StAR in testes by staining tissue slides with specific antibodies.Methods1.Making ethanol-feeding Wistar rats model:To investigate the influence of chronic ethanol feeding on rats testes,Wister rats were randomly divided into four groups and administered intragastrically(i.g.)the different dose of ethanol to each group once a day for twenty weeks totally.After anesthetic executions,rapidly takes the testes into liquid nitrogen or fixed in the 30%formaldehyde,dehydrate and embed in paraffin.2.Show the change of testes tissues affected by ethanol with Hematoxylin and Eosin staining.3.To assay the serum testosterone level by Roche ECL170.4.To explore the influence of ethanol on PBR and StAR in testes of rats, RT-PCR was employed for determination of StAR mRNA and Western-blot was used for assaying the protein of PBR and StAR.5,To determine the location and expression of PBR and StAR in testes by immunohistochemistryResults1.The pathological change in testes:The morphology and structure of testes tissue were shown by Hematoxylin and Eosin(H&E)staining.Compared to the control,seminiferous tubular wall of testes became thin and the layer of germ cells reduced significantly.Moreover,the number of spermatozoon in the seminiferous tubes decreased,and the broken and misshapen spermatozoon's flagella were frequently observed and no integrated spermatozoon produced.These change strongly suggested that ethanol can decrease spermatozoon production,destroy spermatozoon development and damage the function of spermatogenic cells,even the ability of male reproduction.2.Assay the serum testosterone level:As Compared to the control [(259.62±69.80)ng/dl],the serum testosterone level in each ethanol-feeding group decreased significantly,the percent of decline in the small-,middle- and large-dose ethanol feeding groups were 67.2%[(85.1±23.4)ng/dl,P＜0.01],69.4%[(79.6±23.8) ng/dl,P＜0.01]and 90.0%[(25.9±6.8)ng/dl,P＜0.01]respectively.3.Ethanol down-regulated the mRNA expression of StAR in testes:The StAR mRNA level in each ethanol-feeding group decreased with different extent.The descending percent in the small-,middle- and large-dose ethanol feeding group were 45.9%(P＜0.01),60.2%(P＜0.01)and 81.7%(P＜0.01)respectively,compared to the control,and suggested that there was a dose-dependent relationship between ethanol and the inhibition of StAR mRNA expression.4.Ethanol down-regulated the expression of PBR and StAR protein level: Compared to the control,the descending percentage of PBR and StAR in small-dose ethanol feeding group were 13.8%(P＞0.05)å’Œ34.5%(P＜0.01)respectively,the descending percentage of PBR and StAR were 20.9%(P＜0.05)å’Œ37.7%(P＜0.01) respectively in the middle-dose group,and the descending percent of PBR and StAR in large-dose group were 50.4%(P＜0.01)å’Œ95.2%(P＜0.01)respectively.The descending degree in the large-dose group was the most significant in three groups, and even in the StAR large-dose group.Correlation analysis showed that there were middle correlation between ethanol and PBR or StAR(r=0.68,P＜0.01;r=0.74, P＜0.01,n=34),suggesting that the suppression of ethanol to PBR and StAR depend on the dosage of ethanol.5.Decrease the expression of PBR and StAR in Leydig cells:The results of immunohistochemistry suggest that PBR and StAR expressed mainly in the leydig cells of interstitial testes.Quantitative analysis has been taken with the square percentage of positive cell in the total interstitial tissues.Compared to control,the square percentage of PBR and StAR expression in low-,middle-,high-dose ethanol groups reduced to 33.3%(P＜0.01),37.7%(P＜0.01),63.6%(P＜0.01)and 27.1% (P＜0.01),51.8%(P＜0.01),58.4%(P＜0.01)respectively.Correlation analysis show that there were medium positive correlation between ethanol and PBR or StAR (r=0.79,P＜0.01;r=0.71,P＜0.01,n=34)respectively.Conclusions1.The serum testosterone level decreased in the chronic ethanol-feeding rats.2.The expression of PBR and StAR in testes can be suppressed by ethanol feeding rats for 20 weeks and this may be one of the mechanismto responsible for ethanol inhibition to testosterone.3.Ethanol can inhibit the testosterone biosynthesis,affect tissue morphology in testes,decredse the number and the quality of spermatozoon,even damage the ability of reproduction by direct or other indirect pathways.