Experimental Study On The Expression And Function Of IDO In Crescentic Glomerulonephritis And Its Interference In Vivo

Background and objectivesGlomerulonephritis (GN) is an inflammatory disease of the renal glomeruli and is a major cause of end-stage renal failure. The most severe and rapidly progressive type of GN results from infiltration of inflammatory cells into the glomerular tuft and accumulation of cells and fibrin in Bowman's space, these forms, known as crescentic GN. Increasing evidence has suggested that T cell-mediated cellular immunity may be a more important mediator than the function of self-antibody, CD4+ T helper 1(Th1) cell-mediated response has been suspected as the culprit for glomerular injury in crescentic GN. The glomerular influx of CD4+ T cells and macrophages results in local cell proliferation, and formation of glomerular crescents and severe glomerular injury. It is enforced by interrupting B7/CD28 co-stimulation pathway, and attenuated by Th2 cytokines or transfer of CD4+ CD25+ T cell, and independent of autologous antibodies(Ab). Further studies about the exact regulated mechanism of activation and effection of CD4+ T cell in pathogenic mechanisms of crescentic GN are needed, and these researches may lead to the development of novel therapies for crescentic glomerulonephritis.Tryptophan(Trp) is an essential amino acid for mammals. Bacteria which can synthesize tryptophan also obtain it from their environment when available. These presumably tryptophan is a very important consumed amino acid to synthesize. Indoleamine 2,3-dioxygenase (EC 1.13.11.42, IDO) is a heme-containing dioxygenase which catalyzes the first and rate-limiting step in the major pathway of L-tryptophan catabolism in mammals. Either antigen presenting cells (APC) such as dendritic cells (DCs) or tissue cells which express IDO, were shown to suppress the T-cell responses by depriving T-cells of Trp in vitro. The immunosuppressive activity of IDO was then applied to inhibit the T-cell-mediated reject on allografted pancreas islets in mice and such IDO-expressing islets have also been demonstrated to show a significant prolongation of graft survival. Similarly, it has recently been shown that the T-cell-mediated experimental asthma and experimental autoimmune encephalomyelitis were inhibited by the IDO which was up-regulated in pulmonary and microglial cells. Based on these findings in vitro and in experimental animal systems, the concept that cells expressing IDO can suppress the T-cell responses and induce tolerance has thus emerged as a new paradigm in immunology. Richard found IFN-γ–/– mice developed significant crescent formation and an increased cellular infiltrate in both the glomerulus and the interstitium compared with normal mice. But the mechanisms behind the protective effect of IFN-γin this model and in other models of organ-specific autoimmunity are complex and remain incompletely understood. Recently research showed IFN-γstimulation of APC could result in increased IDO expression, and the activity of IDO might be the important mechanisms of IFN-γin tolerance induction in many autoimmunity disease. Those investigation suggested that IDO may play an important role in crescentic GN. MHC class II-expressing renal tubular epithelial cells can function as APC for T cells,and no reports about whether the TEC express the IDO and its role in the crescentic GN. So we study the expression and function of IDO in crescentic GN. So we study the expression and function of IDO in crescentic GN. The best characterized and most widely used model of crescentic GN is nephrotoxic serum nephritis(NTN), in which heterologous anti-GBM antibody(Ab) acts as a planted antigen in glomeruli. We want to first examine the expression and function of IDO on crescentic GN kidney samples as well as on human renal tubular epithelial cells (RTEC) line HK-2 cells. Second, construct a recombinant adenovirus that expresses IDO for gene therapy in vivo. Last, we test the interference effects of enforceing IDO by gene transfection and interrupting IDO by 1-methyl-DL-tryptophan (1-MT) in crescentic GN in a rat model of NTN.Methods and results:We fist examined the expression of IDO on crescentic GN and normal human kidney samples. In immunohistochemical staining revealed no expression of IDO in the normal kidney tissues, but much high expression in renal tubular epithelial cells of crescentic GN. The IDO expression had a negative correlation with the number of PCNA+ cells in tubulointerstitial and with the tubulointerstitial injury. The expression of IDO in RTEC line HK-2 cells also was examined by RT-PCR, immunocytochemistry and high performance liquid chromatography (HPLC),and rusults showed that the IDO is no expression on HK-2 cells in normal culture conditions, but its expression and the activity is dramatically increased by stimulation of IFN-γin a dose and time dependent manner compared with control groups. In vitro, pretreatment of IFN-γ-stimulated HK-2 cells cocultured with Jurkat cells can significantly inhibite the proliferation and promote the apoptosis of Jurkat cells. These results suggest that the activity of IDO negatively regulates T cell activation by TEC, and may play an important inhibitory role in TEC-mediated immune activation and immuno-pathology in the kidney.Next, we construct a recombinant adenovirus that expresses IDO for gene therapy in vivo. Full-length mouse IDO cDNA was subcloned into pAdTrack-CMV shuttle plasmid. The product was linearized to mediate homologous recombination with pAdEasy-1 vector in BJ5183 host bacteria. The positive clone was identified by restriction endonuclease digestion and further confirmed by sequencing. The recombined adenovirus DNA was transfected into 293 cells with LipofectamineTM2000 for packaging and amplification of Ad-IDO virus and the high-titer adenovirus supernatants were harvested. The expression of IDO is monitored by EGFP fluorescence in infected cells. After transfection with adenovirus DNA, infectious virus was only produced to cause cytopathic effect in the permissive cell line AD293 but not in the non-permissive cell line Hela, confirming only replication-defective but not wild type virus was generated. The specific expression of mouse IDO was verified by PCR in AD 293 cells after infection with Ad-IDO, but not Ad-EGFP, a similarly constructed control virus. Ad-IDO, but not Ad-EGFP, can degradate of tryptophan in culture medium. The results showed that we have successfully constructed a recombinant adenovirus Ad-IDO. The virus will be useful to enforce the activity of IDO in animal models of crescentic GN in vivo.The last, we test the inteference effect of enforceing IDO by gene transfection and interrupting IDO by 1-MT in the development of crescentic GN in a rat model of NTN. It was demonstrated that the activity of IDO was increased significantly in NTN rat than in the normal rat, when administration of intravenous injection of Ad-IDO into NTN rat, not only had the IDO transfected on renal tubular epithelial cells, but also the tryptophan catabolism in serum was increased more higher than the control NTN rat, and ameliorated renal injury with decreased glomerular crescent formation, CD4+T cells and PCNA+ cells accumulation in kidney tissue and proteinuria compared with the control NTN rat, and prominent splenocyte cells apotosis(p<0.01).While the administration of 1-MT by intraperitoneally injection resulted in the decereased of IDO activity in NTN rat and accelerates crescentic GN renal pathology injured with significant increased glomerular crescent formation, CD4+T cells and PCNA+ cells accumulation in kidney tissue and proteinuria compared with the control NTN rat. Our results demonstrated the involvement of the role of IDO in the pathogenesis of crescentic GN, and enhancing the activity of IDO in vivo may be beneficial for the treatment of human crescentic GN.Conclusions:Our results suggest:1.The upregulated IDO of renal tubular epithelial cells can suppresse T cell activation, and this might play an inhibited role in T cell mediated immune activation and immunopathology in crescentic GN.2. The activity of IDO is increased in vivo in NTN rat, and which may play an important role in pathogenesis of crescentic GN. While enforced the activity of IDO in vivo might provided a novel therapy method for crescentic GN.