The Roles Of TLR4 In Diabetes Nephropathy Of Type 1 Diabetes And TLR3 In Autoimmune Hepatitis

BackgroundDiabetic nephropathy is the leading cause of end-stage renal failure in young people and affects about 40%of individuals with long-standing type 1 diabetes. Current therapy, which includes treatment for hyperglycemia, hypertension and dyslipidemia can slow the rate of progression of diabetic nephropathy, but eventually end-stage renal failure will still occur in a proportion of patients. Therefore, the effects of currently used treatments must be maximized and identification of new strategies and additional therapeutic targets for treating diabetic nephropathy will be important.Many factors are involved in the pathogenesis of diabetic nephropathy. Metabolic factors, such as the formation of advanced glycation end products and increased flux through the polyol pathways, have been implicated, In addition, growth factors may also play a role. Although the pathogenesis of diabetic nephropathy is not considered to be primarily immune mediated, nevertheless studies examining renal biopsies in patients with type 1 diabetes have noted T cell infiltration of the juxtaglomerular apparatus. Macrophages have also been detected in kidney sections from patients with diabetic nephropathy although no distinction was made between patients with type 1 or type 2 diabetes.Therefore, it's very important to investigate the role of immune reponses in the diabetic nephropathy. TLR play an important role in the innate immune system. TLR4 is expressed on macrophages, airway epithelia, adipose tissue, skeletal muscle and vascular endothelial and smooth muscle cells and is the key receptor for the lipopolysaccharide (LPS) component of Gram-negative bacteria. TLR4 also interacts with other endogenous and exogenous substances, including heat-shock proteins, fibronectin, fibrinogen, free fatty acids and saturated fatty acids. TLR4 links immune stimulation to the induction of the innate immune response. Activated TLR4 induces expression of a spectrum proinflammatory cytokines. Recent studies have shown increased Toll-like receptor 2 (TLR2) and TLR4 expression and signalling in monocytes from type 1 diabetic patients.TLR4 is one of important receptor in the development of type 1 diabetes, obesity and insulin resistance.Research Objectives1. In the present study, we focused on the non-obese diabetic (NOD) mouse model that develops spontaneous autoimmune diabetes similar to human type 1 diabetes.2. To test the hypothesis that immune responses are involved in the development of diabetic nephropathy.3. To investigate cellular and humoral immune parameters in spontaneous diabetic NOD mice.4. To investigate the role of TLR4 in the early stage of diabetic nephropaty.Materials and Methods1. AnimalsFemale NOD/Caj mice and TLR4-/-NOD mice2. Determination of diabetes and diabetic nephropathyNOD mice were observed for diabetes development by weekly screening for glycosuria. Mouse urine was screened for proteinuria by Albustix. Twenty-four hour urine collections were obtained using metabolic cages and quantitation of urine albumin was determined using the Protein Assay Kit from Bio-Rad according to the manufacturer's instructions.3. HistologyMice were perfused with ice-cold PBS and then with buffer containing 10%formalin. Tissues were further fixed in 4%buffered paraformaldehyde for 2 days, embedded in paraffin and processed for sectioning. Extracellular matrix deposition in glomeruli was assessed by periodic acid/Schiff (PAS) staining.4. Immunofluorescent labeling and confocal microscopyPerfused kidney was fixed overnight in periodate-lysine-paraformaldehyde (PLP) fixative, embedded in OCT compound from Tissue-Tek, and snap frozen. Cryosections were rehydrated with PBS followed by blocking with 2%goat serum. The following primary antibodies were used for staining the kidney sections:Kidney sections were examined and photographed using a META510 confocal microscope.3. Electron microscopyThin kidney sections were cut, and then were observed in a Philips CM 10 transmission EM. We performed immunogold labeling to confirm Ig deposition and location of Ig deposition.4. RNA extraction and real-time quantitative PCRTotal RNA was isolated from mouse kidney tissues using TRIzol. Equal amounts of RNA, measured by spectrophotometer and RNA gel, were used for first-strand cDNA synthesis with SuperScriptⅢ-RT kit. cDNA product was then subject to the quantitative PCR (qPCR) amplification. The primers were designed using the Primer Bank and synthesized by Sigma. Real-time quantitative PCR was performed using an iCycler system.5. Dertermination of insulin antibody6. Statistical analysisStatistical analysis was performed using GraphPad Prism software 4.0ResultsWe found that the glomeruli of diabetic NOD mice were infiltrated with T and B cells, as well as CD11c+dendritic cells, which had close contact with CD4+and CD8+T cells in the infiltrates. We also found that Ig deposits in the glomeruli of diabetic NOD mice were accompanied by the presence of complement C3. Moreover, the serum from diabetic mice contained autoantibodies directed towards components of the glomeruli and these antibodies were not present in non-diabetic NOD mice. The immune changes in the kidney occurred together with increasing kidney weight and urinary albumin excretion along with duration of diabetes. We also found Ig depositon and the presence of complement C3 in TLR4-/- mice, which was much less than those in WT mice.ConclusionsWe provide evidence that infiltrating lymphocytes and anti-kidney autoantibodies may be involved in diabetic nephropathy in autoimmune diabetes in the NOD mouse. The deficiency of TLR4 can decrease the deposition of Ig and complement C3, which showed that TLR4 plays a role in the early stage of diabetic nephropathy. Understanding the role that the immune system plays in the pathogenesis of diabetic nephropathy could lead to identification of new strategies and/or additional therapeutic targets for prevention and treatment of diabetic nephropathy. BackgroundsToll-like receptors (TLR) recognise pathogen-associated molecular patterns (PAMPs) to detect the presence of pathogens. In addition to their role in innate immunity, TLR also play a major role in the regulation of inflammation, even under sterile conditions such as injury and wound healing. This involvement has been suggested to depend, at least in part, on the ability of TLR to recognise several endogenous TLR ligands termed damage-associated molecular patterns (DAMPs). The liver not only represents a major target of bacterial PAMPs in many disease states but also upregulates several DAMPs following injury.Toll-like receptor 3 (TLR3) is known to respond to double-stranded RNA from viruses, apoptotic and/or necrotic cells. Dying cells are a rich source of ligands that can activate TLR, such as TLR3. TLR3 expressed in the liver is likely to be a mediator of innate activation and inflammation in the liver. The importance of this function of TLR3 during acute hepatitis has not previously been fully explored.Research ObjectivesWe used the mouse model of concanavalin A (ConA)-induced hepatitis and observed a novel role for TLR3 in hepatocyte damage in the absence of an exogenous viral stimulus. Materials and Methods1. Animals:NOD/Caj mice, C57BL/6 (B6) mice, TLR3-/- B6 mice and TLR3-/- NOD mice.2. Induction of hepatitis:ConA was dissolved in sterile PBS and injected intravenously to mice at a dose of 15mg/kg. Mice were bled 0,8 and 24h following ConA administration. Control mice were injected with PBS. Mice were sacrificed 24h after ConA injection.3. Assessment of liver function:Serum alanine aminotransferase (ALT) was measured using a standard kit.4. Isolation of liver mononuclear cells:Perfused livers were passed through a 200-gauge stainless steel mesh. The cells were resuspended in 30%percoll and 70%percoll was gently placed under the suspension. After gradient centrifugation, liver MNCs were collected from the interface and washed with PBS.5. Flow cytometric analysis:All fluorescence-conjugated antibodies used in this study were purchased from eBioscience unless otherwise specified. After blocking with anti-Fc receptor, surface markers were identified by specific mAbs conjugated with different fluorochromes. To analyze intracellular proteins, cells were first fixed and permeabilized, and then stained with appropriate mAbs using a Cytofix/Cytoperm plus kit. Flow cytometric analysis was performed using Flowjo software.6. Histological examination and immunostaining:Livers were stained with H&E by standard methods. For immunostaining, perfused liver was fixed overnight in periodate-lysine-paraformaldehyde (PLP) fixative. After embedding in OCT compound, the liver tissue was snap frozen. Cryosections were rehydrated with PBS followed by blocking with 2%goat serum. The primary and secondary antibodies were used to stain the liver sections. Liver sections were examined and photographed using a META510 confocal microscope.7. Analysis of apoptosis of hepatocytes:Hepatic cell nuclei positive for DNA strand breaks was determined by TUNEL assay using a fluorescence detection kit.8. Measurement of serum cytokine levels:Serum cytokine levels for IL-6, IFN-y, TNF-a and IL-17 were detected using Luminex technology. IFN-a was measured using an ELISA kit according to the manufacturer's instructions.9. Generation of bone marrow chimeric mice:Bone marrow cells were harvested from WT or TLR3-/- mice by flushing femurs and tibiae with PBS. Chimeric mice were generated by transferring donor BM cells into irradiated recipients.10. Effect of RNA from damaged liver tissue on splenocyte response to ConA stimulation: Splenocytes from WT or TLR3-/- mice were stimulated with ConA in the presence or absence of total cellular RNA isolated from liver tissue damaged by repeated freezing and thawing (DT, damaged tissue).11. Statistical analysis:Data were mostly presented as mean±SEM. Differences between groups were assessed using analysis of variance (ANOVA) followed by Bonferroni post hoc test or student's t-test wherever appropriate. Ap value less than 0.05 was considered statistically significant.ResultsInterestingly, TLR3 expression in liver mononuclear cells and sinus endothelial cells was upregulated after ConA injection and TLR3-/- mice were protected from ConA-induced hepatitis. Moreover, splenocytes from TLR3-/- mice proliferated less to ConA stimulation in the presence of RNA derived from damaged liver tissue compared with wild type (WT) mice. To determine the relative contribution of TLR3 expression by hematopoietic cells or non-hematopoietic to liver damage during ConA-induced hepatitis, we generated bone marrow (BM) chimeric mice. TLR3-/- mice engrafted with WT hematopoietic cells were protected in a similar manner to WT mice reconstituted with TLR3-/- bone marrow, indicating that TLR3 signaling in both non-hematopoietic and hematopoietic cells plays an important role in mediating liver damage.ConclusionsIn summary, our data suggest that TLR3 signaling is necessary for ConA-induced liver damage in vivo and that TLR3 regulates inflammation and the adaptive T cell immune response in the absence of viral infection.