Yogesh Scindia

Activation of innate immune responses through Toll-like receptor 3 causes a rapid loss of salivary gland function

BACKGROUND Recent studies have demonstrated the expression of Toll-like receptor 3 (TLR3) in salivary glands and epithelial cell lines derived from Sjögrens syndrome (SS) patients. As viral infections are considered to be a trigger for SS, in this study we investigated whether in vivo engagement of TLR3 affects salivary gland function. METHODS Female New Zealand Black/WF1 mice were repeatedly injected with polyinosinic:polycytidylic acid [poly(I:C)]. TLR3 expression within submandibular glands was studied using immunohistochemistry. RNA levels of inflammatory cytokines in the submandibular glands were determined by real time polymerase chain reaction. Pilocarpine induced saliva volume was used as an index of glandular function. RESULTS Immunohistochemical analysis of submandibular glands showed TLR3 expression in epithelium of serous and mucous acini, granular convoluted tubules, and ducts. Poly(I:C) treatment rapidly up-regulated the mRNA levels of type I interferon (IFN) and inflammatory cytokines in the submandibular glands. One week after treatment, the saliva volumes in poly(I:C) treated mice were significantly reduced in comparison with the phosphate-buffered saline (PBS) treated mice. Hematoxylin and eosin staining showed that salivary gland histology was normal and lymphocytic foci were not detected. Glandular function recovered after poly(I:C) treatment was stopped. CONCLUSIONS Our results demonstrate that engagement of TLR3 within the salivary glands results in a rapid loss of glandular function. This phenomenon is associated with the production of type I IFN and inflammatory cytokines in the salivary glands. Restoration of glandular function suggests that for viral etiology of SS, a chronic infection of salivary glands might be necessary.

Mesangial pathology in glomerular disease: targets for therapeutic intervention

The glomerulus is the filtration unit of the kidney. Disruption of glomerular function may be caused by primary glomerular pathology or secondary to systemic diseases. The mesangial, endothelial and epithelial cells of the glomerulus are involved in most pathologic processes. Animal models provide an understanding of the molecular basis of glomerular disease. These studies show that mesangial cells are critical players in the initiation and progression of disease. Therefore, modulation of mesangial cell responses offers a novel therapeutic approach. The complex architecture of the kidney, specifically the renal glomerulus, makes targeted drug delivery especially challenging. Targeted delivery of therapeutic agents reduces dose of administration and minimises unwanted side effects caused by toxicity to other tissues. The currently available modalities demonstrating the feasibility of mesangial cell targeting are discussed.

Anti–α8 integrin immunoliposomes in glomeruli of lupus‐susceptible mice: A novel system for delivery of therapeutic agents to the renal glomerulus in systemic lupus erythematosus

OBJECTIVE Glomerular mesangial cells are active participants in the pathogenesis of lupus glomerulonephritis (GN). Thus, targeted delivery of therapeutic agents to mesangial cells would be an attractive approach to treatment. However, lack of known unique mesangial cell surface markers has hampered this process. This study was undertaken in a mouse model of lupus GN to identify mesangial markers and to develop a system for targeted drug delivery to the glomerulus. METHODS Based on previous observations, alpha8 integrin expressed on the surface of glomerular mesangial cells was selected as a target molecule for delivery. Two mouse strains susceptible to lupus GN, NZM2328 and (NZM2328 x NOD)F1, were studied. Glomerular expression of alpha8 integrin in normal and nephritic mice was confirmed by immunofluorescence and quantitative polymerase chain reaction analysis. Liposomes were formulated and conjugated with an anti-alpha8 integrin antibody. These immunoliposomes were loaded with DiI, a red fluorescent dye, to allow tracking in vivo, and injected into the tail vein of female mice at different ages. Specificity of targeting was studied by fluorescence microscopy and flow cytometry. RESULTS Expression of alpha8 integrin was observed in the glomeruli of normal and nephritic mice. Anti-alpha8 integrin immunoliposomes were detected in the glomerulus and glomerular mesangial cells after tail vein injection in normal and nephritic mice. Delivery of DiI by anti-alpha8 integrin immunoliposomes was tissue specific, being observed predominantly in the glomeruli, with some nonspecific uptake by CD11b cells. CONCLUSION These findings are the first demonstration of specific delivery of anti-alpha8 integrin immunoliposomes to the mesangium following tail vein injection in mice. Anti-alpha8 integrin immunoliposomes thus offer a novel approach for targeted drug therapy in lupus and other glomerular diseases.

Activation of innate immunity accelerates sialoadenitis in a mouse model for Sjögren’s syndrome-like disease

OBJECTIVE Sjögrens syndrome is a chronic autoimmune disorder characterized by progressive lymphocytic infiltration within the salivary and lacrimal glands. This study was undertaken to investigate the effects of innate immunity activation on sialoadenitis in a mouse strain genetically susceptible for development of SS-like disease. METHODS Female New Zealand Black X New Zealand White F1 mice were repeatedly treated with toll-like 3 receptor agonist poly(I:C). Submandibular glands were investigated at different time points for sialoadenitis by immunohistochemistry and for gene expression of different chemokines by quantitative PCR. Submandibular gland-infiltrating cells were characterized by flow cytometry. RESULTS Poly(I:C) treatment significantly upregulated the expression of multiple chemokines within the submandibular glands. The severity and incidence of sialoadenitis was considerably higher in poly(I:C)-treated mice. There was a preponderance of dendritic cells and NK cells in the initial inflammatory cell infiltrates, and these were followed by CD4+ T cells. CONCLUSIONS Our data clearly demonstrate that systemic activation of innate immunity accelerates sialoadenitis in a mouse model for SS-like disease. These findings suggest that chronic activation of innate immunity can influence certain features of SS.

Hepcidin Mitigates Renal Ischemia-Reperfusion Injury by Modulating Systemic Iron Homeostasis

Iron-mediated oxidative stress is implicated in the pathogenesis of renal ischemia-reperfusion injury. Hepcidin is an endogenous acute phase hepatic hormone that prevents iron export from cells by inducing degradation of the only known iron export protein, ferroportin. In this study, we used a mouse model to investigate the effect of renal ischemia-reperfusion injury on systemic iron homeostasis and determine if dynamic modulation of iron homeostasis with hepcidin has therapeutic benefit in the treatment of AKI. Renal ischemia-reperfusion injury induced hepatosplenic iron export through increased ferroportin expression, which resulted in hepatosplenic iron depletion and an increase in serum and kidney nonheme iron levels. Exogenous hepcidin treatment prevented renal ischemia-reperfusion-induced changes in iron homeostasis. Hepcidin also decreased kidney ferroportin expression and increased the expression of cytoprotective H-ferritin. Hepcidin-induced restoration of iron homeostasis was accompanied by a significant reduction in ischemia-reperfusion-induced tubular injury, apoptosis, renal oxidative stress, and inflammatory cell infiltration. Hepcidin -: deficient mice demonstrated increased susceptibility to ischemia-reperfusion injury compared with wild-type mice. Reconstituting hepcidin-deficient mice with exogenous hepcidin induced hepatic iron sequestration, attenuated the reduction in renal H-ferritin and reduced renal oxidative stress, apoptosis, inflammation, and tubular injury. Hepcidin-mediated protection was associated with reduced serum IL-6 levels. In summary, renal ischemia-reperfusion injury results in profound alterations in systemic iron homeostasis. Hepcidin treatment restores iron homeostasis and reduces inflammation to mediate protection in renal ischemia-reperfusion injury, suggesting that hepcidin-ferroportin pathway holds promise as a novel therapeutic target in the treatment of AKI.

Deficiency of a Transcriptional Regulator, Inhibitor of Differentiation 3, Induces Glomerulonephritis in Apolipoprotein E-Deficient Mice A Model Linking Hyperlipidemia and Renal Disease

The clinical association between hyperlipidemia and renal disease is well established, yet hyperlipidemia as a cause for renal disease is rare. Apolipoprotein E-deficient (ApoE(-/-)) mice develop hyperlipidemia and are a model for atherosclerosis. Introducing deficiency of inhibitor of differentiation 3 (Id3) in ApoE(-/-) mice further exacerbates atherosclerosis. ID3 is a transcription regulator expressed in multiple cell types. Id3(-/-) mice develop antibodies to self-antigens and salivary gland autoimmunity. This study was undertaken to investigate a link between hyperlipidemia, autoimmunity, and renal disease. ApoE(-/-), Id3(-/-), and ApoE(-/-)Id3(-/-) double-knockout (DKO) mice were studied at different ages for renal pathological features and function. Serum samples were analyzed for the presence of autoantibodies. At 16 weeks, DKO mice developed mesangioproliferative glomerulonephritis (GN), leading to severe proteinuria. GN was associated with glomerular deposition of lipids and immune complexes and with macrophage infiltration. DKO mice had high levels of circulating autoantibodies. Although ApoE(-/-) mice had glomerular lipid deposits and Id3(-/-) mice had circulating autoantibodies, neither group of age-matched single-knockout mice developed GN. These data provide support for the hypothesis that induction of renal disease in hyperlipidemia is dictated by additional factors. Our study shows that some of these factors are regulated by ID3. Thus, ID3 is a novel risk factor linking cardiovascular and renal disease.

Novel therapeutic approaches to lupus glomerulonephritis: translating animal models to clinical practice.

Systemic lupus erythematosus is a chronic autoimmune disease frequently affecting the kidney. Renal involvement is characterized by glomerular immune complex deposits and proliferative glomerulonephritis progressing to glomerulosclerosis and kidney failure. The development of systemic lupus erythematosus is regulated genetically, and lupus susceptibility genes have been linked to immune hyper-responsiveness and loss of immune regulation. In addition to the systemic immune defects, recent studies in animal models show that susceptibility to lupus nephritis is influenced by intrinsic renal factors. Thus, renal cell responses to immune-mediated glomerular injury determine disease outcome. This supports the idea that future treatments for lupus nephritis need to focus on regulating end-organ responses. The feasibility of this approach has been shown in animal models of kidney disease. For more than 50 years, the emphasis in management of lupus nephritis has been suppression of autoimmune responses and systemic control of inflammation. This review describes recently developed targeted drug delivery technologies and potential targets that can regulate glomerular cell responses, offering a novel therapeutic approach for lupus nephritis.

Activated CD4+ T Cells Target Mesangial Antigens and Initiate Glomerulonephritis

Aims: The role of kidney infiltrating T cells in the pathology of lupus nephritis is unclear. This study was undertaken to investigate whether CD4+ T cell responses to a surrogate mesangial antigen can initiate glomerulonephritis. Methods: Ovalbumin (OVA) was deposited in the glomerular mesangium of C57BL/6 (B6) mice using anti-α8-integrin immunoliposomes (α8ILs). This was followed by injection of activated OVA-reactive CD4+ transgenic OT2 T cells. Trafficking of antigen-specific OT2 T cells to kidneys and lymph nodes was studied by flow cytometry. Glomerular pathology and immune cell infiltration was characterized by immunostaining. Role of CCR2 deficiency on T cell-mediated glomerulonephritis was investigated using B6.ccr2–/– mice. Results: α8ILs delivered OVA specifically to the renal glomeruli. Adoptively transferred OT2 T cells preferentially accumulated in renal lymph nodes and in the renal cortex. Kidneys showed glomerular inflammation with recruitment of endogenous T cells, dendritic cells and macrophages. T cell-mediated inflammation induced mesangial cell activation and an increase in glomerular MCP1 and fibronectin. The formation of inflammatory foci was driven by Ly6C monocytes and was CCR2 dependent. Conclusions: The findings from this study show that T cells reactive with antigens in the mesangium are sufficient to initiate glomerular pathology. Antigen-specific CD4 T cells act by inducing glomerular MCP1 production which mediates recruitment of inflammatory monocytes resulting in glomerulonephritis. Thus, downmodulation of T cell responses within the kidneys of lupus patients will be a beneficial therapeutic approach.

Biocompatibility of ferritin-based nanoparticles as targeted MRI contrast agents

Ferritin is a naturally occurring iron storage protein, proposed as a clinically relevant nanoparticle with applications as a diagnostic and therapeutic agent. Cationic ferritin is a targeted, injectable contrast agent to measure kidney microstructure with MRI. Here, the toxicity of horse spleen ferritin is assessed as a step to clinical translation. Adult male mice received cationic, native and high dose cationic ferritin (CF, NF, or HDCF) or saline and were monitored for 3weeks. Transient weight loss occurred in the ferritin groups with no difference in renal function parameters. Ferritin-injected mice demonstrated a lower serum iron 3weeks after administration. In ferritin-injected animals pre-treated with hydrocortisone, there were no structural or weight differences in the kidneys, liver, lung, heart, or spleen. This study demonstrates a lack of significant detrimental effects of horse-derived ferritin-based nanoparticles at MRI-detectable doses, allowing further exploration of these agents in basic research and clinical diagnostics.

Genetic Complementation Results in Augmented Autoantibody Responses to Lupus-Associated Antigens

Lupus-prone female New Zealand Mixed (NZM)2328 mice develop high titers of anti-nuclear and anti-dsDNA autoantibodies. Despite high expression of type I IFNs, these mice do not develop autoantibodies to the small nuclear ribonucleoprotein (snRNP) complex. Thus, additional genetic factors must regulate the generation of anti-snRNP autoantibodies. In contrast, despite much lower expression of type 1 IFNs, the diabetes-prone NOD mice spontaneously make anti-snRNP autoantibodies, albeit at a low incidence. To determine whether combination of high type I IFN response of NZM mice with appropriate susceptibility genes of NOD mice would result in anti-snRNP Ab response, cohorts of (NZM2328 × NOD)F1 mice were generated and characterized for development of autoimmunity. In comparison with parental strains, the PBMCs from F1 mice showed intermediate expression of type I IFN-responsive genes and augmented expression of IL-6 transcripts. TLR7 expression was similar in all strains. The F1 mice had very high incidence and titer of anti-snRNP autoantibodies, anti-nuclear Abs, and anti-dsDNA autoantibodies. The levels of anti-snRNP autoantibody correlated with the expression levels of type I IFN-responsive genes. None of the F1 mice developed diabetes, and only female mice developed severe renal disease. Our data demonstrate that only in presence of appropriate susceptibility genes, anti-snRNP autoantibodies are induced and type I IFNs amplify this response. A synergy between IL-6 and type I IFNs might be critical for amplifying overall autoantibody responses in systemic lupus erythematosus. In NZM/NOD F1 mouse, genetic complementation between NZM and NOD genes leads to expression of phenotypes similar to those seen in certain lupus patients.