Development and function of Foxp3(+) regulatory T cells.

Regulatory T cells (Tregs) have been recognized as having a major role in maintaining peripheral tolerance and preventing and limiting autoimmune and chronic inflammatory diseases. Tregs derive from the thymus and also develop peripherally. In this review, we discuss recent progress in our understanding of the basic mechanisms involved in Treg development and function in protecting against autoimmunity in the periphery, including thymic selection, peripheral induction and the many mechanisms of Treg suppression. Specifically in kidney disease, Tregs have been shown to play a role in limiting injury and may potentially have a therapeutic role.

DNA vaccine encoding CD40 targeted to dendritic cells in situ prevents the development of Heymann nephritis in rats.

The CD40-CD154 costimulatory pathway has been shown to be critical for both T- and B-cell activation in autoimmune disease. Here, we assessed the effects of blocking this pathway using CD40 DNA vaccine enhanced by dendritic cell targeting on the development of active Heymann nephritis, a rat model of human membranous nephropathy. DNA vaccination delivers plasmid DNA encoding the target antigen, either alone or in combination with enhancing elements, to induce both humoral and cellular immune responses. To determine whether CD40 DNA vaccine targeting the encoded CD40 directly to dendritic cells would improve the efficacy of the vaccination against self-protein CD40, we utilized a plasmid encoding a single-chain Fv antibody specific for the dendritic cell-restricted antigen-uptake receptor DEC205 (scDEC), the target gene CD40, and the adjuvant tetanus sequence p30. This vaccine plasmid was compared to a control plasmid without scDEC. Rats vaccinated with scDEC-CD40 had significantly less proteinuria and renal injury than did rats receiving scControl-CD40 and were protected from developing Heymann nephritis. Thus, CD40 DNA vaccination targeted to dendritic cells limits the development of Heymann nephritis.

IL-2/IL-2Ab complexes induce regulatory T cell expansion and protect against proteinuric CKD.

Regulatory T cells (Tregs) help protect against autoimmune renal injury. The use of agonist antibodies and antibody/cytokine combinations to expand Tregs in vivo may have therapeutic potential for renal disease. Here, we investigated the effects of administering IL-2/IL-2Ab complexes in mice with adriamycin nephropathy, a model of proteinuric kidney disease that resembles human focal segmental glomerulosclerosis. Injecting IL-2/IL-2Ab complexes before or, to a lesser extent, after induction of disease promoted expansion of Tregs. Furthermore, administration of this complex was renoprotective, evidenced by improved renal function, maintenance of body weight, less histologic injury, and reduced inflammation. IL-2/IL-2Ab reduced serum IL-6 and renal expression of IL-6 and IL-17 but enhanced expression of IL-10 and Foxp3 in the spleen. In vitro, the addition of IL-2/IL-2Ab complexes induced rapid STAT-5 phosphorylation in CD4 T cells. In summary, these data suggest that inducing the expansion of Tregs by administering IL-2/IL-2Ab complexes is a possible strategy to treat renal disease.

CD8+ regulatory T cells induced by T cell vaccination protect against autoimmune nephritis.

Autoreactive T cells play a pivotal role in the pathogenesis of autoimmune kidney disease. T cell vaccination (TCV) may limit autoimmune disease and induce CD8+ regulatory T cells (Tregs). We used Heymann nephritis (HN), a rat model of human membranous nephritis, to study the effects of TCV on autoimmune kidney disease. We harvested CD4+ T cells from renal tubular antigen (Fx1A) -immunized rats and activated these cells in vitro to express the MHC Class Ib molecule Qa-1. Vaccination of Lewis rats with these autoreactive Fx1A-induced T cells protected against HN, whereas control-primed T cells did not. Rats that underwent TCV had lower levels of proteinuria and serum creatinine and significantly less glomerulosclerosis, tubular damage, and interstitial infiltrates. Furthermore, these rats expressed less IFN-γ and IL-6 in splenocytes, whereas the numbers of Tregs and the expression of Foxp3 were unchanged. In vitro cytotoxicity assays showed CD8+ T cell-mediated elimination of Qa-1-expressing CD4+ T cells. In vivo, TCV abrogated the increase in Qa-1-expressing CXCR5+ TFH cells observed in HN compared with controls. Taken together, these results suggest that TCV protects against autoimmune kidney disease by targeting Qa-1-expressing autoreactive CD4+ cells.

DEC205-DC targeted DNA vaccine against CX3CR1 protects against atherogenesis in mice.

Studies disrupting the chemokine pathway CX3CL1 (fractalkine)/ CX3CR1 have shown decreased atherosclerosis in animal models but the techniques used to interrupt the pathway have not been easily translatable into human trials. DNA vaccination potentially overcomes the translational difficulties. We evaluated the effect of a DNA vaccine, targeted to CX3CR1, on atherosclerosis in a murine model and examined possible mechanisms of action. DNA vaccination against CX3CR1, enhanced by dendritic cell targeting using DEC-205 single chain variable region fragment (scFv), was performed in 8 week old ApoE-/- mice, fed a normal chow diet. High levels of anti-CX3CR1 antibodies were induced in vaccinated mice. There were no apparent adverse reactions to the vaccine. Arterial vessels of 34 week old mice were examined histologically for atherosclerotic plaque size, macrophage infiltration, smooth muscle cell infiltration and lipid deposition. Vaccinated mice had significantly reduced atherosclerotic plaque in the brachiocephalic artery. There was less macrophage infiltration but no significant change to the macrophage phenotype in the plaques. There was less lipid deposition in the lesions, but there was no effect on smooth muscle cell migration. Targeted DNA vaccination to CX3CR1 was well tolerated, induced a strong immune response and resulted in attenuated atherosclerotic lesions with reduced macrophage infiltration. DNA vaccination against chemokine pathways potentially offers a potential therapeutic option for the treatment of atherosclerosis.

The roles of inflammatory cytokines in the pathogenesis of ossification of ligamentum flavum.

Ossification of Ligamentum Flavum (OLF) is associated with serious neurologic symptoms including thoracic myelopathy and spinal stenosis. The pathogenesis of thoracic OLF is mainly due to the localized mechanical stress on the ligament induced enchondral ossification. However, despite numerous epidemiological and basic science studies, the mechanism of this process remains unclear. Studies have suggested that inflammatory cytokines, such as IL-6, TNF-α, seem to play a crucial role in OLF. In this review, we summarise the mechanistic information on the roles of inflammation cytokines in OLF and discuss about several therapeutic methods for OLF. Further studies on the role of cytokines in OLF should provide important insights into the designation of therapeutic strategies in preventing human spinal stenosis caused by OLF.

Daedalic DNA vaccination against self antigens as a treatment for chronic kidney disease.

Chronic kidney disease (CKD) is a major cause of death and morbidity in Australia and worldwide. DNA vaccination has been used for targeting foreign antigens to induce immune responses and prevent autoimmune disease, viral infection and cancer. However, the use of DNA vaccination has been restricted by a limited ability to induce strong immune responses, especially against self-antigens which are limited by mechanisms of self-tolerance. Furthermore, there have been few studies on the potential of DNA vaccination in chronic inflammatory diseases, including CKD. We have established strategies of DNA vaccination targeting specific self-antigens in the immune system including co-stimulatory pathways, T cell receptors and chemokine molecules, which have been effective in protecting against the development of CKD in a variety of animal models. In particular, we find that the efficacy of DNA vaccination is improved by dendritic cell (DC) targeting and can protect against animal models of autoimmune nephritis mimicking human membranous nephropathy. In this review, we summarize several approaches that have been tested to improve the efficacy of DNA vaccination in CKD models, including enhanced DNA vaccine delivery methods, DNA vaccine modifications and new molecular targets for DNA vaccination. Finally, we discuss the specific application of DNA vaccination for preventing and treating CKD.

DEC205-DC targeted DNA vaccines to CX3CR1 and CCL2 are potent and limit macrophage migration.

Monocytes utilise a variety of chemokines to traffic to atherosclerotic plaques. CX3C chemokine ligand 1 (CX3CL1 & Fractalkine) and its receptor CX3CR1 and monocyte chemoattractant protein 1 (CCL2) have been identified as chemokines/receptors that have an important role in the migration and recruitment of monocytes during the pathogenesis of several inflammatory diseases including atherosclerosis. DNA vectors containing single chain variable region fragment (scFv) for DC-targeted receptor DEC205 were cloned with mouse CX3CR1 and CCL2 genes respectively, and vaccinated into C57/BL6 mice weekly for 3 weeks. Induced anti-CX3CR1 and anti-CCL2 in vaccinated mice was examined by ELISA and Western Blot analysis, while the cellular response was examined by ELISPOT. The inhibition of chemotaxis of J774 macrophages to Py-4-1 endothelial cells was examined by in vitro transwell migration assay using serum collected from vaccinated mice. All vaccinated mice generated anti-CX3CR1 and anti-CCL2 Ab and cellular response by 8 weeks after DNA vaccination. Macrophage migration towards TNF-α activated endothelial cells was significantly inhibited by serum containing both anti-CX3CR1 or anti-CCL2 Ab from vaccinated mice. These results demonstrate that DC-targeting of DNA vaccines to self-antigens generates functional immune responses which can inhibit specific key chemotactic targets. This suggests a potential therapeutic role for chemokine/receptor DNA vaccination in atherosclerosis, where chemotaxis has a pivotal role in the inflammatory process.