Digoxin Inhibits Induction of Experimental Autoimmune Uveitis in Mice, but Causes Severe Retinal Degeneration.

PURPOSE: Digoxin, a major medication for heart disease, was recently reported to have immunosuppressive capacity. Here, we determined the immunosuppressive capacity of digoxin on the development of experimental autoimmune uveitis (EAU) and on related immune responses. METHODS: The B10.A mice were immunized with interphotoreceptor retinoid-binding protein (IRBP) and were treated daily with digoxin or vehicle control. On postimmunization day 14, the mouse eyes were examined histologically, while spleen cells were tested for cytokine production in response to IRBP and purified protein derivative. The immunosuppressive activity of digoxin was also tested in vitro, by its capacity to inhibit development of Th1 or Th17 cells. To investigate the degenerative effect of digoxin on the retina, naïve (FVB/N × B10.BR)F1 mice were similarly treated with digoxin and tested histologically and by ERG. RESULTS: Treatment with digoxin inhibited the development of EAU, as well as the cellular response to IRBP. Unexpectedly, treatment with digoxin suppressed the production of interferon-γ to a larger extent than the production of interleukin 17. Importantly, digoxin treatment induced severe retinal degeneration, determined by histologic analysis with thinning across all layers of the retina. Digoxin treatment also induced dose-dependent vision loss monitored by ERG on naïve mice without induction of EAU. CONCLUSIONS: Treatment of mice with digoxin inhibited the development of EAU and cellular immune response to IRBP. However, the treatment induced severe damage to the retina. Thus, the use of digoxin in humans should be avoided due to its toxicity to the retina.

Leucine-Rich Repeat Kinase 2 (Lrrk2) Deficiency Diminishes the Development of Experimental Autoimmune Uveitis (EAU) and the Adaptive Immune Response.

BACKGROUND: Mutations in LRRK2 are related to certain forms of Parkinson's disease and, possibly, to the pathogenesis of Crohn's disease. In both these diseases inflammatory processes participate in the pathogenic process. LRRK2 is expressed in lymphoid cells and, interestingly, Lrrk2 (-/-) mice were reported to develop more severe experimental colitis than their wild type (WT) controls. Here, we examined the possible involvement of LRRK2 in the pathogenesis of experimental autoimmune uveitis (EAU), an animal model for human uveitis, by testing Lrrk2 (-/-) mice for their capacity to develop this experimental eye disease and related immune responses. METHODS: Lrrk2 (-/-) mice and their WT controls (C57Bl/6) were immunized with interphotoreceptor retinoid-binding protein (IRBP) and compared for their development of EAU, delayed type hypersensitivity (DTH) by skin tests, production of cytokines in culture, and expression of interferon (IFN)-γ, interleukin (IL)-17 and FoxP3 by spleen cells, using flow cytometry. Peritoneal macrophages were examined for their production of cytokines/chemokines in culture following stimulation with LPS or the oligodeoxynucleotide CpG. The Lrrk2 (-/-) and WT mice were also compared for their response to bovine serum albumin (BSA). RESULTS: The Lrrk2 (-/-) mice developed lower levels of EAU, DTH responses and cytokine production by lymphocytes than did their WT controls. Intracellular expression of IFN-γ and IL-17, by spleen cells, and secretion of cytokines/chemokines by activated peritoneal macrophages of Lrrk2 (-/-) mice trended toward diminished levels, although variabilities were noted. The expression levels of FoxP3 by Lrrk2 (-/-) spleen cells, however, were similar to those seen in WT controls. Consistent with their low response to IRBP, Lrrk2 (-/-) mice responded to BSA less vigorously than their WT controls. CONCLUSIONS: Lrrk2 deficiency in mice diminished the development of EAU and the related adaptive immune responses to IRBP as compared to the WT controls.

Inflammasomes Induced by 7-Ketocholesterol and Other Stimuli in RPE and in Bone Marrow-Derived Cells Differ Markedly in Their Production of IL-1ß and IL-18.

PURPOSE: The inflammatory process plays a major role in the pathogenesis of AMD, and recent data indicate the involvement of inflammasomes. Inflammasomes are intracellular structures that trigger inflammation by producing mature interleukin-(IL)-1ß and IL-18. This study examined the capacity of 7-ketocholesterol (7KCh), an oxysterol that accumulates in the retinal pigmented epithelium (RPE) and choroid, to initiate inflammasome formation in RPE and bone marrow-derived cells. METHODS: Tested cells included fetal human RPE (fhRPE), human ARPE-19 cells, primary human brain microglia cells, and human THP-1 monocyte cells. 7-Ketocholesterol and other compounds were added to the cell cultures, and their stimulatory effects were determined by quantitative PCR and release of cytokines, measured by ELISA and Western blotting. RESULTS: 7-Ketocholesterol efficiently induced inflammasome formation by all primed cell populations, but secreted cytokine levels were higher in cultures of bone marrow-derived cells (microglia and THP-1 cells) than in RPE cultures. Interestingly, inflammasomes formed in cells of the two populations differed strikingly in their preferential production of the two cytokines. Thus, whereas bone marrow-derived cells produced levels of IL-1ß that were higher than those of IL-18, the opposite was found with RPE cells, which secreted higher levels of IL-18. Importantly, Western blot analysis showed that IL-18, but not IL-1ß, was expressed constitutively by RPE cells. CONCLUSIONS: 7-Ketocholesterol efficiently stimulates inflammasome formation and is conceivably involved in the pathogenesis of AMD. In contrast to bone marrow-derived cells, RPE cells produced higher levels of IL-18 than IL-1ß. Further, IL-18, a multifunctional cytokine, was expressed constitutively by RPE cells. These observations provide new information about stimuli and cells and their products assumed to be involved in the pathogenesis of AMD.

Induced regulatory T-cells (iTregs) generated by activation with anti-CD3/CD28 antibodies differ from those generated by the physiological-like activation with antigen/APC.

Regulatory T-cells (Tregs) are responsible for homeostasis of the immune system, as well as for inhibition of pathogenic autoimmune processes. Induced-(i)-Tregs, can be generated in vitro by activation of CD4 cells in the presence of TGF-ß. A commonly used activation mechanism is by antibodies against CD3 and CD28. The physiological-like activation of T-cells, however, is with the specific target antigen presented by antigen-presenting cells (APC). The two modes of activation have been considered to yield the same populations of iTregs. Here, we compared between iTreg populations generated by either one of the two methods and found differences between their capacities to inhibit T-lymphocyte proliferative response, their expression of cell surface antigens and particularly, in their transcript expression profiles of certain chemokines and chemokine receptors. Our data thus indicate that iTregs generated by activation with anti-CD3/CD28 antibodies cannot be considered identical to iTregs generated by antigen/APC.

ITE, a novel endogenous nontoxic aryl hydrocarbon receptor ligand, efficiently suppresses EAU and T-cell-mediated immunity.

PURPOSE: Ligands for aryl hydrocarbon receptor (AHR), such as dioxins, are highly toxic. One such ligand, TCDD, was found to exert potent immunosuppressive capacities in mice developing pathogenic autoimmune processes, including EAU, but its toxicity makes it unusable for humans. A recently identified endogenous AHR ligand, ITE, is also immunosuppressive, but is nontoxic and could therefore be useful for therapy in humans. Here, we tested ITE for its capacity to inhibit EAU and related immune responses. METHODS: EAU was induced in B10.A mice by immunization with interphotoreceptor retinoid-binding protein (IRBP; 40 µg) in CFA. Treatment with ITE was by daily intraperitoneal injection of 0.2 mg. Disease severity was assessed by both fundoscopy and histological examination. Draining lymph node cells were tested for proliferation by thymidine uptake and for cytokine production and release by ELISA. In addition, the intracellular expression of cytokines and Foxp3 was determined by flow cytometry. Serum antibodies were measured by ELISA. RESULTS: Treatment with ITE efficiently inhibited the development of EAU in mice, as well as the cellular immune responses against IRBP and PPD. ITE treatment inhibited the expansion of both Th1 and Th17 subpopulations, as well as their release of the signature cytokines, IFN-gamma and IL-17. The treatment moderately increased, however, the proportion of Foxp3 expressing T-regulatory cells. Antibody production was not affected by the treatment. CONCLUSIONS: ITE, an endogenous AHR ligand, efficiently inhibits EAU development and related cellular immune responses. Being nontoxic, ITE may be considered for treatment of pathogenic immunity in humans.