CD73 Blockade of Extracellular ATP Effect by Oxidized ATP Effectively Mitigated Induced Mouse Experimental Autoimmune Uveitis (EAU).

Various pathological conditions are accompanied by ATP release from the intracellular to the extracellular compartment. Extracellular ATP (eATP) functions as a signaling molecule by activating purinergic P2 purine receptors. The key P2 receptor involved in inflammation was identified as P2X7R. Recent studies have shown that P2X7R signaling is required to trigger the Th1/Th17 immune response, and oxidized ATP (oxATP) effectively blocks P2X7R activation. In this study we investigated the effect of oxATP on mouse experimental autoimmune uveitis (EAU). Our results demonstrated that induced EAU in B6 mice was almost completely abolished by the administration of small doses of oxATP, and the Th17 response, but not the Th1 response, was significantly weakened in the treated mice. Mechanistic studies showed that the therapeutic effects involve the functional change of a number of immune cells, including dendritic cells (DCs), T cells, and regulatory T cells. OxATP not only directly inhibits the T cell response; it also suppresses T cell activation by altering the function of DCs and Foxp3+ T cell. Our results demonstrated that inhibition of P2X7R activation effectively exempts excessive autoimmune inflammation, which may indicate a possible therapeutic use in the treatment of autoimmune diseases

CD73 Blockade of Extracellular ATP Effect by Oxidized ATP Effectively Mitigated Induced Mouse Experimental Autoimmune Uveitis (EAU).

Various pathological conditions are accompanied by ATP release from the intracellular to the extracellular compartment. Extracellular ATP (eATP) functions as a signaling molecule by activating purinergic P2 purine receptors. The key P2 receptor involved in inflammation was identified as P2X7R. Recent studies have shown that P2X7R signaling is required to trigger the Th1/Th17 immune response, and oxidized ATP (oxATP) effectively blocks P2X7R activation. In this study we investigated the effect of oxATP on mouse experimental autoimmune uveitis (EAU). Our results demonstrated that induced EAU in B6 mice was almost completely abolished by the administration of small doses of oxATP, and the Th17 response, but not the Th1 response, was significantly weakened in the treated mice. Mechanistic studies showed that the therapeutic effects involve the functional change of a number of immune cells, including dendritic cells (DCs), T cells, and regulatory T cells. OxATP not only directly inhibits the T cell response; it also suppresses T cell activation by altering the function of DCs and Foxp3+ T cell. Our results demonstrated that inhibition of P2X7R activation effectively exempts excessive autoimmune inflammation, which may indicate a possible therapeutic use in the treatment of autoimmune diseases

Effects of phosphorylatable short peptide-conjugated chitosan-mediated IL-1Ra and igf-1 gene transfer on articular cartilage defects in rabbits.

Previously, we reported an improvement in the transfection efficiency of the plasmid DNA-chitosan (pDNA/CS) complex by the utilization of phosphorylatable short peptide-conjugated chitosan (pSP-CS). In this study, we investigated the effects of pSP-CS-mediated gene transfection of interleukin-1 receptor antagonist protein (IL-1Ra) combined with insulin-like growth factor-1 (IGF-1) in rabbit chondrocytes and in a rabbit model of cartilage defects. pBudCE4.1-IL-1Ra+igf-1, pBudCE4.1-IL-1Ra and pBudCE4.1-igf-1 were constructed and combined with pSP-CS to form pDNA/pSP-CS complexes. These complexes were transfected into rabbit primary chondrocytes or injected into the joint cavity. Seven weeks after treatment, all rabbits were sacrificed and analyzed. High levels of IL-1Ra and igf-1 expression were detected both in the cell culture supernatant and in the synovial fluid. In vitro, the transgenic complexes caused significant proliferation of chondrocytes, promotion of glycosaminoglycan (GAG) and collagen II synthesis, and inhibition of chondrocyte apoptosis and nitric oxide (NO) synthesis. In vivo, the exogenous genes resulted in increased collagen II synthesis and reduced NO and GAG concentrations in the synovial fluid; histological studies revealed that pDNA/pSP-CS treatment resulted in varying degrees of hyaline-like cartilage repair and Mankin score decrease. The co-expression of both genes produced greater effects than each single gene alone both in vitro and in vivo. The results suggest that pSP-CS is a good candidate for use in gene therapy for the treatment of cartilage defects and that igf-1 and IL-1Ra co-expression produces promising biologic effects on cartilage defects

Administration of oxATP mitigated the autoreactive T cell response in the recipient B6 mice.

<p>A) Splenic T cells were isolated 13 days post-immunization from immunized B6 mice (n = 6) with (right panels) or without (left panels) oxATP administration. Cells obtained from all six mice in the same group were pooled, before the T cells are further enriched, stimulated with Ag/APCs, and subjected for phenotypic and functional analysis. The percentage of IL-17⁺ and IFN-γ⁺ cells among the proliferating T cells was assessed after 5-day in vitro stimulation under Th1 (lower panel) and Th17 (Upper panel) polarizing conditions by intracellular staining with PE-conjugated anti-αβTCR Abs and FITC-conjugated anti-IL-17 Abs (upper panels) or APC-conjugated anti-αβTCR and PE-conjugated anti-IFN-γ Abs (lower panels), followed by FACS analysis. B) IL-17and IFN-γ levels in the supernatant of in vitro cultured T cells after exposure to immunizing Ag and APCs for 48 h. **<i>P</i> < 0.01, ns, not significant. C) oxATP neutralized the enhancing effect of ATP on Th17, but not Th1, response. The responder T cells were prepared from immunized B6 mice, which were stimulated in vitro with the immunizing peptide and APCs, under polarizing T activation conditions, as described in Fig 2A, with or without a prior exposure to ATP (100 μM) and/or oxATP (80 μM). The proliferating T cells were assessed after 5-day in vitro stimulation by intracellular staining. D&E) IRBP-specific T cells isolated from oxATP-treated mice were poorly uveitogenic. After 2-day in vitro stimulation of αβ T cells with the immunizing peptides and APCs, 2 x10⁶ cells were adoptively transferred to naïve B6 via i.p. injection and EAU was scored by fundoscopy as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155953#pone.0155953.g001" target="_blank">Fig 1</a>. Data are from one single experiment, representative of three independent experiments.</p

Blockade of Extracellular ATP Effect by Oxidized ATP Effectively Mitigated Induced Mouse Experimental Autoimmune Uveitis (EAU)

<div><p>Various pathological conditions are accompanied by ATP release from the intracellular to the extracellular compartment. Extracellular ATP (eATP) functions as a signaling molecule by activating purinergic P2 purine receptors. The key P2 receptor involved in inflammation was identified as P2X7R. Recent studies have shown that P2X7R signaling is required to trigger the Th1/Th17 immune response, and oxidized ATP (oxATP) effectively blocks P2X7R activation. In this study we investigated the effect of oxATP on mouse experimental autoimmune uveitis (EAU). Our results demonstrated that induced EAU in B6 mice was almost completely abolished by the administration of small doses of oxATP, and the Th17 response, but not the Th1 response, was significantly weakened in the treated mice. Mechanistic studies showed that the therapeutic effects involve the functional change of a number of immune cells, including dendritic cells (DCs), T cells, and regulatory T cells. OxATP not only directly inhibits the T cell response; it also suppresses T cell activation by altering the function of DCs and Foxp3⁺ T cell. Our results demonstrated that inhibition of P2X7R activation effectively exempts excessive autoimmune inflammation, which may indicate a possible therapeutic use in the treatment of autoimmune diseases.</p></div

Inhibition of oxATP on Foxp3⁺ T cells.

<p>A & B) B6 mice were immunized with IRBP_1-20/CFA with or without administration of oxATP (300 ug/mouse). Thirteen days post-immunization and immediately after separation of αβ T cells. Foxp3⁺ T cells were identified after a dual staining with anti-mouse αβTCR and anti-mouse-Foxp3 antibodies, followed by FACS analysis. C) oxATP has an inhibitory effect on Foxp3⁺ T cell. αβ T cells isolated from immunized mice were cultured for 5 days in the absence or presence of oxATP (80 μM) and proportional numbers of Foxp3⁺ T cells were compared among αβ T cells. D) Foxp3⁺ T cells were isolated from immunized B6 mice. CD3⁺ splenic T cells were cultured in vitro in medium containing very low dose (0.5 ng/ml) of IL-2. Five days later, 89% of the CD25⁺ cells separated using MACS column showed Foxp3⁺CD4⁺. E and F) Foxp3⁺ have a stronger inhibitory effect on Th1 response than on Th17 response. Responder T cells isolated from immunized B6 mice were stimulated with splenic APCs in the presence of the immunizing peptide, with or without adding 10% of Foxp3⁺ T cells. Five days after stimulation, IL-17⁺ (under Th17 polarized conditions) and IFN-γ⁺ (under Th1 polarized conditions) cells among the responders were determined by intracellular staining followed by FACS analysis (6E). IFN-γ and IL-17 amounts in the culture medium were measured by ELISA 48 h after in vitro stimulation (6F). **<i>P</i> < 0.01; ns, not significant.</p

Splenic DCs of the oxATP recipients have decreased ability to produce IL-23 and IL-6 and were poor stimulators for Th17 autoreactive T cells.

<p>A) Splenic CD11c⁺ DCs were isolated from oxATP-treated (300 μg/mouse) and untreated immunized B6 mice 13 days post immunization, using MACS column. They were exposed to LPS (100 ng/ml) for 48h. Cytokines in the cultured cell supernatants were examined by ELISA. **<i>P</i> < 0.01, ns, not significant. B) In a 24-well plate, splenic APCs from oxATP-treated and untreated mice were compared for their effectiveness at stimulating Th1 and Th17 responses, by incubating with responder T cells isolated from immunized B6 mice, in the presence of the immunizing peptide (IRBP1-20). Five days after stimulation, the percentage of IL-17⁺ (under Th17 polarized conditions) and IFN-γ⁺ (under Th1 polarized conditions) cells among the responders was determined by intracellular staining followed by FACS analysis. C) IFN-γ and IL-17 amounts in the culture medium were measured by ELISA 48 h after in vitro stimulation. **<i>P</i> < 0.01, ns, not significant.</p

oxATP inhibited autoreactive T cell responses via both a direct effect on T cells and inhibiting DC function.

<p>A-D) Responder T cells and splenic APCs were isolated from immunized B6 mice, 13 days post immunization. Before co-incubation, T cells and DCs were either treated by oxATP (80 μM) or remained untreated. The stimulatory effect on T cells by the immunizing peptide and APCs were examined by co-culture of untreated responder T cells and APCs (3A); responder T cells treated but APCs untreated (3B), responder T cells untreated but APCs treated (3C), or both responder T cells and APCs were oxATP-treated (3D). After a 5-day co-incubation, the activated T cells were separated by Ficoll gradient centrifugation and staining for IL-17⁺ (among Th17 polarized stimulation, left panels of A-D) and IFN-γ⁺ cells (among Th1 polarized stimulation, right panels of A-D). E) IL-17and IFN-γ levels in the supernatant of cultured T cells after exposure to immunizing Ag and APCs for 48 h, were detected by ELISA in triplicates. * <i>P</i> < 0.05, **<i>P</i> < 0.01, ns, not significant.</p