9 research outputs found
Fingolimod (FTY720) as an Acute Rescue Therapy for Intraocular Inflammatory Disease
No full textObjective To examine the efficacy of the immunomodulatory drug fingolimod (FTY720) as a rescue therapy for noninfectious intraocular inflammation. Methods Experimental autoimmune uveoretinitis, the murine correlate of human uveitis, was induced in B10.RIII mice. The mice were treated with 2 oral doses of fingolimod daily, either during early ocular infiltration or following clinical onset of the disease. At subsequent times, retinal infiltrates were examined and enumerated using flow cytometry, and structural disease was assessed and scored using histology. Results Fingolimod treatment, administered 2 days before disease onset, prevented inflammatory cells from infiltrating the retina, with corroborative suppression of histologic disease. A single dose of fingolimod was sufficient in clearing infiltrating leukocytes from the retina within 2 hours of treatment. Furthermore, a single dose of fingolimod administered after disease onset not only abolished retinal infiltrates but also prevented disease relapse for at least 3 weeks. Conclusions A short-term, high-dose treatment with fingolimod rapidly reduces ocular infiltrates in experimental autoimmune uveoretinitis, leading to a normal myeloid cell count within the retina. When given at the early stages of intraocular inflammation, fingolimod resolves disease. Clinical Relevance This study directly demonstrates the therapeutic potential of fingolimod and an acute rescue intervention for human noninfectious posterior-segment intraocular inflammatory disease. Author Affiliations: Department of Cellular and Molecular Medicine, School of Medical Sciences (Drs Raveney, Nicholson, and Dick) and Unit of Ophthalmology, Department of Clinical Sciences at South Bristol (Mr Copland and Drs Nicholson and Dick), University of Bristol, Bristol, England. Dr Raveney is now with the Department of Immunology, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Tokyo, Japan.Objective To examine the efficacy of the immunomodulatory drug fingolimod (FTY720) as a rescue therapy for noninfectious intraocular inflammation. Methods Experimental autoimmune uveoretinitis, the murine correlate of human uveitis, was induced in B10.RIII mice. The mice were treated with 2 oral doses of fingolimod daily, either during early ocular infiltration or following clinical onset of the disease. At subsequent times, retinal infiltrates were examined and enumerated using flow cytometry, and structural disease was assessed and scored using histology. Results Fingolimod treatment, administered 2 days before disease onset, prevented inflammatory cells from infiltrating the retina, with corroborative suppression of histologic disease. A single dose of fingolimod was sufficient in clearing infiltrating leukocytes from the retina within 2 hours of treatment. Furthermore, a single dose of fingolimod administered after disease onset not only abolished retinal infiltrates but also prevented disease relapse for at least 3 weeks. Conclusions A short-term, high-dose treatment with fingolimod rapidly reduces ocular infiltrates in experimental autoimmune uveoretinitis, leading to a normal myeloid cell count within the retina. When given at the early stages of intraocular inflammation, fingolimod resolves disease. Clinical Relevance This study directly demonstrates the therapeutic potential of fingolimod and an acute rescue intervention for human noninfectious posterior-segment intraocular inflammatory disease. Author Affiliations: Department of Cellular and Molecular Medicine, School of Medical Sciences (Drs Raveney, Nicholson, and Dick) and Unit of Ophthalmology, Department of Clinical Sciences at South Bristol (Mr Copland and Drs Nicholson and Dick), University of Bristol, Bristol, England. Dr Raveney is now with the Department of Immunology, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Tokyo, Japan
Autoimmune induction of NR4A2 in CD4<sup>+</sup> T cells is associated with IL-17-secreting T cells.
No full text<p>EAE or EAU was induced in C57BL/6 mice by immunization with MOG<sub>35–55</sub> or IRBP<sub>1–20</sub> peptide in CFA, respectively. CD4<sup>+</sup> T cells were purified from spleen, blood, or target organ (CNS or retina) on the indicated days and RNA was isolated. <b>A</b> and <b>B:</b> NR4A2 expression was quantified by real time PCR relative to GAPDH for T cells from EAE (A) or EAU (B). Timepoints correspond to a minimum of 5 animals and data are representative of 3 independent experiments. CD4<sup>+</sup> T cells from mice with EAE were restimulated with PMA/ionomycin for 3 hours and 4 populations of cytokine secreting cells (IL-17+IFN-γ-, IL-17+IFN-γ+, IL-17-IFN-γ+, and IL-17-IFN-γ-) were sorted by flow cytometry using IFN-γ and IL-17 cytokine secretion assay kits. <b>C:</b> NR4A2 expression by populations of cytokine-secreting CD4<sup>+</sup> T cells was quantified by real time PCR at day 15 post-EAE induction for lymph nodes (LN) and CNS-infiltrating cells (CNS), and day 25 for blood T cells. <b>D</b> and <b>E:</b> NR4A2 expression by IL-17<sup>+</sup>IFN-γ<sup>−</sup> or IL-17<sup>+</sup>IFN-γ<sup>+</sup> CNS-infiltrating T cells (<b>D</b>) or blood T cells (<b>E</b>) was measured by real time PCR at a range of timepoints. Data are representative of 2 independent experiments. <b>F:</b> Th1-mediated diabetes was induced in C57BL/6 mice by 5 daily low dose STZ treatments. Other groups of C57BL/6 mice were immunized with peptides in CFA plus PTX either OVA<sub>323–339</sub> (OVA/CFA) or MOG<sub>35–55</sub> (MOG/CFA). On day 22, NR4A2 expression was assessed by real time PCR amongst CD4<sup>+</sup> T cells from spleen and leukocytes isolated from the relevant target organ (ND, OVA/CFA; CNS, EAE; pancreas, STZ). Timepoints correspond to a minimum of 5 animals and data are representative of 2 independent experiments.</p
Systemic administration of NR4A2-specific siRNA reduces EAE severity.
No full text<p>siRNA, either NR4A2-specific or control, was stabilized in a collagen matrix and administered <i>i.v.</i> to groups of C57BL/6 mice at the time of EAE induction. EAE was scored clinically (<b>A</b>) and at day 15 post-EAE induction, production of IL-17 and IFN-γ by CNS-infiltrating leukocytes restimulated with 20 µg/ml MOG peptide for 96 hours were assessed by ELISA (<b>B</b>). CNS-infiltrating T cells were also assessed for IL-17 production at a range of timepoints by intracellular flow cytometry (<b>C</b>). Data are representative of 3 independent experiments. Control or NR4A2-specific siRNA was applied to MOG-immunized mice at day 10 post-disease induction and disease was scored clinically (<b>D</b>). Timepoints correspond to a minimum of 5 animals and data are representative of 2 independent experiments. IL-21 and IL-23R expressions amongst CNS-infiltrating T cells were measured by real time PCR (<b>E&F</b>). Data are representative of 2 independent experiments. Clinical scores in panels A) and D) were tested with a two-way ANOVA test. *p<0.01, **p<0.001.</p
Absence of NR4A2 is associated with a lack of IL-21 production by Th17 cells.
No full text<p>Naïve CD4<sup>+</sup> T cells were transfected by electroporation with NR4A2-specific siRNA or scrambled control siRNA and were activated with 5 µg/ml plate-bound CD3-specific mAb and 0.5 µg/ml soluble CD28-specific mAb in the presence of 20 ng/ml IL-6, 20 ng/ml IL-23, and 3 ng/ml TGF-β. <b>A:</b> RNA levels of IL-21, IL-23R, and IL-17 were quantified by real time PCR at the indicated timepoints following activation. Data are representative of 3 independent experiments. <b>B:</b> IL-21 supernatant concentration was measured by ELISA at 96 hours. Data are representative of 3 independent experiments. *p<0.05. <b>C:</b> RNA expression of c-maf quantified by real time PCR. <b>D:</b> RNA expression of IL-22 quantified by real time PCR. Data are representative of 2 independent experiments.</p
TNFR1 signalling is a critical checkpoint for developing macrophages that control of T-cell proliferation
No full textMacrophages (Mp) are professional antigen presenting cells, but when they accumulate at sites of inflammation, they can inhibit T cell proliferation. In experimental autoimmune uveoretinitis (EAU), this limits the expansion of T cells within the target organ. To define requirements for the elaboration of this outcome, we have generated populations of Mp in vitro that could also regulate T cell responses; stimulating CD4+ T cell activation and cytokine production, but simultaneously suppressing T cell proliferation. When T cells are removed from the influence of such cells, normal T cell responses are restored. We show that TNFR1 signalling is a critical checkpoint in the development of such Mp, as TNFR1-/- Mp are unable to suppress T cell proliferation. This deficit in antigen presenting cell results in a lack of production of prostaglandin E2 (PGE2), and nitric oxide, which are critical effector mechanisms that inhibit T cell division. However, TNFR1 signalling is not required for the inhibitory function of Mp, since we could circumvent the requirement for this receptor, by maturing Mp in the presence of exogenous IFN-gamma and PGE2. This produced TNFR1-/- Mp that inhibited T cell proliferation and indicates that TNFR1 delivers a signal that is necessary for the development but not the execution of this function.Macrophages (Mp) are professional antigen presenting cells, but when they accumulate at sites of inflammation, they can inhibit T cell proliferation. In experimental autoimmune uveoretinitis (EAU), this limits the expansion of T cells within the target organ. To define requirements for the elaboration of this outcome, we have generated populations of Mp in vitro that could also regulate T cell responses; stimulating CD4+ T cell activation and cytokine production, but simultaneously suppressing T cell proliferation. When T cells are removed from the influence of such cells, normal T cell responses are restored. We show that TNFR1 signalling is a critical checkpoint in the development of such Mp, as TNFR1-/- Mp are unable to suppress T cell proliferation. This deficit in antigen presenting cell results in a lack of production of prostaglandin E2 (PGE2), and nitric oxide, which are critical effector mechanisms that inhibit T cell division. However, TNFR1 signalling is not required for the inhibitory function of Mp, since we could circumvent the requirement for this receptor, by maturing Mp in the presence of exogenous IFN-gamma and PGE2. This produced TNFR1-/- Mp that inhibited T cell proliferation and indicates that TNFR1 delivers a signal that is necessary for the development but not the execution of this function
NR4A2 knockdown prevents IL-17 secretion but not RORγt upregulation.
No full text<p>Naïve CD4<sup>+</sup> T cells were transfected by electroporation with NR4A2-specific siRNA or scrambled control siRNA. Cells were then activated with 5 µg/ml plate-bound CD3-specific mAb and 0.5 µg/ml soluble CD28-specific mAb. <b>A:</b> IFN-γ production by cells activated in the presence or absence of 10 ng/ml IL-12 after 96 hours of culture. <b>B:</b> IL-17 production by cells activated in the presence of 20 ng/ml IL-6, 20 ng/ml IL-23, and TGF-β at a range of concentrations after 96 hours of culture. Significant differences between control and NR4A2 siRNA-treatments were tested with a student’s t-test, *p<0.05. <b>C:</b> IL-17 and IFN-γ intracellular cytokine staining for transfected T cells (control siRNA, left plots; NR4A2 siRNA, right plots) cultured for 96 hours in the presence of 10 ng/ml IL-12 (Th1 conditions, top row plots) or 20 ng/ml IL-6, 20 ng/ml IL-23, and 3 ng/ml TGF-β (Th17 conditions, bottom row plots). <b>D:</b> RORγt RNA expression as measured by real time PCR by activated T cells cultured under Th17 polarizing conditions at a range of timepoints. Data are representative of 5 independent experiments.</p
Exogenous IL-21 restores IL-17 production in the absence of NR4A2.
No full text<p>Naïve CD4<sup>+</sup> T cells were transfected by electroporation with NR4A2-specific siRNA or scrambled control siRNA and were activated with 5 µg/ml plate-bound CD3-specific mAb and 0. 5 µg/ml soluble CD28-specific mAb in the presence of 20 ng/ml IL-6, 20 ng/ml IL-23, and 3 ng/ml TGF-β. To some wells, recombinant IL-21 was added as indicated. <b>A:</b> IL-17 was measured in the supernatants of control or NR4A2 siRNA-treated T cells by ELISA after 96 hours of culture under Th17 polarizing conditions in the presence or absence of IL-21 at the indicated concentrations. *p<0.05. Data are representative of 3 independent experiments. <b>B:</b> IL-23R expression was assessed by intracellular flow cytometric staining after 96 hours of culture under Th17 polarizing conditions in the presence (right plots) or absence (left plots) of 100 pg/ml recombinant IL-21 for control siRNA-treated T cells (top) and NR4A2 siRNA-treated T cells (bottom row). Data are representative of 2 independent experiments.</p
Autoimmune and autoinflammatory mechanisms in uveitis
No full textThe eye, as currently viewed, is neither immunologically ignorant nor sequestered from the systemic environment. The eye utilises distinct immunoregulatory mechanisms to preserve tissue and cellular function in the face of immune-mediated insult; clinically, inflammation following such an insult is termed uveitis. The intra-ocular inflammation in uveitis may be clinically obvious as a result of infection (e.g. toxoplasma, herpes), but in the main infection, if any, remains covert. We now recognise that healthy tissues including the retina have regulatory mechanisms imparted by control of myeloid cells through receptors (e.g. CD200R) and soluble inhibitory factors (e.g. alpha-MSH), regulation of the blood retinal barrier, and active immune surveillance. Once homoeostasis has been disrupted and inflammation ensues, the mechanisms to regulate inflammation, including T cell apoptosis, generation of Treg cells, and myeloid cell suppression in situ, are less successful. Why inflammation becomes persistent remains unknown, but extrapolating from animal models, possibilities include differential trafficking of T cells from the retina, residency of CD8(+) T cells, and alterations of myeloid cell phenotype and function. Translating lessons learned from animal models to humans has been helped by system biology approaches and informatics, which suggest that diseased animals and people share similar changes in T cell phenotypes and monocyte function to date. Together the data infer a possible cryptic infectious drive in uveitis that unlocks and drives persistent autoimmune responses, or promotes further innate immune responses. Thus there may be many mechanisms in common with those observed in autoinflammatory disorders
The B subunit of Escherichia coli heat-labile enterotoxin inhibits Th1 but not Th17 cell responses in established experimental autoimmune uveoretinitis
No full textPURPOSE. To investigate the efficacy of the B subunit of Escherichia coli heat-labile enterotoxin (EtxB) in the treatment of ocular autoimmune disease. Murine experimental autoimmune uveoretinitis (EAU) is an animal model of autoimmune posterior uveitis initiated by retinal antigen-specific Th1 and Th17 CD4 T cells, which activate myeloid cells, inducing retinal damage. EtxB is a potent immune modulator that ameliorates other Th1-mediated autoimmune diseases, enhancing regulatory T-cell activity. METHODS. EAU was induced in B10.RIII mice by immunization with peptide hIRBP. Disease severity was measured by clinical and histologic assessment, and functional responses of macrophages (Mφs) and T cells were assessed, both in vivo and in cocultures in vitro. EtxB was administered intranasally daily for 4 days, starting either 3 days before or 3 days after EAU induction. RESULTS. Preimmunization treatment with EtxB protected mice from EAU, limiting both the number and the activation status of retinal infiltrating immune cells. Treatment after EAU induction did not alter the disease course, despite suppression of IFN-γ. Although EtxB treatment of in vitro cocultures of T cells and Mφs increased IL-10 production, EtxB treatment in vivo increased the proportion and number of IL-17-producing CD4 cells infiltrating the eye. CONCLUSIONS. EtxB preimmunization protects mice from EAU induction by inhibiting Th1 responses, but the resultant reduction in IFN-γ responses by EtxB does not effect infiltration or structural damage in established EAU, where Th17 responses predominate. These data highlight the critical importance of the dynamics of T-cell phenotype and infiltration during EAU when considering immunomodulatory therapy
