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Expression Analysis of Cytokine and Chemokine Genes during the Natural Course of Murine Experimental Autoimmune Uveoretinitis.

ABSTRACT: C57BL/6 mice were immunized with human interphotoreceptor retinoid-binding protein peptides to induce experimental autoimmune uveoretinitis (EAU). From the day of immunization to 30 days later, RNA was isolated daily from the mouse eyes. Dynamic changes in gene expression during the pathogenesis of EAU were analyzed by TaqMan gene expression assay that contained most chemokines/cytokines and their receptors, and signal transducer and activator of transcription (STAT) family genes, using beta-actin as the endogenous control. Gene clusters based on their expression profiles were analyzed to determine the candidate genes for the pathogenesis of inflammation. Hierarchical cluster analysis showed gene expression during EAU development in seven clustering patterns. Hierarchical cluster analysis also identified four distinct phases in daily expression: entrance, acceleration, deceleration, and remission. Gene expression changes in the EAU active phase showed synergetic upregulation of Th1-type genes (IFN-gamma and CXCL10/IP-10) with elevated Th2-type genes (CCL17/TARC and IL-5). Sequential expression changes of STAT1, STAT6, and STAT3 genes represented the dynamic changes of Th1, Th2, and Th17-type inflammatory genes, respectively. The expression pattern of STAT1 was representative of many gene movements. Our results suggested that coordinated action of Th1, Th2, and Th17 genes and STAT family genes are involved in EAU development and resolution.

SUBMITTER: Hashida N

PROVIDER: S-EPMC3767243 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Expression Analysis of Cytokine and Chemokine Genes during the Natural Course of Murine Experimental Autoimmune Uveoretinitis.

Hashida Noriyasu N Ohguro Nobuyuki N Nishida Kohji K

ISRN inflammation 20120920

C57BL/6 mice were immunized with human interphotoreceptor retinoid-binding protein peptides to induce experimental autoimmune uveoretinitis (EAU). From the day of immunization to 30 days later, RNA was isolated daily from the mouse eyes. Dynamic changes in gene expression during the pathogenesis of EAU were analyzed by TaqMan gene expression assay that contained most chemokines/cytokines and their receptors, and signal transducer and activator of transcription (STAT) family genes, using beta-act ...[more]

PMID: 24049648

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Project description:Despite advances in clinical imaging and grading our understanding of retinal immune responses and their morphological correlates in experimental autoimmune uveoretinitis (EAU), has been hindered by the requirement for post-mortem histology. To date, monitoring changes occurring during EAU disease progression and evaluating the effect of therapeutic intervention in real time has not been possible. We wanted to establish whether optical coherence tomography (OCT) could detect intraretinal changes during inflammation and to determine its utility as a tool for accurate scoring of EAU. EAU was induced in C57BL/6J mice and animals evaluated after 15, 26, 36 and 60 days. At each time-point, contemporaneous Spectralis-OCT scanning, topical endoscopic fundal imaging (TEFI), fundus fluorescein angiography (FFA) and CD45-immunolabelled histology were performed. OCT features were further characterised on retinal flat-mounts using immunohistochemistry and 3D reconstruction. Optic disc swelling and vitreous opacities detected by OCT corresponded to CD45+ cell infiltration on histology. Vasculitis identified by FFA and OCT matched perivascular myeloid and T-cell infiltrates and could be differentiated from unaffected vessels. Evolution of these changes could be followed over time in the same eye. Retinal folds were visible and found to encapsulate mixed populations of activated myeloid cells, T-cells and microglia. Using these features, an OCT-based EAU scoring system was developed, with significant correlation to validated histological (Pearson r(2) = 0.6392, P<0.0001, n = 31 eyes) and TEFI based scoring systems (r(2) = 0.6784, P<0.0001). OCT distinguishes the fundamental features of murine EAU in vivo, permits dynamic assessment of intraretinal changes and can be used to score disease severity. As a result, it allows tissue synchronisation with subsequent cellular and functional assessment and greater efficiency of animal usage. By relating OCT signals with immunohistochemistry in EAU, our findings offer the opportunity to inform the interpretation of OCT changes in human uveitis.

Project description:BACKGROUND: Recent reports show that gene therapy may provide a long-term, safe and effective intervention for human diseases. In this study, we investigated the effectiveness of adeno-associated virus 2 (AAV2) based human interferon-alpha (hIFN-?) gene therapy in experimental autoimmune uveoretinitis (EAU), a classic model for human uveitis. METHODOLOGY/PRINCIPAL FINDINGS: An AAV2 vector harboring the hIFN-? gene (AAV2.hIFN-?) was subretinally injected into B10RIII mice at two doses (1.5×10(6) vg, 1.5×10(8) vg). AAV2 vector encoding green fluorescent protein (AAV2.GFP) was used as a control (5×10(8) vg). The expression of hIFN-? in homogenized eyes and serum was detected by ELISA three weeks after injection. The biodistribution of vector DNA in the injected eyes, contralateral eyes and distant organs was determined by PCR. EAU was induced by immunization with IRBP(161-180) three weeks following vector injections, and evaluated clinically and pathologically. IRBP-specific proliferation and IL-17 expression of lymphocytes from the spleen and lymph nodes were assayed to test the influence of the subretinal delivery of AAV2.hIFN-? on the systemic immune response. hIFN-? was effectively expressed in the eyes from three weeks to three months following subretinal injection of AAV2.hIFN-? vector. DNA of AAV2.GFP was observed only in the injected eyes, but not in the distant organs or contralateral eyes. Subretinal injection of both doses significantly attenuated EAU activity clinically and histologically. For the lower dose, there was no difference concerning lymphocyte proliferation and IL-17 production among the AAV2.hIFN-?, AAV2.GFP and PBS injected mice. However, the higher dose of AAV2.hIFN-? significantly suppressed lymphocyte proliferation and IL-17 production. CONCLUSIONS/SIGNIFICANCE: Subretinal delivery of AAV2.hIFN-? lead to an effective expression within the eye for at least three months and significantly attenuated EAU activity. AAV2.hIFN-? was shown to inhibit the systemic IRBP-specific immune response.

Project description:BACKGROUND: Advances in gene transfer techniques have provided long-term, safe and stable transduction of retinal cells following subretinal injection with adeno-associated viral (AAV) vectors. In this study we investigated whether subretinal injection of AAV2-murine IL-27p28 vector was effective in inhibiting experimental autoimmune uveoretinitis (EAU) induced in B10RIII mice. METHODOLOGY/PRINCIPAL FINDINGS: An AAV2 vector encoding the murine IL-27p28 gene (rAAV2-CMV-mIL-27p28) was prepared and subretinally injected into B10RIII mice (4.35×10(8) vector genome (v.g.)). AAV2 vector mediating green fluorescent protein (rAAV2-CMV-GFP) served as a control (5×10(8) v.g.). The concentration of mIL-27p28 in homogenized eyes and serum was assayed by enzyme linked immunosorbent assay (ELISA) after subretinal injection. Human IRBP(161-180) peptide and Complete Freund's Adjuvant were injected into mice receiving either the rAAV2-CMV-mIL-27p28 or rAAV2-CMV-GFP vector. EAU was evaluated clinically and pathologically. The level of IL-17 and IL-10 in homogenized eyes was measured on day 12 and day 21 following immunization. Delayed type hypersensitivity (DTH) and IRBP(161-180)-specific proliferation of lymphocytes from the spleen and lymph nodes were assayed to examine the influence of the subretinal delivery of rAAV2-CMV-mIL-27p28 on the systemic immune response. IL-27p28 was detectable by ELISA within the eyes from two weeks following subretinal injection of the rAAV2-CMV-mIL-27p28 vector and showed a sustained high expression from day 14 to 9 months with a highest expression at 5 months. Subretinal injection of the vector significantly attenuated the severity of EAU disease clinically and pathologically in association with a significantly decreased IL-17 expression and an increased IL-10 expression. The IL-27p28 vector did not affect the systemic immune response, as determined by DTH and IRBP(161-180)-specific lymphocyte proliferation. CONCLUSIONS/SIGNIFICANCE: A high and stable expression of IL-27p28 was observed for at least 9 months following subretinal delivery of rAAV2-CMV-mIL-27p28. The amelioration of EAU disease severity was associated with a decreased IL-17 expression and an increased IL-10 expression.

Dataset Information

Expression Analysis of Cytokine and Chemokine Genes during the Natural Course of Murine Experimental Autoimmune Uveoretinitis.

Publications

Expression Analysis of Cytokine and Chemokine Genes during the Natural Course of Murine Experimental Autoimmune Uveoretinitis.

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