Small Heat Shock Protein αA-Crystallin Prevents Photoreceptor Degeneration in Experimental Autoimmune Uveitis

The small heat shock protein, αA-crystallin null (αA−/−) mice are known to be more prone to retinal degeneration than the wild type mice in Experimental Autoimmune Uveoretinitis (EAU). In this report we demonstrate that intravenous administration of αA preserves retinal architecture and prevents photoreceptor damage in EAU. Interestingly, only αA and not αB-crystallin (αB), a closely related small heat shock protein works, pointing to molecular specificity in the observed retinal protection. The possible involvement of αA in retinal protection through immune modulation is corroborated by adaptive transfer experiments, (employing αA−/− and wild type mice with EAU as donors and Rag2−/− as the recipient mice), which indicate that αA protects against the autoimmune challenge by modulating the systemic B and T cell immunity. We show that αA administration causes marked reduction in Th1 cytokines (TNF-α, IL-12 and IFN-γ), both in the retina and in the spleen; notably, IL-17 was only reduced in the retina suggesting local intervention. Importantly, expression of Toll-like receptors and their associated adaptors is also inhibited suggesting that αA protection, against photoreceptor loss in EAU, is associated with systemic suppression of both the adaptive and innate immune responses

αA treatment has a significant impact on cytokine expression.

<p><b>A</b> and <b>B</b> are RT-PCR assays (A). Retinal Th1 cytokines (TNF-α, IL-12 and IFN-γ) and TH17 cytokine (IL-17) are up-regulated on day 21 in αA−/− EAU mice compared to the WT EAU controls after a challenge with the IRBP peptide. With αA administration (treated αA−/− EAU) these cytokines show significantly reduced expression compared to the untreated αA−/− EAU mice. (B). In the spleen of αA−/− with EAU, the levels of TNF-α show an increase compared to the WT mice with EAU. IL-12 and IFN-γ expression is similar in knockout mice and the WT mice. αA administration reduces the levels of TNF-α, IL-12 and IFN - γ in the spleen in αA−/−mice compared to the untreated αA−/−mice. Note that IL-17 remains unchanged in all three groups of mice. This is in contrast to increased expression of IL-17 in the retinas of αA −/− EAU mice and its inhibition in animals treated with αA (see A).</p

Intravenous administration of αA protects retinal photoreceptors.

<p>Twelve days after EAU was induced in B10RIII (WT) mice with an IRBP peptide, the animals were injected with various crystallins and the retinas were collected and examined on day 21. <b>A, B and C:</b> αA = αA crystallin, β = β crystallin, αB = αB crystallin, γ = γ crystallin and saline. <b>A.</b> Histology of retina and uvea (Hematoxylin and Eosin stained). Arrows indicate photoreceptor inner segments (PR). Note presence of photoreceptors in αA and their degeneration (absence) in Saline treated animals. <b>B</b>. Retinal architecture as revealed by immunostaining for IRBP with anti-IRBP (green, arrows). <b>C.</b> Apoptosis in EAU retinas of animals treated with various crystallins; αA treated, αB treated, saline treated, positive (+ve) control (DNase I treated retina) and (‘no enzyme’) negative (−ve) control. Note that retinal photoreceptors are preserved (<b>A,</b> αA and <b>B,</b> αA); there is little or no apoptosis (TUNEL positive cells) in αA treated animals (<b>C,</b> αA). PR = Photoreceptors, ONL = Outer nuclear layer, INL = Inner nuclear layer, GCL = Ganglion cell layer.</p

Adaptive transfer demonstrates involvement of αA with immune regulation.

<p>Inflammatory cell infiltration is a hallmark of the progression in EAU. Note inflammatory cells (arrows) in the vitreous in (a) representing the status of early retina in Rag2−/− mice injected with splenocytes and lymphocytes from αA−/− mice with EAU. Note the relative absence of this infiltration in b, which represents the status of retina in Rag2−/− mice injected with splenocytes and lymphocytes derived from the wild type mice (129Sve) with EAU. PR = Photoreceptors, ONL = Outer nuclear layer, INL = Inner nuclear layer, GCL = Ganglion Cell layer.</p

Down regulation of gene activity associated with the innate immune pathways.

<p>Commercial PCR arrays were used. Fold decrease was calculated by comparing data obtained with αA−/− EAU without αA treatment and αA−/− EAU mice treated with αA. A = Toll like receptors B = associated adaptive and signaling proteins and C = the NFkB pathway in αA−/− mice with EAU treated with αA. TLRs = Toll like receptors, MyD88: Myeloid differentiation primary response gene (88), Irak1 = Interleukin-1 receptor-associated kinase 1, CD14 = Cluster of differentiation 14, Ticam 2 = TIR domain-containing adapter molecule 2, Tirap = toll-interleukin 1 receptor (TIR) domain containing adaptor protein, Cox-2 = Cyclooxygenase-2, Irf-1 = Interferon regulatory factor 1, IL-1R1 = Interleukin 1 receptor, type I, Il-6re = IL6 responsive element, Map3K1 = Mitogen-activated protein kinase kinase kinase 1, NFkB = Nuclear factor kappa B, Ccl2 = Chemokine (C-C motif) ligand 2, IFN-γ = Interferon-gamma.</p

αA administration protects retinal phenotype in αA−/− mice in EAU.

<p>Prevention of inflammation by intravenous administration of αA crystalline protein in αA −/− mice with EAU on day 14 post immunization (A) whereas in the untreated αA −/− mice with EAU (B), there was inflammatory cell infiltration (arrows) and retinal damage. GCL = Ganglion cell layer, INL = Inner nuclear layer, ONL = Outer nuclear layer, IS = inner segments, OS = outer segments. Magnification: 400×.</p

αA administration markedly lowers protein levels for various cytokines.

<p>ELISA assays show decreased levels of IL-2, IL-12, IFN-γ and TNF-α and Th17 cytokines IL-6, IL-17, IL-23 and TGF-β in EAU mice treated with αA, while Th2 cytokines IL-4 and IL-10 levels are increased.</p

Pathogenic role of retinal microglia in experimental uveitis. Invest Ophthalmol Vis Sci.

PURPOSE. To devise methods for unequivocal identification of activated retinal microglia in experimental autoimmune uveoretinitis (EAU) and to investigate their role in the development of EAU. METHODS. A group of Lewis rats underwent optic nerve axotomy with the application of N-4-(4-didecylaminostyryl)-N methylpyridinium iodide (4Di-10ASP) at the axotomy site. On days 3, 14, and 38 after axotomy, the rats were killed, the eyes were enucleated, and the retinas were stained for OX42. Another group of such axotomized rats were immunized with S-antigen peptide and were killed on days 7 through 12 after the injection with peptide. The enucleated eyes were stained for OX42 and examined by confocal microscope. After axotomy, bone marrow (Y3 X) chimeric rats were injected with S-antigen peptide and were killed on days 10 and 12 after injection. The retinas were evaluated by PCR with Y-specific primers. Finally, a group of axotomized rats was injected with the S-antigen peptide and killed on days 6, 8, 9, and 10 after injection. Their enucleated eyes were examined for microglial expression of TNF␣ and for generation of peroxynitrite. RESULTS. In the axotomized, non-EAU eyes, 4Di-10ASP-labeled ganglion cells were detectable on days 3 and 14, and 4Di-10ASP-containing OX42-positive cells (microglia) were found in the nerve fiber and other inner retinal layers on days 14 and 38. The S-antigen peptide-injected rats showed migration of the microglia (4Di-10ASP-positive and OX42-positive) to the photoreceptor cell layer on day 9, and these cells increased in number at this site on day 10. No macrophages (OX42-positive and 4Di-10ASP-negative) were present at this early stage of EAU, but such cells appeared in the retina on days 11 and 12. PCR of the chimeric EAU retinas showed an absence of the Y chromosome-amplified product on day 10, but the presence of this product was detected on day 12. The expression of TNF␣ and generation of peroxynitrite were noted in the migrated microglia at the photoreceptor cell layer on days 9 and 10 of EAU. CONCLUSIONS. In the early phase of EAU, the microglia migrate to the photoreceptor cell layer where they generate TNF␣ and peroxynitrite. Such microglial migration and activation take place before infiltration of the macrophages. These findings indicate a novel pathogenic mechanism of EAU, in which retinal microglia may initiate retinitis with subsequent recruitment of circulation-derived phagocytes, leading to the amplification of uveoretinitis. (Invest Ophthalmol Vis Sci. 2003;44: 22-31