Altered Metabolic Phenotype of Immune Cells in a Spontaneous Autoimmune Uveitis Model

As one of the leading causes of blindness worldwide, uveitis is an important disease. The exact pathogenesis of autoimmune uveitis is not entirely elucidated to date. Equine recurrent uveitis (ERU) represents the only spontaneous animal model for autoimmune uveitis in humans. As the metabolism of immune cells is an emerging field in research and gains more and more significance to take part in the pathogenesis of various diseases, we conducted experiments to investigate the metabolism of immune cells of ERU cases and healthy controls. To our knowledge, the link between a deviant immunometabolism and the pathogenesis of autoimmune uveitis was not investigated so far. We showed that PBMC of ERU cases had a more active metabolic phenotype in basal state by upregulating both the oxidative phosphorylation and the glycolytic pathway. We further revealed an increased compensatory glycolytic rate of PBMC and CD4(+) T cells of ERU cases under mitochondrial stress conditions. These findings are in line with metabolic alterations of immune cells in other autoimmune diseases and basic research, where it was shown that activated immune cells have an increased need of energy and molecule demand for their effector function. We demonstrated a clear difference in the metabolic phenotypes of PBMC and, more specifically, CD4(+) T cells of ERU cases and controls. These findings are another important step in understanding the pathogenesis of ERU and figuratively, human autoimmune uveitis

Unraveling the Equine Lymphocyte Proteome: Differential Septin 7 Expression Associates with Immune Cells in Equine Recurrent Uveitis

Equine recurrent uveitis is a spontaneous, lymphocyte-driven autoimmune disease. It affects horses worldwide and presents with painful remitting-relapsing inflammatory attacks of inner eye structures eventually leading to blindness. Since lymphocytes are the key players in equine recurrent uveitis, we were interested in potential changes of their protein repertoire which may be involved in disease pathogenesis. To create a reference for differential proteome analysis, we first unraveled the equine lymphocyte proteome by two-dimensional sodium dodecyl sulfate - polyacrylamide gel electrophoresis and subsequently identified 352 protein spots. Next, we compared lymphocytes from ERU cases and healthy horses with a two-dimensional fluorescence difference in gel electrophoresis approach. With this technique, we identified seven differentially expressed proteins between conditions. One of the significantly lower expressed candidates, septin 7, plays a role in regulation of cell shape, motility and migration. Further analyses revealed T cells as the main cell type with decreased septin 7 abundance in equine recurrent uveitis. These findings point to a possible pathogenetic role of septin 7 in this sight-threatening disease

Expression and Distribution Pattern of Aquaporin 4, 5 and 11 in Retinas of 15 Different Species

Aquaporins (AQPs) are small integral membrane proteins with 13 members in mammals and are essential for water transport across membranes. They are found in many different tissues and cells. Currently, there are conflicting results regarding retinal aquaporin expression and subcellular localization between genome and protein analyses and among various species. AQP4, 7, 9 and 11 were described in the retina of men; whereas AQP6, 8 and 10 were earlier identified in rat retinas and AQP4, 5 and 11 in horses. Since there is a lack of knowledge regarding AQP expression on protein level in retinas of different animal models, we decided to analyze retinal cellular expression of AQP4, 5 and 11 in situ with immunohistochemistry. AQP4 was detected in all 15 explored species, AQP5 and AQP11 in 14 out of 15. Interestingly, AQP4 was unambiguously expressed in Muller glial cells, whereas AQP5 was differentially allocated among the species analyzed. AQP11 expression was Muller glial cell-specific in 50% of the animals, whereas in the others, AQP11 was detected in ganglion cell layer and at photoreceptor outer segments. Our data indicate a disparity in aquaporin distribution in retinas of various animals, especially for AQP5 and 11

Characterization of septin 7 expression intensity on lymphocyte subsets by flow cytometry.

<p>Mean intensity of septin 7 expression decreases in lymphocyte subsets of ERU cases (n = 11, dark grey curve) compared to controls (n = 11, light grey curve). Histograms of representative specimen showed unchanged expression in B cells (A + E, CD21). In T cells, septin 7 expression intensity decreased to 75% (B + E, CD4) and 73% (C + E, CD8) of physiological expression level. Lymphocytes were gated according to forward- and sideward-scatter (D). Respective values of all 11 healthy and 11 ERU specimen used in this study are shown in graph (E).</p

Septin 7 expression differences quantified and verified by Western blot.

<p>Septin 7 expression decreases in PBL of ERU diseased horses (n = 11, grey column, Septin 7 expression reduced to 62%) compared to PBL of healthy controls (n = 12, white column, set to 100%). Signal intensities of septin 7 were normalized to beta-actin abundances obtained after stripping and re-incubation of respective blots. Statistical analysis was performed using Student's <i>t</i> test (* p<0.05). Representative protein signals are shown above respective columns; upper Septin 7 signal was used for quantification, lower signal derived from unspecific binding of the antibody to beta actin and was not included in the analyses.</p

Spot map of equine PBL proteome generated by DeCyder 6.5 software.

<p>Protein spots detected on gel after scanning and processing were encircled in green. Spots with differential abundance between control- and ERU specimen (n = 5 each) were encircled in red and numbered according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091684#pone-0091684-t001" target="_blank">table 1</a>. Spot numbers referring to proteins with higher abundance in ERU were highlighted in grey, those with lower abundance in ERU are shown in white. Septin 7 spot (No. 7) showed diminished expression in ERU PBL proteome.</p

Aquaporin 11, a regulator of water efflux at retinal M&uuml;ller glial cell surface decreases concomitant with immune-mediated gliosis.

BACKGROUND: M&uuml;ller glial cells are important regulators of physiological function of retina. In a model disease of retinal inflammation and spontaneous recurrent uveitis in horses (ERU), we could show that retinal M&uuml;ller glial cells significantly change potassium and water channel protein expression during autoimmune pathogenesis. The most significantly changed channel protein in neuroinflammatory ERU was aquaporin 11 (AQP11). Aquaporins (AQP, 13 members) are important regulators of water and small solute transport through membranes. AQP11 is an unorthodox member of this family and was assigned to a third group of AQPs because of its difference in amino acid sequence (conserved sequence is only 11&nbsp;%) and especially its largely unknown function. METHODS: In order to gain insight into the distribution, localization, and function of AQP11 in the retina, we first developed a novel monoclonal antibody for AQP11 enabling quantification, localization, and functional studies. RESULTS: In the horse retina, AQP11 was exclusively expressed at M&uuml;ller glial cell membranes. In uveitic condition, AQP11 disappeared from gliotic M&uuml;ller cells concomitant with glutamine synthase. Since function of AQP11 is still under debate, we assessed the impact of AQP11 channel on cell volume regulation of primary M&uuml;ller glial cells under different osmotic conditions. We conclude a concomitant role for AQP11 with AQP4 in water efflux from these glial cells, which is disturbed in ERU. This could probably contribute to swelling and subsequent severe complication of retinal edema through impaired intracellular fluid regulation. CONCLUSIONS: Therefore, AQP11 is important for physiological M&uuml;ller glia function and the expression pattern and function of this water channel seems to have distinct functions in central nervous system. The significant reduction in neuroinflammation points to a crucial role in pathogenesis of autoimmune uveitis