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.

Müller 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 Müller 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 %) and especially its largely unknown function. 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. In the horse retina, AQP11 was exclusively expressed at Müller glial cell membranes. In uveitic condition, AQP11 disappeared from gliotic Müller 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 Müller 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. Therefore, AQP11 is important for physiological Müller 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.

Retinal Müller glial cells (RMG) play a crucial role in retinal neuroinflammation, including autoimmune uveitis. Increasing evidence supports their function as active modulators of immune responses and potential atypical antigen-presenting cells (APCs). To further investigate this hypothesis, we conducted a differential proteome analysis of primary equine RMG from healthy controls and horses with equine recurrent uveitis (ERU), a spontaneous model of autoimmune uveitis. This analysis identified 310 proteins with differential abundance. Among these, the Major Histocompatibility Complex (MHC) class II and the enzyme Arginase 1 (ARG1) were significantly enriched in RMG from uveitis-affected horses, whereas Mannose Receptor C-type 2 (MRC2) and its interactor Thrombospondin 1 (THBS1) were more abundant in healthy RMG. The detection of MHC class II in equine RMG, consistent with previous studies, validates the robustness of our approach. Furthermore, the identification of ARG1 and MRC2, together with THBS1, provides new insights into the immunomodulatory and antigen-presenting properties of RMG. Immunohistochemical analyses confirmed the proteomic findings and revealed the spatial distribution of ARG1 and MRC2. ARG1 and MRC2 are thus markers for RMG in the neuroinflammatory or physiological milieu and highlight potential differences in the immune function of RMG, particularly in antigen presentation.

Bovine paratuberculosis is a serious chronic disease of the gastrointestinal tract that causes economic losses and dramatically affects animal health in livestock. The underlying infectious agent, Mycobacterium avium subspecies paratuberculosis (MAP), cannot reliably be detected by standard diagnostic tests due to the long asymptomatic disease stage. The aim of this study was to detect proteomic changes in peripheral blood mononuclear cells (PBMC) from cows of the same herd with different MAP infection status after co-incubation with viable MAP in vitro using label-free LC-MS/MS. In our proteomic discovery experiment, we detected 2631 differentially regulated proteins between cows with negative MAP infection status (so-called MAP-resistant cows) and cows with positive MAP infection status (so-called persistently MAP-infected cows). In MAP-resistant cows, we detected enriched immune-related signaling pathways for TLR2 and MHC class II component proteins, among others, indicating a successful defensive immune response of the cows to MAP. In contrast, persistently MAP-infected cows were not directly enriched in immune-related signaling pathways associated with ITGA2B and KCNMA1, among others. The introduction of these distinct immune responses contributes to a better understanding of the bovine immune response and mechanisms of susceptibility to MAP.

Retinal pigment epithelium (RPE) builds the outer blood-retinal barrier of the eye. Since one typical feature of the autoimmune disease, equine recurrent uveitis (ERU), is the breakdown of this barrier, we recently performed comparative analysis of healthy and uveitic RPE. We identified for the first time peripherin 2, which is responsible for visual perception and retina development, to be localized in RPE. The purpose of this study was therefore to validate our findings by characterizing the expression patterns of peripherin 2 in RPE and retina. We also investigated whether peripherin 2 expression changes in ERU and if it is expressed by the RPE itself. Via immunohistochemistry, significant downregulation of peripherin 2 in uveitic RPE compared to the control was detectable, but there was no difference in healthy and uveitic retina. A further interesting finding was the clear distinction between peripherin 2 and the phagocytosis marker, rhodopsin, in healthy RPE. In conclusion, changes in the expression pattern of peripherin 2 selectively affect RPE, but not retina, in ERU. Moreover, peripherin 2 is clearly detectable in healthy RPE due to both phagocytosis and the expression by the RPE cells themselves. Our novel findings are very promising for better understanding the molecular mechanisms taking place on RPE in uveitis.