Immunological Insights in Equine Recurrent Uveitis

Horses worldwide suffer from equine recurrent uveitis (ERU), an organ-specific, immune-mediated disease with painful, remitting-relapsing inflammatory attacks alternating with periods of quiescence, which ultimately leads to blindness. In course of disease, both eyes can eventually be affected and since blind horses pose a threat to themselves and their surroundings, these animals have to be killed. Therefore, this disease is highly relevant for veterinary medicine. Additionally, ERU shows strong clinical and pathological resemblance to autoimmune uveitis in man. The exact cause for the onset of ERU is unclear to date. T cells are believed to be the main effector cells in this disease, as they overcome the blood retinal barrier to invade the eye, an organ physiologically devoid of peripheral immune cells. These cells cause severe intraocular inflammation, especially in their primary target, the retina. With every inflammatory episode, retinal degeneration increases until eyesight is completely lost. In ERU, T cells show an activated phenotype, with enhanced deformability and migration ability, which is reflected in the composition of their proteome and downstream interaction pathways even in quiescent stage of disease. Besides the dysregulation of adaptive immune cells, emerging evidence suggests that cells of the innate immune system may also directly contribute to ERU pathogenesis. As investigations in both the target organ and the periphery have rapidly evolved in recent years, giving new insights on pathogenesis-associated processes on cellular and molecular level, this review summarizes latest developments in ERU research

Aberrant Migratory Behavior of Immune Cells in Recurrent Autoimmune Uveitis in Horses

The participating signals and structures that enable primary immune cells migrating within dense tissues are not completely revealed until now. Especially in autoimmune diseases, mostly unknown mechanisms facilitate autoreactive immune cells to migrate to endogenous tissues, infiltrating and harming organ-specific structures. In order to gain deeper insights into the migratory behavior of primary autoreactive immune cells, we examined peripheral blood-derived lymphocytes (PBLs) of horses with equine recurrent uveitis (ERU), a spontaneous animal model for autoimmune uveitis in humans. In this study, we used a three-dimensional collagen I hydrogel matrix and monitored live-cell migration of primary lymphocytes as a reaction to different chemoattractants such as fetal calf serum (FCS), cytokines interleukin-4 (IL-4), and interferon-gamma (IFN-gamma), and a specific uveitis autoantigen, cellular retinaldehyde binding protein (CRALBP). Through these experiments, we uncovered distinct differences between PBLs from ERU cases and PBLs from healthy animals, with significantly higher cell motility, cell speed, and straightness during migration of PBLs from ERU horses. Furthermore, we emphasized the significance of expression levels and cellular localization of septin 7, a membrane-interacting protein with decreased abundance in PBLs of autoimmune cases. To underline the importance of septin 7 expression changes and the possible contribution to migratory behavior in autoreactive immune cells, we used forchlorfenuron (FCF) as a reversible inhibitor of septin structures. FCF-treated cells showed more directed migration through dense tissue and revealed aberrant septin 7 and F-actin structures along with different protein distribution and translocalization of the latter, uncovered by immunochemistry. Hence, we propose that septin 7 and interacting molecules play a pivotal role in the organization and regulation of cell shaping and migration. With our findings, we contribute to gaining deeper insights into the migratory behavior and septin 7-dependent cytoskeletal reorganization of immune cells in organ-specific autoimmune diseases

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

Chronic Hyperglycemia Drives Functional Impairment of Lymphocytes in Diabetic INSC94Y Transgenic Pigs

People with diabetes mellitus have an increased risk for infections, however, there is still a critical gap in precise knowledge about altered immune mechanisms in this disease. Since diabetic INSC94Y transgenic pigs exhibit elevated blood glucose and a stable diabetic phenotype soon after birth, they provide a favorable model to explore functional alterations of immune cells in an early stage of diabetes mellitus in vivo. Hence, we investigated peripheral blood mononuclear cells (PBMC) of these diabetic pigs compared to non-diabetic wild-type littermates. We found a 5-fold decreased proliferative response of T cells in INSC94Y tg pigs to polyclonal T cell mitogen phytohemagglutinin (PHA). Using label-free LC-MS/MS, a total of 3,487 proteins were quantified, and distinct changes in protein abundances in CD4(+) T cells of early-stage diabetic pigs were detectable. Additionally, we found significant increases in mitochondrial oxygen consumption rate (OCR) and higher basal glycolytic activity in PBMC of diabetic INSC94Y tg pigs, indicating an altered metabolic immune cell phenotype. Thus, our study provides new insights into molecular mechanisms of dysregulated immune cells triggered by permanent hyperglycemia

Mycobacterium avium subsp. paratuberculosis Proteome Changes Profoundly in Milk

Mycobacterium avium subspecies paratuberculosis (MAP) are detectable viable in milk and other dairy products. The molecular mechanisms allowing the adaptation of MAP in these products are still poorly understood. To obtain information about respective adaptation of MAP in milk, we differentially analyzed the proteomes of MAP cultivated for 48 h in either milk at 37 °C or 4 °C or Middlebrook 7H9 broth as a control. From a total of 2197 MAP proteins identified, 242 proteins were at least fivefold higher in abundance in milk. MAP responded to the nutritional shortage in milk with upregulation of 32% of proteins with function in metabolism and 17% in fatty acid metabolism/synthesis. Additionally, MAP upregulated clusters of 19% proteins with roles in stress responses and immune evasion, 19% in transcription/translation, and 13% in bacterial cell wall synthesis. Dut, MmpL4_1, and RecA were only detected in MAP incubated in milk, pointing to very important roles of these proteins for MAP coping with a stressful environment. Dut is essential and plays an exclusive role for growth, MmpL4_1 for virulence through secretion of specific lipids, and RecA for SOS response of mycobacteria. Further, 35 candidates with stable expression in all conditions were detected, which could serve as targets for detection. Data are available via ProteomeXchange with identifier PXD027444

Identification of Ocular Autoantigens Associated With Juvenile Idiopathic Arthritis-Associated Uveitis

The purpose of the current study was to analyze the binding patterns of serum autoantibodies from juvenile idiopathic arthritis (JIA) and JIA-associated uveitis (JIAU) patients to proteomes from different ocular tissues and to identify potential ocular autoantigens in JIAU. Proteomes from porcine iris, ciliary body, or retina tissue were isolated, separated using 2D-gel electrophoresis, and transferred to a blotting membrane. The binding pattern of serum antibodies from JIA or JIAU patients or healthy controls to ocular proteins was visualized by using anti-human IgG secondary antibodies and chemiluminescence reaction. Selected protein spots were excised from silver-stained 2D gels and subjected to mass spectrometry. Serum antibodies binding to ocular proteins were detected in all patient groups and healthy controls. Irrespective of the patient groups, serum antibodies bound to 49 different protein spots of the retina proteome, to 53 of the ciliary body proteome, and to 44 of the iris proteome. The relative binding frequency of sera to these iris protein spots was significantly higher in JIAU than in JIA patients or healthy controls. Particularly in JIAU patients, cluster analyses indicated a broad range of serum antibodies directed against ocular antigens, mostly in the iris proteome. Iris proteins frequently bound by serum antibodies in all groups were identified as tubulin beta chain, vimentin, ATP synthase subunit beta, actin, and L-lactate dehydrogenase B chain. Iris proteins exclusively bound by JIAU serum antibodies were heat shock cognate 71 kDa protein and keratin. Although serum autoantibody binding to ocular antigens was not disease-specific, a significant diversity of autoantibodies against a broad range of antigens, particularly from the iris tissue, was detected in JIAU patients. As the iris is a major site of inflammation in JIAU, the present data give further evidence that autoantibodies may be involved in JIAU immunopathology

Interaction of septin 7 and DOCK8 in equine lymphocytes reveals novel insights into signaling pathways associated with autoimmunity

The GTP-binding protein septin 7 is involved in various cellular processes, including cytoskeleton organization, migration and the regulation of cell shape. Septin 7 function in lymphocytes, however, is poorly characterized. Since the intracellular signaling role of septin 7 is dependent on its interaction network, interaction proteomics was applied to attain novel knowledge about septin 7 function in hematopoietic cells. Our previous finding of decreased septin 7 expression in blood-derived lymphocytes in ERU, a spontaneous animal model for autoimmune uveitis in man, extended the role of septin 7 to a potential key player in autoimmunity. Here, we revealed novel insights into septin 7 function by identification of DOCK8 as an interaction partner in primary blood-derived lymphocytes. Since DOCK8 is associated with important immune functions, our finding of significantly decreased DOCK8 expression and altered DOCK8 interaction network in ERU might explain changes in immune response and shows the contribution of DOCK8 in pathomechanisms of spontaneous autoimmune diseases. Moreover, our analyses revealed insights in DOCK8 function, by identifying the signal transducer ILK as a DOCK8 interactor in lymphocytes. Our finding of the enhanced enrichment of ILK in ERU cases indicates a deviant influence of DOCK8 on inter-and intracellular signaling in autoimmune disease

Banana Lectin from Musa paradisiaca Is Mitogenic for Cow and Pig PBMC via IL-2 Pathway and ELF1

The aim of the study was to gain deeper insights in the potential of polyclonal stimulation of PBMC with banana lectin (BanLec) from Musa paradisiaca. BanLec induced a marked proliferative response in cow and pig PBMC, but was strongest in pigs, where it induced an even higher proliferation rate than Concanavalin A. Molecular processes associated with respective responses in porcine PBMC were examined with differential proteome analyses. Discovery proteomic experiments was applied to BanLec stimulated PBMC and cellular and secretome responses were analyzed with label free LC-MS/MS. In PBMC, 3955 proteins were identified. After polyclonal stimulation with BanLec, 459 proteins showed significantly changed abundance in PBMC. In respective PBMC secretomes, 2867 proteins were identified with 231 differentially expressed candidates as reaction to BanLec stimulation. The transcription factor “E74 like ETS transcription factor 1 (ELF1)” was solely enriched in BanLec stimulated PBMC. BanLec induced secretion of several immune regulators, amongst them positive regulators of activated T cell proliferation and Jak-STAT signaling pathway. Top changed immune proteins were CD226, CD27, IFNG, IL18, IL2, CXCL10, LAT, ICOS, IL2RA, LAG3, and CD300C. BanLec stimulates PBMC of cows and pigs polyclonally and induces IL2 pathway and further proinflammatory cytokines. Proteomics data are available via ProteomeXchange with identifier PXD027505

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