Mycobacterium avium subsp. paratuberculosis Infected Cows Reveal Divergent Immune Response in Bovine Peripheral Blood Derived Lymphocyte Proteome.

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.

NEU1 is more abundant in uveitic retina with concomitant desialylation of retinal cells.

Desialylation of cell surface glycoproteins carried out by sialidases affects various immunological processes. However, the role of neuraminidase 1 (NEU1), one of the four mammalian sialidases, in inflammation and autoimmune disease is not completely unraveled to date. In this study, we analyzed the retinal expression of NEU1 in equine recurrent uveitis (ERU), a spontaneous animal model for autoimmune uveitis. Mass spectrometry revealed significantly higher abundance of NEU1 in retinal Müller glial cells (RMG) of ERU-diseased horses compared to healthy controls. Immunohistochemistry uncovered NEU1 expression along the whole Müller cell body in healthy and uveitic states and confirmed higher abundance in inflamed retina. Müller glial cells are the principal macroglial cells of the retina and play a crucial role in uveitis pathogenesis. To determine whether higher expression levels of NEU1 in uveitic RMG correlate with the desialylation of retinal cells, we performed lectin-binding assays with sialic acid-specific lectins. Through these experiments, we could demonstrate a profound loss of both α2-3- and α2-6-linked terminal sialic acids in uveitis. Hence, we hypothesize that the higher abundance of NEU1 in uveitic RMG plays an important role in the pathogenesis of uveitis by desialylation of retinal cells. As RMG become activated in the course of uveitis and actively promote inflammation, we propose that NEU1 might represent a novel activation marker for inflammatory RMG. Our data provide novel insights in the expression and implication of NEU1 in inflammation and autoimmune disease.

SurA is a cryptically grooved chaperone that expands unfolded outer membrane proteins.

The periplasmic chaperone network ensures the biogenesis of bacterial outer membrane proteins (OMPs) and has recently been identified as a promising target for antibiotics. SurA is the most important member of this network, both due to its genetic interaction with the ß-barrel assembly machinery complex as well as its ability to prevent unfolded OMP (uOMP) aggregation. Using only binding energy, the mechanism by which SurA carries out these two functions is not well-understood. Here, we use a combination of photo-crosslinking, mass spectrometry, solution scattering, and molecular modeling techniques to elucidate the key structural features that define how SurA solubilizes uOMPs. Our experimental data support a model in which SurA binds uOMPs in a groove formed between the core and P1 domains. This binding event results in a drastic expansion of the rest of the uOMP, which has many biological implications. Using these experimental data as restraints, we adopted an integrative modeling approach to create a sparse ensemble of models of a SurA•uOMP complex. We validated key structural features of the SurA•uOMP ensemble using independent scattering and chemical crosslinking data. Our data suggest that SurA utilizes three distinct binding modes to interact with uOMPs and that more than one SurA can bind a uOMP at a time. This work demonstrates that SurA operates in a distinct fashion compared to other chaperones in the OMP biogenesis network.

CD11d is a novel antigen on chicken leukocytes.

In life sciences, antibodies are among the most commonly used tools for identifying, tracking, quantifying and isolating molecules, mainly proteins. However, it has recently become clear that antibodies often fall short with respect to specificity and selectivity and in many cases target proteins are not even known. When commercial availability of antibodies is scarce, e.g. for targeting proteins from farm animals, researchers face additional challenges: they often have to rely on cross-reactive antibodies, which are poorly characterized for their exact target, their actual cross-reactivity and the desired application. In this study, we aimed at identifying the true target of mouse monoclonal antibody 8F2, which was generated against chicken PBMC and used for decades in research, while it's actual target molecule remained unknown. We used 8F2 antibody for immunoprecipitation in chicken PBMC and subsequently identified its true target as CD11d, which was never described in chicken lymphocytes before, by quantitative LC-MSMS. The most abundant interactor of CD11d was identified as integrin beta 2. The existence of this alpha integrin was therefore clearly proven on protein level and provides a first basis to further assess the role of CD11d in chickens in future studies. Data are available via ProteomeXchange with identifier PXD017248. SIGNIFICANCE: Our studies determined CD11d as the true target of a previously uncharacterized mouse monoclonal antibody 8F2, generated against chicken peripheral blood derived mononuclear cells (PBMC). This is therefore now first member of alpha integrins in chickens, that existence was now clearly identified on protein level. The additional identification of CD11d interactors provides information on integrin-dependent regulation of signaling networks, allowing further functional studies.

Proteome profile of neutrophils from a transgenic diabetic pig model shows distinct changes.

INSC94Y transgenic pigs develop a stable diabetic phenotype early after birth and therefore allow studying the influence of hyperglycemia on primary immune cells in an early stage of diabetes mellitus in vivo. Since immune response is altered in diabetes mellitus, with deviant neutrophil function discussed as one of the possible causes in humans and mouse models, we investigated these immune cells in INSC94Y transgenic pigs and wild type controls at protein level. A total of 2371 proteins were quantified by label-free LC-MS/MS. Subsequent differential proteome analysis of transgenic animals and controls revealed clear differences in protein abundances, indicating a deviant behavior of granulocytes in the diabetic state. Interestingly, abundance of myosin regulatory light chain 9 (MLC-2C) was increased 5-fold in cells of diabetic pigs. MLC-2C directly affects cell contractility by regulating myosin ATPase activity, can act as transcription factor and was also associated with inflammation. It might contribute to impaired neutrophil cell adhesion, migration and phagocytosis. Our study provides novel insights into proteome changes in neutrophils from a large animal model for permanent neonatal diabetes mellitus and points to dysregulation of neutrophil function even in an early stage of this disease. Data are available via ProteomeXchange with identifier PXD017274. SIGNIFICANCE: Our studies provide novel basic information about the neutrophil proteome of pigs and contribute to a better understanding of molecular mechanisms involved in altered immune cell function in an early stage diabetes. We demonstrate proteins that are dysregulated in neutrophils from a transgenic diabetic pig and have not been described in this context so far. The data presented here are highly relevant for veterinary medicine and have translational quality for diabetes in humans.

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-γ (IFN-γ), 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.

Retinopathy with central oedema in an INS C94Y transgenic pig model of long-term diabetes.

AIMS/HYPOTHESIS: Diabetic retinopathy is a severe complication of diabetes mellitus that often leads to blindness. Because the pathophysiology of diabetic retinopathy is not fully understood and novel therapeutic interventions require testing, there is a need for reliable animal models that mimic all the complications of diabetic retinopathy. Pig eyes share important anatomical and physiological similarities with human eyes. Previous studies have demonstrated that INS C94Y transgenic pigs develop a stable diabetic phenotype and ocular alterations such as cataracts. The aim of this study was to conduct an in-depth analysis of pathological changes in retinas from INS C94Y pigs exposed to hyperglycaemia for more than 2 years, representing a chronic diabetic condition. METHODS: Eyes from six INS C94Ypigs and six age-matched control littermates were analysed via histology and immunohistochemistry. For histological analyses of retinal (layer) thickness, sections were stained with H&E or Mallory's trichrome. For comparison of protein expression patterns and vessel courses, sections were stained with different antibodies in immunohistochemistry. Observed lesions were compared with reported pathologies in human diabetic retinopathy. RESULTS: INS C94Ypigs developed several signs of diabetic retinopathy similar to those seen in humans, such as intraretinal microvascular abnormalities, symptoms of proliferative diabetic retinopathy and central retinal oedema in a region that is cone rich, like the human macula. CONCLUSIONS/INTERPRETATION: The INS C94Ypig is an interesting model for studying the pathophysiology of diabetic retinopathy and for testing novel therapeutic strategies.

Formin like 1 expression is increased on CD4+ T lymphocytes in spontaneous autoimmune uveitis.

The membrane protein expression repertoire of cells changes in course of activation. In equine recurrent uveitis (ERU), a spontaneous autoimmune disease in horses with relapsing and ultimately blinding inner eye inflammation, CD4+ T lymphocytes are the crucial pathogenic cells activated in the periphery directly prior to an inflammatory episode. In order to find relevant changes in the membrane proteome associated to disease, we sorted CD4+ lymphocytes and compared protein abundance from the generated proteome datasets of both healthy horses and ERU cases. We detected formin like 1, a key player in actin dependent cellular processes such as phagocytosis, cell adhesion and cell migration, with significantly higher abundance in the CD4+ cell membrane proteome of horses with ERU. In transmigration experiments, we demonstrated higher migration rate of cells originating from diseased animals connecting formin like 1 to the migratory ability of cells. These findings are the first description of formin like 1 in association to processes involved in migration of inflammatory CD4+ T cells across the blood-retinal barrier in a spontaneous ocular autoimmune disease and suggest formin like 1 to play a role in the molecular mechanisms of ERU disease pathogenesis. Data are available via ProteomeXchange with identifier PXD005384. BIOLOGICAL SIGNIFICANCE: This comparative proteomic study of membrane proteins of CD4+ T cells identified a novel protein, formin like 1, with expression on the plasma cell membrane of equine CD4+ T cells and a significant change of abundance on CD4+ T cells of horses with a spontaneous autoimmune disease. Functional studies in a cell culture model for transmigration at the blood-retinal barrier (BRB) unraveled a strong impact of formin like 1 on migratory processes of CD4+ T cells across the BRB, a key event in uveitis pathogenesis. These findings provide novel knowledge about changes in the CD4+ immune cell membrane proteome in a spontaneously and naturally occurring autoimmune disease in horses with high relevance for veterinary medicine. Additionally, this model has proven translational quality for human medicine and provides novel proteomic information on autoimmune uveitis in man.

Immunological Characterization of Intraocular Lymphoid Follicles in a Spontaneous Recurrent Uveitis Model.

PURPOSE: Recently, formation of tertiary lymphoid structures was demonstrated and further characterized in the R161H mouse model of spontaneous autoimmune uveitis. In the horse model of spontaneous recurrent uveitis, intraocular lymphoid follicle formation is highly characteristic, and found in all stages and scores of disease, but in depth analyses of immunologic features of these structures are lacking to date. METHODS: Paraffin-embedded eye sections of cases with equine spontaneous recurrent uveitis (ERU) were characterized with immunohistochemistry to gain insight into the distribution, localization, and signaling of immune cells in intraocular tertiary lymphoid tissues. RESULTS: Ectopic lymphoid tissues were located preferentially in the iris, ciliary body, and retina at the ora serrata of horses with naturally-occurring ERU. The majority of cells in the tertiary lymphoid follicles were T cells with a scattered distribution of B cells and PNA+ cells interspersed. A fraction of T cells was additionally positive for memory cell marker CD45RO. Almost all cells coexpressed CD166, a molecule associated with activation and transmigration of T cells into inflamed tissues. Several transcription factors that govern immune cell responses were detectable in the tertiary lymphoid follicles, among them Zap70, TFIIB, GATA3, and IRF4. A high expression of the phosphorylated signal transducers and activators of transcription (STAT) proteins 1 and 5 were found at the margin of the structures. CONCLUSIONS: Cellular composition and structural organization of these inflammation-associated tertiary lymphoid tissue structures and the expression of markers of matured T and B cells point to highly organized adaptive immune responses in these follicles in spontaneous recurrent uveitis.

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.

Aquaporin 11, a regulator of water efflux at retinal Müller glial cell surface decreases concomitant with immune-mediated gliosis.

BACKGROUND: 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. 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 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. CONCLUSIONS: 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.

Novel localization of peripherin 2, the photoreceptor-specific retinal degeneration slow protein, in retinal pigment epithelium.

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.

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 changes and novel interaction partners of talin 1 in effector cells of autoimmune uveitis.

Autoimmune uveitis is characterized by crossing of blood-retinal barrier (BRB) by autoaggressive immune cells. Equine recurrent uveitis (ERU) is a valuable spontaneous model for autoimmune uveitis and analyses of differentially expressed proteins in ERU unraveled changed protein clusters in target tissues and immune system. Healthy eyes are devoid of leukocytes. In ERU, however, leukocytes enter the inner eye and subsequently destroy it. Molecular mechanisms enabling cell migration through BRB still remain elusive. Previously, we detected decreased talin 1 expression in blood-derived granulocytes of ERU cases, linking the innate immune system to ERU. Because changes in leukocyte protein expression pattern may play a role in pathological abnormalities leading to migration ability, we aimed at identifying interactors of talin 1 in leukocytes with immunoprecipitation, followed by LC-MS/MS for candidate identification. This enabled us to identify CD90 (Thy1) as novel interactor of talin 1 besides several other interactors. In blood-derived granulocytes from healthy individuals, CD90 was highly abundant and significantly reduced in ERU, especially in effector cells. Connection between talin 1 and CD90 and their expression differences in inflammation is an interesting novel finding allowing deeper insight into immune response of innate immune system and granulocyte migration ability in this organ-specific autoimmune disease.

Chicken immunoregulatory Ig-like receptor families: an overview and expression details on ggTREM-A1.

Paired immunoregulatory receptors facilitate the coordination of the immune response at the cellular level. In recent years, our group characterized chicken homologues to mammalian immunoregulatory Ig-like receptor families. The first part of this review focuses on the current progress on chicken immunoregulatory Ig-like receptor families. One of these receptors is gallus gallus TREM-A1, which was described as the only member of the chicken TREM family with activating potential. The second part of this review presents a study initiated to further characterize ggTREM-A1 expression. For this purpose we established real-time RT-PCR and generated a specific mab to analyze the expression profile of ggTREM-A1 on mRNA and protein level, respectively. GgTREM-A1 mRNA was predominantly expressed in macrophages, but was also detected in brain, bone marrow, bursa, thymus, spleen and PBMC. Analyzing ggTREM-A1 surface expression by mab staining validated the expression on macrophages. Additionally, we showed high expression on blood monocytes, heterophils and NK cells and on monocytes isolated from bone marrow. Moreover, we detected ggTREM-A1 protein also on thrombocytes, B and T cell subsets, but antigen expression seemed to be lower and more variable in these cells. Immunohistochemistry of chicken brain tissue, combining ggTREM-A1 mab and various markers specific for various brain cell subsets showed expression of ggTREM-A1 on microglial cells, but also on neurons, astrocytes and oligodendrocytes. In conclusion, ggTREM-A1 is expressed on a variety of cells, relevant for the immune system, possibly combining physiological function of different mammalian TREM.

Retinal glycoprotein enrichment by concanavalin a enabled identification of novel membrane autoantigen synaptotagmin-1 in equine recurrent uveitis.

Complete knowledge of autoantigen spectra is crucial for understanding pathomechanisms of autoimmune diseases like equine recurrent uveitis (ERU), a spontaneous model for human autoimmune uveitis. While several ERU autoantigens were identified previously, no membrane protein was found so far. As there is a great overlap between glycoproteins and membrane proteins, the aim of this study was to test whether pre-enrichment of retinal glycoproteins by ConA affinity is an effective tool to detect autoantigen candidates among membrane proteins. In 1D Western blots, the glycoprotein preparation allowed detection of IgG reactions to low abundant proteins in sera of ERU patients. Synaptotagmin-1, a Ca2+-sensing protein in synaptic vesicles, was identified as autoantigen candidate from the pre-enriched glycoprotein fraction by mass spectrometry and was validated as a highly prevalent autoantigen by enzyme-linked immunosorbent assay. Analysis of Syt1 expression in retinas of ERU cases showed a downregulation in the majority of ERU affected retinas to 24%. Results pointed to a dysregulation of retinal neurotransmitter release in ERU. Identification of synaptotagmin-1, the first cell membrane associated autoantigen in this spontaneous autoimmune disease, demonstrated that examination of tissue fractions can lead to the discovery of previously undetected novel autoantigens. Further experiments will address its role in ERU pathology.

Novel potential interacting partners of fibronectin in spontaneous animal model of interstitial cystitis.

Feline idiopathic cystitis (FIC) is the only spontaneous animal model for human interstitial cystitis (IC), as both possess a distinctive chronical and relapsing character. Underlying pathomechanisms of both diseases are not clearly established yet. We recently detected increased urine fibronectin levels in FIC cases. The purpose of this study was to gain further insight into the pathogenesis by assessing interacting partners of fibronectin in urine of FIC affected cats. Several candidate proteins were identified via immunoprecipitation and mass spectrometry. Considerable changes in FIC conditions compared to physiological expression of co-purified proteins were detected by Western blot and immunohistochemistry. Compared to controls, complement C4a and thioredoxin were present in higher levels in urine of FIC patients whereas loss of signal intensity was detected in FIC affected tissue. Galectin-7 was exclusively detected in urine of FIC cats, pointing to an important role of this molecule in FIC pathogenesis. Moderate physiological signal intensity of galectin-7 in transitional epithelium shifted to distinct expression in transitional epithelium under pathophysiological conditions. I-FABP expression was reduced in urine and urinary bladder tissue of FIC cats. Additionally, transduction molecules of thioredoxin, NF-κB p65 and p38 MAPK, were examined. In FIC affected tissue, colocalization of thioredoxin and NF-κB p65 could be demonstrated compared to absent coexpression of thioredoxin and p38 MAPK. These considerable changes in expression level and pattern point to an important role for co-purified proteins of fibronectin and thioredoxin-regulated signal transduction pathways in FIC pathogenesis. These results could provide a promising starting point for novel therapeutic approaches in the future.

Label-free LC-MSMS analysis of vitreous from autoimmune uveitis reveals a significant decrease in secreted Wnt signalling inhibitors DKK3 and SFRP2.

Equine recurrent uveitis is a severe and frequent blinding disease in horses which presents with auto-reactive invading T-cells, resulting in the destruction of the inner eye. Infiltration of inflammatory cells into the retina and vitreous is driven by currently unknown guidance cues, however surgical removal of the vitreous (vitrectomy) has proven therapeutically successful. Therefore, proteomic analyses of vitrectomy samples are likely to result in detection of proteins contributing to disease pathogenesis. Vitreous from healthy and ERU diseased horses were directly compared by quantitative mass spectrometry based on label-free quantification of peak intensities across samples. We found a significant upregulation of complement and coagulation cascades and downregulation of negative paracrine regulators of canonical Wnt signalling including the Wnt signalling inhibitors DKK3 and SFRP2. Based on immunohistochemistry, both proteins are expressed in equine retina and suggest localisation to retinal Müller glial cells (RMG), which may be the source cells for these proteins. Furthermore, retinal expression levels and patterns of DKK3 change in response to ERU. Since many other regulated proteins identified here are associated with RMG cells, these cells qualify as the prime responders to autoimmune triggers.

Immunophenotyping and characterization of BNP colostra revealed pathogenic alloantibodies of IgG1 subclass with specifity to platelets, granulocytes and monocytes of all maturation stages.

Bovine neonatal pancytopenia (BNP) is mainly characterized by multiple haemorrhages, thrombocytopenia and leukocytopenia as a result of bone marrow depletion. BNP can be induced in healthy calves through application of colostrum from BNP donors, proofing that BNP is mediated to maternal alloantibodies. Alloantibody binding to bovine blood cells is present in sera and colostra of BNP donors and is probably initialized by vaccination with a certain BVD vaccine. To understand etiology and pathomechanisms of BNP, we closely characterized disease inducing antibodies regarding immunoglobulin subclass and binding specificities to peripheral blood derived leukocytes and platelets. By exact phenotyping the targeted blood cell subsets, including platelets for the first time, we investigated that BNP alloantibodies are exclusively of IgG1 subclass. Interestingly, IgG1 of BNP colostra bound to 70% leukocytes and 100% platelets irrespective of different bovine breeds and cellular maturity of all specimens tested. Furthermore, staining pattern on platelets as well as leukocyte subsets by BNP-IgG1 alloantibody exposed 100% reactivity to platelets, granulocytes and monocytes. Interestingly, the main part of T-helper cells was not bound by colostral alloantibodies. Our results point to a crucial role of IgG1 antibodies in BNP and to a target antigen that is expressed by all cells of myeloid lineage, but only partially by the lymphoid lineage.