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.

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.

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.

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.

We recently identified a deviant bovine immune phenotype characterized by hyperproliferation of lymphocytes after polyclonal stimulation. This phenotype was first discovered in dams that responded to PregSure BVD vaccination by producing pathological antibodies, triggering the fatal disease “bovine neonatal pancytopenia” in calves. The aim of the study was to gain deeper insights into molecular processes occurring in lymphocytes of immune phenotypes and the effect on their secretome after immune stimulation. Two discovery proteomic experiments were performed with unstimulated and Pokeweed Mitogen (PWM) stimulated lymphocytes, using label-free LC-MS/MS. In lymphocytes, 2447 proteins were quantified, and 1204 proteins were quantified in the secretome. Quantitative proteome analysis of immune deviant and control samples after PWM stimulation revealed clear differences. The increase in abundance of IL17A, IL17F, IL8, CCL5, LRRC59, and CLIC4 was higher in controls through mitogenic stimulation. In contrast, the abundance of IFNγ, IL2, IL2RA, CD83, and CD200 increased significantly more in immune deviant lymphocytes. Additional pathway enrichment analysis of differentially secreted proteins also yielded fundamental differences between the immune phenotypes. Our study provides a comprehensive dataset, which gives novel insights into proteome changes of lymphocytes from different bovine immune phenotypes. These differences point to the development of diverse immune responses of bovine immune phenotypes after immune stimulation.