Equine keratinocytes in the pathogenesis of insect bite hypersensitivity: Just another brick in the wall?

Equine insect bite hypersensitivity (IBH) is the most common skin disease affecting horses. It is described as an IgE-mediated, Type I hypersensitivity reaction to salivary gland proteins of Culicoides insects. Together with Th2 cells, epithelial barrier cells play an important role in development of Type I hypersensitivities. In order to elucidate the role of equine keratinocytes in development of IBH, we stimulated keratinocytes derived from IBH-affected (IBH-KER) (n = 9) and healthy horses (H-KER) (n = 9) with Culicoides recombinant allergens and extract, allergic cytokine milieu (ACM) and a Toll like receptor ligand 1/2 (TLR-1/2-L) and investigated their transcriptomes. Stimulation of keratinocytes with Culicoides allergens did not induce transcriptional changes. However, when stimulated with allergic cytokine milieu, their gene expression significantly changed. We found upregulation of genes encoding for CCL5, -11, -20, -27 and interleukins such as IL31. We also found a strong downregulation of genes such as SCEL and KRT16 involved in the formation of epithelial barrier. Following stimulation with TLR-1/2-L, keratinocytes significantly upregulated expression of genes affecting Toll like receptor and NOD-receptor signaling pathway as well as NF-kappa B signaling pathway, among others. The transcriptomes of IBH-KER and H-KER were very similar: without stimulations they only differed in one gene (CTSL); following stimulation with allergic cytokine milieu we found only 23 differentially expressed genes (e.g. CXCL10 and 11) and following stimulation with TLR-1/2-L they only differed by expression of seven genes. Our data suggests that keratinocytes contribute to the innate immune response and are able to elicit responses to different stimuli, possibly playing a role in the pathogenesis of IBH

LPS-induced modules of co-expressed genes in equine peripheral blood mononuclear cells

Lists of DEGs between LPS stimulated and unstimulated PBMCs. For every effect studied (LPS, and interaction effects: LPS:Fam1, LPS:Fam2) the list of genes differentially expressed at false discovery rate < 0.001 with log 2 fold changes is given. (XLSX 198 kb

Comparison of Skin Prick Tests (SPT), Intradermal Tests (IDT) and In Vitro Tests in the Characterization of Insect Bite Hypersensitivity (IBH) in a Population of Lusitano Horses: Contribution for Future Implementation of SPT in IBH Diagnosis.

Thirty controls (C) and 30 IBH-affected (T) Lusitano horses were evaluated. T horses were included based on anamnesis and physical examination, supported by questionnaires. All horses were submitted to skin tests, Intrademal (IDT) and Skin Prick Tests (SPT), on the neck with 14 specific allergens, 13 recombinant proteins (r-proteins) from Culicoides nubeculosus (Cul n) and Culicoides obsoletus (Cul o) salivary glands and Culicoides nubeculosus Whole Body Extract (Cul n WBE). Addicionally, a cluster of six T and six C horses were also tested with Cul n 3 and Cul n 4 produced in insect cells and barley, as well as E. coli produced Cul o 3 and Cul o WBE. Allergen concentrations were 10 µg/mL for IDT and 100 µg/mL for SPT, and wheal diameters assessed at 20 min, 6 and 48 h. IDTs were considered positive when wheal diameter was ≥50% of the histamine wheal and SPT's ≥ 0.9 cm. In vitro tests, allergen-specific serum IgE and sulfidoleukotriene (sLT) release assay were also carried out. Results showed that Cul n WBE, Cul n 7, 8, 9, Cul o1P and Cul o 2P were the best performing allergens for SPTs (p ≤ 0.0001) for the 1st allergen panel and Cul o WBE, Cul n 3 Bar and Cul n 4 Bac (p ≤ 0.05) for the 2nd, presenting a higher discriminatory diagnostic potential than IDTs, at a concentration of 100 µg/mL, with readings assessed at 20 min. Regarding in vitro tests overall, the sLT release assay performed best

CD154 Expression Indicates T Cell Activation Following Tetanus Toxoid Vaccination of Horses.

Despite the relevance of adaptive immunity against equine pathogens antigen-specific T cell responses of horses are not well characterized and the lack of insight into T cell responses hampers the understanding of the pathogeneses of important diseases. In this study we used tetanus toxoid (TT) as a well-defined antigen to characterize antigen-reactive T cells. Six healthy adult horses received a routine booster against tetanus with an immune stimulating complex (ISCOM)-based vaccine and were followed for 28 days. TT-specific serum antibodies were quantified by ELISA and increased in all horses by day 7 after vaccination. CD154 is an established indicator of antigen-reactive T helper cells in other species, but has not been characterized in horses. CD154 detection in equine PBMC by an anti-human CD154 antibody (clone 5C8) was confirmed by Western blots and then applied for flow cytometry. As a common indicator of equine T cell activation, cytokine induction was studied in parallel. T cells were analyzed by multicolor flow cytometry of PBMC after re-stimulation with TT in vitro. Reactive T helper (Th) cells were characterized by increased frequencies of CD4+CD154+ lymphocytes in in vitro TT-re-stimulated PBMC on day 14 after vaccination of the horses compared to pre-vaccination. The majority of all CD154+ cells after TT re-stimulation were CD4+ Th cells, but CD154 was also induced on CD4- cells albeit in lower frequencies. CD154+CD4+ Th cells were enriched in cytokine-expressing cells compared to CD154-CD4+ Th cells. Similar to the CD4+CD154+ frequencies, CD4+IL-4+, CD4+IFN-γ+ and CD4+TNF-α+ were increased after vaccination, but IL-4+ increased later than IFN-γ+ and CD4+TNF-α+, which already exceeded pre-vaccination frequencies on day 7. CD4+CD154+ frequencies correlated positively with those of CD4+IL-4+ (Th2) on day 14, and negatively with CD4+IFN-γ+ induction on day 7, but did not correlate with CD4+TNF-α+ frequencies or TT-specific antibody concentrations. CD154 appears to be a useful marker of antigen-reactive equine Th cells in combination with cytokine expression. The T cell analyses established here with TT can be applied to other antigens relevant for infections or allergies of horses and in horse models for translational research

In vitro analysis of expression vectors for DNA vaccination of horses: the effect of a Kozak sequence

One of the prerequisite for developing DNA vaccines for horses are vectors that are efficiently expressed in horse cells

Toll-like receptor-ligand induced thymic stromal lymphopoietin expression in primary equine keratinocytes.

BACKGROUND Thymic stromal lymphopoietin (TSLP) plays a key role in the development of allergic inflammation. Little is known about possible triggers of equine TSLP expression. HYPOTHESIS/OBJECTIVES To investigate TSLP expression in equine insect bite hypersensitivity (IBH) skin lesions. The capacity of TLR 1-8 ligands (L) and of atopic cytokine milieu as potential triggers of TSLP and of interleukin (IL)-6 as a downstream effector molecule of TLR signalling, were examined in primary equine keratinocyte cultures. ANIMALS Lesional skin from IBH-affected and healthy skin from control-horses (n = 9 each group) was sampled. METHODS AND MATERIALS Keratinocyte cultures were established from six healthy horses and stimulated with TLR 1-8-L, and with IL-4 and tumor necrosis factor-α, to mimic an atopic inflammation cytokine milieu. TSLP and IL-6 gene expression was assessed by quantitative real-time PCR. RESULTS Expression of TSLP was significantly greater in IBH lesions compared to healthy skin. TLR 1-8-L significantly upregulated TSLP expression in keratinocytes. The strongest upregulation was induced by TLR 1/2-L and TLR 3-L. Combination of atopic cytokine milieu and TLR 1/2-L or TLR 3-L further increased TSLP expression. TLR-L 1-5 stimulation significantly upregulated IL-6 expression. CONCLUSIONS AND CLINICAL IMPORTANCE The data herein suggest that the upregulation of TSLP expression in lesional skin of IBH-affected horses might play a role in IBH development. Moreover, TSLP expression is induced by TLR-L, in particular by TLR 1/2-L and TLR 3-L, and is further increased by atopic cytokine milieu, indicating a mechanism for TSLP-mediated exacerbation of IBH

Povezanost inbreedinga i melanoma kod lipicanskih konja

The relationship between inbreeding and melanoma status (graded from 0 to 4) was analysed by various regression models. Analysed data referred to 296 grey Lipizzan horses originating from five state-owned studs (Austria, Croatia, Hungary, Slovakia and Slovenia) and with average inbreeding coefficient (F=0.107) calculated from extremely informative pedigrees (98% and 76% of horses had completely full pedigree in generation 10 and 20, respectively). In all regression models, in addition, the effects of stud (fixed) and age (covariate) were included. When all data were treated as one population, the estimates from linear and ancestral inbreeding models were not significant. Total inbreeding effect estimates (at F=0.125 and Fa=0.57) were 0.26 and 0.30 for the ancestral inbreeding and linear regression models, respectively. Heterogeneity among state-owned studs in inbreeding effects was also tested for both models and weak statistical significance was obtained for the interaction model with ancestral inbreeding (P=0.049). However, observed effect in the model with interaction was not consistent, did not yield in better model fitting and the obtained significance is probably just a statistical artefact. In general, although some indications about the relationship between ancestral inbreeding and melanoma were present, inbreeding does not appear to be a factor that substantially influences the expression of melanoma in Lipizzan horses.Regresijskim modelima analizirana je povezanost stadija melanoma (na skali od 0 do 4) i inbreedinga. Analizirani podaci odnose se na 296 sivih lipicanskih konja iz pet državnih ergela (Austrija, Hrvatska, Mađarska, Slovačka i Slovenija). Prosječna razina inbreedinga (F=0.107) izračunata je na temelju izuzetno informativnog pedigreea (98% jedinki imalo je potpuno poznat pedigre u generaciji 10, a 76% jedinki imalo je potpuno poznat pedigree u generaciji 20). U svim modelima uvažen je i utjecaj ergele (fiksni utjecaj) te starosti konja (kovarijabla). Kada se analiza koeficijenta regresije (regresija stadija melanoma na koeficijent inbreedinga) odnosila na populaciju kao jednu cjelinu, procjena linearnog koeficijenta regresije za inbreeding kao i za inbreeding predaka nije bila signifikantna. Kod razine inbreedinga F=0.125 i Fa=0.57, ukupna procjena linearnog utjecaja inbreedinga bila je 0.30, a utjecaja inbreedinga predaka bila je 0.26. Analizirana je i heterogenost utjecaja inbreedinga između državnih ergela i za interakcijski model sa inbreedingom predaka dobivena je slaba signifikantna interakcija (P=0.049). Ipak, dobiveni utjecaj u model sa interakcijom nije bio konzistentan, nije bolje objašnjavao nerazjašnjenu varijabilnost modela te je dobivena signifikantnost vjerojatno posljedica slučajnosti. Iz navedenog se može zaključiti, premda postoji indicija o povezanosti inbreedinga predaka i melanoma, da kod lipicanskih konja inbreeding nije čimbenik koji utječe na pojavu melanoma

Kinetics of Plasma Cytokines, Angiopoietin-2, and C-Reactive Protein in Dogs With Gastric Dilatation Volvulus.

Background: The degree of systemic inflammation, reperfusion injury and endothelial activation are potentially important determinants of clinical outcomes in dogs with gastric dilatation volvulus (GDV). Objective: To evaluate plasma concentrations and kinetics of inflammatory markers in dogs with GDV over a time frame of 48 h, and to compare to healthy dogs. Design and Setting: Prospective, observational cohort study in client-owned dogs with GDV. Materials and Methods: Fifteen dogs with GDV and 9 healthy control dogs were enrolled. Plasma concentrations of interleukin (IL)-6, IL-7, IL-8, IL-10, IL-15, IL-18, interferon gamma (IFN-γ), keratinocyte chemotactic-like, monocyte chemotactic protein (MCP)-1, Angiopoietin (Ang)-2, and C-reactive protein (CRP) were measured at admission (prior any therapeutic intervention, (T0), immediately after surgery (T1), 24 ± 4 h (T24), and 48 ± 4 h (T48) post-surgery. Cytokines were measured using multiplex magnetic bead assay. Plasma Ang-2 was measured with a commercial human ELISA test kit validated for dogs. Results: Dogs with GDV had significantly higher plasma concentrations of IFN-γ and IL-10 compared to healthy control dogs at all time points. Concentrations of IL-6 were significantly higher at T1 and T24, concentrations of MCP-1 at T24, and concentrations of CRP at T24 and T48. A significant increase between T0 and T1 was found for IL-6, IL-10, and CRP, between T1 and T24 for IL-8, IFN-γ, MCP-1, and CRP, and between T24 and T48 for IL-15, Ang-2, and CRP. A significant decrease between T0 and T1 was found for IL-7, IL-8, IL-15, IL-18, and Ang-2; between T1 and T24 for IL-6 and KC-like; and between T24 and T48 for IL-6. Conclusion: In GDV dogs, a mild pro-inflammatory reaction was present at admission, which peaked immediately after and up to 24 h post-surgery, mainly represented by IL-6, IFN-γ, MCP-1, and CRP, and which decreased at T48. In addition, the anti-inflammatory IL-10 was increased in GDV dogs at all time points

Development of a comprehensive protein microarray for immunoglobulin E profiling in horses with severe asthma

Background: Severe asthma in horses, known as severe equine asthma (SEA), is a prevalent, performance-limiting disease associated with increased allergen-specific immunoglobulin E (IgE) against a range of environmental aeroallergens. Objective: To develop a protein microarray platform to profile IgE against a range of proven and novel environmental proteins in SEA-affected horses. Animals: Six SEA-affected and 6 clinically healthy Warmblood performance horses. Methods: Developed a protein microarray (n = 384) using protein extracts and purified proteins from a large number of families including pollen, bacteria, fungi, and arthropods associated with the horses, environment. Conditions were optimized and assessed for printing, incubation, immunolabeling, biological fluid source, concentration techniques, reproducibility, and specificity. Results: This method identified a number of novel allergens, while also identifying an association between SEA and pollen sensitization. Immunolabeling methods confirmed the accuracy of a commercially available mouse anti-horse IgE 3H10 source (R2 = 0.91). Biological fluid source evaluation indicated that sera and bronchoalveolar lavage fluid (BALF) yielded the same specific IgE profile (average R2 = 0.75). Amicon centrifugal filters were found to be the most efficient technique for concentrating BALF for IgE analysis at 40-fold. Overnight incubation maintained the same sensitization profile while increasing sensitivity. Reproducibility was demonstrated (R2 = 0.97), as was specificity using protein inhibition assays. Arthropods, fungi, and pollens showed the greatest discrimination for SEA. Conclusions and clinical importance: We have established that protein microarrays can be used for large-scale IgE mapping of allergens associated with the environment of horses. This technology provides a sound platform for specific diagnosis, management, and treatment of SEA