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

Abstract

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.

Fig 1. Primary equine keratinocyte culture: Primary equine keratinocytes were stained with anti-cytokeratin.

(A) and (C) Staining of primary keratinocyte cultures with anti-cytokeratin: Cytoplasmic cytokeratin is shown in pink; nuclei were counterstained with Hoechst and are shown in blue; (A) 20x magnification; (C) 40x magnification. (B) and (D) Staining of keratinocytes with anti-vimentin: No staining was observed with this antibody. Nuclei are shown in blue; (B) 20x magnification; (D) 40x magnification.

Fig 2. Summary of the experimental setup.

Fig 3

A-F. Volcano plots of significant DEGs in following comparisons: Non-stimulated (NS) vs. recombinant allergen pool stimulated, in IBH-KER (A) and H-KER (B). NS vs. C. nubeculosus WBE stimulated in IBH-KER (C) and H-KER (D). NS vs. ACM stimulated in IBH-KER (E) and H-KER (F).

Fig 4

A-B Hierarchical clustering gene expression of top 30 genes of allergic cytokine milieu-stimulated and non-stimulated samples from the two compared conditions in both IBH-KER (A) and H-KER (B). Lower mean counts are shown in light green and higher mean counts in dark blue.

Fig 5

A-B. Volcano plots of significant DEGs in following comparisons: Non-stimulated (NS) vs.TLR 1/2 ligand (Pam3CSK4) of IBH-KER (A) and H-KER (B).

Fig 6

A-B. Hierarchical clustering gene expression of top 30 genes of TLR 1/2 ligand (Pam3CSK4)-stimulated and non-stimulated samples from following conditions: IBH-KER (A) and H-KER (B). Lower mean counts are shown in light green and higher mean counts in dark blue.