Peptidylarginine deiminase 2 (PAD2/PADI2) has been implicated in various inflammatory diseases and, more recently, cancer. The goal of this study was to test the hypothesis that PAD2 promotes oncogenesis using a transgenic mouse model. We found that about 37% of transgenic mice overexpressing human FLAG-PAD2 downstream of the MMTV-LTR promoter develop spontaneous neoplastic skin lesions. Molecular and histopathologic analyses of the resulting lesions find that they contain increased levels of markers for invasion, inflammation, and epithelial-to-mesenchymal transition (EMT) and that a subset of the lesions progress to invasive squamous cell carcinoma (SCC). We then stably overexpressed FLAG-PAD2 in the human SCC cell line, A431, and found that the PAD2-overexpressing cells were more tumorigenic in vitro and also contained elevated levels of markers for inflammation and EMT. Collectively, these studies provide the first genetic evidence that PAD2 functions as an oncogene and suggest that PAD2 may promote tumor progression by enhancing inflammation within the tumor microenvironment. Cancer Res; 74(21); 6306-17. Ó2014 AACR.

Insect bite hypersensitivity (IBH) is a Th-2, IgE-mediated dermatitis of horses caused by bites of insects of the genus Culicoides that has common features with human atopic dermatitis. Together with Th-2 cells, the epithelial barrier plays an important role in development of type I hypersensitivities. In order to elucidate the role of the epithelial barrier and of the skin immune response in IBH we studied the transcriptome of lesional whole skin of IBH-horses (IBH-LE; n = 9) in comparison to non-lesional skin (IBH-NL; n = 8) as well as to skin of healthy control horses (H; n = 9). To study the "baseline state" of the epithelial barrier, we investigated the transcriptome of non-lesional epidermis in IBH-horses (EPI-IBH-NL; n = 10) in comparison with healthy epidermis from controls (EPI-H; n = 9). IBH-LE skin displayed substantial transcriptomic difference compared to H. IBH-LE was characterized by a downregulation of genes involved in tight junction formation, alterations in keratins and substantial immune signature of both Th-1 and Th-2 types with particular upregulation of IL13, as well as involvement of the hypoxic pathway. IBH-NL shared a number of differentially expressed genes (DEGs) with IBH-LE, but was overall more similar to H skin. In the epidermis, genes involved in metabolism of epidermal lipids, pruritus development, as well as IL25, were significantly differentially expressed between EPI-IBH-NL and EPI-H. Taken together, our data suggests an impairment of the epithelial barrier in IBH-affected horses that may act as a predisposing factor for IBH development. Moreover, these new mechanisms could potentially be used as future therapeutic targets. Importantly, many transcriptional features of equine IBH skin are shared with human atopic dermatitis, confirming equine IBH as a natural model of skin allergy.

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.

Purpose of Review Treatment of equine insect bite hypersensitivity (IBH) needs to be improved. Allergen-specific immunotherapy (ASIT), the only curative treatment of allergy, currently has only a limited efficacy for treatment of IBH. This review highlights the latest findings in prophylactic and therapeutic strategies. Recent Findings Prophylactic vaccination against IBH using recombinant Culicoides allergen has been developed in unexposed Icelandic horses and is ready to be tested. Therapeutic virus-like particle (VLP)-based vaccines targeting equine interleukin-(IL-) 5 or IL-31 improved clinical signs of IBH by induction of anti-cytokine antibodies thus reducing eosinophil counts or allergic pruritus, respectively. Summary First studies for development of ASIT using pure r-Culicoides allergens have yielded promising results and need now to be tested in clinical studies for both prevention and treatment of IBH. Therapeutic vaccines inducing neutralizing antibodies against IL-5 or IL-31 will be valuable future treatments for reduction of clinical signs of IBH.

Background: Canine intervertebral disc disease (IVDD) represents a significant clinical problem in veterinary medicine, with similarities to the human pathology. Host-derived damage-associated molecular patterns like fibronectin fragments (FnF) that develop during tissue dysfunction may be of specific relevance to IVD pathologies by inducing an inflammatory response in resident cells. Aim: This project aimed to determine the presence and pathobiological role of FnF during IVD herniation in dogs, with a focus on inflammation. Methods: Herniated nucleus pulposus (NP) material from five dogs as well as non-herniated adjacent NP material from three dogs was collected during spinal surgery required due to acute IVD herniation. The presence of different types of FnF were determined by Western blot analysis. NP cells isolated from six herniated canine IVDs were then exposed to 30 kDa FnF. NP cell inflammation and catabolism was examined by investigating the expression of IL-1β, IL-6, IL-8, and COX-2, as well as MMP-1 and MMP-3 by qPCR (all targets) and ELISA (IL-6, PGE 2 ). Results: Amongst multiple sized FnF (30, 35, 45, and >170kDa), N-terminal fragments at a size of ~30 kDa were most consistently expressed in all five herniated IVDs. Importantly, these fragments were exclusively present in herniated, but not in non-herniated IVDs. Exposure of canine NP cells to 500 nM 30 kDa FnF caused a significant upregulation of IL-6 (62.5 ± 79.9, p = 0.032) and IL-8 (53.0 ± 75.7, p = 0.031) on the gene level, whereas IL-6 protein analysis was inconclusive. Donor-donor variation was observed in response to FnF treatment, whereby this phenomenon was most evident for COX-2, with three donors demonstrating a significant downregulation (0.67 ± 0.03, p = 0.003) and three donors showing upregulation (6.9 ± 5.5, p = 0.21). Co-treatment with Sparstolonin B, a TRL-2/TRL-4 antagonist, showed no statistical difference to FnF treatment alone in all tested target genes. Conclusion: Given the presence of the 30 kDa FnF in canine herniated IVDs and the proinflammatory effect of 30 kDa FnF on NP cells, we concluded that the accumulation of FnF may be involved in the pathogenesis of canine IVDD. These results correspond to the findings in humans with IVDD.