Interleukin-17 pathway activation inEquus caballussupporting limb laminitis

Abstract

22 Supporting Limb Laminitis (SLL) is a painful and crippling secondary complication of 23 orthopedic injuries and infections in horses, often resulting in euthanasia. Due to altered weight 24 bearing, SLL causes structural alternations and inflammation of the interdigitating layers of 25 specialized epidermal and dermal tissues, the lamellae, which suspend the equine distal phalanx 26 from the hoof capsule. Activation of the interleukin-17 (IL-17)-dependent inflammatory pathway 27 is an epidermal stress response that contributes to physiologic cutaneous wound healing as well 28 as pathological skin conditions. To test the hypothesis that IL-17 pathway activation is involved 29 in equine epidermal lamellae in SLL, we analyzed the expression of the IL-17 receptor subunit A 30 and 11 genes upregulated by IL-17 in lamellar tissue isolated from Thoroughbreds euthanized 31 due to naturally occurring SLL and in age and breed matched non-laminitic controls. The IL-17 32 Receptor A subunit was expressed in both non-laminitic and laminitic tissues. In severe acute 33 SLL (n=7) compared to non-laminitic controls (n=8), quantitative PCR demonstrated ~20-100 34 fold upregulation of ß defensin 4 (E. caballus gene DEFB4B) and S100A9 genes. DEFB4B was 35 also upregulated in developmental (n=8), moderate acute (n=7), and severe chronic (n=5) 36 samples. By RT-PCR, S100A8, MMP9, and PTSG2 (COX2) expression was upregulated in most 37 or all severe acute SLL samples, whereas several other genes, CCL2, CxCL8, TNF, IL6 and 38 MMP1 were detected in some, but not all, severe acute samples. PTGS2, CCL2, TNF and IL6 39 were also expressed in some, but not all, developmental and moderate acute disease stages.40 Moreover, expression of DEFB4 by in situ hybridization and calprotectin (S100A9/S100A8) 41 protein by immunofluorescence was detected in keratinocytes, primarily in suprabasal cell 42 layers, from SLL samples. These data support the hypothesis that the IL-17 inflammatory 3 43 pathway is active in equine SLL, and that similarities exist between equine and human epidermal 44 tissue responses to stresses and/or damage. 4 45 Introduction 46 47 Equine laminitis is a common, progressive, crippling and currently incurable disease 48 often necessitating euthanasia to end suffering from the painful loss of limb support. Healthy 49 lamellar tissue connects the inner hoof wall to the distal phalanx (DP), forming a major 50 component of the suspensory apparatus of the DP, a unique adaptation of equids that allows for 51 suspension of almost the entire weight of the animal through the hoof capsule [1; 2]. The hoof is 52 a modified epidermal appendage integrated into the musculoskeletal system by the epidermal and 53 dermal lamellae, homologous to the nail bed, which are extensively folded to form primary and 54 secondary lamellae to increase the surface area of attachment (Fig 1; [3,4]). Similar to skin, 55 keratin 14 is expressed within lamellar epidermal basal cells [5-7]; however equine epidermal 56 lamellae have several architectural and ...