| We hypothesized inflammatory responses in the sole corium of bovine claws disturbed epidermal-dermal interactions. The objectives of this investigation were to (1) determine the effect of sterile and septic sole ulcer lesions on transcriptional expression of growth factors, signaling elements, AP-1 transcription factors and cell-cycle transition elements in epidermal-dermal tissues of the bovine sole corium, (2) determine the effect of sterile and septic sole ulcer lesions on transcriptional expression of markers of keratinocyte differentiation in the sole corium, (3) compare and contrast gene expression of products that orchestrate proliferative and differentiation programs in dermalepidermal elements of the ulcerated lateral claw sole corium, the lesion free medial claw sole corium from lame limbs, and the lateral claw sole corium of the completely normal limbs, and (4) determine the effect of sterile and septic sole ulcer lesions on inflammasome platform transcription, inflammasome body activation and induction of an interleukin 1 response. Tissues were obtained from mature, lactating, Holstein cows from the abattoir. Sole tissues from ulcerated lateral claws, the lesionless medial claw off the ulcerated limbs, and lateral claws of lesion-free hind limbs were excised. Groups included lesions associated with grades 2/3 or grades 4/5 ulcers. Transcripts were quantified by real-time polymerase chain reaction (RT-PCR) and data normalized to endogenous ubiquitin expression. No bacteria were recovered from ulcer lesions (n=6) of lateral claws with an intact sole horn and declared sterile. Cell-cycle transition elements cyclin D, cdk2, cdk4 and cdk6, the stem cell transcription factor p63, the tumor suppressor p21 and cell-cycle inhibitor sigma 14-3-3 were down-regulated in the ulcerated lateral claw and lesion free medial claw of the ulcerated limb. Similarly, PDGF, VEGF, GMCSF, KGF, IGF1 and IGF2, HB-EGF, and TGF-beta and their receptors PDGFR, KGFR, IGF1R, IGF2R and EGFR were down-regulated while GMCSFR was elevated in ulcers and lesion free medial sole coriums. The signal elements c-myc, fos, c-jun, junB, and junD were down-regulated across the ulcerated corium as well as the lesionless medial claw sole corium. Loricrin and involucrin were down-regulated in the ulcer and matching lesion-free medial claw. Cytokeratin 10 expression was diminished in the lesion while serine palmatoyl CoA transferase was diminished only in the medial claw. Cytokeratin 6 and beta1 integrin were elevated across the lesion and the medial claw. Disturbances in proliferative and differentiation mRNAs were associated with increased expression of metalloproteinase 2, metalloproteinase 9, IL-1beta, and IL-1 receptor antagonist in the ulcer. The biologically active cleavage products of both caspase-1 and IL-1beta were elevated in the lesion as well as the medial claw. Ulcer lesions of the corium associated with breached sole horn were contaminated with manure and considered septic. Expression of cdk4, cdk6, p21, and p63 were diminished across the center, margin and the surrounding uninvolved tissue of the septic ulcer as well as the medial claw. Cyclin D decreased across all three regions of the ulcer lesion and cdk2 was elevated across all zones of the lesion. Cyclin B increased in the ulcer center and the lesionless medial claw corium. KGF increased in all zones of the wound as well as in the medial claw. IGF1 increased in the uninvolved tissues proximal to the ulcer. VEGF, IGF2, HBEGF and receptors VEGFR, IGF1R, IGF2R, KGFR and EGFR were diminished across all regions of the ulcer as well as in the medial claw. TGF-alpha decreased in all regions of the lesion and medial claw except in tissue proximal to the wound. PDGF decreased in the uninvolved tissues next to the ulcer and the medial claw corium. GMCSFR increased across all regions of the ulcer and the medial claw. C-myc, fos, c-jun, and jun D decreased across all regions of the ulcer as well as the medial claw. JunB decreased in the medial claw. Involucrin, cytokeratin 14 ,cytokeratin 10 and transglutaminase decreased across all three regions of the ulcer. Similarly, serine palmatoyl CoA transferase 1 and 2 were also down-regulated in the medial claw as well as across the wound. Ceramide glycosyl transferase and loricrin decreased in the tissues adjacent to the ulcer and the medial claw. Bulbous pemphigus protein 180 (BP 180) was down-regulated in the center and margin while alpha6, beta1, and beta4 were decreased across all regions of the ulcer and the medial claw. Disturbances in proliferative and differentiation gene expression patterns were associated with increased metalloproteinase 2, IL-1RI and IL-1RII across all regions of the ulcer. Metalloproteinase 9 and IL-1alpha increased in the ulcer center while IL-1beta, pycard and caspase-1 increased in the ulcer center and margin. Septic ulcers demonstrated increased amounts of the biologically active caspase-1 and IL-1beta. These data indicate ulcer lesions in coriums of claws with intact sole horn are sterile inflammatory responses associated with inflammasome body and IL-1 activation, metalloproteinase expression and depressed proliferative and differentiative processes orchestrating wound repair and sole horn production. Ulcer progression leads to breached claw horn and septic inflammatory responses. Septic ulcers showed characteristic similar to that of non-healing, chronic wounding. Moreover, the lesion-free corium from medial claws showed patterns of expression in the same direction and often same magnitude as in the ulcer. Sole ulceration appears to begin with sterile inflammatory responses that sufficiently disturb epidermal-dermal interactions orchestrating claw horn to generate open, septically inflamed, non-healing wounds. Similar patterns in the lesion-free medial claw indicate corium epidermal-dermal interactions are disturbed across both claws of the limb. |
