Dynamic expression of epidermal caspase 8 simulates a wound healing response

Abstract

Tissue homeostasis and regeneration are regulated by an intricate balance of seemingly competing processes-proliferation versus differentiation, and cell death versus survival. Here we demonstrate that the loss of epidermal caspase 8, an important mediator of apoptosis, recapitulates several phases of a wound healing response in the mouse. The epidermal hyperplasia in the caspase 8 null skin is the culmination of signals exchanged between epidermal keratinocytes, dermal fibroblasts and leukocytic cells. This reciprocal interaction is initiated by the paracrine signalling of interleukin 1alpha (IL1alpha), which activates both skin stem cell proliferation and cutaneous inflammation. The non-canonical secretion of IL1alpha is induced by a p38-MAPK-mediated upregulation of NALP3 (also known as NLRP3), leading to inflammasome assembly and caspase 1 activation. Notably, the increased proliferation of basal keratinocytes is counterbalanced by the growth arrest of suprabasal keratinocytes in the stratified epidermis by IL1alpha-dependent NFkappaB signalling. Altogether, our findings illustrate how the loss of caspase 8 can affect more than programmed cell death to alter the local microenvironment and elicit processes common to wound repair and many neoplastic skin disorders.

Figure 1. Effect of caspase-8 downregulation in the epidermis

A. In situ hybridization of Caspase-8 RNA on 0, 1, 7 and 14 days after excisional wounds. Arrows denote wound sites. Distal (>5mm) and proximal (<1mm) refer to the distance from the wound site. B. Differentiation in the wild type (WT) and knockout (KO) epidermis. K5, K1, and loricrin stain the basal, spinous, and granular layers respectively. Dotted line denotes the basement membrane. C. Hyperproliferation of the epidermis in the KO skin is revealed by increased expression of Ki67.

Figure 2. Characterization of the inflammatory response in the KO skin

A. Increased number of granulocytes (Gr-1) and macrophages (MAC-1) are detected in KO skin. B. Pan (CD3), helper (CD4), cytotoxic (CD8), and γδ (γδTCR) T-cell markers are increased in the KO skin and infiltrate the epidermis (arrowheads). Insets are magnified views of γδT-cell morphology. Epidermal derived pool of γδT cells is stained with Vγ3. C. Quantitative RT-PCR of pro-inflammatory NFkB target genes in WT (orange) and KO (blue) skin of 10-day old mice and wounded skin (purple) samples three days after wounding.

Figure 3. Control of keratinocyte proliferation through epithelial-mesenchymal interactions

A. Expression of phospho-ERK (p-ERK), loricrin and K5 in serial sections from P10 WT and KO skin and skin three days post wounding. B. Extracellular IL-1α is detected by staining in the absence of detergent (denoted as unpermeabilized [unperm.] samples). Detergent was incorporated in the staining protocol to visualize total IL-1α levels in the skin (denoted as permeabilized [perm.] samples). C. Primary keratinocyte growth rate was measured over four days after incubation in caspase-8 WT dermal conditioned media (der CM) in blue, KO der CM (red) or normal growth media (green). D. Keratinocyte growth was measured after incubation in CM from dermal fibroblasts (df) that were primed with either wild type epidermis exposed media (WT epi + df CM; light blue), media incubated with caspase-8 null epidermis (KO epi + df CM; red [***]), normal media (media; green), normal media supplemented with recombinant human IL-1α (media + rhIL-1α; orange), df CM primed with WT epidermal media treated with rhIL-1α (WT epi + rhIL-1α + df CM; dark blue [*]). *-p<0.05; ***-p<0.001 compared to the media which was used as a control. E. Keratinocyte growth rate after incubation in normal media (green), df CM primed with KO epi CM and either control antibody (KO epi + df + con Ab; red), IL-1α inhibitory antibody (KO epi + df + inh. Ab; purple) or recombinant human IL-1α (which is not inhibited by the antibody) added (KO epi + df + inh. Ab + rhIL-1α; pink). F. WT epi CM (light blue), KO epi CM (dark blue) or normal media (green) was added to primary keratinocytes and growth rate measured. Addition of recombinant human IL-1α to normal media (media + rhIL-1α; red) is sufficient to inhibit growth. G. Skin sections from 10 day old caspase-8 WT and KO mice and adult mouse skin three days after excisional wounding were stained for pNFkB, its target gene Bcl-XL (green) and K5 (red). Arrowhead marks wound site. H. WT epi CM and KO epi CM were added to primary keratinocytes and stained for NFκB nuclear translocation. Inhibitory IL-1α antibody (Inh. Ab) which specifically neutralizes the mouse cytokine and/or rhIL-1α was added to the KO epi CM. Keratinocytes were also treated with KO epi CM, WT der CM, KO der CM or a neutralizing antibody specific for mouse IL-1β. Results are representative of three independent experiments.

Figure 4. Mechanism regulating IL-1α secretion

A. Quantitative RT-PCR for caspase-1 in P10 WT and KO skin, and wounded skin (wound) three days after excision. Immunofluorescence of caspase-1 in P10 wild type (WT) and knockout (KO) skin, and wounded skin (wound) three days after excision. B. Expression of caspase-1 in wild type, knockout, and wounded skin. C. IL-1α ELISA from media conditioned by wild type (orange) or knockout (blue) epidermal sheets treated with either DMSO, or inhibitors for caspase-1, p38MAPK, or calpain. Note: the efficacy of the calpain inhibitor was verified by its ability to inhibit processing of filaggrin in differentiating keratinocytes (Supp. Fig. 7). D. Expression of phosphorylated p38 MAPK [(P)-p38] in P10 WT and KO skin and three days post wounding. E. Western blot of caspase-1 and β-actin from cell lysates generated from the epidermal sheets in (C). F. In situ hybridization of NALP3 RNA on P10 WT, KO, and 3 day post-wounded skin sections. G. Quantitative RT-PCR of inflammasome components using RNA isolated from epidermal sheets of WT and KO skin treated with DMSO vehicle control or p38 MAPK inhibitor (p38 inh) for 16 hours. H. Quantitative RT-PCR on RNA from WT epidermal sheets treated with DMSO vehicle control, anisomycin (aniso), or anisomycin + p38 MAPK inhibitor (aniso + p38 inh) for two hours. I. Measurement of IL-1α secretion into conditioned media from WT epidermal sheets treated with DMSO, anisomycin (aniso), p38 inhibitor (p38 inh), and/or caspase-1 inhibitor (casp1 inh). KO epidermal sheets treated with DMSO (KO DMSO) is used as comparison.