Heat shock protein B1-deficient mice display impaired wound healing

Abstract

There is large literature describing in vitro experiments on heat shock protein (hsp)B1 but understanding of its function in vivo is limited to studies in mice overexpressing human hspB1 protein. Experiments in cells have shown that hspB1 has chaperone activity, a cytoprotective role, regulates inflammatory gene expression, and drives cell proliferation. To investigate the function of the protein in vivo we generated hspB1-deficient mice. HspB1-deficient fibroblasts display increased expression of the pro-inflammatory cytokine, interleukin-6, compared to wild-type cells, but reduced proliferation. HspB1-deficient fibroblasts exhibit reduced entry into S phase and increased expression of cyclin-dependent kinase inhibitors p27(kip1) and p21(waf1). The expression of hspB1 protein and mRNA is also controlled by the cell cycle. To investigate the physiological function of hspB1 in regulating inflammation and cell proliferation we used an excisional cutaneous wound healing model. There was a significant impairment in the rate of healing of wounds in hspB1-deficient mice, characterised by reduced re-epithelialisation and collagen deposition but also increased inflammation. HspB1 deficiency augments neutrophil infiltration in wounds, driven by increased chemokine (C-X-C motif) ligand 1 expression. This appears to be a general mechanism as similar results were obtained in the air-pouch and peritonitis models of acute inflammation.

Figure 1. HspB1 deficiency increases IL-1-induced IL-6 expression and inhibits proliferation in fibroblasts.

A, Primary wild-type and hspB1 ^del/del MEF were treated with IL-1 (20 ng/ml) for 4 h or left untreated. Graph shows the concentration of IL-6 in culture medium as determined by ELISA and normalised against values for IL-1-treated wild-type MEF for three separate batches of cells (*P<0.05). B, MEF were treated as in (A), lysed, RNA extracted and IL-6 and GAPDH mRNAs quantified by qRT-PCR. Plot shows IL-6 mRNA/GAPDH mRNA normalised to the value for wild-type cells treated with IL-1 for 1 h. C, Growth curve analysis (means±SEM) of wild-type and hspB1 ^del/del MEF determined by MTT assay at different days post-seeding (n = 3; *P<0.05, **P<0.01, ***P<0.001) or by counting trypan-blue excluded cells (n = 2). D, Plot of mean (%) TUNEL-positive (± SEM) cells for three different batches of MEF per genotype. E, MEF were treated with different concentrations of doxorubicin (as indicated) for 8 h to induce apoptosis, or left untreated, cells lysed and lysates analysed by western blot for the full-length and the cleaved form of PARP. Similar results were obtained in three independent experiments.

Figure 2. HspB1 deficiency inhibits entry into S phase and increases the expression of p21^waf1 and p27 ^kip1.

A, BrdU incorporation following a 2-positive cells (mean % ± SEM) from three independent experiments performed; **P<0.01. C, Western blot of asynchronous MEF lysates for p21^waf1, p27^kip1, hspB1 and GAPDH as a loading control with molecular weights (kDa) of markers indicated. Western blots are representative of three independent experiments.

Figure 3. Expression of hspB1 protein and mRNA is controlled by the cell cycle.

A, MEF were synchronized by serum starvation for 48% FCS-containing medium and analysed by western blot for hspB1, PCNA, p27^kip1 and actin. B, Comparison of the expression of mRNAs for hspB1 and the cell cycle-regulated genes, Myc, and Cyclin E1 determined by qRT-PCR and normalized to GAPDH in a representative synchronized MEF serum release time course. C, Western blot for hspB1, PCNA and loading control, actin, in lysates of MEF following release from nocodazole G2/M block (40 ng/ml). All western blots shown are representative of at least two independent experiments.

Figure 4. HspB1 deficiency impairs excisional cutaneous wound healing.

A, Four 4–14 week-old female wild-type and hspB1 ^del/del mouse. Digital images were taken of each wound at d0, d3, d5 and d7 post-wounding (n = 3–4 mice). Individual wound areas were tracked over time and plotted as a percentage of the intial individual lesion areas±SD (*P<0.05, **P<0.01, ****P<0.0001). B, Digital images of wounds (representative of ≥14 wounds); bar = 2 mm.

Figure 5. HspB1 protein expression in unwounded and wounded murine skin.

A, Unwounded female wild-type skin was stained with Masson’s trichrome; (bar = 100 µm). B, HspB1 was detected by IHC in unwounded female wild-type. Inset: High power image showing hspB1 staining in cells with fibroblast-like morphology in connective tissue beneath panniculus carnosus. C, Unwounded female hspB1 ^del/del skin showing lack of staining with anti-hspB1 antibody. Inset as for (B); (bar = 10 µm). D, HspB1 staining at d3 post-wounding showing expression in epithelial tongues and cells in granulation tissue (representative wounds from 11 mice in three experiments) E, High power image of boxed region indicated in (D) showing hspB1 expressing cells with fibroblast-like morphology in granulation tissue. F, hspB1 staining at d7 showing expression in newly formed muscle, and epithelium (representative wounds from 7–8 mice in two experiments); (bar = 500 µm). G, High power image of boxed region indicated in (F) showing expression of hspB1 in newly formed skeletal muscle and microvasculature; (bar = 10 µm). 11–14 week age-matched female wild-type mice were used.

Figure 6. Histological analysis showing reduced re-epithelialisation, impaired collagen deposition and increased cellular infiltration in hspB1 ^del/del relative to wild-type wounds.

A, Masson’s trichrome staining of wild-type and hspB1 ^del/del d3 and B, d7 wounds as in Fig. 5; bar = 500 µm. C, High power images of cellular infiltrate from boxed regions indicated in (A) are shown (bar = 100 µm). D, as for (C) but at higher magnification showing cells with multi-lobed nuclei characteristic of neutrophils; (bar = 20 µm).E, Plot showing mean areas of incomplete collagen deposition in d7 wild-type and hspB1 ^del/del wounds (n = 5); **P<0.01. F, Plot of mean distance±SEM (n = 3 mice per group) between re-epithelialisation margins (re-ep) in wild-type and hspB1 ^del/del mice in d3 wounds; (*P<0.05 calculated by Student’s t-test of 12 individual wounds per group from 3 experiments). 11–14 week age-matched female wild-type and hspB1 ^del/del mice were used.

Figure 7. HspB1 deficiency results in increased neutrophil but slightly decreased macrophage infiltration of wounds.

A, Neutrophil infiltration in d1 wound granulation tissue assessed by IHC for neutrophil elastase (NE) and macrophage infiltration at d3 detected by F4/80 staining. B, Plot shows elastase positive neutrophils (NE+) per high power field (hpf); 7 fields/wound; 2 wounds per mouse; n = 4 mice; **P<0.01. B, Plot as for (B) showing F4/80-positive cells/hpf; *P<0.05.

Figure 8. HspB1 deficiency promotes cytokine expression at 6-wounding.

A, Plots of cytokine protein expression in homogenised 6(wounded) and distal unwounded tissue (distal) measured by ELISA and expressed as amount of cytokine protein/total protein in wound tissue sample analysed (**P<0.01; *P<0.05). B, Plot of CCL2 protein expression as in (A) but for d1 and d2 post-wounding.

Figure 9. HspB1 deficiency increases neutrophil infiltration and chemokine expression in the zymosan-induced air-pouch and peritonitis models of acute inflammation.

A, Air-pouches were created on the dorsal surfaces of 10–12 week old male wild-type (closed circles) hspB1 ^del/del (open circles) mice and were injected with 100 µl of 1 mg/ml zymosan. Exudates were retrieved at the indicated times. Plot of number of trypan blue-negative infiltrating cells counted with a haemocytometer (n = 5–6 mice per group for 2 h post-zymosan; n = 9–14 from two experiments each for all other times). Plots of CXCL-1, and CXCL-2 protein in exudate supernatants measured by ELISA at 1 h post-zymosan (n = 9 from two experiments). Graphs show mean±SEM. B, 1 ml of zymosan (1 mg/ml) was injected into the peritoneal cavities of 10–12 week-old male wild-type and hspB1 ^del/del mice. Plot of number of infiltrating cells with time of zymosan treatment indicated (n = 4–6) and CXCL-1 and CXCL-2 protein in exudates at as for (A) (n = 10–12 from two experiments); *P<0.05, **P<0.01, ***P<0.001.