Psoriatic skin inflammation induces a pre-diabetic phenotype via the endocrine actions of skin secretome

Abstract

Objective: Psoriasis is a chronic inflammatory skin disease that is thought to affect ∼2% of the global population. Psoriasis has been associated with ∼30% increased risk of developing type 2 diabetes (T2D), with numerous studies reporting that psoriasis is an independent risk-factor for T2D, separate from underlying obesity. Separately, studies of skin-specific transgenic mice have reported altered whole-body glucose homeostasis in these models. These studies imply a direct role for skin inflammation and dysfunction in mediating the onset of T2D in psoriasis patients, potentially via the endocrine effects of the skin secretome on key metabolic tissues. We used a combination of in vivo and ex vivo mouse models and ex vivo human imiquimod (IMQ) models to investigate the effects of psoriasis-mediated changes in the skin secretome on whole-body metabolic function.

Methods: To induce psoriatic skin inflammation, mice were topically administered 75 mg of 5% IMQ cream (or Vaseline control) to a shaved dorsal region for 4 consecutive days. On day 5, mice were fasted for glucose and insulin tolerance testing, or sacrificed in the fed state with blood and tissues collected for analysis. To determine effects of the skin secretome, mouse skin was collected at day 5 from IMQ mice and cultured for 24 h. Conditioned media (CM) was collected and used 1:1 with fresh media to treat mouse explant subcutaneous adipose tissue (sAT) and isolated pancreatic islets. For human CM experiments, human skin was exposed to 5% IMQ cream for 20 min, ex vivo, to induce a psoriatic phenotype, then cultured for 24 h. CM was collected, combined 1:1 with fresh media and used to treat human sAT ex vivo. Markers of tissue inflammation and metabolic function were determined by qPCR. Beta cell function in isolated islets was measured by dynamic insulin secretion. Beta-cell proliferation was determined by measurement of Ki67 immunofluorescence histochemistry and BrDU uptake, whilst islet apoptosis was assessed by caspase 3/7 activity. All data is expressed as mean ± SEM.

Results: Topical treatment with IMQ induced a psoriatic-like phenotype in mouse skin, evidenced by thickening, erythema and inflammation of the skin. Topical IMQ treatment induced inflammation and signs of metabolic dysfunction in sub-cutaneous and epidydimal adipose tissue, liver, skeletal muscle and gut tissue. However, consistent with islet compensation and a pre-diabetic phenotype, IMQ mice displayed improved glucose tolerance, increased insulin and c-peptide response to glucose, and increased beta cell proliferation. Treatment of sAT with psoriatic mouse or human skin-CM replicated the in vivo phenotype, leading to increased inflammation and metabolic dysfunction in mouse and human sAT. Treatment of pancreatic islets with psoriatic mouse skin-CM induced increases in beta-proliferation and apoptosis, thus partially replicating the in vivo phenotype.

Conclusions: Psoriasis-like skin inflammation induces a pre-diabetic phenotype, characterised by tissue inflammation and markers of metabolic dysfunction, together with islet compensation in mice. The in vivo phenotype is partially replicated by exposure of sAT and pancreatic islets to psoriatic-skin conditioned media. These results support the hypothesis that psoriatic skin inflammation, potentially via the endocrine actions of the skin secretome, may constitute a novel pathophysiological pathway mediating the development of T2D.

Keywords: Adipose tissue; Diabetes; Inflammation; Pancreatic islets; Psoriasis; Skin.

Figure 1

IMQ induced an inflammatory phenotype in mouse skin. C57Bl/6 mice were treated with IMQ (3.75 mg IMQ/day) or Vaseline control for 4 days. (A) Representative Day 4 images displaying erythema and scaling (B) daily double-fold skin thickness, n = 45, (C) post-sacrifice spleen weight (n = 6) and (D) Representative H&E staining of mouse skin. (E) Epidermal thickness of mouse skin calculated from H+E staining. 6 sections per mouse were stained and n = 3–4 mice were used. Four measurements were taken per image. (F–G) qPCR analysis of inflammatory gene expression in mouse skin, n = 6–8. Data is expressed as mean ± SEM. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001, vs. Vaseline control.

Figure 2

Topical IMQ application induces inflammation and markers of metabolic dysfunction in mice. Tissue and serum were obtained from C57Bl/6 mice treated with IMQ (3.75 mg IMQ/day) or Vaseline control for 4 days. (A–H) Gene expression of inflammatory and metabolic markers was assessed by qPCR in sAT (A–B), eAT (C–D), liver (E–F) and skeletal muscle (G–H), n = 4–10; (I) Triglyceride levels were assessed in liver and skeletal muscle by colorimetric assay; (J–M) Serum cytokine concentrations were measured by MSD U-plex assay; (J) TNFα, (K) IL6, (L) IL1β and (M) IL17A (n = 12). Black bars = Vaseline control, blue bars = IMQ. Data is expressed as mean fold change from controls ± SEM for gene expression and mean ± SEM for serum and tissue TG analysis. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 vs. Vaseline control.

Figure 3

Glucose tolerance and β-cell proliferation are enhanced in IMQ mice. C57Bl/6 mice were treated with IMQ (3.75 mg IMQ/day) or Vaseline control for 4 days. Glucose response to IPGTT over (A) 2-hours and (B) 30 min. (C) Plasma insulin and (D) plasma C-peptide levels from terminal blood samples taken at 30 min post-glucose administration, (n = 6–7). (E) Representative immunofluorescence images of pancreata stained for insulin (red), Ki67 (green) and DAPI (blue) (F) percentage of Ki67 positive β-cells, (G) β-cell area, and (H) β-cell number, n = 7. (I) Caspase-Glo® 3/7 apoptosis assay using IMQ- and Vaseline-mouse islets, n = 4–5. (J) Dynamic glucose-stimulated insulin secretion using isolated islets from IMQ- and Vaseline-mice (four channels were used per treatment using islets pooled from six mice for each experiment, results are pooled from n = 2 experiments), and (K) area under the curve for the first 30 min, n = 5. Data is expressed as mean ± SEM. ∗P < 0.05, ∗∗P < 0.01, ∗∗P < 0.01, ∗∗∗P < 0.001.

Figure 4

Effects of skin-derived factors on sAT inflammation and dysfunction and pancreatic beta-cell functional mass. Mouse sAT was incubated in mouse skin CM for 48 h. Human sAT was incubated in human skin CM for 24 h, and then in serum-free DMEM for a further 24 h. Gene expression of inflammatory and metabolic markers was assessed by qPCR in (A) mouse sAT and (B–C) human sAT. (D) Phospho(Ser47) AKT levels were assessed using a whole-cell lysate kit (Mesoscale Discovery) in human sAT. For mouse sAT, n = 4 mice. For human results n = 4 collections of sAT from four different donors. (E–K) Mouse islets were incubated for 24 h with skin CM collected from cultured Vaseline or IMQ treated mouse skin. (E) Islet apoptosis, n = 11; (F–H) Beta-cell BrDU uptake, 150–200 islets pooled from six mice were used for each treatment group. (F) Islets were stained for insulin (red) and BrDU (green); (G) percentage of BrDU positive β-cells and (H) β-cell area. Representative images for each treatment group are also shown (F). (I) Dynamic glucose-stimulated insulin secretion using isolated islets treated with IMQ-mouse skin CM for 24 h. Four channels were used per treatment using islets pooled from six mice. Perifusion results are presented from two pooled experiments (J) Area under the curve calculated from (I). (K) Islet insulin content, n = 3–6 replicates per treatment group. Data is expressed as mean ± SEM. White bars = Vaseline control, blue bars = IMQ. Data is expressed as mean fold change vs controls ± SEM. ∗∗P < 0.01, ∗∗∗P < 0.001 vs Control.

Figure 5

Schematic of proposed hypothesis showing how skin inflammation contributes to whole-body glucose metabolism.