Psychological stress downregulates epidermal antimicrobial peptide expression and increases severity of cutaneous infections in mice

Abstract

The skin is the first line of defense against microbial infection, and psychological stress (PS) has been shown to have adverse effects on cutaneous barrier function. Here we show that PS increased the severity of group A Streptococcus pyogenes (GAS) cutaneous skin infection in mice; this was accompanied by increased production of endogenous glucocorticoids (GCs), which inhibited epidermal lipid synthesis and decreased lamellar body (LB) secretion. LBs encapsulate antimicrobial peptides (AMPs), and PS or systemic or topical GC administration downregulated epidermal expression of murine AMPs cathelin-related AMP and beta-defensin 3. Pharmacological blockade of the stress hormone corticotrophin-releasing factor or of peripheral GC action, as well as topical administration of physiologic lipids, normalized epidermal AMP levels and delivery to LBs and decreased the severity of GAS infection during PS. Our results show that PS decreases the levels of 2 key AMPs in the epidermis and their delivery into LBs and that this is attributable to increased endogenous GC production. These data suggest that GC blockade and/or topical lipid administration could normalize cutaneous antimicrobial defense during PS or GC increase. We believe this to be the first mechanistic link between PS and increased susceptibility to infection by microbial pathogens.

Figure 1. PS downregulates epidermal AMP expression.

Normal hairless mice (n = 3 each for immuno­histochemistry and RT-PCR studies; 3–4 replications for each experiment in these and subsequent experiments) were exposed to insomnia- and crowding-induced PS (B and E) for 72 h, while littermate controls (A and D) were not stressed. Frozen sections (8 μm) were stained with primary antibodies to CRAMP and mBD3 and processed as described in Methods (for controls, see Supplemental Figure 1). Throughout the figures, white arrows indicate normal levels of positive immunostaining (green); white arrowheads indicate reduced staining; asterisks indicate positive immunostaining of pilosebaceous follicles; and “d” and “e” indicate dermis and epidermis, respectively. (C and F) mRNA was extracted from PS and control mouse epidermis, followed by quantitative RT-PCR (see Methods). Normalization in this and all subsequent studies was to 18S mRNA, with 2–3 replicates per sample (n = 3 per cohort). Scale bars: 50 μm. *P = 0.007.

Figure 2. Systemic steroid-induced downregulation of epidermal AMPs mimics effects of PS.

Cohorts of normal hairless mice (n = 3 per cohort) received intraperitoneal dexamethasone (dex; 450 μg/cell/kg daily for 3 d, C and F) or vehicle alone (daily for 3 d, B and E) or were left untreated (A and D). Biopsies were obtained for immunostaining for CRAMP and mBD3 (see Methods). Scale bar: 50 μm.

Figure 3. Superpotent topical steroid also downregulates epidermal AMPs.

Normal hairless mice (n = 3 per group) were treated topically with either clobetasol (0.05%, B and D) or vehicle (60 μl to a 3-cm² area, A and C) once daily for 3 d. Frozen sections (8 μm) were immunostained for either CRAMP (A and B) or mBD3 (C and D) (see Methods). Scale bar: 50 μm.

Figure 4. PS-induced downregulation of epidermal AMPs is reversed by blockade of endogenous GC production and/or action.

Hairless mice (n = 3 per group) were exposed to PS with concurrent intraperitoneal administration of either RU-486 (C and G), antalarmin (ant; D and H), or vehicle (B and F). For dosage, timing, and drug concentrations, see Methods. Frozen sections (8 μm) were immunostained with CRAMP (A–D) or mBD3 (E–H) primary antibodies (see Methods). Samples A and E were from untreated control littermates. Scale bar: 50 μm.

Figure 5. PS-induced decrease in AMP delivery to epidermal LBs is reversed by RU-486.

(A and B) Ultrathin sections labeled with CRAMP (A) and mBD3 (B) primary antibodies followed by a 10-nm colloidal gold–tagged secondary antibody after embedding for electron microscopy. Sections were postfixed in osmium tetroxide and embedded in LR White medium. Black arrows denote unlabeled LBs; circles indicate label in cytosol. (A) CRAMP was labeled (black arrowheads) in nonstressed normal controls (Co), and CRAMP labeling reappeared in PS mice treated with RU-486 (PS+Ru), but not with exogenous lipids (PS+L). (B) Exogenous lipids restored labeling of mBD3 in LB in PS mice (black arrowheads). Scale bars: 100 nm. (C–F) Quantitative data for immunolabeling of CRAMP and mBD3 in LB in nonstressed control or PS mice plus either RU-486 (C and D) or lipid (E and F) cotreatment. *P < 0.05; **P < 0.001.

Figure 6. Endogenous GCs downregulate AMP expression in normal mouse epidermis.

Immunostaining for CRAMP (A–D) and mBD3 (E–H) mRNA expression was assessed in biopsies from normal hairless mice (n = 3 per group) treated with RU-486 (B and F), antalarmin (C and G), or vehicle (A and E) and from cohorts of adrenalectomized (adrex; D, H, I, and J) and sham-operated (I and J) hairless Skh1 mice (n = 3 per group). Scale bar: 50 μm. *P = 0.04; **P = 0.006.

Figure 7. Coapplication of topical physiologic lipids partially normalizes mBD3 expression in the face of PS or increased GCs.

Hairless mice (n = 4 or 5 per cohort) received either an equimolar mixture of ceramides, cholesterol, and free fatty acids (1:1:1 molar ratio; 2% final concentration) in propylene glycol/ethanol (7:3 v/v) vehicle (60 μl to a 3-cm² area) or vehicle alone, while being cotreated with either PS (E and F) or topical clobetasol (GC, B and C). Frozen sections were immunostained for mBD3 (see Methods). Controls treated only with vehicle are shown in A and D. Scale bars: 50 μm.

Figure 8. PS increases the severity of cutaneous GAS infection.

(A) Female 8- to 10-wk-old Skh1/Hr mice were subjected to normal conditions or PS for 72 h, and then subsequently injected intradermally with 4.8 x 10⁸ CFU/ml GAS (n = 6 per group). Mice were then photographed daily for 4 d to monitor lesion size. (A) Representative lesions at day 4 from nonstressed and PS mice. (B) Lesion size (mean ± SEM) was calculated ± SEM for day 1 and day 4 lesions. *P < 0.05 versus nonstressed. (C) Representative lesions of PS mice (n = 5–6 per group) immediately prior to and 72 h after IP injection with either vehicle or RU-486 (6 mg/kg). After 72 h of PS or nonstressed conditions, mice were injected intradermally with 4.8 x 10⁸ CFU/ml GAS. A representative photograph of day 4 lesions from each group is shown. (D) Lesion size (mean ± SEM) was calculated for day 4 lesions. ^†P < 0.05 versus PS and RU-486; ^#P < 0.05 versus PS and vehicle.

Figure 9. Divergent mechanisms for PS-induced downregulation of epidermal CRAMP and mBD3.