Episodic binge ethanol exposure impairs murine macrophage infiltration and delays wound closure by promoting defects in early innate immune responses

Abstract

Background: Exacerbation of cutaneous wound infections and delayed wound closure are frequent complications seen in alcohol exposed subjects who sustain injuries. We previously reported that acute alcohol exposure alters the early dermal inflammatory phase of wound healing and also several parameters of the proliferative wound healing phase in wounds from ethanol (EtOH)-treated mice for several days or weeks after EtOH exposure. Hence, it is likely that the cumulative defects arising in the early phases of the wound healing process directly contribute to the increased complications observed in intoxicated patients at the time of injury.

Methods: C57BL/6 mice were given intraperitoneal EtOH (2.2 g/kg body weight) or vehicle (saline) EtOH using our episodic binge EtOH exposure protocol (3 days EtOH, 4 days off, 3 days EtOH) to yield a blood alcohol concentration (BAC) of 300 mg/dl at the time of wounding. Mice were subjected to six 3 mm full-thickness dorsal wounds and immediately treated topically with 10 μl of sterile saline (control) or diluted Staphylococcus aureus corresponding to 1 × 10(4) CFU/wound. Wounds were harvested at 24 hours post injury to evaluate wound area, neutrophil and macrophage accumulation, and the protein levels of cytokines, interleukin-6 (IL-6), IL-1β, and IL-10, and chemokines, macrophage inflammatory protein-2 (MIP-2) and MIP-1α, monocyte chemotactic protein-1 (MCP-1), and keratinocyte-derived chemokine (KC). The abundance and localization of cathelicidin-related antimicrobial peptide (CRAMP) and the kallikrein epidermal proteases (KLK5 and KLK7) were also determined.

Results: Compared to control mice, EtOH-treated mice exhibited delayed wound closure, decreased macrophage accumulation, and impaired production of MIP-1α. Furthermore, skin from EtOH-treated mice demonstrated a reduction in the abundance of epidermal CRAMP and KLK7.

Conclusions: These findings suggest that EtOH exposure hinders several distinct components of the innate immune response, including phagocyte recruitment and chemokine/cytokine and AMP production. Together, these effects likely contribute to delayed wound closure and enhanced infection severity observed in intoxicated patients.

Keywords: Antimicrobial Peptides; Cathelicidin; Inflammation; Macrophages; Proteases; Wound Healing.

Figure 1. Ethanol exposure increases the percentage of open wound area

(A) Representative images of wounds from saline and ethanol-treated mice (n=18 per group). Scale bars = 1 cm. (B) Percentage of open wound area quantification. % open wound area = (average # pixels per wound 24hours after injury/ average # pixels per wound immediately following injury) *100. Data are represented as mean ± SEM. * p<0.0001 by Student's t-test.

Figure 2. Ethanol exposure decreases macrophage infiltration into infected wounds, but does not affect neutrophil infiltration

(A) H&E histological staining. Left image: Low-power magnification of skin sections. The boxed area indicates the wound margin. Right image: High-power magnification of the wound margin demonstrating large numbers of infiltrating cells. (B) IHF of wound margin from saline (n=9) or ethanol (n=5) treated mice. Arrows (→) indicate Gr-1⁺ neutrophils, stained green. Arrowheads (▶) indicate Moma-2⁺ macrophages, stained red. DAPI nuclear stain is blue. No antibody control did not show any staining (data not shown). (C) Quantification of Gr-1⁺ neutrophils. (D) Quantification of Moma-2⁺ macrophages. (E) Quantification of Mean Fluorescence Intensity of Moma-2⁺ cells. Data are represented as mean ± SEM. * = p < 0.05 by Student's t-test.

Figure 3. Ethanol exposure specifically decreases the production of the chemokine, MIP1-α, the pro-inflammatory cytokine, IL-1β, and the anti-inflammatory cytokine, IL-10

Chemokine levels were determined by ELISA. (A-D), Cytokine levels were determined by multiplex bead array (E-H). (A) MIP-2 levels in wounds from saline (n=13) or ethanol (n=12) treated mice. (B) KC levels in wounds from saline (n=13) or ethanol (n=12) treated mice. (C) MCP-1 levels in wounds from saline (n=13) or ethanol (n=11) treated mice. (D) MIP-1α levels in wounds from saline (n=18) or ethanol (n=17) treated mice. (E-H) (E) IL-6, (F) IL-1β, (G)TNFα, and (H) IL-10 levels in wounds from saline (n=18) or ethanol (n=16) treated mice. Data are represented as geometric mean with 95% Cl.* = p < 0.05 by Student's t-test.

Figure 4. Ethanol exposure decreases epidermal CRAMP and KLK7 production, but not KLK5

(A,B) IHF using rabbit-anti-CRAMP 1° antibody and Alexafluor 488-donkey-anti-rabbit 2° antibody. Whole rabbit sera negative control showed minimal background staining (data not shown). CRAMP levels in the epidermis are higher in saline-treated (A) mice as compared to ethanol-treated (B) mice. (C,D) IHF using goat anti-human KLK5 1° antibody and Cy3-donkey-anti-goat 2° antibody. Goat IgG control showed minimal background staining (data not shown). KLK5 levels in saline-treated (C) and ethanol-treated (D) mice. (E,F) IHF using goat anti-human KLK7 1° antibody and Cy3-donkey-anti-goat 2° antibody. Goat IgG control showed minimal background staining (data not shown). KLK7 levels in saline-treated (E) and ethanol-treated (F) mice. The wound edge is located on the right side of all images.