Chronic ethanol feeding increases the severity of Staphylococcus aureus skin infections by altering local host defenses

Abstract

Alcoholics are at increased risk of Staphylococcus aureus skin infection and serious sequelae, such as bacteremia and death. Despite the association between alcoholism and severe S. aureus skin infection, the impact of EtOH on anti-S. aureus cutaneous immunity has not been investigated in a model of chronic EtOH exposure. To test the hypothesis that EtOH enhances the severity of S. aureus skin infection, mice were fed EtOH for ≥12 weeks via the Meadows-Cook model of alcoholism and inoculated with S. aureus following epidermal abrasion. Evidence of exacerbated staphylococcal disease in EtOH-fed mice included: skin lesions that were larger and contained more organisms, greater weight loss, and increased bacterial dissemination. Infected EtOH-fed mice demonstrated poor maintenance and induction of PMN responses in skin and draining LNs, respectively. Additionally, altered PMN dynamics in the skin of these mice corresponded with reduced production of IL-23 and IL-1β by CD11b(+) myeloid cells and IL-17 production by γδ T cells, with the latter defect occurring in the draining LNs as well. In addition, IL-17 restoration attenuated S. aureus-induced dermatopathology and improved bacterial clearance defects in EtOH-fed mice. Taken together, the findings show, in a novel model system, that the EtOH-induced increase in S. aureus-related injury/illness corresponds with defects in the IL-23/IL-17 inflammatory axis and poor PMN accumulation at the site of infection and draining LNs. These findings offer new information about the impact of EtOH on cutaneous host-defense pathways and provide a potential mechanism explaining why alcoholics are predisposed to S. aureus skin infection.

Keywords: IL-17; PMNs; T cells.

Figure 1.. Chronic EtOH feeding increases the severity of S. aureus-induced illness.

EtOH-fed or H₂O control mice were infected with 5.0 × 10⁷ S. aureus organisms. (A) Lesion area following S. aureus skin infection. (B, top) Representative images of H₂O control mouse receiving mock infection via sandpaper and DPBS on days 1 and 3 after treatment. (Middle and bottom) Representative images of skin lesions in H₂O control mice and EtOH-fed mice (indicated on the left) on days 1 and 3 after infection. (C) Weight loss following S. aureus skin infection. (D) Bacterial load of skin lesions, 1 and 3 days postinfection (calculated as described in Materials and Methods) and bacterial load in kidney, 3 days postinfection. (E) Bacterial load of skin lesions, 7 days postinfection. Error bars represent sem. Post-test, *P ≤ 0.05; **P ≤ 0.01; n ≥ 8 mice/treatment group.

Figure 2.. PMN and monocyte accumulation in infected skin rapidly declines, whereas PMN infiltration into the draining LNs is reduced persistently in EtOH-fed mice.

(A and B, left) Flow cytometric gating strategy for PMNs and monocytes from a representative H₂O control mouse is shown for skin (A) and LN (B). (Middle and right) Graphs indicate density (A) or number (B) of infiltrating cells in these tissues from EtOH-fed or age-matched H₂O controls at 1 and 3 days post-S. aureus skin infection. Density was calculated as described in Materials and Methods. Day 1 versus day 3 EtOH mice, P < 0.01. Despite the significant growth of skin lesions from day 1 to day 3 in EtOH-fed mice (P < 0.01; Fig. 1A), there was a decrease in the density of PMNs in skin preparations from EtOH-fed mice, P < 0.01. Error bars represent sem. Post-test, *P ≤ 0.05; n ≥ 8 mice/treatment group.

Figure 3.. Chronic EtOH feeding selectively decreases PMN function by reducing TLR2 and CRAMP expression, leaving in vivo phagocytosis of S. aureus intact.

(A, left) Gating strategy for evaluating phagocytosis of FITC-labeled S. aureus by PMNs from a representative H₂O control mouse. (Right) Density of phagocytic PMNs isolated from skin lesions of EtOH-fed or H₂O controls, 1 day postinfection. Error bars represent sem; n ≥ 8 mice/treatment group. (B, left) Representative images of nonphagocytic and phagocytic PMNs. Internalized S. aureus organisms are labeled by arrows. (Right) The percentage of phagocytic PMNs within cytospin preparations from EtOH-fed or H₂O controls, 3 days postinfection. Error bars represent sem; n ≥ 8 mice/treatment group. (C) CRAMP and TLR2 mean fluorescence intensity (MFI) values for Ly6C⁺ Ly6G⁺ PMNs (gated as in Fig. 2A) in lesional skin preparations from EtOH-fed or H₂O controls, 1 and 3 days postinfection. Error bars represent sem. Post-test, *P ≤ 0.05; **P ≤ 0.01. Data are representative of 2 independent experiments; n ≥ 4 mice/treatment group (day 1), and n ≥ 6 mice/treatment group (day 3).

Figure 4.. EtOH impairs early IL-23 and IL-1β responses at the site of infection.

Skin cell suspensions were generated from S. aureus-infected EtOH-fed or age-matched H₂O controls, and IL-23 and IL-1β were assessed by intracellular cytokine staining. Gating strategies from a representative H₂O control mouse are shown in the contour plots. Bar graphs show the density of cytokine-producing myeloid cells, 1 and 3 days postinfection. Error bars represent sem. Post-test, *P ≤ 0.05; n ≥ 6 mice/treatment group.

Figure 5.. EtOH impairs T cell-mediated IL-17 responses in the skin and draining LN.

Skin and skin draining LN cell suspensions were generated from EtOH-fed or age-matched H₂O controls, and IL-17 production by CD4 and γδ T cells was assessed by intracellular cytokine staining and RORγt expression. Gating strategies from a representative H₂O control mouse are shown in the contour plots. Bar graphs show the density (A) and number (B) of cytokine-producing cells in the skin (A) and LN (B) at 3 and 7 days post-infection. Error bars represent sem. Post-test, *P ≤ 0.05; **P ≤ 0.01; n ≥ 8 mice/treatment group.

Figure 6.. i.d. Administration of rIL-17 improves lesion size and S. aureus clearance.

(A) Diagram of the rIL-17 intervention strategy (see Materials and Methods for further details). (B) Lesion area following S. aureus skin infection following indicated treatments. Statistical significance corresponds to comparisons of EtOH-fed mice receiving rIL-17 administration versus vehicle alone (DPBS). (C) Bacterial load of skin lesions, 7 days postinfection from EtOH-fed and age-matched H₂O controls receiving rIL-17 administration or vehicle alone (DPBS). Error bars represent sem. *P < 0.05. Data are representative of 2 independent experiments; n ≥ 4 mice/treatment group.