Excessive localized leukotriene B4 levels dictate poor skin host defense in diabetic mice

Abstract

Poorly controlled diabetes leads to comorbidities and enhanced susceptibility to infections. While the immune components involved in wound healing in diabetes have been studied, the components involved in susceptibility to skin infections remain unclear. Here, we examined the effects of the inflammatory lipid mediator leukotriene B4 (LTB4) signaling through its receptor B leukotriene receptor 1 (BLT1) in the progression of methicillin-resistant Staphylococcus aureus (MRSA) skin infection in 2 models of diabetes. Diabetic mice produced higher levels of LTB4 in the skin, which correlated with larger nonhealing lesion areas and increased bacterial loads compared with nondiabetic mice. High LTB4 levels were also associated with dysregulated cytokine and chemokine production, excessive neutrophil migration but impaired abscess formation, and uncontrolled collagen deposition. Both genetic deletion and topical pharmacological BLT1 antagonism restored inflammatory response and abscess formation, followed by a reduction in the bacterial load and lesion area in the diabetic mice. Macrophage depletion in diabetic mice limited LTB4 production and improved abscess architecture and skin host defense. These data demonstrate that exaggerated LTB4/BLT1 responses mediate a derailed inflammatory milieu that underlies poor host defense in diabetes. Prevention of LTB4 production/actions could provide a new therapeutic strategy to restore host defense in diabetes.

Keywords: Bacterial infections; Dermatology; Eicosanoids; Inflammation; Macrophages.

Figure 1. Increased lesion size and bacterial loads in diabetic mice correlates with higher LTB₄ levels than nondiabetic infected mice.

(A) STZ-diabetic and control (CT) mice were infected s.c. with 3 × 10⁶ CFU and infection areas were measured every other day for 9 days. Data represent mean ± SEM (*P < 0.05, 2-way ANOVA with 2-way ANOVA followed by Tukey’s multiple comparison corrections). (B) Photographs of CT and STZ mice at days 3 and 9 after infection. (C) Bacterial CFUs measured from skin biopsy homogenates at day 9 after infection. (D) Alox5 mRNA expression in skin from diabetic and CT mice on day 1 after infection detected by quantitative PCR (qPCR). (E) Eicosanoids measured by mass spectrometry from skin biopsy homogenates from the naive skin and from samples taken on day 1 and 9 after infection from diabetic and nondiabetic mice. (F) LTB₄ in the skin of diabetic and nondiabetic mice at 1 day after infection and measured by EIA. (G) Ltb4r1 mRNA expression in skin from diabetic and CT mice on day 1 after infection detected by qPCR. (H) Ltb4r2 mRNA expression in skin from diabetic and CT mice on day 1 after infection detected by qPCR. Data are mean ± SEM of 5–10 mice from 2–5 experiments. *P < 0.05 vs. naive mice. ^#P < 0.05 vs. CT mice.

Figure 2. Inhibition of BLT1, but not BLT2, improves host defense in diabetic mice during MRSA skin infection.

(A) WT CT, STZ-treated WT, STZ-treated Alox5^–/–, and STZ-treated Ltb4r1^–/– mice were infected s.c. with MRSA, and infection areas were measured for 9 days. (B) Bacterial CFUs in the skin at day 9 after infection. Data are mean ± SEM of 5–10 mice. *P < 0.05 vs. WT CT mice. ^#P < 0.05 vs. STZ-treated WT mice. (C) CT and STZ-treated mice were infected s.c. with MRSA. STZ-treated mice were treated daily with topical ointments, vehicle-control, 0.001% BLT1 antagonist (U-75302), or 0.001% BLT2 antagonist (LY255283), and infection area was measured as in A. (D) Bacterial CFUs from mice in C at day 9 after infection. Data are mean ± SEM of 4–6 mice. *P < 0.05 vs. CT mice. ^#P < 0.05 vs. STZ-treated mice treated with vehicle control ointment. (E) Mice were infected s.c. with bioluminescent-expressing MRSA. Infection areas were measured every other day in CT and STZ-treated mice treated daily with vehicle control or 0.001% BLT1 antagonist (U-75302) ointments. (F) Bioluminescence imaging (BLI) to quantify bacterial burden in the skin at days 1, 3, 4, 7, and 9 after infection. (G) Representative images of bioluminescent MRSA infection in control and diabetic mice that were treated or not with BLT1 antagonist scanned by BLI. (H) Lesion size of nondiabetic NOD (ctNOD) and diabetic NOD (dbNOD) mice infected with MRSA by s.c. injection. Infected dbNOD mice were treated daily with vehicle-control or 0.001% BLT1 antagonist (U-75302) ointments as in C. Data are mean ± SEM of 5–10 mice. *P < 0.05 vs. CT mice. ^#P < 0.05 vs. STZ-treated mice treated with vehicle control. (I) Gram stains of CT and diabetic mice that were infected and treated with the BLT1 antagonist with MRSA for 1 and 9 after infection. Top panels show 100× and bottom panels show 1,000× magnification with an inset of a cropped zoomed view of 5 ,000× magnification. Arrows indicate bacteria.

Figure 3. Diabetic mice have compromised abscess morphology.

(A) CT and STZ-treated mice were infected with MRSA by s.c. injection. STZ-treated mice were treated daily with topical 0.001% BLT1 antagonist (U-75302) ointment or vehicle control, and biopsies were collected at day 1 after MRSA skin infection or from naive skin and sectioned for histology and H&E staining. The black dotted line outlines the abscess edge. Black arrows indicate cell recruitment. (B) Representative images for slides stained with Masson’s trichrome blue stain of control and diabetic mice infected and treated as above. White arrows and dotted lines show abscess edge. In all circumstances, stained images are representative of 3–5 mice/group from 2–4 experiments. Top panels show 40× magnification, and bottom panels show 400× magnification.

Figure 4. Excessive LTB₄ production drives poor neutrophil organization and altered chemokine/cytokine production at the site of infection of diabetic mice.

(A) Biopsies were collected from CT and diabetic naive mice and infected mice, and they were treated daily with the BLT1 antagonist on days 1 and 9 after MRSA skin infection and sectioned for histology. Representative images of IHC for neutrophils (Ly6G/C) are stained in brown with blue counterstain from 3–5 mice from 2–4 experiments. Top panels show 4× magnification. Bottom panels show 40× magnification. Black arrows indicate neutrophils. (B) Intravital imaging from the infection site of Lys^EGFP CT and diabetic mice with frames taken at 0, 10, 20, and 30 minutes. The 30-minute frames include track paths of individual cells. The lines show the cell path and the color refers to the median velocity of the cell (red is fast, blue is slow). (C) Left graph: Median velocity of GFP⁺ cells. Middle graph: Track displacement of GFP⁺ cells. Right graph: Ratio of displacement/track duration to determine the directionality of GFP⁺ cells. (D) Tissue homogenate from CT and diabetic mice that were infected or not and treated with the BLT1 antagonist once a day for 9 days were subjected to a multiplex assay, and the heatmap of analytes from 3–8 mice are shown. (E–G)Skin homogenates were processed and subjected to ELISA for RAGE, ICAM-1, and CXCL2 abundance. Data are mean ± SEM of 3–8 mice from 2–4 experiments. *P < 0.05 vs. naive mice. ^#P < 0.05 vs. CT mice. ^{^}P < 0.05 vs. STZ mice treated with vehicle control ointment.

Figure 5. Skin-macrophages drive detrimental host defense actions in diabetic mice.

(A) CT and STZ mice were infected and treated with the BLT1 antagonist once a day for 9 days, and skin biopsies from days 1 and 9 after infection were subjected to IHC staining for macrophages (F4/80) in brown with blue counterstain. Top panels show 40× magnification, and bottom panels show 400× magnification from 3–5 mice from 2–4 experiments. Arrows indicate macrophages. (B) Monocytes and macrophages were depleted in diabetic MMDTR mice as described in the Methods prior to MRSA skin infection. After 6 hours, skin biopsies were collected and sectioned for immunofluorescence staining for neutrophils (Ly6G) shown in red and DAPI counterstain shown in blue. Representative images of 20× magnification from 3–4 mice. White arrows indicate neutrophils. (C) Infection areas of CT, STZ, and DT-treated STZ-MMDTR mice on day 2 after infection with MRSA. (D) Bacterial CFU in the skin CT, STZ, and DT-treated STZ-MMDTR determined 2 days after infection. (E) Percentage of neutrophils (Ly6G⁺) in the skin of CT, STZ, and DT-treated STZ-MMDTR determined by flow cytometry at day 2 after infection. (F) LTB₄ ELISA on tissue homogenates detected 2 days after infection from CT, STZ, and DT-treated STZ-MMDTR mice. Data are mean ± SEM of 3–7 mice from 1–2 experiments. *P < 0.05 vs. CT mice. ^#P < 0.05 vs. STZ-treated mice. (G–J) Multiplex assay on skin biopsy homogenates 2 days after infection of CT, STZ, and DT-treated STZ-MMDTR CXCL2 (G), MMP8 (H), CCL2 (I), and P-selectin (J). Data are mean ± SEM of 3–7 mice from 1–2 experiments. *P < 0.05 vs. naive mice. ^#P < 0.05 vs. CT mice. ^{^}P < 0.05 vs. STZ-treated mice.

Figure 6. Summary.

Left panel: MRSA skin infection induces macrophage activation, which leads to LTB₄ production and neutrophil recruitment. Both of these outcomes are elevated in diabetic animals compared with control animals. Middle panel: Excessive LTB₄/BLT1 activity in the skin during MRSA infection in diabetic mice results in uncontrolled neutrophil recruitment, poor abscess formation, uncontrolled bacterial burden, and exaggerated inflammation. Right panel: BLT1 antagonist treatment of infected diabetic mice results in reduced neutrophil recruitment, restoration of neutrophil direction, proper abscess formation, and improved bacterial clearance. Our model suggests that BLT1 antagonist treatment dampens the cycle of chronic inflammation during infection.