Control of Pseudomonas aeruginosa skin infections in mice is mast cell-dependent

Abstract

Mast cells (MCs) have recently been shown to be essential for the elicitation of efficient immune responses in murine sepsis. To explore whether MCs also contribute to the control of bacterial skin infections, we studied skin lesions induced by Pseudomonas aeruginosa (PA) in genetically MC-deficient Kit(W)/Kit(W-v) mice, normal Kit(+/+) mice, and MC-reconstituted Kit(W)/Kit(W-v) mice. PA injections resulted in strikingly (>2-fold) larger skin lesions in Kit(W)/Kit(W-v) mice than in Kit(+/+) mice, which exhibited pronounced MC degranulation at infection sites. In addition, neutrophil recruitment following PA injections and bacterial clearance from sites of infection was significantly impaired in Kit(W)/Kit(W-v) mice compared with Kit(+/+) mice. Notably, the adoptive transfer of MCs to the skin of Kit(W)/Kit(W-v) mice before PA infection resulted in normal neutrophil accumulation as well as skin lesions comparable with those in Kit(+/+) mice in both bacterial burden and size. These findings demonstrate for the first time that activated MCs are crucial for the induction of protective innate immune responses to bacterial skin infections.

Figure 1

P. aeruginosa infections cause markedly larger lesions in the absence of MCs. Skin lesion size in MC-deficient Kit^W/Kit^W-v mice (n = 27) and normal Kit^+/+ mice (n = 32) after subcutaneous injection of PA (6.5 × 10⁵ CFU) in 100 μl. Data were pooled from six independent experiments and expressed as means ± SEM. Statistical analyses were performed by using the unpaired two-tailed Student’s t-test for single point comparison; time course data were tested by multiple analysis of variance for repeated measurements. *P < 0.05, **P < 0.01, ***P < 0.005.

Figure 2

MCs are activated at sites of P. aeruginosa skin infections. A: Degranulation of skin MCs in vivo after subcutaneous injection of defined amounts of PA. Data were pooled from two independent experiments (n = 5–12). *P < 0.01, ***P < 0.005. B: Activation/serotonin release of skin MCs ex vivo after incubation with defined amounts of PA. Data were obtained from of three to six independent experiments and expressed as means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.005. C: ET-1 skin levels were assessed from 6-mm punch biopsies, virtually identical in size and weight, harvested at defined time points after subcutaneous injections of PA and calculated as fmol/mm² skin surface. Data were pooled from two independent experiments (n = 6–11) and expressed as means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.005.

Figure 3

Neutrophil accumulation and bacterial clearance at sites of P. aeruginosa infection are impaired in the absence of MCs. A: MPO skin levels (reflecting cutaneous neutrophil accumulation) after PA infection of MC-deficient Kit^W/Kit^W-v mice and normal Kit^+/+ mice. Data were derived from three independent experiments (n = 9–14) and expressed as means ± SEM. ⁺P < 0.05, ⁺⁺⁺P < 0.005 versus vehicle; ***P < 0.005 versus Kit^W/Kit^W-v mice. B: Bacterial clearance from sites of PA infections in Kit^W/Kit^W-v mice. Data were pooled from three independent experiments (n = 14–15). ***P < 0.005.

Figure 4

Control of P. aeruginosa skin infections including polymorphonuclear neutrophil influx and bacterial clearance are MC-dependent. Skin lesion size (A), neutrophil recruitment (B), and bacterial clearance from sites of infection (C) in MC-reconstituted Kit^W/Kit^W-v mice (Kit^W/Kit^W-v mice + MC; n = 9–14), Kit^+/+ mice (n = 9–32), and Kit^W/Kit^W-v mice (n = 9–27) injected subcutaneously with PA. Skin was harvested 24 hours after subcutaneous infection with PA. Histological sections were processed for H&E staining and imaged at ×50 (D–F) and ×400 (G–I) magnification. Data were pooled from three to six independent experiments and expressed as means ± SEM. Statistical analyses were performed by using the unpaired two-tailed Student’s t-test for single point comparison; time course data were tested by multiple analysis of variance for repeated measurements. ⁺P < 0.05, ⁺⁺P < 0.01, ⁺⁺⁺P < 0.005 versus Kit^W/Kit^W-v mice + MC; *P < 0.05, **P < 0.01, ***P < 0.005 versus Kit^+/+ mice; n.s., not significant.