The role of CD1d-restricted NKT cells in the clearance of Pseudomonas aeruginosa from the lung is dependent on the host genetic background

Abstract

Pseudomonas aeruginosa is an important human opportunistic pathogen, accounting for a significant fraction of hospital-acquired lung infections. CD1d-restricted NKT cells comprise an unusual innate-like T cell subset that plays important roles in both bacterial and viral infections. Previous reports have differed in their conclusions regarding the role of NKT cells in clearance of P. aeruginosa from the lung. Since there is significant strain-dependent variation in NKT cell number and function among different inbred strains of mice, we investigated whether the role of NKT cells was dependent on the host genetic background. We found that NKT cells did indeed play a critical role in the clearance of P. aeruginosa from the lungs of BALB/c mice but that they played no discernible role in clearance from the lungs of C57BL/6 mice. We found that the strain-dependent role of NKT cells was associated with significant strain-dependent differences in cytokine production by lung NKT cells and that impaired clearance of P. aeruginosa in BALB/c CD1d(-/-) mice was associated with an increase in neutrophil influx to the lung and increased levels of proinflammatory cytokines and chemokines after infection. Finally, we found that the role of alveolar macrophages was also dependent on the genetic background. These data provide further support for a model in which the unusually high level of variability in NKT cell number and function among different genetic backgrounds may be an important contributor to infectious-disease susceptibility and pathology.

FIG 1

Strain-dependent role of NKT cells in clearance of Pseudomonas aeruginosa. Wild-type (WT), B6 CD1d^−/−, B6 Jα18^−/−, B6 γδ^−/−, or B6 CD1d^−/−γδ^−/− mice were infected oropharyngeally with 2 × 10⁷ CFU per mouse. (A) The number of live colonies in lungs 24 h after infection. Each symbol represents a single mouse. Lines denote the mean CFU. (B) Strain-dependent difference in BAL fluid leukocytic infiltrate 24 h after infection. (Left) Representative Giemsa staining of BAL fluid leukocytic infiltrate that is predominantly neutrophils. (Right) Cell counts of BAL fluid leukocytic infiltrate. Data represent means ± standard errors of the means (SEM); n = 5 to 9 mice/strain. Results are representative of three separate experiments. ****, P < 0.0001. (C) No effect of γδ T cells in Pseudomonas clearance on the B6 background. Data represent numbers of live colonies in lungs 24 h after infection. Each symbol represents a single mouse. Lines denote the mean CFU.

FIG 2

Strain-dependent response to αGalCer by lung leukocytes. Lung and liver leukocytes were prepared from 8-week-old C57BL/6J and BALB/cJ mice. (A) NKT and conventional T cells as a percentage of CD45⁺ lung leukocytes. A representative contour plot is shown. (B) Cytokine production by CD45⁺ lung leukocytes stimulated in vitro with αGalCer. Data represent means ± SEM; n = 6 BALB/c, 8 C57BL/6J mice, and 2 BALB/c CD1d^−/− mice. (C) Cytokine production by CD45⁺ liver leukocytes stimulated in vitro with αGalCer. Data were normalized based on the frequency of TCR⁺CD1dtet/PBS57⁺ NKT cells, as assessed by flow cytometry. Data represent means ± SEM; n = 4 mice. The data represent the combined results of two separate experiments.

FIG 3

The strain-dependent difference in lung NKT cell function is NKT cell intrinsic. NKT cells from 8-week-old C57BL/6J and BALB/cJ mice were subjected to FACS analysis using CD45-enriched lung leukocytes based on TCR and CD1dtet/PBS57 staining. Equal numbers of NKT cells were incubated in anti-CD3 coated plates for 96 h, after which cytokine and chemokine production was assessed using BioPlex. Data represent means ± standard deviations (SD) of cytokine and chemokine production from duplicate wells.

FIG 4

Role of CD1d and NKT cells in neutrophil influx to the airways early in infection. (A) Time course of PAO1 clearance. BALB/cJ mice were infected with 2 × 10⁷ CFU, and live colonies in lungs were counted 24 h later. Each symbol represents the CFU from a single mouse. Lines denote the mean CFU. (B) Comparison of levels of PAO1 clearance in the lungs of different strains 6 h after infection. Lines denote the mean CFU. WT and CD1d-deficient mice were infected with 2 × 10⁷ PAO1 o.p., and live colonies in lungs were counted 6 h later. (C) Increased neutrophil numbers in the BAL fluid of BALB/c CD1d^−/− mice 6 h after infection. Data represent means ± SEM; n = 4 mice per strain. *, P < 0.05.

FIG 5

Enhanced inflammatory response in the absence of CD1d. Results represent cytokine and chemokine profiles of BAL fluid 6 h after infection with PAO1. Data represent means ± SEM; n = 4 mice per strain. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

FIG 6

Strain-dependent effect of alveolar macrophage depletion. Mice were depleted of alveolar macrophages using clodronate (Clod)-loaded liposomes and infected with P. aeruginosa 24 h later. The numbers of live colonies in lungs 24 h after infection are shown. Each symbol represents a single mouse. Lines denote the mean CFU. The combined results of two separate depletions and infections are shown. *, P < 0.05; **, P < 0.01; ***, P < 0.001.