Role of neutrophils in response to Bordetella pertussis infection in mice

Abstract

Pertussis is an acute respiratory disease caused by the bacterium Bordetella pertussis, for which humans are the only known reservoir. During infection, B. pertussis releases several toxins, including pertussis toxin (PT) and adenylate cyclase toxin (ACT), which have both been shown to play roles in promoting bacterial growth during early infection in a mouse model. Furthermore, in vitro and in vivo studies suggest that PT and ACT affect neutrophil chemotaxis and/or function, thereby altering the innate immune response. In this study we depleted animals of neutrophils to investigate whether neutrophils play a protective role during B. pertussis infection in mice. In addition, by infection with toxin-deficient strains, we investigated whether neutrophils are the main targets for PT and/or ACT activity in promoting bacterial growth. Surprisingly, we found no role for neutrophils during B. pertussis infection in naïve mice. However, in previously infected (immune) mice or in mice receiving immune serum, we observed a significant role for neutrophils during infection. Furthermore, in this immune mouse model our evidence indicates that neutrophils appear to be the main target cells for ACT, but not for PT.

FIG. 1.

Effects of neutrophil depletion and inhibition of neutrophil chemotaxis on bacterial load. (A and B) Numbers of neutrophils in the airways on days 1 and 2 after B. pertussis WT infection (5 × 10⁶ CFU) in mice treated with the indicated dose of RB6 or control rat IgG (complete inhibition of neutrophil recruitment to the airways was observed at both time points when using 1 mg of RB6) (A) or with the indicated dose of anti-CXCR2 polyclonal antibody or control goat serum. (C and D) Numbers (CFU) of bacteria recovered from the lower respiratory tract (trachea plus lungs) are shown for mice treated with either RB6 or control rat IgG and infected with B. pertussis WT or the indicated mutant strains on day 4 postinoculation (C) or for mice treated with either anti-CXCR2 polyclonal antibody or control goat serum and infected with B. pertussis WT or ΔPT on days 1 and 2 postinoculation (D). n = three to four mice/treatment group. The results represent one of three separate experiments. *, P < 0.05 versus control; , significantly different from the WT strain.

FIG. 2.

Clearance of B. pertussis infection in neutropenic mice. Numbers (CFU) of bacteria recovered from the lower respiratory tract of mice treated with either RB6 or control rat IgG and infected with B. pertussis WT or ΔPT on days 7 and 14 postinoculation are shown. n = four mice/treatment group. *, P < 0.005; **, P < 0.001.

FIG. 3.

Bacterial loads, neutrophil recruitment, and KC gene expression during B. pertussis WT and ΔPT infection in immune mice. (A) Numbers (CFU) of bacteria recovered from the lower respiratory tract of immune mice (previously infected with ΔPT) that were challenged with 5 × 10⁶ CFU of WT or ΔPT at the indicated times postchallenge. (B) Numbers of neutrophils in the airways of immune mice challenged with 5 × 10⁶ CFU of WT or ΔPT at the indicated time postchallenge. (C) Kinetics of KC gene expression (measured by real-time RT-PCR) over 7 days after challenge of immune mice with 5 × 10⁶ CFU of WT or ΔPT. Data are mean fold increases relative to samples from PBS control-treated mice. (D) Numbers (CFU) of bacteria recovered from the lower respiratory tract of immune mice treated with either RB6 or control rat IgG and challenged with B. pertussis WT or the indicated mutant strains (5 × 10⁶ CFU) on day 4 postchallenge. n = three to four mice/treatment group. The results represent one of two separate experiments. *, P < 0.05; **, P < 0.02.

FIG. 4.

Bacterial loads after B. pertussis infection in mice after transfer of naïve or immune serum. Numbers (CFU) of bacteria recovered from the lower respiratory tract of mice that received either immune or naïve serum and treated with either RB6 or control rat IgG and infected with B. pertussis WT or ΔPT (5 × 10⁵ CFU) on day 4 postinfection are shown. n = four mice/treatment group. *, P < 0.02; **, P < 0.002; , significantly different from the WT strain.