image_1_Azithromycin Clears Bordetella pertussis Infection in Mice but Also Modulates Innate and Adaptive Immune Responses and T Cell Memory.jpeg

<p>Treatment with the macrolide antibiotic azithromycin (AZM) is an important intervention for controlling infection of children with Bordetella pertussis and as a prophylaxis for preventing transmission to family members. However, antibiotics are known to have immunomodulatory effects independent of their antimicrobial activity. Here, we used a mouse model to examine the effects of AZM treatment on clearance of B. pertussis and induction of innate and adaptive immunity. We found that treatment of mice with AZM either 7 or 14 days post challenge effectively cleared the bacteria from the lungs. The numbers of innate immune cells in the lungs were significantly reduced in antibiotic-treated mice. Furthermore, AZM reduced the activation status of macrophages and dendritic cells, but only in mice treated on day 7. Early treatment with antibiotics also reduced the frequency of tissue-resident T cells and IL-17-producing cells in the lungs. To assess the immunomodulatory effects of AZM independent of its antimicrobial activity, mice were antibiotic treated during immunization with a whole cell pertussis (wP) vaccine. Protection against B. pertussis induced by immunization with wP was slightly reduced in AZM-treated mice. Antibiotic-treated wP-immunized mice had reduced numbers of lung-resident memory CD4 T cells and IL-17-production and reduced CD49d expression on splenic CD4 T cells after challenge, suggestive of impaired CD4 T cell memory. Taken together these results suggest that AZM can modulate the induction of memory CD4 T cells during B. pertussis infection, but this may in part be due to the clearance of B. pertussis and resulting loss of components that stimulate innate and adaptive immune response.</p

Relative Contribution of Th1 and Th17 Cells in Adaptive Immunity to <i>Bordetella pertussis</i>: Towards the Rational Design of an Improved Acellular Pertussis Vaccine

<div><p>Whooping cough caused by <i>Bordetella pertussis</i> is a re-emerging infectious disease despite the introduction of safer acellular pertussis vaccines (Pa). One explanation for this is that Pa are less protective than the more reactogenic whole cell pertussis vaccines (Pw) that they replaced. Although Pa induce potent antibody responses, and protection has been found to be associated with high concentrations of circulating IgG against vaccine antigens, it has not been firmly established that host protection induced with this vaccine is mediated solely by humoral immunity. The aim of this study was to examine the relative contribution of Th1 and Th17 cells in host immunity to infection with <i>B. pertussis</i> and in immunity induced by immunization with Pw and Pa and to use this information to help rationally design a more effective Pa. Our findings demonstrate that Th1 and Th17 both function in protective immunity induced by infection with <i>B. pertussis</i> or immunization with Pw. In contrast, a current licensed Pa, administered with alum as the adjuvant, induced Th2 and Th17 cells, but weak Th1 responses. We found that IL-1 signalling played a central role in protective immunity induced with alum-adsorbed Pa and this was associated with the induction of Th17 cells. Pa generated strong antibody and Th2 responses, but was fully protective in IL-4-defective mice, suggesting that Th2 cells were dispensable. In contrast, Pa failed to confer protective immunity in IL-17A-defective mice. Bacterial clearance mediated by Pa-induced Th17 cells was associated with cell recruitment to the lungs after challenge. Finally, protective immunity induced by an experimental Pa could be enhanced by substituting alum with a TLR agonist that induces Th1 cells. Our findings demonstrate that alum promotes protective immunity through IL-1β-induced IL-17A production, but also reveal that optimum protection against <i>B. pertussis</i> requires induction of Th1, but not Th2 cells.</p> </div

IL-1RI signalling is required for induction of Th17 responses and Pa-induced protection against <i>B. pertussis</i>.

<p>IL-1RI^−/− and WT mice were immunized i.p. twice (0 and 28 days) with Pa. 14 days after the second immunization, mice were challenged by exposure to an aerosol of live <i>B. pertussis</i>. (A) The number of CFU in the lungs were quantified at intervals after challenge. (B, C) <i>B. pertussis</i>-specific cytokine production by spleen cells on the day of challenge (B) or <i>B. pertussis</i>-specific cytokine production by lung mononuclear cells 3, 7 and 10 days post challenge (C) was determined by ELISA. (D) <i>B. pertussis</i>-specific antibody in serum on the day of challenge (Co: control; KO: IL-1RI^−/−). *−/− versus WT. Results are mean values for 4 mice per group at each time point and each panel is representative of 3 independent experiments.</p

Induction of protective Th17 cells is associated with neutrophil recruitment and killing of <i>B. pertussis</i>.

<p>(A, B) WT, IL-4^−/− and IL-17A^−/− mice were immunized i.p. with Pa and challenged with <i>B. pertussis</i> as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003264#ppat-1003264-g003" target="_blank">Figure 3</a>. Recruitment of GR1⁺ neutrophils and F4/80⁺ macrophages in the lungs (A) and CXCL1 and CCL3 concentrations in lung homogenates (B) following aerosol challenge with live <i>B. pertussis</i>. p<0.05, **p<0.01, ***p<0.001 WT + Pa or IL-4^−/− + Pa versus WT + PBS; +p<0.05, ++ p<0.01, +++ p<0.001 WT + Pa or IL-4^−/− +Pa versus IL-17A^−/− +Pa. (C) Effect of recombinant IL-17A, IL-17F or IFN-γ, in the presence of mouse serum from naive or immune mice (containing <i>B. pertussis</i> antibodies from Pa-immunized mice) on neutrophil-mediated killing of <i>B. pertussis in vitro</i>. *p<0.05, **p<0.01 versus control. Results in A and B are mean values for 4 mice per group at each time point and each panel is representative of 3 independent experiments. Results in C are mean values for triplicate assays and are representative of 3 experiments.</p

Th17 and Th1 cells mediate host immunity to <i>B. pertussis</i> in the respiratory tract of naive mice.

<p>(A–C) Naive C57BL/6 mice were exposed to an aerosol infection with <i>B. pertussis</i> and groups of 4 mice were sacrificed at the indicated time points. (A) Lung mononuclear cells were stimulated with heat-killed <i>B. pertussis</i> and after 3 days of culture IL-17A was quantified in supernatants by ELISA. (B–C) lung mononuclear cells were incubated with brefeldin-A for 1 h and intracellular cytokine staining for IL-17A, together with surface staining for CD4 was performed, followed by FACS analysis. Results are expressed as mean frequencies of IL-17A⁺CD4⁺ cells (B), with sample FACS plots (C) (D–E) C57BL/6 WT and IL-17A^−/− mice were aerosol challenged with <i>B. pertussis</i> and groups of 4 mice were sacrificed at the indicated time points. CFU counts were performed on lung homogenates (D) ** p<0.01 IL-17A^−/− versus WT. Neutrophil recruitment was determined by FACS analysis on lung lavage (E). (F) Spleen cells from IFN-γ^−/− or WT mice that had cleared a respiratory infection with <i>B. pertussis</i> were stimulated <i>in vitro</i> with killed <i>B. pertussis</i> and IL-12 (Th1) or IL-1β and IL-23 (Th17) respectively. After 4 days of culture surviving cells were harvested and <i>B. pertussis</i>-specific Th1, Th17 or both (10×10⁶) were transferred to naive mice, which were aerosol challenged with live <i>B. pertussis</i> 24 hours later. Naive mice that did not receive a cell transfer and mice injected with T cells from a naive mouse were used as controls. The course of infection was followed by performing CFU counts on the lungs at intervals after challenge. +p<0.05, +++ p<0.001 Th1+Th17 versus control; ** p<0.01, *** p<0.001 Th17 versus control. Results (except panel C) are mean values for 4 mice per group at each time point and each panel is representative of either 3 to 4 independent experiments.</p

Protective immunity induced with Pa is dependent on IL-17A but not IL-4.

<p>WT, IL-17A^−/−, IL-4^−/− or IFN-γ^−/− mice were immunized i.p. twice (0 and 28 days) with Pa. 14 days after the second immunization, mice were challenged by exposure to an aerosol of live <i>B. pertussis</i>. (A) The number of CFU in the lungs were quantified at intervals after challenge. (B) <i>B. pertussis</i>-specific cytokine production by spleen cells on day of challenge. (C) <i>B. pertussis</i>-specific antibody in serum on the day of challenge. *p<0.05, **p<0.01, ***p<0.001 knockout versus WT. Results are mean values for 4 mice per group at each time point and each panel is representative of 2 independent experiments.</p

Substitution of CpG for alum promotes induction of Th1 cells, which enhances the efficacy of a laboratory-prepared pertussis vaccine.

<p>Mice were immunized i.p. twice (0 and 28 days) with PBS, laboratory-prepared Pa in PBS (Ag) or formulated with alum (Al) or CpG. Mice were challenged by exposure to an aerosol of live <i>B. pertussis</i> 14 days after the second immunization. (A) The number of CFU in the lungs were quantified at intervals after challenge. (B) <i>B. pertussis</i>-specific cytokine production by spleen cells on day of challenge. (C) <i>B. pertussis</i>-specific antibody in serum on the day of challenge. +p<0.05, ++p<0.01, +++p<0.001 CpG versus alum; *p<0.05, **p<0.01, ***p<0.001 versus antigen in PBS. (D) WT, IL-17A^−/− or IFN-γ^−/− mice were immunized i.p. twice with a laboratory-prepared Pa formulated with CpG. Mice were challenged by exposure to an aerosol of live <i>B. pertussis</i> 14 days after the second immunization. The number of CFU in the lungs were quantified at intervals after challenge. Results are mean values for 4 mice per group at each time point and each panel (except D) is representative of 2 independent experiments.</p