Cytokine responses to two common respiratory pathogens in children are dependent on interleukin-1β

Abstract

Protracted bacterial bronchitis (PBB) in young children is a common cause of prolonged wet cough and may be a precursor to bronchiectasis in some children. Although PBB and bronchiectasis are both characterised by neutrophilic airway inflammation and a prominent interleukin (IL)-1β signature, the contribution of the IL-1β pathway to host defence is not clear. This study aimed to compare systemic immune responses against common pathogens in children with PBB, bronchiectasis and control children and to determine the importance of the IL-1β pathway. Non-typeable Haemophilus influenzae (NTHi) stimulation of peripheral blood mononuclear cells (PBMCs) from control subjects (n=20), those with recurrent PBB (n=20) and bronchiectasis (n=20) induced high concentrations of IL-1β, IL-6, interferon (IFN)-γ and IL-10. Blocking with an IL-1 receptor antagonist (IL-1Ra) modified the cellular response to pathogens, inhibiting cytokine synthesis by NTHi-stimulated PBMCs and rhinovirus-stimulated PBMCs (in a separate PBB cohort). Inhibition of IFN-γ production by IL-1Ra was observed across multiple cell types, including CD3⁺ T cells and CD56⁺ NK cells. Our findings highlight the extent to which IL-1β regulates the cellular immune response against two common respiratory pathogens. While blocking the IL-1β pathway has the potential to reduce inflammation, this may come at the cost of protective immunity against NTHi and rhinovirus.

FIGURE 1

Non-typeable Haemophilus influenzae (NTHi)-stimulated cytokine production. Peripheral blood mononuclear cells from healthy control children (n=17), children with protracted bacterial bronchitis (PBB) (n=19) and children with bronchiectasis (BE) (n=20) were cultured ex vivo in the presence of NTHi. Supernatant was collected at 24 h for the innate response cytokines (interleukin (IL)-1β, interleukin-1 receptor antagonist (IL-1Ra), IL-18 and IL-6) and at 72 h for the adaptive response cytokines (interferon (IFN)-γ and IL-10). a) IL-1β concentration. b) IL-1Ra concentration. c) IL-18 concentration. d) IL-6 concentration. e) IFN-γ concentration. f) IL-10 concentration. Box and whisker plots display median, interquartile ranges and range. **: p<0.01 by Wilcoxon matched-pairs signed rank test; ***: p<0.001 by Wilcoxon matched-pairs signed rank test.

FIGURE 2

Effects of interleukin-1 receptor antagonist (IL-1Ra) on peripheral blood mononuclear cell (PBMC) responses to non-typeable Haemophilus influenzae (NTHi). PBMCs were cultured ex vivo with NTHi in the presence or absence of the IL-1Ra anakinra. Data are from 14–15 healthy control children, 17–19 children with protracted bacterial bronchitis (PBB) and 12–13 children with bronchiectasis (BE). a) Interleukin (IL)-1β concentration. b) IL-6 concentration. c) IL-18 concentration. d) IFN-γ concentration. e) IL-10 concentration. ns: nonsignificant. *: p<0.05 by Wilcoxon matched-pairs signed rank test; **: p<0.01 by Wilcoxon matched-pairs signed rank test; ***: p<0.001 by Wilcoxon matched-pairs signed rank test.

FIGURE 3

Rhinovirus (RV)1B-stimulated cytokine production. Peripheral blood mononuclear cells from children with protracted bacterial bronchitis (PBB) were cultured ex vivo in the presence of RV1B. Supernatant was collected at 24 h for the innate response cytokines (interleukin (IL)-1β, interleukin-1 receptor antagonist (IL-1Ra), IL-18 and IL-6) and at 72 h for the adaptive response cytokines (interferon (IFN)-γ and IL-10). a) IL-1β concentration (n=10). b) IL-1Ra concentration (n=10). c) IL-18 concentration (n=6). d) IL-6 concentration (n=10). e) IFN-γ concentrations (n=8). f) IL-10 concentration (n=8). Box and whisker plots display median, interquartile ranges and range. *: p<0.05 by Wilcoxon matched-pairs signed rank test; **p<0.01 by Wilcoxon matched-pairs signed rank test.

FIGURE 4

Effects of interleukin-1 receptor antagonist (IL-1Ra) on peripheral blood mononuclear cell (PBMC) responses to rhinovirus (RV)1B. PBMCs from children with protracted bacterial bronchitis (PBB) were cultured ex vivo with RV1B in the presence or absence of the IL-1Ra anakinra. a) Interleukin (IL)-1β concentration (n=11). b) IL-6 concentration (n=11). c) Interferon (IFN)-γ concentration (n=12). d) IL-10 concentration (n=12). *: p<0.05 by Wilcoxon matched-pairs signed rank test.

FIGURE 5

Effects of interleukin (IL)-1β on peripheral blood mononuclear cell (PBMC) responses to non-typeable Haemophilus influenzae (NTHi) and rhinovirus (RV)1B. PBMCs from healthy adults were cultured with NTHi or RV1b in the presence or absence of recombinant IL-1β. Data are from 7–14 experiments. a) IL-6 concentration (n=7). b) Interferon (IFN)-γ concentration. c) IL-10 concentration. Mean and standard deviations are shown. *: p<0.05 by Wilcoxon matched-pairs signed rank test; ***: p<0.001 by Wilcoxon matched-pairs signed rank test.

FIGURE 6

Interleukin-1 blockade effects interferon (IFN)-γ production by multiple cell types. a) Gating strategy for flow cytometry: CD56⁺ natural killer (NK) cells (CD3⁻CD56⁺), CD3⁺ T-cells (CD3⁺CD56⁻) and other cells (CD3⁻CD56⁻) were identified within the total gated lymphocytes. The percentage of IFN-γ-producing cells was then evaluated in each of the cell subtypes. b) Percentage of non-typeable Haemophilus influenzae (NTHi)-stimulated IFN-γ-producing cells in the absence and presence of interleukin-1 receptor antagonist (IL-1Ra) at 24 h post-stimulation (n=9). *: p<0.05 by Wilcoxon matched-pairs signed rank test; **: p<0.01 by Wilcoxon matched-pairs signed rank test.