Figures
- FIGURE 1
Interleukin (IL)-1β-producing cells in peripheral blood mononuclear cells (PBMCs). Gating strategy for flow cytometry: monocytes (CD14+), NK cells (CD56+) and T-cells (CD3+) were obtained from total gated lymphocytes. Pro-IL-1β+ cells were then evaluated in each of the cell subtypes. a) Frequency of pro-IL-1β+ cells at 24 h post nontypeable Haemophilus influenzae (NTHi) stimulation (adult PBMCs, n=7). b) Percentage of each pro-IL-1β+ cell type in total lymphocytes. *: p<0.05 by Wilcoxon matched-pairs signed rank test.
- FIGURE 2
Expression of genes related to inflammasome activation in peripheral blood mononuclear cell (PBMC) and bronchoalveolar lavage (BAL) macrophages. PBMC or lung macrophages were cultured ex vivo for 24 h in the presence of nontypeable Haemophilus influenzae (NTHi). a) Expression of interleukin (IL)-1β in i) control PBMCs (n=16), ii) protracted bacterial bronchitis (PBB) PBMCs (n=16) and iii) BAL macrophages (n=18); b) expression of NLRC4 in i) control PBMCs (n=11) ii) PBB PBMCs (n=14) and iii) BAL macrophages (n=15). Data are presented as individual data points, median and interquartile ranges. *: p<0.05; **: p<0.01; ***: p<0.001 by Wilcoxon matched-pairs signed rank test.
- FIGURE 3
Effects of a caspase-1 inhibitor and NLRP3 inhibitor on nontypeable Haemophilus influenzae (NTHi)-induced interleukin (IL)-1β production. a) Peripheral blood mononuclear cells (PBMCs) and bronchoalveolar lavage (BAL) cells were cultured ex vivo for 24 h with NTHi in the presence or absence of the caspase-1 inhibitor (C1I) Z-YVAD-FMK. IL-1β produced by healthy control (HC) PBMCs (n=20), protracted bacterial bronchitis (PBB) PBMCs (n=20), BAL macrophages (PBB BAL Mac) (n=9) and BAL neutrophils (PBB BAL Neut) (n=9). b) BAL macrophages (n=9) were cultured ex vivo for 24 h in the presence of NTHi and NLRP3 inhibitor MCC950. Data are presented as mean±sd. *: p<0.05; **: p<0.01; ***: p<0.001 by Wilcoxon matched-pairs signed rank test.
- FIGURE 4
Visualisation of the NLRP3 inflammasome in bronchoalveolar lavage (BAL) macrophages. Representative confocal microphotos of cells cultured on chamberslides a) in control medium or b) stimulated with nontypeable Haemophilus influenzae (NTHi), both revealing specks of NLRP3 (red) and cleaved caspase-1 (green) in colocalisation (yellow/orange). Blue is the pseudocolour of DAPI (4',6-diamidino-2-phenylindole). Scale bars=40 μm (main images) and 16 μm (inset images). c) Quantitative analysis of NLRP3 specks in control and NTHi-stimulated macrophages (n=8) showed a significant difference between the groups. ***: p<0.001 by Mann–Whitney U-test.
- FIGURE 5
Colocalisation of NLRP3 specks with cleaved interleukin (IL)-1β in bronchoalveolar lavage (BAL) macrophages, and nontypeable Haemophilus influenzae (NTHi)-stimulated monocyte-derived macrophages. BAL macrophages cultured for 24 h in a) control medium or b) in presence of NTHi both revealed specks of NLRP3 (red) and cleaved IL-1β (green) in colocalisation (yellow/orange; arrows). Monocyte-derived macrophages showed c) only homogenous fluorescence when cultured in control medium, but d) revealed numerous NLRP3/cleaved IL-1β specks (arrows) when stimulated with NTHi. Blue is the pseudocolour of DAPI (4',6-diamidino-2-phenylindole). Scale bars=20 μm. Images are representative confocal microphotos of cells from at least four donors.
- FIGURE 6
Nontypeable Haemophilus influenzae (NTHi)-induced increased specks of AIM2 in bronchoalveolar lavage (BAL) macrophages. Representative confocal images of AIM2 (red) in chamberslides of BAL macrophages cultured for 24 h in a) control medium or b) in presence of NTHi. Blue is the pseudocolour of DAPI (4',6-diamidino-2-phenylindole). Scale bars=40 μm (main images) and 16 μm (inset images). c) Quantitative analysis of AIM2 specks in control and NTHi-stimulated samples (n=4) showed a significant difference between the groups. *: p<0.05 by Mann–Whitney U-test.
- FIGURE 7
Nontypeable Haemophilus influenzae (NTHi)-induced inflammasome specks in peripheral blood mononuclear cell (PBMC) and blood monocytes. Specks of NLRP3 (red) were not detected in a) PBMCs cultured in control medium, but were readily detected in b) the presence of NTHi. Specks of AIM2 (red) were not detected in PBMCs c) cultured in control medium, but were readily detected in d) the presence of NTHi. Specks of NLRP3 (red) were e) not detected in monocytes cultured in control medium, but f) were readily detected in the presence of NTHi. g) and h) Dual labelling of NTHi-stimulated monocytes revealed colocalised specks of AIM2 (red) and cleaved interleukin (IL)-1β (green). Yellow is merged colour of red and green. Blue is the pseudocolour of DAPI (4',6-diamidino-2-phenylindole). Scale bars=20 μm (main images) and 8 μm (inset images). Images are representative confocal microphotos of cells from of at least four donors.
Tables
- TABLE 1
Subject demographics
PBMCs BAL cells Controls PBB Subjects 20 20 35 Age years 2.2 (0.3–4.5) 1.6 (0.9–2.8) 2.6 (1.3–4.4) Males 15 (75) 16 (80) 20 (57) Culturable bacterial organisms n N/A 3 (3–4) 3 (3–4) Differential cell count ×106·L-1 Total cell count 8.90 (8.18–10.20) 11.0 (9.05–12.2) N/A Neutrophils 2.88 (1.69–3.72) 3.63 (2.49–4.96) N/A Monocytes 0.80 (0.59–1.02) 1.09 (0.78–1.16) N/A Eosinophils 0.34 (0.12–0.67) 0.38 (0.19–0.59) N/A Lymphocytes 4.82 (3.81–5.87) 5.32 (4.04–6.60) N/A Immune cells % Neutrophils N/A N/A 23.0 (5.00–41.7) Macrophages N/A N/A 65.0 (32.6–89.0) Lymphocytes N/A N/A 7.60 (3.80–12.0) Eosinophils N/A N/A 0.00 (0.00–0.30) Data are presented as n, median (interquartile range) or n (%). PBMCs: peripheral blood mononuclear cells; BAL: bronchoalveolar lavage; PBB: protracted bacterial bronchitis; N/A: not applicable.
Supplementary Material
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Supplementary material 00130-2017_supp
