Figures
- FIGURE 1
Increased bacterial DNA in bronchoalveolar lavage fluid (BALF) of neutrophilic asthma subjects. a) Bacterial DNA levels in BALF of normal subjects, non-neutrophilic asthma subjects and neutrophilic asthma subjects. b) Correlations between bacterial DNA and neutrophils and c) interleukin (IL)-8 in BALF of non-neutrophilic asthma subjects (black open circles) and neutrophilic asthma subjects (red open circles). The horizontal bars represent medians.
- FIGURE 2
Bacterial DNA amplified interleukin (IL)-8 production in IL-17-exposed human airway epithelial cells and lung macrophages. a) Primary normal human small airway epithelial cells (n=5 subjects) grown at air–liquid interface. b) Primary normal human tracheobronchial epithelial cells under submerged culture (n=4 subjects). c) Primary normal human alveolar macrophages (n=5 subjects). Cells were stimulated with or without IL-17 for 24 h and then treated with or without bacterial DNA for 48 h. (−) indicates controls. The horizontal bars represent medians.
- FIGURE 3
Bacterial DNA increased neutrophilic inflammation in interleukin (IL)-17-exposed mouse lungs. Wild-type C57BL/6 mice (n=10 to 12 mice per group) from two independent experiments were intranasally challenged with IL-17 (3 µg/mouse) for 24 h, followed by DNA (1 µg/mouse) derived from nontypeable Haemophilus influenzae. After 24 h, bronchoalveolar lavage fluid (BALF) was analysed for a) % of neutrophils, b) total numbers of neutrophils and c) neutrophil chemoattractant LIX. (−) indicates controls. The horizontal bars represent medians.
- FIGURE 4
Role of DNA signalling in bacterial DNA-mediated amplification of neutrophil chemokines. a) Primary normal human small airway epithelial cells (n=4 subjects) under submerged culture were treated with interleukin (IL)-17 for 24 h, and then exposed to bacterial DNA±A151 or A151 negative control (ctrl) for 48 h. b) Human tracheobronchial epithelial cells transduced with lentivirus containing the STING sgRNA or scramble (SCR) control sgRNA plasmid construct were treated with IL-17 for 24 h, and then exposed to bacterial DNA under the submerged condition for 48 h. c) Mouse tracheal epithelial cells (mTEC) isolated from wild-type (C57BL/6) and Toll-like receptor 9 (TLR9) knockout (KO) mice were grown at air–liquid interface and stimulated with or without IL-17 for 24 h and then treated with/without bacterial DNA for 48 h. d) Bone marrow-derived macrophages from wild-type and TLR9 KO mice were cultured and stimulated with or without IL-17 and exposed to either bacterial DNA (bDNA) or mammalian DNA (mDNA) extracted from mTEC. n=3 to 4 replicates. (−) indicates controls. The horizontal bars represent means.
- FIGURE 5
Toll-like receptor 9 (TLR9) conditional knockout in myeloid cells downregulates airway neutrophilic inflammation in mice exposed to interleukin (IL)-17 and bacterial DNA. LysMCre+TLR9fl/fl and LysMCre–TLR9fl/fl mice were challenged with IL-17 (3 µg/mouse) in 0.01% bovine serum albumin (BSA) or 0.01% BSA (control) for 24 h, followed by bacterial DNA (1 µg/mouse) challenge or Tris-EDTA buffer via intranasal inoculation. After 24 h of DNA exposure, bronchoalveolar lavage fluid (BALF) was collected and analysed for a,b) neutrophils and c) neutrophil chemoattractant LIX. (−) indicates controls. The horizontal bars represent medians.
- FIGURE 6
Interleukin (IL)-36γ in human bronchoalveolar lavage fluid (BALF) and cultured airway epithelial cells. a) Representative Western blot images and densitometry of IL-36γ protein in BALF from healthy control subjects, non-neutrophilic asthma (NNA) and neutrophilic asthma (NA) subjects. Each dot represents one subject. b) IL-36γ Western blot images and densitometry in supernatants of cultured primary normal human small airway epithelial cells (n=5 subjects) exposed to IL-17 for 24 h, and then bacterial DNA for 48 h. c) Primary normal human small airway epithelial cells were stimulated with or without IL-17 for 24 h and then treated with/without bacterial DNA in the presence of an IgG isotype control or an IL-36γ neutralising antibody for 48 h. (−) indicates controls. The horizontal bars represent medians.
- FIGURE 7
Recombinant human DNase I reduces lung neutrophilic inflammation in interleukin (IL)-17-challenged and nontypeable Haemophilus influenzae (NTHi)-infected mice. Wild-type C57BL/6 mice were challenged with IL-17 (3 µg/mouse), NTHi (107 CFU/mouse) and DNase I (5 µg/mouse) via intranasal inoculation. After 24 h, bronchoalveolar lavage fluid (BALF) was collected for analysing total numbers of a) neutrophils, b) neutrophil chemoattractant LIX, c) count of colony forming units (CFU) in the right lung tissue homogenate and d) IL-36γ protein using Western blot. (−) indicates controls. The horizontal bars represent medians.
- FIGURE 8
Proposed mechanisms by which bacterial DNA and interleukin (IL)-17 cooperate to amplify airway neutrophilic inflammation. IL-17 binds to its receptor and induces IL-36γ expression. IL-36γ binds to its heterodimeric receptor complex, subsequently recruits MyD88, activates MAPK and NF-κB signalling cascades, and induces IL-8 and neutrophilic inflammation. Bacterial DNA binds to TLR9, leading to activation of MAPK and NF-κB signalling pathways. Activation of IL-36γ signalling also results in TLR9 translocation, leading to further activation of TLR9 signalling and amplification of neutrophilic inflammation. MyD88: adaptor protein myeloid differentiated protein 88; IRAK1: interleukin-1 receptor-associated kinase 1; MAPK: mitogen-activated protein kinase; NF-κB: nuclear transcription factor kappa B; TLR9: Toll-like receptor 9.
Tables
- TABLE 1
Characteristics of healthy and asthma subjects
Healthy subjects Non-neutrophilic asthma (NNA) Neutrophilic asthma (NA) p-value Subjects n 20 14 16 Age years 33.6±2.0 42.2±3.7 51.8±3.9 <0.01 NA versus healthy, >0.05 NA versus NNA Sex M/F n 7/13 6/8 8/8 0.33 NA versus healthy and NNA FEV1 % predicted 96.0±2.7 75.4±5.5 61.2±5.0 <0.01 NA versus healthy,
0.07 NA versus NNABMI kg·m−2 29.7±1.3 27.5±1.5 29.3±1.5 0.55 NA versus healthy and NNA FENO ppb 23.2±3.3 36.3±5.9 52.7±13.1 0.02 NA versus healthy, >0.05 NA versus NNA ACT score N/A 17.5±1.3 17.1±1.6 0.84 NA versus NNA Inhaled corticosteroid use None High dose (3), low dose (11) High dose (9), low dose (5), medium dose (1), unknown (1) 0.11 NA versus NNA Neutrophils % in BALF 1.9±0.3 1.4±0.2 15.3±4.6 <0.01 NA versus healthy and NNA Eosinophils % in BALF 0.5±0.2 1.8±0.7 3.0±0.9 <0.01 NA versus healthy, >0.05 NA versus NNA Macrophages % in BALF 91.5±1.0 90.4±2.7 72.4±4.9 <0.01 NA versus healthy and NNA Lymphocytes % in BALF 6.1±0.8 6.4±1.3 9.9±1.7 0.11 NA versus healthy and NNA IL-8 pg·mL−1 in BALF 24.8±1.5 41.8±5.8 138.5±42.7 <0.01 NA versus healthy and NNA Data expressed as mean±sem unless otherwise stated. All subjects were nonsmokers. M: male; F: female; FEV1: forced expiratory volume in 1 s; BMI: body mass index; FENO: fractional exhaled nitric oxide; ACT: asthma control test; BALF: bronchoalveolar lavage fluid; IL-8: interleukin-8; N/A: not applicable.
Supplementary Material
Please note: supplementary material is not edited by the Editorial Office, and is uploaded as it has been supplied by the author.
Supplementary materials and methods 00474-2022.SUPPLEMENT
Supplementary figures S1, S2, S3 and S4 00474-2022.SUPPLFIGURES
