Collagen coated flasks

Primary human nasal epithelial cells (HNECs) (3 males, 1 female, non-smoker, aged 45–65 years, non-allergic) were harvested from nasal polyps by gentle brushing in a method described by Ramezanpour et al.³⁹ . Extracted cells were suspended in Bronchial Epithelial Growth Media (BEGM, CC-3170, Lonza, Walkersville, MD, USA), supplemented with 2% Ultroser G (Pall Corporation, Port Washington, NY, USA). The cell suspension was depleted of macrophages using anti-CD68 (Dako, Glostrup, Denmark) coated culture dishes, and HNECs were maintained with B-ALI™ growth medium (Lonza, Walkersville, USA) in collagen coated flasks (Thermo Scientific, Walthman, MA, USA) in a cell incubator at 37 °C with 5% CO₂.

Primary HNECs were harvested from nasal mucosa by gentle brushing. Extracted cells were suspended in PneumaCultTM- Ex Plus medium (StemCell Technologies, Vancouver, Canada).
Macrophages were removed by treating the cells with anti-CD68 (Dako, Glostrup, Denmark) coated petri dishes for 20 min at 37 ℃. HNECs were maintained with PneumaCultTM- Ex Plus medium in collagen coated flasks (Thermo Scientific, Walthman, MA, USA) at 37 °C with 5% CO₂ until confluence. 5*10⁵ cells were seeded in collagen coated 24 well plates (Corning, NY, USA) and 8 well chamber slides (Corning, NY, USA) respectively. Cells were treated with 5% bacterial planktonic supernatants for different times followed by RNA extraction, immunofluorescence staining or protein extraction as described below. Cells treated with purified SpA (50 μg /ml) and 5% TSB were used as positive and negative control, respectively.

This study was approved by the Institutional Review Board for Clinical Research of Hokkaido University Hospital, Sapporo, Japan (019–0242) and by The Queen Elizabeth Hospital Human Research Ethics Committee (reference HREC/15/TQEH/132), and was conducted in accordance with the Declaration of Helsinki. Written informed consent was obtained from study participants prior to tissue or cell collection. Primary human nasal epithelial cells (HNECs) were taken from the nasal mucosa of the inferior turbinates of seven patients treated for deviation of the nasal septum as previously described (Cooksley et al., 2015 (link); Suzuki et al., 2018 (link); Ramezanpour et al., 2019 (link)). The cells were suspended in Bronchial Epithelial Growth Medium (BEGM, CC-3170, Lonza, Walkersville, MD, USA) supplemented with Amphotericin B (Fujifilm, Osaka, Japan) and Penicillin/Streptomycin (Fujifilm, Osaka, Japan). Monocytes were depleted from the cell suspension using anti-CD68 antibody (Dako, Glostrup, Denmark) -coated cell culture dishes. HNECs were incubated at 37°C in humidified conditions under 5% CO₂ in collagen-coated flasks (Thermo Scientific, Waltham, MA, USA) until passage 2.

HNECs were harvested from the inferior turbinate using a nasal brush and cultured as described (Ramezanpour et al., 2016 (link), 2017 (link)). Briefly, extracted HNECs were suspended in Bronchial Epithelial Growth Media (BEGM, CC-3170, Lonza, Walkersville, MD, USA), supplemented with 2% Ultroser G (Pall Corporation, Port Washington, NY, USA). The cell suspension was depleted of monocytes using anti-CD68 (Dako, Glostrup, Denmark) coated culture dishes, and HNECs expanded in routine cell culture conditions of 37°C humidified air with 5% CO₂ in collagen coated flasks (Thermo Scientific, Walthman, MA, USA). HNECs were tested at passage two and confirmed to be of epithelial lineage via reactivity to pan-Cytokeratin and CD45 antibodies (both from Abcam, Cambridge, MA, USA), and a Diff-Quick staining method used in the assessment of cell morphology by professional cytologists (IMVS, The Queen Elizabeth Hospital, Woodville, Australia).

Immortalised wild‐type C57BL/6 bone marrow‐derived macrophages (iBMDMs) and NLRP3‐deficient iBMDMs reconstituted with NLRP3‐Flag and ASC‐Cerulean (termed ASC‐cerulean iBMDMs) were grown in DMEM supplemented with 10% heat‐inactivated FBS and 2 mm glutamine supplied by Thermo Fisher (Melbourne, Australia). Bone marrow cells from C57BL/6 wild‐type mice were differentiated for 7 days in DMEM supplemented with 10% (v/v) FCS and 1% (v/v) penicillin/streptomycin solution (Thermo Fisher) and macrophage colony‐stimulating factor (20% v/v L929 mouse fibroblast supernatant).
Primary human airway cells⁵⁹ were obtained from normal subjects who were non‐smokers or had not smoked for > 15 years; none had a diagnosis of asthma or COPD (normal FEV₁ measurements). Studies were approved by the Monash Health and Monash Medical Centre Human Research Ethics Committee, consent was obtained from all subjects and studies were conducted in accordance with the approved guidelines. Primary bronchial epithelial cells (pBEC) were obtained from bronchial brushings and cultured under submerged conditions on collagen‐coated flasks (Thermo Fisher) in supplemented bronchial epithelial growth medium (Lonza, Melbourne, Australia). All bronchial brushings were obtained from the same anatomical regions (bronchial generations 4–7) and were used within five passages.

Ethical approval for nasal brushing from CRS patients was granted from the Queen Elizabeth Hospital Human Ethics Committee and only consented patients were included in the study. Exclusion criteria included active smoking, age less than 18 years, and systemic disease. Primary human nasal epithelial cells (HNECs) were harvested from nasal polyps by gentle brushing in a method as described in [16 (link)]. Extracted cells were suspended in Bronchial Epithelial Growth Media (BEGM, CC-3170, Lonza, Walkersvill, MD, USA), supplemented with 2% Ultroser G (Pall Corporation, Port Washington, NY, USA). The cell suspension was depleted of monocytes using anti-CD68 (Dako, Glostrup, Denmark) coated culture dishes, and HNECs expanded in routine cell culture conditions of 37°C humidified air with 5% CO₂ in collagen coated flasks (Thermo Scientific, Walthman, MA, USA). HNECs were tested at passage two and confirmed to be of epithelial lineage via reactivity to pan-Cytokeratin and CD45 antibodies (both from Abcam, Cambridge, MA, USA), and a Diff-Quick staining method used in the assessment of cell morphology by professional cytologists (IMVS, The Queen Elizabeth Hospital, Woodville, Australia).