Fig 2: WNT signalling regulates expression of antimicrobial peptides (AMPs) in small airway epithelial cells. mRNA expression of a) AXIN2 and b) S100A8 and S100A9 in small airway epithelial cells exposed to WNT inducer Chir99021 (CT) or WNT inhibitors FH535 (FH) or WNT5A for 24 h, relative to vehicle-exposed basal cells. Gene expression comparative ??Cp is presented as a mean (n=9–14). The impact of CT, WNT5A and FH was calculated using a paired t-test; *: p<0.05; **: p<0.01; ****: p<0.0001.

Fig 3: Chronic obstructive pulmonary disease (COPD) small airway epithelial cells (SAECs) exert a delayed antimicrobial peptide (AMP) response to non-typeable Haemophilus influenzae (NTHi). mRNA expression of AMPs (DEFB1, DEFB4A (gene name for ß-defensin 2 protein), CAMP, SLPI, S100A7, S100A8, S100A9, PI3) in non-COPD-derived (nC, grey) and COPD-derived (C, red) SAECs exposed to NTHi for 3 h or 24 h relative to PBS-exposed SAECs. Gene expression comparative crossing point change (??Cp) is presented as a mean (n=11–18). Differences between NTHi and corresponding PBS-exposed SAECs were calculated with paired t-test; *: p<0.05, **: p<0.01, ***: p<0.001, ****: p<0.0001. Differences between means of non-COPD and COPD SAECs were calculated with a Mann–Whitney test; #: p<0.05.

Fig 4: S100 calcium binding protein A8/S100 calcium binding protein A9 (S100A8/A9) and ß-defensin 2 (BD2) proteins are differentially regulated by non-typeable Haemophilus influenzae (NTHi) in non-chronic obstructive pulmonary disease (non-COPD) and COPD small airway epithelial cell (SAEC) secretions. Expression of a) BD2 and b) S100A8/A9 proteins in supernatants (SNs) obtained from SAEC air–liquid-interface cultures from apical (left) and basolateral (right) compartment measured by ELISA. Data are presented as an expression of the protein in SN obtained from SAECs exposed to NTHi, relative to the expression of protein in SNs obtained from SAEC exposed to PBS for 3 h (PBS-exposed condition as 100%; BD2 n=9; S100A8/A9 apical non-COPD SN n=11, apical COPD SN n=10, basolateral non-COPD SN n=6, basolateral COPD SN n=4). Impact of NTHi on protein expression in non-COPD and COPD SAEC SN was calculated using a one-sample t-test; *: p<0.05.

Fig 5: S100 calcium binding protein A8/S100 calcium binding protein A9 (S100A8/A9) modulates macrophage phenotype and metabolism. a) Phosphorylation status of signalling molecules upon 30 min S100A8/A9 (5 µg·mL-1) treatment depicted as fold-difference relative to PBS-induced phosphorylation (n=3). Influence of S100A8/A9 on phosphorylation was calculated using a one-sample t-test. b) mRNA expression of FCGR1A, FCGR2A, MARCO, TLR2 and TLR4 in human monocyte-derived macrophages (MDMs) exposed to PBS or S100A8/A9 (5 µg·mL-1) for 6 h. Relative gene expression, expressed as crossing point change (?Cp=Cp for GAPDH-Cp for gene of interest) is presented as a mean (n=11). c) Expression of surface markers (CD64, CD332, MARCO, CD163, CD206, CD11C, CD40, CD80 and CD38) presented as geometric mean fluorescence intensity (MFI) on MDMs exposed for 24 h to PBS or S100A8/A9, measured by flow cytometry (n=5). d) Expression of cytokines (pg·mL-1) in supernatants from MDMs exposed to PBS or S100A8/A9 for 24 h. Data are presented as a mean (n=5). e) Glycolysis stress test: glycolysis, glycolytic capacity and glycolytic reserve in MDMs upon PBS or S100A8/A9 exposure for 24 h, presented as a mean (n=3). Impact of S100A8/A9 calculated using paired t-test; *: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.0001.