CSE induces greater NETosis of neutrophils derived from patients with COPD; CSE-NETs contain chDNA and mtDNA

CSE induces NETosis in human peripheral blood neutrophils (hPBNs);22,23 CS exposure also triggers chDNA and mtDNA release from lung tissue. We asked whether hPBNs from healthy participants and patients with COPD (supplementary Tables S1, S2) exhibit the different capacities of NETosis upon CSE stimulation, and whether the mtDNA:chDNA ratio differs between CSE-induced NETs and those produced by PMA or lomomycin, other known NETosis inducers. We quantified NETosis percentage in hPBNs treated with escalating doses of CSE and varying incubation time, then measured the mtDNA:chDNA ratio within NETs generated by CSE, PMA and iomomycin, respectively, by using next-generation sequencing, and assessing the 16s (mtDNA marker) to 18s (chDNA marker) ratio. We observed that hPBNs derived from patients with COPD (COPD-neutrophils) display a higher potency to release NETs upon stimulation with increasing CSE (Fig. 1a–f, Supplementary Figs. S2d–j, S3a and Supplementary Movie S1). CSE-induced NETs contained both mtDNA and chDNA at a ratio of approximately 1.3:1, whereas those stimulated by lomomycin and PMA contained a higher ratio of mtDNA to chDNA (Fig. 1g, h). These results were further corroborated by the quantification cycle (Cq) value ratio of 16s to 18s (Fig. 1i, j). Together, COPD-neutrophils show increased NETosis upon CSE stimulation, and CSE-NETs contain lower ratios of mtDNA:chDNA compared to those induced by PMA and lomomycin.

Fig. 1

Cigarette smoke extract (CSE) induces human peripheral blood neutrophils (hPBNs) to release neutrophil extracellular traps (NETs) in a dose- and time-dependent manner; CSE-induced NETs contain both mitochondrial DNA (mtDNA) and chromatin DNA (chDNA). Statistical analysis: n = 3–11 for each point or each bar in (b–j) from 3–11 healthy participants or 3–10 patients with COPD, respectively, data were presented as the mean ± standard deviation; Differences are assessed by the (b–f) two-way or (g–j) one-way ANOVA analysis of variance, followed Tukey’s honest significant test; *P < 0.05, ***P < 0.001 and ****P < 0.0001 represent a significant difference from the group of blank or group of healthy participants, the scattered samples and the p values are displayed in (g–j). a Representative immunofluorescence co-staining images display the NETs stimulated by 50 nM of phorbol-12-myristate-13-acetate (PMA) for 4 h and 5% CSE for 18 h; The NETs are costained with DNA (blue), MPO (myeloperoxidase, green) and histone H3 (red), and indicated by yellow arrows, scale bar: 30 μm. b, c The percentage of hPBNs, derived from b healthy participants and c patients with COPD, to release NETs upon stimulation with increasing dose of CSE and time of incubation, as charcterized by immunofluorescence staining of NETs components (supplementary Method 7); the stimulation of 50 nM of PMA as a positive control. d–f hPBNs derived from the patients with COPD release a higher percentage of NETs than those of healthy participants under the stimulation of d) 5% CSE, e 25% CSE, and f 50% CSE. g, h The ratio of the reads per kilobase per million mapped reads (RPKM) of mtDNA to the RPKM of chDNA in NETs induced by ionomycin (4 μM, incubated for 4 h), PMA (50 nM, for 4 h), 5% CSE (for 18 h) and 50% CSE (for 4 h) derived from g healthy participants and h patients with COPD, as assessed by next-generation sequencing (supplementary Method 10). i, j The ratio of the quantification cycle (Cq) value of 16 s to Cq of 18 s in spontaneous NETs (Blank) and those induced by ionomycin, PMA, 5% CSE and 50% CSE, derived from i healthy participants and j patients with COPD, as assessed by quantitative real-time polymerase chain reaction (qPCR) (supplementary Method 13)

CSE-induced NETosis requires mitochondrial ROS, but not NOX

NETosis triggered by PMA is nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX)-dependent, but the mechanism of NETosis induced by CSE remains elusive. Given that CSE-NETs exhibit heightened mtDNA content compared to PMA-NETs, we asked whether mitochondrial reactive oxygen species (mtROS) or NOX played a pivotal role in CSE-induced NETosis. We thus evaluated the inhibitory effects on NETosis induced by 5% CSE or PMA using respective inhibitors for mtROS (mitoTEMPO), mitochondrial respiration (thenoyltrifluoroacetone, TTFA), and NOX (diphenyleneiodonium chloride [DPI] and VAS2870 [VAS]). Inhibiting mtROS and mitochondrial respiration using mitoTEMPO and TTFA selectively suppressed 5% CSE-induced, but not PMA-induced, NETosis (Fig. 2a, b) in hPBNs derived from healthy participants, whereas NOX inhibitors DPI and VAS specifically attenuated PMA-induced, but not 5% CSE-induced NETosis (Fig. 2c, d); Deoxyribonuclease-I (DNase-I, endonuclease for single/double-stranded DNA) and GW311616A (GW, inhibitor of neutrophil elastase [NE]) treatments were effective against both 5% CSE- and PMA-induced NETosis (Fig. 2e, f and Supplementary Fig. S3b), with comparable results observed for COPD patient-derived hPBNs (Supplementary Fig. S4a–f). Treatment with 5% CSE resulted in elevated mtROS levels, particularly in hPBNs from COPD patients (Fig. 2g–i), with co-staining suggesting that mitochondria are the primary source of cellular ROS (supplementary Fig. S4g). Additionally, 5% CSE exposure triggered co-localisation of oxidative DNA damage marker 8-OHdZG (8-hydroxy-2’-deoxyguanosine) with mitochondrial membrane protein TOMM20 (outer mitochondrial membrane complex subunit 20) on the hPBNs (Fig. 2j) and enhanced 8-OHdZG staining in CSE-NETs derived from both healthy participants and patients with COPD (Fig. 2k and supplementary Fig. S4h). Together, CSE-induced NETosis relies on mtROS and mitochondrial respiration; CSE also provokes DNA damage within the mitochondrial membrane of hPBNs and in the formed CSE-NETs.

Fig. 2

Cigarette smoke extract (CSE)-induced NETosis requires mitochondrial reactive oxygen species (ROS) and mitochondrial respiratory chain, but not nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Statistical analysis: n = 3–10 for each bar in (a–f, h, i, k), from at least three healthy participants or three patients with COPD, data were presented as the mean ± standard deviation; Differences are assessed by the (a–f, k) one-way or (h, i) two-way ANOVA analysis of variance, followed Tukey’s honest significant test; P < 0.05 represents a significant difference, the scattered samples and the p values are displayed in figures. a–f The effects of several chemicals on the percentage of NETosis of human peripheral blood neutrophils (hPBNs, derived from healthy participants), stimulated by 5% CSE and 50 nM of PMA for 18 h: a 50 μM of mitoTEMPO (a mitochondrially targeted antioxidant), b 50 μM of thenoyltrifluoroacetone (TTFA, a mitochondrial respiration inhibitor), c 50 μM of diphenyleneiodonium chloride (DPI, a NADPH oxidase inhibitor), d 50 μM of VAS2870 (VAS, a NADPH oxidase inhibitor), e 200 IU/mL of deoxyribonuclease-I (DNase-I, an endonuclease for single- and double-stranded DNA) and f 50 μM of GW311616A (a selective human neutrophil elastase inhibitor), as characterized by immunofluorescence staining of NETs components (supplementary Method 7). g–i The incubation of 5% CSE for 2 or 4 h induces the release of mitochondrial ROS (stained by mitoSOX Red, supplementary Method 8, 17) by hPBNs from both healthy participants and patients with COPD, as assessed by i flow cytometry and fluorescence staining (scale bar: 50 μm), and summarised in g the percentage of positive events and h the mean intensity of mitoSOX Red. j The immunofluorescence colocalization of 8-hydroxy-2’-deoxyguanosine (8-OHdG, a marker of oxidative stress to DNA, green) and translocase of the outer mitochondrial membrane complex subunit 20 (TOMM20, a mitochondrial outer membrane marker, red), with and without the treatment of 5% CSE (supplementary Method 8). Note: oxidative-damaged DNA are presented on the mitochondrial membrane following the treatment of 5% CSE for 2 h (scale bar: 10 μm). k The fluorescence intensity (mean) of 8-OHdZG on hPBNs treated with 5% CSE and 50% CSE from healthy participants and patients with COPD

CSE-NETs increase the proliferation and production of both NF-κB-dependent cytokines and type-I IFNs in human AECs (hAECs), and the maturation of human DCs (hDCs)

Previous studies have shown that PMA, IL-8, UA, and MSU-induced NETs can exert cytotoxic effects or modulate the expression of IL-8 and IL-6 on AECs;25,26,27,28,29 however, the impact of CSE-induced NETs on primary human AECs remains uncertain. To address this, we prepared 5% CSE-induced NETs from healthy participants (Supplementary Method 9 and Supplementary Fig. S5a), and treated hAECs with escalating doses of NETs-DNA. We found that both 6 μg/mL and 12 μg/mL, but not 24 μg/mL, CSE-NETs promoted hAECs proliferation (Fig. 3a–c). In hAEC, the NETs treatment led to a dose-dependent increase in gene expression and soluble protein levels of NF-κB-dependent cytokines (CXCL5, IL-8, TNFα and IL-1β), as well as type-I IFNs (IFN-β1 and IL-12, Fig. 3d–l). Additionally, NETs treatment facilitated NF-κB (P65 and P50) activation in hAECs (Supplementary Figs. S5b, S6a–e), and maturation of hDCs (Fig. 3m, n). Together, CSE-NETs promote the proliferation and production of NF-κB-dependent cytokines and type-I IFNs in hAECs, and enhance the maturation of hDCs.

Fig. 3

Neutrophil extracellular traps (NETs) induced by cigarette smoke extract (CSE-NETs) dose-dependently promote the proliferation and the gene expressions of both nuclear factor kappa B (NF-κB)-dependent inflammatory cytokines and type-I interferons (IFNs) in the human airway epithelial cells (hAECs); CSE-NETs promote the maturation of human dendritic cells (DCs). Statistical analysis: n = 3–16 for each bar in (a–m) from at least three independent experiments, data were presented as the mean ± standard deviation; Differences are assessed by the (a–l) one-way or (m) two-way ANOVA analysis of variance, followed Tukey’s honest significant test; P < 0.05 represents a significant difference, the scattered samples and the p values are displayed in figures. a–c Human peripheral neutrophils were stimulated by 5% CSE for 18 h to induce NETs, which were subsequently collected and quantified for DNA concentrations. Effects of 6, 12, and 24 μg/mL NETs (incubated for 48 h) on the proliferation of hAECs, as assessed by the a EdU proliferation assay (supplementary Method 11) and mRNA expressions of b MKI67 and c PCNA (both are markers of proliferation, supplementary Method 13). d–l Effects of 6, 12, and 24 μg/mL NETs on the mRNA expressions and soluble levels of NF-κB-dependent inflammatory cytokines and type-I IFNs of hAECs (supplementary Method 13, 26): d mRNA expression of CXCL5 (C-X-C motif chemokine ligand 5), e mRNA expression of CXCL8, f mRNA expression of TNFα (tumour necrosis factor-alpha), g mRNA expression of IL-1β (interleukin 1β), h soluble levels of IL-1β in cell-culture supernatants, i soluble levels of CXCL8 in cell-culture supernatants, j mRNA expression of IFN-β1, k mRNA expression of IL-12 and l soluble levels of IFN-β in cell-culture supernatants. m, n 12 μg/mL of NETs promote the maturation of dendritic cells (differentiated from peripheral blood monocytes) from both healthy participants and patients with COPD (supplementary Method 15); the maturation is evaluated by m the percentage of CD86+ CD40+ cells, as assessed by flow cytometry with a gating strategy illustrated in (n) (Supplementary Method 17)

cGAS/TLR9 is required for CSE-NETs-induced proliferation and production of both NF-κB-dependent cytokines and type-I IFNs in hAECs, and maturation of hDCs

cGAS/TLR9 senses mtDNA/chDNA to activate downstream signalling cascades. Given the presence of mtDNA and chDNA in CSE-NETs, we asked whether these receptors contribute to the proliferation and production of cytokines in hAECs treated with CSE-NETs. We found that silencing both cGAS and TLR9 reduced NETs-mediated hAECs proliferation (Supplementary Fig. S5c–f and Fig. 4a–c, m–o). cGAS silencing ameliorated the NETs-induced gene expression and/or soluble protein levels of CXCL8, IL-1β, IFN-β1, and IL-12 (Fig. 4d–l), whereas TLR9 silencing ameliorated NETs-induced gene expression and/or soluble protein levels of CXCL5, TNFα, IL-1β and IL-12 (Fig. 4p–x). Furthermore, NETs-induced NF-κB (P65 and P50) activation in hAECs was inhibited upon cGAS and TLR9 silencing (Supplementary Fig. S6a–e). The inhibitors RU.521 and ODN 2088, targeting cGAS and TLR9, respectively, also decreased NETs-mediated DC maturation (Fig. 4y, z). Together, DNA sensor cGAS/TLR9 is essential for CSE-NETs-induced proliferation, production of NF-κB-dependent cytokines and type-I IFNs in hAECs, and hDCs maturation.

Fig. 4

Cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS), and toll-like receptor 9 (TLR9) are required for the neutrophil extracellular traps (NETs)-stimulated proliferation, expressions of both nuclear factor kappa B (NF-κB)-dependent inflammatory cytokines and type-I interferons (IFNs) in human airway epithelial cells (hAECs), and maturation of human dendritic cells (hDCs). Statistical analysis: n = 6–10 for each bar in (a–x), n = 3 for each bar in y from at least three independent experiments, data were presented as the mean ± standard deviation; Differences are assessed by the a–y two-way ANOVA analysis of variance, followed Tukey’s honest significant test; P < 0.05 represents a significant difference, the scattered samples and the p values are displayed in figures. Effects of a–c cGAS and m–o TLR9 silencing on 12 μg/mL of NETs-stimulated proliferation of hAECs, as assessed by the a, m EdU proliferation assay (supplementary Method 11), and the mRNA expression of b, n MKI67 and c, o PCNA (both are markers of proliferation, supplementary Method 13). Effects of d–l cGAS and p, x TLR9 silencing on 12 μg/mL of NETs-induced mRNA expression and soluble levels of NF-κB-dependent inflammatory cytokines and type-I interferons (IFNs) in hAECs (supplementary Method 13, 26): d, p mRNA expression of CXCL5 (C-X-C motif chemokine ligand 5), e, q mRNA expression of CXCL8, f, r mRNA expression of TNFα (tumour necrosis factor-alpha), g, s mRNA expression of IL-1β (interleukin 1β), h, t soluble levels of IL-1β in cell-culture supernatants, i, u soluble levels of CXCL8 in cell-culture supernatants, j, v mRNA expression of IFN-β1, k, w mRNA expression of IL-12 and l, x soluble levels of IFN-β in cell-culture supernatants. y The inhibition of cGAS and TLR9 by 5 μM of RU.521 and 2 μM of ODN 2088, respectively, reduces 12 μg/mL of NETs-mediated maturation of hDCs (supplementary Method 16). z Representative flow cytometry images display the gating strategy and maturation of DCs treated with either RU.521 or ODN 2088, as evaluated by the percentage of CD86+ CD40+ cells (supplementary Method 17)

Knockout of cGAS and TLR9, respectively alleviates airway inflammation, NETs infiltration and production of NF-κB-dependent cytokines, but not type-I IFNs, in a COPD mouse model

We then investigated whether the knockout of NETs-DNA sensor cGAS/TLR9 could mitigate long-term CS exposure-induced airway inflammation in an established COPD mouse model (Supplementary Method 19). We exposed cGAS and TLR9 knockout mice (cGAS−/− and TLR9−/−) and corresponding littermate to CS, and evaluated their severity of airway inflammation and changes in lung functions (Fig. 5a and Supplementary Fig. S7). Compared to CS-littermate (CS-littermate) mice, CS-treated cGAS−/− mice (CS-cGAS−/−) displayed reduced total cells, neutrophils and lymphocytes in bronchoalveolar lavage fluid (BALF, Fig. 5b–d), decreased NF-κB-dependent cytokines, including CXCL5, TNFα, IL-1β in BALF (Fig. 5e–h), CXCL5, GM-CSF, IL-1β in serum (Supplementary Fig. S9n–p), and CXCL5, IL-1β in lung tissue slices (Supplementary Fig. S9c, d, h–k), suppressed lung tissue NF-κB P65 activation (Supplementary Fig. S9q), improved lung function indicated by reduced FRC/BW (functional residual capacity/body weight) and increased FEV100/FVC (forced expiratory volume at 100 ms/forced vital capacity, Fig. 5k, l), and reduced histological score, mucin stain score, and mean linear intercept (MLI) of the alveoli (Fig. 5m, n and Supplementary Fig. S9a, b, f, g). The severity of NETs infiltration, as assessed by immunofluorescence co-staining of main components of NETs (Supplementary Method 24 and Supplementary Fig. S8), decreased in the CS-cGAS−/− mice (Fig. 5o, p), and was correlated with neutrophil count, histological score, and FEV100/FVC in CS-treated mice (Supplementary Fig. S9t–v). However, no change in type-I IFNs was observed in either CS-littermates or CS-cGAS−/− mice (Fig. 5i, j and Supplementary Fig. S9e, l, m, r, s), potentially due to impaired expression of IFN-α/β receptor subunit-1 (IFNAR1) in hAECs upon CS exposure (Supplementary Fig. S6f). A similar amelioration pattern was seen in CS-TLR9−/− mice (Supplementary Figs. S10, S11). Collectively, cGAS and TLR9 Knockout independently mitigated long-term CS-induced NF-κB (but not typer-I IFNs)-related airway inflammation.

Fig. 5

Guanosine monophosphate-adenosine monophosphate synthase (cGAS) knock-out (cGAS−/−) mice treated with cigarette smoke (CS) exposure display decreased productions of nuclear factor kappa B (NF-κB)-dependent inflammatory cytokines, but not type-I interferons (IFNs), alleviated airway inflammation, infiltration of neutrophil extracellular traps (NETs) and improved lung functions, as compared with CS-treated littermate. Statistical analysis: n = 9–20 for each bar in (b–l, n, p), data were presented as the mean ± standard deviation; Differences are assessed by the b–l, n, p two-way ANOVA analysis of variance, followed by Tukey’s honest significant test; P < 0.05 represents a significant difference, the scattered samples and the p values are displayed in figures. a A brief outline for the experiments of the COPD mouse model (supplementary Method 18, 19). b–d CS-treated cGAS−/− mice display alleviated airway inflammation as reflected by: b total cell counts, c neutrophil counts and d lymphocyte counts in the bronchoalveolar lavage fluid (BALF, supplementary Method 22). e–h CS-treated cGAS−/− mice reveal overall reduced productions of NF-κB-dependent inflammatory cytokines in the BALF (supplementary Method 22, 26): e C-X-C motif chemokine ligand 5 (CXCL5), f granulocyte-macrophage colony-stimulating factor (GM-CSF), g tumour necrosis factor-alpha (TNFα) and h interleukin 1β (IL-1β). i, j No significant changes of type-I IFNs are shown in BALF of either CS-treated littermate or cGAS−/− mice: i IFN-β, j IL-12. k, l CS-treated cGAS−/− mice reveal alleviated emphysema and airflow limitation in lung function tests (supplementary Method 21) as evaluated by: k functional residual capacity/body weight (FRC/BW), l forced expiratory volume at 100 ms/forced vital capacity (FEV100/FVC). m Representative images of hematoxylin-eosin (H&E)-stained lung slices display the decreased severity of airway inflammation in the CS-treated cGAS−/− mice, compared with CS-treated littermate (scale bar: 100 μm, Supplementary Method 23), as summarised in n histological score. o Representative immunofluorescence images display the decreased infiltration of NETs (co-stained with DNA, myeloperoxidase, and histone H3) in the lung slices of CS-treated cGAS−/− mice, compared with CS-treated littermate (scale bar: 100 μm, supplementary Method 24), as summarised in (p) normalised area of NETs

Inhibition of NETosis by mitoTEMPO alleviates airway inflammation, NETs infiltration and production of NF-κB-dependent cytokines, but not type-I IFN, in the COPD mouse model

To further investigate the contribution of NETs to long-term CS exposure-induced airway inflammation, we treated the COPD mouse model with mitoTEMPO, the specific mtROS inhibitor that inhibits CSE-induced NETosis as shown above. Compared to CS-treated controls, CS-treated mice receiving intraperitoneal injection of mitoTEMPO (CS-MT i.p mice, Fig. 6a) displayed reduced total cells, neutrophils, and lymphocytes in BALF (Fig. 6b–d), decreased NF-κB-dependent cytokines (CXCL5, GM-CSF and IL-1β) in both BALF (Fig. 6e–h), serum (Supplementary Fig. S12n–p) and lung tissue slices (Supplementary Fig. S12c, d, h–k), suppressed lung tissue NF-κB P65 activation (Supplementary Fig. S12q), improved lung function indicated by reduced FRC/BW and increased FEV100/FVC (Fig. 6k, l), and reduced histological score, mucin stain score, and MLI of alveoli (Fig. 6m, n and Supplementary Fig. S12a, b, f, g). The severity of NETs infiltration decreased in the CS-MT i.p mice (Fig. 6o, p), and was correlated with IL-1β and CXCL5 levels, but not IFN-β1 level, in the BALF of CS-treated mice (Supplementary Fig. S12t–v). No changes in type-I IFNs were observed (Fig. 6i, j and Supplementary Fig. S12e, l, m, r, s). Overall, mitoTEMPO alleviated long-term CS-induced NETs infiltration and NF-κB (but not typer-I IFNs)-related airway inflammation.

Fig. 6

Cigarette smoke (CS)-exposed wild-type mice treated with an intraperitoneal injection (i.p) of mitoTEMPO (MT) reveal decreased productions of nuclear factor kappa B (NF-κB)-dependent inflammatory cytokines, but not type-I interferons (IFNs), alleviated airway inflammation, infiltration of neutrophil extracellular traps (NETs), and improved lung functions, as compared with CS-treated saline i.p mice. Statistical analysis: n = 7–16 for each bar in (b–l, n, p), data were presented as the mean ± standard deviation; Differences are assessed by the (b–l, n, p) two-way ANOVA analysis of variance, followed Tukey’s honest significant test; P < 0.05 represents a significant difference, the scattered samples and the p values are displayed in figures. a A brief outline for the experiments of the COPD mouse model (Supplementary Method 18, 19). b–d CS-treated MT i.p mice reveal alleviated airway inflammation as reflected by the b total cell counts, c neutrophil counts and d lymphocyte counts in the bronchoalveolar lavage fluid (BALF, Supplementary Method 22). e–h CS-treated MT i.p mice reveal overall reduced production of NF-κB-dependent inflammatory cytokines in the BALF (supplementary Method 22, 26): e C-X-C motif chemokine ligand 5 (CXCL5), f granulocyte-macrophage colony-stimulating factor (GM-CSF), g tumour necrosis factor-alpha (TNFα) and h interleukin 1β (IL-1β). i, j CS-treated MT i.p mice reveal reduced levels of j IL-12, but not i IFN-β, in the BALF, compared with CS-treated saline i.p mice. k, l CS-treated MT i.p mice reveal alleviated emphysema and airflow limitation in lung function tests (supplementary Method 21) as evaluated by k functional residual capacity/body weight (FRC/BW) and l forced expiratory volume at 100 ms/forced vital capacity (FEV100/FVC). m Representative images of hematoxylin-eosin (H&E)-stained lung slices display the decreased severity of airway inflammation in CS-treated MT i.p mice, compared with CS-treated saline i.p mice (scale bar: 100 μm, supplementary Method 23), as summarised in n histological score. o Representative immunofluorescence images reveal the decreased infiltration of NETs (co-stained with DNA, myeloperoxidase and histone H3) in lung slices of CS-treated MT i.p mice, compared with CS-treated saline i.p mice (scale bar: 100 μm, supplementary Method 24), as summarised in (p) normalised area of NETs

Degradation of NETs-DNA by DNase-I alleviates NETs infiltration and emphysema-phenotype in the COPD mouse model

DNase-I has been shown to reduce airway inflammation induced by acute CS exposure.23 To explore the therapeutic potential of targeting NETs-DNA to mitigate airway inflammation induced by long-term CS exposure, we employed DNase-I to degrade NETs-DNA in the COPD mouse model. Compared with CS-treated control, CS-treated mice receiving nebulised DNase-I (DNase-I Neb. mice, Fig. 7a) showed alleviated emphysema-phenotype, as indicated by reduced FRC/BW (Fig. 7k) and MLI of alveoli (Fig. S13a, f), although there were only downward trends for total and different cell counts in BALF (Fig. 7b–d), histological scores (Fig. 7m, n), and mucin staining scores (Supplementary Fig. S13b, g) were observed in DNase-I Neb. mice. Nevertheless, NF-κB-dependent cytokines CXCL5 and IL-1β levels in BALF (Fig. 7e–h) and lung tissue slices (Supplementary Fig. S13c, d, h–k), IL-1β level in serum (Supplementary Fig. S13n–p), and NF-κB P65 activation in lung tissues (Supplementary Fig. S13q) significantly decreased in DNase-I Neb. mice. The severity of NETs infiltration also decreased in CS-DNase-I Neb. mice (Fig. 7o, p), and was correlated with MLI of alveoli and FRC/BW in CS-treated mice (Supplementary Fig. S13t, u). No changes in type-I IFNs were observed (Fig. 7i, j and Supplementary Fig. S13e, l, m, r, s). Together, DNase-I treatment alleviates NETs infiltration and emphysema features in the COPD mouse model. Supplementary Table S3 summarises the improved indicators seen in CS-cGAS−/−, TLR9−/−, MT i.p, and DNase-I Neb. mice.

Fig. 7

Cigarette smoke (CS)-exposed wild-type mice treated with the nebulization (Neb.) of deoxyribonuclease-I (DNase-I) reveal the decreased infiltration of neutrophil extracellular traps (NETs) and improved lung function, as compared with CS-treated saline Neb. mice. Statistical analysis: n = 7–15 for each bar in (b–l, n, p), data were presented as the mean ± standard deviation; Differences are assessed by the (b–l, n, p) two-way ANOVA analysis of variance, followed Tukey’s honest significant test; P < 0.05 represents a significant difference, the scattered samples and the p values are displayed in figures. a A brief outline for the experiments of the COPD mouse model (Supplementary Method 18, 19). b–d No significant changes of cell counts in bronchoalveolar lavage fluid (BALF) of CS-treated DNase-I Neb. mice are observed (Supplementary Method 22): b total cell counts, c neutrophil counts and d lymphocyte counts. e–h CS-treated DNase-I Neb. mice reveal the decreased levels of e C-X-C motif chemokine ligand 5 (CXCL5) and h interleukin 1β (IL-1β), but not that of f granulocyte-macrophage colony-stimulating factor (GM-CSF) or g TNFα (tumour necrosis factor-alpha) in the BALF (supplementary Method 22, 26). i, j No significant changes of type-I interferons (IFNs) level in the BALF of either CS-treated saline Neb. or DNase-I Neb. mice are observed: i IFN-β, j IL-12. k, l CS-treated DNase-I Neb. mice reveal alleviated emphysema, but not airflow limitation, in lung function tests (Supplementary Method 21) as evaluated by: k functional residual capacity/body weight (FRC/BW), l forced expiratory volume at 100 ms/forced vital capacity (FEV100/FVC). m Representative images of hematoxylin-eosin (H&E)-stained lung slices reveal that significant changes in the severity of airway inflammation are absent in the CS-treated DNase-I Neb. mice (scale bar: 100 μm, Supplementary Method 23), as summarised in n histological score. o Representative immunofluorescence images display the decreased infiltration of NETs (co-stained with DNA, MPO and Histone H3) in lung slices of the CS-treated DNase-I Neb. mice, compared with CS-treated saline Neb. mice (scale bar: 100 μm, Supplementary Method 24), as summarised in (p) normalised area of NETs

Disordered NF-κB-dependent cytokines, but not type-I IFNs, are correlated with MPO and NE activity in the BALF of patients with COPD

Patients with COPD, both stable and exacerbated, exhibit increased NETs in induced sputum17,18 that correlate with airflow limitation severity and microbiota diversity.19,20,21 However, the relationship between NETs components, NF-κB-dependent cytokines, and type-I IFNs in BALF of COPD patients remains unexplored. We collected BALF from 34 healthy participants (13 without and 21 with smoking history) and 32 COPD patients with smoking history (Supplementary Methods 1–3 and Supplementary Tables S1,