Dna of neutrophil extracellular traps promote nf-κb-dependent autoimmunity via cgas/tlr9 in chronic obstructive pulmonary disease

ABSTRACT Chronic obstructive pulmonary disease (COPD) is characterised by persistent airway inflammation even after cigarette smoking cessation. Neutrophil extracellular traps (NETs) have

been implicated in COPD severity and acute airway inflammation induced by short-term cigarette smoke (CS). However, whether and how NETs contribute to sustained airway inflammation in COPD

remain unclear. This study aimed to elucidate the immunoregulatory mechanism of NETs in COPD, employing human neutrophils, airway epithelial cells (AECs), dendritic cells (DCs), and a

long-term CS-induced COPD mouse model, alongside cyclic guanosine monophosphate-adenosine monophosphate synthase and toll-like receptor 9 knockout mice (_cGAS__-−/−_, _TLR9__−/−_);

Additionally, bronchoalveolar lavage fluid (BALF) of COPD patients was examined. Neutrophils from COPD patients released greater cigarette smoke extract (CSE)-induced NETs (CSE-NETs) due to

mitochondrial respiratory chain dysfunction. These CSE-NETs, containing oxidatively-damaged DNA (NETs-DNA), promoted AECs proliferation, nuclear factor kappa B (NF-κB) activation,

NF-κB-dependent cytokines and type-I interferons production, and DC maturation, which were ameliorated/reversed by silencing/inhibition of cGAS/TLR9. In the COPD mouse model, blocking

NETs-DNA-sensing via _cGAS_−_/−_ and _TLR9_−_/−_ mice, inhibiting NETosis using mitoTEMPO, and degrading NETs-DNA with DNase-I, respectively, reduced NETs infiltrations, airway inflammation,

NF-κB activation and NF-κB-dependent cytokines, but not type-I interferons due to IFN-α/β receptor degradation. Elevated NETs components (myeloperoxidase and neutrophil elastase activity)

in BALF of COPD smokers correlated with disease severity and NF-κB-dependent cytokine levels, but not type-I interferon levels. In conclusion, NETs-DNA promotes NF-κB-dependent autoimmunity

via cGAS/TLR9 in long-term CS exposure-induced COPD. Therefore, targeting NETs-DNA and cGAS/TLR9 emerges as a potential strategy to alleviate persistent airway inflammation in COPD. SIMILAR

CONTENT BEING VIEWED BY OTHERS N6-METHYLADENOSINE-MODIFIED CIRCSAV1 TRIGGERS FERROPTOSIS IN COPD THROUGH RECRUITING YTHDF1 TO FACILITATE THE TRANSLATION OF IREB2 Article 24 February 2023 A

DISINTEGRIN AND METALLOPROTEINASE DOMAIN-15 DEFICIENCY LEADS TO EXAGGERATED CIGARETTE SMOKE-INDUCED CHRONIC OBSTRUCTIVE PULMONARY DISEASE (COPD)-LIKE DISEASE IN MICE Article 20 July 2020

NEUTROPHIL EXTRACELLULAR TRAPS PROMOTE IMMUNOPATHOGENESIS OF VIRUS-INDUCED COPD EXACERBATIONS Article Open access 09 July 2024 INTRODUCTION Chronic obstructive pulmonary disease (COPD) is a

leading cause of global morbidity and mortality,1 and is characterised by persistent airflow limitation owing to progressive airway remodelling and alveolar destruction. Cigarette smoke (CS)

is the primary risk factor for COPD. CS induces an abnormal inflammatory response, characterised by increased neutrophils, macrophages and T and B lymphocytes recruited from the

circulation. These inflammatory cells, together with lung structural cells, including airway epithelial cells (AECs), secrete numerous proinflammatory cytokines to induce chronic airway

inflammation, which essentially drives tissue injury in COPD.2 Anti-inflammation is considered the mainstream strategy for COPD therapy. Bronchodilators, including long-acting muscarinic

antagonists (LAMAs) and long-acting beta-2 agonists (LABAs), either as standalone agents or in combination with anti-inflammation agents (inhaled corticosteroids, ICS), constitute the

cornerstone of pharmacological treatment for patients with COPD. Nevertheless, ICS administration alone does not decelerate the rate of lung function decline in patients with COPD.3

Moreover, in contrast to the transient acute airway inflammation induced by short-term CS exposure, the airway inflammation observed in COPD persists even after smoking cessation,4,5

suggesting that the efficacy of the current therapy is limited by persistent airway inflammation. However, researchers have not completely elucidated the underlying mechanism. A key aspect

of persistent airway inflammation lies in the delayed immunological reactions caused by a complex interplay between innate and adaptive immune systems, particularly in response to

pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs). DAMPs are notably augmented in COPD due to hazardous substances from CS exposure, oxidative

stress-induced apoptosis, and impaired phagocytosis by alveolar macrophages.6 Pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs) and cyclic guanosine

monophosphate-adenosine monophosphate synthase (cGAS), recognise these DAMPs and initiate signalling cascades, often culminating in the nuclear factor kappa B (NF-κB) activation in COPD.7,8

Once activated, NF-κB translocates to the nucleus within AECs and alveolar macrophages,9,10 stimulating transcription of inflammatory cytokines genes, including C-X-C motif chemokine ligands

(CXCL5), interleukins (such as IL-8, IL-1β), granulocyte-macrophage colony-stimulating factor (GM-CSF), tumour necrosis factor-alpha (TNF-α), and type-I interferons (IFNs, such as IFN-β).

These cytokines and IFNs further perpetuate and exacerbate airway inflammation by recruiting and activating neutrophils, macrophages, and lymphocytes (including B cells, T cells and Natural

Killer Cells).2,11 Additionally, viral infections, such as those caused by adenovirus, can amplify inflammation through their persistent DNA recognised by TLRs and cGAS, to further stimulate

cytokines production through NF-κB pathway.12,13 On the other hand, there is evidence of autoimmune elements within the COPD pathology, where self-DNA released from apoptosis and necrosis

of lung tissue may trigger an inappropriate immune response via TLRs and cGAS activation, thereby contributing to sustained airway inflammation.14,15 Recently, research has increasingly

highlighted neutrophil extracellular traps (NETs), a web-like structure composed of self-DNA released by the neutrophils, as an active contributor to COPD pathogenesis16: (1) Clinically, the

induced sputum of patients with both stable and exacerbated COPD shows increased level of NETs,17,18 which have been associated with the severity of airflow limitation and microbiota

diversity;19,20,21 (2) In vitro, the CS extract (CSE) is capable of inducing NETosis, a releasing process of NETs, on both human22,23 and mouse24 neutrophils; (3) NETs stimulated by

phorbol-12-myristate-13-acetate (PMA)25,26 and IL-827 cause cytotoxicity in the human primary AECs and AECs cell lines; (4) PMA-,25,28 uric acid (UA)-, and monosodium urate crystal (MSU)-29

induced NETs promote, whereas IL-8-induced NETs prohibit,27 the expressions of IL-8 and IL-6 on AECs; (5) CSE-induced NETs (CSE-NETs) activate the maturation of myeloid dendritic cells (DCs)

to initiate a T-cell-mediated immune response;22,24 and (6) In vivo, NETs or self-DNA have been implicated in the development of acute airway inflammation in short-term CS-treated

mice.23,30 Therefore, NETs participate in short-term CS-induced acute airway inflammation by affecting the function of AECs and DCs. However, researchers have not yet investigated the

contribution of DNA components in NETs (NETs-DNA) to persistent airway inflammation in COPD by initiating an autoimmune response. In this study, we investigated whether and how NETs

contribute to long-term CS-induced COPD, with a focus on the immunoregulatory effects of NETs-DNA on AECs, and the role of NETs and targeted pathways as therapeutic targets to alleviate

persistent airway inflammation in COPD. RESULTS 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. 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. 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. 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. 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. 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. 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. 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, S2). In comparison to healthy non-smokers and smokers, patients with COPD showed increased

levels of NF-κB-dependent cytokines (IL-1β, CXCL8 in BALF, Fig. 8a, d, and CXCL5, IL-1β in lung tissue slices, Supplementary Fig. S16e–j) and NETs components (MPO, NE activity in BALF, Fig.

8j, Supplementary Fig. S15a), but decreased the level of type-I IFN (IFN-β in BLAF, Fig. 8g, and in lung tissue slices, Supplementary Fig. S16k–m). The levels of IL-1β, CXCL8, and MPO

correlated negatively with FEV1/FVC and FEV1%Pred (the ratio of tested FEV1 to predicted FEV1), respectively, after adjusting for age, sex, body mass index (BMI), and smoking history (Fig.

8b, c, e, f, k, l). NE activity showed a negative correlation with FEV1/FVC (supplementary Fig. S15b, c). Conversely, IFN-β level did not correlate with either FEV1/FVC or FEV1%Pred (Fig.

8h, i). Notably, MPO level positively correlated with CXCL8 level, and NE activity correlated with IL-1β and CXCL8 levels, respectively (Fig. 8m, n and Supplementary Fig. S15d, e); however,

neither MPO nor NE activity correlated with IFN-β level (Fig. 8o and Supplementary Fig. S15f). Additionally, we observed increased nuclear expression of NF-κB P65 protein in airway

epithelial cells of COPD patient lung slices, compared with that of non-smokers and smokers in the healthy group (Supplementary Table S4 and Supplementary Fig. S16a–d). Together, NETs

components are linked to dysregulated NF-κB-dependent cytokines, but not type-I IFNs, in BALF of COPD patients. DISCUSSION NETs are initially recognised to entrap and kill pathogens via a

DNA framework comprising abundant antimicrobial granular proteins, such as MPO and NE.31 However, they are immunogenic and have been implicated in several autoimmune and non-infectious

diseases.25,32,33 In COPD, augmented self-DNA from increased lung cell apoptosis acts as DAMPs, impeding inflammation resolution. NETs, rich in DNA content, have been implicated in the loss

of microbiota diversity among patients with COPD20 and CS-induced acute airway inflammation in murine models.23,30 Nevertheless, the role of NETs in mediating innate and adaptive immune

responses to sustain airway inflammation in COPD remains unknown. Herein, we demonstrated the immunomodulatory role of NETs in long-term CS-induced COPD as follows: (1) hPBNs derived from

patients with COPD exhibited a stronger ability to release CSE-NETs, which contained high levels of oxidatively-damaged mtDNA and chDNA; (2) CSE-NETs not only stimulated the proliferation

and production of both NF-κB-dependent cytokines and type-I IFNs on hAECs, but also promoted the maturation of hDCs; (3) these effects were ameliorated by the silencing/inhibition of cGAS

and TLR9 in vitro; (4) in vivo, the knockout of DNA sensors (_cGAS__−/−_ and _TLR9__−/−_ mice), the inhibition of NETs (by mitoTEMPO i.p), and the degradation of NETs (by DNase-I _Neb_.)

respectively resulted in alleviated airway inflammation, reduced production of NF-κB-dependent cytokines (but not type-I IFNs), suppressed NF-κB P65 activation, and improved lung function in

the long-term CS-induced COPD mouse model; (5) the severity of NETs infiltration was correlated with that of airway inflammation in the CS-treated mice; and (6) In COPD patients, the levels

or activity of NETs components (MPO and NE activity) in BALF correlate with disease severity and the abundance of NF-κB-dependent cytokines (IL-1β and CXCL8), but not those of type-I IFN

(IFN-β). Taken together, the DNA components in CSE-NETs promoted NF-κB-, but not type-I IFNs-, dependent autoimmunity via cGAS/TLR9 in long-term CS-induced COPD. The cause of excessive

NETosis in COPD is largely unknown;34 despite spontaneous NETosis regulated by CXCR2 in patients with COPD,35 NETosis induced by CS exposure might be a more likely contributor. Notably, we

observed a stronger capacity for NETosis in COPD-neutrophils upon CSE stimulation. Consistently, COPD-neutrophils demonstrated increased NETosis following exposure to diesel exhaust,36 thus

suggesting greater susceptibility to undergo NETosis following exposure, potentially linked to augmented mtROS release in COPD-neutrophils. Unlike PMA-induced NETosis that relies on NOX,

CSE-triggered NETosis responds primarily to mtROS. Consistently, ribonucleoprotein (RNP)-induced NETosis required mtROS instead of NOX in patients with systemic lupus erythematosus,37

thereby indicating that NETosis induced by different inducers may rely on dissimilar mechanisms.38 Moreover, CSE-NETs consist of abundant oxidatively-damaged DNA, which is more capable of

promoting autoimmune reactions33 and the formation of autologous NETs.39 Furthermore, CSE-NETs comprised a lower ratio of mtDNA to chDNA compared to PMA- and iomomycin-NETs, since mtDNA

exhibits different immunoregulatory features from chDNA,40 NETs induced by different stimuli may exert diverse cellular effects. This variability could account for the controversial effects

of PMA- and IL-8-induced NETs on IL-8/IL-6 expression in hAECs.25,27,28 Accordingly, we investigated the effects of CSE-NETs on the function of hAECs, which are essential for maintaining

immune homoeostasis in the lungs.41 CSE-NETs stimulated proliferation, production of both NF-κB-dependent cytokines and type-I IFNs, and NF-κB (P65 and P50) activation in hAECs. Moreover,

both cGAS and TLR9 were required to facilitate these effects, thus emphasising the importance of NETs-DNA sensing on hAECs to mediate an autoimmune response. Previously, CSE-NETs were shown

to promote DC maturation to prime T-cell-mediated immune response,22,24 and we further showed that inhibition of cGAS and TLR9 suppressed the CSE-NETs-induced maturation of hDCs. Taken

together, CSE-NETs triggered hAEC and hDC dysfunction, and the sensing of NETs-DNA by both airway structural cells (such as hAECs) and immune cells (such as hDCs) promoted autoimmune

reactions. TLR4 and TLR9 are critical PPRs involved in COPD pathogenesis.42,43,44 TLR4 recognises exogenous ligands (such as lipopolysaccharide), respiratory syncytial virus fusion protein,

and endogenous ligands like heat shock proteins (HSP60, HSP70) and high mobility group box 1 (HMGB1), whereas TLR9 specialises in the detection of unmethylated CpG motifs prevalent in

bacterial and viral DNA, as well as in human mtDNA.45 cGAS is a crucial innate immune sensor that detects cytoplasmic DNA from pathogens or cellular injury. Both cGAS and TLR9 are reported

DNA sensors capable of sensing cytosolic/extracellular mtDNA/chDNA.46,47 The recognition of self-DNA by cGAS and TLR9 not only activates the NF-κB signalling pathway to release

NF-κB-dependent cytokines but also induces the robust production of type-I IFNs.15 These cytokines and IFNs serve as prominent immunomodulators in autoimmune contexts.48,49 Excessive

activation of cGAS/TLR9 has been linked to allergic and autoimmune diseases.50,51 For example, sensing of cytosolic DNA by cGAS in hAECs52 and TLR9 in innate lymphoid cells53 plays a role in

asthma pathogenesis. However, the role of NETs-DNA recognition by cGAS/TLR9 in eliciting NF-κB-dependent cytokines and type-I IFNs response in COPD is unclear. Interestingly, our in vitro

data showed that CSE-NETs stimulated type-I IFNs production in hAECs, while in vivo data suggested that CS exposure resulted in decreased type-I IFNs levels in both CS-treated mice and

patients with COPD; moreover, only levels of NF-κB-dependent cytokines, but not type-I IFNs, were correlated with disease severity in COPD mouse models and patients with COPD. This

controversy between in vitro and in vivo results might stem from the impairment of IFNAR1 expression on hAECs, a crucial receptor of type-I IFNs to facilitate the downstream signalling

cascade,54 by long-term CS exposure (supplementary Fig. S6f),55 thus hindering the overall amplification and production of type-I IFNs. Notably, the decreased expression of type-I IFNs has

been previously reported in COPD contexts, the suppressed type-I IFNs in COPD might compromise the antiviral defence ability, thus contributing to persistent airway inflammation and even

acute exacerbations.56,57 These findings emphasised the key role of NETs-DNA and the associated NF-κB signalling, but not type-I IFNs response, in sustained airway inflammation in COPD. The

interplay and differences between cGAS and TLR9 in their downstream signalling upon DNA recognition remain poorly understood. In hDCs, the pathways regulated by cGAS and TLR9 may exist in

parallel and display interdependencies to suppress each other.58 Notably, mitochondrial DNA itself has been shown to activate NETosis via the cGAS-stimulator of interferon genes (STING)

axis, with enhancing NE production and extracellular DNA release within NETs during sterile inflammation,39 suggesting a functional involvement of cGAS-STING in NETosis. Therefore, it is

possible that NETs-DNA could stimulate a feedback loop in neutrophils by further promoting NETosis under CS exposure conditions. Consistently, we observed significantly less NETs

infiltration in CS-treated cGAS−/− (but not TLR9−/−) mice, indicating that cGAS, but not TLR9, participates in CS-induced NETosis. Consequently, _cGAS_ deficiency might not only reduce

NETs-DNA sensing but also suppress CS-induced NETosis, thus providing potentially superior protection against CS exposure than _TLR9_ deficiency. Regarding NETs-DNA sensing in hAECs,

although silencing _cGAS_ or _TLR9_ individually led to varied reductions in CSE-NETs-induced cytokines/IFNs expressions/productions, no significant differences emerged from two-way ANOVA

analysis (supplementary Fig. S14). Nevertheless, further investigation into the intricate regulatory roles that cGAS and TLR9 play in DNA sensing and response is warranted, to better clarify

their differences in downstream signalling upon DNA recognition. MitoTEMPO is a superoxide inhibitor that specifically targets the mitochondria.59 We observed a desirable control of

CS-induced COPD following mitoTEMPO treatment probably owing to the following reasons: (1) mitoTEMPO efficiently inhibits CSE-induced NETosis; (2) mitoTEMPO helps in restoring the

equilibrium of oxidant and antioxidant molecules,60 which are characterised in CS-induced COPD;61 (3) oxidatively-damaged mtDNA/chDNA is more capable of inducing the disordered release of

cytokines, which is likely controlled by mitoTEMPO. Previously, DNase-I was shown to protect mice from type-2 immunopathology33 and CS-induced acute airway inflammation,23 we further showed

that the degradation of NETs-DNA by DNase-I treatment alleviated NF-κB-dependent cytokines and emphysema-phenotype in the long-term CS-treated mice, suggesting that DNase-I could offer

modest protective effects against the sustained airway inflammation and subsequent alveolar destruction. It should be noted that the therapeutic potential of DNase-I might be limited by the

suboptimal nebulised administration methods used in this study. Nevertheless, we further showed that the severity of NETs infiltration was correlated with that of airway inflammation and

emphysema, as well as the levels of NF-κB-dependent cytokines in the CS-treated mice, thus supporting the importance of NETs-DNA in CS-induced airway inflammation and alveolar destruction.

In summary, our in vivo data demonstrate that sensing-blockage/inhibition/degradation of NETs-DNA confers therapeutic benefits in the long-term CS-induced COPD mouse model. However, when

translating these observations to clinical practice, it is essential to acknowledge the complexity of COPD pathophysiology, where targeting NETs or cGAS/TLR9 alone might not be sufficient to

fully address the intricate interactions between innate and adaptive immunity, DAMPs, cytokines, and concurrent pathogen challenges. Potential synergistic benefits in reducing inflammation

and improving lung function could be achieved through combination therapies targeting multiple aspects of COPD, including but not limited to NETosis and cGAS/TLR9 pathways. Moreover, given

the critical role of cGAS/TLR9 signalling in pathogen recognition and antiviral responses, as well as NETs’ function in pathogen clearance and lung microbiota maintenance, targeted

interventions must be judiciously designed to minimise disruption of host defence mechanisms. Our study has limitations: while we establish correlations between NETs components, disease

severity, and NF-kB-dependent cytokines levels in BALF of patients with COPD adhering to standard LAMA or ICS + LABA therapies, we lack information on individual pharmacological regimens

that might impact MPO and NE levels. This is particularly pertinent given previous studies suggesting an association between NETs abundance and ICS resistance or ICS regular use in patients

with asthma,62,63 implying potential interactions between NETosis and ICS therapy. Future research could benefit from incorporating detailed clinical records to elucidate how current

therapeutic strategies influence CS-induced NETosis. Herein, we present evidence that CS-induced NETs-DNA sensed by cGAS and TLR9 in AECs and DCs promotes NF-κB-, but not type-I IFNs,

dependent autoimmunity in long-term CS-induced COPD. Despite their roles in eliminating pathogens and maintaining microbiota diversity in the lungs, NETs contribute self-DNA as a significant

DAMP, promoting disordered production of inflammatory cytokines and thus sustaining airway inflammation in an autoimmune fashion in COPD. Consequently, NETs-DNA and its sensing receptors

(cGAS/TLR9) represent potential therapeutic targets to reduce persistent airway inflammation in COPD. Supplementary Fig. S17 illustrates the proposed mechanism. MATERIALS AND METHODS STUDY

APPROVAL AND ETHICS STATEMENTS The protocol of human study was reviewed and approved by the Chinese Ethics Committee for Registering Clinical Trials (approval number: ChiCTR900022271). All

animal experiments were approved and conducted in accordance with the guidelines of the Animal Ethics Committee of the West China Hospital (approval number: 2018049A). PATIENTS AND SAMPLES

COPD diagnosis adhered to the Global Strategy for the Diagnosis, Management, and Prevention of COPD (GOLD) criteria. From January 2016 to March 2020, the study recruited 45 healthy

individuals and 42 COPD patients at West China Hospital, all providing informed consent. Among them, 11 healthy volunteers and 10 COPD patients contributed peripheral blood samples for NETs

and DCs experiments. Additionally, 34 healthy participants (divided into 13 non-smokers and 21 smokers) and 32 COPD patients with a history of smoking underwent BALF testing (Supplementary

Tables S1, S2). The collections of BALF, circulating neutrophils, and monocytes were described in the supplementary materials. NEUTROPHILS ISOLATION AND NETS STIMULATION Neutrophils from

COPD patients and healthy participants were isolated using the MACSxpress Neutrophil Isolation Kit (Miltenyi Biotec, Cat No. 130-104-434) according to the manufacturer’s instructions, with

purity confirmed by flow cytometry for CD15 and CD16. Freshly isolated cells (1.0 × 106) were seeded on fibrinogen-coated coverslips and incubated in serum-free RPMI 1640 for adhesion.

Subsequently, NETs were induced by varying concentrations of CSE at 37 °C for 4 to 24 h to assess CSE-induced NETosis; PMA and ionomycin stimulation were used as positive control. We

administered mitoTEMPO, TTFA, DPI, VAS, DNase-I, and GW as described in the supplementary materials. QUANTIFICATION OF NETS-RELEASING NEUTROPHILS The evaluation of NETs-releasing neutrophils

percentage was conducted by immunofluorescence co-staining of the following NETs components: myeloperoxidase (MPO), histone H3, and DNA. Briefly, NETs on coverslips were washed twice with

phosphate buffer saline (PBS), permeabilized with ice-cold acetone:methanol (−20 °C), fixed in neutral formalin, then blocked and incubated overnight at 4 °C with primary antibodies for MPO

and histone H3. After PBS washes, secondary antibodies were applied for 2 h. Following mounting with DAPI medium, samples were imaged on whole-filed fluorescence microscopy and evaluated as

described in the supplementary materials. COLLECTION OF NETS-CONTAINING SUPERNATANTS CSE-induced NETs were prepared under 5% CSE for 18 h, which induces approximately 50% NETosis without

neutrophil necrosis. After CSE exposure, NETs were thoroughly washed and detached with MNase (Thermo Fisher Scientific, Cat No. 88216, 20 units/mL, 37 °C, 30 min). DNA concentration was

quantified using the QuantiFluor dsDNA system (Promega, Cat No. E2670). NETs were enriched and sequenced by next-generation sequencing as described in the supplementary materials. CULTURE

AND SIMULATION OF HAECS Primary hAECs were obtained from Lifeline Cell Technology (Cat No. FC-0016) and maintained in BrochiaLife Medium Complete Kit (Cat No. LL-0023) on collagen-coated

plates. Cells at passages 3–5 were subjected to 6, 12 or 24 μg/mL NETs for 48 or 72 h. RNA and protein extraction ensued using E.Z.N.A. HP Total RNA Isolation Kit (Omega Bio-Tek, Cat No.

R6812) and Minut Total Protein Extraction Kit (Invent Biotechnologies, Cat No. SD-001/SN-002), respectively. Proliferation was assessed via Click-iT EdU Assay (Invitrogen, C10499). cGAS and

TLR9 expression in hAECs was silenced using small interfering RNA (siRNA) (Life Technologies) transfected with TransIT-TKO Reagent (Mirus Bio, _Cat No_. MIR 2150), with transfection

efficiency and cytotoxicity monitored using BLOCK-iT Alexa Fluor Red Control (Invitrogen, Cat No. 14750100). Optimal siRNA sequences (Cat No. S28872 for cGAS, Cat No_._ S41746 for TLR9) were

applied prior to NETs stimulation. The supplementary materials described the detailed methods of cell culture, stimulation, siRNA knockdown of _cGAS_ and _TLR9_, quantitation of

proliferation, reverse-transcription quantitative PCR (RT-qPCR), western blotting on primary hAECs. DIFFERENTIATION AND STIMULATION OF PERIPHERAL MONOCYTES-DERIVED DCS Monocytes were

isolated from fresh peripheral blood of healthy donors or COPD patients via lymphoprep density gradient medium (StemCell Technologies, _Cat No_. 07801). Briefly, blood diluted with PBS

containing 2% FBS (StemCell Technologies, Cat No. 07905) was centrifuged at 800×_g_ for 30 min. Monocytes were harvested and differentiated into DCs using the ImmunoCult Dendritic Cell

Culture Kit (StemCell Technologies, Cat No. 10985) at 37 °C for 3 days, and further differentiated for 2 days with fresh medium to yield immature DCs. These cells were then stimulated with

12 μg/mL NETs for 24 h at 37 °C for subsequent flow cytometry analysis. ImmunoCult Dendritic Cell Maturation Supplement (StemCell Technologies, Cat No. 10985) was employed as a positive

control for DC maturation. To explore cGAS and TLR9’s role in NETs-induced DC maturation, RU.521 (Aobious Inc., _Cat No_. AOB37877, a selective cGAS inhibitor), and ODN 2088 (InvivoGen,

tlr1-2088, a recognised TLR9 antagonist disrupting CpG ODNs and TLR9 interaction) were employed. The detailed methods of differentiation, stimulation, and flow cytometry assessment of

monocyte-derived DCs were described in the supplementary materials. ANIMAL EXPERIMENTS We obtained cGAS knockout mice (_cGAS__−/−_, _Cat No_. 026554) and TLR9 knockout mice (_TLR9__−/−_, Cat

No. 034329) from Jackson Laboratories. The COPD mouse model was established using an established nose-only CS exposure method.64 Briefly, 8-week-old, sex-matched mice were placed in custom

nose-only exposure tubes (China pattern No. ZL201821367875.5) for smoke inhalation. Smoke from Marlboro cigarettes (1.0 mg nicotine, 11 mg tar) was generated using a smoking machine (CH

Technologies) and diluted with fresh air. Mice were exposed to smoke from ~30 cigarettes in two 75-min sessions per day, 5 days a week, for 12 weeks, interspersed with recovery periods.

MitoTEMPO (Sigma-Aldrich, Cat No. SML0737) was prepared at 0.5 mg/mL in sterile saline, filtered and administered intraperitoneally (50 μg in 100 μL) to 25 g mice on smoking days, with

saline as control. DNase-I (Sigma-Aldrich, Cat No. 11284932001) was dissolved to 2 mg/mL; 4 mL dissolved DNase-I containing 16,000 units was aerosolized over 15 min on smoking days, and

saline was used for controls. We then assessed lung function, NETs infiltration, histology, cell counts in BALF, and levels of proinflammatory cytokines in serum and BALF as described in the

supplementary materials. QUANTIFICATION OF NETS INFILTRATION IN LUNG SLICE OF MOUSE The NETs in lung sections of mice were immunofluorescence co-stained with MPO, histone H3 and DNA. Lung

sections underwent dewaxing, hydration, and antigen retrieval in citrate buffer at 95 °C. Autofluorescence reduction was achieved with sodium borohydride incubation, followed by PBS washing.

Post-blocking, primary antibodies for MPO and histone H3 were applied overnight at 4 °C, followed by secondary antibodies (Alexa Fluor 555 and 647) for 2 h. Slides were washed, mounted with

DAPI medium, and stored at 4 °C. Negative controls were included. Imaging was conducted on a Zeiss Imager Z2 microscope. ImageJ was used to analyse fluorescence images. Briefly, the lung

areas in images were measured, followed by demarcation of NETs areas and background areas from negative controls. Normalised NETs areas were calculated as the average percentage of NETs

areas minus background areas across three random views per slice, with detailed methods described in the supplementary materials. ASSESSMENT OF NF-ΚB-DEPENDENT-CYTOKINES, TYPE-I IFNS LEVELS

Cytokine and interferon levels in mouse serum and BALF were determined using the Bio-Plex Pro Mouse Chemokine Luminex Assay Kit (BioRad, Lot No. 17005875). Human BALF analysis was executed

with the Magnetic Luminex Screening Assay Kit (R&D systems, Cat No. LXSAHM) on a Bio-Plex 200 system (BioRad) following standard procedures as detailed in the supplementary materials. In

vitro, hAECs were stimulated with TLR9/cGAS siRNA and NETs, and culture supernatants post-centrifugation were stored at −80 °C. ELISA (R&D Systems, Cat No. VAL101, VAL103 and VAL137)

quantified IL-1β, CXCL8 and IFN-β levels per manufacturer instructions. DNA-BINDING ELISAS FOR ACTIVATED NF-ΚB P65 AND P50 Nuclear extracts of hAECs stimulated with TLR9/cGAS siRNA and/or

NETs were prepared with a nuclear extract kit (Active Motif, Cat No. 40410). Whole-cell extracts of frozen lung tissues from _TLR9__−/−_, _cGAS__−/_− and control mice post-CS exposure were

also obtained. Protein content was quantified via Bradford assay (BioRad, Cat No. 5000201). NF-κB P65 and P50 activities were assessed using NF-κB P65 (or P50) Activity Assay (Active Motif,

Cat No. 40096 and 41096) according to the manufacturer’s instructions as described in the supplementary materials. STATISTICAL ANALYSIS Data are expressed as mean ± SD. Statistical details,

including analysis methods, significance thresholds (_P_ < 0.05), _n_ values, scatter plots, and _p_ values, are presented within figure legends and supplementary materials. Briefly, for

two-group comparisons, data normality was assessed via Shapiro–Wilk or Kolmogorov–Smirnov tests; normally distributed datasets were analysed by unpaired _t_-tests, while non-normal datasets

underwent Mann–Whitney _U_-tests. For multiple-group comparisons, one-way or two-way ANOVA, complemented by Tukey’s honest significant test, were employed. For partial correlation analysis,

Pearson’s partial correlation tests controlled for covariates (age, sex, BMI, smoking history of COPD patients), succeeded by multiple linear regression analyses. DATA AVAILABILITY The raw

sequence data have been deposited in the Genome Sequence Archive in National Genomics Data Center, China National Center for Bioinformation/Beijing Institute of Genomics, Chinese Academy of

Sciences, under accession number HRA001059 that are publicly accessible at https://ngdc.cncb.ac.cn/gsa-human. Other data were available from the supplemental material, or from the

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smoking-induced mouse model of COPD. _Ann. Med._ 51, 314–329 (2019). Article  CAS  PubMed  PubMed Central  Google Scholar  Download references ACKNOWLEDGEMENTS This work was supported by the

1•3•5 project for disciplines of excellence, West China Hospital, Sichuan University [grant numbers ZYGD23009]; the National Natural Science Foundation of China (NSFC) [grant numbers

81830001, 81670038, 81800015 and 31871157]; the Natural Science Foundation of Sichuan Province [Grant number 2022NSFSC0603]; the China Postdoctoral Science Foundation [Grant numbers

2019T120851 and 2018M643501]; the Fundamental Research Funds for the Central Universities (the Postdoctoral Foundation of Sichuan University) [Grant number 2018SCU12028]. AUTHOR INFORMATION

AUTHORS AND AFFILIATIONS * Department of Respiratory and Critical Care Medicine, West China Hospital, West China School of Medicine, and Division of Pulmonary Diseases, State Key Laboratory

of Biotherapy, Sichuan University, Chengdu, Sichuan, 610041, China Jun Chen, Tao Wang, Xiaoou Li, Lijuan Gao, Ke Wang, Mengxin Cheng, Zijian Zeng, Lei Chen, Yongchun Shen & Fuqiang Wen

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search for this author inPubMed Google Scholar CONTRIBUTIONS Conceptualization: J.C., X.L. and F.W.; Methodology: J.C., T.W. and X.L.; Acquisition of data: J.C., L.G., K.W., M.C. and Z.Z.;

Analysis and interpretation of data: J.C., Y.S. and L.C.; Project administration: T.W.; Drafting the article and illustrator: J.C.; Revising the article critically for important intellectual

content: J.C., T.W. and F.W. All authors have read and approved the article. CORRESPONDING AUTHOR Correspondence to Fuqiang Wen. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare

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