Self-DNA release and STING-dependent sensing drives inflammation to cigarette smoke in mice

Cigarette smoke exposure is a leading cause of chronic obstructive pulmonary disease (COPD), a major health issue characterized by airway inflammation with fibrosis and emphysema. Here we demonstrate that acute exposure to cigarette smoke causes respiratory barrier damage with the release of self-dsDNA in mice. This triggers the DNA sensor cGAS (cyclic GMP-AMP synthase) and stimulator of interferon genes (STING), driving type I interferon (IFN I) dependent lung inflammation, which are attenuated in cGAS, STING or type I interferon receptor (IFNAR) deficient mice. Therefore, we demonstrate a critical role of self-dsDNA release and of the cGAS-STING-type I interferon pathway upon cigarette smoke-induced damage, which may lead to therapeutic targets in COPD.

Chronic obstructive pulmonary disease (COPD) is a severe chronic inflammatory disease associated with impaired lung functions. This disease is characterized by chronic lung inflammation which can lead to critical tissue destruction in some cases. The major cause of COPD is cigarette smoking. However other triggers such as air pollution are known contributors. Available treatments display only limited efficacy attempting to inhibit chronic inflammation characterized by the recruitment and activation of both innate (neutrophils and macrophages) and adaptive (T and B lymphocytes) immune cells in the small airways 1 . Immune and tissue cells, including epithelial and endothelial cells as well as fibroblasts, secrete a variety of proinflammatory mediators, in particular chemokines, cytokines and others mediators 2 .
Inflammation observed in COPD patients, smokers and sometimes even ex-smokers, is often driven by oxidative stress characterized by excessive reactive oxygen species production leading to DNA damage, cell death and subsequent pulmonary inflammation 1,3 . Indeed, cigarette smoke-induced cell death is characterized by danger associated molecular patterns (DAMPs) release contributing to persistent neutrophilic airway inflammation characteristic of COPD. Double-stranded (ds) DNA was shown to strongly correlate with neutrophilic inflammation 4 , causing inflammation and emphysema 5 . Nucleic acid sensing is a common and effective strategy to recognize microorganisms and initiate immune responses. However, it could also be an important feature of COPD 6 . Growing evidence demonstrates that pathogen receptors can also recognize self-nucleic acids in particular mitochondrial DNA (mtDNA) or nuclear DNA abnormally present in the cytosol 7,8 . Detection of these mislocalized self-nucleic acids can promote sterile inflammation and autoimmune diseases 9 . Among pattern recognition receptors (PRRs) involved in self-nucleic acid recognition, the dsDNA sensor TLR9 has been shown to participate to the development of emphysema caused by cigarette smoking in both mouse 10 and human 11 . An increasing interest has emerged recently for stimulator of interferon genes (STING), an endoplasmic reticulum membrane-expressed protein. STING is activated by cyclic dinucleotides (CDNs), second messengers derived from microorganisms or synthesized by the enzyme cyclic GMP-AMP synthase (cGAS) after its binding to pathogen or self-derived dsDNA, including nuclear DNA and mtDNA 8,[12][13][14][15] . DNA binding triggers cGAS to convert ATP and GTP into cGAMP, a CDN driving STING activation and cytokine production including type I interferons (type I IFN) 8,16,17 . STING is an adapter protein of nucleic acid sensors, located at the crossroads of several intracellular signaling pathways. In addition to cGAS, other cytosolic receptors (e.g. DDX41, IFI16) can sense DNA or CDNs and activate STING 18,19 . Its activation triggers transcription factors such as interferon regulatory factor 3 (IRF3) or nuclear factor κB (NF-κ B) and cytokine production including type I IFN involved in host immune responses. The type I IFNs (IFN I) family is a multi-gene cytokine family comprising 13 subtypes of the IFN-α family in human (14 in mouse) and a single IFN-β subtype 20 . While primarily involved in responses against viral and bacterial infections, the role of IFN I in other disease settings such as autoimmunity is well established 21,22 . IFNs I interact with the specific IFNAR membrane receptor, leading to the transcription of Interferons  Stimulated Gene (ISG) 23 . More recently, it was shown that STING activation also leads to the secretion of type III IFNs 24,25 and IL-1β 26 .
The immune consequences of self-nucleic acid release and detection by the cGAS/STING signaling pathway in pulmonary chronic inflammation and COPD is largely unknown. We showed previously that IL-1β and the inflammasome adaptor ASC are essential to cigarette smoke-induced inflammation, or elastase-induced emphysema in mice indicating that the inflammasome is involved in the establishment of COPD 27,28 . We identified uric acid as an endogenous DAMP released upon elastase-induced lung injury and activating the NLRP3/ASC inflammasome, driving IL-1β-dependent lung inflammation and emphysema 28 . Here we report a critical role of the cGAS/STING/type I interferon pathway in cigarette smoke-induced lung inflammation.

Results
Cigarette smokeexposure induces self-DNA release in the bronchoalveolar space. Cigarette smoke (CS)-exposure was shown to induce cell damage and death with free DNA release 4 and we first analyzed the DNA-content in the airways of acutely CS-exposed mice. C57BL/6 wild-type (WT) mice were exposed three times a day and euthanized 16 h after 1, 2, 3 or 4 days of exposure (Fig. 1A). Self-dsDNA increase in bronchoalveolar lavage fluid (BALF) was detectable only on day 4, together with neutrophil recruitment in the airways (Fig. 1B). Importantly, self-dsDNA level and neutrophil influx into the BALF are correlated suggesting that CS-induced self-DNA may act as a proinflammatory signal for neutrophil recruitment (Fig. 1B). A kinetic analysis of DNA release and inflammation was performed at 1, 4, 6, 12 or 20 h after the last exposure of a 4 days CS-exposure protocol (Fig. 1C). The data show that self-dsDNA is detectable at 6 h after the last exposure on day 4, preceding neutrophil recruitment which is visible between 12 h and 20 h (Fig. 1D). The neutrophil attracting chemokine CXCL1 (C-X-C motif chemokine)/KC is increased in BALF between 4 h to 12 h after the last CS-exposure (Fig. 1D). In addition, neutrophil influx into the BALF is confirmed by the increased level of myeloperoxidase (MPO) in the BALF at 12 and 20 h after the last CS-exposure, thus after CXCL1/KC production (Fig. 1D). These results indicate that 4 days CS-exposure induce self-dsDNA release, with kinetics preceding neutrophil recruitment into the broncho-alveolar space. Nevertheless, high self-dsDNA levels persist 20 h after the last exposure at time where BALF neutrophil influx was the most important, suggesting that neutrophils may also release their own DNA as neutrophil extracellular traps (NETs). To evaluate NET formation in response to CS-exposure, we performed immunofluorescence staining of bronchoalveolar lavage (BAL) cells collected from CS-exposed WT mice showed a specific signal for citrullinated histone H3, a specific marker of NETs. This signal colocalized with DAPI, suggesting that neutrophils recruited upon CS-exposure also release their DNA as citrullinated histone H3-containing NETs (Fig. 1E). In conclusion, CS-induced self-DNA release from damaged lung cells, promotes the recruitment of neutrophils that amplify airway inflammation through NET-mediated DNA release.
Acute CS-exposure increases cGAS and STING expression in the lung. We then asked whether the DNA sensor cGAS and the adaptor protein STING are involved in the sensing of self-dsDNA released upon CS-exposure, and analysed their expression in acutely CS-exposed mice. CS-exposed WT mice overexpressed cGAS (Mb21d1) ( Fig. 2A), and STING (Tmem173) (Fig. 2B) mRNA in the lungs at 4 days of CS-exposure. Furthermore, CS-exposure increased pulmonary expression of the cGAS and STING proteins in WT mice over control air-exposed mice while STING −/− mice or cGAS −/− mice did not express cGAS or STING, as expected ( Our data indicate that self-dsDNA released is accompanied by the overexpression of the DNA sensor cGAS and the adaptor protein STING after acute CS-exposure. These results suggest that early dsDNA release induces cGAS and STING expression which is in line with an engagement of the cGAS/STING pathway in pulmonary inflammatory response to CS-exposure.

Cigarette smoke-induced lung inflammation is STING dependent. Since STING is overexpressed
in the lung of CS-exposed mice, we next investigated whether the STING pathway is required for CS-induced lung inflammation. We exposed wild-type or STING deficient (STING −/− ) mice to CS for 4 days and analysed the pulmonary inflammation. The increase of dsDNA levels in BALF observed in CS-exposed WT mice was significantly reduced in the BALF of CS-exposed STING −/− mice (Fig. 3A), suggesting that self-dsDNA is released de novo dependent on STING. In addition, CS-exposure induced an increase in protein extravasation in the BALF of WT mice, but not in STING −/− mice indicating a reduced respiratory barrier damage in the absence of STING (Fig. 3B). Total inflammatory cell and neutrophil counts recovered in the BALF were decreased in STING −/− CS mice as compared to WT CS mice (Fig. 3C,D). Among immune cells, neutrophils are known to play a major role in response to CS 29,30 . As a correlate of neutrophil recruitment, the neutrophil marker MPO was significantly reduced in the BALF and lungs of CS-exposed STING −/− mice as compared to WT mice (Fig. 3E,F). Analyzing the expression of the neutrophil attracting chemokines, we observed that BALF and lung levels of CXCL1/KC (Fig. 3G,H), CXCL5/LIX (Fig. 3I,J) and CXCL15/Lungkine (Fig. 3K,L) were significantly lower after CS-exposure in STING −/− mice as compared to WT mice. In addition, BALF and lung levels of the IFN I downstream CXCL10/ IP-10 chemokine were not increased in STING −/− CS mice after exposure as compared to CS-exposed WT mice (Fig. 3M,N). Finally, levels of the remodeling factors matrix metalloproteinase (MMP)-9 ( Fig. 3O,P) and tissue inhibitor of metalloproteinases (TIMP)-1 (Fig. 3Q,R) in lungs were reduced in CS-exposed STING −/− mice in DNA sensor cGAS, but not TLR9, is required for CS-induced lung inflammation. To investigate whether the cGAS sensor is involved in CS-induced DNA sensing and lung inflammation, we exposed cGAS deficient mice (cGAS −/− ) to CS for 4 days. Compared to WT mice, cGAS −/− CS-exposed mice presented less self-dsDNA Figure 1. Acute cigarette smoke-(CS) exposure induces DNA release and neutrophil influx in BALF. Mice were exposed to CS three times a day and euthanized 16h after 1, 2, 3 or 4 days of exposure (A). Self-dsDNA content in BALF of WT mice exposed to CS after 1, 2, 3 or 4 days, neutrophil count and, correlation between self-dsDNA and neutrophils were shown. (B). Mice were exposed during 4 days and euthanized 1, 4, 6, 12 or 20 h after the last exposure (C). Self-dsDNA and CXCL1 levels, neutrophils and MPO in BALF were shown (D). Immunostaining of citrullinated histone H3 (CitH3) in green and DNA in blue was performed on BAL cells of WT-CS mice at day 4, 16 h after the last exposure. Scale bars = 100 µm (E). Bar graph are expressed ± SEM.
in BALF (Fig. 4A) and a slight reduction in protein extravasation in BALF which did not reach statistical significance (Fig. 4B), suggesting a cGAS-dependent barrier injury. In addition, cGAS −/− mice exposed to CS presented a reduced recruitment of total cells, neutrophils (Fig. 4C,D) and MPO levels in BALF and lung (Fig. 4E,F) as compared to CS-exposed WT mice. Moreover, there was some reduction in CXCL1/KC, CXCL5/LIX, CXCL15/Lungkine and CXCL10/IP-10 in the BALF of CS-exposed cGAS −/− mice (Fig. 4G-J). The levels of remodeling factors MMP-9 and TIMP-1 were reduced in the BALF of CS-exposed cGAS −/− mice as compared to WT mice (Fig. 4K,L). Since the expression of TLR9, another self-dsDNA sensor, has been reported in CS-induced emphysema in mice 10 and in humans 11 , we also exposed TLR9 deficient mice (TLR9 −/− ) to CS during 4 days and analysed the inflammatory response. CS-exposed TLR9 −/− mice exhibited similar self-DNA levels ( Supplementary Fig. S1A), total cells, macrophages and neutrophils in the BALF (Supplementary Fig. S1B-D), as compared to CS-exposed WT mice, together with similar MPO, CXCL1 and CXCL5 levels in the BALF (Supplementary Fig. S1E-G), indicating that self-dsDNA release and pulmonary inflammation to acute 4 days CS-exposure are independent of TLR9 signaling. All together these results indicate that cGAS but not TLR9, is involved in self-dsDNA sensing and lung inflammation after CS-exposure, likely through the STING signaling pathway.

Cigarette smoke-exposure induced pulmonary inflammation is mediated by type I interferons.
We next investigated the role of type I IFNs in CS-induced lung inflammation. Type I IFNs expression was www.nature.com/scientificreports www.nature.com/scientificreports/ increased in the lung of WT mice exposed to CS as measured by the overexpression of Ifnα4 mRNA (Fig. 5A). We exposed WT mice and mice deficient for the type I IFN receptor (IFNAR −/− ) to CS. IFNAR −/− mice presented attenuated total cell (Fig. 5B), neutrophil (Fig. 5C) recruitment and MPO levels (Fig. 5D) in the BALF. There was a great reduction of CXCL1/KC in the BALF or lung of CS-exposed IFNAR −/− mice (Fig. 5E,F) as well as the chemokine CXCL5 (Fig. 5G,H). CS-exposed IFNAR −/− mice also produced reduced levels of CXCL15/Lungkine in the BALF (Fig. 5I) as compared to CS-exposed WT mice. In addition, BALF levels of the downstream CXCL10 chemokine were reduced in IFNAR −/− mice (Fig. 5J). Altogether, these results demonstrate that type I IFN signaling through the IFNAR pathway plays a critical role in the inflammatory response to CS exposure in mice through cGAS/STING signaling.

Discussion
We show that mouse CS-exposure promotes self-dsDNA release which correlates with neutrophil influx into the broncho-alveolar space. dsDNA may act as a proinflammatory signal for pulmonary inflammation 4 . Self-dsDNA may originate from damaged cells such as alveolar epithelial cells and macrophages, but also from neutrophilic death, possibly through NETosis which was shown to be an integral part of CS-induced experimental inflammation and human COPD, amplifying airway inflammation 31,32 . Importantly, defective repair of DNA damage was implicated in the pathogenesis of COPD, suggesting a key role for DNA release and sensing 33,34 . In addition, incomplete tobacco combustion generates carbon black nanoparticles (nCB). Nanoparticles accumulation in pulmonary dendritic cells and macrophages initiates and sustains lung inflammation, promoting emphysema development 35 . In mice and COPD patients, nCB uptake by macrophages induces DNA repair enzymes leading to dsDNA breaks 5 . The nucleus or the mitochondria can be a source of self-dsDNA triggering DNA sensors activation after CS-exposure, as shown for silica particles 36 .
Importantly, we report that CS-exposure triggers cGAS and STING expression at both mRNA and protein levels. In addition, cGAS/STING pathway is involved in pulmonary inflammation and remodeling upon CSexposure. Furthermore, part of self-dsDNA release is dependent on cGAS and STING indicating that pulmonary damage induced de novo cell death and self-dsDNA dependent lung inflammation through an amplification loop 37 . We performed an immunostaining of BAL cells using a specific citrullinated histone H3 antibody. We observed that cells from CS-exposed mice displayed a citrullinated histone H3 signal that co-localizes with specific DNA marker DAPI indicating the presence of NETs. These results suggest that self-DNA measured in the BALF may also originate from dying recruited neutrophils through NET formation. In addition, using gene deficient mouse strains we show that the absence of cGAS or STING leads to attenuated lung inflammation including reduction of type I IFN/IFNAR-dependent secretion of the interferon inducible gene (ISG) CXCL10. Even if other pathways may lead to type I IFN production, STING activation is widely recognized in the literature as a type I IFN inducer. Therefore, our results support the concept that the cGAS/STING/type I interferon pathway plays an important role in CS-induced pulmonary inflammation even if other pathways exist. www.nature.com/scientificreports www.nature.com/scientificreports/ We identify cGAS as a critical cytosolic DNA sensor upstream of STING implicated in lung inflammation to CS, whereas TLR9 receptor, another DNA sensor protein, is dispensable. Other molecules contributing to cytosolic DNA sensing and type I IFN pathway activation in a STING-dependent manner may also be involved in particular DDX41, IFI16 or mouse IFI204, DAI or others [38][39][40][41][42][43] . In addition, other danger signal receptors independently of STING or TLR9 may be activated leading to type I IFN/IFNAR-dependent pulmonary inflammation including MDA5/RIG-l, TLR4/MyD88, TLR7MyD88 or TLR8/MyD88,). Indeed, during CS-induced cell death or stress, several signaling pathways may be activated, converging in type I interferon secretion. However, our results showing a clear decrease in CXCL1, neutrophil influx and inflammation in cGAS and STING deficient mice demonstrate that cGAS/STING pathway participates in the establishment of lung inflammation upon CSexposure. Open questions remain and include how dsDNA released upon injury is transported into the cytosol to interact with cGAS.
Our study emphasizes that viruses and inorganic particles share similarities in regards to the signaling pathways involved and especially here the cGAS/STING/IFNAR pathway. In line with this statement, it was shown that CS increases poly (I:C)-induced airway inflammation, possibly via an increased expression of the poly (I:C) sensor TLR3 44 . However, others studies indicated that intratracheal CS extract administration or whole body CS exposure can cause antiviral immunosuppression in these mice, inhibiting RIG-I induction as well as IFN-β and CXCL10 expression in vivo 45 . Importantly, CXCL10 was shown to control the secretion of elastolytic MMPs in www.nature.com/scientificreports www.nature.com/scientificreports/ lung tissue macrophages in former smokers with emphysema devoid of infection, suggesting autoinflammatory mechanisms in COPD 35,46 .
We showed previously that type I IFN production with IFNAR signaling 47 and more recently self-DNA release and cGAS/STING-dependent type I IFN secretion 36 drive silica-induced lung inflammation confirming the important role of this pathway in particle-induced lung inflammation and pathology. We demonstrate here that self-dsDNA sensing and cGAS/STING pathway are crucial in CS-driven lung inflammation. We reported that CS-and silica-induced inflammation depend on the NLRP3 inflammasome 27,28 and showed that STING may also participate to NLRP3 inflammasome activation and induced pulmonary inflammation 36 .
Interestingly, a recent study showed that in human myeloid cells cytosolic DNA sensing by cGAS/STING triggers potassium efflux that activates NLRP3 inflammasome, leading to IL-1β secretion independently of type I IFN secretion 48 . These results illustrate the complexity of innate immune regulation to cell death and damage.
In conclusion, we show here that cigarette smokeexposure causes cell injury with self-dsDNA release triggering cGAS/STING pathway and leading to type I IFN secretion and pulmonary inflammation. Thus, STING might represent a potential therapeutic target in order to control lung inflammation upon cigarette smoking and prevent COPD development.

Materials and Methods
Mice. Wild-type C57BL/6J (WT) male mice were purchased from the Janvier laboratory (Janvier Laboratory, Mice were placed in a smoke chamber of InExpose system (EMKA Technologies, Paris, France) and exposed to the smoke of four cigarettes per exposure, three times a day, for four consecutive days. Bronchoalveolar lavage (BAL) and lung tissue were harvested 16 hours after the last exposure. Bronchoalveolar lavage (BAL). BAL was performed as previously described 27  Quantitative RT-PCR. RNA was purified from lung homogenates by using Tri-Reagent (Sigma-Aldrich, Saint-Louis, MO) extraction protocol. Reverse transcription of RNA into cDNA was carried out with GoTaq qPCR-Master Mix (Promega, Madison, WI). RT-qPCR was performed with Fast SYBR Green Master mix (Promega) on an ARIA MX (Agilent Technologies, Santa Clara, CA). Primers for Tmem173 (#QT00261590), Mb21d1 (#QT00131929) and Ifnα4 (#QT01774353) were purchased from Qiagen (Qiagen, Hilden, Germany). RNA expression was normalized to Gapdh (#QT00166768) expression and analysed using the ΔΔ Ct method.