Aim: Postresuscitation hemodynamics are associated with hospital mortality/functional outcome. We sought to determine whether low-dose steroids started during and continued after cardiopulmonary resuscitation (CPR) affect postresuscitation hemodynamics and other physiological variables in vasopressor-requiring, in-hospital cardiac arrest. Methods: We conducted a two-center, randomized, double-blind trial of patients with adrenaline (epinephrine)-requiring cardiac arrest. Patients were randomized to receive either methylprednisolone 40 mg (steroids group) or normal saline-placebo (control group) during the first CPR cycle post-enrollment. Postresuscitation shock was treated with hydrocortisone 240 mg daily for 7 days maximum and gradual taper (steroids group), or saline-placebo (control group). Primary outcomes were arterial pressure and central-venous oxygen saturation (ScvO(2)) within 72 hours postROSC. Results: Eighty nine of 98 controls and 80 of 86 steroids group patients with ROSC were treated as randomized. Primary outcome data were collected from 100 patients with ROSC (control, n = 54; steroids, n = 46). In intention-to-treat mixed-model analyses, there was no significant effect of group on arterial pressure, marginal mean (95% confidence interval) for mean arterial pressure, steroids vs. control: 74 (68-80) vs. 72 (66-79) mmHg] and ScvO(2) [71 (68-75)% vs. 69 (65-73)%], cardiac index [2.8 (2.5-3.1) vs. 2.9 (2.5-3.2) L/min/m(2)], and serum cytokine concentrations [e.g. interleukin-6, 89.1 (42.8-133.9) vs. 75.7 (52.1-152.3) pg/mL] determined within 72 hours post-ROSC (P = 0.12-0.86). There was no between-group difference in body temperature, echocardiographic variables, prefrontal blood flow index/cerebral autoregulation, organ failure free days, and hazard for poor in-hospital/functional outcome, and adverse events (P = 0.08->0.99). Conclusions: Our results do not support the use of low-dose corticosteroids in in-hospital cardiac arrest.

Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods Appendix 1 Data availability References Decision letter Author response Article and author information Metrics Abstract Lung cancer and chronic lung diseases impose major disease burdens worldwide and are caused by inhaled noxious agents including tobacco smoke. The cellular origins of environmental-induced lung tumors and of the dysfunctional airway and alveolar epithelial turnover observed with chronic lung diseases are unknown. To address this, we combined mouse models of genetic labeling and ablation of airway (club) and alveolar cells with exposure to environmental noxious and carcinogenic agents. Club cells are shown to survive KRAS mutations and to form lung tumors after tobacco carcinogen exposure. Increasing numbers of club cells are found in the alveoli with aging and after lung injury, but go undetected since they express alveolar proteins. Ablation of club cells prevents chemical lung tumors and causes alveolar destruction in adult mice. Hence club cells are important in alveolar maintenance and carcinogenesis and may be a therapeutic target against premalignancy and chronic lung disease. https://doi.org/10.7554/eLife.45571.001 eLife digest The deadliest form of lung cancer is called lung adenocarcinoma, or LUAD. Tobacco chemicals often cause the disease by damaging the genetic information of lung cells. The damage leads to harmful changes in the DNA sequence which prompt the cells to form tumors. For instance, the most common of these changes takes place in a gene called KRAS. However, it is still unclear exactly which type of lung cells are more likely to develop into a tumor. In the lungs, airway epithelial cells cover the inside of the passages that bring the air inside little sacks called alveoli, which are lined by alveolar cells. Previous studies have used genetic methods to switch on the KRAS mutation in different compartments of the mouse lung. This showed that groups of airway cells, of alveolar cells, and of a class of cells located at the junction between airways and alveoli could all give rise to cancer. However, these experiments did not examine how tobacco chemicals could give rise to tumors in different groups of lung cells. Here, Spella et al. triggered LUAD in adult mice by exposing them to the toxic chemicals found in tobacco smoke, but without making any change to the KRAS gene. These mice also had genetically engineered reporters that could be used to deduce where the resulting tumors came from. DNA sequencing showed that the airway epithelial cells gained KRAS mutations after the chemical treatment. When the airway epithelial cells were experimentally removed before the treatments with tobacco chemicals, these mice did not get LUAD tumors. Spella et al. also observed that the tobacco-induced tumors came from the epithelial cells in the airways, and not from the cells in the alveoli. Moreover, when the lung was damaged, airway cells could move to the alveoli and start adopting the identity of alveolar cells, thereby replenishing this population. Together, these experiments imply that tobacco-induced LUAD starts in the airway epithelial cells. These findings suggest that airway epithelial cells could be targeted to stop lung cancer early on. Further studies should also examine how airway epithelial cells can transition to look more like alveolar cells when the lungs get harmed. https://doi.org/10.7554/eLife.45571.002 Introduction Chronic lung diseases present tremendous health burdens attributed to dysfunctional alveolar repair (Barnes et al., 2015; Lozano et al., 2012; Spella et al., 2017). Lung adenocarcinoma (LUAD), the leading cancer killer worldwide, is mainly caused by chemical carcinogens of tobacco smoke that induce mutations of the Kirsten rat sarcoma viral oncogene homologue (KRAS) in yet unidentified pulmonary cells (Torre et al., 2015; Forbes et al., 2011; Hecht, 1999; Campbell et al., 2016; Cancer Genome Atlas Research Network, 2014). The discovery of the cellular lineages and the transcriptional programs that underlie lung regeneration and carcinogenesis is extremely important, since epithelial developmental pathways are intimately related with oncogenic signaling to jointly regulate stemness and drug resistance (Barbie et al., 2009; Seguin et al., 2014). To this end, lineage-specific genes encoding epithelial proteins that support the physiological functions of the lungs were recently shown to suffer non-coding insertions and deletions in LUAD, lending further support to the longstanding notion that epithelial cells that express lung-restricted proteins are the cellular sources of LUAD (Imielinski et al., 2017). However, these cells of origin of LUAD remain only partially charted. Previous pulmonary lineage tracing studies that utilized noxious insults and ectopic expression of oncogenes in the respiratory epithelium incriminated both airway and alveolar cells as progenitors of newly formed alveoli and/or LUAD in adult mice (Zuo et al., 2015; Kim et al., 2005; Cho et al., 2011; Xu et al., 2012; Sutherland et al., 2014; Mainardi et al., 2014; Desai et al., 2014). To this end, airway epithelial cells (AEC) line the bronchi and include ciliated, basal, goblet, and Clara or club cells; alveolar type II cells (ATII) and alveolar macrophages (AMΦ) are distributed across the distal lung parenchyma; and bronchoalveolar stem cells (BASC) with dual AEC/ATII properties are located at the bronchoalveolar junctions. Established markers currently used to label these pulmonary lineages include acetylated tubulin (TUBA1A) for ciliated cells, keratin 5 (KRT5) for basal cells, forkhead box J1 (FOXJ1) for goblet cells, Clara cell secretory protein (CCSP) for club cells, surfactant protein C (SFTPC) and lysozyme 2 (LYZ2) for ATII cells, and LYZ2 for AMΦ, are summarized in Figure 1A and Figure 1—figure supplement 1, and are extensively studied in Desai et al. (2014) and Treutlein et al. (2014). However, existing mouse models for lineage tracing feature incomplete and/or promiscuous lung cell labeling, that is cellular markings fail to identify all cells of a target lineage (false negative marking) or wrongfully identify other cells outside of the target lineage (false positive marking) (Zuo et al., 2015; Kim et al., 2005; Cho et al., 2011; Xu et al., 2012; Sutherland et al., 2014; Mainardi et al., 2014; Desai et al., 2014). In addition, all studies that attempted to address the cellular origins of LUAD to date employed overexpression of oncogenes such as KRASG12D in the lungs, to conclude that ATII cells or BASC are the most probable culprits of the disease (Kim et al., 2005; Cho et al., 2011; Xu et al., 2012; Sutherland et al., 2014; Mainardi et al., 2014; Desai et al., 2014). However, it was recently shown that oncogenic KRASG12D-driven mouse lung tumors do not imitate the mutational landscape of human LUAD as closely as tobacco carcinogen-induced LUAD do (Campbell et al., 2016; Cancer Genome Atlas Research Network, 2014; Westcott et al., 2015). Figure 1 with 19 supplements see all Download asset Open asset Airway cells in urethane-induced lung tumors. (A) Cartoon of the different lung epithelial lineages, their distribution in the airways (club, goblet, ciliated, and basal cells) and the alveoli (alveolar type I and II cells), their permanent fluorescent genetic labeling in the reporter mice used in this study (green color), and the protein markers used for their identification. See also Figure 1—figure supplements 1–5. (B) Lung sections from naïve 6-week-old GFP;CCSP.CRE mice (n = 22), in which all airway cells bear permanent genetic GFP+ (green arrows) and all other cells TOMATO+ (red arrows) labels, counterstained with nuclear Hoechst33258 dye (top) or immunostained for the club cell marker CCSP and the alveolar type II cell marker SFTPC (bottom). a, alveoli; b, bronchi; v, vein. See also Figure 1—figure supplements 6–8. (C) Proliferating cell nuclear antigen (PCNA; brown) and hematoxylin (blue)-stained (top) and CCSP (green) and Hoechst33258 (blue)-stained (bottom) lung tumor sections of urethane-treated C57BL/6 mice six months post-treatment (n = 5/group), depicting endobronchial lung adenocarcinomas (white arrows). See also Figure 1—figure supplements 9–11. (D) Lung sections of GFP;CCSP.CRE mice (n = 10) at six months post-urethane treatment bearing hyperplasias and tumors (dashed outlines, top), and immunostained for the club cell marker CCSP (bottom left) and the alveolar type II cell marker SFTPC (bottom right). Note the GFP-labeled lesions of airway origin that have lost CCSP and have acquired SFTPC immunoreactivity. See also Figure 1—figure supplements 12–19. CCSP, Clara cell secretory protein; TUBA1A, acetylated α-tubulin; SFTPC, surfactant protein C; LYZ2, lysozyme 2; FOXJ1, forkhead box J1; KRT5, keratin 5. https://doi.org/10.7554/eLife.45571.003 Here we aimed at identifying the cell lineage(s) that give rise to human-relevant tobacco carcinogen-triggered LUAD in mice and that regenerate adult murine alveoli after injury. For this, we combined mouse models of genetic labeling and ablation of airway and alveolar epithelial cells with noxious and tumorigenic insults to the adult lung. To achieve this, we adapted multi-hit chemical carcinogen exposure protocols to the murine C57BL/6 strain that is resistant to chemical tumor induction (Miller et al., 2003; Malkinson et al., 1997; Stathopoulos et al., 2007), and corroborated the findings with the FVB strain that is susceptible to single-hit carcinogenesis (Westcott et al., 2015; Stathopoulos et al., 2007; Vreka et al., 2018). We show that aging, toxic, and carcinogen insults to the adult mouse lung cause expansion of airway-marked cells to the alveolar parenchyma, where they express the alveolar marker SFTPC and facilitate alveolar repair and carcinogenesis. In addition, we report how airway cells preferentially sustain chemical-induced KRAS mutations leading to LUAD that are spatially linked with neighboring bronchi. Moreover, genetic ablation of airway cells is shown to hinder alveolar maintenance and carcinogenesis in mice, indicating a central role for these cells in alveolar regeneration and LUAD triggered in response to environmental challenges. Results Accurate genetic labeling of the airway lineage To evaluate the contribution of different epithelial lung cell lineages to chemical-induced LUAD, we crossed a CRE-reporter strain that switches somatic cells from membranous tdTomato (mT; hereafter TOMATO) to membranous GFP (mG; hereafter GFP) fluorescence upon CRE-mediated recombination (mT/mG; hereafter TOMATO mice) (Muzumdar et al., 2007) to six different CRE-driver strains on the C57BL/6 background (Desai et al., 2014; Oikonomou et al., 2012; Okubo et al., 2005; Hayashi et al., 2002; Ogilvy et al., 1998; Tronche et al., 1999). This permitted the permanent genetic GFP-labeling of different lung cell lineages (mouse strains are listed in Figure 1A and Figure 1—figure supplement 2, and in Materials and methods and in Appendix 1). Double heterozygote offspring at six postnatal weeks (i.e., after mouse lung development is complete [Zuo et al., 2015; Desai et al., 2014]) were examined for GFP-labeling (results are shown in Figure 1A, Figure 1—figure supplements 3 and 4, and in Figure 1—figure supplement 4—source data 1). This approach labeled permanently all AEC of GFP;CCSP.CRE mice, some AEC and all ATII of GFP;SFTPC.CRE mice, some ATII and all AMΦ of GFP;LYZ2.CRE mice, and various other cells in the remaining intercrosses (Figure 1A, Figure 1—figure supplements 3–5, and Figure 1—figure supplement 5—source data 1). Co-localization of GFP-labeling with lineage protein markers (listed in Figure 1A and Figure 1—figure supplement 1) revealed that genetic GFP-labeling in GFP;CCSP.CRE mice marked all airway epithelial cells including club and ciliated cells, in GFP;SFTPC.CRE mice most airway and all alveolar epithelial type II cells, and in GFP;LYZ2.CRE mice some alveolar epithelial type II cells and all alveolar macrophages (Figure 1B, Figure 1—figure supplements 6–8, Figure 1—figure supplement 7—source data 1, Figure 1—figure supplement 8—source data 1).These findings show precise airway epithelial lineage labeling in GFP;CCSP.CRE mice and non-specific airway/alveolar/myeloid lineage labeling in GFP;SFTPC.CRE and GFP;LYZ2.CRE mice. Airway cells in chemical-induced lung adenocarcinoma We next triggered LUAD in GFP;CCSP.CRE, GFP;SFTPC.CRE, and GFP;LYZ2.CRE mice on the C57BL/6 background using repetitive exposures to the tobacco carcinogens urethane (ethyl carbamate, EC; stand-alone mutagen and tumor promoter) (Westcott et al., 2015; Miller et al., 2003; Stathopoulos et al., 2007; Vreka et al., 2018) or 3-methylcholanthrene followed by butylated hydroxytoluene (MCA/BHT; a two-hit mutagen/tumor promoter regimen) (Malkinson et al., 1997) (Figure 1C, Figure 1—figure supplements 9 and 10, and Figure 1—figure supplement 10—source data 1). In both models, preneoplastic (airway epithelial hyperplasias and atypical alveolar hyperplasias) and neoplastic (adenoma and LUAD) lesions classified according to established guidelines (Nikitin et al., 2004) were located both in the airways and the alveolar regions. However, established lung tumors were most frequently located near or inside the airways (Figure 1C and Figure 1—figure supplement 11). All hyperplasias and tumors of GFP;SFTPC.CRE and some of GFP;LYZ2.CRE mice were GFP-labeled, but this was not informative, since baseline marking of GFP;SFTPC.CRE and GFP;LYZ2.CRE mice were non-specific. Interestingly, all hyperplasias and tumors of GFP;CCSP.CRE mice contained GFP-labeled airway cells that did not express the club cell marker CCSP anymore, but had acquired expression of the alveolar epithelial markers SFTPC with or without LYZ2 (Figure 1D, Figure 1—figure supplements 12–15, and Figure 1—figure supplement 13—source data 1). Identical results were recapitulated using single urethane hits to GFP;CCSP.CRE, GFP;SFTPC.CRE, and GFP;LYZ2.CRE mice backcrossed >F12 to the susceptible FVB strain, which result in human LUAD-like mutations including KrasQ61R (Westcott et al., 2015; Vreka et al., 2018; Kanellakis et al., 2019) (Figure 1D and Figure 1—figure supplements 16–19). Collectively, these data support that airway cells contribute to chemical-induced LUAD, shifting from airway to alveolar marker expression during carcinogenesis. Airway cells sustain KrasQ61R mutations and give rise to juxtabronchial tumors We next used digital droplet PCR (ddPCR) to determine the lung lineages that suffer KrasQ61R driver mutations at early time-points after single urethane hits (Westcott et al., 2015; Vreka et al., 2018; Kanellakis et al., 2019). For this, GFP;CCSP.CRE and GFP;LYZ2.CRE mice backcrossed >F12 to the susceptible FVB strain received urethane and duplexed ddPCR designed to single-copy-co-amplify Kras and RosamT was performed one and two weeks later. Interestingly, GFP-labeled cells of both mouse strains had KrasQ61R mutations at one week post-urethane, but KrasQ61R mutations selectively persisted in GFP-labeled airway cells in the lungs of GFP;CCSP.CRE mice at two weeks (Figure 2A, Figure 2—figure supplement 1, and Figure 2—source data 1). In addition, three-dimensional reconstruction of tumor-bearing lungs of FVB mice at 6 months post-urethane using high-resolution micro-computed tomography (μCT) revealed that most lung tumors were spatially linked with the airways, in accord with pathology results (Figure 2B and C, and Figure 2—source data 2). These results support the involvement of airway cells in chemical-induced lung adenocarcinoma formation in mice. Figure 2 with 1 supplement see all Download asset Open asset Airway cells sustain KrasQ61R mutations inflicted by urethane and give rise to juxtabronchial lung adenocarcinomas. (A) DNA was extracted from the lungs of GFP;CCSP.CRE and GFP;LYZ2.CRE mice (FVB strain) one and two weeks post-urethane treatment (n = 5/group). Summary of duplexed digital droplet PCR (ddPCR) results using primers and probes specific for the RosamT and the KrasWT sequences. Note that all cell types equally suffer initial KrasQ61R mutations, but only GFP-labeled cells of GFP;CCSP.CRE mice (i.e. airway cells) maintain the KrasQ61R mutation after two weeks. See also Figure 2—figure supplement 1. Data are shown as violin plot. P, overall probability, two-way ANOVA. ***: p<0.001 compared with all other groups, Bonferroni post-tests. (B) Representative high-resolution micro-computed tomography (μCT) lung sections (top) and three-dimensional reconstructions (bottom) from urethane-treated FVB mice six months after treatment (n = 10). Note lung tumors attached to (green arrows) or contained within (blue arrows) the airways, as well as lung tumors with no obvious link to a bronchus (red arrows). (C) Summary of results from μCT (data from Figure 2B) and pathology (data from Figure 1C) shown as violin plot. P, probability, two-way ANOVA.*, ***, and ****: p<0.05, p<0.001, and p<0.0001, respectively, compared with airway-attached tumors, Bonferroni post-tests. Shown are also Spearman’s correlation coefficient (ρ) and probability (P) for correlation of μCT and pathology results. https://doi.org/10.7554/eLife.45571.029 Figure 2—source data 1 Quantification ofKrasmutant droplets in duplexed digital droplet PCR (ddPCR). https://doi.org/10.7554/eLife.45571.031 Download elife-45571-fig2-data1-v2.xlsx Figure 2—source data 2 Quantification of tumor airway link. https://doi.org/10.7554/eLife.45571.032 Download elife-45571-fig2-data2-v2.xlsx Alveolar dissemination of airway-labeled cells during carcinogenesis Since airborne carcinogens act globally on the respiratory field (Franklin et al., 1997), we examined non-neoplastic alveolar areas of carcinogen-treated GFP;CCSP.CRE mice, to discover markedly increased numbers of GFP-labeled cells in the alveoli of carcinogen-treated mice compared with saline-treated or naïve controls (Figure 3A, Figure 3—figure supplements 1 and 2, and Figure 3—figure supplement 2—source data 1). Immunostaining revealed that juxtabronchial GFP-labeled cells still expressed CCSP, but lost CCSP and acquired SFTPC expression when located in alveoli and tumors (Figure 3B and Figure 3—figure supplements 3 and 4). The expansion of airway cells after urethane exposure was also documented using bioluminescent imaging of double heterozygote offspring of CCSP.CRE intercrosses with Luciferase-expressing (LUC) mice (Safran et al., 2003), a strain emitting light specifically from airway epithelia (Figure 3—figure supplement 5, and Figure 3—figure supplement 5—source data 2). In addition, co-staining of human LUAD (Giopanou et al., 2015) for the alveolar marker SFTPC and the airway markers CCSP and KRT5 showed co-localization of SFTPC with KRT5 but not with CCSP (Figure 3C and Figure 3—figure supplement 6). These results suggest that airway epithelial cells expand to alveolar regions during field cancerization by tobacco carcinogens, a process involving either direct alveolar cell recycling by airway epithelial cells or transient CCSP expression by alveolar cells during carcinogenesis. Moreover, that human and murine LUAD carry airway imprints although their location and protein expression suggests an alveolar origin (Desai et al., 2014; Aberle et al., 2011; Mason et al., 2000; Lindskog et al., 2014; Sutherland and Berns, 2010). Figure 3 with 6 supplements see all Download asset Open asset Expansion of airway cells in the tumor-initiated lung. (A) Non-neoplastic alveolar regions from lung sections of saline-, urethane (ethyl carbamate, EC)-, and 3-methyl-1,2-dyhydrobenzo[j]aceanthrylene/butylated hydroxytoluene (MCA/BHT)-treated GFP;CCSP.CRE mice at six months into treatment (n = 8 mice/group). Note the few GFP-labeled cells of saline-treated mice and their increased numbers in carcinogen-treated mice (arrows). See also Figure 3—figure supplements 1 and 2. (B) Juxtabronchial region from lung section of urethane-treated GFP;CCSP.CRE mouse at six months into treatment (n = 22) stained for the alveolar type II cell marker SFTPC. Arrows and legend indicate different phenotypes of extrabronchial GFP-labeled cells. See also Figure 3—figure supplements 3–5. (C) Merged high-power image of SFTPC and KRT5 co-staining of human lung adenocarcinoma (n = 10) shows significant co-localization of the two markers in a subset of tumor cells (arrows). See also Figure 3—figure supplement 6. CCSP, Clara cell secretory protein; SFTPC, surfactant protein C; KRT5, keratin 5. https://doi.org/10.7554/eLife.45571.033 Airway cells in the aging and injured adult alveolus We next examined the kinetics of lineage-labeled cells during aging, injury, and repair. While the number of GFP-labeled cells in the alveoli of aging GFP;SFTPC.CRE and GFP;LYZ2.CRE mice was stable, GFP-labeled airway cells in the alveoli of aging GFP;CCSP.CRE mice progressively increased and expressed SFTPC protein (Figure 4A and B and Figure 4—source data 1). Bleomycin treatment, which depletes alveolar type II cells (Lawson et al., 2005), accelerated the accumulation of GFP-labeled airway cells in the alveoli and in urethane-triggered LUAD (Figure 4C and D, Figure 4—figure supplements 1 and 2, Figure 4—source data 2, and Figure 4—figure supplement 2—source data 1). GFP-labeled airway cells expressing the alveolar marker SFTPC also increased in the alveoli of GFP;CCSP.CRE mice exposed to perinatal hyperoxia that damages forming alveoli (Rawlins et al., 2009), and in the alveoli of GFP;CCSP.CRE mice treated with naphthalene that kills airway epithelial cells (Sutherland and Berns, 2010; Rawlins et al., 2009), but were not identified within the airways of naphthalene-treated GFP;CCSP.CRE mice; these appeared to be repopulated by GFP-labeled airway cells that express the club cell marker CCSP (Figure 4E–4H, Figure 4—figure supplements 3 and 4, Figure 4—source datas 3 and 4, and Figure 4—figure supplement 4—source data 2). In line with the latter finding, no GFP-labeled alveolar cells were identified in the airways of GFP;LYZ2.CRE mice recovering from naphthalene-induced injury (Figure 4G and H). Taken together, the data indicate that airway-originated cells repopulate both the airways and the alveoli during aging and recovery from injury, while alveolar cells do not reconstitute the airways, in line with previous findings (Desai et al., 2014; Rawlins et al., 2009). The observed alveolar spread of airway-labeled cells was explained by either peripheral migration of airway cells or transient CCSP expression by regenerating alveolar cells. Figure 4 with 4 supplements see all Download asset Open asset Airway cells in alveolar repair. (A) Non-neoplastic alveolar regions from lung sections of aging GFP;CCSP.CRE mice (bottom right section is also SFTPC-immunostained) show increasing numbers of alveolar GFP-labeled cells with age (arrows). Green arrows: genetically GFP-labeled, SFTPC-immunoreactive airway cells in alveolus of 15-month-old GFP;CCSP.CRE mouse. (B) Data summary (n = 5 mice/time-point) from (A) shown as violin plot. Color-coded boxes indicate time windows of experiments in (C-H). P, probability, one-way ANOVA. ns, ***, and ****: p>0.05, p<0.001, and p<0.0001, respectively, for comparison with time-point zero by Bonferroni post-tests. (C) SFTPC-immunostained lung sections of GFP;CCSP.CRE mice show accelerated increase of alveolar GFP-labeled SFTPC-immunoreactive airway cells after bleomycin treatment (arrows). See also Figure 4—figure supplement 1 and Figure 4—figure supplement 2. (D) Data summary from (C) shown as violin plots (n = 4 mice/time-point). P, probabilities, one-way ANOVA. ns, *, **, ***, and ****: p>0.05, p<0.05, p<0.01, p<0.001, and p<0.0001, respectively, for comparison with day zero by Bonferroni post-tests. (E) SFTPC-stained lung sections of GFP;CCSP.CRE mice at two months after perinatal exposure to 98% O2 show enlarged alveoli (evident by increased mean linear intercept) enriched in GFP-labeled SFTPC-immunoreactive airway cells (arrows) compared with 21% O2. (F) Data summary from (E) shown as violin plots (n = 6 mice/group). P, probabilities, t-test. (G) Lung sections (top) of GFP;CCSP.CRE mice (n = 5 mice/group) show enrichment of alveoli in GFP-labeled cells post-naphthalene treatment (arrows). Lung sections (bottom) of GFP;LYZ2.CRE mice (n = 5 mice/group) at six weeks post-naphthalene show no bronchial (b) GFP-labeled cells. See also Figure 4—figure supplements 3 and 4. (H) Data summary from (G) shown as violin plot (n = 5 mice/time-point). P, probability, two-way ANOVA. ns and ****: p>0.05 and p<0.0001, respectively, for comparison with corn oil by Bonferroni post-tests. CCSP, Clara cell secretory protein; SFTPC, surfactant protein C; LYZ2, lysozyme 2. https://doi.org/10.7554/eLife.45571.042 Figure 4—source data 1 Quantification of alveolar GFP+ cells in GFP;CCSP.CRE mice during aging. https://doi.org/10.7554/eLife.45571.047 Download elife-45571-fig4-data1-v2.xlsx Figure 4—source data 2 Quantification of SFTPC+ and GFP+ cells in GFP;CCSP.CRE mice after bleomycin treatment. https://doi.org/10.7554/eLife.45571.048 Download elife-45571-fig4-data2-v2.xlsx Figure 4—source data 3 Data of mean linear intercept and GFP+/SFTPC+cells in GFP;CCSP.CRE mice after hyperoxia treatment. https://doi.org/10.7554/eLife.45571.049 Download elife-45571-fig4-data3-v2.xlsx Figure 4—source data 4 Data of GFP+/SFTPC+ cells in GFP;CCSP.CRE and GFP;LYZ2.CRE mice after naphthalene treatment. https://doi.org/10.7554/eLife.45571.050 Download elife-45571-fig4-data4-v2.xlsx Airway cells maintain alveoli and foster tumors To further examine the role of airway and alveolar cells in alveolar homeostasis and lung carcinogenesis, we ablated them by crossing CCSP.CRE, SFTPC.CRE, and LYZ2.CRE mice to mice expressing Diphtheria toxin in somatic cells upon CRE-mediated recombination (DTA mice) (Voehringer et al., 2008). Triple transgenic GFP;DRIVER.CRE;DTA intercrosses were also generated to evaluate ablation efficiency. As expected, SFTPC.CRE;DTA and GFP;SFTPC.CRE;DTA mice were fetal lethal (no double or triple heterozygote offspring was obtained by n > 3 intercrosses,>10 litters, and >60 off-springs for each genotype; p<0.0001, Fischer’s exact test). However, all other ablated mice survived till adulthood. Airway epithelial ablation was complete in GFP;CCSP.CRE;DTA mice, while some GFP-labeled alveolar macrophages persisted in GFP;LYZ2.CRE;DTA mice, presumably freshly recruited monocytes initiating LYZ2 expression. Immunostaining revealed that the denuded airway epithelium of 12-week-old GFP;CCSP.CRE;DTA mice contained few flat CCSP+SFTPC+LYZ2+ immunoreactive cells, while the apparently intact alveolar spaces of GFP;LYZ2.CRE;DTA mice harbored only some CCSP-SFTPC-LYZ2+immunoreactive alveolar macrophages (Figure 5A, Figure 5—figure supplements 1 and 2, and Figure 5—figure supplement 2—source data 1). Remarkably, morphometric and functional analyses of 12-week-old DTA control, CCSP.CRE;DTA, and LYZ2.CRE;DTA mice showed that LYZ2.CRE;DTA mice displayed normal airway caliper and mean linear intercept (measures of airway and alveolar structure), normal number of CD45+ CD11b+ myeloid cells in bronchoalveolar lavage (BAL; measure of airspace inflammation), and normal airways resistance and static compliance (measures of airway and alveolar function) compared with DTA controls. However, CCSP.CRE;DTA mice displayed widened airway and alveolar dimensions with inflammatory interalveolar septal destruction evident by increased mean linear intercept, CD45+ CD11b+ cells in BAL, and static compliance (Figure 5B and C and Figure 5—source data 1), mimicking human chronic obstructive pulmonary disease (Barnes et al., 2015). Finally, we exposed control and ablated mice to ten consecutive weekly urethane exposures. All mice survived six months into carcinogen treatment, and CCSP.CRE;DTA and LYZ2.CRE;DTA mice were equally protected from LUAD development compared with controls (Figure 5D and E, and Figure 5—source data 2). Taken together, these results show that the CCSP+ airway lineage maintains postnatal alveolar structure and function, and, together with the LYZ2+ alveolar lineage, are required for lung adenocarcinoma development. Figure 5 with 2 supplements see all Download asset Open asset Airway cell-ablated mice display alveolar destruction and are protected from carcinogenesis. (A) Lineage marker-immunostained lung sections of 12-week-old GFP;CCSP.CRE;DTA and GFP;LYZ2.CRE;DTA mice (n = 6/group) show increased bronchial and alveolar size and flat CCSP+ SFTPC+ LYZ2+ cells in the airways of GFP;CCSP.CRE;DTA mice (green arrows), and CCSP-SFTPC-LYZ2+ alveolar macrophages in the airspaces of GFP;LYZ2.CRE;DTA mice (blue arrows). See also Figure 5—figure supplements 1 and 2. (B) Hematoxylin and eosin-stained lung sections (n = 6/group) from 12-week-old DTA (controls), CCSP.CRE;DTA (airway epithelial suicide model), and LYZ2.CRE;DTA (alveolar epithelial suicide model) mice. (C) Data summaries of mean linear intercept, bronchoalveolar lavage (BAL) myeloid cells, pressur

Lung adenocarcinoma (LUAD) and chronic lung diseases caused by smoking and environmental noxious agents are the deadliest diseases worldwide, sharing a partially charted pathobiology of dysfunctional alveolar repair. Here we sought to identify the respiratory epithelial dynamics and molecular signatures participating in adult lung maintenance and chemical carcinogenesis. We employed novel mouse models of respiratory epithelial marking and ablation, a battery of pulmonary toxins and carcinogens, experimental protocols of carcinogen-induced LUAD, tobacco carcinogen-induced LUAD cell lines, and human transcriptomic data and identified a prominent involvement of airway molecular programs in alveolar maintenance and carcinogen-induced LUAD. The airway-specific transcriptomic signature was redistributed to the alveoli after toxic and carcinogenic insults and resulted in marked contributions of airway-labeled cells to injury-recovered alveoli and LUAD. Airway cells maintained Kras mutations and therefore possibly contributed to lung cancer initiation, while LUAD were spatially linked to neighboring airways. Transcriptomic profiling of carcinogen-induced murine and human LUAD revealed enrichment in airway signatures, while ablation of airway cells distorted alveolar structure and function and protected mice from LUAD development. Collectively, these results indicate that airway cells and/or transcriptomic signatures are essential for alveolar maintenance and LUAD development.

Lung cancer and chronic lung diseases impose major disease burdens worldwide and are caused by inhaled noxious agents including tobacco smoke. The cellular origins of environmental-induced lung tumors and of the dysfunctional airway and alveolar epithelial turnover observed with chronic lung diseases are unknown. To address this, we combined mouse models of genetic labeling and ablation of airway (club) and alveolar cells with exposure to environmental noxious and carcinogenic agents. Club cells are shown to survive KRAS mutations and to form lung tumors after tobacco carcinogen exposure. Increasing numbers of club cells are found in the alveoli with aging and after lung injury, but go undetected since they express alveolar proteins. Ablation of club cells prevents chemical lung tumors and causes alveolar destruction in adult mice. Hence club cells are important in alveolar maintenance and carcinogenesis and may be a therapeutic target against premalignancy and chronic lung disease.

Chronic obstructive pulmonary disease is a chronic inflammatory disorder with an increased incidence of lung cancer. The emphysema component of chronic obstructive pulmonary disease confers the greatest proportion to lung cancer risk. Although tumors create inflammatory conditions to escape immunity, the immunological responses that control growth of nascent cancer cells in pre-established inflammatory microenvironments are unknown. In this study, we addressed this issue by implanting OVA-expressing cancer cells in the lungs of mice with cigarette smoke–induced emphysema. Emphysema augmented the growth of cancer cells, an effect that was dependent on T cytotoxic cells. OVA-specific OTI T cells showed early signs of exhaustion upon transfer in emphysema tumor hosts that was largely irreversible because sorting, expansion, and adoptive transfer failed to restore their antitumor activity. Increased numbers of PD-L1– and IDO-positive CD11c+ myeloid dendritic cells (DCs) infiltrated emphysema tumors, whereas sorted emphysema tumor DCs poorly stimulated OTI T cells. Upon adoptive transfer in immunocompetent hosts, T cells primed by emphysema tumor DCs were unable to halt tumor growth. DCs exposed to the emphysema tumor microenvironment downregulated MHC class II and costimulatory molecules, whereas they upregulated PD-L1/IDO via oxidative stress–dependent mechanisms. T cell activation increased upon PD-L1 blockade in emphysema DC–T cell cocultures and in emphysema tumor hosts in vivo. Analysis of the transcriptome of primary human lung tumors showed a strong association between computed tomography–based emphysema scoring and downregulation of immunogenic processes. Thus, suppression of adaptive immunity against lung cancer cells links a chronic inflammatory disorder, emphysema, to cancer, with clinical implications for emphysema patients to be considered optimal candidates for cancer immunotherapies.

Electronic cigarettes (e-cigs) are advertised as a less harmful nicotine delivery system or as a new smoking cessation tool. We aimed to assess the in vivo effects of e-cig vapor in the lung and to compare them to those of cigarette smoke (CS). We exposed C57BL/6 mice for either 3 days or 4 wk to ambient air, CS, or e-cig vapor containing 1) propylene glycol/vegetable glycerol (PG: VG-Sol; 1:1), 2) PG:VG with nicotine (G:VG-N), or 3) PG:VG with nicotine and flavor (PG:VG-N + F) and determined oxidative stress, inflammation, and pulmonary mechanics. E-cig vapors, especially PG:VG-N + F, increased bronchoalveolar lavage fluid (BALF) cellularity, Muc5ac production, as well as BALF and lung oxidative stress markers at least comparably and in many cases more than CS. BALF protein content at both time points studied was only elevated in the PG:VG-N + F group. After 3 days, PG: VG-Sol altered tissue elasticity, static compliance, and airway resistance, whereas after 4 wk CS was the only treatment adversely affecting these parameters. Airway hyperresponsiveness in response to methacholine was increased similarly in the CS and PG:VG-N + F groups. Our findings suggest that exposure to e-cig vapor can trigger inflammatory responses and adversely affect respiratory system mechanics. In many cases, the added flavor in e-cigs exacerbated the detrimental effects of e-cig vapor. We conclude that both e-cig vaping and conventional cigarette smoking negatively impact lung biology.

Malignant pleural mesothelioma is resistant to currently used treatment. Angiopoieitn-1 directly promotes mesothelioma cell growth in a Tie-2-dependent fashion. Angiopoietin/Tie-2 axis may thus be valid targets for therapeutic interventions against mesothelioma. We hypothesized that a soluble angiopoietin inhibitor (Murine Tek-deltaFc) would halt mesothelioma progression in vivo by enhancing mesothelioma cell proliferation and inhibiting tumor angiogenesis. Our hypothesis was challenged on two syngeneic mesothelioma in vivo models (AB1 cells-Balb/c mice and AE17 cells-C57BL/6 mice. Even though both mesothelioma cell lines express the Angiopoietin-1/-2 and Tie-2, murine Tek-deltaFc hampered AB1 but not AE17 mesothelioma growth in vivo by enhancing tumor cell apoptosis and limiting tumor angiogenesis. Neither angiopoietins (Angs)-1 and -2 nor the inhibitor affected mesothelioma cell growth in vitro. AB1 (responding) tumors were more vascularized and displayed higher endothelial Tie-2 and lower tumor Ang-1 expression than the (non-responding) AE17 tumors. Angiopoietins-1 and -2 are expressed in tumors and pleural cavity of mesothelioma patients demonstrating the clinical relevance of our experimental observations. In conclusion, disrupting Ang-Tie-2 signaling limits mesothelioma angiogenesis and halts tumor progression. Tumor vascularity, endothelial Tie-2 expression and tumor Ang-1 expression may predict mesothelioma response to Tek-deltaFc.

Inspiratory resistive breathing (IRB), a hallmark of obstructive airway diseases, is associated with strenuous contractions of the inspiratory muscles and increased negative intrathoracic pressures that act as an injurious stimulus to the lung. We have shown that IRB induces pulmonary inflammation in healthy animals. p38 kinase is activated in the lung under stress. We hypothesized that p38 is activated during IRB and contributes to IRB-induced pulmonary inflammation. Anesthetized, tracheostomized rats breathed spontaneously through a two-way valve. Resistance was connected to the inspiratory port to provoke a peak tidal inspiratory pressure 50% of maximum. Following 3 and 6 h of IRB, respiratory system mechanics were measured and bronchoalveolar lavage (BAL) was performed. Phosphorylated p38, TNF-α, and MIP-2α were detected in lung tissue. Lung injury was estimated histologically. SB203580 (p38 inhibitor) was administered prior to IRB (1 mg kg−1). Six hours of IRB increased phosphorylated p38 in the lung, compared with quietly breathing controls (p = 0.001). Six hours of IRB increased the numbers of macrophages and neutrophils (p = 0.01 and p = 0.005) in BAL fluid. BAL protein levels and lung elasticity increased after both 3 and 6 h IRB. TNF-α and MIP-2α increased after 6 h of IRB (p = 0.01 and p < 0.001, respectively). Increased lung injury score was detected at 6 h IRB. SB203580 administration blocked the increase of neutrophils and macrophages at 6 h IRB (p = 0.01 and p = 0.005 to 6 h IRB) but not the increase in BAL protein and elasticity. TNF-α, MIP-2α, and injury score at 6 h IRB returned to control. p38 activation contributes to IRB-induced pulmonary inflammation.

Inspiratory resistive breathing (IRB), a hallmark of obstructive airway diseases, is associated with large negative intrathoracic pressures, due to strenuous contractions of the inspiratory muscles. IRB is shown to induce lung injury in previously healthy animals. Src is a multifunctional kinase that is activated in the lung by mechanical stress. ERK1/2 kinase is a downstream target of Src. We hypothesized that Src is activated in the lung during IRB, mediates ERK1/2 activation and IRB-induced lung injury.

INTRODUCTION:Resistive breathing (RB), a hallmark of obstructive airway diseases, is characterized by strenuous contractions of the inspiratory muscles that impose increased mechanical stress on the lung. RB is shown to induce pulmonary inflammation in previous healthy animals. Tiotropium bromide, an anticholinergic bronchodilator, is also shown to exert anti-inflammatory effects. The effect of tiotropium on RB-induced pulmonary inflammation is unknown.METHODS:Adult rats were anesthetized, tracheostomized and breathed spontaneously through a two-way non-rebreathing valve. Resistances were connected to the inspiratory and/or expiratory port, to produce inspiratory resistive breathing (IRB) of 40% or 50% Pi/Pi,max (40% and 50% IRB), expiratory resistive breathing (ERB) of 60% Pe/Pe,max (60% ERB) or combined resistive breathing (CRB) of both 40% Pi/Pi,max and 60% Pe/Pe,max (40%/60% CRB). Tiotropium aerosol was inhaled prior to RB. After 6 h of RB, mechanical parameters of the respiratory system were measured and bronchoalveolar lavage (BAL) was performed. IL-1β and IL-6 protein levels were measured in lung tissue. Lung injury was estimated histologically.RESULTS:In all, 40% and 50% IRB increased macrophage and neutrophil counts in BAL and raised IL-1β and IL-6 lung levels, tissue elasticity, BAL total protein levels and lung injury score. Tiotropium attenuated BAL neutrophil number, IL-1β, IL-6 levels and lung injury score increase at both 40% and 50% IRB. The increase in macrophage count and protein in BAL was only reversed at 40% IRB, while tissue elasticity was not affected. In all, 60% ERB raised BAL neutrophil count and total protein and reduced macrophage count. IL-1β and IL-6 levels and lung injury score were increased. Tiotropium attenuated these alterations, except for the decrease in macrophage count and the increase in total protein level. In all, 40%/60% CRB increased macrophage and neutrophil count in BAL, IL-1β and IL-6 levels, tissue elasticity, total protein in BAL and histological injury score. Tiotropium attenuated the aforementioned alterations.CONCLUSION:Tiotropium inhalation attenuates RB-induced pulmonary inflammation.

P001 - Sepsis impairs the capillary response within hypoxic capillaries and decreases erythrocyte oxygen-dependent ATP efflux

Combined resistive breathing (CRB) is the hallmark of obstructive airway disease pathophysiology.We have previously shown that severe inspiratory resistive breathing (IRB) induces acute lung injury in healthy rats.The role of expiratory resistance is unknown.The possibility of a load-dependent type of resistive breathing-induced lung injury also remains elusive.Our aim was to investigate the differential effects of IRB, expiratory resistive breathing (ERB), and CRB on healthy rat lung and establish the lowest loads required to induce injury.Anesthetized tracheostomized rats breathed through a two-way valve.Varying resistances were connected to the inspiratory, expiratory, or both ports, so that the peak inspiratory pressure (IRB) was 20%-40% or peak expiratory (ERB) was 40%-70% of maximum.CRB was assessed in inspiratory/expiratory pressures of 30%/50%, 40%/50%, and 40%/60% of maximum.Quietly breathing animals served as controls.At 6 hours, respiratory system mechanics were measured, and bronchoalveolar lavage was performed for measurement of cell and protein concentration.Lung tissue interleukin-6 and interleukin-1β levels were estimated, and a lung injury histological score was determined.ERB produced significant, load-independent neutrophilia, without mechanical or permeability derangements.IRB 30% was the lowest inspiratory load that provoked lung injury.CRB increased tissue elasticity, bronchoalveolar lavage total cell, macrophage and neutrophil counts, protein and cytokine levels, and lung injury score in a dose-dependent manner.In conclusion, CRB load dependently deranges mechanics, increases permeability, and induces inflammation in healthy rats.ERB is a putative inflammatory stimulus for the lung.

Introduction: Density of tertiary lymphoid structures and numbers of tumor-infiltrating CD8+ve T cells confer positive prognostic value in lung cancer. However, in the majority of lung cancer patients the anti-tumor immune responses are poor, underscoring the existence of immune escape mechanisms used by cancer cells. Immunoglobulin-like transcripts (ILTs) are a group of membrane inhibitory receptors which negatively regulate the functions of immune cells. ILT3 expression has been observed in gastric cancer cells and associated with cancer immune escape. Aims: To analyze ILT3 expression and its association with lung cancer tolerance. Methods: We evaluated cellular expression of ILT3, density of intratumoral lymphoid follicles and numbers of tumor-infiltrating CD8+ve cells, by immunohistochemistry on paraffin-embedded tissue specimens of 39 lung adenocarcinoma patients, TNM stage IA-IIIA, who underwent thoracotomy from 2009 to 2011. ILT3 expression was related to density of lymphoid follicles and CD8+ve T cells. Results: ILT3 expression was observed on the surface and cytoplasm of lung cancer cells of 46% of the patients. Patients with ILT3+ve cancer cells showed a significantly lower density of intratumoral lymphoid follicles, (median, range 17916, 0-109937 μm2 / optical field) than those with ILT3-ve cancer cells (64005, 0-155585 μm2 / optical field), p=0.018. There were no differences observed in CD8+ve T cytotoxic cells and Disease Specific Survival of the patients. Conclusions: We may have discovered a novel immune checkpoint in human lung adenocarcinoma, which could offer untapped opportunities for therapeutic intervention.

Introduction: COPD increases the incidence of lung cancer independently of cigarette smoking. A hallmark of cancer is its remarkable ability to evade immunity. Cigarette smoke (CS) exposure has been associated with suppressed T cell immunity against pathogens. T cells are the frontline of defense against tumors. AIMS. To establish an orthotopic model of lung cancer in fully immunocompetent mice and investigate whether CS suppresses anti-tumor T cell responses. Methods: C57BL/6J wild type mice were either exposed to CS or sham. On day 30 syngeneic Lewis Lung Cancer (LLC) cells (or cancer-free media) were inoculated on the left lung lobe. On day 40 animals were sacrificed. Intratumoral and draining lymph node T cells were analyzed by 8-colour flow-cytometry. Results: A higher number of T helper and T cytotoxic (Tc) was observed in the lungs and draining lymph nodes of both CS-exposed and sham-exposed mice after inoculation of lung cancer cells compared to cancer free media, showing that cancer cell inoculation triggers adaptive immune responses in our mice models. However, intratumoral and lymph node T cells of CS-exposed mice with cancer showed a less activated/cytotoxic phenotype and produced higher levels of the inhibitory molecules TIM3, CTLA4, CD39, CD73 and of the immunosuppressive cytokine IL-10 than those of sham-exposed mice with cancer. Conclusions: Short-term CS-exposure suppresses early anti-tumor T cell responses in vivo. Impaired anti-cancer specific immunity in smokers could be a critical determinant of lung cancer escape from immunesurveillance mechanisms.

Inspiratory resistive breathing (IRB) is characterized by large negative intrathoracic pressures and was shown to induce pulmonary inflammation in previously healthy rats. Matrix metalloproteinases (MMP)-9 and -12 are induced by inflammation and mechanical stress in the lung. We hypothesized that IRB induces MMP-9 and -12 in the lung. Anesthetized, tracheostomized rats breathed spontaneously through a two-way valve, connected to an inspiratory resistance, with the tidal inspiratory tracheal pressure set at 50% of the maximum. Quietly breathing animals served as controls. After 3 and 6 h of IRB, respiratory mechanics were measured, bronchoalveolar lavage (BAL) was performed, lung injury score was estimated, and lung MMP-9 was estimated by zymography and ELISA. MMP-9 and MMP-12 immunohistochemistry was performed. Isolated normal alveolar macrophages were incubated with BAL from rats that underwent IRB. After 18 h, MMP-9 and -12 levels were measured in supernatants, and immunocytochemistry was performed. Macrophages were treated with IL-1β, IL-6, or TNF-α, and MMP-9 in supernatants was measured. After 6 h of IRB, leukocytes in BAL increased, and IL-1β and IL-6 levels were elevated. Elasticity and injury score were increased after 3 and 6 h of IRB. Lung MMP-9 levels increased after 6 h of IRB. MMP-9 and MMP-12 were detected in alveolar macrophages and epithelial (bronchial/alveolar) cells after 3 and 6 h of IRB. MMP-9 and MMP-12 were found in supernatants after treatment with 6 h of IRB BAL. Cytosolic immunostaining was detected after treatment with 3 and 6 h of IRB BAL. All cytokines induced MMP-9 in culture supernatants. In conclusion, IRB induces MMP-9 and -12 in the lung of previously healthy rats.

Combined inspiratory and expiratory resistive breathing (CRB) is the landmark of obstructive airway disease. Inspiratory loading and CRB via tracheal banding are known to induce acute lung injury in previously healthy animals. The role of expiratory resistance remains unknown.Aim: To elucidate the differential effect of inspiratory, expiratory and combined resistive loading on healthy rat lung mechanics and inflammatory status and establish the lowest loads required to induce injury.Anesthetized tracheostomized wistar rats breathed through a two-way valve. A resistance was connected to the inspiratory, expiratory or both ports, so that measured peak inspiratory pressure was modulated to (IRB) 20-40% of maximum or peak expiratory to (ERB) 30-70%. Combined resistive breathing was studied in inspiratory/expiratory pressures of 30%/50%, 40%/50% and 40%/60% of maximum. Quietly breathing animals served as controls. At six hours, respiratory system mechanics were measured and bronchoalveolar lavage (BAL) was performed for cell and protein concentration counting. Both IRB and CRB increased tissue elasticity, BAL total cell, macrophage and neutrophil counts and protein content in a dose dependent manner. IRB 30% and CRB 40%/50% were identified as the lowest loads able to induce significant mechanical distortion and lung inflammation. ERB failed to induce significant changes, with the exception of BAL fluid neutrophilia. IRB and CRB, above 30% and 40%/50% respectively, dose-dependently derange mechanics, increase permeability and induce inflammation in healthy rats. ERB is a putative per se inflammatory stimulus for the lung.

Introduction: During exacerbations of obstructive lung diseases, inflammation and the accompanying bronchoconstriction lead to significant airway narrowing, i.e. resistive breathing (RB). In order to distinguish between the effects of the mechanical stressor and the underlying inflammation we developed a RB model via tracheal banding (TB), and investigated the effects of RB in mice with pre-existing lung inflammationinduced by endotoxin inhalation. Since smooth muscle tone is a crucial determinant of bronchoconstriction and involves nitric oxide (NO) signaling through soluble guanylyl cyclase (sGC) activation, the expression of sGC was studied. Materials and Methods: Adult C57BL/6 mice underwent resistive breathing for 24 hours by placing a nylon band around the extrathoracic trachea, providing a 50% tracheal occlusion. The following groups were studied: 1. TB mice and quietly breathing sham operated mice (controls) 2. Mice treated with inhaled lipopolysaccharide (LPS) pseudomonas aeruginosa 10mg/3ml via nebulization and controls. 3. Mice treated with LPS 24 hours prior TB. Mice were sacrificed 24h post operation. Results: Mice subjected to resistive breathing via TB, exhibited a more pronounced increase in bronchoalveolar lavage fluid cellularity and protein content compared with mice treated with LPS alone. By using forced oscillation technique, TB further increased tissue elasticity and airway resistance. The most pronounced decrease in the expression of sGCα1 and β1 subunits was observed in the group subjected to LPS inhalation and TB. Conclusion: TB aggravates pre-existing lung inflammation and sGC downregulation implicating new insights into the mechanisms during obstructive lung disease exacerbations.