Blunt chest trauma may cause acute lung injury (ALI) and/or its most severe manifestation, i.e., the Acute Respiratory Distress Syndrome (ARDS). The leading cause of chronic obstructive pulmonary disease (COPD) is cigarette smoke (CS). Both active and passive smoking are independent risk factors for ALI following blunt chest trauma, while active smoking alone promotes the liability to develop an ARDS (1). Lung contusion following blunt chest trauma promotes oxidative stress and apoptosis, but also both pulmonary and systemic hyperinflammation (2, 3), all these aggravates are also elements of COPD. Prechallenged CS exposure aggravated ALI induced by injurious mechanical ventilation (4), and we recently showed that CS exposure prior to blunt chest trauma is associated with more severe posttraumatic inflammation and nitrosative stress, ultimately leading to aggravated organ dysfunction and damage (3).
There is ample evidence in rodent models that the maintenance of hydrogen sulfide (H2S) availability (5), in particular resulting from the expression and activity of cystathionine-γ-lyase (CSE), assumes major protective importance in asthma and COPD (6). In contrast, equivocal data are available on the role of CSE during ALI, in that both protective (2), and deleterious (7), properties have been described. None of the aforementioned models, however, investigated animals with pre-existing chronic pulmonary diseases, and moreover, these models did not integrate any standard intensive care measures, e.g., hemodynamic monitoring, fluid resuscitation, and lung-protective mechanical ventilation. We previously demonstrated that inhaling H2S attenuated systemic hyperinflammation in resuscitated murine septic shock (8). Similarly, in a murine model of blunt chest trauma, infusing sodium sulfide (Na2S) reduced posttraumatic lung tissue apoptosis, stress protein expression, and histological damage (2). The protective effect of Na2S in combination with hypothermia coincided with reduced CSE expression, thus suggesting a critical role of CSE in the posttraumatic adaptive stress response (2, 9). Given the protective impact of CSE on CS-induced COPD and ALI, we tested the hypothesis whether CSE knockout would aggravate pulmonary dysfunction after blunt chest trauma in CS-exposed mice.
MATERIALS AND METHODS
This study was authorized by the federal authorities for animal research of the Regierungspräsidium Tübingen (approved animal experimentation number: 1130), Baden-Württemberg, Germany, and the Animal Care Committee of the University of Ulm, Baden-Württemberg, Germany, and performed in adherence with the National Institutes of Health Guidelines on the Use of Laboratory Animals and the European Union “Directive 2010/63 EU on the protection of animals used for scientific purposes.” The experiments were implemented using C57BL/6J mice that were received from Charles River laboratories Germany (Sulzbach, Germany) and homozygous (CSE−/−) mutant mice (C57BL/6J.129SvEv) bred in-house (10). Animals were kept under standardized conditions, and were equally distributed in terms of gender, age, and body weight (each of the four groups comprised four male and four female animals; 10–25 weeks, 26 ± 3 g). Two additional C57BL/6J mice that did not undergo CS exposure, anesthesia, chest trauma, and surgery served as native controls for immunoblotting and electrophoretic mobility shift assays (EMSA).
Cigarette smoke inhalation procedure
The CS exposure was performed on 16 mice for 5 days per week over a period of 3 to 4 weeks using a standardized protocol, as described previously (3). Prior to the blast wave procedure, mice were allowed to recover for 1 week to avoid acute stress effects induced by the CS procedure per se.
Implementation of general anesthesia, blast wave, and surgery
All animals received a blunt chest trauma and were grouped according to wild type (WT) and CSE−/− with (CS) or without (nonCS) CS exposure. Prior to chest trauma mice C57BL/6J (nonCS, CS: n = 8 each) and CSE−/− mice (nonCS, CS: n = 8 each) were anesthetized with sevoflurane (2.5%; Sevorane, Abbott, Wiesbaden, HE, Germany) and buprenorphine (1.5 μg·g−1; Temgesic, Reckitt Benckiser, Slough, UK), as described previously (3). Blunt chest trauma was induced by a single blast wave positioned on the middle of the thorax, as described previously (2). Briefly, a Mylar polyester film (Du Pont de Nemur, Bad Homburg, Germany) was rapidly ruptured by compressed air, thereby releasing a reproducible single blast wave to the murine midsternal chest to reproducibly produce a lung contusion without serious organ damage. Immediately afterwards, mice received ketamine (120 μg·g−1; Ketanest-S, Pfizer, New York City, NY), midazolam (1.25 μg.g−1; Midazolam-ratiopharm, Ratiopharm, Ulm, BW, Germany) and fentanyl (0.25 μg.g−1; Fentanyl-hameln, Hameln Pharma Plus GmbH, Hameln, NI, Germany), and were placed on a procedure bench incorporating a closed-loop-system for body temperature control (2, 8). Lung-protective mechanical ventilation using a small animal ventilator (FlexiVent, Scireq, MO, Canada) was performed via a tracheostomy (3, 8). Surgical instrumentation comprised catheters in the jugular vein, the carotid artery, and the bladder (3). General anesthesia was titrated to guarantee complete tolerance against noxious stimuli and was sustained by continuous intravenous administration of ketamine, midazolam, and fentanyl to reach deep sedation, fluid resuscitation comprised hydroxyethyl starch 6% (Tetraspan, Braun Medical, Melsungen, HE, Germany) (3). Animals were mechanically ventilated with a nearly identical respiratory strategy as in our previous study investigating a combination of CS exposure and blunt chest trauma-induced ALI (3), i.e., using a pressure-controlled mode with initial ventilator settings being FiO2 = 0.21, respiratory rate 150 min−1, tidal volume 6 μL·g−1, inspiratory/expiratory time ratio 1:2, and positive end-expiratory pressure (PEEP) 3 cmH2O (3). The respiratory rate was modified to maintain an arterial PaCO2 between 30 mm Hg and 40 mm Hg, and the PEEP was varied according to the arterial PaO2 (if PaO2/FiO2 < 300 mm Hg: PEEP = 5 cmH2O; if PaO2/FiO2 < 200 mm Hg: PEEP = 8 cmH2O) (3). The recruitment manoeuvers (5 s hold at 18 cmH2O) were repeated hourly to avoid an impairment of thoracopulmonary compliance due to anesthesia- and/or supine position-induced atelectasis. At the end of the experiment the animals were exsanguinated, blood and lung tissue were taken immediately thereafter, and prepared for further analyses (3). All lung tissue was utilized due to organ size, and to be more exact the left lung was harvested for IHC, whereas the right lung served for immunoblotting, EMSA, and cytokine and chemokine evaluation.
Parameters of lung mechanics, hemodynamics, gas exchange, and metabolism
Systemic hemodynamics, body temperature, and static thoracopulmonary compliance were recorded hourly, blood gas tensions, acid-base status, glycemia, and lactatemia were assessed at the end of the 4 h period of mechanical ventilation (3).
Cytokine and chemokine concentrations
As described previously (2, 3, 8), lung tissue and plasma cytokine and chemokine levels were determined by using a mouse multiplex cytokine kit (Bio-Plex Pro Cytokine Assay, Bio-Rad, Hercules, Calif) according to the manufacturers’ instructions.
Single cell gel electrophoresis assay (comet assay)
The degree of DNA single strand breaks in whole blood was determined using the alkaline version of the comet assay (11), as described previously.
Immunoblotting and EMSA
Immunoblotting for anti-B-cell lymphoma-extra-large (Bcl-XL; Cell Signaling, Danvers, Mass), anti-heme oxygenase-1 (HO-1; Abcam, Cambridge, UK), anti-inducible isoform of the nitric oxide synthase (iNOS; Cell Signaling), antihypoxia-inducible factor 1-α (HIF-1α; Cell Signaling), and anti-NFκB inhibitor α (IκBα; Thermo Fisher Scientific, Waltham, Mass) was performed, as described previously (2, 3, 8). Primary antibodies were detected by using horseradish peroxidase-conjugated secondary antibodies (Cell Signaling or Santa Cruz, Dallas, Tex). Both anti-β-actin (Santa Cruz) and anti-vinculin (Santa Cruz) served as loading controls. Densitometry measurements were performed using NIH Image J software (http://rsb.info.nih.gov/nih-image), results are presented as densitometric sum relative to the two C57BL/6J control animals (2, 3).
The activation of the NFκB (32P-labeled double stranded oligonucleotide containing the NFκB (HIVκB-site) (5′-AGT TGA GGG GAC TTT CCC AGG C-3′, Biomers, Ulm, BW, Germany)) was determined using an EMSA (3, 8). The data are displayed as fold increase over the mean of two C57BL/6J control animals that did not undergo anesthesia, surgical instrumentation, nor trauma (3, 8).
Immunohistochemistry (IHC) for extravascular anti-albumin (Alb; Santa Cruz) accumulation, nitrotyrosine formation (antinitrotyrosine, Merck Millipore, Darmstadt, BW, Germany), and the expression of anti-angiopoietin-1 (Ang-1; R&D systems, Minneapolis, Minn), antivascular endothelial growth factor (VEGF; Abcam, Cambridge, UK), anticleaved caspase-3 (Cell Signaling) and anticystathionine-β-synthase (CBS; Santa Cruz) was performed as described previously (3). Primary antibodies were detected by secondary antibodies conjugated to AP (Alkaline Phosphatase-conjugated antibody; Jackson, ImmunoResearch, West Grove, Pa) and visualized with a red chromogen (Darko REAL Detection System Chromogen Red), and Mayers hematoxylin (Sigma, Taufkirchen, Germany). Visualization was performed using the Zeiss Axio Imager A1 microscope (Zeiss, Jena, TH, Germany). Four distinct 800,000 μm2 regions were quantified for intensity of signal by using the Axio Vision 4.8 software. Results are presented as densitometric sum (2, 3).
Histological analyses were independently performed by two experienced pathologists (AS and PM) blinded for group assignment. Similar to a previous report (3), analyzed criteria comprised dystelectasis, thickening of alveolar membranes, emphysematous overdistension, and inflammatory cell (macrophages and lymphocytes) infiltration (12). Macrophages and lymphocytes were included into our scoring system, as our pathologists, after taking into account distinct staining patterns of PMNs, were only able to identify a very small amount of PMNs. We did not consider additional stainings as necessary, because enzyme histochemical analyses are only feasible in unfixed tissue. Our findings are in line with a previous study revealing mice to have fewer circulating neutrophils (10%–25%) compared with human (50%–70%) (12). That study further highlights the difficulty to distinguish between granulocytes and mononuclear round cells in murine lung tissue. The analyzed parameters were scored from 0 (absent), 1 (hardly detectable), 2 (rare), 3 (minor), 4 (moderate), to 5 (extensive); macrophages were counted and scored as number per high power field. These scores were summed up to a total histopathology score. Typical examples from each group of the histological items analyzed are presented in Figure 1.
Unless stated otherwise, all data are presented as median (quartiles). The sample sizes were based on our previous experiments (2, 3): a statistical power analysis using the Horovitz-index (PaO2/FiO2 ratio), thoracopulmonary compliance, and lung tissue NFκB activation as main criteria were performed, and based on two-sided testing, α = 0.05, power 80%, and non-parametric analysis of variance, which had yielded a minimum of n = 8 to 10 for eight experimental groups. We first excluded normal distribution by using the Kolmogorov–Smirnov test and analyzed intergroup differences using the Kruskal–Wallis one-way ANOVA on ranks test and subsequently the Dunn test for two-tailed multiple comparison. Gender-dependent differences were calculated by using the Mann–Whitney test. The significance level was set to P < 0.05. Quantitative graphical presentations and statistical analyses were accomplished by using GraphPad Prism 5 (GraphPad Software Inc, La Jolla, Calif).
Effects of CSE knockout alone on trauma response: Table 1 shows that CSE−/− nonCS mice had higher heart rate and static thoracopulmonary compliance and lower blood glucose levels as compared with WT nonCS, whereas blood gas tensions and lactate levels were comparable in the two groups. CSE knockout increased local and systemic proinflammatory chemokine and cytokine concentrations (Table 2), and the tail moment in the alkaline comet assay of whole blood samples (Fig. 2). Figures 3 and 4 demonstrate in lung tissue homogenates that in comparison to WT nonCS, CSE−/− nonCS mice had increased HO-1, and decreased HIF-1α and IκBα formation, while iNOS and Bcl-XL expression remained unaffected. NFκB activation showed the opposite response to IκBα formation (Fig. 4). Immunohistochemistry (Fig. 5) revealed that knocking out CSE alone did not significantly affect albumin extravasation, nitrotyrosine formation, and the expression of Ang-1, VEGF, cleaved caspase-3, and CBS. The quantitative analyses of the histopathological evaluation are presented in Table 3: CSE−/− was associated with significantly more posttraumatic dystelectasis and alveolar inflammatory cell infiltration.
Effects of CSE knockout following CS exposure: CSE−/− mice had the highest values for heart rate, blood pressure, diuresis, and static thoracopulmonary compliance values and both the lowest levels of lactatemia and glycemia; metabolic acidosis was less severe than in their WT normal counterparts (Table 1). With respect to hemodynamic parameters, male CSE−/− mice revealed a significant increase of MAP compared with the female gender, and in line an increase in urinary excretion and anesthesia rate (Supplement data Table 1, https://links.lww.com/SHK/A471). Table 2 shows that CSE−/− CS mice had the overall highest KC, plasma MCP-1, and lung tissue IL-18 levels. The inflammatory response patterns were particularly increased in the male animals (Supplement data Table 2–3, https://links.lww.com/SHK/A472 and https://links.lww.com/SHK/A473). Figure 2 reveals that CSE knockout was not associated with further changes of the tail moment in the alkaline comet assay of whole blood samples when compared with the WT CS mice. Figures 3 and 4 show that CSE−/− CS had unchanged levels of HO-1, iNOS, NFκB expression, and IκBα activation comparable to respective levels in WT CS mice, but decreased Bcl-XL and HIF-1α formation in lung tissue. Similar to the nonCS groups, Figure 5 demonstrates that CSE−/− combined with CS exposure neither affected tissue albumin extravasation, cleaved caspase-3, CBS, nitrotyrosine formation nor the expression of VEGF when compared with the WT CS mice. However, the least expression of Ang-1 was found in the CSE−/− CS group. While CS exposure in WT mice was associated with significantly more pronounced posttraumatic thickening of the alveolar membranes, dystelectasis and inflammatory cell accumulation, pretraumatic CS exposure of CSE−/− mice caused about the same changes except for alveolar membrane thickening, which was attenuated compared with WT animals (Table 3). In contrast to the physiological and parameters and the cytokine and chemokine levels, neither tail moment, inflammatory protein expression nor histopathological evaluation showed any major gender-related effect (Supplement data Table 4–7, https://links.lww.com/SHK/A474, https://links.lww.com/SHK/A475, https://links.lww.com/SHK/A476, and https://links.lww.com/SHK/A477).
In this study, we tested the hypothesis whether genetic CSE knockout would exacerbate pulmonary dysfunction caused by blunt chest trauma-induced ALI in CS-exposed mice. The main findings were that CSE knockout alone substantially increased local and systemic cytokine and chemokine release, aggravated posttraumatic histological damage, and ultimately increased lung compliance, most likely reflecting lung hyperinflation. We also confirmed previous data that pretraumatic CS exposure alone increased posttraumatic local and systemic inflammation and thereby pulmonary histological damage, also leading to higher lung compliance documenting emphysematous overdistension. Finally, after pretraumatic CS exposure, CSE knockout further enhanced both the posttraumatic hyperinflammatory response and the impairment of lung mechanics, while pulmonary histological damage was not significantly worsened. Hence, maintenance of CSE expression is critically important not only for adaptive response to ALI per se, but also for CS-induced COPD.
Posttraumatic CSE−/− effects without CS exposure
CSE−/− mice showed a more pronounced posttraumatic local and systemic inflammatory response, which coincided with more severe dystelectasis and higher static thoracopulmonary compliance (Tables 1–3). Hence, even without pretraumatic CS exposure, CSE−/− mice revealed an immunological, histological, and physiological profile similar to WT mice with pretraumatic CS exposure. Our findings are in good agreement with the protective effects of well-maintained CSE expression in models of COPD and/or asthma (5), in which the otherwise pronounced pulmonary hyperinflammation was attenuated. Despite the comparable histological evidence of emphysematous lung overdistension in our experiments, the higher pulmonary compliance in the CSE−/− mice also confirms the following conclusion: COPD is associated with a hyperinflation-induced increase of the static compliance, even when total lung capacity is still normal (10). Our findings further suggest the crucial role of CSE activity during ALI. This is, however, in contrast to previous reports demonstrating that its deletion rather than maintaining its activity protected against pancreatitis-induced ALI (13) and acute liver failure (14). These previous studies, however, investigated CSE−/− mice that did not receive standard intensive care treatment: in fact, in our resuscitated murine model of blunt chest trauma investigating animals undergoing lung-protective mechanical ventilation (2, 3), higher CSE expression coincided with attenuation of ALI, suggesting that CSE is crucially involved in the posttraumatic adaptive stress response, in analogy to its pivotal role in preserving organ function during acute kidney injury (9). The importance of CSE expression for the adaptive stress response is further supported by the significantly higher tail moment in the blood cell comet assay (Fig. 2), a marker of oxidative stress, and the higher lung tissue HO-1 expression in the CSE−/− mice when compared with their WT counterparts, which, declined upon pretraumatic CS exposure (Fig. 3): Murine ALI induced by blunt chest trauma or sepsis resulting from cecal-ligation-and-puncture was associated with increased pulmonary HO-1 expression, and in these models, inhalation of exogenous H2S attenuated this effect (2, 8). Finally, the rational that CSE is crucial for the posttraumatic adaptive stress response is also supported by our findings that CSE knockout reduced IκBα expression and increased NFκB activation when compared with the WT animals (Fig. 4). In contrast, it markedly decreased HIF-1α expression (Fig. 3). In fact, HIF-1α and NFκB are referred to as “two extremes in the oxygen related gene expression” (15).
At the end of the 4 h posttraumatic observation period CSE−/− mice had lower glycemia values (Table 1). This observation fits with previous reports: CSE knockout was associated with (at least partial) inhibition of glycogenolysis and gluconeogenesis (16). Moreover, in mice, gluconeogenesis is suppressed by sepsis (17), and endotoxin-induced (18) hyperinflammation; in other words, the rate of gluconeogenesis is a marker of liver metabolic activity (11). In addition to its role in the lungs, our findings, hence, suggest that constitutive CSE expression is also crucial to maintain the hepatic metabolic capacity.
Posttraumatic CSE−/− effects following CS exposure
In good agreement with our previous study (3), pretraumatic CS exposure per se was not associated with a major posttraumatic increase of lung tissue and systemic pro-inflammatory cytokine and chemokine release, whereas both nitrotyrosine formation and histological evidence of inflammatory cell infiltration was enhanced, and HIF-1α expression decreased. Overall, CSE knockout did not further modify this response, albeit CSE−/− mice with pre-traumatic CS exposure exhibited the highest posttraumatic KC, MCP-1, and lung tissue IL-18 levels (Table 2).
All experimental groups comprised identical numbers of male and female mice, i.e., n = 4 of each per group. Interestingly, despite this small number of individuals, the blood and lung tissue cytokine and chemokine response was more pronounced in male CSE−/− mice after CS exposure (Supplement data Table 2–3, https://links.lww.com/SHK/A472 and https://links.lww.com/SHK/A473). While it is well established that estrogens can per se markedly improve outcome after trauma and hemorrhage by modulating the immune function (19), we can only speculate why the attenuated inflammatory response in female mice was only present in the CSE−/− mice after CS exposure. However, Zhu et al. (20) demonstrated that estrogen injection increased myocardial CSE expression in ovariectomized rats, which was associated with decreased oxidative stress and pro-inflammatory cytokine release.
Strikingly, pretraumatic CS exposure in CSE−/− mice was associated with an attenuation rather than an aggravation of alveolar membrane thickening when compared with CS-exposed WT animals (Table 3). We can only speculate on this observation: there was no histopathological evidence of further increased emphysematous overdistension, but CSE−/− mice with pretraumatic CS exposure had the highest static thoracopulmonary compliance, i.e., signs of lung hyperinflation. It is well established that evaluation of alveolar septal thickening highly depends on the degree and uniformity of pulmonary inflation (12).
In WT mice, pretraumatic CS exposure was associated with increased extravascular lung albumin staining, suggesting aggravated alveolar endothelial barrier dysfunction (21) (Fig. 5). Interestingly, this was not reflected in the response of tissue VEGF, but in fact, for Ang-1 expression. The latter is referred to as a marker of tight rather than damaged endothelial barrier (21). Notably, the lowest Ang-1 expression of all experimental groups was found in the CSE−/− mice with pretraumatic CS exposure. This observation now also suggests a role for endogenous H2S in maintaining a tight endothelial barrier: exogenous administration of the H2S-releasing salt Na2S was already shown to attenuate the particular matter-induced barrier disruption of pulmonary endothelial cells in vitro(22). CSE−/− mice with pretraumatic CS exposure also showed the least posttraumatic lung tissue VEGF expression of all groups, which may represent further support of the role of CSE for barrier (dys)function during ALI with underlying chronic lung disease, i.e., an acute “second hit” in addition to CS exposure: in the bronchoalveolar lavage fluid of smokers challenged with endotoxin inhalation, the increased levels of biomarkers of lung hyperinflammation and barrier disruption coincided with the lowest VEGF content (23). Finally, the marked posttraumatic depression of VEGF expression in CS-exposed CSE−/− mice might be related to the well-established role of H2S as an oxygen sensor (24): Clearly, exogenous H2S decreased HIF-1α and VEGF accumulation under hypoxic conditions (25), but after ischemia exogenous H2S enhanced tissue VEGF and HIF-1α (26).
Pretraumatic CS exposure in CSE−/− mice went along with the highest values for heart rate and blood pressure (Table 1). It is well established that CSE knockout leads to arterial hypertension (27); however, it did not affect heart rate in otherwise healthy mice (28). CS exposure alone without any additional acute challenge did not affect heart rate in mice either (29), but increased overall sympathetic activity (30). Hence, the posttraumatic hemodynamic response in the CS-exposed CSE−/− mice was presumably due to an addition of the two former effects, which in turn, also resulted in the significantly higher needs for anesthesia drugs and diuresis when compared with the other experimental groups. Again, this effect was more pronounced in the male mice, which is well in line with the abolished estrogen-induced inhibition of vascular smooth muscle cell proliferation in CSE−/− mice (Supplement data Table 1, https://links.lww.com/SHK/A471) (31). It is tempting to speculate that this hyperdynamic circulation and the subsequent effect on urine output caused less metabolic acidosis when compared with the WT animals, and the lowest levels of lactatemia of all experimental groups: in all groups, lactatemia was in the normal range, and, moreover, the level of lactatemia did not account for changes in the anion gap responsible for the metabolic acidosis; in other words, the latter was due to other fixed acids than lactic acid.
CS-exposed CSE−/− mice also had the lowest blood glucose levels of all experimental groups (Table 1). Several mechanisms may be responsible for this observation: increased sympathetic activity and the more pronounced hyperdynamic circulation may have caused increased glucose turnover (32); a higher degree of systemic hyperinflammation as suggested by the cytokine and chemokine plasma levels may have caused enhanced glycolytic activity (33); increased glucose oxidation as described for patients with COPD (34); right heart dysfunction due to increased right ventricular afterload induced by lung hyperinflation may have impaired liver metabolic activity (35), ultimately decreasing gluconeogenesis. Glycemia values alone did not allow differentiating these phenomena, which would have only been possible using more detailed quantitative analyses of the various glucose pathways.
Limitations of the study
Clearly, we cannot provide direct proof that the effects of CS exposure and CSE knockout are interrelated, since no “rescue therapy data” with exogenous H2S administration in CSE−/− mice is available. Moreover, due to lacking measurements of H2S formation, we cannot directly relate the observed effects of CSE knockout to its function for H2S production. It should be noted in this context, however, that so far no gold standard is available for the measurement of H2S concentrations, the values reported in the literature varying by two to three orders of magnitude (9), and that we and others demonstrated that there is no direct relation between measured H2S concentrations and the biological effect observed (9). Finally, the pattern of the lung tissue expression of the (HO-1, iNOS, Bcl-XL, HIF-1α, IκBα, and NFκB) was less consistent than the response of the blood and tissue cytokine and chemokines. Hence, both CS exposure and CSE knockout may have differential effects on various mediator proteins studied.
In the present study, we tested the hypothesis whether the genetically engineered CSE knockout would aggravate pulmonary dysfunction after blunt chest trauma-induced ALI in CS-exposed mice. Mice lacking the CSE gene, in particular the male gender, revealed substantially more pronounced local and systemic cytokine and chemokine release, ultimately leading to aggravated posttraumatic histological damage and deterioration of lung mechanics. The effects of CSE knockout were quantitatively comparable to those of pretraumatic CS exposure alone, and the combination of CSE knockout and pretraumatic CS exposure further enhanced both the posttraumatic hyperinflammatory response and the impairment of lung mechanics. Taken together, in this model of acute-on-chronic pulmonary injury, the presented data suggests that CSE expression is important for the adaptive response to ALI, in particular in CS-induced COPD.
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