Smoke inhalation injury (SII) is associated with a mortality of approximately 30% and is characterized by airway inflammation mediated by the chemical irritants in smoke, which often leads to acute respiratory distress syndrome (ARDS) (1–5). An estimated 20% to 30% of patients affected by burn injuries also suffer SII, which is one of the major risk factors associated with high mortality in burn victims (2, 3).
In the pathogenesis of ARDS, the inflammation in the airway causes the migration of neutrophils and monocytes into the lung followed by the release of cytotoxic enzymes, reactive oxygen species (ROS), and other oxidative agents (6–8). The accumulation of these toxic substances is an important contributor to the damage to the airway epithelium and endothelium of the lung parenchyma. As a consequence, gas exchange by the injured alveoli is impaired, leading to hypoxemia and hypercapnia (6, 8, 9).
Club cell protein 10-kDa (CC10) is the major protein secreted by Club cells. These CC10-expressing cells have been shown to play a critical role in maintaining the integrity of the airway epithelium and in facilitating epithelial repair (10). CC10 is a potent anti-inflammatory protein with multiple mechanisms of action, including inhibition of phospholipase A2 (PLA2) (11–13), inhibition of neutrophil chemotaxis (14, 15), and suppression of NF-κB signaling (16, 17).
A decrease in native CC10 protein has been found in broncho-alveolar lavage fluid in ARDS (14, 18, 19), while chronic respiratory conditions, such as asthma (20), chronic obstructive pulmonary disease (COPD) (21, 22), and cigarette smoking-induced bronchial dysplasia (23) result in a decrease in circulating CC10. CC10 is also deficient in respiratory distress of prematurity, leading to development of neonatal bronchopulmonary dysplasia (BPD) in severely preterm infants (24, 25). Decreased circulating CC10 has been proposed as a biomarker for some of these inflammation-associated pathologies and is suggested to play a role in the pathophysiology of these conditions (19, 20).
The efficacy of recombinant human CC10 (rhCC10) has been evaluated in several animal models of acute lung injury (ALI), in which reduction in pulmonary inflammatory mediators, protection and preservation of pulmonary architecture, mechanical lung function, gas exchange, and pulmonary vascular barrier have been observed (26–31). Importantly, rhCC10 has been shown to decrease airway inflammation in preterm neonates with respiratory distress (32).
ROS are generated during acute and severe inflammatory responses in ALI and respiratory distress (7), including SII. CC10 is oxidized in tracheal aspirates of preterm infants experiencing respiratory distress and the presence of oxidatively modified CC10 isoforms correlates with greater incidence of death or BPD (24, 33).
In the present study, we assessed dose effects of rhCC10 on lung function in a well-characterized and clinically relevant ovine model of ARDS induced by smoke inhalation. Specifically, the potential of rhCC10 protein to control the inflammatory response associated with high morbidity and mortality following smoke inhalation and to preserve pulmonary function was evaluated. In contrast to previously published studies, the type of ALI is very different, having been induced by traumatic smoke inhalation in otherwise fully developed healthy adult lungs in the present study.
Thirty-six adult female sheep (30 kg–40 kg; merino; ∼3 years) were surgically prepared as indicated (Supplement (SDC1, http://links.lww.com/SHK/A876)). On the day of the study, following the collection of baselines for hemodynamics and blood samples, smoke inhalation injury was induced under deep anesthesia and pain control as previously shown (34). Injury consisted of insufflation of 48 breaths of cool cotton smoke. Following injury, animals were awakened, placed on a mechanical ventilator (Servo Ventilator 900C; Siemens, Elema, Sweden), and randomized into one of four groups, including the Control group (injured and treated with 60 mL of normal saline every 12 h, n = 7), CC10-1 group (injured and treated with rhCC10 at a dose of 1 mg/kg/d (0.5 mg/kg every 12 h), n = 8), CC10-3 group (injured and treated with rhCC10 at a dose of 3 mg/kg/d (1.5 mg/kg every 12 h), n = 7) and CC10-10 group (injured and treated with rhCC10 at a dose of 10 mg/kg/d (5 mg/kg every 12 h), n = 8). CC10 doses were selected based on efficacy observed in different models of ALI (26, 27, 30). Sham animals (n = 6) were instrumented but received sham injury and saline as treatment. All doses of rhCC10 or saline were administered as intravenous infusion via the central venous catheter every 12 h starting 1 h after injury (administered at 1, 13, 25, and 37 h postinjury). Treatment was started at 1 h postinjury because it is sufficient time for the injury to be established and because it was thought to be a reasonable time to intervention for human smoke exposures. Determination of the duration of the therapeutic intervention window after 1 h was beyond the scope of this initial study. rhCC10 was provided by Therabron Therapeutics, Inc. (Rockville, Md). Hemodynamics, pulmonary function, and blood gases were recorded every 6 h. Urine and plasma samples were collected every 6 h. Animals were monitored for 48 h postinjury, then sacrificed, and lungs harvested as previously described (35). Criteria for euthanasia prior to 48-h endpoint were PaO2 < 50 mm Hg or PaCO2 > 90 mm Hg for 1 h regardless of adjustments of FiO2, respiratory rate, and pulmonary toilet. Euthanasia was done by chemical cardiac arrest under deep anesthesia by IV administration of Xylazine 0.2 mg/kg and ketamine 10 mg/kg to 15 mg/kg, followed by 1 mL/kg saturated KCl solution. All the experimental procedures were approved by the UTMB IACUC and by the USAMRMC ACURO prior to initiation.
Fluid resuscitation, assessment of fluid balance, and plasma protein
After the injury, the animals were resuscitated with Ringer lactate using the formula (4 mL × 40 × kg × day, with half of the daily fluid requirement delivered in the first 8 h). The majority of patients suffering from smoke inhalation injury are affected by large cutaneous burn injuries; therefore, we provided a fluid plan close to what a burn patient receives. To accurately monitor fluid balance during the study, the sheep had free access to food, but water was restricted. Urinary output was assessed with a urinary bladder catheter (Foley Catheter, 14Fr; BARDEX, Covington, Ga). Total protein concentration in fresh plasma was evaluated using a refractometer (National Instrument, Baltimore, Md), as it has shown to correlate well with colloid osmotic pressure (35).
Please refer to Online Supplement (SDC1, http://links.lww.com/SHK/A876) for details regarding animal preparation, cardiopulmonary management and analysis, plasma and lung tissue assessment, and including CC10 gene expression.
Calculations were performed using SPSS Version 19.0 (SPSS Inc, Chicago, Ill) and GraphPad Prism version 6 (GraphPad Software, San Diego, Calif). Sampling distribution of the collected data was assessed by the Shapiro–Wilk normality test. Analysis of the treatment effect of one parameter over time was evaluated using Linear Mixed Model, and adjusted pairwise comparisons by Fisher test were used to determine which of the treatments had a different effect. Comparison between treatment groups at different time points was performed using a two-way analysis of variance, including group and time as independent variables, followed by adjusted pairwise comparison. Sets of data for a single time point; such as lung tissue samples, were analyzed using one-way analysis of variance followed by adjusted pairwise comparison. The survival time and mortality among groups was evaluated with log-rank test adjusted for multiple comparisons, and Fisher exact test, respectively. Correlation coefficient between two non-normally distributed variables was assessed by Spearman rank test. Values reported are expressed as mean ± SEM. The differences were considered significant when the P value was smaller than 0.05.
The degree of injury was comparable among injured groups as indicated by the COHb levels following smoke inhalation, the percent of bronchial exfoliation, and the development of hypoxemia
Refer to Online Supplement (SDC1, http://links.lww.com/SHK/A876) for details.
Effect of rhCC10 on 48-h survival time
All animals survived the first 24 h. The 48 h survival was five out of seven in the Control group (71.4%), six out of eight in the CC10-1 group (75%), seven out of seven in the CC10-3 group (100%), seven out of eight in the CC10-10 group (87.5%), and six out of six in Sham group (100%) (Fig. 1). The small sample size in our experiment failed to demonstrate a significant difference in 48-h survival using a log-rank survival analysis, although both CC10-3 and CC10-10 groups exhibited an trend in increased survival versus the Control group.
Deterioration of gas exchange was attenuated with the highest doses of rhCC10
The decrease of PaO2/FiO2 ratio below the ARDS reference value of 300 mm Hg was evident in all SII-induced animals. The decline of this parameter was significantly attenuated in the CC10-10 treatment group, showing the equivalent of mild ARDS (between 200 mm Hg and 300 mm Hg) by the end of the study compared with the CC10-1 (P = 0.007) and Control (P = 0.02) groups that exhibited a mean PaO2/FiO2 ratio equivalent to moderate ARDS (between 100 mm Hg and 200 mm Hg) (Fig. 2A).
The oxygenation index (OI) was significantly higher in all injured groups compared with the Sham group. However, the increase in OI was significantly attenuated in the CC10-3 and CC10–10 groups (Fig. 2B). Likewise, the pulmonary shunt fraction (Qs/Qt) was increased in all injured groups versus the Sham group, although no difference in Qs/Qt was observed between the treatment groups and the Control group (Fig. 2C).
High-dose rhCC10 attenuated the increase in airway pressure and airway obstruction
The peak inspiratory pressure (PIP) was significantly increased in all the injured groups versus the Sham group. Treatment with 10 mg/kg/d rhCC10 showed a significant decrease in PIP versus Control; likewise, a decreasing trend (P = 0.07) in PIP was observed in the CC10–3 group (Fig. 3A). Lung compliance was also significantly decreased in all injured groups versus the Sham group and the CC10–1 group demonstrated an attenuation versus the Control group (Fig. 3B). The bronchial obstruction score (determined by histopathology analysis) showed that the large airways were obstructed in all the injured groups versus the Sham group. However, the increase in bronchial obstruction was significantly decreased in the CC10–10 group versus the Control group (Fig. 3C). Accordingly, the bronchiolar obstruction score showed that the small airways were significantly obstructed in the Control and CC10–1 groups versus the Sham group. Though in the CC10–10 group, the bronchiolar obstruction was significantly reduced versus the Control group and had no significant difference versus the Sham group (Fig. 3D). Spearman correlation demonstrated a significant correlation between the last PIP before euthanasia and the bronchial obstruction score (r = 0.6, P<0.0001) as well as between the last recording of lung compliance and the bronchiolar obstruction score (r = −0.68, P<0.0001).
rhCC10 decreased systemic vascular hyperpermeability to proteins and water
Statistical analysis indicated that the Control group had an increase in fluid accumulation (determined by the balance of fluid input and urinary output) during the 48 h postinjury time, indicating fluid retention. However, fluid accumulation was statistically lower in the CC10–1, CC10–3, and CC10–10 treatment groups versus the Control group. Fluid balance at 48 h in surviving animals was 2.2 ± 0.4, 1.4 ± 0.4, 1.7 ± 0.5, and 1.1 ± 0.4 L in the Control, CC10–1, CC10–3, and CC10–10 groups, respectively. By comparison, the Sham group had a fluid balance of 0.7 ± 0.3 L (Fig. 4A). Similarly, the Control and the CC10–1 groups had a decrease in plasma protein concentration versus the Sham group. At 48 h compared with baseline, the plasma protein was reduced by 13.7 ± 0.4% in the Control group and 17 ± 1%, 5.3 ± 3.6%, 2.3 ± 4.3% in the CC10–1, CC10–3, and CC10–10 groups, respectively. By comparison, the Sham group had a 2 ± 1.1% reduction in plasma protein levels at the 48-h time-point (Fig. 4B). The hemoglobin level at 48 h was similar in all five groups demonstrating that the fluid resuscitation was comparable among groups.
rhCC10 reduced pulmonary edema
Alveolar edema determined by histopathology analysis indicated that the leakage of plasma content was increased in the Control and CC10–1 group versus the Sham group. The CC10–3 and CC10–10 groups were statistically similar to the Sham group and statistically lower than the Control group (Fig. 4C). Likewise, the pulmonary hemorrhage score was significantly reduced in all CC10 treatment groups compared with the Control group (Fig. 4D).
rhCC10 reduced neutrophil infiltration, myeloperoxidase (MPO) activity, and oxidative stress in lung tissue
Histopathology analysis of lung tissue indicated that neutrophil accumulation in the lungs was elevated in all injured groups compared with Sham, but was significantly reduced in all rhCC10-treated groups compared with the Control group (Fig. 5A). In lung tissue homogenate, the levels of MPO activity in the Control group increased versus the Sham group (3.2 ± 0.7-fold), and this increase was significantly attenuated in the CC10–3 and CC10–10 groups (Fig. 5B). Generation of ROS by MPO results in protein oxidation that can be assessed by measuring the protein carbonyl content in proteins recovered from lung tissue. The CC10–1, CC10–3, and CC10–10 treatment groups showed a dose-dependent decrease in protein carbonyl concentration compared with the Control group (Fig. 5C). Representative images of lung tissue for the Sham, Control, and CC10–10 are shown in Figure 5D.
In vitro ROS-mediated modification of rhCC10
ROS-mediated modification of rhCC10 in vivo can be simulated in vitro using MPO + H2O2, sodium hypochlorite (NaOCl), and meta-chloroperbenzoic acid (mCPBA) and reaction progress can be monitored using reverse phase high performance liquid chromatography (see Supplemental Digital Content for detailed reaction conditions, analytical methods (SDC1, http://links.lww.com/SHK/A876), and figures (SDC2, http://links.lww.com/SHK/A877)). Isoelectric focusing confirmed the presence of CC10 isoforms in the reactions (SDC3, http://links.lww.com/SHK/A878). Unreacted rhCC10 shows a major band at pI = 4.8 with a minor band at 4.7. Multiple new bands were observed in ROS-reacted samples, including one new isoform at pI 5.5 that was generated in all three reactions and appears to be the same as the pI 5.5 native CC10 isoform found in infant tracheal aspirates as reported previously (24, 33).
CC10 pharmacokinetics and pulmonary gene expression
Total CC10 (combination of native ovine CC10 and rhCC10) was measured in plasma by competitive enzyme-linked immunosorbent assay (ELISA), as previously described (32). Measurement of CC10 concentration in plasma at 3 h postinjury (2 h after intravenous infusion) showed that the concentration of CC10 increased significantly in the CC10–3 and CC10–10 groups (24.4 and 66.1-folds respectively) compared with the Control groups. CC10–1 group had 4.7-fold increase without statistical significance (Fig. 6A). In lung tissue, the level of CC10 protein was significantly reduced in all injured groups versus the Sham group. The CC10 levels in lung in all rhCC10 treatment groups were not significantly higher than the Control group (Fig. 6C). Because the antihuman CC10 antibody used in the competitive ELISA is cross-reactive with ovine CC10 (Fig. 6D), these results reflect a combination of native ovine CC10 and rhCC10. Similar to CC10 protein levels, CC10 mRNA in lung was statistically reduced in the Control and CC10–1 groups versus the Sham group. In the CC10–3 and CC10–10 treatment groups, the CC10 gene expression was not different compared with the Sham group and was slightly higher than the Control group (Fig. 6B).
Smoke inhalation injury induces a severe, often lethal, lung injury characterized by severe pulmonary inflammation, epithelial exfoliation, airway obstruction, pulmonary hemorrhage, and pulmonary edema (1, 36). The ovine SII model was established not only to simulate pulmonary injury and dysfunction, but also to simulate clinical practice in critically ill patients experiencing acute lung injury and ARDS (34, 37).
The main finding in this study is that treatment with intravenously administered high-dose rhCC10 (given 1 h postinjury and every 12 h thereafter) reduced pulmonary inflammation, protected pulmonary architecture, improved gas exchange and lung biomechanics, and improved fluid balance. The improvement in lung function is consistent with previous findings in which intravenously delivered rhCC10 protected pulmonary architecture, reduced pulmonary inflammation, and improved lung function in a rabbit model of ARDS (27). As a PLA2 inhibitor, rhCC10 may also prevent the degradation of lung surfactant, thus preserving lung function (28, 30). As an inhibitor of NF-kB signaling in airway epithelial cells, rhCC10 may suppress the downstream inflammatory response in ALI (17), as observed in several other models of ALI in which rhCC10 was evaluated (26–31). Intratracheally administered rhCC10 has also been reported to significantly reduce pulmonary inflammation in premature infants with respiratory distress (32).
CC10, also known as secretoglobin 1A1, CCSP, CC16, uteroglobin, and urine protein-1, is the primary secretory product of non-ciliated respiratory epithelial cells lining the airways, including Club cells. Club cell subpopulations include progenitor stem cells in the airways that repopulate the epithelium following injury. In addition to its anti-inflammatory and anti-fibrotic properties (38, 39), the CC10 protein is thought to play a role in the repair of the respiratory epithelium postinjury (40). Administration of rhCC10 has been shown to facilitate repair of the pulmonary epithelium following naphthalene injury in a mouse model, specifically increasing the number of Club cells (41).
The histopathology assessment of large and small airway obstruction in the present study suggests that rhCC10 not only suppressed damaging inflammation in the lung, but also may have facilitated repair of the airway epithelium postinjury. Although the mechanistic aspects of CC10 are not completely understood, the overall improvement in lung biomechanics mediated by rhCC10 may be a result of the decreased inflammatory response and/or accelerated epithelial repair, resulting in decreases in epithelial sloughing, mucus obstruction, and bronchospasm. Whether due to decreased inflammation, accelerated epithelial repair, or a combination of the two, preservation of pulmonary architecture, including airway integrity, and lung compliance mediated by rhCC10 directly correlated with improved gas exchange. PaO2/FiO2 ratio, the main indicator of the gas exchange, improved with rhCC10 treatment in a dose-dependent manner. The improvement of this outcome by itself is particularly relevant, as it is primarily correlated with the mortality of SII-ARDS patients (2). The deterioration in OI was also attenuated by rhCC10 with the two higher CC10 doses. Together, these parameters integrate both gas exchange and lung biomechanics (35, 42), which contribute to the overall effect of high-dose rhCC10 in attenuating the severity of ARDS.
These improvements in lung function are consistent with the effects of rhCC10 in previous studies using various animal models of ALI (26, 27, 28, 30), particularly in a rabbit model of ARDS using intratracheal and intravenous routes of administration for rhCC10 (27), and in a preterm lamb model of infant respiratory distress syndrome which showed that intratracheal instillation of rhCC10, administered shortly after lung surfactant, resulted in improvement of lung compliance and PIP, compared with surfactant alone (26, 28).
Consistent with previous observations, rhCC10 treatment decreased neutrophil MPO in lung tissue, as well as the number of neutrophils in lung tissue (29, 30) in response to lung injury. In the absence of CC10, knockout mice exhibit elevated neutrophil responses to various respiratory pathogens and inhaled insults (31, 43–46). Thus, the property of native CC10 and rhCC10 to inhibit neutrophil infiltration in the lungs in ALI is well documented. rhCC10 treatment also decreased the number of neutrophils in the tracheal aspirate fluid in a small phase I clinical trial in premature infants that received a single intratracheal dose of rhCC10 (32), but did not appear to increase the incidence or severity of bacterial infection in these fragile patients (32). Since uterine infection is often a cause of premature birth, resulting in exposure of infant lungs to bacterial pathogens, these observations suggest that rhCC10 was relatively safe as used. However, the effect of rhCC10 treatment on PMN recruitment to the lungs in bacterial pneumonia in a non-knockout model has not been directly studied and it is not known whether rhCC10 could blunt the host response to a bacterial infection and increase mortality (47).
Carbonyl groups on proteins are generated through interactions with ROS, which are in turn generated by MPO plus H2O2 during ALI (48, 49). Therefore, the rhCC10-mediated dose-dependent decrease in protein carbonyl content observed in lung tissue is highly consistent with the decrease in neutrophils and MPO activity in lung tissue. It is also consistent with our observation that rhCC10 can react with oxygen radicals (see Supplement SDC1–3, http://links.lww.com/SHK/A876, http://links.lww.com/SHK/A877, http://links.lww.com/SHK/A878), rendering them inert, thereby revealing a novel anti-inflammatory and protective mechanism of rhCC10 as a scavenger of ROS. Lack of understanding of CC10's mechanism (s) of action has limited the resources applied to its development as an ALI therapeutic and this new information may help encourage new avenues of research.
Another compelling and novel observation was that the rhCC10 decreased systemic vascular leak of both fluid and protein. rhCC10 suppressed pulmonary vascular leak in previously reported models, and in human preterm infants, as measured by total protein in term amniotic fluid or brochioalveolar lavage, but none of those studies evaluated systemic vascular permeability, and it is unclear whether rhCC10 also affected systemic vascular permeability in previous studies (26, 27, 32, 50, 51). Sheep lung histopathology scores for alveolar edema and pulmonary hemorrhage assessed at 48 h demonstrated a rhCC10-mediated reduction in pulmonary edema and vascular leak in SII, which was significant in the high-dose rhCC10 group.
The pulmonary edema characteristic of ARDS is primarily attributed to the impairment of endothelial barrier integrity and only partially attributable to a defect in the epithelial barrier that maintains homeostasis in the alveoli (9); therefore, the rhCC10-mediated attenuation in systemic vascular leak and the attenuated alveolar edema likely share a common pathway in SII.
Multiple studies have also shown that CC10 is lower in the tracheal aspirates or broncho-alveolar lavage fluid of patients who died of respiratory failure compared with those who recover, including respiratory distress of prematurity (24), pneumonia (52), and ARDS (14). Other studies found that CC10 in plasma or serum increases during ALI (53–56). In contrast, decreases in circulating CC10 correlate with the loss of lung function and loss of Club cells with progressive airway remodeling in chronic lung diseases such as COPD and bronchiolitis obliterans syndrome (16, 57–61). The dose-dependent trend toward increased CC10 in lung tissue confirmed that rhCC10 can be delivered to the lung using intravenous administration in ALI, as previously observed in rabbits (27). Treatment with high doses of rhCC10 also attenuated the decrease in lung CC10 mRNA caused by SII, suggesting that the rhCC10 protected Club cells in the airway epithelium. It is not clear whether the elevated CC10 protein content in lung in the high-dose rhCC10 groups is due to the presence of ovine CC10 or human CC10, since the two could not be distinguished in our ELISA.
With respect to overall effects mediated by rhCC10, these results demonstrate that the optimal dose tested is 10 mg/kg/d for this type of lung injury. This dose conferred the greatest potential benefit in the measured outcomes, with no apparent adverse effects. Suboptimal doses of 1 mg/kg/d and 3 mg/kg/d also provided some benefit. A novel potential mechanism of action for rhCC10 was also identified; scavenging of ROS and augmenting other known mechanisms and activities such as PLA2 inhibition, inhibition of NF-κB signaling in airway epithelia, and decreasing pulmonary neutrophil influx; a potent and protective combination. Perhaps more significant, from a clinical management perspective, was the finding that high doses of rhCC10 normalized systemic fluid balance in SII. In summary, based on the current results and in correlation with previous findings, rhCC10 mediated a significant anti-inflammatory effect in the airways and reduced systemic vascular permeability which attenuated lung dysfunction and the severity of ARDS.
The authors thank Daniel and Lillian Traber for their contributions to the conception and design of this study.
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