Club cell protein (CC)16 as potential lung injury marker in a porcine 72 h polytrauma model

Despite achievements to reduce injury-related mortality and morbidity in the last decades, the number of polytraumatized patients admitted to hospitals remains high with approximately 4.8 million human deaths worldwide caused by traumatic injuries every year [1]. In polytraumatized patients blunt thoracic trauma is common, and around 20–25% of deaths in severely injured patients treated in emergency departments are caused by chest injuries [2]. Furthermore, thoracic trauma has been shown to be associated with increased posttraumatic inflammation compared to polytrauma patients who did not undergo thoracic trauma, increased rates of pneumonia and Acute Respiratory Distress Syndrome (ARDS) and lower survival rates of patients [3‐5]. It is of utmost importance for the medical community to detect changes in the lungs as early as possible to prevent acute lung injury (ALI).

Club cell protein (CC)16 is a 15.8 kDa homodimeric protein and the major protein secreted in airways by the non-ciliated bronchiolar Club cells [6, 7]. It occurs in very high concentrations in the epithelial lining where it appears to play an antioxidant and anti-inflammatory role [7]. Its role as a potential biomarker to detect pulmonary damage in polytraumatized patients or to identify polytraumatized patients at risk for secondary pulmonary complications has been extensively studied during the last decades. Wutzler et al. reported that in patients who have sustained multiple injuries, the early elevation of CC16 serum levels can be used to accurately identify patients with versus those without lung injury [8]. Furthermore, serum CC16-levels have been shown to correlate with the volume of lung contusion in polytraumatized patients [8], and were proposed to serve as a biomarker to monitor the progression of pulmonary contusion [9]. Negrin et al. investigated CC16 levels in polytraumatized patients with severe thoracic injury and reported that CC16 levels exceeding 30.51 ng/ml on day two after trauma may allow a firmer diagnosis for the development of pneumonia [10]. Another study reported a second peak in CC16 levels in patients developing pneumonia after polytrauma with severe thoracic trauma [11]. Xu et al. investigated ex vivo in vitro association of CC16 and polymorphonuclear leukocytes (PMNL) in polytraumatized patients with or without pneumonia and observed significant effects of CC16 on PMNL in patients with pneumonia [12]. The authors hypothesized that CC16 might reduce the migratory capacity of PMNL, and thus, modulate their function in patients with respiratory complications after trauma [12]. Several factors affecting circulating CC16 have been identified as, e.g. on the one hand chronic exposure to tobacco smoke has been reported to cause a decrease in CC16 levels [13], while on the other hand acute exposure of firefighters to fire smoke has been reported to increase CC16 serum levels [14]. Following the hypothesis that CC16 might indicate lung epithelial injury during polytrauma in a large animal model with high translational aspects, we investigated the biomarker character of CC16 in a standardized porcine trauma model consisting of lung contusion, liver laceration, tibial fracture, and hemorrhagic shock followed by fluid resuscitation and fracture fixation with external fixator.

Elevated systemic CC16 levels in conditions of acute as well as chronic lung damage have been shown before [8, 14, 21]. Nevertheless, there are still scientific debates about which markers are reliable, especially in multiple injuries, to assessing lung injury. Many of these markers were not specific enough or were subject to major changes in their expression levels regarding the overall damage, a disease state/pre-existing conditions, or the etiology and have been influenced by the time of measurement [22‐26].

Our results clearly indicated that CC16 has been significantly changed both locally in the lung tissue as well as systemically after polytrauma with thoracic injury. Interestingly, the systemic levels of CC16 increased very early after polytrauma. These findings are in direct agreement with data from Wutzler et al. and Lin et al. [11, 27]. In these studies, an early increase within the first hours after trauma was detected reaching baseline levels within 24 h again. In addition, Wutzler et al. have shown a second CC16 increase in sera obtained from traumatized patients who developed pulmonary complications [11]. The time course of the CC16 increase and the renewed decrease to baseline level within 24 h was reproduced in the underlying polytrauma model. Due to multiple sampling time points of plasma before and directly after trauma induction, resulting in a higher temporal resolution, it has been shown that the concentrations’ peak was around 3 h after trauma with baseline values being reached again after 24 h. Whether this was dependent on the trauma severity, or the intensity of lung damage remains to be investigated in further studies. The secondary increase of CC16 serum levels in clinically later occurring respiratory problems has been described by Wutzler et al., and was not reproduced in our 72 h model, but is also might not be expected within this short ventilation period in our model [11]. Prolonged observational time points are necessary to validate the clinically observed relevance of CC16 as a potential indicator for the secondary lung complications after trauma.

Another interesting effect regarding the different concentrations of CC16 over time in the different compartments might have occurred due to a short-term high, but then decreasing and persisting release of CC16. While CC16 increased very quickly systemically and decreased after a short peak, CC16 was only significantly increased in the BAL at 72 h after polytrauma. Furthermore, CC16 was present in lower concentrations in the lung of polytraumatized animals after 72 h compared to sham animals. These differences may have occurred due to an initial storage dependent release, which was possibly followed by a synthesis and expression dependent release.

Lung damage or respiratory complications are often driven by inflammatory changes involving the proinflammatory cytokine IL-8. The above described significantly increased values of IL-8 in the BAL obtained from polytraumatized animals have been associated with the loss of pulmonary barrier function, and a CC16 increase found in polytraumatized animals. In parts this has been confirmed by Reynolds et al. who demonstrated in their mouse model over-expressing transgenic human IL-8 that the prolonged presence of high IL-8 concentrations, as observed in trauma, exerted a damaging effect to the lung [28]. The authors demonstrated modifications in the context of long-term structural damage, with damaged and leaky epithelial tight junctions. Yang et al. showed that this might have been based on surfactant A and B expression depending on IL-8 occurrence [29]. In addition, Lin et al. have demonstrated that CC16 levels at ICU admission directly correlated with the severity of ARDS, but they only provided limited information for prognostic purposes [27]. In the present study, lung injury has been confirmed by increased total protein content in the BAL, which indicated functional disruption of the lung barrier. This is in line with the findings of Herrero et al. who found a direct correlation between ZO-1, loss of barrier function and edema formation in their mouse model [30]. In our study, sham animals have shown significantly higher values of membrane-bound ZO-1, which represented a more intact and functional lung barrier here. Taken together, our findings indicate that CC16 may constitute as an early biomarker for the pulmonary barrier loss that is associated with increased lung injury in this large animal polytrauma model, thus suggesting that this model reflected the pathophysiological modulations that have been observed in the human situation of polytrauma. Furthermore, this study provides insights into changes of CC16 levels in lung tissue after polytrauma, which is not possible to be investigated in human clinical studies.

Since the measurements were made exclusively in comparison of a polytrauma to a sham group and no isolated thoracic trauma group was included, the influence of other organ injuries, cannot be completely excluded. The characteristics as a lung epithelial injury marker can only be drawn by comparison to other animal models. Therefore, to be able to make a definitive statement, further investigations would have to be carried out taking into account an isolated thoracic trauma group including different degrees of trauma and outcome-oriented longer time courses. Furthermore, further studies need to be undertaken to evaluate the informative value of CC16 and its expression patterns under pre-disease conditions. Nevertheless, our study indicates that CC16 might also be used as a marker for lung injury in the porcine model of polytrauma. The potentially lung-protective inhalation of CC16 would be a worthy further study to consider if this is a therapeutic target. The temporal limitations to 72 h of the monitoring phase do not allow conclusions to be drawn about unaffected outcome at this time, or the development of later occurring organ and/or multiple organ failure in this model. Further studies would need to pay more attention to important cell populations such as e.g. neutrophils and other immune-related cells.

Our data demonstrated that CC16 may be used as a potential marker of lung injury in this porcine polytrauma model. Early increased systemic CC16 levels indicated lung damage after polytrauma, while the local CC16 increase in BAL may be used as a later marker even 3 days after polytrauma to indicate the lung injury which is associated with the barrier breakdown and inflammatory changes.