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Introduction

Blunt chest (thoracic) trauma (TxT) is common in polytraumatized patients, and 20-25% of deaths in severely injured patients treated in emergency departments are caused by chest injuries (1). Typical complications in these patients include disseminated intravascular coagulation, pneumonia, acute lung injury (ALI), or its more severe form, acute respiratory distress syndrome (ARDS) (2,3). While the role of mechanical damage in the pathogenesis of post-traumatic complications associated with lung injury is undisputed, current research indicates that lung damage is driven by local and systemic inflammatory reactions (4).

Hoth et al demonstrated increased levels of pro-inflammatory CXCL1 in bronchoalveolar lavage fluid at 3 h post-injury in a murine model of pulmonary contusion (5). Furthermore, the recruitment of neutrophils to injured lung tissue, which is primarily responsible for pulmonary dysfunction following lung contusion, has been shown to be dependent on the expression of CXCL1, among other pro-inflammatory factors (6). Recently, the authors demonstrated that a 'double-hit' model of TxT, followed by cecal ligation and puncture (CLP) as the second hit, which was also applied in the underlying study, increased the expression of pro-inflammatory chemokines and cytokines, including CXCL1 (7). Thus, in line with the two-hit hypothesis, a traumatic insult as the first hit, followed by secondary surgeries or complications, may lead to detrimental outcomes. Consistent with the findings of other researchers, it was found that isolated experimental lung trauma induced a profound inflammatory reaction; however, it was insufficient to establish the ongoing pathological pulmonary changes associated with ALI (7,8). However, secondary CLP following TxT, as applied herein, reflected the human etiology of indirect lung damage ending in ALI (7,9-11). Another murine 'double-hit' model that includes CLP, followed by inducing pneumonia via Pseudomonas aeruginosa 4-7 days later, significantly improved survival when pneumonia was induced after 7 days. Improved survival was associated with the restoration of interferon (IFN)-γ by stimulated splenocytes (12). These findings underline the importance of timing of the second hit, and thus also for therapeutic interventions. IFN-γ plays a key role in the regulation of both innate and acquired antimicrobial immunity by inter alia, stimulating macrophage functions, such as phagocytosis, respiratory burst activity, antigen presentation and cytokine secretion (13,14). The functional significance of IFN-γ in antimicrobial defense has been demonstrated by the increased susceptibilities of IFN-γ−/− and IFN-γ-R−/− mice to a variety of infections (15). Furthermore, IFN-γ seemed to facilitate systemic inflammation during CLP-induced abdominal sepsis in mice (16).

Club cell protein (CC)16 is an anti-inflammatory protein derived from epithelial club cells in the lungs (17,18). Its systemic concentrations are associated with the extent of lung contusion and with pulmonary complications in traumatized patients, which underscores its biomarker characteristics (19,20). Although its exact functions in vivo remain unclear, CC16 plays an important protective role in the respiratory tract against oxidative stress and inflammatory responses due to its anti-inflammatory properties (21). With regard to the two-hit hypothesis, the inflammatory response following trauma is essential for host defense; however, it can cause further tissue damage if triggered by a secondary stimulus (22,23). Since reducing inflammation attenuates pathological injury and the survival of mice with ALI (24), as a pulmonary anti-inflammatory protein, CC16 may play an important regulatory role during the development ALI following trauma. However, CC16 may also play a pathological role in immunoparalysis with concomitant post-traumatic infectious complications (25). This role was addressed in a recent study by the authors, which demonstrated the anti-inflammatory effects of CC16 on CLP-induced ALI following TxT in mice (9). While the early intrapulmonary inhibition of CC16 following TxT reduced lung damage, the later inhibition of CC16 deteriorated lung damage after 24 h. However, it remains questionable whether the latter may have been followed by reduced lung damage in a prolonged observational period. Since this question and the one regarding the pathophysiological relevance of these observations to survival remain unanswered, the present study evaluated whether the local neutralization of CC16 affects mortality after sepsis-induced ALI following blunt chest trauma in a murine model.

Materials and methods

Animals and experimental model

All experiments were conducted in accordance with German federal laws regarding the protection of animals. The experiments were performed at Goethe University Hospital in Frankfurt, Germany. The present study was approved by the responsible government authority, the Veterinary Department of the Regional Council in Darmstadt, Germany (Regierungspräsidium Darmstadt; AZ: FK 1068), and the study was performed under ARRIVE guidelines (26).

For the survival analysis, 48 male CL57BL/6N mice (weighing 25±5 g at 6-8 weeks of age) were included, and 72 male CL57BL/6N mice (6-8 weeks old, weighing 25±5 g) were included for experiments with sacrifice after 6 or 24 h post-CLP. All animals were purchased from Janvier Labs. The animals were provided with free access to water and food ad libidum before and after the experimental procedures. At 30 min prior to the experiment, buprenorphine (Indivior Eu Ltd., 0.1 mg/kg body weight) was administered to all mice subcutaneously. This process was repeated every 12 h. For the survival analysis, buprenorphine (0.1 mg/kg body weight) was administered every 6 h for 48 h after CLP. From 48-96 h post-CLP, buprenorphine (0.1 mg/kg body weight) was administered every 12 h. Drinking water provided to the mice was enriched with metamizole (Ratiopharm) to maintain adequate analgesia during the experiment.

For TxT, mice underwent general mask anesthesia with 3% isoflurane (Baxter Deutschland GmbH), according to partly-modified standardized protocols described previously (7,9,10,27). Briefly, the mice were placed in the supine position under a cylinder. The cylinder was separated by a Mylar polyester film with a thickness of 0.05 mm [DuPont de Nemours (Deutschland) GmbH] and placed 2.5 cm above the sternum. Delivering compressed air to the upper part of the cylinder ruptured the membrane and led to a standardized pressure wave. This resulted in standardized, bilateral, blunt chest trauma. According to their group allocation, mice received an intratracheal application of either a CC16 antibody or a control antibody immediately following TxT. Uteroglobin/SCGB1A1 (CC16 Ab; LS Biosciences) or IgG control (IgG) antibody (10 µg/ml, R&D Systems) were administered either immediately following the induction of thoracic trauma (early application) or following CLP (late application). For antibody administration, mice were placed in the supine position, and the tongue was kept aside. A buttoned cannula was placed at the beginning of the trachea, and 50 µl were carefully administered. Mice were then kept in the reverse Trendelenburg position for 30 sec to ensure proper antibody distribution inside the lungs. After 24 h, mice underwent intraperitoneal anesthesia with ketamine (100 mg/kg body weight, Zoetis Deutschland GmbH) and xylazine (10 mg/kg body weight, Bayer Leverkusen), and depending on group allocation, the following procedures were performed. Either median laparotomy and CLP were performed as previously described (7,28) or median laparotomy and eversion of the caecum without any further manipulation was performed. Briefly, the abdominal cavity was opened by midline laparotomy (approximately 1 cm), and the cecum was carefully exposed. Subsequently, 7 mm of the distal caecum were ligated using Premilene 5-0 suture (B. Braun Melsungen AG) and perforated with a single through-and-through puncture using a 25G cannula (BD Biosciences).

For survival experiments, a 18G cannula (BD Biosciences) was used. After this procedure, a small amount of feces was extruded, and the cecum was restored to the abdominal cavity. Abdominal closure was performed by a two-layer suture. According to group allocation, mice received an intratracheal application of either a CC16 antibody or control IgG antibody, as described above, immediately following thoracic trauma or following CLP. The sham-operated (sham) control group underwent the identical anesthesia procedures described above, but without TxT, as well as with median laparotomy and eversion of the caecum, but without CLP. Immediately following CLP, 1 ml of prewarmed G5% solution (B. Braun Melsungen AG) was administered intraperitoneally. Depending on group allocation, either 6 or 24 h post-CLP, mice received anesthesia as described above and sacrifice was performed, or mice were observed for 6 days after CLP and euthanized via blood withdrawal by a puncture of the heart under general anesthesia with 3% isoflurane (Baxter Deutschland GmbH). During the survival analysis, mice were euthanized when they reached one of the following conditions: Permanently closed eyes, lateral position, severe dyspnea with mouth breathing, cyanosis, body weight loss >20% or apathy. An overview of the experimental design is presented in Fig. 1.

Figure 1 Experimental design. Mice underwent TxT and CLP and were assigned to the ALI group. According to group allocation, a CC16 antibody (Ab) or IgG antibody was administered immediately after either TxT (early Ab) or CLP (late Ab). Animals were then euthanized for sampling (n=6) after 6 or 24 h following CLP. Survival (S) was assessed for 6 days following CLP (n=8). TxT, thoracic trauma; CLP, cecal ligation and puncture; ALI, acute lung injury.

Group allocation

The animals were randomly allocated to experimental groups, as described below. For survival analyses, all mice, apart from those in the sham group received TxT followed by CLP (ALI) after 24 h. In total, 8 mice received an intratracheal application of CC 16 antibody, and 8 mice received an intratracheal application of the control antibody (IgG), as described above, immediately following TxT. A total of 16 mice received an intratracheal an application of a CC 16 or control antibody (i.e., 8 mice received CC 16 Ab, and 8 mice received IgG) immediately following CLP. In total, 8 mice were allocated to the control group without any application of antibodies, while 8 mice were allocated to the sham group, which received median laparotomy and eversion of the caecum, but not CLP or TxT. The same group allocation was used for the groups of mice sacrificed at 6 and 24 h (n=6 in each group). Mice received a CC 16 or control antibody (IgG) immediately after TxT (early Ab) and were sacrificed after 6 h (6 h_early Ab) or 24 h (24 h_early Ab) after CLP or received a CC 16 or control antibody (IgG) immediately after CLP (late Ab), followed by sacrifice after 6 h (6 h_late Ab) or 24 h (24 h_late Ab).

Systemic IFN-γ determination

The caval vein was punctured by a heparinized syringe for blood withdrawal at 6 or 24 h after CLP. Subsequently, plasma was isolated from blood samples by centrifugation (1,164 × g for 15 min at 4°C) and stored at −80°C for further IFN-γ measurements. IFN-γ was measured with a BD CBA Mouse Inflammation kit (BD Biosciences) according to the manufacturer's instructions. Briefly, 50 μl of capture beads were added to polystyrene FACS tubes (BD Pharmingen™) to 50 μl serum. Subsequently, 50 μl of Mouse Inflammation PE Detection Reagent were added followed by incubating for 2 h at room temperature. Subsequently, samples were washed with 1 ml of the wash buffer and centrifuged at 200 × g for 5 min at room temperature. The supernatant was discarded, and the pellets were resuspended in 300 µl of the wash buffer. Analyses were performed using a BD FACSCanto 2™ and FCAP Array™ Software (BD Biosciences). Cytometric Bead Array (CBA) was performed as previously described (29).

Quantification of CXCL1 expression levels in the lungs

Following blood withdrawal, the animals were perfused with 20 ml PBS via the inferior caval vein. The lungs were then removed and snap-frozen in liquid nitrogen. Lung tissue was homogenized in a protein lysis buffer at 4°C, followed by centrifugation for 30 min at 4°C and 20,000 × g. Subsequently, supernatants were stored at −80°C. CXCL1 concentrations in the lungs were determined using a Mouse CXCL1/KC ELISA kit from R&D Systems according to the manufacturer's instructions (Wiesbaden-Nordenstadt). ELISA was performed using an Infinite M200 microplate reader (Tecan Deutschland GmbH).

RNA isolation and reverse transcription (RT) semi-quantitative polymerase chain reaction (RT-qPCR)

Following sacrifice, snap-frozen specimens from the lungs were stored at −80°C, and RNA isolation was applied using an RNeasy-system (Qiagen GmbH), as previously described (30). Residual DNA was removed using an RNase-free DNase kit (Qiagen GmbH). The amount and quality of RNA were measured photometrically using a NanoDrop ND-1000 device (NanoDrop Technologies, Inc.). Subsequently, RNA was reverse transcribed into cDNA using an AffinityScript QPCR cDNA Synthesis kit (Stratagene) according to the manufacturer's instructions. A specific primer for mouse CXCL1 was used to evaluate CXCL1 gene expression (NM_008176, UniGene no. Mm.21013; cat no. PPM03058A), and GAPDH (NM_008084, UniGene no. Mm.343110; cat no. PPM02946E) (both from SA Biosciences) gene expression measurements served as a reference. According to the manufacturer's instructions, the PCR reaction was set up in a 25 µl volume with 1X RT2 SYBR-Green/Rox qPCR Master Mix (SA Biosciences) and measured on a Stratagene Mx3005p QPCR system (Stratagene). The two-step amplification protocol was as follows: Initial denaturation at 95°C for 10 min, followed by 40 cycles at 95°C with 15 sec of denaturation and 60 sec of annealing/extension at 60°C. The comparative threshold-cycle (CT) method (2−ΔΔCq method) was used to calculate the target gene's relative expression (31). Sham-operated animals served as 100% expression reference following normalization to GAPDH. The method was applied using a previously described protocol (30).

Statistical analyses

Statistical analysis was performed using GraphPad Prism 6 (GraphPad Software, Inc.). Based on the D'Agostino-Pearson normality test, differences between the groups were analyzed using the non-parametric Kruskal-Wallis test, followed by Dunn's post hoc test for the correction of multiple comparisons. The data are presented as the means ± standard error of the mean. Survival was analyzed via the log-rank test, and the Bonferroni correction was applied to correct for multiple comparisons. A P-value <0.05 was considered to indicate a statistically significant difference.

Results

IFN-γ concentrations in the blood

At 6 or 24 h following thoracic trauma and CLP, a significant increase in the levels of pro-inflammatory IFN-γ compared to the sham group was detected (P<0.05; Fig. 2). Early antibody application did not significantly alter the IFN-γ levels between the ALI groups after 6 or 24 h (Fig. 2). Whereas late antibody application led to comparable IFN-γ levels among all the ALI groups after 6 h, a significant increase in the IFN-γ levels vs. the sham group was observed only in the control group after ALI was compared with the sham group (P<0.05; Fig. 2C and D).

Figure 2 Systemic levels of IFN-γ are shown. A CC16 antibody (Ab) or IgG antibody was administered immediately following either thoracic trauma (early Ab, A and B) or CLP (late Ab, C and D). Animals were euthanized for sampling after 6 or 24 h. Data are presented as the means ± standard error of the mean; #P<0.05 vs. sham (n=6). IFN-γ, interferon-γ; CC16, club cell protein 16; CLP, cecal ligation and puncture; ALI, acute lung injury.

CXCL1 gene expression and protein concentration

Although CXCL1 gene expression was significantly elevated in the control and IgG groups at 6 h following thoracic trauma and CLP compared to the sham group (P<0.05; Fig. 3A and C), no significant differences were observed after 24 h among these groups (Fig. 3B and D). The early application of the CC16 antibody significantly increased CXCL1 gene expression after 6 h compared to all other groups (P<0.05; Fig. 3A). However, after 24 h, no significant changes among the groups were observed (Fig. 3B). Whereas late CC16 antibody application led to levels comparable to those of the other groups undergoing thoracic trauma and CLP after 6 h, a significant increase in CXCL1 gene expression following late CC16 antibody application vs. all other groups was observed after 24 h (P<0.05; Fig. 2D).

Figure 3 Pulmonary (A-D) CXCL1 gene expression and (E-F) CXCL1 protein expression levels are shown. A CC16 antibody (Ab) or IgG antibody was administered to the lungs immediately following either thoracic trauma (early Ab) (A and B, and E and F) or CLP (late Ab) (C and D, and G and H). After 6 or 24 h, the animals were euthanized for sampling. Data are presented as the means ± standard error of the mean; #P<0.05, vs. sham group; *P<0.05, CC16 Ab ALI group vs. all other groups (n=6). CC16, club cell protein 16; CLP, cecal ligation and puncture; ALI, acute lung injury.

CXCL1 protein concentration was significantly elevated in all 3 ALI groups at 6 and 24 h following thoracic trauma and CLP compared to the sham group (P<0.05; Fig. 3E-H). Whereas early CC16 antibody application led to levels comparable to those of the other groups undergoing thoracic trauma and CLP after 6 h, a significant increase in CXCL1 protein concentration following early CC16 antibody application vs. all the other groups was observed after 24 h (P<0.05; Fig. 3F). The late application of the CC16 antibody following thoracic trauma and CLP led to a slight increase in the CXLC1 protein concentrations compared to the control and IgG groups upon ALI after 6 and 24 h; however, this difference was insignificant (Fig. 3G and H).

Body weight changes during the 7-day observation period

Every 24 h for 7 days, the weight of the mice was documented. During the first 48 h, the weight of the animals was stable in all groups. After 48 h, all groups undergoing thoracic trauma and CLP exhibited a decrease in weight compared with the sham group (Fig. 4).

Figure 4 Bodyweight during a 6-day observational period following sepsis induction. According to group allocation, a CC16 antibody (Ab) or IgG antibody was administered immediately after either thoracic trauma (early Ab) or CLP (late Ab). Data are presented as the means ± standard error of the mean. CC16, club cell protein 16; TxT, thoracic trauma; CLP, cecal ligation and puncture; ALI, acute lung injury.

Survival rates after sepsis-induced ALI following TxT

All animals in the sham group survived during the observation period (Fig. 5). Within 48 h, the survival rate decreased among the groups subjected to TxT and CLP compared to the sham group (Fig. 5A). Within 60 h, the survival rate in the ALI control group significantly decreased compared to the sham group (P<0.05; Fig. 5B). The group receiving early CC16 Ab treatment exhibited a significantly higher survival rate compared to the control group following TxT and CLP (P<0.05; Fig. 5B). After 72 h, no marked differences in survival were observed between the 3 groups undergoing TxT and CLP (Fig. 5C). However, all ALI groups exhibited significantly decreased survival rates compared to the sham group (P<0.05; Fig. 5C). Comparable data were observed until the end of the observational period of 6 days post-CLP (Fig. 5D).

Figure 5 Survival was assessed during a 6-day observational period following sepsis induction. Mice underwent TxT and CLP, and were assigned to the ALI. The sham group underwent identical anesthesia and surgical procedures without performing TxT or CLP. The control group underwent ALI without an antibody application. Interventions with a CC16 antibody (Ab) or IgG antibody were performed immediately after either TxT (early Ab) or CLP (late Ab). Data are presented as Kaplan-Meier estimates for different time periods (A, 0-48 h; B, 24-60 h; C, 24-72 h; D, 24-168 h). *P<0.05 vs. sham group (n=8). CC16, club cell protein 16; TxT, thoracic trauma; CLP, cecal ligation and puncture; ALI, acute lung injury.

Discussion

The present study investigated whether the intratracheal neutralization of CC16 affects survival following 'double-hit' trauma consisting of TxT followed by CLP in mice. Recently, the anti-inflammatory potential of CC16 24 h post-CLP in this model was confirmed. The local anti-inflammatory characteristic of CC16 is supported by the present data and by results previously demonstrated in non-traumatic lung injury (32,33). A recent study by the authors demonstrated that upon the early intratracheal neutralization of CC16 following chest trauma, reduced lung injury at 24 h post-CLP was observed (9). Notably, the late neutralization of CC16 following CLP aggravated lung injury during the same observational period (9). Thus, although the anti-inflammatory potential of CC16 in the underlying model was clearly confirmed, previous studies did not determine whether the results are caused by the timing of antibody application or disease kinetics or whether the observed effects affect survival in this model. The present study demonstrated that although the overall mortality rate was not significantly altered upon early CC16 neutralization, delayed mortality within 60 h after the second hit was present in this group.

Blunt chest trauma triggers a potent inflammatory response in lung tissue and the airways, implying the (intrapulmonary) release of pro-inflammatory chemokines and cytokines, which promote the activation of alveolar macrophages, chemotaxis, and pulmonary neutrophil infiltration (34,35). Since several studies have suggested that the pro-inflammatory response is a key driver of lung damage during ALI, a pathologically-relevant role of the anti-inflammatory CC16 has been postulated. Miller et al confirmed that increased pulmonary IL-8 (CXCL8) levels in patients suffering from ARDS were associated with an increased influx of neutrophils, key drivers of organ damage (36). Similar findings were confirmed in vivo via interleukin (IL)-8 neutralization, which significantly reduced lung damage in a rabbit model of lung injury induced by acid aspiration (37). The recruitment of neutrophils to the lungs is a key event in the early development of ALI and is mediated by CXCL1 (38). Reutershan et al reported significantly reduced neutrophilic migration to the lungs in CXCL1 receptor-deficient CXCR2−/− mice in a murine model of LPS-induced ALI (38). Similarly, CXCL1 receptor neutralization reduced neutrophilic infiltration and, moreover, attenuated lung damage in a mouse model of ventilator-induced lung injury (39). Consistent with these findings, the important role of CXCL1 in local tissue inflammation was confirmed by its suppressed expression, which was concurrent with reduced LPS-triggered pulmonary inflammation in mice (40). Furthermore, in sepsis-induced lung injury, the reduced extravasation of neutrophils into alveolar cavities was associated with reduced pulmonary CXCL1 levels (41). Similarly, the pathophysiologically relevant role of CXCL1 was confirmed in other models of lung injury (42,43). The data of the present study indicate that CC16 may have a direct effect on the pulmonary level of CXCL1 in a 'double-hit' model of sepsis-induced ALI following thoracic trauma. Since the early neutralization of CC16 immediately following thoracic trauma increased CXCL1 concentrations after 24 h, this finding confirms the anti-inflammatory effect of CC16. However, the results prompt a conflictive discussion. The above-mentioned studies suggest that CXCL1 plays a detrimental role in such a model, yet the early increase in CXCL1 in our study was associated with delayed mortality. Thus, other factors must also be pathophysiologically relevant.

It is well known that the development of post-traumatic lung injury depends on several factors, including damage- or pathogen-associated molecular patterns (9,22). During post-traumatic ALI/ARDS development, numerous mediators released from tissue-resident cells act as chemoattractants for invading immune cells and further stimulate local cells to build a pro-inflammatory micromilieu (44). While it is widely assumed that this type of hyperinflammatory reaction contributes to subsequent lung damage, Störmann et al observed less lung damage following the early neutralization of CC16 immediately after trauma, which was associated with an enhanced influx of neutrophilic granulocytes (9). This group demonstrated prolonged survival following CC16 neutralization within the early post-traumatic phase of post-blunt chest trauma and CLP. However, survival rates will be discussed below. Of note, while local effects have been observed, in line with the previous study by the authors, intrapulmonary CC16 neutralization did not influence systemic inflammation, as assessed via IFN-γ levels (9). CC16 can function as an anti-cytokine and a natural immunosuppressor by inhibiting the production and biological activity of IFN-γ (45). However, the interplay between IFN-γ and CC16 was demonstrated in the lungs, and no systemic data were provided (46). The authors concluded that IFN-γ may act as a potent regulator of CC16/CC10 gene expression and that increased IFN-γ in lung epithelial cells may be modulated by increasing CC16 production, stimulated by a regulatory feedback loop (46,47), which potentially maintains the balance between the local pro- and anti-inflammatory processes. This remains to be determined in future studies.

Adjacent to its role as a biomarker for pulmonary injury and lung complications following trauma, several studies have demonstrated that CC16 has significant pathophysiological effects on the development of pulmonary complications (9,20,48). Recently, the authors demonstrated that CC16 exerts anti-inflammatory effects in sepsis-induced ALI following thoracic trauma in vivo (9). Furthermore, early and local CC16 inhibition decreased pulmonary injury after 24 h, indicating that CC16 may exert protective effects in early inflammation during ALI (9). However, CC16 inhibition after sepsis-induction aggravated lung damage after 24 h (9). Thus, although CC16 neutralization increased early inflammation and was associated with reduced pulmonary damage, the long-term outcome remains unclear. Therefore, the pathological relevance of CC16 to survival in our sepsis-induced trauma model was evaluated. In general, the approach of influencing inflammation after traumatic insult with the aim of preventing infectious and organ complications is highly challenging since the post-traumatic inflammatory response, with its numerous associated mechanisms, is very complex. Although the underlying pathomechanisms of trauma-induced ALI are unclear, in the present study, it was confirmed that CC16 exerts significant local anti-inflammatory effects. Furthermore, lung inflammation induced by cigarette smoke and adverse outcomes were ameliorated by CC16 in a murine COPD model (32,33). Of note, in patients with pulmonary morbidities, including COPD and asthma, CC16 plays an important role in pathology and performs protective functions during exposure to cigarette smoke (33,49). Although reduced lung damage following CC16 neutralization was observed, the general mortality rate did not change in the current model. The present study demonstrates that early CC16 inhibition may delay mortality within a defined timeframe after sepsis-induction; however, no evidence of it benefitting long-term survival was observed. The highest mortality rates were observed between 48 and 72 h in the present model. Similar data from other studies reveal that the majority of deaths following CLP-induced polymicrobial sepsis occur within 24-48 h after CLP or within 24-96 h (50,51). In contrast to later therapeutic interventions, early antibody application improves survival rates (52). Thus, the relevance of delayed mortality to CC16 neutralization within a defined timeframe, as shown in the present study, remains to be further elucidated.

Considering that the timeline and protracted inflammation following sepsis favor hospital-acquired infections and worsen patient outcomes through immunosuppression (53), the data of this and our recent study remain intriguing (9). Throughout their medical care, which may include surgery, severely injured patients are subjected to damaging endogenous or exogenous molecules, which mediate systemic inflammation or immunosuppression and have clinical consequences, such as infections and organ failure (53). In a recent study by the authors, it was discussed how, in post-traumatic immunosuppression, it appears reasonable that the anti-inflammatory effects of CC16 may be detrimental in the underlying model (9). However, considering hyperinflammation and the second-hit hypothesis, which suggests the presence of a detrimental hyperinflammatory state following trauma, CC16 may be expected to exert beneficial effects. The lack of significant changes in survival rates following CC16 neutralization for 6 days post-sepsis indicates that there are other, possibly more decisive, factors affecting final outcomes in this model. However, it remains to be further elaborated whether delayed mortality observed following early anti-inflammatory therapy can be improved in long-term outcomes. This effect could be evaluated in future studies by alternatively supplementing the original therapy with a second dose of either anti-CC16 Ab or recombinant CC16 at a later timepoint.

A significant limitation of the present study remains the lack of mechanical ventilation, as all animals were consistently spontaneously breathing. Furthermore, fluid management comparable to the clinical setting was not included. These two major contributors to ALI/ARDS were not taken into account in the present study. Moreover, by means of animal welfare, the number of animals in each group was limited. Interspecies variability in the innate immune response must be considered when in vivo experimental results are applied to humans (54). Furthermore, some interesting phenomena, such as decreased bodyweight in the early Ab treatment group, were observed, although this decrease was insignificant between the groups. In the present study, a sufficient explanation cannot be provided, and it remains undetermined whether a longer observational period may have provided evidence of further alterations or significant differences in survival benefits between the groups.

The present study confirmed the anti-inflammatory effects of CC16 following CLP-induced ALI after TxT in mice. As reported in a recent study by the authors (9), the early therapeutic strategy of CC16 inhibition (after TxT) increased inflammation, and it was further concomitant with delayed mortality within a defined time window after CLP; however, no long-term survival benefits were observed. In the previous study by the authors (9), later-stage CC16 inhibition resulted in poor pulmonary outcomes, and no effects on short- or long-term survival were observed. Thus, it remains to be determined whether, in post-traumatic ALI cases, a rapid local pro-inflammatory reaction may be necessary, which should be followed by another therapeutic inflammation-modulating step at a later timepoint.

Acknowledgments

The authors would like to thank Katrin Jurida, Kerstin Kontradowitz, and Alexander Schaible from the Department of Trauma, Hand and Reconstructive Surgery, Goethe University, Frankfurt for their outstanding technical assistance.

Funding

The present study was supported by grants from the DFG WU 820/2-1, HI 820/5-1 and RE 3304/8-1. The funders had no role in the design of the study, in the collection, analyses, or interpretation of data, in writing the manuscript, or in the decision to publish the results.

Availability of data and materials

All data generated or analyzed during this study are included in this published article or are available from the corresponding author on reasonable request.

Authors' contributions

BR, SW, FH and IM were involved in the conceptualization of the study. JTV, PS, NB and BR were involved in the study methodology. JTV was involved in data validation. BR and JTV were involved in the formal analysis. JTV, PS and NB were involved in the investigative aspects of the study. JTV and PS were involved in data curation. JTV was involved in the writing and preparation of the original draft. JTV, SW, FH, IM and BR were involved in the writing, reviewing and editing of the manuscript. JTV and BR were involved in visualization of the results. BR supervised the study. JTV and BR were involved in project administration. BR, FH and SW were involved in funding acquisition. All authors read and approved the final manuscript.

Ethics approval and consent to participate

All experiments were conducted in accordance with German federal laws regarding the protection of animals. The experiments were performed at Goethe University Hospital in Frankfurt, Germany. The present study was approved by the responsible government authority, the Veterinary Department of the Regional Council in Darmstadt, Germany (Regierungspräsidium Darmstadt, Hessen, Germany; AZ: FK 1068), and the study was performed under ARRIVE guidelines (26).

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References