In this study, we observed that in pigs, challenged into the oropharynx with P. aeruginosa, the lateral-Trendelenburg position reduced systemic inflammation through the prevention of VAP. Also, this study demonstrated that in a validated animal model of VAP, serum IL-10 and TNF-α were the only cytokines that varied during VAP development. Yet, culture of tracheal secretions still outperformed all evaluated diagnostic parameters.
Effects of study interventions on systemic inflammation
We consistently demonstrated in previous studies in sheep [16, 30] and pigs [17, 31] that the Trendelenburg position avoided VAP, but to the best of our knowledge, this is the first comprehensive report regarding its effects on systemic inflammation. Our study adds to previous literature and suggests that during mechanical ventilation, the Trendelenburg position might limit systemic inflammation. In particular, we found that IL-1β, IL-1RA, IL-4, and IL-8 were consistently lower in the Trendelenburg group. In contrast, modifying duty cycle and PEEP did not have any effect on systemic inflammation and, as previously reported , on VAP. Importantly, our study primarily focused on cytokines that might variate during the development of VAP, thus it is plausible that the association of aforementioned cytokines with the Trendelenburg position might have been related to the prevention of VAP. Indeed, previous findings confirmed higher levels of IL-1β, specifically in bronchoalveolar lavage fluids [24, 26], of patients with VAP, whereas an association between systemic and pulmonary IL-1RA and VAP [10, 26] has not been established. As for IL-4, this biomarker has not been tested in VAP and preliminary studies have found in IL-4-knockout mice resistance to P. aeruginosa pulmonary infection and increased TNF-α production . Also in pediatric patients with pneumonia, IL-4 was a reliable marker of severity of the disease . Finally, clinical studies have confirmed a surge in IL-8 with VAP .
Considering that in clinical settings VAP still lacks of a diagnostic gold standard, an additional purpose of our study was assessing accuracy of several diagnostic parameters. In line with previous reports , clinical variables, such as body temperature, WBC, and PaO2/FIO2, were highly unspecific. As for systemic cytokines, previous clinical studies [10, 24, 25, 33,34,35,36] have appraised biomarkers in bloodstream, lungs, and pleural space to find the best diagnostic marker. Yet, discriminatory inflammatory markers that could reliably diagnose VAP are difficult to be identified in clinical settings, because at the time of VAP development, ICU patients often present other infections. Furthermore, ICU patients might be in an immune-paralysis state [37,38,39], which increases the risk of developing VAP , while hindering patient’s inflammatory response. Given the abovementioned challenges, the use of a reliable animal model of VAP , developed in healthy animals without concomitant illnesses, could facilitate identification of diagnostic markers and redirect on the most promising.
We found that only IL-10 and TNF-α were independently associated with the development of VAP. IL-10 is an anti-inflammatory cytokine that inhibits activation and effector function of T cells, monocytes, and macrophages . Millo and collaborators  did not find any variation in plasma and BAL IL-10 in patients who developed VAP. Similarly, Conway Morris et al. confirmed that IL-10 did not have potential value for discriminating VAP from non-infected patients . Whereas, Martin-Loeches and collaborators found significant differences in IL-10 concentration between VAP and no-VAP patients ; nevertheless, multivariate analyses failed to corroborate diagnostic value of IL-10. TNF-α is predominately produced by macrophages and exert various effects such as fever, cachexia, and inhibition of tumorigenesis and viral replication. Millo et al. found higher levels of TNF-α in bronchoalveolar lavage fluids of VAP patients .
Of note, we found that P. aeruginosa endotracheal aspirate (ETA) concentration overcame diagnostic accuracy of all cytokines, yielding an AU-ROC higher than 80% (Fig. 3). A clinical trial  tested diagnostic value of culture of tracheal secretions vs. bronchoalveolar lavage fluids and it did not find any difference between study groups. Thus, latest European  and American  guidelines for the management of patients with VAP recommended obtaining samples of respiratory secretions to diagnose VAP. Our findings support this recommendation; yet, it is important to emphasize that tracheal secretions culture requires 1 to 3 days before definitive results, ultimately limiting initial therapeutic options. Also, as reported in Fig. 4, we found a marginal association between P. aeruginosa ETA concentration and lung colonization. This could be related to the limited number of animals or to specific features of our model; indeed, following oropharyngeal challenge, the animals consistently developed colonization of the proximal airways, irrespective of the subsequent colonization of the lungs and VAP development.
Our preliminary findings should be interpreted in light of the potential clinical applications. First, clinical feasibility of inverse-ratio ventilation is challenging, and given the marginal results reported in our latest analysis and previous studies , it should not be recommended in clinical settings. Second, in our studies, we failed to find efficacy of PEEP in reducing systemic inflammation or VAP, but these findings are in contrast with a previous clinical study that found lower incidence of VAP in patients ventilated with PEEP of 5 vs. 0 cm H2O . This study was discontinued earlier for low recruitment rate, thus future clinical corroborations are essential to further explore the value of PEEP. Third, the recent results of the Gravity-VAP Trial  confirmed preventive benefits associated with the lateral-Trendelenburg position, but the study was discontinued earlier, due to a very low incidence of VAP and marginal effects in secondary outcomes. Of note, in the Gravity-VAP trial, the lateral-Trendelenburg position was applied for only 2 days following intubation, and higher safety was reported in patients who did not present pulmonary infiltrates. Considering the positive results from experimental studies [17, 30, 31], but the limitations of the latest randomized trial, risks and benefits of such intervention should be carefully pondered, carefully examining timing and duration of the intervention, which should exclusively be applied to patients who are not intubated for pulmonary causes. Furthermore, pulmonary and systemic inflammation should be monitored. Finally, although our results confirm diagnostic accuracy of ETA, the delay for culture results often causes overtreatment or inappropriate treatment of multi-drug resistant pathogens. Thus, development of novel rapid molecular assays, custom-made for pathogen specific for VAP and for drug resistance genes, are needed. In addition, given the variability in biomarkers concentration among different patient populations and courses of treatment, a comprehensive evaluation of the dynamics of these markers, rather than the absolute cut-off values should be prioritized.
First, although we conducted a 72-h study, in clinical settings, VAP may develop after several days of MV; therefore, in our model, some pathogenic mechanisms and the inflammatory response could somehow diverge in comparison with the critically ill, ventilated patient. Second, our animals were healthy at the beginning of the study and inflammatory changes were specifically related to the new iatrogenic infection. Nevertheless, in the early phase of the experiment, inflammatory markers could have been affected by the surgical interventions performed during animal preparation. Third, considering the complexity of cytokine signaling pathways in critically ill patients and potential inter-species differences, our results require further validation in humans. Fourth, this was an analysis of animals included in a previous trial  and inferences should primary assist for future confirmatory analyses. Fifth, our findings should be discussed critically, because pigs are quadruped and were maintained prone, due to inherent risks of lung dysfunction when maintained in the supine position for prolonged period of times. Patients are normally maintained in the supine semirecumbent position, and the auto-regulation mechanisms in pulmonary and hemodynamic physiology, which may have played a role in our findings, could be different in pigs and humans. Finally, this study did not encompass the entire range of inflammatory biomarkers that could vary during the course of VAP. For instance, due to methodological limitations of the porcine assay, we did not measure procalcitonin, which was evaluated in previous clinical studies .