Mechanical ventilation (MV) is a lifesaving therapy in respiratory failure. During conventional MV, positive pressure is used to overcome the resistive and elastic properties of the respiratory system, leading to stretch, and strain of lung units. Stress and strain are crucial to keep the homeostasis of the lungs (Hubmayr and Kallet, 2018). Stretching of alveolar epithelial cells (AECs), also during MV, is a trigger of surfactant turnover and release (Abreu et al., 2008). However, MV may also cause injury, amplifying the lung-specific inflammatory response in both healthy and injured lungs, with release of cytokines, and damage to the alveolar integrity (Xuan et al., 2015; Rentzsch et al., 2017).

Lung injury due to MV, so-called ventilator-induced lung injury (VILI), has different pathophysiological mechanisms (Loza et al., 2015; Pelosi et al., 2018; Abreu et al., 2019). These include tidal overdistension, or volutrauma, and cyclic opening and closing of atelectatic lung units, termed atelectrauma (Rocco et al., 2012). Excessive cell stretching increases the release of surfactant proteins (Bartolák-Suki et al., 2017), production of inflammatory cytokines, and other pro-inflammatory molecules through different pathways by modulation of gene expression (Santos and Slutsky, 2000). Thereby, such events also cause numerous cellular and biochemical events in the pathogenesis of acute respiratory distress syndrome (ARDS) (Sipahi, 2014).

Mechanisms of mechano-transduction constitute the basis of the epiphenomenon generally referred to as biotrauma. In clinical practice, positive end expiratory pressure (PEEP) is used in an attempt to protect lungs from atelectrauma (Dreyfuss et al., 1988; Vlahakis et al., 1999; Huhle et al., 2018). However, PEEP strategies have yielded conflicting results regarding lung protection (Spieth et al., 2009; Bugedo et al., 2017). While the effect of PEEP on lung macrostructure is better defined, i.e., the stabilization of lung units and a more even distribution of mechanical stress (Rentzsch et al., 2017), little is known about how static stretch, resulting from use of PEEP, impacts the inflammatory response of AECs under dynamic stretching. Studies revealed that, in cultured AECs, non-variable cyclic stretch, in contrast to non-stretched resting conditions, induces the production of cytokines (Vlahakis et al., 1999; Li et al., 2003), reactive oxygen species (Chapman et al., 2005) and promotes cytoskeleton remodeling (DiPaolo et al., 2010), rendering these cells a valuable model for studying these aspects in vitro. Therefore, in the present study, we aimed to investigate the inflammatory response of rat type 2 AECs cell line to different conditions of stretching. We hypothesized that static stretch increases the pro-inflammatory response of AECs to dynamic stretch.

The detailed methodology of the experiments is described in the Supplementary Figure S1.

The main findings of this study were that, in LPS-stimulated, compared to non-stimulated rat type 2 AECs: 1) dynamic stretch increased the expression of AREG, IL-6, MCP-1, MIP-2, as well as the protein concentration of IL-6 and MCP-1 in the medium; 2) static stretch increased gene expression of MCP-1 and MIP-2, but not AREG, and resulted in higher protein secretion of IL-6, but not MCP-1. To our knowledge, this is the first study addressing the effects of static stretch on the pro-inflammatory response of in vitro L2 AECs submitted to dynamic stretch.

Our experiments combining static stretch with different dynamic stretch conditions were conducted in type L2 AECs, because they can easily grow under controlled conditions, and are the main cell type involved in the immune response of the lung alveolar epithelium (Kang and Kim, 2017). A major strength of our study is that neither dynamic nor static stretch, nor the LPS used for stimulation, generated any significant changes in cell survival, thus allowing us to directly assess the pro-inflammatory response. Importantly, static and dynamic stretch were designed to mimic the mechanical stress patterns that AECs are submitted to in vivo, during MV, since there is a considerable body of evidence that mechano-transduction plays a key role in VILI (Chen et al., 2018). Changes in alveolar epithelial cell deformation during modifications of lung volume have been described in vivo (Bartolák-Suki et al., 2017). Studies in isolated lungs suggest that, if the lung volume increases by 40–100% of the total lung capacity, the basal surface area of alveolar epithelial cells increases by 34–35% (Tschumperlin and Margulies, 1999; Tschumperlin et al., 2000; Wirtz and Dobbs, 2000).

On the other hand, if a single alveolus or even the whole lung is modelled as a sphere, it can be shown that an increase of alveolar surface area (SA) by factor a, yields an increase of corresponding volume V by a ^3/2. Therefore, during spontaneous breathing in humans, at a functional residual capacity (FRC) of 30 ml/kg body weight, a tidal volume of 7 ml/kg body weight results in an increase of lung volume of 23% and an increase of ∼15% of ASA. To increase lung volume from FRC to lung total capacity (TLC) (80 ml/kg body weight) would correspond to a 166% volume increase and a 92% increase of SA. In rats, however during anesthesia a FRC of 11 ml/kg has been reported (Schulz and Muhle, 2000). Ventilation with a tidal volume of 6 ml/kg body weight results in a 54% increase of LV and a 30% increase of SA. Accordingly, an increase of lung volume from FRC to TLC (42 ml/kg body weight in rats) yields a volume increase of 270% and an increase of SA of 140%. Thus, in our experiments a maximal total increase of SA of 45% mirrored dynamic and static mechanical strain occurring during low to low tidal volume ventilation in rats at low PEEP.

Mechanical ventilation may induce an inflammatory response in the lung tissue (Santos and Slutsky, 2000), leading to ARDS (Birukova et al., 2012; Sipahi, 2014). In ARDS patients, high levels of PEEP are used to reduce VILI (Briel et al., 2010). Mechanical ventilation with low tidal volumes and higher PEEP reduced the incidence of pulmonary complications in patients without acute lung injury (Neto et al., 2012). In contrast, other clinical studies have indicated that the level of PEEP is unrelated to the mortality of the ARDS patients (Walkey et al., 2017; Group et al., 2020). Recent data have also shown that higher PEEP promoted higher lung inflammation than lower PEEP, at comparable low tidal volumes and driving pressures (Ding et al., 2013; Ochiai, 2015; Hamlington et al., 2016; Silva et al., 2016). For this reason, we have asked if static stretch (which in vivo is increased by PEEP) modulates the inflammatory response. To mimic in AECs the pathophysiology of clinical ARDS (Costa et al., 2017), we used LPS, frequently utilized to induce inflammation in vitro (Dreyfuss et al., 1988; McRitchie et al., 2000; Birukova et al., 2012; Wong and Johnson, 2013) or ARDS, in rats, in vivo (Pugin et al., 1998; Ding et al., 2013).

We found that, neither dynamic stretch, static stretch, nor LPS significantly affected cell junctions and cell monolayer organization, as previously shown (Rentzsch et al., 2017; Knudsen and Ochs, 2018). Actin filament organization was not modified at lower dynamic stretch (Rentzsch et al., 2017; Knudsen and Ochs, 2018) but only at a 30% dynamic stretch, which corresponds to a high increase in total lung capacity. Previous studies have shown that MV modulates the lung inflammatory response, either through direct cell rupture or through the so-called “mechano-transduction” mechanism, which is not yet fully understood (Santos and Slutsky, 2000; Sutherasan et al., 2014; Tian et al., 2016). Recent data (Tian et al., 2016) suggest that long term (28–72 h) stretching or LPS exposure of cultured human pulmonary artery endothelial cells affect cell junctions and disrupted cell monolayers, a process mediated by RhoA kinase (Marshall et al., 2010). This is in agreement with the changes in actin filament organization we observed at a high dynamic stretch, changes that could be mediated by Rho GTPase (i.e., RhoA) signaling, and could involve crosstalk with extracellular matrix–dependent integrin signaling via SRC (Tehrani et al., 2007). Future studies are necessary to clarify these aspects.

AREG, a protein with tissue protective effects during inflammation (Zaiss et al., 2013), and is involved in the response to mechanical loading in various mesodermal-derived tissues (Deacon and Knox, 2010; Marshall et al., 2010; Shao and Sheng, 2010). We found that AREG expression was increased in LPS-stimulated cells submitted to different dynamic stretch conditions, at 1 h. However, further stimulation (4 h) reduced AREG expression, suggesting that its tissue protective effects may be reduced after long-term stretching. This decrease in AREG expression may be due to reduced transcription, and/or increased mRNA degradation (Nakayama et al., 2013), controlled by cell signaling pathways, as previously observed in other systems (Zhang et al., 2009; Terry et al., 2010; Keren, 2011; Latasa et al., 2012; Zaiss et al., 2013).

The focus of our study was to understand if and how PEEP modulated the inflammatory response (Rentzsch et al., 2017; Knudsen and Ochs, 2018). We have found that, in LPS-stimulated L2 AECs, dynamic and static stretch levels influenced both the expression and secretion of specific cytokines (i.e. IL-6, MCP-1, and MIP-2). This is in agreement with in vivo studies in mice, showing increased expression and release of IL-6 (Tremblay et al., 2002; Heise et al., 2011; Goldman et al., 2014), MCP-1 or MIP-2 (Hegeman et al., 2010; Hawwa et al., 2011; Herbert et al., 2016; Jia et al., 2016; Zhu et al., 2016) during MV. Specifically, in LPS-stimulated L2 AECs, the expression of IL-6, MCP-1, and MIP-2 was significantly increased after static stretch, compared to non-LPS treated cells. Our data are in agreement with in vivo studies showing that the expression of several cytokines (including IL-6, MCP-1, or MIP-2) was significantly enhanced after exposure to LPS (Heise et al., 2011) and that, mechanical ventilation synergistically amplified the release of IL-6, MIP-2, IL-1β, and TNF-α in rats treated with LPS (Pugin et al., 1998; Ding et al., 2013). In addition, our data shows that MCP-1 behaves differently than the other cytokines, i.e., its release from LPS-treated cells was enhanced by dynamic, but not by static stretch, suggesting that the two types of stretch may differentially influence the immune response. Moreover, these findings support previous evidence that plasma membrane tension affects the release of inflammatory cytokines, by orchestrating complex aspects of cell trafficking and motility (Keren, 2011), and modulating cell signaling (Tremblay et al., 1997; Ranieri et al., 1999).

Static stretch increased the pro-inflammatory response of dynamically stretched LPS-stimulated type L2 AECs which is suggestive of a potential pro-inflammatory effect of PEEP during mechanical ventilation at the cellular level.