This study aimed to examine the role of RAGE induced autophagy in ATII cells in response to endotoxin stimulation. In this study, an increase of autophagy activity in the lung, particularly in ATII cells, was observed, which was associated with RAGE signal activation in ALI. Specific knockdown of RAGE improved LPS-induced ATII cells dysfunction and reduced autophagy activation, implying a harmful role of autophagy in pro-inflammatory cytokine-induced ALI/ARDS. Our findings suggest that the overactivation of autophagy via a RAGE-dependent pathway contributes to ALI upon endotoxin exposure. To the best of our knowledge, this is the first study to demonstrate that epithelial cell autophagy activation under LPS stimulation occurred via a RAGE-dependent pathway.
Type I and type II AECs together make up the epithelium of alveoli and play critical roles in lung physiology and pathology. The alveolar epithelium provides protection against environmental insults, regulates water and ion transport and produces pulmonary surfactants to maintain alveolar homeostasis [
24]. Whether the injury of type I alveolar epithelial cells, which account for more than 90% of the alveolar area, or the injury of other cells in the lungs or vascular endothelium, they are the factors of the occurrence and development of ARDS [
25]. Alveolar epithelial damage impairs surfactant release in primary ARDS, inhibits alveolar fluid clearance, and lowers lung compliance [
9]. As shown in our results, when type I cells are lost due to lung injury, type II cells act as progenitor cells, multiply and replace those cells. And that plays a crucial part in epithelial healing and the development of lung fibrosis in ARDS [
2]. Therefore, protecting AECs from damage is a potential therapeutic strategy against ALI.
In the present study, we found that injury of the alveolar epithelium was accompanied by autophagy enhancement and we observed that overexpression of autophagy-related factors resulted in apoptosis of ATII cells. Although autophagy was initially described as a non-apoptotic pathway of programmed cell death, it now appears that autophagy plays a highly context-specific part in mediating cell death [
26]. Activation of autophagy may be a common signaling mechanism in the pathogenesis of ALI. This was supported by a study suggesting that autophagy was activated in LPS-induced ALI mice models [
27]. Autophagic cell death in the lungs is an acute phase response that contributes to the development of ARDS [
28]. Levels of autophagy proteins (Beclin1 and LC3 II) and inflammatory factors (IL-6 and TNF- α) were all increased in mice AEC cells under endotoxin stimulation, confirming that autophagy is a way of stress response of lung epithelial cells [
29]. In vitro
, it was shown that LPS may lead to autophagic death of A549 cells through PERK-dependent unfolded protein reactions (UPR) [
30]. Consistently, the autophagy in ATII cells was correlated with the stimulation intensity of LPS in our study; however, when 100 µg/mL LPS was used, the activity of autophagy in ATII cells was decreased, but apoptosis was still evident. It was considered that autophagy was further uncontrolled and that it exacerbated cell damage. Whether in inhalation lung injury mouse models or LPS-treated A549 cells or LPS-treated primary ATII cells, when autophagy activity was inhibited by 3-MA, the vigor of cells was recovered, the apoptosis of cells and lung tissue injury was reduced compared with the uninhibited group, suggesting that uncontrollable autophagy led to AECs damage and subsequently developed into ALI or even ARDS; inhibition of autophagy activity may be an effective measure to protect from lung injury. Remarkably, we noted that autophagy activation under endotoxin stimulation was correlated with the RAGE/STAT3 pathway, and mapped directly to the ability of ATII cells to survive cytotoxic insult, suggesting that RAGE may be a potential mediator of enhanced autophagy in the inflammation microenvironment.
ALI/ARDS secondary to pulmonary infection developed in the stepwise process of the overwhelming inflammatory response [
12]. As a receptor of many proinflammatory ligands, the role assigned to RAGE is to initiate the inflammatory response, which is primarily based on the following findings: first, RAGE is highly expressed in inflammatory lesions and produces pro-inflammatory mediators in numerous inflammatory illnesses; second, blockage of RAGEs restrained inflammatory response by arresting central inflammation signaling pathways [
31]. In patients with infection-related ARDS, it was found that both serum and BALF levels of sRAGE were much higher than those in control subjects, and they were positively correlated with levels of IL-6 and IL-8 [
9], which was consistent with our findings. In addition, another study revealed that sRAGE enhanced IL-6 release in the absence of S100B in alveolar type I-like cells and that sRAGE could have proinflammatory properties [
32]. sRAGE has been traditionally considered a sink for proinflammatory RAGE ligands and as such has been associated with protection from inflammatory stress and disease [
5]. In addition to behaving as a decoy receptor, sRAGE may transduce proinflammatory signals, thereby inducing leukocyte recruitment to sites of injury or inflammation [
33]. High levels of sRAGE in circulation indicated that cell surface RAGE has been overstimulation, which if it persists might intensify proinflammatory processes and exacerbate pathological states [
34]. Consistent with this role, the bacterial burden and neutrophil infiltration was shown to worsen following sRAGE administration in a mouse model of bacterial lung infection, indicating that sRAGE may indeed sustain inflammation in acute settings [
35]. However, despite having a positive connection with inflammatory factors in LPS-induced mice, we found that sRAGE could not superimpose on LPS-induced autophagy and apoptosis in ATII cells. So, further investigation is required to fully comprehend the intricate biological characteristics of sRAGE on different types of AT cells and their contribution to LPS-induced inflammatory response. In addition, we found that RAGE deficiency significantly reduced pulmonary inflammatory infiltration and pulmonary edema either by reducing cytokine release or by inhibiting autophagy activity. In general, LPS is frequently used to induce sepsis [
18]. Intriguingly, a study reported that the increased serum levels of sRAGE are associated with ALI but not sepsis or septic shock, suggesting that RAGE is a biomarker of lung injury rather than sepsis [
36]. It was verified that
Ager−/− mice were also partially protected from injury following gram-negative (
E. coli) or gram-positive (
Streptococcus pneumoniae) bacterial challenges [
37,
38]. Thus, we speculate that RAGE is the key mediator of ATII cells injury underlying inflammation. In this study, LPS induced the activation of RAGE and autophagy, accompanied by STAT3 phosphorylation, and RAGE deletion leads to weakened activation of downstream protein STAT3. STAT3 activation may be a common signaling mechanism in the pathogenesis of LPS-induced ALI. A study verified that the STAT3 transcription factor is activated in lung injury and promotes macrophage and inflammatory cell infiltration in the lung and BALF, the inhibition of the STAT3 signaling pathway protects the lungs from damage [
39]. The RAGE signaling pathway may directly or indirectly lead to the production of pro-inflammatory cytokines, which can also induce endoplasmic reticulum (ER) stress of chronic inflammation, suggesting that RAGE may be a key mediator of ER stress [
40]. RAGE activation is even found to prolong and excessive UPR in some disease models [
41]. Autophagy is related to ER stress at many levels, autophagy activation occurs under ER stress [
42]. However, there is no relevant study on whether RAGE induced autophagy is related to endoplasmic reticulum stress, which is also the direction of our next exploration.