Early activation of pro-fibrotic WNT5A in sepsis-induced acute lung injury

Acute respiratory distress syndrome (ARDS) is a severe inflammatory process caused by pulmonary or systemic insults to the lung alveolar-capillary barrier [1]-[3]. Sepsis is the most common predisposing factor underlying ARDS and is characterized by systemic inflammation in response to circulating microbes or microbial toxins such as lipopolysaccharide (LPS), also termed endotoxin, a component of the cell wall of gram-negative bacteria. Sepsis and sepsis-induced ARDS are common syndromes associated with high morbidity and mortality [1],[2],[4]. Effective repair of the alveolar epithelium requires proliferation and migration of type-II alveolar epithelial cells, and their differentiation into type-I alveolar cells [1],[5]. In addition, lung fibroblast migration and proliferation occur early after lung injury and are necessary for ongoing lung healing [6]-[8]. Damage to the alveolar epithelium can lead to abnormal repair that culminates in a vigorous fibroblastic response, leading to uncontrolled extracellular matrix deposition and destruction of lung parenchymal architecture [8],[9].

The role of β-catenin-mediated wingless integration (Wnt) signaling is proving to be central to mechanisms of lung healing and fibrosis [10],[11]. Tissue repair involves re-epithelialization, in which injured cells are replaced by cells of the same type and normal parenchyma may be replaced by connective tissue leading to fibrosis [11]. Königshoff et al. [10] showed that WNT ligands induce lung epithelial cell proliferation, fibroblast activation, and collagen synthesis, and is upregulated in a bleomycin-induced lung injury model and also in humans with idiopathic pulmonary fibrosis. Wnt binding to cognate Frizzled receptors results in cytosolic accumulation of β-catenin, which then translocates to the nucleus and participates in gene transcription [11]-[13]. Wnt/β-catenin signaling stimulates tissue remodeling and wound closure, or tissue remodeling and destruction through matrix metallopeptidases (MMPs) and other gene products [14]. This activation stimulates many of the pro-inflammatory cytokines participating in inflammation-mediated lung destruction and hyaline membrane formation [12], and induces expression of growth-associated genes such as cyclin D1 and vascular endothelial growth factor (VEGF) [15]. MMP7 (also known as matrilysin) is a target gene of the Wnt signaling pathway found on the surface of lung epithelial cells and is a key regulator of pulmonary fibrosis [16].

In the present study, we examined the hypothesis that the Wnt/β-catenin pathway is activated in the lungs very early after sepsis and plays a role in initiating the lung repair process. To test this hypothesis we used a well-established LPS-induced cell injury model using human lung cells based on the first steps in the development of sepsis and sepsis-induced ARDS [17]-[21]. Then, we validated the main gene targets of this pathway in a clinically relevant murine model of sepsis-induced ARDS by cecal ligation and perforation (CLP), and in lung biopsies obtained from patients who died within the first 24 h of septic ARDS.

We examined the translational impact of the WNT/β-catenin pathway in an LPS-induced human lung cell injury model and validated the main gene targets of this pathway in the lungs of septic experimental animals and in human lungs from autopsies. The major findings of our study are: (1) WNT5A is expressed very early by human airway epithelial cells and lung fibroblasts in response to LPS; (2) upregulation of WNT5A expression and non-phospho Ser33/37/Thr41 β-catenin are associated with upregulation of downstream target genes that are involved in profibrotic transformation of injured tissues, such as MMP7, cyclin D1 and VEGF; and (3) pulmonary fibrosis is induced very early during sepsis-induced ARDS, both experimentally and clinically. These findings suggest that WNT5A and β-catenin contribute very early to repair the damage to lung tissue and may play a role in restructuring lung architecture during sepsis-induced ARDS.

We selected BEAS-2B and MRC-5 cell lines as representative human airway epithelial cells and lung fibroblasts because these cells have been implicated in the pathogenesis of sepsis-induced ARDS [18]-[24] and subsequent fibrosis [31]. These cell models provide a powerful translational in vitro approach for recapitulating human ARDS. LPS-treated human BEAS-2B cells are an accepted and validated in vitro cell injury model of the acute lung inflammatory response based on the first steps in the development of sepsis and sepsis-induced ARDS [18]. Lung airway epithelial cells and fibroblasts generate various immune effectors such as cytokines, chemokines, and several peptides in response to inflammatory stimuli [23],[32], which control lung inflammation, lung injury and lung repair [9],[12],[31],[33]. We selected E. coli LPS because it has been used in most endotoxin-induced lung injury models [21],[34] and LPS is a key pathogen recognition molecule for sepsis [33],[34]. Because previous in vitro studies using LPS-stimulated airway epithelial cells and fibroblasts focused on activation of pro-inflammatory mediators and increased cytokine release [20],[35],[36], we examined the modulation of WNT5A, β-catenin, MMP7, cyclin D1 and VEGF molecules that contribute to lung repair and fibrosis [12],[16],[37].

We extended our in vitro findings by confirming that collagen synthesis and the main target gene products of this pathway (WNT5A, MMP7) increased in a clinically relevant model of sepsis-induced lung injury and in lungs from patients who died with severe sepsis and ARDS. We used CLP as a clinically relevant and well characterized animal model to explore the fibrotic transformation in the lungs during the first 24 h of sepsis. CLP induced a reproducible and consistent septic and sepsis-induced ARDS condition in accordance with previous studies [17],[28]. Histopathological features of CLP-induced ARDS in animals included atelectasis, pulmonary edema, and acute inflammatory infiltrates. Lung tissue damage is observed in 90% of patients dying from sepsis [38]. Moreover, lung cells can activate mechanisms for initiating tissue repair, a process which involves re-epithelialization; injured cells are replaced by cells of the same type, but in some cases, normal parenchyma is replaced by connective tissue leading to fibrosis [11]. There is evidence of fibrotic changes in the earliest stages of ARDS [26],[39],[40]. β-catenin signaling stimulates tissue remodeling, cell migration, and wound closure through MMPs, but if the process is uncontrolled, it can drive tissue destruction through MMPs and other mediators [11]. Wnt ligands induce lung epithelial cell proliferation, fibroblast activation and collagen synthesis [16]. Collagen and other matrix extracellular molecules are the main components of the extracellular matrix, and MMP7 is a key mediator of pulmonary fibrosis [16].

Several Wnt genes are expressed in the developing and adult lung. Of these, Wnt5a and Wnt7b are expressed at high levels in the airway epithelium [14]. We chose to examine the modulation of WNT5A because it has been implicated in several pulmonary disorders [11] and has not been studied in the context of sepsis and LPS-induced ARDS. In our study, WNT5A was detected with moderate intensity in alveolar walls and septa in the lungs of CLP rats and in the lungs of humans who died with early septic ARDS. Blumenthal et al. [41] reported that the expression of WNT5A required Toll-like receptor signaling and NF-κB activation. In a previous report by our group, and using the same epithelial cell injury model as in the present study, we showed that LPS modulated the NF-κB activation through the Toll-like receptor signaling [22]. The fact that β-catenin is rapidly upregulated in our epithelial/fibroblast cell injury model suggests that the WNT/β-catenin pathway could be continuously stimulated during ARDS and it could be a mechanism for perpetuating lung injury or for initiating lung repair. Thus, the activation of Wnt signaling after sepsis-induced ARDS likely represents a regenerative signal of the damaged epithelium [42]. Using expression microarrays, Vuga et al. [43] showed that WNT5A was significantly increased in fibroblasts isolated from lung tissues of patients with lung fibrosis compared with fibroblasts from normal lung tissues. They also reported increased cell proliferation when normal lung fibroblasts were treated with WNT5A.

Our findings parallel those of Chilosi et al. [44] who found aberrant WNT/β-catenin pathway activation in lungs from patients with idiopathic pulmonary fibrosis, suggesting that this pathway could be responsible for dysfunctional lung repair processes leading to severe and irreversible pulmonary remodeling. This is a relevant translational finding because the development of pulmonary fibrosis has been found to have a direct correlation with severity of lung injury and mortality in ARDS patients [45]. The cell cycle regulatory molecule cyclin D1 gene is one of the target genes for the Wnt/β-catenin signaling pathway, and VEGF is required for maintenance of adult lung alveolar structures. Any tissue repair involves coordinated cellular infiltration together with extracellular matrix deposition and re-epithelialization. Proteolytic degradation of the extracellular matrix requires MMPs which are regulated by Wnt signaling. It is uncertain why ARDS resolution involves fibrosis in some patients but not in others. Using western blot analysis of Wnt target gene products cyclin D1 and MMP7, Königshoff et al. [16] demonstrated increased functional Wnt/β-catenin signaling in pulmonary fibrosis compared with patients without pulmonary fibrosis. Zuo et al. [46] analyzed samples from patients with pulmonary fibrosis using microarray technology and found that Mmp7 was the most upregulated gene, a finding that was confirmed by immunohistochemistry. The increased expression of cyclin D1, VEGF, and MMP7 in our study supports the importance of Wnt signaling in perpetuating lung inflammation and provides insights into the early development of a pro-fibrotic response during sepsis-induced ARDS. A greater understanding of modulators of WNT expression and the effects of WNT proteins in similar models will be paramount for clarifying the role of this pathway in lung inflammation and repair.

Our study does have some limitations. First, although the animal model used in the present investigation was CLP, we have examined autopsies from patients with different types of septic ARDS. However, there are no data suggesting that there is anything specific about pulmonary versus non-pulmonary insults in terms of different pulmonary fibrotic responses during severe sepsis. In an acid aspiration lung injury model, we found a similar fibrotic transformation as in our septic model [26]. A recent study [40] has shown that pulmonary fibrosis represents an early pathologic response in patients with ARDS, independent of the pulmonary or extrapulmonary nature of its cause. Second, we did not explore the effects of inhibitors of the Wnt pathway to irrefutably demonstrate that activation of Wnt pathway in the lung by a septic insult is responsible for the upregulation of downstream target genes (such as MMP7, cyclin D1, VEGF) that are involved in the pro-fibrotic transformation of injured tissues. However, studies by other investigators on selective inhibition of the Wnt/β-catenin signaling pathway [44],[47],[48] have indicated that the WNT/β-catenin pathway is a target for anti-inflammatory and anti-fibrotic actions.