Severe acute respiratory syndrome (SARS)-CoV-2 pathogenesis, characterized by clinical phenotypes spanning from asymptomatic infection to mild disease with symptoms related to airways tract implication, severe pneumonia, acute respiratory distress syndrome (ARDS) and multiple organ failure, remains largely obscure (Hu et al., 2021). Asymptomatic, mild, and severe diseases have been correlated to different cytokine signatures and to differences in innate and adaptive responses to SARS-CoV-2 infection (Bindoli et al., 2020; Carsetti et al., 2020).

It is now well assessed that SARS-CoV-2 first infects epithelial cells of the upper respiratory tract (nasal passages and throat) and especially lungs (bronchi and alveoli), where alveolar type I and type II cells (AT1 and AT2, respectively) are believed to mediate the first encounter with the virus; infection of alveolar macrophages is determinant in mediating the amplification of the inflammatory and immune responses (Grant et al., 2021).

Mechanistically, cell entry is mediated by the engagement of the receptor angiotensin-converting enzyme (ACE2) (Grant et al., 2021). ACE2 is also expressed by cells in the kidney, blood vessels, heart, whose infection by SARS-CoV-2 may mediate the characteristic multi-organ pathology (Grant et al., 2021). Viral uptake is also promoted by transmembrane protease serine (TMPRSS)2, disintegrin and metallopeptidase domain (ADAM)17 cleaving ACE2, in addition to activating the S protein of the virus for membrane fusion (Heurich et al., 2014; Hoffmann et al., 2020). Neuropilin-1 (NRP1) also potentiates SARS-CoV-2 cell entry and infectivity (Cantuti-Castelvetri et al., 2020).

Similarly to the other serosal membranes (i.e., peritoneum, and pericardium), pleura is lined by a monolayer of mesothelial cells (MCs) arranged on a basement membrane, which separates it from the submesothelial stroma (Mutsaers et al., 2015). MCs differentiation is controlled by the transcription factor Wilms’ tumor (WT)1, which is commonly used for lineage-tracing experiments (Gulyas and Hjerpe, 2003; Karki et al., 2014). The main function of MCs resides in the creation of a slippery non-adhesive surface allowing frictionless movements between adjacent parietal and visceral surfaces (Mutsaers et al., 2015). Moreover, MCs favor leukocyte recirculation and regulate the development of immune responses (Terri et al., 2021).

With respect to pathological conditions such as infections, MCs are a key factor in driving the immune response through the production of large quantities of extracellular mediators such as inflammatory cytokines and chemokines (Terri et al., 2021). Moreover, inflammatory and infectious stimuli may promote a complex multistep phenomenon called mesothelial to mesenchymal transition (MMT) (Lopez-Cabrera, 2014). In this context, MCs progressively lose epithelial-like features, acquiring new mesenchymal-like invasive and profibrotic abilities. Lineage-tracing of WT1-positive MCs in a context of fibrotic lung disease provided evidence of MMT induction in vivo (Sontake et al., 2018).

Due to its anatomic localization, pleura may be affected by many viral infections of the respiratory tract including influenza, coxsackievirus, respiratory syncytial virus (RSV), cytomegalovirus (CMV) (Nestor et al., 2013).

In spite of the conceivable MCs participation in COVID-19 pathogenesis, so far only indirect evidence has been reported. In this study, we highlight that SARS-CoV-2 causes structural modifications in the pleura with disruption of the mesothelial monolayer and the generation of WT1/cytokeratin-positive cells infiltrating the sub-mesothelial stroma. When analysing cellular/molecular mechanisms underlying this event, we found that MeT5A cells (a pleura non tumorigenic mesothelial cell line) express specific receptors and coreceptors for SARS-CoV-2 and produce infectious viral particles. Moreover, MeT5A cells infection resulted in the production of a broad repertoire of interferons, pro- and anti-inflammatory cytokines, and metalloproteases (MMPs).

Overall, this study provides a first evidence on a specific involvement of pleura MCs in SARS-CoV-2 pathology.

Our observations provide a first evidence of SARS-CoV-2-infection/replication and inflammatory cytokine secretion by pleura MCs. Moreover, immunohistochemical analysis of samples from COVID-19 patients demonstrated a specific alteration of pleura characterized by disruption of the MC monolayer and invasion of the sub-mesothelial stroma by spindle-like MCs.

These data fall in a context where pleura-specific role in SARS-CoV-2 pathology has not yet been clarified. In particular, no evidence so far pointed to a direct role of pleura MCs in the COVID-19 pathogenesis. This in spite of the fact that around 10% of patients develop pleural effusions, have higher incidence of severe/critical illness, mortality rate, and longer hospital stay time compared to their counterparts without pleural effusion (Luo et al., 2020; Mo et al., 2020; Zhan et al., 2021). While there are reports linking fibrosis onset with disease severity, the cellular and molecular mechanisms have been incompletely studied so far.

Indeed, circulating ECM components have been correlated with prognosis (Ding et al., 2020). In COVID-19 patients, radiographic evidence of a wide range of pulmonary alterations associated to fibrotic damage and lung functional impairment are commonly observed (Shi et al., 2020). In fatal cases, increased expression of ECM-specific proteins and ECM regulators has been reported (Skibba et al., 2020). These data suggest that lung fibrosis onset is a pathogenic mechanism of severe SARS-CoV-2 infections. Accordingly, post infectious pulmonary fibrosis is also a known outcome in survivors of SARS, an infection with the closely related virus, SARS-CoV (Hui et al., 2005).

In this study, the observation of MCs loss with disruption of the MCs monolayer in biopsies of visceral pleura from autopsies of COVID-19 patients leads to hypothesize a direct or cell-mediated cytopathic effect of the virus. In particular, WT1-and cytokeratin-positive cells with a fibroblastoid morphology (having undergone bona fide MMT) were found the sub-mesothelial stroma.

Since MCs form in normal conditions a continuous monolayer above the basal membrane, the observation of MCs in the sub-mesothelial stroma during SARS-CoV-2 infection implicates the onset of an invasive program in these cells. However, the limited number of patients analyzed in this study made impossible to make correlations with clinical data and to further characterize the underlying invasive process.

Events linked with MCs plasticity may influence the fibrotic process in different ways. MMT in particular, has been demonstrated as a common mechanism of fibrosis in serosal membranes exposed to biomechanical, inflammatory and infectious stimuli (Ruiz-Carpio et al., 2017; Namvar et al., 2018; Raby et al., 2018; Strippoli et al., 2020).

Once transdifferentiated, MCs produce abundant amounts of TGFβ1, fibronectin, collagens (Mutsaers et al., 2015; Rossi et al., 2018). These cells may also rearrange the ECM through the expression of contractile proteins (i.e., αSMA) as well as the production of MMPs, such as MMP-2, -9 and -14 or MMP inhibitors such as TIMP1 and PAI1 (Ma et al., 1999; Strippoli et al., 2020).

So far, only indirect evidence pointed to an ability of SARS-CoV-2 to infect MCs. Viral load has been occasionally demonstrated in pleural fluid of infected patients; in this case, pleura MCs were found with large multiple nuclei, consistent with a cytopathic effect of the virus, although an infection of MCs by SARS-CoV-2 was not been formally proved (Bennett et al., 2020; Malik et al., 2020; Mei et al., 2020). Furthermore, an indirect evidence of a role of MCs is the fact that pleural effusions are linked with the onset of pleural fibrosis, which, along with pericarditis, is frequently found during COVID-19 (Falasca et al., 2020; Zeng et al., 2020). Indeed, MCs are key determinant of lung fibrotic diseases (Mutsaers et al., 2015; Liu et al., 2020).

In this study, we demonstrated that MeT5A cells express the main entry factors implicated in SARS-CoV-2 infection, i.e. ACE2, TMPRRS2, ADAM17 and NRP1. Cleavage of ACE2 has been demonstrated to impact on viral entry (Hoffmann et al., 2020). While it is already known that MCs express high levels of NRP1, a coreceptor of VEGFR with pro-fibrotic activity, the expression of ACE2, TMPRSS2 and ADAM17 has not been reported so far (Perez-Lozano et al., 2013; Cantuti-Castelvetri et al., 2020).

ACE2 levels in airway epithelial cells have been demonstrated to increase during SARS-CoV-2 infection, potentially rendering these patients even more vulnerable to SARS-CoV-2 (Chua et al., 2020). Other events, such as cigarette smoke may also increase ACE2 levels provoking SARS-CoV-2 increased infectivity in these individuals (Liu et al., 2021).

Our data suggest that ACE2 is already expressed in MCs in basal conditions, and the modulation of its expression is not a major mechanism regulating SARS-CoV-2 infection in these cells; in fact we found an high expression of ACE2 in uninfected MeT5A cells, and a tendential increase upon infection with SARS-CoV-2.

SARS-CoV-2 has a strong specificity in terms of cellular effects. For instance, SARS-CoV-2 promotes apoptosis in VERO3, but not in CALU3 cells (Park et al., 2021). In these cell lines, the induction of apoptosis has been related to high virus production and limited induction of interferon response. Moreover, cell culture conditions are extremely relevant to study SARS-CoV-2 effects in the lung and other organs. For instance, freshly isolated primary alveolar cultures from healthy individuals were found only minimally susceptible to SARS-CoV-2 (Hou et al., 2020).

Our data suggest that apoptosis is not induced in infected MeT5A cells. It seems conceivable that in our experimental conditions MeT5A survival is linked to the induction of Interferon response, which provokes the progressive clearance of infection over time. Apoptosis observed in vitro in other cell types (e.g., AT2 cells) may be influenced by specific experimental systems used, such as the use of feeder layer or organoids (Mulay et al., 2021; Katsura et al., 2020; Van Der Vaart et al., 2021). In these latter soft ECM-3D cultures often used to prevent AT2 cell differentiation, the induction of apoptosis may be relatively favored (Zhang et al., 2011; Halder et al., 2012).

By means of multiple approaches, we demonstrated that MCs sustain SARS-CoV-2 infection. Moreover, with respect to the cellular response to infection, we found an early (1.5 h post inoculum) induction of IFN-α and -β mRNA upon treatment with SARS-CoV-2. While the induction of a rapid IFN response is an indirect proof of MC infection, it also witnesses the ability of these cells to effectively clear SARS-CoV-2 infection at later time points. Interestingly, SARS-CoV-2 is sensitive to IFN-β treatment, and it may modulate the onset of the type I IFN response in infected cells through the activity of many viral proteins (Lei et al., 2020).

Differently from in vitro studies, autoptic examination of pleura failed to demonstrate infection of MCs by SARS-CoV-2. The almost complete loss of the MC monolayer, the relatively small number of patients examined or the timing of analysis may explain these negative results. To this respect, further study is warranted.

It is known that cytokine production has both a pathogenic and a prognostic role in SARS-CoV-2 infection. Cytokine storm is responsible for multiorgan pathology and eventually death, and inflammatory cytokine signatures may predict COVID-19 severity and patient survival (Del Valle et al., 2020; Mangalmurti and Hunter, 2020).

With respect to cytokine production by MCs, it should be considered these cells are key players in surveying the composition of the fluids covering the serosal membranes and in leukocyte recirculation. In coordination with resident macrophages and other immune cells, MCs secrete inflammatory cytokines and chemokines during bacterial and viral infections due to specific TLR activation, contributing in the shaping of the subsequent immune response (Terri et al., 2021) (Raby et al., 2018).

The analysis of cytokines secreted by SARS-CoV-2-infected MCs highlighted a predominance of anti-inflammatory (i.e., IL-10, sTNF-Rs) over pro-inflammatory (IL-1β, TNFα, IL-6) response. Indeed, while inflammatory cytokine production was negligible (IL-1β, TNFα) or not significantly increased (IL-6), the production of IL-10 and of TNFRs was significantly enhanced upon viral infection. MCs are known to secrete high amounts of IL-6 in vitro even in unstimulated conditions, while production of TNF-alpha and IL-1 is more restricted to specific pro-inflammatory stimuli (Topley et al., 1993; Douvdevani et al., 1994; Chen et al., 2015).

Of note, increased expression of sTNF-Rs has been previously reported in septic pleural effusions (Marie et al., 1997). The increase of interferons (IFNα−β−γ and IL-28-29) corresponds to an increase of cytokines with anti-inflammatory/immunomodulatory activity (IL-10, IL-11 and IL-20). During SARS-CoV-2 infection, anti-inflammatory mediators are secreted at the same time with pro-inflammatory mediators, and in particular, IL-10 and IL-6 expression both correlate with disease severity (Han et al., 2020). However, IL-10 appears to have a “double edge” activity during inflammation: this cytokine is a key negative regulator of T cell mediated responses, but is also endowed with pro-inflammatory effects, including stimulation of IFNγ production (Lu et al., 2021).

Moreover, MCs produced significantly increased levels of IL-2 and IL-12 and IL-27, which may both activate NK and Th1 lymphocytes and promote antigen presentation. Interestingly, IL-27 is both an IFNγ-induced cytokine and an activator of IL-10 synthesis (Murugaiyan et al., 2009; Blahoianu et al., 2014).

The production of MMPs by MCs is potentially relevant for induction of the pleura fibrotic response observed during SARS-CoV-2 infection. Finally, another significantly increased cytokine was osteocalcin. This cytokine implicated in ECM remodeling promotes bone formation and may counteract osteoporosis observed in SARS-CoV-2 patients. A loop with IL-6, abundantly expressed by MCs, promotes osteocalcin expression (Chowdhury et al., 2020).

It is conceivable that due to the considerable extension of pleura surface (2000 cm² in an average adult male), the serous fluid recycling and the high vascularization of this organ, the predominant production of cytokines with anti-inflammatory activity by MCs may have a systemic effect of homeostatic dampening of the inflammatory response during infection. On the other hand, pleura may contribute to macrophage/lymphocyte activation and, through the secretion of mediators of ECM remodeling such as MMPs, play a role in lung fibrosis.

Of note, biologic drugs interfering the activity of some of the same cytokines here demonstrated to be produced by infected MCs are being currently analyzed for their therapeutic potential to dampen the noxious inflammatory response (Mangalmurti and Hunter, 2020).

Overall, our results suggest that a transient MCs infection, in combination with other local signals released by stromal/immune cells, may promote both cell death with pleura disruption, and induction of MMT with the acquisition of an invasive abilities. At the same time, SARS-CoV-2 infection promotes the secretion of cytokines and other extracellular mediators which may both modulate the inflammatory response and induce ECM remodeling. A comprehensive experimental model of MCs response to SARS-CoV-2 infection is shown in Figure 5.

Thus, MCs are candidates for cellular interventions aimed at restoring the continuity of the monolayer, at modulating the anti-viral immune response and at limiting the insurgence of fibrosis. These discoveries warrant more study to better define the role of MCs and their potential implication for therapy.