SARS-CoV2 at the Alveolar Epithelial-Endothelial Interface – A Hypothesis for the Pathogenesis of COVID-19

Severe COVID-19 is associated with viraemia and multiple organ disease. Similar clinicopathological features have been previously seen in SARS and MERS. Clinically, the severity of SARS, MERS and COVID-19 has been associated with the presence of SARSCoV, MERS-CoV or SARS-CoV2 viraemia in affected patients. In vitro work has looked at the pattern of viral entry and release from polarised epithelial cells infected by coronaviruses. This work has demonstrated a correlation between the severity of a coronavirus infection and the ability of the virus to reach and infect the basal surface of host cells. It has been postulated that this ability helps the virus invade the bloodstream of the host, resulting in a systemic infection with multiple organ involvement. Here we propose that basal surface release and entry of COVID-19 into and out of cells at epithelial-endothelial interface plays a key pathogenic role in severe COVID-19 disease. Background The breach of the epithelial-endothelial barrier at the alveoli appears to be a critical step for the development of severe COVID-19 infection. It has been previously suggested that immune and endothelial dysfunction may play an important role in this process. It has been postulated that the breach of the epithelial-endothelial barrier begins with inflammation and the enrolment immune cells into the alveolar cavity. From there, it is hypothesised, that SARS-CoV2 infected immune cells invade the bloodstream through a leaky endothelium (1,2). Thus, providing a pathway through which viraemia and systemic disease may occur in severe COVID-19. Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 31 May 2020 doi:10.20944/preprints202005.0480.v1 © 2020 by the author(s). Distributed under a Creative Commons CC BY license. However, little has been said about the pattern of SARS-CoV2 entry and release from infected cells at alveolar epithelial-endothelial interface. Here, based on clinical, pathological, cellular and molecular evidence we suggest that in severe COVID-19 disease critical pathogenic events may occur at the alveolar supportive tissue where epithelium and endothelium meet. Viraemia and multiple organ involvement are associated with severe SARS, MERS and COVID-19 Previously, the human coronaviruses SARS-CoV and MERS-CoV have caused the international SARS and MERS epidemics, respectively (3,4). Clinically COVID-19 resembles SARS and MERS, with most cases of these infections showing a self-limiting respiratory infection and a minority of cases showing severe multi-organ disease (3–6). Like COVID-19, SARS and MERS are associated with viraemia (7–10). Interestingly, viraemia in SARS, MERS and COVID-19 correlates with poorer clinical outcomes. The extent of SARS-CoV2 RNAaemia is closely correlated with level of the proinflammatory cytokine IL-6, a marker of COVID-19 disease, and poorer prognosis (11). Similarly, SARS-CoV RNAaemia was previously found to be associated with prolonged illness (12) and admission to an intensive care unit (9). Furthermore, MERS-CoV RNAaemia has been correlated with markedly increased risk of death (8). It is important to note, that MERS infection is associated with a greater degree of mortality and viraemia compared to COVID19. Thus, While SARS-CoV2 RNAaemia was found in 15% of hospitalised COVID-19 patients (11), MERS-CoV2 RNAaemia was reported in 30% of cases (8). Thus, evidence from SARS, MERS and Covid-19 clinical research suggests that viraemia may play an important pathogenic role in severe disease. Histology of the alveolar wall facilitates the infection of different cell types The alveolar wall consists of three elements: surface epithelium, supporting tissue and blood vessels. Surface epithethilium is made up of type I and II alveolar epithelial cells. The supporting tissue is formed by the basement membrane (fine reticular, collagenous and elastic fibres) and occasional fibroblasts. Blood vessels are mainly capillaries made up of endothelial cells (13). The alveolar walls are defended locally by alveolar macrophages that reside in the alveolar cavity. Blood born dendritic cells also play a role in detecting antigens in the alveolar cavity but to do this they extend processes from the supporting tissue layer where they can temporarily reside (14). In addition, neutrophils, macrophages and lymphocytes can be recruited to the alveolar wall through the vasculature during infection (2). Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 31 May 2020 doi:10.20944/preprints202005.0480.v1

However, little has been said about the pattern of SARS-CoV2 entry and release from infected cells at alveolar epithelial-endothelial interface. Here, based on clinical, pathological, cellular and molecular evidence we suggest that in severe COVID-19 disease critical pathogenic events may occur at the alveolar supportive tissue where epithelium and endothelium meet.
Viraemia and multiple organ involvement are associated with severe SARS,
Interestingly, viraemia in SARS, MERS and COVID-19 correlates with poorer clinical outcomes. The extent of SARS-CoV2 RNAaemia is closely correlated with level of the proinflammatory cytokine IL-6, a marker of COVID-19 disease, and poorer prognosis (11).
Similarly, SARS-CoV RNAaemia was previously found to be associated with prolonged illness (12) and admission to an intensive care unit (9). Furthermore, MERS-CoV RNAaemia has been correlated with markedly increased risk of death (8). It is important to note, that MERS infection is associated with a greater degree of mortality and viraemia compared to COVID-19. Thus, While SARS-CoV2 RNAaemia was found in 15% of hospitalised COVID-19 patients (11), MERS-CoV2 RNAaemia was reported in 30% of cases (8).
Thus, evidence from SARS, MERS and Covid-19 clinical research suggests that viraemia may play an important pathogenic role in severe disease.

Histology of the alveolar wall facilitates the infection of different cell types
The alveolar wall consists of three elements: surface epithelium, supporting tissue and blood vessels. Surface epithethilium is made up of type I and II alveolar epithelial cells. The supporting tissue is formed by the basement membrane (fine reticular, collagenous and elastic fibres) and occasional fibroblasts. Blood vessels are mainly capillaries made up of endothelial cells (13).
The alveolar walls are defended locally by alveolar macrophages that reside in the alveolar cavity. Blood born dendritic cells also play a role in detecting antigens in the alveolar cavity but to do this they extend processes from the supporting tissue layer where they can temporarily reside (14). In addition, neutrophils, macrophages and lymphocytes can be recruited to the alveolar wall through the vasculature during infection (2).
The supporting tissue in the alveolar walls forms an attenuated layer beneath the epithelium and surrounding blood vessels. In most of the alveolar wall, the basement membrane which supports the capillary endothelium is directly applied to the basement membrane supporting the alveolar epithelium (13). At these points, only 100nm separates the basal surface of epithelial cells and endothelial cells in the alveoli (15).
Post-mortem studies of SARS cases looking at the cellular distribution of SARS-CoV infection have shown that the main target of the virus is alveolar epithelial cells (16,17). Type II alveolar epithelial cells appear to be preferentially affected compared to type I (16). However, other cell types, including alveolar macrophages, lymphocytes and fibroblast, also demonstrate evidence of SARS-CoV replication within them. Importantly, endothelial cells are positive for intracellular SARS-CoV RNA demonstrating that, despite not been in direct contact with the air rich alveolar cavity, they have been infected by the virus (7). Interestingly, although there is post-mortem evidence that SARSC-CoV2 is primarily infects in alveolar epithelial cells (18) The S-proteins in CoVs bind to specific cell surface receptors in target cells to initiate infection.
The pattern of ACE2 and DPP4 expression is consistent with the potential for COVID-19,  pathology. In this vein, recent work has suggested that the non-structural protein nsp7 interacts with Rab proteins, a family of intracellular transport regulators, in SARS-CoV2 infected cells (47). Targeting therapies to nsp7 may be a novel avenue of treatment.
Finally, focusing research on the interface between epithelial and endothelial may draw our attention to therapies that target the basement membrane and supportive tissue in the alveoli such as those used for pulmonary fibrosis (48). Targeting pharmacological therapies to the critical areas of pathology may result in more effective treatment for COVID-19.

Contributors
ADAS carried out the literature search and drafted the paper. ADAS and FCAS reviewed the evidence and formed the hypothesis. FCAS revised the manuscript. ADAS and FCAS have reviewed the paper prior to submission. This manuscript has not been currently accepted or published elsewhere.

Declaration of interests
We declare no competing interests.