ACE2 and the Inflammatory Response to SARS-CoV-2
Differently from other flu-like viral infections, COVID19 showed a relatively high lethality, although the latter was quite different in different geographical areas. The appearance of ARDS and acute severe lung injury was initially regarded as the main insult, and patients underwent the typical treatment developed for ARDS. However, more recent evidence from autopsy series revealed that COVID-19 is a systemic disease, in which interstitial pneumonia is associated with extensive microvascular damage, which is not limited to the lungs and leads to fibrin deposition, neutrophils trapping in microvasculature, and arteriolar microthrombi. Moreover, cardiovascular involvement has been observed, occasionally leading to myocardial injury and sudden death after weeks from the initial infection. These findings are crucial to optimize the treatment of COVID-19 patients, hopefully improving the prognosis (Buja et al., 2020; Carsana et al., 2020; Magro et al., 2020).
Another determinant of the clinical picture and a negative prognostic factor is an aggressive proinflammatory response of the host to the infection. Huang described the clinical features of 41 patients admitted to their Institute in January of 2020 with confirmed COVID-19 and pneumonia. In 29% (n = 12) of patients ARDS occurred and in 10% invasive mechanical ventilation was required. These patients clearly showed increased production of IL-1β, IFN-γ, IP-10, MCP-1, IL-4, and IL-10 compared to healthy people. Moreover, the patients who required access to the intensive care unit had significantly higher levels of proinflammatory cytokines compared to those who did not require intensive care, suggesting that this “cytokine storm” may trigger the development of the most severe forms of the disease (Huang C. et al., 2020).
The molecular mechanisms involved in the cytokine storm and the role of ACE2 are currently still not fully understood. As discussed above, virus binding to the ACE2 is the event that initiates viral replication in susceptible cells, such as alveolar epithelial cells, vascular cells and immune system cells (macrophages, monocytes). This has been suggested to trigger the primary inflammatory response, which involves apoptosis and pyroptosis (Fu et al., 2020). The apoptosis pattern indices cell death to avoid viral replication in the absence of overt inflammation, whereas, pyroptosis is a violent form of programmed cell death, followed by an inflammatory storm (Cookson and Brennan, 2001; Liu et al., 2016). In the standard pyroptosis model, when the pathogen enters the host cell, some specific structures on the pathogen surface (PAMPs–pathogen associated molecular patterns) are identified by pattern recognition receptors (PRRs) on the host membrane. One common PRR is NOD-like receptors protein 3 (NLRP3), which forms together with a protein caspase activating protein (ASC or apoptosis-associated speck-like protein containing a caspase recruitment domain) and pro-caspase 1, the inflammasome unit capable of recruiting proinflammatory cytokines and inducing cell lysis with further inflammation signals (Fernandes-Alnemri et al., 2007; Schroder and Tschopp, 2010; Wree et al., 2014; Figure 9).
FIGURE 9  Possible role of ACE2 in the molecular mechanisms involved in the “cytokine storm” of SARS-CoV-2. In the SARS-CoV-2 infection, pyroptosis has been demonstrated to be active in macrophages, being viroporin 3a a trigger for NLRP3 with subsequent production of proinflammatory cytokines, such as IL-1β (Chen et al., 2019). In COVID-19 patients, the pyroptosis activation has been indirectly demonstrated by the high serum level of IL-1β (Huang C. et al., 2020). Moreover, the severe leucopoenia and lymphopenia, commonly showed in COVID-19 pneumonia and associated with poor prognosis, are likely due to lymphocyte injury by pyroptosis (Yang, 2020).
Another proposed mechanism that triggers the proinflammatory response to SARS-CoV-2 is ACE/ACE2 imbalance in the RAAS system. It has been speculated that when ACE2 is occupied by the virus, its effects in limiting the amount and activity of Ang-II is decreased; Ang-II is consequently increased and it has been reported to induce a proinflammatory effect via the AT1R receptor (Marchesi et al., 2008; Eguchi et al., 2018). The Ang-II/AT1R system activates disintegrin and the metalloprotease ADAM 17 (also known as TNFα cleavage enzyme TACE), which lead to the intracellular production of epidermal growth factor ligands (EGFR) and TNFα production, with subsequent stimulation of the transcription factor NF-K B, a pivotal player in proinflammatory cytokines release. Additionally, ADAM 17 activation induces the production of the soluble form of IL-6Rα, active on IL-6-STAT3 pathway, which in turn amplifies NF-K B signaling (Murakami et al., 2019; Takimoto-Ohnishi and Murakami, 2019; Palau et al., 2020). The convergence on hyperactivation of NF-K B seems to be crucial in inducing the cytokine storm. The mechanism is self-feeding, given that NF-K B induces the expression of the angiotensinogen gene, amplifying the Ang-II inflammatory response (Costanzo et al., 2003; Hirano and Murakami, 2020).
ACE2 shedding is an additional mechanism contributing to inflammation (Fu et al., 2020). ACE2 shedding consists in proteolytic cleavage of the protein ectodomain and leads to the release of enzymatically active soluble ACE2 (sACE), whose action is not completely understood, but might enhance the pro-inflammatory response (Guy et al., 2005). ADAM 17 and disintegrin are able to shed not only TNFα but also a variety of membrane-anchored enzymes, including ACE2 (Black et al., 1997; Palau et al., 2020), as demonstrated by Lambert in 2005 (Guy et al., 2005). In their experiment, human embryonic kidney cells (HEK293) expressing human ACE2 (HEK-ACE2) were treated with phorbol myristate acetate stimulation and the production of a polypeptide of 105 kDa was demonstrated by western blot analysis, suggesting the occurrence of a proteolytic shedding in the ectodomain of ACE2. Subsequently, they demonstrated that the cleavage was performed by ADAM17, incubating cells with a specific hydroxamic acid-based metalloproteinase inhibitor, namely TAPI-1 (TNFα protease inhibitor 1) (Guy et al., 2005).
ACE2 shedding seems to be inducible by specific stimuli produced either by a viral infection or by the immune system. Jia et al. (2009) confirmed ACE2 shedding in differentiated primary airway epithelial cells and Calu-3-cell line. Furthermore, they demonstrated that the incubation with IL-1β at the dosage of 100 ng/ml and TNF α 100 ng/ml induced ACE2 shedding and sACE release, with a maximum after 18 h of incubation (Jia et al., 2009). These findings suggest that an inflammatory response is likely to modulate ACE2 expression and shedding, also during a viral infection. Following this data, ADAM17 inhibition was proposed as a possible pharmacological target in SARS-CoV-2, but further studies are needed to evaluate this hypothesis (Palau et al., 2020).
Other conditions have been assumed to be involved in the susceptibility to SARS-CoV-2 and among them vitamin D deficiency was proposed as a credible candidate. The interesting candidate could be identified as a modifiable risk factor (hypovitaminosis D) and a potential tool in COVID-19 prevention or ancillary treatment. The rationale has been summarized by Grant et al. in a recent review on the evidence supporting a possible correlation between vitamin D and SARS-CoV-2 risk: (i) the seasonal flare of SARS-CoV-2 which coincides with the nadir of vitamin D levels, (ii) the association between hypovitaminosis D and pulmonary infections together with the demonstrated protective role in acute respiratory infections, in adults (iii) the anti-inflammatory role of vitamin D which could be of benefit against the so called “cytokine storm,” which seems to be a major player in SARS-CoV-2 morbidity and mortality (Martineau et al., 2017; Zhou et al., 2019; Grant et al., 2020).