Although initially considered relatively harmless pathogens, human coronaviruses (HCoVs) are nowadays known to be associated with more severe clinical complications. Still, their precise pathogenic potential is largely unknown, particularly regarding the most recently identified species HCoV-NL63 and HCoV-HKU1. HCoVs need host cell proteins to successively establish infections. Proteases of the renin–angiotensin system serve as receptors needed for entry into target cells; this article describes the current knowledge on the involvement of this system in HCoV pathogenesis.
Role of the RAS in SARS-CoV & HCoV-NL63 infection
The high mortality rate (approaching 10%) following the SARS-CoV epidemic in 2003 is primarily attributable to respiratory failure caused by development of ARDS. To infect its target host cells, SARS-CoV utilizes ACE2, the RAS component now known to orchestrate protection from acute lung failure/ARDS. Notably, after engagement of viral spike proteins with ACE2, the amount of cell surface-expressed ACE2 is reduced. This phenomenon of ACE2 receptor downregulation has been shown to provoke a worsening of lung failure in a SARS-CoV-infected mouse model. By possessing a remarkable higher level of systemic Ang II, SARS-CoV-treated wild-type mice resemble the phenotype observed in ace2 knockout mice, once again emphasizing the key role of ACE2 during ARDS. The worsened ARDS symptoms observed in infected mice could be partially reversed by AT1 receptor blocker treatment, proving that continuous Ang II binding to AT1 receptors promotes exacerbation of lung injury during SARS-CoV infection. Thus, SARS-CoV can deteriorate acute lung failure through dysregulation of pulmonary RAS activities. It is worth noting that studies analyzing the role of human ace2 gene polymorphisms in progression of lung injury during SARS-CoV infections did not confirm such a correlation. Other unknown factors are possibly involved in the overall mechanism of lung damage induced by SARS-CoV.
At present, there are some indications that downregulation of myocardial ACE2 expression by SARS-CoV induces symptoms of cardiac damage and dysfunction in some SARS patients, including arrhythmias, sudden cardiac death and systolic as well as diastolic dysfunction. Similarly to respiratory infection, SARS-CoV infection of the heart induces a downregulation of cellular ACE2 expression. As an essential regulator of heart function, ACE2 has well established roles in the development of cardiovascular diseases. A reduction in cardiac ACE2 expression most likely results in locally elevated levels of Ang II and might, therefore, also account for these pathophysiological processes, by Ang II-mediated activation of AT1 receptors and loss of the protective effects of Ang (1–7). However, direct evidence for this hypothesis is still lacking.
Although a possible molecular explanation for the severe clinical outcome during SARS-CoV infections is now available, questions remain about the precise pathogenic mechanisms of HCoV-NL63, which also utilizes ACE2 to infect human respiratory cells. In contrast to SARS-CoV, clinical symptoms during HCoV-NL63 infections are usually mild to moderate and alveolar damage is rarely seen. More severe respiratory disease is only observed in immunocompromised patients, the elderly and children. Several scenarios might explain this discrepancy of ACE2 utilization with absence of ARDS symptoms. As discussed, this might be, in part, caused by different cell entry strategies of both CoVs after ACE2 binding. Furthermore, varying binding efficiency to ACE2 could be a feasible explanation. HCoV-NL63 seems to bind ACE2 with a lower affinity compared with SARS-CoV. Moreover, HCoV-NL63 might lack a pathogenicity factor that is present in SARS-CoV. This factor could possibly be encoded by an accessory gene of the SARS-CoV genome. While SARS-CoV possesses an unusually high number of these genes (eight) with a still unknown function, only one is detected in the HCoV-NL63 genome.
The performance of the RAS is well known to decrease during normal aging processes, exemplified by a progressive decrease in circulating renin and plasma renin activity. In this respect, it is important to note that a recent study in post-mortem lung tissue of deaths attributed to chest infection or pneumonia described a high percentage of HCoV-NL63 infection (5.3%), most of whom were elderly, indicating that with increasing age, HCoV-NL63 might lead to significant mortality. To date, only one HCoV-NL63-related death has been described in the literature, namely a 92-year-old man. Aging seems to be involved in SARS-CoV infections as well, since the strongest predictor of poor disease outcome appears to be an advanced age (>60 years). Thus, a HCoV infection may cause more RAS-related damage in the elderly because of an impaired overall RAS activity.
Involvement of the RAS in HCoV-229E infection
Aminopeptidase N, which converts Ang III into Ang IV within the RAS cascade, functions as a receptor for HCoV-229E. Similar to ACE2 downregulation, an abrogation of APN expression could also give rise to potential pathophysiological consequences. Unfortunately, knowledge about APN and its putative role in HCoV-229E pathogenesis is insufficient and far from complete. This is probably, in part, attributable to the absence of severe symptoms during HCoV-229E infections in healthy adults, which are generally associated with mild and self-limiting upper respiratory tract diseases or ‘common colds’. An increasing body of evidence is, however, pointing towards more severe upper and lower respiratory tract illnesses, such as pneumonia, in young children, elderly and immunocompromised individuals. Moreover, certain studies advocate for a putative neuroinvasive and neurovirulent capacity for HCoV-229E. Unraveling a possible contribution of RAS in HCoV-229E infections, in particular through functioning of its enzymatic component APN, might therefore still provide important novel insights in pathogenic mechanisms of this human pathogen. Putative physiological consequences after APN-mediated RAS deregulation might be explained by several distinct scenarios. Here, some of the most interesting theories are discussed, with an emphasis on possible mechanisms evoking or dampening respiratory tract pathologies.
Increased levels of Ang III
Within the RAS, APN is involved in conversion of Ang III to Ang IV. This process can be blocked using a specific APN inhibitor (PC-18), which increases the half-life of Ang III by 3.9-fold. Similarly, APN dowregulation during HCoV-229E infections will most probably contribute to an elevated Ang III expression level at sites of HCoV-229E infection. It is tempting to speculate that, like Ang II upregulation, increased Ang III levels might imbalance RAS and subsequently initiate pathological processes. Most of the current knowledge involving RAS-mediated harmful processes, however, only provide specific evidence for Ang II as a key player and not much is demonstrated for Ang III thus far. Ang II is considered to be a growth factor that regulates cell proliferation/apoptosis and fibrosis, as well as a proinflammatory mediator that attracts inflammatory cells to sites of tissue injury. Since it shares many of its physiological properties with Ang II, Ang III is currently postulated to participate in certain harmful processes as well. In particular, Ang III has been reported to participate in initiation and progression of kidney injury. In mesangial and renal interstitial fibroblast, Ang III binding to AT1 receptors is associated with overexpression of growth-related, profibrotic and proinflammatory genes, a prominent one being TGF-β. TGF-β is known to be a key player in development of morphological alterations, including fibrosis and atrophy, by stimulating synthesis of extracellular matrix components and reducing collagenase production. Also, at sites of human lung tissue injury, TGF-β expression is detected and inhibition of TGF-β in animal models attenuates development of fibrosis. In contrast to a possible role for Ang III overexpression in kidney fibrotic processes, lung tissue injury exacerbation is assumed to be predominantly controlled by Ang II, again by activation of AT1 receptors. Ang II is thought to exert its fibrotic effects by inducing TGF-β1 production, and triggering fibroblast proliferation and differentiation into collagen-secreting cells. AT1 receptor binding by Ang III might be an organ-specific event and in some organs, including the pulmonary system, Ang II is more important in activating AT1 receptors. Results of several studies are indeed pointing towards Ang II as a main effector peptide in lung tissue. Locally produced Ang II in the lung, for example, has recently been demonstrated to be a critical factor in governing bronchial smooth muscle contraction through activation of AT1 receptors. Moreover, Ang II is involved in exacerbation of acute lung injury. However, research regarding Ang III function is still in its infancy and a putative role for Ang III-mediated pathophysiological processes in the respiratory tract is currently unknown and yet to be defined. As a consequence, a role for Ang III in HCoV-229E pathogenesis is far from confirmed.
Reduced levels of Ang IV
Besides a putative elevated expression of Ang III, downregulation of APN may equally result in a reduced formation of Ang IV proteins. In this scenario, AT4 receptors are insufficiently activated. Presence of Ang IV-specific binding sites has been identified in various tissues and cells, including brain, spinal cord, colon, heart, kidney and vascular endothelial cells. With regard to the pulmonary system, Ang IV plays a role in the regulation of blood flow by stimulating release of nitric oxide, a potent vasoconstrictor.Furthermore, Ang IV seems to contribute to proliferation of lung vascular endothelial cells. Most likely, these effects are attributable to systemic Ang IV, which is produced by the ‘peripheral RAS’ and released into the vascular system. Data regarding local pulmonary production of Ang IV and AT4 receptor expression are unfortunately unavailable. Therefore, uncertainty remains as to what extent Ang IV is an actual pulmonary RAS effector peptide, and whether it could be involved in HCoV-229E pathogenesis.
With respect to apn knockout mice, nothing relevant for the mechanism of HCoV-229E pathogenesis has been reported thus far. Despite the broad range of APN functions, mice deficient in APN expression are not severely attenuated, and support only a role for APN in regulation of arterial blood pressure and the pathogenesis of hypertension. This does certainly not exclude a role for APN in HCoV-229E pathogenesis, since initial investigations regarding ace2-/- mice also did not reveal the present knowledge regarding ACE2 and acute lung injury exacerbation. As a consequence, simultaneous induction of acute lung injury/ARDS in this animal model is necessary to elucidate the actual involvement of pulmonary APN in HCoV-229E pathogenesis.
In fact, severe clinical symptoms are generally not observed during HCoV-229E infection and this does not correspond to the previously described potential harmful consequences following modulation of APN expression. With respect to this, an attenuated APN expression at sites of HCoV-229E entrance might also give rise to effects that are beneficial for the host. Most striking is the recent suggestion that APN might actually be involved in regulation of chronic inflammation in the lung, by initiating chemokine production. A continuous influx of inflammatory cells into the lung is not always beneficial for the host and might initiate fibrotic processes and organ-damaging processes. Systemic application of a specific APN inhibitor (actinonin) in a silica-induced murine model of lung fibrosis reduced chemokine secretion (e.g., IL-6 and monocyte chemoattractant protein-1) in lung and bronchoalveolar lavage fluid, and resulted in a decreased level of pulmonary fibrosis.Other systemic effects during these APN inhibitor treatment experiments were not observed. In addition, reduced chemokine secretion after APN inhibitor treatment has been confirmed in vitro, in cultured human lung epithelial cells, advocating for an important role for APN in orchestrating pulmonary chemokine production. A putative involvement of APN in chronic lung inflammation has also been suggested by another study. The activity of APN in bronchoalveolar lavage fluid was significantly higher in patients with sarcoidosis compared with control individuals, correlating to the number of infiltrating T lymphocytes. APN is therefore thought to participate in inflammatory processes by orchestrating lymphocyte chemotaxis.
The rapid accumulation of proinflammatory cytokines (hypercytokinemia or ‘cytokine storm’) and chemokines in the respiratory tract is regarded as a prominent mediator in the pathogenesis of viral infections. Concentrations of IL-6 and -8 in nasal secretions, for instance, correlate with the severity of symptoms observed during upper respiratory tract infections. SARS-CoV, murine hepatitis virus and feline infectious peritonitis virus infections are also characterized by excessive and local invasion of activated immune cells and cytokines. Therefore, it is interesting to speculate that APN is a contributor in the host response against coronaviral infections of nasal mucosa, and that its downregulation might also be accompanied by a dampening of disease pathogenesis. On the contrary, a stark reduction of cytokine and chemokine production is certainly not at all beneficial for the host and will give rise to serious adverse effects, including failure to efficiently combat the viral infection. Maintenance of a balance between host defenses and respiratory tract injury (e.g., fibrosis) is therefore essential.
It is debatable to what extent these putative APN-mediated inflammatory effects (reduction of chemokine production and lymphocyte chemotaxis) are attributable to an imbalance within pulmonary RAS pathways, since a definite mechanistic explanation is not provided by these preliminary studies. Hypothetically, APN may exert its inflammatory potential through formation of the RAS effector peptide Ang IV. On the contrary, it might also be a result of initiation of intracellular signaling and subsequent gene activation by APN, a RAS-independent activity. When APN-mediated intracellular signaling mechanisms are indeed essential in these putative lung protective processes, triggering of such cascades upon HCoV-229E binding should happen as well. It is unknown whether HCoV-229E possesses the capacity to activate intracellular signaling pathways after association with APN. Nonetheless, an abolished APN expression level seems to attenuate development of chronic lung inflammation and pulmonary fibrosis, which is in direct contrast to ACE2 downregulation, which results in exacerbation of lung disease. Notwithstanding the fact that more evidence is definitely needed to demonstrate this APN-induced chemokine production and enhancement of pulmonary disease pathogenesis, it might provide an explanation for the lack of severe clinical symptoms during most HCoV-229E infections. A reduced level of APN expression following HCoV-229E binding would in fact decrease the amount of pulmonary damage after initiation of infection. However, this theory encompassing a major role of APN in pulmonary inflammation might very well be over simplistic since inflammatory responses are generally very complex.
RAS as a highly dynamic system
Besides a potential beneficial effect of downregulation of APN expression, there are several alternative explanations for the general absence of severe clinical complications during HCoV-229E infections. APN is not unique in its ability to convert Ang III into Ang IV. APB is involved in this process as well. This raises the possibility that APB might compensate for an abolished activity of APN, at the same time implicating that Ang III degradation is not severely attenuated. This theory is supported by the notion that RAS is a highly dynamic and multilayered system. For instance, genetically engineered mice that do not express endothelial ACE and therefore lack ACE within the lung, are still capable of maintaining normal physiology. Lung chymase or an increased generation of Ang II by nonendothelial ACE may counterbalance the lack of local ACE expression, revealing a compensatory mechanism within the RAS. It is, therefore, not unlikely that besides APB, additional bypass mechanisms exist that maintain normal physiology after virus-induced APN internalization.
Increasing evidence is pointing towards an important role for the RAS during HCoV pathogenesis. Here, we addressed the specific interplay of three HCoVs with the RAS, in order to obtain novel clues regarding their pathogenicity in the human host. Clearly, pulmonary RAS negatively affects severe acute lung injury during SARS-CoV infection. Nevertheless, a lot of issues regarding ACE2 involvement in CoV pathogenesis still remain unsolved. Particularly, the lack of severe lung injury after infection with the newly identified HCoV-NL63, which also targets ACE2 as its primary entry receptor, is remarkable. The absence of a compensatory mechanism within RAS for the abolished pulmonary ACE2 expression is also highly interesting and will require further investigation.
Although far from confirmed, an interesting and putative role for a second RAS component, APN, in HCoV infection is reviewed as well. Preliminary data provide an attractive explanation for the general absence of severe clinical symptoms during infections with HCoV-229E. Disturbances in APN expression levels might protect from an overactivated inflammatory response (‘cytokine storm’), a process linked to initiation of organ fibrosis and failure, by orchestrating lymphocyte chemotaxis into HCoV-229E-infected lung tissue. Important clues might be obtained by lung injury induction studies with animal models lacking APN expression, since current knowledge is strictly based on APN inhibitor experiments.
Coronaviral pathogenesis is a highly complex process in which interactions between host and pathogen determine the outcome of virus-induced disease. It is becoming evident that the RAS is involved in HCoV infections, and presumably of high importance for their pathogenicity. In addition, these findings once more confirm the hypothesis that severe imbalances within the tightly regulated RAS provoke a broad spectrum of pathologies. Elucidation of the exact pathophysiological roles of the pulmonary RAS certainly contributes to clarification of the strategies used by HCoVs to elicit specific diseases, and might provide a definite demonstration of their etiology. Eventually, a better understanding of HCoV pathogenesis might lead to development of new therapeutic strategies and/or vaccination protocols.
Deregulation of the RAS may very well be a part of the pathogenicity of HCoVinfections. A therapy aimed at restoring the RAS equilibrium provides the opportunity to treat the symptoms of an infection. Especially in elderly patients, this treatment might be beneficial as the aged population is most vulnerable to deregulation of the RAS. ACE inhibitors and AT1 receptor antagonists are in use to decrease high blood pressure. Future research may elucidate the benefit of local administration of these drugs to neutralize the effects of HCoV-NL63- or SARS-CoV-induced ACE2 downregulation in the lungs.
Terminology of renin–angiotensin system-involved aminopeptidases.
Aminopeptidases constitute a diverse set of peptidases, and serve to proteolytically process amino acid residues from the N-terminus of protein and bioactive peptides. Since these enzymes have been identified using several different characteristics, including number of removed amino acids; residue preference; cellular location; metal ion content and pH at which maximum activity is observed, a particularly complex labyrinth of aminopeptidase classification systems currently exists. Aminopeptidase N (EC184.108.40.206) shows a preference for cleavage of neutral amino acid residues. Since Ala is the amino acid most efficiently broken down by this peptidase, it is also named alanyl aminopeptidase. Furthermore, the enzyme is known as microsomal leucine aminopeptidase or aminopeptidase M, reflecting its close association with microsomal membrane fractions in pig kidney from which it was purified. Sequence comparisons revealed that aminopeptidase N is identical to CD13, a cell surface glycoprotein originally defined on subsets of normal and malignant myeloid cells. Aminopeptidase B (EC220.127.116.11) preferentially cleaves N-terminal basic amino acids. Since many enzymatic assays have been performed using Arg-naphthylamide, the enzyme is also known as arginine aminopeptidase. In addition, aminopeptidase B is indicated as arylamidase II or cytosol aminopeptidase IV. Aminopeptidase A (EC18.104.22.168) hydrolyzes N-terminal acidic amino acids. Since glutamyl derivates are most efficiently hydrolyzed, the enzyme is also designated glutamyl aminopeptidase. Alternative names include angiotensinase A, aspartate aminopeptidase, glutamyl peptidase and membrane aminopeptidase II.
Coronaviruses are considered important human pathogens
Human coronaviruses (HCoVs) initially seemed to be associated primarily with self-limiting upper respiratory tract infections in healthy adults, characterized by mild clinical symptoms.
SARS-CoV has been proven to cause severe lower respiratory tract infections, causing high morbidity and mortality during the SARS epidemic in 2003.
At present, all HCoV isolates are being recognized as causative agents of more severe respiratory tract complications, including pneumonia, especially in weakened patients: infants, elderly and immunocompromised individuals.
Precise pathogenic potential of HCoV species remains largely unconfirmed
Since studies needed to unravel a causal link with a specific disease are hampered by a lack of a suitable animal model and/or cell culture system, current knowledge has been primarily obtained through population-based studies.
Elucidation of the physiologic consequences following virus–host interaction processes might provide alternative insights into the complex process of HCoV pathogenesis.
The renin–angiotensin system is involved in human coronavirus pathogenesis
Two integral proteases of the renin–angiotensin system (RAS) are entry receptors for HCoVs: neutral aminopeptidase and angiotensin-converting enzyme (ACE)2.
By means of downmodulation of cellular ACE2 expression, SARS-CoV directly impairs normal physiologic function of ACE2 within the RAS. As ACE2 is a prominent negative regulator and protects from worsening of acute lung injury, a loss of ACE2 expression is thought to provoke the severe symptoms observed during infection with SARS-CoV. The potency of HCoV-NL63 to unbalance the RAS needs further investigation.
Although unconfirmed, abolished cellular expression of aminopeptidase N might prevent over-activation of pulmonary inflammation and organ damage, which might explain the general absence of severe clinical symptoms during HCoV-229E infections.
Reference & source information: https://www.futuremedicine.com/
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