La Trobe University researchers are the first in the world to characterize precisely how COVID-19 attacks lung tissues – an important step in preventing long-term damage.
Publishing their results today in Communications Biology Journal the researchers produced atomic-level images of the interplay between a protein found in the virus, and those in human tissues, including lungs.
Study co-author, Professor Marc Kvansakul from La Trobe University, said understanding how the virus attacks lung tissue is critical if we are to prevent long-term lung damage in some COVID-19 patients – including those with few or no risk factors.
“We’ve seen many patients around the world recover from the acute phase of the disease, only to discover that they have long-term damage to lungs and other organs,” Professor Kvansakul said.
“Pinpointing exactly how this damage occurs brings us an important step closer to developing treatments that can be administered while patients are still in intensive care.
“The ultimate aim is to help people recover faster and more completely, and prevent any lingering respiratory issues,” Professor Kvansakul said.
Using powerful beams of light at the Australian Synchrotron, the researchers were able to produce images of how the SARS-CoV-2 E binds to and hijacks Pals1, a key protein found in human tissue.
This then creates a gap for the virus to enter the delicate lung tissue, leading to irreversible scarring.
Study co-author, Professor Patrick Humbert from La Trobe University, said although new COVID-19 vaccines are being administered around the world, finding treatments to combat its long-term effects remains critical.
“We’ve already seen how this virus can mutate into new strains, meaning our current vaccines won’t always be effective,” Professor Humbert said.
“There are also many countries around the world – including Australia – that are unlikely to achieve high vaccination levels for some time.
“The virus is going to be with us for a very long time, so helping people achieve a fast and complete recovery is absolutely vital,” Professor Humbert said.
Professor Kvansakul said the next step is to develop drugs to target this virus-host interaction – treatments that could potentially reduce infectivity and viral spread, as well as lung damage.
This would be especially important for people who have not been vaccinated or who have poor responses to the current vaccines.
The worldwide pandemic of coronavirus disease 2019 (COVID-19) caused by infection with severe acute respiratory syndrome (SARS) coronavirus (CoV)-2, a member the Betacoronavirus genus in the subfamily Orthocoronavirinae, which has led to more than 2.5 million deaths (https://coronavirus.jhu.edu), urges a focused search for the underlying pathogenic mechanisms and treatment options.1–3
The lung is the primary organ affected in patients with COVID-19, wherein the primary cause of mortality is hypoxic respiratory failure, resulting from acute respiratory distress syndrome (ARDS), with severe hypoxemia, often requiring assisted ventilation.4 While similar, in some ways, to ARDS secondary to other causes, lungs of patients with COVID-19 exhibit distinct vascular involvement features, including severe endothelial injury and cell death via apoptosis and/or pyroptosis, widespread capillary inflammation, and thrombosis.5,6
Furthermore, the pulmonary pathology of COVID-19 is characterized by focal inflammatory cell infiltration, impeding alveolar gas exchange and resulting in areas of local tissue hypoxia, consistent with the idea that a feedback loop exists between viral infection and hypoxia in disease progression. Vascular endothelial cells play an essential role in both innate and adaptive immune responses, and are considered to be “conditional innate immune cells” centrally participating in various inflammatory and immune pathologies.7,8
Activated endothelial cells produce cytokines/chemokines, dynamically recruit and activate inflammatory cells and platelets, and centrally participate in pro-thrombotic processes (microangiopathy).9–11 Endothelial cells are also actively engaged in a cross-talk with the complement system, an essential arm of innate immunity.
Recent reports present pathological findings of localized direct infection of vascular endothelial cells with SARS-CoV-2, yet widespread endothelial cell dysfunction and inflammation exists, raising the possibility of indirect activation, through infection, of other susceptible cell types, which cause hyperinflammation and aberrant anti-viral responses.6,12–15 Furthermore, recent reports present evidence for complement deposition in SARS-CoV-2-damaged endothelium, further strengthening the idea that, in severe cases of COVID-19, complement activation is an essential component, generating destructive hemorrhagic, capillaritis-like tissue damage, clotting, and hyperinflammation.12,16,17 Thus, complement-targeted therapies are actively in development.18
Based on our work on the role of complement in pulmonary hypertension (PH) and respiratory virus infections, we herein review evidence and examine the possibility that localized SARS-CoV-2 infection and hypoxia act synergistically on microvascular endothelial cells to drive widespread and excessive complement-dependent pro-inflammatory and microthrombotic responses in COVID-19-induced lung injury.19–23 These ideas are summarized in Fig. 1. Herein, we review evidence for a potential role of complement in COVID-19-induced lung injury.
Evidence that COVID-19-induced severe ARDS is a distinct state of endotheliitis
The leading cause of mortality in patients with COVID-19 is hypoxemic respiratory failure from ARDS.31 Pathological studies, primarily based on an autopsy of patients with the disease, provided evidence for a set of distinct features of COVID-19-associated lung injury, which cannot be accounted for by the cellular processes underlying classical ARDS.12,14,32–34
One recent study revealed that the lungs from COVID-19 patients have three pathological features distinct from those of H1N1/influenza lung injury:14 (1) evidence of severe endothelial injury associated with intracellular SARS-CoV-2 virus and disrupted endothelial membranes; (2) widespread vascular thrombosis with microangiopathy and occlusion of alveolar capillaries; (3) a unique form of intussusceptive angiogenesis, not traditionally observed in ARDS. This study further suggested direct SARS-CoV-2 infection of lung vascular endothelial cells (previously seen in kidney endothelial cells of the glomerular capillary loops),13 with accompanying infiltration of inflammatory cells, evidence of endothelial and inflammatory cell death, disruption of intracellular endothelial junctions, cell swelling, and loss of contacts with the basement membrane.
Other studies also support significant local inflammation, alveolar hemorrhage, and thrombosis4,35–37 and indirectly implicate endothelial infection.5,8,11,13,37 It should be noted, however, that, following these reports, many investigators have raised serious questions regarding whether pulmonary vascular endothelial cells are directly infected with the virus.28,38
The SARS-CoV-2 spike protein binds to host cells via angiotensin-converting enzyme 2 (ACE2) and viral entry is facilitated by cell surface proteases, including TMPRSS2, the liposomal cysteine proteases cathepsins B and, L (CTSB, CTSL), the furin protease present in the secretory pathway and endocytic compartments, and other factors, such as neuropilin-1.39–43 In the absence of ACE2 receptor and these proteases, it has been suggested that, in some cells, an alternative route of entry exists, whereby SARS-CoV-2 binds to cells via basigin (BSG, also known as CD147).44
A recent study evaluated these signaling pathways regarding the possibility of supporting infection in three different types of endothelial cells: blood outgrowth, lung microvascular, and aortic. Compared to nasal epithelial cells or Vero-E6 cells (African green monkey kidney fibroblast cell line), endothelial cells expressed low to undetectable levels of ACE2 and TMPRSS2 but comparable levels of BSG.45 Endothelial cells showed no susceptibility to live SARS-CoV-2 or SARS-CoV-2 pseudo-virus entry or infection.45,46 However, these cells did display susceptibility to both Ebola and vesicular stomatitis virus infection.
Even in the presence of inflammation, where an endothelial cell was pretreated with IL-1β, the cells remained refractory to SARS-CoV-2 infection. A separate study demonstrated that primary human endothelial cells lack ACE2 receptors at the RNA and protein levels, and that SARS-CoV-2 is incapable of directly infecting endothelial cells from pulmonary, cardiac, kidney, or brain tissues, even after stimulation with a variety of factors (e.g., IL-1β, TNF-α, IL-6).27 In contrast, pulmonary endothelial cells transfected with ACE2 receptors can be infected, indicating that endothelial cells are permissive for SARS-CoV-2 replication.27
The failure of these monoculture systems to identify a potential for endothelial cell infection by SARS-CoV-2 could be due to the nature and deficiencies of the endothelial monolayer culture systems. Therefore, more complex ex vivo systems have also been used to investigate the cellular tropism of human CoVs, including SARS-CoV-2. Hui et al. compared virus tropism and replication competence of SARS-CoV-2 with SARS-CoV, Middle East respiratory syndrome (MERS)-CoV and H1N1 influenza A virus in ex vivo cultures of the human bronchus and lung.25
Immunohistochemical staining showed that SARS-CoV-2 extensively infected ciliated cells, non-ciliated mucus-secreting (goblet) cells, and club cells, but not basal cells, of the bronchial epithelium. In cultured lung parenchymal cells, there was positive staining for SARS-CoV-2 antigen in the spindled, morphologically epithelial type 1 pneumocytes. In this study, double staining showed no co-localization of viral antigen in macrophages. Importantly, there was no evidence of infection of vascular endothelium in blood vessels of the lung, in contrast with that previously reported for MERS-CoV.25,47 Similarly, Hou et al. used high-sensitivity RNA in situ mapping to show: (1) the highest ACE2 expression in epithelial cells of the nose with decreasing expression throughout the lower respiratory tract, which was (2) paralleled by a striking gradient of SARS-CoV-2 infection in proximal (high) versus distal (low) pulmonary epithelial cultures. COVID-19 autopsied lung studies identified focal disease and, congruent with culture data, SARS-CoV-2-infected ciliated and type 2 pneumocyte cells in airway and alveolar regions, respectively.24
Described animal model, the human-lung only mice, which are immune-deficient mice implanted with authentic human lung tissue, allow for the study of SARS-CoV-2 in a single platform with direct comparisons of experimental outcomes.48,26 In this model system, the human lung tissue displayed robust virus replication, and sustained activation of the innate host immune response. SARS-CoV-2 was noted predominately in the epithelium. No viral antigen was detected in the human CD34 expressing endothelial cells. Viral antigen was clearly identified in cells, which express pro-SP-C (alveolar type II pneumocytes), acetylated alpha-tubulin IV (ciliated cells), and a few vimentin-expressing (mesenchymal) cells, but was not detected in alveolar type I cells or club cells. Studies in animal models of SARS-CoV-2 infection have also failed to identify any obvious signs of endothelial infection despite clear evidence of endothelial dysfunction and thrombosis.49,50
Another potential target cell of SARS-CoV-2, that could participate in the endothelial dysfunction observed during infection, is the pericyte, specialized cells embedded in the basement membrane of the vessel wall. Recent studies have suggested that ACE2 is highly expressed in pericytes of certain tissues, especially the brain and heart, making them targets for infection.51,52 At present, there is no histologic confirmation of pericyte infection in patients with fatal SARS-CoV-2 infection. Reports have suggested pericyte loss or detachment in lung tissue, consistent with the idea that infection of the cells leads to endothelial instability and endothelium-mediated thrombosis.28 Fig. 2 summarizes current data on SARS-CoV-2 lung cellular tropism.
Collectively, these observations support the idea that the vascular dysfunction and thrombosis, observed in severe COVID-19, could be the result of factors released either by adjacent infected cells, i.e. epithelial cells, or by circulating systemic inflammatory mediators.45,53 What seems clear is that SARS-CoV-2 pathogenicity involves amplification of cellular damage via the activation of immunological and cellular injury systems not exclusively accounted for by SARS-CoV-2 direct cytopathic effects. Thus, mechanistic insights into the origin(s) of the high degree of endothelial dysfunction and pro-inflammatory activation (endotheliitis) in COVID-19 patients could lead to a better understanding of the progression of pulmonary involvement, with a potential impact on the morbidity and mortality of the disease.
Activation of the complement cascade is involved in COVID-19 lung vascular disease
The complement system is a critical part of the host immune response to bacterial and viral infections.54 In the late 19th century, it was identified as a heat-sensitive, non-specific “complement” to the more specific humoral immunity pathways, i.e., its name—complement system.55 The complement system has been described as one way the innate immune system can detect and respond to foreign antigens. It is now recognized that the complement system comprises nearly 60 proteins, including several receptors and regulatory proteins.56 Complement activation has direct cytotoxic effects, and it can amplify danger signals and augment inflammatory responses.
These protein components of the cascade are inactive in their native state, but activation of the pathway causes proteolytic cleavage of several pathway proteins. These cleaved proteins, in turn, form enzymes (“convertases”) that lead to further protein cleavage. Activation of the system occurs through three major pathways.57 The classical and mannose binding lectin (MBL) pathways are canonically activated by IgM/G and carbohydrate moieties on pathogens, respectively.
They converge at C2aC4b, which acts as a C3 protease or convertase that mediates the cleavage of C3 into C3a and C3b fragments.58 There is also the Alternative pathway, which can act as a C3 amplification loop, that can contribute significantly to complement activation from the classical and MBL pathways, and can itself be triggered by altered/injured surfaces, such as damaged or foreign tissue.59
The resulting activation fragment C3b then joins C2aC4b from the classical/MBL pathways, or C3bBb from the Alternative pathway to form a C5 convertase, which cleaves C5 into C5a and C5b fragments, the latter of which, wherein C5b can associate with C6, C7, C8, and C9 to form C5b-9, the so-called membrane attack complex (MAC).59 It is also clear that a pathway, now termed the Extrinsic pathway, exists, and comprises a collection of proteases, including thrombin, kallikrein, elastases, that possess certain convertase activity, some of which are part of the coagulation cascade.60 Once the complement cascade is triggered, many effectors are generated, including (1) the opsonins (C3b, C4b), which can mark cells or foreign invaders for phagocytosis; (2) the anaphylatoxins (i.e., C5a, C3a, C4a), which are broad-spectrum immune activators known to promote a variety of immune processes including immune cell chemotaxis, NETosis, production of cytokines, inflammasomes, reactive oxygen species (ROS), and eicosanoids, all of which are known to participate in a variety of vascular injuries; and (3) the MAC (C5b-9), which causes cell lysis and other forms of collateral damage, that are observed when complement is activated, including the release of damage-associated molecular patterns (DAMPs; e.g., hyaluronan and ATP) that can further activate complement in a self-perpetuating cycle.61,62
Because of this vast array of proteins with inflammatory and destructive capabilities, the complement system is very tightly regulated through a number of inhibitory proteins that are constitutively present in the serum, local tissue microenvironments, and on cell surfaces.63 Endothelial cells are continually exposed to high concentrations of complement proteins in plasma. They express several different complement regulatory proteins on their outer membranes, however, and are ordinarily very efficient at regulating complement activity on the surface (Fig. 3).64 The soluble complement regulator factor H also adheres to ligands on endothelial cells, thereby providing an additional mechanism of protection. Nevertheless, congential or injury-associated impairments in complement regulation can leave endothelial cells susceptible to complement-mediated injury. This association is well established in atypical hemolytic uremic syndrome, a cause of thrombotic microangiopathy.65
reference link : https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8138299/
More information: Airah Javorsky et al, Structural basis of coronavirus E protein interactions with human PALS1 PDZ domain, Communications Biology (2021). DOI: 10.1038/s42003-021-02250-7