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Lung Fibrosis: Post-COVID-19 Complications and Evidences

Background COVID 19, a lethal viral outbreak that devastated lives and the economy across the globe witnessed non-compensable respiratory illnesses in patients. As been evaluated in reports, patients receiving long-term treatment are more prone to acquire Pulmonary Fibrosis (PF). Repetitive damage and repair of alveolar tissues increase oxidative stress, inflammation and elevated production of fibrotic proteins ultimately disrupting normal lung physiology skewing the balance towards the fibrotic milieu. Aim In the present work, we have discussed several important pathways which are involved in post-COVID PF. Further, we have also highlighted the rationale for the use of antifibrotic agents for post-COVID PF to decrease the burden and improve pulmonary functions in COVID-19 patients. Conclusion Based on the available literature and recent incidences, it is crucial to monitor COVID-19 patients over a period of time to rule out the possibility of residual effects. There is a need for concrete evidence to deeply understand the mechanisms responsible for PF in COVID-19 patients.

which was proven to be more sensitive in determining the severity of PF 13 . CT scan images could also be used to differentiate between ILD and PF relying on the data of ground glass opacities 14 . PF usually arises after acute respiratory distress syndrome (ARDS). ARDS is divided into two phases: an immediate exudative inflammatory phase and a proliferative phase marked by alveolar epithelial cell hyperplasia 12 Along the line of understanding the unknown post-COVID-19 consequences, short follow-ups have demonstrated fibrotic changes in the lungs at various durations considering the severity spectrum 15,16 . There is a need to unveil the incomplete mechanistic pathway of PF interlinking post COVID observations in patients lungs. Our review majorly focuses on the important pathways that can form a base in an attempt to identify the crucial targets and minimise the risk of post COVID PF for severely affected patients undergoing long term treatment.

Structure & pathogenesis
The coronavirus pertains to the order of nidovirales belonging to the family; coronaviridae, which are further categorized into 4 different genera namely: α, β, γ and δ coronaviruses 17 . Amongst the aforementioned viruses, α and β coronaviruses are more pathogenic and infect mammals, whereas γ and δ coronavirus afflict avians more than mammals 18 . The SARS-CoV-2 structure consists of a clustered envelope, positive-sense, single-strand RNA comprising of 30,000 bases along with a 5'cap structure and a 3'poly A tail 19 . The genomic sequence of SARS-CoV-2 is 89% similar to that of Bat-CoV which possibly connotes bat origin 20 .
The major structural proteins assembled in the viruses which are held accountable to penetrate host cells and initiate replications are Surface spike (S) protein, Membrane (M) protein, Envelope (E) protein, Nucleocapsid (N) protein. Surface spike (S) protein: The SARS-CoV-2 entry is mediated by S-protein 21 which consists of 2 subunits specifically S1 and S2 subunits. The S1 subunit consists of the N-terminal domain and receptor binding domain, both the domains help in binding with the hACE2 receptor. While S2 subunit is made up of seven components notably, Fusion peptide (FP), Heptad Repeat 1 (HR1), Central Helix (CH), Connector Domain (CD), Heptad Repeat 2 (HR2), Transmembrane Domain (TMD) and Cytoplasmic tail (CT) as its main function is to initiate fusion between host-viral cell membrane 22 . The S protein undergoes 2 step cleavage events, the initial splitting of S1 and S2 followed by splitting of S2 into FP and S2' which is common in both SARS-CoV-1 and SARS-CoV-2, however SARS-CoV-2 additionally contains furin cleavage. In particular, the furin cleavage initiates cell-cell union, which facilitates efficient viral entrance and propagation from one cell to another in human lung cells 23 . Membrane (M) Protein: It consists of three major domains; N-Terminal ectodomain, three transmembrane helices and C-Terminal domain, it plays a major role in the congregation of new virions by interacting with S, E & N proteins which are necessary for initiating membrane budding 24 . Envelope (E) protein: The E protein has the smallest transmembrane and includes 3 domains, an N-terminal hydrophilic ectodomain, a domain of hydrophobic transmembrane lastly a long hydrophilic C-terminal endodomain. This protein is responsible for causing loss of membrane potential and actuates host inflammasomes 25 . Nucleocapsid (N) protein: The N protein acts as a package of viral proteins that converts viral RNA into helical ribonucleocapsid proteins (RNP) which allows alteration with other structural proteins during virion assembly further encompassing genome encapsidation 26 . There are two domains assembled into the N protein, an N-terminal RNA binding domain and a C terminal domain that helps in the formation of dimers, as reported by Qiaozhen et al. N protein also possesses self-assemble properties and the N2b domain is relatively similar to beta coronavirus structure 27 .
The S, M and E protein invades the endoplasmic reticulum whereas the N protein gets attached to the genomic RNA (+) strand to produce nucleoprotein complex 26 . Additionally, the virus also possesses 16 non-structural proteins (nsp 1-16) 28 and 5-8 accessory proteins along with 14 open reading frames (ORF) that encompass 2/3 rd part of the genome which is more prone to mutation. Repetitive mutations in the SARS-CoV-2 genome resulted in numerous variants which have altered its pathogenic ability, in due triggering new symptoms like conjunctivitis, blurred vision, itchiness, tingling in peripheral limbs, etc.
leaving the physicians perplexed. A list of important mutations in SARS-CoV-2 is shown in table 1.

Pathogenesis
The SARS-CoV-2 virus primarily binds to type-2 pneumocyte cells in the alveoli of the lungs which further invades into cell surfaces of specific receptors like hACE2 and CD90L 44 . The two subunits of S proteins S1 and S2 have a significant and specific role; on the binding of the virus, the Receptor Binding Domain (RBD) present in the S1 subunit fosters down-regulation of ACE2 receptors, and the S2 subunit assimilates binding with ACE2 receptors and illustrates the fusion between host-viral cell membrane 45 . The ACE2 receptors play a key role in the regulation of the Renin-Angiotensin System (RAS) by maintaining a balance between homeostasis and fluidic ions. The Angiotensin Converting Enzyme (ACE) processes and converts Angiotensin I (AT1) to Angiotensin II (AT2), 46,47 further, the increased production of AT2 activates myriads of signalling pathways. Moreover, AT2 helps in releasing aldosterone and induces vasoconstriction due to imbalance between the sodium ions and fluid, causing excess fluid retention as well as exacerbating pulmonary vascular permeability inducing profibrotic effects. AT II is also responsible to activate the collagen 1 gene through MAPK/ERK pathway and promoting TGF-β, being essential factors for provoking fibrotic responses. Apart from binding to ACE2 receptors, a transmembrane serine protease 2 (TMPRSS2) provides direct entry for the S protein of SARS-CoV-2 48 and permits penetration into the targeted cells 49 .
Upon invading the host's cell, the virus enters into the Endoplasmic Reticulum (ER), and amplifies ER modification through promoting synthesis of viral RNA via orf1a and orf1ab proteins. These viral proteins are transported to the cell surfaces and get released through exocytosis. This transport is greatly involved in the replication and proliferation of the virus. Certain host kinases such as Casein Kinase II (CK2), Cyclic Dependent Kinase (CDK) and Protein Kinase C (PKC) phosphorylate the host receptors and play a pivotal role during replication of the SARS-CoV-2 virus 50 . The aforementioned host kinases regulate the cell cycle and are also responsible for detaining cell function that abides cell death. Once the cell cycle is impaired, the viral proteins incorporated into the structural N protein of the SARS-CoV-2 are elevated and become liable for impeding host translation.
During the infection, the virus gets replicated employing mitosis and facilitates migration assisting deeper invasion into the respiratory cells through a process known as cytoskeletal rearrangement. This rearrangement induces internal as well as external mechanical forces, recognized by integrins and is further transduced through activating the mechano-sensitive pathways. The integrins act as a linker between the cytoskeleton and extracellular matrix (ECM) which provides a biochemical mechanism, commencing vascular contraction and compelling rigidity to the matrix present inside the alveolar lung epithelial cells, leading to fibroblast activation, macrophage phagocytosis and transmuting the endothelial barrier function 51 . Integrins such as α v β 3 , α v β 5 , α v β 6 not only induces profibrotic pathway but also directly activate TGF-β. The rise of TGF-β levels results in the actuation of fibroblasts, cellular contraction, stiffening of matrix and aggravated collagen deposition, these conformational changes instigate the host's fibrinogenic mechanism ultimately causing the lung injury 52,47 . Considering the antigen-presenting cells (APC) in the SARS-CoV-2 virus also stimulate the release of pro-inflammatory cytokines [IL-6, IL-1β, IL-12, IL-18, IL-33, TNF-α, TGF-β] and chemokines like IL-8, CCL2, CCL3, CCL5, CXCL8, CXCL9, CXCL10, etc. instigating cytokine storm, henceforth mutilates the lining of epithelial cells and reaches into the blood circulation causing multi-organ injuries 44,53,54 .
A retrospective cohort study analysed the chemokine levels of 72 patients infected from SARS-CoV-2. The report illustrated levels of different inflammatory cytokines including IL-6, IL-8, IL-10, TNF-α, etc. during the stay period right after hospitalisation till the unprecedented death. It was observed that IL-8 levels always remained high in severely affected COVID-19 patients stating it as one of the prognostic marker for untimely death 55 . Similar conclusions were drawn by Francesco and associates inclusive of 14 days. Observations were compared between IL-6, IL-8, IL-10 and other pro-inflammatory factors, out of which levels of IL-8 constantly surged during the hospital stay in severely ill patients. Thus, emphasising on the fact that IL-8 can be considered as one of the prognostic markers based on the evaluations 56 . Along the line of understanding the unknown post-COVID-19 consequences, short follow-ups have demonstrated fibrotic changes in the lungs at various durations considering the severity spectrum 15,16 . The direct viral mechanism involved in induction of lung fibrosis in recovered COVID-19 patients is still unknow

COVID-19 and PF
PF (PF) is a sequela of the tissue repair responses which prevail following multiple tissue injuries during chronic inflammatory conditions. PF usually arises after acute respiratory distress syndrome (ARDS).
ARDS is a severe, acute form of microvascular injuries present in lungs which can be divided into two phases: an immediate exudative inflammatory phase and a proliferative phase marked by alveolar epithelial cell hyperplasia 12  Usually, myofibroblasts are not circulated in tissue in normal conditions 76 . As a study done by Hinz, during the restoration of scars present on lung tissue, myofibroblasts emerge for a brief period and are released in a controlled manner for maintaining the normal tissue framework utilizing intense collagen formation on the scars 77 . Various progenitors that activate myofibroblasts are smooth muscle cells in the arterial wall, chondrocytes present in cartilage, osteoblasts in bones and pericytes in vascularized tissues 78 . Apart from these pro-activators, cells of the epithelium and/or endothelium also generate myofibroblasts upon activation via a process known as an epithelial mesenchymal transition (EMT) 79 . Abhijeet P. D and associates exemplified cases and suggested a detailed study considering cytokine TGF-β1 as a critical key factor inducing cellular and molecular changes that interfere with EMT signalling. According to the data obtained it was observed that SARS-CoV-2 induced viral infection could deplete the epithelial and endothelial cellular activity and restrain the easy permeability of spike proteins of SARS-CoV-2. The enhanced permeation via cellular changes could make respiratory cells more prone to edema and increase the risk of PF 80 .

Molecular mechanism of PF
To understand the molecular mechanism of PF various studies were put forth, it was noted that transdifferentiation of epithelial cells into mesenchymal is a major contributor for progression of PF. EMT itself is an intricate mechanism that has both protective and detrimental effects in normal and pathological lung conditions, respectively. It has been demonstrated that EMT plays a necessary role in embryogenesis, gastrulation, and during the formation of the neural crest, heart, and other organs 81

Pathways that may interlink COVID-19 and PF
In most cases, COVID-19 death is a result of lung failure due to severe acute respiratory distress syndrome.
This syndrome is particularly attributed as an uncontrolled inflammatory response by a "cytokine storm", ultimately leading to edema and fibrosis in the lungs at later stages. Hypothesizing the below pathways may allow us to understand the connections between pro-inflammatory factors and other signalling mechanisms giving an undue rise of deaths even after complete treatment.

TGF-β Signalling Pathway:
According to a few pieces of evidence reported during COVID-19 infection, the infection cases were associated with a sharp rise in inflammatory and immune response, resulting in activation of various mediators particularly, TGF-β, right after declined lung function 94

WNT-signalling pathway:
WNT plays a crucial part in controlling cell proliferation and migration during inflammation and PF 103 . This pathway is distorted in various pathological conditions such as CNS disorders, disturbed psychological behaviour, improper metabolism, etc. 104 In presence of TGF-β, the WNT pathway is upregulated and as reported by COVID-19 survivors, some clinical findings exhibited increased WNT expression 105,106 . WNT is encoded by a large family of secreted protein factors in humans 107,108 . WNT signal transduction can be classified as 1. Canonical pathway or (WNT/ β-Catenin pathway), 2. WNT/PCP (Planar Cell Polarity pathway), 3. WNT/Ca +2 pathway 109 . Amongst these pathways, the canonical pathway influences majorly during inflammation and helps in the activation of the fibroblasts [109][110][111] . In the present scenario, there is some evidence which points out WNT canonical pathway being somehow linked to cytokine storm syndrome which is the considerable factor responsible for inducing ARDS in severe COVID-19 infected patients 107,112 .
To explore more, researchers analysed reports of COVID-19 patients who were severely infected, observed higher WNT levels than that of normal, specifically WNT5a in blood samples, which if over stimulated induces ARDS and makes one prone to PF in short span 107 . A study forecasted on WNT and TGF-β showed synchronized positive feedback to each other 113,114 . WNT ligands play a very crucial role in repairing injured tissues, by several mechanisms, including signal control, embryogenesis, cell proliferation, apoptosis, organogenesis, migration and sustains polarity 103

YAP/TAZ Signalling pathway:
Yes-Associated Proteins (YAP)/Transcriptional co-activator with PDZ-binding motif (TAZ) combinedly is transcriptional co-activator that acts as a key regulator in maintaining organ size and as a mechanical property it also activates fibroblasts, through down-streaming hippo pathway 118

Can Post COVID-19 PF resolve with time?
In a wake of the existing quo, the rise of new cases is being witnessed in some countries, the strains of the mutated virus didn't spare embarking the new symptoms along with unknown multiple complications even after being successfully treated 142 . One of the major threats still wandering is PF; an unavoidable consequence emerging just after ARDS in patients undergoing long COVID-19 treatment, the repetitive detriment of capillaries causes plasma protein to leak, which implies an inevitable risk of ARDS 143 . A proportion of acute ARDS survivors are likely to develop a fibroproliferative response, which comprises fibroblast accumulation, collagen deposition, and perhaps other pulmonary ECM components 144 , the severity of which has been linked to the duration of the disease 145 . Furthermore, despite the fact that invasive mechanical ventilation has profoundly altered ARDS care, ventilator-associated lung damage is thought to be a significant contributor to PF in ventilated ARDS patients 146