Alveolar Epithelial Cell Type II as main target of SARS-CoV-2 virus and COVID-19 development via NF-Kb pathway deregulation.

Maurizio Carcaterra (  [email protected] ) Azienda Sanitaria Locale Vieterbo Belcolle Hospital Cristina Caruso Azienda Ospedaliera San Giovanni Addolorata

Research Article

Keywords: COVID-19, SARS-CoV-2, COVID-19 pathogenesis, NF-kB pathway activation, Alveolar Epitehlial Cell Type 2, AEC-II, ARDS.

Posted Date: September 16th, 2020

DOI: https://doi.org/10.21203/rs.3.rs-77539/v1

License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License

Version of Record: A version of this preprint was published on November 23rd, 2020. See the published version at https://doi.org/10.1016/j.mehy.2020.110412. Inflammation an d Regeneration Alveolar Epithelial Cell Type II as main target of SARS-CoV-2 virus and COVID-19 development via NF-Kb pathway deregulation. --Manuscript Draft--

Manuscript Number: IREG-D-20-00045 Full Title: Alveolar Epithelial Cell Type II as main target of SARS-CoV-2 virus and COVID-19 development via NF-Kb pathway deregulation. Article Type: Research article Section/Category: Inflammation Funding Information: Abstract: Background: The Corona Virus Disease (COVID-19) caused by Severe Acute Respiratory Syndrome Corona Virus 2 (SARS-CoV-2) requires a rapid solutionand global collaborative efforts in order to define preventive and treatment strategies. Methods: One of the major challenges of this disease is the high number of patients needing advanced respiratory support due to the Acute Respiratory Distress Syndrome (ARDS) as the lung is the major –although not exclusive-target of the virus. The molecular mechanisms, pathogenic drivers and the target cell type(s) in SARS- CoV-2 infection are still poorly understood, but the development of a “hyperactive” immune response is proposed to play a role in the evolution of the disease and it is envisioned as a major cause of morbidity and mortality. Results: Here we propose a theory by which the main targets for SARS-CoV-2 are the Type II Alveolar Epithelial Cells and the clinical manifestations of the syndrome are a direct consequence of their involvement. We hypotize the existence of a vicious cycle by which once alveolar damage starts in AEC II cells, the inflammatory state is supported by macrophage pro- inflammatory polarization (M1), cytokines release and by the activation of the NF-κB pathway. Conclusions: If this theory is confirmed, future therapeutic efforts can be directed to target Type 2 alveolar cells and the molecular pathogenic drivers associated with their dysfunction with currently available therapeutic strategies. Corresponding Author: MAURIZIO CARCATERRA Ospedale di Belcolle Viterbo, Lazio ITALY Corresponding Author E-Mail: [email protected];[email protected] Corresponding Author Secondary Information: Corresponding Author's Institution: Ospedale di Belcolle Corresponding Author's Secondary Institution: First Author: MAURIZIO CARCATERRA First Author Secondary Information: Order of Authors: MAURIZIO CARCATERRA Cristina Caruso Order of Authors Secondary Information: Opposed Reviewers: Additional Information: Question Response

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Powered by Editorial Manager® and ProduXion Manager® from Aries Systems Corporation Title 1 Alveolar Epithelial Cell Type II as main target of SARS-CoV-2 virus and COVID-19 2 development via NF-Kb pathway deregulation. 3 4 Running Title 5 6 COVID-19 sustained by NF-Kb pathway deregulation 7 8 Keywords 9 10 COVID-19, SARS-CoV-2, COVID-19 pathogenesis, NF-kB pathway activation, 11 Alveolar Epitehlial Cell Type 2, AEC-II, ARDS. 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

1 Abstract: 2 3 Background: The Corona Virus Disease (COVID-19) pandemic caused by Severe 4 5 6 Acute Respiratory Syndrome Corona Virus 2 (SARS-CoV-2) requires a rapid solution 7 8 and global collaborative efforts in order to define preventive and treatment strategies. 9 10 11 Methods: One of the major challenges of this disease is the high number of patients 12 13 needing advanced respiratory support due to the Acute Respiratory Distress Syndrome 14 15 (ARDS) as the lung is the major –although not exclusive- target of the virus. The 16 17 18 molecular mechanisms, pathogenic drivers and the target cell type(s) in SARS-CoV-2 19 20 infection are still poorly understood, but the development of a “hyperactive” immune 21 22 response is proposed to play a role in the evolution of the disease and it is envisioned as 23 24 25 a major cause of morbidity and mortality. 26 27 Results: Here we propose a theory by which the main targets for SARS-CoV-2 are the 28 29 Type II Alveolar Epithelial Cells and the clinical manifestations of the syndrome are a 30 31 32 direct consequence of their involvement. We hypotize the existence of a vicious cycle 33 34 by which once alveolar damage starts in AEC II cells, the inflammatory state is 35 36 supported by macrophage pro-inflammatory polarization (M1), cytokines release and by 37 38 39 the activation of the NF-κB pathway. 40 41 Conclusions: If this theory is confirmed, future therapeutic efforts can be directed to 42 43 target Type 2 alveolar cells and the molecular pathogenic drivers associated with their 44 45 46 dysfunction with currently available therapeutic strategies. 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

1 2 3 4 5 6 7 8 9 10 Graphical Abstract 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 Introduction: 38 39 In the past twenty years there have been precedents for epidemic clusters sustained by 40 41 viruses that have bats as their main hosts. The dynamic of the transmission from bat- 42 43 44 borne viruses to humans is very complex and often involves the presence of additional 45 46 intermediate hosts [1–3]. The human Coronavirus (hCoV) family belongs to a well- 47 48 recognized bat-borne family of viruses that infects humans causing predominant 49 50 51 damage to the respiratory system strictly connected with the development of ARDS [4, 52 53 5]. Previous Coronavirus-mediated diseases are Severe Acute Respiratory Syndrome 54 55 56 Coronavirus (SARS-Co-V) or Middle East Respiratory Syndrome CoV (MERS CoV) 57 58 [6–8] closely related to the new SARS-CoV-2 responsible for Coronavirus Disease 19 59 60 (COVID-19) [1, 9–11]. ARDS is a manifestation of lung injury, therefore not a disease 61 62 63 64 65

itself, rather a syndrome induced by different insults and characterized by substantial 1 2 heterogeneity [4, 5]. We propose the hypothesis that the main target of the virus is the 3 4 Alveolar Epithelial Cell Type II and that a dysregulation of the Nuclear Factor kappa- 5 6 light-chain-enhancer of activated B cells pathway (NF-κB pathway) drives ARDS 7 8 9 leading to Multiple Organ Failure (MOF), one of the most frequent causes of death. By 10 11 correlating the clinical data and examining the pathophysiology of the various diseases 12 13 present in COVID-19, we focused our attention on the intra and extra cellular signaling 14 15 16 mechanisms that are activated following damage at the alveolar level and on how these 17 18 signals are distantly propagated and amplified. 19 20 21 Discussion: 22 23 When a pathogen like a virus infects humans, the immune system could clear the 24 25 infection by limiting viral spread (effective antiviral response), or cause an excessive 26 27 28 inflammation and tissue destruction by cytotoxic cells and consequent development of 29 30 immuno-pathological damage. The epithelial cells are critical for balancing these two 31 32 extreme situations [12]. The upper respiratory tract provides the first line of defense in 33 34 35 that it activates a very complex system of signaling and recruitment of immune cells, in 36 37 order to prevent pathogens from reaching the alveoli, the most important part of the 38 39 respiratory system because responsible of gas exchange. Nevertheless, coronaviruses 40 41 42 (especially SARS-CoV and MERS) are able to reduce or delay the expression of 43 44 cytokines in human lung epithelial cell lines. This is an effective system to escape 45 46 immune recognition by innate receptors in the infected cell [13, 14], which could 47 48 49 therefore facilitate the progression of the virus into the lower respiratory airways (i.e. 50 51 the alveolar space) (Fig.1). 52 53 54 55 56 57 58 59 60 61 These observations are coherent with some clinical evidence of CoVID-19 as well: 1) A 62 63 64 65

high number of asymptomatic patients are positive at the nasopharyngeal swab for virus 1 2 detection with real-time reverse transcription (RT)-PCR, which strongly suggests that 3 4 despite viral load is present, no symptoms occur due to the lack of innate immune 5 6 response; 2) On the other hand, in mild symptomatic patients –some time negative with 7 8 9 nasopharyngeal swab-, early alveolar damage can still be detected with CT scan [15], 10 11 thus demonstrating that the first and most important site of damage is the lung 12 13 parenchyma. 14 15 16 The alveolar epithelium is composed by two different Alveolar Epithelial Cells (AEC): 17 18 Alveolar Epithelial Cell Type I (AEC-I) that represents 85-90% of all alveolar cells and 19 20 21 Alveolar Epithelial Cell Type II (AEC-II), 10-15%[16]. The latter, despite the small 22 23 number, is fundamental for many functions, that we can summarize as follows: 1) 24 25 production of the surfactant (S-protein SP); 2) stabilization of the airway epithelial 26 27 28 barrier (AEB); 3) immune-defense; 4) airway regeneration during lung injury, a process 29 30 carried out by progenitors for AEC-I cells [17]. Moreover AEC-II is the only lung cell 31 32 that produces all of the components of the surfactant complex and aids in the 33 34 35 biosynthesis, storage and release of the entire production cycle which is fundamental to 36 37 lung integrity [18, 19] because responsible of lowering the superficial tension, thereby 38 39 preventing alveolar collapse. Once the alveolar damage starts, in the absence of an 40 41 42 effective response, the pathogenesis progresses to ARDS. 43 44 At least three different phases have been identified in ARDS: 1) The exudative phase; 2) 45 46 The proliferative phase; 3) The fibrotic phase [4]. In the exudative phase the lung 47 48 49 responds to insults by activating innate cell-mediated immunity that leads to an 50 51 alteration of both the vascular endothelium and the alveolar epithelium. In this phase 52 53 there is a ”cytokine storm” with an intense promotion of inflammation supported by the 54 55 56 recruitment of neutrophils, monocytes and macrophages and by the activation of 57 58 alveolar epithelial cells [20, 21]. The second proliferative phase is the most important 59 60 for the recovery of normal lung function and for the restoration of normal alveolar- 61 62 63 64 65

endothelial architecture. The third fibrotic phase is associated with an increase in 1 2 mortality and the need for longer [4, 5, 22]. In the COVID-19 3 4 syndrome, the high mortality rate in critically ill patients suggests that there could be an 5 6 imbalance between these phases. Furthermore, pulmonary fibrosis may be one of the 7 8 9 most severe complications and a cause of permanent lung damage after patients recover 10 11 from CoV-2 infection, thus prevention of this complication is an issue that urgently 12 13 needs to be addressed. 14 15 16 SARS-CoV-2 is an enveloped virion containing one positive-strand RNA genome 17 18 whose main sequence has been reported [23]. We also know that the Angiotensin 19 20 Converting Enzyme 2 (ACE2) receptors are the main point of entry for coronaviruses 21 22 23 through the binding with glycoprotein (GP) spikes proteins [24]. Relative to other 24 25 coronaviruses, CoV-2 has a greater affinity (10-20 fold) for the receptor probably due to 26 27 a different conformation of GP spikes within the subunit [25]. After binding the virus 28 29 30 envelope is fused to target cells via a Trans Membrane Protease Serin 2, TMPRSS2 [26, 31 32 27]. Into the alveoli the ACE2 receptors are present on the AEC-II cell membrane and 33 34 recent evidence suggests that one of the highest expression of both ACE2 and 35 36 37 TMPRSS2 is in AEC-II [28, 29] and in transient secretory cell in more central bronchial 38 39 branches [29], where the latter could act as a transporter to the more peripheral alveoli. 40 41 42 This line of evidence lends strong support to our theory of the main role played by 43 44 AEC-II. 45 46 Previous studies have shown that in the case of SARS-CoV and MERS the virus 47 48 49 downregulates the expression of ACE2 [30, 31], and recently confirmed in SARS-CoV2 50 51 [32].This is not a contradiction because Ace2 receptors counteract inflammation. Indeed 52 53 ACE 2 plays a fundamental role in the Renin Angiotensin System (RAS), having a 54 55 56 protective function through the conversion of Angiotensin II to Angiotensin 1-7, that 57 58 counteracts the effects of Angiotensin II, promoting vasodilatory, anti-inflammatory, 59 60 antioxidant activities. Moreover, in murine models, the ACE2 knockout displayed more 61 62 63 64 65

severe symptoms of the disease compared to wild-type mice, while ACE2 1 2 overexpression appeared protective [31, 33–35]. ACE2 is also known to play a crucial 3 4 role in the apoptosis of alveolar epithelial cells and is downregulated in lung fibrosis 5 6 [36], and its actions on peptide signals balance and offset those of ACE [37]. 7 8 9 Intriguingly, a decreased glomerular expression of ACE2 has been described in rodent 10 11 models of diabetes [38, 39] [40]. These observations are consistent with some clinical 12 13 aspects, also common to other coronavirus infections that correlate the severity of the 14 15 16 disease with the presence of comorbidities such as diabetes, hypertension and 17 18 cardiovascular diseases. This line of evidence supports the hypothesis that ACE2 is 19 20 actually reduced as already suggested by some authors [32, 41] and plays a possible 21 22 23 protective role, also explaining why angiotensin converting enzyme inhibitors (ACEI) 24 25 and angiotensin receptor blocker (ARB) [33, 42], may also be protective. 26 27 The role of the endothelium and the interplay with macrophages and neutrophils. 28 29 30 A characteristic element of ARDS is the increase in capillary permeability resulting 31 32 from the damage of the AEC cells and the endothelium which includes an accumulation 33 34 of liquid rich in proteins in the alveoli and consequent release of proinflammatory 35 36 37 cytokines: TNF, IL1 e IL6[43]. This corresponds to the exudative phase mentioned 38 39 above [4], during which resident macrophages, with the M1 phenotype, release 40 41 42 additional proinflammatory cytokines, in a series of events also called "cytokine storm" 43 44 , ultimately leading to the apoptosis of the AEC-II and AEC-I cells with further damage 45 46 to the alveolus-capillary barrier, to the reduction of surfactant and to the formation of 47 48 49 intravascular –and microvascular-clots. 50 51 The pulmonary endothelium plays a leading role in lung damage, so much so that some 52 53 authors have called it " the orchestra conductor in respiratory diseases “[44]. In the lung 54 55 56 at rest the endothelium assumes an inhibitory effect on inflammation and coagulation, 57 58 whereas after cytokines stimulation it converts to a pro-inflammatory phenotype [45]. 59 60 Neutrophil activation is fundamental in this process, because their accumulation 61 62 63 64 65

enhances the progression of inflammation and alveolar damage. The NF-κB pathway is 1 2 fundamental to regulate many important cellular behaviors, in particular, inflammatory 3 4 responses, cellular growth and apoptosis, as well as in regulating the survival, activation 5 6 and differentiation of innate immune cells and of inflammatory T cells[46]. 7 8 9 Furthermore, NF-κB prevents the pro-inflammatory cytokine TNF-alpha from inducing 10 11 the apoptosis of neutrophils in response to inflammation or viral-infections and exposes 12 13 ligands for neutrophils by promoting the degradation of endothelial glycocalyx[45]. We 14 15 16 envision that the result of this chain of events substantially leads to: 1) Apoptosis of 17 18 AEC-II cells; 2) NF-κB pathway hyper-activation; 3) Enhanced survival of neutrophils 19 20 and their accumulation, 4) Downregulation of ACE2, 5) Surfactant reduction due to 21 22 23 apoptosis of AEC-II. (Fig.2). NF-κ B is activated by viruses, such as HIV-1, HTLV-1, 24 25 hepatitis B virus (HBV), hepatitis C virus (HCV), Ebstein Barr Virus (EBV), and 26 27 influenza virus [47]. The role of NF-Kb pathway in SARS-CoV infection has already 28 29 30 been postulated by other authors in the past[48] 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

The reduction of the surfactant is a major driver of the collapse of the alveolus, 1 2 therefore all the pathologies that involve its reduction aggravate the damage. It is known 3 4 that diabetes for example involves a reduction in the production of surfactant as well as 5 6 a reduction in ACE2 [49]; this explains why diabetes represents an aggravating 7 8 9 condition for ARDS and for COVID-19 patients as well. The transition to the next 10 11 restoration or proliferative phase is essential for tissue homeostasis after damage. This 12 13 phase is governed by the phagocytosis of apoptotic neutrophils (efferocytosis), by 14 15 16 resident macrophages that eventually switch towards the M2 phenotype [4]. However, 17 18 we believe that this latter phase is delayed by SARS-CoV2 by NF-κB pathway 19 20 amplification and inflammation sustained in a vicious circle manner (Fig.3). Recently, 21 22 23 in an Italian study on more than 40 autopsies, pathological alterations of the exudative 24 25 phase and the early proliferative phase were found, where fibrotic phase is reported as 26 27 focal and not widespread; therefore, the authors suggest that none of the patients had 28 29 30 progressed to the fibrotic phase, possibly because of the short duration of the disease 31 32 [50]. Otherwise they describe mural fibrosis in 65% of patients. It is important to 33 34 underline that the three phases of ARDS do not behave exactly as described and that not 35 36 37 every patient will develop fibrosis. These steps may overlap and occur in a 38 39 heterogenous way regarding time and different lung region. Thus, inflammatory and 40 41 42 repair mechanisms initiate in parallel and not in a serial way. This evidence is also 43 44 supported by a recent review [51]. Furthermore, we believe that the early fibrotic 45 46 pattern showed before the onset of symptoms could be COVID-19 related. 47 48 49 Macrophages are key regulators of both inflammation and its resolution, and NF-κB 50 51 plays a key role in macrophage responses and differentiation into pro-inflammatory 52 53 (M1) or anti-inflammatory (M2) phenotype, which is strongly influenced by cytokines 54 55 56 [52]. In the inactive form NF-κB is sequestered in the cytoplasm through the combined 57 58 action of inhibitory protein IκB and its subunits (IκBα/β/γ). One possible mechanism 59 60 that could play an important role in the inhibition of IkB protein family members and 61 62 63 64 65

therefore upholds activation of NF-κB pathway, is the involvement of microRNA(s) 1 2 that physiologically regulate the inflammatory response. Micro RNAs (miRNAs) are 3 4 small, non-coding RNAs that regulate the expression of protein-coding genes [53]. 5 6 Recently, miRNA levels have been used as a novel non-invasive biomarker for the 7 8 9 diagnosis of various diseases [54, 55]. Expression of some miRNAs, such as miR 132 10 11 and miR 146a, have the capacity to dampen the inflammatory response in alveolar 12 13 macrophages [56]. 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 Results: 48 49 50 During this pandemic, various therapies were tested, with some effects on 51 52 improvement, where we find confirmation of our hypotheses on the dysregylation of the 53 54 NF-κB pathway as a potential key driver of disease progression. 55 56 57 It has been noted that disease severity is not only attributed to direct viral damage, but 58 59 also by immune-mediated injury induced by SARS-CoV-2, where distinctive features 60 61 62 63 64 65

have been noticed in severe and critical patients with COVID-19: progressive increase 1 2 of inflammation and an unusual trend of hypercoagulation[57]. Indeed, 3 4 thromboprophylaxis with Low Weight Molecular Heparin (LMWH) in COVID-19 5 6 patients has been implemented in several centers worldwide, where it has demonstrated 7 8 9 a clear overall benefit in hospitalized patients on mechanical ventilation, reducing the 10 11 death rate in this particular patient population [58, 59]. Currently, no study has 12 13 methodically evaluated the benefit of LMWH at earlier stages of the disease. However, 14 15 16 given that the endothelial damage and consequent micro-thrombi formation are likely 17 18 key pathogenic triggers of downstream events[60], including of inflammation, studies in 19 20 this direction could be very useful; It is further particularly interestingly to note that 21 22 23 heparin also acts on macrophages reducing pro-inflammatory cytokine secretion and, 24 25 most importantly, inhibits the NF-κB pathway in AEC-II cells reducing 26 27 inflammation[61]. Failure to downregulate NF- 28 κB was indeed observed in other, 29 30 unrelated chronic inflammatory diseases, such as in rheumatoid arthritis [62]. 31 32 Furthermore, one of the first drugs used in COVID-19 was Tocilizumab, a monoclonal 33 34 antibody directed to the IL6 receptor that blocks the interaction with its ligand IL-6, one 35 36 37 of the main pro-inflammatory cytokines involved in the ” cytokines storm ” [63, 64]. A 38 39 reduction in IL-6 produce a negative feedback on NF-Kb pathway [65]. 40 41 42 Recently some authors have reported on suspect Kawasaki Syndrome induced by 43 44 SARS-CoV2 in children [66]. This finding, previously already reported for others 45 46 coronavirus infections [67], further supports our hypothesis of the essential role of NF- 47 48 49 Kb pathway. In fact, and strikingly, recent studies have highlighted the importance of 50 51 the NF-κB pathway activation as a direct and cause-effect factor in the 52 53 immunopathology underlying Kawasaki syndrome [68], and the role of the miRNAs 54 55 56 was also hypothesized for this pathology [69]. Therefore, we suppose that Kawasaki 57 58 Syndrome is likely to be secondarily triggered, rather than directly caused by SARS- 59 60 CoV2 and could explain even other occasional reporting of this association in the 61 62 63 64 65

past[67, 70], and may be our findings could be useful to better understand the Kawasaki 1 2 Syndrome etiopathogenesis. 3 4 Conclusion: 5 6 In essence, we propose the following. 7 8 9 1. The main target of SARS-CoV-2 is the AEC-II cell and the NF-κB activation herein 10 11 is a key driver of severe ARDS in Covid 19 patients. The first colonization of the upper 12 13 airways is a necessary but not sufficient condition for the development of COVID-19 14 15 16 syndrome, because of an initial immuno-escape strategy of the virus from the Innate 17 18 Immune response. We believe that the greater affinity of SARS-CoV-2 for ACE2 19 20 relatively to other coronaviruses enables it to more efficiently reach the main target, 21 22 23 namely the AEC-II cells which though represent only 10% of all alveolar cells, play a 24 25 fundamental role in the mediation of inflammatory responses and, ultimately in the 26 27 remodeling of the lung and its homeostasis. The infection of these cells subsequently 28 29 30 leads to the activation and amplification of the inflammatory process in other cell types, 31 32 including AEC-I and the endothelium, during which the activation of the NF-κB 33 34 pathway is amplified by an excessive transcription mediated by the virus with a 35 36 37 mechanism to be ascertained. 38 39 Two different mechanisms could explain the NF-κB pathway involvement, namely the 40 41 42 activation of Interferons during the course of the Innate Immune Response; 43 44 alternatively, but not mutually exclusive, miRNAs could also be directly or indirectly 45 46 involved, as shown in other pathological conditions. A similarly important question is 47 48 49 whether the endothelium itself, which also expresses ACE2 receptors, is a direct port of 50 51 entry for the virus and direct contributor to the pathogenesis of the coagulopathy, as 52 53 speculated recently [57]. The direct endothelial damage theory could certainly explain 54 55 56 the hyper-coagulation state observed in many Covid 19 patients and could also 57 58 contribute to the enhanced pathology seen in hypertensive patients, given that 59 60 hypertension promotes endothelial damage and a hyper-coagulative state. 61 62 63 64 65

2. The early Alveolar Damage and delayed of the proliferative phase contributes to 1 2 severe and permanent damage. We think that there is a protracted phase of exudation 3 4 with destruction of the alveolus and the endothelium and Diffuse Alveolar Damage 5 6 (DAD). All these features are the consequence of a pro-inflammatory state, in which the 7 8 9 main role is played by the M1 polarized alveolar macrophages. Moreover, macrophages 10 11 are identified as predominant cells in the intra-alveolar cavity of COVID-19 12 13 patients[51]. The endothelium is exposed to injury and therefore contributes to 14 15 16 development of ARDS, by widespread and extensive deposition of extracellular matrix 17 18 leading to early interstitial and intra-alveolar fibrosis, and non-pulmonary syndrome 19 20 (e.g., Immune Thrombocytopenic Purpura) [71]. The timing of macrophage polarization 21 22 23 changing from M1 to M2 is furthermore relevant and dependant from miRNAs 24 25 expression [52, 72, 73] (Fig. 4), and could be justify the overlapping of epithelial 26 27 vascular and fibrotic pattern of lung injury. 28 29 30 After the ARDS started a Multi Organ Failure (MOF) could be develop, probably by 31 32 endothelium infection via ACE2 receptors, as recently reported [74]. 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 Some co-morbidities of patients may favor the entry mechanism of SARS-CoV2 into 58 59 60 AEC-II cells, such as scarce presence of surfactant (like in diabetes). Another possible 61 62 63 64 65

enhancing cause of viral infection is the reduced expression of ACE2 in diabetes, 1 2 hypertension and cardiac pathologies. In addition, some gender differences can also be 3 4 explained by the influence of hormonal factors in the production of surfactant: female 5 6 hormones are known to have a function favoring the production of surfactant. This may 7 8 9 explain why women have a less severe disease course. This observation is also 10 11 supported by the fact that, regardless of the course of the disease, the incidence of the 12 13 disease in women remains lower in all age groups up to menopause, from there onwards 14 15 16 the incidence is comparable to that of men. Additionally, there could be a hormonal 17 18 influence that could also justify the lower incidence in children under 10 years of age. 19 20 If this hypothesis were to be confirmed by clinical and molecular finding, the following 21 22 23 interventions could be attempted (Fig. 5): 24 25 1. NF-kB Inhibitors: the NF-κB signaling pathway has become a potential target 26 27 for pharmacological intervention for a wide range of pathologies, such as cancer 28 29 30 (angiogenesis and metastases) and chronic inflammation. Numerous inhibitors 31 32 of NF-κB have been identified that act on different mechanism of NF-kB 33 34 activation. In a review by Gilmore and Hersovitch reported over 750 inhibitors 35 36 37 of NF-κB activation, and summarized their activity essentially in three 38 39 mechanisms: (1) blockage of the incoming stimulating signal at an early stage; 40 41 42 (2) interference with a cytoplasmic step in the NF-κB activation pathway by 43 44 blockage of a specific component of the cascade (e.g., the activation of the IKK 45 46 complex, or degradation of IκB); or (3) blockage of NF-κB nuclear activity[75]. 47 48 49 Two examples of these inhibitors are: Denosumab, a humanized monoclonal 50 51 antibody that binds Receptor Activator of Nuclear factor Kappa B Ligand 52 53 (RANKL), used in metastatic bone cancer and in Rheumatoid Arthritis , a well- 54 55 56 known and tolerate drug with a potent NF-Kb pathway inhibition activity; as to 57 58 Bortezonib, a drug used in multiple mieloma, that inhibits the 26S subunit of the 59 60 61 62 63 64 65

proteasome which leads to the inability of IκB degradation and NF-κB activation 1 2 [76]. 3 4 2. Macrophage control of polarization to promote the switching from 5 6 proinflammatory (M1) to anti-inflammatory (M2) phenotype. [52, 72, 77]. 7 8 9 3. Surfactant (Replacement/Improvement) 10 11 a. Replacement by Liquid Ventilation (Partial/Total): is a technique of 12 13 mechanical ventilation in which the lungs are insufflated with an 14 15 16 oxygenated perfluorochemical liquid (PFC). Some studies suggest that 17 18 PFC reduced neutrophil adhesion, activation, and migration[78]; 19 20 moreover, inhibit inflammatory cytokine expression and NF-kB pathway 21 22 23 activation[79]. However, although fascinating and promising, to date it is 24 25 not clear which patients with ARDS can benefit from this type of 26 27 treatment, which still requires mechanical ventilation (at least in the 28 29 30 Total Liquid Ventilation technique) [[80]. Furthermore, is to establish the 31 32 potential virus diffusion risk with this technique by aerosol of PFC 33 34 elimination. 35 36 37 b. Improvement: ACE2 Human Recombinant use demonstrates in 38 39 preliminary study that Surfactant protein D concentrations were 40 41 42 increased, and interleukin-6 concentrations were decreased[81–83]. 43 44 Moreover, in a recent study, human recombinant soluble ACE2 45 46 (hrsACE2) are used to blocks growth of SARS-CoV-2 in VERO cells 47 48 49 [83, 84]. 50 51 c. Special consideration for diabetic patient: Recently a study demonstrated 52 53 the potential role of Liraglutide, a drug used in diabetes, to enhance 54 55 56 ACE2 expression in the lung and to reduce the lung and cardiovascular 57 58 impairment induced by diabetes, moreover improving the surfactant 59 60 production (SP-A and SP-B)[85]. 61 62 63 64 65

4. Endothelium protection: Low Weight Molecular Heparin, already widely used, 1 2 but with early administration before the onset of clinical symptoms and at 3 4 therapeutic dosage. 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 List of abbreviations: 30 31 AEC I: Alveolar Epithelial Cell Type I 32 33 AEC II: Alveolar Epithelial Cell Type II 34 ACE2: Angiotensin Converting Enzyme 2 35 36 ARDS: Acute Respiratory Distress Syndrome 37 38 EBV: Ebstein Barr Virus 39 40 HCoV: The human Coronavirus 41 HIV-1: Human Immunodeficiency Virus 1 42 43 HTLV-1: human T-cell lymphotropic virus 44 45 HBV: hepatitis B virus, 46 47 HCV: hepatitis C virus 48 49 IL1: Interleukin 1 50 IL6: Interleukin 6 51 52 LMWH: Low Weight Molecular Heparin 53 54 MERS CoV: Middle East Respiratory Syndrome CoV 55 56 MOF: Multiple Organ Failure 57 NF-κB pathway: Nuclear Factor kappa light chain enhancer of activated B cells 58 59 pathway 60 61 (RT)-PCR real-time reverse transcription polymerise chain reaction 62 63 64 65

SARS: Severe Acute Respiratory Syndrome 1 SARS-CoV-2: Severe Acute Respiratory Syndrome Corona Virus 2 2 3 SARS-Co-V: Severe Acute Respiratory Syndrome Coronavirus 4 5 SP: surfactant protein 6 TMPRSS2: Trans Membrane Protease Serin 2 7 8 TNF: Tumor Necrosis Factor 9 10 COVID-19: Corona Virus Disease 2019 11 12 13 Declarations: 14 15 • Ethical Approval and Consent to participate: not applicable 16 17 • Consent for publication: yes 18 19 • Availability of supporting data: not applicable 20 21 • Competing interests: none 22 23 • Funding: no fundings received 24 25 • Authors' contributions: Maurizio Carcaterra had the main idea of this paper, 26 27 Cristina Caruso contributed to collecting data and writing of the paper. 28 • Acknowledgements: I'm grateful to Associate Professor Maria Laura 29 30 Avantaggiati (Department of Oncology Georgetown University. Lombardi 31 32 Cancer Center, Washington D.C.) for helpful suggestion and proofreading 33 34 35 Authors' information: 36 37 M. Carcaterra 1, C. Caruso2 38 39 1 40 Radiation Oncologist at Ospedale di Belcolle, Viterbo (VT) Italy. 41 42 Radiation Oncologist at Azienda Ospedaliera San Giovanni Addolorata, Rome 43 44 2 45 (RM) Italy. 46 47 1)Maurizio Carcaterra, 01100 Viterbo (VT) Italy (Belcolle Hospital) Radiation 48 49 Oncology Department, [email protected] (institutional), 50 51 carcaterra.mauriziogmail.com (personal) 52 2)Cristina Caruso, 00100 Rome (RM) Italy (San Giovanni Addolorata Hospital) 53 54 Radiation Oncology Department, [email protected] (institutional), 55 56 [email protected] (personal) . 57 58 59 60 61 62 63 64 65

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Figure 1

SARS-CoV-2 viral progression pathway: main steps

Figure 2

NF-Kb pathway upregulation mechanism Figure 3

Role of M1 macrophage phenotype in sustaining vicious circle of inammation Figure 4

Impaired Proliferative phase of Acute Respiratory Distress Syndrome in SARS-CoV-2 Figure 5

Scheme of the pathogenetic mechanisms and possible therapeutic interventions

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