key: cord-0894448-6yvcsntm
authors: Kamle, Suchitra; Ma, Bing; He, Chuan Hua; Akosman, Bedia; Zhou, Yang; Lee, Chang Min; El-Deiry, Wafik S.; Huntington, Kelsey; Liang, Olin; Machan, Jason T.; Kang, Min-Jong; Shin, Hyeon Jun; Mizoguchi, Emiko; Lee, Chun Geun; Elias, Jack A.
title: Chitinase 3-like-1 is a Therapeutic Target That Mediates the Effects of Aging in COVID-19
date: 2021-02-16
journal: bioRxiv
DOI: 10.1101/2021.01.05.425478
sha: ff5c65d7a95f7e2e8db0299449f4180f189319c6
doc_id: 894448
cord_uid: 6yvcsntm

COVID-19 is caused by the SARS-CoV-2 (SC2) virus and is more prevalent and severe in the elderly and patients with comorbid diseases (CM). Because chitinase 3-like-1 (CHI3L1) is induced during aging and CM, the relationships between CHI3L1 and SC2 were investigated. Here we demonstrate that CHI3L1 is a potent stimulator of the SC2 receptor ACE2 and viral spike protein priming proteases (SPP), that ACE2 and SPP are induced during aging and that anti-CHI3L1, kasugamycin and inhibitors of phosphorylation, abrogate these ACE2- and SPP-inductive events. Human studies also demonstrated that the levels of circulating CHI3L1 are increased in the elderly and patients with CM where they correlate with COVID-19 severity. These studies demonstrate that CHI3L1 is a potent stimulator of ACE2 and SPP; that this induction is a major mechanism contributing to the effects of aging during SC2 infection and that CHI3L1 coopts the CHI3L1 axis to augment SC2 infection. CHI3L1 plays a critical role in the pathogenesis of and is an attractive therapeutic target in COVID-19.

It is highly transmissible and has caused a global pandemic killing >1.5 million people and infecting > 80 million people worldwide as of December, 2020 (1) (2) (3) (4) (5) (6) (7) . The disease caused by SC2, COVID-19, was initially noted to manifest as a pneumonia with fever, cough, fatigue and dyspnea as major manifestations (8) . However, it is now known to be a systemic disease with manifestations in many organs (9, 10) . In addition, it is now known that there is a spectrum of disease severity with patients that are asymptomatic, 10-20% of patients that require hospitalization (4, 5, 11) . and patient with respiratory failure that require intensive care (12) .

Unique clinical features have also been described including pulmonary fibrosis, an increased frequency of vascular thrombotic events, a coagulopathy and pulmonary angiitis (13, 14) .

However, the cellular and molecular events that account for this impressive clinical and pathologic heterogeneity are poorly understood.

One of the most unique features of COVID-19 is its impressive relationship to aging and comorbid disorders. The elderly is at increased risk of contracting COVID-19 and experience higher complication and case fatality rates (15, 16) . COVID-19 has also been devastating effects on and elderly and residents of congregate care facilities (17, 18) . Similarly, approximately 50% of hospitalized COVID-19 patients have preexisting medical conditions including diabetes, hypertension, obesity and metabolic syndrome, cardiovascular disease and chronic lung diseases like COPD and asthma. These co-morbidities associate with enhanced susceptibility to SC2 infection, more severe disease and a higher mortality (1-3, 5, 15, 19, 20) . Surprisingly, the cellular and molecular events that underlie the effects of aging and comorbid diseases in COVID- 19 have not been defined.

SC2 interacts with cells via its spike (S) protein which binds to its host cell receptor angiotensin converting enzyme 2 (ACE2) which mediates viral entry (21) (22) (23) (24) . After binding, the S protein is processed by the S priming proteases (SPP) TMPRSS2, Cathepsin L (CTSL) and in some cases FURIN into S1 and S2 subunits. The latter mediates the fusion of the viral envelope and cell plasma membrane which is required for virus-cell entry (15, 19, 23) . In the human disease, SC2 passes through the mucus membranes of the upper and lower respiratory tracts and infects epithelial cells via this ACE2-S protein-protease mechanism (15, 19, 25) . The virus can then enter the bloodstream and, via this viremia, infect other cells and organs that express ACE2 and SPP (26) . It has been proposed that the levels of expression of ACE2 and the SPP play key roles in determining the magnitude and organ location of the infection and the severity of the disease (15, 19, 27) . However, the mechanisms that SC2 uses to generate its effects in different tissues have not been defined. Furthermore, although ACE2 is expressed widely in human tissues (15) , the critical processes that regulate ACE2 expression and activation have only recently begun to be investigated.

Virus infection of mammalian cells leads to innate and adaptive immune responses that restrict viral replication, augment viral clearance and limit tissue damage and disease severity (28) . To allow infection to occur many viruses have developed strategies to evade and or suppress these immune responses (28) (29) (30) . They can also co-opt host responses to augment viral replication (28) (29) (30) . These events can drastically influence disease pathogenesis, the course of the infection, disease severity and viral persistence in the host (28) (29) (30) . Compared to other positive stranded (+) RNA viruses, coronaviruses have exceptionally large genomes and employ complex genome expression strategies (28) . Many of the coronavirus genes participate in virushost interplay to create an optimal environment for viral replication (28) . However, the host pathways that SC2 co-opts to foster infection and replication have not been adequately defined and the possibility that SC2 utilization of host responses contributes to the mechanisms by which aging and comorbid diseases augment the prevalence and severity of COVID-19 has not been addressed.

Chitinase 3-like-1 (CHI3L1; also called YKL-40 in human and Chil1 or BRP-39 in mouse)

is an evolutionarily conserved member of the 18 glycosyl hydrolase (GH 18) gene family that is produced by a spectrum of cells in response to a variety of injury and cytokine stimuli (31) (32) (33) (34) (35) . It is the cornerstone of a critical pathway that is activated during injury and inflammation, regulates innate and adaptive immunity and heals and protects (32, 34, (36) (37) (38) . The latter is mediated by its ability to inhibit apoptosis and other forms of cell death while driving fibroproliferative repair (36, 38) . CHI3L1 is readily detected in the circulation of normals and the levels of circulating and tissue CHI3L1 are increased in diseases characterized by inflammation, injury, remodeling and repair (31-33, 37, 39-46) . Interestingly CHI3L1 is also expressed in an exaggerated manner in aging and in the same comorbid diseases that are risk factors for COVID-19 (47) (48) (49) (50) (51) (52) (53) (54) . CHI3L1 and ACE2 are also both mediators of pulmonary protective responses (36, (55) (56) (57) (58) . In light of these impressive similarities we hypothesized that the induction of ACE2 and SPP is part of the CHI3L1induced healing and repair response. We also hypothesized that SC2 co-opts the host CHI3L1 axis to augment its ability to infect, spread and generate disease. To address these hypotheses, we used genetically modified mice, in vitro approaches and human cohorts. These studies demonstrated that (a) CHI3L1 is a potent stimulator of ACE2 and SPP in pulmonary epithelial cells and vascular cells; (b) that the expression of ACE2 and SPP are increased during murine aging and that these aging-induced inductive events are mediated by CHI3L1; and (c) that interventions that alter CHI3L1 effector responses are also potent inhibitors of ACE2 and SPP and viral infection. Lastly, human studies demonstrated that the levels of circulating CHI3L1 are increased in COVID-19 (+) patients that are elderly, have comorbid diseases and manifest severe COVID-19 disease. These findings support the concept that CHI3L1 plays a critical role in the pathogenesis of and is an attractive therapeutic target in COVID-19. They also provide a mechanistic explanation for how aging and comorbid diseases contribute to the pathogenesis of COVID-19.

To begin to address the relationship(s) between CHI3L1 and SC2, we characterized the effects of CHI3L1 on the expression and accumulation of ACE2 and SPP in vivo. In these studies, we compared the expression of Ace2 and SPP in lungs from inducible CC10 promoter-driven CHI3L1 overexpressing Tg mice (CHI3L1 Tg) and wild type (WT) controls. These studies revealed the induction of Ace2, Tmprss2 and Ctsl in lungs from CHI3L1 Tg mice (Fig. 1, A and B ). Ace2 protein was most prominent in airway epithelium where it colocalized with CC10 ( Fig. 1C and Supplemental Fig. S1 ). Ace2 was also seen in alveoli where it co-localized with pro-SP-C ( Fig.   1C and Supplemental Fig. S1 ). Tmprss2 and Ctsl were also prominently expressed in airway epithelial cells (Fig. 1 , C and D and Fig. S1 ) and TMPRSS2 was also seen in alveolar macrophages (Fig. 1D ).

Tg CHI3L1 also augmented the expression and accumulation of Ace2 and SPP in pulmonary blood vessels. Immunohistochemistry demonstrated that Ace2 and Tmprss2 colocalized with CD-31 on endothelial cells and transgelin (+) vascular smooth muscle cells ( Fig. 1E and Supplemental Fig. S1 ). When viewed in combination these studies demonstrate that CHI3L1 is a potent stimulator of epithelial and vascular cell ACE2 and SPP in vivo.

In vitro experiments were next undertaken to determine if rCHI3L1 regulated the expression and or accumulation of ACE2 and SPP. These studies demonstrated that rCHI3L1 stimulated ACE2 and SPP (TMPRSS2, Cathepsin L) gene expression and protein accumulation in human Calu-3 epithelial cells in a time-and dose-dependent manner (Fig. 2, A and B) . These effects were not Calu-3 cell specific because similar results were obtained with A549 epithelial cells, primary human small airway epithelial cells (HSAEC cells) and lung fibroblasts (Supplemental Figs. S2 and S3). When viewed in combination, these studies demonstrate that CHI3L1 stimulates ACE2 and SPP in a variety of cells in vitro.

To understand the consequences of CHI3L1 induction of ACE2 and SPP, studies were undertaken to determine if this induction augmented the ACE2 receptor binding of Spike (S) protein and or the metabolism of S into its S1 and S2 subunits. In these experiments lysates were prepared from Calu-3 cells incubated with rCHI3L1 or vehicle control and recombinant (r) spike protein was added. To assess S-receptor binding, immunoprecipitation and immunoblots were serially undertaken with anti-ACE2 and anti-S respectively. To assess S metabolism, the cell lysates were incubated for 24 hours with rS and Western blots were undertaken with S1 and S2 specific antibodies. As can be seen in Figs. 2C and 2D, CHI3L1 stimulation was associated with enhanced ACE2-S binding and enhanced metabolism of S into its respective subunits.

To further address the consequences of these inductive events, Pseudovirus moieties were generated by incorporating the SC2 "S" protein into lentivirus moieties which contained a GFP marker. The infectious capacity of these pseudovirus S moieties was then evaluated using cells incubated with rCHI3L1 or control vehicle. In keeping with the findings noted above, rCHI3L1 induction of ACE2 and SPP augmented pseudovirus S incorporation into lung epithelial cells. (Fig.   2E ). FACS evaluations further confirmed these observations by demonstrating that rCHI3L1 enhanced the cellular integration of S-protein containing pseudovirus and that treatment with anti-CHI3L1 monoclonal antibody (FRG) ameliorated rCHI3L1-stimulated pseudovirus cell integration ( Fig. 2F ). CHI3L1 similarly enhanced the cellular integration of pseudovirus containing S sequence with D416 or G416 and FRG treatment inhibited pseudoviral infection regardless of S sequence (Fig. 2F ). When viewed in combination, these studies demonstrate that CHI3L1 stimulation of ACE2 and SPP increases S-ACE2 binding, S metabolism and pseudovirus S infection.

The finding that CHI3L1 is a potent stimulator of ACE2 and SPP raises the interesting possibility that CHI3L1 may be a useful therapeutic target in COVID-19. To begin to address this possibility, we characterized the effects of a humanized monoclonal antibody against CHI3L1 developed in our laboratory (called FRG), and a small molecule Chitinase 1 and CHI3L1 inhibitor called kasugamycin in vitro and in vivo. The monoclonal inhibitor of CHI3L1 (FRG) was a potent inhibitor of CHI3L1 stimulation of epithelial cell ACE2 and SPP in vitro (Fig. 3, A and B) . FRG was also a powerful inhibitor of Tg CHI3L1 induction of ACE2 and SPP in vivo (Fig. 3C ).

Interestingly, kasugamycin was a similarly powerful inhibitor of CHI3L1 induction of ACE2 and SPP in vitro and in vivo (Fig. 3, D&E) . These studies highlight antibody-based and small molecule CHI3L1 inhibitors that control ACE2 and SPP and have promise as therapeutics in COVID-19.

To date, all studies of CHI3L1 have assumed that it is not post-translationally modified.

However, phosphorylation site prediction analysis using the NetPhos program (v2.0) suggests that CHI3L1 is a phosphoprotein with high potential for serine/threonine phosphorylation. Further investigation also provided a number of lines of evidence that suggest that CHI3L1 is effective as a phosphoprotein. This included (a) sequence mining of CHI3L1 which revealed a cyclin binding domain that is highly predictive of CDK phosphorylation (Supplemental Fig. S4 ); (b) sites of serine phosphorylation of rCHI3L1 confirmed with immunoblot assays with an anti-phosphoserine antibody (EMD Millipore, AB1603) (Fig. 4A) ; (c) the demonstration that CHI3L1 phosphorylation is dependent on CDK activity based on its inhibition by the broad spectrum CDK inhibitor Flavopiridol. As a result we hypothesized that phosphorylation in this region is essential for CHI3L1 effector responses. To address this hypothesis we used in vitro and in vivo approaches.

In the former, we generated WT CHI3L1 and mutant forms of rCHI3L1 that could not be phosphorylated and compared their ability to induce epithelial cell ACE2 and SPP. As can be seen in Fig. 4B , WT CHI3L1 was a powerful stimulator of epithelial ACE2 and SPP and this effect was abrogated by mutations at AA 230 (Serine to Arginine mutation at site of amino acid 230 of CHI3L1). Mutations at AA 235 or 237 did not have similar effects (data not shown). In the in vivo experiments, we compared the effects of transgenic CHI3L1 in mice treated with the broad spectrum CDK inhibitor Flavopiridol (Fig. 4C ) or the selective CDK 1 and 2 inhibitor (BMS 265246) and their vehicle controls (Fig. 4D ). In both cases, the CDK inhibitors abrogated the ability of CHI3L1 to stimulate epithelial cells ACE2 and SPP. These studies demonstrate that CHI3L1 is effective as a phosphoprotein and highlight the ability of CDK inhibitors, particularly inhibitors of CDK 1 and 2, to control this phosphorylation and, in turn, serve as a therapeutic in COVID-19.

The elderly is at increased risk of contracting COVID-19 and experience higher complication and case fatality rates (15, 16) . These unique features have interesting parallels in the CHI3L1 axis where the levels of circulating CHI3L1 increase with aging (59) . To further understand these relationships, we measured the levels of circulating CHI3L1 in WT mice as they age. As can be seen in figure 5A , the levels of circulating CHI3L1 and the levels of downstream ACE2, and SPP increased significantly in comparisons of 6 and 12-month-old WT mice.

Importantly, treatment of WT mice twice a week with the monoclonal antibody FRG from 6 months to 12 months of age was remarkably effective in inhibiting the expression and accumulation of CHI3L1, ACE2 and SPP in aged mice (Fig. 5B ). These studies demonstrate that the increase in circulating CHI3L1 that is seen with aging stimulates the expression and accumulation of ACE2 and SPP. They also suggest that these changes in CHI3L1, ACE2 and SPP contribute to the pathogenesis of the heightened COVID-19 responses in the elderly.

As noted above, SC2 infections are more common and more severe in the elderly and patients with comorbid diseases (1-3, 5, 15, 19, 20) . Studies from our laboratory and others have also demonstrated that the levels of circulating CHI3L1 are also increased in the diseases that are COVID-19 risk factors (47) (48) (49) (50) (51) (52) (53) (54) . Thus, to further understand the relationships between CHI3L1 and the COVID-19 risk factors, we measured the levels of CHI3L1 in the serum of normal healthy individuals and patients presenting to the emergency department (ED) at Rhode Island Hospital for medical evaluations that prompted a COVID-19 diagnostic evaluation. The demographics of these cohorts can be seen in supplemental Table S1 . In keeping with reports from our laboratory and others the majority of the normal healthy individuals had levels of circulating CHI3L1 between 15 and 60 ng/ml (Fig. 6A) . Interestingly, the levels of circulating CHI3L1 in the individuals in the normal healthy cohort was not significantly different than the levels in patients presenting to the ED who did not have comorbid diseases (hypertension, diabetes, arthritis, neurologic disease, cancer, stroke, obesity and or chronic lung disease) (Fig. 6A ). They were however, significantly lower than the levels in the circulation of patients presenting to the ED with existing comorbid diseases ( Fig. 6A ) and patients that tested positive for COVID-19 (Fig. 6B ). When the COVID-19

(-) and (+) ED patients were evaluated together the levels of circulating CHI3L1 were increased in the patients that were greater than 50 years of age ( Fig. 6C ) and or had hypertension (Fig. 6D ).

The levels of circulating CHI3L1 correlated with COVID-19 disease severity. This can be seen in Figures 6E and 6F which demonstrate that the levels were significantly increased in patients that were admitted to the hospital compared those that were discharged to home (Fig. 6E ) and that the circulating levels of CHI3L1 were significantly increased in patients with higher COVID Severity Scores (CSS) ( Fig. 6F and Table S2 ). When only COVID-19 (+) patients were evaluated, the levels of circulating CHI3L1 were increased in patients with hypertension versus those without hypertension; patients that were admitted versus those that were sent home from the ED; patients that were greater than 50 years old versus younger individuals and patients with comorbid diseases versus COVID-19 (+) patients without concurrent comorbid disorders (Fig. 6 , G-J).

Overall, these studies demonstrate that the levels of circulating CHI3L1 are increased in the elderly and patients with comorbid disease like hypertension. They also demonstrate that the levels of circulating CHI3L1 are increased in COVID-19 (+) patients that are elderly, have comorbid diseases and manifest severe COVID-19. These observations support the concept that SC2 co-opts the CHI3L1 axis to stimulate ACE2 and SPP which augment viral infection and foster SC2 disease manifestations. They also suggest that the effects of the COVID-19 risk factors are mediated, at least in part, by their ability to stimulate CHI3L1.

The CHI3L1 axis is a major endogenous healing and repair response and ACE2 is a protective moiety in acute lung injury (ALI) (38, (55) (56) (57) . To determine if they are related to one another, we characterized the effects of CHI3L1 on ACE2 and SPP in vivo and in vitro. In our initial studies, we compared the expression of Ace2 and SPP in lungs from CC10-driven CHI3L1 overexpressing Tg mice and WT controls. These studies demonstrated induction of Ace2, Tmprss2 and CtsL in lungs from Tg (+) mice. Ace2 was most prominent in airway epithelium. It was also seen in alveoli where it co-localized with SP-C and CC10 and stained blood vessels where it co-localized on endothelial and smooth muscle cells. Tmprss2 and Ctsl were also seen in airway epithelial cells and Tmprss2 was seen in alveolar macrophages. In the in vitro experiments rCHI3L1 impressively stimulated the levels of mRNA encoding ACE2, TMPRSS2 and CTSL in human lung epithelial cell lines (Calu-3, A549) and primary human small airway epithelial cells (HSAEC) and lung fibroblasts. Importantly, these inductive responses were markedly decreased by treatment with moieties that alter CHI3L1-induced effector responses such as monoclonal anti-CHI3L1 (FRG), kasugamycin and inhibitors of CHI3L1 phosphorylation.

These findings support the concept that SC2 co-opts the CHI3L1 healing and repair response to increase the number of cellular targets for viral entry. They also suggest that the host's attempt to heal and repair via CHI3L1 is complicit in SC2 viral infection and COVID-19 morbidity and mortality.

In the time since the outbreak of the COVID-19 pandemic unique features of its epidemiology have been appreciated. This includes the impressive increase in the prevalence and severity of the disease in the elderly, particularly those in congregate care settings (16, 17) .

The prevalence and severity of COVID-19 are also increased in patients with comorbid diseases including diabetes, hypertension, obesity and metabolic syndrome, cardiovascular disease and chronic lung diseases like COPD where they associate with enhanced susceptibility to SC2 infection, more severe disease and a higher mortality (1-3, 5, 15, 19, 20) . These unique features have interesting parallels in CHI3L1. Specifically, the levels of circulating CHI3L1 increase with aging. In fact, they have been reported to be the best predictor of all-cause mortality in octogenarians (59) . In addition, studies from our laboratory and others have demonstrated that the levels of circulating CHI3L1 are also increased in the very diseases that are COVID-19 risk factors (47) (48) (49) (50) (51) (52) (53) (54) where they correlate with disease severity (49, 50, 53) . ACE2 expression and viral loads have been suggested to increase with aging and it has been proposed that these changes can explain the impressive severity of COVID-19 in the elderly and patients with comorbid diseases (16, 17) . Our studies support this contention and provide novel insights into the mechanisms that may underlie these associations by demonstrating that the levels of circulating CHI3L1 increase during murine aging, that these inductive events are abrogated by treatment with anti-CHI3L1 and that CHI3L1 stimulates ACE2 and SPP which increases ACE2-S protein-cell interaction, S protein activation and pseudovirus-S infection.

COVID-19-associated vasculitis and vasculopathy are now considered defining features of the systemic disease caused by SC2 (10, 60) . The importance of vascular alterations can also be appreciated in recent studies comparing the lungs of patients infected with SC2 and influenza (13) . Both manifest diffuse alveolar damage and perivascular infiltrating lymphocytes. There were, however, distinctive vascular manifestations in the COVID-19 tissues. They included (a) severe endothelial injury with intra-endothelial SC2 virus and disrupted endothelial membranes;

(b) widespread vascular thrombosis; and (c) elevated levels of intussusceptive angiogenesis.

They also noted increased numbers of ACE2+ endothelial cells in infected patients versus controls (13) . These and other studies have led to the belief that endothelial injury plays a key role in these responses and that the injury is caused by direct viral infection and perivascular inflammation (10, 13) . Our studies support this concept by demonstrating, for the first time, that CHI3L1 is a powerful stimulator of endothelial cell and vascular smooth muscle cell ACE2 and SPP. They also raise the intriguing possibility that the CHI3L1 is a key stimulator of the pulmonary and systemic manifestations of COVID-19 and that it does this, at least in part, by enhancing the susceptibility of endothelial cells and smooth muscle cells to infection with the virus.

Studies from our lab and others have demonstrated that CHI3L1 is a critical regulator of inflammation and innate immunity and a stimulator of Type 2 immune responses, fibroproliferative repair and angiogenesis (34, 37, 38, (61) (62) (63) (64) (65) . These studies also demonstrated that CHI3L1 is dysregulated in a variety of diseases characterized by injury, inflammation and or tissue remodeling (31-33, 37, 39-46) . In keeping with these findings, we focused our recent efforts on the development of CHI3L1-based interventions that could serve as therapeutics for CHI3L1dependent diseases. These studies have defined an exciting therapeutic platform based on CHI3L1. Our initial studies focused on biologic approaches and the generation of a panel of monoclonal antibodies against CHI3L1 using full length and peptide immunogens. These antibodies were assessed in a variety of murine models. The most impressive responses were obtained with a monoclonal antibody against a peptide that contained the sequence between AA 223-234 of human CHI3L1. This antibody is now called FRG. It was originally a mouse IgG2b kappa. A humanized version has been generated on an IgG1 backbone. There are a number of reasons to believe that FRG can be an effective therapeutic in COVID-19. First, FRG is a potent inhibitor of the basal levels and the CHI3L1-stimulated expression and accumulation of ACE2 and SPP. It is also important to point out that studies from our lab and others have demonstrated that CHI3L1 drives inflammation in a type 2 direction and augments type 2 and decreases type 1 T cell differentiation (34, 66) . This is problematic for COVID-19 because, in contrast to type 2 immunity, type1 immune responses have potent antiviral effects (67) . Thus, FRG treatment would abrogate CHI3L1 stimulation of type 2 responses and augment type 1 antiviral responses in a number of cells and tissues. FRG also inhibits fibroproliferative repair which could ameliorate the pathologic fibrosis that occurs in patients with respiratory failure due to COVID-19 who are intubated for an extended interval (68) . Based on these observations one can see how FRG could be used as a prophylactic to diminish the chances of an individual getting infected after exposure to a SC2-infected person. One can also see how FRG, alone or in combination with antivirals such as remdisivir, could be therapeutically useful in patients with established SC2 infection. Our studies demonstrate that CHI3L1 has an activated state that is phosphorylated and that this activation is mediated by CDKs. They also demonstrate that AA 230 plays a critical role in CHI3L1 effector responses. In keeping with these findings Flavopiridol, a broad spectrum CDK inhibitor, proved to be a powerful inhibitor of CHI3L1 stimulation of ACE2 and SPP in vitro and in vivo. This is likely due, in great extent, to the inhibition of CDK 1 and or 2 because BMS 265246 had similar effects. It is also tempting to speculate that, because the monoclonal antibody FRG was generated against a site of phosphorylation, that its impressive potency is the result of its ability to function as a blocking antibody and its ability to interfere with this critical phosphorylation site. These findings suggest that Flavopiridol, BMS265246 and possibly other CDK inhibitors could be useful therapeutics in COVID-19. This could be an exciting example of drug repurposing because Flavopiridol, which is also called Alvocidib, has already received orphan drug designation from the FDA and EMA for the treatment of CLL (73) .

RNA viruses often trigger the RIG like helicase (RLH) innate immune system. In this response, antiviral immunity is triggered by the dsRNA and 5' triphosphate bearing molecules that arise in the cytosol as viral replication intermediates. These moieties are recognized by widely expressed cytoplasmic sensors from the RIG-I-like receptor family including retinoic acidinducible gene I (RIG-I) and melanoma differentiation-associated protein 5 (mda-5) (28). The

(mitochondrial antiviral signaling molecule), the central integrator of this pathway, to triggers a type I interferon (IFN)-based antiviral response (28) . The RLH pathway plays a major role in viral control and clearance. Interestingly, studies from our laboratory have also demonstrated that the RLH pathway is also a powerful inhibitor of CHI3L1 expression and accumulation (74) . Based on these observations one would expect that SC2 infection and subsequent RLH activation would decrease the levels of circulating CHI3L1. This would decrease the expression of ACE2 and SPP which would decrease viral infection and spread and subsequent tissue injury. In keeping with the appreciation that viruses have evolved strategies that suppress or evade antiviral immunity (75) , the levels of induction of type I IFNs are decreased in patients with COVID-19 (76, 77) . We believe this decreased production of type I IFNs is a major contributor to the prevalence and severity of COVID-19 because it would increase the levels of circulating Chi3l, increase the expression and accumulation of ACE2 and SPP and augment viral infection and spread.

Early strains of SARS-CoV-2 from Wuhan China showed limited genetic diversity (78) .

However, genetic epidemiologic investigations conducted in late February 2020 have identified an emerging D614G mutation of the SARS-CoV-2 spike protein in viral strains from Europe (78, 79) . Patients infected with D614G associated SARS-C0V-2 manifest enhanced viral loads.

Pseudotyped virus with G614 mutations also manifest enhanced viral infectivity based on a spike protein that is more likely to assume an "open" configuration and bind to ACE2 with enhanced avidity (78) . One can envision how S protein mutations can lead to viruses that are increasingly problematic from a therapeutic perspective. However, one can also see how CHI3L1-based therapeutics which inhibit ACE2 and SPP can inhibit SARS-CoV-2 infection regardless of the S protein sequence.

Our studies demonstrate that CHI3L1 is a major stimulator of ACE2 and SPP that enhances SC2 "S" protein-receptor binding and activation and augments SC2 infection and spread. They also led us to appreciate that the levels of circulating CHI3L1 play a major role in defining the propensity for and severity of SC2 infection and contribute to the mechanisms by which aging and comorbid diseases contribute to the pathogenesis of COVID-19. To further understand the interactions between CHI3L1 and SC2, we measured the levels of circulating CHI3L1 in a cohort of normal individuals and in a cohort of patients presenting to the ED with symptoms suggestive of COVID-19. These studies support our speculations in a number of ways.

First, the levels of circulating CHI3L1 in patients in the ED cohort that were infected with SC2 and patients with comorbid diseases were significantly greater than in the normal healthy controls. In accord with what has been reported in the literature (59, 80) , when the ED cohort patients were evaluated as a group, the levels of circulating CHI3L1 were increased in patients that were elderly, had hypertension, had comorbid diseases or required hospitalization. Importantly, within the COVID-19 positive ED patients the levels of circulating CHI3L1 were statistically increased in the patients that were elderly, had hypertension or other comorbid diseases and or required hospitalization. These observations support the concept that SC2 co-opts the CHI3L1 axis to augment viral infection and foster its disease manifestations. They also raise the interesting possibility that quantification of the levels of circulating CHI3L1 can be useful in assessing the severity and need for hospital admission of patients presenting with COVID-19. Additional experimentation will be required to assess this speculation and further understand the relationships between CHI3L1 and COVID-19.

Lung-specific CHI3L1 overexpressing transgenic mice in which CHI3L1 was targeted to the lung with the CC10 promoter (Chi3l1 Tg) have been generated and characterized by our laboratory as previously described (34, 81) . These mice were between 6-12 weeks old when used in these studies.

All animals were humanely anesthetized with Ketamine/Xylazine (100mg/10mg/kg/mouse) before any intervention. The protocols that were used in these studies were evaluated and approved by the Institutional Animal Care and Use Committee (IACUC) at Brown University.

Protein lysates from macrophages and whole mouse lungs were prepared with RIPA lysis buffer (ThermoFisher Scientific, Waltham, MA, USA) containing protease inhibitor cocktail (ThermoFisher Scientific) as per the manufacturer's instructions. 20 to 30 µg of lysate protein was subjected to electrophoresis on a 4-15% gradient mini-Protean TGX gel (Bio-Rad, Hercules, CA, USA). It was then transferred to a membrane using a semidry method with a Trans-Blot Turbo

Transfer System (Bio-Rad). The membranes were then blocked with Tris-buffered saline with Tween20 (TBST) with 5% non-fat milk for 1 hour at room temperature. After blocking, the membranes were incubated with the primary antibodies overnight at 4 0 C in TBST and 5% BSA.

Th primary antibodies used in this study are: a-ACE2 (Abcam, ERR4435(2), ab108252), a-TMPRSS2 (Abcam, EPR3861, ab92323) , a-CTSL (R&D Systems, AF1515). b-actin (Santa Cruz

Biotechnology, Sc47778). The membranes were then washed 3 times with TBST and incubated with secondary antibodies in TBST, 5% non-fat milk for 1 hour at room temperature. After 3 additional TBST washes, Supersignal West Femto Maximum Sensitivity Substrate Solution (Thermofisher Scientific) was added to the membrane and immunoreactive bands were detected by using a ChemiDoc (Bio-Rad) imaging system.

Total cellular RNA was obtained using TRIzol reagent (ThermoFisher Scientific) followed by RNA extraction using RNeasy Mini Kit (Qiagen, Germantown, MD) according to the manufacturer's instructions. mRNA was measured and used for real time (RT)-PCR as described previously (34, 36) . The primer sequences used in these studies are summarized in supplemental Table S3 . Ct values of the test genes were normalized to internal housekeeping genes such as b-actin, GAPDH or RPL13a.

Formalin-fixed paraffin embedded (FFPE) lung tissue blocks were serially sectioned at 5 μm thickness and mounted on glass slides. After deparaffinization and dehydration, heat-induced epitope retrieval was performed by boiling the samples in a steamer for 30 minutes in antigen unmasking solution (Abcam, antigen retrieval buffer, 100x citrate buffer pH:6.0). To prevent nonspecific protein binding, all sections were blocked in a ready-to-use serum free protein blocking solution (Dako/Agilent, Santa Clara, CA) for 10 minutes at room temperature. The sections were then incubated with primary antibodies (a-Ace2 (R&D Systems, AF3437), a-Tmprss2 (Abcam, ab92323), a-Ctsl (R&D Systems, AF1515), a-CC10 (Santa Cruz

Biotechnology, sc-365992), a-SPC (Abcam, ab90716), a-CD31 (BD Pharmingen, 550274), atransgelin (Abcam, ab14106)) overnight at 4°C. After three washings, fluorescence-labeled secondary antibodies were incubated for 1 hour at room temperature. The sections were then counterstained with DAPI and cover slips were added.

Double label immunohistochemistry was employed as previously described by our lab (34)

The murine monoclonal anti-CHI3L1 antibody (FRG) was generated using peptide antigen (amino acid 223-234 of human CHI3L1) as immunogen through Abmart Inc (Berkeley Heights, NJ). This monoclonal antibody specifically detects both human and mouse CHI3L1 with high affinity (kd »1.1x10 -9 ). HEK-293T cells were transfected with the FRG construct using Lipofectamine TM 3000 (Invitrogen, # L3000015). Supernatant was collected for 7 days and the antibody was purified using a protein A column (ThermoFisher scientific, # 89960). Ligand binding affinity and sensitivity were assessed using ELISA techniques.

Pseudotyped SARS-CoV-2 virus which has a lentiviral core expressing GFP but with the SARS-CoV-2 spike protein (expressing D614 or G614 S protein) on its envelope were obtained from 

The effects of CHI3L1 on the binding of ACE2 and S protein were evaluated using Calu-3 cells and recombinant S protein. In brief, Calu-3 cells were incubated with vehicle (PBS) or rCHI3L1 (500ng/ml) for 24 hrs, recombinant S protein from SARS-COV2 (GeneScript, # Z03481-100) was added to the cells and further incubated for 2hrs. The cells were then harvested and subjected to co-immunoprecipitation (Co-IP) and immunoblot assays as described by our laboratory (65) .

The impact of CHI3L1 on the cellular processing of S-protein was also assessed using Calu-3 cells in vitro. In these experiments, cells were incubated with vehicle (PBS) or rCHI3L1 (500ng/ml) for 24 hrs, harvested and lysates were produced. The lysates were then incubated with recombinant S protein for 2 hrs and the un-cleaved S and cleaved S1 and S2 proteins were evaluated by Western immunoblot analysis using anti-SARS-CoV-2 Spike antibody (Proscience, #3525).

WT CHI3L1-histidine tagged pcDNA was obtained from the MedImmune Inc (Gaithersburg, MD).

The putative serine or tyrosine phosphorylation sites of CHI3L1 located between 230-239 were This included their comorbid diseases, symptoms, discharge or admission, oxygen treatment or ICU transfer as outlined in Table S2 .

Statistical evaluations were undertaken with SPSS or GraphPad Prism software. As appropriate, groups were compared with 2-tailed Student's t test or with nonparametric Mann-Whitney U test.

Values are expressed as mean ± SEM. One-way ANOVA or nonparametric Kruskal-Wallis tests were used for multiple group comparisons. Statistical significance was defined as a level of P < 0.05.

The 

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Disclosures: Jack A Elias is a cofounder of Elkurt Pharmaceuticals that develops therapeutics based on the 18 glycosyl hydrolase gene family.