Vitamin D Regulation of Immune Function.

PURPOSE OF REVIEW: To review the mechanisms by which vitamin D and its metabolites regulate the immune system to facilitate the ability of the body to prevent and/or treat SARS-CoV2 and other respiratory infections and encourage further research into the role that vitamin D supplementation plays in preventing/treating such infections. RECENT FINDINGS: Vitamin D deficiency is associated with an increased risk of SARS-CoV2 and other respiratory infections. Clinical trials in general demonstrate that correction of vitamin D deficiency reduces the risk of hospitalization, ICU admission, and death from SARS-CoV2 infection. The airway epithelium and alveolar macrophages express the enzyme, CYP27B1, that produces the active metabolite of vitamin D, 1,25(OH)2D, and the vitamin D receptor, VDR. Vitamin D and its metabolites promote the innate immune response, which provides the first line of defense against viral and bacterial infections while restricting the adaptive immune response, which if unchecked promotes the inflammatory response leading to the acute respiratory distress syndrome and death. The rationale for treating vitamin D deficiency to reduce the risk of SARS-CoV2 infection and supplementing patients with vitamin D early in the course of SARS-CoV2 infection rests primarily on the ability of vitamin D metabolites to promote an effective immune response to the infection.

Vitamin D regulation of immune function during covid-19.

Covid-19 has to date infected a confirmed 275 million people with 5.4 million, now dead, with the count rising every day. Although the virus, SARS-CoV2, causing Covid-19 infects many cells in the body, its infection of the upper and lower respiratory tract (upper airway epithelia and pulmonary alveolar pneumocytes and macrophages) causing what is now called a cytokine storm in the lungs is the major cause of morbidity and mortality. This results from a dysregulation of the innate immune system with an outpouring of proinflammatory cytokines and chemokines leading to abnormal activation of the adaptive immune pathway. Airway epithelia constitutively expresses CYP27B1, the enzyme producing the active vitamin D metabolite, 1,25(OH)2D, and the vitamin D receptor (VDR) for which 1,25(OH)2D is the ligand. Pulmonary alveolar macrophages, on the other hand, are induced to express both CYP27B1 and VDR by various pathogens including viruses and cytokines released from infected epithelia and other immune cells. Although not demonstrated for corona viruses like SARS-CoV2, for other viruses and other respiratory pathogens activation of innate immunity leading to increased local 1,25(OH)2D production has been shown to enhance viral neutralization and clearance while modulating the subsequent proinflammatory response. Whether such will be the case for SARS-CoV2 remains to be seen, but is currently being proposed and investigated. This mini review will discuss some of the mechanisms by which vitamin D may help reduce morbidity and mortality in this devastating pandemic.

Intersubject Variation in ACE2 Protein Expression in Human Airway Epithelia and Its Relationship to Severe Acute Respiratory Syndrome Coronavirus 2.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2 ) initiates entry into airway epithelia by binding its receptor, angiotensin-converting enzyme 2 (ACE2). METHODS: To explore whether interindividual variation in ACE2 abundance contributes to variability in coronavirus disease 2019 (COVID-19) outcomes, we measured ACE2 protein abundance in primary airway epithelial cultures derived from 58 human donor lungs. RESULTS: We found no evidence for sex- or age-dependent differences in ACE2 protein expression. Furthermore, we found that variations in ACE2 abundance had minimal effects on viral replication and induction of the interferon response in airway epithelia infected with SARS-CoV-2. CONCLUSIONS: Our results highlight the relative importance of additional host factors, beyond viral receptor expression, in determining COVID-19 lung disease outcomes.

The TMPRSS2 Inhibitor Nafamostat Reduces SARS-CoV-2 Pulmonary Infection in Mouse Models of COVID-19.

The coronavirus disease 2019 (COVID-19) pandemic has caused significant morbidity and mortality on a global scale. The etiologic agent, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), initiates host cell entry when its spike protein (S) binds to its receptor, angiotensin-converting enzyme 2 (ACE2). In airway epithelia, the spike protein is cleaved by the cell surface protease TMPRSS2, facilitating membrane fusion and entry at the cell surface. This dependence on TMPRSS2 and related proteases suggests that protease inhibitors might limit SARS-CoV-2 infection in the respiratory tract. Here, we tested two serine protease inhibitors, camostat mesylate and nafamostat mesylate, for their ability to inhibit entry of SARS-CoV-2 and that of a second pathogenic coronavirus, Middle East respiratory syndrome coronavirus (MERS-CoV). Both camostat and nafamostat reduced infection in primary human airway epithelia and in the Calu-3 2B4 cell line, with nafamostat exhibiting greater potency. We then assessed whether nafamostat was protective against SARS-CoV-2 in vivo using two mouse models. In mice sensitized to SARS-CoV-2 infection by transduction with human ACE2, intranasal nafamostat treatment prior to or shortly after SARS-CoV-2 infection significantly reduced weight loss and lung tissue titers. Similarly, prophylactic intranasal treatment with nafamostat reduced weight loss, viral burden, and mortality in K18-hACE2 transgenic mice. These findings establish nafamostat as a candidate for the prevention or treatment of SARS-CoV-2 infection and disease pathogenesis. IMPORTANCE The causative agent of COVID-19, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), requires host cell surface proteases for membrane fusion and entry into airway epithelia. We tested the hypothesis that inhibitors of these proteases, the serine protease inhibitors camostat and nafamostat, block infection by SARS-CoV-2. We found that both camostat and nafamostat reduce infection in human airway epithelia, with nafamostat showing greater potency. We then asked whether nafamostat protects mice against SARS-CoV-2 infection and subsequent COVID-19 lung disease. We performed infections in mice made susceptible to SARS-CoV-2 infection by introducing the human version of ACE2, the SARS-CoV-2 receptor, into their airway epithelia. We observed that pretreating these mice with nafamostat prior to SARS-CoV-2 infection resulted in better outcomes, in the form of less virus-induced weight loss, viral replication, and mortality than that observed in the untreated control mice. These results provide preclinical evidence for the efficacy of nafamostat in treating and/or preventing COVID-19.

The SARS-CoV-2 Transcriptome and the Dynamics of the S Gene Furin Cleavage Site in Primary Human Airway Epithelia.

The spike (S) polypeptide of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) consists of the S1 and S2 subunits and is processed by cellular proteases at the S1/S2 boundary that contains a furin cleavage site (FCS), 682RRAR↓S686 Various deletions surrounding the FCS have been identified in patients. When SARS-CoV-2 propagated in Vero cells, it acquired deletions surrounding the FCS. We studied the viral transcriptome in Vero cell-derived SARS-CoV-2-infected primary human airway epithelia (HAE) cultured at an air-liquid interface (ALI) with an emphasis on the viral genome stability of the FCS. While we found overall the viral transcriptome is similar to that generated from infected Vero cells, we identified a high percentage of mutated viral genome and transcripts in HAE-ALI. Two highly frequent deletions were found at the FCS region: a 12 amino acid deletion (678TNSPRRAR↓SVAS689) that contains the underlined FCS and a 5 amino acid deletion (675QTQTN679) that is two amino acids upstream of the FCS. Further studies on the dynamics of the FCS deletions in apically released virions from 11 infected HAE-ALI cultures of both healthy and lung disease donors revealed that the selective pressure for the FCS maintains the FCS stably in 9 HAE-ALI cultures but with 2 exceptions, in which the FCS deletions are retained at a high rate of >40% after infection of ≥13 days. Our study presents evidence for the role of unique properties of human airway epithelia in the dynamics of the FCS region during infection of human airways, which is likely donor dependent.IMPORTANCE Polarized human airway epithelia at an air-liquid interface (HAE-ALI) are an in vitro model that supports efficient infection of SARS-CoV-2. The spike (S) protein of SARS-CoV-2 contains a furin cleavage site (FCS) at the boundary of the S1 and S2 domains which distinguishes it from SARS-CoV. However, FCS deletion mutants have been identified in patients and in vitro cell cultures, and how the airway epithelial cells maintain the unique FCS remains unknown. We found that HAE-ALI cultures were capable of suppressing two prevalent FCS deletion mutants (Δ678TNSPRRAR↓SVAS689 and Δ675QTQTN679) that were selected during propagation in Vero cells. While such suppression was observed in 9 out of 11 of the tested HAE-ALI cultures derived from independent donors, 2 exceptions that retained a high rate of FCS deletions were also found. Our results present evidence of the donor-dependent properties of human airway epithelia in the evolution of the FCS during infection.

ColdZyme Maintains Integrity in SARS-CoV-2-Infected Airway Epithelia.

SARS-CoV-2 infection causing the COVID-19 pandemic calls for immediate interventions to avoid viral transmission, disease progression, and subsequent excessive inflammation and tissue destruction. Primary normal human bronchial epithelial cells are among the first targets of SARS-CoV-2 infection. Here, we show that ColdZyme medical device mouth spray efficiently protected against virus entry, excessive inflammation, and tissue damage. Applying ColdZyme to fully differentiated, polarized human epithelium cultured at an air-liquid interphase (ALI) completely blocked binding of SARS-CoV-2 and increased local complement activation mediated by the virus as well as productive infection of the tissue model. While SARS-CoV-2 infection resulted in exaggerated intracellular complement activation immediately following infection and a drop in transepithelial resistance, these parameters were bypassed by single pretreatment of the tissues with ColdZyme mouth spray. Crucially, our study highlights the importance of testing already evaluated and safe drugs such as ColdZyme mouth spray for maintaining epithelial integrity and hindering SARS-CoV-2 entry within standardized three-dimensional (3D) in vitro models mimicking the in vivo human airway epithelium.IMPORTANCE Although our understanding of COVID-19 continuously progresses, essential questions regarding prophylaxis and treatment remain open. A hallmark of severe SARS-CoV-2 infection is a hitherto-undescribed mechanism leading to excessive inflammation and tissue destruction associated with enhanced pathogenicity and mortality. To tackle the problem at the source, transfer of SARS-CoV-2, subsequent binding, infection, and inflammatory responses have to be avoided. In this study, we used fully differentiated, mucus-producing, and ciliated human airway epithelial cultures to test the efficacy of ColdZyme medical device mouth spray in terms of protection from SARS-CoV-2 infection. Importantly, we found that pretreatment of the in vitro airway cultures using ColdZyme mouth spray resulted in significantly shielding the epithelial integrity, hindering virus binding and infection, and blocking excessive intrinsic complement activation within the airway cultures. Our in vitro data suggest that ColdZyme mouth spray may have an impact in prevention of COVID-19.