Mucosal Innate Immunity of Human Surfactant Protein A Genetic Variants against SARS-CoV-2 Infection

Abstract

More than 7 million people have died of the coronavirus disease-2019 (COVID-19) since first reported in December of 2019. Infection in some patients manifests as life-threatening ALI/ARDS, multi-organ dysfunctions, and/or death characterized by active viral replication and profound inflammatory cell influx into tissues/organs. SARS coronavirus-2 (SARS-CoV-2) infects human angiotensin-converting enzyme 2 (hACE2)-expressing cells through its spike protein (S protein). The S protein is highly glycosylated and could be a target for lectins. Surfactant protein A (SP-A) is a collectin, expressed by lung alveolar type II cells and other mucosal epithelial cells; it plays a crucial role in innate immunity and inflammatory regulation. SP-A modulates pathogenic infection and disease severity by binding to microbial and host glycoproteins to alter infectivity and regulate host inflammation. The human SP-A gene is located on chromosome 10q22-23, which contains two functional genes SP-A1 and SP-A2 (gene names: SFTPA1 and SFTPA2), and a pseudogene. SP-A1 and SP-A2 are highly polymorphic and consist of several genetic variants, such as SP-A1 (variants 6A2, 6A4) and SP-A2 (variants 1A0, 1A3). It has been demonstrated that these variants have differential antiviral and immunoregulatory capacities in response to various viral infections. The goal of this study was to investigate the mechanistic role of human SP-A variants in response to SARS-CoV-2 infection and COVID-19 susceptibility and severity. The results from this study showed that native human SP-A can bind SARS-CoV-2 S protein, receptor-binding domain (RBD), and hACE2 in a dose-dependent manner. A decrease in S protein and RBD binding was observed in the presence of EDTA and sugars, indicating that the SP-A carbohydrate-recognition domain (CRD) mediates S protein binding in a calcium-dependent manner. We further showed that human SP-A can attenuate viral infectivity in susceptible host cells, evidenced by the dose-dependent reduction in viral load in infected cells. These results suggest that human SP-A can bind SARS-CoV-2 S protein, RBD, and hACE2 to attenuate SARS-CoV-2 infectivity in susceptible host cells. Next, we examined the variations in antiviral and immunoregulatory roles of human SP-A variants in response to SARS-CoV-2 infection. The binding studies showed that in vitro-expressed SP-A variants differentially interact with S protein. Moreover, cells inoculated with SARS-CoV-2 pretreated with the 1A0 variant had a more reduced virus titer than those pretreated with the 6A2 variant, indicative of their differential antiviral capacities. These findings from in vitro studies demonstrated that human SP-A and their genetic variants directly interact with viral S protein to differentially modulate SARS-CoV-2 infectivity. To perform in vivo study, six genetically modified double-hTG mouse lines, expressing both hACE2 and the respective SP-A variants: (hACE2/6A2 (6A2), hACE2/6A4 (6A4), hACE2/1A0 (1A0), and hACE2/1A3 (1A3), one SP-A knockout (hACE2/SP-A KO (KO) and one hACE2/mouse SP-A (K18) mice, were generated and challenged intranasally with 103 PFU SARS-CoV-2 (Delta) or saline (Sham). We observed that these infected mice had differential COVID-19 severity. Infected KO and 1A0 mice had more mortality and lung injury compared to other mouse lines, and disease severity correlated with enhanced upregulations of inflammatory genes that play vital roles in host immunity such as MyD88 and Stat3 in the lungs of KO and 1A0 mice. Furthermore, pathway analysis identified several important signaling pathways involved in lung defense, including pathogen-induced cytokine storm, NOD1/2, toll-like receptor, neuroinflammation, and Trem1 signaling pathways. Consistent with the transcriptomic data, expressions of inflammatory mediators such as G-CSF, IL-6, and IL-1β were comparatively higher in the lungs and sera of KO and 1A0 mice with the highest mortality rate. We further examined other organ injuries (kidney, intestine, and brain) in the infected mice; we found a more severe acute kidney injury (AKI) and intestinal damage in KO and 6A4 mice compared to other double-hTG mice. Viral titers were generally lower in the kidneys and brains of infected double-hTG mice relative to KO mice. Inflammatory mediators like TNF-α, IL-6, IL-1β, and MCP-1 were comparatively higher in KO and 6A4 mice with the most severe AKI. High virus presence and inflammatory markers were also observed in the brain and hippocampus of all infected mice. The results from in vivo studies suggest that SP-A variants differentially protect against severe COVID-19. Furthermore, the human COVID-19 patient studies revealed increased SP-A levels in the saliva of COVID-19 patients compared to healthy controls and highlighted the potential use of SP-A levels as a biomarker for COVID-19 severity. Collectively, these findings underscore the importance of host innate immune collectins and contribute to our understanding of the roles of host genetic variations in the observed population-level differences in COVID-19 susceptibility and severity.