Ephrin Ligands are Upregulated in the Saliva of SARS‐CoV‐2 Infected Patients

Abstract

BackgroundPatients infected with SARS‐CoV‐2 may develop severe lung disease characterized by respiratory distress, systemic thrombosis, or death. Molecular mechanisms responsible for severe cases of COVID‐19 remain unclear. We investigated the role of the Eph family receptor‐interacting ligands (ephrin‐A1 and ephrin‐B2) in COVID‐19 patients. Ephrin binding to its tyrosine kinase Eph receptor plays important roles in injury and inflammation. Serum levels of ephrin‐A1 ligand correlates positively with COVID‐19 severity (Mendoza et al, CellPress 2021). Based on this recent report, and the emerging role of Ephrin‐Eph signaling in lung injury, we hypothesize that ephrin‐mediated cytokine signaling is upregulated in epithelial and immune cells assayed from saliva samples obtained from seriously ill COVID‐19 patients seeking emergency evaluation and treatment.MethodsSaliva samples were collected from patients (± SARS‐CoV‐2) in the University of Utah Emergency Department (ED) with IRB‐approved written consent. Saliva samples were incubated 1:1 in Streck Preservative (prior to EpCAM and CD45 labeling) or with 0.008% glutaraldehyde (following ELISA and multiplex immunoassays) in order to inactivate virus.ResultsFlow cytometric evaluation of saliva for EpCAM+ and CD45+ cells indicated that SARS‐CoV‐2 positive samples contain both epithelial and immune cell types ( see Fig 1 ). Uninfected saliva samples (control) primarily contained EpCAM+ cells. COVID‐19 samples (n=67 patients) expressed higher levels of type 1 interferon [IFNγ1 and IFNα2 (p< 0.001)] vs control (n=64 patients). Interferon induced protein [IP‐10] was also upregulated in COVID‐19 patients (p<0.001), as well as several cytokines in the interleukin family [IL‐1β, IL‐6, IL‐8 (p<0.001) and IL‐12p70 (p<0.05)]. TNF‐α did not differ significantly between the ± SARS‐CoV‐2 samples evaluated. Using a subset of the +SARS‐CoV‐2 samples (n=20) and uninfected controls obtained from the ED (n=20), we show that both ephrin‐A1 and ephrin‐B2 ligands are significantly elevated in +SARS‐CoV‐2 samples vs. ED control samples (p<0.001). Ephrin‐B2 levels correlated negatively with IL‐12p70 (Pearson Correlation =‐0.53, p=.01).ConclusionSaliva is low risk to obtain and is a biologically relevant sample that can be used to reliably measure changes in epithelial and immune cell responses of COVID‐19 patients. Specifically, our study showed that ephrin‐A1 and ephrin‐B2 ligands are upregulated in the saliva of COVID‐19 patients in a TNF‐α independent manner and that ephrin‐B2 correlates negatively with IL‐12p70 (a potent pro‐inflammatory cytokine). Future studies are aimed at establishing ephrin ligands as a biomarker for severity of lung injury and improving understanding the patho‐physiology of SARS‐CoV‐2 infection.