Examination of gene expression in saliva samples from COVID-19 patients to study the host defense response against SARS-CoV-2 in the oral cavity

To the editor

Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19), which presents with respiratory symptoms including fever, sore throat, cough, and congestion, suggesting that infection of the oral cavity and nasopharynx (ONP) is an obligate step in its pathogenesis. Consistent with this role in the initial infection, oral and nasopharyngeal epithelial cells express the SARS-CoV-2 spike protein receptor, angiotensin-converting enzyme 2 (ACE2) (Sakaguchi et al., 2020), and RT-PCR based testing can routinely detect SARS-CoV-2 RNA within both the nasal and buccal tissues. Local innate and adaptive immune responses in the oral epithelia are almost certainly involved in the defense against SARS-CoV-2 infection, yet little is known about these defenses.

In an initial approach to elucidate the antiviral defense in the oral cavity, we identified the expression of the antiviral type I interferon (IFN) and IFN-stimulated genes (ISGs) expressed in oral epithelial cells (Brice et al., 2019). In addition, we demonstrated the vitamin D-mediated induction of cathelicidin (McMahon et al., 2011; Menzel et al., 2019) in the oral epithelium. Cathelicidin is a host defense peptide that inactivates another enveloped, orally transmitted virus through membrane disruption (Brice et al., 2018). However, our understanding of the dynamic antiviral defense mechanisms of the oral cavity is poorly developed. In order to help combat this major public health emergency, we must devote a significant effort to characterization of the antiviral immune defenses in the oral cavity.

A sample of gingival epithelium has been examined by single cell RNA sequencing (scRNAseq), and shows the wide variety of cell types, including several that play important roles in antiviral defense, including plasmacytoid dendritic cells (pDC), innate lymphoid cells (ILC), T cells and NK cells (Byrd2020). An analysis of this database indicates the expression of numerous genes, including ACE2 and proinflammatory cytokine genes in several cell types. This supports the hypothesis that SARS-CoV-2 can infect the oral epithelium as part of an initial infection, and that antiviral defenses are found in the oral cavity.

To examine the response of oral tissues to SARS-CoV-2 infection, we obtained saliva and buccal samples from patients with COVID-19 and uninfected control individuals (details on subject recruitment, demographics and sample isolation are provided in Supplementary Information). Quantification of ACE2 mRNA by QRT-PCR shows that there is a significant reduction in mean ACE2 mRNA levels in the COVID-19 patients compared to uninfected controls (Figure 1a). The related virus, SARS-CoV, also utilizes ACE2 as its receptor, and infection of epithelial cells with this virus leads to a downregulation of ACE2 mRNA (Kuba et al., 2005). Our result suggests that SARS-CoV-2 acts similarly in infected oral tissues, to downregulate ACE2 expression. This could negatively affect the body’s ability to regulate the renin-angiotensin pathway, and lead to more severe disease.

FIGURE 1.

Expression of ACE2 (a) and IL-6 (b) in oral samples from patients with COVID-19 and uninfected controls. Samples were collected by a sterile flocked collection swab and placed in RNAIater for subsequent total RNA isolation. ACE2 and IL-6 mRNA levels were quantified by QRT-PCR relative to β-actin using SYBR Green and the 2^−ΔΔCq method. Data are shown with mean ± SEM, and are significant as determined by two-tailed t test (Graph Pad Prism version 8.0) [Colour figure can be viewed at wileyonlinelibrary.com]

We also examined the expression of the pro-inflammatory cytokine IL-6, and discovered that surprisingly, its expression was reduced in COVID-19 patients (Figure 1b). However, Chen et al. have recently shown that SARS-CoV-2 infection inhibits the JAK-STAT pathway that regulates IL-6 production (Chen et al., 2020). These results from clinical samples confirm the prior experimental results, and further demonstrate that we can observe the virus-mediated inhibition of innate antiviral defense mechanisms in oral tissues.

Together, our results show that the oral cavity represents an important interface for the infection of SARS-CoV-2, and easily obtained buccal swabs and saliva samples can be used to study the host response to its infection. We urge the oral microbiology and immunology community to partner with infectious disease clinicians and other medical professionals as we have done, and utilize these important resources to assist the broader scientific community in its effort to battle this public health emergency. Essential studies include (but are not limited to) changes in the oral microbiota in infection, the function of innate antiviral immune mediators in the oral cavity, and the effect of infection on oral pathogenic conditions.