Cytokine-Induced Modulation of SARS-CoV2 Receptor Expression in Primary Human Nasal Epithelial Cells

Abstract

Background: Viral entry of severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) via the spike protein enables endocytosis into host cells using the ACE2 receptor and TMPRSS2. The frequent upper respiratory tract symptoms of COVID-19 and the localization of the virus to the nasopharynx, the most common site of swabbing, indicate that the sinonasal mucosa may play an important role in SARS-CoV2 infection and viral replication. Methods: This paper investigates the presence of ACE2 receptor and TMPRESS2 expression in the primary human nasal epithelial cells (HNECs) from the following: chronic rhinosinusitis without nasal polyps (CRSsNP), CRS with nasal polyps (CRSwNP) and control (non-CRS) patients, and maps the expression changes when exposed to Th1, Th2, Th17-associated cytokines. Results: We found that ACE2 and TMPRSS2 expression was higher in control HNECs than CRSwNP HNECs, and that both ACE2 and TMPRSS2 were downregulated further by Th2 cytokines in CRSwNP HNECs. Conclusions: This indicates an immune dysregulated state of CRSwNP mucosa, which normally contributes to a chronic inflammatory state, and might support an altered susceptibility to SARS-CoV2 infection and transmission.

Keywords: ACE2; TMPRSS2; chronic rhinosinusitis; human nasal epithelial cells.

Figure 1

Boxplot showing ACE2 (A) and TMPRSS2 (B) expression in 3 groups (control, CRSsNP and CRSwNP). The patient cohort consisted of 12 control patients, 18 with CRSsNP and 12 with CRSwNP. (A) The mRNA expression of ACE2 does not show any significant difference between groups. (B) The TMPRSS2 is significantly different between the groups (1-way ANOVA p-value = 0.03). * p < 0.05. ns = not significant.

Figure 2

Boxplot showing ACE2 (A) and TMPRSS2 (B) expression in CRS and control Cases. The patient cohort consisted of 42 patients, including 30 CRS and 12 control cases. (A) CRS status is not significantly correlated with ACE2 mRNA expression (Student t-test p-value = 0.12). (B) CRS is significantly correlated with a reduced TMPRSS2 expression (Student t-test p-value = 0.014). * p < 0.05. ns = not significant.

Figure 3

Boxplot of asthma status versus ACE2 (A) and TMPRSS2 (B) expression. The patient cohort consisted of 42 patients including 23 asthma and 19 non-asthma cases. (A) Asthma is not significantly correlated with ACE2 expression (Student t-test p-value = 0.18). (B) Asthma patients show significant reduction of TMPRSS2 mRNA expression compared with non-asthma patients (Student t-test p-value = 0.01). * p < 0.05. ns = not significant.

Figure 4

Boxplot log of ACE2 (A) and TMPRSS2 (B) expression of human nasal epithelial cells treated with IFN-α, IFN-γ, IL-1β, TNF-α for 24 h. The patient cohort consisted of 4 patients with CRSwNP, 7 with CRSsNP and 4 control cases. (A) IFN-α and IFN-γ show significantly higher ACE2 mRNA expression compared with untreated cells and the other treatment groups (Tukey HSD p-values all < 0.001). (B) There are no significant effects on TMPRESS2 expression for HNECs treated with IFN-α, IFN-γ, IL-1β and TNF-α within each of the control, CRSsNP and CRSwNP groups (2-way ANOVA p-value = 0.162).

Figure 5

ACE2 protein expression (A) and TMPRSS2 protein expression (B) in human nasal epithelial cells was determined using immunofluorescence staining for ACE2 (red), TMPRSS2 (green) and nuclei (blue) in the absence (no treatment control-NC) and presence of IFN-α or IFN-γ. The white bar is 50 µm and 20 × objective. ACE2 (C) and TMPRSS2 (D) fluorescence intensity of HNEC cultures (harvested form inferior turbinates from CRS) with IFN-α and IFN-γ treatments. The values are shown as means ± SEM, * p < 0.05, ** p < 0.01, *** p < 0.001, n = 6.

Figure 6

Boxplot log of ACE2 (A) and TMPRSS2 (B) expression of human nasal epithelial cells after treating with TH2 and TH17 families for 24 h. The patient cohort consisted of 4 patients with CRSwNP, 5 with CRSsNP and 4 control cases. (A) ACE2 mRNA expression differed significantly between CRSwNP group (1-way ANOVA p-value = 0.00463) though not between the TH2 and TH17 cytokines (2-way ANOVA p-value = 0.557). (B) TMPRSSS expression differed between CRS groups (2-way ANOVA p-value = 0.0378) and treatment groups (2-way ANOVA p-value = 2.05 × 10⁻⁷).