Antiviral innate immunity is diminished in the upper respiratory tract of severe COVID-19 patients

Abstract

Understanding early innate immune responses to coronavirus disease 2019 (COVID-19) is crucial to developing targeted therapies to mitigate disease severity. Severe acute respiratory syndrome coronavirus (SARS-CoV)-2 infection elicits interferon expression leading to transcription of IFN-stimulated genes (ISGs) to control viral replication and spread. SARS-CoV-2 infection also elicits NF-κB signaling which regulates inflammatory cytokine expression contributing to viral control and likely disease severity. Few studies have simultaneously characterized these two components of innate immunity to COVID-19. We designed a study to characterize the expression of interferon alpha-2 (IFNA2) and interferon beta-1 (IFNB1), both type-1 interferons (IFN-1), interferon-gamma (IFNG), a type-2 interferon (IFN-2), ISGs, and NF-κB response genes in the upper respiratory tract (URT) of patients with mild (outpatient) versus severe (hospitalized) COVID-19. Further, we characterized the weekly dynamics of these responses in the upper and lower respiratory tracts (LRTs) and blood of severe patients to evaluate for compartmental differences. We observed significantly increased ISG and NF-κB responses in the URT of mild compared with severe patients early during illness. This pattern was associated with increased IFNA2 and IFNG expression in the URT of mild patients, a trend toward increased IFNB1-expression and significantly increased STING/IRF3/cGAS expression in the URT of severe patients. Our by-week across-compartment analysis in severe patients revealed significantly higher ISG responses in the blood compared with the URT and LRT of these patients during the first week of illness, despite significantly lower expression of IFNA2, IFNB1, and IFNG in blood. NF-κB responses, however, were significantly elevated in the LRT compared with the URT and blood of severe patients during peak illness (week 2). Our data support that severe COVID-19 is associated with impaired interferon signaling in the URT during early illness and robust pro-inflammatory responses in the LRT during peak illness.

Keywords: COVID-19; Gene Expression; Inflammation; Innate Immune Response; Interferons.

Figure 1.

Study design diagram.

Figure 2.. Higher ISGs and NF-κB in the URT in mild COVID-19 patients.

ISG Score and NF-kB score measured by NanoString in upper respiratory samples collected in the first 10 days from the post-symptom onset in mild (n=6) and severe COVID patients (n=4) compared to healthy control (n=5). A) ISGs score measured by NanoString in upper respiratory samples collected in the first 10 days from the post-symptom onset. B) NF-κB score measured by NanoString in upper respiratory samples collected in the first 10 days from the post-symptom onset. For both scores, mild and severe COVID-19 patients were compared to healthy control (n=5). Kruskal Wallis test is used to compare the three groups and corrected Dunn’s test for multiple comparisons was used to compare mild and severe COVID patients to HC. *P<0.05, **P<0.01, NS P<0.05

Figure 3.. Thirteen type I IFN response genes are increased in mild COVID-19 in the URT.

The 13 type I IFN response gene transcriptions were measured by NanoString in the upper respiratory tract of patients in the first 10 days from the post-symptom onset. mild (n=6) and severe COVID (n=4) patients. Data for both groups are shown as fold change from healthy controls. Kruskal Wallis test is used to compare mild and severe COVID patients, *p<0.05,

Figure 4.. Viral load in the URT of mild and severe patients.

Viral load was measured by ddPCR from patients that had samples before and after 10 days post-symptoms onset. A) Shows viral load in the mild subjects first 10 days post-symptom onset (n=5, 7 samples) compared with after day 10 post-symptom onset (n=3, 3 samples), p=0.1629. B) Shows viral load in the severe subjects for the first 10 days post-symptom onset (n=4, 13 samples) compared with after day 10 post-symptom onset (n=4, 20 samples), p=0.2527. Individual dots indicate independent samples, solid line indicates the mean and SEM. Unpaired T test was used to compared between groups.

Figure 5.. Increased expression of CGAS/STING INFB1 pathway in the URT of severe COVID-19 patients.

A – F) The IFNA2, IFNB1, IFNG, STING-1, and CGAS gene transcriptions were measured by NanoString in the URT of patients in the first 10 days following symptom onset. Mild (n=6) and severe COVID (n=4) patients. Data for both groups are shown as fold change from healthy controls. Kruskal Wallis test is used to compare mild and severe COVID patients *P<0.05, NS P>0.05

Figure 6.. IRFs are differentially regulated in mild and severe COVID-19 patients.

A-F) The IRF3, IRF5, IRF7, IRF8 and IRF9 gene transcriptions were measured by NanoString in the URT of patients in the first ten days following symptom onset. Mild (n=6) and Severe COVID (n=4) patients. Data for both groups are shown as fold change from healthy controls. Kruskal Wallis test is used to compare Mild and severe COVID patients *P<0.05

Figure 7.. ISGs and NF-κB scores differ across anatomical compartment in severe COVID-19 patients.

A-C) IFNA2, INFB1 and IFNG expression measured by NanoString in upper respiratory samples collected in the first three weeks following symptom onset in severe COVID-19 patients compared to healthy control (n=5). D-E) ISGs and NF-κB score were measured by NanoString in Blood (Week 1 n=3, Week 2 n=11, Week3 n=22), URT (Week 1 n=5, Week 2 n=23, Week3 n=39), and LRT (Week 2 n=10, Week3 n=15). Kruskal Wallis test is used to compare the three groups and corrected Dunn’s test for multiple comparisons was used to compare mild and severe COVID patients to HC. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001