key: cord-0892072-suv9gj0l
authors: Kim, Myung-Ho; Salloum, Shadi; Wang, Jeffrey Y; Lai Ping, Wong; Regan, James; Lefteri, Kristina; Manickas-Hill, Zachary; Gao, Ce; Li, Jonathan Z; Sadreyev, Ruslan I; Yu, Xu G; Chung, Raymond T
title: Type I, II, and III interferon signatures correspond to COVID-19 disease severity
date: 2021-05-24
journal: J Infect Dis
DOI: 10.1093/infdis/jiab288
sha: 4e42de3c8f9ee3438fd0b0763838ee6f1f3a3ce3
doc_id: 892072
cord_uid: suv9gj0l

We analyzed the plasma levels of interferons and cytokines, and the expression of interferon-stimulated genes in peripheral blood mononuclear cells in COVID-19 patients with different disease severity. Mild patients exhibited transient type I interferon responses, while ICU patients had prolonged type I interferon responses with hyper-inflammation mediated by interferon regulatory factor 1. Type II interferon responses were compromised in ICU patients. Type III interferon responses were induced in the early phase of SARS-CoV-2 infection, even in convalescent patients. These results highlight the importance of type I and III interferon responses during the early phase of infection in controlling COVID-19 progression.

M a n u s c r i p t 4 Background Infection with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) results in diverse clinical outcome of coronavirus disease 2019 (COVID-19). Most COVID-19 patients experience mild clinical course, but approximately 5% of the SARS-CoV-2 positive patients experience severe disease [1] , acute respiratory distress syndrome, which necessitates supplemental oxygen therapy or intensive care unit (ICU) care. Patients with mild cases, who do not need either hospitalization or ICU care, recover within around 14 days after symptom onset with viral clearance [2] . However, patients who need ICU care experience mild to moderate symptoms followed by a secondary respiratory worsening with prolonged viral load. Although the vaccination against SARS-CoV-2 has been feasible, still therapeutic options for COVID-19 patients are limited: SARS-CoV-2 neutralizing antibodies, antiviral agents, immunosuppressive agents.

Virus recognition by innate immune sensors of host cell induces type I and III IFN production. Type I and Type III IFNs induce the expression of interferon-stimulated genes (ISGs) which have the antiviral capacity [3] . Type I IFN, not Type III IFN, induces proinflammatory genes' expression by selective induction of the transcription factor interferon regulatory factor 1 (IRF-1) [4] . In contrast to virally induced Type I and III IFN, Type II IFN is produced predominantly by T and NK cells upon stimulation with antigens and cytokines. Type II IFN stimulates antigen-specific adaptive immunity and activates innate immunity, particularly through the activation of macrophages.

However, SARS-CoV-2 evades the IFN responses of host by escaping immune recognition, suppressing the functions of IFNs and ISGs, and interfering antigen presentation process [3] . IFN responses modulated by both viral and host factors determine the clinical outcome of COVID-19 patients. Several studies have reported impaired type I and II IFN responses in patients with severe COVID-19 during the early phase of infection [5, 6] ; however, the dynamic IFN responses during SARS-CoV-2 infection need to be defined. Here, we A c c e p t e d M a n u s c r i p t 5   comprehensively investigated type I, II, and III IFN signatures in COVID-19 with different   disease severity. We analyzed the plasma levels of IFNs and IRF-1 regulated cytokines/chemokines, and the expression of ISGs in peripheral blood mononuclear cells (PBMCs). 

Interferon-stimulated genes (ISGs), previously reported to be relevant in viral infection [4] , were selected for analyzing mRNA expression of ISGs. mRNA expression of ISGs was quantified by the NanoString platform (NanoString Technologies, Table 3 ).

Grouped data are generally presented as median ± IQR, with groups compared by the Kruskal-Wallis test with Dunn's multiple comparisons test for non-parametric data using Prism 9.0 (Graphpad)

Patients confirmed positive for SARS-CoV-2 were subdivided into three groups based on disease severity during their clinical encounters: Outpatient (Out, n=23), Hospitalization under non-ICU conditions (Mild, n=21), Hospitalization in the ICU (ICU, n=23). Convalescent patients, who were recovered from COVID-19 and confirmed negative for SARS-CoV-2, were included as a control group (Conv, n=19). The blood samples were collected typically between 5 to 44 days after symptom onset (DfSO). (Supplementary Table 1 and Supplementary Figure 1A ).

The plasma levels of the type I IFN IFN-α were the highest in the ICU patients, followed by the Mild and Out patients. However, when evaluated by duration after symptom onset, some Table 2 ). The levels of IL-12, CCL7, and TRAIL were higher in Out and Mild patients, as with IFN-γ (Supplementary Figure 2B) .

Heatmap clustering of plasma cytokines and chemokines yielded two major clusters: one consisting of IFN-γ, IL-12, CCL7, and TNF-α (IFN-γ cluster) and another cluster consisting of IRF-1 regulated cytokines and chemokines (IRF-1 cluster). IFN-γ cluster was upregulated in the Out and Mild patients, but not in the ICU patients ( Figure 1D ).

IRF-1 regulated genes in PBMCs were upregulated in only the ICU patients, while antiviral genes were upregulated in both Mild and ICU patients (Figure 2A Figure   2D ).

It is known that older adults and men are at higher risk of hospitalization and death if they are diagnosed with COVID-19. Our study further affirmed this finding since the Mild and ICU patients were older than Out patients (Supplementary Figure 1B) . However, Mild and ICU patients displayed differences in IFN signatures despite sharing comparable ages. We performed a multivariable regression analysis with each of 15 cytokines and chemokines as dependent variables and age, gender, race/ethnicity, sample collection day as independent variables (Supplementary Table 2 ). Based on this analysis, we confirmed that our findings A c c e p t e d M a n u s c r i p t 8 were not significantly affected by the independent variables. Therefore, we concluded that our observations were not explained by age, gender, or race/ethnicity.

Several studies have reported that the type I and III IFN responses in patients with severe COVID-19 are suppressed during the early phase of infection [5, 7] . However, other studies have shown that patients with severe COVID-19 have robust type I IFN responses [6, 8] . In Several studies have reported the reduction of the plasma type II IFN in patients with severe COVID-19 similar to our findings [5] . A series of analyses on the immune cells have suggested that IFN-γ producing CD4+T, CD8+T, and NK cells are exhausted and depleted in patients with severe COVID-19 [9, 10] , which could plausibly explain the decreased plasma IFN-γ levels in ICU patients. However, there are conflicting data suggesting that PD-1-expressing SARS-CoV-2 specific CD8+ T cells are not truly exhausted in COVID-19 patients [11] .

The levels of IFN-γ stimulated genes were somewhat diminished in Mild patients compared with Out and Conv patients, they were still substantially upregulated compared to ICU patients. In this regard, these findings were similar to the pattern observed in plasma IFN-γ A c c e p t e d M a n u s c r i p t 9 levels. Viruses including coronaviruses, MERS-CoV and H5N1 influenza virus, interfere with the antigen presentation process through MHC molecules. The ORF8 protein of SARS-CoV-2 downregulates MHC class I molecules [12] , although the evidence for the interference of antigen presentation by SARS-CoV-2 is still lacking. Several studies have demonstrated the downregulation of MHC class I and II molecules in antigen presenting cells of COVID-19 patients, regardless of disease severity [13, 14] . Thus, decreased expression of MHC molecules in PBMCs and reduced plasma IFN-γ could synergistically subvert adaptive immunity in ICU patients.

Type III IFN is induced earlier than type I IFN upon virus infection, and suppresses initial viral spread without activating inflammation. Type I IFN response is triggered later to enhance antiviral activity and induce IRF-1 mediated inflammatory responses [3] .

Interestingly, some of the Conv patients showed increased IFN-λ1/3 levels ( Figure 1C ) and the upregulated antiviral genes, while not inducing IRF-1 related genes (Figure 2A and B) , even though the patients were confirmed negative for SARS-CoV-2. While it is remotely possible that these patients may have been re-infected with SARS-CoV-2 and not developed detectable viral RNA, it would appear much more likely that the virus was rapidly cleared by type III IFN responses prior to engagement of a type I IFN responses.

Type III IFN therapy could be a novel therapeutic strategy against COVID-19. Early use of type I IFNs has benefits in virus clearance and clinical outcomes in COVID-19 patients.

However, later use of type I IFNs could potentially delay recovery and increase mortality.

This could be attributed to IRF-1 related hyper-inflammation. While the expression of type I IFN receptors is ubiquitous, the expression of type III IFN receptors is limited to epithelial cells [3] . Thus, type III IFN therapy could be an effective alternative to type I IFN therapy A c c e p t e d M a n u s c r i p t 14 Figure 2 

Transmission, Diagnosis, and Treatment of Coronavirus Disease 2019 (COVID-19): A Review

Clinical and epidemiological characteristics of 1420 European patients with mild-to-moderate coronavirus disease 2019

Type I and Type III Interferons -Induction, Signaling, Evasion, and Application to Combat COVID-19

Differential Activation of the Transcription Factor IRF1 Underlies the Distinct Immune Responses Elicited by Type I and Type III Interferons

Impaired type I interferon activity and inflammatory responses in severe COVID-19 patients

Longitudinal analyses reveal immunological misfiring in severe COVID-19

Imbalanced Host Response to SARS-CoV-2 Drives Development of COVID-19

Immunophenotyping of COVID-19 and influenza highlights the role of type I interferons in development of severe COVID-19

Functional exhaustion of antiviral lymphocytes in COVID-19 patients

Clinical and immunological features of severe and moderate coronavirus disease 2019

PD-1-Expressing SARS-CoV-2-Specific CD8(+) T Cells Are Not Exhausted, but Functional in Patients with COVID-19

The ORF8 Protein of SARS-CoV-2 Mediates Immune Evasion through Potently Downregulating MHC-I

Peripheral immunophenotypes in children with multisystem inflammatory syndrome associated with SARS-CoV-2 infection

A single-cell atlas of the peripheral immune response in patients with severe COVID-19

Peginterferon lambda for the treatment of outpatients with COVID-19: a phase 2, placebo-controlled randomised trial