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

Background: Coronavirus disease 2019 (Covid-19) is a major global threat that has already caused more than 100,000 deaths worldwide. It is characterized by distinct patterns of disease progression implying a diverse host immune response. However, the immunological features and molecular mechanisms involved in Covid-19 severity remain so far poorly known. Methods: We performed an integrated immune analysis that included in-depth phenotypical profiling of immune cells, whole-blood transcriptomic and cytokine quantification on a cohort of fifty Covid19 patients with a spectrum of disease severity. All patient were tested 8 to 12 days following first symptoms and in absence of anti-inflammatory therapy. Results: A unique phenotype in severe and critically ill patients was identified. It consists in a profoundly impaired interferon (IFN) type I response characterized by a low interferon production and activity, with consequent downregulation of interferon-stimulated genes. This was associated with a persistent blood virus load and an exacerbated inflammatory response that was partially driven by the transcriptional factor NFκB. It was also characterized by increased tumor necrosis factor (TNF)-α and interleukin (IL)-6 production and signaling as well as increased innate immune chemokines. Conclusion: We propose that type-I IFN deficiency in the blood is a hallmark of severe Covid-19 and could identify and define a high-risk population. Our study provides a rationale for testing IFN administration combined with adapted anti-inflammatory therapy targeting IL-6 or TNF-α in most severe patients. These data also raise concern for utilization of drugs that interfere with the IFN pathway.


Introduction
Early clinical descriptions of the first SARS-CoV-2 coronavirus disease  cases at the end of 2019 rapidly highlighted distinct patterns of disease progression 1 . Although most patients experience mild-to-moderate disease, 10 to 20% progress to severe or critical disease, including pneumonia and acute respiratory failure 2 . Based on data from patients with laboratory-confirmed Covid-19 from mainland China, admission to intensive care unit (ICU), invasive mechanical ventilation or death occurred in 6.1%of cases 1 . This proportion of critical cases is higher than that estimated for seasonal Influenza 3 . Additionally, relatively high rates of respiratory failure were reported in young adults (aged 50 years and lower) with previously mild comorbidities (hypertension, diabetes mellitus, overweight) 4 . In severe cases, clinical observations typically describe a two-step disease progression, starting with a mild-to-moderate presentation followed by a secondary respiratory worsening 9 to 12 days after onset of first symptoms 2,5,6 . Respiratory deterioration is concomitant with extension of ground-glass lung opacities on chest computed tomography (CT) scans, lymphocytopenia, high prothrombin time and D-dimer levels 2 . This biphasic evolution marked by a dramatic increase of acute phase reactants in the blood suggests a dysregulated inflammatory host response resulting in an imbalance between pro-and anti-inflammatory mediators. This leads to the subsequent recruitment and accumulation of leukocytes in tissues causing acute respiratory distress syndrome (ARDS) 7 . However, little is known about the immunological features and the molecular mechanisms involved in Covid-19 severity.
To test the hypothesis of a virally-driven hyperinflammation leading to severe disease, we employed an integrative approach based on clinical and biological data, in-depth phenotypical analysis of immune cells, standardized whole-blood . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

(which was not certified by peer review)
The copyright holder for this preprint this version posted April 23, 2020. . https://doi.org/10.1101/2020.04. 19.20068015 doi: medRxiv preprint 5 transcriptomic analysis and cytokine measurements on a group of fifty Covid-19 patients with variable severity from mild to critical.
. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

Study participants
Fifty patients with diagnosis of COVID-19 according to WHO interim guidance and positive SARS-CoV-2 RT-PCR testing on a respiratory sample (nasopharyngeal swab or invasive respiratory sample) were included in this non-interventional study.
Inpatients with pre-existing unstable chronic disorders (such as uncontrolled diabetes mellitus, severe obesity defined as body mass index greater than 30, unstable chronic respiratory disease or chronic heart disease) were excluded. Since median duration from onset of symptoms to respiratory failure was previously shown to be 9.5 (interquartile range, 7.0-12.5) days 8 , we analyzed immune signatures between 8 to 12 days after first symptoms for all patients and before the initiation of any antiinflammatory treatment. Healthy controls were asymptomatic adults, matched with cases on age and with a negative SARS-CoV-2 RT-PCR testing at time of inclusion.
The study conforms to the principles outlined in the Declaration of Helsinki, and received approval by the appropriate Institutional Review Board (Cochin-Port Royal Hospital, Paris, France).
The severity of COVID-19 was classified based on the adaptation of the Sixth

Revised Trial Version of the Novel Coronavirus Pneumonia Diagnosis and Treatment
Guidance. Mild cases were defined as mild clinical symptoms (fever, myalgia, fatigue, diarrhea) and no sign of pneumonia on thoracic computed tomography (CT) scan. Moderate cases were defined as clinical symptoms associated with dyspnea and radiological findings of pneumonia on thoracic CT scan, and requiring a maximum of 3 L/min of oxygen. Severe cases were defined as respiratory distress requiring more than 3 L/min of oxygen and no other organ failure. Critical cases were defined as respiratory failure requiring mechanical ventilation, shock and/or other . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted April 23, 2020.

Peripheral blood leukocytes phenotyping
Patients' characteristics are detailed in the Supplementary Appendix and depicted in Table 1  is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted April 23, 2020. . https://doi.org/10.1101/2020.04. 19.20068015 doi: medRxiv preprint 8 was found in infected patients, especially critical patients, and NK cells displayed a significant increase in Tim-3 expression ( Figure 1G). Furthermore, expression of exhaustion-related genes, such as BATF, IRF4 and CD274, significantly increased with disease severity (Supplementary Figure 5C).
Finally, high annexin-V expression by flow cytometry and upregulation of apoptosisrelated genes in the blood from severe and critical patients supported that lymphocytopenia could be partly explained by increased T cell apoptosis (Supplementary Figure 6).

Activation of innate and inflammatory pathways in severe and critical cases
To investigate the immunological transcriptional signatures that characterize disease severity, we quantified the expression of 594 immunology-related genes in blood cells (Figure 2A). We identified differentially expressed genes as a function of severity grades ( Figure 2B). Unsupervised principal component analysis (PCA) separated patients with high disease severity on principal component 1 (PC1), driven by inflammatory and innate immune response encoding genes (GSEA enrichment score with q-value <0.2) ( Figure 2C). PC2, that was enriched in genes encoding proteins involved in both type I and type II interferon (IFN) responses, distinguished mild-to-moderate patients from the other groups. Collectively, these data suggest a severity grade-dependent increase in activation of innate and inflammatory pathways; in contrast, the IFN response is high in mild-to-moderate patients while it is reduced in more severe patients.

Impaired type I interferon antiviral response
. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted April 23, 2020. . https://doi.org/10.1101/2020.04. 19.20068015 doi: medRxiv preprint Type I IFNs are essential for antiviral immunity 12 . Multiplex gene expression analysis showed an upregulation of genes involved in type I IFN signaling (such as IFNAR1, JAK1, TYK2) contrasting with a striking downregulation of interferon-stimulated genes (ISGs) (such as MX1, IFITM1, IFIT2) in critical patients ( Figure 3A).
Accordingly, ISG score, based on the expression of 6 ISGs defining a type I IFN signature 13 , was significantly reduced in critical compared to mild-to-moderate patients ( Figure 3B, Supplementary Figure 7A). Consistently, plasma levels of IFN-α2 protein measured by Simoa digital ELISA 14 were significantly lower in critical than in mild-to-moderate patients ( Figure 3C) and correlated with ISG (R 2 =0.27; Figure 7B), while IFN-β was undetectable in all of the patients (data not shown). To further assess the global type I IFN activity, an in vitro cytopathic assay was used 15 . IFN activity in serum was significantly lower in severe and critical than in mild-to-moderate patients ( Figure 3D). Of note, plasmacytoid dendritic cells, the main source of IFN-α 16 , were reduced in infected patients compared to controls, but there was no difference between groups ( Figure 3E).

P=0.0005) (Supplementary
Finally, we evaluated the response of whole blood cells to an IFN-α stimulation. An increase in ISG score upon IFN-α stimulation was observed, that was similar in infected patients of any severity and controls ( Figure 3F), indicating that the potential for response to type I IFN was not impacted in critically ill patients. As a possible consequence of impaired IFN-α production, we observed an increased plasma viral load, a possible surrogate marker of uncontrolled lung infection, in severe and critical compared to mild-to-moderate patients, while viral load in nasal swabs was comparable between groups ( Figure 3G). Overall, these data suggest that patients with severe and critical Covid-19 have an impaired type I IFN production and a lower viral clearance.
. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

(which was not certified by peer review)
The copyright holder for this preprint this version posted April 23, 2020. . https://doi.org/10.1101/2020.04. 19.20068015 doi: medRxiv preprint Dissecting the mechanisms of hyperinflammation in severe and critical patients Severe Covid-19 was reported to induce a cytokine storm 7,17 . Cytokine and chemokine-related genes were found to be increasingly expressed as a function of disease severity in the study cohort ( Figure 4A, Supplementary Figure 8A).
Interestingly, cytokine whole blood RNA levels did not always correlate with protein plasma levels. IL-6, a key player of the cytokine storm in Covid-19 18 Figure 8F), the active form of IL-1β protein was low (Figure 4D), suggesting that pro-IL-1β was poorly cleaved and secreted, and that inflammasome activation could not be the main player in the cytokine storm. Circulating IL-1α was also not detected (data not shown). These findings contrast with the detection of high levels of circulating IL-1 receptor antagonist (IL-1RA) and upregulation of IL1R1 transcripts, . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

(which was not certified by peer review)
The copyright holder for this preprint this version posted April 23, 2020. The cytokine storm has been associated with massive influx of innate immune cells, namely neutrophils and monocytes, which may aggravate lung injury and precipitate ARDS 19 . We therefore analyzed expression of chemokines and chemokines receptors involved in the trafficking of innate immune cells ( Figure 4A). While the neutrophil chemokine CXCL2 was detected in the serum but with no difference between groups, its receptor CXCR2 was significantly upregulated in severe and critical patients ( Figure 4H). Accordingly, severe disease was accompanied with higher neutrophilia. Monocyte chemotactic factor CCL2 was increased in the blood of . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

(which was not certified by peer review)
The copyright holder for this preprint this version posted April 23, 2020. . https://doi.org/10.1101/2020.04. 19.20068015 doi: medRxiv preprint 12 infected patients, as well as the transcripts of its receptor CCR2 and was associated with low circulating inflammatory monocytes (Figure 4I), suggesting a role for the CCL2/CCR2 axis in the monocyte chemoattraction into the inflamed lungs. These observations are in accordance with recently published studies in bronchoalveolar fluids from Covid-19 patients, describing the key role of monocytes 19 . Overall, these results support a framework whereby an ongoing inflammatory cascade, initiated by impaired type I IFN production, may be fueled by both PAMPs and DAMPs.
. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

(which was not certified by peer review)
The copyright holder for this preprint this version posted April 23, 2020.

Discussion
In this study, we identified an impaired type I IFN response associated with high blood viral load in severe and critical Covid-19 patients, that inversely correlated with an excessive NFκB-driven inflammatory response associated with increased TNF-α and IL-6. Coronaviruses can also inhibit multiple stages of translational initiation 27 . Several hypotheses may be proposed to explain interindividual variability in IFN response to . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

(which was not certified by peer review)
The copyright holder for this preprint this version posted April 23, 2020. Our study confirmed that IL-6 plays an important role in pathogenesis and severity of Covid-19. Based on promising case series 39 , randomized trials are . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

(which was not certified by peer review)
The copyright holder for this preprint this version posted April 23, 2020. . https://doi.org/10.1101/2020.04. 19.20068015 doi: medRxiv preprint currently evaluating the benefits of IL-6 targeted therapy. Our study also provides a case for the inhibition of the TNF axis. Indeed, TNF is highly expressed in alveolar macrophages and anti-TNF does not block immune response in animal models of virus infection 40 . Moreover, TNF blockade induces a significant IL-6 expression reduction 40 . Other targets could also be considered such as chemokines antagonists that block migration of monocytes and neutrophils to the inflamed lungs.
Based on our study, we propose that type I IFN deficiency is a hallmark of severe Covid-19 and infer that severe and critical Covid-19 patients could be potentially relieved from the IFN deficiency by IFN administration and from exacerbated inflammation by adapted anti-inflammatory therapies targeting IL-6 or TNF-α.
. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

(which was not certified by peer review)
The copyright holder for this preprint this version posted April 23, 2020. . https://doi.org/10.1101/2020.04. 19.20068015 doi: medRxiv preprint . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

(which was not certified by peer review)
The copyright holder for this preprint this version posted April 23, 2020. . https://doi.org/10.1101/2020.04. 19.20068015 doi: medRxiv preprint . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

(which was not certified by peer review)
The copyright holder for this preprint this version posted April 23, 2020.  . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

(which was not certified by peer review)
The copyright holder for this preprint this version posted April 23, 2020. . https://doi.org/10.1101/2020.04. 19.20068015 doi: medRxiv preprint Contributors JH, NY, DD, FRL, SK and BT had the idea for and designed the study and had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. JH, NY, AF, DD, FRL, SK and BT drafted the paper. JH, NY, LB, AC, JB, DD, FRL, SK and BT did the analysis, and all authors critically revised the manuscript for important intellectual content and gave final approval for the version to be published. All authors agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Declaration of interests
We declare no competing interests.

Acknowledgments
This study was supported by the Fonds IMMUNOV, for Innovation in Immunopathology. The study was also supported by the Institut National de la Santé is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

(which was not certified by peer review)
The copyright holder for this preprint this version posted April 23, 2020. Legendre, Jonathan Marey and Alexis Régent. We thank Dr Y. Gaudin for his advices on viral mechanism. We thank all the patients, supporters and our families for their confidence in our work.
. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

(which was not certified by peer review)
The copyright holder for this preprint this version posted April 23, 2020.

Median interval from first symptoms on admission (IQR), days
10 (9-11) 9 (9-11) 10 (9-11.5) 10 (9-11)     CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

Fever on admission
The copyright holder for this preprint this version posted April 23, 2020. . https://doi.org/10.1101/2020.04. 19.20068015 doi: medRxiv preprint as cells/ml according to severity group. (F) ISG score before and after stimulation of whole blood cells by IFN-α (10 3 UI/mL for 3 hours). (G) Viral loads in nasal swab estimated by RT-PCR and expressed in Ct and blood viral load evaluated by digital PCR (B-F) Results represent the fold-increased expression compared to the mean of unstimulated controls and are normalized to GAPDH. P values were determined by the Kruskal-Wallis test, followed by Dunn's post-test for multiple group comparisons with median reported; *P < 0.05; **P < 0.01; ***P < 0.001. is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted April 23, 2020. . https://doi.org/10.1101/2020.04. 19.20068015 doi: medRxiv preprint