Innate Immune Response in COVID-19

Functional Innate Immune Response
A balance between successful evasion of the virus from host cell sensing pathways and the counter mechanisms developed by the host cells to overcome these inhibitory effects determines whether an early immune response could be generated or not (Liang et al., 2020). Though most of the studies point towards the successful evasion mechanisms employed by CoVs, emerging evidence suggests that an adequate early antiviral response could be mounted (Park and Iwasaki, 2020). That early response may hold the key for limiting the viral propagation in the majority of the COVID-19 patients (approx 80%) who are asymptomatic or develop mild symptoms and successfully clear the virus. Considered the recent work on COVID-19, here we provide a detailed molecular and clinical understanding of the innate immune response. We specifically discuss how these immune responses dictate the recovery from disease or development of the immunopathological state.

Interferon Response
By initiating an early antiviral response, signaling via IFNs and ISGs is critical for the viral clearance and an impediment for the development of the pathological state. Several in vitro and animal studies have established the central role of these signaling pathways in SARS-CoV infection. STAT1 knockout mice infected with SARS-CoV exhibited severe disease symptoms, conferred by increased viral replication and propagation and was further associated with reduced survival rate (Hogan et al., 2004; Frieman et al., 2010). Similarly, SARS-CoV propagation increases in IFNR1-/- and ILFNLR1/- double knockout mice, suggesting an essential role of these signaling pathways in mitigating antiviral response (Mahlakõiv et al., 2012).
Recent in vitro studies point to a more robust IFN response generated by SARS-CoV-2 compared to its predecessor. Epithelial cells infected with SARS-CoV-2 displayed better IFN response than cells infected with SARS-CoV. This IFN response was STAT1 phosphorylation-dependent with subsequent expression of antiviral ISGs (Lokugamage et al., 2020). In line with these in vitro findings, transcriptome data from bronchial alveolar lavage fluid (BALF) taken from 8 COVID-19 patients revealed extensive upregulation of about 83 ISGs, suggesting robust IFN response generated against SARS-CoV-2 (Zhou Z. et al., 2020). Further, a study by Ziegler et al. (2020) suggested that ACE2 may also act as a type of ISG in some respiratory epithelial cells; this may point towards using ACE2 modulators as viable therapeutic options for SARS-CoV-2.
Based on the recent clinical data on COVID-19 patients, we can infer that mild/moderate patients should possess optimal early IFN response. Whereas, weak or delayed IFN response may be the tipping point in eliciting hyperinflammatory state, allowing extensive viral propagation. Previous studies in animal models have shown that early IFN response was the determining factor in inhibiting viral propagation and attenuating disease condition (Channappanavar et al., 2016). In line with this, a recent study has shown that COVID-19 patients with mild/moderate conditions possess functional type I and type III IFN response. Specifically, patients with mild/moderate symptoms have adequate levels of IFNA transcript and protein in the plasma. The presence of detectable IFN levels in these subsets of patients was also associated with the expression of downstream signaling receptors and molecules like IFNAR1, JAK1, and TYK2, suggesting functional IFN response. However, no IFNB mRNA or protein was detected, while optimal levels of IFN-λ were detected both at the mRNA and protein levels. Expectedly, the levels of type I and type III IFNs positively correlated with the viral load and severity of the disease (Hadjadj et al., 2020).
In agreement with the critical role of early IFN response in attenuating infectious state, another study finds that cells pre-treated with IFN-β or IFN-λ exhibit resistance to SARS-CoV-2 infection by significantly decreasing the virus copy number. Similarly, 3D culture organoids pre-treated with either IFN-β or IFN-λ led to reduced viral infection. Cells depleted for either IFNAR1 or IFNLR1 had an overall increase in the number of SARS-CoV-2 infected cells, suggesting the integral role of IFN signaling in attenuating viral propagation (Stanifer et al., 2020). Further, IFN response was adequate in younger patients compared to older ones, which may partly explain the higher risk of infection in older people (Wei et al., 2020). Additionally, people with comorbid conditions like diabetes – a condition associated with impaired IFN response, are more susceptible to SARS-CoV-2 infection, which further points toward the critical role of IFN signaling in the early clearance of the virus (Erener, 2020). However, a comprehensive and longitudinal analysis of the IFN response in mild/moderate patients is warranted to understand the functional consequence of this immune response throughout the disease and recovery. Overall, considering the relatively better IFN response and ISG expression induced by SARS-CoV-2, one can argue that this functional immune response is a probable reason for the relatively lower mortality rate seen in COVID-19, compared to previous SARS-CoV and MERS infections (Meo et al., 2020). However, these early findings warrant further proof.

Early Immune Response by Alveolar Epithelial Cells (ATII)
Activated alveolar macrophages (AM) and recruited inflammatory monocytes/macrophages are majorly responsible for the secretion of cytokines and chemokines in early phases of infection, with a substantial contribution from infected ATII cells as well. This early response is necessary to recruit and activate the adaptive immune system and hence drive the clearance of the virus without inflicting immunopathological state. While the levels of cytokines and chemokines are well-regulated during this phase of infection, a check on the activation profile and recruitment of these innate immune to the sites of infection is critical. Thus, a regulated and controlled release of cytokines and chemokines in the early phase of infection is not necessarily proinflammatory but drives the successful viral clearance and the probable reason behind the limited propagation of infection as seen in the majority of the COVID-19 cases exhibiting mild symptoms (Song et al., 2020; Tay et al., 2020).
Among the cytokines secreted by virus-infected airway epithelial cells, IL-6 plays a prominent role in the early recruitment and differentiation of monocytes, neutrophils, and lymphocytes which express the corresponding IL-6 receptor (IL-6R). Though IL-6 is chiefly secreted by macrophages (activated AMs and inflammatory macrophages) in the lungs, secretion of IL-6 by ATII is also significant. In vitro studies on SARS-CoV have shown the release of IL-6 by ATII in response to RIG-I and TLR signaling via activation of NF κB pathway (Ndlovu et al., 2009; Tanaka et al., 2012). Additionally, proinflammatory cytokines TNF-α and IL-1β secreted by macrophages act on ATII cells to cause the release of IL-6 (Crestani et al., 1994; Schwingshackl et al., 2013).
Transcriptional profiling in normal human bronchial epithelial (NHBE) infected with SARS-CoV-2 shows upregulation of IL-6, suggesting that these lung epithelial cells may contribute to early IL-6 response seen in non-severe COVID-19 patients (Blanco-Melo et al., 2020). However, more conclusive studies like tissue immunohistochemistry or single-cell immuno-profiling of the lung epithelial cells will clarify their contribution in IL-6 secretion in vivo.

Early Immune Response by Alveolar Macrophages
Lung resident macrophages like AM are generally present in the terminal airways where they serve a regulatory function to maintain normal cellular homeostasis. Previous studies have defined a critical role of these cells in successful viral clearance (Hartwig et al., 2014). Depletion of these cells in animals infected with mouse hepatitis virus type 1 (MHV-1) resulted in a marked reduction of antiviral response. AMs have also been shown indispensable during SARS-CoV infection. The depletion of these cells was associated with worsened disease outcomes in a mouse model of SARS-CoV (Page et al., 2012).
Further, BALF fluid analysis of SARS-CoV infected patients revealed an increase in AM population, which persisted over two months and significantly correlated with viral clearance (Wang et al., 2005). In addition to their activation by the secondary response during viral infection, few in vitro studies have shown that these cells can also be directly targeted by SARS-CoV (Mossel et al., 2008; Joel Funk et al., 2012), though contradictory reports are available (Yip et al., 2014). Overall, the data supporting the antiviral response by AMs cells is largely based on other respiratory infections like influenza virus and MERS, with a few reports on SARS-CoV (Mossel et al., 2008; Joel Funk et al., 2012).
Studying these responses in COVID-19 patients may be challenging due to technical limitations (like difficulty in obtaining the optimal number of these cells from the lungs and their rapid functional and phenotypic changes during cell culture). However, we can draw inferences from other cell types and correlate specific markers from cells directly obtained from the lung tissue. One such recent elegant study using scRNA-seq and cluster analysis revealed the activation status of AMs in BALF fluid derived from COVID-19 patients. The analysis is based on the signature genes expressed by these cells, which are markedly different from recruited inflammatory macrophages (Liao et al., 2020). Surprisingly, the number of these cells declined in patients with severe disease symptoms, and the presence of proinflammatory macrophages can take their place (Liao et al., 2020).
A recent study (pre-print, not yet peer-reviewed) has shown infection and propagation of SARS-CoV-2 in macrophages present in lymph nodes and spleen (Chen Y. et al., 2020). However, direct infection and replication of the virus was not explored in detail, specifically under in vitro settings. Previous studies on SARS-CoV suggest low replication in these cells, probably due to phagocytosis (Yilla et al., 2005). Thus, these results suggest that AMs’ response to SARS-CoV-2 may be complicated but necessary for the activation and recruitment of other innate cells like monocytes, dendritic cells, neutrophils, natural killer (NK) cells, and essential in the regulation of the adaptive immune system (Soroosh et al., 2013; Hartwig et al., 2014; Meischel et al., 2020).

Dysfunctional Innate Immune Response
On average, about 15% of the COVID-19 patients exhibit severe disease symptoms whereas 5% become critical, but the figures are subject to change owing to the ongoing increase in the number of cases (Berlin et al., 2020). By looking at the immunological trajectories of these patients, it has become evident that impaired early IFN response followed by hyperactivated innate and a dysfunctional adaptive immune response is the vital pathological factors contributing to disease severity in COVID-19 patients (Blanco-Melo et al., 2020; Mathew et al., 2020). However, there are also reports, suggesting a more complex interplay in these immune responses, which needs a thorough understanding of developing effective immunotherapy-based interventions and for successful vaccine development.

Impaired Interferon Response
Based on previous molecular and clinical studies on SARS-CoV and the recent data on SARS-CoV-2, it is becoming evident that the delay in primary IFN response may be due to multiple factors such as (1) poor overall immune function of a patient with a compromised adaptive response as in older people, (2) patients with comorbidity, (3) genetic factors or epigenetic changes associated with crucial genes and transcriptional factors involved in IFN signaling, and (4) age and sex of the patient, probably making the older individuals and males more susceptible to COVID-19 (Bastard et al., 2020; Li M.Y. et al., 2020; Nguyen et al., 2020; Verdecchia et al., 2020; Zhou F. et al., 2020). Thus, overall these factors may compromise the host cell immune system and delay the early antiviral response. Especially in the case of RNA viruses, evasion of host immune response is managed by interfering with PRRs, PLRs, TLRs, and IFN signaling (Kikkert, 2020). Additionally, inhibition is also conferred by hijacking host cell biosynthetic machinery and eventually inducing host cell apoptosis as discussed above.
Previous studies have unequivocally demonstrated poor IFN response to SARS-CoV during severe infection, which is also apparently the case with SARS-CoV-2, reviewed recently by Park and Iwasaki (2020). In vitro culture of the primary lung, epithelial cells infected with the SARS-CoV-2 generated inadequate IFN response (Blanco-Melo et al., 2020). By looking at the clinical samples, a large body of data suggests impaired IFN signaling in severe and critically ill COVID-19 patients. Blood analysis from across the studies reveals low or undetectable levels of IFN-β and IFN-λ levels in patients exhibiting severe disease symptoms or patients admitted to the ICU with in a critical condition (Hadjadj et al., 2020). Of note, an elegant study was conducted to explore the functional role of IFN signaling during various stages of COVID-19 disease severity. The study found robust impairment of IFN signaling in critically ill and severe patients in comparison to mild/moderate and healthy individuals. IFN-β mRNA and protein were undetectable in all patients, whereas IFN-α2 protein was highly reduced in the plasma of severe and critically ill patients, corroborated with reduced IFN activity. In line with the impaired IFN signaling, robust downregulation of some of the ISGs (MX1, IFITM1, IFIT2) observed in severe and critically ill patients suggest an overall reduced IFN response (Hadjadj et al., 2020).
Consistent with the low circulating levels of IFNs, transcriptional analysis of post-mortem lung samples further confirmed these observations and revealed no detectable type I or Type III IFNs. Among the SARS-CoV-2 proteins which directly interfere with IFN response, ORF6, ORF8, and N protein inhibit IFN-β and NF-κB signaling (Li J.Y. et al., 2020). Further, Konno et al. (2020) have identified a more extended variant of ORF3b with presumably more vigorous anti-IFN activity. Thus, these early observations may point towards an impaired early IFN response by the host cells against SARS-CoV-2
Adding to the essential role of IFN in early antiviral response, two recent studies have shown that genetic changes are associated with inadequate IFN response. In the first study, the presence of IFN neutralizing auto-antibodies found in patients who exhibited more severe disease condition (Bastard et al., 2020). These auto-antibodies were more prevalent in men than women, that partly explains the susceptibility of men to COVID-19. None of the asymptomatic or mild cases had detectable auto-antibodies. In the other study, mutations in 13 key genes implicated in TLR3- and IRF7-dependent exhibit loss-of-function (Zhang Q. et al., 2020). Patients or the cells derived from these patients with loss-of-function in these genes had inadequate IFN response and vulnerable to SARS-CoV-2 infection. In a similar study on four patients with severe disease symptoms, the whole exome-sequencing revealed loss-of-function of TLR7, which is essentially involved in IFN signaling. These patients exhibited decreased expression of IRF7, IFNB1, and ISG15, along with reduced production of IFN-γ (Van Der Made et al., 2020). Thus, impaired IFN signaling, mediated either directly by the virus by interfering at various steps in the IFN signaling, or genetic predisposition of some individuals to inadequate IFN response and presence of IFN neutralizing auto-antibodies are some of the significant factors which determine the COVID-19 disease severity. The dysfunctional IFN response in conjunction with other innate and adaptive immune responses may thus decide the path to recovery or progression to more severe form of the disease (Hadjadj et al., 2020). Impaired type I interferon activity and exacerbated inflammatory responses in severe COVID-19 patients (Hadjadj et al., 2020; Park and Iwasaki, 2020). A comprehensive understanding of the molecular mechanisms by which SARS-CoV-2 causes impaired IFN response is still lacking, and future studies may help us to understand this.
Nevertheless, these initial reports, along with the previous findings on SARS-CoV, are the basis behind exploring the therapeutic efficacy of IFN treatment for COVID-19 patients. Currently, there are ongoing clinical trials with IFN-β1a (NCT04350671), which is in phase II, and IFN-l (NCT04388709) for the treatment of COVID-19. The preliminary results with these drugs have been encouraging as of now (Davoudi-Monfared et al., 2020).

Release of Damage-Associated Molecular Patterns and Proinflammatory Molecules
The impaired early IFN response results in high viral propagation that subsequently leads to the induction of a robust proinflammatory response (Davidson et al., 2015). The cytopathic nature of these viruses induces substantial death in infected ATII cells (apoptotic as well as necrotic) which leads to the release of a wide range of damage-associated molecular patterns (DAMPs) and cytotoxic molecules. Similarly, activated AMs also respond to the released DAMPs and act concurrently with PAMPs to amplify the proinflammatory response. A list and role of potential PAMPs, DAMPs, and their respective PRRs have been reviewed previously (Leiva-Juárez et al., 2018).
Circulating nuclear and mitochondrial DNA, and histones serve as potential DAMPs during viral infections. These molecules signal via the TLR pathway and induce robust expression of proinflammatory molecules. Among the DAMPs secreted by virus-infected and damaged epithelial cells, the role of high-mobility group box one protein (HMGB1) and S100 are well known (Leiva-Juárez et al., 2018; Gong et al., 2020). HMGB1 after binding to TLR4 induces activation of NF-κB signaling and release of proinflammatory molecules. Additionally, HMGB1 also activates receptors like TREM1/2, and receptors for advanced glycation end products (RAGE) which are also involved in NF-κB activation (Yang and Tracey, 2010). S100 initiates similar downstream signaling after binding with TLR4 and RAGE receptors (Ma et al., 2017), these studies were recently reviewed by Gong et al. (2020). Previous animal studies with other respiratory viruses have shown a close correlation of increased serum HMGB1 levels with lung injury and disease severity (Patel et al., 2018). Similarly, elevated expression of S100A9 was present in patients during acute lung injury mediated by the respiratory syncytial viral (RSV; Foronjy et al., 2016). Although as of now, presence of HMGB1 has no report in COVID-19 patients, the damage in the lung parenchyma in post-mortem biopsies suggests that it is highly likely that this protein may implicate in disease pathogenesis and hyperinflammation (Andersson et al., 2020; Zhang Q. et al., 2020).
Increased expressions of S100A8, S100A9, and S100A12 calgranulins found in the BALF fluid from COVID-19 patients indicate their potential role in generating the proinflammatory response (Zhou Z. et al., 2020). Further, Zou et al. (2020) showed increased presence of cell-free DNA and citrullinated histones in blood samples obtained from 50 COVID-19 patients. Studies on other inflammatory diseases have shown a close correlation between the presence of these molecules with disease severity (Resman Rus et al., 2016). However, their functional role is yet unexplored, but the increased expression of some of these DAMPs in COVID-19 patients suggests their potential implication in disease pathogenesis. Future studies will clarify the involvement of various other DAMPs in perpetuating the proinflammatory state, and specifically the role of HMGB1.
In addition to the secretion of DAMPs, AM and virus infected ATII cells secrete a range of pro-inflammatory molecules (Hussell and Bell, 2014; Glaser et al., 2019). Among these, increased IL-6 levels are consistently detected in cultured cells infected with SARS-CoV and SARS-CoV-2 (Ye et al., 2018; Herold et al., 2020; Liu J. et al., 2020; Liu T. et al., 2020). Notably, levels of TNF-α, IL-8, IL-10, GM-CSF, CXCL10, and CCL5 secreted by infected ATII and activated AMs were also consistently shown to increase during SARS-CoV and SARS-CoV-2 infections (Ward et al., 2005; Huang C. et al., 2020; Patterson et al., 2020). Transcriptional profiling of cytokines and chemokines in normal human lung epithelial cells (NHBE) infected with SARS-CoV-2 revealed increased levels of CCL20, CXCL1, IL-1B, IL-6, CXCL3, CXCL5, CXCL6, CXCL2, CXCL16, and TNF-α by primary lung epithelial cells in response to SARS-CoV-2 infection (Blanco-Melo et al., 2020). Thus, lung resident ATII and AM cells besides being integral to the antiviral response also participate in generating a profound proinflammatory state.

Proinflammatory Molecules Released by Infiltrating Myeloid Cells

Circulating inflammatory monocytes/macrophages
A detailed account of the role of inflammatory macrophages in the pathogenesis of SARS-CoV is reported by He et al. (2007). Animal studies have demonstrated extensive recruitment and accumulation of these cells in the lungs, which correlated with the release of TNF-α, IL-1β, and IL-6 and the development of ARDS, reviewed by Gralinski and Baric (2015). Interestingly, depletion of these inflammatory macrophages in animals infected with SARS-CoV was associated with a high recovery rate, thus suggesting their critical role in disease pathogenesis (Channappanavar et al., 2016). Similarly, SARS-CoV infection in animals with STAT1 knockout in alternatively activated macrophages displayed attenuated lung damage and protection from disease (Page et al., 2012). Besides, a large number of clinical studies support an integral role of IMMs in SARS-CoV infected patients (Wong et al., 2004; Tisoncik et al., 2012; Liu et al., 2019). Recent studies from BALF from COVID-19 patients have also demonstrated the critical role of circulating monocyte-derived macrophages in the induction of robust proinflammatory reaction (Liao et al., 2020). Blood cell analysis of 18 COVID-19 patients revealed an activated status of inflammatory macrophages (Zhang D. et al., 2020). In line with these findings, scRNA-seq followed by immune cell profiling of blood cells revealed an increased number of CD14++ monocytes (Wen et al., 2020). Severe and critically ill patients also exhibit macrophage activation syndrome (MAS) in some cases (Giamarellos-Bourboulis et al., 2020). Thus, all the evidence directs towards a critical role of inflammatory macrophages in disease severity during COVID-19 and a potential therapeutic target. Intervention which reduces the impetus to induce MAS like antibodies directed against IL-6 and IL-1β has shown promising clinical outcomes, reviewed by Otsuka and Seino (2020).

Proinflammatory neutrophils
Like other innate immune cells, neutrophils are protective in the early phases of infection by neutralizing the viral particles and release of protective molecules to interfere with the viral propagation (Drescher and Bai, 2013). However, in severe cases, the number of these cells increases at the sites of infection and they become the leading damage-causing cells. Excessive infiltration of these cells in the lungs is associated with secretion of TNF-α, IL-6, IL-1β, IL-7, IL-23, and IL-36, along with a broad range of other cytokines and damage-causing neutrophil extracellular traps (NETs; Tecchio et al., 2014). Additionally, these neutrophils also secrete a range of chemokines like CCL2/3/4, CXCL1-13 to attract more neutrophils and monocytes from the circulation (Sokol and Luster, 2015).
Emerging evidence suggests a pivotal role of neutrophils in the pathogenesis of COVID-19. Immune cell profiling revealed activated status of these cells which was associated with increased levels of NETs and correlated with acute-phase reaction (Chen G. et al., 2020; Qin et al., 2020; Zuo et al., 2020). Similarly, an increase in the number of activated neutrophils was present in the BALF of COVID-19 patients (Liao et al., 2020; Xiong et al., 2020). Thus, based on these recently published studies, the neutrophil number in the blood can be used as a predictive marker for disease severity (Zhang et al., 2020a).

Natural killer cells
Natural killer cells are essential in the early phase of viral infection to assist in the clearance of the virus by interacting with death receptors expressed on the infected cells (Vidal et al., 2011). Previous clinical studies have shown decreased NK cell number in SARS-CoV patients, which was more pronounced in severe cases (Wang and Xia, 2004). A recent blood profile of COVID-19 patients suggested a similar decline in the number of NK cells in severe cases, along with an increased expression of exhaustion markers (Chen X. et al., 2020; Tan L. et al., 2020b; Zheng H.Y. et al., 2020). On the contrary, no significant difference was found in the number of total NK cells, in non-ICU vs 10 ICU admitted patients (Zhou et al., 2020a). This discrepancy in number could probably be due to differential temporal immune response and the underlying prevailing disease conditions in some patients. Immune cell profiling data from early recovery stage (ERS) and late recovery stage (LRS) COVID-19 patients revealed a biphasic effect, with fewer NK cells during early recovery ERS, which recovered during LRS (Wen et al., 2020). Thus, besides the underlying disease state, the NK cell number may also be sensitive to the time of sample collection and hence may not serve as a potential disease marker. Further, these studies could also suffer from the limitation of the variation in the age of the patients studied which may make it difficult to provide a definite role of these cells concerning COVID-19 disease severity (Nikolich-Zugich et al., 2020), necessitating more conclusive studies.

Lung resident and monocyte-derived dendritic cells
Lung resident dendritic cells majorly have a protective role during the early onset of the disease by activating the adaptive immune cell response. Under the influence of PAMPs, DAMPs, and inflammatory cytokine signaling, lung resident dendritic cells are conditioned and migrate to the draining lymph node under the influence of CCR7 where they prime naïve CD4+ and CD8+ T cells (Braun et al., 2011; Thaiss et al., 2011). In contrast, monocyte-derived dendritic cells generate under the influence of GM-CSF, IFN-γ, and IL-4, along with other proinflammatory signals (Qu et al., 2014). Previous studies have shown elevated secretions of CCL3, CCL5, MCP-1, IP-10, TNF-α, and IL-6 by activated inflammatory dendritic cells (DCs) in response to SARS-CoV (Law et al., 2005). Recent reports also suggest the presence of activated dendritic cells in COVID-19 patients. Notably, meta-transcriptomic sequencing of BALF obtained from 8 COVID-19 patients revealed an activated status of these cells along with neutrophils, as compared to other innate and adaptive immune cells (Yang A.P. et al., 2020; Zhou Z. et al., 2020). Thus, based on previous clinical studies on SARS-CoV infection and recent emerging studies on SARS-CoV-2, it is evident that hyperinflammatory immune response in severe and critically ill COVID-19 patients is mainly mounted by infiltrated innate immune cells at the site of infection with a substantial contribution by the adaptive immune cells as discussed below in the section on the dysfunctional adaptive immune response.