Antiviral Response and Immunopathogenesis of Interleukin 27 in COVID-19

doi:10.21203/rs.3.rs-2514034/v1

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Antiviral Response and Immunopathogenesis of Interleukin 27 in COVID-19

https://doi.org/10.21203/rs.3.rs-2514034/v1

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published 13 Jun, 2023

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The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, is associated with a high mortality rate. The clinical course is attributed to the severity of pneumonia and systemic complications. In COVID-19 patients and murine models of SARS-CoV-2 infection, the disease may be accompanied by over-exuberant production of cytokines, leading to accumulation of immune cells in affected organs such as lungs. Previous reports have shown that SARS-CoV-2 infection antagonizes interferon (IFN)-dependent antiviral response, thereby preventing the expression of IFN-stimulated genes (ISGs). Lower IFN levels have been linked to more severe COVID-19. Interleukin 27 (IL27) is a heterodimeric cytokine composed of IL27p28 and EBI3 subunits that induce both pro- and anti-inflammatory responses. Recently, we and others have reported that IL27 also induces a strong antiviral response in an IFN-independent manner. Here, we investigated transcription levels of both IL27 subunits in COVID-19 patients. Results show that SARS-CoV-2 infection modulates TLR1/2-MyD88 signaling in PBMCs and monocytes, and induces NF-κB activation and robust pro-inflammatory response-dependent NF-κB-target genes expression, including EBI3; as well as it activates IRF1 signaling, that induces IL27p28 mRNA expression. Results suggest that IL27 induces a robust STAT1-dependent pro-inflammatory and antiviral response in an IFN-independent manner in COVID-derived PBMCs, and Monocytes as a function of severe COVID-19 clinical course. Similar results were observed in SARS-CoV-2 Spike protein-stimulated macrophages. Thus, IL27 can trigger host antiviral response suggesting the possibility of novel therapeutics against SARS-CoV-2 infection in humans.

SARS-CoV-2

COVID-19

Innate immune response

Toll-like receptors

Inflammatory response

Antiviral response

Interleukin 27

Interferon

Coronavirus disease 2019 (COVID-19) induced by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a single-stranded positive-sense RNA (ssRNA+) virus associated with high mortality rate, attributed to the severity of pneumonia and systemic complications (reviewed in: [1]). Most SARS-CoV-2 infected individuals have mild flu-like symptoms, nevertheless, about 20% of infected individuals develop a more severe illness due to viral pneumonia, often resulting in need for hospitalization [2]. The clinical manifestations of COVID-19 can vary from asymptomatic infection to mild-moderate, severe, and ultimately critical respiratory illness with multi-organ dysfunctions that can lead to death [3]. Studies of COVID-19 patients and murine models of SARS-CoV-2 infection indicate that severe disease may be triggered by over-exuberant production of cytokines (cytokine storm), leading to the accumulation of immune cells in affected organs such as lungs [4][5]. Hyperinflammation is related to exacerbation of symptoms in severe and critical COVID-19 patients [6]. Huang et al., (2020) reported that SARS-CoV-2 infection generates a delayed but overactive production of interleukin (IL) 1β (IL1β), IL6, CXCL8/IL8, IL10, tumor necrosis factor alpha (TNFα), and monocyte chemoattractant protein 1 (CCL2/MCP1), inducing a dysregulated innate immune response [3]. Further, numerous studies have shown that markers, including IL6, CXCL8/IL8, or patterns from multiomics-based approach, might be associated with COVID-19 severity and lethality [7] [8][9]. However, although severe disease presentation show elevated pro-inflammatory cytokines [10], given the clinical diversity and variable characteristics of the disease, it is unclear how dysregulation of innate and adaptive immune responses is linked to severity of COVID-19.

Innate immune response is the first line of host defense following viruses infection through activation of pattern recognition receptors (PRRs), including Toll-like receptors (TLRs) [11], which are the central mediators of the innate and adaptive immune responses. TLRs recognize conserved pathogen-associated molecular patterns (PAMPs) of microbes to induce pro-inflammatory and antiviral response [11] [12][13][14]. Among cell surface TLRs, TLR2 forms homodimer or heterodimer with TLR1 or TLR6 (TLR2 complex) [15]. TLR2 complex play essential role in sensing diverse microbial PAMPs, including lipoproteins, lipoteichoic acid (Gram-positive bacteria), and chitin (fungi) [16]. Thus, upon recognition of PAMPs, TLR2 recruits adaptor proteins, including myeloid differentiation primary response 88 (MyD88) and Interleukin 1 receptor-associated kinases (IRAK) to form the Myddosome, and initiate signaling pathways that culminate in activation of nuclear factor-kB (NF-κB), and transcription factors, including NF-κB1, NF-κB2, RELA, RELB, c-REL and IκBα (negative regulator) [11][17, 18]. Activation of NF-κB results in the transcription of many NF-κB-target genes that promote secretion of cytokines and chemokines, implicated in pro-inflammatory response [19][20] [21][22][23][24] [17]. Additionally, TLR2 complex recognize structural PAMPs of several viruses, including human immunodeficiency virus type 1 (HIV-1) [25], Herpes simplex virus 1 [26], Hepatitis C virus (HCV) [27], Dengue virus [28], and Chikungunya virus (CHIKV) [17]. Furthermore, it has been reported that TLR2 recognize the SARS-CoV-2 Envelope (E) and Spike (S) glycoproteins to induce pro-inflammatory response [29] [30] [31]. While there is one study showing that TLR4 recognize SARS-CoV-2 Spike protein to induce strong immune response [32], another study reports that SARS-CoV-2 by itself is not recognized by TLR4, in both TLR4-HEK293 cells and primary human dendritic cells [33].

RNA sensors such as TLR3 and TLR7 also plays a crucial role in recognition of SARS-CoV-2 RNAs, and the activation of signaling pathways that culminate in induction of NF-κB and interferon response factors (IRFs), including IRF1, IRF3 and IRF7 [34][31] [17]. NF-κB and IRFs regulate expression of pro-inflammatory mediators as well as interferons (IFNs) in many cell types [35][36] [11].

The antiviral response is closely related to transcription of interferon (IFN) genes, their inhibition can lead to increased susceptibilities to viral infections [37]. Based on genetic, structural, and functional characteristics, and receptor usage to signaling in cells, three types of IFNs have been identified [38][39][40]. Type I IFN (IFNα, β, ε, κ, ω) bind to IFN alpha/beta receptor complex [IFNAR (IFNAR1/IFNAR2)] [39][41], type II IFN (IFNγ)] signals through IFN-gamma receptor complex [IFNGR (IFNGR1/IFNGR2)] [38], and type III IFN (IFNλ1–4) bind to the interferon-Lambda receptor complex [IFNLR (IFNLR1/ IL10RB) [42]. The IFN-I/IFNAR and IFN-III/IFNLR are the most important IFNs/IFNRs expressed by many cell types and are secreted from infected cell. Thus, these two types of IFNs triggers relevant gene expression via the Janus kinase (JAK) signaling pathway, which phosphorylates and activates signal transducer and activator of the transcription 1 (STAT1) and STAT2, that together with IRF9 forms a transcription factor complex known as IFN stimulated gene factor 3 (ISGF3) [43][37]. ISGF3 is translocated into the nucleus and binds to IFN-stimulated response elements (ISRE) to upregulated the expression of numerous IFN-stimulated genes (ISGs) encoding antiviral proteins (AVPs), cytokines, and chemokines that orchestrate antiviral state.

In infected cells, SARS-CoV-2 non-structural proteins inhibit innate immune receptor signaling and IFN production [44]. Furthermore, SARS-CoV-2 is highly sensitive to exogenously administered type I/III IFNs [45]. Hadjadj et al. (2020) reported low levels of IFN-I among COVID-19 patients, which correlated with poor outcomes and prognosis [46]. Moreover, suppression of IFN-I and IFN-III response in patients is associated with severe COVID-19 [46][47] [48]. It has been suggested that the low IFN-dependent antiviral response in COVID-19 patients may be due to the failure of innate immune cells to mount appropriate IFN-I response to infection, as evidenced by reduced frequency and activity of plasmacytoid dendritic cells, the main source of IFNα during viral infections, in COVID-19 patients [49]. Additionally, it has been reported that IFN-I-dependent antiviral response in lungs is stronger in patients with moderate disease as compared to those with severe COVID-19 [49]. As well, IFN-III plays important role in host defense against SARS-CoV-2 infection, since the production of IFNλ1 and IFNλ3 in upper airways induce the expression of protective ISGs in mild COVID-19 cases [50]. However, [51] reported that SARS-CoV-2 infection appears to result in disruption of IFN response, which is linked to loss-of-function of genes that encode regulators of IFN induction, including TLR3, IRF3, IRF9, and IFN-I signaling [52]. Loske et al. reported that basal levels of several PRRs and ISG were pre-activated in children but, upon SARSCoV-2 infection, their expression was higher in children than in adults [53]; however, expression of the IFN coding genes was not reported in these studies. Together, the reports suggest that SARS-CoV-2 has evolved escape mechanisms to inhibit IFN and down-stream signaling targets to escape host immunity. Thus, identification of factors that enhance innate antiviral response, that are independent on IFN signaling, may contribute to the development of potentially novel therapeutics against COVID-19.

Interleukin 27 (IL27) is a heterodimeric cytokine, member of the IL6/IL12 family of cytokines, composed of IL27p28 and Epstein-Barr virus-induced 3 (EBI3) subunits [54]. Its interaction with a heterodimeric IL27Rα and Gp130 [55] activates JAK-STAT signaling which plays critical role in the induction of the cellular response. IL27 is expressed by antigen-presenting cells (APC) exposed to inflammatory stimuli that causes both pro-inflammatory and anti-inflammatory responses with diverse influence on the immune system [55][56, 57]. In monocytes, IL27 signals through STAT1, STAT3, and NF-kB [58], whereas in macrophages it signals through STAT1 and STAT3 [59]. IL27 has received significant attention due to studies showing that IL27 suppresses replication of viruses including HIV-1 [60], HBV [61], HCV [62], Influenza A virus (IAV) [63], cytomegalovirus (CMV) [64], and CHIKV [17, 65]. Recently, we reported that IL27 induces STAT1-depedent proinflammatory and antiviral response in CHIKV-infected macrophages through two signal pathways; an early signal dependent on recognition of CHIKV-PAMPs by TLR1/2-MyD88 to activate NF-κB, which in turn induces the mRNA expression of EBI3, and second signal dependent on the recognition of CHIKV replication intermediate (dsRNA) by TLR3-TRIF, to activate IRF1, which induces IL27p28 mRNA expression [17]. Both signaling pathways are required for the production of functional IL27 protein involved in the induction of ISGs, including AVPs, cytokines, CC- and CXC- chemokines, in an IFN-independent manner.

Since we and others have reported that IL27 induces strong antiviral response against viral infections in an IFN-independent manner, in the present study we investigated transcription levels of IL27 in COVID-19 patients to determine whether IL27 might induce antiviral response against SARS-CoV-2 infection. For this, we reanalyzed two mRNA datasets, first one was performed in peripheral blood mononuclear cells (PBMCs) from healthy individuals and patients with moderate, severe, and critical COVID-19; and the second in monocytes (Mon) from healthy individuals and patients with moderate and severe COVID-19. Additionally, we reanalyzed the mRNA dataset of SARS-CoV-2 Spike protein-stimulated macrophages. Our analysis focused on IL27 signaling components and ISG-encoding genes, which should facilitate better understanding of protective and pathogenic immune response in COVID-19 patients.

2.1. Bioinformatic analysis of previously published mRNA datasets and prediction of cell signaling pathways activation.

To investigate the transcription level of IL27 in COVID-19 patients, we reanalyzed two RNA-Seq datasets. The first RNA-Seq was published by Hadjadj et al., (2020) [46] (kindly donated by Terrier B) and performed in PBMCs from healthy individuals (n = 13) and patients with moderate (n = 11), severe (n = 10), and critical (n = 11) COVID-19. The second RNA-Seq dataset (GSE176290) was downloaded from the Genome Expression Ommnibus (GEO) [66] performed in monocytes (Mon) from healthy individuals (n = 9) and patients with moderate (n = 9) and severe (n = 11) COVID-19. We also reanalyzed the publicly available RNA-Seq GSE173488 (GEO) [67] in which included human monocyte-derived macrophages (MDMs) from healthy donors that were stimulated or not with 1 µg/ml SARS-CoV-2 Spike protein for 4 hours (n = 4). Gene expression (mRNA) was expressed as RNA counts and the counts matrix of RNA-Seq datasets was normalized to counts per million (CPM). Then, mRNA was further normalized by calculating Reads per kilobase per million mapped reads (RPKM) [CPM/Gene size (Kbs)]. We predict the activation of cell signaling pathways by quantifying mRNA expression (RNA counts or RPKM) of immune biomarkers. These biomarkers are associated with myeloid markers, migration markers, inductors of antiviral response, TLR signaling pathway, IL27 signaling pathway, IRFs, NF-κB complex, NF-κB target genes, and ISGs, including antiviral proteins (AVPs), STAT1-dependent cytokines, and STAT1-dependent CC- and CXC-chemokines. This approach was previously reported [23].

3.1. Expression of myeloid and migratory markers in human PBMCs and Monocytes are associated with COVID-19 severity

Monocytes are phagocytic cells of the innate immune system implicated in immunopathogenesis of viral infection. Zheng et al., (2021) reported significant reduction of Mon frequency in PBMCs from COVID-19 patients [30], suggesting possible migration of Mon into the tissues. However, role of human Mon in immunopathogenesis of SARS-CoV-2 infection is poorly understood. In this study, we evaluated expression of myeloid and migration markers in both COVID-derived PBMCs and COVID-derived Mon. We observed that both PBMCs and Mon up-regulated expression of mRNAs of myeloid markers, including CD14, α-integrins (CD11b and CD11c), FCγ receptors (CD64A), and macrophage scavenger receptors (CD163), as a function of the severity of COVID-19 patients (Figs. 1A and C). Further, COVID-derived PBMCs and Mon up-regulated the expression of migration markers, including endothelial adhesion molecules, including L-selectin and Intercellular Adhesion Molecule 1 (ICAM1), and CC/CXC chemokine receptors (CCR1, CXCR1), as a function of the severity of COVID-19 patients (Figs. 1B and D). Together, the results show that up-regulation of myeloid and migration markers is consistent in both COVID-derived PBMCs and COVID-derived Mon, suggesting that monocytes could be the main subset into PBMCs that respond to SARS-CoV-2 infection in humans, suggesting a possible immunopathogenic role of Mon activation in SARS-CoV-2 infection.

3.2. Interleukin 27 subunits mRNAs are highly induced and correlated with disease severity, both in PBMCs and Monocytes from COVID-19 patients.

Previous reports have shown that SARS-CoV-2 infection antagonizes IFN-dependent antiviral response [46][47] [48]. Additionally, Hadjadj et al., (2020) reported that PBMCs from COVID-19 patients did not express significantly high levels of any of the 3 types of IFN [46]. Here, we explore the mRNA expression of IFNs and IL27 subunits in both COVID-19 infection derived PBMCs and Mon. Transcriptomic analysis showed that neither COVID-derived PBMCs nor COVID-derived Monocytes express IFN-I, including IFNα1 and IFNβ1 (Figs. 2A-B and 3A-B, respectively). Neither IFN-II such as IFNγ (Figs. 2C and 3C, respectively), nor IFN-III, except for IFNλ1, which was expressed in COVID-derived PBMCs from critical COVID-19 patients (Fig. 2D), but not in COVID-derived Monocytes (Fig. 2D). Next, we investigated whether IL27 positively correlated with COVID-19 severity. The transcriptomic analysis shows that the expression of both IL27 subunits, IL27p28 and EBI3, increased with severity of COVID-19 in both PBMCs and Mon (Fig. 2E-F and 3E-F, respectively). Collectively, the data suggest that an increase in IL27 expression correlated with COVID-19 indicating that IL27 might be a marker of antiviral response to SARS-CoV-2 infection or, possible biomarker of COVID-19 progression.

3.3. Interleukin 27 signaling components are expressed in COVID-derived PBMCs and Monocytes

Considering that COVID-derived PBMCs and Monocytes show high levels of both IL27 subunits, we evaluated mRNA expression levels of IL27 signaling components in these cells. We observed that COVID-derived PBMCs and Monocytes showed significantly increased levels of receptor subunit Gp130, the receptor-associated kinase JAK2, STAT1 and STAT3, and the negative regulator SOCS3 (Fig. 4A and 5A, respectively), as a function of COVID-19 disease severity. As shown in the Fig. 4B, we observed a significant positive correlation between mRNA expression level of IL27p28 subunit and IL27 signaling components in COVID-derived PBMCs, including Gp130 (R = 0.2779, P = 0.0002), JAK2 (R = 0.4997, P < 0.0001), STAT1 (R = 0.3676, P < 0.0001), STAT3 (R = 0.3299, P < 0.0001), and SOCS3 (R = 0.4619, P < 0.0001).

Since we and others have reported that IL27 is essential for antiviral immunity [68][65], we proceeded to determine ISG mRNA levels in both COVID-derived PBMCs and monocytes. While the expression of Guanylate-binding Protein 1 (GBP1), Interferon-gamma Inducible Protein 16 (IFI16), Interferon Induced Protein with Tetratricopeptide Repeats 2 (IFIT2), Interferon Induced Transmembrane Protein 1 (IFITM1), and MX Dynamin Like GTPase 1 (MX1) was increased in COVID-19-derived PBMCs (Fig. 4C), the expression of APOBEC3A, GBP1, IFIT1, IFITM1, ISG15, MX1, and OAS1 was increased in COVID-19-derived monocytes (Fig. 5B). Similarly, we found that the expression pattern of STAT1-dependent cytokines and chemokines, including IL15, TNF-related Apoptosis-inducing Ligand (TRAIL), and B-cell Activating Factor (BAFF), CCL2, CCL7, CXCL10, and CXCL11 were increased with severity of COVID-19 both in PBMC and Mon, while CCL8 mRNA was significantly expressed in moderate but decreased in severe and critical COVID-19 patients; CXCL9 was significantly expressed in critical COVID-19 patients, but not in other groups (Fig. 4E). As shown in Fig. 4F, a moderate-high positive correlation between mRNA expression of STAT1 and their target genes, including ISGs such as GBP1 (R = 0.8809, P < 0.0001), IFITM1 (R = 0.7820, P < 0.0001), MX1 (R = 0.7156, P < 0.0001); cytokines such as BAFF (R = 0.6665, P < 0.0001) and TRAIL (R = 0.7639, P < 0.0001), and chemokines, including CCL2 (R = 0.4078, P < 0.0001), and CXCL10 (R = 0.4341, P < 0.0001) was observed in COVID-derived PBMCs. The data suggest that COVID-derived PBMCs and Monocytes activated IL27 signaling induce a robust STAT1-dependent pro-inflammatory and antiviral response dependent of ISGs expression.

3.4. Expression levels of TLRs, TLR adapter, as well as TLRs downstream signaling components, is associated with the severity of COVID-19.

TLRs activation drives transcriptional expression of both IL27p28 and EBI3 genes through the induction of IRF1 and NF-κB, respectively [17]. To determine whether any TLRs were positively correlated with COVID-19 severity, as IL27, we analyzed the dataset for TLR expression in patients with differing severities of COVID-19 (Figs. 6 and 7). We found that the expression of TLR2 was increased with severity of COVID-19 in both PBMCs and Mon (Figs. 6A and 7A, respectively), similar to that observed with IL27 subunits (Figs. 2E and F). We note that TLR1 was also increased with severity of CODIV-19 in Mon (Fig. 7A). As well, expression of TLR1, TLR4, TLR5, and TLR8 was significantly elevated in patients with severe and critical COVID-19 and the expression of TLR7 was increased in patients with moderate and critical (Fig. 6A). By contrast, expression of TLR4, TLR5, TLR6, and TLR7 was not altered in COVID-19-derived Mon (Fig. 7A). The expression of TLR3 did not show changes with the progression of COVID-19 in both PBMC and Mon (Figs. 6A and 7A). The data suggest a link of IL27 and certain TLRs with disease progression in patients with COVID-19.

MyD88 is important for proinflammatory cytokine production during SARS-CoV-1 infection [69]. To determine whether MyD88 or other TLR adapter TRIF (TIR-domain-containing adapter-inducing interferon-β), plays a role in SARS-CoV-2-induced inflammatory responses, we analyzed mRNA expression of these factors in PBMC and Mon from patients with differing severities of COVID-19. We found that the expression of MyD88 was increased with severity of COVID-19 in both PBMC and Mon (Figs. 6B and 7B). By contrast, the expression of TRIF was significantly increased only in PBMC from patients with severe and critical COVID-19 but not on Mon (Figs. 6B and 7B). Furthermore, as observed in Fig. 6B, mRNA levels of key mediators of TLR signaling, including IRAK2, and IRAK3 were increased in PBMC from patients with severe and critical COVID-19. Also, while IRAK2 was elevated in Mon from patients with moderate and severe COVID-19, IRAK3 was only increased in severe COVID-19 (Fig. 7B).

Although our transcriptomic analysis showed no expression of IFNs, we proceeded to evaluate the expression of IRFs, since they are induced by signaling via PRRs, including TLRs. We found that the expression pattern of IRF7 was increased with severity of COVID-19 in both PBMC and Mon (Figs. 6A and 7A). While the expression of IRF1 was significantly increased in patients with severe and critical COVID-19-derived PBMC (Fig. 6B), its expression in Monocytes was increased only in patients with severe COVID-19 (Fig. 7B), as was observed with IL27p28 and EBI3 (Figs. 2E and F, and 3E and 3F). IRF3 expression did not show any changes with the disease progression either in PBMC or in Mon of COVID-19 patients (Fig. 6A and 7B, respectively). The mRNA expression of various components of the NF-κB complex, including NF-κB1, NF-κB2, RELB, and IκBα, was significantly induced in PBMC from patients with severe and critical COVID-19 (Fig. 6C). In contrast, these components were increased only in Monocytes from patients with severe COVID-19 (Fig. 7C). Similarly, mRNA levels of NF-κB-target genes, including IL1β and IL10 were significantly increased independently of severity of COVID-19; CXCL1 and cyclooxygenase 2 (COX2) mRNA expression was significantly higher in PBMC from patients with severe and critical COVID-19 (Fig. 6D). TNFα mRNA expression level was significantly increased only in critical COVID-19-derived PBMC (Fig. 6D). In COVID-19-derived Mon, TNFα, IL1β, IL6, IL10, CXCL8/IL8, and COX2 were also significantly increased depending on the severity (Fig. 7D). Collectively, the results shown here suggest an association of TLRs and pathogenesis of COVID-19.

3.5. SARS-CoV-2 Spike protein modulates expression of TLR1/2-MyD88, downstream signaling components and induces pro-inflammatory and antiviral response IL27-dependent in human macrophages.

Both TLR2 and MyD88 expression have been associated with severity of COVID-19 disease since they induced production of pro-inflammatory cytokines by sensing structural SARS-CoV-2-PAMPs [30]. Further, activation of TLR1/2-MyD88 signaling induces a robust NF-κB-dependent pro-inflammatory response and a low but significant IL27-dependent antiviral response in both human and murine macrophages [17]. To determine whether TLR1/2-MyD88 induction by SARS-CoV-2 Spike protein (S-protein) plays a role in the establishment of pro-inflammatory and antiviral response IL27-dependent, in macrophages, we reanalyze a publicly available RNA-seq GSE173488 (GEO) [67]. As shown in Fig. 8A, transcriptomic analysis shows that human MDMs stimulated with SARS-CoV-2 S-protein significantly up-regulate mRNA expression of TLR1 and TLR2, as well as TLR adapters and mediators, including MyD88, TRIF, IRAK2, and IRAK3, as found in COVID-derived PBMCs and Mon. These results are consistent with significantly high expression levels of NF-κB-complex, including NF-κB1, NF-κB2, RELA, RELB, and IκBα (Fig. 8B), as well as genes targeted by NF-κB (TNFα, IL1β, IL6, IL10, CXCL1, CXCL8/IL8, and COX2; Fig. 8C)..Additionally, we found that IRF1 and IRF7 mRNA expression was significantly increased in MDMs stimulated with SARS-CoV-2 S-protein; in contrast, expression of IRF3 was downregulated (Fig. 8D). Interestingly, while MDMs stimulated with SARS-CoV-2 S-protein do not express the IFN-I (IFNα1 and IFNβ1), IFN-II (IFNγ), and IFN-III (IFNλ1) mRNAs, significantly increased levels of IL27p28 and EBI3 mRNA expression was observed (Fig. 8E). Also, we found that the expression of IL27 signaling components (Gp130, JAK1, STAT1, STAT3, and SOCS3) was significantly elevated in MDMs stimulated with SARS-CoV-2 S-protein (Fig. 8F). As expected, we found significantly increased expression of AVP-encoding ISG mRNAs, including GBP1, IFIT2, IFITM1, ISG15, MX1, OAS2, and Viperin, as well as STAT1-dependent cytokines such as IL7, IL15, and TRAIL in MDMs stimulated with SARS-CoV-2 S protein (Fig. 8G and H). As expected, we found significantly increased expression of AVP-encoding ISG mRNAs, including GBP1, IFIT2, IFITM1, ISG15, MX1, OAS2, and Viperin, as well as STAT1-dependent cytokines such as IL7, IL15, and TRAIL in MDMs that were stimulated with SARS-CoV-2 S protein (Fig. 8I). Overall, the data suggest that stimulation of human MDMs with SARS-CoV-2 S-protein induces TLR1/2-MyD88 signaling, which in turn promotes mRNA expression of both IL27 subunits, IL27p28 and EBI3, leading to a strong IL27-dependent pro-inflammatory and antiviral response, in IFN-independent manner.

Recognition of viral components by innate immune receptors such as PRRs, activates both IFN-I and IFN-III responses [70]. Both TLRs and RIG-I/MDA5 are activated in response to the virus, and initiate signaling cascades to induce IFNα/β and IFNλ [71] [70], which induces ISGs, the main effectors of antiviral protection [72]. There is an emerging body of evidence showing that SARS-CoV-2 infection elicits weaker induction of IFN-I and robust inflammatory response in COVID-19 patients [46]. Loss of IFN-I related immunity could cause a severe symptom in patients with COVID-19 [53]. Other studies suggest that elevated IFNs levels correlate with and contribute to severe COVID-19 [73][74] [75]. It has been proposed that severe COVID-19 infection might drive an uncontrolled expression of the IFN-I response leading to pathology instead of viral containment [76]. Although SARS-CoV2 has evolved multiple mechanisms to block the induction and action of IFNs [77][78], and despite the fact that IFN-I levels in patients' sera are below detection levels, ISGs expression is detected [79][46]. Thus, the results suggest that either a limited amount of IFNs is sufficient to induce ISGs or, alternatively, the expression of ISGs is activated in an IFN-independent manner in COVID-19 patients. In current study, we performed a transcriptomic analysis of PBMCs and Monocytes from COVID-19 patients classified as moderate, severe, or critical. We did not observe changes in IFN-I (IFNα and IFNβ), IFN-II (IFNγ) and IFN-III (IFNλ1) mRNA levels in COVID-derived PBMCs and Monocytes, except for IFNλ1, which was significantly increased in PBMCs from patients with critical COVID-19. Importantly, we noted that the mRNA expression levels of IL27p28 and EBI3, both IL27 subunits, increased significantly with severity of COVID-19, as compared to the healthy group. Thus, the PBMCs from COVID-19 patients in critical conditions or Monocytes from COVID-19 patients in severe conditions showed highest expression levels of the two IL27 subunits. In agreement with our results, Zamani et al., (2022) reported that the levels of IL27 was significantly higher in COVID-19 patients than healthy subjects and disease severity was associated with IL27 level [80]. While IL27 was significantly reduced in severe COVID-19 patients who needed to undergo intensive care unit therapy, the IL27 level was significantly higher in severe COVID-19 survivors [80]. In addition, it was previously reported that IL27 levels in serum was higher in patients with chikungunya fever than in acute or subacute stage of the disease [81]. In influenza A virus infection, delivery of recombinant IL27 limited the recruitment of inflammatory cells, including monocytes and neutrophils into the lung, without affecting the T cell response, suggesting that IL27 can control the immune response to influenza [82]. By contrast, low levels of IL27 was reported in COVID-19 patients at admission [83]. When the authors compared mild with moderate or severe disease levels in patients, they did not observe statistically significant changes in the cytokines including IL27. The authors proposed IL27 as an early biomarker of COVID-19 severity, as was proposed by Cavalcanti et al., (2019) in patients with chikungunya fever [81]. In agreement with our transcriptomic analyses, other studies described high levels of IL27 in COVID-19 patients. Huang et al., (2022) reported significant elevation of IL27 in multisystem inflammatory syndrome in children, an acute, febrile and severe SARS-CoV-2-associated syndrome, as compared to controls [84]. Further, a study performed in COVID-19 patients also reported increase in IL27 production [85]. In vitro studies using human aortic endothelial cells treated with recombinant SARS-CoV-2 S-protein resulted in enhanced secretion of inflammatory cytokines, including IL27 [86]. Based on these observations, and our results showing significant increase depending on the severity of the disease, we propose that IL27 can be useful marker of COVID-19 disease progression.

Both PBMCs and Monocytes from COVID-19 patients classified according to their severity express high levels of the two subunits of IL27, and the mRNA expression of IL27 dependent on the severity of the disease. mRNA expression level of STAT1 and STAT3 is among the highest in COVID patients, regardless of disease severity. Results suggest that the IL27 signaling is activated in response to SARS-CoV-2 infection. The observation could be of importance in altering the inflammatory response, as it has been reported that IL27 acts in an pro- and anti-inflammatory manner, downregulating Th17 response via STAT1, and may signal T naïve cells in a STAT3-dependent manner [87]. Likewise, in COVID-derived PBMCs and COVID-derived Mon, the mRNA expression levels of STAT1-dependent cytokines, as well as CC- and CXC-chemokines were significantly increased as a function of severity of disease consistent with the development of inflammation observed in COVID-19 patients.

A comprehensive understanding of the host inflammatory and antiviral response during SARS-CoV-2 infection is needed that allows us to identify the association of COVID-19 pathogenesis versus protection from SARS-CoV-2 infection. Such an information is also critical to identify alternative pathways that could suppress SARS-CoV-2 infection; given the dysfunction of the IFNs signaling pathway described in COVID-19 patients. SARS-CoV-2 evades the IFN-mediated antiviral response with the help of several viral proteins [reviewed in: [88]]. In addition, nsp16 protein, in conjunction with nsp10, methylates viral mRNAs in the 5′-end to mimic host’s mRNA and inhibit the activation and function of RIG-I/MDA5 involved in the recognition of SARS-CoV-2 RNAs [89][90]. Further, viral protein such as nsp1, nsp3, nsp6, nsp13, orf3a, orf6, orf7a, orf7b, orf8, and M inhibit ISGs expression by blocking the activation and nuclear translocation of ISGF3 complex [reviewed in: [88]. We observed that although there is no modulation of mRNA expression of type I and II IFNs, there are significantly higher levels of ISGs expression, depending on severity of the disease in both COVID-derived PBMCs and Monocytes, and in MDMs treated with SARS-CoV-2 S protein. In agreement with our results, an IL27-dependent ISGs expression was reported in other studies [68] [65]. Thus, generation of innate AVPs, including OAS1, OAS2, OASL, and MX1, in response to IL27 protected against Zika virus infection in human keratinocytes [68]. This antiviral response occurs in STAT1- and IRF3-dependent but STAT2-, TYK2-, and IFNAR1-independent manner. Recently we described that the IL27 signaling is activated in CHIKV-infected MDMs and that kinetics of IL27p28/EBI3 mRNA expression and IL27 protein production correlated with the expression of AVPs in CHIKV-infected MDMs [65]. Furthermore, we showed that stimulation of THP-1-derived macrophages with recombinant-human IL27 induced the JAK-STAT signaling and robust pro-inflammatory and antiviral response in IFN-independent manner [65]. The current study has shown that expression of ISGs, including APOBEC3A, GBP1, IFIT2, IFITM1, ISG15, MX1, OAS2, and Viperin is driven by IL27 in a STAT1-dependent manner, which was induced through the activation of TLR1/2-MyD88 signaling, although it has previously been shown to be induced primarily by type I IFNs [91]. In agreement with our results, Patel et al., (2018) reported that IL27 is expressed by fibroblasts and induced IDO1, APOBEC3G, and MxA expression [92]. Consistent with these results, Pratumchai et al., (2022) describe that B cell-derived IL27 promotes control of persistent lymphocytic choriomeningitis virus (LCMV) infection, and B cell-derived IL27 drive antiviral immunity and antibody response by regulating virus-specific CD4 + T cells and Tfh cell functions during late stages of persistent LCMV infection [98]. Consequently, we propose that IL27 also constitutes an alternative line of defense against SARS-CoV-2, but also against other viruses. Our literature review on COVID-19/SARS-CoV-2 revealed a gap in knowledge regarding the potential involvement of IL27 in the induction of ISGs expression. Building upon this gap, we hypothesized that IL27 may play a role in controlling SARS-CoV-2 infection through ISGs expression. To the best of our knowledge, this hypothesis has not been previously studied or reported in the literature.

We observed increased mRNA expression levels of TLR1/TLR2 (MyD88-dependent TLRs), as well as the TLR1/2-MyD88 signaling components, NF-kB complex, and IRFs in COVID-derived PBMCs, COVID-derived Mon, and S-protein-stimulated MDMs. Based on this observation, we hypothesize that recognition of SARS-CoV-2-PAMPs by TLR1/2-MyD88 induces IL27 expression. This, in turn, activates a robust IL27-STAT1-dependent pro-inflammatory and antiviral response in COVID-19 patients, as a function of disease severity. This hypothesis is supported by reports showing that TLR2 recognize SARS-CoV-2 Envelope and Spike proteins to induce NF-κB-dependent pro-inflammatory response [29] [30] [31]. Further, we reported that following stimulation of TLR1/2-MyD88 with their respective agonists, human MDMs and murine BMDM induce IL27 production that orchestrate both pro-inflammatory and antiviral response [17]. Additionally, Wirtz et al. (2005) reported that stimulation of murine DCs via TLR2, TLR4, and TLR9 (MyD88-dependent TLRs) transactivated the EBI3 promoter via MyD88 and NF-κB [93]. Similarly, it has been reported that IL27p28 is highly expressed in murine DCs and peritoneal macrophages in response to TLR3 and TLR4 activation [94][95]. Further, Zhang et al., (2010) reported that IRF1 and IRF8 regulate IL27p28 gene transcription in murine macrophages in response to TLR4 activation, by specifically binding to the IRF1 response element in the IL27p28 promoter [96]. Collectively, the results suggest that regulation of IL27 expression in monocytes, macrophages and DC is dependent on TLR signaling and activation of NF-κB and IRF1 to induce transcriptional expression of EBI3 and IL27p28 genes. In current study, we report that TLR1/2-MyD88 signaling components, NF-κB complex, and IRF1 were significantly increased as a function of the severity of COVID-19, and results were consistent with expression levels of IL27 subunits in COVID-19 patients. Thus, IL27 could play a novel host-defensive role to subvert viral inhibition of IFNs and may take over infection control.

In current study, we reanalyzed the RNA-Seq data performed on MDMs stimulated with SARS-CoV-2 S-protein [67]. These authors have shown that SARS-CoV‐2 S‐protein primes inflammasome formation and the release of mature IL1β in macrophages derived from COVID‐19 patients, but not in macrophages from healthy SARS‐CoV‐2 naïve individuals. Interestingly, our transcriptomic analysis of these MDMs from healthy individuals, stimulated with SARS-CoV-2 S-protein also yielded similar results to those we observed in PBMCs and Mon from COVID-19 patients. In brief, we found that stimulation of MDMs with SARS-CoV-2 S-protein leads to the expression of TLR1/2-MyD88 signaling components, NF-κB-complex, NF-κB-target genes, IRFs, and both subunits of IL27 (IL27p28 and EBI3), as well as the induction of ISGs, including STAT1-dependent AVPs, cytokines and chemokines, but not the expression of any type of interferon. Additionally, Barreda et al., (2021) reported that DCs interaction with the SARS-CoV-2 S-protein promotes activation of key signaling molecules involved in inflammation, including MAPK, AKT, STAT1, and NF-κB, which correlates with the production of pro-inflammatory cytokines [97]. Furthermore, the immunopathology induced by SARS-CoV-2 S-protein in macrophages was triggered by STAT1 phosphorylation [98]. Collectively, our findings with PBMCs and Mon derived from COVID‐19 patients, and S-protein-stimulated MDMs suggests that SARS-CoV-2 infection causes profound changes in transcription program that drives an unrecognized mechanism which induces inflammatory response and acts in host antiviral response, that is IL27-dependent and IFN-independent. Although our study adds to the list of cytokines essential for the defense against the viruses produced by APCs, further study is needed to delineate the mechanisms behind why and how IL27 signaling protects the host from SARS-CoV-2 infection.

We report transcriptomic analysis of COVID-derived PBMCs, COVID-derived monocytes, and SARS-CoV-2 S-protein-stimulated MDMs. We observed significative increases in mRNA levels of TLR1 and TLR2 (MyD88-dependent TLRs), as well as the TLR1/2-MyD88 signaling components, NF-kB complex, NF-kB-target genes, IRFs, the two IL27 subunits (IL27p28 and EBI3), IL27 signaling components, and ISGs including STAT1-dependent AVPs, cytokines, CC- and CXC-chemokines, but not IFNs genes expression in COVID-derived PBMCs, COVID-derived Mon, and SARS-CoV-2 S-protein-stimulated MDMs, we suggest that recognition of structural SARS-CoV-2-PAMPs by TLR1/2-MyD88 (Fig. 9A) induces NF-kB-complex activation (Fig. 9B) and a robust pro-inflammatory response dependent of NF-kB-target genes expression (Fig. 9C), including EBI3. Further, induction of TLR1/2-MyD88 signaling by SARS-CoV-2 infection activate IRF1 to induces IL27p28 mRNA expression (Fig. 9D) and produces a functional IL27 which induces a robust IL27-STAT1-dependent pro-inflammatory and antiviral response associate with induction of antiviral state in COVID-19 patients (Fig. 9E), as a function of disease severity. Thus, IL27 may be able to trigger host antiviral response in an IFN-independent manner (Fig. 9F). Therefore, identification of innate antiviral immune factors that are IFN signaling independent may lead to the development of novel therapeutics against SARS-CoV-2 infection in humans.

Limitations of the study

A major limitation of this work was that the results obtained thanks to the transcriptomic analysis of 2 mRNA datasets obtained from PBMCs and monocytes from COVID-19 patients classified according to the severity grade were not confirmed experimentally, although the functionality of these findings were indirectly obtained thanks to the results obtained with macrophages stimulated with SARS-CoV-2 S-protein. Nevertheless, our experimental approach allowed us to dissect an alternate mechanism to might counteract the ability acquired by the SARS-CoV-2 to interfere with the IFN signaling pathway and induction of ISGs expression that should be subsequently validated in an in vivo animal model.

Authorship contribution statement

JFVL: Conceptualization, Formal analysis, Investigation, Methodology, Writing-original draft, Writing-review and Analyzed data. SUI: Conceptualization, Methodology, Resources, Writing-original draft, Writing-review and editing, Supervision and Analyzed data.

Authorship contribution statement

Conflict of interest. The authors declare that they have no competing interests.

Acknowledgments

The authors thank Ajit Kumar for reading the manuscript and his valuable comments. This research was supported by Minciencias/Colciencias [grant No. 111584467188], and Universidad de Antioquia-CODI, acta 2020-34065. The funders played no role in the study design, data collection, and analysis, decision to publish, or preparation of the manuscript.

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Antiviral Response and Immunopathogenesis of Interleukin 27 in COVID-19

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Abstract

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1. Introduction

2. Materials And Methods

2.1. Bioinformatic analysis of previously published mRNA datasets and prediction of cell signaling pathways activation.

3. Results

3.3. Interleukin 27 signaling components are expressed in COVID-derived PBMCs and Monocytes

4. Discussion

Conclusions

Declarations

References

Status:

Journal Publication

Version 1