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Elevated Expression Levels of Lung ComplementAnaphylatoxin, Neutrophil ChemoattractantChemokine IL-8, and RANTES in MERS-CoV-InfectedPatients: Predictive Biomarkers for Disease Severityand MortalityMaaweya Awadalla 

King Saud University https://orcid.org/0000-0002-1270-2216Asif Naeem 

King Fahad Medical CityHaitham Alkadi 

King Fahad Medical CityAref A. Alamri 

Ministry of HealthAhmad S. AlYami 

King Fahad Medical CityAbdullah AlJuryyan 

King Fahad Medical CityWael Alturaiki 

Majmaah UniversityMushira Enani 

King Fahad Medical CitySamia Towfeek Al-Shouli 

King Saud UniversityAbdullah M. Assiri 

Ministry of HealthBandar Alosaimi  ( balosaimi@kfmc.med.sa )

King Fahad Medical City

Research Article

Keywords: MERS-CoV infection, complement system, immunopathology, lung, cytokine, chemokine

Posted Date: March 15th, 2021

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DOI: https://doi.org/10.21203/rs.3.rs-285036/v1

License: This work is licensed under a Creative Commons Attribution 4.0 International License.  Read Full License

Version of Record: A version of this preprint was published at Journal of Clinical Immunology on July 7th,2021. See the published version at https://doi.org/10.1007/s10875-021-01061-z.

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AbstractThe complement system represents an innate immune response consisting of a protein network. Over-activation of the complement system plays an important role in in�ammation, tissue damage, andinfectious disease severity. The prevalence of MERS-CoV in Saudi Arabia remains signi�cant and casesare still being reported. The role of complement in Middle East Respiratory Syndrome coronavirus (MERS-CoV) pathogenesis and complement‐modulating treatment strategies has received limited attention, andstudies involving MERS-CoV-infected patients have not been reported. This study offers the �rst insightinto the pulmonary expression pro�le including Seven complement proteins including complementregulatory factors during MERS-CoV infection. We also measured the expression of lung neutrophilchemoattractant chemokine IL-8 (CXCL8) and RANTES (CCL5). Our results signi�cantly indicate highexpression levels of complement anaphylatoxins (C3a and C5a), IL-8, and RANTES in the lungs of MERS-CoV-infected patients. The upregulation of lung complement anaphylatoxins, C5a and C3a, waspositively correlated with IL-8, RANTES and the fatality rate. Our results also showed upregulation of thepositive regulatory complement factor P (properdin), suggesting positive regulation of the complementduring MERS-CoV infection. In addition, we also demonstrated that a high viral load in all patients withMERS-CoV correlated with C5a and C3a levels. Pulmonary complement mediators, disease severity, andan increased fatality rate may be linked to the degree of complement activation against MERS-CoV. Highlevels of lung C5a, C3a, factor P, IL-8 and RANTES may contribute to the immunopathology, diseaseseverity, ARDS development, and a higher fatality rate in MERS-CoV-infected patients. These �ndingshighlight the potential prognostic utility of C5a, C3a, IL-8 and RANTES as biomarkers for MERS-CoVdisease severity and mortality. To further explore the functional partners (protiens) prediction of highlyexpressed proteins (C5a, C3a, factor P, IL-8 and RANTES), the computational protein–protein interaction(PPI) network was constructed, and six proteins (hub nodes) were identi�ed.  

IntroductionThe COVID-19 pandemic caused by severe respiratory syndrome coronavirus 2 (SARS-CoV-2) has gainedsigni�cant attention in the medical and scienti�c communities. The Middle East Respiratory Syndromecoronavirus (MERS-CoV) has caused considerable medical and health issues in many countries,particularly in Saudi Arabia. The prevalence of MERS-CoV in the Kingdom of Saudi Arabia (KSA) remainssigni�cant. MERS-CoV cases are still being reported in Saudi Arabia, and a high prevalence of MERS-CoVin dromedary camels and direct contact with infected camels have been linked to human infections(Alosaimi et al., 2020; Assiri et al., 2016; Azhar et al., 2014; Rabaan et al., 2021; WHO, 2021). MERS-CoV isa single-standard RNA virus of the Betacoronavirus genus. It was �rst reported in the KSA (Jeddah City) in2012. As of December 27, 2020, 2564 laboratory-confirmed cases and 881 associated deaths (case-fatality ratio, 34.4%) were reported in 27 countries worldwide, of which 2121 cases (82.7%) were reportedin Saudi Arabia. The majority of the fatalities (37.1%, 788 deaths) also occurred in Saudi Arabia (WHO,2021). An excessive in�ammatory response is a prominent phenotype associated with MERS-CoVinfection, which leads to lung immunopathology, disease progression, and poor clinical outcome. MERS-

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CoV infections are characterized by dysregulation in both the innate and adaptive immune systems [5],[6]. Several in�ammatory cytokines and chemokines (IL-1β, IL-2, IL-6, IL-7, IL-8, IL-10, G-CSF, GM-CSF, IP-10, MCP-1, MIP-1α, IFN-γ, TNF-α, CCL2, and CCL3) are signi�cantly associated with severe MERS-CoVinfection and higher fatality rates [5], [7], [8]. Elevated in�ammatory cytokine and chemokine levels duringSARS‐CoV-1, MERS‐CoV, and SARS‐CoV‐2 infections are signi�cantly associated with massive in�ltrationof immune cells into the lungs and poor disease outcome [5], [8]–[10]. The complement system consistsof a multiprotein network belonging to both the innate and adoptive immune systems [11]. Depending onthe manner of activation, the complement cascade operates by three pathways: the classical pathway,the lectin pathway, and the alternative pathway [12], [13]. Cross talk between the complement andcoagulation systems plays a crucial role in vascular endothelial damage and thromboin�ammation [14].A number of viral infections are associated with complement activation and coagulation dysfunction [11],[15], [16]. Over-activation of pulmonary and systemic complement plays a key role in in�ammation,endothelial cell damage, thrombus formation, and intravascular coagulation, which results in multipleorgan failure and eventually death [12], [15], [17]. This over-stimulation leads to the formation of thecomplement anaphylatoxins, C3a and C5a. C5a is a chemoattractant for neutrophils, monocytes,eosinophils, and T cells [15], [18]. Following infection, complement anaphylatoxins stimulate phagocyticcells and enhance the production of TNF-α, IL‐1β, IL‐6, IL‐8, granular enzymes, and free radicals. Thesemediators promote vascular dysfunction, �brinolysis, and microvascular thrombosis formation [11], [12],[14], [15], [17], [18]. C5a also plays an important role in stimulating the expression of P‐selectin,intercellular adhesion molecule‐1, �bronectin, and �brinogen. The upregulation of adhesion moleculesactivates different cell signaling and pro‐in�ammatory pathways [15], [16].

The role of complement in MERS-CoV disease immunopathology and complement‐modulating treatmentstrategies during MERS-CoV infection has received limited attention. There are many importantunanswered questions regarding complement, MERS-CoV interactions, and disease outcome. In addition,little is known regarding pulmonary complement activation during MERS-CoV infection, the manner inwhich complement activation affects disease severity, or the association of complement response withviral load and mortality.

In this study, we performed a comprehensive investigation of the pulmonary complement proteins, IL-8(CXCL8) and CCL5 (RANTES) expression in MERS-CoV-infected patients in addition to viral loaddetermination. We also assessed the correlation between these factors and the fatality rate. To ourknowledge, this is the �rst study demonstrating a relationship between lung complement proteins andcomplement regulatory factors in MERS-CoV-infected patients.

Materials And MethodsPatient Selection, Sample Collection, and Preparation and Analysis

A total of 31 MERS-CoV-positive patients and 15 healthy matched controls were enrolled in this study.Lower respiratory samples (bronchoalveolar lavage (BAL) or tracheal aspirate (TA)) were collected. To

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exclude the effects of antiviral therapy on the expression of complement proteins, IL-8 (CXCL8) and CCL5(RANTES), all samples were collected before the administration of any antiviral treatment. Samples werecentrifuged at 1000 rpm at 4°C for 5–10 min. The cell-free supernatants were used for the analysis ofcomplement proteins, in�ammatory chemokines, RANTES (CCL5), and MERS-CoV viral load. Theexclusion criteria were as follows: 1) patients coinfected with other respiratory pathogens, 2)immunocompromised patients, 3) patients under treatment with anti-in�ammatory and/orimmunosuppressive drugs, and 4) patients with preexisting autoimmune disease. These exclusion criteriawere selected to exclude any possible effects on the expression of the clinicopathological factors listedabove. This study was reviewed and approved by the Institutional Review Board at King Fahad MedicalCity (IRB register number 019-053).

Measurement of Complement In�ammatory MediatorsAnaphylatoxins (C3a/C3b and C5a) and Classical PathwayComplement Component (C1q) and C2 LevelsPulmonary levels of human complement C3 and C5 fragments, as well as C3b, C1q, and C2, weremeasured using ELISA kits (HCA39-K01-Eagle Biosciences, Inc., Columbia, USA; ab193695, Abcam,Cambridge, UK; ab195461, Abcam, Cambridge, UK; ab170246, Abcam, Cambridge, UK; ab154132, Abcam,Cambridge, UK) following the manufacturer’s instructions. The concentrations were calculated usingstandard curves.

Quantitation of Serum Regulatory Complement Component (Factor) Levels

The concentrations of four human complement regulatory factors, factors P (properdin), I, C4-bindingprotein (C4-BP), and H, were quanti�ed in the lung using ELISA kits (ab222864, Abcam, Cambridge, UK;ab195460, Abcam, Cambridge, UK; ab222866, Abcam, Cambridge, UK; HK342 Hycult Biotech, Uden,Netherlands). The ELISAs were done following the manufacturer’s instructions. The concentrations ofeach factor were calculated using standard curves.

Quanti�cation of Serum Chemokine RANTES (CCL5) LevelsWe determined the local RANTES chemokine levels in MERS-CoV-infected patients (n = 30) and healthyindividuals (n = 18). Lung RANTES levels were quanti�ed using the human RANTES ELISA Kit (R&DSystems, Minneapolis, USA) following the manufacturer’s protocol. RANTES concentrations werecalculated using standard curves, and the results were expressed as pg/ml.

Measurement of Pulmonary Pro-In�ammatory Cytokine andChemokine Pro�les Using ELISArray

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The concentrations of major human pro-in�ammatory cytokines and chemokines were measured in therespiratory samples of 30 MERS-CoV-infected patients and 18 healthy individuals using the multi-analyteELISArray (Qiagen, Germantown, MD, USA) following the manufacturer’s protocol. The absorbance of theELISArray was measured at 450 nm. The concentrations were calculated using a standard curve.Cytokine/chemokine levels were expressed as pg/ml.

Protein-protein interaction (PPI) Network Construction andIdenti�cation of Hub ProteinsTo further explore the potential interplay among the differentially expressed proteins (C3a, C5a, factor P(properdin) IL-8 and RANTES) in the lung of MERS-CoV infected patients with the potential interactors.Protein-protein interaction networks was constructed using bioinformatics resources of the Search Toolfor the Retrieval of Interacting Genes/Proteins database (STRING version 11.0). This bioinformaticsresources provides known and predicted protein-protein interactions networks. We used multiple proteins(C3a, C5a, factor P (properdin) IL-8 and RANTES) as an input (seed proteins). Active interaction sources,includes; databases, co-occurrence of genes, homology of proteins, experiments (biochemical/geneticdata), co-expression of gene and text mining as well as species limited to “Homo sapiens”, con�dencescore > 0.4 and maximum interactors (= 20) were used to construct the STRING networks. The STRINGresource is available online at https://string-db.org/. The constructed STRING PPI network was exportedto Cytoscape software (version 3.8.2 http://apps.cytoscape.org/apps/mcode) to visualize and additionalanalysis of functional protein-protein interaction.

Statistical AnalysisStatistical analyses were performed using the GraphPad 5.0 software (GraphPad Software, San Diego,CA, USA). Data were assessed using a t-test. The correlations between complement proteins,in�ammatory factors, and chemokines were assessed using the Pearson’s correlation test. The resultswere presented as means ± standard deviation (SD), unless otherwise speci�ed. A p-value of <0.05 wasconsidered statistically signi�cant.

Results3.1 Basic Patient Characteristics

A total of 31 MERS-CoV-infected patients (22 male and 9 female) and 15 healthy controls (9 male and 6female) were included in this study (Figure 1). The ages ranged from 31 to 100 years. The overall meanand median age were 68.26 ± 16.04 and 73 years, respectively. The mean ages in the non-survival andsurvival patients were 74.5 ± 11.3 and 53± 15.8 years, respectively. Twenty-two (70.97%) patients died, 9recovered (29.03%), and 25 (80.6 %) required intensive care unit (ICU) admission (Fig 1).

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3.2 Activation of Complement Is Associated with MERS-CoV Infection

3.2.1 Complement Anaphylatoxin Expression Levels Are Signi�cantlyIncreased in MERS-CoV-Infected Patients Compared with HealthyControlsOver-activation of the complement system is associated with immunopathology and tissue damage.Therefore, we hypothesized that the elevated lung complement protein levels occurring during MERS-CoVinfection are involved in the massive in�ltration of immune cells into the lungs and result in poor diseaseoutcome. To assess whether lung complement anaphylatoxins (C3a and C5a) were increased in MERS-CoV-infected patients, we examined complement components in lower respiratory lung samples usingELISA. As shown in (Fig 2), the levels of complement anaphylatoxins (C3a and C5a) and C1q weresigni�cantly higher in the lungs of MERS-CoV-infected patients compared with healthy controls (Fig 2A,2B, and 2G). Together, these results show that the high levels of complement anaphylatoxins in the lungsof MERS-CoV-infected patients are signi�cantly increased, suggesting a signi�cant pulmonarycomplement activation. In addition, the elevated levels of lung complement anaphylatoxins suggest arole for these factors in lung tissue damage, immunopathology, ARDS development, and mortality ofMERS-CoV-infected patients.

3.2.2 MERS-CoV Infection Is Associated with Positive Regulation of the Complement Response

Elevated levels of complement anaphylatoxins prompted us to evaluate the changes in otherimmunoregulatory proteins. Complement regulatory proteins function by inhibiting complement over-activation to avoid in�ammation and tissue damage. We quanti�ed factor P (properdin) levels in the lungof MERS-CoV-infected patients. As shown in (Fig 2C), the levels of positive regulatory factor P (properdin)were signi�cantly higher in the MERS-CoV-infected patients compared with the healthy control group,suggesting that the levels of factor P may enhance complement activation during MERS-CoV infection.Factor P measurement in serum may provide evidence for the involvement of the alternative complementpathway since factor P (properdin) represents an important factor in the activation of the alternativepathway.

3.2.3 MERS-CoV Infection Is Associated with Distinct Levels of Negative Regulatory Complement Proteins

The measurement of negative regulatory proteins in the lung of MERS-CoV-infected patients may provideevidence for complement system regulation. The quantitation of several complement negative regulatoryfactors (factor I, C4-BP, and factor H) revealed that the levels of factors I and H were increased in thelower lung respiratory samples (Fig 2D and 2E). These negative regulatory proteins play an important rolein complement system regulation. In contrast, the levels of C4-BP were unchanged, and there was nostatistical signi�cance between the levels in the MERS-CoV-infected patients compared with healthycontrols (Fig 2F). This result indicates that MERS-CoV may suppress and inhibit the function of C4-B.

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3.3 Pulmonary Neutrophil Chemoattractant Chemokine IL-8 (CXCL8) and RANTES (CCL5) Levels AreElevated in MERS-CoV-Infected Patients

To examine the levels of chemokines in the lung during MERS-CoV infection, we quanti�ed IL-8 andRANTES in the lower respiratory tract of MERS-CoV-infected patients and the healthy control group. Asshown in Figure 2, IL-8 and RANTES levels were signi�cantly higher in the lungs of MERS-CoV-infectedpatients compared with that in the healthy control group (Fig 2H and 2I).

3.2 Activation of Complement Is Associated with MERS-CoV Infection

3.2.1 Complement Anaphylatoxin Expression Levels Are Signi�cantlyIncreased in MERS-CoV-Infected Patients Compared with HealthyControlsOver-activation of the complement system is associated with immunopathology and tissue damage.Therefore, we hypothesized that the elevated lung complement protein levels occurring during MERS-CoVinfection are involved in the massive in�ltration of immune cells into the lungs and result in poor diseaseoutcome. To assess whether lung complement anaphylatoxins (C3a and C5a) were increased in MERS-CoV-infected patients, we examined complement components in lower respiratory lung samples usingELISA. As shown in (Fig 2), the levels of complement anaphylatoxins (C3a and C5a) and C1q weresigni�cantly higher in the lungs of MERS-CoV-infected patients compared with healthy controls (Fig 2A,2B, and 2G). Together, these results show that the high levels of complement anaphylatoxins in the lungsof MERS-CoV-infected patients are signi�cantly increased, suggesting a signi�cant pulmonarycomplement activation. In addition, the elevated levels of lung complement anaphylatoxins suggest arole for these factors in lung tissue damage, immunopathology, ARDS development, and mortality ofMERS-CoV-infected patients.

3.2.2 MERS-CoV Infection Is Associated with Positive Regulation of the Complement Response

Elevated levels of complement anaphylatoxins prompted us to evaluate the changes in otherimmunoregulatory proteins. Complement regulatory proteins function by inhibiting complement over-activation to avoid in�ammation and tissue damage. We quanti�ed factor P (properdin) levels in the lungof MERS-CoV-infected patients. As shown in (Fig 2C), the levels of positive regulatory factor P (properdin)were signi�cantly higher in the MERS-CoV-infected patients compared with the healthy control group,suggesting that the levels of factor P may enhance complement activation during MERS-CoV infection.Factor P measurement in serum may provide evidence for the involvement of the alternative complementpathway since factor P (properdin) represents an important factor in the activation of the alternativepathway.

3.2.3 MERS-CoV Infection Is Associated with Distinct Levels of Negative Regulatory Complement Proteins

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The measurement of negative regulatory proteins in the lung of MERS-CoV-infected patients may provideevidence for complement system regulation. The quantitation of several complement negative regulatoryfactors (factor I, C4-BP, and factor H) revealed that the levels of factors I and H were increased in thelower lung respiratory samples (Fig 2D and 2E). These negative regulatory proteins play an important rolein complement system regulation. In contrast, the levels of C4-BP were unchanged, and there was nostatistical signi�cance between the levels in the MERS-CoV-infected patients compared with healthycontrols (Fig 2F). This result indicates that MERS-CoV may suppress and inhibit the function of C4-B.

3.3 Pulmonary Neutrophil Chemoattractant Chemokine IL-8 (CXCL8) and RANTES (CCL5) Levels AreElevated in MERS-CoV-Infected Patients

To examine the levels of chemokines in the lung during MERS-CoV infection, we quanti�ed IL-8 andRANTES in the lower respiratory tract of MERS-CoV-infected patients and the healthy control group. Asshown in Figure 2, IL-8 and RANTES levels were signi�cantly higher in the lungs of MERS-CoV-infectedpatients compared with that in the healthy control group (Fig 2H and 2I).

3.5 Lung Complement Anaphylatoxins (C3a and C5a), IL-8, and RANTES Levels Are Associated withARDS Development and the Fatality Rate

The Pearson correlation analysis revealed a signi�cant association of C3a, C5a, IL-8, and RANTES withARDS development and a higher fatality rate (Table 1). Higher levels of these mediators were signi�cantlypositively correlated with ARDS and a higher fatality rate. These immune mediators increase the risk ofdeveloping ARDS and mortality in MERS-CoV-infected patients. Elevation of lung C3a, C5a, IL-8, andRANTES may represent biomarkers for ARDS development and a predictor for inhospital mortality.

Table 1. Correlation between lung complement C3a, C5a, IL-8, and RANTES with ARDS development andin hospital mortality

Variable Pearson (r) P-value

C3a Death (r = 0.5249)

ARDS (r = 0.5249)

0.0024

0.0024

C5a Death (r = 0.6845)

ARDS (r = 0.6845)

<0.0001

<0.0001

RANTES Death (r = 0.7392)

ARDS (r = 0.7392)

<0.0001

<0.0001

IL-8 Death (r = 0.7836)

ARDS (r = 0.7836)

<0.0001

<0.0001

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3.6 Lung Complement Anaphylatoxins (C3a and C5a) and Their Correlation with Factor P, IL-8, andRANTES

We further examined whether there were correlations between lung complement anaphylatoxins (C3a andC5a), factor P, IL-8, and RANTES. The results indicated that lung complement C5a was positivelycorrelated with factor P, IL-8, and RANTES, whereas complement C3a was positively correlated with IL-8and RANTES (Table 2). A positive correlation between RANTES, IL-8, and complement factor P was alsoobserved (Table 2). Similarly, IL-8 was positively correlated with complement factor P (Table 2).

Table 2. Lung complement anaphylatoxins (C3a and C5a) and theircorrelation with factor P, IL-8, and RANTES

Variable Pearson (r) P-value

C3a RANTES (r = 0.3806)

IL-8 (r = 0. 0.4710)

0.0347

0.0075

C5a RANTES (r = 0.5486)

IL-8 (r = 0.3762)

Factor P (r = − 0.3949)

0.0014

0.0370

0.0279

RANTES Factor P (r = 0.5563)

IL-8 (r = 0.5141)

0.0012

0.0031

IL-8 Factor P (r = 0.5939) 0.0004

3.7 Protein-protein interaction (PPI) Network Analysis and Hub Proteins

To understand the protein-protein interaction between the complement proteins andcytokines/chemokines and predicted interactors, we performed data mining and curation to constructand map the network of protein-protein interactions using the STRING databases. C3a, C5a, factor P(properdin) IL-8 and RANTES were used as seeds for network analysis. The PPI network showed 20proteins that have the highest interaction scores with C3a, C5a, factor P (properdin), IL-8 and RANTES(Fig 4).For C3a, C5a, IL-8 and RANTES, individual protein-protein interaction was also created (Fig 5,6 and7). The name of proteins, predicted functional partners and actions are shown in (Table S1). This networkcomprises several types of interactions including; gene neighborhood, gene fusions, co-occurrence, co-expression, text-mining, protein homology and experiments (biochemical/genetic data). The physical(direct) or functional (indirect) interactions also was shown. The constructed network contained 25nodes, 149 edges, average node degree 11.9 and the PPI enrichment p-value was < 1.0e-16. Cytoscapeanalysis were shown in the (Table S2). CytoHubba analysis identi�ed top 6 proteins (hub nodes) basedon Betweenness, Closeness, Degree, EcCentricity, EPC, MCC and MNC calculation methods (Table S3 and

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S4). These hub nodes are involved in in�ammatory response and molecular binding interaction as well asligand-receptor interaction. These results indicate that predicted proteins (interactors) and hub proteinsmay also have a role in MERS-CoV disease severity.

DiscussionAn excessive in�ammatory response is a major characteristic of MERS-CoV infections and results indisease progression and poor clinical outcomes. MERS-CoV infections are characterized by dysregulationof the innate and adaptive immunity systems. Several in�ammatory cytokines/chemokines and over-activation of complement proteins are signi�cantly associated with severe MERS-CoV infections and ahigher fatality rate [5], [6], [8]. In this study, we sought to determine the levels of pulmonary complementproteins, neutrophil chemoattractant chemokine IL-8 (CXCL8), and RANTES (CCL5), as well as viral load,in MERS-CoV-infected patients and assessed their association with mortality. This study is the �rst toevaluate the pulmonary complement protein expression pro�le during MERS-CoV infection. Variousinnate immune markers in respiratory samples collected from MERS-CoV-infected patients and healthysubjects were evaluated. The results showed high levels of complement anaphylatoxins (C3a and C5a),positive complement regulatory proteins, neutrophil chemoattractant chemokine IL-8 (CXCL8), RANTES(CCL5), and viral load (data not shown) in MERS-CoV-infected patients. A number of studies have foundthat high viral load, duration of viral shedding, and direct cytopathic effects were associated with severecomplications during SARS-CoV-1, SARS-CoV-2, and MERS-CoV infections [19]–[22]. MERS-CoV infectionis characterized by persistent viral load, and in severe cases of MERS-CoV infection, viral shedding isdetected beyond 21 days [23]. Our results showed that all MERS-CoV-infected patients exhibited high viralloads (Ct values). Further studies revealed that MERS-CoV-infected patients requiring ICU admission hadhigh MERS-CoV RNA levels [24]. In this study, 70% of the MERS-CoV-infected patients developedpneumonia and required ICU admission.

Over-activation of the immune response, including the complement system, is believed to be an importantfactor for the high fatality rate of in�uenza pandemic (1918); H1N1, H5N1, and H7N9; SARS-CoV-1,MERS-CoV; Ebola infection; and, most recently, COVID-19 (SARS-CoV-2) infection [5], [10], [25]–[32].Complement activation leads to the production of several effector pro-in�ammatory molecules, includinganaphylatoxins C3a and C5a. Over-stimulation of the complement system or inadequate inhibitioncauses tissue damage [33] and formation of high levels of anaphylatoxins C3a and C5a. C5a is achemoattractant for neutrophils, monocytes, eosinophils, and other in�ammatory cells. C5a and C3a alsoplay important roles in in�ammatory cascades [34], [35]. Following infection, complement anaphylatoxinsstimulate phagocytic cells and the production of high levels of in�ammatory cytokines (cytokine storm),granular enzymes, and free radicals. These mediators may eventually contribute to vascular dysfunction,�brinolysis, microvascular thrombosis formation, or tissue damage [11], [12], [14], [15], [17], [18]. C5a is akey complement protein in the activation and bridging of the innate and adaptive immune responses [35].In this study, the expression levels of the pulmonary neutrophil chemoattractant chemokine, IL-8 (CXCL8),C5a, and C3a were increased in MERS-CoV-infected patients and associated with a higher fatality rate. Anumber of viral infections are associated with complement activation and coagulation dysfunction [11],

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[15], [16]. Our results are consistent with previous findings [36], [37] showing excessive complementactivation, particularly anaphylatoxins C3a, C5a, and C5b-9, during MERS-CoV and SARS-CoV-1 infection,which, in turn, contribute to lung tissue damage, a hyper-in�ammatory response, and severecomplications from infection. Furthermore, several studies have revealed that C5a was associated withacute lung diseases, severe pneumonia, and immunopathology during highly pathogenic viral infectionwith H1N1, H5N1, H7N9, and SARS-CoV-1 [31], [32], [36], [38]. Similar to SARS-CoV-1 and MERS-CoV,patients with COVID-19 (SARS-CoV-2) are characterized by complement activation with high levels ofcomplement protein [11], [26], [39], [40]. Moreover, one study found that high levels of C3a and C5a inlower respiratory tract samples were associated with severe complications from H5N1 infection [41]. Thisresult con�rms that the pulmonary complement is activated and anaphylatoxins C3a and C5a arereleased upon infection with in�uenza. Many studies have reported high levels of pulmonary andsystematic C5a in highly pathogenic in�uenza-infected patients [16], [31]. Likewise, respiratory samplesfrom mice infected with the highly pathogenic avian in�uenza, H5N1, exhibited signi�cant increases ofC5a [42]. A rapid elevation of C3a variants (C3a, C3b, iC3b, C3c, C3d) was observed in mice infected withSARS-CoV-1, which contributed to the systemic pro-in�ammatory response and lung injury [36]. C3activation has also been shown to be elevated in cells infected with SARS-CoV-2 [43]. In addition, in vitroand in vivo experiments on human respiratory syncytial viral infections showed complement-mediatedlung damage induced by high levels of C3a [44]. These �ndings clearly demonstrate that C5a and C3arecruit and activate in�ammatory immune cells and play a central role in lung injury andimmunopathology during respiratory viral infection. In contrast, complement C5a and C3a are involved inARDS pathogenesis, neutrophil recruitment and activation, and lung endothelial and epithelial injury [35],[45]. Previous studies have demonstrated that highly pathogenic viral infections are characterized by arapid progression to ARDS [46]–[51]. In addition, MERS-CoV infection is associated with a more severepneumonia compared with SARS-CoV-1 infection [47]. ARDS was shown to be the main cause ofmortality among patients infected with MERS-CoV, SARS-CoV-1, and highly pathogenic in�uenza virus[46], [52], [53]. In this study, we observed that almost all of the MERS-CoV-infected patients rapidlydeveloped ARDS and required ICU admission. Therefore, we hypothesize that complement-induced ARDSmay contribute to the disease severity and high mortality rate of MERS-CoV-infected patients observed inthis study.

We observed that the expression levels of pulmonary neutrophil chemoattractant chemokine IL-8 (CXCL8)and C5a were signi�cantly higher in MERS-CoV-infected patients compared with the healthy controlgroup. C5a contributes to the formation of neutrophil extracellular traps (NETosis) and in�ammatorycytokine induction. IL-8 and C5a are important chemoattractants for neutrophil recruitment, activation,and neutrophil accumulation, and both induce NETosis. Excess NETosis contributes to the in�ammation,pathological cellular damage, and acute lung injury in mice infected by in�uenza virus [54]–[56].Similarly, high numbers of neutrophils and NETs were induced by C5a and contributed to theimmunopathology, alveolar damage, and acute lung injury during in�uenza A H1N1 infection [54]. Anumber of in vitro and in vivo studies have found that C5a was associated with macrophage andendothelial cell activation, endothelial damage, NETosis, and increases in alveolar-capillary barrier

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permeability, as well as vascular leakage [34], [57]. Thus, we hypothesize that high C5a, C3a, and IL-8levels may play a vital role in lung tissue damage, immunopathology, ARDS development, ICU admission,and mortality in MERS-CoV-infected patients.

During SARS-CoV-1 infection, C5a induced several pro-in�ammatory cytokines and chemokines includingIL-8 [58]. C5a can also establish a positive feedback loop consisting of IL-8 induction, which results infurther IL-8 production. Thus, a loop of continued IL-8 production may result in further in�ammatory cellrecruitment to the lung and subsequently contribute to lung damage and pathological changes [59]. Wefound that high levels of the lung complement anaphylatoxins, C5a and C3a, were closely associatedwith the overexpression of pulmonary IL-8 in MERS-CoV-infected patients. There was a signi�cantcorrelation between C5a, C3a, and IL-8. The overproduction of C5a and IL-8 may be responsible for moredamage to the host tissue compared with MERS-CoV. Previous studies have also shown that the levels ofIL‐8 signi�cantly correlate with neutrophil numbers and airway in�ammation, as well as lung dysfunction,in patients with chronic obstructive pulmonary disease and asthma [18], [60]. Therefore, we conclude thatthe high levels of C5a might be a direct mediator of neutrophil chemoattraction or an indirect inducer ofIL-8 production during MERS-CoV infection.

C5a is also known to activate in�ammatory cells to release reactive oxygen species (ROS). ROSoverproduction leads to oxidative stress that subsequently contributes to airway and lung damage [61]–[63]. A previous in vivo study demonstrated that ROS are strongly associated with lung damage andpneumonia in mice infected with in�uenza virus [64]. Mice treated with an antioxidant resulted insigni�cantly reduced mortality, lung damage, and pathogenesis following in�uenza virus infection [65].Several studies have observed that anti-C5a and a C5aR receptor antagonist signi�cantly blocked andinhibited neutrophil oxidative burst formation [18], [66], [67]. These studies suggest a critical role of theC5a/C5aR/ROS axis in lung pathology and virus-induced immunopathology. We hypothesize that highlevels of C5a induce ROS, which results in a more severe MERS-CoV infection, ARDS, andimmunopathology.

Several studies have shown that targeted therapy with C5a and C5aR represents a signi�cant anti-in�ammatory approach to control in�ammatory cell recruitment, immunopathology, and acute lungdamage induced by highly pathogenic viral infections [31], [68], [69]. One study showed that the blockadeof the C5a–C5aR axis in mice infected with MERS-CoV markedly reduced pathological changes in lungand spleen tissue, viral load, lymphopenia, and systemic and pulmonary in�ammatory responses [37].Another study showed that complement-de�cient mice infected with SARS-CoV-1 were associated withdecreased lung neutrophilia, a pro-in�ammatory response, and viral replication [36]. Likewise, micede�cient in C3aR infected with HRSV exhibited reduced airway hyper-responsiveness; mucus secretion;and reduced lymphocyte, neutrophil, macrophage, and eosinophil in�ltration [44]. A recent study indicatedthat high levels of C3 mediated periodontal in�ammation and an increased risk for periodontitis inpatients with diabetes [70]. The same study found that the C3 knockout mouse model inhibitedin�ammatory cytokines in diabetic mice and reduced the risk of periodontal disease.

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Blocking and targeting C5a/C5aR and C3a/C3aR during respiratory viral infections signi�cantly reducedand controlled pathology, lung damage, and mortality and inhibited in�ammatory cell in�ltration in thelung, T-lymphocyte apoptosis, and NETosis [31], [71]. In addition, in vivo experiments con�rmed thatantihuman C5a antibody treatment in H5N1- and H7N9-infected African green monkeys resulted inreduced in�ltration of in�ammatory cells in the lung and decreased levels of pro-in�ammatory cytokinesand chemokines [32], [71]. Furthermore, studies have shown similar associations between the inhibitionof the complement system during highly pathogenic viral infection and decreased acute lung injury and arobust immune response [12], [36], [71], [72].

A recent study demonstrated that severe COVID-19 cases are associated with high levels of plasma C5aand soluble membrane attack complex (sC5b-9), signifying the C5a blockade as a potential interventionstrategy [73], [74]. Several randomized controlled trials showed that anti-C5 therapy increased survival insevere COVID-19 cases and signi�cantly reduced in�ammatory marker production [73], [75]–[77]. Thesestudies con�rmed that the inhibition of an over-activated complement response and its signalingpathways signi�cantly affected immunopathology, respiratory disease severity, and viral replication.Accordingly, it is reasonable to speculate that targeting the C5a/C5aR and C3a/C3aR axes may beeffective at controlling MERS-CoV infection-induced immunopathology as MERS-CoV infection similarlyleads to acute lung injury.

Over-activation of the complement system is controlled by a series of proteins known as the regulators ofcomplement activation. These proteins play a key role in preventing complement-mediated tissue and celldamage and dysregulation of one or more of the complement regulatory proteins, which results in tissueinjury, immunopathology, and in�ammation-associated disease [78], [79]. The complement system isnegatively regulated by several complement proteins, including factor I, C1-inhibitor (CI-INH), factor H, andC4-BP [80]–[83]. In contrast, factor P (properdin) is a positive regulatory complement protein. In this study,the levels of positive regulatory factor P (properdin) were signi�cantly higher in the lungs of MERS-CoV-infected patients compared with healthy controls, suggesting that the levels of factor P may positivelyregulate complement activation during MERS-CoV infection [81]. Factor P (properdin) has beenassociated with complement-mediated organ injury in various human diseases. Therapy targeting factorP (properdin) showed bene�cial outcomes and prevented complement-mediated tissue damage [84]–[90].The measurement of factor P may provide evidence for the involvement of the alternative complementpathway since factor P (properdin) is an important factor in alternative pathway activation [91]. A recentstudy demonstrated that SARS-CoV-2 can directly activate the alternative complement pathway [74]. Incontrast, the levels of negative regulatory proteins, factor I and C4-BP, were decreased in the lung ofMERS-CoV-infected patients when compared with healthy subjects. These negative regulatory proteinsplay an important role in regulating the complement system. The low levels of factor I and C4-BP suggestthat patients with MERS-CoV have a reduced capacity to control and regulate complement activation.These results indicate that MERS-CoV somehow suppressed and inhibited negative regulatorycomplement proteins during infection [80], [81], [92], [93]. A number of viral infection-mediated chronicin�ammatory and autoimmunity diseases are associated with complement regulatory proteindysfunction [79], [81], [94], [95]. Over-activation of pulmonary and systemic complement plays a key role

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in in�ammation, endothelial cell damage, thrombus formation, and intravascular coagulation andultimately leads to multiple-organ failure and death [12], [18]. In this study, high levels of pulmonarycomplement mediators, disease severity, and increased mortality appear to be linked to the degree ofcomplement activation against MERS-CoV. Complement C3a and C5a may be an independent risk factorfor death in MERS-CoV-infected patients.

RANTES (CCL5) is a key proin�ammatory chemokine produced during respiratory viral infection. Virus-infected lung and epithelial cells secret high amount of RANTES (in addition to immune cells). Also,RANTES (CCL5) has a critical role in the platelet activation and initiation of the coagulation cascade [96].In this study, we detected high levels of RANTES (CCL5), this in�ammatory chemokine was signi�cantlycorrelated with death and ARDS among MERS-CoV infected patients. In an in vitro experiment, infectionof human monocyte-derived macrophages (MDMs) and dendritic cells (MDDCs) with MERS-CoV showeda high levels and upregulation of RANTES (CCL5) and other in�ammatory chemokines such as MIP-1α,IP-10 and IL-8 [97]. Recent studies showed that the sever and mild COVID-19 patients had elevated levelsof RANTES (CCL5) [97]–[99]. Recent data form SARAS-CoV studies suggested targeting CCR5 could be atherapeutic strategy for COVID-19 [99]–[101].Previous studies also showed increased levels of RANTES(CCL5) contributed to exacerbation of allergic airway in�ammation and severe human respiratorysyncytial infection [102]–[104].

Studying the protein-protein interaction networks and the predicted protein interactors provides asigni�cance data to explore the functions of proteins [105], [106]. In PPI networks, proteins that havedirect interaction with several other proteins are called hub proteins (hub nodes). Proteins with moreinteraction partners may become targets for follow-up investigation [107]. In this study, the constructedPPI network (25 nodes and 149 edges) demonstrated that C3, C5, CCL5, CR1, CXCL8, IL10, IL-4 andCXCR1 were the hub proteins (nodes). Most of these hub proteins are involved in the in�ammatoryresponse and molecular binding interaction as well as ligand-receptor interaction. These results indicatethat predicted proteins (interactors) may also have a role in MERS-CoV disease severity. The function andexact role of hub proteins identi�ed in our PPI network in MERS-CoV infection require furtherinvestigation.

Here, we propose a mechanism to explain the role of pulmonary complement anaphylatoxins (C3a andC5a) IL-8 and RANTES (CCL5) in severe MERS-CoV infection. High levels of complement anaphylatoxinsC5a and IL-8 recruit and activate neutrophils. Subsequently, activated neutrophils undergo NETosis andROS production, which results in oxidative stress. Increased levels of C5a and IL-8 may cause anin�ammatory loop that contributes to extensive cellular damage and pathological changes. Also,elevation of complement anaphylatoxins C5a and C3a may induce cytokine storm and in�ammatorycascades. On the other hand, RANTES (CCL5) recruit and activate macrophage while, C3a recruit, activeand degranulate mast cells and eosinophils which results in airway smooth muscle contraction. Thesemeditators eventually contribute to airway and lung damage, more severe MERS-CoV infection, ARDS,and immunopathology. The overexpression of lung C5a during MERS-CoV infection may establish anampli�cation loop of IL-8 induction. Thus, a loop of continued IL-8 production leads to increased

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in�ammatory cell activation and recruitment to the lung, subsequently contributing to the lungpathological features. Our results and hypothesis may explain the events regulating complementactivation during MERS-CoV infection.

The main limitation of this study is that, we measured complement protiens, IL-8 (CXCL8), RANTES(CCL5) levels and viral load at a single time point; measuring this mediator expression levels and viralload at different time points to create a kinetic pro�le might provide additional information.

We conclude that the high levels of complement anaphylatoxins, C5a and C3a; positive regulatorycomplement protein (factor P); IL-8 (CXCL8); and CCL5 in the lower respiratory tracts of MERS-CoV-infected patients are associated with immunopathology, higher fatality rates, more severe disease, andARDS development. High levels of complement mediators, disease severity, and increased mortalityappear to be linked to the degree of complement activation against MERS-CoV. Furthermore, the levels ofC3a, C5a, IL-8 and CCL5 in the lung may be useful biomarkers to predict a more severe MERS-CoVinfection and mortality. targeting any of these mediators may offer an effective treatment for MERS-CoV.As such, future large studies characterizing components of the complement system at different stages ofMERS-CoV infection may offer an effective immunotherapeutic strategy.

DeclarationsAcknowledgments

The authors thank the Deanship of Scienti�c Research and RSSU at King Saud University for theirtechnical support.

Funding

The authors thank the Research Center at King Fahad Medical City for funding this study (Grant No 019-053).

Ethics approval

This study was reviewed and approved by the Institutional Review Board at King Fahad Medical City (IRBregister number 019-053). This study procedures were in accordance with the 1964 Helsinki Declarationand later versions.

Declaration of Competing Interest

The authors declare that they have no con�ict of interest.

Availability of data and materials

The datasets generated during and/or analysed during the current study are available from thecorresponding author on reasonable request.

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Con�ict of Interest

The authors declare that they have no competing interests

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Figures

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Figure 1

Characteristics and classi�cation of MERS-CoV -infected patients and clinical disease outcomes

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Figure 2

Lung complement proteins, IL-8 and RANTES, in MERS-CoV-infected patients and healthy controls. (A)The concentration of C3a was signi�cantly higher in MERS-CoV-infected patients compared with that inthe healthy control group (P < 0.0001). (B) Complement C5a was signi�cantly higher in the MERS-CoV-infected patients compared with that in the healthy control group (P < 0.0001). (C) The levels of factor Pwere signi�cantly elevated in the MERS-CoV-infected patients compared with that in the healthy control

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group (P < 0.0001). (D) The levels of factor H were elevated in the MERS-CoV-infected patients comparedwith that in the healthy control group (P < 0.0236). (E) Complement factor I was elevated in the MERS-CoV-infected patients compared with that in the healthy control group (P < 0.0065). (F) There was nostatistical signi�cance (ns) in the lung concentrations of C4BP in MERS-CoV-infected patients comparedwith that in the healthy control group. (G) The levels of C1q were signi�cantly elevated in the MERS-CoV-infected patients compared with that in the healthy control group (P < 0.0001). (H) The concentrations ofIL-8 were signi�cantly higher in the MERS-CoV-infected patients compared with that in the healthy controlgroup (P < 0.0001). (I) The levels of RANTES were signi�cantly elevated in the MERS-CoV-infectedpatients compared with that in the healthy control group (P < 0.0001).

Figure 3

Lung complement proteins, IL-8 and RANTES, in MERS-CoV-infected patients. Patients were divided intonon-survival (n = 22) and survival (n = 9) groups. (A) The concentration of C3a was elevated in the non-survival group compared with that in the survival group (P < 0.0024). (B) Complement C5a was higher inthe non-survival group compared with that in the survival group (P < 0.0001). (C) The levels of factor Pwere signi�cantly elevated in the non-survival group compared with that in the survival group (P <0.0001). (D) The levels of C4BP were signi�cantly elevated in the survival group compared with that inthe non-survival group (P < 0.0113). (E) The concentration of IL-8 was signi�cantly higher in the non-survival group compared with that in the survival group (P < 0.0001). (F) The levels of RANTES weresigni�cantly elevated in the non-survival group compared with that in the survival group (P < 0.0001).

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Figure 4

Actions of C3a, C5a, factor P (properdin) IL-8 and RANTES protein-protein interaction network usingCytoscape software. PPI network contains 25 nodes, 149 edges, average node degree 11.9 and the PPIenrichment p-value is < 1.0e-16. A node represents a protein, and a line represents an interaction. The linethickness represents the degree of con�dence prediction of the interaction.

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Figure 5

Actions of C3a protein-protein interaction network and the 21 predicted functional partners (proteins). Anode represents a protein, and a line represents an interaction between interactors. The line thicknessrepresents the degree of con�dence prediction of the interaction.

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Figure 6

Actions of C5a protein-protein interaction network and the 18 predicted functional partners (proteins). Anode represents a protein, and a line represents an interaction between interactors. The line thicknessrepresents the degree of con�dence prediction of the interaction.

Figure 7

Actions of IL-8 protein-protein interaction network and the 8 predicted functional partners (proteins). Anode represents a protein, and a line represents an interaction between interactors. The line thicknessrepresents the degree of con�dence prediction of the interaction.

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