Abstract
The most recently discovered interferon (IFN) family, type III IFNs or lambda IFNs (IFN-λs) are caused by viral infection and act in mucosal barriers, such as the respiratory tract. In this study, we assessed the serum levels of IFN-λs in new coronavirus disease-2019 (COVID-19) patients. Sixty-four COVID-19 patients were enrolled in this study. All cases were divided into the intensive care unit (ICU) and non-ICU groups according to their symptoms. Fourteen samples of healthy controls were also included. The serum levels of IFN-λ1 and IFN-λ2 were analyzed by specific enzyme-linked immunosorbent assay (ELISA) kits. The concentrations of IFN-λ1 and IFN-λ2 induced in the serum of non-ICU patients (836.7 ± 284.6 and 798.8 ± 301.5 pg/mL, respectively) were higher than found in ICU patients (81.57 ± 34.25 and 48.32 ± 28.13 pg/mL, respectively) (P = 0.004 and P = 0.006, respectively) and healthy controls (85.57 ± 33.63 and 65.82 ± 21.26 pg/mL, respectively) (P = 0.03 and P = 0.04, respectively). Meanwhile, no significant differences were found in the concentration of both cytokines between the ICU patients and healthy controls. We conclude that higher levels of IFN-λs are associated with decreased clinical manifestations in COVID-19 patients. These cytokines could be a promising therapeutic agent to avoid the overwhelming consequences of COVID-19.
Introduction
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of new coronavirus disease-2019 (COVID-19), is closely related to the SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV) (Corman and others 2018). SARS-CoV-2 can be effectively transmitted among humans, and the World Health Organization (WHO) announced the COVID-19 outbreak as a pandemic. It will also continue to have significant public health effects for years to come (Andreakos and Tsiodras 2020; Huang and others 2020; Lu and others 2020). Typical clinical symptoms of COVID-19 are fever, cough, myalgia, and shortness of breath (Huang and others 2020). Severe cases often develop acute lung injury or acute respiratory distress syndrome (ARDS), which is the fatal form (Chen and others 2020).
The components of the innate immune system act as first responders for the detection and clearance of viral infections. Interferons (IFNs), key components of the innate immunity, play a crucial role in the immune response to viral infections (Prokunina-Olsson and others 2020). IFNs are categorized into 3 groups (type I IFNs, type II IFNs, and type III IFNs). Meanwhile, lambda IFN (IFN-λ) family, also known as type III IFNs, was found decades after type I IFNs; they were also characterized initially as innate antiviral components with signaling pathways and expression profiles similar to type I IFNs (Kotenko and others 2019).
In humans, the IFN-λ family comprises 4 members [i.e., IFN-λ1/interleukin-29 (IL-29), IFN-λ2/IL-28A, IFN-λ3/IL-28B, and IFN-λ4] all of which signal through a unique heterodimeric receptor complex consisting of IFN-λ receptor 1 (IFN-λR1) (IFN-λRA, IL-28RA) and IL-10R2 (IL-10RB). The main function of IFN-λ is to prevent viral infection by establishing an antiviral state and, if infected, to slow viral replication and dissemination (Andreakos and others 2019).
The expression of IFN-λR1 is primarily limited to the epithelial cells of the respiratory tract. As a result, it is widely suspected that IFN-λs have effective first line immunological protection against viral respiratory tract infections (Davidson and others 2016; Galani and others 2017; Klinkhammer and others 2018). Viruses have developed several strategies to interfere with IFN expression, and it appears to be particularly true of coronaviruses.
In a related earlier study, experimental infection with a MERS-CoV strain (human coronavirus EMC) did not induce expression of IFNs type I and type III in respiratory tissue cultures, whereas infection with influenza virus induced large amounts of both types (Chan and others 2013). However, there are limited studies regarding the role of IFN-λs in COVID-19. Therefore, we aimed to evaluate the serum levels of IFN-λ1 and IFN-λ2 in intensive care unit (ICU) and non-ICU COVID-19 patients and compare them with healthy controls.
Materials and Methods
Study populations
This study was approved by the Ethics Committee of Babol University of Medical Sciences (No:
The clinical classifications are based on the following manifestations: (1) non-ICU group: fever, imaging finding of pneumonia, respiratory symptoms, oxygen saturation (SpO2) ≤93%, and oxygen partial pressure (PaO2)/fraction of inspired oxygen (FiO2) in arterial blood ≤300 mmHg; and (2) ICU group: respiratory failure and requirement of mechanical ventilation, shock, combined organ failure, and need of ICU admission. Subjects (n = 14) without any infection symptoms and negative for the SARS-CoV-2 test were enrolled as a control group.
Collection of blood, separation of serum, and cytokine quantitation
In this study, 5 mL of venous blood was obtained from each of the subjects. Serum samples were separated by 1,000 × gravitational units (g)/10 min centrifugation of clotted blood samples. The sera were stored at −80°C until the assessment of IFN-λ1 and IFN-λ2 levels. The concentration of these cytokines was measured by enzyme-linked immunosorbent assay (ELISA) kits (Boster Bio, Pleasanton, CA). All assays were performed in duplicate according to the manufacturer's instructions. The sensitivity of IFN-λ1 and IFN-λ2 ELISA kits is 10 pg/mL.
Statistical analysis
Continuous variables were described by mean ± standard error or median (interquartile range). Categorical variables are shown as numbers and percentages. Baseline characteristics and IL levels of subjects were compared with the Kruskal–Wallis test and Dunn's multiple comparisons post-test. Statistical analyses were performed on GraphPad Prism 8.0 for Windows (GraphPad Software, Inc., San Diego, CA). All statistical tests were 2-tailed at the significance level of P value <0.05.
Results
Clinical findings
Fourteen healthy controls, 14 patients in ICU, and 50 non-ICU patients were consecutively enrolled in this case–control study. On admission, the majority of patients had leukocytosis (14.29% in ICU versus 24% in moderate/severe), lymphopenia (42.86% in ICU versus 12% in moderate/severe), abnormalities of C-reactive protein, and aspartate aminotransferase as described in Table 1. The mean age in ICU and non-ICU patients was 53.42 (18.04) and 58.72 (19.16) years, respectively, which was significantly higher than healthy controls (38.28 [9.46]; P = 0.001). At least 1 comorbidity was seen more commonly in ICU cases than healthy controls (71.43% versus 7.14%; P < 0.001), but not compared with the non-ICU patients (71.43% versus 62%; P = 0.515).
Background Characteristics of Participants
Continuous variables compared with independent t-test, categorical variables compared with chi-square test or Fisher's exact test.
WBC: white blood cells, BUN: blood urea nitrogen, AST: aspartate aminotransferase, ALT: alanine aminotransferase, NLR: neutrophil-lymphocyte ratio, PLR: platelet-lymphocyte ratio
Concentration of IFN-λ1 and IFN-λ2 in controls and 2 case groups
The concentration of IFN-λ1 and IFN-λ2 in controls and 2 case groups is shown in Table 2. The serum levels of IFN-λ1 and IFN-λ2 varied a great deal between different individuals. The mean values of IFN-λ1 in ICU patients and healthy controls were 81.57 ± 34.25 and 85.57 ± 33.63 pg/mL, respectively (P = 0.9). The serum level of IFN-λ1 was significant between non-ICU patients and healthy subjects (836.7 ± 284.6 versus 81.57 ± 34.25 pg/mL; P = 0.03). Also, there was a significant difference between ICU and non-ICU patients regarding the serum levels of IFN-λ1 (81.57 ± 34.25 versus 836.7 ± 284.6 pg/mL; P = 0.004).
The Concentration of Interferon-λ1 and Interferon-λ2 Between Patient Groups and Healthy Controls
Data in bold represent statistically significant and other data represent nonsignificant.
Significant at P value <0.05.
Significant at P value <0.01.
ICU, intensive care unit; IFN, interferon.
As shown in Table 2, the concentration of IFN-λ2 in the serum of non-ICU patients (798.8 ± 301.5 pg/mL) was higher than in the healthy controls (65.82 ± 21.26 pg/mL; P = 0.04). A significant difference was also observed for IFN-λ2 between non-ICU and ICU groups (798.8 ± 301.5 versus 48.32 ± 28.13 pg/mL; P = 0.006). However, the mean values of IFN-λ2 were not significantly different in the ICU patients (48.32 ± 28.13 pg/mL) compared with healthy controls (65.82 ± 21.26 pg/mL; P = 0.9).
Discussion
Although a rapid and fine-tuned immune response is the first line of defense against viral infection, an enormous inflammatory innate response and compromised adaptive immunity can contribute to tissue damage both locally and systemically. Therefore, given the essential role of the innate immune system in viral infections, further knowledge of the mechanism behind the innate immunity might give us clues about the clinical management of COVID-19 patients and prevention of the transition from moderate to critical stages.
IFN-λs promote innate immunity after viral infections, mainly by inducing an antiviral state in epithelial cells. Respiratory mucosa is central to the IFN-λ-mediated antiviral defense. This is the area where IFN-λs are initially induced and mainly act, reducing the spread of the virus (Galani and others 2017). Furthermore, IFN-λ can elicit the release of immunomodulatory cytokines from epithelial cells, and thus enhancing adaptive immune responses to viruses that target mucosal barriers (Ye and others 2019b).
In this study, we found that the levels of IFN-λ1 and IFN-λ2 were elevated in the serum of non-ICU COVID-19 patients compared with the healthy controls and ICU COVID-19 subjects. A previous similar study showed that type I IFN is induced in COVID-19 patients and also its concentration may be decreased in critically ill patients (Hadjadj and others 2020). In contrast, another study reported that in COVID-19 patients, type I and III IFNs are not produced as they were not found in the serum of a small COVID-19 cohort (Blanco-Melo and others 2020). This inconsistency could be as a result of transient illustrations of a probably heterogeneous disease process, which were the focuses of these studies. When IFN-λs are produced in high levels, this is enough to confront SARS-CoV-2 infection and accelerate the recovery of the infected subjects. In contrast, when IFN-λs levels are low, type I IFNs are activated to strengthen the antiviral defenses of the patients.
However, type I IFNs cause excessive proinflammatory responses defined by the upregulation of various cytokines, including IL-6, IL-1b, and tumor necrosis factor alpha (TNF-α) (Andreakos and others 2017). Decreased production of IFN-λs, accompanied by induction of type I IFNs, gives rise to the “cytokine storm,” which results in severe pneumonia and ARDS. A previous study has also shown that animals, lacking a functional IFN-λ receptor, present similar immunopathology (Galani and others 2017). The type I IFN receptor is expressed ubiquitously in the body, whereas the type III IFN receptor is found mainly on epithelial cells and neutrophils (Stanifer and others 2020). Although the genes triggered by type I and type III IFN signaling are identical, the responses are different due to differences in cell type specificity and signaling kinetics. The response of IFN type I is stronger, fast, and short-lived, whereas the IFN type III response is less strong, sluggish, and long-lasting (Lazear and others 2019).
Interestingly, IFN-λs can induce the release of immunoregulatory cytokines from epithelial cells. Therefore, this type of IFN is more tissue-protective and anti-inflammatory than others. Moreover, IFN-λs can improve adaptive immune responses by stimulating cytotoxic T cell, T helper 1 (Th1), and antibody responses, which are central to the long-term immunity (Ye and others 2019a, 2019b). It seems that IFN-λs act in co-operation with type I IFNs to enhance the antiviral immunity for balance between fine-tuning protection and minimal side effects. Thus, the administration of IFN-λs might be an optional treatment for COVID-19 patients by inhibiting the cytokine storm and its side effects.
Conclusion
Increased levels of IFN-λs were associated with decreased clinical manifestations in COVID-19 patients. As SARS-CoV-2 primarily infects epithelial cells of the respiratory tract and IFN-λs triggers antiviral gene expression in these cells without hyperinflammation, IFN-λs might be a potential therapy for COVID-19 patients.
Footnotes
Acknowledgments
We thank the staff members of the Ayatollah Rouhani, Shahid Beheshti, and Shahid Yahya Nezhad Hospitals (Babol, Iran) for the collection of blood samples. We also thank the staff members of the Immunoregulation Research Center, Immunology Department, Babol, Iran, for co-operation in experimental procedures.
Author Disclosure Statement
No competing financial interests exist
Funding Information
No funding was received.