The Role of IL-6 in RNA Virus Infection

Abstract

IL-6 is a pleiotropic cytokine produced in response to tissue damage and infections. This up-regulation was observed during infection with a highly virulent VSV strain. There was potential association between IL-6 levels and virus virulence. In this chapter we would like to explore in more detail the biological functions of IL-6 in different virus models. We also discuss the debatable role of IL-6 during viral infections. Previous studies show the potential role of IL-6 to mount a proper immune response during some viral infections, others link this cytokine with exacerbation of viral disease. These latter findings lend support to the hypothesis that up-regulation of IL-6 during certain viral infections may promote virus survival and/or exacerbation of clinical disease. Previous experimental evidences also suggest potential negative consequences that increased levels of IL-6 might have on the cellular immune response against viruses.

1. Introduction

The pandemic of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has set a major healthcare issues and economic burden worldwide. Like other RNA viruses, SARS-CoV-2, while adapting to their new human hosts, is prone to genetic evolution with the development of mutations over time, resulting in mutant variants that may have different characteristics than its ancestral strains. Currently, treatments of COVID-19 are mainly repurposing drugs or symptomatic with no definitive treatment directed against the virus [1].

Coronaviruses (CoVs), enveloped positive-sense RNA viruses, are characterized by club-like spikes that project from their surface, an unusually large RNA genome, and a unique replication strategy. In the absence of specific treatment or antiviral drugs been proven against SARS-CoV-2, researchers have proposed many therapeutics agents used as adjunctive treatments for COVID-19 patients apart from supplemental oxygen therapy or mechanical ventilation [1, 2].

In Coronavirus infection, viral surface glycoproteins, double-stranded RNA, and intracellular viral proteins all have the capacity to activate signal transduction pathways leading to the expression of cytokines and chemokines. Cytokine storm is one of the main mechanisms of the disease and is believed to trigger an exaggerated immune response in the host and has been observed more frequently in severe COVID-19 patients associated with complications, such as acute respiratory distress syndrome (ARDS) and other multiple organ injuries [2].

An important consequence of RNA virus infection and COVID-19 disease is cell-free DNA (cfDNA) found in body fluids such as serum or plasma. cfDNA originates from nuclear or mitochondrial DNA released from dead/dying cells, DNA released from live cells, and foreign DNA from invading viruses [3]. The other interesting points are the fact that monoclonal antibody against IL-6 receptors or IL-6 inhibitor has shown to be an effective agent in COVID-19 patients with severe illness. Initially, it is frequently used for the treatment of rheumatoid arthritis patients. These drugs targeting IL-6 as inflammatory mediators will decrease inflammatory response in cytokine storm, hence minimizing the incidence of jeopardize complications, such as ARDS. Tocilizumab and other anti-IL-6 receptors antagonist has been recommended as an immunotherapy in severely ill patients and improved the clinical outcomes as well as decrease in mortality rate [3, 4]. The aim of this review is describe the role of IL-6 in RNA virus infection.

2. Method

This article is a narrative review study include IL-6 and RNA virology, clinical impact, diagnosis, and treatment. The search was conducted using five keywords “IL-6,” “RNA Virus,” “IL-6 inhibitors” in combination with “human” in PubMed, Scopus, and ScienceDirect among articles between 2000 and April 2022. We focused on publications post-year 2000, with emphasis on the past 10 years, but we did not exclude commonly referenced, relevant, and influential older publications. The clinical trial, case–control, review, and a meta-analysis study of 20 years; 2000–2022 articles, case series, cohort, and cross-sectional studies were reviewed. We also reviewed the references of each article to include further other studies or reports not identified by the search. We excluded articles considering the expert viewpoints and letters to the editor. We limit our search to English written articles and articles on human study.

3. Discussion

3.1 Inflammatory cytokine (IL-6) and RNA virus infection

After RNA virus entered the host. The innate immune system is the first line defensive mechanism for this virus. The response will be responsible for detecting pathogen-associated molecular patterns (PAMPs). Viral RNA is a potent inducer of antiviral innate immune signaling. It will provokes an antiviral state by directing expression of interferons (IFNs) and pro-inflammatory cytokines. The +RNA viruses developed various methods to avoid detection and downstream signaling. This mechanism includes isolation of viral RNA replication in membranous viral replication organelles (ROs) [5].

The defense mechanism of the host includes rapid production interferons and other pro-inflammatory cytokines. This is an important consequence of virus detection. This condition contributes to an antiviral state in both the infected host cell and other surrounding cells. The next phases showed that interferons will play an essential role in coordinating the antiviral adaptive response system [5].

One of the most studied cytokines is IL-6. In RNA virus infection IL-6 is considered one of the most important cytokines during an infection, along with interleukin 1 (IL-1) and tumor necrosis factor alpha (TNF-a). IL-6 is a pleotropic cytokine produced in response many types of tissue damage including fibroblasts, keratinocytes, mesangial cells, vascular endothelial cells, mast cells, macrophages, dendritic cells, and T and B cells. After targeting its specific receptor, IL-6 starts a cascade of signaling events mainly associated with the JAK/STAT3 (Janus kinase (JAK)/signal transducer and activator of transcription 3) activation pathway. This cascade will promote the transcription of multiple downstream genes associated with cellular signaling processes, including cytokines, receptors, adaptor proteins, and protein kinases. It will also regulate the production of proteins implicated in regulation of many gene expression. The biological consequences of IL-6 production have been associated with pro inflammatory effects [5, 6].

As an immediate answer after RNA virus infections, different immune cellular pathogen recognition receptors, including toll-like receptors (TLR:2, 3, 4, 7, 8, and 9), nucleotide-binding oligomerization domain-like receptors, and retinoic acid-inducible gene-1-like receptors, are able to sense a variety of pathogen-associated molecular patterns displayed by viruses (envelope glycoproteins, single and double-stranded RNA), which stimulate transcription of IL-6 among other proinflammatory cytokines [5].

IL-6 play significant role either in positive or negative ways. The animal studies showed its ability to repress the replication of CSFV (classical swine fever virus) in swine peripheral blood mononuclear cells. However, experimental scientific evidence also suggests the negative impact of increasing IL-6 level. The potential role of IL-6 increase is the establishment of a viral persistent state in infected hosts. The animal studies showed that overexpression of IL-6 during the viral immune response might induce viral persistence by impairing the polarization and functionality of Th1 cells and the lytic capacity of CD8 T-cells through different mechanisms, leading to chronic infections. As a consequence of the constant antigen stimulation, CD8 T-cells become unresponsive and fail to develop into memory CD8 T-cells, a situation that limits viral clearance. The other negative impact of upregulated IL-6 is increasing inflammation followed by cytokine secretion and cellular recruitment as described during autoimmune diseases. This inflammation state may be an advantage for some RNA viruses by providing new targets for subsequent infections [6, 7].

Previous report and reviews showed that high levels of interleukin 6 (IL-6) and Interleukin 8 (IL 8) were found in the very acute stage associated with lung lesions in SARS-CoV-1 patients. The IL-6 can induce the hyper-innate inflammatory response. In the cases of SARS-CoV-1, very high levels of IL-6 were associated with significant and severe inflammation state, and its correlated with high mortality. Some observational retrospective and systematic review/meta-analysis showed that high IL-6 and C-reactive protein (CRP) were significantly correlate with mortality and severity of the disease. Some recent evidences also proved that critically ill patients with severe respiratory failure and SARS-CoV-2 have either immune dysregulation or macrophage-activation syndrome, both of which are characterized by pro-inflammatory cytokines (IL-6). The immune dysregulation, in particular, is driven by the Interleukin-6 (IL-6). The most significant impact of this condition features of this immune dysregulation are: (1) over-production of pro-inflammatory cytokines by monocytes, and (2) lymphocyte dysregulation with CD4 lymphopenia [7, 8].

After the pandemic of COVID-19, some recent evidences showed that there are of many similarities between Macrophage Activation Syndrome (MAS) disease and COVID-19 pneumonia. This phenomenon is a pathological condition that called over production of cytokine secretion. The loss of first line anti-viral defense mechanism may be responsible for this activation. It will cause prolonging and sustained IL-6 secretion. The Sustained IL-6 secretion was also correlated with the serum viral RNA load [7, 8, 9].

3.2 The up regulation of IL-6 in RNA virus infection

In common, pathogen-associated molecular patterns (PAMPs) recognized by pathogen recognition receptors (PRRs) in RNA virus infected lesions. The damage-associated molecular patterns (DAMPs) released from damaged cells in non-infectious inflammation will provoke IL-6 synthesis in many cells such as immune-competent cells, mesenchymal cells, fibroblasts, endothelial cells, and epithelial cells [10, 11]. The IL-6 initiates warning signals to the entire body, and many experiments have shown that serum IL-6 levels are elevated in patients RNA virus infection. Some studies in Hepatitis B virus infection showed that IL-6 is also a good marker for HBV-related disease progression [12]. The levels of IL-6 are significantly higher in chronic hepatitis B (CHB) patients than in healthy individuals [13]. The IL-6 is also significantly higher in patients with advanced liver disease (LC or HCC) compared to the CHB groups [14].

The IL-6 is an important proinflammatory cytokines during RNA virus infection onset, especially, at the mucosal sites. However, the impact of IL-6 on the disease outcome may vary significantly. The IL-6-dependent Th17 activation and differentiation are important for effective neutrophil migration, IL-6 together with IL-15 modulate cytolytic capacity of CD8⁺ T cells [15]. In this part, IL-6, as a pyrogenic cytokine, contributes to thermostatic regulation that is very important for effective anti-viral response [16].

On the other hand, the upregulation of IL-6 has been implicated in the progression of RNA viral infections. In this part, IL-6 synergizes with IL-1b and TNF to upregulate trypsin expression, trypsin activates matrix metalloproteinases and causes the breakdown of basal membrane and extracellular matrix, that cause increased tissue permeability and edema [17]. The upregulation of IL-6 promotes Th17 cell differentiation and IL-17A secretion, which, in turn, activates the expression of anti-apoptotic molecules, such as Bcl-X_L, favoring survival of virus-infected cells in the model of persistent viral infection [18]. In the COVID-19 infection, the increase of IL-17 that mediated by IL-6 promotes the migration of neutrophils whichcontribute to the pathogenesis of ARDS [19, 20].

• IL-6 promotes highly specific reaction of adaptive immunity by stimulating of CD8⁺ T cells and B cells, which is balanced by T regulatory cells.

• IL-6 facilitates survival of phagocytic neutrophils.

• IL-6 can provide unfavorable Th2 and Th17 over Th1 helper differentiation and facilitate tissue injury by dysregulation of extracellular matrix and attraction of neutrophils and pro-inflammatory macrophages (Figure 1).

Figure 1.

The IL-6 demonstrates opposing effects during the immune response to viral infections [6].

3.3 Clinical implication

What are the implications of this findings? There is a fact that some viral strains can cross the barrier of the immune response and induce the over-production of IL-6. This condition correlated with the advancement of viral activity. This condition consequently followed by an up-regulation in the production of IL-6, this polymorphisms in the region of the IL-6 gene stimulate the overexpression of IL-6 that also correlated with viral progression. This loop correlated with the increase of the viruses virulence that damaging Th1 cell polarization and functionality. This condition caused viremia and the loss of CD8 T-cells ability to develop memory cells, thus reducing the capacity to fight viral load. Constant replication of the virus fails to grow into long-out plasma cells, limiting their ability to clear the virus Prolonged RNA virus infections increase levels of IL-6, that further makes accumulation of this pathologies state (inflammation plus cytokines and cellular presence). This state may be advantageous for several RNA viruses, mainly because it offers some opportunities for near-future infections because there are new target cells to choose from. There were a debate on how is the inflammatory cytokine, IL-6, in viral infections can be used as a biomarker for prognosis. An exploration of the IL-6 function as well as that of IL-6 inhibition in treating persistent infections might aid in developing its therapeutic benefit may reveal information about its utility [8, 9].

The study from Saji et al. showed that IL-6 is important prognosis biomarker. The study from 102 patients with moderate to severe COVID-19 showed that the 30-day was significantly higher in patients with high IL-6 (> 49 pg./mL) and SARS-CoV-2 RNAaemia (> 1.5 copies/μL) compared to those with high IL-6 or RNAaemia or without high IL-6 and RNAaemia (88% vs. 22% or 8%, log-rank test P = 0.0097 or P < 0.0001, respectively) [17].

The other study in showed similar result. Patients with hypoxemia had significantly higher concentrations of IL-6, C-reactive protein, procalcitonin, fibrinogen, total bilirubin, aspartate aminotransferase and alanine aminotransferase at initial screening. ROC analyses identified IL-6 as the most robust predictor of hypoxemia. The concentration of IL-6 > 24 pg./mL predicted the development of hypoxemia with the sensitivity of 100% and specificity of 88.9%. The positive and negative predictive values were 76.9, and 100% respectively [18].

The prognostic value of clinical severity also been showed. The study in the 140 COVID-19 patients, the levels of IL-6, CRP, and PCT increased in 95 (67.9%), 91 (65.0%), and 8 (5.7%) patients on admission, respectively. The proportion of patients with increased IL-6, CRP, and PCT levels was significantly higher in the severe patients than in the mild one. Cox proportional hazard model showed that IL-6 and CRP could be used as independent factors to predict the severity of COVID-19. Furthermore, patients with IL-6 > 32.1 pg./mL or CRP > 41.8 mg/L were more likely to have severe complications [19].

3.4 IL-6 inhibitor as promising treatment

IL-6 is produced by dendritic cells, macrophages, mast cells, and other innate immune cells. Several previous studies showed that IL-6 has long been considered a marker of inflammation. The increased levels of IL-6 significantly noted in number of diseases that related with ongoing inflammatory cell activation. The IL-6 can also be produced by non-immune cells such as epithelial cells, endothelial cells, keratinocytes, and fibroblasts among others, in response to specific stimuli [4].

The presence of IL-6 may not necessarily correlate with the production of other inflammatory cytokines and it may not be just a marker of ongoing inflammation, but a direct player in the immune response. A number of studies have shown a role of IL-6 in the adaptive immune response, primarily on the differentiation fate of CD4 T cells, but IL-6 can also modulate aspects of the innate immune response [17, 18].

Elevated levels of IL-6 in the lung and in serum have been found in patients infected with the influenza virus, including the 2009 H1N1 pandemic influenza [21, 22]. IL-6- and IL-6-mediated signals are essential for survival to a non-lethal dose of influenza H1N1 virus infection. Deficiency of IL-6 or IL-6R prevents clearance of the H1N1 virus in association with low numbers of neutrophils present in the lungs of infected individuals. We also show that IL-6 provides survival signals to protect neutrophils from influenza virus-triggered apoptosis. Impaired virus clearance caused by the lack of IL-6 or IL-6R signals leads to emphysema-like destruction of the lung and, ultimately, death [23, 24]. Thus, IL-6 is a protective factor against primary infection with the influenza H1N1 virus by promoting the innate phase of the immune response and virus clearance (Figure 2) [23, 25, 26].

Figure 2.

Overexpression of IL6 and its negative impact [15].

With coronavirus disease 2019 (Covid-19), the role of localized inflammation was evident. Patients with severe symptoms has high interleukin-6, a cytokine produced by macrophages that induces a proinflammatory response and is often elevated in patients with Covid-19. Some studies showed the benefit of IL-6 inhibitors.

The REMACAP trials, which had an adaptive design, approximately 800 patients in need of respiratory or blood-pressure support or both were randomly assigned to placebo or a single injection of an interleukin-6 receptor blocker, tocilizumab or sarilumab. The primary outcome was a composite of in-hospital death and days free of respiratory or blood-pressure support to day 21. The group receiving an interleukin-6 receptor blocker had an in-hospital mortality of 27%, as compared with 36% in the control group, and those receiving the receptor blocker had a median of 10 to 11 organ support–free days, as compared with 0 days for control [27].

Conflicting result was shown in CONVACTA trial. This randomized, controlled trial include 452 patients with Covid-19 (oxygen saturation, ≤93%) were randomly assigned in a 2:1 ratio to receive one dose of tocilizumab or placebo. The primary outcome was clinical status at day 28; mortality was a secondary outcome. The group receiving tocilizumab had a median clinical status of 1 (discharged or ready for discharge), and the control group had a median clinical status of 2 (out of intensive care and not receiving supplemental oxygen). Mortality was 19.7% in the tocilizumab group and 19.4% in the control group [28].

The meta-analysis of 27 randomized trials of IL-6ra that included 10,930 patients with COVID-19indicate that all-cause mortality was reduced in patients hospitalized for COVID-19 and treated with IL-6ra compared with those treated with placebo or usual care. By day 28 after randomization, 1407 deaths occurred among 6449 patients randomized to receive IL-6 antagonists and 1158 deaths occurred among 4481 patients randomized to usual care or placebo (summary odds ratio [OR], 0.86 [95% CI, 0.79–0.95]; P = .003 based on a fixed-effects meta-analysis). Importantly, a significant mortality benefit was only found when IL-6 inhibitor were coadministered with glucocorticoids (summary OR for the association of IL-6 antagonist treatment with 28-day all-cause mortality, 0.78 with concomitant glucocorticoid administration vs. 1.09 without glucocorticoid administration). The benefits of IL-6 inhibitor were most evident among patients who received respiratory support with oxygen by nasal cannula, face mask, high-flow nasal oxygen (OR for death, 0.81 [95% CI, 0.67–0.98]), or noninvasive ventilation (OR, 0.83 [95% CI, 0.72–0.96]) vs. those who required invasive mechanical ventilation (IMV) (OR, 0.95 [95% CI, 0.78–1.16]) [29].

The review showed that, IL-6 receptor antagonist hold promise for patients hospitalized for COVID-19 with progressive disease and substantial oxygen requirements but are not yet merited for widespread use among patients with mild disease nor with prolonged invasive mechanical ventilation [30].

4. Conclusion

There are evidences supporting a significant role of IL-6 during viral infections. IL-6 production that may be detrimental to the cellular immune response during viral infections. The change in IL-6 production during the immune response to viral infection are (i) the increased ability of some viral strains to overcome the immune response using a variety of evasion strategies, and consequently up-regulate the production of IL-6 as a result of increased viral loads, and (ii) polymorphisms in the IL-6 gene promoter stimulating overexpression of IL-6 during the immune response. The increased levels of IL-6 significantly related with ongoing inflammatory cell activation and poor prognosis.