Does Interleukin-6 Bridge SARS-CoV-2 With Virus-Associated Cancers?

Abstract

To date SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2), a member of the Coronaviridae family, has infected more than 40 million people worldwide. A second wave of SARS-CoV-2 infection is aggressively surging. The clinical worsening of SARS-CoV-2 infection appears to be strictly associated with comorbidities, which can be used to establish an intrinsic patient network whose molecular profile is pivotal for identifying and successfully treating populations at risk. Herein, we focus on the direct interaction between SARS-CoV-2 and virus-associated cancers, exploring the critical role of interleukin-6 (IL-6) as a mediator of this complex cross talk. IL-6 production is enhanced in diverse viral infections ranging from human papilloma virus (HPV) to hepatitis B virus (HBV), human immunodeficiency virus (HIV), and SARS-CoV-2 infection. High systemic levels of IL-6 are associated with viral persistence and poor clinical outcomes in SARS-CoV-2–infected patients. Blockade of IL-6/IL-6R, using specific molecules, is under investigation in active clinical trials for the treatment of patients with SARS-CoV-2. Although the data are as yet inconclusive, they pave the way for selective targeting of crucial cytokine-activated aberrant signaling in SARS-CoV-2 infection.

INTRODUCTION

The SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) pandemic opens a challenging debate for basic, translational, and clinical scientists regarding the impact of COVID-19 (coronavirus disease 2019) on patients with cancer, mainly dealing with cancer management and the risk of SARS-CoV-2 infection. Indeed, patients undergoing treatment with chemotherapy, radiation therapy, immunotherapy, and targeted therapies for cancer are considered high-risk subjects for infection, as indicated in numerous specific guidelines regarding the management of these patients during the COVID-19 pandemic. In addition, patients receiving immunotherapy with checkpoint inhibitors are markedly susceptible to severe forms of COVID-19.[1] Thus, interactions between SAR-CoV-2 and cancer have mostly been highlighted to evaluate the risks associated with intrinsic disease characteristics, patient comorbidities, physiological features, and administered treatment.

The focus of this review is to draw attention to a lesser explored area regarding the potential direct interaction between SARS-CoV-2 and virus-associated cancers. Principally, this interaction involves the immunologic profile of the patient, considering the pivotal role the immune system plays in the anti-COVID response as well as in cancer. We hypothesize that cytokine IL-6 (interleukin-6) may bridge the gap between COVID-19 and virus-mediated cancers.

SARS-CoV-2, THE LATEST CORONAVIRUS TO JUMP TO HUMANS

Coronaviridae is a family of single-stranded RNA viruses that also includes SARS-CoV-2. Several members of the Coronaviridae family circulate continuously in humans, causing only mild respiratory disease. Unlike these members, both SARS-CoV-2 and the Middle East respiratory syndrome coronavirus (MERS-CoV) are transmitted from animals to humans and cause severe respiratory diseases. The World Health Organization named the disease caused by SARS-CoV-2 as COVID-19. SARS-CoV-2 contains four structural proteins, namely S (spike), E (envelope), M (membrane), and N (nucleocapsid). N protein stores genomic RNA, while S, E, and M constitute the viral envelope. Since the spike protein is crucial for the binding of SARS-CoV-2 to the host cell membrane, it has thoroughly been investigated and its 3D structure has been solved. The binding of the surface unit S1 of the S protein to a cellular receptor favors viral attachment to the host cell. Furthermore, SARS-CoV-2 entry into cells requires protease-mediated S protein priming. This leads to cleavage at the S1-S2 site and fusion of viral and cellular membranes. The process is driven by the S2 subunit. Furthermore, angiotensin-converting enzyme 2 (ACE2) is a known SARS-CoV-2 receptor for cellular entry. Upon S protein–receptor engagement, the cellular serine protease TMPRSS2 primes S protein. The interface of SARS-S/ACE2, which plays a critical role for SARS-CoV transmissibility, has been elucidated at the atomic level.

It has been proposed that the coronavirus disease can evolve into 3 worsening clinical stages. Stage 1 involves initial infection where patients show poor or no symptoms; stage 2 entails the possibility of developing lung disease with or without hypoxia; finally, in a minority of patients there is systemic evolution (stage 3) associated with a state of generalized inflammation (cytokine storm).[2]

SARS-CoV-1 and SARS-CoV-2 infections trigger a burst of proinflammatory cytokines, dramatically fostering a virulent infection. For example, high serum levels of IL-6 have been associated with acute and advanced stages of SARS-CoV-2 infection in patients with lung lesions.[3]

INTERLEUKIN-6 (IL-6)

IL-6 is a 4-helix bundle pleiotropic cytokine with both proinflammatory and anti-inflammatory functions. Among the first of several activities of IL-6 to be identified—which also led to its isolation and cloning in 1986 by Kishimoto[4] in Japan—is the stimulation of proliferation and differentiation of B cells. However, this cytokine has also several other functions. This is why various names have been used in the past for this single molecule, such as hybridoma/plasmacytoma growth factor, interferon-beta-2 (IFN-β-2), and hepatocyte-stimulating factor. However, all these terms have been abandoned and the molecule is now universally known as IL-6.

Since its initial discovery rapid progress has been made in understanding the activities of IL-6, the IL-6 receptor system, and the IL-6 signal transduction mechanism. The most characterized functions of IL-6 include the following:

1. Stimulation of acute phase response in the liver;

2. Induction of fever through its ability to cross the blood-brain barrier and to initiate the synthesis of Prostaglandin E2;

3. Energy mobilization in muscle and fat;

4. Stimulation of osteoclast formation and increased bone resorption; and

5. Production of neutrophils.

IL-6 is produced by a variety of cell types including T cells, fibroblasts, and macrophages. In the latter, IL-6 is induced in response to specific microbial molecules, referred to as pathogen-associated molecular patterns (PAMPs). These PAMPs bind to an important group of detection molecules of the innate immune system, called pattern recognition receptors, including Toll-like receptors (TLRs), which are present on the cell surface and intracellular compartments and induce intracellular signaling cascades that give rise to inflammatory cytokine production.[5] This is considered to be the main mechanism leading to excessive IL-6 production during infection by SARS-CoV-2. IL-6 signals through a cell-surface type I cytokine receptor complex consisting of the ligand-binding IL-6Rα chain and the signal-transducing molecule gp130 (Fig. 1).[3,6]

Figure 1.

Interleukin-6 (IL-6) signaling pathways. In the classical IL-6 signaling pathway, induced by binding of IL-6 to membrane-bound IL-6R, the activation of JAK/STAT, AKT, and MAPK pathways determines the release of cytokines and consequent feedback inhibition through SOCS. In the trans-signaling pathway, in which the IL-6/IL-6R complex activates gp130, there is a shift toward greater JAK/STAT signaling than MAPK signaling through suppression of SOCS, leading to greater transcription of cytokines and a proinflammatory state. AKT: protein kinase B; ERK: extracellular regulated kinases; gp130: glycoprotein 130; IL-6: interleukin 6; IL-6R: interleukin 6 receptor; JAK: Janus kinase; MAPK: mitogen-activated protein kinase; NFkB: nuclear factor kappa B; PI3K: phosphatidylinositol 3-kinases; sIL-6R: soluble IL-6 receptor; SOCS: suppressor of cytokine signalling; STAT: signal transducer and activator of transcription.

Gp130 is the common signal transducer for several cytokines of the same family, called the IL-6 family or the gp130 family of cytokines, which include leukemia inhibitory factor, ciliary neurotropic factor, oncostatin M, IL-11, and cardiotrophin-1, and is almost ubiquitously expressed in most tissues. Conversely, the expression of IL-6Rα is restricted to certain cell types. Upon initial binding of IL-6 to its receptor, the complex recruits gp130 to form a 2:2:2 stoichiometric hexameric complex, which leads to gp130 dimerization and activation of JAK/STAT cytoplasmic signaling (Fig. 1).[7]

In addition to the membrane-bound receptor, a soluble form of IL-6R (sIL-6R) has been purified from human serum and urine. Many cells are unresponsive to IL-6 stimulation alone, but because they express gp130, they can be made responsive through the sIL-6R/IL-6 complex. This phenomenon has been shown, for example, to promote nerve regeneration through remyelination or also clonal hemopoiesis.

IL-6 is involved in several diseases such as rheumatoid arthritis and Castleman disease as well as diabetes, atherosclerosis, depression, Alzheimer disease, systemic lupus erythematosus, multiple myeloma, and in particular cancer, where its production has often been associated with the presence of cancer stem cells. More than a decade ago, this evidence led to the development of anti–IL-6 targeting agents including the prototypical tocilizumab, a humanized monoclonal antibody targeting IL-6 receptor, initially approved for the treatment of Castelman disease and rheumatoid arthritis.[8]

More recently, IL-6 has been shown to be pivotal in driving the cytokine release syndrome (CRS), a form of systemic hyperinflammatory response that can be triggered by several factors such as infections, like SARS-CoV-1, and certain therapies such as adoptive T-cell therapies with chimeric antigen receptor T-cell (CAR-T). It occurs when large numbers of white blood cells are activated and release inflammatory cytokines including IL-6 (one of the most abundant), which in turn activates more white blood cells. The inflammatory cascade induces endothelial activation, increased coagulation, and capillary leak, which together cause circulatory collapse and shock. It is for these reasons that tocilizumab has currently been approved for the treatment of CRS, which is one of the most frequent and serious adverse events following CAR-T administration.[9]

Also, because IL-6 production strongly increases during COVID-19—with its levels increasing proportionally with disease gravity—tocilizumab and other IL-6/IL-6R blockade molecules have been at the center of several clinical trials with promising, although inconclusive, results so far.[10]

IL-6 AND ACUTE/CHRONIC VIRAL INFECTION

High levels of IL-6 in COVID-19 are not surprising, as many viruses can alter IL-6 levels during acute and chronic infection. One of the essential roles of IL-6 in producing a proper immune response during certain viral infections has been put forward, and a link to this cytokine with exacerbation of viral disease was reported.[11] Thus, IL-6 seems to exert a bimodal effect on viral infection by inhibiting or promoting virus survival, as suggested by the exacerbation of clinical disease in certain viral infections.

This pleiotropic cytokine produced by multiple cell types in response to tissue damage and infection[12,13] exerts a key role along with a few others (ie, IL-1 and TNF-α) in modulating the host immune response.[14] However, the first evidence of IL-6′s role in the progression of viral infection stems from animal studies. Disruption of the IL6 gene impairs specific cytotoxic T-cell response and production of specific immunoglobulin G (IgG) antibodies in mice infected with vaccine virus and vesicular stomatitis virus, respectively.[15] In another mouse model, in which IL-6 activity was blocked by monoclonal antibodies, T-helper and B-cell responses were reduced during the late stages of infection by lymphocyte choriomeningitis virus, impairing viral clearance.[16] In addition, in IL-6–deficient mice, IL-6 is essential for mouse survival upon influenza virus infection. Among the pleiotropic functions of this cytokine are many prosurvival activities. These include optimal promotion of T-cell response, IgG isotype switching, lung tissue repair, inflammatory resolution, and prevention of virus-induced apoptosis in lung epithelial cells.[17,18]

Viruses can display a variety of pathogen-associated molecular patterns that may stimulate IL-6 together with other proinflammatory cytokines. These patterns can be discerned through warning signals displayed by infected cells with different immune cellular pathogen recognition receptors (TLRs, DNA receptors, retinoic acid-inducible gene-1–like receptors, nucleotide-binding and oligomerization domain–like receptors).[12,19] Proof of the importance of these interactions comes from studies on NS4B protein of flavivirus. In particular, it has been reported that specific mutations in a TLR-like structure within NS4B of classical swine fever virus (CSFV) can attenuate virulence in pigs. In addition, sustained accumulation of IL-6 in infected pig tonsils was observed. In this model, exogenous IL-6 inhibited CSFV replication in peripheral blood mononuclear cells, the natural target cell of this flavivirus. It is worthy to note that hepatitis C virus, belonging to the flavivirus family, is tightly regulated by IL-6 levels, and progression to chronic infection seems to be linked to IL-6 promoter polymorphisms.[20] The same antiviral effect of IL-6 was also reported for hepatitis B virus (HBV), as shown in hepatoma cell lines where IL-6 suppresses virus replication and decreases the number of viral genome–containing nucleocapsids.[21] In addition, the repressive effect of IL-6 on HBV replication is mediated by loss of HNF1α and HNF4α binding to covalently closed circular DNA (cccDNA) and redistribution of STAT3 binding from the cccDNA to IL-6 cellular target genes, leading to the alteration of epigenetic control of the nuclear cccDNA mini-chromosome and HBV transcription.[22] IL-6 can block HBV infection by inhibiting HBV receptor expression in hepatocytes of the human liver.[23] It is currently thought that IL-6 increases the activity of Enh1 to control HBx expression and HBV replication through activation of the IL-6R/gp130/STAT3 signaling pathway.[24] On the other hand, HBx (in combination with IRAK-1, p38/ERKs, or NF-kB) can activate the IL6 gene, thus stimulating IL-6 protein synthesis.[25,26]

HPV infections elicit production and release of several inflammatory cytokines from the main target cell types, namely keratinocytes and skin fibroblasts, as well as different components of the innate and adaptive immune response including macrophages, natural killer cells, and lymphocytes. In a study on cytokine expression in HPV-immortalized cells, human keratinocytes immortalized with E6 and E7 genes from carcinogenic alpha HPV16 and beta HPV38 showed high levels of mRNA and protein IL-6 expression,[27] indicating that the presence of E6/E7 from high-risk HPVs can increase IL-6 expression. Interestingly, the target proteins of high-risk E6 and E7—p53, and Rb—regulate the expression of IL-6 negatively, raising the question of whether the degradation of these proteins may be the main mechanism of IL-6 induction by high-risk HPVs. Recent evidence suggests that this process could be modulated by E6 through induction of p53 degradation but also by a mechanism independent from p53.[28] However, IL-6 (as a 2-faced Janus) might have potential negative consequences on the cellular immune response against viruses.

In this setting, different potential mechanisms involving IL-6 might affect viral clearance, ultimately favoring the establishment of a persistent-state viral infection and, in the case of carcinogenic viruses (ie, hepatitis B and C or HPV), neoplastic transformation (Fig. 2). One mechanism could be represented by the ability of IL-6 to negatively regulate effector CD8 T-cell response after T-cell activation. This inhibition is orchestrated by IL-6–mediated CD8⁺ T-cell stimulation, by upregulation of phospho-STAT3 and by the suppressor of cytokine signaling (SOCS3), along with STAT4 phosphorylation and T-bet downregulation.[29] These data were confirmed in vivo by using a monoclonal antibody against IL-6 during acute infection in mice using a murine leukemia virus with reduced viral loads, with increased production of IFN-γ and granzyme B.[29] This negative regulation of virus-specific CD8 T-cell activity is also achieved by the synergistic interaction between IL-6 and interleukin 17 (IL-17), which has been associated with viral persistence in the experimental model of Theiler murine encephalomyelitis virus (TMEV) infection. The synergistic interaction between IL-6 and IL-17 leads to induction of anti-apoptotic molecules (Bcl-2 and Bcl-xL), inhibiting the destruction of TMEV-infected cells by virus-specific CD8⁺ T cells (Fig. 1).[30] The inhibition of apoptosis is also linked to Fas-FasL pathway blockage by IL-17.[31] In addition, Th17 cells may exert potential detrimental effects (ie, prevention of Th1 cells; inhibition of IL-2 and IFN-γ production) during viral infections caused by a variety of viruses including coxsackie virus, hepatitis C virus (HCV), herpes simplex virus, and influenza virus.[32] Finally, in the above-cited model of experimental TMEV infection, excessive production of IL-6 resulted in upregulation of PD-1 and PDL-1 molecules, which may reduce CD8⁺ cytolytic function.[33] Indeed, T-cell ligation of PD-1 by PDL-1, expressed on chronically infected cells, alters immunity against viruses by preventing T-cell expansion (Fig. 2).[34]

Figure 2.

Schematic representation of interleukin-6 (IL-6)–mediated connection between SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) and cancer-associated virus infections. The release of IL-6, induced by SARS-CoV-2 infection, could promote the establishment of a viral persistent state in different ways: 1) through a negative regulation of CD8⁺ T-cell response determined by the upregulation of phospo-STAT3 and SOCS and by the downregulation of phospo-STAT4 and T-bet; 2) through the induction of apoptosis in the infected cells promoted by IL-6 and IL-17 synergistic interaction; 3) through the promotion of Th17 cells in the Th1/Th17 axis; and 4) through the increase of PD-1 and PDL-1 expression, leading to the inhibition of T-cell expansion. Bcl-2: B-cell lymphoma 2; Bcl-xL: B-cell lymphoma-extra large; HBV: Hepatitis B virus; HCV: Hepatitis C virus; HPV: Human Papilloma Virus; IL-6: Interleukin 6; IL-17: interleukin 17; JAK: Janus kinase; PDL1: programmed death-ligand 1; SOCS: suppressor of cytokine signalling; STAT: signal transducer and activator of transcription; T-bet: T-box transcription factor TBX21; Th1: T helper 1; Th17: T helper 17.

Besides SARS-CoV-1[35] and CoV-2,[36] other preclinical and clinical studies have already reported that an increase in systemic IL-6 levels exacerbate clinical outcomes during viral infections. High serum levels of IL-6 have been detected in patients chronically affected by hepatitis B and C viruses,[37,38] HIV,[39] Chikungunya virus,[40] influenza virus,[41] Andes virus,[42] and Crimean-Congo hemorrhagic fever virus.[43] However, the high IL-6 levels observed may be a result of ongoing active infection that fails to be cleared or the consequence, rather than the cause, of viral persistence. Nevertheless, even local production of IL-6 increases during viral infection. In patients with HIV, high levels of IL-6 in ectocervical tissues are correlated with enhanced transcriptional levels of HIV-1.[44] Moreover, a transcriptome analysis of persistently infected pharyngeal tissues collected from cattle with foot-and-mouth disease virus showed a local increase of IL-6 expression,[45] suggesting that overexpression of IL-6 might be a mechanism favoring virus persistence. Interestingly, the tonsils are a hot spot of HPV persistence and cancer transformation, where the role of IL-6 in head and neck squamous cell carcinoma progression is well established.[46] However, studies on the direct relationship between IL-6 and HPV in tonsils are still lacking. Experimental evidence suggests that viral chronic infections might be induced by overexpression of IL-6, which impairs the polarization of Th1 cells and lytic capacity of CD8 T cells. The constant stimulation of viral antigens during viral persistence may in turn produce unresponsive CD8 T cells that fail to develop into memory CD8 T cells, thus limiting viral clearance.[47]

Notably, this experimental evidence has been clearly confirmed in a recent large study conducted on COVID-19 patients where high IL-6 serum levels correlated with low CD8⁺ T-cell counts and mortality.[48]

In summary, at least three major mechanisms may be advocated to explain the potential role of IL-6 in impairing viral clearance and favoring chronic infections: 1) reduction of apoptosis in infected cells by increased IL-17 production from the cellular environment that, in turn, may induce production of anti-apoptotic Bcl-2 and Bcl-xL proteins, thus favoring their survival; 2) reduction of CD8 and NK activation by decreased IFN-γ production via SOCS3 induction, which affects STAT4 phosphorylation; and 3) unbalanced Th1/Th2 stimulation favoring Th2 polarization of CD4 cells via stimulation of the STAT3 pathway, which increases IL-4 production and SOCS1 expression. SOCS1 may affect STAT1 phosphorylation, leading to impaired IFN-γ production. Importantly, these hypothetical mechanisms have to be experimentally validated. Further studies must be carried out to provide a confirmatory role for IL-6 in the genesis and/or maintenance of chronic viral infections. Finally, increased levels of IL-6 might also trigger immunopathology mechanisms during chronic infections through increased inflammation signals,[49] providing new cellular targets for subsequent viral infections.[50] Furthermore, it is assumed that viral IL-6 upregulation is functional to immune evasion strategy, although there is no scientific evidence for the causal relationship between IL-6 levels and virulence.

SUMMARY

In conclusion, there are several sets of data supporting the significant role IL-6 plays during viral infections. However, some viruses overcome the immune response by using different evasion strategies,[51] consequently upregulating IL-6 production as a result of increased viral loads, whereas polymorphisms in the IL6 gene promoter may explain overexpression of IL-6 during the immune response, leading to the progression of infection as reported for hepatitis B and C viruses.[52] Thus, different conditions may alter IL-6 production, which in turn may be detrimental to the cellular immune response during viral infections.

FINAL REMARKS

The key concepts derived from the data herein are reported below.

1. Owing to its pleiotropic effects, IL-6 alters in many—and in some instances, opposite—ways the fate of infections caused by several viruses including cancer-associated viruses (eg, HCV, HPV, HBV).

2. During the COVID-19 pandemic, clear sets of data have highlighted IL-6 as one of the main players of the cytokine storm triggered by SARS-CoV-2; its inhibition for therapeutic purposes through monoclonal antibodies (tocilizumab) may be responsible for the reactivation of chronic hepatitis virus infections. Even short periods of IL-6 deregulation can alter the body's response to chronic infections, which may develop into cancer.[53] In patients with COVID-19 treated with tocilizumab, HBV and herpes simplex virus 1 infections were reactivated.[54]

3. IL-6 may favor chronic infection by cancer-associated viruses by altering the balance of the inflammatory response.

Future studies are required to address and clarify whether patients with COVID-19 could be at risk of virus-associated cancers, and therefore would possibly need dedicated follow-up. One possible answer to this hypothetical question may come from epidemiologic evaluation of the postpandemic rise in chronic viral pathologies (hepatitis, cirrhosis, warts), and above all of precancerous lesions (cervical intraepithelial cancer, anal intraepithelial neoplasia, vulvar intraepithelial neoplasia) and/or virus-associated neoplasms (liver cancer, cervix, anal, or oropharynx). While awaiting these data, patients with COVID-19 should be monitored on a long-term basis for other additional conditions including malignancies.

Funding Statement

Source of Support: Supported in part by grant of Research Project Regione Lazio - Lazio-Innova, funded under L.R. 13/08.