Association of IL-13 in respiratory syncytial virus-induced pulmonary disease: still a promising target

Respiratory syncytial virus: a problem for the young & old

Respiratory syncytial virus (RSV) is a negative-strand RNA virus and a member of the Paramyxoviridae family, genus Pneumovirus. It infects nearly all infants by the age of 2 years and is the leading cause of bronchiolitis in children worldwide. It is estimated by the CDC that up to 125,000 pediatric hospitalizations in the USA each year are due to RSV, at an annual cost of over US$300 million [1–3]. However, the elderly also succumb to RSV infections, often leading to pneumonia [4,5]. Thus, while RSV is especially detrimental in very young infants whose airways are small and easily occluded, it is also still recognized as an important pathogen in patients with asthma, transplant recipients, patients with chronic obstructive pulmonary disease (COPD), the elderly, as well as other patients with chronic lung disease. During asthmatic exacerbations, RSV can cause prolonged episodes of illness and recent data suggest that episodes of acute exacerbations of COPD due to RSV cause approximately 10,000 deaths in persons over the age of 65 years in the USA per year [3]. The combined annual average mortality due to RSV is predicted to be over 17,000 in the USA. These numbers are probably grossly underestimated, as RSV has not been examined in adults in a consistent manner. Thus, RSV not only causes significant exacerbated lung disease in young and old patients, but it is also directly associated with significant levels of mortality.

One clinically relevant feature of RSV disease that may predispose children to later childhood pulmonary disease is the inability to acquire protective immunity due to an altered immune response [4,5]. Individuals previously infected with RSV can subsequently be reinfected with the same clinical isolate at a later time. This may be due to something inherently dysfunctional with the programming of the memory response to RSV. With repeated infections, protective immunity can develop but this may also come at a cost of profoundly altering the immune environment in the lung. Patient populations with compromised lung function (especially COPD patients) are at a high risk of severe complications due to RSV infection. This risk is nearly as prevalent as those associated with influenza infections [6–10]. RSV compounds these complications further due to its ability to persist for long periods in the lung even after the acute disease has been resolved. This is thought to be associated with the development of an altered immune environment that clears the virus less efficiently [11–13].

Some approaches utilizing antibodies and vaccines have been attempted in the past to alleviate RSV infection-associated disease but have offered very limited success. Anti-RSV antibodies are available and appear to alleviate severe disease; however, they perform best when given by a prophylactic protocol. On the contrary, palivizumab prevents the development of severe RSV illness in certain high-risk infants and children but is unable to prevent infection with RSV [14]. Importantly, the cost associated with prophylaxis treatment with palivizumab far exceeds the cost savings from reduced hospitalization [15,16]. Moreover, no safe and effective vaccine is available [17,18]. The repeated attempts of vaccine development against RSV have been hampered by the problems inherent in neonatal immunization [19] as well as a history of vaccine-enhanced illness upon reinfection with the virus. The latter incident occurred during the late 1960s when children were vaccinated with alum-precipitated formalin-inactivated RSV vaccine (FI-RSV) [20,21]. The clinical manifestations were a result of priming with FI-RSV that led to the enhanced Th2 lymphocyte response, mucus production and eosinophilia following challenge. Moreover, steroid treatment of children with severe RSV disease showed no difference in cytokine levels in tracheal aspirates [22]. Thus, there is also a lack of clinical benefit of steroid treatment in children with severe RSV bronchiolitis.

Antiviral immune responses to RSV

The innate immune system has evolved mechanisms to detect characteristics of viral nucleic acids that are either distinct in structure (dsRNA) or in subcellular location (ssRNA). Since RSV is an ssRNA negative-sense virus, both ssRNA and dsRNA species are formed and can be targets for the innate immune system. Recognition of the virus induces two distinct responses. First, the viral nucleic acids activate the innate immune system via Toll-like receptors (TLRs) triggering the induction of type 1 interferons (IFNs). Type 1 IFNs elicit an antiviral state in virally infected cells and activate immunoregulatory functions in nearby cells. Second, TLR activation induces important early mediators such as chemokines (RANTES, also known as CCL5, and monocyte chemotactic protein 1) and cytokines (IL-6 and IL-12), which tailor the adaptive immune T-cell response to the invading pathogen. However, RSV-specific T cells can be both protective and pathogenic. Studies in mice with depleted CD4 and CD8 T cells have shown that RSV persists for several weeks but no overt disease symptoms are observed, suggesting a pathogenic role for T cells during the infectious disease response [23]. This latter observation rests on the fact that although individually both CD4 and CD8 contribute to terminating RSV replication and increasing viral clearance, it comes at a cost of significant immunopathology. However, CD8 T-cell depletion studies have shown enhanced Th2 responses during RSV infection, demonstrating that CD8 T cells may also provide important regulatory and protective signals, such as IFN-γ.

As previously mentioned, during RSV infection the activation of TLRs on dendritic cells triggers IL-12 production and induces the differentiation of IFN-γ-producing Th1 cells. However, pathogen-induced Th2 (IL-4 and IL-13) responses tend to arise in the absence of TLRs, leading some to suggest that Th2 differentiation is a pathway that is triggered in the absence of IL-12. Not all Th1-inducing stimuli default to Th2 responses in the absence of IL-12, indicating that other signals may exist on antigen-presenting cells to elicit a Th1 response. The strongest evidence for this possibility comes from recent studies showing that the expression of Notch ligands delta-like and jagged can provide these previously unknown instructional signals for the development of Th1 and Th2 cells, respectively [24]. Recently, our laboratory has shown that RSV infection of dendritic cells upregulates the expression of notch ligand Dll4 that modulates the Th2 response without affecting the Th1 response both in vitro and in vivo[25]. In addition, neutralization of Dll4 enhanced Th2 responses with increased IL-4 and IL-13 in the presence of RSV, resulting in mucus overproduction. Thus, it is likely that multiple mechanisms control the outcome of the immune responses during RSV infection.

Data from our laboratory support IL-17 production during RSV infection in mice, as has been reported previously by Hashimoto et al. IL-17 is produced by another subset of T helper cells known as Th17 cells [26]. The intensity of IL-17 production during RSV infection may also depend upon Dll4 as under primary skewing conditions Dll4 augments IL-17 production [27]. Previous studies have demonstrated an association of RSV-induced IL-17 production during dysregulated exacerbation of allergic airway responses in mice, further supporting a role for IL-17 in the pathogenesis of RSV-induced disease [26]. Although IL-17 has been implicated in the induction of mucin genes, its role in mucus production during RSV disease pathogenesis is unclear, but recent unpublished data from our laboratory indicate that IL-17 may contribute to mucus hypersecretion. Thus, it may be that the most devastating immune environment that can be elicited by RSV is one that has both IL-13 and IL-17 induced and leads to an intense mucus secretion profile. The overproduction of mucus, while in itself complicating, would greatly exacerbate the disease condition in patients with underlying pulmonary problems, such as asthma and COPD patients.

The different shades of IL-13 during RSV infection

IL-13-induced airway hyperreactivity

Several cytokines have been identified in the last decade that orchestrate the progression of lung damage by setting up the appropriate inflammatory milieu after RSV infection, leading to long-term pulmonary problems. One of these cytokines in particular is IL-13. It has been associated with increased mucus production, goblet cell hypertrophy and airway hyperreactivity (AHR) in animal models [28,29]. These pathophysiologic outcomes and the augmented pulmonary dysfunction are the end result of increased IL-13 in the lung acting in concert with other interacting factors such as tissue damage and airway remodeling response during the resolution phase of RSV infection [30–32]. IL-13-induced overproduction of mucus in itself can be a pathologically devastating event that can lead to airway congestion and lung dysfunction [33,34]. This observation was supported by studies utilizing IL-13-transgenic mice with targeted IL-13 overproduction in the lung that resulted in an overabundance of mucus production, severe AHR and end-stage fibrosis around the airway [35]. Subsequently, studies with a neutralizing antibody for IL-13 showed significant inhibition in mucus production and AHR post-RSV infection [36]. These data further demonstrate that IL-13 may not only be a contributing factor, but also the primary cytokine that initiates RSV-induced lung dysfunction. Although historically IL-4 is associated with severe Th2 responses, during a primary RSV infection very little IL-4 is produced in the lung. Interestingly, physiological changes observed during RSV infection can also be independent of IL-4 [37]. An important characteristic of RSV is its strong propensity for inducing airway responses that exacerbate or may even promote asthma-like inflammation. To this end, IL-13 is also a major activating factor mediating exacerbation of allergic responses [36].

Immunomodulatory role of IL-13

In addition to its role in causing a severe immune response to RSV, IL-13 also has an immunomodulatory role. In animal studies, neutralization of IL-13 using an IL-13-specific polyclonal antibody resulted in a concomitant increase in IL-12 production, suggesting a regulatory effect of IL-13 on IL-12 production. IL-12 has been linked to antiviral responses to RSV and other viruses and is known to effect viral antigen clearance. Thus, a potential mechanism could be alteration of IL-12 production as a result of altered IL-13 production, which in turn results in an alteration in viral antigen clearance and leads to prolonged persistence of viral antigens. This is specifically harmful to the host as the continued presence of RSV-specific antigens can damage epithelial cell barriers in the airways, resulting in AHR [38–40]. These observations were supported by studies demonstrating a decrease in IL-12 production in children who developed RSV-induced bronchiolitis [41]. The function of IL-12 during RSV infection may be both direct for activation of natural killer cells and cytotoxic T-cell responses, as well as indirect for induction of IFN-γ, which has been shown to directly activate antiviral responses. Interestingly, while IL-13 neutralization in animal studies reduces several pathophysiologic parameters, it does not alter IFN-γ production and therefore IL-13 may not directly alter this aspect of antiviral immunity. Together, these data suggest that the IL-12/IL-13 axis may be critical in determining the severity of lung dysfunction associated with AHR, goblet cell hyperplasia and mucus production during RSV infections. By contrast, Zhou et al. demonstrated that IL-13 is associated with reduced illness in terms of weight loss of mice that were infected with RSV during the primary response and had favorable effects on viral clearance [42]. The differences in the results were attributed to the strains of virus that were utilized to induce RSV infections [42]. Later studies confirmed the detrimental role of IL-13 in RSV disease [43]. A study using neonatal RSV infection followed by subsequent reinfection later in life with RSV demonstrated that early RSV infection exacerbates RSV-induced disease in adults. The study further established that enhancement of AHR, pulmonary eosinophilia and mucus hyperproduction during reinfection were dependent on IL-13 [44].

IL-13: mouse to humans

In humans, two critical syndromes have been associated with RSV infections that suggest a role for type 2 cytokines in RSV pathogenesis: airway mucus production and the presence of eosinophilic cationic proteins in the nasal secretions. These disease-contributing parameters have been carefully studied using animal models of RSV disease. Studies in rodent models of RSV infections have established that there is increased IL-13 production during the development of the disease [29,36,45,46]. Focusing upon eosinophilia, Johnson et al. demonstrated that IL-13 is causative for RSV-G glycoprotein-induced eosinophilia after RSV infection [47]. It was further reaffirmed by Castilow et al. that IL-13 is required for eosinophil entry into the lung during RSV vaccine-enhanced disease [48]. These observations were extended to demonstrate that the recruitment of eosinophils into the lung parenchyma was shown to be dependent on IL-13-induced chemokines CCL22 and CCL11. The studies highlight the contribution of IL-13 during a secondary response to RSV in mice previously immunized with the G protein [48].

In humans, IL-13 and Th2 responses have also been closely linked to disease severity. Sera of RSV-infected children have skewed Th2 cytokines with reported increases of IgE, IL-5 and IL-13 [49]. To this end, increased RSV-specific T-cell response in infants with severe RSV bronchiolitis causes episodes of wheezing and the development or exacerbation of asthma later in childhood, especially in infants with a family history of atopy [50,51]. A genetic abnormality due to functional IL-13 polymorphism can also contribute to complications later in life [52]. Studies with Gambian children at 5 years of age have shown that severe RSV infection in early life is associated with a higher production of type 2 cytokines [53]. A cohort study of children with severe RSV infection during the first year of life with a 5-year follow-up reported that patients with severe RSV disease produced significantly higher IL-13 concentrations in response to tuberculin, and had skin response to allergens, airway reactivity and increased serum IgE concentrations. In another study, Sigurs et al. compared 47 children hospitalized with RSV bronchiolitis and 93 healthy children at the mean age of 1 and 3 years [50]. The same group of children were studied at 7.5 years of age and the study concluded that RSV bronchiolitis had a higher risk for the development of asthma and also a significantly elevated risk ratio for allergic sensitization. Studies investigating age-related differences in cytokine responses to RSV infection have reported increased levels of IL-13 levels in children over 12 months of age compared with children aged younger than 6 months. Thus, very early in life, age-related differences in immune responses to RSV infection can be observed and developmental changes in cytokine responses to RSV infection may be considered in the control of RSV bronchiolitis in young children [54]. To establish the course of the immune development, the RSV Bronchiolitis in Early Life cohort study offered a unique opportunity to follow the changes in Th1 and Th2 cytokine production during the first 6 years of life after severe RSV bronchiolitis, and to investigate the subsequent development of asthma, allergic sensitization and eczema. The study found that Th1 cytokines in peripheral blood mononuclear cells tend to decrease and Th2 cytokines tend to increase over time after initial severe RSV bronchiolitis [55]. Thus, while there are clearly multiple mediators that are involved in the generation of RSV-induced disease, evidence in both humans and animal models indicates that a major pathogenic factor in acute and long-term disease sequelae is IL-13.

Concluding remarks

The role of IL-13 during RSV disease progression in mouse models correlates with the presence of IL-13 in human disease phenotypes, suggesting it to be a putative target during disease. In addition to the association of IL-13 with AHR and mucus production in the airways, its immunomodulatory role during RSV infections may also compound the detrimental immune environment that develops, further complicating long-term disease during RSV infection. It is also important to recognize that other mechanisms such as regulation of IL-13 by notch and the association of IL-13 with IL-17 during RSV infections are additional unexplored areas that will further define the pathogenic mechanisms involved in pulmonary disease. Thus, the therapeutic potential of targeting IL-13 to alter the detrimental physiological and cytokine responses in the lung may help alleviate the long-term lung damage that initiates and/or maintains chronic lung problems following RSV infections.

Financial & competing interests disclosure

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

No writing assistance was utilized in the production of this manuscript.