When defense backfires: inflammatory determinants of RSV-induced disease severity

Abstract

Respiratory syncytial virus (RSV) poses a widespread global burden that typically causes mild, cold-like symptoms in infected healthy adults. However, in vulnerable populations, such as the very young or older adults, RSV infection can lead to severe and potentially fatal disease. Both viral and host factors play a dynamic role in determining disease severity. In the host, dysregulation of both the innate and adaptive immune responses can result in unrestrained viral replication and excessive inflammation. Insight has been gained into the host factors that drive more severe disease following RSV infection using animal models and clinical studies. In this review, we discuss both clinical and in vivo animal model findings to elucidate host immune responses that contribute to disease severity, highlighting pathways that may serve as beneficial therapeutic targets. PubMed, ScienceDirect and Google Scholar were accessed to search for articles up to June 2026. Eligibility criteria included in vitro, in vivo, and clinical studies evaluating the factors associated with RSV disease severity, published in peer-reviewed journals.

1. Introduction

Respiratory syncytial virus (RSV) is a single-stranded, negative-sense RNA virus that exhibits high prevalence in the first year of life [1]. By age two, approximately 90% of children have been infected by RSV and 40% of these cases result in lower respiratory tract infection (LRTI) [2]. It is estimated that approximately 10% of RSV-induced LRTIs will require hospitalization [3]. RSV is a leading cause of mortality of children under 5 years of age. By age 3, virtually all children have been infected by RSV at least once, though reinfection is common due to waning of mucosal IgA along with suboptimal T cell immunity [4–7]. While RSV infection causes an enormous pediatric burden, it is also recognized as a source of severe disease in adults over 65 years of age with an annual mortality rate that approaches that of influenza A [8,9]. RSV symptoms commonly include fever, middle-ear effusions, and lower respiratory illness such as bronchiolitis, tracheobronchitis, and wheezing. In the most severe cases, patients may develop pneumonia, hypoxia, apnea, and acute respiratory failure [10,11].

Several host factors may contribute to RSV disease severity. Age is a key factor that influences the severity of RSV disease. This is highlighted when examining hospitalization trends in children under five years of age which show the highest numbers of hospitalization in the six months and under age group [12]. Premature birth also increases the risk of severe RSV infection as preterm infants are more likely to develop RSV-induced bronchiolitis than full-term infants [13,14]. Additional host factors, especially in young children, also include chronic medical conditions such as lung disease and congenital heart disease [15,16]. Hypersensitivity of the airways is another significant factor, as an increased presence of inflammatory cells in the airways contributes to chronic airway inflammation and structural changes. Chronic pulmonary inflammation can lead to the rapid loss of lung function over time, such as permeability of the protective barrier and damage to the airway epithelium, as well as increased RSV replication in mucus-secreting goblet cells which are more prevalent in hypersensitive airways [17]. Preexisting chronic diseases, such as asthma, can also influence RSV disease severity, and it has been proposed that genetic predisposition to asthma, even if presentation of asthmatic symptoms has yet to occur, may increase RSV disease severity [18,19]. Thus, there are many host factors that can alter the host response to infection which may combine to impact RSV disease severity.

2. The initial battle: innate sensing and early antiviral defense

As RSV enters the host, the virus is initially detected by sentinel cells of the innate immune system. RSV expresses multiple pathogen-associated molecular patterns (PAMPs) recognized by innate pattern recognition receptors (PRRs) that are expressed both on the outside and inside of cells. Upon activation of these PRRs early innate cell responders, such as dendritic cells (DCs), macrophages, neutrophils, and natural killer (NK) cells, produce cytokines and chemokines that promote an inflammatory response and immune cell infiltration to the infection site. Although the initial inflammatory response is critical for controlling viral replication, the host inflammatory response must be tightly regulated to limit tissue damage to the host. Thus, the host immune response to RSV infection plays a vital role in determining RSV disease severity.

2.1. Type I and type III interferon response

Type I interferons (IFNs) consist of the many subtypes of IFN-α as well as IFN-β. Upon recognition of RSV viral proteins, type I IFNs are induced as one of the initial host inflammatory responses. Recent clinical studies indicate that type I IFN levels peak early after infection even prior to the onset of symptoms [20,21]. Low IFN-α levels are associated with more severe illness, bronchiolitis, and respiratory failure following RSV infection in infants [22]. Nasopharyngeal samples from children under 2 years of age displayed lower type I IFN levels which correlated with more severe disease [23,24]. Thus, lower mucosal interferon concentrations were associated with more severe RSV disease in infants, suggesting type I IFNs play an important protective role during RSV infection.

One of the major family of PRRs involved in type I IFN production following RSV infection is Toll-like receptors (TLRs). Early studies demonstrated activation of TLR4 by the RSV fusion (F) protein, leading to the production of type I IFNs via Myeloid Differentiation Primary Response 88 (MyD88) signaling [25]. Along with TLR4, TLR7 has also been identified to activate the MyD88 adaptor in response to RSV, leading to downstream type I IFN production. TLR3 is also activated during RSV infection but primarily utilizes the Toll/IL-1R-domain-containing adaptor-inducing interferon-β (TRIF) adaptor protein to induce the production of type I IFNs [24,26]. A previous report identified that infants hospitalized with severe RSV bronchiolitis were found to have polymorphisms in TLR2, TLR4, and TLR9 which correlated with severity of disease and mortality rate [27]. These data indicate that dysregulation of TLR signaling dramatically alters RSV-induced disease severity. Additional investigation examining the impact of these TLR polymorphisms on the production of type I IFNs will further inform the connection between RSV-mediated TLR signaling and type I IFNs. In addition to TLRs, the RIG-I-like receptor (RLR) is also activated during RSV infection, and signals through the Mitochondrial Antiviral Signaling (MAVS) adapter protein, leading to the induction of type I IFNs. In the absence of either MAVS or RIG-I, mice exhibit increased viral replication during RSV infection [28,29]. The increased viral replication is likely due to the reduction in type I IFN production in the absence of either MAVS or RIG-I signaling, thereby further demonstrating the importance of the type I IFN response in controlling RSV infection.

Type I IFNs bind and signal through the alpha/beta IFN receptor (IFNAR) leading to inhibition of viral replication and subsequent direction of the host immune response. Activation of IFNARs induces the downstream activation of the Janus kinases/signal transducers and activators of transcription (JAK/STAT) signaling cascade, triggering the expression of interferon stimulated genes (ISGs). ISGs protect host cells against viral infection and help facilitate the initiation of an anti-viral CD8 T cell response [30,31]. Studies have shown that RSV-infected IFNAR1-deficient mice exhibit a marked decrease in expression of IFN-inducible genes as well as increased viral loads in the lungs, though both weight loss and airway obstruction, indicators of disease severity in mice, were ameliorated [32,33]. Therefore, while type I IFNs are critical in inhibiting viral replication, RSV-mediated activation of IFNAR signaling may contribute to increased disease severity.

Type III IFNs are made up of four IFN-λ subtypes that are also induced upon RSV infection. Production of type III IFNs is dependent on TLR3 as well as RLR activation and downstream signaling. While lung epithelial cells are known producers of type III IFNs, nasal epithelial cells as well as DCs and macrophages can also secrete type III IFNs [34,35]. RSV infection of the upper respiratory tract (URT) was shown to induce type III IFNs but not type I IFNs [35]. Samples taken from RSV-infected infants displayed an association between lower type III IFN expression levels and more severe disease [23,36]. The higher susceptibility of infants and young children to RSV infection may be due to impaired expression of type III IFNs [37]. Recently the type III IFN, IFN-λ4, was shown to be induced following RSV infection in a human airway epithelial cell line [38]. Recombinant IFN-λ4 treatment induced expression of ISGs and limited RSV infection. Moreover, IFN-λ signaling has been shown to play a vital role in RSV resistance, as mice lacking both the IFN-λ and IFNAR receptors showed enhanced susceptibility to infection [39]. Inhibition of IFN-λ resulted in increased lung inflammation, suggesting a protective role during RSV infection.

2.2. Master of innate immune inflammation: NLRP3 inflammasome

Inflammasome activation contributes to both viral clearance and the innate immune system’s response to viral infection. The Nod-like receptor family, pyrin domain-containing 3 (NLRP3) inflammasome is activated in response to RSV infection. Inflammasome activation is a two-step process that requires a priming signal followed by an activation signal. Priming results in transcription of inflammasome components, while activation leads to the formation of a multi-protein complex and subsequent cleavage and release of IL-1β and IL-18 from the cell. RSV activates multiple TLRs, which can serve as the priming signal. TLR2 and TLR4 are the primary TLRs implicated in RSV-induced inflammasome priming. Very recently, soluble RSV glycoprotein (G) was shown to prime the NLRP3 inflammasome through TLR2 signaling [40]. Alternatively, TLR4 was activated in response to stimulation with the RSV F protein [41]. The priming signal may vary depending on the host species, cell type studied, strain of RSV and soluble vs membrane bound viral proteins. This dynamic interplay between virus and host factors warrants further study and clarification. The RSV small hydrophobic (SH) protein, that serves as a viroporin, has been shown to act as signal two and activate the inflammasome complex by inducing membrane permeability [42]. Infection with an RSV strain lacking expression of the SH gene or chemical inhibition of the ion channel activity of the SH protein resulted in failure to induce secretion of IL-1β [42]. Both reactive oxygen species (ROS) and potassium efflux following RSV infection were also shown to drive NLRP3 inflammasome activation [43]. However, a specific RSV protein was not connected to inflammasome activation in this study.

Inflammasome activation has been shown to enhance the Th2 response and mucus production during RSV infection. NLRP3 inhibitor treatment decreased IL-1β levels in the bronchial alveolar lavage (BAL) as well as diminished production of Th2-associated cytokines such as IL-4, IL-5 and IL-13, as well as CCL2, IL-33, and Gob5 mucus gene expression [44]. NLRP3 activation also impacts innate cell recruitment, as inhibition of the inflammasome resulted in reduced total numbers of interstitial macrophages, DCs, neutrophils, and group 2 innate lymphoid cell (ILC2) but showed no difference in T cell numbers [44]. In concert with this data, inhibition of IL-1β signaling through blockade of the IL-1R also reduced mucus, cellular infiltrates and the Th2 response following RSV infection [45].

The inflammatory responses induced by NLRP3 inflammasome activation lead to increased production of the pro-inflammatory cytokines, IL-1β and IL-18, both of which contribute to lung inflammation and damage during RSV infection [46,47]. In clinical studies, IL-1β levels have been observed to be highest in the most severe cases of RSV. Studies have shown increased IL-1β expression in nasopharyngeal and tracheal samples from RSV-infected infants, though, to date no direct evidence of NLRP3 activation in clinical samples has been reported [36,45]. Considering the impact that NLRP3 inflammasome activation has on immune cell recruitment, cytokine production, and pathology during RSV infection, targeting the NLRP3 pathway may represent an appropriate target for therapeutic intervention.

2.3. RSV genes and genetic variation: impacts on inflammation and immune evasion

Evading activation of the host immune system allows viruses to replicate and spread to new hosts. The type I and type III interferon responses are critical for early host defense against viral infection. To replicate and transmit efficiently, RNA viruses, like RSV, must evade the host interferon response. RSV nonstructural proteins 1 and 2 (NS1 and NS2) have been shown to inhibit type I and III IFN production and signaling. NS1 has been shown to bind to transcriptional regulatory elements leading to the disruption of host immune response genes and results in downregulation of IFN induction and signaling [48]. NS1 and NS2 promote the degradation of important IFN signaling pathway elements such as RIG-I, interferon regulatory factor 3 (IRF3), signal transducer and activator of transcription 2 (STAT2), and TNF receptor-associated factor 3 (TRAF3) through the formation of a “degradasome” [49]. Furthermore, NS2 has been shown to bind to the N-terminal caspase recruitment domain (CARD) of RIG-I, inhibiting its interaction with MAVS and preventing downstream signaling [50].

In addition to antagonizing a type I IFN response, NS1 and NS2 may also affect Th17 lymphocyte proliferation, impair DC maturation, and promote a Th2 polarized response [51,52]. Infection of immature DCs with a recombinant RSV strain lacking expression of both NS1 and NS2 resulted in upregulation of activation, maturation and adhesion molecules including: CD38, CD54, CD80, CD83, and CD86. Additionally, in the absence of NS1/2 cells secreted significantly more TNF, IL-6, IL-10, CCL4 and CCL5 [53]. These findings demonstrate that NS1/2 suppress the host immune response during RSV infection. Expression of the NS1 protein was also shown to inhibit CD8 T cell activation and skew the CD4 T cells toward a Th2 phenotype [52]. RSV-mediated dysregulation of type I and III IFNs through NS1/2 may therefore play a multifaceted role in the severity of RSV disease by modulating both the innate and adaptive immune response.

NS1 and NS2 play a significant role in inhibiting the type I IFN response, however there are additional RSV proteins that exhibit immune-modulating activity. During infection, the RSV nucleoprotein (N) colocalizes with melanoma differentiation-associated protein 5 (MDA5) and MAVS in inclusion bodies, which are typically sites for viral replication [54]. The sequestration of these innate immune receptors with the RSV N protein leads to a decreased type I IFN response. Viral inclusion bodies have also been shown to colocalize with type I IFN signaling proteins such as TANK binding kinase 1 (TBK1) and IRF3 during infection with influenza A, Ebola and phlebovirus. To date, this has not been shown in RSV infection; however, the sequestration of TBK1 and IRF3 during infection with other RNA viruses highlights the need for further investigation. The RSV G protein, in its soluble form, alters the hosts cytokine response by mimicking the chemokine CX3C and outcompeting CX3C for binding to the CX3CR1 receptor. Impaired signaling through CX3CR1 led to a reduction in monocyte migration and adhesion following RSV infection [55]. Another major function of CX3CR1 singling is T cell recruitment. For that reason, the reduction in CX3C binding driven by the RSV G protein may offer RSV a replicative edge due to a dampened immune response. The RSV matrix (M) protein has also been implicated in the suppression of the host interferon response. The matrix protein was shown to bind to receptor of activated C kinase 1 (RACK1) leading to a reduction in IFN-β production [56]. Taken together, there are multiple RSV proteins capable of inhibiting the host cell immune response and cytokine production.

Both subtypes of RSV, A and B, exhibit significant genetic variability and diversity each year. Genetic variations can widely impact viral fitness and the host immune response. Mutations and even duplications frequently occur in the RSV G protein, which resides on the surface of the virion. Prior studies demonstrated that the G protein is not necessary for in vitro viral replication, however mutations in the G protein can impact disease severity and neutralizing antibody binding [57]. Moreover, the high mutation frequency makes G a difficult target for vaccination and monoclonal antibody therapy. The RSV F protein, although more highly conserved than the G protein, has also exhibited mutations at various antigenic sites. Studies examining these mutations mainly focus on the impact that these mutations have on antibody binding and less on changes in inflammation. However, given that both the RSV G and F proteins are known to activate host cell TLRs and impact inflammatory signaling pathways, it is reasonable to hypothesize that mutations in these proteins could dampen or enhance inflammation. Additional study of RSV’s genetic variability and its influence on host inflammatory responses will offer deeper insight into the mechanisms that drive increased disease severity.

3. Inflammation: bridging the innate and adaptive responses

Following innate inflammation and activation is the rise of the adaptive immune response. Antigen-presenting cells activate specific and potent CD4 and CD8 T cell responses against pathogens. Cytokine production can also influence the adaptive immune response and is essential to T cell function. The role of NF-κB, a major regulator of cytokine production, in shaping the T cell response has been well established. Innate cell types also utilize cytokine expression to induce tissue-environmental pressures to shape the T cell response via inflammation.

3.1. Group 2 innate lymphoid cells (ILC2)

ILC2s are a population of innate cells known to promote Th2 responses. They perform a key role in lung homeostasis and the initiation of inflammation as well as help facilitate the transition from an innate to an adaptive response. While ILC2s have been primarily characterized in studies of patients with allergic asthma, ILC2s have been shown to be potent promoters of airway inflammation and hyperresponsiveness in RSV-induced bronchiolitis [58,59]. Moreover, an elevated frequency of ILC2s in nasal aspirates has been associated with an increase RSV disease severity in infants [59].

ILC2s are activated by release of alarmins (IL-33 and thymic stromal lymphopoitin [TSLP]) by infected epithelial cells [58]. TSLP released by the infected epithelial cells enhances ILC2 proliferation and activation, leading to production of IL-13, which activates goblet cells resulting in mucus production and airway hyperresponsiveness (AHR) [58]. ILC2s are activated by IL-33 during RSV infection and are crucial for IL-13-mediated airway inflammation [60,61]. ILC2s may also promote RSV-induced Th2 CD4 T cell expansion and cytokine expression via OX40/OX40L interaction [62]. Recently, pulmonary ILC2s have been suggested to function as antigen-presenting cells to induce the activation of Th2 cells via the major histocompatibility complex (MHC) II pathway during RSV infection [63]. Thus, ILC2 activation of the Th2 immune response may contribute to more severe disease during RSV infection. Taken together, ILC2s are proinflammatory mediators capable of directing the adaptive immune response and have a substantial impact on RSV disease severity.

3.2. Nuclear factor-kappa beta (NF-κB)

NF-κB is a key transcription factor that is critical for the production of IFNs and has been shown to be essential for NLRP3 activation during RSV infection [46]. Nasal swabs taken from hospitalized infants with RSV-induced bronchiolitis displayed increased expression of NF-κB at admission compared to levels at discharge [64]. Other studies examining the transcriptome of airway neutrophils and monocytes collected from infants with severe RSV disease revealed an inflammatory phenotype characterized by elevated NF-κB signaling [65,66]. Airway epithelial cells isolated from nasal aspirates of infant donors expressed high IFN-λ levels when exposed to RSV which was regulated by NF-κB signaling [67]. Exacerbated production of proinflammatory mediators driven by NF-κB signaling is a significant factor in increased RSV disease severity.

RSV infection triggers TLR3 activation leading to downstream activation of NF-κB-dependent inflammatory gene regulatory network (GRN) by inducing bromodomain-containing protein (BRD)-4 to form a complex with the transcription factors NF-κB and RelA (also known as p65) [68]. All other TLRs recruit MyD88 to activate transcriptional gene regulators such as NF-κB and IFN regulatory factors [69]. TLR2 has been demonstrated to be activated by RSV infection, leading to NF-κB activation [43,69]. It has also been shown that RSV-activated NF-κB and IL-4/IL-13 activated STAT6 bind to the thymus and activation-regulated chemokine (TARC) promoter region to induce expression of CCL17 [70]. CCL17 recruits Th2 and regulatory T cells (Tregs) into the lungs and is associated with eosinophilic inflammation and disease [70–72]. NF-κB has also been shown to activate CCL5 gene expression during RSV infection of the respiratory epithelial cells [73]. CCL5 levels were found to be increased in both the lung and BAL, peaking first at 24 hours post-infection, with a secondary peak at day 7 post-infection [74]. CCL5 acts as a potent chemoattractant for T cells, macrophages and natural killer cells to the site of inflammation [74,75]. Thus, NF-κB activation during RSV infection results in the transcription of multiple inflammatory target genes.

RSV infection has also been shown to impair p53 transcriptional activity during the infection cycle via proteasome-dependent degradation and induces phosphorylation of p65, leading to the up-regulation of NF-κB and dysregulation of several cytokines and chemokines [76]. RSV has also been shown to sequester the p65 subunit to inclusion bodies, impeding translocation to the nucleus and inhibiting interferon signaling, which may play a role in enhancing viral replication [77]. Inhibition of NF-κB signaling using an IκB inhibitor reduced chemokine gene expression and airway inflammation following RSV infection [78]. RSV-induced NF-κB activation elicits the release of inflammatory cytokines and chemokines that drive immune responses and causes damage to the respiratory tract which can contribute to increased RSV disease severity.

4. The adaptive immune dichotomy: protection vs. pathology

T cell-mediated protection versus pathology represents a delicate balance between controlling pathogens and preventing tissue damage as depicted in Figure 1. A protective response clears infection and builds memory; however, a pathogenic response can occur when the immune response is dysregulated or excessive. Proper regulation of the T cell response is important to avoid inducing immunopathology following RSV infection. Induction of different T cell subsets can alternatively help or hinder RSV clearance and their role in disease severity has been a source of debate. The overall balance between a Th1 and Th2 response is a critical determinant of RSV disease severity, as a response more skewed toward a Th2 phenotype has been associated with more severe disease. Likewise, the presence and function of Tregs is necessary to avoid excess inflammation and tissue damage as well as prevent the induction of a pathogenic Th17 response. Thus, there are many factors that play a role in inflammation during RSV infection.

Figure 1.

Imbalanced immune responses to RSV drive immunopathology. From left to right: Pathogenic – Th2 Driven. An immune response that is heavily skewed toward Th2 during RSV infection leads to immunopathology. RSV NS1 and NS2 proteins inhibit early type I IFN signaling in addition to impairing DC maturation and function. Th2-associated IL-4 delays viral clearance and contributes to airway hyperresponsiveness (AHR). NS1/NS2 also inhibit T cell activation and production of IFN-γ, leading to an imbalance of the Th1 and Th2 response and inhibiting viral clearance. Protective – Balanced Th1/Th2. In a protective response against RSV there is early induction of the type I IFN response. This early induction leads to control of viral replication, maturation of DCs and activation of alveolar macrophages. Inflammatory cytokines, including IL-1β, are secreted by alveolar macrophages and other innate immune cells. Th1 cells and cytotoxic CD8 T cells are primed, produce IFN-γ and aid in viral clearance. Regulatory T cells limit CD8 T cell activation and cytokine production thereby limiting immunopathology. Pathogenic – Inflammation. A pathogenic response to RSV can also be driven by excessive inflammation. Factors that can contribute to excessive inflammation are viral strain, host genetics and age. Secretion of IL-33 by RSV infected epithelial cells enhances ILC2 activation and proliferation. IL-13 production by ILC2s cells increases mucus production further contributing to AHR. Robust TLR and inflammasome activation in alveolar macrophages results in significant levels of IL-1β and IL-6, which are all correlated with severe disease. In addition, IL-6 polarizes CD4 T cells to a Th17 phenotype. IL-17 production by Th17 cells also leads to increased mucus production. CD8 T cells are a significant contributor to immunopathology through the secretion of IFN-γ and TNF.

4.1. Th1 immune response

CD4 Th1 differentiation is driven by the production of IL-12 and maintained by the cytokines IFN-γ and IL-2. These cells readily secrete IFN-γ and aid in clearance of intracellular pathogens, including viruses. Clinical studies have suggested that weak Th1 polarizing signals and increased Th2-associated cytokines, such as IL-4, promotes severe disease. Studies have shown that infants with more severe illness exhibit decreased levels of IFN-γ both systematically and in the respiratory tract compared to infants with mild illness. When examining the need for ventilation, lower levels of IFN-γ were associated with an increased need for oxygen support [79,80]. Multiple studies have demonstrated that decreased IFN-γ production is associated with more severe disease, though other studies claim a combination of decreased Th1 and increased Th2 responses contribute to increased disease severity [22,36,79]. Thus, IFN-γ plays a critical role in balancing the immune response and preventing severe disease during RSV infection.

Animal studies have also highlighted the importance of the Th1 response in reducing disease during RSV infection. IFN-γ has been associated with viral clearance following RSV infection. Intranasal treatment with IFN-γ demonstrated accelerated viral clearance while antibody depletion of IFN-γ preceding RSV infection resulted in elevated viral titers in the lung [81,82]. IL-12, which is necessary for development of a Th1 response was also shown to regulate disease following RSV infection. Mice treated with IL-12 neutralizing antibodies had increased airway hyperreactivity and mucus production, in addition to increases in the Th2-associated cytokine IL-13 [83]. This data suggests that a Th1 response is necessary for curbing a Th2 response during RSV infection.

4.2. Th2 immune response

There is a clear established link between Th2-associated cytokines and severe disease during RSV infection. Clinical studies have shown a positive correlation between increased levels of Th2 cytokines and children with more severe symptoms compared to children experiencing mild disease [80,84]. Sera of RSV-infected children and older adults have been reported to exhibit skewed Th2 responses, including an increase in IL-13 and IL-6 production [85,86]. IL-6 is regarded as one of the cytokines that regulates the Th1/Th2 balance, as it both suppresses Th1 differentiation and promotes a Th2 response by induction of IL-4 production via nuclear factor of activated T cells (NFAT) signaling [87,88]. Both increased levels of IL-4 and IL-6 have been observed in RSV-infected infants [89,90]. Furthermore, the use of mechanical ventilation and oxygen supplementation for infants with severe RSV disease may be driven by IL-4-induced asthma [79,91–93]. Consequently, an elevated Th2 response is strongly associated with more severe RSV disease.

Th2-associated cytokines have been linked with increased airway hyperresponsiveness. IL-4 overexpression in RSV infected mice has been shown to cause a delay in viral clearance and inhibition of IL-4 using antibody treatment diminished illness [94,95]. IL-4 and IL-5 deficient mice infected with RSV exhibited decreased eosinophilia and AHR [96]. This data correlates with the established role of IL-5 in eosinophil infiltration into the lung. The chemokine CCL5 may also impact the Th2 response during RSV infection. Murine studies have shown increased levels of CCL5, a known ligand of CC chemokine receptor 1 (CCR1), during RSV infection. CCR1 deficient mice infected with RSV exhibited decreased airway sensitivity as well as decreased production of the Th2-associated cytokine IL-13, further demonstrating that CCL5 signaling through CCR1 drives a Th2 response during RSV infection [97]. When examining clinical correlations, increased CCR1 levels were found to be positively correlated with duration of infant hospital and ICU stay. Therefore, CCR1 signaling may be involved in the recruitment of Th2 cells to the lung resulting in Th2 cytokine production that enhances RSV disease severity. Furthermore, CCR1 signaling may be a key factor in determining if CCL5 will promote a Th2 response as opposed to a Th1 response. Future studies will be necessary to determine the exact mechanisms driving CCL5-mediated Th1 vs Th2 bias.

4.3. Th17 immune response

RSV-induced respiratory distress and asthma have also been shown to be associated with a Th17 response [98,99]. Children with severe RSV infection exhibit increased IL-17 levels in tracheal aspirates and elevated plasma concentrations of IL-17 [99]. An increased frequency of IL-17 producing CD4 T cells was also observed in the peripheral blood of infants during RSV infection, along with the presence of elevated Th17 polarizing and maintenance cytokines IL-6 and IL-23, compared to healthy controls [100]. Murine studies have also shown that RSV infection promotes Th17 cell differentiation and IL-17 secretion [101,102]. When examining possible mechanisms driving the Th17 response during RSV infection, one study determined that the activation of tachykinins and complement anaphylatoxin C3a induced a Th17 response [103].

Recently, an association between Th17 and AHR in RSV-infected animals has also been reported [104]. IL-10 and IL-17A levels in the lung were shown to be increased in RSV-infected mice leading to AHR and increased airway inflammation [104]. It has been shown that high RSV-induced IL-17 levels suppress IL-27, a known down-regulator of Th17 lineage-specific transcription factor ROR-γt as well as being involved in modulating the Th1/Th2 response [105]. IL-27 receptor knockout mice revealed decreased IFN-γ and increased Th2 secreted IL-4, IL-5, and IL-13 in the lungs, although antibody neutralization of IL-17 returned Th2 cytokine levels back to WT levels while IFN-γ levels were not affected [103]. CD8 T cells suppress IL-17 via IFN-γ to protect against RSV-mediated allergic airway disease, and neutralization of IL-17 resulted in a decrease in the number of CD4 and CD8 T cells in the lung [106]. Th17 lymphocytes have been reported to express IL-13 receptor alpha (IL-13Rα), suggesting that these cells may be directly modulated by IL-13, a Th2 cytokine. IL-17 has been shown to increase mucus production through enhancing IL-13 production, as neutralization of IL-17 suppressed expression of Muc5ac and Gob5 as well as IL-13 [102,107]. Th17-induced mucus production and a heightened Th2 response contribute to increased disease severity by worsening lung inflammation and airway obstruction.

4.4. Regulatory T cells

Regulatory T cells (Tregs) are fundamental in dampening inflammation and maintaining self-tolerance. During viral infections, Treg function ensures that the immune system responds in a controlled manner while allowing for viral clearance. Upon RSV infection, Tregs accumulate in the lungs and the mediastinal lymph node to limit RSV-specific T cell responses and suppress inflammation by inhibiting the RSV-specific CD8 T cell response and TNF production [108]. Insufficient control of the regulatory T cell response is thought to contribute to RSV disease severity. Infants with severe RSV disease display a reduction of activated Tregs compared to healthy infants [109]. The reduction in Tregs correlated with an increase in TNF and the Th1-associated cytokine IFN-γ [109]. Another study found no significant difference in the presence of Tregs in infants with recurrent wheezing compared to non-wheezing infants, however impaired production of TNF was observed in the recurrent wheezing group suggesting impaired function of Tregs [110]. Tregs thus play a protective role in RSV disease severity.

A decrease in the number of Tregs has been observed in mice with an acute RSV infection and Treg depletion correlates with more severe disease [108,111]. It has been shown in mice that the RSV NS1 protein stimulates TSLP and OX40L production in DCs, activating the AKT-mTOR signaling pathways in naïve CD4 T cells, leading to inhibition of Tregs and Th1 cells and an increase in Th2 and Th17 cells [112]. IL-6 was also observed to be highly elevated by the NS1 protein which is known to inhibit Tregs [112]. Additionally, Bettelli et al. demonstrated that CD4 T cells differentiate into Tregs in the presence of TGF-β and into Th17 cells in the presence of TGF-β and IL-6 [113]. Taken together, elevated IL-6 expression in RSV infection may contribute to skewing the differentiation of CD4 T cells toward a Th17 subset and the inhibition of Tregs, resulting in more severe disease.

Several murine studies have determined the effects of Treg depletion during RSV infection. Delayed viral clearance occurred when Tregs were depleted, suggesting that Tregs may facilitate viral clearance [108,114]. Furthermore, when the Treg response is inhibited, CD8 T cell migration is delayed resulting in slower viral clearance [114]. When Tregs were depleted, there was a significant increase and persistence in CD4 and CD8 T cells as well as a greater influx of eosinophils into the airways [108,115]. The abundant presence of CD8 T cells resulted in an increased and unrestrained production of TNF and IFN-γ, suggesting the importance of Tregs in modulating a systemic inflammatory response [108,115]. Lastly, the development of Th2-associated pathologies were also described in Treg depleted mice, highlighting the role Tregs play in limiting pathologic Th2 immune responses, along with restricting lung inflammation and disease severity [115]. Previous studies have determined that Tregs play an indispensable role in inhibiting the proliferation and function of Th2 and Th17 effector T cells. In Treg depleted mice during an RSV infection, CD4 T cells significantly increased IL-13 expression [108]. As IL-13 is known to promote mucus production and airway hyperresponsiveness, it stands to reason that depletion of Tregs, which regulate the inflammatory response, results in airway obstruction and respiratory failure.

IL-10 receptor (IL-10R) signaling also plays an important role in the function and stability of Tregs through maintaining immune homeostasis and suppressing a Th17 response [102,116]. Mice that were administered anti-IL-10R antibodies exhibited a significant increase in weight loss, indicating more severe disease. Blocking IL-10R during RSV infection resulted in a decrease in Treg number and an increase in Th17 cells as well as an increase in IFN-γ and IL-13 producing T cells [116,117]. Furthermore, a small population of IL-10 producing Tregs have also been identified during RSV infection [117]. Treg-produced IL-10 may thus suppress overactive effector T cell subsets which contribute to increased disease severity. It has also been previously reported in other disease models that IL-10 acts directly on Tregs to support regulatory function and inhibit inflammation. IL-10R-induced activation of the STAT3 signaling pathway leads to binding of regulatory elements in the IL-10 gene, thus inducing production of IL-10. The positive feedback loop may thus enhance the ability of Tregs to suppress proinflammatory responses. These data together suggest that Tregs may promote their regulatory function via autocrine signaling of IL-10. Stimulation of Treg cells via IL-10R signaling may thus limit lung inflammation and RSV disease severity.

4.5. Cytotoxic CD8 T cells

CD8 T cells play an important role in the adaptive immune response to RSV and are crucial for viral clearance. In a highly translational yet still preclinical model, CD8 T cells were shown to be important in controlling RSV viral burden. In this model immunodeficient mice were implanted with human lung tissue resulting in the formation of lung structures over time. When mice were infected with RSV, they were unable to clear the virus. However, adoptive transfer of primed autologus human CD8 T cells reduced viral titers in the lungs [118]. In a human RSV challenge study the presence of RSV memory CD8 T cells in the airway strongly correlated with an overall reduction in disease severity and viral load [6]. These studies further demonstrate the importance of CD8 T cells in RSV clearance. Analysis of cytokines and CD8 T cells in infants with severe disease revealed low levels of cytokines, including IFN-γ, and the near absence of CD8 T cells [119]. These data would also suggest a protective role of CD8 T cells during RSV infection. In contrast, when examining levels of cytotoxic granules produced by CD8 T cells, an increase in granzyme A and B levels in tracheal aspirates of children with severe RSV was observed compared to controls [120].

Studies performed in murine models indicate CD8 T cells can also contribute to immunopathology during RSV infection. Early studies examining the role of CD8 T cells demonstrated that adoptive transfer of RSV-specific cytotoxic T lymphocytes (CTL) caused increased pulmonary disease while also accelerating viral clearance [121]. In concert with this data, another study found that depletion of CD8 T cells in mice reduced lung pathology and illness scores but increased viral burdens in the lung [122]. One study also showed that increased neutrophil recruitment via CXCL1 to the lung prior to RSV infection correlated with susceptibility to RSV infection. In CXCL1 treated mice, there were increased CD8 T cells in the lung, leading to severe clinical symptoms during the adaptive immune timepoint [7]. CD8 T cell immunopathology is not confined to the initial effector response but is also driven by memory CD8 T cells. To study this, a robust RSV-specific memory CD8 T cell response was generated using a dendritic cell immunization model. Mice were immunized with dendritic cells loaded with the RSV immunodominant epitope M2_82–90 and boosted one week later with an attenuated recombinant listeria monocytogenes (LM) strain expressing the M2_82–90 epitope. Upon infection with RSV memory CD8 T cells rapidly cleared virus, however mice exhibited severe disease and reduced survival [123]. These studies demonstrate the role CD8 T cells can play in RSV-mediated immunopathology.

Upon activation CD8 T cells secrete cytotoxic granules and cytokines such as IFN-γ and TNF as effector mechanisms for killing target cells. Antibody depletion of TNF during RSV infection resulted in reduced weight loss and illness scores in mice, but did not impact viral clearance from the lungs [124]. IFN-γ-deficient CD8 CTLs were unable to control viral titers following RSV infection, however mice displayed a faster recovery of weight compared to control mice [125]. In the DC immunization model memory CD8 T cell-mediated immunopathology was significantly reduced by neutralizing IFN-γ in the airways of mice [123]. Therefore the function of CD8 T cells, specifically cytokine production, is essential for development of severe disease. The role of CD8 T cells highlights the dichotomy between viral clearance and pathology. When CD8 T cell-mediated pathology is high, viral titers are low. This further suggests that viral replication does not directly correlate with the level of pathology during RSV infection.

5. Vaccination and therapeutics: finding a safe balance

In rare instances, vaccination can lead to enhanced host immunopathology following subsequent infection. This phenomenon is termed vaccine-enhanced disease and has hindered RSV vaccine development since the 1960s. The first developed RSV vaccine, utilizing formalin-inactivated virus, caused vaccinated children to exhibit enhanced respiratory disease following their first RSV infection after vaccination, resulting in hospitalization and 2 deaths. Clinical findings from this tragic incident identified increased levels of CCL5, a decreased type I IFN response, non-neutralizing antibodies and a shift toward type 2 polarization, all of which correspond with severe RSV disease and demonstrate the fragile balance between protective and pathogenic immune responses [126].

The FDA has recently approved RSV vaccines for older adults and pregnant women for the protection of newborns during the first few months following birth. A key discovery in the development of the current approved vaccines is the use of the RSV F protein stabilized in its prefusion form. The prefusion F (preF) protein has multiple antigen sites that are critical for mounting a robust neutralizing antibody response, which likely contributes to the success of the vaccine. However, trials using an mRNA vaccine encoding either the RSV preF or a combination of RSV preF and human metapneumovirus (hMPV) F protein in children ages 5 to 23 months were halted due to safety concerns [127]. Both vaccines successfully mounted neutralizing antibody responses, unlike the formalin-inactivated vaccine. However, upon infection with RSV or hMPV a small number of children developed severe lower respiratory tract infections resulting in cessation of the trial. The question of what is necessary to generate a safe and effective RSV vaccine for children remains. RSV vaccination trials highlight the impact that age can have on the immune response to vaccination and infection. Critically examining the differences in the immune response between these age groups may be the key to determining safe vaccination strategies in children. Until a safe vaccine for infants and children is developed, RSV-specific monoclonal antibodies represent the only preventative therapeutic currently available. Although administration of these monoclonals has proven to be safe and effective, current limitations include age, cost and availability.

Every year, hundreds of thousands of children are infected with RSV and many are hospitalized due to severe disease with current therapies limited in their ability to reduce RSV disease severity. Ribavirin is currently the only drug in clinical use for the treatment of severe RSV infection, though several other F protein inhibitors to block viral entry are in clinical trials. As such there is still a pressing need for treatments that can alleviate severe RSV disease in cases involving hospitalization and oxygen administration. There are several key pathways that can be targeted for potential treatment. NF-κB inhibition has been a topic of discussion within the RSV field, as proinflammatory cytokines greatly contribute to lung damage. Stimulation of IκBα, an NF-κB inhibitor, greatly decreased RSV induction of IFN-β and CXCL10 but did not increase viral mRNA or replication in human airway epithelial cells [128]. Another inhibitor, Bay11–7082, has been shown to decrease MUC1 expression in airway epithelial cells [129]. Acute bronchiolitis induced by RSV in a mouse model was successfully attenuated by inhibiting the NF-κB pathway, using dimethyl fumarate, and reduced IL-33 expression, leading to a significant decrease in IL-4 and IL-10 cytokine expression [130]. While these inhibitors have been used in murine studies, they have not been approved for human therapeutic use, though many other NF-κB inhibitors have been approved for other therapies, including PI3K inhibitors, which modulate activation and NF-κB-dependent gene expression, such as Idelalisib, Alpelisib, Copanlisib, and Duvelisib. To date, these have not been used in RSV clinical trials but may prove to be viable options for NF-κB inhibition to alleviate severe RSV disease.

Antibody treatments that target the actions of cytokines may be another therapeutic approach to consider for severe RSV cases. IL-33 has been shown to be highly expressed in infants with severe RSV disease; however, it has been more well studied in asthma cases. Itepekimab, an IL-33 antibody, is in clinical trials for moderate-to-severe asthma. Targeting IL-33 in RSV may help manage inflammation in the lung as well as manage symptoms and airway remodeling. Another antibody, Mepolizumab against IL-5, is also in clinical studies and has shown to reduce asthma exacerbations in children. As asthma and RSV-induced acute bronchiolitis share many mechanistic causes, it is possible that this therapy may ameliorate RSV-associated airway hyperresponsiveness. Another potential target is the proinflammatory cytokine IL-1β and the IL-1 signaling pathway. The IL-1R antagonist Anakinra is approved by the FDA and been examined in severe cases of SARS-CoV-2 to restore the inflammatory balance. Exploration of Anakinra or other inflammasome pathway inhibitors may prove beneficial in treatment of RSV immunopathology and reduction of severe disease. Therapeutic targets must be carefully chosen given the propensity of cytokines and chemokines to either dimmish or enhance disease during RSV infection. Furthermore, targeting inflammatory cytokines and signaling pathways may prevent the host from controlling viral replication.

6. Conclusion and future perspectives

RSV infections are a significant global burden and present a substantial risk for all infants, elderly and immunocompromised individuals. Several host factors play into the immune system’s response to infection which in turn can impact the severity of the disease. It is becoming increasingly recognized that host inflammatory responses to RSV infection play a critical role in determining disease severity. Release of inflammatory mediators, such as TNF, IL-1β and IL-6 which are crucial for viral clearance, both drive inflammation and recruit immune cells to the lung. Both human and murine studies have yielded sometimes contradictory evidence with several studies demonstrating that lower levels of proinflammatory cytokines result in increased disease severity, while others show that increased levels correlated to more severe disease. It is possible that both sides are correct however, as it may be necessary to have just the right amount of expression to be protective, with too little leading to more severe disease by not being able to control viral replication and excessive proinflammatory cytokine and chemokine expression as well as mucus production leading to increased host tissue damage and more severe disease. This can manifest as cytokine dysregulation resulting in increased inflammation and worsening lung function.

RSV continues to cause severe disease in both very young children and the elderly. Despite our current understanding of the virus and host immune response to RSV, there is still much to discover. RSV-induced inflammation depends on various host and viral factors, many of which are still being studied to understand the underlying mechanisms and the downstream effects that result from the interactions between viral and host proteins. RSV continues to evolve genetically, which impacts the immune response to the virus. Continued clinical surveillance and viral genome sequencing efforts will be essential in understanding how RSV continues to evolve and the potential impact of that evolution on the host immune response.

Article highlights.

• RSV disease severity is dependent on the host immune response.

• The host immune response can be either protective or pathogenic depending on several factors.

• TNF, IL-1β, and IL-6 are key cytokines which contribute to inflammation, cell recruitment, and viral clearance.

• The immune response of young children must be better understood to aid in the development of safer treatment and vaccination strategies.