Leukemia inhibitory factor protects the lung during respiratory syncytial viral infection

Respiratory syncytial virus (RSV) is a major respiratory pathogen, which typically infects the airway epithelium [1]. Those infected with RSV, usually infants, the elderly and immunocompromised patients but also healthy adults [2],[3], develop mild to severe cough and dyspnea [4]. Wheezing and asthma symptoms are observed following severe RSV lower respiratory tract infection [5]. RSV infection occur frequently in the respiratory tract of individuals with an underlying lung disease, such chronic obstructive pulmonary disease (COPD) [6]. A range of inflammatory responses are associated with RSV infection, such as the induction of proinflammatory cytokines, chemokines and growth factors, such as GROα/CXCL1, IL-1β, IL1-RA, IL-2, IL-6, IL-7, CXCL8/IL-8, IL-9, CXCL10/IP-10, CXCL11, IL-15, IL-18, CCL2/MCP-1, CCL3/MIP-1β, CCL4/MIP-1β, IFN-γ, CCL5/RANTES, epidermal growth factor (EGF), hepatocyte growth factor (HGF), granulocyte colony-stimulating factor (G-CSF), GM-CSF, fibroblast growth factor (FGF), vascular endothelial cell growth factor (VEGF) and tumor necrosis factor-α (TNF-α) [1],[7]-[16]. The importance of this cytokine response is underscored by the fact that increased levels of IL-1β, IL1-RA, IL-6, IL-7, IL-8, G-CSF, EGF and HGF are associated with RSV infection severity [10],[15]. Others have found that SNPs in IL-19, IL-20, MUC5AC, TNFRSF1B, C3, CTLA4, CXCL9, IL4R, and IL-7 genes are associated with recurrent wheezing following RSV infections [17]. Although RSV-induced cytokines have been studied extensively, the signaling mechanisms that regulate their expression and whether specific host cytokines contribute to airway injury or inflammation resolution remain incompletely understood.

Pathological studies demonstrate that RSV primarily infects airway epithelial cells [18]; therefore it is conceivable that a significant component of immune signaling associated with RSV infections originates from airway epithelial responses. Cytokines, chemokines and growth factors are expressed in these cells in response to microbial stimuli [19],[20] with numerous pathogen recognition receptors playing a major role in cytokine responses. Pathogen recognition receptors, such as TLRs and retinoic acid-inducible gene-1 (RIG-I)-like receptors [21], induce major signaling cascades following viral stimulation [22]. Indeed, the viral load of RSV correlates with RIG-I mRNA levels [23]. TLR3, –7, –8 and –9 recognize nucleic acid ligands but TLR4 proteins also bind to a major viral antigen of RSV F (fusion) protein [24]. Laboratory of genetics and physiology 2 (LGP2) and melanoma differentiation-associated protein-5 (MDA5) equally could be associated with RSV-induced immune responses [25]. Additionally, ligands for nucleotide-binding oligomerization domain-containing protein (NOD)-like receptors (NLR), such as NOD2, enhance TLR-ligand-induced activation and triggering such signaling cascades are plausible means of developing RSV-specific immunity [26]. Therefore, RSV can induce several pathogen recognition receptors resulting in multiple host immune responses.

In this study we employed in vitro and in vivo approaches to evaluate the cytokine/chemokine response to RSV infection. Normal human small airway epithelial (SAE) cells and A549 cells were utilized to examine 42 cytokine responses following RSV stimuli. Several newly identified RSV-inducible cytokines (leukemia inhibitory factor (LIF), migration inhibitory factor (MIF), stem cell factor (SCF), CCL27, CXCL12 and stem cell growth factor beta (SCGF-β)) were induced in airways cells in vitro and in mouse lungs during a viral infection in vivo. The influence of TIR-domain-containing adapter-inducing interferon-β (Trif) and mitochondrial antiviral-signaling protein (MAVS) signaling pathways were examined on RSV-induced cytokine levels in mice and A549 cells. The significance of LIF, an IL-6 cytokine family member, expression during RSV was also determined. LIF is primarily recognized for its ability to preserve the totipotency of embryonic stem cells [27] and is detected in acute respiratory distress syndrome (ARDS) [28] but recently LIF was described to protect the lung from injury during pneumonia [29]. Utilizing neutralizing anti-LIF IgG, LIF was observed to play a critical role in protecting the lung from injury during RSV infection. Our findings indicate that RSV infection induces a significant host cytokine response associated with viral clearance and airway protection.

To our knowledge, these findings represent the first evidence that MIF, LIF, CTAK/CCL27, SDF-1α/CXCL12, SCGF-β and SCF are expressed during an RSV infection and LIF signaling is critical to prevent lung damage. Others and we demonstrate that a spectrum of cytokines is released to counter RSV infection [1],[7]-[10],[34] but further analysis is required to determine the role of these immune responses in RSV pathology. The results of this study demonstrate that RLRs and Trif signaling pathways play major roles in the immune response to RSV infection for both inflammation induction and resolution. Interestingly, 34 of the 42 cytokines examined in A549 and SAE cells were released upon RSV stimulation. This study highlights the robust epithelial responses to RSV infection, characterizes two major signaling cascade needed to counter viral infectivity and identified the role of LIF signaling during an RSV infection.

Many of the RSV-inducible cytokines described in this study have already been described in clinical samples, in vitro epithelial cell culture or in animals models [1],[7]-[16], such as elevated MIG/CXCL9 levels in BALB/c mice inoculated with RSV [35]. Importantly, increases in media or mouse BALF IL1-RA, IL-6, IL-7, CXCL10/IP-10, CXCL8/IL-8, CCL2/MCP-1, CCL3/MIP-1β, CCL4/ MIP-1β, TNF-α, IFN-γ and CCL5/RANTES following RSV infection reflects similar observations from clinical studies [10],[36]. To our knowledge, we are the first to identify that RSV infection in epithelial cells and mice can induce MIF, LIF, CTAK/CCL27, SDF-1α/CXCL12, SCGF-β and SCF expression and secretions. This is an important finding as each new target can potentially play a different role in RSV clearance. MIF activates lymphocytes, granulocytes and monocytes/macrophages and plays a key role in several host immune cell responses [37]. CTAK/CCL27 is a cytokine associated with T cell activation and migration [38] and is elevated in severe tuberculosis cases [39]. Recombinant protein SDF-1α/CXCL12 does not prevent RSV infection of Hep2 cells [40] but SDF-1α/CXCL12 is strongly chemotactic for lymphocytes [41] and contributes to pulmonary fibrosis [42]. The potential role of SCGF-β in RSV infections is not as straightforward to elucidate but SCGF-β can support growth of primitive hematopoietic cells [43] and can promotes proliferation of erythroid or myeloid progenitors. SCF and its soluble receptor c-kit correlate with asthma severity [44]. This study identified an important role for LIF in RSV infection. Therefore, identifying these new RSV inducible targets in clinical samples and their role in viral clearance may provide important insights into host responses to RSV infection.

Other investigators have demonstrated the therapeutic potential of LIF expression in other models of lung injury, as it protects against lung injury [29],[32],[33]. Recombinant LIF reduces cytokine expression [29] and alters alveolar neutrophil numbers [29],[32]. Lif KO animals have increased expression of CXCL1, GM-CSF, RANTES/CCL5 and MIP-1β/CCL3 early during an experimental autoimmune encephalomyelitis model and reduced CCL2, CCL3, and CXCL10 at a later stage of the disease [45]. In this study neutralizing LIF signaling enhanced CXCL1, RANTES/CCL5, CXCL10/IP-10, MIP-1β/CCL3 and MCP-1/CCL2 at day 7 of an RSV infection. Therefore LIF expression has the potential to regulate key factors in the immune system. Targeted overexpression of LIF in mouse airway epithelial cells significantly protects the airways during hyperoxia, with improved survival and decreased pulmonary edema [33]. Other IL-6 family members (IL-6 [46] and IL-11 [47]) have similar functions, which suggest that IL-6 family cytokines may collectively protect lungs from injury. LIF is a prominent STAT3-activating cytokine that facilitates tissue protection during pneumonia [29] and plays a similar airway protective role during RSV infection. The significance of LIF expression and STAT3 activation in RSV infection represents an intriguing area for further study. LIF has been shown to regulate apoptosis with some investigators suggesting LIF acts as a pro-apoptotic mediator [48],[49] while others have demonstrated LIF to have anti-apoptotic potential [50],[51]. In this study increased apoptotic BAL cells were observed in animals administered anti-LIF IgG prior to RSV infection. Enhanced Fas, Fap, Il24, and Tnfsf15 expression is observed following LIF depletion in animals with bacterial pneumonia [29], which could contribute to the increased apoptosis observed here. Whether this induction of apoptosis contributes to the pathology observed in this study is unknown and requires further investigation. Epithelial damage correlates with airway hyperreactivity in asthma patients [52] and the increased protein levels in the BALF of animals treated with anti-LIF IgG prior to RSV infection could also contribute to the enhanced airway resistance observed in these animals. LIF enhances the maintenance of stem cells [27], which may directly impact lung tissue regeneration and repair [53], similar to IL-6 activation of STAT3 [54]. These data reveal the vast biological processes of LIF signaling during RSV infection. Whether the other cytokines identified in this study also play such major roles on disease pathology needs to be addressed.

Our study also reveals that Trif and RLR signaling regulate a significant proportion of the cytokines induced by RSV infection, with LIF expression sensitive to Trif activation. Prior to undertaking this study, several pathogen recognition receptors were associated with regulating the immune response against RSV infection, with RIG-I [55], TLR7 [56] and TLR3 [57] known to affect numerous cytokine responses. Several investigators characterized the RIG-I-like family proteins as critical for the detection of RSV, using human cell lines [30] and knockout mice [58]. RIG-I principally recognizes dsRNA [59]; however Sendai virus defective interfering RNA [60] and the genomic "panhandle" structure of influenza virus [61] activate RLR signaling. These "panhandle" structures allow negative-strand RNA viral genomes to achieve partially double-stranded RNA [62], which may allow RSV to induce RLRs. Our study observed early cytokine responses following RSV infection and identified a pronounced RIG-I response, which could be lost over time following NS1 or NS2 binding to RIG-I/MDA5 as reported by others [63],[64]. Also, RSV induction of TLR3 is regulated by RIG-I-dependent IFN-γ secretions from infected epithelial cells, which is mediated by both IFN response-stimulated element (ISRE) and signal transducer and activator of transcription (STAT) sites in its proximal promoter [65]. Therefore there are multiple levels of RLRs regulation in RSV infections that can both trigger RLRs responses or subsequently adapt to block RLR-mediated signaling.

Recently, several investigators have utilized well-defined primary cell culture techniques to investigate viral-host interactions [1],[66],[67], which may be a closer representation of human RSV infections compared to the approach we employed here. IFN-–/– are not secreted by RSV-infected well-defined bronchial epithelial cells [66], suggesting that there could be major signaling differences in well-defined primary cell culture techniques and monolayers. However, our overall findings suggest that utilizing cell monolayers in combination with mouse models is an acceptable approach as our cytokine profile are comparable to that observed in the human disease state [10]. In addition, our study has exposed new RSV inducible host immune response cytokines. Since we only explored epithelial cell responses in vitro, our in vivo parallel approach also validates our monolayer results and suggests that the epithelium is a significant source of initial immune responses following RSV infection.

Our studies demonstrate that RSV exposure stimulates a cascade of early immune response in epithelial cells and a large number of these responses act in an RLR/Trif dependent manner. These findings also identified new cytokines that are triggered by RSV lung infection, with LIF signaling critical for the protection of the lung from injury during RSV infection. Profiling the loss and gain of the other identified cytokines (MIF, CTAK/CCL27, SDF-1α/CXCL12, SCGF-β and SCF) over the course of an RSV infection may help to decipher the relevancy of these cytokines in viral clearance and lung integrity, and represents important questions to be addressed by future studies.

PG designed and conducted the cell and animal experiments, compiled and interpreted data, and wrote the paper. AJD and NC aided PG in animal treatments and tissue collection. RFF co-wrote the paper with PG. All authors were involved in revising the manuscript critically for important intellectual content, read and approved the final manuscript.