Leukemia inhibitory factor protects the lung during respiratory syncytial viral infection

Abstract

Background: Respiratory syncytial virus (RSV) infects the lung epithelium where it stimulates the production of numerous host cytokines that are associated with disease burden and acute lung injury. Characterizing the host cytokine response to RSV infection, the regulation of host cytokines and the impact of neutralizing an RSV-inducible cytokine during infection were undertaken in this study.

Methods: A549, primary human small airway epithelial (SAE) cells and wild-type, TIR-domain-containing adapter-inducing interferon-β (Trif) and mitochondrial antiviral-signaling protein (Mavs) knockout (KO) mice were infected with RSV and cytokine responses were investigated by ELISA, multiplex analysis and qPCR. Neutralizing anti-leukemia inhibitory factor (LIF) IgG or control IgG was administered to a group of wild-type animals prior to RSV infection.

Results and discussion: RSV-infected A549 and SAE cells release a network of cytokines, including newly identified RSV-inducible cytokines LIF, migration inhibitory factor (MIF), stem cell factor (SCF), CCL27, CXCL12 and stem cell growth factor beta (SCGF-β). These RSV-inducible cytokines were also observed in the airways of mice during an infection. To identify the regulation of RSV inducible cytokines, Mavs and Trif deficient animals were infected with RSV. In vivo induction of airway IL-1β, IL-4, IL-5, IL-6, IL-12(p40), IFN-γ, CCL2, CCL5, CCL3, CXCL1, IP-10/CXCL10, IL-22, MIG/CXCL9 and MIF were dependent on Mavs expression in mice. Loss of Trif expression in mice altered the RSV induction of IL-1β, IL-5, CXCL12, MIF, LIF, CXCL12 and IFN-γ. Silencing of retinoic acid-inducible gene-1 (RIG-I) expression in A549 cells had a greater impact on RSV-inducible cytokines than melanoma differentiation-associated protein 5 (MDA5) and laboratory of genetics and physiology 2 (LGP2), and Trif expression. To evaluate the role of LIF in the airways during RSV infection, animals were treated with neutralizing anti-LIF IgG, which enhanced RSV pathology observed with increased airspace protein content, apoptosis and airway hyperresponsiveness compared to control IgG treatment.

Conclusions: RSV infection in the epithelium induces a network of immune factors to counter infection, primarily in a RIG-I dependent manner. Expression of LIF protects the lung from lung injury and enhanced pathology during RSV infection.

Figure 1

RSV replication induces host cytokines production in airway epithelial cells. A549 cells were treated with mock, RSV or UV-inactivated RSV (UV-RSV) for 24 hours. (A-B) Secreted cytokines were analyzed by multiplex or ELISA. Graphs are represented as mean cytokine concentration (pg/ml) ± S.E.M, with each measurement performed 3 times on 6 replicates/group. Samples were collected from 3 experiments performed on separate days. * and ** represent a p value less than 0.05 compared to mock or RSV treated cells for each cytokine target, respectively.

Figure 2

RSV challenge induces MIF, LIF, CCL27, CXCL12, SCGF-β and SCF airway expression. FVB/NJ mice were infected with 1x10⁶ pfu of RSV, UV-RSV or mock and animals were euthanized on days 0, 1, 3, 5, 7 and 9-post challenge. MIF, LIF, CTAK/CCL27, SDF-1/CXCL12, SCGF-β and SCF (A) lung gene expression and (B) BALF protein levels were analyzed by qPCR and multiplex, respectively. Graphs are represented as relative quantification (RQ) or BALF concentration of the mean ± S.E.M, with each measurement performed 3 times on 10 animals/group. *Represents a p value less than 0.05 compared to mock treated mice on each corresponding day.

Figure 3

RSV induces multiple pathogen recognition receptors in A549 and SAE cells. (A) Representative immunofluorescence staining of SAE and A549 cells treated with mock or RSV and stained 24 hours later with RSV F-specific monoclonal antibody. Cell lysates were examined for viable viruses by plaque assays. (B) qPCR and (C) immunblots were performed to examine levels of pathogen recognition receptors and downstream signaling in cells 1-day post RSV infection. Graphs are represented as (B) PFU (Log₁₀) or (C) RQ of the mean ± S.E.M, where n = 10 replicates/group and each assay was performed in triplicate. Each experiment was performed on samples obtained from 3 experiments from separate days. *Represents a p value less than 0.05 compared to mock treated mice.

Figure 4

Loss of Trif or Mavs expression enhances RSV viral titer in the lungs of mice. (A) RSV infection resulted in a drop in body weight in all animal groups and (B) a significant increase in the viral load 9 days post RSV challenge in the Mavs KO mouse group compared to mock treated mice. Graphs are represented as mean ± S.E.M., where each measurement was performed 3 times on 10 animals/group. *Represents a p value less than 0.05 comparing Mavs KO mice to wild-type mice 9 days post RSV challenge.

Figure 5

Silencing RIG-I subdues RSV induced cytokine release from A549 cells. (A-B) Multiplex or ELISA analysis was performed on secreted cytokines from A549 cells transfected with siRNA targeting RIG-I (DDX58), LGP2 (DHX58), MDA5 (IFIH1), Trif or a negative sequence control. Graphs are represented as mean cytokine concentration ± S.E.M, where n = 10 replicates/group and each assay were performed in triplicate. Each experiment was performed on samples obtained from 3 experiments from separate days. *Represents a p value less than 0.05 compared to control siRNA treated cells following RSV infection.

Figure 6

RSV induced airway injury is enhanced following LIF neutralization. FVB/NJ mice were IP injected with control IgG or anti-LIF IgG prior to intranasal infected with 1x10⁶ pfu of RSV and animals were euthanized 7-days post infection. (A) Animal body weight, (B) BALF cellularity and BALF cells undergoing apoptosis were determined. (C) Airway hyperresponsiveness to increasing doses of methacholine was assessed in each animal group. (D) RSV infected animals receiving anti-LIF IgG have enhanced protein content in BALF compared to mice administered control IgG. (E) Neutralizing LIF levels did not impact on RSV viral multiplicity, demonstrated by qPCR for RSV N. (F) CXCL1, RANTES, CCL5, CXCL10/IP-10, MIP-1α/CCL3 and MCP-1/CCL2 gene expression in lung tissue was analyzed by qPCR. Graphs are represented as mean ± S.E.M., where each measurement was performed 3 times on 10 animals/group. * represents a p value less than 0.05 comparing control IgG and anti-LIF IgG treated mice both infected with RSV.