Brian D. Rudd

Differential Role for TLR3 in Respiratory Syncytial Virus-Induced Chemokine Expression

ABSTRACT Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infection in young infants worldwide. Previous studies have reported that the induction of interleukin-8/CXCL8 and RANTES/CCL5 correlates with disease severity in humans. The production of these chemokines is elicited by viral replication and is NF-κB dependent. RSV, a negative-sense single-stranded RNA virus, requires full-length positive-sense RNA for synthesis of new viral RNA. The aim of our studies was to investigate whether active viral replication by RSV could evoke chemokine production through TLR3-mediated signaling pathways. In TLR3-transfected HEK 293 cells, live RSV preferentially activated chemokines in both a time- and dose-dependent manner compared to vector controls. RSV was also shown to upregulate TLR3 in human lung fibroblasts and epithelial cells (MRC-5 and A549). Targeting the expression of TLR3 with small interfering RNA decreased synthesis of IP-10/CXCL10 and CCL5 but did not significantly reduce levels of CXCL8. Blocking the expression of the adapter protein MyD88 established a role for MyD88 in CXCL8 production, whereas CCL5 synthesis was found to be MyD88 independent. Production of CCL5 by RSV was induced directly through TLR3 signaling pathways and did not require interferon (IFN) signaling through the IFN-α/β receptor. TLR3 did not affect viral replication, since equivalent viral loads were recovered from RSV-infected cells despite altered TLR3 expression. Taken together, our studies indicate that TLR3 mediates inflammatory cytokine and chemokine production in RSV-infected epithelial cells.

Plasmacytoid dendritic cells inhibit pulmonary immunopathology and promote clearance of respiratory syncytial virus

Respiratory syncytial virus (RSV) infection is widely spread and is a major cause of bronchiolitis in infants and high-risk adults, often leading to hospitalization. RSV infection leads to obstruction and inflammation of the airways and induction of innate and acquired immune responses. Because dendritic cells (DCs) are essential in the elicitation of these immune responses, we investigated the presence and the role of dendritic cell subtypes upon RSV infection in the lung. Here, we report that RSV infection increased the number of both conventional and plasmacytoid dendritic cells in the lung and the lung-draining lymph nodes. In particular, the increase in plasmacytoid dendritic cell numbers was sustained and lasted until 30 d after infection. Depletion of plasmacytoid dendritic cells resulted in decreased RSV clearance. In addition, depletion of plasmacytoid dendritic cells resulted in an exacerbation of all manifestations of immune-mediated pathology caused by RSV infection. In conclusion, this study demonstrates that both conventional and plasmacytoid dendritic cells are attracted to the site of RSV infection. It is demonstrated that plasmacytoid dendritic cells play a protective role during RSV infection by modulation of local immune responses.

Deletion of TLR3 Alters the Pulmonary Immune Environment and Mucus Production during Respiratory Syncytial Virus Infection

The detection of a viral infection by pattern recognition receptors (PAMPs) is an integral part of antiviral immunity. In these studies we have investigated the role of TLR3, which recognizes dsRNA, in Respiratory Syncytial virus (RSV) infection using B6 background mice with a TLR3 deletion. Although we observed no changes in viral growth, we did find that TLR3−/− mice demonstrated significant increases in mucus production in the airways of RSV-infected mice. The qualitative assessment was observed by examining differentially stained lungs, followed by immunohistochemical staining for gob5, a mucus-associated protein. The histopathologic observations were verified using quantitative gene expression analyses examining gob5 gene expression. Changes in pulmonary mucus production were accompanied by an increase in pulmonary IL-13 as well as IL-5 expression and eosinophils in the airways of TLR3−/− mice. Examining leukocytes in the airway indicated an accumulation of eosinophils in TLR3−/− mice, but not wild-type mice, after RSV infection. Isolated lung draining lymph node cells from TLR3−/− mice produced significant increases in Th2-type cytokines, IL-5, and IL-13, compared with wild-type TLR3+/+ mice only after RSV infection. To demonstrate a causative link, we depleted TLR3−/− mice of IL-13 during RSV infection and found that mucus and gob5 expression in the lungs was attenuated. Together, these studies highlight that although TLR3 may not be required for viral clearance, it is necessary to maintain the proper immune environment in the lung to avoid developing pathologic symptoms of disease.

Notch ligand Delta-like 4 regulates disease pathogenesis during respiratory viral infections by modulating Th2 cytokines

Recent data have indicated that an important instructive class of signals regulating the immune response is Notch ligand–mediated activation. Using quantitative polymerase chain reaction, we observed that only Delta-like 4 (dll4) was up-regulated on bone marrow–derived dendritic cells after respiratory syncytial virus (RSV) infection, and that it was dependent on MyD88-mediated pathways. Using a polyclonal antibody specific for dll4, the development of RSV-induced disease was examined. Animals treated with anti-dll4 had substantially increased airway hyperresponsiveness compared with control antibody-treated animals. When the lymphocytic lung infiltrate was examined, a significant increase in total CD4+ T cells and activated (perforin+) CD8+ T cells was observed. Isolated lung CD4+ T cells demonstrated significant increases in Th2-type cytokines and a decrease in interferon γ, demonstrating an association with increased disease pathogenesis. Parellel in vitro studies examining the integrated role of dll4 with interleukin-12 demonstrated that, together, both of these instructive signals direct the immune response toward a more competent, less pathogenic antiviral response. These data demonstrate that dll4-mediated Notch activation is one regulator of antiviral immunity.

MyD88-Mediated Instructive Signals in Dendritic Cells Regulate Pulmonary Immune Responses during Respiratory Virus Infection

Respiratory syncytial virus (RSV) is the leading cause of respiratory disease in infants worldwide. The induction of innate immunity and the establishment of adaptive immune responses are influenced by the recognition of pathogen-associated molecular patterns by TLRs. One of the primary pathways for TLR activation is by MyD88 adapter protein signaling. The present studies indicate that MyD88 deficiency profoundly impacts the pulmonary environment in RSV-infected mice characterized by the accumulation of eosinophils and augmented mucus production. Although there was little difference in CD4 T cell accumulation, there was also a significant decrease in conventional dendritic cells recruitment to the lungs of MyD88−/− mice. The exacerbation of RSV pathophysiology in MyD88−/− mice was associated with an enhanced Th2 cytokine profile that contributed to an inappropriate immune response. Furthermore, bone marrow-derived dendritic cells (BMDC) isolated from MyD88−/− mice were incapable of producing two important Th1 instructive signals, IL-12 and delta-like4, upon RSV infection. Although MyD88−/− BMDCs infected with RSV did up-regulate costimulatory molecules, they did not up-regulate class II as efficiently and stimulated less IFN-γ from CD4+ T cells in vitro compared with wild-type BMDCs. Finally, adoptive transfer of C57BL/6 BMDCs into MyD88−/− mice reconstituted Th1 immune responses in vivo, whereas transfer of MyD88−/− BMDCs into wild-type mice skewed the RSV responses toward a Th2 phenotype. Taken together, our data indicate that MyD88-mediated pathways are essential for the least pathogenic responses to this viral pathogen through the regulation of important Th1-associated instructive signals.