Respiratory syncytial virus inhibits granulocyte apoptosis through a phosphatidylinositol 3-kinase and NF-kappaB-dependent mechanism.

Respiratory syncytial virus (RSV) is a common cause of lower respiratory tract disease in children. It is associated with increased neutrophil numbers in the airway. In this study, we assessed whether this ssRNA virus can directly influence granulocyte longevity. By culturing RSV with granulocytes, it was observed that virus delays both constitutive neutrophil and eosinophil apoptosis. Using pharmacological inhibitors, the RSV-induced delay in neutrophil apoptosis was found to be dependent on both PI3K and NF-kappaB, but not p38 MAPK or MEK1/MEK2 activation. Using blocking Abs and a reporter cell line, we were able to exclude TLR4 as the receptor responsible for mediating RSV-induced delay in neutrophil apoptosis. The antiapoptotic effect was abrogated by preincubation with the lysosomotropic agent chloroquine, indicating the requirement for endolysosomal internalization. Furthermore, addition of ssRNA, a ligand for the intracellular TLR7/TLR8, also inhibited neutrophil apoptosis, suggesting that intracellular TLRs could be involved in induction of the antiapoptotic effect. Using the BioPlex cytokine detection assay (Bio-Rad), we found that IL-6 was present in supernatants from RSV-exposed neutrophils. IL-6 was found to inhibit neutrophil apoptosis, suggesting that there is an autocrine or paracrine antiapoptotic role for IL-6. Finally, RSV treatment of neutrophils resulted in increased expression of the antiapoptotic Bcl-2 protein Mcl-1. Taken together, our findings suggest involvement of multiple intracellular mechanisms responsible for RSV-induced survival of granulocytes and point toward a role for intracellular TLRs in mediating these effects.

Characterization of the CD8+ T cell responses directed against respiratory syncytial virus during primary and secondary infection in C57BL/6 mice.

The BALB/c mouse model for human respiratory syncytial virus infection has contributed significantly to our understanding of the relative role for CD4+ and CD8+ T cells to immune protection and pathogenic immune responses. To enable comparison of RSV-specific T cell responses in different mouse strains and allow dissection of immune mechanisms by using transgenic and knockout mice that are mostly available on a C57BL/6 background, we characterized the specificity, level and functional capabilities of CD8+ T cells during primary and secondary responses in lung parenchyma, airways and spleens of C57BL/6 mice. During the primary response, epitopes were recognized originating from the matrix, fusion, nucleo- and attachment proteins, whereas the secondary response focused predominantly on the matrix epitope. C57BL/6 mice are less permissive for hRSV infection than BALB/c mice, yet we found CD8+ T cell responses in the lungs and bronchoalveolar lavage, comparable to the responses described for BALB/c mice.

Respiratory syncytial virus infection of monocyte-derived dendritic cells decreases their capacity to activate CD4 T cells.

Respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract infections in children, the elderly, and immune-compromised individuals. CD4 and CD8 T cells play a crucial role in the elimination of RSV from the infected lung, but T cell memory is not sufficient to completely prevent reinfections. The nature of the adaptive immune response depends on innate immune reactions initiated after interaction of invading pathogens with host APCs. For respiratory pathogens myeloid dendritic cell (DC) precursors that are located underneath the epithelial cell layer lining the airways may play a crucial role in primary activation of T cells and regulating their functional potential. In this study, we investigated the role of human monocyte-derived DC in RSV infection. We showed that monocyte-derived DC can be productively infected, which results in maturation of the DC judged by the up-regulation of CD80, CD83, CD86, and HLA class II molecules. However, RSV infection of DC caused impaired CD4 T cell activation characterized by a lower T cell proliferation and ablation of cytokine production in activated T cells. The suppressive effect was caused by an as yet unidentified soluble factor produced by RSV-infected DC.

Human CD8(+) T cell responses against five newly identified respiratory syncytial virus-derived epitopes.

CD8(+) T lymphocytes play a major role in the clearance of respiratory syncytial virus (RSV) infections. To be able to study the primary CTL response in RSV-infected children, epitopes presented by a set of commonly used HLA alleles (HLA-A1, -A3, -B44 and -B51) were searched for. Five epitopes were characterized derived from the matrix (M), non-structural (NS2) and second matrix (M2) proteins of RSV. All epitopes were shown to be processed and presented by RSV-infected antigen-presenting cells. HLA-A1 tetramers for one of these epitopes derived from the M protein were constructed and used to quantify and phenotype the memory CD8(+) T cell pool in a panel of healthy adult donors. In about 60 % of the donors, CD8(+) T cells specific for the M protein could be identified. These cells belonged to the memory T cell subset characterized by expression of CD27 and CD28, and down-regulation of CCR7 and CD45RA. The frequency of tetramer-positive cells varied between 0.4 and 3 per 10(4) CD8(+) T cells in PBMC of healthy asymptomatic adult donors.

HLA-DP4 presents an immunodominant peptide from the RSV G protein to CD4 T cells.

CD4 T cells play a crucial role during virus infections by producing antiviral cytokines and by regulating humoral and cellular immune responses. Unfortunately however, exaggerated CD4 T cell responses can cause significant immune-mediated disease as was observed during RSV infections in children previously vaccinated with a formalin-inactivated virus in the 1960s. It has been observed that vaccination with the G protein of RSV tends to prime mice for a similar Th2-mediated enhanced disease. Whether the G protein may play a role in enhanced disease in man is unclear. In the present study, we identified an immunodominant epitope in the conserved region of the G protein encompassing amino acid residues 162-175. This epitope is presented in the context of HLA-DPB1*0401 and DPB1*0402, the most prevalent HLA class II alleles. Importantly, in some patients, a mixed Th1/Th2 response against this epitope was found in bronchoalveolar lavage samples during primary RSV infections.