Abstract
BackgroundPrevious studies have demonstrated that DC differentially regulate influenza A virus (IAV)–specific CD8 T cell responses in vivo during high and low dose IAV infections. Furthermore, in vitro infection of DC with IAV at low versus high multiplicities of infection (MOI) results in altered cytokine production and a reduced ability to prime naïve CD8 T cell responses. Flow cytometric detection of IAV proteins within DC, a commonly used method for detection of cellular IAV infection, does not distinguish between the direct infection of these cells or their uptake of viral proteins from dying epithelial cells.Methods/Principal FindingsWe have developed a novel, sensitive, single-cell RT-PCR–based approach to assess the infection of respiratory DC (rDC) and lymph node (LN)-resident DC (LNDC) following high and low dose IAV infections. Our results show that, while a fraction of both rDC and LNDC contain viral mRNA following IAV infection, there is little correlation between the percentage of rDC containing viral mRNA and the initial IAV inoculum dose. Instead, increasing IAV inoculums correlate with augmented rDC MOI.Conclusion/SignificanceTogether, our results demonstrate a novel and sensitive method for the detection of direct IAV infection at the single-cell level and suggest that the previously described ability of DC to differentially regulate IAV-specific T cell responses during high and low dose IAV infections could relate to the MOI of rDC within the LN rather than the percentage of rDC infected.
Highlights
Following influenza A virus (IAV) infection, respiratory dendritic cells mature, upregulate costimulatory molecules, and migrate to the regional lymph nodes (LN) where they participate in the induction of virus-specific CD8 T cell responses [1,2,3]
IAV infection of pulmonary respiratory dendritic cells (rDC) at high, but not low multiplicities of infection (MOI), resulted in increased production of IL-6, MIP-1a and IFNa by certain DC subsets [10]. Similar to these in vitro studies, we have demonstrated that rDC and LNDC produce an increased concentration of IL-12p40 during a high-dose IAV infection setting that results in LNDCmediated killing of CD8 T cells, a blunted virus-specific CD8 T cell response in the lungs, and a lethal outcome [11]
While cells infected at a 0.01 MOI showed only a faint band for NS2 mRNA expression (0.068 intensity relative to cyclophilin), those infected at a 10 MOI showed a more intense band (0.865 intensity relative to cyclophilin), suggesting increased viral loads in cells infected at high MOI relative to those infected at low MOI
Summary
Following influenza A virus (IAV) infection, respiratory dendritic cells (rDC) mature, upregulate costimulatory molecules, and migrate to the regional lymph nodes (LN) where they participate in the induction of virus-specific CD8 T cell responses [1,2,3]. IL-12p70 has been shown to promote increased CD8 T cell survival, CD25 upregulation and cytolytic activity [4,5,6] Because of their essential role in regulating the induction of adaptive immune responses, it is not surprising that migratory rDC and LNDC have been implicated as key players in governing the phenotype and magnitude of adaptive immune responses following lethal vs sublethal dose IAV infections. Previous studies have shown that in vitro infection of bonemarrow derived DC [7,8,9] or isolated murine respiratory DC [10] with IAV can influence subsequent DC cytokine production and priming of virus-specific CD8 T cell responses. In vitro infection of DC with IAV at low versus high multiplicities of infection (MOI) results in altered cytokine production and a reduced ability to prime naıve CD8 T cell responses. Flow cytometric detection of IAV proteins within DC, a commonly used method for detection of cellular IAV infection, does not distinguish between the direct infection of these cells or their uptake of viral proteins from dying epithelial cells
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