In this report, we examined the responses of total lung CD11c+ cells enriched for RDC and specific RDC subsets to influenza virus infection. We demonstrated that the pattern of infection of total unseparated RDC (CD11c+ SiglecF− cells) by influenza virus was not uniform. A fraction of this total cell population was readily infected by influenza virus and expressed high levels of influenza virus nucleocapsid protein as early as 4 h p.i., particularly at the highest MOI employed (i.e., 10). A second fraction of cells appeared more resistant to infection and expressed nucleocapsid protein over a more prolonged time interval. This second subpopulation also upregulated cell surface costimulatory ligands and sustained high-level expression of these ligands throughout the course of in vitro infection. Based on these findings, we went on to examine whether the apparent heterogeneity in the response to influenza virus infection, and in particular the apparent differences in sensitivity to infection by the virus, reflected the responses of distinct RDC subsets present in the culture to infection. We demonstrated that different RDC subsets showed differential susceptibilities to influenza virus infection in vitro: CD103+ DC were the most susceptible to infection, with CD11bhi DC demonstrating intermediate susceptibility to infection and pDC the least. These in vitro observations were corroborated by in vivo studies on the susceptibilities of these three RDC subsets to infection. Finally, the differences in the responses of these RDC subsets to infection were also reflected in the MOI-dependent production of at least one cytokine, IL-12, by the different RDC subsets in response to virus infection.
Several different DC subsets have been identified in the lungs, each with distinct properties indicating functional specialization (12
). In the mouse, RDC express CD11c, which serves as a conventional marker for the identification and isolation of this lung cell type. RDC can be further subdivided based on the level of MHC class II expression; the expression of various cell surface markers, most notably the myeloid cell marker CD11b; and more recently the differential expression of the CD103 integrin alpha chain (36
). The expression of the last two markers is of considerable interest, as they distinguish the anatomical localization of these two RDC subsets. Our initial analysis of the susceptibilities of isolated CD11c+
lung cells to infection with influenza virus in vitro (Fig. ) suggested that different subsets of RDC may be differentially susceptible to infection with type A influenza virus. Two sets of observations supported this hypothesis. First, in our analysis of the susceptibilities of purified CD103+
RDC, and pDC to influenza virus infection, a clear difference in susceptibility was noted (Fig. ), with CD103+
RDC representing the most susceptible RDC subset and pDC the least susceptible subset. Second, when isolated from the lungs of influenza virus-infected mice and analyzed directly ex vivo, the CD103+
RDC subset also demonstrated the greatest percentage of influenza virus-infected (NP-expressing) cells (Fig. ). While in vitro infection of RDC at an MOI as high as 10 may appear to be artificially high, in vivo exposure of RDC (particularly the airway-localized CD103+
RDC to an MOI as high as 10) is possible, if not likely, because of the very high local lung virus titers achieved early in the infected RT of mice (24
). Nonetheless, it is noteworthy that, in spite of infection of RDC in vitro with virus at an MOI as high as 10 or after high-dose infection in vivo, only a fraction of the RDC were susceptible to infection. The mechanistic basis for this resistance to infection remains to be defined.
The finding that the CD103+
cells are the RDC subset most susceptible to infection among those examined is particularly intriguing, as these cells have been demonstrated to localize to the epithelial mucosa of the large (conducting) and small airways, forming a dense intraepithelial/subepithelial web (36
). The close anatomical proximity of this RDC subset to the respiratory epithelium, the major target of productive influenza virus infection in the RT, make this RDC subset a likely candidate for efficient infection by influenza virus. Indeed, among the CD11c+
cells, which migrate from the lungs to the draining mediastinal lymph nodes in response to influenza virus infection (23
), only the CD103+
RDC subset is infected by the virus and is a potent stimulator of naïve virus-specific CD8+
T cells (T. S. Kim and T. J. Braciale, unpublished data). While these findings might argue for a prominent/dominant role of CD103+
RDC in the orchestration of the adaptive immune response to influenza virus because of their susceptibility to infection and proximity in vivo to epithelial cells, such an interpretation should be considered with caution. As this report demonstrates, the three RDC subsets analyzed are each susceptible to infection with influenza virus in vitro and in vivo. Furthermore, both the CD103+
DC and the CD11bhi
RDC subsets can trigger the induction of response by antigen-specific CD8+
T cells after in vitro infection of the RDC and coculture with naïve T cells (X. Hao and T. J. Braciale, unpublished data). At present, the precise role of each of these RDC subsets in the induction of adaptive immune responses to influenza virus is uncertain and remains to be elucidated, although recent studies suggest that the CD103+
DC and the CD11bhi
RDC may have distinct and complementary roles in the induction of T-cell responses to soluble antigen introduced into the RT (5
The unseparated CD11c+ RDC (Fig. ) produced several different inflammatory mediators. In the cases of several mediators (e.g., the murine cytokine CXCL1 [KC] and MIP-1α), the time courses of chemokine secretion by uninfected RDC undergoing maturation in vitro and RDC infected at different MOIs were compatible (Fig. ) or at least did not reach statistical significance (Fig. ). It appears, therefore, that direct ex vivo infection of these mature lung tissue DC by influenza virus provides only a modest stimulus for the production of some proinflammatory mediators beyond that observed with uninfected RDC. This could reflect suppression of the innate host response in the infected cells by influenza virus or, perhaps more provocatively, mucosally localized DC like RDC may only weakly respond in situ to inflammatory stimuli, like virus infection, in order to prevent the development of excessive local inflammation in the RT. In keeping with this concept, we found that elevated IL-6 production (Fig. ) was only observed after infection at the highest MOI (also observed for IFN-α). By contrast, the secretion of IL-12p40 was suppressed after infection at the highest MOI employed (Fig. ), suggesting an immunosuppressive effect of influenza virus infection on the production of certain critical mediators.
Analysis of the cytokine responses of individual RDC subsets (Fig. ) revealed the cellular sources of these mediators. As expected, lung pDC were the major producers of IFN-α among the RDC, and production of this cytokine was dependent upon the MOI. pDC were also prominent producers of most of the other mediators examined, in keeping with their likely role as early innate immune effectors critical in the early host response to virus infection (9
). Although the CD11bhi
RDC subset has been implicated as an important producer of a variety of proinflammatory mediators in the RT (5
), the chemokine RANTES was the predominant inflammatory mediator produced by this RDC subset (Fig. ).
IL-12 is believed to play a critical role in the induction of adaptive immune responses through its ability to drive the differentiation of activated T cells, and in particular CD8+
T cells, into fully functional effector cells (10
). As noted above, there was a progressive decrease in the secretion of the IL-12p40 molecule with increasing MOIs in both the unseparated RDC (Fig. ) and the CD103+
RDC subset (Fig. ). The MOI-dependent decrease in IL-12p40 production by CD103+
RDC is most easily explained by influenza virus-induced inhibition of host protein synthesis, as this RDC subset is most susceptible to infection. If CD103+
RDC serve as the dominant APC for the induction of a CD8+
T-cell response to influenza virus, then infection of this cell type at a high MOI in the RT could impair the cells' capacity to effectively present antigen and activate antiviral CD8+
T cells after their migration into the lymph nodes from the infected lungs through dysregulation of IL-12 production, as previously suggested by us (24
). Exposure of RDC to the rapid and high-level lung virus replication after a high-inoculum-dose infection in vivo could mimic the impact of in vitro infection at a high MOI on the function/antigen-presenting capability of CD103+
RDC and result in the defective CD8+
T-cell response to high-dose influenza virus infection. A test of this possibility awaits isolation and detailed characterization of this and other RDC subsets isolated from mice infected with different virus inocula.
As demonstrated here (Fig. ) and elsewhere (36
), the alveolar macrophages are the most highly represented CD11c+
cell subset in the normal murine lung, with total RDC and individual RDC subsets representing smaller fractions of the total CD11c+
cells. Isolation of sufficient numbers of individual RDC subsets from normal mouse lungs to carry out the analyses described here was not feasible. We therefore developed a strategy to increase the numbers of RDC isolated from the lung by relying on the DC growth factor Flt3L to generate expanded numbers of RDC in the lungs for subsequent analysis and in vitro characterization. Although treatment with Flt3L has been reported to increase the total number of RDC in the lungs (27
), exposure to Flt3L may also increase the maturation/activation state of the RDC. This could be particularly problematic using the plasmid-based approach employed here to express this growth factor. However, we did not note any significant difference in the level of maturation/costimulatory ligand expression among total RDC or individual RDC subsets isolated from the lungs of healthy and Flt3L-treated mice (Fig. and ). Thus, for the three RDC subsets analyzed in vitro in this report, we believe that the use of RDC isolated from the lungs after Flt3L-induced expansion closely mimicked the corresponding RDC subsets isolated from the normal lungs. We did, however, identify a population of CD11blow/intermediate
-expressing cells with variable levels of MHC class II expression that were increased in the lungs of Flt3L-treated mice (Fig. ). We do not know the nature of the cells, but they likely represent precursors to the more mature RDC subsets. These lung CD11c+
cells likely were recently mobilized from the bone marrow to the lungs as a result of growth factor treatment.
In summary, in this report, we have shown that RDC can be efficiently infected by influenza virus. Importantly, for the first time, we provided evidence that different RDC subsets display differential susceptibilities to influenza virus infection and differential cytokine production. Our results suggest a complex interaction among RDC subsets and other innate immune cells responding to influenza virus infection.