Understanding both innate and adaptive immune responses against a vaccine strain of F. tularensis
is of interest for basic immunology as well as for vaccine development for intracellular pathogens. Studies using LVS illustrate the potential of a live attenuated vaccine, and at the same time, LVS is an excellent tool for studying immune responses to intracellular pathogens in animal models. In this report, we focused our attention on the characterization of IFN-γ-producing cells during the early stage of F. tularensis
LVS vaccination, particularly those expressing NK cell markers, given previous unexpected results regarding the minimal impact of NK cell depletion on LVS vaccination (31
). In order to obtain a dynamic evaluation of IFN-γ responses, we determined IFN-γ production over time, using several complementary approaches. By 3 days after sublethal i.d. LVS infection, bacteria in the spleen and liver reached peak levels (Fig. ); concurrently, IFN-γ mRNA, IFN-γ secreted protein, and a substantial number of IFN-γ-producing cells, as determined by ICS, were detectable (Fig. and ). By day 5, a surge of secreted IFN-γ was accompanied by increased total cell numbers (Fig. ) and IFN-γ+
cells (Fig. ) in both organs. Finally, by day 7, innate immune responses appeared to be effective, since bacterial organ burdens started to decline, while IFN-γ-producing cells as well as IFN-γ secretion were still present at substantial levels and T cells were activated.
Following initial qualitative characterization of the time course of IFN-γ production in spleens and livers from LVS-infected mice (Table ), we selected 4 days of infection for further detailed studies. At this time point, all of the different IFN-γ+ cell types detected throughout the first week were in some stage of IFN-γ production. Studies with separated splenocyte subpopulations clearly established that B cells (CD19+ enriched cells) did not contribute (Fig. ). Detailed analysis at day 4, using unmanipulated total numbers of splenic and liver lymphocytes for multicolor flow cytometry, permitted calculation of the total number of IFN-γ+ cells of each cell type in each organ (Fig. to ). This analysis revealed that conventional NK cells, NK T cells, and NK DCs as well as different myeloid cells, including numerous DCs, were involved in IFN-γ production during innate immune responses (Table ; Fig. to ), in a T-cell-independent manner (Fig. ). The results were remarkable for illustrating functional redundancy that ensures many sources of this critical cytokine, with a large variety of cells involved, as well as the evolution of their engagement over time. Not surprisingly, production by different cell types peaked at different time points. Although neutrophils and macrophages producing IFN-γ in vivo following LVS infection were reproducibly detectable, their numerical contribution to the total production was minimal throughout the week studied.
One protein clearly important in regulating IFN-γ production is MyD88, part of the signal transduction cascade engaged upon the activation of Toll-like receptors (TLRs) secondary to the recognition of pathogens. The absence of MyD88 renders mice susceptible to many intracellular pathogens (19
), including LVS (10
), at least in part because of poor production of IFN-γ. Thus, it will be important to next assess the role of MyD88 and associated TLRs in initiating IFN-γ production by individual cell types. In addition to being critical for survival of initial infection with virtually any intracellular pathogen, the sources of innate, early IFN-γ production are of interest because IFN-γ regulates the expression of such a large variety of genes. At least 200 genes, many involved in antigen presentation, antimicrobial functions, and leukocyte trafficking, are regulated by IFN-γ (44
). Given the global importance of this mediator, it is perhaps not surprising that so many innate immune cell types are endowed with the ability to respond and to produce it following infection; this redundancy in function likely ensures a sufficient IFN-γ response that leads to gene activation, the associated functional cascade, and ultimately, survival.
NK cells and T lymphocytes have commonly been considered the principal sources of IFN-γ during infections with intracellular bacteria. In classic studies, NK cells were identified as those responding to Listeria
exposure in vitro (52
), and in vivo or in vitro depletion of NK cells followed by Listeria
exposure reduced IFN-γ production at day 1 but not day 7 or 14 after infection (50
). Similarly, using Listeria monocytogenes
-infected mice, NK cells were described as the main source of IFN-γ in spleens and livers of RAG mice about 18 h after infection; major histocompatibility complex class II-positive IFN-γ+
cells were not detected (51
). Most recently, murine cells bearing both NK markers and CD11c, which may more closely resemble NK cells than DCs in function (6
) and which produce abundant IFN-γ upon Listeria
infection, have been described (40
). Indeed, these NK DCs may be the most important cell type contributing to control of Listeria
infection. The relationship between NK DCs and another unusual population, dubbed “IK DCs,” for IFN-producing killer DCs, remains to be fully clarified (8
). In this study, we were readily able to recognize LVS-stimulated IFN-γ production by several NK-related subpopulations, including traditional NK cells (defined as CD45+
cells), NK T cells (defined as CD45+
cells), and NK DCs (defined as CD45+
Other studies showed clearly that CD8α+
DCs can be a major source of IFN-γ production upon in vitro culture of splenocytes with Listeria
). Furthermore, mice lacking the cytokine receptor common gamma subunit (γC−
mice), which are severely deficient in NK cells, exhibited normal early IFN-γ production and control of Listeria
), and transfer of DCs into Listeria
mice greatly increased IFN-γ production and control of infection (48
). Similarly, in the present study, CD11c+
cells which clearly lacked NK markers were the dominant IFN-γ+
cell type found in LVS-infected spleens as well as in livers. Given the importance and complexity of the many DC subpopulations now being defined, future studies will concentrate on delineating the DC subpopulations responding to Francisella
in greater detail.
Another interesting alternative source of IFN-γ production quickly after infection is “bystander” CD8+
T cells, which are apparently non-antigen specific. Thus, when murine spleen cells were incubated in vitro with Burkholderia pseudomallei
, both NK cells and CD8+
cells produced IFN-γ within 24 to 48 h (32
), and CD8+
T cells in mice infected with Burkholderia pseudomallei
produced IFN-γ within 16 h of infection (32
). However, such cells were not readily detectable in either spleens or livers of LVS-infected mice.
Indeed, for LVS infection, thus far only NK cells have been implicated in innate IFN-γ production. Mouse liver lymphocytes produced IFN-γ in vitro when they were stimulated with LVS or type A Francisella
, and at least half of those lymphocytes were NK cells (54
). Similarly, 72 h after intranasal LVS infection, CD11b+
lung cells harvested and then cultured in vitro with phorbol myristate acetate and ionomycin appeared to be the major producers of IFN-γ (33
). Furthermore, NK cell depletion followed by intranasal LVS infection reduced serum IFN-γ levels compared to those of control mice. It is important, however, that strong polyclonal stimulation, while useful for increasing IFN-γ detection, may facilitate the expansion and functions of one cell type over another during in vitro culture, thereby altering the observed pattern. In this study, we instead elected to study cells obtained from LVS-infected mice by ICS without further deliberate stimulation during the in vitro culture period required for brefeldin addition beyond that provided by the numbers of bacteria present in vivo and carried over into the in vitro cultures. Strikingly, this approach revealed a much greater variety of cell types involved in innate IFN-γ production following LVS infection in vivo than that obtained by in vitro studies or using ex vivo phorbol myristate acetate stimulation. Indeed, by 4 days after i.d. LVS infection, the numbers of DCs, traditional NK cells, and NK DCs producing IFN-γ were roughly equivalent. If the principal function of NK cells during intracellular infections is the production of IFN-γ, the identification of large numbers of non-NK-related cells producing IFN-γ provides an explanation as to why NK cell depletion has apparently little impact during LVS vaccination.
Another understudied aspect of infections that disseminate to organs of the reticuloendothelial system is the role of liver leukocytes. The liver, as a peripheral site of infection, is an important site of cell-mediated immune surveillance and effector functions (34
) and is rich in NK cells and unconventional T cells, such as NK T cells (24
). LVS infects livers of mice within 3 days of infection by any route and replicates in both Kupffer cells and hepatocytes (12
). As noted above, mouse liver lymphocytes cultured with LVS in vitro produced IFN-γ. At least half of the production was attributed to NK1.1+
NK cells, but the balance of the cells were not characterized (54
). Here we demonstrated qualitatively similar results between liver leukocytes and splenocytes (Fig. ), although some quantitative differences were evident. The numbers of leukocytes, particularly T cells, in LVS-infected livers increased at an even higher rate than did the numbers of splenocytes (Fig. ); it remains to be determined whether this resulted from local proliferation and/or migration from professional immune system organs. Bacterial organ burden data for livers were always slightly higher than those for spleens at all time points, and bacterial clearance was somewhat slower in livers (Fig. ). Most notably, the proportion of leukocytes producing IFN-γ consistently appeared to be higher in livers than in spleens (Fig. and ; Table ).
The production of IFN-γ from some cells may depend on signals released by other cell types, and the lack of those cells therefore alters IFN-γ production. To address this possibility, we compared the response of LVS-infected, T- and B-cell-deficient RAG mice to that of WT mice. In the absence of B and T cells, the numbers of IFN-γ-producing cells were comparable between the two strains of mice (Fig. ), indicating that myeloid cells did not require the presence of lymphocytes to respond properly to LVS infection. This is consistent with the observation that IFN-replete but lymphocyte-deficient SCID
mice infected with LVS i.d. survive for up to 3 weeks after infection (23
). The lack of lymphocytes that might otherwise confound flow cytometry analyses or separation studies also served to further confirm the predominance of the numbers of IFN-γ+
DCs in LVS-infected mice (Fig. ).
These analyses are, of course, subject to the limitations of the techniques applied. Only cells from infected organs that were amenable to preparation in single-cell suspensions were included in the flow cytometry analyses. Furthermore, detection of intracellular cytokine does not necessarily result in secreted protein. Nonetheless, the concordance between the presence of IFN-γ mRNA, IFN-γ+
cells by ICS, and IFN-γ protein secretion in vitro following in vivo LVS infection lends confidence to the interpretation that the flow analyses accurately detected at least a large proportion of the cells involved. Obviously, the limited sensitivity of ICS may still fail to detect some cells, even in single-cell preparations. Our intent here was to evaluate IFN-γ production in vivo by using an approach that resembles clinical vaccination and infection as closely as possible. Despite such acknowledged limitations, this approach has nonetheless served to reveal a complex innate immune response and, in particular, has emphasized the unexpected contribution of DCs as a major IFN-γ-producing cell type in both spleens and livers from LVS-infected mice. Other studies have shown that subtypes of DCs, particularly plasmacytoid DCs, produce IFN-α in response to viral infection or stimulation (14
). The present study firmly establishes DCs as a major source of IFN-γ production after intracellular bacterial infection. The specific relationship between subsets of cytokine-producing DCs, infection of DCs, and antigen presentation is currently unknown (17
) and is a subject of intense current study. Further studies to completely characterize the subtypes and functions of DCs that respond to LVS infection, as well as the infection status of DCs that produce cytokines, are under way. We expect such studies to have significant implications for immunotherapeutic strategies and for vaccine development.