The results of this study confirm and extend a recent study that demonstrated that TLR2 is critical for protection of mice against a primary pulmonary infection with F. tularensis
. However, in contrast to a previous report 
, we show that the requirement for TLR2 is independent of the route of infection, since TLR2−/−
mice had significantly decreased survival rates compared to controls following infection by either the intranasal or the intradermal route. MyD88 also contributes significantly to survival of a primary pulmonary LVS infection, consistent with its reported role in the intradermal infection 
. The results of this study further demonstrate that the host protective response to primary infection with F. tularensis
is also dependent on TLR2/MyD88 signaling. Importantly, TLR2−/−
macrophages are impaired in their ability to express pro-inflammatory cytokines and chemokines in response to F. tularensis
LVS infection and pro-inflammatory cytokine expression is significantly impaired in vivo
in the lungs of infected TLR2-deficient mice. Finally, this study revealed that neither TLR1−/−
mice were any more susceptible to LVS infection than control mice, although a previous study reported an exclusive role for TLR6 in the pro-inflammatory cytokine response of BM-derived dendritic cells to LVS in vitro 
As stated above, our conclusion that TLR2−/−
mice are more susceptible to primary infection with LVS regardless of the route of infection differs from conclusions drawn by Collazo et al. 
. It should be noted, however, that the survival rate reported by Collazo et al.
for i.d. LVS infection of TLR2−/−
mice (68%, n
19) is not very different from the overall survival rate of 56% (n
16) observed in this study, suggesting that the somewhat different results and the distinct conclusions may be due to differences in the infectious doses given, or simply due to different statistical treatment of the data. It is also important to point out that in our studies, as well as those of Collazo et al.
, mice were infected intradermally with doses of LVS that are significantly below (1/40th
, respectively) the reported i.d. LD50
for B6 mice (~2×106
. It seems very likely therefore that infection with higher doses of LVS would result in an even larger difference in survival rates between control mice and TLR2−/−
mice, as was observed by Malik et al.
for intranasal LVS infections 
, and supporting the conclusion that TLR2 is important in both i.d. and i.n. infections. An accurate understanding of the role of particular TLRs in host responses to infections by different routes is important because of the implications for the rational design of vaccine-enhancing adjuvants.
mice both demonstrated decreased survival rates compared to wild-type controls, the MyD88−/−
mice were significantly more susceptible to i.n. and i.d. LVS infection than were the TLR2−/−
mice. The reduced survival time for the MyD88−/−
mice also correlated with a greater increase in bacterial burden compared to TLR2−/−
mice beginning on day 3 post-infection. Notable also was the complete lack of detectable TNF in the lungs of the MyD88−/−
mice at days 1–5 post-infection. This is not surprising given the role of MyD88 as a critical signaling adaptor in numerous pro-inflammatory signaling pathways, including those activated via the IL-1β and IL-18 receptors, and in other TLR signaling pathways, e.g.
. Indeed, Mariathasan et al.
recently demonstrated that F. tularensis
grew to higher titers in the organs of mice depleted of IL-1-β or IL-18 by treatment with neutralizing antibodies 
. In that regard, however, Collazo et al.
recently reported that survival of TLR9−/−
mice after i.d. LVS infection was no different than for wild-type control mice 
. Thus the basis for the difference in susceptibility to infection observed between MyD88−/−
mice requires further study.
The survival studies reported herein indicate that neither TLR1 nor TLR6 is required exclusively to pair with TLR2 in recognition of F. tularensis
LVS ligands. These results would appear inconsistent with a recent study that demonstrated a complete abrogation of TNF secretion by dendritic cells from TLR6-deficient mice but normal TNF secretion by TLR1-deficient cells 
. We have also recently compared the TNF response to LVS by bone marrow-derived dendritic cells and have found no difference in the response between cells derived from TLR1−/−
mice; however, the response from both were impaired relative to wild-type control dendritic cells (unpublished data). The reason for the discrepancy in results is not clear, but recently Re and colleagues have demonstrated that either TLR1 or TLR6 expressed in HEK-293 cells can recognize and mediate a response to LVS ligands 
and the same group has recently identified specific ligands for TLR2/TLR1 in LVS 
. Thus, the observation that deficiencies in either TLR1 or TLR6 have no impact on the survival of mice whereas TLR2 deficiency has a profound impact suggests that TLR1 and TLR6 may be redundant in the ability to recognize F. tularensis
ligands in concert with TLR2. Interestingly, in our studies, macrophages from TLR6−/−
mice expressed significantly higher levels of TNF, IL-6, and MCP-1 than wild-type macrophages. It is not known if the absence of TLR6 leads to aberrant or increased activation through TLR2/TLR1 or if TLR6 normally functions to negatively regulate a signaling pathway.
Our studies and those of others 
indicate that TLR4 plays no protective role in the host immune response to F. tularensis
infection. This has been a somewhat surprising finding since F. tularensis
is a Gram-negative bacterium and because a previous study reported that TLR4-defective mice (C3H/HeJ strain) were more susceptible to intradermal infection with LVS 
. However, the LPS produced by F. tularensis
has very little endotoxin activity compared to the LPS produced by E. coli
species and has recently been shown to bind poorly, if at all, to TLR4 
. Moreover, other Gram-negative bacteria with atypical LPS have been reported to signal primarily through TLR2 rather than TLR4 
. It is interesting to note that the host inflammatory response to F. tularensis
infection in the lungs of wild-type mice appears to be significantly delayed 
, suggesting the possibility that the absence of a potent TLR4 ligand in F. tularensis
plays an important role in immune evasion. Indeed, recent studies have observed decreased virulence and enhanced innate immune responses for F. tularensis
mutants with altered lipid A moieties 
. Although we have shown that TLR2−/−
mice are more susceptible to i.n. infection with F. tularensis
, a strain with a distinct, more biologically active LPS, and which is more highly virulent in mice than the LVS strain 
, TLR4 does not contribute significantly to host protection against this strain either.
TNF is well-known to play an important role in the immune response to F. tularensis
infection and TNF-deficient mice succumb quickly to LVS infection, as do IL-12-deficient and IFN-γ-deficient mice 
. Such studies support a model in which early expression of TNF, IL-12 and IFN-γ by diverse myeloid and lymphoid cell types in response to F. tularensis
infection induces recruitment of inflammatory cells and IFN-γ production primarily from NK cells and dendritic cells 
that in turn further activate macrophages and dendritic cells and induce Th1 immunity. The significant reduction in the expression of TNF by TLR2-deficient macrophages infected in vitro
and the significant reduction of TNF expression in the lungs of TLR2−/−
mice infected with LVS is striking and suggests that the increased susceptibility of TLR2-deficient mice to infection can perhaps be accounted for solely by the impairment of TNF expression in vivo
early in the infection. However, other TLR2-dependent pro-inflammatory cytokines and chemokines may also play important roles in the protective host immune response as illustrated by the TLR2-dependent induction of IL-6 and MCP-1 in infected macrophages. Although our data do not reveal which cells harbor LVS in the lungs or which cells are producing proinflammatory cytokines, others have shown that alveolar macrophages, dendritic cells, and neutrophils are the primary cells in the lung that are infected with F. tularensis 
. Future studies will attempt to delineate the role of the expression of individual cytokines by specific cell types in the TLR-dependent host response to F. tularensis
Finally, the components of F. tularensis
that are responsible for activating innate immune responses in the host are just beginning to be identified 
. TLR2 is expressed on many cell types and has been reported to bind to a broad array of microbial components 
, most notably lipoproteins 
, but also peptidoglycan 
, and recently a bacterial porin 
. TLR2 has also been shown to play a role in the host response to a number of infections by both Gram-positive and Gram-negative bacteria, including Staphylococcus aureus 
, Streptococcus pneumonia 
, Legionella pneumophila 
, and Porphyromonas gingivalis 
; it also plays an important role in the responses to a number of bacteria that express an atypical LPS 
. Identification of the TLR2 ligands responsible for activating host protective responses to F. tularensis
, as well as other possible F. tularensis
PAMPs, will be important for a complete understanding of F. tularensis
pathogenesis and therefore an important goal of future studies.