infections can be characterized by a biphasic inflammatory response. At the outset of infection, when the bacteria are primarily confined to the site of inoculation, there is little evidence of inflammation or production of detectable proinflammatory cytokines. However, shortly after the bacteria have disseminated and have begun to replicate in peripheral tissues, inflammation becomes evident and, in the setting of lethal disease, contributes to the death of infected hosts (20
). The mechanisms by which Francisella
evades initiating early proinflammatory responses are not fully understood. Similarly, the direct contribution of Francisella
in the induction of inflammation at the end of infection is equally poorly characterized.
In this report, we demonstrate that evasion of induction of early inflammatory responses by virulent F
in both primary target cells in vitro and pulmonary tissues in vivo is partially dependent on the absence of CD14. Supplementation of CD14 in vitro to target cells most likely to be present at the site of infection, i.e., dendritic cells, or in vivo directly at the site of inoculation, i.e., the lung, resulted in increased production of several proinflammatory cytokines. Importantly, CD14-mediated production of proinflammatory cytokines in vivo correlated with modest, but significant, control of bacterial replication and dissemination. The lack of control of bacterial replication in vitro may be explained by the absence of additional effector cells capable of responding to the initial wave of proinflammatory cytokines via production of other soluble mediators that stimulate killing in target cells. For example, preliminary data generated in our laboratory suggest that F
SchuS4 inhibits the ability of infected cells to respond to certain proinflammatory cytokines (e.g., TNF-α) and undergo activation. Thus, control of F
SchuS4 may rely on indirect activation via other, uninfected effector cells. In support of this hypothesis, previous reports suggest that efficient killing of intracellular F
requires production of IFN-γ and that the source of IFN-γ during the critical early stages of infection is NK cells (42
). However, optimal production of IFN-γ by NK cells typically requires IL-12p70 secreted from resident macrophages and/or DC (reviewed in reference 57
). Thus, it is reasonable to speculate that while production of proinflammatory cytokines, such as TNF-α and IL-12, following addition of CD14 to cells or tissues is the first step toward killing SchuS4, it is not sufficient on its own. Rather, early control of SchuS4 infection requires induction of IFN-γ by other, uninfected host cells responding to IL-12. Our in vitro culture consisted only of DC or monocytes and lacked IFN-γ-producing “effector” cells. Thus, although CD14 facilitated increased production of several proinflammatory cytokines, cells capable of responding to these cytokines via secretion of more potent stimulating cytokines were not present and no control of SchuS4 replication was observed. In vivo, the full complement of cells, both target and effector, are present at the site of infection. Therefore, it is possible that the IL-12 present in CD14-treated, SchuS4-infected mice activated resident NK cells to produce IFN-γ, resulting in modest control of SchuS4 replication.
The production of cytokines following pulmonary F
SchuS4 infection is tightly correlated with the recruitment of effector cells, such as granulocytes and monocytes (21
). However, this recruitment does not typically occur until 3 days after infection (21
). Rather, at the outset of infection, the primary pulmonary cell types infected are alveolar macrophages and dendritic cells, both of which lack CD14 (11
) (Fig. ). In fact, in one report, SchuS4 was not even detected in resident granulocytes during the first day of infection (30
). Thus, an additional explanation for the early control of SchuS4 in the lungs and spleens of sCD14-treated animals is that there was early recruitment of effector cells, such as granulocytes and monocytes, into these organs. However, we did not observe recruitment of granulocytes or monocytes following administration of sCD14 into either the lung or spleen. Thus, our data indicate that the cells responsible for producing proinflammatory cytokines in the presence of CD14 are resident alveolar macrophages and dendritic cells.
Our studies also suggest that the dependence on CD14 for elicitation of early inflammatory responses in vivo may be unique to F
. Previous reports examining the role of CD14 in other pulmonary bacterial infections have suggested that the presence of this receptor plays a deleterious role in control of bacterial replication and dissemination. For example, mice lacking CD14 controlled pulmonary Burkholderia pseudomallei
infections significantly better than wild-type mice did (63
). Similarly, CD14 was found to contribute to mortality and development of pulmonary pathology observed in mice infected with Mycobacterium tuberculosis
). However, it should be noted that in both of these examples the bacteria typically elicit a strong inflammatory response during the early stages of infection and that this response is thought to contribute to the overall pathology of disease mediated by these microorganisms. To better assess the contribution of CD14 in infections mediated by Gram-negative bacteria that do not typically elicit strong inflammatory responses, we examined the effects CD14 has on production of cytokines and chemokines by human dendritic cells and monocytes following infection with B
. In contrast to F
SchuS4, addition of sCD14 to human DC infected with B
failed to elicit production of cytokines and chemokines from these mammalian cells (see Fig. S1 in the supplemental material). However, the presence of CD14 did appear to play a role in the recognition of B
by human monocytes (see Fig. in the supplemental material). Thus, the role of CD14 in recognition of B
is cell type dependent. This suggests that the requirement for CD14 in the recognition of strain SchuS4 by both dendritic cells and monocytes is unique to this virulent bacterium.
Given the effect CD14 had on the induction of proinflammatory cytokines during the first few days of infection, it is possible that this receptor contributes to the overwhelming production of cytokines that is typically associated with mortality in F
SchuS4-infected hosts (20
). However, SchuS4-infected CD14−/−
mice were not more susceptible to SchuS4 infection and did not have significant differences in the amount of cytokine detected in target tissues at the end stage of disease compared to wild-type controls (Tables and ). The disparity of these data with results examining the role of CD14 at the outset of infection is similar to a phenomenon described in other bacterial infections. Earlier reports examining the roles for cell surface receptors in the recognition of soluble and particulate bacterial antigens demonstrated that CD14 was capable of recognizing both soluble and particulate antigens. In contrast, another cell surface receptor complex known to interact with bacterial antigens, CD11b/CD18, responded only to particulate antigen (26
). These observations were extended by examining the contribution of CD14 and CD11b/CD18 during different stages of infection with Neisseria meningitidis
. In that study, stimulation of host cells by small numbers of bacteria (representing the early phase on infection) was dependent on CD14. In contrast, activation of host cells by large numbers of bacteria (representing the terminal stage of disease) was CD14 independent (31
). Additional reports have shown that CD11b/CD18 enhances responses against intact bacteria and can compensate for the absence of CD14 in the recognition of these microorganisms (43
). Thus, while CD14 appears to be critical for recognition of F
SchuS4 at the outset of infection, production of proinflammatory cytokines at the end of disease, when high numbers of bacteria are present in a variety of tissues, was CD14 independent and may require signaling through other receptors, such as CD11b/CD18.
CD14 acts as a coreceptor for both TLR2 and TLR4/MD-2 (25
). Thus, it is likely that the CD14-dependent secretion of cytokines from human and mouse cells following F
SchuS4 infection is due to enhanced interaction of SchuS4 antigens with either TLR2 or TLR4/MD-2. Further, since the contribution CD14 makes in elicitation of cytokine production occurs early after infection with SchuS4, it was also likely that the antigens interacting with this molecule are present on the surface of the bacterium. Specific TLR agonists present in SchuS4 have not been formally reported. However, recent reports examining the ability of intact, attenuated subspecies of Francisella
, e.g., F
LVS, and their associated antigens to stimulate TLR-dependent responses have revealed several TLR2 and TLR4/MD-2 agonists in the membranes of these bacteria.
The two TLR4/MD-2 agonists found in F
LVS are LPS and the heat shock protein, DnaK (HSP70) (4
). LPS associated with both F
LVS and SchuS4 is tetraacylated and, therefore, is a very poor TLR4 agonist (46
). Initially, it was assumed that poorly acylated LPS failed to interact with CD14, ultimately interfering with efficient delivery of LPS to the TLR4/MD-2 complex. However, it has been shown that the number of acyl groups present on LPS molecules does not impact their ability to interact with CD14 (44
). Rather, the presence of six or more acyl groups is essential for interaction of LPS with the MD-2 portion of the TLR4/MD-2 complex (44
). Thus, the weak activity tetraacylated LVS and SchuS4 LPS have in TLR4/MD-2 stimulation is not dependent on CD14 but on the absence of acylation required for triggering TLR4/MD-2 responses. Therefore, a predominate role for Francisella
LPS in mediating inflammatory responses via CD14 and TLR4 seems unlikely.
The other TLR4/MD-2 agonist present in attenuated subspecies of Francisella tularensis
is the heat shock protein DnaK, also known as HSP70 (4
). Heat shock proteins, e.g., HSP60 and HSP70, are present in both pro- and eukaryotic systems and have been widely recognized as TLR4/MD-2 and TLR2 agonists that require CD14 for efficient delivery to the TLRs (53
). Both HSP60 and HSP70 are secreted by F
into culture medium, but only HSP60 has been found to be secreted during intracellular infection (41
). Further, HSP60, but not HSP70, is located in the periplasmic membrane of F
). Thus, although HSP70 has been formally identified as a TLR4 agonist, the absence of secretion of this antigen during intracellular infections suggest that its role in elicitation of early inflammatory responses may be fairly minimal. Rather, HSP60 represents a more likely candidate antigen for stimulation of CD14-dependent TLR4/MD-2 and/or TLR2 responses following infection with viable, intact bacteria.
Earlier studies have shown that live, intact F
LVS induces inflammatory responses primarily through TLR2 (19
). Since that initial observation, several lipoproteins from F
LVS have been identified as TLR2 agonists (55
). These lipoproteins include Tul4 and FTT1103 and appear to require heterodimerization of TLR2 and TLR1 for optimal elicitation of proinflammatory cytokines from mammalian cells. Interestingly, these studies did not address the requirement for CD14 in elicitation of cytokines by Francisella
lipoproteins. Thus, both Tul4 and FTT1103 could contribute to the production of cytokines and chemokines we observed herein, but the specific contribution of CD14 will need to be determined.
The failure of F. tularensis SchuS4 to elicit production of proinflammatory cytokines and chemokines early after infection is believed to contribute to the overall virulence of this bacterium. The mechanisms by which F. tularensis SchuS4 evades this early detection are not well characterized and represent a significant hurdle in the development of novel therapeutics and vaccines. Data presented herein demonstrate, for the first time, that elicitation of early inflammatory responses in the lung strongly correlates with modest control of SchuS4 replication and dissemination. Furthermore, we show that induction of these early proinflammatory responses in vitro and in vivo was dependent on CD14. Together, our data suggest that identification of host molecules, such as CD14, that contribute toward the early detection of F. tularensis SchuS4 may aid in development of novel therapeutics for treatment of this aggressive, virulent bacterium.