DCs are a central component of the mammalian immune system and serve as both responders during innate immunity and a bridge to effective and long-lasting memory immune responses. Thus, it is unsurprising that many pathogens have developed mechanisms to modulate and disable DCs (1
). One way in which pathogens interfere with host immune responses, including those mediated by DCs, is manipulation of normal host systems meant to limit hyperresponsiveness to microbial products, resulting in control of inflammation. For example, the temporary state of refractoriness to endotoxin and other microbial products following sepsis (referred to as “microbial tolerance”) tempers the inflammatory response and development of septic shock (as reviewed in reference 35
). However, this state of tolerance also leaves the host susceptible in a hyporesponsive state and, thus, more susceptible to secondary infection (23
). It has been postulated that some pathogens may use induction of tolerance as a mechanism by which to evade the immune response to successfully infect and replicate in the host (28
). The specific mechanism(s) of microbial tolerance is varied and ill defined and has been suggested to operate at multiple levels from alteration of receptor expression to impairment of cell signaling pathways.
Data presented herein suggest that virulent F. tularensis may utilize microbial tolerance as a mechanism to evade elicitation of proinflammatory cytokines in human DCs, ultimately dysregulating the immune response in favor of the bacterium. Using GFP-Schu S4, we also observed that cells negative for this bacteria responded poorly to secondary stimulation with E. coli LPS. In contrast to previous reports with more attenuated strains of F. tularensis, we demonstrate that direct infection of DCs was not required to successfully interfere with DC responsiveness to multiple TLR agonists. It is possible that these cells harbored small numbers of GFP-Schu S4 cells that were below the threshold of detection. However, due to the magnitude of nonresponsiveness in Schu S4-negative cells and the ability of sterile, S4CM to inhibit DC responsiveness, we do not believe that small (1 to 2 CFU) numbers of undetectable GFP-Schu S4 cells were the primary contributors to tolerance in Schu S4-negative cells. Rather, our data suggest that in addition to modulation of infected cells, Schu S4 secreted or shed molecules that induced a refractory state in uninfected bystander human DCs. The specific mechanism of immunosuppression may be different among bystander and directly infected cells. However, both result in poor DC responsiveness and may contribute to the overall pathogenesis of Schu S4-mediated disease. Our observations of DC suppression represent an important advance in understanding how pathogens might manipulate these critical host cells. As described, the induction of microbial tolerance in human cells following exposure to microbial products is a well-established phenomenon. However, in the case of traditional tolerance, the pathogen stimulates an initial wave of proinflammatory cytokines which may serve to alert the host to the invading pathogen. Our data with Schu S4 suggest that highly virulent pathogens may have developed mechanisms by which they can effectively suppress cell responsiveness in the complete absence of proinflammatory cytokine production, thus evading both the initial wave of host effector responses as well as tempering the ability of the host to mobilize additional defenses. Furthermore, little is understood about how human DCs are modulated by virulent pathogens. Thus, data presented herein provide an important concept concerning modulation of primary DCs by highly virulent pathogens and should be considered when analyzing the interaction of these cells with other infectious agents.
The suppression of bystander human DCs presented here correlates with our previous findings in the murine model of pneumonic tularemia. In those studies, inhalation of Schu S4 inhibited the recruitment of proinflammatory cells throughout the lung in response to LPS (7
). Importantly, this occurred at a point when infection was restricted to the airways, suggesting that fulminant infection of the lung was not required to exert organ-wide tolerance to LPS. Although we did not define the mechanism of unresponsiveness in that study, it is possible that direct modulation and induction of tolerance by Francisella
products contributes to the unresponsiveness. Indeed, it has been observed that humans experimentally infected with virulent F. tularensis
develop in vivo tolerance to LPS from other gram-negative bacteria (24
). The specific mechanism of tolerance in humans infected with Francisella
was not defined at the time of that study. However, with the recognition of TLRs as key receptors for recognition of LPS and other pathogen-associated molecular products, it is possible that Francisella
is directly modifying TLR-mediated activity. Inhibition of TLR signaling and the resulting cytokine production may occur at several levels including decreased expression of TLRs on the cell surface, inhibition of phosphorylation of specific signaling molecules required for induction of cytokine production, and induction of TLR-negative regulators such as Tollip, suppressor of cytokine signaling 1 (SOCS1) to SOCS3 and IRAK-M (as reviewed in reference 29
). The direct effect of Francisella
on each of these steps in TLR signaling is currently being examined in our laboratory. Thus, although the murine lung environment is quite different from that of in vitro cultured human DCs, it is tempting to speculate that identification of the specific mechanisms involved in the inhibition of human DCs may also be at work in the murine lung. Thus, the data presented herein may provide important new clues as to how virulent Francisella
, and potentially other important human pulmonary pathogens, might be influencing both directly infected cells and uninfected bystanders.
Although a number of microbial products are capable of inducing modulating immune responses, the most well-studied molecule is LPS (as reviewed in references16
). F. tularensis
is a gram-negative pathogen and, thus, possesses LPS. While high concentrations of purified Schu S4 LPS could suppress human DC responsiveness, further depletion of LPS from S4CM failed to restore responsiveness of human DCs to unrelated TLR agonists (E. coli
LPS) (Fig. and ). The poor stimulatory capacity of Schu S4 LPS is in agreement with previous reports examining immunogenicity and the tolerogenic capacity of LPS derived from the more attenuated F. tularensis
strain LVS (2
). However, it is well established that optimal induction of signaling via E. coli
LPS in mammalian cells requires both CD14 and LPS binding protein (LBP). Although E. coli
LPS is capable of stimulating primary human DCs in the absence of these two proteins (Fig. to , , , and ), it is possible that CD14 and LBP are required for cellular activation or suppression mediated by Schu S4 LPS. Both CD14 (cell membrane bound and soluble) and LBP are present in abundance in vivo. Thus, while Francisella
LPS does not play a predominant role in the induction of tolerance in host macrophages and DCs in vitro, it may exert a more profound effect in the in vivo setting. Furthermore, there may be other heat-stable Schu S4 components capable of mediating tolerance to multiple microbial TLR agonists in vitro and in vivo that were not identified in this study. Identification of these components is currently being pursued in our laboratory. This identification will enable us to develop novel therapeutics and vaccines against tularemia. Additionally, given the strong suppressive activity mediated by these molecules, they may also represent a useful class of anti-inflammatory agents capable of dampening destructive inflammatory responses present during other diseases.
FIG. 11. Depletion of Schu S4 LPS in S4CM does not restore DC responsiveness to E. coli LPS. S4CM was incubated with anti-LPSFt(anti-Ft LPS) antibodies. Schu S4 LPS/anti-LPSFtcomplexes were then removed as described in Materials and Methods. S4CM treated with (more ...)
In addition to microbial tolerance, there are other potential bacterial components and mechanisms that may contribute to the suppression of human DC activity. F. tularensis
encodes a pathogenicity island (FPI) that is critical for survival and replication of the bacterium (36
). Specifically, several of these genes have been shown to be important for F. tularensis
escape from the phagosome, enabling replication of the bacterium in host cell cytoplasm (43
). Interestingly, one gene and the resulting protein have been implicated in both phagosomal escape (resulting in replication of the bacterium) and induction of tolerance in murine and human macrophages. LVS bacteria lacking the iglC
gene failed to inhibit responsiveness to E. coli
LPS following infection of J774.1 macrophages (50
). Furthermore, a spontaneous LVS mutant that failed to produce IglC induced both proinflammatory cytokines and was unable to suppress responsiveness of human macrophages to E. coli
). We detected IglC protein in S4CM. However, depletion of iglC
from S4CM failed to restore responsiveness of human DCs to other TLR agonists (data not shown). This suggests that IglC alone is not responsible modulation of human DC.
The FPI also encodes a type VI secretion system. In other bacteria, type VI secretion systems are responsible for secretion of molecules that are capable of disabling the host immune response. For example, proteins secreted via the type VI secretion system described in Vibrio cholera
are responsible for cytotoxicity in mammalian macrophages (38
). Porphyromonas gingivalis
utilizes type VI secretion to expel gingipain proteases. Interestingly, an important function of these proteases is the induction of LPS tolerance in macrophages (18
). Type VI secretion systems have also been identified in Yersinia
). While their presence is associated with virulence, the specific effectors and their mechanisms of action on a cellular level have not been defined. A similar phenomenon may be at work in Francisella
. That is, in Francisella
species, it is possible that while IglC may have a role in phagosomal escape, it is not the effector molecule mediating hyporesponsiveness. Rather, IglC may act as part of a type VI secretion apparatus that aides in the delivery of a yet to be defined effector molecule ultimately responsible for inhibiting host cell responsiveness to inflammatory stimuli. Additional studies examining the specific role of IglC and other proteins encoded in the FPI in the induction of Francisella
-mediated tolerance are currently under way in our laboratory.
As a facultative intracellular pathogen, F. tularensis
must contend with the host immune system from both inside and outside individual host cells. It is likely that the mechanisms by which F. tularensis
accomplishes this are complex and involve a number of strategies. Here, we show for the first time that one way in which virulent F. tularensis
interferes with host immunity is by suppressing responsiveness of both directly infected cells and uninfected bystander cells. Interestingly, in contrast to the production of an array of proinflammatory cytokines observed in cells exposed to other microbially derived TLR agonists such as LPS or peptidoglycan prior to the onset of unresponsiveness, the Schu S4 LPS and other components present in the culture medium were only weakly inflammatory. Thus, the profound suppression mediated by F. tularensis
in DCs was not obtained at the cost of an initial burst of cytokines that might alert the immune system. Importantly, the broad suppression of DC responsiveness (in terms of cytokine secretion) is similar to that observed in the lungs of mice following aerosol infection with Schu S4 (7
). Together, these data suggested that Schu S4 suppresses the host immune response in a global sense; that is, interference with host responsiveness is not restricted to infected cells but extends to uninfected cells that may aid in the resolution of infection. These data also have broad implications for how both human DCs and the murine pulmonary environment are manipulated by virulent pathogens and represent an important consensus of global dysregulation by a pathogen in murine and human cells.