Tularemia is a disease that can be divided into phases. The early, critical stages of infection are marked by a striking absence of inflammatory responses despite exponentially replicating bacteria (36
). Further, virulent F. tularensis
not only evades early detection in the host, but also actively suppresses inflammation within the first few days of disease (4
). During the last phase of infection the host rapidly transitions from a quiescent inflammatory response to sepsis paired with massive inflammation and cell death (36
). In correlation with these in vivo observations, F. tularensis
has been shown to both suppress and exacerbate inflammatory responses among cells infected in vitro (6
). Generation of these contradictory outcomes appears to depend on the subspecies and strain of F. tularensis
used, the cell type analyzed, the species from which the cell was derived and the activation status of the cell. Given the dichotomy of host responses during different stages of tularemia and the cell types targeted by the bacterium during these stages, the success of novel therapeutics to treat this disease may greatly depend on the phase of infection and type of cell harboring bacteria. Thus, it is critical to clarify and understand virulence mechanisms used by F. tularensis
to cause disease in the context of the cell type at different stages of infection.
Resting, immature dendritic cells serve as sentinels of the immune system, capable of activating both innate and adaptive immune responses (40
). Thus, successful modulation of this cell population by virulent microbes can be an essential component in mediating microbial pathogenesis. Previous reports have demonstrated that pulmonary dendritic cells and alveolar macrophages represent the primary, initial targets of F. tularensis
during lung infections (42
). Identification of how F. tularensis
suppresses function of these cells may lead to important insights into the pathogenesis of tularemia and other pulmonary pathogens.
In this report, we provided evidence of at least one mechanism of virulence utilized by virulent SchuS4, but not attenuated LVS, following infection of human cells. We confirmed one disparity between LVS and SchuS4 is their ability to induce inflammatory responses in hDC and defined one mechanism by which virulent F. tularensis suppresses function of immature hDC. Specifically, we found that induction of IFN-β by SchuS4 played a central role in early suppression of a critical aspect of hDC activation. We also observed that induction and secretion of IFN-β was restricted to infection of hDC with virulent F. tularensis strain SchuS4. We extended these observations by demonstrating that internalization of viable SchuS4 and endosomal acidification were required for induction of IFN-β. Surprisingly, induction of IFN-β was not associated with propagation of host inflammatory responses during SchuS4 infection. IFN-β was not correlated with secretion of IL-1β in F. tularensis infected hDC nor was it correlated with strong activation of caspase-1 in hDC infected with F. tularensis. Furthermore, unlike IFN-β primed hDC infected with LVS, treatment of hDC with recombinant IFN-β failed to restrict replication of SchuS4. Finally, rather than acting as a signal for inflammation, SchuS4 induced IFN-β suppressed production of IL-12p40 in hDC.
We first confirmed and extended previous observations that described differences in the ability of LVS and SchuS4 to induce secretion of inflammatory cytokines in hDC. In agreement with published data examining secretion of pro-inflammatory cytokines in hDC by serum opsonized LVS, we observed consistent production of pro-inflammatory cytokines following infection of hDC with attenuated, non-opsonized LVS (11
). The production of pro-inflammatory cytokines following LVS infection of hDC correlated well with the fact that LVS is an attenuated vaccine strain in humans. Indeed, in the setting of vaccination a modest inflammatory response would serve to aid in controlling growth of the vaccine strain as well as promote development of effective adaptive immunity. In contrast to LVS, we observed that virulent SchuS4 failed to induce secretion of pro-inflammatory cytokines and actively suppressed responsiveness of infected cultures to other stimuli (). Together these data support the hypothesis that one primary mechanism of virulence employed by fully virulent strains of F. tularensis
in humans is the evasion and inhibition of inflammation.
In contrast to this manuscript, we had previously reported that SchuS4 failed to induce IFN-β in hDC 24, 48 and 72 hours after infection (6
). However, at that time we did not appreciate the extreme potency of this protein or the sensitivity of this cytokine to denaturation. Human IFN-β can exert anti-viral activity when present in quantities as small as 5 U/mL, which (depending on the source of IFN-β) represents as little as 20 pg/mL of hIFN-β (44
). This concentration is well below the level of detection of most ELISA kits designed to detect free IFN-β. Additionally, data presented herein suggest that increased gene expression of IFN-β occurs very early after infection with SchuS4, i.e. within 8–12 hours after infection. Thus, it is possible that our failure to detect IFN-β in cultures of SchuS4 infected hDC in our earlier report was due to both poor sensitivity of human IFN-β ELISAs and the time point at which we examined culture supernatants for IFN-β. Recently, an ELISA with vastly improved sensitivity for human IFN-β has been developed. Indeed, when we utilized this highly sensitive ELISA for detection of IFN-β at earlier time points after infection, e.g. 12hours, we routinely detected small, but significant amounts of IFN-β in culture supernatants of SchuS4 infected hDC ().
To elucidate if there was a contribution of IFN-β in our system we neutralized the activity of this cytokine using polyclonal and monoclonal neutralizing antibodies directed against this protein. We found that neutralization of IFN-β partially restored the ability of hDC infected with SchuS4 to produce IL-12p40, but not TNF-α, in response to inflammatory stimuli (). Addition of polyclonal or monoclonal antibodies directed against human IFN-β partially restored responsiveness of SchuS4 infected hDC to LPS and had no effect on induction of IL-12p40 in response to SchuS4 alone. The lack of complete restoration of the IL-12p40 response may be attributable to additional, undefined mechanisms by which SchuS4 interferes with host cell cytokine production. Similarly, the absence of IL-12p40 production in response to SchuS4 alone in the presence of neutralizing anti-IFN-β antibodies may also suggest that there are multiple mechanisms by which SchuS4 modulates production of IL-12p40. Alternatively, the absence of IL-12p40 in SchuS4 infected hDC may also be attributable to the fact that wild type SchuS4 may not possess ligands that are capable of inducing inflammatory responses on their own. For example, SchuS4 does not appear to effectively stimulate pro-inflammatory responses in hDC upon engagement of the host cell in the absence of specific co-receptors or throughout the infection ( and 6, 16, 45). Additionally, SchuS4 does not provoke production of inflammatory cytokines in mice during the first three days of infection (4
). Regardless of the inability of SchuS4 to provoke an inflammatory response on its own, our data clearly show that in the context of SchuS4 infection of hDC, IFN-β acts as an anti-inflammatory cytokine by selectively targeting production of IL-12p40.
Initially, this anti-inflammatory role for IFN-β in F. tularensis
infections was unexpected. Several recent reports have shown that activation of the host inflammasome by attenuated strains of F. tularensis
, as indicated by release of IL-1β and cell death, was dependent on production of IFN-β following infection of mouse macrophages with attenuated F. tularensis
). Importantly, IFN-β dependent activation of the inflammasome in these studies was directly correlated with control of bacterial replication. Therefore, it was proposed that IFN-β might represent an attractive therapeutic for treatment of pneumonic tularemia . Alternatively, in other settings Type I IFNs have been shown to act as a potent anti-inflammatory in human cells capable of suppressing production of cytokines such as IL-12p40 (46
). Given these important and contrasting implications, we examined the possibility that SchuS4 mediated IFN-β might be facilitating activation of the inflammasome and the suppression of IL-12p40 in hDC.
To determine if IFN-β induced during SchuS4 infection contributed to activation of the inflammasome we examined both secretion of mature IL-1β as well as presence of active caspase-1. We did not detect mature IL-1β in supernatants of hDC infected with either SchuS4 or LVS, which suggested that the inflammasome was not active in these cells. However, we observed minimal amounts of pro-IL-1β in cell lysates of LVS infected hDC. Since pro-IL-1β is required to generate mature, cleaved IL-1β and we failed to detect large quantities of intracellular pro-IL-1β, we could not use secretion of this cytokine as a reliable read-out for inflammasome activation. Thus, we directly assessed activation of caspase-1 in hDC. Using this technique we found that activated caspase-1 was present in minimal numbers of cells and only at late time points in infection among LVS and SchuS4 infected cultures. However, since LVS failed to induce IFN-β in hDC, the modest induction of active caspase-1 in hDC infected with F. tularensis was not dependent on IFN-β.
As discussed above, other laboratories have demonstrated a protective role for IFN-β in which addition of recombinant IFN-β resulted in control of LVS replication in mouse cells (13
). Thus, we tested whether addition of rhIFN-β had an effect on replication of LVS and SchuS4 in hDC. Similar to previous studies in mouse cells, addition of rhIFN-β to hDC cultures resulted in control of LVS replication. However, a similar effect of IFN-β on the replication of SchuS4 in hDC was not observed. There are a number of possibilities that might explain why IFN-β was unable to control SchuS4 infection. First, IFN-β has been tied to induction of the inflammasome resulting in both secretion of IL-1β and cell death (48
). Either of these inflammasome mediated activities may aid in the control of bacterial replication. Thus, it is possible that following treatment with rhIFN-β SchuS4 either failed to activate the inflammasome or that the bacterium interfered with inflammasome activity. A second possibility for the inability of IFN-β to contribute to control of SchuS4 may lie in sub-optimal activation of reactive oxygen and reactive nitrogen species. We have previously demonstrated that both reactive oxygen and reactive nitrogen are required for control of SchuS4 in human cells (49
). Although IFN-β can activate these pathways, optimal induction of the oxidative burst can be dependent on the presence of IFN-β in combination with other pro-inflammatory cytokines (50
). Unlike LVS, SchuS4 does not induce secretion of these pro-inflammatory cytokines following infection of hDC (). Therefore, it is possible that IFN-β failed to optimally activate specific antimicrobial pathways in hDC that contribute to control of bacterial replication. The specific mechanism by which SchuS4 evades IFN-β mediated control of bacterial replication is currently being examined by our laboratory.
In the present manuscript, we used a combination of chemical compounds and SchuS4 mutants to explore the mechanism by which SchuS4 induced IFN-β. Using these approaches, we demonstrated that induction of IFN-β did not occur following engagement of the bacteria with receptors at the host cell surface. Rather, active phagocytosis of SchuS4 followed by endosomal acidification was required for induction of IFN-β. F. tularensis briefly transits through a host endosome before escaping into the cytosol where the bacterium undergoes replication. It has been suggested that escape of attenuated F. tularensis into the cytosol is sufficient for induction of IFN-β. However, experiments conducted with a defined mutant of SchuS4 (SchuS4Δ0369c) that displays similar kinetics for endosomal escape and early replication, clearly demonstrated that endosomal escape and replication of the bacterium during the first 8 hours of infection were not sufficient to induce IFN-β in human cells ().
FTT0369c is a protein unique to F. tularensis
species and is required for virulence of SchuS4 both in vitro and in vivo (28
). The specific function of FTT0369c in F. tularensis
physiology has not been identified. A homolog of FTT0369c is present in LVS and is designated FTL1306. Given the dramatic difference in the ability of SchuS4 and LVS to induce IFN-β in hDC and the apparent contribution of FTT0369c toward induction of this cytokine, it was initially surprising that LVS failed to provoke IFN-β in hDC. However, comparison of FTT0369c and FTL1306 sequences revealed 4 amino acid differences between these two proteins. Thus, a possible explanation for the difference between SchuS4 and LVS to induce IFN-β in hDC is that these amino acid substitutions lie in areas that are important for the structure and, by extension, specific function of FTL1306 in LVS.
Additionally, FTT0369c may act to regulate expression of other genes essential for virulence in SchuS4. In LVS, FTL1306 contributes to the expression of RipA (personal communication, Dr. Thomas Kawula, University of North Carolina). Similar to FTT0369c in SchuS4, RipA in LVS was required for both intracellular replication and suppression of pro-inflammatory responses in mouse macrophages (51
). SchuS4 possesses a homolog of RipA, but neither the function of this protein nor the contribution FTT0369c makes toward its expression have been thoroughly explored. Thus, it is possible that the failure of SchuS4Δ0369c to induce IFN-β and suppress IL-12p40 may not solely be attributed to the absence of FTT0369c, but rather an indirect effect via the down regulation of RipA. Finally, since the sequence of FTL1306 is conserved among the F. tularensis
holarctica subspecies, it is possible that the contribution this protein makes toward induction of IFN-β may reflect both the heightened attenuation of LVS as a vaccine strain and the moderate virulence observed in the holarctica subspecies (8
Type I IFNs, and specifically IFN-β, are cytokines with pleiotropic activity. Type I IFN can enhance antiviral immunity and promote strong inflammatory responses. This inflammation can lead to both resolution and exacerbation of infection. Conversely, IFN-β has been tightly associated with suppressing inflammatory responses in humans. For example, the anti-inflammatory action of IFN-β is believed to be a critical element in the resolution of inflammation in multiple sclerosis and lupus (53
). Data provided herein shows that, in the context of infection with virulent F. tularensis
in resting human dendritic cells, IFN-β acts as an anti-inflammatory cytokine to suppress IL-12p40 production. IL-12p40 is essential for control of in vivo replication of attenuated strains of F. tularensis
). Furthermore, recent work in our laboratory demonstrates an absolute requirement for IL-12p40 in survival of intranasal SchuS4 infection (Bosio CM, unpublished observations). Therefore, the ability of SchuS4 induced IFN-β to negatively modulate production of IL-12p40 brings to light an important mechanism of virulence utilized by these bacteria. Further characterization of the specific host and bacterial components that participate in IFN-β mediated suppression following F. tularensis
infection will provide critical information for development of novel vaccines and therapeutics directed against this pathogen, as well as to the understanding of how successful, highly virulent, intracellular bacteria modulate human cells to cause lethal disease.