The AIM2 inflammasome induces maturation of the proinflammatory cytokines IL-1β and IL-18. Using AIM2-deficient mice, Fitzgerald and colleagues and Alnemri and colleagues show that the AIM2 inflammasome is essential for host defense against cytosolic bacteria and DNA viruses. Inflammasomes regulate the activity of caspase-1 and the maturation of interleukin 1β (IL-1β) and IL-18. AIM2 has been shown to bind DNA and engage the caspase-1-activating adaptor protein ASC to form a caspase-1-activating inflammasome. Using Aim2-deficient mice, we identify a central role for AIM2 in regulating caspase-1-dependent maturation of IL-1β and IL-18, as well as pyroptosis, in response to synthetic double-stranded DNA. AIM2 was essential for inflammasome activation in response to Francisella tularensis, vaccinia virus and mouse cytomegalovirus and had a partial role in the sensing of Listeria monocytogenes. Moreover, production of IL-18 and natural killer cell–dependent production of interferon-γ, events critical in the early control of virus replication, were dependent on AIM2 during mouse cytomegalovirus infection in vivo. Collectively, our observations demonstrate the importance of AIM2 in the sensing of both bacterial and viral pathogens and in triggering innate immunity.

Francisella tularensis (Ft), the causative agent of tularemia, elicits a potent inflammatory response early in infection, yet persists within host macrophages and can be lethal if left unchecked. We report in this study that Ft live vaccine strain (LVS) infection of murine macrophages induced TLR2-dependent expression of alternative activation markers that followed the appearance of classically activated markers. Intraperitoneal infection with Ft LVS also resulted in induction of alternatively activated macrophages (AA-Mφ). Induction of AA-Mφ by treatment of cells with rIL-4 or by infection with Ft LVS promoted replication of intracellular Ftn, in contrast to classically activated (IFN-γ plus LPS) macrophages that promoted intracellular killing of Ft LVS. Ft LVS failed to induce alternative activation in IL-4Rα^−/− or STAT6^−/− macrophages and prolonged the classical inflammatory response in these cells, resulting in intracellular killing of Ft. Treatment of macrophages with anti-IL-4 and anti-IL-13 Ab blunted Ft-induced AA-Mφ differentiation and resulted in increased expression of IL-12 p70 and decreased bacterial replication. In vivo, Ft-infected IL-4Rα^−/− mice exhibited increased survival compared with wild-type mice. Thus, redirection of macrophage differentiation by Ft LVS from a classical to an alternative activation state enables the organism to survive at the expense of the host.

Francisella tularensis(Ft), a Gram-negative intracellular bacterium, is the etiologic agent of tularemia. Infection of mice with <10FtLive Vaccine Strain (FtLVS) organisms i.p. causes a lethal infection that resembles human tularemia. Here, we show that immunization with as little as 0.1 ngFtLVS lipopolysaccharide (Ft-LPS), but notFtlipid A, generates a rapid antibody response that protects wild-type (WT) mice against lethalFtLVS challenge. Protection is not induced inFt-LPS-immunized B cell-deficient mice (μMT or JhD), malexidmice, or Ig transgenic mice that produce a single IgH (not reactive withFt-LPS). Focusing on the cellular mechanisms that underlie this protective response, we show thatFt-LPS specifically stimulates proliferation of B-1a lymphocytes that bind fluorochrome-labeledFt-LPS and the differentiation of these cells to plasma cells that secrete antibodies specific forFt-LPS. This exclusively B-1a antibody response is equivalent in WT, T-deficient (TCRαβ^−/−, TCRγδ^−/−), and Toll-like receptor 4 (TLR4)-deficient (TLR4^−/−) mice and thus is not dependent on T cells or typical inflammatory processes. Serum antibody levels peak ≈5 days afterFt-LPS immunization and persist at low levels for months. Thus, immunization withFt-LPS activates a rare population of antigen-specific B-1a cells to produce a persistent T-independent antibody response that provides long-term protection against lethalFtLVS infection. These data support the possibility of creating effective, minimally invasive vaccines that can provide effective protection against pathogen invasion.

Francisella tularensis (Ft), a Gram-negative intracellular bacterium, is the etiologic agent of tularemia. Although attenuated for humans, i.p. infection of mice with 10⁶ organisms. To examine the immunological consequences of Ft LVS infection on the innate immune response, the inflammatory responses of mice infected i.p. or intradermally were compared. Mice infected i.p. displayed greater bacterial burden and increased expression of proinflammatory genes, particularly in the liver. In contrast to most LPS, highly purified Ft LVS LPS (10 μg/ml) was found to be only minimally stimulatory in primary murine macrophages and in HEK293T cells transiently transfected with TLR4/MD-2/CD14, whereas live Ft LVS bacteria were highly stimulatory for macrophages and TLR2-expressing HEK293T cells. Despite the poor stimulatory activity of Ft LVS LPS in vitro, administration of 100 ng of Ft LVS LPS 2 days before Ft LVS challenge severely limited both bacterial burden and cytokine mRNA and protein expression in the absence of detectable Ab at the time of bacterial challenge, yet these mice developed a robust IgM Ab response within 2 days of infection and survived. These data suggest that prior administration of Ft LVS LPS protects the host by diminishing bacterial burden and blunting an otherwise overwhelming inflammatory response, while priming the adaptive immune response for development of a strong Ab response.

Francisella tularensis, an aerobic, non-spore-forming, gram-negative coccobacillus, is the causative agent of tularemia. We reported previously thatF. tularensislive vaccine strain (LVS) elicited strong, dose-dependent NF-κB reporter activity in Toll-like receptor 2 (TLR2)-expressing HEK293T cells and proinflammatory gene expression in primary murine macrophages. Herein, we report thatF. tularensisLVS-induced murine macrophage proinflammatory cytokine gene and protein expression are overwhelmingly TLR2 dependent, as evidenced by the abrogated responses of TLR2^−/−macrophages.F. tularensisLVS infection also increased expression of TLR2 both in vitro, in mouse macrophages, and in vivo, in livers fromF. tularensisLVS-infected mice. Colocalization of intracellularF. tularensisLVS, TLR2, and MyD88 was visualized by confocal microscopy. Signaling was abrogated if theF. tularensisLVS organisms were heat or formalin killed or treated with chloramphenicol, indicating that the TLR2 agonist activity is dependent on new bacterial protein synthesis.F. tularensisLVS replicates in macrophages; however, bacterial replication was not required for TLR2 signaling because LVSΔguaA, anF. tularensisLVSguanine auxotroph that fails to replicate in the absence of exogenous guanine, activated NF-κB in TLR2-transfected HEK293T cells and induced cytokine expression in wild-type macrophages comparably to wild-typeF. tularensisLVS. Collectively, these data indicate that the primary macrophage response toF. tularensisLVS is overwhelmingly TLR2 dependent, requires de novo bacterial protein synthesis, and is independent of intracellularF. tularensisreplication.

The macrophage proinflammatory response to Francisella tularensis (Ft) live vaccine strain (LVS) was shown previously to be TLR2 dependent. The observation that intracellular Ft LVS colocalizes with TLR2 and MyD88 inside macrophages suggested that Ft LVS might signal from within the phagosome. Macrophages infected with LVSΔiglC, a Ft LVS mutant that fails to escape from the phagosome, displayed greatly increased expression of a subset of TLR2-dependent, proinflammatory genes (e.g., Tnf) but decreased expression of others (e.g., Ifnb1). This latter subset was similarly mitigated in IFN-β^−/− macrophages indicating that while Ft LVS-induced TLR2 signaling is necessary, cytosolic sensing of Ft to induce IFN-β is required for full induction of the macrophage proinflammatory response. Although LVSΔiglC greatly increased IL-1β mRNA in wild-type macrophages, protein secretion was not observed. IL-1β secretion was also diminished in Ft LVS-infected IFN-β^−/− macrophages. rIFN-β failed to restore IL-1β secretion in LVSΔiglC-infected macrophages, suggesting that signals in addition to IFN-β are required for assembly of the inflammasome and activation of caspase-1. IFN-β plays a central role in controlling the macrophage bacterial burden: bacterial recovery was greater in IFN-β^−/− than in wild-type macrophages and treatment of Ft LVS-infected macrophages with rIFN-β or 5,6-dimethylxanthenone-4-acetic acid, a potent IFN-β inducer, greatly decreased the intracellular Ft LVS burden. In toto, these observations support the hypothesis that the host inflammatory response to Ft LVS is complex and requires engagement of multiple signaling pathways downstream of TLR2 including production of IFN-β via an unknown cytosolic sensor and activation of the inflammasome.