Anaplasma phagocytophilum is an obligate intracellular pathogen that resides within neutrophils and can cause fever, pancytopenia, or death. IFN-γ plays a critical role in the control of A. phagocytophilum; however, the mechanisms that regulate IFN-γ production remain unclear. In this study, we demonstrate that apoptotic specklike protein with a caspase-activating recruiting domain (ASC)/PYCARD, a central adaptor molecule in the Nod-like receptor (NLR) pathway, regulates the IL-18/IFN-γ axis during A. phagocytophilum infection through its effect on caspase-1. Caspase-1- and asc-null mice were more susceptible than control animals to A. phagocytophilum infection due to the absence of IL-18 secretion and reduced IFN-γ levels in the peripheral blood. Moreover, caspase-1 and ASC deficiency reduced CD4+ T cell-mediated IFN-γ after in vitro restimulation with A. phagocytophilum. The NLR family member IPAF/NLRC4, but not NALP3/NLRP3, was partially required for IFN-γ production in response to A. phagocytophilum. Taken together, our data demonstrate that ASC and caspase-1 are critical for IFN-γ-mediated control of A. phagocytophilum infection.

Anaplasma phagocytophilum is a Gram-negative, obligate intraneutrophilic bacterium carried by ticks and is the cause of human anaplasmosis (1). Symptomatic infection is variable, and often includes pancytopenia, splenomegaly, and vascular lesions of the gastrointestinal tract and liver (1). The bacterium A. phagocytophilum is one of the few bacteria known to survive and replicate within the hostile environment of the neutrophil. A. phagocytophilum encodes a type IV secretion system and lacks genes for lipid A, peptidoglycan biosynthesis, and bacterial flagellin, the main monomeric subunit of the flagella that provide bacterial motility (2).

A. phagocytophilum infection studies have shown little evidence for a role of TLRs in pathogen clearance (3, 4). However, the control of A. phagocytophilum replication depends on the activation of lymphocytes, and both Abs and T cell-mediated IFN-γ have been shown to play an important role in the eradication process (5, 6). The fact that A. phagocytophilum can effectively elicit adaptive immunity despite its pronounced lack of TLR engagement suggests that another line of defense is critical to control A. phagocytophilum infection.

We previously demonstrated that IL-1β and IL-18 were up-regulated when A. phagocytophilum infects neutrophils and promyelocytic cells (3, 7, 8). Some Nod-like receptors (NLR)3 have been shown to regulate IL-1β and IL-18 secretion (9, 10, 11, 12, 13). NLR proteins associate with the adaptor molecule apoptotic specklike protein with a caspase-activating recruiting domain (ASC)/PYCARD, and recruit caspase-1 and caspase-5 (via NALP1/NLRP1) or cardinal and caspase-1 (via NALP3/NLRP3) forming a large intracellular complex coined the inflammasome. The inflammasome induces the activation of caspase-1, resulting in subsequent processing and secretion of IL-1β and IL-18 (9, 10, 11, 12, 13). IL-18 plays an important role in driving the production of IFN-γ. In this study, we demonstrate that ASC and caspase-1 are critical for production of IL-18 in response to A. phagocytophilum, which in turn is required for the IFN-γ-dependent phase of A. phagocytophilum clearance.

The promyelocytic cell line (HL-60) was acquired from the American Type Culture Collection. Uninfected cells were sustained with medium replacement once per week. A. phagocytophilum-infected cells were maintained, as previously described, with an equal volume of uninfected cells added to infected cells and diluted 1/5 with fresh medium (14, 15). Cell-free A. phagocytophilum was collected from 95% A. phagocytophilum-infected HL-60 cells. Infected cells were centrifuged for 10 min at 4000 × g. The cell pellet was resuspended in culture medium and lysed by six passages through a 25-gauge needle, followed by six passages through a 27-gauge needle. The cell lysate was centrifuged at 1200 × g for 3 min, and the supernatant was used for infection.

The NCH-1 isolate of A. phagocytophilum was used throughout these studies (16). Blood from rag1-null mice chronically infected with A. phagocytophilum NCH-1 strain (25% neutrophil infection) was used to inoculate immunocompetent C57BL/6 or gene-deficient mice. Bacterial infection in rag1-null mice was determined using the percentage of granulocytes containing morulae (A. phagocytophilum aggregates) (5). To quantify A. phagocytophilum load in the peripheral blood, 100 μl of anticoagulated peripheral blood from gene-deficient and wild-type mice was incubated twice with 900 μl of erythrocyte lysis buffer (Sigma-Aldrich) at room temperature for 20 min. DNA was extracted with DNeasy Tissue Kit (Qiagen), according to the manufacturer’s recommendation. DNA samples were mixed with the iQ SYBR Green I Supermix (Bio-Rad) in the iCycler Thermal Cycler (Bio-Rad). Quantitative real-time PCR was performed, as previously described (3). DNA levels were normalized to the mouse β-actin gene (GenBank accession X03672). A. phagocytophilum 16s rRNA (GenBank accession M73224) gene was then quantified. The relative bacterial load difference in the peripheral blood of wild-type and gene-deficient mice was calculated by normalizing the bacterial copy numbers of wild-type mice to 1 after determining the fold difference when compared with gene-deficient mice.

C57BL/6, il-18-null, and rag1-null mice were purchased from The Jackson Laboratory. Caspase-1-null, nalp3-null, asc-null, and ipaf-null mice have been previously described (17, 18, 19) and were backcrossed onto the C57BL/6 background for six (caspase-1-null mice), nine (asc-null mice), eight (nalp3-null mice), and four (ipaf-null mice) generations. All mice used for in vivo infection were 4–12 wk of age, sex matched, and maintained under specific pathogen-free conditions. The experiments were done in accordance with the Yale University Institutional Animal Care and University Committee guidelines.

Recombinant murine protein standards and Ab pairs for IL-12p40/p70, IFN-γ, IL-1β, and IL-18 were purchased from BD Pharmingen and MBL, respectively. Retro-orbital bleeding was performed at indicated time points from gene-sufficient and gene-deficient mice, and cytokine levels were assessed in sandwich ELISA, as previously described (5).

Wild-type and gene-deficient mice were euthanized, and spleens were removed 6 days postinfection with A. phagocytophilum. Following spleen removal, the splenocytes from wild-type and gene-deficient mice were restimulated with A. phagocytophilum for 18 h and surface stained with PE-Cy5-conjugated rat anti-mouse CD3 (17A2; rat (SD) IgG2b), allophycocyanin-conjugated rat anti-mouse CD4 (RM4-5; rat (DA) IgG2a), allophycocyanin-Cy7-conjugated rat anti-mouse CD8a (53-6.7; rat IgG2a), and PE-conjugated hamster anti-mouse CD69 (H1.2F3; Armenian hamster IgG1) (BD Pharmingen). For the intracellular staining, brefeldin A was added 4 h before harvesting, and FITC-conjugated rat anti-mouse IFN-γ (XMG1.2; rat IgG1) was used. For the NK and NK T cell experiments, splenocytes from the wild-type and gene-deficient mice were euthanized and spleens were removed 9 days postinfection with A. phagocytophilum and surfaced stained with allophycocyanin-conjugated anti-mouse pan-NK cells (CD49b) (DX5; rat IgM), FITC-conjugated anti-mouse NKG2A/C/E (20d5; rat IgG2a), PE-Cy5-conjugated rat anti-mouse CD3 (17A2; rat (SD) IgG2b), and PE-conjugated anti-mouse IFN-γ (XMG1.2; rat IgG1). Splenocytes were activated in the presence of PMA (5 ng/ml; Calbiochem) and ionomycin (250 ng/ml; CalBiochem), as previously described (20). Flow cytometry was performed using the BD LSR II System (BD Biosciences), and data were analyzed using FlowJo software (Tree Star).

Nalp3-sufficient and nalp3-null mice were i.p. injected with LPS (30.0 mg/kg); 5 h after LPS injection, mice were euthanized and blood was collected by cardiac puncture. LPS from Escherichia coli serotype 0111:B4 was purchased from Invitrogen Life Technologies.

We performed statistical analysis by using unpaired Student’s t test. A. phagocytophilum genome copy numbers present in the peripheral blood of wild-type and gene-deficient mice were compared, and values of p < 0.05 were considered statistically significant.

We sought to understand the IFN-γ-dependent pathway that contributes to A. phagocytophilum clearance. IL-18 is a proinflammatory cytokine that plays an important role in both innate and adaptive immune responses against intracellular pathogens (21, 22). In addition, IL-18 activation increases Th1-mediated immune responses (21, 22), which are important for A. phagocytophilum eradication. IL-18 played an important role in A. phagocytophilum clearance in mice during early infection (Fig. 1 A). A. phagocytophilum load was increased in the peripheral blood of il-18-null mice by 2 (p = 0.56)-, 390 (p = 0.03)-, and 5-fold (p = 0.25) when compared with il-18-sufficient mice at days 2, 4, and 6 postinfection, respectively. The bacterial load in il-18-null mice was similar to control mice at day 9 postinfection when substantial Ab responses that facilitate pathogen clearance have developed against A. phagocytophilum (6). A. phagocytophilum was not detected in the peripheral blood of il-18-sufficient and il-18-null mice at day 14 postinfection (data not shown).

FIGURE 1.

Il-18-null mice have an increased bacterial load and decreased IFN-γ levels upon A. phagocytophilum infection. A, 100 μl of infected blood from rag1-null mice was used to infect 6- to 12-wk-old mice. The bacterial load in the peripheral blood of il-18-sufficient and il-18-null mice at days 2 (n = 4 per group), 4, 6, and 9 (n = 25 and n = 23 per group) was measured using quantitative real-time PCR. The relative bacterial load difference in the blood of il-18-sufficient and il-18-null mice was calculated by normalizing the bacterial copy numbers of il-18-sufficient mice to 1 after determining the fold difference when compared with il-18-null mice. Experiments were repeated four times, and one representative experiment is shown. Pooled sera concentrations of IL-18 (B), IFN-γ (C), and IL-12p40/p70 (D) were measured by ELISA at indicated times. Data represent the mean and SD. ND, no relative bacterial load difference in the blood between il-18-sufficient and il-18-null mice.

FIGURE 1.

Il-18-null mice have an increased bacterial load and decreased IFN-γ levels upon A. phagocytophilum infection. A, 100 μl of infected blood from rag1-null mice was used to infect 6- to 12-wk-old mice. The bacterial load in the peripheral blood of il-18-sufficient and il-18-null mice at days 2 (n = 4 per group), 4, 6, and 9 (n = 25 and n = 23 per group) was measured using quantitative real-time PCR. The relative bacterial load difference in the blood of il-18-sufficient and il-18-null mice was calculated by normalizing the bacterial copy numbers of il-18-sufficient mice to 1 after determining the fold difference when compared with il-18-null mice. Experiments were repeated four times, and one representative experiment is shown. Pooled sera concentrations of IL-18 (B), IFN-γ (C), and IL-12p40/p70 (D) were measured by ELISA at indicated times. Data represent the mean and SD. ND, no relative bacterial load difference in the blood between il-18-sufficient and il-18-null mice.

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Comparison of A. phagocytophilum infection kinetics in several immunocompetent mouse strains has shown that infection is almost undetectable at days 1–2, peaks at day 4–7, and then rapidly declines at days 8–10, with some slight variations in C3H/HeN, C3H/HeJ, C57BL/6, BALB/c, DBA/2, and CD1 mice (5, 23, 24, 25, 26, 27, 28, 29, 30). IL-18 secretion kinetics followed a similar pattern in the wild-type mice (Fig. 1,B). Because IL-12 and IL-18 play a synergistic effect on IFN-γ production, we examined IL-12 and IFN-γ levels in the peripheral blood of il-18-null mice (21, 22). Interestingly, IFN-γ production in il-18-null mice infected with A. phagocytophilum infection was almost completely abolished (Fig. 1,C). IL-12 secretion, however, was not affected during A. phagocytophilum infection (Fig. 1 D). These data suggest that the host response against A. phagocytophilum is IL-18/IFN-γ dependent.

The activity of IL-18 (but not IL-12) is dependent on the intracellular cysteine protease caspase-1 (9, 10, 11, 12, 13). Therefore, we investigated whether caspase-1-null mice had a defect in IL-18/IFN-γ secretion. Caspase-1-null mice had increased susceptibility to A. phagocytophilum at days 2 (18-fold; p = 0.08), 4 (385-fold; p = 0.02), and 6 (14-fold; p = 0.04) postinfection, and the bacterial infection kinetics was comparable to that described for the il-18-null mice (Fig. 2 A). No differences were detected at later time points.

FIGURE 2.

Caspase-1 deficiency ablates caspase-1-driven IL-18 secretion, reduces IFN-γ levels, and increases A. phagocytophilum load in infected mice. A, 100 μl of infected blood from rag1-null mice was used to infect 6- to 12-wk-old mice. The bacterial load in the peripheral blood of caspase-1-sufficient and caspase-1-null mice at days 2 (n = 4 per group), 4, 6, and 9 (n = 17 per group) was measured using quantitative real-time PCR. The relative bacterial load difference in the blood of caspase-1-sufficient and caspase-1-null mice was calculated by normalizing the bacterial copy numbers of caspase-1-sufficient mice to 1 after determining the fold difference when compared with caspase-1-null mice. Experiments were repeated four times, and one representative experiment is shown. Levels of IL-18 (B) and IFN-γ (C) in pooled sera of caspase-1-sufficient and caspase-1-null mice were measured using ELISA during the course of infection. Data represent the mean and SD. ND, no relative bacterial load difference in the blood between caspase-1-sufficient and caspase-1-null mice.

FIGURE 2.

Caspase-1 deficiency ablates caspase-1-driven IL-18 secretion, reduces IFN-γ levels, and increases A. phagocytophilum load in infected mice. A, 100 μl of infected blood from rag1-null mice was used to infect 6- to 12-wk-old mice. The bacterial load in the peripheral blood of caspase-1-sufficient and caspase-1-null mice at days 2 (n = 4 per group), 4, 6, and 9 (n = 17 per group) was measured using quantitative real-time PCR. The relative bacterial load difference in the blood of caspase-1-sufficient and caspase-1-null mice was calculated by normalizing the bacterial copy numbers of caspase-1-sufficient mice to 1 after determining the fold difference when compared with caspase-1-null mice. Experiments were repeated four times, and one representative experiment is shown. Levels of IL-18 (B) and IFN-γ (C) in pooled sera of caspase-1-sufficient and caspase-1-null mice were measured using ELISA during the course of infection. Data represent the mean and SD. ND, no relative bacterial load difference in the blood between caspase-1-sufficient and caspase-1-null mice.

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We used these mice to further evaluate the influence of IL-18 in IFN-γ production (Fig. 2, B and C). As expected from our previous results (Fig. 1), levels of IL-18 in the peripheral blood were impaired and IFN-γ was drastically reduced in caspase-1-null mice during A. phagocytophilum infection (Fig. 2, B and C). In contrast, IL-12 levels in the bloodstream of caspase-1-null mice during the course of pathogen infection were comparable to those of caspase-1-sufficient mice (Fig. 3). These results demonstrate that caspase-1 is required for secretion of IL-18 and IFN-γ, which in turn play a pivotal role in A. phagocytophilum clearance.

FIGURE 3.

IL-12p40/p70 secretion is not affected in wild-type and gene-deficient mice. A total of 100 μl of infected blood from rag1-null mice was used to infect 6- to 12-wk-old C57BL/6 (n = 15), ipaf-null (n = 7), caspase-1-null (n = 12), and asc-null (n = 12) mice. Pooled sera concentrations of IL-12p40/p70 were measured by ELISA at indicated times. Data represent the mean plus SD.

FIGURE 3.

IL-12p40/p70 secretion is not affected in wild-type and gene-deficient mice. A total of 100 μl of infected blood from rag1-null mice was used to infect 6- to 12-wk-old C57BL/6 (n = 15), ipaf-null (n = 7), caspase-1-null (n = 12), and asc-null (n = 12) mice. Pooled sera concentrations of IL-12p40/p70 were measured by ELISA at indicated times. Data represent the mean plus SD.

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Recent studies have demonstrated the importance of ASC in activating caspase-1 (9, 10, 11, 12, 13). To test whether ASC is required for IL-18 maturation and IFN-γ production, we infected asc-null and asc-sufficient mice with A. phagocytophilum. Asc-null mice showed similar bacterial infection kinetics to the il-18- and caspase-1-null mice with increased bacterial load at days 2 (49-fold; p = 0.04), 4 (1351-fold; p < 0.01), and 6 (109-fold; p = 0.02) postinfection (Fig. 4,A). ASC deficiency completely abolished IL-18 secretion (Fig. 4,B), and IFN-γ was dramatically reduced during A. phagocytophilum infection (Fig. 4 C), which supports the notion that ASC actively recruits caspase-1 for IL-18 maturation.

FIGURE 4.

ASC deficiency impairs IL-18 secretion, reduces IFN-γ levels in the peripheral blood, and increases A. phagocytophilum load in infected mice. A, 100 μl of infected blood from rag1-null mice was used to infect 6- to 12-wk-old mice. The bacterial load in the peripheral blood of asc-sufficient and asc-null mice at days 2 (n = 4 per group), 4, 6, and 9 (n = 17 per group) was measured using quantitative real-time PCR. The relative bacterial load difference in the blood of asc-sufficient and asc-null mice was calculated by normalizing the bacterial copy numbers of asc-sufficient mice to 1 after determining the fold difference when compared with asc-null mice. Experiments were repeated four times, and one representative experiment is shown. Pooled sera concentrations of IL-18 (B) and IFN-γ (C) were measured by ELISA at indicated times. Data represent the mean plus SD. ND, no relative bacterial load difference in the blood between asc-sufficient and asc-null mice.

FIGURE 4.

ASC deficiency impairs IL-18 secretion, reduces IFN-γ levels in the peripheral blood, and increases A. phagocytophilum load in infected mice. A, 100 μl of infected blood from rag1-null mice was used to infect 6- to 12-wk-old mice. The bacterial load in the peripheral blood of asc-sufficient and asc-null mice at days 2 (n = 4 per group), 4, 6, and 9 (n = 17 per group) was measured using quantitative real-time PCR. The relative bacterial load difference in the blood of asc-sufficient and asc-null mice was calculated by normalizing the bacterial copy numbers of asc-sufficient mice to 1 after determining the fold difference when compared with asc-null mice. Experiments were repeated four times, and one representative experiment is shown. Pooled sera concentrations of IL-18 (B) and IFN-γ (C) were measured by ELISA at indicated times. Data represent the mean plus SD. ND, no relative bacterial load difference in the blood between asc-sufficient and asc-null mice.

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To test whether splenocytes from asc-null, caspase-1-null, and il-18-null mice produce less IFN-γ compared with cells from wild-type mice, we infected wild-type and gene-deficient mice with A. phagocytophilum for 6 days, harvested the spleens, and determined IFN-γ production from splenocytes after A. phagocytophilum restimulation. We did not detect IFN-γ in the supernatants of nonrestimulated splenocytes (Fig. 5,A). IFN-γ production in wild-type cells greatly increased after A. phagocytophilum restimulation for 18 h (Fig. 5,A). In contrast, the production of IFN-γ was drastically reduced in caspase-1-null, il-18-null, and asc-null cells when compared with wild-type splenocytes. These results suggest that splenocytes from caspase-1-null, il-18-null, and asc-null mice have a defect in IFN-γ production after A. phagocytophilum infection (Fig. 5 A).

FIGURE 5.

ASC/caspase-1 regulates CD4+ T cell-mediated IFN-γ during A. phagocytophilum immunity. Wild-type and gene-deficient mice infected with A. phagocytophilum for 6 days had their spleens removed and were euthanized. Wild-type (n = 6), caspase-1-null (n = 6), asc-null (n = 6), and il-18-null (n = 6) splenocytes (106 cells) were cultured in vitro for 18 h in the presence of live A. phagocytophilum (108 bacterial cells). A. phagocytophilum was prepared and purified, as described in Materials and Methods. Uninfected host cells were used as a negative control. A, IFN-γ was determined by ELISA. Wild-type and gene-deficient cells were restimulated with 108 live A. phagocytophilum, and mouse spleen cells were gated on CD4+/CD69+ (B) and CD8+/CD69+ (C) for intracellular IFN-γ analysis. The percentage of cells producing IFN-γ after A. phagocytophilum restimulation is shown in parentheses. The black, blue, and red lines indicate the isotype control, nonrestimulated, and restimulated cells, respectively.

FIGURE 5.

ASC/caspase-1 regulates CD4+ T cell-mediated IFN-γ during A. phagocytophilum immunity. Wild-type and gene-deficient mice infected with A. phagocytophilum for 6 days had their spleens removed and were euthanized. Wild-type (n = 6), caspase-1-null (n = 6), asc-null (n = 6), and il-18-null (n = 6) splenocytes (106 cells) were cultured in vitro for 18 h in the presence of live A. phagocytophilum (108 bacterial cells). A. phagocytophilum was prepared and purified, as described in Materials and Methods. Uninfected host cells were used as a negative control. A, IFN-γ was determined by ELISA. Wild-type and gene-deficient cells were restimulated with 108 live A. phagocytophilum, and mouse spleen cells were gated on CD4+/CD69+ (B) and CD8+/CD69+ (C) for intracellular IFN-γ analysis. The percentage of cells producing IFN-γ after A. phagocytophilum restimulation is shown in parentheses. The black, blue, and red lines indicate the isotype control, nonrestimulated, and restimulated cells, respectively.

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To determine whether ASC/caspase-1-driven IL-18 secretion regulates the production of IFN-γ in CD4+ and CD8+ T cells during A. phagocytophilum infection, we quantified the production of IFN-γ using intracellular staining. Wild-type CD4+ T cells produced more IFN-γ in response to A. phagocytophilum when compared with gene-deficient cells (Fig. 5,B). We did not detect differences in CD8+ T cell-mediated IFN-γ production from wild-type and gene-deficient cells (Fig. 5 C). Taken together, these results strongly suggest that ASC/caspase-1 partially regulates Th1-mediated immune responses during A. phagocytophilum infection.

NK and NK T cells may be involved in early IFN-γ production during A. phagocytophilum infection because these innate immune cells produce high levels of IFN-γ in the first days postinfection against various pathogens (31, 32) and a recent study has suggested that NK and NK T cells are major components in the early pathogenesis of A. phagocytophilum infection (33). Therefore, we sought to characterize whether the ASC/caspase-1 pathway affects NK and NK T cell-mediated IFN-γ production during A. phagocytophilum infection. Our results suggested that only a small percentage of NK T (NK1.1+ CD3+) cells produced IFN-γ when wild-type, asc-, caspase-1-, and il-18-null splenocytes were stimulated with A. phagocytophilum (Fig. 6 A). PMA/Ionomycin stimulation showed an increased production of IFN-γ mediated by NK T cells in all mouse groups, suggesting that the ASC/caspase-1 pathway does not influence IFN-γ production mediated by NK T cells in the A. phagocytophilum model.

FIGURE 6.

The caspase-1 pathway does not affect IFN-γ production mediated by NK and NK T cells during A. phagocytophilum stimulation. Splenocytes from the wild-type and gene-deficient mice were euthanized, and spleens were removed 9 days postinfection with A. phagocytophilum. Wild-type and gene-deficient splenocytes (106 cells) were cultured in vitro for 18 h in the presence of live A. phagocytophilum (108 bacterial cells). A. phagocytophilum was prepared and purified, as described in Materials and Methods. Uninfected host cells were used as a negative control. Isotype controls were added in each treatment. PMA (5 ng/ml)- and ionomycin (250 ng/ml)-stimulated cells were used as positive control. A, NK T (NK1.1+ CD3+) cells were stained, and intracellular IFN-γ analysis was performed. The percentage of NK T cells producing IFN-γ is shown. B, NK (CD49b+ NKG2A/C/E+ CD3) cells were stained, and intracellular IFN-γ analysis was performed. The percentage of NK cells producing IFN-γ is shown. NR, nonrestimulated cells.

FIGURE 6.

The caspase-1 pathway does not affect IFN-γ production mediated by NK and NK T cells during A. phagocytophilum stimulation. Splenocytes from the wild-type and gene-deficient mice were euthanized, and spleens were removed 9 days postinfection with A. phagocytophilum. Wild-type and gene-deficient splenocytes (106 cells) were cultured in vitro for 18 h in the presence of live A. phagocytophilum (108 bacterial cells). A. phagocytophilum was prepared and purified, as described in Materials and Methods. Uninfected host cells were used as a negative control. Isotype controls were added in each treatment. PMA (5 ng/ml)- and ionomycin (250 ng/ml)-stimulated cells were used as positive control. A, NK T (NK1.1+ CD3+) cells were stained, and intracellular IFN-γ analysis was performed. The percentage of NK T cells producing IFN-γ is shown. B, NK (CD49b+ NKG2A/C/E+ CD3) cells were stained, and intracellular IFN-γ analysis was performed. The percentage of NK cells producing IFN-γ is shown. NR, nonrestimulated cells.

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NK (CD49b+ NKG2A/C/E+ CD3) cells from wild-type, asc-, and caspase-1 mice restimulated with A. phagocytophilum produced similar levels of IFN-γ when compared with PMA/ionomycin-stimulated cells (Fig. 6 B). However, il-18-null NK cells did not produce IFN-γ in the presence of A. phagocytophilum. The biological significance of this finding needs to be further explored; however, our results clearly confirm previous findings (33) and suggest that NK cells participate in A. phagocytophilum eradication. Moreover, similar to NK T cells, the ASC/caspase-1 pathway does not play a role in IFN-γ production mediated by NK cells because asc- and caspase-1-null NK cells stimulated with A. phagocytophilum produced similar levels of IFN-γ when compared with the wild-type treatment. Similar results were obtained when NK cells were defined with the phenotypic markers NK1.1+ CD3 (data not shown).

NALP3, along with the adaptor molecule ASC, has been shown to play a central role in caspase-1 activation in response to a number of stimuli, such as pore-forming toxins, ATP, uric acid, and the pathogens Staphylococcus aureus and Listeria monocytogenes (9, 10, 11, 12, 13). We have assessed the role of NALP3 in the immune response against A. phagocytophilum and found that NALP3 was not required for IL-18 and IFN-γ secretion during A. phagocytophilum infection (Fig. 7, B and C). Bacterial load analysis verified that no differences in A. phagocytophilum burden were detected in the blood of nalp3-null and nalp3-sufficient mice at both days 4 (4-fold; p = 0.28) and 8 (3-fold; p = 0.06) postinfection (Fig. 7,A). Serum IL-12 levels, as expected, were also unchanged between wild-type and nalp3-null mice (Fig. 7,D). Notably, serum IL-1β and IL-18 in response to in vivo challenge with LPS was greatly reduced in nalp3-null mice after 5-h stimulation (Fig. 7, E and F) (17). These data suggest that NALP3 does not have any protective effect against A. phagocytophilum, and also suggest a divergence in the roles of NALP3 with those of ASC and caspase-1.

FIGURE 7.

NALP3 is not required for IFN-γ production during A. phagocytophilum infection. A, 100 μl of infected blood from rag1-null mice was used to infect 6- to 12-wk-old nalp3-sufficient (n = 10) and nalp3-null (n = 10) mice, and the bacterial load was measured in the peripheral blood using quantitative real-time PCR. The relative bacterial load difference in the blood of nalp3-sufficient and nalp3-null mice was calculated by normalizing the bacterial copy numbers of nalp3-sufficient mice to 1 after determining the fold difference when compared with nalp3-null mice. Pooled sera concentrations of IL-18 (B), IFN-γ (C), and IL-12 (D) were measured by ELISA at indicated times. Nalp3-sufficient (n = 5) and nalp3-null (n = 5) mice were i.p. injected with LPS (30.0 mg/kg); 5 h after LPS injection, mice were euthanized and blood was collected by cardiac puncture. Pooled sera were used to detect IL-1β (E) and IL-18 (F) levels. ND, no relative bacterial load difference in the blood between nalp3-sufficient and nalp3-null mice. Data represent the mean plus SD.

FIGURE 7.

NALP3 is not required for IFN-γ production during A. phagocytophilum infection. A, 100 μl of infected blood from rag1-null mice was used to infect 6- to 12-wk-old nalp3-sufficient (n = 10) and nalp3-null (n = 10) mice, and the bacterial load was measured in the peripheral blood using quantitative real-time PCR. The relative bacterial load difference in the blood of nalp3-sufficient and nalp3-null mice was calculated by normalizing the bacterial copy numbers of nalp3-sufficient mice to 1 after determining the fold difference when compared with nalp3-null mice. Pooled sera concentrations of IL-18 (B), IFN-γ (C), and IL-12 (D) were measured by ELISA at indicated times. Nalp3-sufficient (n = 5) and nalp3-null (n = 5) mice were i.p. injected with LPS (30.0 mg/kg); 5 h after LPS injection, mice were euthanized and blood was collected by cardiac puncture. Pooled sera were used to detect IL-1β (E) and IL-18 (F) levels. ND, no relative bacterial load difference in the blood between nalp3-sufficient and nalp3-null mice. Data represent the mean plus SD.

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We then examined the role of IPAF in A. phagocytophilum clearance. IPAF has previously been shown to be important for caspase-1 activation in response to the Gram-negative bacteria Salmonella typhimurium and Legionella pneumophila (34, 35, 36). Ipaf-null mice had increased susceptibility to A. phagocytophilum, which was observed at days 2 (99-fold; p < 0.01), 4 (357-fold; p = 0.02), and 6 (26-fold; p = 0.03) postinfection (Fig. 8,A). We did not observe any difference in bacterial load at day 9 postinfection between ipaf-sufficient and ipaf-null mice. IL-18 and IFN-γ secretion were reduced at day 4, but not completely impaired, as observed in A. phagocytophilum-infected caspase-1- and asc-null mice (Fig. 8, B and C). These results suggest that IPAF partially regulates IL-18 and IFN-γ production during infection with A. phagocytophilum. It is also likely that other NLR molecules, through the adaptor molecule ASC, contribute to A. phagocytophilum-driven production of IL-18/IFN-γ.

FIGURE 8.

IPAF is partially required for IFN-γ production upon A. phagocytophilum infection. A, Bacterial load was measured in the peripheral blood of ipaf-sufficient and ipaf-null mice at days 2 (n = 4 per group), 4 (n = 16 and n = 8 per group), 6 (n = 6 per group), and 9 (n = 16 and n = 8 per group) using quantitative real-time PCR. The relative bacterial load difference in the blood of ipaf-sufficient and ipaf-null mice was calculated by normalizing the bacterial copy numbers of ipaf-sufficient mice to 1 after determining the fold difference when compared with ipaf-null mice. Pooled sera concentrations of IL-18 (B) and IFN-γ (C) were measured. Data represent the mean plus SD. ND, no relative bacterial load difference in the blood between ipaf-sufficient and ipaf-null mice.

FIGURE 8.

IPAF is partially required for IFN-γ production upon A. phagocytophilum infection. A, Bacterial load was measured in the peripheral blood of ipaf-sufficient and ipaf-null mice at days 2 (n = 4 per group), 4 (n = 16 and n = 8 per group), 6 (n = 6 per group), and 9 (n = 16 and n = 8 per group) using quantitative real-time PCR. The relative bacterial load difference in the blood of ipaf-sufficient and ipaf-null mice was calculated by normalizing the bacterial copy numbers of ipaf-sufficient mice to 1 after determining the fold difference when compared with ipaf-null mice. Pooled sera concentrations of IL-18 (B) and IFN-γ (C) were measured. Data represent the mean plus SD. ND, no relative bacterial load difference in the blood between ipaf-sufficient and ipaf-null mice.

Close modal

A Th1-mediated immune response, evidenced by IFN-γ production, is important for clearance of A. phagocytophilum infection (5). How the innate immune response against A. phagocytophilum is initiated remains the question of much debate, and independent studies have shown little evidence for a role of TLRs in clearance of this pathogen (3, 4). We demonstrate in this study that the NLR family of proteins plays a central role in initiating innate immune responses against A. phagocytophilum and partially regulates the development of a Th1 response during A. phagocytophilum infection. NLRs are structurally related to plant resistance proteins that detect specific avirulence gene products produced by pathogens (37). We found that the adaptor molecule ASC was essential for IL-18 and IFN-γ production in vivo following infection with A. phagocytophilum. Deficiency of the downstream molecules caspase-1 and IL-18 resulted in very similar in vivo phenotypes, in that A. phagocytophilum infection resulted in defective IFN-γ production paralleled by increased early susceptibility to infection. We did not detect measurable serum IL-1β following infection with A. phagocytophilum, suggesting that the effects of ASC and caspase-1 deficiency were predominantly mediated by defects in IL-18 secretion.

We investigated whether the ASC/caspase-1 pathway affects IFN-γ production mediated by NK and NK T cells because these innate immune cells respond rapidly to foreign Ags without the need of immunization or preactivation (31, 32), and also a recent study has demonstrated that NK and NK T cells play a role in A. phagocytophilum clearance during the early stages of infection (33). NK cells prominently secreted IFN-γ upon A. phagocytophilum stimulation, as previously suggested (33). However, we found no evidence that the ASC/caspase-1 pathway instructs NK and NK T cells to produce IFN-γ during A. phagocytophilum infection. To the contrary, NK cells from asc- and caspase-1-null mice produced levels of IFN-γ similar to PMA/ionomycin-activated cells. Taken together, one should conclude that the source of IFN-γ regulated by the ASC/caspase-1/IL-18 axis during the early phase of A. phagocytophilum infection remains unresolved, considering that the effect of ASC/caspase-1 pathway is only partial on Th1 cells.

It has been previously demonstrated that active IL-18 may be generated through a caspase-1-independent mechanism (38, 39, 40). Our results suggest that NK cell regulation of IFN-γ upon A. phagocytophilum stimulation is entirely dependent on IL-18; yet, IL-18 secretion may be regulated in an ASC/caspase-1-independent manner. Further experimentation needs to be done to examine this mechanism. However, caspase-1-independent cleavage of IL-18 leading to NK-mediated IFN-γ production as a mechanism of cytokine regulation may be possible upon A. phagocytophilum stimulation.

Although the adaptor molecule ASC is required for the induction of IL-18/IFN-γ secretion during A. phagocytophilum infection, it is unclear whether the high susceptibility of asc-null mice is solely due to these cytokines. Mice lacking caspase-1 and IL-18 harbored ∼400-fold more bacteria in the bloodstream when compared with wild-type mice. In contrast, asc-null mice harbor a 1400-fold bacterial increase in the peripheral blood when compared with the wild-type mice, suggesting that IL-18/IFN-γ release only partially accounts for A. phagocytophilum susceptibility. The high susceptibility of asc-null mice during A. phagocytophilum infection could also be due to a defect in host cell death. It is worth noting that asc-null macrophages are significantly more resistant to cell death when infected with Francisella (41), and ASC plays a caspase-1-independent role in cell death through Bax and caspase-9 (42, 43). Therefore, neutrophil death in asc-null mice may account for differences in bacterial load because delay in normal neutrophil apoptosis increases A. phagocytophilum load (3, 44, 45, 46). The role of caspase-1-independent, ASC-dependent signaling during A. phagocytophilum infection needs to be further explored.

It is unclear which NLR molecule triggers IL-18/IFN-γ secretion. We have evaluated two NLR molecules, NALP3 and IPAF, which have been implicated in the activation of caspase-1 (9, 10, 11, 12). The NALP3 inflammasome has been shown to be required for caspase-1 activation in response to microbial pore-forming toxins, endogenous danger signals such as ATP and uric acid, and the pathogens L. monocytogenes and S. aureus (9, 10, 11, 12). However, our data indicate that NALP3 is not involved in the recognition of A. phagocytophilum, because nalp3-null mice infected with A. phagocytophilum do not have any defect in their ability to clear the infection and produce IL-18 and IFN-γ. These data support the hypothesis that NALP3 regulation of the inflammasome is independent of an operational bacterial type III or type IV secretion system or pore-forming proteins, as previously suggested (47). A. phagocytophilum has a functional type IV secretion system (2).

The IPAF inflammasome has been demonstrated to play a crucial role in caspase-1 activation in response to Salmonella typhimurium and Legionella pneumophila (34, 35, 36, 48, 49, 50, 51). A common feature of these pathogens is the presence of a type III or type IV secretion system, which is required for their ability to activate caspase-1. IPAF contributes to host resistance to A. phagocytophilum because ipaf-null mice display increased susceptibility to in vivo infection. However, it is unclear how IPAF is mediating this effect, because IL-18 and IFN-γ secretion was only marginally affected. Our findings support the trimeric interactions among IPAF, ASC, and caspase-1 because ASC may stabilize the interaction between caspase-activating recruiting domains of IPAF and caspase-1 (48). However, we cannot rule out that an unknown NALP3-independent molecule may also interact with ASC and caspase-1 during A. phagocytophilum recognition and clearance. This model may be similar to the L. pneumophila assembly of the inflammasome in which at least two host cytosolic receptors, NAIP5/Birc1e and Ipaf, activate caspase-1 (36, 52). Immunoprecipitation studies should indicate whether NAIP5/Birc1e and Ipaf interact during A. phagocytophilum infection.

The molecular mechanisms of A. phagocytophilum pathogenesis are relatively unknown. A. phagocytophilum enters neutrophils via caveola-mediated endocytosis and replicates in the cytosol in membrane-bound inclusions called morulae (53, 54). Two type IV secretion system proteins from A. phagocytophilum, VirB9 and VirB6, are thought to be important during the obligatory intracellular trafficking and evasion of the lysosome pathway in neutrophils (2). NLR sensing of type IV proteins from A. phagocytophilum is a tempting hypothesis to explore because neutrophils have the ability to form the inflammasome (55) and our experiments suggest that inflammasome formation occurs during neutrophil colonization by A. phagocytophilum (data not shown).

The present data define signaling pathways that lead to the integration of innate and adaptive host immune response against A. phagocytophilum. However, much remains to be investigated. For example, it will be important to determine which cells secrete IL-18 during A. phagocytophilum infection. Signaling events that trigger IL-18 should contrast with IL-1β because there is a constitutive pool of intracellular pro-IL-18 in the cell (22, 56). Considerable emphasis should be put on the identification and characterization of A. phagocytophilum components that trigger the ASC/caspase-1 pathway. A. phagocytophilum does not carry genes that code for lipid A, peptidoglycan biosynthesis, and bacterial flagellin (2). The absence of components that are recognized by TLRs may provide means of avoiding the host cell innate recognition. Finally, it is unknown how A. phagocytophilum-infected neutrophils assist other cells for Ag presentation. As in other infections, dendritic cells are most likely responsible for priming T cell immune responses. The interactions between neutrophils and dendritic cells in initiating Ag-specific immune responses need to be defined.

In summary, we have shown that the NLR pathway is the arm of the innate immune system that recognizes A. phagocytophilum upon infection in mice. Moreover, we have demonstrated that NLRs can effectively elicit a strong Th1-mediated immune response, evidenced by IFN-γ production upon A. phagocytophilum infection. These results should permit a deeper understanding of how A. phagocytophilum and other rickettsia trigger immune responses, and such knowledge should contribute to rational approaches to designing novel antirickettsial therapies.

We thank Debbie Beck and Nancy Marcantonio for excellent technical assistance.

The authors have no financial conflict of interest.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

This work was supported by Public Health Service Grant 5R01AI041440-08 (to E.F.) from the National Institute of Infectious Diseases and 5P30DK034989-22, Yale University Digestive Disease Research Core Center (to J.H.F.P.) from the National Institute of Digestive Diseases and Kidney and Ellison Foundation (to R.A.F.), and by the Northeast Biodefense Center-Lipkin (to E.F.). J.H.F.P. and F.S.S. are Brown-Coxe Fellow in Medical Sciences and Pfizer Fellow in Infectious Diseases, respectively. R.A.F. is an investigator with the Howard Hughes Medical Institute.

3

Abbreviations used in this paper: NLR, Nod-like receptor; ASC, apoptotic specklike protein with a caspase-activating recruiting domain.

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