Nod1/Nod2-mediated recognition plays a critical role in induction of adaptive immunity to anthrax after aerosol exposure

Abstract

Toll-like receptors and Nod-like receptors (NLR) play an important role in sensing invading microorganisms for pathogen clearance and eliciting adaptive immunity for protection against rechallenge. Nod1 and Nod2, members of the NLR family, are capable of detecting bacterial peptidoglycan motifs in the host cytosol for triggering proinflammatory cytokine production. In the current study, we sought to determine if Nod1/Nod2 are involved in sensing Bacillus anthracis infection and eliciting protective immune responses. Using mice deficient in both Nod1 and Nod2 proteins, we showed that Nod1/Nod2 are involved in detecting B. anthracis for production of tumor necrosis factor alpha, interleukin-1 alpha (IL-1 alpha), IL-1 beta, CCL5, IL-6, and KC. Proinflammatory responses were higher when cells were exposed to viable spores than when they were exposed to irradiated spores, indicating that recognition of vegetative bacilli through Nod1/Nod2 is significant. We also identify a critical role for Nod1/Nod2 in priming responses after B. anthracis aerosol exposure, as mice deficient in Nod1/Nod2 were impaired in their ability to mount an anamnestic antibody response and were more susceptible to secondary lethal challenge than wild-type mice.

FIG. 1.

TNF-α secretion by J774A.1 macrophages after exposure to B. anthracis spores. (A) J774A.1 macrophages were untreated (circles) or treated with 3 μg CytD (squares) for 45 min and subsequently exposed to Sterne spores as described in Materials and Methods. Intracellular bacteria were enumerated at 0, 2.5, 5, 7.5, and 10 h after spore exposure. (B) Supernatants collected from J774A.1 treated with (+CytD) or without (−CytD) CytD after exposure to medium alone (C), irradiated spores (I-Ba), or viable spores (Ba) and assayed for TNF-α. Data are expressed as the mean ± standard error of the mean from three samples. Data are representative of two independent experiments. A Student t test was used for statistical analysis, and P values of <0.05 are indicated (*).

FIG. 2.

Characterization of pulmonary adherent cells. Pulmonary adherent cells were collected from BL/6 and Nod1/Nod 2 KO mice as described in Materials and Methods and subsequently stained for CD11b and CD11c markers. Three lung cell populations, i.e., interstitial macrophages (intMac) (CD1lc^low/CD11b^high), DC (CD1lc^high/CD11b^high), and AMac (CD1lc^high/CD11b^low), were identified, and the percentage of each population was different at 1 h (A) and 24 h (B) following culture. Cells that did not stain for either CD11b or CD11c at each time point are also indicated (Neg). Each dot represents a single animal, and a bar is drawn at the mean. A Student t test was used for statistical analysis. No statistically significant difference was observed between BL/6 and Nod/Nod2 cells for any cell population except for AMac at 1 h (P = 0.003).

FIG. 3.

Nod1/Nod2 proteins are involved in recognition of live B. anthracis for induction of proinflammatory mediators. Pulmonary cells from WT BL/6 and Nod1/Nod2 KO mice were collected and exposed to medium alone (C), irradiated Sterne strain spores (I-Ba), or viable Sterne strain spores (Ba). (A) At 2, 8, and 18 h postexposure, supernatant was collected and assayed for TNF-α. (B) At 8 h after spore exposure, mRNA levels of the indicated proinflammatory mediators were measured. Real-time data are expressed as a fold increase in expression over the result for cells treated with medium alone after normalizing to β-actin using the 2^−ΔΔCT method. Data are representative of two independent experiments and are presented as the mean ± standard deviation. A Student t test was used for statistical analysis, and P values of <0.05 are indicated (*).

FIG. 4.

BMDMs from WT and Nod1/Nod2 KO mice were stimulated with 10 ng/ml LPS in the presence or absence of 10 μg/ml MDP for 24 h. Cell-free supernatants were analyzed by ELISA for production of IL-6 (A) and TNF-α (B). ** and *** indicate significant differences between cultures with and without MDP at P values of <0.01 and <0.001, respectively. The statistical significance of differences between two groups was determined by a two-tailed t test with unequal variance (Aspin-Welch's t test; Microsoft Excel). Error bars indicate standard deviations.

FIG. 5.

Nod1/Nod2 plays a significant role in induction of protective immunity after primary aerosol exposure to B. anthracis. (A) BL/6, TLR2 KO, Nod1/Nod2 KO, and A/J mice were exposed to aerosolized spores and survival followed for 10 days. (B) BL/6, TLR2 KO, and Nod1/Nod2 KO mice that survived aerosol challenge were rechallenged with a lethal dose of Sterne strain spores 30 days after primary challenge and survival followed for 10 days. Naïve BL/6 mice were included as controls. (C) Sera were collected from BL/6 and Nod1/Nod2 KO mice 14 and 28 days after primary aerosol challenge and assayed for anti-PA IgG titers. Results are pooled from three independent experiments.

FIG. 6.

Nod1/Nod2 plays a significant role in induction of anamnestic immunity after primary aerosol exposure to B. anthracis. BL/6 and Nod1/Nod2 KO mice were exposed to aerosolized spores. Mice that survived aerosol challenge were rechallenged with a lethal dose of Sterne strain spores 30 days after primary challenge. Sera were collected from BL/6 and Nod1/Nod2 KO mice 28 days after primary aerosol challenge and 2 and 4 days after the secondary lethal challenge. Sera were assayed for anti-PA IgG titers. Results are pooled from two independent challenges. A Student t test was used for statistical analysis. For the BL/6 mice and the Nod1/Nod2 mice, titers at day 2 postchallenge and day 4 postchallenge were compared to titers on day 28. Only the BL/6 titers at day 4 were significantly different from those at day 28.

FIG. 7.

Impaired expansion of lymphocytes in draining LNs of Nod1/Nod2 KO mice after aerosol challenge with Sterne strain B. anthracis. Seven days after primary aerosol challenge, cervical LNs were collected from mice and assessed for total LN cell numbers. Values for unchallenged mice are also included (day 0). Using flow cytometric analysis, the percentage and numbers of CD4⁺ T cells and CD8⁺ T cells were also measured. Data are representative of two independent experiments. A Student t test was used for statistical analysis, and P values of <0.05 are indicated (*). Error bars indicate standard deviations.

FIG. 8.

Nod1/Nod2 recognition of spores by pulmonary cells is required for optimal IL-12p70 production. Pulmonary cells from WT BL/6 and Nod1/2 KO mice were collected and exposed to medium alone (C), irradiated Sterne strain spores (I-Ba), or viable Sterne strain spores (Ba). Twenty-four hours following stimulation, supernatants were collected and assayed for IL-10 (A) and IL-12p70 (B). A Student t test was used for statistical analysis, and P values are indicated. Error bars indicate standard deviations.