Role of Bacillus anthracis spore structures in macrophage cytokine responses

Abstract

The innate immune response of macrophages (Mphi) to spores, the environmentally acquired form of Bacillus anthracis, is poorly characterized. We therefore examined the early Mphi cytokine response to B. anthracis spores, before germination. Mphi were exposed to bacilli and spores of Sterne strain 34F2 and its congenic nongerminating mutant (DeltagerH), and cytokine expression was measured by real-time PCR and an enzyme-linked immunosorbent assay. The exosporium spore layer was retained (exo+) or removed by sonication (exo-). Spores consistently induced a strong cytokine response, with the exo- spores eliciting a two- to threefold-higher response than exo+ spores. The threshold for interleukin-1beta (IL-1beta) production by wild-type Mphi was significantly lower than that required for tumor necrosis factor alpha expression. Cytokine production was largely dependent on MyD88, suggesting Toll-like receptor involvement; however, the expression of beta interferon in MyD88-/- Mphi suggests involvement of a MyD88-independent pathway. We conclude that (i) the B. anthracis spore is not immunologically inert, (ii) the exosporium masks epitopes recognized by the Mphi, (iii) the Mphi cytokine response to B. anthracis involves multiple pattern recognition receptors and signaling pathways, and (iv) compared to other cytokines, IL-1beta is expressed at a lower spore concentration.

FIG. 1.

B. anthracis spores elicit an early cytokine mRNA response in primary Mφ. B. anthracis spores (filled bars) or heat-killed bacilli (open bars) were added at different MOIs to a culture of primary Mφ (3 × 10⁶) that were placed in serum-free RPMI 1640 for the duration of the infection (1 h). The mRNA levels for IL-1β (A), TNF-α (B), and IFN-β (C) were quantified. **, P ≤ 0.01; *, P ≤ 0.05.

FIG. 2.

The spore form of ΔgerH B. anthracis elicits a stronger cytokine protein response than the vegetative form in murine Mφ. Primary murine Mφ were exposed to the heat-killed vegetative or the exo⁺ form of the ΔgerH B. anthracis spores at an MOI of 0.2:1 in serum-free RPMI 1640. The Mφ were viable after 24 h, when the supernatants were harvested and analyzed by ELISA for amounts of IL-1β (A), TNF-α (B), IL-6 (C), and RANTES protein (D) made by the Mφ. **, P ≤ 0.01.

FIG. 3.

Exosporium-deficient spores elicit a stronger cytokine message response than spores covered with exosporium. Primary murine Mφ were exposed to the exo⁺ or exo⁻ forms of the Sterne spores at an MOI of 0.2:1, and cytokine message levels were quantified 1 h after exposure by real-time PCR. The exo⁻ spores induced a significantly higher response for IFN-β (A), IL-1β (B), TNF-α (C), and IL-6 (D). **, P ≤ 0.01; *, P ≤ 0.05.

FIG. 4.

IL-1β is expressed at lower concentrations of spores than TNF-α. Primary murine Mφ plated at 1 × 10⁶ cells/well were challenged overnight with increasing amounts of the ΔgerH exo⁺ spores, and the supernatants were assayed by ELISA for IL-1β (A) and TNF-α (B) production. **, P ≤ 0.01; *, P ≤ 0.05; n.s., not significant, in comparison to medium control levels.

FIG. 5.

The Mφ mRNA cytokine response is dependent on MyD88. Primary murine Mφ from MyD88^−/− or wild-type (WT) control C57/BL6 mice were exposed to Sterne exo⁺ spores of B. anthracis at an MOI of 0.2:1 for 1 h, and their cytokine message levels for IFN-β (A), IL-1β (B), TNF-α (C), and IL-6 (D) were determined by real-time PCR. **, P ≤ 0.01; *, P ≤ 0.05, for comparison to medium control levels.

FIG. 6.

The IFN-β response is not mediated by TLR4 or due to endotoxin contamination. (A) B. anthracis spores (MOI, 0.2:1) or LPS (100 ng/ml) was preincubated for 30 min with recombinant ENP (ENP) (3 μg/ml) or phosphate-buffered saline prior to being added to the culture of primary murine Mφ. The IFN-β message level of the Mφ 1 h poststimulation was determined by real-time PCR. (B) Mφ from TLR4-defective (C3H/HeJ) or control (C3H/HeN) mice were challenged with Sterne spores at an MOI of 0.2:1, and their IFN-β message levels 1 hour after challenge were determined by real-time PCR.