cmi12232-sup-0001-figs1.tif744KFig. S1. Circulating LT does not decrease viability of bone marrow PMNs. C57Bl/6 mice were injected with 6.25 μg of LT and PMNs were isolated from the bone marrow of intoxicated and non-intoxicated mice 24 h later. Cells were then stained for flow cytometry. PMNs were gated for by Ly-6G and side scatter, the bar graph represents % viability of cells as determined by fixable Aqua Dead Cell Stain in that subset. n = 3 mice per condition.

Fig. S2. Three defined stages of B. anthracis infection. (A–C) Black and white photos of a single A/J mouse injected subcutaneously in the left ear with 1 × 105 luminescent B. anthracis Sterne strain (BIG23) spores, overlaid with a false colour representation of photon emission intensity as indicated by the scale on the right in p s−1 cm−2 sr−1 at different stages of infection.

A. Early stage of infection defined by vegetative bacilli, represented by luminescence, being confined to the initial site of spore inoculation.

B. Mid-stage of infection defined by vegetative bacilli spread to the regional draining lymph node.

C. Late-stage infection defined by bacterial spread to the kidneys.

cmi12232-sup-0003-figs3.tif762KFig. S3. Ability to produce LT makes B. anthracis more virulent. A/J mice were infected with 1 × 106 cfu of vegetative LF or EF knockout BIG23. Once light was detected in the kidneys, representing dissemination, mice were euthanized according to ACUC protocol. There was a significant defect in ability to disseminate in LF knockout. B. anthracis significance determined by log rank test (**P < 0.01). n = 4 mice for each experimental condition.

Fig. S4. A/J PMNs kill vegetative TKO similarly to C57Bl/6 PMNs. (A–C) PMNs isolated from A/J and C57Bl/6 mice were inoculated with vegetative B. anthracis at an moi of 1:1 and incubated at 37°C. At indicated time points samples were put on ice and mixed with 0.1% Saponin for 10 min. Samples were then serially diluted and plated for enumeration on LB agar.

A. Comparison between PMN ability to kill vegetative TKO between C57Bl/6 and A/J PMNs. No statistical difference was seen at any time.

B. Effects of LT on ability of A/J PMN to kill vegetative TKO. A/J mice were i.p. injected with 6.25 μg of LT and PMNs were isolated 24 h later. Significant differences were seen between LT intoxicated and non-intoxicated PMNs at 0.5 h (*P < 0.05), 1 h (**P < 0.01) and 1.5 h (**P < 0.01).

C. A/J and C57Bl/6 PMN ability to kill vegetative TKO was determined after i.p. injection of 6.25 μg LT 24 h prior to PMN isolation. No significant differences in killing ability were seen at any time point. n = 3 mice for each experimental group.


Fig. S5. PMN accumulation at sites of inflammation can be measured in real time.

A and B. NECre Luc mouse with 2.5 μg PMA in 5 μl DMSO pipetted onto the ear had net luminescence measured over the course of 48 h in both the non-inflamed right ear (A) and the inflamed left ear (B), where a significant increase was found from time zero to 48 h (*P = 0.0213) where n = 6 mice per condition.

C. Luminescence was measured in both ears of a NECre Luc mouse over the course of 48 h. Results are representative of three separate mice.

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