FIG 5.

Anti-MPO immunohistochemistry and Gram stainings of subcutaneous MGAS2221 infection sites showing inhibition of neutrophil infiltration, neutrophil necrosis, and GAS persistence. C57BL/6J mice were subcutaneously inoculated with 5.6 × 10⁷ CFU of MGAS2221, and skin containing infection sites were collected at days 1, 2, and 4 after inoculation. Sections of the skin infection sites were stained with anti-MPO antibody and hematoxylin counterstain and examined by IHC. In these MPO IHC/hematoxylin stains, the nucleus (if it was not lost) and MPO in neutrophils were blue and red, respectively. (A, C, and E) Images of MPO IHC/hematoxylin stains for sections of infected lung at days 1 (A), 2 (C), and 4 (E). The braces indicate the layers of neutrophils that surrounded the infection sites and exhibited nuclear staining with hematoxylin. The boxes in panels C and E indicate the locations of the infection sites shown in the Gram stain images in panels D and F, respectively. (B) Relative blue-channel intensities of MPO IHC staining at skin infection sites as estimates of relative levels of recruited neutrophils. The data points are differences in the average intensities of whole infection sites between uninfected and infected mice (5 mice per group). The panels also contain the MPO IHC intensities of MGAS2221 Δsse infection described for Fig. 7. (D and F) Images of Gram staining of the lung sections showing parts of the infection sites located at the places indicated by the boxes in panels C and E. The braces in panels D and F indicate the layers of neutrophils that had intact nuclei, lacked stained GAS bacteria, and surrounded the GAS sites. The circle indicates one of many stained GAS clumps, and numerous GAS organisms as individual bacteria or in chains can be seen after zooming in by 400%. The rest of the infection sites that are not shown exhibited stained GAS. Scale bars: 100 μm (A, C, and E) and 20 μm (D and F).