Figure 4.

MPO fluorescence molecular imaging in infection. (A) Mice were injected with salmonella intracerebrally to induce cerebritis. Fluorescence reflectance imaging (FRI) of coronal brain slices was performed, showing increased fluorescence signal consistent with MPO activity in the ipsilateral but not contralateral hemisphere. Saline injection did not trigger significant MPO activity (N = 6, *** p < 0.0002, student's t-test, experiment was performed twice). (B) Immunofluorescence microscopy of fresh-frozen tissue sections. Staining for MPO activity (MABS, red) and MPO protein (as detected with an MPO-antibody, green) revealed increased MPO protein both within and close to the regions of cerebritis, while elevated MPO activity was only detected within the region of cerebritis (bar = 100 μm, confocal microscopy at 25X). (C) Wildtype and MPO-KO mice were injected subcutaneously with Streptococcus pneumoniae (SPn) to induce bacterial cellulitis with formation of neutrophil extracellular traps (NETs), or with saline as negative control. FRI was performed. Yellow circles outline sites of SPn or vehicle injections. Fluorescence images of MPO activatable biotinylated sensor (MABS) (MPO activity, red) and Sytox Green (extracellular DNA, green) as well as a merged fluorescence image (MABS plus Sytox Green) demonstrate co-localization of MPO activity with extracellular DNA, consistent with NET formation at the site of infection in wildtype mice (top row). In MPO-KO mice, extracellular DNA but not MPO activity is seen at the site of infection (bottom row). Increased levels of MPO activity and extracellular DNA are seen in the SPn injected thigh but not the saline injected thigh (*** p < 0.001, N=4 mice per group, student's t-test, experiment was replicated three times). Increased levels of extracellular DNA but not MPO activity are seen in MPO-KO mice injected with SPn or saline (*** p < 0.001, N=3 mice per group, student's t-test, experiment was replicated twice).