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. 2013 Mar 5;4:56. doi: 10.3389/fimmu.2013.00056

Figure 3.

Figure 3

Partial degradation of NETs and bacterial entrapment within NETs associated with S. pneumoniae 19F. (A) Lungs of mice infected with influenza alone (FLU) exhibit continuous staining (arrows) of MPO (red) and histone H2B (green). However, lungs of mice with viral and bacterial co-infection (FLU + SP) revealed large clusters of NETs often filled with degraded DNA, appearing as punctate staining (asterisks) of MPO and histone H2B. (B) To ascertain whether S. pneumoniae and other bacterial species cause NETs degradation, neutrophils were isolated from the lungs of influenza-infected mice (Narasaraju et al., 2011), incubated with S. pneumoniae, K. pneumoniae, P. aeruginosa, or S. aureus at a neutrophil:bacterial ratio of 1:10, and stained with DAPI. Extensive NETs were notable when neutrophils were incubated with S. pneumoniae (PMN + SP), compared with incubation with the other 3 bacterial species (data not shown). NETs were negligible for the untreated neutrophils (PMN) serving as negative control. Partially degraded NETs were observed when incubated with S. pneumoniae, suggesting DNA degradation in NETs by endonucleases produced by S. pneumoniae. Arrow indicates bacteria trapped inside the NETs, while arrowheads depict partially degraded NETs. (C) Experiments were performed to study NETs interactions with bacteria. Normal mouse bone marrow-derived neutrophils (PMN) served as negative control. These neutrophils were also incubated with BALF from influenza-infected mice (PMN + BALF). Neutrophils with infected BALF were also incubated with S. pneumoniae at various MOI (PMN + BALF + SP). Immunostaining for NETs and bacteria was performed for tests and controls. S. pneumoniae bacteria (red) were found entrapped within the DNA fibers (blue) and histone H2B (green) depending on MOI. Higher MOI of 100 displayed greater accumulation and entrapment of bacteria in the vicinity of NETs compared to lower MOI of 0.1. (D) Bacterial entrapment on NETs was significantly enhanced with increasing MOI. Values represent the means ± SE of at least 3 independent experiments. *Denotes P < 0.05 vs. MOI of 0.01.