Fig 1.
Cytocentrifuge preparation of alveolar macrophages after 7 days of culture.
Bronchoalveolar lavage fluid cells were allowed to adhere for 4 hours, non-adherent cells were removed, and the remaining cells were cultured for 7 days. At that time point the cultures consisted of a uniform population of macrophages. Modified Wright’s stain, 400X magnification.
Fig 2.
Electrophoresis and Western blot.
Lanes 1 and 2: Protein electrophoresis. Lane 1: Eluate in buffer containing 0.2% sodium dodecyl sulfate and 0.1% Tween-20 in 50 mM Tris HCl pH 8 yielded a single band ~240 kDa, consistent with SALSA. Lane 2: Equine duodenal tissue lysate. Equine duodenal tissue lysate showed a prominent band of similar size. Lanes 1 and 2 were present on the same gel as the ladder, and these lanes were spliced together to only show the relevant lanes.
Fig 3.
A: Phagocytes, which were mostly neutrophils, were gated based on high forward and side scatter. Lymphocytes were readily identified by low forward and side scatter. B: Fluorescence in three different cell populations; histograms. Neutrophils not incubated with bacteria (grey) were used to set the threshold for fluorescence. After incubation with bacteria, >90% of neutrophils (red) were fluorescent. Lymphocytes incubated with bacteria (yellow) did not fluoresce except for a small proportion (<4%), which may reflect inclusion of some phagocytes (monocytes or neutrophils) in the gate. C: Cytocentrifuge preparation of a sample incubated with 20 μg/mL SALSA at 37°C. Most neutrophils (thick arrows) contained multiple phagocytosed bacterial cocci in the cytoplasm. These bacteria were also noted in clusters extracellularly (thin arrow). Lymphocytes (arrowheads) did not contain bacteria. The background contains many lysed and a few intact erythrocytes, an expected finding in a sample that underwent erythrocyte lysis during sample preparation. Modified Wright’s stain, 1000X magnification.
Table 1.
Neutrophil phagocytosis of bacteria after incubation with different concentrations of SALSA.
Fig 4.
Neutrophil phagocytosis of bacteria after incubation with different concentrations of SALSA: A: The highest concentration of SALSA (red line, 20 μg/mL) resulted in fewer fluorescent and more non-fluorescent cells, consistent with decreased phagocytosis, compared to the other incubation groups. B: Ridgeline plot of fluorescence in cells incubated with different concentrations of SALSA or PBS.
Table 2.
P-values comparing fluorescent neutrophil percentages in samples incubated with different concentrations of SALSA.
Fig 5.
Mean or median cytokine concentration in alveolar macrophage supernatant.
Macrophages were incubated for 24 hours with 20, 10, or 5 μg/mL of SALSA / 1 μg/mL LPS, with only LPS, or with media alone. Error bars represent the 95% confidence intervals. Red asterisks indicate samples incubated with SALSA which had a significant treatment effect compared to samples incubated with LPS alone.
Table 3.
Cytokine concentrations (pg/mL) in supernatant of alveolar macrophages.