Dynamics of Increasing IFN-γ Exposure on Murine MH-S Cell-Line Alveolar Macrophage Phagocytosis of Streptococcus pneumoniae

Abstract

Previous investigations have demonstrated that activation with the type II interferon, IFN-γ, downregulates alveolar macrophage (AM) phagocytosis of Streptococcus pneumoniae. While these studies have shown clear effects at discrete time points, the kinetics of the macrophage response to IFN-γ over time, with respect to pneumococcal phagocytosis, have not been shown. Here, we describe these kinetics in the murine MH-S AM cell-line, a well-established model useful for investigations of AM phenotype and function. We measure binding and internalizing rates of S. pneumoniae following exposure to increasing durations of physiologic levels of IFN-γ. When MH-S murine alveolar macrophage (mAM) were exposed to IFN-γ for increasing durations of time, from 0 to 6 days before inoculation with the type II S. pneumoniae, D39, exposure for 6 h transiently reduced bacterial binding by 50%, which was temporarily restored at 2 and 3 days of exposure. Bacterial internalization was also reduced shortly following initial exposure, however, internalization continued to fall to less than 5% that of IFN-γ naïve controls after 6 days of exposure. These data may help explain otherwise contradictory reports from the literature regarding timing between infections and reductions in macrophage function.

FIG. 1.

Nonlinear effects of IFN-γ exposure on bacterial binding. MH-S cells were seeded and grown in media (changed daily) for 7 days, with varying durations of exposure to IFN-γ (25 ng/mL/day) lasting between 0 and 6 days before inoculation with the type II Streptococcus pneumoniae D39. Bacterial binding was measured as the difference between total bacterial colony forming units (CFUs) internalized and bound (bacteria released from MH-S cells with saponin treatment only) versus bacterial CFUs internalized (gentamicin protection assay to kill extracellular-bound cells followed by saponin treatment to release internalized bacteria). Bacterial CFUs reflect bacteria bound per macrophage (ie, bound CFU/viable MH-S cells). Binding in each condition are then reported as a proportion of the mean CFUs bound to IFN-γ unexposed control cells. Each bar represents pooled results from 3 independent experiments and each experiment examining 3 distinct wells for each condition for a total of 9 data points per condition (depicted as gray filled circles). Boxes represent the interquartile range (IQR) and whiskers extend to 1.5× IQR on either side. Horizontal lines represent the median and the connecting line connects the means. Asterisks represent P<0.05 based on a 2-tailed Dunnett's test, relative to IFN-γ naïve controls.

FIG. 2.

Bacterial internalization as a function of exposure to IFN-γ. MH-S cells were seeded and grown in media (changed daily) for 7 days, with varying durations of exposure to IFN-γ (25 ng/mL/day) lasting between 0 and 6 days before inoculation with the type II Streptococcus pneumoniae D39. Bacterial internalization was measured as the number of internal bacterial CFUs released from MH-S cells after treatment with gentamicin and saponin. Bacterial internalization was quantified as bacteria internalized per macrophage and are reported here as proportions of the mean CFUs internalized in IFN-γ unexposed cells. Figure layout is as described in Figure 1 except that asterisks represent P values <0.01.

FIG. 3.

Changes in the phagocytic index (PI) with extended IFN-γ exposure. MH-S cells were seeded and grown in media (changed daily) for 7 days, with varying durations of exposure to IFN-γ (25 ng/mL/day) lasting between 0 and 6 days before inoculation with a green-fluorescence protein (GFP) expressing D39 pneumococcus [multiplicity of infection (MOI) of 15]. Fifty minutes following bacterial inoculation, cells were gently washed thrice with 1 mL ice-cold phosphate-buffered saline (PBS) to remove unbound bacteria immediately before fixing with 4% paraformaldehyde. The PI was calculated as the fraction of GFP-positive MH-S cells per field (eg, % MH-S cells associated with at least 1 bacterium) multiplied by the mean fluorescence intensity (MFI; representing both bound and internalized bacteria) per cell. Data are representative of 2 independent experiments, and each experimental result calculated by enumerating at least 5 randomly selected fields from each of 3 replicate wells. Data were normalized for each experiment to the PI of the control cells and plotted (filled gray dots). Boxes represent the IQR and whiskers extend to 1.5× IQR on either side. Horizontal lines represent the medians and the connecting line connects the means. Asterisks represent P<0.00; two-tailed single sample t-test with Bonferroni correction for multiple comparisons.