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Clinical Trial
. 2014 May 15;192(10):4709-17.
doi: 10.4049/jimmunol.1302692. Epub 2014 Apr 11.

Phagocytosis of Staphylococcus aureus by human neutrophils prevents macrophage efferocytosis and induces programmed necrosis

Mallary C Greenlee-Wacker  1 , Kevin M Rigby, Scott D Kobayashi, Adeline R Porter, Frank R DeLeo, William M Nauseef
Affiliations
Clinical Trial

Phagocytosis of Staphylococcus aureus by human neutrophils prevents macrophage efferocytosis and induces programmed necrosis

Mallary C Greenlee-Wacker et al. J Immunol. .

Abstract

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) pose a significant threat to human health. Polymorphonuclear leukocytes (PMN) are the first responders during staphylococcal infection, but 15-50% of the initial ingested inoculum survives within the PMN phagosome and likely contributes directly or indirectly to disease pathogenesis. We hypothesize that surviving intracellular CA-MRSA undermine effective phagocyte-mediated defense by causing a decrease in macrophage uptake of PMN containing viable S. aureus and by promoting PMN lysis. In support of this hypothesis, PMN harboring viable CA-MRSA strain USA300 (PMN-SA) upregulated the "don't eat me" signal CD47, remained bound to the surface, and were inefficiently ingested by macrophages. In addition, coculture with PMN-SA altered the macrophage phenotype. Compared to macrophages fed USA300 alone, macrophages challenged with PMN-SA produced more IL-8 and less IL-1 receptor antagonist, TNF-α, activated caspase-1, and IL-1β. Although they exhibited some features of apoptosis within 3 h following ingestion of S. aureus, including phosphatidylserine exposure and mitochondrial membrane depolarization, PMN-SA had sustained levels of proliferating cell nuclear Ag expression, absence of caspase activation, and underwent lysis within 6 h following phagocytosis. PMN lysis was dependent on receptor-interacting protein 1, suggesting that PMN-SA underwent programmed necrosis or necroptosis. These data are the first demonstration, to our knowledge, that bacteria can promote sustained expression of proliferating cell nuclear Ag and that human PMN undergo necroptosis. Together, these findings demonstrate that S. aureus surviving within PMN undermine the innate immune response and may provide insight into the pathogenesis of S. aureus disease.

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Figures

Figure 1
Figure 1. PMN challenged with S. aureus upregulate “don’t eat me” signal CD47
PMN were in buffer alone or fed USA300 or RN6390 at an MOI of 1:1 and monitored for 60-180 minutes. Cells were stained for annexin V-FITC and propidium iodide (PI) to distinguish apoptotic and necrotic cells, respectively (A). Shown on the left axis and connected by a solid line are annexinV+ cells, and on the right axis and connected by a dashed line are annexinV+PI+ cells. Symbols represent the mean of at least five experiments +/− SEM (A). PMN were stained after 60 minutes with JC-1 dye to measure mitochondrial depolarization and analyzed by flow cytometry. P-values were determined using a repeated measures one-way analysis of variance (ANOVA) and Dunnett’s posttest (B, n = 5 +/− SEM, *P<0.05). PMN were isolated and either aged for 18-24 hours (Aged) or challenged with USA300 at an MOI of 1:1 for 60 minutes. Cells were stained for CD47 and analyzed by flow cytometry. Shown is a representative of three experiments (C), and average MFI from the five individual experiments ± SEM (D). P-values were determined using one-way analysis of variance (ANOVA) and Tukey’s posttest. *P<0.05 vs. PMN and #P<0.05 vs. Aged PMN.
Figure 2
Figure 2. Interactions between PMN containing viable S. aureus and human macrophages
Aged or freshly isolated PMN were stained with CTFR and freshly isolated PMN were either left in buffer or fed sGFP-expressing USA300 at an MOI of 1:1 for 60 minutes (PMN-SA). Cell suspensions (Aged PMN, fresh PMN, PMN-SA) were then fed to human monocyte-derived macrophages (HMDMs) and monitored for 15, 30, and 60 minutes. Cells were stained for CD14 and CD15 and analyzed by flow cytometry. % surface bound (A), % internalized (B), and % of SA associated with CD14+ macrophages and CTFR+ PMN (C) were calculated as described in Materials and Methods. Symbols represent the mean of four experiments ± SEM. P-values were determined using a repeated measures one-way ANOVA and Tukey’s posttest for each time point (*P<0.05 PMN-SA vs PMN and Aged PMN and #P <0.05 Aged PMN vs PMN and PMN-SA).
Figure 3
Figure 3. Macrophage cytokine production is skewed by PMN-SA
HMDMs were left alone (Mac), fed USA300 (SA) at an MOI of 1:1, or fed PMN-SA at a ratio of 5 PMN-SA per macrophage or 15 PMN-SA per macrophage. Following a 6-hour incubation, supernatants were analyzed by Luminex or ELISA. Bars represent the mean of three individual experiments ± SEM. Statistical analyses were performed using a oneway ANOVA and Tukey’s posttest. *P<0.05 vs macrophages and #P<0.05 vs macrophages + SA.
Figure 4
Figure 4. Inflammasome activation is dampened by PMN-SA
HMDMs were treated with buffer or primed with LPS for 2 hours prior to treatment buffer alone, silica, USA300, or PMN-SA. Supernatants were analyzed for IL-1β production by Luminex for following a 6- or 12-hour incubation (A), or by ELISA following a 6-hour incubation (B). Caspase-1 cleavage was analyzed by immunoblotting following a 6-hour incubation (C). Bars represent the mean of five experiments ± SEM (B) or three experiments (C). For A P-values were determined using a one-way ANOVA and Turkey’s posttest, *P<0.05 vs macrophages and ^P<0.05 vs macrophages + SA. For B, an outlier experiment was identified using Prism software and was excluded from the analysis. P-values were then determined using a repeated measures one-way ANOVA, *P<0.05 vs macrophages + LPS, #P<0.05 vs macrophages + LPS+ Silica and ^P<0.05 vs macrophages + SA.
Figure 5
Figure 5. Analysis of PCNA and caspase-3 in human PMNs following phagocytosis
Lysates from PMN that were in buffer alone, challenged with USA300 at an MOI of 1:1, or treated with anti-Fas antibody as indicated and were analyzed by immunoblot for PCNA, caspase-3, and actin. Shown is a representative of three experiments (A). To quantify band intensity, immunoblots were scanned and analyzed using a phosphorimager. Data were plotted as the signal above background vs. Time 0 for the indicated bands ± SEM (B, n = 4).
Figure 6
Figure 6. Caspase activity in human PMNs following phagocytosis
(A-D) Human neutrophils were cultured with IgG and serum complement-coated latex beads (Beads, filled squares, 10 beads per PMN), USA300 (red circles, MOI of 10:1), or anti-FAS antibody (αFAS, filled triangles) as indicated. At each time point, samples were clarified by centrifugation and caspase activity in culture supernatants and stimulated PMNs was determined with an ApoAlert Caspase Assay Plate kit according to the manufacturer’s instructions. Data for PMNs are the mean ± standard error of 2-3 experiments for all assays except the 2 h time point, in which case there is a single data point. The level of caspase activity for FAS-stimulated PMNs at 6 h is set as 100% (positive control) and all other data are expressed relative to the αFAS data points at 6 h. Statistical analyses were performed using a one-way analysis of variance (ANOVA) and Tukey’s posttest. *P<0.05 for αFAS vs. USA300; #P<0.05 for Beads vs. USA300; ^P<0.05 for αFAS vs. Beads.
Figure 7
Figure 7. Necrostatin-1-dependent inhibition of PMN lysis
Human neutrophils were cultured with USA300 at an MOI of 10:1 in the presence of necrostatin-1, or vehicle (0 μM Nec-1) or without bacteria (DMSO) and cell lysis was determined by release of LDH. Data for PMNs are the mean ± standard error of 4-8 experiments. Statistical analyses were performed using a one-way analysis of variance (ANOVA) and Dunnett’s posttest. *P<0.05 for PMNs + USA300 treated with necrostatin-1 vs. vehicle control (0 μM). ns, not significant.
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