Of Men Not Mice: Bactericidal/Permeability-Increasing Protein Expressed in Human Macrophages Acts as a Phagocytic Receptor and Modulates Entry and Replication of Gram-Negative Bacteria

Abstract

Macrophages as immune cells prevent the spreading of pathogens by means of active phagocytosis and killing. We report here the presence of an antimicrobial protein, bactericidal/permeability-increasing protein (BPI) in human macrophages, which actively participates in engulfment and killing of Gram-negative pathogens. Our studies revealed increased expression of BPI in human macrophages during bacterial infection and upon stimulation with various pathogen-associated molecular patterns, viz., LPS and flagellin. Furthermore, during the course of an infection, BPI interacted with Gram-negative bacteria, resulting in enhanced phagocytosis and subsequent control of the bacterial replication. However, it was observed that bacteria which can maintain an active replicating niche (Salmonella Typhimurium) avoid the interaction with BPI during later stages of infection. On the other hand, Salmonella mutants, which cannot maintain a replicating niche, as well as Shigella flexneri, which quit the endosomal vesicle, showed interaction with BPI. These results propose an active role of BPI in Gram-negative bacterial clearance by human macrophages.

Keywords: Gram-negative bacteria; antimicrobial protein; bacterial niche; innate immunity; macrophage evolution; phagocytic receptor.

Figure 1

Bacterial permeability-increasing protein is expressed in human monocyte/macrophages. Human or mouse monocytes/macrophages were exposed to indicated experimental conditions. Total RNA was extracted from (A) human and mouse monocyte and macrophage cell lines, (B) human peripheral blood monocytes (hPBMCs) and BPI expression were analyzed by semiquantitative RT-PCR. PCR products were purified and sent for sequencing for further confirmation. (C) Human PBMCs were PFA fixed and stained for BPI (red), CD11b (green), and nuclei (blue) and visualized by confocal microscopy.

Figure 2

Regulation of BPI expression in human monocytes/macrophages. U937 monocytes/macrophages were exposed to indicated inflammatory stimuli/pathogen. (A,B) Total RNA was isolated and BPI expression was investigated by real-time PCR [n = 5 (SD)]. (C) BPI expression was checked by flow cytometry [n = 3 (SD)]. (D) Total protein was isolated from U937 macrophage, and BPI levels were analyzed by western blot (n = 4). Bottom: quantitative evaluation of the relative BPI expression normalized to β-actin intensity. (E) Cells were PFA fixed and stained for BPI (red) and nuclei (blue). Cells were imaged by confocal microscopy (n = 6). Key: ***p < 0.001, **p < 0.005, *p < 0.05; ns, not significant.

Figure 3

Human macrophages expressed BPI and kills intracellular bacteria. (A) Fold proliferation and percentage survival of bacteria after knocking down of BPI in U937 macrophages. Statistical significance was calculated with respect to untransfected control [n = 5 (SD)]. (B) Fold proliferation of STM after knocking down of BPI in human PBMCs-derived macrophages. Statistical significance was calculated with respect to scrambled dsRNA-transfected control [n = 6 (SD)]. (C) Total protein was isolated from BPI dsRNA-transfected and untransfected control U937 macrophages, and BPI levels were checked by western blot (n = 3). (D) RAW 264.7 macrophages were transfected with either pcDNA empty vector (pcDNA EV) or pcDNA carrying the expression sequence of human BPI (pcDNA hBPI). Twenty-four hours post-transfection, the cells were infected with STM and bacterial proliferation was quantified [n = 4 (SD)]. (E) Total protein was isolated from pcDNA EV-transfected and pcDNA hBPI-transfected RAW 264.7 macrophages, and BPI levels were quantified by western blot (n = 3). Key: ***p < 0.001, **p < 0.005, *p < 0.05; ns, not significant.

Figure 4

BPI enhances bacterial uptake in human macrophages. (A) U937 macrophages were infected with STM–GFP (green) at an MOI of 10. Thirty minutes post-infection, cells were fixed with paraformaldehyde and stained for BPI (red) and nuclei (blue) (n = 4 experiments). (B,D) Percentage phagocytosis of bacteria after knocking down of BPI in U937 macrophages. U937 macrophages were infected with the indicated bacteria at an MOI of 10. Bacterial entry was quantified by plating cell lysates after 30 min post-infection [n = 6 (SD)]. For experiments with Salmonella Typhi (STY), percentage phagocytosis of BPI dsRNA transfected cells was compared to untransfected control and was normalized to STY (WT). (C) Percentage phagocytosis of STM in hPBMC-derived macrophages. Statistical significance was calculated with respect to scrambled dsRNA-transfected control [n = 6 (SD)]. Key: ***p < 0.001, **p < 0.005, *p < 0.05; ns, not significant.

Figure 5

Time-course analysis of BPI interaction with Salmonella Typhimurium in U937 macrophages during the course of infection. (A) U937 macrophages were infected with STM–GFP (green) at an MOI of 50. Cells were fixed with PFA at the indicated time points and stained for BPI (red) and nuclei (blue) (n = 3). Panel shows representative bright field and merged images for each time point. (B) Quantification of colocalization of BPI with STM–GFP [n = 3 (SD)]. (C) U937 macrophages were infected with STM–GFP at an MOI of 10. Bacterial entry was quantified by plating the cell lysates after 30 min post-infection. Bacterial replication was quantified by plating the cell lysates at indicated time points and was normalized to the CFU at 1 h post-infection. Fold proliferation and percentage survival were calculated [n = 3 (SD)]. Key: ***p < 0.001, **p < 0.005, *p < 0.05; ns, not significant.

Figure 6

BPI interaction with Gram-negative bacteria inside macrophages. (A) U937 macrophages were infected with GFP-tagged (green) Gram-negative bacteria [STM14028, PFA fixed STM, E. coli DH5α, and Shigella flexneri (SHG)] at an MOI of 50. Two hours post-infection, cells were fixed with PFA and stained for BPI (red) and nuclei (blue). White arrows indicate GFP-positive bacteria. The boxed area in the SHG infected set is magnified to view BPI around the bacteria (n = 3). (B) Quantification of colocalization of BPI with the bacteria. (C) Quantification of MFI of BPI at ROI was done by the Zen Blue edition software provided by Zeiss [n = 3 (SD)]. Key: ***p < 0.001, **p < 0.005, *p < 0.05.

Figure 6

BPI interaction with Gram-negative bacteria inside macrophages. (A) U937 macrophages were infected with GFP-tagged (green) Gram-negative bacteria [STM14028, PFA fixed STM, E. coli DH5α, and Shigella flexneri (SHG)] at an MOI of 50. Two hours post-infection, cells were fixed with PFA and stained for BPI (red) and nuclei (blue). White arrows indicate GFP-positive bacteria. The boxed area in the SHG infected set is magnified to view BPI around the bacteria (n = 3). (B) Quantification of colocalization of BPI with the bacteria. (C) Quantification of MFI of BPI at ROI was done by the Zen Blue edition software provided by Zeiss [n = 3 (SD)]. Key: ***p < 0.001, **p < 0.005, *p < 0.05.

Figure 7

STM avoids interaction with BPI inside macrophages by maintaining an actively replicating niche (SCV). U937 macrophages were infected with (A) STM, (B) STM ΔsifA, (F) STM EV (empty vector), or (G) STM LLO at an MOI of 50. Two hours post-infection, the cells were fixed with PFA at the indicated time points and stained for BPI (red) and LAMP2 (green). Nuclei and bacteria were labeled with 4′,6-diamidino-2-phenylindole (DAPI) (blue). White arrows indicate DAPI-positive bacteria. The boxed area in each set is magnified to view BPI around the bacteria. (C,H) Quantification of colocalization of BPI with bacteria. (D,I) Quantification of MFI of BPI at ROI. (E) Quantification of colocalization of LAMP2 with bacteria at ROI. All images were quantified by using the Zen Blue edition software provided by Zeiss [n = 3 (SD)]. Key: ***p < 0.001, **p < 0.005.

Figure 7

STM avoids interaction with BPI inside macrophages by maintaining an actively replicating niche (SCV). U937 macrophages were infected with (A) STM, (B) STM ΔsifA, (F) STM EV (empty vector), or (G) STM LLO at an MOI of 50. Two hours post-infection, the cells were fixed with PFA at the indicated time points and stained for BPI (red) and LAMP2 (green). Nuclei and bacteria were labeled with 4′,6-diamidino-2-phenylindole (DAPI) (blue). White arrows indicate DAPI-positive bacteria. The boxed area in each set is magnified to view BPI around the bacteria. (C,H) Quantification of colocalization of BPI with bacteria. (D,I) Quantification of MFI of BPI at ROI. (E) Quantification of colocalization of LAMP2 with bacteria at ROI. All images were quantified by using the Zen Blue edition software provided by Zeiss [n = 3 (SD)]. Key: ***p < 0.001, **p < 0.005.