Bacteriophages immunomodulate the response of monocytes

Abstract

Bacteriophages are present in fluids from cirrhosis patients. However, their effect on the immune response is unknown. In this work, we explore the role of phages in the phenotype, function, and cytokine production of monocytes. We stimulated healthy monocytes with five different butanol-purified phage suspensions infective for Gram-negative and Gram-positive bacteria. We studied the expression of the monocyte markers involved in lipopolysaccharide recognition (LPS; CD14), antigen presentation (HLA-DR) and co-stimulation (CD86), and the concentration of induced cytokines (TNF-α, IFN-α, and IL-10) by phages. To confirm the direct role of phages without the interference of contaminating soluble LPS in phage suspensions, polymyxin B was added to the cell cultures. Phagocytosis experiments were assessed by flow cytometry using labeled phage suspensions. We observed that butanol-purified phages reduced the surface levels of CD14 and CD86 in monocytes and increased the secreted levels of TNF-α and IL-10 compared with the control sample containing only butanol buffer. All phage suspensions showed downregulation of HLA-DR expression but only Staphylococcus aureus phage contaminated with Escherichia coli reached statistical significance. The addition of polymyxin B did not restore the monocytic response induced by phages, suggesting that the effect was not caused by the presence of LPS. Monocytes were able to phagocyte phages in a dose- and time-dependent manner. To conclude, the phagocytosis of butanol-purified phages altered the phenotype and cytokine production of monocytes suggesting they become tolerogenic.

Keywords: Bacteriophages; cirrhosis; immunity.

Figure 1.

Changes in monocyte markers expression induced by butanol-purified Gram-negative and Gram-positive phages. (a) Percentage of CD14⁺, (b) CD14⁺HLA-DR⁺ , and (c) CD14⁺CD86⁺ cells from PBMCs stimulated with 1/100 butanol buffer and 1/100 butanol-purified phages without adding polymixin B (polyB; solid bar) or after adding polyB (stripe pattern). (d) Representative flow cytometry image of CD14⁺CD86⁺ cells from PBMCs stimulated with 1/100 butanol buffer or 1/100 SOM1 phage with or without polyB. *<0.05; **<0.01; ***<0.001; *Buffer vs Phage; ^#Buffer with polyB vs. Phage with polyB; ^$Phage vs. Phage with polyB. (A color version of this figure is available in the online journal.)

Figure 2.

Inflammation induced by butanol-purified Gram-negative and Gram-positive phages. (a) TNF-α and (b) IL-10 levels measured by ELISA assays in the supernatants of PBMCs stimulated with 1/100 butanol-purified phages without (solid bar) or after adding polyB (stripe pattern). *<0.05; **<0.01; ***<0.001; *Buffer vs Phage; ^#Buffer with polyB vs. Phage with polyB; ^$Phage vs. Phage with polyB. (A color version of this figure is available in the online journal.)

Figure 3.

Phagocytosis of Gram-negative and Gram-positive phages by monocytes. (a) Kinetic assay of phagocytosis: Percentage of phagocytic monocytes cultured with 1/5, 1/10, and 1/20 of SYBR-Gold-labeled Gram-negative (SOM4) and Gram-positive (ɸ11) phage for 20 and 40 min at 37°C. Phagocytosis was determined using flow cytometry and was expressed as the percentage of phagocytic monocytes stained with anti-CD14. ^*<0.05, Phagocytosis 1/5 vs. 1/10 or 1/20. (b) Temperature assay to determine the non-specific phagocytosis of phages: monocytes were cultured with 1/5, 1/10, and 1/20 of SYBR-Gold-labeled Gram-negative (SOM4) and Gram-positive (ɸ11) phage for 40 min at 4°C to inhibit the mechanism of phagocytosis. ^*<0.05, ^**<0.01, Phagocytosis at 37°C vs. phagocytosis at 4°C. (A color version of this figure is available in the online journal.)