Pro- and anti-inflammatory responses of peripheral blood mononuclear cells induced by Staphylococcus aureus and Pseudomonas aeruginosa phages - PubMed (original) (raw)
Pro- and anti-inflammatory responses of peripheral blood mononuclear cells induced by Staphylococcus aureus and Pseudomonas aeruginosa phages
Jonas D Van Belleghem et al. Sci Rep. 2017.
Abstract
The ability of bacteriophages to kill bacteria is well known, as is their potential use as alternatives to antibiotics. As such, bacteriophages reach high doses locally through infection of their bacterial host in the human body. In this study we assessed the gene expression profile of peripheral blood monocytes from six donors for twelve immunity-related genes (i.e. CD14, CXCL1, CXCL5, IL1A, IL1B, IL1RN, IL6, IL10, LYZ, SOCS3, TGFBI and TNFA) induced by Staphylococcus aureus phage ISP and four Pseudomonas aeruginosa phages (i.e. PNM, LUZ19, 14-1 and GE-vB_Pae-Kakheti25). The phages were able to induce clear and reproducible immune responses. Moreover, the overall immune response was very comparable for all five phages: down-regulation of LYZ and TGFBI, and up-regulation of CXCL1, CXCL5, IL1A, IL1B, IL1RN, IL6, SOCS3 and TNFA. The observed immune response was shown to be endotoxin-independent and predominantly anti-inflammatory. Addition of endotoxins to the highly purified phages did not cause an immune response comparable to the one induced by the (endotoxin containing) phage lysate. In addition, the use of an intermediate level of endotoxins tipped the immune response to a more anti-inflammatory response, i.e. up-regulation of IL1RN and a strongly reduced expression of CXCL1 and CXCL5.
Conflict of interest statement
The authors declare that they have no competing interests.
Figures
Figure 1
Gene expression analysis of 12 immunity-related genes, assessed by means of RT-qPCR, after 20 h stimulation of PBMCs with S. aureus strain ATCC 6538 or S. aureus phage ISP. Comparison of the immune response of the PBMCs, expressed as log2 fold change, after normalization with reference gene ACTB, as induced by the phage lysate, highly purified phage (undiluted and diluted hundredfold) or its bacterial host (i.e. S. aureus strain ATCC 6538). The immune response induced by the highly purified phage preparation is stronger than the one induced by the phage lysate. Diluting of the highly purified phage led to an almost complete reduction of the observed immune response, with the exception of CXCL5 and IL1B. A strong difference is present between the immune response induced by the phage compared to its bacterial host (i.e. S. aureus strain 6538).
Figure 2
Gene expression analysis of 12 immunity-related genes, assessed by means of RT-qPCR, after 20 h of stimulation of PBMCs with S. aureus phage ISP in combination with endotoxins (i.e. 0.1, 10−3 or 10−7 EU/ml). (A,B,C) 103 pfu/PBMC in combination with (A) 0.1 EU/PBMC, (B) 10−3 EU/PBMC or (C) 10−7 EU/PBMC leads to an immune response more similar to the one induced by the phage alone. (D,E,F) Reducing the phage titer to 10 pfu/PBMC causes the immune response to tilt in the direction of the endotoxin induced immune response instead of the phage induced immune response.
Figure 3
Effect of endotoxins in the presence P. aeruginosa phage PNM. (A) Gene expression analysis of 12 immunity-related genes by means of RT-qPCR after 20 h of stimulation of PBMCs. PBMCs were stimulated with P. aeruginosa phage PNM, either a phage lysate (105 pfu/PBMC; 0.1 EU/PBMC) or a highly purified phage preparation (105 pfu/PBMC; 10−5 EU/PBMC) in combination with 0.1 EU/PBMC. The pro-inflammatory markers IL1A, IL1B, CXCL1 and CXCL5, and the anti-inflammatory markers SOCS3, IL10, IL1RN and IL6 are upregulated The addition of 0.1 EU/PBMC to the highly purified P. aeruginosa phage preparation does not revert the observed immune response to that of the phage lysate for IL10, IL1A, IL6, TNFA and CXCL1. (B) Principal components analysis of P. aeruginosa phage PNM with or without the addition of endotoxins. The immune response induced by the highly purified phage PNM (
) differs from the one induced by the phage PNM lysate (
), as these two groups are visibly separated. When endotoxins are added to a final concentration of 0.1 EU/PBMC to the highly purified phage (
), the immune response is similar to the highly purified phage (
) and not towards the phage lysate (
), indicating that the observed difference is not due to the presence of LPS but due to bacterial proteins present in the phage lysate.
Figure 4
Gene expression analysis of 12 immunity-related genes by means of RT-qPCR after 20 h stimulation of PBMCs. PBMCs were stimulated with either (A) P. aeruginosa phage PNM, (B) P. aeruginosa phage LUZ19, (C) P. aeruginosa phage 14-1 or (D) P. aeruginosa phage GE_vB_Pae-Kakheti25. The pro-inflammatory markers IL1A, IL1B, CXCL1 and CXCL5 and the anti-inflammatory markers SOCS3, IL10, IL1RN and IL6 are up-regulated. Only phages PNM (A) and 14-1 (C) slightly up-regulate TNFA, whereas phages LUZ19 (B) and GE_vB_Pae-Kakheti25 (D) cause a slight down-regulation of TNFA.
Figure 5
Principal component analysis (PCA). Comparison of the immune response of PBMCs induced by all five tested phages (Highly purified or phage lysate; magenta) and their bacterial host strain (blue). The PCA clearly shows that the bacteria (blue) and phages (magenta) for two separate groups, indicating that the immune response induced is different between these two types of stimuli. There is no clear distinction between the different phages, indicating that these five different phages induced similar responses. Moreover the gene expression of IL1RN, IL1B and SOCS3, IL6 and IL1A, TNFA and IL10, and LYZ and TGFBI are correlated.
Figure 6
Hypothetical view of the interaction of bacteriophages with mammalian immune cells. Phages are able to interact with (currently unknown) immune receptors and induce corresponding immune responses. The immune responses induced by the bacteriophages can either be pro- of anti-inflammatory. For instance, the tested phages are able to induce the pro-inflammatory cytokines IL1α and IL1β. Through the induction of IL1RN by the phage, the phage is able to inhibit the pro-inflammatory responses that are otherwise induced by these cytokines. Pro-inflammatory cytokines are marked in red (i.e. TNF-α, IFN-γ,IL1α, IL1β, IL-6, IL-17, CXCL1 and CXCL5), anti-inflammatory cytokines are depicted in green (i.e. TGF-β, IL-4, IL-10 and IL1RN). Dark orange depicts proteins that play a key role in the removal or perception of bacterial pathogens (i.e. CD14 and LYZ).
References
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