Staphylococcus aureus Biofilm-Conditioned Medium Impairs Macrophage-Mediated Antibiofilm Immune Response by Upregulating KLF2 Expression

Abstract

Staphylococcus aureus infections associated with the formation of biofilms on medical implants or host tissue play a critical role in the persistence of chronic infections. One critical mechanism of biofilm infection that leads to persistent infection lies in the capacity of biofilms to evade the macrophage-mediated innate immune response. It is now increasingly apparent that microorganisms exploit the negative regulatory mechanisms of the pattern recognition receptor (PRR)-mediated inflammatory response to subvert host cell functions by using various virulence factors. However, the detailed molecular mechanism, along with the identity of a target molecule, underlying the evasion of the macrophage-mediated innate immune response against S. aureus infection associated with biofilm formation remains to be elucidated. Here, using an in vitro culture model of murine macrophage-like RAW 264.7 cells, we demonstrate that S. aureus biofilm-conditioned medium significantly attenuated the capacity for macrophage bactericidal and proinflammatory responses. Importantly, the responses were associated with attenuated activation of NF-κB and increased expression of Kruppel-like factor 2 (KLF2) in RAW 264.7 cells. Small interfering RNA (siRNA)-mediated silencing of KLF2 in RAW 264.7 cells could restore the activation of NF-κB toward the bactericidal activity and generation of proinflammatory cytokines in the presence of S. aureus biofilm-conditioned medium. Collectively, our results suggest that factors secreted from S. aureus biofilms might exploit the KLF2-dependent negative regulatory mechanism to subvert macrophage-mediated innate immune defense against S. aureus biofilms.

Keywords: KLF2; S. aureus biofilm; macrophages.

FIG 1

Effects of S. aureus biofilm-conditioned medium on the phagocytic and bactericidal capacities of RAW 264.7 macrophages. (A) Effect of S. aureus biofilm on the phagocytic activity of macrophages. RAW 264.7 cells were treated with conditioned medium from either a planktonic culture (PLK-CM) or a biofilm culture (BIO-CM) of S. aureus for 2 h. Next, the extent of bacterial phagocytosis by RAW 264.7 cells was assessed by measuring the fluorescence intensity from the phagocytized bacteria (opsonized Alexa Fluor 488-conjugated E. coli). (Left) Quantification of phagocytosis (n = 8). (Right) Representative fluorescence images of RAW 264.7 cells. (B) Effect of S. aureus biofilm on the bactericidal activity of macrophages. RAW 264.7 cells were exposed to PLK-CM or BIO-CM for 2 h and then incubated with live S. aureus bacteria for 1 h, followed by an antibiotic protection assay to count the number of intracellular S. aureus bacteria that survived within macrophages (n = 5 per group). (C) Effect of S. aureus biofilm on secretion of nitric oxide from RAW 264.7 macrophages. RAW 264.7 cells were treated with normal culture medium (control [CTR]), PLK-CM, or BIO-CM for 8 h, and nitric oxide concentrations in the supernatant of the cell culture were measured. (D) qPCR analysis of iNOS mRNA from RAW 264.7 cells 2 and 8 h following treatment with PLK-CM or BIO-CM (n = 8). *, P < 0.05.

FIG 2

Effects of S. aureus biofilm-conditioned medium on activation of NF-κB and expression of proinflammatory cytokines in RAW 264.7 macrophages. (A) Transcriptional activation of NF-κB quantified by NF-κB luciferase expression in RAW 264.7 cells transfected with the NF-κB luciferase reporter following treatment with normal culture medium (CTR), PLK-CM, or BIO-CM for 8 h (n = 4 per group). RLU, relative luciferase units. (B) qPCR analysis of mRNA expression of the proinflammatory cytokines IL-1β and IL-6 from RAW 264.7 cells following treatment with PLK-CM or BIO-CM for 2 or 8 h (n = 6 to 8). *, P < 0.05.

FIG 3

Effects of S. aureus biofilm-conditioned medium on PRR-mediated expression of iNOS mRNA in RAW 264.7 macrophages. RAW 264.7 cells were treated with normal culture medium (Control) or BIO-CM in the presence or absence of the PRR agonists Pam2CSK4 (Pam2) (agonist for TLR2), CpG (agonist for TLR9), and MDP (agonist for NOD2). Treatment was conducted for 8 h, and the expression of iNOS mRNA was quantified by qPCR analysis (n = 4). *, P < 0.05; **, P < 0.01.

FIG 4

Effects of S. aureus biofilm-conditioned medium on expression of KLF2 in RAW 264.7 macrophages. (A) qPCR analysis of expression of KLF2 mRNA from RAW 264.7 cells treated with PLK-CM or BIO-CM for 2 or 8 h (n = 6 per group). *, P < 0.05. (B) Representative Western blot images of expression of the KLF2 protein from RAW 264.7 cells treated with PLK-CM or BIO-CM for 2 h. β-Actin expression was used as an internal control. (C) Fluorescence images of RAW 264.7 cells expressing KLF2 protein. Cells were treated with PLK-CM or BIO-CM for 30 min and then immunostained with anti-KLF2 antibody (green). Bar = 40 μm.

FIG 5

Effects of siRNA-mediated knockdown of the KLF2 gene on bactericidal activity and nitric oxide secretion in RAW 264.7 macrophages. siRNA-mediated knockdown of the KLF2 gene was induced in RAW 264.7 cells, and cells were exposed to normal culture medium (CTR-CM), PLK-CM, or BIO-CM. (A) Bactericidal activity of RAW 264.7 cells with siRNA knockdown of KLF2 (KLF2 siRNA) or scrambled control siRNA (CTR siRNA) following treatment with CTR-CM, PLK-CM, or BIO-CM for 2 h (n = 4 to 5 per group). (B) Nitric oxide concentrations measured from the supernatant of RAW 264.7 cells transfected with CTR siRNA or KLF2 siRNA following treatment with PLK-CM or BIO-CM for 8 h (n = 5). (C) qPCR analysis of iNOS mRNA from RAW 264.7 cells transfected with CTR siRNA or KLF2 siRNA following treatment with PLK-CM or BIO-CM for 2 or 8 h (n = 8). *, P < 0.05.

FIG 6

Effects of siRNA-mediated knockdown of the KLF2 gene on activation of NF-κB and expression of proinflammatory cytokines in RAW 264.7 macrophages. (A) Transcriptional activation of NF-κB quantified by NF-κB luciferase expression in RAW 264.7 cells transfected with the NF-κB luciferase reporter and KLF2 siRNA (or scrambled siRNA [CTR siRNA]), following treatment with PLK-CM or BIO-CM for 8 h (n = 4 to 6 per group). (B) Effect of siRNA-mediated knockdown of the KLF2 gene on expression of proinflammatory cytokines in RAW 264.7 cells. siRNA-mediated knockdown of the KLF2 gene was induced in RAW 264.7 cells, and the cells were treated with BIO-CM for 2 or 8 h. The expression levels of IL-1β and IL-6 mRNAs were then quantified by qPCR analysis. Results were compared with those for RAW 264.7 cells transfected with scrambled siRNA (CTR siRNA) (n = 6 to 8 per group). *, P < 0.05.