Fig 1.
Surgical operation and S. aureus biofim formation in a rat PJI model.
(a) Rats were divided into three groups including the sham group, the S. aureus-infected group and the S. aureus-infected group receiving vancomycin treatment. All rats were sacrificed 7 days after surgery and the treated femurs were taken. Notably, severe bone osteolytic lesion with pus formation was observed in the S aureus-infected femur, but less osteolytic lesion was detected in the vancomycin-treated infection femur. (b) Scanning electron microscopy images of biofilm formation in femurs at day 7 post-infection. Biofilm formation and S. aureus cocci were more evident in the femur lumen of the S. aureus-infected group than those of the vanomycin-treated infection group (original magnification ×10,000).
Fig 2.
Expansion of MDSCs, total macrophages, and M2-macrophages during S. aureus infection in rats.
The sham group, the S. aureus-infected group, and the S. aureus-infected group receiving vancomycin were included in the experiments and each group contained 4 rats. Blood samples were collected at different time points after operation. After lysis of the red blood cells, the remaining leukocytes were analyzed for the proportions of CD11bc+His48+ MDSCs (a), CD68+ macrophages (b), and CD68+CD206+ M2-macrophages (c) by flow cytometry. Symbol # indicates significant difference vs. day 0 (p < 0.01) and symbol * indicates significant difference vs. the shame group (p < 0.01).
Fig 3.
S. aureus biofilm triggers expansion of different immune cell types from mouse bone marrow cells in vitro.
After coculture of mouse bone marrow cells (BMCs) with increasing concentrations (0.2, 0.6 and 2.0 mg/ml) of S. aureus biofilm for 48 hr, the proportions of CD11b+Gr1+ MDSCs (a and b), MDSC subsets including M-MDSCs (CD11b+Ly6ChighLy6G-) and G-MDSCs (CD11b+Ly6ClowLy6G+) (c and d), F4/80+ macrophages (e), as well as F4/80+CD206+ M2-macrophages (f) were evaluated by flow cytometry (N = 3). As noted, to examine the proportions of M-MDSCs and G-MDSCs, the CD11b+ cell populations were first gated from BMCs and then the proportions of Ly6C and Ly6G cells in the CD11b+ cell population were evaluated (c and d). Values in parentheses (c) represent the normalized percentages of individual MDSC subsets in BMCs. *p < 0.01, **p < 0.001.
Fig 4.
S. aureus biofilm is capable of stimulating the conversion of the CD11b-positive MDSCs into macrophages or M2-macrophgates in vitro.
The isolated CD11b-positive MDSCs were cocultured with elevated amounts (0.2, 0.6 and 2.0 mg/ml) of S. aureus biofilm for 48 hr. The proportions of total macrophages (F4/80+) (a) and M2-macrophages (F4/80+CD206+) (b) in the CD11b-positive cell population were measured by flow cytometry. **p < 0.001.
Fig 5.
S. aureus biofilm promotes differentiation of M-MDSCs but not G-MDSCs into macrophages or M2-macrophages in vitro.
(a) Morphological characterization of the sorted M-MDSCs (CD11b+Ly-6C+Ly-6G-) and G-MDSCs (CD11b+Ly-6ClowLy-6G+). Giemsa-stained M-MDSCs (P1) and G-MDSCs (P2) were examined by light microscopy. (b) M-MDSCs and G-MDSCs were individually co-cultured with S. aureus biofilms for 72 hr. The biofilm-treated cells were subsequently analyzed for the expression of F4/80 and CD206 by flow cytometry. (c) Changes in the proportion of F4/80-positive macrophages in the sorted M-MDSCs after treatment with S. aureus biofilm (N = 3). (d) Changes in the proportion of M2-macrophages (F4/80+CD206+) in the sorted M-MDSCs after biofilm treatment (N = 3). (e and f) Treatment of G-MDSCs with S. aureus biofilm failed to affect the proportions of F4/80+ cells (macrophages) and F4/80+CD206+ cells (M2-macrophages) (N = 3). **p < 0.001.
Fig 6.
S. aureus biofilm augments the immunosuppressive activity of the cultured BMCs and the CD11b-positive population in vitro.
(a) Mouse BMCs left untreated or treated with S. aureus biofilm (0.2 mg/ml) for 48 hr were cocultured with activated CFSE-labeled T cells at different ratios (1:1, 0.5:1 and 0.25:1). The proliferation of CFSE-labeled T cells was examined by flow cytometry (N = 3). Proliferation of T cell control stimulated by anti-CD3/CD28 beads only was set as 100% (naive) in the experiment. (b) The CD11b-positive cell populations isolated from the untreated or biofilm-treated BMCs were used to coculture with activated CFSE-labeled T cells at different ratios (1:1, 0.5:1 and 0.25:1). The proliferation of CFSE-labeled T cells was examined by flow cytometry (N = 3). *p < 0.01, **p < 0.001.
Fig 7.
Expression of Arginase-1, iNOS, IL-10 and IL-6 in BMCs or in the CD11b-positive MDSCs after exposure to S. aureus biofilm.
(a-d) Mouse BMCs were treated with different amounts (0.2, 0.6 and 2.0 mg/ml) of S. aureus biofilm for 48 hr. RNA samples extracted from the untreated or biofilm-treated BMCs were used for analyzing the expression levels of Arginase-1, iNOS, IL-10 and IL-6 by quantitative RT-PCR. (e-h) The expression levels of Arginase-1, iNOS, IL-10 and IL-6 mRNAs were quantified among the CD11b-positive cell populations isolated from the biofilm-treated BMCs as described above. *p < 0.01, **p < 0.001.
Fig 8.
S. aureus biofilm is able to promote expansion of CD4+CD25+Foxp3+ Tregs from CD4+ lymphocytes through modulation of MDSC function in vitro.
(a and b) The CD11b-positive MDSC fractions were individually isolated from BMCs that were untreated or treated with different amounts (0.2, 0.6 and 2.0 mg/ml) of biofilm. The isolated CD11b-positive MDSCs were subsequently cocultured with spleen T cells (activated by anti-CD3/CD28 beads) at a 1:1 ratio. After 24- or 48-hr coculture, the frequency of CD4+CD25+Foxp3+ T cells was analyzed by flow cytometry using a Treg Flow kit. (c and d) Bars indicate the percentages of CD4+CD25+Foxp3+ T cells out of CD4+ lymphocytes in coculture experiments as described above (N = 2).
Fig 9.
Recruitment and polarization of exogenous EGFP-expressing, CD11b-positive cells during S. aureus biofilm infection in mice.
(a and b) Representative confocal microscopic images showing the recruitment and polarization of the EGFP-expressing cells in tissues near the S. aureus infection sites. Tissues near the S. aureus infected site were taken, frozen in -80°C, cryosectioned and stained with F4/80-PE (a and b, iii) and CD206-PerCP antibody (a and b, iv). White boxes indicate the EGFP+F4/80+CD206+ cells, and dashed boxes indicate the EGFP+F4/80-CD206- cells (probably MDSCs). Notably, only a very small proportion (<15%) of EGFP-positive cells were detected as the EGFP+F4/80-CD206- or EGFP+F4/80+CD206- phenotype in the infected sites. Moreover, cells double positive for F4/80 and CD206 (arrows; endogenous M2-macrophages) were commonly detected in the infected sites. (c) A model for the development and polarization of MDSCs during S. aureus biofilm stimulation.