Selected variables in 28 RA patients with PJI treated with two-stage exchange.

<p><b>NOTE.</b> Data are no. (%) of joints, unless otherwise indicated.</p><p>NS: Not significant; CRP: C-reactive protein; ESR: erythrocyte sedimentation rate.</p>*<p>Statistical significance (p<0.05).</p

Flow chart of treatment modalities for 46 episodes of PJI in RA patients treated between 2002 and 2008.

<p>Flow chart of treatment modalities for 46 episodes of PJI in RA patients treated between 2002 and 2008.</p

<i>Staphylococcus aureus</i> biofilm elicits the expansion, activation and polarization of myeloid-derived suppressor cells <i>in vivo</i> and <i>in vitro</i>

<div><p><i>Staphylococcus aureus</i> (<i>S</i>. <i>aureus</i>) is one of the most common causes of biofilm infections in periprosthetic joint infections (PJIs). Accumulating evidence has shown that the immunosuppressive environment established by <i>S</i>. <i>aureus</i> biofilm infection in PJIs involves the presence of myeloid-derived suppressor cells (MDSCs) and M2-macrophages. Due to the diversity of MDSCs, little is known about whether <i>S</i>. <i>aureus</i> biofilm preferentially expands specific MDSC subsets or whether MDSCs can further differentiate into M2-macrophages during <i>S</i>. <i>aureus</i> biofilm infection. Here, we show that in agreement with the results from an established rat PJI model, <i>S</i>. <i>aureus</i> biofilm cocultured with freshly isolated bone marrow cells (BMCs) <i>in vitro</i> significantly increases the proportions of MDSCs, total macrophages and M2-macrophages. Interestingly, we find that treatment of the BMCs <i>in vitro</i> with <i>S</i>. <i>aureus</i> biofilm preferentially promotes the expansion of monocytic MDSCs but not granulocytic MDSCs. Biofilm treatment also substantially enhances the overall MDSC immunosuppressive activity in addition to the MDSC expansion <i>in vitro</i>. Importantly, we provide evidence that <i>S</i>. <i>aureus</i> biofilm is capable of further stimulating the conversion of monocytic MDSCs into M2-macrophages <i>in vitro</i> and <i>in vivo</i>. Collectively, our studies reveal a direct link between MDSCs and M2-macrophages occurring in <i>S</i>. <i>aureus</i>-associated PJIs.</p></div

<i>S</i>. <i>aureus</i> biofilm triggers expansion of different immune cell types from mouse bone marrow cells <i>in vitro</i>.

<p>After coculture of mouse bone marrow cells (BMCs) with increasing concentrations (0.2, 0.6 and 2.0 mg/ml) of <i>S</i>. <i>aureus</i> biofilm for 48 hr, the proportions of CD11b⁺Gr1⁺ MDSCs (a and b), MDSC subsets including M-MDSCs (CD11b⁺Ly6C^highLy6G^-) and G-MDSCs (CD11b⁺Ly6C^lowLy6G⁺) (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.</p

Surgical operation and <i>S</i>. <i>aureus</i> biofim formation in a rat PJI model.

<p>(a) Rats were divided into three groups including the sham group, the <i>S</i>. <i>aureus</i>-infected group and the <i>S</i>. <i>aureus</i>-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 <i>S aureus</i>-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 <i>S</i>. <i>aureus</i> cocci were more evident in the femur lumen of the <i>S</i>. <i>aureus</i>-infected group than those of the vanomycin-treated infection group (original magnification ×10,000).</p

<i>S</i>. <i>aureus</i> biofilm is capable of stimulating the conversion of the CD11b-positive MDSCs into macrophages or M2-macrophgates <i>in vitro</i>.

<p>The isolated CD11b-positive MDSCs were cocultured with elevated amounts (0.2, 0.6 and 2.0 mg/ml) of <i>S</i>. <i>aureus</i> 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.</p

<i>S</i>. <i>aureus</i> biofilm augments the immunosuppressive activity of the cultured BMCs and the CD11b-positive population <i>in vitro</i>.

<p>(a) Mouse BMCs left untreated or treated with <i>S</i>. <i>aureus</i> 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.</p

<i>S</i>. <i>aureus</i> biofilm is able to promote expansion of CD4⁺CD25⁺Foxp3⁺ Tregs from CD4⁺ lymphocytes through modulation of MDSC function <i>in vitro</i>.

<p>(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).</p

Expansion of MDSCs, total macrophages, and M2-macrophages during <i>S</i>. <i>aureus</i> infection in rats.

<p>The sham group, the <i>S</i>. <i>aureus</i>-infected group, and the <i>S</i>. <i>aureus</i>-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).</p

<i>S</i>. <i>aureus</i> biofilm promotes differentiation of M-MDSCs but not G-MDSCs into macrophages or M2-macrophages <i>in vitro</i>.

<p>(a) Morphological characterization of the sorted M-MDSCs (CD11b⁺Ly-6C⁺Ly-6G^-) and G-MDSCs (CD11b⁺Ly-6C^lowLy-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 <i>S</i>. <i>aureus</i> 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 <i>S</i>. <i>aureus</i> 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 <i>S</i>. <i>aureus</i> biofilm failed to affect the proportions of F4/80⁺ cells (macrophages) and F4/80⁺CD206⁺ cells (M2-macrophages) (N = 3). **p < 0.001.</p