Aberrant Expression of SLC7A11 Impairs the Antimicrobial Activities of Macrophages in Staphylococcus Aureus Osteomyelitis in Mice

Staphylococcus aureus (S. aureus) persistence in macrophages, potentially a reservoir for recurrence of chronic osteomyelitis, contributes to resistance and failure in treatment. As the mechanisms underlying survival of S. aureus in macrophages remain largely unknown, there has been no treatment approved. Here, in a mouse model of S. aureus osteomyelitis, we identified significantly up-regulated expression of SLC7A11 in both transcriptomes and translatomes of CD11b+F4/80+ macrophages, and validated a predominant distribution of SLC7A11 in F4/80+ cells around the S. aureus abscess. Importantly, pharmacological inhibition or genetic knockout of SLC7A11 promoted the bactericidal function of macrophages, reduced bacterial burden in the bone and improved bone structure in mice with S. aureus osteomyelitis. Mechanistically, aberrantly expressed SLC7A11 down-regulated the level of intracellular ROS and reduced lipid peroxidation, contributing to the impaired bactericidal function of macrophages. Interestingly, blocking SLC7A11 further activated expression of PD-L1 via the ROS-NF-κB axis, and a combination therapy of targeting both SLC7A11 and PD-L1 significantly enhanced the efficacy of clearing S. aureus in vitro and in vivo. Our findings suggest that targeting both SLC7A11 and PD-L1 is a promising therapeutic approach to reprogram the bactericidal function of macrophages and promote bacterial clearance in S. aureus osteomyelitis.


Supplementary Figures
Figure S1.Pathogenesis of S. aureus-induced osteomyelitis is accompanied by an immunosuppressive state in macrophages.(A and B) Representative images of H&E staining and histological scores of the femurs from the mice model of implant-associated S. aureus osteomyelitis and control ones.Blue stars show the position of the implant in bone marrow cavity.Dark arrows show abscess in bone marrow cavity, and blue arrows show reactive new bone formation.D3, D7, and D14 represent time points of sample collection by days 3, 7, and 14 after surgery, respectively.Scale bar, 200 µm.n = 5/group.(C) Representative images of H&E staining of the femurs from the mice model of implant-associated S. aureus osteomyelitis and control ones.The upper and lower images respectively illustrate the medullary cavity structure around the metaphyseal trabecular bone and implantation site at high magnification.Green arrows show neutrophils, red stars show fibrosis,

Figure S2 .
Figure S2.SLC7A11 expression is up-regulated in macrophages after S. aureus infection.(A and B) Volcano plots of the DEGs in the transcriptomes and translatomes of CD11b + F4/80 + cells from the femurs of S. aureus osteomyelitis mice and control ones by day 14 after surgery.The red dots indicate genes or proteins with significantly up-regulated expression (Up), the blue dots indicate genes or proteins with significantly down-regulated expression (Down), and the black dots indicate genes or proteins with non-significant differential expression (N.S).(C) GO analysis of up-regulated DEGs in both the transcriptomes and translatomes.(D and E) Representative images of flow cytometry and quantification of SLC7A11 levels in CD11b + Ly6G + neutrophils from S. aureus osteomyelitis mice and control ones.D3, D7, and D14 represent time points of sample collection by days 3, 7, and 14 after surgery, respectively.n = 5/group.(F and G) Representative images of flow cytometry and quantification of SLC7A11 levels in CD11b + Ly6C + monocytes from S. aureus osteomyelitis mice and control ones.D3, D7, and D14 represent time points of

Figure S4 .
Figure S4.Erastin treatment ameliorates the pathogenesis of S. aureus osteomyelitis in mice.Representative immunohistochemical images of SLC7A11 in the femurs from S. aureus osteomyelitis mice treated with vehicle or erastin and control ones (A).Quantification of the number of SLC7A11 + cells per mm 2 tissue area (N.SLC7A11 + cells) is shown in (B).Mice were euthanized and the right femurs were collected by day 14 after implant-associated S. aureus osteomyelitis surgery.Scale bar, 250 µm.n = 5/group.Data are shown as means ± SEM.One-way ANOVA with Dunnett's T3 post hoc test was used.*P < 0.05, **P < 0.01, ***P < 0.001.

Figure S5 .
Figure S5.Macrophage-specific knockout of Slc7a11 ameliorates the pathogenesis of S. aureus osteomyelitis in mice.(A) A schematic diagram of the breeding strategy for Lyz2Cre-Slc7a11 f/f mice and Slc7a11 f/f mice.(B) Representative images of mouse genotype identification.(C and D) Representative images and quantification of western blots for SLC7A11 in BMDMs that were isolated from Slc7a11 f/f mice and Lyz2Cre-Slc7a11 f/f mice and treated with S. aureus (MOI = 10) or an equal volume of PBS for 12 hours.n = 3/group.(E and F) Representative immunohistochemical images of SLC7A11 in the femurs from Lyz2Cre-Slc7a11 f/f mice and Slc7a11 f/f mice by day 14 after surgery.Quantification of the number of SLC7A11 + cells per mm 2 tissue area (N.SLC7A11 + cells) is shown in (F).Scale bar, 250 µm.n = 5/group.Data are shown as means ± SEM.Two-way ANOVA with Dunnett's T3 post hoc test (D) or unpaired two-tailed Student's t-test (F) was used.*P < 0.05, **P < 0.01, ***P < 0.001.

Figure S6 .
Figure S6.Blocking SLC7A11 promotes PD-L1 expression via the ROS-NF-κB axis in macrophages after S. aureus infection.(A) GO analysis of DEGs in the translatomes of CD11b + F4/80 + cells from femurs of S. aureus infected mice and control ones.GO items with an adjusted P-value < 0.05 were considered significantly enriched.(B) Heatmap of the DEGs in the GO item "negative regulation of cell activation".(C) The mRNA expression level of Slc7a11 in BMDMs after 12 hours of S. aureus infection, with or without si-Slc7a11 transfection.After 48 hours of transfection with siRNA fragments targeting Slc7a11 (si-Slc7a11) or negative control ones (si-NC), BMDMs were treated with S. aureus at a MOI of 10 or an equal volume of PBS.n = 3/group.(D and E) Representative images and quantification of immunofluorescence staining for PD-L1 in BMDMs after 12 hours of S. aureus infection, with or without si-Slc7a11 transfection.Scale bar, 20 µm.n = 3/group.Data