EGFR-MEK1/2 cascade negatively regulates bactericidal function of bone marrow macrophages in mice with Staphylococcus aureus osteomyelitis

The ability of Staphylococcus aureus (S. aureus) to survive within macrophages is a critical strategy for immune evasion, contributing to the pathogenesis and progression of osteomyelitis. However, the underlying mechanisms remain poorly characterized. This study discovered that inhibiting the MEK1/2 pathway reduced bacterial load and mitigated bone destruction in a mouse model of S. aureus osteomyelitis. Histological staining revealed increased phosphorylated MEK1/2 levels in bone marrow macrophages surrounding abscess in the mouse model of S. aureus osteomyelitis. Activation of MEK1/2 pathway and its roles in impairing macrophage bactericidal function were confirmed in primary mouse bone marrow-derived macrophages (BMDMs). Transcriptome analysis and in vitro experiments demonstrated that S. aureus activates the MEK1/2 pathway through EGFR signaling. Moreover, we found that excessive activation of EGFR-MEK1/2 cascade downregulates mitochondrial reactive oxygen species (mtROS) levels by suppressing Chek2 expression, thereby impairing macrophage bactericidal function. Furthermore, pharmacological inhibition of EGFR signaling prevented upregulation of phosphorylated MEK1/2 and restored Chek2 expression in macrophages, significantly enhancing S. aureus clearance and improving bone microstructure in vivo. These findings highlight the critical role of the EGFR-MEK1/2 cascade in host immune defense against S. aureus, suggesting that S. aureus may reduce mtROS levels by overactivating the EGFR-MEK1/2 cascade, thereby suppressing macrophage bactericidal function. Therefore, combining EGFR-MEK1/2 pathway blockade with antibiotics could represent an effective therapeutic approach for the treatment of S. aureus osteomyelitis.


Introduction
Osteomyelitis, characterized by progressive inflammation and destruction in bone and surrounding tissue caused by microbial pathogens, is a challenging condition in orthopedics [1].Staphylococcus aureus (S. aureus) is the most prevalent causative pathogen isolated in cases of osteomyelitis.Due to its resilience, S. aureus is closely associated with recurrent osteomyelitis [2][3][4].Even after surgical debridement, implant removal, and a full course of antibiotics, treatment failure rates can reach 37.9%, with a recurrence rate of 25.9% [3,5].Therefore, new therapeutic strategies are needed to overcome S. aureus therapy resistance in osteomyelitis.
The ability of S. aureus to evade host immune surveillance is critical for its persistence and relapse despite antibiotic treatment.This pathogen employs various strategies, such as producing toxins to hijack host cell signaling, forming biofilms on implants and in the osteocyte lacuno-canalicular network of cortical bone, and internalizing into both phagocytic and nonphagocytic cells [6][7][8][9][10].Macrophages and neutrophils are recruited to infection sites to eradicate bacteria, forming the first line of host defense against S. aureus [11,12].However, accumulating evidence suggests that S. aureus can survive intracellularly within macrophages, contributing to bacterial dissemination and pathogenesis [13,14].Understanding the mechanisms that enable S. aureus survival within macrophages is essential.
The extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinases (MAPKs), a classical MAPKs cascade comprising MAPKK kinase, MAPK kinase 1/2 (MEK1/ 2), and ERK1/2, is well known for its role in cellular proliferation, differentiation, and survival [15].Accumulating evidence suggests that aberrant activation of the MEK1/2-ERK1/2 pathway supports the pathogenic process during viral infection and is closely associated with the progression of inflammatory tissue injury [16][17][18].A recent study showed that combining antibiotic treatment with MEK1/2-p-ERK1/2 inhibition reduced excessive inflammation and restored bone union in a murine MRSA-infected fracture model [19].However, the role of the MEK1/2 pathway in the immune defense against S. aureus remains unclear.
Here, we investigated the role of MEK1/2 pathway in the pathogenesis of S. aureus osteomyelitis and explored how its overactivation might suppress macrophage bactericidal activity.We found phosphorylated MEK1/2 (p-MEK1/2) levels were upregulated in bone marrow

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EGFR-MEK1/2 cascade and Staphylococcus aureus osteomyelitis macrophages during S. aureus infection both in vivo and in vitro.Blocking this pathway rescued bone destruction and reduced the bacterial burden in infected bones.In addition, we identified a novel function of the overactivated epidermal growth factor receptor (EGFR)-MEK1/2 cascade in suppressing mitochondrial ROS (mtROS) levels, thereby impairing macrophage bacterial clearance capacity.Our findings suggest that targeting the MEK1/2 pathway could be a promising strategy for overcoming resistance to therapy in S. aureus-induced osteomyelitis.

Pharmacological blockade of MEK1/2 pathway alleviates bone destruction and bacterial load in femurs of mice with S. aureus osteomyelitis
Recent studies have documented that activation of the MEK1/2 pathway represents a crucial signal transducer in response to viral or bacterial infection, leading to inflammatory responses and tissue injury [18,19].To investigate whether pharmacological blocking of the MEK1/2 pathway can inhibit the pathogenesis of S. aureus osteomyelitis, we treated mice with S. aureus osteomyelitis using PD0325901, an MEK1/2 inhibitor, in combination with gentamicin.As revealed by micro-CT imaging, PD0325901-treated mice exhibited significantly reduced cortical bone loss compared with vehicle-treated mice (Fig 1A and 1B).In addition, PD0325901-treated mice showed significant improvements in bone mineral density (BMD) and bone volume/tissue volume (BV/TV) in the distal femora compared with vehicle-treated mice (Fig 1C and 1D).Further analysis showed that the improved BV/TV in PD0325901-treated mice was mainly due to rescued trabecular number (Tb.N) and a significantly decreased trabecular bone pattern factor (Tb.Pf), with no obvious changes in the trabecular thickness (Tb.Th) (Fig 1E -1G).Consistent with these microstructural changes, H&E staining showed severe loss of trabecular bone density, empty lacunae in remaining trabeculae in distal femoral metaphysis, fibroblastic hyperplasia in peri-implant bone marrow, and extensive reactive new bone formation around cortical bone in femurs of vehicle-treated mice.In contrast, PD0325901-treated mice exhibited significantly improved bone morphology and structure with limited pathological changes around the implant (Fig 1H).Blind scoring of bone sections confirmed notably rescued bone destruction in S. aureus-infected femurs in PD0325901-treated mice compared with vehicle-treated mice (Fig 1I).
The observation that PD0325901 treatment alleviated bone destruction in mice with S. aureus osteomyelitis suggested that blocking the MEK1/2 pathway may reduce bacterial burden in bone.In support of this hypothesis, immunofluorescence assays showed significantly lower amounts of S. aureus-positive staining in bone marrow of PD0325901-treated mice compared with vehicle-treated mice (Fig 1J and 1K).In addition, classical culture-based analysis revealed a significant decrease in bacterial CFU per gram of bone tissue in infected femurs in PD0325901-treated mice compared with vehicle-treated mice (Fig 1L and 1M).Taken together, these results suggest that overactivation of the MEK1/2 pathway may impair antibacterial defense.

Phosphorylation of MEK1/2 in macrophages is upregulated in mouse femurs following S. aureus osteomyelitis
MEK1/2 is typically activated via serine phosphorylation by upstream activator kinases [20].To assess the effect of S. aureus infection on MEK1/2 activity, we analyzed the levels of phosphorylated MEK1/2 (p-MEK1/2) in mouse femurs 14 days post-infection.Immunostaining results revealed a notably elevated level of p-MEK1/2, mainly located in cells surrounding the

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EGFR-MEK1/2 cascade and Staphylococcus aureus osteomyelitis  and 2B).Since neutrophils are the main immune cells in infectious nidus, surrounded by macrophages [21], we examined p-MEK1/2 expression in F4/ 80 + cells using immunofluorescence staining.Our data clearly showed a significant increase in F4/80 + p-MEK1/2 + cells in bone marrow away from abscess in S. aureus-infected right femurs compared with control mice (Fig 2C and 2D).Notably, p-MEK1/2 levels were predominantly upregulated in F4/80 + macrophages surrounding the abscess (Fig 2C and 2D).Moreover, the levels of F4/80 + p-MEK1/2 + cells in the left femoral bone marrow (the contralateral side of surgery and infection) of S. aureus-infected mice were comparable to those in control mice (S1 Fig).These data suggest that the MEK1/2 pathway is primarily activated in F4/80 + macrophages surrounding the abscess.

Activation of the MEK1/2 pathway is associated with reduced mitochondrial ROS (mtROS) levels and suppressed bactericidal activity in macrophages
We next investigated whether S. aureus might activate the MEK1/2 pathway in primary cultures of BMDMs.Western blot analysis revealed that S. aureus upregulated p-MEK1/2 levels in BMDMs in a concentration-dependent manner after 30 min (Fig 3A and 3B).This observation suggests that the MEK1/2 pathway in macrophages may be rapidly activated in response to S. aureus infection.
We then assessed whether overactivation of the MEK1/2 pathway affects macrophage function during S. aureus infection.Blocking the MEK1/2 pathway with PD0325901 did not affect the phagocytic ability of BMDMs (Figs 3C and S2A).Importantly, the bacterial killing assay demonstrated that PD0325901 significantly reduced intracellular bacterial burden in BMDMs at indicated time points post-infection (Figs 3D and S2B), indicating enhanced bactericidal function of macrophages with MEK1/2 pathway inhibition.
Given the critical role of mtROS in the bactericidal function of macrophages [22], and our recent finding of a notable reduction in mtROS levels 12 h and 24 h post-infection [23], we determined whether inhibiting MEK1/2 pathway affects mtROS levels in macrophages.Mito-Tracker Green and MitoSOX staining revealed that PD0325901 treatment for 12 h or 24 h significantly increased mtROS levels in BMDMs (Fig 3E and 3F).These findings suggest that overactivation of the MEK1/2 pathway suppresses mtROS production, thereby impairing the bactericidal function of macrophages.
Considering the crucial role of phagocyte NADPH oxidase in host defense by generating superoxide anion and other ROS molecules [24,25], we evaluated the effect of PD0325901 on total protein levels of NADPH oxidase components, including p22phox, NOX2/gp91phox, p47phox, p67phox, and p40phox.Surprisingly, the levels of these proteins remained unchanged after 12 and 24 h of S. aureus infection (S2C-S2H Fig) .However, PD0325901 treatment significantly upregulated NOX2 levels after 12 h and 24 h of S. aureus infection (Fig 3G and 3H).We inferred that the ability of BMDMs to produce ROS from NADPH oxidase in phagocyte decreased to basal levels, insufficient to restrict invasion of S. aureus after 12 h of persistent infection.Thus, overactivation of MEK1/2 may not only suppress mtROS changes using the scoring system established by Smeltzer et al [51].n = 8/group, Mann-Whitney test.(J) Representative images and (K) Quantification of immunofluorescence for S. aureus-positive staining area in femurs in S. aureus osteomyelitis mice treated with PD0325901 (0.2 mg/kg/day) or the same volume of vehicle (5% DMSO).Scale bars, 200 μm.n = 5/group.(L) Representative images and (M) Quantification of S. aureus colonies growing on an agar plate after being recovered from the femur.10 μl of S. aureus suspension diluted in PBS (1: 500) was placed and cultured on LB agar plates for 24    production but also disrupt phagocytic ROS production in BMDMs under prolonged S. aureus infection.
To understand the molecular mechanism by which S. aureus activates the MEK1/2 pathway, we analyzed transcriptome data from femurs of mice with S. aureus-induced osteomyelitis and control samples on day 14 post-infection (GEO: GSE166522) from our previous work [27].We overlapped MAPK cascade-related differentially expressed genes (DEGs) and DNA repair-related DEGs in Gene Ontology (GO) enrichment, and MAPK signaling pathwayrelated DEGs in Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment using the Venn diagram.Results identified EGFR as a central candidate DEGs (Fig 4A).We then assessed EGFR signaling activity in BMDMs in response to S. aureus infection.Results showed that the phosphorylation levels of EGFR were significantly upregulated by S. aureus at MOI of 1 and 10 (Fig 4B and 4C), with activation peaking at 30 min (S4A and S4B Fig).
Observing that S. aureus activated both EGFR signaling and the MEK1/2 pathway, we hypothesized that S. aureus might activate the MEK1/2 pathway through EGFR signaling.Indeed, treating BMDMs with erlotinib, an EGFR inhibitor, partially abolished MEK1/2 phosphorylation induced by S. aureus (Fig 4D and 4E).We then determined whether overactivation of EGFR signaling was associated with macrophage dysfunction.Erlotinib treatment did not affect the phagocytic ability of BMDMs (Figs 4F and S4C), but notably enhanced the bacterial killing rate of cells after 12 h of infection (Figs 4G and S4D).Consistent with functional rescue, erlotinib treatment also restored mtROS to higher levels after 12 h and 24 h of S. aureus infection (S4E and S4F Fig).
Next, we evaluated whether combining erlotinib and PD0325901 might further enhance the bactericidal function of BMDMs after 12 h of infection.Unexpectedly, we did not observe a synergistic effect (Figs 4H and S4G).Additionally, MitoSOX staining showed that both PD0325901 and erlotinib treatment upregulated mtROS levels, but their combination did not further increase mtROS levels after 12 h of S. aureus infection (Fig 4I and 4J).Thus, persistent S. aureus infection may suppress mtROS production, thereby restricting the bactericidal function of macrophages through the EGFR-MEK1/2-dependent pathway.
While mtROS is essential for fighting infection, uncontrolled production can trigger excessive inflammation [28,29].We found that blocking the MEK1/2 pathway did not block the mRNA expression of IL-1β, IL-6, or TNF-α induced by S. aureus challenge, but strikingly upregulated IL-6 and TNF-α mRNA levels compared with vehicle-treated macrophages (S5A-S5C  Considering that TLR2 is essential for optimal innate immune defense against S. aureus infection [30], we evaluated the effect of C29, a TLR2 inhibitor, on EGFR-MEK1/2 cascade activity.Western blot results showed that C29 treatment significantly blocked MEK1/2 phosphorylation as early as 15 min after S. aureus challenge but had no effect on EGFR activation (Fig 5A -5C).These data indicate that TLR2 signaling contributes to MEK1/2 pathway activation but is not responsible for EGFR signaling activation in response to S. aureus challenge.Furthermore, phagocytosis assays revealed that C29 treatment did not affect the phagocytotic activity of BMDMs (Figs 5D and S6A).However, the bacterial killing assay showed that C29 treatment did not significantly affect bactericidal function at earlier time points (1 h and 4 h), but decreased bacterial killing rate and increased bacterial burden in BMDMs after 12 h of infection (Figs 5E and S6B), indicating that TLR2 signaling is critical for bacterial killing activity of macrophages after persistent S. aureus infection.Consistent with this finding, C29 treatment further decreased mtROS levels in BMDMs after 12 h of S. aureus infection (Fig 5F and  5G).These data demonstrate that the EGFR-MEK1/2 cascade might play a negative feedback role in TLR2 signaling-mediated mtROS production and bacterial killing during S. aureus infection.

EGFR-MEK1/2 activation suppresses mtROS by inhibiting Chek2 expression
To further discover how EGFR-MEK1/2 activation suppresses mtROS production upon S. aureus challenge, we treated BMDMs with S. aureus for 12 h for transcriptome analysis.Analysis of DEGs associated with cellular responses to environmental stimuli and regulation of DNA damage checkpoints showed that three genes were significantly downregulated (Fig 6A and 6B).Chek2, a central effector of DNA damage response, mediates DNA checkpoint activation under stress conditions [31].We confirmed that S. aureus infection suppressed Chek2 mRNA expression in BMDMs as early as 4 h post-infection (Fig 6C).The suppressed effect on Chek2 mRNA expression was alleviated by either PD0325901 or erlotinib treatment (Fig 6D).Immunofluorescence staining showed strikingly reduced levels of total and subcellular Chek2 in BMDMs after 12 h of S. aureus infection (Fig 6E -6H).Notably, both PD0325901 and erlotinib treatments significantly increased Chek2 intensity in the cytoplasm and nucleus (Fig 6E -6G), with enhanced Chek2 localization in mitochondria (Fig 6H ).Consistent with the effect on bactericidal function, combined treatment with PD0325901 and erlotinib did not further increase Chek2 protein levels (Fig 6E -6H).Considering that Chek2 activation contributes to high mtROS production [32], we evaluated the function of Chek2 in mtROS production in response to S. aureus infection.Our results showed that inhibition of Chek2 by BML-277 blocked mtROS activation induced by PD0325901 or erlotinib treatment (Figs 6I and 6J).Taken together, these results demonstrate that EGFR and MEK1/2 activation by S. aureus infection reduces mtROS levels by suppressing Chek2 expression, thereby impairing macrophages' bactericidal function.
Next, we investigated whether blocking EGFR signaling could reduce bacterial load and rescue bone destruction in vivo.Mice with implant associated osteomyelitis were treated with mtROS in BMDMs detected using MitoTracker Green and MitoSOX Red.Cells were infected with S. aureus (MOI = 10) for 1 h.After removing non-phagocytosed extracellular bacteria, cells were treated with 10 μM erlotinib, 1 μM PD0325901, a combination of 10 μM erlotinib and 1 μM PD0325901, or or vehicle (DMSO) for  To confirm the role of EGFR signaling in the MEK1/2 pathway, we evaluated its expression in vehicle-and erlotinib-treated mice with S. aureus osteomyelitis.Immunofluorescence staining showed that erlotinib treatment substantially decreased p-MEK1/2 levels in F4/80 + cells (Fig 7K and 7L).We then evaluated whether inhibiting EGFR signaling could rescue Chek2 expression in macrophages.Results showed that erlotinib treatment markedly restored decreased Chek2 expression in S. aureus-infected femurs (Fig 7M and 7N).Together, our findings indicate that S. aureus infection may activate EGFR-MEK1/2 signaling, thereby suppressing mitochondrial ROS levels by downregulating Chek2 expression, leading to the progression of S. aureus osteomyelitis in mice.

Discussion
Macrophages are essential components of the first line of host defense against S. aureus infection.However, S. aureus employs multiple self-defensive strategies to survive within and escape macrophages, the mechanism of which remains largely unexplored.Here, we demonstrate that blocking the EGFR-MEK1/2 pathway may be a promising strategy to strengthen host defense against S. aureus osteomyelitis.Importantly, we uncover a previously unknown aspect of the EGFR-MEK1/2 cascade involved in intracellular survival of S. aureus in macrophages: suppression of mtROS production through downregulation of Chek2 expression, which reduces cell bactericidal activity.This study therefore identifies the EGFR-MEK1/2 cascade in macrophages as a critical target for the treatment of S. aureus osteomyelitis.
Recent studies have highlighted the important role of the MEK1/2 pathway in sustaining the replication of Salmonella and viruses in cells [33][34][35], emphasizing the critical function of MEK1/2 overactivation in infection propagation.Similarly, our current work further reveals the critical role of the MEK1/2 pathway in suppressing the bactericidal activity of macrophages.We show that overactivation of the MEK1/2 pathway leads to reduced mtROS levels, impairing the bactericidal function of macrophages.Moreover, blocking the MEK1/2 pathway reduces bacterial load and mitigates bone destruction in mice with osteomyelitis, suggesting that targeting the MEK1/2 pathway may be a potential therapeutic approach to restore compromised immunity in chronic S. aureus osteomyelitis.It is important to note that abnormal activation of this pathway has also been linked to inflammatory tissue lesions, and blocking field of view (FOV) (F), the FI of Chek2 in the nucleus (G) and mitochondria (H) per cell.n = 8/group, **p < 0.01, *** p < 0.001, one-way ANOVA with Dunnett's post-hoc test.(I) Representative confocal images and (J) Quantification of mitochondrial ROS (mtROS) levels detected using MitoTracker Green and MitoSOX Red in BMDMs.Nuclei were stained with Hoechst 33342.After phagocytosis for 1 h and removal of the extracellular S. aureus, cells were treated with 5 μM BML-277 or vehicle (DMSO) with or without 1 μM PD0325901 or 10 μM erlotinib.mtROS levels were detected after 12 h of treatment.Scale bars, 20 μm.***p < 0.01, one-way ANOVA with Tukey's post-hoc test.
https://doi.org/10.1371/journal.ppat.1012437.g006MEK1/2 is believed to alleviate pro-inflammatory responses [17,35,36].Contrary to this belief, we observed that blocking the MEK1/2 pathway led to further upregulation of inflammatory factors in S. aureus-infected BMDMs in vitro.Therefore, the notable improvement in bone structure by blocking the MEK1/2 pathway in the femurs of mice with S. aureus osteomyelitis is likely due to the enhanced bactericidal activity of macrophages in the bone.
Our finding that EGFR signaling is activated by S. aureus in BMDMs is both surprising and interesting.EGFR, a transmembrane protein with tyrosine kinase activity, is ubiquitously expressed in various cells and aberrantly activated in lung cancer, colorectal cancer, and other diseases [37].Increasing evidence supports the pivotal roles of EGFR signaling in pathogenic bacterial infection, such as invasion of Serratia proteamaculans into m-Hela cells [38], induction of inflammation in epithelia cells in response to Candida albicans infection [39], and activation of inflammatory responses in macrophages by Helicobacter pylori infection [40].Our current findings reveal a novel role of EGFR signaling upon S. aureus infection: its overactivation is associated with reduced bacterial killing activities in macrophages.Additionally, our studies demonstrate that activation of EGFR signaling by S. aureus may lead to downregulation of mtROS levels and reduction of macrophage bactericidal function via the MEK1/2 pathway.However, the MEK1/2 pathway is also a key mediator of TLRs signaling-mediated innate immune responses [41].Supporting this, our data indicate that TLR2 signaling is required to activate the MEK1/2 pathway in the context of S. aureus infection.Although studies have reported mutual activation between EGFR and TLR signaling in the presence of microbe [42,43], we have not observed inhibition of EGFR signaling by blocking TLR2 during S. aureus challenge.Therefore, it is reasonable to presume that EGFR and TLR2 signaling may independently regulate the MEK1/2 pathway in response to S. aureus infection.
Accumulating evidence indicates that mtROS is a critical component of antimicrobial responses in macrophages [22,44,45].Supporting this, our previous study showed that prolonged infection (12 h or longer) leads to a dramatic decline in mtROS levels and reduced bactericidal activity in macrophages [23].Our current data further identify Chek2, a positive regulator of mtROS production [29], is negatively regulated by the EGFR-MEK1/2 cascade during S. aureus infection.Despite the pivotal role of mtROS in the antimicrobial functions of innate immune cells, a strong mtROS signal is critical for the proinflammatory response, which can damage cells and tissues during infection [46][47][48].Our data reveal that EGFR signaling and MEK1/2 may collaboratively regulate mtROS levels and bactericidal function of macrophages via Chek2 while differentially control inflammatory responses upon S. aureus infection.It is important to note that while blocking TLR2 with C29 significantly suppresses p-MEK1/2 levels, it also decreases mtROS production and impairs the bactericidal function of macrophages during persistent S. aureus infection.This indicates that TLR2 is crucial for maintaining macrophage antimicrobial function through pathways other than MEK1/2.

Conclusion
The current study has extended our understanding of the critical role of EGFR-MEK1/2 cascade in the persistent infection of S. aureus in macrophages.By blocking the MEK1/2 pathway both in vivo and in vitro, we identified MEK1/2 as a hub pathway in negatively regulating mtROS levels and compromising the bactericidal function of bone marrow macrophages aureus-infected femurs of mice treated with erlotinib and vehicle.Scale bars, 50μm.n = 5/group.(M) Representative images and (N) Quantitative analysis of F4/80 + (red) and Chek2 + (green) cells in S. aureus-infected femurs of mice treated with erlotinib and vehicle.Scale bars, 50μm.n = 5/group.** p < 0.01, *** p < 0.001.Statistical significance was analyzed by Student's t test.https://doi.org/10.1371/journal.ppat.1012437.g007

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EGFR-MEK1/2 cascade and Staphylococcus aureus osteomyelitis during S. aureus infection.Although the precise mechanism by which the MEK1/2 pathway inhibits Chek2 expression and suppresses mtROS production requires further investigation, our data suggest that downregulation of Chek2 expression in cells contributes to reduced mtROS levels and subsequently suppressed bactericidal function of macrophages.Taken together, our findings indicate that over activation of the EGFR-MEK1/2 cascade may be a core event in suppressing bactericidal function during S. aureus infection.Therefore, targeting this pathway may represent a promising avenue for breakthrough therapies for S. aureus osteomyelitis.

Bacteria culture
The S. aureus used in this study was isolated from an osteomyelitis patient, as previously described [27].S. aureus strains were maintained at -80˚C in tryptic soy broth (TSB) containing 25% (v/v) glycerol.They were cultured from cold storage by plating on tryptic soy agar (TSA) at 37˚C.A single clone of S. aureus was selected and cultured in TSB by shaking at 200 rpm for 16-18 h before being collected by centrifugation at 2500g for use in vitro and in vivo infection experiments.After resuspension in phosphate buffered saline (PBS), S. aureus strains were adjusted to 1×10 8 CFU/ml by measuring optical density at 600nm (OD 600 ) of 0.5.

Implant-associated S. aureus osteomyelitis mouse model and treatments
All mouse care and treatment procedures followed guidelines approved by the Animal Care and Use Committee of Southern Medical University Nanfang Hospital.Male C57BL/6 mice (8-10 weeks) were purchased from Southern Medical University Animal Center (Guangzhou, China).All mice were housed under standard conditions (23 ± 2˚C, 12 hours light-dark cycle) with free access to water and food.
The mouse model of implant-associated S. aureus osteomyelitis was established as previously described [27].Briefly, male C57BL/6 mice aged 10-12 weeks were anaesthetized with tribromoethanol (250 mg/kg).After exposing the right femur by blunt dissection, a small hole was drilled in the mid-shaft with a 27-gauge needle.A sterilized stainless pin, 2 mm in length, was inserted through the hole, followed by slow injection of 2 μl S. aureus suspension (1×10 5 CFU/ml) or PBS (control group) into the intramedullary canal.The hole was seamed with bone wax, the incision was sutured and disinfected.
To evaluate the effect of MEK1/2 blocking on the progression of S. aureus osteomyelitis in mice, S. aureus-infected mice were randomly assigned to treatment with PD0325901 (0.2 mg/ kg/day, i.p., #S1036, Selleck) or vehicle (5% dimethyl sulfoxide (DMSO) in normal saline) from day 5 post-infection.To assess the role of EGFR signaling in the pathogenesis of S. aureus osteomyelitis in mice, S. aureus-infected mice were randomly treated with erlotinib (10 mg/ kg/day, i.p., #HY-1208, MCE) or vehicle (10% DMSO in corn oil) from day 5 post-infection.Gentamicin at 10 mg/kg had a poor killing effect on intracellular S. aureus in a mouse model of peritonitis [49], and our recent work showed that a substantial amount of S. aureus resides in Lyz2 + macrophages in the bone marrow of osteomyelitis mice treated with gentamicin at a dose of 20 mg/kg/d [50].To investigate the role and mechanism of intracellular S. aureus in pathogenesis of osteomyelitis, all mice were treated with gentamicin (20 mg/kg/day, i.p.) from day 1 post-surgery to reduce the extracellular burden of S. aureus in infected femurs.The mice were euthanized on day 14 after surgery, and their implanted right femurs were collected for further analysis.

Immunohistochemistry and immunofluorescence
For immunohistochemistry staining, femurs were fixed in 4% paraformaldehyde overnight, decalcified in 10% ethylenediamenetetraacetic acid (EDTA) solution for 10 days, and processed for paraffin-embedding.Four-μm-thick sections were placed on glass slide, deparaffinized, and rehydrated before antigen retrieval.Endogenous peroxidase activity was blocked by incubating sections in a 0.3% (v/v) hydrogen peroxide solution for 15 min.After blocking with goat serum for 1 h at room temperature, sections were incubated with phospho-MEK1/2 antibody (AF3385, Affinity, China) overnight at 4˚C.Samples were then incubated in HRP-conjugated goat anti-rabbit secondary antibody (HA1001, Huabio, China) for 1 h.Peroxidase activity was revealed using a 3,3'-diaminobenzidine (DAB) kit (ZLI-9018, ZSGB-BIO, China) and nuclei were counterstained with hematoxylin (H-3404-100, Vector, USA).Image pro plus 6.0 was used to analyze the integrated optical density of p-MEK1/2 in the bone marrow.

Microcomputed tomography (micro-CT)
Bone destruction and new bone formation were assessed using micro-CT imaging with Skyscan (Bruker, New York, USA).Each femur was imaged at a pixel size of 9 μm, with a voltage of 70 kV, a current of 145 mA, and an integration time of 300 ms.The 3D reconstruction images were processed using Image Processing Language V5.15 software.To evaluate cortical bone destruction and reactive bone formation, the region of interest (ROI) was centered in the middle of the femur, where the pin was implanted.To analyze changes in trabecular bone microstructure in the distal femur, the lower border was set 0.5 mm away from the growth plate, and the upper border was defined as 2 mm from this position longitudinally.Parameters such as cortical bone loss, reactive new bone formation, bone mineral density (BMD), bone

Histological analyses
Tibias and femurs were fixed in 4% paraformaldehyde for 48 h, decalcified in 10% EDTA solution for 10 days, and processed for paraffin-embedding.Hematoxylin-eosin (H&E) staining was performed following standard procedures.Histopathological signs of osteomyelitis were scored using the methods described by Smeltzer et al. [51], based on the presence of acute intraosseous inflammation, chronic intraosseous inflammation, periosteal inflammation, and bone necrosis, each graded on a scale of 0-4.

Bacterial burdens in implanted-femurs
Fourteen days post-infection, mice were anaesthetized with tribromoethanol (250 mg/kg) and euthanized by cervical dislocation, implanted femurs were dissected free of soft tissue, and after removal of intramedullary implants, femurs were homogenized in 1 ml PBS.S. aureus burdens were determined by plating serial dilutions of homogenate on TSA plates.After 12 h of incubation at 37˚C, bacterial colonies were imaged and counted using ImageJ software (V1.8.0, National Institute of Health).

Isolation and culture of bone marrow-derived macrophages (BMDMs)
BMDMs were prepared from 8-10 weeks old C57BL/6 mice following established protocols [23].Mice were euthanized by cervical dislocation, and bone marrow cells were flushed from the femora and tibiae.After lysis of red blood cells with ACK lysis buffer, bone marrow cells were cultured and differentiated in RPMI-1640 medium supplemented with 30% L929 conditioned medium, 10% fetal bovine serum, and 1% penicillin/streptomycin.Cells were seeded at a density of 2.0×10 6 /ml in 12-well or 6-well plates for 7 days, with medium refresh every 2 days.On day 8 of differentiation, the cells were treated for further analysis.
For evaluating intracellular bacterial killing capacity, after the removal of extracellular bacteria, infected BMDMs were cultured in medium containing 1% penicillin & streptomycin for 1, 4 or 12 h, with or without 1 μM PD0325901, 10 μM erlotinib, or 100 μM C29.At the indicated time points, cells were lysed, and diluted aliquots were spread on TSA plates.Bacterial colonies were counted after overnight incubation at 37˚C.

RNA sequencing and data analysis
Our previous RNA sequencing data from femurs of mice with S. aureus osteomyelitis and controls, deposited in Gene Expression Omnibus (GEO) database (assessing number: GSE166522) were analyzed.Differentially expressed genes (DEGs) (p value < 0.05, and │Log 2 (fold change)│>1) were subjected to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses using clusterProfiler R package (Version 3.6.2).GO and KEGG terms with adjusted p values of < 0.05 were considered significant enrichment.
For RNA sequencing in BMDMs, cells were infected with S. aureus at a MOI of 10 for 1 h, followed by treatment with 20 μg/mL lysostaphin and 50 μg/mL gentamicin to remove extracellular bacteria.After three washes with PBS, BMDMs were cultured for an additional 24 h.Afterwards, cells were washed with PBS, lysed in Trizol (9108, Takara, Japan), and sent to OmicShare (Guangzhou, China) for next-generation sequencing.RNA was quality-assessed with Agilent 2100 (Agilent, USA) using RNA 6000 Nano kit (5067-1511, Agilent, USA), with RNA integrity number above 9 for library construction.RNA samples from three independent experiments were used for library construction following the protocol of Illumina NovaSeq 6000 platform and sequenced using a Thermal Cycler (Eastwin, Suzhou, China) with HighSensitivity DNA assay Kit (5067-4626, Agilent, USA).FASTq sequencing files were aligned to the mouse reference genome (GRCm38, mm10) using STAR aligner for further analysis.DEGs between S. aureus infected and control BMDMs were identified using limma package (3.54.0).GO analysis of DEGs (p value < 0.05, and │Log 2 (fold change)│>1) was performed with the clusterProfiler package (Version 4.7.1) in R platform (Version 4.2.3, https:// www.r-project.org/).Terms with adjusted p values < 0.05 were considered significantly enriched.Heatmap was plotted using the pheatmap R package (version 1.0.12).

Statistical analysis
Statistical analyses were performed using GraphPad Prism 8 (GraphPad Software, California, USA) or SPSS 26.0 (IBM, New York, USA).Quantifications were based on at least three independent experimental groups.Data were expressed as mean ± standard error of mean (SEM).Significant differences between two groups were evaluated by the Student's t test or Mann-Whitney U test.One-way ANOVA with Tukey's or Dunnett post-hoc test was used to compare multiple groups.A value of p < 0.05 was considered statistically significant.

Fig 5 .Fig 6 .
Fig 5. TLR2 signaling is not responsible for EGFR-MEK1/2 pathway-mediated bactericidal function in BMDMs.(A) Representative images and quantification of western blot for p-MEK1/2 and total MEK1/2 (B), p-EGFR and total EGFR (C) levels in BMDMs.After pretreatment with 100 μM C29 or vehicle (DMSO) for 1 h, cells were infected with S. aureus at MOI of 10, and whole cell lysates were collected at the indicated time points for western blot analysis.**p < 0.01, ***p < 0.001, one-way ANOVA with Tukey's post-hoc test.(D) Quantification of intracellular colonies of S. aureus for phagocytosis assay.BMDMs were pretreated with 100 μM C29 or vehicle (DMSO) for 1 h, followed by S. aureus infection at a MOI of 10 for 1 h.n = 8/group.(E) Quantification of intracellular colonies of S. aureus for the bacterial killing assay.After removing the extracellular bacteria, BMDMs were cultured in growth media with presence or absence of 100 μM C29 for 12 h.n = 8/group, *** p < 0.001, Student's t test.(F) Representative images and (G) Quantification of mitochondrial ROS (mtROS) levels in BMDMs.n = 8/group, * p < 0.05, Student's t test.After removal of the extracellular S. aureus and additional 12 h of culture with 100 μM C29 or vehicle (DMSO), mtROS levels were detected using MitoTracker Green and MitoSOX Red.Nuclei were stained with Hoechst 33342.https://doi.org/10.1371/journal.ppat.1012437.g005