Murine Osteomyelitis Model Research Articles

Longitudinal intravital imaging of the bone marrowfor analysis of the race for the surface in a murine osteomyelitis model.

Critical knowledge gaps of orthopedic infections pertain to bacterial colonization. The established dogma termed the Race for the Surface posits that contaminating bacteria compete with host cells for the implant post-op, which remains unproven without real-time in vivo evidence. Thus, we modified the murine longitudinal intravital imaging of the bone marrow (LIMB) system to allow real-time quantification of green fluorescent protein(GFP+) host cells and enhanced cyan fluorescent protein(ECFP+) or red fluorescent protein(RFP+) methicillin-resistant Staphylococcus aureus (MRSA) proximal to a transfemoral implant. Following inoculation with ~105 CFU, an L-shaped metal implant was press-fit through the lateral cortex at a 90° angle ~0.150 mm below a gradient refractive index (GRIN) lens. We empirically derived a volume of interest (VOI) = 0.0161 ± 0.000675 mm3 during each imaging session by aggregating the Z-stacks between the first (superior) and last (inferior) in-focus LIMB slice. LIMB postimplantation revealed very limited bacteria detection at 1 h, but by 3 h,56.8% of the implant surface was covered by ECFP+ bacteria, and the rest were covered by GFP+ host cells. 3D volumetric rendering of the GFP+ and ECFP+ or RFP+ voxels demonstrated exponential MRSA growth between 3 and 6 hin the Z-plane, which was validated with cross-sectional ex vivo bacterial burden analyses demonstrating significant growth by ~2 × 104 CFU/hon the implant from 2 to 12 hpost-op (p < 0.05; r2 > 0.98). Collectively, these results show the competition at the surface is completed by 3 hin this model and demonstrate the potential of LIMB to elucidate mechanisms of bacterial colonization, the host immune response, and the efficacy of antimicrobials.

Global Transcriptomic Study of Circular-RNA Expression Profile in Osteoclasts Infected by Intracellular Staphylococcus aureus.

Osteomyelitis is difficult to cure, and the rapidly rising morbidity is a thorny problem accompanied by a large number of joint replacement applications. Staphylococcus aureus is the main pathogen of osteomyelitis. Circular RNAs (circRNAs), as emerging noncoding RNAs, play important roles in multiple physiopathological processes which could provide novel insights into osteomyelitis. However, little is known about the roles of circRNAs in the pathogenesis of osteomyelitis. Osteoclasts, considered bone sentinels, are the resident macrophages in bone and may play the immune defense roles in osteomyelitis. It has been reported that S. aureus can survive in osteoclasts, but the function of osteoclast circRNAs in response to intracellular S. aureus infection remains unclear. In this study, we investigated the profile of circRNAs in osteoclasts infected by intracellular S. aureus through high-throughput RNA sequencing. In total, 24 upregulated and 62 downregulated differentially expressed circRNAs were identified and subsequently analyzed to demonstrate their potential functions. On this basis, three circRNAs (chr4:130718154-130728164+, chr8:77409548-77413627-, and chr1:190871592-190899571-) were confirmed as potential novel biomarkers for the diagnosis of osteomyelitis through the murine model of osteomyelitis. Most importantly, we verified that the circRNA chr4:130718154-130728164+ named circPum1 could regulate the host autophagy to affect the intracellular infection of S. aureus through miR-767. In addition, circPum1 could serve as a promising serum biomarker in osteomyelitis patients caused by S. aureus infection. Taken together, this study provided the first global transcriptomic profile analysis of circRNAs in osteoclasts infected by intracellular S. aureus and first proposed a novel perspective for the pathogenesis and immunotherapy of S. aureus-induced osteomyelitis from the term of circRNAs.

The major role of sarA in limiting Staphylococcus aureus extracellular protease production in vitro is correlated with decreased virulence in diverse clinical isolates in osteomyelitis

ABSTRACT We previously demonstrated that MgrA, SarA, SarR, SarS, SarZ, and Rot bind at least three of the four promoters associated with genes encoding primary extracellular proteases in Staphylococcus aureus (Aur, ScpA, SspA/SspB, SplA-F). We also showed that mutation of sarA results in a greater increase in protease production, and decrease in biofilm formation, than mutation of the loci encoding any of these other proteins. However, these conclusions were based on in vitro studies. Thus, the goal of the experiments reported here was to determine the relative impact of the regulatory loci encoding these proteins in vivo. To this end, we compared the virulence of mgrA, sarA, sarR, sarS, sarZ, and rot mutants in a murine osteomyelitis model. Mutants were generated in the methicillin-resistant USA300 strain LAC and the methicillin-sensitive USA200 strain UAMS-1, which was isolated directly from the bone of an osteomyelitis patient during surgical debridement. Mutation of mgrA and rot limited virulence to a statistically significant extent in UAMS-1, but not in LAC, while the sarA mutant exhibited reduced virulence in both strains. The reduced virulence of the sarA mutant was correlated with reduced cytotoxicity for osteoblasts and osteoclasts, reduced biofilm formation, and reduced sensitivity to the antimicrobial peptide indolicidin, all of which were directly attributable to increased protease production in both LAC and UAMS-1. These results illustrate the importance of considering diverse clinical isolates when evaluating the impact of regulatory mutations on virulence and demonstrate the significance of SarA in limiting protease production in vivo in S. aureus.

Staphylococcus aureus internalization impairs osteoblastic activity and early differentiation process

Staphylococcus aureus is the most frequent aetiology of bone and joint infections (BJI) and can cause relapsing and chronic infections. One of the main factors involved in the chronicization of staphylococcal BJIs is the internalization of S. aureus into osteoblasts, the bone-forming cells. Previous studies have shown that S. aureus triggers an impairment of osteoblasts function that could contribute to bone loss. However, these studies focused mainly on the extracellular effects of S. aureus. Our study aimed at understanding the intracellular effects of S. aureus on the early osteoblast differentiation process. In our in vitro model of osteoblast lineage infection, we first observed that internalized S. aureus 8325-4 (a reference lab strain) significantly impacted RUNX2 and COL1A1 expression compared to its non-internalized counterpart 8325-4∆fnbAB (with deletion of fnbA and fnbB). Then, in a murine model of osteomyelitis, we reported no significant effect for S. aureus 8325-4 and 8325-4∆fnbAB on bone parameters at 7 days post-infection whereas S. aureus 8325-4 significantly decreased trabecular bone thickness at 14 days post-infection compared to 8325-4∆fnbAB. When challenged with two clinical isogenic strains isolated from initial and relapse phase of the same BJI, significant impairments of bone parameters were observed for both initial and relapse strain, without differences between the two strains. Finally, in our in vitro osteoblast infection model, both clinical strains impacted alkaline phosphatase activity whereas the expression of bone differentiation genes was significantly decreased only after infection with the relapse strain. Globally, we highlighted that S. aureus internalization into osteoblasts is responsible for an impairment of the early differentiation in vitro and that S. aureus impaired bone parameters in vivo in a strain-dependent manner.

CHI3L1 promotes Staphylococcus aureus-induced osteomyelitis by activating p38/MAPK and Smad signaling pathways

AimsStaphylococcus aureus (S. aureus) is the most common causative bacterial pathogen involved in promoting infection-induced osteomyelitis, a disease resulting in severe bone degradation. In this study, we aimed to identify the mechanism behind inhibition of osteoclast survival and differentiation by CHI3L1, a lectin previously reported to regulate S. aureus-induced osteomyelitis. Main methodsThe role of CHI3L1 in osteoclast survival, proliferation, and differentiation was studied ex vivo using primary human bone marrow derived stem cells (HBMSCs) and transducing them with lentiviral expression vectors or shRNA knockdown constructs. Cell apoptosis was analyzed by flow cytometry using annexin V-fluorescein isothiocyanate/propidium iodide staining. Cell proliferation was assessed using alkaline phosphatase, Alcian Blue, and TRAP staining. The qRT-PCR was used to measure mRNA levels of osteoclast maturation markers, and western blotting was used to analyze protein expression. An in vivo murine model for osteomyelitis and microcomputed tomography analyses of infected femurs were used to study the effects of CHI3L1 on bone erosion. Key findingsOverexpression of CHI3L1 significantly reduced HBMSC cell viability, proliferation, and differentiation, and knockdown improved these effects in the presence of S. aureus infection. More specifically, CHI3L1 constitutively activated the p38/MAPK pathway to promote apoptosis. Furthermore, CHI3L1 induced activation of the Smad pathway by promoting phosphorylation of Smad-1/5 proteins. Finally, overexpression of CHI3L1 significantly induced bone erosion upon S. aureus infection in a murine osteomyelitis model, and knockdown of CHI3L1 significantly alleviated this effect. SignificanceCHI3L1 played a vital role in osteoblast differentiation and proliferation by regulating the p38/MAPK and Smad signaling pathways to promote S. aureus-induced osteomyelitis.

Concurrent Local Delivery of Diflunisal Limits Bone Destruction but Fails To Improve Systemic Vancomycin Efficacy during Staphylococcus aureus Osteomyelitis.

Staphylococcus aureus osteomyelitis is a debilitating infection of bone. Treatment of osteomyelitis is impaired by the propensity of invading bacteria to induce pathological bone remodeling that may limit antibiotic penetration to the infectious focus. The nonsteroidal anti-inflammatory drug diflunisal was previously identified as an osteoprotective adjunctive therapy for osteomyelitis, based on the ability of this compound to inhibit S. aureus quorum sensing and subsequent quorum-dependent toxin production. When delivered locally during experimental osteomyelitis, diflunisal significantly limits bone destruction without affecting bacterial burdens. However, because diflunisal's "quorum-quenching" activity could theoretically increase antibiotic recalcitrance, it is critically important to evaluate this adjunctive therapy in the context of standard-of-care antibiotics. The objective of this study is to evaluate the efficacy of vancomycin to treat osteomyelitis during local diflunisal treatment. We first determined that systemic vancomycin effectively reduces bacterial burdens in a murine model of osteomyelitis and identified a dosing regimen that decreases bacterial burdens without eradicating infection. Using this dosing scheme, we found that vancomycin activity is unaffected by the presence of diflunisal in vitro and in vivo Similarly, locally delivered diflunisal still potently inhibits osteoblast cytotoxicity in vitro and bone destruction in vivo in the presence of subtherapeutic vancomycin. However, we also found that the resorbable polyester urethane (PUR) foams used to deliver diflunisal serve as a nidus for infection. Taken together, these data demonstrate that diflunisal does not significantly impact standard-of-care antibiotic therapy for S. aureus osteomyelitis, but they also highlight potential pitfalls encountered with local drug delivery.

Host nutrient milieu drives an essential role for aspartate biosynthesis during invasive Staphylococcus aureus infection

The bacterial pathogen Staphylococcus aureus is capable of infecting a broad spectrum of host tissues, in part due to flexibility of metabolic programs. S. aureus, like all organisms, requires essential biosynthetic intermediates to synthesize macromolecules. We therefore sought to determine the metabolic pathways contributing to synthesis of essential precursors during invasive S. aureus infection. We focused specifically on staphylococcal infection of bone, one of the most common sites of invasive S. aureus infection and a unique environment characterized by dynamic substrate accessibility, infection-induced hypoxia, and a metabolic profile skewed toward aerobic glycolysis. Using a murine model of osteomyelitis, we examined survival of S. aureus mutants deficient in central metabolic pathways, including glycolysis, gluconeogenesis, the tricarboxylic acid (TCA) cycle, and amino acid synthesis/catabolism. Despite the high glycolytic demand of skeletal cells, we discovered that S. aureus requires glycolysis for survival in bone. Furthermore, the TCA cycle is dispensable for survival during osteomyelitis, and S. aureus instead has a critical need for anaplerosis. Bacterial synthesis of aspartate in particular is absolutely essential for staphylococcal survival in bone, despite the presence of an aspartate transporter, which we identified as GltT and confirmed biochemically. This dependence on endogenous aspartate synthesis derives from the presence of excess glutamate in infected tissue, which inhibits aspartate acquisition by S. aureus Together, these data elucidate the metabolic pathways required for staphylococcal infection within bone and demonstrate that the host nutrient milieu can determine essentiality of bacterial nutrient biosynthesis pathways despite the presence of dedicated transporters.

MyD88 and IL-1R signaling drive antibacterial immunity and osteoclast-driven bone loss during Staphylococcus aureus osteomyelitis.

Staphylococcus aureus is able to infect virtually all organ systems and is a frequently isolated etiologic agent of osteomyelitis, a common and debilitating invasive infection of bone. Treatment of osteomyelitis requires invasive surgical procedures and prolonged antibiotic therapy, yet is frequently unsuccessful due to extensive pathogen-induced bone damage that can limit antibiotic penetration and immune cell influx to the infectious focus. We previously established that S. aureus triggers profound alterations in bone remodeling in a murine model of osteomyelitis, in part through the production of osteolytic toxins. However, staphylococcal strains lacking osteolytic toxins still incite significant bone destruction, suggesting that host immune responses are also major drivers of pathologic bone remodeling during osteomyelitis. The objective of this study was to identify host immune pathways that contribute to antibacterial immunity during S. aureus osteomyelitis, and to define how these immune responses alter bone homeostasis and contribute to bone destruction. We specifically focused on the interleukin-1 receptor (IL-1R) and downstream adapter protein MyD88 given the prominent role of this signaling pathway in both antibacterial immunity and osteo-immunologic crosstalk. We discovered that while IL-1R signaling is necessary for local control of bacterial replication during osteomyelitis, it also contributes to bone loss during infection. Mechanistically, we demonstrate that S. aureus enhances osteoclastogenesis of myeloid precursors in vitro, and increases the abundance of osteoclasts residing on bone surfaces in vivo. This enhanced osteoclast abundance translates to trabecular bone loss, and is dependent on intact IL-1R signaling. Collectively, these data define IL-1R signaling as a critical component of the host response to S. aureus osteomyelitis, but also demonstrate that IL-1R-dependent immune responses trigger collateral bone damage through activation of osteoclast-mediated bone resorption.