Staphylococcus aureus Panton-Valentine Leukocidin worsens acute implant-associated osteomyelitis in humanized BRGSF mice.

Staphylococcus aureus is the most common pathogen that causes implant-associated osteomyelitis, a clinically incurable disease. Immune evasion of S. aureus relies on various mechanisms to survive within the bone niche, including the secretion of leukotoxins such as Panton-Valentine leukocidin (PVL). PVL is a pore-forming toxin exhibiting selective human tropism for C5a receptors (C5aR1 and C5aR2) and CD45 on neutrophils, monocytes, and macrophages. PVL is an important virulence determinant in lung, skin and soft tissue infections. The involvement of PVL in S. aureus pathogenesis during bone infections has not been studied extensively yet. To investigate this, humanized BALB/c Rag2-/-Il2rg-/-SirpaNODFlk2-/- (huBRGSF) mice were subjected to transtibial implant-associated osteomyelitis with community-acquired methicillin-resistant S. aureus (CA-MRSA) USA300 wild type strain (WT), an isogenic mutant lacking lukF/S-PV (Δpvl), or complemented mutant (Δpvl+pvl). Three days post-surgery, Δpvl-infected huBRGSF mice had a less severe infection compared to WT-infected animals as characterized by 1) improved clinical outcomes, 2) lower ex vivo bacterial bone burden, 3) absence of staphylococcal abscess communities (SACs) in their bone marrow, and 4) compromised MRSA dissemination to internal organs (liver, kidney, spleen, heart). Interestingly, Δpvl-infected huBRGSF mice had fewer human myeloid cells, neutrophils, and HLA-DR+ monocytes in the bone niche compared to WT-infected animals. Expectedly, a smaller fraction of human myeloid cells were apoptotic in the Δpvl-infected huBRGSF animals. Taken together, our study highlights the pivotal role of PVL during acute implant-associated osteomyelitis in humanized mice.

Longitudinal intravital imaging of the bone marrow for 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 h in the Z-plane, which was validated with cross-sectional ex vivo bacterial burden analyses demonstrating significant growth by ~2 × 104 CFU/h on the implant from 2 to 12 h post-op (p < 0.05; r2 > 0.98). Collectively, these results show the competition at the surface is completed by 3 h in this model and demonstrate the potential of LIMB to elucidate mechanisms of bacterial colonization, the host immune response, and the efficacy of antimicrobials.

The 2023 Orthopaedic Research Society's International Consensus Meeting on musculoskeletal infection: Summary from the host immunity section.

Musculoskeletal infections (MSKI), which are a major problem in orthopedics, occur when the pathogen eludes or overwhelms the host immune system. While effective vaccines and immunotherapies to prevent and treat MSKI should be possible, fundamental knowledge gaps in our understanding of protective, nonprotective, and pathogenic host immunity are prohibitive. We also lack critical knowledge of how host immunity is affected by the microbiome, implants, prior infection, nutrition, antibiotics, and concomitant therapies, autoimmunity, and other comorbidities. To define our current knowledge of these critical topics, a Host Immunity Section of the 2023 Orthopaedic Research Society MSKI International Consensus Meeting (ICM) proposed 78 questions. Systematic reviews were performed on 15 of these questions, upon which recommendations with level of evidence were voted on by the 72 ICM delegates, and another 12 questions were voted on with a recommendation of "Unknown" without systematic reviews. Two questions were transferred to another ICM Section, and the other 45 were tabled for future consideration due to limitations of available human resources. Here we report the results of the voting with internet access to the questions, recommendations, and rationale from the systematic reviews. Eighteen questions received a consensus vote of ≥90%, while nine recommendations failed to achieve this threshold. Commentary on why consensus was not achieved on these questions and potential ways forward are provided to stimulate specific funding mechanisms and research on these critical MSKI host defense questions.

Multi-omics analysis identifies IgG2b class-switching with ALCAM-CD6 co-stimulation in joint-draining lymph nodes during advanced inflammatory-erosive arthritis.

Introduction: Defective lymphatic drainage and translocation of B-cells in inflamed (Bin) joint-draining lymph node sinuses are pathogenic phenomena in patients with severe rheumatoid arthritis (RA). However, the molecular mechanisms underlying this lymphatic dysfunction remain poorly understood. Herein, we utilized multi-omic spatial and single-cell transcriptomics to evaluate altered cellular composition (including lymphatic endothelial cells, macrophages, B-cells, and T-cells) in the joint-draining lymph node sinuses and their associated phenotypic changes and cell-cell interactions during RA development using the tumor necrosis factor transgenic (TNF-Tg) mouse model. Methods: Popliteal lymph nodes (PLNs) from wild-type (n=10) and TNF-Tg male mice with "Early" (5 to 6-months of age; n=6) and "Advanced" (>8-months of age; n=12) arthritis were harvested and processed for spatial transcriptomics. Single-cell RNA sequencing (scRNAseq) was performed in PLNs from the TNF-Tg cohorts (n=6 PLNs pooled/cohort). PLN histopathology and ELISPOT along with ankle histology and micro-CT were evaluated. Histopathology of human lymph nodes and synovia was performed for clinical correlation. Results: Advanced PLN sinuses exhibited an increased Ighg2b/Ighm expression ratio (Early 0.5 ± 0.1 vs Advanced 1.4 ± 0.5 counts/counts; p<0.001) that significantly correlated with reduced talus bone volumes in the afferent ankle (R2 = 0.54, p<0.001). Integration of single-cell and spatial transcriptomics revealed the increased IgG2b+ plasma cells localized in MARCO+ peri-follicular medullary sinuses. A concomitant decreased Fth1 expression (Early 2.5 ± 0.74 vs Advanced 1.0 ± 0.50 counts, p<0.001) within Advanced PLN sinuses was associated with accumulation of iron-laden Prussian blue positive macrophages in lymph nodes and synovium of Advanced TNF-Tg mice, and further validated in RA clinical samples. T-cells were increased 8-fold in Advanced PLNs, and bioinformatic pathway assessment identified the interaction between ALCAM+ macrophages and CD6+ T-cells as a plausible co-stimulatory mechanism to promote IgG2b class-switching. Discussion: Collectively, these data support a model of flare in chronic TNF-induced arthritis in which loss of lymphatic flow through affected joint-draining lymph nodes facilitates the interaction between effluxing macrophages and T-cells via ALCAM-CD6 co-stimulation, initiating IgG2b class-switching and plasma cell differentiation of the expanded Bin population. Future work is warranted to investigate immunoglobulin clonality and potential autoimmune consequences, as well as the efficacy of anti-CD6 therapy to prevent these pathogenic events.

Staphylococcus aureus isolates from children with clinically differentiated osteomyelitis exhibit distinct transcriptomic signatures.

There is substantial genomic heterogeneity among Staphylococcus aureus isolates of children with acute hematogenous osteomyelitis (AHO) but transcriptional behavior of clinically differentiated strains has not been previously described. This study evaluates transcriptional activity of S. aureus isolates of children with AHO that may regulate metabolism, biosynthesis, or virulence during bacterial growth and pathogenesis. In vitro growth kinetics were compared between three S. aureus clinical isolates from children with AHO who had mild, moderate, and severe illness. Total RNA sequencing was performed for each isolate at six separate time points throughout the logarithmic phase of growth. The NASA RNA-Sequencing Consensus Pipeline was used to identify differentially expressed genes allowing for 54 comparisons between the three isolates during growth. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment pathways were used to evaluate transcriptional variation in metabolism, biosynthesis pathways and virulence potential of the isolates. The S. aureus isolates demonstrated differing growth kinetics under standardized conditions with the mild isolate having higher optical densities with earlier and higher peak rates of growth than that of the other isolates (p<0.001). Enrichment pathway analysis established distinct transcriptional signatures according to both sampling time and clinical severity. Moderate and severe isolates demonstrated pathways of bacterial invasion, S. aureus infection, quorum sensing and two component systems. In comparison, the mild strain favored biosynthesis and metabolism. These findings suggest that transcriptional regulation during the growth of S. aureus may impact the pathogenetic mechanisms involved in the progression of severity of illness in childhood osteomyelitis. The clinical isolates studied demonstrated a tradeoff between growth and virulence. Further investigation is needed to evaluate these transcriptional pathways in an animal model or during active clinical infections of children with AHO.

Systemic IL-27 administration prevents abscess formation and osteolysis via local neutrophil recruitment and activation.

Interleukin-27 is a pleiotropic cytokine whose functions during bacterial infections remain controversial, and its role in patients with S. aureus osteomyelitis is unknown. To address this knowledge gap, we completed a clinical study and observed elevated serum IL-27 levels (20-fold higher, P < 0.05) in patients compared with healthy controls. Remarkably, IL-27 serum levels were 60-fold higher in patients immediately following septic death than in uninfected patients (P < 0.05), suggesting a pathogenic role of IL-27. To test this hypothesis, we evaluated S. aureus osteomyelitis in WT and IL-27Rα-/- mice with and without exogenous IL-27 induction by intramuscular injection of rAAV-IL-27p28 or rAAV-GFP, respectively. We found that IL-27 was induced at the surgical site within 1 day of S. aureus infection of bone and was expressed by M0, M1 and M2 macrophages and osteoblasts but not by osteoclasts. Unexpectedly, exogenous IL-27p28 (~2 ng·mL-1 in serum) delivery ameliorated soft tissue abscesses and peri-implant bone loss during infection, accompanied by enhanced local IL-27 expression, significant accumulation of RORγt+ neutrophils at the infection site, a decrease in RANK+ cells, and compromised osteoclast formation. These effects were not observed in IL-27Rα-/- mice compared with WT mice, suggesting that IL-27 is dispensable for immunity but mediates redundant immune and bone cell functions during infection. In vitro studies and bulk RNA-seq of infected tibiae showed that IL-27 increased nos1, nos2, il17a, il17f, and rorc expression but did not directly stimulate chemotaxis. Collectively, these results identify a novel phenomenon of IL-27 expression by osteoblasts immediately following S. aureus infection of bone and suggest a protective role of systemic IL-27 in osteomyelitis.

Evidence of Neutralizing and Non-Neutralizing Anti-Glucosaminidase Antibodies in Patients With S. Aureus Osteomyelitis and Their Association With Clinical Outcome Following Surgery in a Clinical Pilot.

Staphylococcus aureus osteomyelitis remains a very challenging condition; recent clinical studies have shown infection control rates following surgery/antibiotics to be ~60%. Additionally, prior efforts to produce an effective S. aureus vaccine have failed, in part due to lack of knowledge of protective immunity. Previously, we demonstrated that anti-glucosaminidase (Gmd) antibodies are protective in animal models but found that only 6.7% of culture-confirmed S. aureus osteomyelitis patients in the AO Clinical Priority Program (AO-CPP) Registry had basal serum levels (>10 ng/ml) of anti-Gmd at the time of surgery (baseline). We identified a small subset of patients with high levels of anti-Gmd antibodies and adverse outcomes following surgery, not explained by Ig class switching to non-functional isotypes. Here, we aimed to test the hypothesis that clinical cure following surgery is associated with anti-Gmd neutralizing antibodies in serum. Therefore, we first optimized an in vitro assay that quantifies recombinant Gmd lysis of the M. luteus cell wall and used it to demonstrate the 50% neutralizing concentration (NC50) of a humanized anti-Gmd mAb (TPH-101) to be ~15.6 µg/ml. We also demonstrated that human serum deficient in anti-Gmd antibodies can be complemented by TPH-101 to achieve the same dose-dependent Gmd neutralizing activity as purified TPH-101. Finally, we assessed the anti-Gmd physical titer and neutralizing activity in sera from 11 patients in the AO-CPP Registry, who were characterized into four groups post-hoc. Group 1 patients (n=3) had high anti-Gmd physical and neutralizing titers at baseline that decreased with clinical cure of the infection over time. Group 2 patients (n=3) had undetectable anti-Gmd antibodies throughout the study and adverse outcomes. Group 3 (n=3) had high titers +/- neutralizing anti-Gmd at baseline with adverse outcomes. Group 4 (n=2) had low titers of non-neutralizing anti-Gmd at baseline with delayed high titers and adverse outcomes. Collectively, these findings demonstrate that both neutralizing and non-neutralizing anti-Gmd antibodies exist in S. aureus osteomyelitis patients and that screening for these antibodies could have a value for identifying patients in need of passive immunization prior to surgery. Future prospective studies to test the prognostic value of anti-Gmd antibodies to assess the potential of passive immunization with TPH-101 are warranted.

Low albumin level is more strongly associated with adverse outcomes and Staphylococcus aureus infection than hemoglobin A1C or smoking tobacco.

Postsurgical deep musculoskeletal infections are a major clinical problem in Orthopaedic Surgery. A serum-based nomogram, which can objectively risk-stratify patients, and aid surgeons in delineating infection risk associated with orthopedic surgical interventions, would be immensely helpful. Here, we constructed a multi-parametric nomogram based on serum anti-Staphylococcus aureus antibody responses, patient characteristics including demographics and standard clinical tests. This nomogram was formally tested in a prospective cohort study comparing 303 hospitalized patients with culture-confirmed S. aureus infection compared with a cohort of 223 healthy screened preoperative patients. Serum anti-S. aureus antibody responses, standard of care clinical tests, and patient demographic data were utilized to perform multivariate logistic regression analysis to quantify the presence of infection and adverse outcome using odds ratios (OR) and to assess predictive ability via area under the ROC curve (AUC). At enrollment, high anti-S. aureus IgG titers were predictive of infection. Remarkably, low serum albumin was found to be significantly associated with infection (OR = 479.963, 95% CI 61.59 - 3740.33, p < 0.0001) and this finding was surprisingly higher than BMI or HbA1c-associations. Combining all risk factors in the nomogram yielded a diagnostic AUC of 0.949 for predicting S. aureus infection. Our results indicate that a serum-based multi-parametric nomogram can be useful in diagnosing S. aureus infections, and importantly, malnourishment is significantly associated with these infections.

Skeletal infections: microbial pathogenesis, immunity and clinical management.

Osteomyelitis remains one of the greatest risks in orthopaedic surgery. Although many organisms are linked to skeletal infections, Staphylococcus aureus remains the most prevalent and devastating causative pathogen. Important discoveries have uncovered novel mechanisms of S. aureus pathogenesis and persistence within bone tissue, including implant-associated biofilms, abscesses and invasion of the osteocyte lacuno-canalicular network. However, little clinical progress has been made in the prevention and eradication of skeletal infection as treatment algorithms and outcomes have only incrementally changed over the past half century. In this Review, we discuss the mechanisms of persistence and immune evasion in S. aureus infection of the skeletal system as well as features of other osteomyelitis-causing pathogens in implant-associated and native bone infections. We also describe how the host fails to eradicate bacterial bone infections, and how this new information may lead to the development of novel interventions. Finally, we discuss the clinical management of skeletal infection, including osteomyelitis classification and strategies to treat skeletal infections with emerging technologies that could translate to the clinic in the future.

Probiotic induced synthesis of microbiota polyamine as a nutraceutical for metabolic syndrome and obesity-related type 2 diabetes.

The gut microbiota regulates multiple facets of host metabolism and immunity through the production of signaling metabolites, such as polyamines which are small organic compounds that are essential to host cell growth and lymphocyte activation. Polyamines are most abundant in the intestinal lumen, where their synthesis by the gut microbiota is influenced by microbiome composition and host diet. Disruption of the host gut microbiome in metabolic syndrome and obesity-related type 2 diabetes (obesity/T2D) results in potential dysregulation of polyamine synthesis. A growing body of evidence suggests that restoration of the dysbiotic gut microbiota and polyamine synthesis is effective in ameliorating metabolic syndrome and strengthening the impaired immune responses of obesity/T2D. In this review, we discuss existing studies on gut microbiome determinants of polyamine synthesis, polyamine production in obesity/T2D, and evidence that demonstrates the potential of polyamines as a nutraceutical in obesity/T2D hosts.

StaphAIR: A Label-Free Antigen Microarray Approach to Detecting Anti-Staphylococcus aureus Antibody Responses in Orthopedic Infections.

Arrayed imaging reflectometry (AIR) is an optical biosensor platform for simple, multiplex measurement of antigen-specific antibody responses in patient blood samples. Here, we report the development of StaphAIR, an 8-plex Staphylococcus aureus antigen array on the AIR platform for profiling antigen-specific anti-S. aureus humoral immune responses. Initial validation experiments with mouse and humanized monoclonal antibodies against the S. aureus autolysin glucosaminidase (Gmd) domain, and subsequent testing with dilution series of pooled positive human serum confirmed analytically robust behavior of the array, with all antigens displaying Langmuir-type dose-response curves. Testing a cohort of 82 patients with S. aureus musculoskeletal infections (MSKI) and 30 healthy individuals enabled discrimination of individual patient responses to different S. aureus antigens, with statistical significance between osteomyelitis patients and controls obtained overall for four individual antigens (IsdA, IsdB, Gmd, and SCIN). Multivariate analyses of the antibody titers obtained from StaphAIR revealed its utility as a potential diagnostic tool for detecting S. aureus MSKI (area under the receiver operating characteristic curve (AUC) > 0.85). We conclude that StaphAIR has utility as a high-throughput immunoassay for studying and diagnosing osteomyelitis in patients.

Staphylococcus aureus Cell Wall Biosynthesis Modulates Bone Invasion and Osteomyelitis Pathogenesis.

Staphylococcus aureus invasion of the osteocyte lacuno-canalicular network (OLCN) is a novel mechanism of bacterial persistence and immune evasion in chronic osteomyelitis. Previous work highlighted S. aureus cell wall transpeptidase, penicillin binding protein 4 (PBP4), and surface adhesin, S. aureus surface protein C (SasC), as critical factors for bacterial deformation and propagation through nanopores in vitro, representative of the confined canaliculi in vivo. Given these findings, we hypothesized that cell wall synthesis machinery and surface adhesins enable durotaxis- and haptotaxis-guided invasion of the OLCN, respectively. Here, we investigated select S. aureus cell wall synthesis mutants (Δpbp3, Δatl, and ΔmreC) and surface adhesin mutants (ΔclfA and ΔsasC) for nanopore propagation in vitro and osteomyelitis pathogenesis in vivo. In vitro evaluation in the microfluidic silicon membrane-canalicular array (µSiM-CA) showed pbp3, atl, clfA, and sasC deletion reduced nanopore propagation. Using a murine model for implant-associated osteomyelitis, S. aureus cell wall synthesis proteins were found to be key modulators of S. aureus osteomyelitis pathogenesis, while surface adhesins had minimal effects. Specifically, deletion of pbp3 and atl decreased septic implant loosening and S. aureus abscess formation in the medullary cavity, while deletion of surface adhesins showed no significant differences. Further, peri-implant osteolysis, osteoclast activity, and receptor activator of nuclear factor kappa-B ligand (RANKL) production were decreased following pbp3 deletion. Most notably, transmission electron microscopy (TEM) imaging of infected bone showed that pbp3 was the only gene herein associated with decreased submicron invasion of canaliculi in vivo. Together, these results demonstrate that S. aureus cell wall synthesis enzymes are critical for OLCN invasion and osteomyelitis pathogenesis in vivo.

Development of Bisphosphonate-Conjugated Antibiotics to Overcome Pharmacodynamic Limitations of Local Therapy: Initial Results with Carbamate Linked Sitafloxacin and Tedizolid.

The use of local antibiotics to treat bone infections has been questioned due to a lack of clinical efficacy and emerging information about Staphylococcus aureus colonization of the osteocyte-lacuno canalicular network (OLCN). Here we propose bisphosphonate-conjugated antibiotics (BCA) using a "target and release" approach to deliver antibiotics to bone infection sites. A fluorescent bisphosphonate probe was used to demonstrate bone surface labeling adjacent to bacteria in a S. aureus infected mouse tibiae model. Bisphosphonate and hydroxybisphosphonate conjugates of sitafloxacin and tedizolid (BCA) were synthesized using hydroxyphenyl and aminophenyl carbamate linkers, respectively. The conjugates were adequately stable in serum. Their cytolytic activity versus parent drug on MSSA and MRSA static biofilms grown on hydroxyapatite discs was established by scanning electron microscopy. Sitafloxacin O-phenyl carbamate BCA was effective in eradicating static biofilm: no colony formation units (CFU) were recovered following treatment with 800 mg/L of either the bisphosphonate or α-hydroxybisphosphonate conjugated drug (p < 0.001). In contrast, the less labile tedizolid N-phenyl carbamate linked BCA had limited efficacy against MSSA, and MRSA. CFU were recovered from all tedizolid BCA treatments. These results demonstrate the feasibility of BCA eradication of S. aureus biofilm on OLCN bone surfaces and support in vivo drug development of a sitafloxacin BCA.

Interleukin-27 and Its Diverse Effects on Bacterial Infections.

Innate and adaptive immune responses against pathogens are known to be carefully orchestrated by specific cytokines that initiate and down regulate immune cell functions from the initial infection through tissue repair and homeostasis. However, some cytokines, including interleukin-27, are expressed at multiple phases of the infection, such that their pro and anti-inflammatory functions have been difficult to interpret. As elucidation of specific cytokine functions throughout infection is central to our understanding of protective vs. susceptible immunity and return to homeostasis vs. prolonged inflammation leading to septic shock, here we review the literature on IL-27 signaling and the various functions of this heterodimeric ligand member of the IL-12 cytokine family. Canonically, IL-27 is produced by antigen-presenting cells, and is thought of as an immunostimulatory cytokine due to its capacity to induce Th1 differentiation. However, many studies have also identified various immunosuppressive effects of IL-27 signaling, including suppression of Th17 differentiation and induction of co-inhibitory receptors on T cells. Thus, the exact role of IL-27 in the context of infectious diseases remains a topic of debate and active research. Additionally, as recent interest has focused on clinical management of acute vs. chronic infections, and life-threatening "cytokine storm" from sepsis, we propose a hypothetical model to explain the biphasic role of IL-27 during the early and late phases of immune responses to reconcile its known pro and anti-inflammatory functions, which could be therapeutically regulated to improve patient outcomes of infection.

Increased myocellular lipid and IGFBP-3 expression in a pre-clinical model of pancreatic cancer-related skeletal muscle wasting.

BACKGROUND: Skeletal muscle wasting (SMW) in cancer patients is associated with increased morbidity, mortality, treatment intolerance and discontinuation, and poor quality of life. This is particularly true for patients with pancreatic ductal adenocarcinoma (PDAC), as over 85% experience SMW, which is responsible for ~30% of patient deaths. While the established paradigm to explain SMW posits that muscle catabolism from systemic inflammation and nutritional deficiencies, the cause of death, and the cellular and molecular mechanisms responsible remain to be elucidated. To address this, we investigated the relationship between tumour burden and survival in the KCKO murine PDAC model. METHODS: Female C57BL/6J mice 6-8 weeks of age underwent orthotopic injection with KCKO-luc tumour cells. Solid tumour was verified on Day 5, post-tumour inoculation. In vivo, longitudinal lean mass and tumour burden were assessed via dual-energy X-ray absorptiometry and IVIS imaging, respectively, and total body weight was assessed, weekly. Animals were sacrificed at a designated end point of 'failure to thrive'. After sacrifice, lower limb hind muscles were harvested for histology and RNA extraction. RESULTS: We found a strong correlation between primary tumour size and survival (r2  = 0.83, P < 0.0001). A significant decrease in lower limb lean mass was first detected at Day 38 post-implantation vs. no tumour controls (NTCs) (P < 0.0001). SMW was confirmed by histology, which demonstrated a 38%, 32.7%, and 39.9% decrease in fibre size of extensor digitorum longus, soleus, and tibialis anterior muscles, respectively, in PDAC mice vs. NTC (P < 0.002). Histology also revealed a 67.6% increase in haematopoietic cells within the muscle of PDAC mice when compared with NTC. Bulk RNAseq on muscles from PDAC mice vs. NTC revealed significant increases in c/ebpß/Δ, il-1, il-6, and tnf gene expression. Pathway analyses to identify potential upstream factors revealed increased adipogenic gene expression, including a four-fold increase in igfbp-3. Histomorphometry of Oil Red-O staining for fat content in tibialis anterior muscles demonstrated a 95.5% increase in positively stained fibres from PDAC mice vs. NTC. CONCLUSIONS: Together, these findings support a novel model of PDAC-associated SMW and mortality in which systemic inflammation leads to inflammatory cell infiltration into skeletal muscle with up-regulated myocellular lipids.

Humanized Mice Exhibit Exacerbated Abscess Formation and Osteolysis During the Establishment of Implant-Associated Staphylococcus aureus Osteomyelitis.

Staphylococcus aureus is the predominant pathogen causing osteomyelitis. Unfortunately, no immunotherapy exists to treat these very challenging and costly infections despite decades of research, and numerous vaccine failures in clinical trials. This lack of success can partially be attributed to an overreliance on murine models where the immune correlates of protection often diverge from that of humans. Moreover, S. aureus secretes numerous immunotoxins with unique tropism to human leukocytes, which compromises the targeting of immune cells in murine models. To study the response of human immune cells during chronic S. aureus bone infections, we engrafted non-obese diabetic (NOD)-scid IL2Rγnull (NSG) mice with human hematopoietic stem cells (huNSG) and analyzed protection in an established model of implant-associated osteomyelitis. The results showed that huNSG mice have increases in weight loss, osteolysis, bacterial dissemination to internal organs, and numbers of Staphylococcal abscess communities (SACs), during the establishment of implant-associated MRSA osteomyelitis compared to NSG controls (p < 0.05). Flow cytometry and immunohistochemistry demonstrated greater human T cell numbers in infected versus uninfected huNSG mice (p < 0.05), and that T-bet+ human T cells clustered around the SACs, suggesting S. aureus-mediated activation and proliferation of human T cells in the infected bone. Collectively, these proof-of-concept studies underscore the utility of huNSG mice for studying an aggressive form of S. aureus osteomyelitis, which is more akin to that seen in humans. We have also established an experimental system to investigate the contribution of specific human T cells in controlling S. aureus infection and dissemination.

Emerging electron microscopy and 3D methodologies to interrogate Staphylococcus aureus osteomyelitis in murine models.

Recent breakthroughs in our understanding of orthopaedic infections have come from advances in transmission electron microscopy (TEM) imaging of murine models of bone infection, most notably Staphylococcus aureus invasion and colonization of osteocyte-lacuno canalicular networks of live cortical bone during the establishment of chronic osteomyelitis. To further elucidate this microbial pathogenesis and evaluate the mechanism of action of novel interventions, additional advances in TEM imaging are needed. Here we present detailed protocols for fixation, decalcification, and epoxy embedment of bone tissue for standard TEM imaging studies, as well as the application of immunoelectron microscopy to confirm S. aureus occupation within sub-micron canaliculi. We also describe the first application of the novel Automated-Tape-UltraMicrotome system with three-dimensional reconstruction and volumetric analyses to quantify S. aureus occupation within the osteocyte-lacuno canalicular networks. Reconstruction of the three-dimensional volume broadened our perspective of S. aureus colonization of the canalicular network and, surprisingly, revealed adjacent noninfected canaliculi. This observation has led us to hypothesize that viable osteocytes of the osteocyte-lacuno canalicular networks respond and resist infection, opening future research directions to explain the paradox of adjacent uninfected canaliculi and life-long deep bone infection in patients with chronic osteomyelitis.

Distinct expression trend of signature antigens of Staphylococcus aureus osteomyelitis correlated with clinical outcomes.

The major limitations of clinical outcome predictions of osteomyelitis mediated by Staphylococcus aureus (S. aureus) are not specific and definitive. To this end, current studies aim to investigate host immune responses of trend changes of the iron-regulated surface determinant (Isd) of IsdA, IsdB, IsdH, cell wall-modifying proteins of amidase (Amd) and glucosaminidase (Gmd), and secreted virulence factor of chemotaxis inhibitory protein S. aureus (CHIPS) and staphylococcal complement inhibitor (SCIN) longitudinally to discover their correlationship with clinical outcomes. A total of 55 patients with confirmed S. aureus infection of the long bone by clinical and laboratory methods were recruited for the study. Whole blood was collected at 0, 6, 12 months for the serum that was used to test IsdA, IsdB, IsdH, Gmd, Amd, CHIPS, and SCIN using a customized Luminex assay after clinical standard care parameters were collected. The patients were then divided into two groups: (1) infection controlled versus (2) adverse outcome based on clinical criteria for statistical analysis. We found that standard clinical parameters were unable to distinguish therapeutic outcomes. Significant overexpression of all antigens was confirmed in infection patients at 0-, 6-, and 12-month time points. A distinct expression trend and dynamic changes of IsdB, Amd, Gmd, and CHIPS were observed between infection controlled and adverse outcome patients, while the IsdA, IsdH, SCIN remained demonstrated no statistical significance. We conclude that dynamic changes of specific antigens could predict clinical outcomes of S. aureus osteomyelitis. Clinical Relevance: The trend changes of host immune responses to S. aureus specific antigens of IsdB, Gmd, Amd, and CHIPS could predict clinical outcomes of S. aureus osteomyelitis.

Serum antibodies against Staphylococcus aureus can prognose treatment success in patients with bone infections.

Prognosing life-threatening orthopedic infections caused by Staphylococcus aureus remains a major clinical challenge. To address this, we developed a multiplex assay to assess the humoral immune proteome against S. aureus in patients with musculoskeletal infections. We found initial evidence that antibodies against some antigens (autolysins: Amd, Gmd; secreted immunotoxins: CHIPS, SCIN, Hla) were associated with protection, whereas antibodies against the iron-regulated surface determinant (Isd) proteins (IsdA, IsdB, IsdH) were aligned with adverse outcomes. To formally test this, we analyzed antibody levels and 1-year clinical outcomes of 194 patients with confirmed S. aureus bone infections (AO Trauma Clinical Priority Program [CPP] Bone Infection Registry). A staggering 20.6% of the enrolled patients experienced adverse clinical outcomes (arthrodesis, reinfection, amputation, and septic death) after 1-year. At enrollment, anti-S. aureus immunoglobulin G (IgG) levels in patients with adverse outcomes were 1.35-fold lower than those in patients whose infections were successfully controlled (p < 0.0001). Overall, there was a 51%-69% reduction in adverse outcome risk for every 10-fold increase in initial IgG concentration against Gmd, Amd, IsdH, CHIPS, SCIN, and Hla (p < 0.05). Notably, anti-IsdB antibodies remained elevated in patients with adverse outcomes; for every 10-fold change in the ratio of circulating anti-Isd to anti-Atl IgG at enrollment, there was a trending 2.6-fold increased risk (odds ratio = 2.555) of an adverse event (p = 0.105). Moreover, antibody increases over time correlated with adverse outcomes and decreases with positive outcomes. These studies demonstrate the potential of the humoral immune response against S. aureus as a prognostic indicator for assessing treatment success and identifying patients requiring additional interventions.

Lack of Humoral Immunity Against Glucosaminidase Is Associated with Postoperative Complications in Staphylococcus aureus Osteomyelitis.

BACKGROUND: Glucosaminidase (Gmd) is known to be a protective antigen in animal models of Staphylococcus aureus osteomyelitis. We compared the endogenous anti-Gmd antibody levels in sera of patients with culture-confirmed S. aureus bone infections to their sera at 1 year after operative treatment of the infection. METHODS: A novel global biospecimen registry of 297 patients with deep-wound culture-confirmed S. aureus osteomyelitis was analyzed to assess relationships between baseline anti-Gmd serum titers (via custom Luminex assay), known host risk factors for infection, and 1-year postoperative clinical outcomes (e.g., infection control, inconclusive, refracture, persistent infection, septic nonunion, amputation, and septic death). RESULTS: All patients had measurable humoral immunity against some S. aureus antigens, but only 20 patients (6.7%; p < 0.0001) had high levels of anti-Gmd antibodies (>10 ng/mL) in serum at baseline. A subset of 194 patients (65.3%) who completed 1 year of follow-up was divided into groups based on anti-Gmd level: low (<1 ng/mL, 54 patients; 27.8%), intermediate (<10 ng/mL, 122 patients; 62.9%), and high (>10 ng/mL, 18 patients; 9.3%), and infection control rates were 40.7%, 50.0%, and 66.7%, respectively. The incidence of adverse outcomes in these groups was 33.3%, 16.4%, and 11.1%, respectively. Assessing anti-Gmd level as a continuous variable showed a 60% reduction in adverse-event odds (p = 0.04) for every tenfold increase in concentration. No differences in patient demographics, body mass index of >40 kg/m, diabetes status, age of ≥70 years, male sex, Charlson Comorbidity Index of >1, or Cierny-Mader host type were observed between groups, and these risk factors were not associated with adverse events. Patients with low anti-Gmd titer demonstrated a significant 2.68-fold increased odds of adverse outcomes (p = 0.008). CONCLUSIONS: Deficiency in circulating anti-Gmd antibodies was associated serious adverse outcomes following operative treatment of S. aureus osteomyelitis. At 1 year, high levels of anti-Gmd antibodies were associated with a nearly 3-fold increase in infection-control odds. Additional prospective studies clarifying Gmd immunization for osteomyelitis are needed. LEVEL OF EVIDENCE: Prognostic Level IV. See Instructions for Authors for a complete description of levels of evidence.