RESUMO
Healthy skin maintains a diverse microbiome and a potent immune system to fight off infections. Here, we discovered that the epithelial-cell-derived antimicrobial peptides defensins activated orphan G-protein-coupled receptors (GPCRs) Mrgpra2a/b on neutrophils. This signaling axis was required for effective neutrophil-mediated skin immunity and microbiome homeostasis. We generated mutant mouse lines lacking the entire Defensin (Def) gene cluster in keratinocytes or Mrgpra2a/b. Def and Mrgpra2 mutant animals both exhibited skin dysbiosis, with reduced microbial diversity and expansion of Staphylococcus species. Defensins and Mrgpra2 were critical for combating S. aureus infections and the formation of neutrophil abscesses, a hallmark of antibacterial immunity. Activation of Mrgpra2 by defensin triggered neutrophil release of IL-1ß and CXCL2 which are vital for proper amplification and propagation of the antibacterial immune response. This study demonstrated the importance of epithelial-neutrophil signaling via the defensin-Mrgpra2 axis in maintaining healthy skin ecology and promoting antibacterial host defense.
Assuntos
Infecções Bacterianas , Neutrófilos , Receptores Acoplados a Proteínas G , Animais , Camundongos , Antibacterianos , Proteínas de Transporte , Defensinas/genética , Disbiose , Queratinócitos , Receptores Acoplados a Proteínas G/metabolismo , Staphylococcus aureusRESUMO
Staphylococcus aureus skin colonization and eosinophil infiltration are associated with many inflammatory skin disorders, including atopic dermatitis, bullous pemphigoid, Netherton's syndrome, and prurigo nodularis. However, whether there is a relationship between S. aureus and eosinophils and how this interaction influences skin inflammation is largely undefined. We show in a preclinical mouse model that S. aureus epicutaneous exposure induced eosinophil-recruiting chemokines and eosinophil infiltration into the skin. Remarkably, we found that eosinophils had a comparable contribution to the skin inflammation as T cells, in a manner dependent on eosinophil-derived IL-17A and IL-17F production. Importantly, IL-36R signaling induced CCL7-mediated eosinophil recruitment to the inflamed skin. Last, S. aureus proteases induced IL-36α expression in keratinocytes, which promoted infiltration of IL-17-producing eosinophils. Collectively, we uncovered a mechanism for S. aureus proteases to trigger eosinophil-mediated skin inflammation, which has implications in the pathogenesis of inflammatory skin diseases.
Assuntos
Dermatite Atópica , Eosinofilia , Infecções Estafilocócicas , Animais , Camundongos , Eosinófilos/metabolismo , Staphylococcus aureus/metabolismo , Peptídeo Hidrolases/metabolismo , Pele/metabolismo , Dermatite Atópica/metabolismo , Infecções Estafilocócicas/metabolismo , Celulite (Flegmão)/metabolismo , Celulite (Flegmão)/patologia , Inflamação/metabolismoRESUMO
Neutrophil extracellular traps (NETs) are a key antimicrobial feature of cellular innate immunity mediated by polymorphonuclear neutrophils (PMNs). NETs counteract microbes but are also linked to inflammation in atherosclerosis, arthritis, or psoriasis by unknown mechanisms. Here, we report that NET-associated RNA (naRNA) stimulates further NET formation in naive PMNs via a unique TLR8-NLRP3 inflammasome-dependent pathway. Keratinocytes respond to naRNA with expression of psoriasis-related genes (e.g., IL17, IL36) via atypical NOD2-RIPK signaling. In vivo, naRNA drives temporary skin inflammation, which is drastically ameliorated by genetic ablation of RNA sensing. Unexpectedly, the naRNA-LL37 'composite damage-associated molecular pattern (DAMP)' is pre-stored in resting neutrophil granules, defining sterile NETs as inflammatory webs that amplify neutrophil activation. However, the activity of the naRNA-LL37 DAMP is transient and hence supposedly self-limiting under physiological conditions. Collectively, upon dysregulated NET release like in psoriasis, naRNA sensing may represent both a potential cause of disease and a new intervention target.
Assuntos
Alarminas , Catelicidinas , Armadilhas Extracelulares , Inflamação , Neutrófilos , Armadilhas Extracelulares/metabolismo , Neutrófilos/metabolismo , Neutrófilos/imunologia , Inflamação/metabolismo , Inflamação/genética , Animais , Humanos , Camundongos , Alarminas/metabolismo , Inflamassomos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Queratinócitos/metabolismo , RNA/genética , RNA/metabolismo , Psoríase/genética , Psoríase/metabolismo , Psoríase/patologia , Transdução de Sinais , Ativação de Neutrófilo/genética , Imunidade Inata/genéticaRESUMO
Biofilm-associated bacterial infections are the major reason for treatment failure in many diseases including burn trauma infections. Uncontrolled inflammation induced by bacteria leads to materiality, tissue damage, and chronic diseases. Specialized proresolving mediators (SPMs), including maresin-like lipid mediators (MarLs), are enzymatically biosynthesized from omega-3 essential long-chain polyunsaturated fatty acids, especially docosahexaenoic acid (DHA), by macrophages and other leukocytes. SPMs exhibit strong inflammation-resolving activities, especially inflammation provoked by bacterial infection. In this study, we explored the potential direct inhibitory activities of three MarLs on Gram-positive (Staphylococcus aureus) and Gram-negative (Pseudomonas aeruginosa and Escherichia coli) bacteria in their biofilms that are leading bacteria in burn trauma-related infections. We also examined the effects of MarLs on the bactericidal activities of a typical broad-spectrum antibiotic, carbenicillin (carb), on these bacteria in their preformed biofilms. The results revealed that MarLs combined with carbenicillin can inhibit the survival of Gram-positive and Gram-negative bacteria in their biofilms although MarLs alone did not exhibit bactericidal activity. Thus, our findings suggest that the combination of MarLs and carbenicillin can lower the antibiotic requirements to kill the bacteria in preformed biofilms.
Assuntos
Queimaduras , Doenças Transmissíveis , Infecções Estafilocócicas , Infecção dos Ferimentos , Humanos , Antibacterianos/farmacologia , Carbenicilina/farmacologia , Bactérias Gram-Negativas , Bactérias Gram-Positivas , Biofilmes , Bactérias , Escherichia coli , Inflamação , Testes de Sensibilidade MicrobianaRESUMO
Phosphodiesterase 4 (PDE4) is highly expressed in keratinocytes and immune cells and promotes pro-inflammatory responses upon activation. The activity of PDE4 has been attributed to various inflammatory conditions, leading to the development and approval of PDE4 inhibitors as host-directed therapeutics in humans. For example, the topical PDE4 inhibitor, crisaborole, is approved for the treatment of mild-to-moderate atopic dermatitis and has shown efficacy in patients with psoriasis. However, the role of crisaborole in regulating the immunopathogenesis of inflammatory skin diseases and infection is not entirely known. Therefore, we evaluated the effects of crisaborole in multiple mouse models, including psoriasis-like dermatitis, AD-like skin inflammation with and without filaggrin mutations, and Staphylococcus aureus skin infection. We discovered that crisaborole dampens myeloid cells and itch in the skin during psoriasis-like dermatitis. Furthermore, crisaborole was effective in reducing skin inflammation in the context of filaggrin deficiency. Importantly, crisaborole reduced S. aureus skin colonization during AD-like skin inflammation. However, crisaborole was not efficacious in treating S. aureus skin infections, even as adjunctive therapy to antibiotics. Taken together, we found that crisaborole reduced itch during psoriasis-like dermatitis and decreased S. aureus skin colonization upon AD-like skin inflammation, which act as additional mechanisms by which crisaborole dampens the immunopathogenesis in mouse models of inflammatory skin diseases. Further examination is warranted to translate these preclinical findings to human disease.
Assuntos
Dermatite Atópica , Inibidores da Fosfodiesterase 4 , Psoríase , Infecções Estafilocócicas , Humanos , Animais , Camundongos , Staphylococcus aureus , Proteínas Filagrinas , Modelos Animais de Doenças , Dermatite Atópica/tratamento farmacológico , Inibidores da Fosfodiesterase 4/uso terapêutico , Prurido/tratamento farmacológico , Psoríase/tratamento farmacológico , Infecções Estafilocócicas/tratamento farmacológico , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/uso terapêutico , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4 , Inflamação/tratamento farmacológicoRESUMO
T cell cytokines contribute to immunity against Staphylococcus aureus, but the predominant T cell subsets involved are unclear. In an S. aureus skin infection mouse model, we found that the IL-17 response was mediated by γδ T cells, which trafficked from lymph nodes to the infected skin to induce neutrophil recruitment, proinflammatory cytokines IL-1α, IL-1ß, and TNF, and host defense peptides. RNA-seq for TRG and TRD sequences in lymph nodes and skin revealed a single clonotypic expansion of the encoded complementarity-determining region 3 amino acid sequence, which could be generated by canonical nucleotide sequences of TRGV5 or TRGV6 and TRDV4 However, only TRGV6 and TRDV4 but not TRGV5 sequences expanded. Finally, Vγ6+ T cells were a predominant γδ T cell subset that produced IL-17A as well as IL-22, TNF, and IFNγ, indicating a broad and substantial role for clonal Vγ6+Vδ4+ T cells in immunity against S. aureus skin infections.
Assuntos
Interleucina-17/fisiologia , Infecções Estafilocócicas/imunologia , Staphylococcus aureus/patogenicidade , Subpopulações de Linfócitos T/imunologia , Linfócitos T/imunologia , Animais , Modelos Animais de Doenças , Humanos , Linfonodos/imunologia , Camundongos , Infecções Estafilocócicas/microbiologiaRESUMO
Fibrosis is a major health burden across diseases and organs. To remedy this, we study wound-induced hair follicle neogenesis (WIHN) as a model of non-fibrotic healing that recapitulates embryogenesis for de novo hair follicle morphogenesis after wounding. We previously demonstrated that TLR3 promotes WIHN through binding wound-associated dsRNA, the source of which is still unclear. Here, we find that multiple distinct contexts of high WIHN all show a strong neutrophil signature. Given the correlation between neutrophil infiltration and endogenous dsRNA release, we hypothesized that neutrophil extracellular traps (NETs) likely release nuclear spliceosomal U1 dsRNA and modulate WIHN. However, rather than enhance regeneration, we find mature neutrophils inhibit WIHN such that mice with mature neutrophil depletion exhibit higher WIHN. Similarly, Pad4 null mice, which are defective in NET production, show augmented WIHN. Finally, using single-cell RNA sequencing, we identify a dramatic increase in mature and activated neutrophils in the wound beds of low regenerating Tlr3-/- mice. Taken together, these results demonstrate that although mature neutrophils are stimulated by a common pro-regenerative cue, their presence and NETs hinder regeneration.
Assuntos
Armadilhas Extracelulares , Neutrófilos/imunologia , Neutrófilos/metabolismo , Regeneração , Animais , Biomarcadores , Imunofluorescência , Perfilação da Expressão Gênica , Humanos , Imunofenotipagem , Camundongos , Camundongos Knockout , Infiltração de Neutrófilos , Análise de Célula Única/métodos , Pele/metabolismo , Cicatrização/genética , Cicatrização/imunologiaRESUMO
IL-17 is a potent proinflammatory cytokine that drives pathogenesis of multiple autoimmune diseases, including psoriasis. A major source of pathogenic IL-17 is a subset of γδ T cells (Tγδ17) that acquires the ability to produce IL-17 while developing in the thymus. The mechanisms that regulate homeostasis of Tγδ17 cells and their roles in psoriasis, however, are not fully understood. In this paper, we show that the heparan sulfate proteoglycan syndecan-1 (sdc1) plays a critical role in regulating homeostasis of Tγδ17 cells and modulating psoriasis-like skin inflammation in mice. sdc1 was predominantly expressed by Tγδ17 cells (but not IL-17- Tγδ cells) in the thymus, lymph nodes, and dermis. sdc1 deficiency significantly and selectively increased the frequency and absolute numbers of Tγδ17 cells by mechanisms that included increased proliferation and decreased apoptosis. Adoptive transfer experiments ruled out a significant role of sdc1 expressed on nonhematopoietic cells in halting expansion and proliferation of sdc1-deficient Tγδ17 cells. When subjected to imiquimod-induced psoriasiform dermatitis, Tγδ17 cells in sdc1KO mice displayed heightened responses accompanied by significantly increased skin inflammation than their wild-type counterparts. Furthermore, transferred sdc1-deficient γδ T cells caused more severe psoriasiform dermatitis than their sdc1-sufficient counterparts in TCR-ßδ KO hosts. The results uncover a novel role for sdc1 in controlling homeostasis of Tγδ17 cells and moderating host responses to psoriasis-like inflammation.
Assuntos
Dermatite/imunologia , Interleucina-17/imunologia , Psoríase/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Sindecana-1/imunologia , Linfócitos T/imunologia , Animais , Dermatite/genética , Dermatite/patologia , Modelos Animais de Doenças , Inflamação/genética , Inflamação/imunologia , Inflamação/patologia , Interleucina-17/genética , Camundongos , Camundongos Knockout , Psoríase/genética , Psoríase/patologia , Receptores de Antígenos de Linfócitos T gama-delta/genética , Sindecana-1/genética , Linfócitos T/patologiaRESUMO
Infection is a major complication of implantable medical devices, which provide a scaffold for biofilm formation, thereby reducing susceptibility to antibiotics and complicating treatment. Hematogenous implant-related infections following bacteremia are particularly problematic because they can occur at any time in a previously stable implant. Herein, we developed a model of hematogenous infection in which an orthopedic titanium implant was surgically placed in the legs of mice followed 3 wk later by an i.v. exposure to Staphylococcus aureus This procedure resulted in a marked propensity for a hematogenous implant-related infection comprised of septic arthritis, osteomyelitis, and biofilm formation on the implants in the surgical legs compared with sham-operated surgical legs without implant placement and with contralateral nonoperated normal legs. Neutralizing human monoclonal antibodies against α-toxin (AT) and clumping factor A (ClfA), especially in combination, inhibited biofilm formation in vitro and the hematogenous implant-related infection in vivo. Our findings suggest that AT and ClfA are pathogenic factors that could be therapeutically targeted against Saureus hematogenous implant-related infections.
Assuntos
Anticorpos Antibacterianos/farmacologia , Anticorpos Neutralizantes/farmacologia , Artrite Infecciosa , Biofilmes/efeitos dos fármacos , Implantes Experimentais/microbiologia , Osteomielite , Infecções Estafilocócicas , Staphylococcus aureus/fisiologia , Animais , Artrite Infecciosa/tratamento farmacológico , Artrite Infecciosa/etiologia , Artrite Infecciosa/microbiologia , Modelos Animais de Doenças , Humanos , Masculino , Camundongos , Osteomielite/tratamento farmacológico , Osteomielite/etiologia , Osteomielite/microbiologia , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/etiologia , Infecções Estafilocócicas/microbiologia , TitânioRESUMO
BACKGROUND: Atopic dermatitis (AD) is associated with epidermal barrier defects, dysbiosis, and skin injury caused by scratching. In particular, the barrier-defective epidermis in patients with AD with loss-of-function filaggrin mutations has increased IL-1α and IL-1ß levels, but the mechanisms by which IL-1α, IL-1ß, or both are induced and whether they contribute to the aberrant skin inflammation in patients with AD is unknown. OBJECTIVE: We sought to determine the mechanisms through which skin injury, dysbiosis, and increased epidermal IL-1α and IL-1ß levels contribute to development of skin inflammation in a mouse model of injury-induced skin inflammation in filaggrin-deficient mice without the matted mutation (ft/ft mice). METHODS: Skin injury of wild-type, ft/ft, and myeloid differentiation primary response gene-88-deficient ft/ft mice was performed, and ensuing skin inflammation was evaluated by using digital photography, histologic analysis, and flow cytometry. IL-1α and IL-1ß protein expression was measured by means of ELISA and visualized by using immunofluorescence and immunoelectron microscopy. Composition of the skin microbiome was determined by using 16S rDNA sequencing. RESULTS: Skin injury of ft/ft mice induced chronic skin inflammation involving dysbiosis-driven intracellular IL-1α release from keratinocytes. IL-1α was necessary and sufficient for skin inflammation in vivo and secreted from keratinocytes by various stimuli in vitro. Topical antibiotics or cohousing of ft/ft mice with unaffected wild-type mice to alter or intermix skin microbiota, respectively, resolved the skin inflammation and restored keratinocyte intracellular IL-1α localization. CONCLUSIONS: Taken together, skin injury, dysbiosis, and filaggrin deficiency triggered keratinocyte intracellular IL-1α release that was sufficient to drive chronic skin inflammation, which has implications for AD pathogenesis and potential therapeutic targets.
Assuntos
Dermatite Atópica/metabolismo , Inflamação/metabolismo , Interleucina-1alfa/metabolismo , Proteínas de Filamentos Intermediários/deficiência , Queratinócitos/metabolismo , Animais , Dermatite Atópica/imunologia , Dermatite Atópica/microbiologia , Disbiose/imunologia , Disbiose/metabolismo , Proteínas Filagrinas , Inflamação/imunologia , Inflamação/microbiologia , Interleucina-1alfa/imunologia , Queratinócitos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos KnockoutRESUMO
Staphylococcus aureus wound infections delay healing and result in invasive complications such as osteomyelitis, especially in the setting of diabetic foot ulcers. In preclinical animal models of S. aureus skin infection, antibody neutralization of alpha-toxin (AT), an S. aureus-secreted pore-forming cytolytic toxin, reduces disease severity by inhibiting skin necrosis and restoring effective host immune responses. However, whether therapeutic neutralization of alpha-toxin is effective against S. aureus-infected wounds is unclear. Herein, the efficacy of prophylactic treatment with a human neutralizing anti-AT monoclonal antibody (MAb) was evaluated in an S. aureus skin wound infection model in nondiabetic and diabetic mice. In both nondiabetic and diabetic mice, anti-AT MAb treatment decreased wound size and bacterial burden and enhanced reepithelialization and wound resolution compared to control MAb treatment. Anti-AT MAb had distinctive effects on the host immune response, including decreased neutrophil and increased monocyte and macrophage infiltrates in nondiabetic mice and decreased neutrophil extracellular traps (NETs) in diabetic mice. Similar therapeutic efficacy was achieved with an active vaccine targeting AT. Taken together, neutralization of AT had a therapeutic effect against S. aureus-infected wounds in both nondiabetic and diabetic mice that was associated with differential effects on the host immune response.
Assuntos
Anticorpos Monoclonais/farmacologia , Anticorpos Neutralizantes/farmacologia , Toxinas Bacterianas/antagonistas & inibidores , Diabetes Mellitus Experimental/imunologia , Proteínas Hemolisinas/antagonistas & inibidores , Infecções Cutâneas Estafilocócicas/tratamento farmacológico , Cicatrização/efeitos dos fármacos , Ferimentos não Penetrantes/tratamento farmacológico , Animais , Carga Bacteriana/efeitos dos fármacos , Toxinas Bacterianas/imunologia , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/microbiologia , Armadilhas Extracelulares/efeitos dos fármacos , Armadilhas Extracelulares/microbiologia , Proteínas Hemolisinas/imunologia , Humanos , Imunidade Inata/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/efeitos dos fármacos , Monócitos/microbiologia , Neutrófilos/efeitos dos fármacos , Neutrófilos/imunologia , Neutrófilos/microbiologia , Pele/efeitos dos fármacos , Pele/imunologia , Pele/microbiologia , Infecções Cutâneas Estafilocócicas/complicações , Infecções Cutâneas Estafilocócicas/imunologia , Infecções Cutâneas Estafilocócicas/microbiologia , Vacinas Antiestafilocócicas/farmacologia , Cicatrização/imunologia , Ferimentos não Penetrantes/complicações , Ferimentos não Penetrantes/imunologia , Ferimentos não Penetrantes/microbiologiaRESUMO
Infections with Staphylococcus aureus are a continuing and growing problem in community and hospital settings. Preclinical animal modeling of S. aureus relies on experimental infection, which carries some limitations. We describe here a novel, spontaneous model of oral staphylococcal infection in double knockout mice, deficient in the receptors for IL-17 (IL-17RA) and interferon (IFN)-γ (IFNγRI), beginning at 6 to 8 weeks of age. IFNγRI(-/-)IL17RA(-/-) (GRAKO) mice developed progressive oral abscesses. Cytometric methods revealed extensive neutrophilic infiltration of oral tissues in GRAKO mice; further investigation evidenced that IL-17 predominated neutrophil defects in these mice. To investigate the contribution of IFN-γ signaling to this native host defense to S. aureus, we observed perturbations of monocyte recruitment and macrophage differentiation in the oral tissues of GRAKO mice, and CXCL9/chemokine ligand receptor (CXCR)3-driven recruitment of T-cell oral tissues and draining lymph nodes. To address the former finding, we depleted macrophages and monocytes in vivo from IL17RA(-/-) mice using liposomes loaded with clodronate. This treatment elicited oral abscesses, recapitulating the phenotype of GRAKO mice. From these findings, we propose novel collaborative functions of IL-17 and IFN-γ, acting through neutrophils and macrophages, respectively, in native mucocutaneous host defenses to S. aureus.
Assuntos
Interferon gama/imunologia , Interleucina-17/imunologia , Mucosa Bucal/imunologia , Mucosa Bucal/microbiologia , Infecções Estafilocócicas/imunologia , Staphylococcus aureus , Animais , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Transdução de Sinais/imunologiaRESUMO
Approximately 20% of the population is persistently colonized by Staphylococcus aureus in the nares. Th17-like immune responses mediated by the interleukin-17 (IL-17) family of cytokines and neutrophils are becoming recognized as relevant host defense mechanisms for resolution of S. aureus mucocutaneous infections. Since antimicrobial peptides are regulated by the IL-17 cytokines, we sought to determine the role of IL-17 cytokines in production of antimicrobial peptides in a murine model of S. aureus nasal carriage. We discovered that nasal tissue supernatants have antistaphylococcal activity, and mice deficient in both IL-17A and IL-17F lost the ability to clear S. aureus nasal colonization. IL-17A was found to be sufficient for nasal mBD-3 production ex vivo and was required for CRAMP, mBD-3, and mBD-14 expression in response to S. aureus colonization in vivo These data were confirmed in a clinical study of nasal secretions in which elevated levels of the human forms of these antimicrobial peptides were found in nasal secretions from healthy human subjects when they were colonized with S. aureus but not in secretions from noncolonized subjects. Together, these data provide evidence for the importance of IL-17A regulation of antimicrobial peptides and IL-17F in the clearance of S. aureus nasal carriage.
Assuntos
Peptídeos Catiônicos Antimicrobianos/metabolismo , Portador Sadio/imunologia , Interleucina-17/metabolismo , Nariz/microbiologia , Staphylococcus aureus , Adulto , Animais , Peptídeos Catiônicos Antimicrobianos/genética , Regulação da Expressão Gênica/fisiologia , Humanos , Interleucina-17/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Regulação para Cima , beta-DefensinasRESUMO
Skin homeostasis relies on a delicate balance between host proteases and protease inhibitors along with those secreted from microbial communities, as disruption to this harmony contributes to the pathogenesis of inflammatory skin disorders, including atopic dermatitis and Netherton's syndrome. In addition to being a prominent cause of skin and soft tissue infections, the gram-positive bacterium Staphylococcus aureus is a key player in inflammatory skin conditions due to its array of 10 secreted proteases. Herein we review how S. aureus proteases augment the development of inflammation in skin disorders. These mechanisms include degradation of skin barrier integrity, immune dysregulation and pruritis, and impairment of host defenses. Delineating the diverse roles of S. aureus proteases has the potential to reveal novel therapeutic strategies, such as inhibitors of proteases or their cognate target, as well as neutralizing vaccines to alleviate the burden of inflammatory skin disorders in patients.
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INTRODUCTION: The U.S. Military members experiencing combat-related injuries have a higher chance of developing infections by multidrug-resistant (MDR) bacteria at admission to military hospitals. MDR wound infections result in higher amputation rates and greater risks for subsequent or chronic infections that require readmission or extended stay in the hospital. Currently, there is no FDA-clear, deployable early diagnostic system for suitable field use.We are reporting our efforts to improve a previously developed Rapid Label-free Pathogen Identification (RAPID) system to detect viable MDR bacteria in wound infections and perform antibiotic susceptibility testing (AST). Specifically, we added multiplex and automation capability and significantly simplified the sample preparation process. A functional prototype of the improved system was built, and its performance was validated using a variety of lab-prepared spiked samples and real-world samples. MATERIALS AND METHODS: To access the baseline performance of the improved RAPID system in detecting bacteria presence, we selected 17 isolates, most of them from blood or wound infections, and prepared mono-strain spiked samples at 104 to 106 cfu/mL concentration. These samples were processed and analyzed by the RAPID system. To demonstrate the AST capability of the system, we selected 6 strains against 6 different antibiotics and compared the results from the system with the ones from the gold standard method.To validate the system's performance with real-world samples, we first investigated its performance on 3 swab samples from epicutaneous methicillin-resistant Staphylococcus aureus-exposed mouse model. The AST results from our system were compared with the ones from the gold standard method. All animal experiments were approved by the Johns Hopkins University Animal Care and Use Committee (Protocol No. MO21M378). Then, we obtained swab samples from 7 atopic dermatitis (AD) patients and compared our AST results with the ones from the gold standard method. The human subject protocol was approved by the Johns Hopkins Medicines Institutional Review Boards (Study No. CR00043438/IRB00307926) and by USAMRDC (Proposal Log Number/Study Number 20000251). RESULTS: High-quality data were obtained from the spiked samples of all 17 strains. A quantitative analysis model built using these data achieved 94% accuracy in predicting the species ID in 8 unknown samples. The AST results on the spiked samples had shown 100% matching with the gold standard method. Our system successfully detects the presence/absence of viable bacteria in all 3 mouse and 7 AD patient swab samples. Our system shows 100% and 85.7% (6 out of 7) accuracy when compared to the oxacillin susceptibility testing results for the mouse and the AD patient swabs, respectively. CONCLUSIONS: Our system has achieved excellent performance in detecting viable bacteria presence and in performing AST in a multiplex, automated, and easy-to-operate manner, on both lab-prepared and real samples. Our results have shown a path forward to a rapid (sample-to-answer time ≤3 hours), accurate, sensitive, species-specific, and portable system to detect the presence of MDR combat-related wound infections in the field environment. Our future efforts involve ruggedizing the RAPID system and evaluating performance under relevant environmental conditions.
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Staphylococcus aureus is the leading cause of skin and soft tissue infections (SSTIs) in the U.S. as well as more serious invasive diseases, including bacteremia, sepsis, endocarditis, surgical site infections, osteomyelitis, and pneumonia. These infections are exacerbated by the emergence of antibiotic-resistant clinical isolates such as methicillin-resistant S. aureus (MRSA), highlighting the need for alternatives to antibiotics to treat bacterial infections. We have previously developed a multi-component toxoid vaccine (IBT-V02) in a liquid formulation with efficacy against multiple strains of Staphylococcus aureus prevalent in the industrialized world. However, liquid vaccine formulations are not compatible with the paucity of cold chain storage infrastructure in many low-to-middle income countries (LMICs). Furthermore, whether our IBT-V02 vaccine formulations are protective against S. aureus isolates from LMICs is unknown. To overcome these limitations, we developed lyophilized and spray freeze-dried formulations of IBT-V02 vaccine and demonstrated that both formulations had comparable biophysical attributes as the liquid formulation, including similar levels of toxin neutralizing antibodies and protective efficacy against MRSA infections in murine and rabbit models. To enhance the relevancy of our findings, we then performed a multi-dimensional screen of 83 S. aureus clinical isolates from LMICs (e.g., Democratic Republic of Congo, Palestine, and Cambodia) to rationally down-select strains to test in our in vivo models based on broad expression of IBT-V02 targets (i.e., pore-forming toxins and superantigens). IBT-V02 polyclonal antisera effectively neutralized toxins produced by the S. aureus clinical isolates from LMICs. Notably, the lyophilized IBT-V02 formulation exhibited significant in vivo efficacy in various preclinical infection models against the S. aureus clinical isolates from LMICs, which was comparable to our liquid formulation. Collectively, our findings suggested that lyophilization is an effective alternative to liquid vaccine formulations of our IBT-V02 vaccine against S. aureus infections, which has important implications for protection from S. aureus isolates from LMICs.
Assuntos
Staphylococcus aureus Resistente à Meticilina , Infecções Estafilocócicas , Animais , Camundongos , Coelhos , Staphylococcus aureus , Países em Desenvolvimento , Antibacterianos , Vacinas Bacterianas , ToxoidesRESUMO
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.
Assuntos
Procedimentos Ortopédicos , Ortopedia , Humanos , Consenso , Antibacterianos/uso terapêutico , ImunoterapiaRESUMO
The anterior nares of humans are the major reservoir for Staphylococcus aureus colonization. Approximately 20% of the healthy human population is persistently and 80% is intermittently colonized with S. aureus in the nasal cavity. Previous studies have shown a strong causal connection between S. aureus nasal carriage and increased risk of nosocomial infection, as well as increased carriage due to immune dysfunction. However, the immune responses that permit persistence or mediate clearance of S. aureus on the nasal mucosa are fundamentally undefined. In this study, we developed a carriage model in C57BL/6J mice and showed that clearance begins 14 days postinoculation. In contrast, SCID mice that have a deficient adaptive immune response are unable to eliminate S. aureus even after 28 days postinoculation. Furthermore, decolonization was found to be T cell mediated but B cell independent by evaluating carriage clearance in T-cell receptor ß/δ (TCR-ß/δ) knockout (KO) and IgH-µ KO mice, respectively. Upregulation of the cytokines interleukin 1ß (IL-1ß), KC (also termed CXC ligand 1 [CXCL1]), and IL-17A occurred following inoculation with intranasal S. aureus. IL-17A production was crucial for clearance, since IL-17A-deficient mice were unable to effectively eliminate S. aureus carriage. Subsequently, cell differential counts were evaluated from nasal lavage fluid obtained from wild-type and IL-17A-deficient colonized mice. These counts displayed IL-17A-dependent neutrophil migration. Antibody-mediated depletion of neutrophils in colonized mice caused reduced clearance compared to that in isotype-treated controls. Our data suggest that the Th17-associated immune response is required for nasal decolonization. This response is T cell dependent and mediated via IL-17A production and neutrophil influx. Th17-associated immune responses may be targeted for strategies to mitigate distal infections originating from persistent S. aureus carriage in humans.
Assuntos
Interleucina-17/metabolismo , Neutrófilos/fisiologia , Nariz/microbiologia , Infecções Estafilocócicas/tratamento farmacológico , Staphylococcus aureus/fisiologia , Linfócitos T/fisiologia , Animais , Linfócitos B , Portador Sadio/imunologia , Portador Sadio/microbiologia , Regulação Bacteriana da Expressão Gênica , Humanos , Interleucina-17/genética , Interleucina-23/genética , Interleucina-23/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Nariz/imunologia , Infecções Estafilocócicas/imunologia , Staphylococcus aureus/imunologiaRESUMO
In this issue of Cell Host & Microbe, Kashaf et al. and Key et al. examine isolates of Staphylococcus aureus among individuals with atopic dermatitis, revealing insights into evolution, antibiotic resistance, transmission mechanisms, skin colonization, and virulence factors. These findings further our understanding of disease pathogenesis and potential treatments.
Assuntos
Dermatite Atópica , Infecções Estafilocócicas , Humanos , Dermatite Atópica/patologia , Staphylococcus , Pele/patologia , Staphylococcus aureusRESUMO
Staphylococcus aureus is a leading cause of bacteremia, further complicated by the emergence of antibiotic-resistant strains such as methicillin-resistant S. aureus (MRSA). A better understanding of host defense mechanisms is needed for the development of host-directed therapies as an alternative approach to antibiotics. The levels of IL-1, IL-17, and TNF-α cytokines in circulation have been associated with predictive outcomes in patients with S. aureus bacteremia. However, their causative role in survival and the cell types involved in these responses during bacteremia is not entirely clear. Using a mouse model of S. aureus bacteremia, we demonstrated that IL-17A/F and TNF-α had no significant impact on survival, whereas IL-1R signaling was critical for survival during S. aureus bacteremia. Furthermore, we identified that T cells, but not neutrophils, monocytes/macrophages, or endothelial cells were the crucial cell type for IL-1R-mediated survival against S. aureus bacteremia. Finally, we determined that the expression of IL-1R on γδ T cell, but not CD4+ or CD8+ T cells was responsible for survival against the S. aureus bacteremia. Taken together, we uncovered a role for IL-1R, but not IL-17A/F and TNF-α in protection against S. aureus bacteremia. Importantly, γδ T cell-intrinsic expression of IL-1R was crucial for survival, but not on other immune cells or endothelial cells. These findings reveal potential cellular and immunological targets for host-directed therapies for improved outcomes against S. aureus bacteremia.