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
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
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
Surgical site infections (SSIs) are commonly caused by Staphylococcus aureus We report that a combination of three monoclonal antibodies (MEDI6389) that neutralize S. aureus alpha-toxin, clumping factor A, and four leukocidins (LukSF, LukED, HlgAB, and HlgCB) plus vancomycin had enhanced efficacy compared with control antibody plus vancomycin in two mouse models of S. aureus SSI. Therefore, monoclonal antibody-based neutralization of multiple S. aureus virulence factors may provide an adjunctive perioperative approach to combat S. aureus SSIs.
Assuntos
Antibacterianos/farmacologia , Anticorpos Monoclonais/farmacologia , Infecções Estafilocócicas/tratamento farmacológico , Infecção da Ferida Cirúrgica/tratamento farmacológico , Animais , Anticorpos Monoclonais Humanizados/farmacologia , Proteínas de Bactérias/imunologia , Anticorpos Amplamente Neutralizantes/farmacologia , Coagulase/imunologia , Leucocidinas/imunologia , Staphylococcus aureus Resistente à Meticilina/patogenicidade , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Infecções Estafilocócicas/microbiologia , Infecção da Ferida Cirúrgica/microbiologia , Vancomicina/farmacologiaRESUMO
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
C-C motif chemokine receptor 2 (CCR2) is an important mediator of myeloid cell chemotaxis during inflammation and infection. Myeloid cells such as monocytes, macrophages, and neutrophils contribute to host defense during orthopedic implant-associated infections (OIAI), but whether CCR2-mediated chemotaxis is involved remains unclear. Therefore, a Staphylococcus aureus OIAI model was performed by surgically placing an orthopedic-grade titanium implant and inoculating a bioluminescent S. aureus strain in knee joints of wildtype (wt) and CCR2-deficient mice. In vivo bioluminescent signals significantly increased in CCR2-deficient mice compared with wt mice at later time points (Days 14-28), which was confirmed with ex vivo colony-forming unit enumeration. S. aureus γ-hemolysin utilizes CCR2 to induce host cell lysis. However, there were no differences in bacterial burden when the OIAI model was performed with a parental versus a mutant γ-hemolysin-deficient S. aureus strain, indicating that the protection was mediated by the host cell function of CCR2 rather than γ-hemolysin virulence. Although CCR2-deficient and wt mice had similar cellular infiltrates in the infected joint tissue, CCR2-deficient mice had reduced myeloid cells and γδ T cells in the draining lymph nodes. Taken together, CCR2 contributed to host defense at later time points during an OIAI by increasing immune cell infiltrates in the draining lymph nodes, which likely contained the infection and prevented invasive spread.
Assuntos
Infecções Estafilocócicas , Staphylococcus aureus , Animais , Proteínas Hemolisinas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores CCR2 , Receptores de QuimiocinasRESUMO
Signal transducer and activator of transcription 3 (STAT3) is important for psoriasis pathogenesis because STAT3 signaling downstream of IL-6, IL-21, IL-22, and IL-23 contributes to T helper type 17 cell development and because transgenic mice with keratinocyte (KC) STAT3 expression (K14-Stat3C mice) develop psoriasis-like dermatitis. In this study, the relative contribution of STAT3 signaling in KCs versus in T cells was evaluated in the imiquimod model of psoriasis-like dermatitis. Mice with STAT3-inducible deletion in KCs (K5-Stat3-/- mice) had decreased psoriasis-like dermatitis and epidermal STAT3 phosphorylation compared with wild-type mice, whereas mice with constitutive deletion of STAT3 in all T cells were similar to wild-type mice. Interestingly, mice with KC-inducible deletion of IL-6Rα had similar findings to those of K5-Stat3-/- mice, identifying IL-6/IL-6R as a predominant upstream signal for KC STAT3-induced psoriasis-like dermatitis. Moreover, psoriasis-like dermatitis inversely associated with type 1 immune gene products, especially CXCL10, whereas CXCL10 limited psoriasis-like dermatitis, suggesting that KC STAT3 signaling promoted psoriasis-like dermatitis by restricting downstream CXCL10 expression. Finally, treatment of mice with the pan-Jak inhibitor, tofacitinib, reduced psoriasis-like dermatitis and epidermal STAT3 phosphorylation. Taken together, STAT3 signaling in KCs rather than in T cells was a more important determinant for psoriasis-like dermatitis in a mechanism that involved upstream KC IL-6R signaling and downstream inhibition of type 1 immunityâassociated CXCL10 responses.
Assuntos
Dermatite , Psoríase , Animais , Quimiocina CXCL10 , Dermatite/patologia , Modelos Animais de Doenças , Interleucina-6/metabolismo , Queratinócitos/metabolismo , Camundongos , Camundongos Transgênicos , Receptores de Interleucina-6 , Fator de Transcrição STAT3/metabolismo , Linfócitos T/metabolismoRESUMO
Staphylococcus aureus is an important cause of various infections in humans, including bacteremia, skin and soft tissue infections, and infections associated with implanted medical devices. The emergence of hospital- and community-acquired methicillin-resistant Staphylococcus aureus (MRSA) underscores the urgent and unmet need to develop novel, safe, and effective antibiotics against these multidrug-resistant clinical isolates. Oxazolidinone antibiotics such as linezolid have excellent oral bioavailability and provide coverage against MRSA infections. However, their widespread and long-term use is often limited by adverse effects, especially myelosuppression. TBI-223 is a novel oxazolidinone with potentially reduced myelosuppression, compared to linezolid, but its efficacy against MRSA infections is unknown. Therefore, the preclinical efficacy of TBI-223 (80 and 160 mg/kg twice daily) was compared with that of linezolid (40 and 80 mg/kg twice daily) and sham treatment in mouse models of MRSA bacteremia, skin wound infection, and orthopedic-implant-associated infection. The dosage was selected based on mouse pharmacokinetic analysis of both linezolid and TBI-223, as well as measurement of the MICs. In all three models, TBI-223 and linezolid had comparable dose-dependent efficacies in reducing bacterial burden and disease severity, compared with sham-treated control mice. Taken together, these findings indicate that TBI-223 represents a novel oxazolidinone antibiotic that may provide an additional option against MRSA infections. Future studies in larger animal models and clinical trials are warranted to translate these findings to humans. IMPORTANCE Staphylococcus aureus is the predominant cause of bloodstream, skin, and bone infections in humans. Resistance to commonly used antibiotics is a growing concern, making it more difficult to treat staphylococcal infections. Use of the oxazolidinone antibiotic linezolid against resistant strains is hindered by high rates of adverse reactions during prolonged therapy. Here, a new oxazolidinone named TBI-223 was tested against S. aureus in three mouse models of infection, i.e., bloodstream infection, skin infection, and bone infection. We found that TBI-223 was as effective as linezolid in these three models. Previous data suggest that TBI-223 has a better safety profile than linezolid. Taken together, these findings indicate that this new agent may provide an additional option against MRSA infections. Future studies in larger animal models and clinical trials are warranted to translate these findings to humans.
Assuntos
Bacteriemia , Staphylococcus aureus Resistente à Meticilina , Oxazolidinonas , Infecções Estafilocócicas , Animais , Camundongos , Acetamidas/farmacologia , Acetamidas/uso terapêutico , Antibacterianos/efeitos adversos , Bacteriemia/tratamento farmacológico , Linezolida/efeitos adversos , Testes de Sensibilidade Microbiana , Oxazolidinonas/efeitos adversos , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/microbiologia , Staphylococcus aureusRESUMO
Staphylococcus aureus causes most skin infections in humans, and the emergence of methicillin-resistant S. aureus (MRSA) strains is a serious public health threat. There is an urgent clinical need for nonantibiotic immunotherapies to treat MRSA infections and prevent the spread of antibiotic resistance. Here, we investigated the pan-caspase inhibitor quinoline-valine-aspartic acid-difluorophenoxymethyl ketone (Q-VD-OPH) for efficacy against MRSA skin infection in mice. A single systemic dose of Q-VD-OPH decreased skin lesion sizes and reduced bacterial burden compared with vehicle-treated or untreated mice. Although Q-VD-OPH inhibited inflammasome-dependent apoptosis-associated speck-like protein containing caspase activation and recruitment domain (ASC) speck formation and caspase-1-mediated interleukin-1ß (IL-1ß) production, Q-VD-OPH maintained efficacy in mice deficient in IL-1ß, ASC, caspase-1, caspase-11, or gasdermin D. Thus, Q-VD-OPH efficacy was independent of inflammasome-mediated pyroptosis. Rather, Q-VD-OPH reduced apoptosis of monocytes and neutrophils. Moreover, Q-VD-OPH enhanced necroptosis of macrophages with concomitant increases in serum TNF and TNF-producing neutrophils, monocytes/macrophages, and neutrophils in the infected skin. Consistent with this, Q-VD-OPH lacked efficacy in mice deficient in TNF (with associated reduced neutrophil influx and necroptosis), in mice deficient in TNF/IL-1R and anti-TNF antibody-treated WT mice. In vitro studies revealed that combined caspase-3, caspase-8, and caspase-9 inhibition reduced apoptosis, and combined caspase-1, caspase-8, and caspase-11 inhibition increased TNF, suggesting a mechanism for Q-VD-OPH efficacy in vivo. Last, Q-VD-OPH also had a therapeutic effect against Streptococcus pyogenes and Pseudomonas aeruginosa skin infections in mice. Collectively, pan-caspase inhibition represents a potential host-directed immunotherapy against MRSA and other bacterial skin infections.
Assuntos
Caspases , Staphylococcus aureus Resistente à Meticilina , Animais , Caspase 1 , Inibidores de Caspase/farmacologia , Imunoterapia , Inflamassomos , Interleucina-1beta , Camundongos , Inibidores do Fator de Necrose TumoralRESUMO
IgE induced by type 2 immune responses in atopic dermatitis is implicated in the progression of atopic dermatitis to other allergic diseases, including food allergies, allergic rhinitis, and asthma. However, the keratinocyte-derived signals that promote IgE and ensuing allergic diseases remain unclear. Herein, in a mouse model of atopic dermatitis-like skin inflammation induced by epicutaneous Staphylococcus aureus exposure, keratinocyte release of IL36α along with IL-4 triggered B cell IgE class-switching, plasma cell differentiation, and increased serum IgE levels-all of which were abrogated in IL-36R-deficient mice or anti-IL36R-blocking antibody-treated mice. Moreover, skin allergen sensitization during S. aureus epicutaneous exposure-induced IL-36 responses was required for the development of allergen-specific lung inflammation. In translating these findings, elevated IL36 cytokines in human atopic dermatitis skin and in IL36 receptor antagonist-deficiency patients coincided with increased serum IgE levels. Collectively, keratinocyte-initiated IL36 responses represent a key mechanism and potential therapeutic target against allergic diseases.
Assuntos
Dermatite Atópica/imunologia , Imunoglobulina E/imunologia , Interleucina-1/imunologia , Queratinócitos/imunologia , Plasmócitos/imunologia , Staphylococcus aureus/imunologia , Animais , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Dermatite Atópica/genética , Dermatite Atópica/microbiologia , Humanos , Switching de Imunoglobulina , Imunoglobulina E/genética , Interleucina-1/genética , Interleucina-4/genética , Interleucina-4/imunologia , Queratinócitos/microbiologia , Camundongos , Camundongos Knockout , Plasmócitos/patologiaRESUMO
BACKGROUND: Although preclinical data suggested that tumor necrosis factor-alpha (TNF) neutralization in heart failure (HF) would be beneficial, clinical trials of TNF antagonists were paradoxically negative. We hypothesized that TNF induces opposing inflammatory and remodeling responses in HF that are TNF-receptor (TNFR) specific. METHODS AND RESULTS: HF was induced in wild-type (WT), TNFR1(-/-), and TNFR2(-/-) mice via coronary ligation. Compared with WT HF, 4-week postinfarction survival was significantly improved in both TNFR1(-/-) and TNFR2(-/-) HF. Compared with sham, WT HF hearts exhibited significant remodeling with robust activation of nuclear factor (NF)-kappaB, p38 mitogen-activated protein kinase, and JNK2 and upregulation of TNF, interleukin (IL)-1beta, IL-6, and IL-10. Compared with WT HF, TNFR1(-/-) HF exhibited (1) improved remodeling, hypertrophy, and contractile function; (2) less apoptosis; and (3) diminished NF-kappaB, p38 mitogen-activated protein kinase, and JNK2 activation and cytokine expression. In contrast, TNFR2(-/-) HF showed exaggerated remodeling and hypertrophy, increased border zone fibrosis, augmented NF-kappaB and p38 mitogen-activated protein kinase activation, higher IL-1beta and IL-6 gene expression, greater activated macrophages, and greater apoptosis. Oxidative stress and diastolic function were improved in both TNFR1(-/-)and TNFR2(-/-) HF. In H9c2 cardiomyocytes, sustained NF-kappaB activation was proapoptotic, an effect dependent on TNFR1 signaling, whereas TNFR2 overexpression attenuated TNF-induced NF-kappaB activation. CONCLUSIONS: TNFR1 and TNFR2 have disparate and opposing effects on remodeling, hypertrophy, NF-kappaB, inflammation, and apoptosis in HF: TNFR1 exacerbates, whereas TNFR2 ameliorates, these events. However, signaling through both receptors is required to induce diastolic dysfunction and oxidative stress. TNFR-specific effects in HF should be considered when therapeutic anti-TNF strategies are developed.
Assuntos
Insuficiência Cardíaca/fisiopatologia , NF-kappa B/fisiologia , Receptores Tipo II do Fator de Necrose Tumoral/fisiologia , Receptores Tipo I de Fatores de Necrose Tumoral/fisiologia , Fator de Necrose Tumoral alfa/fisiologia , Remodelação Ventricular/fisiologia , Animais , Apoptose , Cardiomegalia/etiologia , Cardiomegalia/fisiopatologia , Células Cultivadas , Insuficiência Cardíaca/complicações , Insuficiência Cardíaca/metabolismo , Interleucinas/biossíntese , Interleucinas/genética , Masculino , Camundongos , Camundongos Knockout , Proteína Quinase 9 Ativada por Mitógeno/metabolismo , Contração Miocárdica , Miocardite/etiologia , Miocardite/fisiopatologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Estresse Oxidativo , Receptores Tipo I de Fatores de Necrose Tumoral/deficiência , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Receptores Tipo II do Fator de Necrose Tumoral/deficiência , Receptores Tipo II do Fator de Necrose Tumoral/genética , Proteínas Recombinantes de Fusão/fisiologia , Regulação para Cima , Remodelação Ventricular/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
In vivo whole-animal optical (bioluminescence and fluorescence) imaging of Staphylococcus aureus infections has provided the opportunity to noninvasively and longitudinally monitor the dynamics of the bacterial burden and ensuing host immune responses in live anesthetized animals. Herein, we describe several different mouse models of S. aureus skin infection, skin inflammation, incisional/excisional wound infections, as well as mouse and rabbit models of orthopedic implant infection, which utilized this imaging technology. These animal models and imaging methodologies provide insights into the pathogenesis of these infections and innate and adaptive immune responses, as well as the preclinical evaluation of diagnostic and treatment modalities. Noninvasive approaches to investigate host-pathogen interactions are extremely important as virulent community-acquired methicillin-resistant S. aureus strains (CA-MRSA) are spreading through the normal human population, becoming more antibiotic resistant and creating a serious threat to public health.
Assuntos
Staphylococcus aureus Resistente à Meticilina/metabolismo , Imagem Óptica , Infecções Cutâneas Estafilocócicas , Animais , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Coelhos , Infecções Cutâneas Estafilocócicas/diagnóstico , Infecções Cutâneas Estafilocócicas/metabolismo , Infecções Cutâneas Estafilocócicas/patologiaRESUMO
Orthopedic implant-associated infection (OIAI) is a major complication that leads to implant failure. In preclinical models of Staphylococcus aureus OIAI, osteomyelitis and septic arthritis, interleukin-1α (IL-1α), IL-1ß, and tumor necrosis factor (TNF) are induced, but whether they have interactive or distinctive roles in host defense are unclear. Herein, a S. aureus OIAI model was performed in mice deficient in IL-1α, IL-1ß, or TNF. Mice deficient in IL-1ß or TNF (to a lesser extent) but not IL-1α had increased bacterial burden at the site of the OIAI throughout the 28-day experiment. IL-1ß and TNF had a combined and critical role in host defense as mice deficient in both IL-1R and TNF (IL-1R/TNF-deficient mice) had a 40% mortality rate, which was associated with markedly increased bacterial burden at the site of the OIAI infection. Finally, IL-1α- and IL-1ß-deficient mice had impaired neutrophil recruitment whereas IL-1ß-, TNF-, and IL-1R/TNF-deficient mice all had impaired recruitment of both neutrophils and monocytes. Therefore, IL-1ß and TNF contributed to host defense against S. aureus OIAI and neutrophil recruitment was primarily mediated by IL-1ß and monocyte recruitment was mediated by both IL-1ß and TNF.
Assuntos
Interleucina-1beta/metabolismo , Infiltração de Neutrófilos , Infecções Relacionadas à Prótese/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Interleucina-1alfa/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Infecções Relacionadas à Prótese/imunologia , Infecções Estafilocócicas/imunologia , Infecções Estafilocócicas/metabolismoRESUMO
Acute and chronic wounds affect millions and are associated with billions of dollars in healthcare costs. The use of healing markers, biochemical cues from biocompatible matrices and materials, and their correlation with wound healing has the potential to generate valuable diagnostic, prognostic, and therapeutic information. In this study, we developed a collagen-dextran oxygen-sensing biocomposite scaffold membrane in which a phosphorescent oxygen sensor was incorporated to monitor physiological oxygen using in vivo phosphorescence imaging in a preclinical mouse model of wound healing. The oxygen-sensing biocomposite scaffold membrane enabled the noninvasive and longitudinal monitoring of oxygenation changes in vivo in an approach compatible with commercially available preclinical in vivo imaging system instruments. This study provides a new and novel capability where a biocomposite material can serve as a biocompatible, biodegradable theranostic platform to promote and assess tissue oxygenation during wound healing.
Assuntos
Materiais Biocompatíveis/química , Nanomedicina Teranóstica , Alicerces Teciduais/química , Animais , Corantes/química , Dendrímeros/síntese química , Dendrímeros/química , Diabetes Mellitus Experimental/patologia , Membranas , Camundongos , Oxigênio/análise , Polietilenoglicóis/química , CicatrizaçãoRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
BACKGROUND: In vivo bioluminescence imaging (BLI) provides noninvasive monitoring of bacterial burden in animal models of orthopaedic implant-associated infection (OIAI). However, technical limitations have limited its use to mouse and rat models of OIAI. The goal of this study was to develop a larger, rabbit model of OIAI using in vivo BLI to evaluate the efficacy of an antibiotic-releasing implant coating. METHODS: A nanofiber coating loaded with or without linezolid-rifampin was electrospun onto a surgical-grade locking peg. To model OIAI in rabbits, a medial parapatellar arthrotomy was performed to ream the femoral canal, and a bright bioluminescent methicillin-resistant Staphylococcus aureus (MRSA) strain was inoculated into the canal, followed by retrograde insertion of the coated implant flush with the articular surface. In vivo BLI signals were confirmed by ex vivo colony-forming units (CFUs) from tissue, bone, and implant specimens. RESULTS: In this rabbit model of OIAI (n = 6 rabbits per group), implants coated without antibiotics were associated with significantly increased knee width and in vivo BLI signals compared with implants coated with linezolid-rifampin (p < 0.001 and p < 0.05, respectively). On day 7, the implants without antibiotics were associated with significantly increased CFUs from tissue (mean [and standard error of the mean], 1.4 × 10 ± 2.1 × 10 CFUs; p < 0.001), bone (6.9 × 10 ± 3.1 × 10 CFUs; p < 0.05), and implant (5.1 × 10 ± 2.2 × 10 CFUs; p < 0.05) specimens compared with implants with linezolid-rifampin, which demonstrated no detectable CFUs from any source. CONCLUSIONS: By combining a bright bioluminescent MRSA strain with modified techniques, in vivo BLI in a rabbit model of OIAI demonstrated the efficacy of an antibiotic-releasing coating. CLINICAL RELEVANCE: The new capability of in vivo BLI for noninvasive monitoring of bacterial burden in larger-animal models of OIAI may have important preclinical relevance.
Assuntos
Antibacterianos/administração & dosagem , Linezolida/administração & dosagem , Infecções Relacionadas à Prótese/prevenção & controle , Rifampina/administração & dosagem , Infecções Estafilocócicas/prevenção & controle , Análise de Variância , Animais , Preparações de Ação Retardada , Modelos Animais de Doenças , Combinação de Medicamentos , Medições Luminescentes , Masculino , Staphylococcus aureus Resistente à Meticilina , CoelhosRESUMO
Industrial hog operation (IHO) workers are at increased risk of carrying Staphylococcus aureus in their nares, particularly strains that are livestock-associated (LA) and multidrug-resistant. The pathogenicity of LA-S. aureus strains remains unclear, with some prior studies suggesting reduced transmission and virulence in humans compared to community-associated methicillin-resistant (CA-MRSA) S. aureus. The objective of this study was to determine the degree to which LA-S. aureus strains contracted by IHO workers cause disease relative to a representative CA-MRSA strain in a mouse model of skin and soft tissue infection (SSTI). Mice infected with CC398 LA-S. aureus strains (IHW398-1 and IHW398-2) developed larger lesion sizes with higher bacterial burden than mice infected with CA-MRSA (SF8300) (p < 0.05). The greatest lesion size and bacterial burden was seen with a CC398 strain that produced a recurrent SSTI in an IHO worker. The LA-S. aureus infected mice had decreased IL-1ß protein levels compared with CA-MRSA-infected mice (p < 0.05), suggesting a suboptimal host response to LA-S. aureus SSTIs. WGSA revealed heterogeneity in virulence factor and antimicrobial resistance genes carried by LA-S. aureus and CA-MRSA strains. The observed pathogenicity suggest that more attention should be placed on preventing the spread of LA-S. aureus into human populations.
Assuntos
Infecções Comunitárias Adquiridas/veterinária , Gado/microbiologia , Staphylococcus aureus Resistente à Meticilina/patogenicidade , Doenças das Aves Domésticas/epidemiologia , Dermatopatias Infecciosas/veterinária , Infecções dos Tecidos Moles/veterinária , Infecções Estafilocócicas/veterinária , Animais , Infecções Comunitárias Adquiridas/epidemiologia , Infecções Comunitárias Adquiridas/microbiologia , Modelos Animais de Doenças , Camundongos , Doenças das Aves Domésticas/microbiologia , Dermatopatias Infecciosas/epidemiologia , Dermatopatias Infecciosas/microbiologia , Infecções dos Tecidos Moles/epidemiologia , Infecções dos Tecidos Moles/microbiologia , Infecções Estafilocócicas/epidemiologia , Infecções Estafilocócicas/microbiologia , VirulênciaRESUMO
In vivo bioluminescence imaging has been used to monitor Staphylococcus aureus infections in preclinical models by employing bacterial reporter strains possessing a modified lux operon from Photorhabdus luminescens. However, the relatively short emission wavelength of lux (peak 490 nm) has limited tissue penetration. To overcome this limitation, the gene for the click beetle (Pyrophorus plagiophtalamus) red luciferase (luc) (with a longer >600 emission wavelength), was introduced singly and in combination with the lux operon into a methicillin-resistant S. aureus strain. After administration of the substrate D-luciferin, the luc bioluminescent signal was substantially greater than the lux signal in vitro. The luc signal had enhanced tissue penetration and improved anatomical co-registration with infected internal organs compared with the lux signal in a mouse model of S. aureus bacteremia with a sensitivity of approximately 3 × 104 CFU from the kidneys. Finally, in an in vivo mixed bacterial wound infection mouse model, S. aureus luc signals could be spectrally unmixed from Pseudomonas aeruginosa lux signals to noninvasively monitor the bacterial burden of both strains. Therefore, the S. aureus luc reporter may provide a technological advance for monitoring invasive organ dissemination during S. aureus bacteremia and for studying bacterial dynamics during mixed infections.
Assuntos
Bacteriemia/microbiologia , Coinfecção/microbiologia , Besouros/enzimologia , Luciferases/metabolismo , Infecções por Pseudomonas/microbiologia , Infecções Estafilocócicas/microbiologia , Infecção dos Ferimentos/microbiologia , Animais , Bacteriemia/diagnóstico por imagem , Bacteriemia/metabolismo , Coinfecção/diagnóstico por imagem , Coinfecção/metabolismo , Besouros/genética , Diagnóstico por Imagem/métodos , Feminino , Genes Reporter , Luciferases/genética , Medições Luminescentes , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Infecções por Pseudomonas/diagnóstico por imagem , Infecções por Pseudomonas/metabolismo , Pseudomonas aeruginosa/isolamento & purificação , Pseudomonas aeruginosa/metabolismo , Coelhos , Infecções Estafilocócicas/diagnóstico por imagem , Infecções Estafilocócicas/metabolismo , Staphylococcus aureus/isolamento & purificação , Staphylococcus aureus/metabolismo , Infecção dos Ferimentos/diagnóstico por imagem , Infecção dos Ferimentos/metabolismoRESUMO
Staphylococcus aureus infections are a major threat in healthcare, requiring adequate early-stage diagnosis and treatment. This calls for novel diagnostic tools that allow noninvasive in vivo detection of staphylococci. Here we performed a preclinical study to investigate a novel fully-human monoclonal antibody 1D9 that specifically targets the immunodominant staphylococcal antigen A (IsaA). We show that 1D9 binds invariantly to S. aureus cells and may further target other staphylococcal species. Importantly, using a human post-mortem implant model and an in vivo murine skin infection model, preclinical feasibility was demonstrated for 1D9 labeled with the near-infrared fluorophore IRDye800CW to be applied for direct optical imaging of in vivo S. aureus infections. Additionally, 89Zirconium-labeled 1D9 could be used for positron emission tomography imaging of an in vivo S. aureus thigh infection model. Our findings pave the way towards clinical implementation of targeted imaging of staphylococcal infections using the human monoclonal antibody 1D9.