RESUMEN
IL-17 contributes to the pathogenesis of certain autoimmune diseases, but conversely is essential for host defense against fungi. Ab-based biologic drugs that neutralize IL-17 are effective in autoimmunity but can be accompanied by adverse side effects. Candida albicans is a commensal fungus that is the primary causative agent of oropharyngeal and disseminated candidiasis. Defects in IL-17 signaling cause susceptibility to candidiasis in mice and humans. A key facet of IL-17 receptor signaling involves RNA-binding proteins, which orchestrate the fate of target mRNA transcripts. In tissue culture models we showed that the RNA-binding protein AT-rich interaction domain 5A (Arid5a) promotes the stability and/or translation of multiple IL-17-dependent mRNAs. Moreover, during oropharyngeal candidiasis, Arid5a is elevated within the oral mucosa in an IL-17-dependent manner. However, the contribution of Arid5a to IL-17-driven events in vivo is poorly defined. In this study, we used CRISPR-Cas9 to generate mice lacking Arid5a. Arid5a -/- mice were fully resistant to experimental autoimmune encephalomyelitis, an autoimmune setting in which IL-17 signaling drives pathology. Surprisingly, Arid5a -/- mice were resistant to oropharyngeal candidiasis and systemic candidiasis, similar to immunocompetent wild-type mice and contrasting with mice defective in IL-17 signaling. Therefore, Arid5a-dependent signals mediate pathology in autoimmunity and yet are not required for immunity to candidiasis, indicating that selective targeting of IL-17 signaling pathway components may be a viable strategy for development of therapeutics that spare IL-17-driven host defense.
Asunto(s)
Productos Biológicos , Candidiasis , Encefalomielitis Autoinmune Experimental , Animales , Autoinmunidad , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Humanos , Interleucina-17/metabolismo , Ratones , ARN Mensajero/genética , Proteínas de Unión al ARN/metabolismo , Receptores de Interleucina-17/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
P75 pan-neurotrophin receptor (p75NTR) is an important receptor for the role of neurotrophins in survival and death of neurons during development and after nerve injury. Our previous research found that the precursor of brain-derived neurotrophic factor (proBDNF) regulates pain as an inflammatory mediator. The current understanding of the role of proBDNF/p75NTR signaling pathway in inflammatory arthritis pain and rheumatoid arthritis (RA) is unclear. We recruited 20 RA patients, 20 healthy donors (HDs), and 10 osteoarthritis (OA) patients. Hematoxylin and eosin (H&E) staining and immunohistochemistry (IHC) of proBDNF and p75NTR in synovial membrane were performed and evaluated. We next examined the mRNA and protein expression of proBDNF/p75NTR signaling pathway in peripheral blood mononuclear cells (PBMCs) and synovial tissue. ELISA and flow cytometry were assessed between the blood of RA patients and HD. To induce RA, collagen-induced arthritis (CIA) were induced in mice. We found over-synovitis of RA synovial membrane compared to OA controls in histologic sections. P75NTR and sortilin mRNA, and proBDNF protein level were significantly increased in PBMCs of RA patients compared with the HD. Consistently, ELISA showed that p75NTR, sortilin, tumor necrosis factor α (TNF-α), interleukin-1ß (IL-1ß), interleukin-6 (IL-6), and interleukin-10 (IL-10) levels in the serum of RA patients were increased compared with HD and p75NTR, sortilin were positively correlated with Disease Activity Score in 28 joints (DAS28). In addition, using flow cytometry we showed that the increased levels of proBDNF and p75NTR characterized in CD4+ and CD8+ T cells of RA patients were subsequently reversed with methotrexate (MTX) treatment. Furthermore, we found pathological changes, inflammatory pain, upregulation of the mRNA and protein expression of proBDNF/p75NTR signaling pathway, and upregulation of inflammatory cytokines in spinal cord using a well-established CIA mouse model. We showed intravenous treatment of recombinant p75ECD-Fc that biologically blocked all inflammatory responses and relieved inflammatory pain of animals with CIA. Our findings showed the involvement of proBDNF/p75NTR pathway in the RA inflammatory response and how blocking it with p75ECD-Fc may be a promising therapeutic treatment for RA.
Asunto(s)
Artritis Reumatoide/metabolismo , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Interleucinas/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Receptores de Factor de Crecimiento Nervioso/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Adulto , Animales , Femenino , Humanos , Interleucinas/sangre , Leucocitos Mononucleares/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Precursores de Proteínas/metabolismo , Membrana Sinovial/metabolismo , Linfocitos T/metabolismo , Factor de Necrosis Tumoral alfa/sangreRESUMEN
The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has led to more than 5 million deaths worldwide to date. Due to the limited therapeutic options so far available, target-based virtual screening with LC/MS support was applied to identify the novel and high-content compounds 1-4 with inhibitory effects on SARS-CoV-2 in Vero E6 cells from the plant Dryopteris wallichiana. These compounds were also evaluated against SARS-CoV-2 in Calu-3 cells and showed unambiguous inhibitory activity. The inhibition assay of targets showed that compounds 3 and 4 mainly inhibited SARS-CoV-2 3CLpro, with effective Kd values. Through docking and molecular dynamics modeling, the binding site is described, providing a comprehensive understanding of 3CLpro and interactions for 3, including hydrogen bonds, hydrophobic bonds, and the spatial occupation of the B ring. Compounds 3 and 4 represent new, potential lead compounds for the development of anti-SARS-CoV-2 drugs. This study has led to the development of a target-based virtual screening method for exploring the potency of natural products and for identifying natural bioactive compounds for possible COVID-19 treatment.
Asunto(s)
Antivirales/farmacología , Productos Biológicos/farmacología , Pruebas de Sensibilidad Microbiana/métodos , Floroglucinol/farmacología , SARS-CoV-2/efectos de los fármacos , Terpenos/farmacología , Cromatografía Líquida de Alta Presión , Cromatografía Liquida , Cristalografía por Rayos X , Sistemas de Liberación de Medicamentos , Dryopteris/química , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Simulación del Acoplamiento Molecular , Estructura Molecular , Realidad VirtualRESUMEN
Osthole is a natural coumarin that exhibits wide biological and pharmacological activities such as neuroprotective, osteogenic, immunomodulation, antitumor, and anti-inflammatory effects. In this study, we investigated the antifungal effects of osthole in vitro A checkerboard microdilution assay showed that osthole has significant synergistic effect with fluconazole against fluconazole-resistant Candida albicans Similar results were obtained from a growth curve assay. Meanwhile, XTT reduction assay demonstrated the synergism of fluconazole and osthole against C. albicans biofilm formation. Microarray results showed that the expression of genes involved in the oxidation-reduction process, energy metabolism, and transportation changed significantly after the combined treatment with fluconazole and osthole, and further results showed that endogenous reactive oxygen species (ROS) was significantly increased in the combination group. In conclusion, these results demonstrate the synergism of fluconazole and osthole against fluconazole-resistant C. albicans and indicate that endogenous ROS augmentation might contribute to the synergism of fluconazole and osthole.
Asunto(s)
Antifúngicos/farmacología , Candida albicans/efectos de los fármacos , Candidiasis/tratamiento farmacológico , Cumarinas/farmacología , Fluconazol/farmacología , Especies Reactivas de Oxígeno/metabolismo , Candida albicans/genética , Candidiasis/microbiología , Farmacorresistencia Fúngica/genética , Sinergismo Farmacológico , Quimioterapia Combinada , Regulación Fúngica de la Expresión Génica/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Estrés Oxidativo/efectos de los fármacosRESUMEN
Nitric oxide (NO) is a small molecule with a wide range of biological activities in mammalian and bacteria. However, the role of NO in fungi, especially Candida albicans, is not clear. In this study, we confirmed the generation of endogenous NO in C. albicans, and found that the production of endogenous NO in C. albicans was associated with nitric oxide synthase pathway. Our results further indicated that the production of endogenous NO in C. albicans was reduced under oxidative stress such as menadione or H2O2 treatment. Meanwhile, exogenous NO donor, sodium nitroprusside (SNP), synergized with H2O2 against C. albicans. Interestingly, SNP could inhibit the antifungal effect of azoles against C. albicans in vitro, suggesting that NO might be involved in the resistance of C. albicans to antifungals. Collectively, this study demonstrated the production of endogenous NO in C. albicans, and indicated that NO may play an important role in the response of C. albicans to oxidative stress and azoles.
RESUMEN
Pterostilbene (PTE) is a stilbene-derived phytoalexin that originates from several natural plant sources. In this study, we evaluated the activity of PTE against Candida albicans biofilms and explored the underlying mechanisms. In 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) reduction assays, biofilm biomass measurement, confocal laser scanning microscopy, and scanning electron microscopy, we found that ≤16 µg/ml PTE had a significant effect against C. albicans biofilms in vitro, while it had no fungicidal effect on planktonic C. albicans cells, which suggested a unique antibiofilm effect of PTE. Then we found that PTE could inhibit biofilm formation and destroy the maintenance of mature biofilms. At 4 µg/ml, PTE decreased cellular surface hydrophobicity (CSH) and suppressed hyphal formation. Gene expression microarrays and real-time reverse transcription-PCR showed that exposure of C. albicans to 16 µg/ml PTE altered the expression of genes that function in morphological transition, ergosterol biosynthesis, oxidoreductase activity, and cell surface and protein unfolding processes (heat shock proteins). Filamentation-related genes, especially those regulated by the Ras/cyclic AMP (cAMP) pathway, including ECE1, ALS3, HWP1, HGC1, and RAS1 itself, were downregulated upon PTE treatment, indicating that the antibiofilm effect of PTE was related to the Ras/cAMP pathway. Then, we found that the addition of exogenous cAMP reverted the PTE-induced filamentous growth defect. Finally, with a rat central venous catheter infection model, we confirmed the in vivo activity of PTE against C. albicans biofilms. Collectively, PTE had strong activities against C. albicans biofilms both in vitro and in vivo, and these activities were associated with the Ras/cAMP pathway.
Asunto(s)
Antifúngicos/farmacología , Antifúngicos/uso terapéutico , Biopelículas/efectos de los fármacos , Candida albicans/efectos de los fármacos , Candida albicans/fisiología , Estilbenos/farmacología , Estilbenos/uso terapéutico , Animales , Candida albicans/metabolismo , Femenino , Proteínas Fúngicas/metabolismo , Pruebas de Sensibilidad Microbiana , Microscopía Electrónica de Rastreo , Ratas , Ratas Sprague-DawleyRESUMEN
Autoantibody-mediated glomerulonephritis (AGN) arises from dysregulated renal inflammation, with urgent need for improved treatments. IL-17 is implicated in AGN and drives pathology in a kidney-intrinsic manner via renal tubular epithelial cells (RTECs). Nonetheless, downstream signaling mechanisms provoking kidney pathology are poorly understood. A noncanonical RNA binding protein (RBP), Arid5a, was upregulated in human and mouse AGN. Arid5a-/- mice were refractory to AGN, with attenuated myeloid infiltration and impaired expression of IL-17-dependent cytokines and transcription factors (C/EBPß, C/EBPδ). Transcriptome-wide RIP-Seq revealed that Arid5a inducibly interacts with conventional IL-17 target mRNAs, including CEBPB and CEBPD. Unexpectedly, many Arid5a RNA targets corresponded to translational regulation and RNA processing pathways, including rRNAs. Indeed, global protein synthesis was repressed in Arid5a-deficient cells, and C/EBPs were controlled at the level of protein rather than RNA accumulation. IL-17 prompted Arid5a nuclear export and association with 18S rRNA, a 40S ribosome constituent. Accordingly, IL-17-dependent renal autoimmunity is driven by Arid5a at the level of ribosome interactions and translation.
Asunto(s)
Autoanticuerpos , Proteínas de Unión al ADN , Glomerulonefritis , Interleucina-17 , Ratones Noqueados , Factores de Transcripción , Animales , Interleucina-17/metabolismo , Glomerulonefritis/inmunología , Glomerulonefritis/genética , Glomerulonefritis/metabolismo , Glomerulonefritis/patología , Humanos , Ratones , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Autoanticuerpos/inmunología , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Proteína beta Potenciadora de Unión a CCAAT/metabolismo , Proteína beta Potenciadora de Unión a CCAAT/genética , Proteína delta de Unión al Potenciador CCAAT/metabolismo , Proteína delta de Unión al Potenciador CCAAT/genética , Ratones Endogámicos C57BL , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/inmunología , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
It was found in our previous study that berberine (BBR) and fluconazole (FLC) used concomitantly exhibited a synergism against FLC-resistant Candida albicans in vitro. The aim of the present study was to clarify how BBR and FLC worked synergistically and the underlying mechanism. Antifungal time-kill curves indicated that the synergistic effect of the two drugs was BBR dose dependent rather than FLC dose dependent. In addition, we found that BBR accumulated in C. albicans cells, especially in the nucleus, and resulted in cell cycle arrest and significant change in the transcription of cell cycle-related genes. Besides BBR, other DNA intercalators, including methylene blue, sanguinarine, and acridine orange, were all found to synergize with FLC against FLC-resistant C. albicans. Detection of intracellular BBR accumulation by fluorescence measurement showed that FLC played a role in increasing intracellular BBR concentration, probably due to its effect in disrupting the fungal cell membrane. Similar to the case with FLC, other antifungal agents acting on the cell membrane were able to synergize with BBR. Interestingly, we found that the efflux of intracellular BBR was FLC independent but strongly glucose dependent and associated with the drug efflux pump Cdr2p. These results suggest that BBR plays a major antifungal role in the synergism of FLC and BBR, while FLC plays a role in increasing the intracellular BBR concentration.
Asunto(s)
Antifúngicos/farmacología , Berberina/farmacología , Candida albicans/efectos de los fármacos , Fluconazol/farmacología , Proteínas Fúngicas/genética , Regulación Fúngica de la Expresión Génica , Naranja de Acridina/farmacología , Benzofenantridinas/farmacología , Transporte Biológico , Candida albicans/genética , Candida albicans/metabolismo , Puntos de Control del Ciclo Celular/efectos de los fármacos , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Relación Dosis-Respuesta a Droga , Farmacorresistencia Fúngica/genética , Sinergismo Farmacológico , Proteínas Fúngicas/metabolismo , Sustancias Intercalantes/farmacología , Isoquinolinas/farmacología , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Azul de Metileno/farmacología , Pruebas de Sensibilidad MicrobianaRESUMEN
Candida albicans has become the fourth leading pathogen of nosocomial bloodstream infections largely due to biofilm formation on implanted medical devices. Previous microarray data indicated that almost all genes in methionine (Met)/cysteine (Cys) biosynthesis pathway were up-regulated during biofilm formation, especially during the adherence period. In this work, we studied the role of Met/Cys biosynthesis pathway by disrupting ECM17, a gene encoding sulfite reductase in C. albicans. It was found that the ecm17Δ/Δ mutant failed to catalyze the biochemical reaction from sulfite to H(2)S and hardly grew in media lacking Met and Cys. NaSH, the donor of H(2)S, dose-dependently improved the growth of ecm17Δ/Δ in media lacking a sulfur source. Sufficient Met/Cys supply inhibited the expression of ECM17 in a dose-dependent manner. These results validated the important role of ECM17 in Met/Cys biosynthesis. Interestingly, the ecm17Δ/Δ mutant showed diminished ability to form biofilm, attenuated adhesion on abiotic substrate and decreased filamentation on solid SLD medium, especially under conditions lacking Met/Cys. Further results indicated that ECM17 affected the expressions of ALS3, CSH1, HWP1 and ECE1, and that the cAMP-protein kinase A (PKA) pathway was associated with ECM17 and Met/Cys biosynthesis pathway. These results provide new insights into the role of Met/Cys biosynthesis pathway in regulating cAMP-PKA pathway and benefiting biofilm formation.
Asunto(s)
Biopelículas/crecimiento & desarrollo , Candida albicans/enzimología , Candida albicans/fisiología , Cisteína/biosíntesis , Metionina/biosíntesis , Sulfito Reductasa (NADPH)/metabolismo , Candida albicans/genética , Adhesión Celular , Medios de Cultivo/química , Perfilación de la Expresión Génica , Regulación Fúngica de la Expresión Génica , Técnicas de Inactivación de Genes , Sulfuro de Hidrógeno/metabolismo , Hifa/crecimiento & desarrollo , Sulfito Reductasa (NADPH)/genética , Sulfitos/metabolismoRESUMEN
Candida albicans is the most common fungal pathogen. Galleria mellonella is widely used as an infection model host. Nevertheless, the G. mellonella-C. albicans infection model had not been optimized for drug evaluation before this study. In this work, we revealed that 5 × 10(5) colony forming unit (CFU)/larva was a suitable inoculum to optimize the G. mellonella-C. albicans infection model in order to evaluate antifungal agents. Using our optimized model, the antifungal effect of fluconazole, amphotericin B and flucytosine, and the synergy between amphotericin B and flucytosine were successfully verified. Thus, this study provides a rapid, inexpensive and reliable way to evaluate antifungals in vivo.
Asunto(s)
Antifúngicos/farmacología , Candida albicans/efectos de los fármacos , Modelos Animales de Enfermedad , Mariposas Nocturnas/microbiología , Anfotericina B/farmacología , Animales , Candida albicans/patogenicidad , Fluconazol/farmacología , Flucitosina/farmacología , Larva/microbiologíaRESUMEN
Combating fungal pathogens poses metabolic challenges for neutrophils, key innate cells in anti-Candida albicans immunity, yet how host-pathogen interactions cause remodeling of the neutrophil metabolism is unclear. We show that neutrophils mediate renal immunity to disseminated candidiasis by upregulating glucose uptake via selective expression of glucose transporter 1 (Glut1). Mechanistically, dectin-1-mediated recognition of ß-glucan leads to activation of PKCδ, which triggers phosphorylation, localization, and early glucose transport by a pool of pre-formed Glut1 in neutrophils. These events are followed by increased Glut1 gene transcription, leading to more sustained Glut1 accumulation, which is also dependent on the ß-glucan/dectin-1/CARD9 axis. Card9-deficient neutrophils show diminished glucose incorporation in candidiasis. Neutrophil-specific Glut1-ablated mice exhibit increased mortality in candidiasis caused by compromised neutrophil phagocytosis, reactive oxygen species (ROS), and neutrophil extracellular trap (NET) formation. In human neutrophils, ß-glucan triggers metabolic remodeling and enhances candidacidal function. Our data show that the host-pathogen interface increases glycolytic activity in neutrophils by regulating Glut1 expression, localization, and function.
Asunto(s)
Candidiasis , Transportador de Glucosa de Tipo 1 , Neutrófilos , beta-Glucanos , Animales , Proteínas Adaptadoras de Señalización CARD/metabolismo , Candida albicans , Candidiasis/inmunología , Glucosa/metabolismo , Transportador de Glucosa de Tipo 1/metabolismo , Ratones , Neutrófilos/inmunología , beta-Glucanos/metabolismoRESUMEN
Antibody-mediated glomerulonephritis (AGN) is a clinical manifestation of many autoimmune kidney diseases for which few effective treatments exist. Chronic inflammatory circuits in renal glomerular and tubular cells lead to tissue damage in AGN. These cells are targeted by the cytokine IL-17, which has recently been shown to be a central driver of the pathogenesis of AGN. However, surprisingly little is known about the regulation of pathogenic IL-17 signaling in the kidney. Here, using a well-characterized mouse model of AGN, we show that IL-17 signaling in renal tubular epithelial cells (RTECs) is necessary for AGN development. We also show that Regnase-1, an RNA binding protein with endoribonuclease activity, is a negative regulator of IL-17 signaling in RTECs. Accordingly, mice with a selective Regnase-1 deficiency in RTECs exhibited exacerbated kidney dysfunction in AGN. Mechanistically, Regnase-1 inhibits IL-17-driven expression of the transcription factor IκBξ and, consequently, its downstream gene targets, including Il6 and Lcn2. Moreover, deletion of Regnase-1 in human RTECs reduced inflammatory gene expression in a IκBξ-dependent manner. Overall, these data identify an IL-17-driven inflammatory circuit in RTECs during AGN that is constrained by Regnase-1.
Asunto(s)
Enfermedades Autoinmunes/metabolismo , Glomerulonefritis , Proteínas I-kappa B/metabolismo , Interleucina-17/metabolismo , Túbulos Renales , Proteínas Proto-Oncogénicas/metabolismo , Ribonucleasas , Animales , Células Epiteliales/metabolismo , Glomerulonefritis/inmunología , Glomerulonefritis/fisiopatología , Inmunidad Innata , Inflamación/metabolismo , Túbulos Renales/inmunología , Túbulos Renales/patología , Ratones , Insuficiencia Renal/inmunología , Insuficiencia Renal/metabolismo , Ribonucleasas/deficiencia , Ribonucleasas/inmunología , Transducción de Señal/inmunologíaRESUMEN
Infections are the second major cause of mortality in patients with kidney disease and accompanying uremia. Both vascular access and non-access-related infections contribute equally to the infection-related deaths in patients with kidney disease. Dialysis is the most common cause of systemic infection by Candida albicans in these patients. C albicans also reside in the gastrointestinal tract as a commensal fungus. However, the contribution of gut-derived C albicans in non-access-related infections in kidney disease is unknown. Using a mouse model of kidney disease, we demonstrate that uremic animals showed increased gut barrier permeability, impaired mucosal defense, and dysbiosis. The disturbance in gut homeostasis is sufficient to drive the translocation of microbiota and intestinal pathogen Citrobacter rodentium to extraintestinal sites but not C albicans Interestingly, a majority of uremic animals showed fungal translocation only when the gut barrier integrity is disrupted. Our data demonstrate that uremia coupled with gut mucosal damage may aid in the translocation of C. albicans and cause systemic infection in kidney disease. Because most of the individuals with kidney disease suffer from some form of gut mucosal damage, these results have important implications in the risk stratification and control of non-access-related opportunistic fungal infections in these patients.
Asunto(s)
Candida albicans/fisiología , Citrobacter rodentium/fisiología , Mucosa Intestinal/microbiología , Intestinos/microbiología , Uremia/microbiología , Animales , Citrobacter rodentium/crecimiento & desarrollo , Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades , Interacciones Huésped-Patógeno , Humanos , Mucosa Intestinal/patología , Ratones , Ratones Endogámicos C57BL , SimbiosisRESUMEN
Intestinal fungi are critical for modulating host immune homeostasis and underlying mechanisms remain unclear. We show that dendritic cell (DC)-specific deficiency of casitas B-lineage lymphoma (c-Cbl) renders mice susceptible to dextran sodium sulfate (DSS)-induced colitis. Mechanistically, we identify that c-Cbl functions downstream of Dectin-2 and Dectin-3 to mediate the ubiquitination and degradation of noncanonical nuclear factor κB subunit RelB. Thus, c-Cbl deficiency in DCs promotes α-mannan-induced activation of RelB, which suppresses p65-mediated transcription of an anti-inflammatory cytokine gene, il10, thereby aggravating DSS-induced colitis. Moreover, suppressing fungal growth with fluconazole or inhibition of RelB activation in vivo attenuates colitis in mice with DC-specific deletion of c-Cbl. We also demonstrate an interaction between c-Cbl and c-Abl tyrosine kinase and find that treatment with DPH, a c-Abl agonist, synergistically increases fungi-induced c-Cbl activation to restrict colitis. Together, these findings unravel a previously unidentified fungi-induced c-Cbl/RelB axis that sustains intestinal homeostasis and protects against intestinal inflammation.
Asunto(s)
Colitis , FN-kappa B , Proteínas Proto-Oncogénicas c-cbl/metabolismo , Animales , Colitis/inducido químicamente , Hongos/metabolismo , Inflamación , Ratones , FN-kappa B/metabolismo , Ubiquitina-Proteína LigasasRESUMEN
The morphological switch between yeast and hyphae of Candida albicans is essential for its interaction with the host defense system. However, the lack of understanding of host-pathogen interactions during C. albicans infection greatly hampers the development of effective immunotherapies. Here, we found that priming with the C. albicans FLO8-deficient (flo8) mutant, locked in yeast form, protected mice from subsequent lethal C. albicans infection. Deficiency of Dectin-2, a fungus-derived α-mannan recognition receptor, completely blocked flo8 mutant-induced protection. Mechanistically, the flo8 mutant-induced Dectin-2/CARD9-mediated IL-10 production in DCs and macrophages to block thymus atrophy by inhibiting the C. albicans-induced apoptosis of thymic T cells, which facilitated the continuous output of naive T cells from the thymus to the spleen. Continuous recruitment of naive T cells to the spleen enhanced Th1-biased antifungal immune responses. Consequently, depletion of CD4+ T cells or blockade of IL-10 receptor function using specific antibodies in mice completely blocked the protective effects of flo8 mutant priming against C. albicans infection. Moreover, mannans exposed on the surface of the flo8 mutant were responsible for eliciting protective immunity by inhibiting the C. albicans-induced apoptosis of thymic T cells to sustain the number of naive T cells in the spleen. Importantly, priming with the flo8 mutant extensively protected mice from polymicrobial infection caused by cecal ligation and puncture (CLP) by enhancing Th1-biased immune responses. Together, our findings imply that targeting FLO8 in C. albicans elicits protective immune responses against polymicrobial infections and that mannans extracted from the flo8 mutant are potential immunotherapeutic candidate(s) for controlling infectious diseases.
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Candidiasis , Sepsis , Animales , Proteínas Adaptadoras de Señalización CARD , Candida albicans/fisiología , Hifa , Mananos/farmacología , RatonesRESUMEN
Excessive cytokine activity underlies many autoimmune conditions, particularly through the interleukin-17 (IL-17) and tumor necrosis factor-α (TNFα) signaling axis. Both cytokines activate nuclear factor κB, but appropriate induction of downstream effector genes requires coordinated activation of other transcription factors, notably, CCAAT/enhancer binding proteins (C/EBPs). Here, we demonstrate the unexpected involvement of a posttranscriptional "epitranscriptomic" mRNA modification [N6-methyladenosine (m6A)] in regulating C/EBPß and C/EBPδ in response to IL-17A, as well as IL-17F and TNFα. Prompted by the observation that C/EBPß/δ-encoding transcripts contain m6A consensus sites, we show that Cebpd and Cebpb mRNAs are subject to m6A modification. Induction of C/EBPs is enhanced by an m6A methylase "writer" and suppressed by a demethylase "eraser." The only m6A "reader" found to be involved in this pathway was IGF2BP2 (IMP2), and IMP2 occupancy of Cebpd and Cebpb mRNA was enhanced by m6A modification. IMP2 facilitated IL-17-mediated Cebpd mRNA stabilization and promoted translation of C/EBPß/δ in response to IL-17A, IL-17F, and TNFα. RNA sequencing revealed transcriptome-wide IL-17-induced transcripts that are IMP2 influenced, and RNA immunoprecipitation sequencing identified the subset of mRNAs that are directly occupied by IMP2, which included Cebpb and Cebpd Lipocalin-2 (Lcn2), a hallmark of autoimmune kidney injury, was strongly dependent on IL-17, IMP2, and C/EBPß/δ. Imp2-/- mice were resistant to autoantibody-induced glomerulonephritis (AGN), showing impaired renal expression of C/EBPs and Lcn2 Moreover, IMP2 deletion initiated only after AGN onset ameliorated disease. Thus, posttranscriptional regulation of C/EBPs through m6A/IMP2 represents a previously unidentified paradigm of cytokine-driven autoimmune inflammation.
Asunto(s)
Adenosina/análogos & derivados , Proteínas Potenciadoras de Unión a CCAAT/inmunología , Interleucina-17/inmunología , Proteínas de Unión al ARN/inmunología , Factor de Necrosis Tumoral alfa/inmunología , Adenosina/inmunología , Animales , Autoinmunidad/inmunología , Proteínas Potenciadoras de Unión a CCAAT/genética , Línea Celular , Femenino , Humanos , Inflamación/inmunología , Interleucina-17/genética , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas de Unión al ARN/genéticaRESUMEN
Commensal fungi are an important part of human microbial community, among which Candida albicans and Candida glabrata are two common opportunistic pathogens. Unlike the high pathogenicity of C. albicans, C. glabrata is reported to show low pathogenicity to the host. Here, by using a Galleria mellonella infection model, we were able to confirm the much lower virulence of C. glabrata than C. albicans. Interestingly, pre-exposure to live C. glabrata (LCG) protects the larvae against subsequent various lethal fungal infections, including C. albicans, Candida tropicalis, and Cryptococcus neoformans. Inconsistently, heat-inactivated C. glabrata (HICG) pre-exposure can only protect against C. albicans or C. tropicalis re-infection, but not C. neoformans. Mechanistically, LCG or HICG pre-exposure enhanced the fungicidal activity of hemocytes against C. albicans or C. tropicalis. Meanwhile, LCG pre-exposure enhanced the humoral immunity by modulating the expression of fungal defending proteins in the cell-free hemolymph, which may contribute to the protection against C. neoformans. Together, this study suggests the important role of C. glabrata in enhancing host immunity, and demonstrates the great potential of G. mellonella model in studying the innate immune responses against infections.
Asunto(s)
Candida glabrata/inmunología , Hongos/inmunología , Hongos/patogenicidad , Mariposas Nocturnas/inmunología , Mariposas Nocturnas/microbiología , Micosis/inmunología , Micosis/prevención & control , Animales , Hongos/clasificación , Hemocitos/inmunología , Hemocitos/microbiología , Inmunidad Humoral , Larva/microbiología , VirulenciaRESUMEN
Disseminated candidiasis caused by the fungus Candida albicans is a major clinical problem in individuals with kidney disease and accompanying uremia; disseminated candidiasis fatality is twice as common in patients with uremia as those with normal kidney function. Many antifungal drugs are nephrotoxic, making treatment of these patients particularly challenging. The underlying basis for this impaired capacity to control infections in uremic individuals is poorly understood. Here, we show in multiple models that uremic mice exhibit an increased susceptibility to systemic fungal infection. Uremia inhibits Glut1-mediated uptake of glucose in neutrophils by causing aberrant activation of GSK3ß, resulting in reduced ROS generation and hence impaired killing of C. albicans in mice. Consequently, pharmacological inhibition of GSK3ß restored glucose uptake and rescued ROS production and candidacidal function of neutrophils in uremic mice. Similarly, neutrophils isolated from patients with kidney disease and undergoing hemodialysis showed similar defect in the fungal killing activity, a phenotype rescued in the presence of a GSK3ß inhibitor. These findings reveal a mechanism of neutrophil dysfunction during uremia and suggest a potentially translatable therapeutic avenue for treatment of disseminated candidiasis.
Asunto(s)
Candidiasis , Enfermedades Renales , Animales , Candida albicans , Candidiasis/complicaciones , Candidiasis/tratamiento farmacológico , Glucosa , Humanos , Ratones , NeutrófilosRESUMEN
AIM: To assess the function of Ngg1 in Candida albicans and reveal the role of NGG1 in the morphological conversion and virulence of C. albicans. MATERIALS & METHODS: C. albicans NGG1 gene was deleted in the wild-type strain SC5314 and the function of Ngg1 was assessed by western blot analysis. The phenotypes and the virulence of the ngg1 mutants were examined. Microarray analysis was performed to explore the mechanism. RESULTS: The ngg1 mutants attenuated acetylated histone H3, obviously reduced filamentous growth and showed significantly diminished pathogenicity in all the infection models. CONCLUSION: This study suggested the histone acetyltransferase activity of C. albicans Ngg1 and revealed the important role of NGG1 in morphological conversion and virulence of C. albicans. [Formula: see text].
Asunto(s)
Candida albicans/crecimiento & desarrollo , Candida albicans/patogenicidad , Proteínas Fúngicas/metabolismo , Histona Acetiltransferasas/metabolismo , Hifa/crecimiento & desarrollo , Animales , Western Blotting , Candida albicans/citología , Candida albicans/genética , Candidiasis/microbiología , Candidiasis/patología , Modelos Animales de Enfermedad , Proteínas Fúngicas/genética , Eliminación de Gen , Perfilación de la Expresión Génica , Histona Acetiltransferasas/genética , Lepidópteros , Ratones , Análisis por Micromatrices , Análisis de Supervivencia , VirulenciaRESUMEN
BACKGROUND: Cyclic acyldepsipeptides (ADEPs) are a novel class of antibacterial agents, some of which (e.g., ADEP 4) are highly active against Gram-positive bacteria. The focus of these in vivo studies is ADEP B315, a rationally designed compound that has the most potent in vitro activity of any ADEP analog reported to date. METHODS: In vivo efficacy experiments were performed using lethal intraperitoneal mice infection models with a methicillin-sensitive S. aureus (MSSA) and a methicillin-resistant (MRSA) strain. The infected mice were treated with ADEP B315, a des-methyl analog of ADEP 4, vancomycin, or the vehicle used for the ADEPs and their survival was assessed daily. A subset of MSSA-infected mice was sacrificed soon after inoculation and the bacterial burden was measured in their livers and spleens. The toxicity of ADEP B315 was assessed in viability assays using human whole blood cultures. RESULTS: In the MSSA experiments, all mice treated with the vehicle succumbed to the infection within 24 hours. All tested compounds were effective in prolonging survival of infected mice (p<0.001). Mice treated with ADEP B315 had a 39% survival rate by 10 days compared to 7% survival in mice treated with a des-methyl ADEP 4 analog (p = 0.017). Survival of the infected mice treated with ADEP B315 was comparable to those treated with vanocmycin (p = 0.12) at the same dose. Further, bacterial burden in the liver and spleen was significantly lower in mice treated with ADEP B315 compared to controls. In the MRSA experiments, ADEP B315 was able to significantly prolong survival compared to mice treated with either the vehicle (p = 0.001) or vancomycin (p = 0.007). ADEP B315 exhibited no significant toxicity in human whole blood cultures at concentrations up to 25 µg/ml. CONCLUSIONS: ADEP B315 is safe and can cure mice that have lethal infections of methicillin-sensitive and -resistant strains of S. aureus.