RESUMEN
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.
Asunto(s)
Infecciones Bacterianas , Neutrófilos , Receptores Acoplados a Proteínas G , Animales , Ratones , Antibacterianos , Proteínas Portadoras , Defensinas/genética , Disbiosis , Queratinocitos , Receptores Acoplados a Proteínas G/metabolismo , Staphylococcus aureusRESUMEN
Shigella is a Gram-negative bacterium that causes bacillary dysentery worldwide. It invades the intestinal epithelium to elicit intense inflammation and tissue damage, yet the underlying mechanisms of its host selectivity and low infectious inoculum remain perplexing. Here, we report that Shigella co-opts human α-defensin 5 (HD5), a host defense peptide important for intestinal homeostasis and innate immunity, to enhance its adhesion to and invasion of mucosal tissues. HD5 promoted Shigella infection in vitro in a structure-dependent manner. Shigella, commonly devoid of an effective host-adhesion apparatus, preferentially targeted HD5 to augment its ability to colonize the intestinal epithelium through interactions with multiple bacterial membrane proteins. HD5 exacerbated infectivity and Shigella-induced pathology in a culture of human colorectal tissues and three animal models. Our findings illuminate how Shigella exploits innate immunity by turning HD5 into a virulence factor for infection, unveiling a mechanism of action for this highly proficient human pathogen.
Asunto(s)
Adhesión Bacteriana/fisiología , Disentería Bacilar/inmunología , Interacciones Huésped-Patógeno/fisiología , Shigella/patogenicidad , alfa-Defensinas , Animales , HumanosRESUMEN
In the mammalian intestine, flagellar motility can provide microbes competitive advantage, but also threatens the spatial segregation established by the host at the epithelial surface. Unlike microbicidal defensins, previous studies indicated that the protective activities of human α-defensin 6 (HD6), a peptide secreted by Paneth cells of the small intestine, resides in its remarkable ability to bind microbial surface proteins and self-assemble into protective fibers and nets. Given its ability to bind flagellin, we proposed that HD6 might be an effective inhibitor of bacterial motility. Here, we utilized advanced automated live cell fluorescence imaging to assess the effects of HD6 on actively swimming Salmonella enterica in real time. We found that HD6 was able to effectively restrict flagellar motility of individual bacteria. Flagellin-specific antibody, a classic inhibitor of flagellar motility that utilizes a mechanism of agglutination, lost its activity at low bacterial densities, whereas HD6 activity was not diminished. A single amino acid variant of HD6 that was able to bind flagellin, but not self-assemble, lost ability to inhibit flagellar motility. Together, these results suggest a specialized role of HD6 self-assembly into polymers in targeting and restricting flagellar motility.
Asunto(s)
Antiinfecciosos , Células de Paneth , Animales , Humanos , Células de Paneth/metabolismo , Flagelina/metabolismo , Antiinfecciosos/metabolismo , Bacterias/metabolismo , Flagelos/metabolismo , MamíferosRESUMEN
SignificanceWe report the development of peptidomimetic antibiotics derived from a natural antimicrobial peptide, human α-defensin 5. By engaging multiple bacterial targets, the lead compound is efficacious in vitro and in vivo against bacteria with highly inducible antibiotic resistance, promising a useful therapeutic agent for the treatment of infections caused by antibiotic-resistant bacteria.
Asunto(s)
Antibacterianos/química , Defensinas/química , Descubrimiento de Drogas/métodos , Peptidomiméticos/química , Antibacterianos/farmacología , Defensinas/farmacología , Bacterias Gramnegativas/efectos de los fármacos , Humanos , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Peptidomiméticos/farmacología , Relación Estructura-ActividadRESUMEN
Metastatic (as well as tumor) microenvironments contain both cancer-promoting and cancer-restraining factors. The balance between these opposing forces determines the fate of cancer cells that disseminate to secondary organ sites. In search for microenvironmental drivers or inhibitors of metastasis, we identified, in a previous study, the beta subunit of hemoglobin (HBB) as a lung-derived antimetastatic factor. In the present study, exploring mechanisms regulating melanoma brain metastasis, we discovered that brain-derived factors restrain proliferation and induce apoptosis and necrosis of brain-metastasizing melanoma cells. Employing various purification procedures, we identified a heterodimer composed of hemoglobin alpha and beta chains that perform these antimetastatic functions. Neither the alpha nor the beta subunit alone was inhibitory. An alpha/beta chain dimer chemically purified from human hemoglobin inhibited the cell viability of primary melanomas, melanoma brain metastasis (MBM), and breast cancer cell lines. The dimer-induced DNA damage, cell cycle arrest at the SubG1 phase, apoptosis, and significant necrosis in four MBM cell lines. Proteomic analysis of dimer-treated MBM cells revealed that the dimer downregulates the expression of BRD4, GAB2, and IRS2 proteins, playing crucial roles in cancer cell sustainability and progression. Thus, we hypothesize that the hemoglobin dimer functions as a resistance factor against brain-metastasizing cancer cells.
Asunto(s)
Antineoplásicos , Neoplasias Encefálicas , Melanoma , Humanos , Melanoma/genética , Proteínas Nucleares , Proteómica , Factores de Transcripción , Neoplasias Encefálicas/genética , Hemoglobinas , Antineoplásicos/farmacología , Necrosis , Línea Celular Tumoral , Microambiente Tumoral , Proteínas que Contienen Bromodominio , Proteínas de Ciclo CelularRESUMEN
The HIV-1 matrix protein p17 (p17) is a pleiotropic molecule impacting on different cell types. Its interaction with many cellular proteins underlines the importance of the viral protein as a major determinant of human specific adaptation. We previously showed the proangiogenic capability of p17. Here, by integrating functional analysis and receptor binding, we identify a functional epitope that displays molecular mimicry with human erythropoietin (EPO) and promotes angiogenesis through common beta chain receptor (ßCR) activation. The functional EPO-like epitope was found to be present in the matrix protein of HIV-1 ancestors SIV originated in chimpanzees (SIVcpz) and gorillas (SIVgor) but not in that of HIV-2 and its ancestor SIVsmm from sooty mangabeys. According to biological data, evolution of the EPO-like epitope showed a clear differentiation between HIV-1/SIVcpz-gor and HIV-2/SIVsmm branches, thus highlighting this epitope on p17 as a divergent signature discriminating HIV-1 and HIV-2 ancestors. P17 is known to enhance HIV-1 replication. Similarly to other ßCR ligands, p17 is capable of attracting and activating HIV-1 target cells and promoting a proinflammatory microenvironment. Thus, it is tempting to speculate that acquisition of an epitope on the matrix proteins of HIV-1 ancestors capable of triggering ßCR may have represented a critical step to enhance viral aggressiveness and early human-to-human SIVcpz/gor dissemination. The hypothesis that the p17/ßCR interaction and ßCR abnormal stimulation may also play a role in sustaining chronic activation and inflammation, thus marking the difference between HIV-1 and HIV-2 in term of pathogenicity, needs further investigation.
Asunto(s)
Eritropoyetina/genética , Antígenos VIH/metabolismo , VIH-1/metabolismo , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/metabolismo , Células Cultivadas , Epítopos/inmunología , Eritropoyetina/metabolismo , Evolución Molecular , Antígenos VIH/genética , Seropositividad para VIH , VIH-1/genética , VIH-2 , Humanos , Imitación Molecular , Virus de la Inmunodeficiencia de los Simios , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
Mouse α-defensins, better known as cryptdins, are host protective antimicrobial peptides produced in the intestinal crypt by Paneth cells. To date, more than 20 cryptdin mRNAs have been identified from mouse small intestine, of which the first six cryptdins (Crp1 to Crp6) have been isolated and characterized at the peptide level. We quantified bactericidal activities against Escherichia coli and Staphylococcus aureus of the 17 cryptdin isoforms identified by Ouellette and colleagues from a single jejunal crypt (A. J. Ouellette et al., Infect Immun 62:5040-5047, 1994), along with linearized analogs of Crp1, Crp4, and Crp14. In addition, we analyzed the most potent and weakest cryptdins in the panel with respect to their ability to self-associate in solution. Finally, we solved, for the first time, the high-resolution crystal structure of a cryptdin, Crp14, and performed molecular dynamics simulation on Crp14 and a hypothetical mutant, T14K-Crp14. Our results indicate that mutational effects are highly dependent on cryptdin sequence, residue position, and bacterial strain. Crp14 adopts a disulfide-stabilized, three-stranded ß-sheet core structure and forms a noncanonical dimer stabilized by asymmetrical interactions between the two ß1 strands in parallel. The killing of E. coli by cryptdins is generally independent of their tertiary and quaternary structures that are important for the killing of S. aureus, which is indicative of two distinct mechanisms of action. Importantly, sequence variations impact the bactericidal activity of cryptdins by influencing their ability to self-associate in solution. This study expands our current understanding of how cryptdins function at the molecular level.
Asunto(s)
alfa-Defensinas , Ratones , Animales , Secuencia de Aminoácidos , Escherichia coli/genética , Staphylococcus aureus , Intestino Delgado , Isoformas de ProteínasRESUMEN
Alternative antibacterial therapies refractory to existing mechanisms of antibiotic resistance are urgently needed. One such attractive therapy is to inhibit bacterial adhesion and colonization. Ser O-heptosylation (Ser O-Hep) on autotransporters of Gram-negative bacteria is a novel glycosylation and has been proven to be essential for bacterial colonization. Herein, we chemically synthesized glycopeptides containing this atypical glycan structure and an absolute C6 configuration through the assembly of Ser O-Hep building blocks. Using glycopeptides as haptens, we generated first-in-class poly- and monoclonal antibodies, termed Anti-SerHep1a and Anti-SerHep1b, that stereoselectively recognize Ser O-heptosylation (d/l-glycero) with high specificity in vitro and in vivo. Importantly, these antibodies effectively blocked diffusely adhering Escherichia coli 2787 adhesion to HeLa cells and in mice in a dose- and Ser O-Hep-dependent manner. Together, these antibodies represent not only useful tools for the discovery of unknown serine O-heptosylated proteins bearing various C6 chiral centers but also a novel class of antiadhesion therapeutic agents for the treatment of bacterial infection.
Asunto(s)
Anticuerpos Monoclonales , Polisacáridos , Humanos , Animales , Ratones , Células HeLa , Glicosilación , Polisacáridos/química , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales/uso terapéutico , Escherichia coli , Glicopéptidos/químicaRESUMEN
Defensins are short cationic, amphiphilic, cysteine-rich peptides that constitute the front-line immune defense against various pathogens. In addition to exerting direct antibacterial activities, defensins inactivate several classes of unrelated bacterial exotoxins. To date, no coherent mechanism has been proposed to explain defensins' enigmatic efficiency toward various toxins. In this study, we showed that binding of neutrophil ?-defensin HNP1 to affected bacterial toxins caused their local unfolding, potentiated their thermal melting and precipitation, exposed new regions for proteolysis, and increased susceptibility to collisional quenchers without causing similar effects on tested mammalian structural and enzymatic proteins. Enteric ?-defensin HD5 and ?-defensin hBD2 shared similar toxin-unfolding effects with HNP1, albeit to different degrees. We propose that protein susceptibility to inactivation by defensins is contingent to their thermolability and conformational plasticity and that defensin-induced unfolding is a key element in the general mechanism of toxin inactivation by human defensins.
Asunto(s)
Toxinas Bacterianas/metabolismo , Exotoxinas/metabolismo , alfa-Defensinas/metabolismo , alfa-Defensinas/farmacología , beta-Defensinas/metabolismo , Antígenos Bacterianos/metabolismo , Proteínas Bacterianas/metabolismo , Línea Celular , Quimotripsina/metabolismo , Enterotoxinas/metabolismo , Humanos , Unión Proteica , Conformación Proteica , Desplegamiento Proteico , Proteolisis , Proteínas Represoras/metabolismo , Termolisina/metabolismo , alfa-Defensinas/inmunologíaRESUMEN
Currently, there are no approved drugs for the treatment of flavivirus infection. Accordingly, we tested the inhibitory effects of the novel θ-defensin retrocyclin-101 (RC-101) against flavivirus infection and investigated the mechanism underlying the potential inhibitory effects. First, RC-101 robustly inhibited both Japanese encephalitis virus (JEV) and Zika virus (ZIKV) infections. RC-101 exerted inhibitory effects on the entry and replication stages. Results also indicated that the nonstructural protein NS2B-NS3 serine protease might serve as a potential viral target. Furthermore, RC-101 inhibited protease activity at the micromolar level. We also demonstrated that with respect to the glycoprotein E protein of flavivirus, the DE loop of domain III (DIII), which is the receptor-binding domain of the E protein, might serve as another viral target of RC-101. Moreover, a JEV DE mutant exhibited resistance to RC-101, which was associated with deceased binding affinity of RC-101 to DIII. These findings provide a basis for the development of RC-101 as a potential candidate for the treatment of flavivirus infection. IMPORTANCE Retrocyclin is an artificially humanized circular θ-defensin peptide, containing 18 residues, previously reported to possess broad antimicrobial activity. In this study, we found that retrocyclin-101 inhibited flavivirus (ZIKV and JEV) infections. Retrocyclin-101 inhibited NS2B-NS3 serine protease activity, suggesting that the catalytic triad of the protease is the target. Moreover, retrocyclin-101 bound to the DE loop of the E protein of flavivirus, which prevented its entry.
Asunto(s)
Antivirales/farmacología , Encefalitis Japonesa/tratamiento farmacológico , Péptidos/farmacología , Proteínas no Estructurales Virales/antagonistas & inhibidores , Infección por el Virus Zika/tratamiento farmacológico , Animales , Chlorocebus aethiops , Cricetinae , Defensinas/química , Virus de la Encefalitis Japonesa (Especie)/crecimiento & desarrollo , Humanos , Dominios Proteicos/genética , Células Vero , Proteínas del Envoltorio Viral/metabolismo , Internalización del Virus/efectos de los fármacos , Replicación Viral/efectos de los fármacos , Virus Zika/crecimiento & desarrolloRESUMEN
The design of bioactive supramolecular chirality is always hampered by the lack of feasible schemes to assigned specific biological activities. Herein, we developed a "mirror-image peptide grafting" method to graft the epitopes of bioactive d-peptide onto the miniprotein template to construct a self-assembled supraparticle. Grafting DPMIß, a 12-mer d-enantiomeric peptide functioned as the p53 agonist, onto Apamin, we successfully constructed a self-assembled d-enantiomeric miniprotein supermolecule nanoparticle, termed DMSN. This chiral supraparticle possesses a favorable pharmaceutical profile including the passive tumor targeting, cell membrane penetration, intracellular reductive responsiveness, and endosome escaping. DMSN showed in vitro and in vivo p53-dependent antiproliferative activity and augmented antitumor immunity elicited by anti-PD1 therapy. This enabling strategy will allow us to fabricate a class of peptide/protein-derived supramolecular chirality with predictable biological activities and will likely have a broad impact on the chiral nanotechnology at the service of prevention and treatment of human diseases.
Asunto(s)
Nanopartículas , Nanotecnología , Humanos , Inmunoterapia , Péptidos , EstereoisomerismoRESUMEN
Human neutrophil peptides (HNPs), also known as human myeloid α-defensins degranulated by infiltrating neutrophils at bacterial infection loci, exhibit broad antomicrobial activities against bacteria, fungi, and viruses. We have made a surprising recent finding that Shigella, a highly contagious, yet poorly adhesive enteric pathogen, exploits human α-defensins including HNP1 to enhance its adhesion to and invasion of host epithelial cells. However, the critical molecular determinants responsible for HNP1-enhanced Shigella adhesion and invasion have yet to be investigated. Using cultured epithelial cells and polarised Caco2 cells as an in vitro infection model, we demonstrated that HNP1 promoted Shigella infection in a structure- and sequence-dependent manner, with two bulky hydrophobic residues, Trp26 and Phe28 important for HNP1 self-assembly, being most critical. The functional importance of hydrophobicity for HNP1-enhanced Shigella infection was further verified by substitutions for Trp26 of a series of unnatural amino acids with straight aliphatic side chains of different lengths. Dissection of the Shigella infection process revealed that bacteria-rather than host cells-bound HNP1 contributed most to the enhancement. Further, mutagenesis analysis of bacterial surface components, while precluding the involvement of lipopolysaccharides (LPS) in the interaction with HNP1, identified outer membrane proteins and the Type 3 secretion apparatus as putative binding targets of HNP1 involved in enhanced Shigella adhesion and invasion. Our findings provide molecular and mechanistic insights into the mode of action of HNP1 in promoting Shigella infection, thus showcasing another example of how innate immune factors may serve as a double-edged sword in health and disease.
Asunto(s)
Adhesión Bacteriana , Células Epiteliales/microbiología , Shigella flexneri/patogenicidad , alfa-Defensinas/metabolismo , Aminoácidos/química , Animales , Células CACO-2 , Disentería Bacilar , Células Epiteliales/metabolismo , Cobayas , Células HCT116 , Células HeLa , Interacciones Huésped-Patógeno , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Lipopolisacáridos/metabolismo , Microscopía Electrónica de Rastreo , Mutagénesis , Neutrófilos/inmunología , Shigella flexneri/ultraestructura , alfa-Defensinas/químicaRESUMEN
Intracellular protein-protein interactions (PPIs) are a vital and yet underexploited class of therapeutic targets for their crucial roles in cellular processes and involvement in disease initiation and progression. Although some successful chemistry and nanotechnologies have been introduced into peptide PPI modulators to allow cell and tissue permeability, significant challenges remain with regard to the efficient and precise modulation of PPIs within specific cells of diseased tissues, such as solid tumors. Herein, an intratumoral transformable hierarchical framework, termed iPLF, was fabricated via a two-step self-assembly between peptides and lanthanide-doped nanocrystals. In this proof-of-concept study, using NanoEL effect, TME response, and tumor marker targeting, iPLF in vivo delivered the p53-MDM2 modulator DPMI into tumor cells and ß-catenin-Bcl9 modulator Bcl9p into tumor stem cells. This crafted programmed nanomedicine with triple-stage delivery and responsiveness accurately modulated the specific intracellular protein-protein interactions, resulting in the suppression of tumor growth and metastasis in vivo, while maintaining a highly favorable safety profile. iPLF reached the goal of accurate, potent, and hazard-free intracellular PPI modulation, thereby providing a means to improve current knowledge of PPI networks and a novel therapeutic strategy for a great variety of diseases.
Asunto(s)
Antineoplásicos/farmacología , Elementos de la Serie de los Lantanoides/farmacología , Neoplasias/tratamiento farmacológico , Péptidos/farmacología , Mapas de Interacción de Proteínas/efectos de los fármacos , Animales , Antineoplásicos/química , Antineoplásicos/uso terapéutico , Preparaciones de Acción Retardada/química , Diseño de Fármacos , Células HCT116 , Humanos , Elementos de la Serie de los Lantanoides/química , Elementos de la Serie de los Lantanoides/uso terapéutico , Ratones , Nanomedicina , Nanopartículas/química , Neoplasias/metabolismo , Neoplasias/patología , Péptidos/química , Péptidos/uso terapéutico , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Factores de Transcripción/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , beta Catenina/metabolismoRESUMEN
Human α-defensins are 3- to 5-kDa disulfide-bridged peptides with a multitude of antimicrobial activities and immunomodulatory functions. Recent studies show that human enteric α-defensin 5 (HD5), a host defense peptide important for intestinal homeostasis and innate immunity, aids the highly infectious enteropathogen Shigella in breaching the intestinal epithelium in vitro and in vivo Whether and how HD5 influences Shigella infection of resident macrophages following its invasion of the intestinal epithelium remain poorly understood. Here, we report that HD5 greatly promoted phagocytosis of Shigella by macrophages by targeting the bacteria to enhance bacterium-to-cell contacts in a structure- and sequence-dependent fashion. Subsequent intracellular multiplication of phagocytosed Shigella led to massive necrotic cell death and release of the bacteria. HD5-promoted phagocytosis of Shigella was independent of the status of the type 3 secretion system. Furthermore, HD5 neither inhibited nor enhanced phagosomal escape of Shigella Collectively, these findings confirm a potential pathogenic role of HD5 in Shigella infection of not only epithelial cells but also macrophages, illuminating how an enteropathogen exploits a host protective factor for virulence and infection.
Asunto(s)
Disentería Bacilar/microbiología , Disentería Bacilar/patología , Interacciones Huésped-Patógeno , Shigella/patogenicidad , alfa-Defensinas/metabolismo , Animales , Adhesión Bacteriana , Células Cultivadas , Células Epiteliales/microbiología , Humanos , Mucosa Intestinal/microbiología , Mucosa Intestinal/patología , Macrófagos/microbiología , Ratones , FagocitosisRESUMEN
Clinical translation of therapeutic peptides, particularly those targeting intracellular protein-protein interactions (PPIs), has been hampered by their inefficacious cellular internalization in diseased tissue. Therapeutic peptides engineered into nanostructures with stable spatial architectures and smart disease targeting ability may provide a viable strategy to overcome the pharmaceutical obstacles of peptides. This study describes a strategy to assemble therapeutic peptides into a stable peptide-Au nanohybrid, followed by further self-assembling into higher-order nanoclusters with responsiveness to tumor microenvironment. As a proof of concept, an anticancer peptide termed ß-catenin/Bcl9 inhibitors is copolymerized with gold ion and assembled into a cluster of nanohybrids (pCluster). Through a battery of in vitro and in vivo tests, it is demonstrated that pClusters potently inhibit tumor growth and metastasis in several animal models through the impairment of the Wnt/ß-catenin pathway, while maintaining a highly favorable biosafety profile. In addition, it is also found that pClusters synergize with the PD1/PD-L1 checkpoint blockade immunotherapy. This new strategy of peptide delivery will likely have a broad impact on the development of peptide-derived therapeutic nanomedicine and reinvigorate efforts to discover peptide drugs that target intracellular PPIs in a great variety of human diseases, including cancer.
RESUMEN
Neutrophils are the first and most numerous cells to arrive at the site of an inflammatory insult and are among the first to die. We previously reported that alpha defensins, released from apoptotic human neutrophils, augmented the antimicrobial capacity of macrophages while also inhibiting the biosynthesis of proinflammatory cytokines. In vivo, alpha defensin administration protected mice from inflammation, induced by thioglychollate-induced peritonitis or following infection withSalmonella entericaserovar Typhimurium. We have now dissected the antiinflammatory mechanism of action of the most abundant neutrophil alpha defensin, Human Neutrophil Peptide 1 (HNP1). Herein we show that HNP1 enters macrophages and inhibits protein translation without inducing the unfolded-protein response or affecting mRNA stability. In a cell-free in vitro translation system, HNP1 powerfully inhibited both cap-dependent and cap-independent mRNA translation while maintaining mRNA polysomal association. This is, to our knowledge, the first demonstration of a peptide released from one cell type (neutrophils) directly regulating mRNA translation in another (macrophages). By preventing protein translation, HNP1 functions as a "molecular brake" on macrophage-driven inflammation, ensuring both pathogen clearance and the resolution of inflammation with minimal bystander tissue damage.
Asunto(s)
Macrófagos/metabolismo , Biosíntesis de Proteínas , ARN Mensajero/metabolismo , Infecciones por Salmonella/metabolismo , Salmonella typhimurium , alfa-Defensinas/metabolismo , Animales , Humanos , Macrófagos/patología , Ratones , Infecciones por Salmonella/patología , alfa-Defensinas/farmacologíaRESUMEN
α-defensins are abundant antimicrobial peptides with broad, potent antibacterial, antifungal, and antiviral activities in vitro. Although their contribution to host defense against bacteria in vivo has been demonstrated, comparable studies of their antiviral activity in vivo are lacking. Using a mouse model deficient in activated α-defensins in the small intestine, we show that Paneth cell α-defensins protect mice from oral infection by a pathogenic virus, mouse adenovirus 1 (MAdV-1). Survival differences between mouse genotypes are lost upon parenteral MAdV-1 infection, strongly implicating a role for intestinal defenses in attenuating pathogenesis. Although differences in α-defensin expression impact the composition of the ileal commensal bacterial population, depletion studies using broad-spectrum antibiotics revealed no effect of the microbiota on α-defensin-dependent viral pathogenesis. Moreover, despite the sensitivity of MAdV-1 infection to α-defensin neutralization in cell culture, we observed no barrier effect due to Paneth cell α-defensin activation on the kinetics and magnitude of MAdV-1 dissemination to the brain. Rather, a protective neutralizing antibody response was delayed in the absence of α-defensins. This effect was specific to oral viral infection, because antibody responses to parenteral or mucosal ovalbumin exposure were not affected by α-defensin deficiency. Thus, α-defensins play an important role as adjuvants in antiviral immunity in vivo that is distinct from their direct antiviral activity observed in cell culture.
Asunto(s)
Infecciones por Adenoviridae/inmunología , Adenoviridae/inmunología , Antiinfecciosos/inmunología , Anticuerpos Neutralizantes/inmunología , Antivirales/inmunología , Defensinas/inmunología , Animales , Femenino , Humanos , Íleon/inmunología , Intestino Delgado/inmunología , Intestinos/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Células de Paneth/inmunología , alfa-Defensinas/inmunologíaRESUMEN
Although in decline after successful anti-HIV therapy, B-cell lymphomas are still elevated in HIV-1-seropositive (HIV+) persons, and the mechanisms are obscure. The HIV-1 matrix protein p17 persists in germinal centers long after HIV-1 drug suppression, and some p17 variants (vp17s) activate Akt signaling and promote growth of transformed B cells. Here we show that vp17s derived from four of five non-Hodgkin lymphoma (NHL) tissues from HIV+ subjects display potent B-cell growth-promoting activity. They are characterized by amino acid insertions at position 117-118 (Ala-Ala) or 125-126 (Gly-Asn or Gly-Gln-Ala-Asn-Gln-Asn) among some other mutations throughout the sequence. Identical dominant vp17s are found in both tumor and plasma. Three of seven plasma samples from an independent set of NHL cases manifested multiple Ala insertions at position 117-118, and one with the Ala-Ala profile also promoted B-cell growth and activated Akt signaling. Ultradeep pyrosequencing showed that vp17s with C-terminal insertions are more frequently detected in plasma of HIV+ subjects with than without NHL. Insertion of Ala-Ala at position 117-118 into reference p17 (refp17) was sufficient to confer B-cell growth-promoting activity. In contrast, refp17 bearing the Gly-Asn insertion at position 125-126 did not, suggesting that mutations not restricted to the C terminus can also account for this activity. Biophysical analysis revealed that the Ala-Ala insertion mutant is destabilized compared with refp17, whereas the Gly-Asn form is stabilized. This finding provides an avenue for further exploration of structure function relationships and new treatment strategies in combating HIV-1-related NHL.
Asunto(s)
Transformación Celular Viral , Antígenos VIH/metabolismo , Infecciones por VIH/metabolismo , VIH-1/metabolismo , Linfoma de Células B/metabolismo , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/metabolismo , Adulto , Linfocitos B/metabolismo , Linfocitos B/patología , Línea Celular Tumoral , Femenino , Antígenos VIH/genética , Infecciones por VIH/genética , Infecciones por VIH/patología , VIH-1/genética , Humanos , Linfoma de Células B/genética , Masculino , Persona de Mediana Edad , Mutagénesis Insercional , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/genética , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
West Nile virus (WNV) is a re-emerging pathogen and the leading cause of epidemic encephalitis in the United States. Inflammatory monocytes are a critical component of the cellular infiltrate found in the CNS during WNV encephalitis, although the molecular cues involved in their migration are not fully understood. In mice, we previously showed that WNV infection induces a CCR2-dependent monocytosis that precedes monocyte migration into the CNS. Currently, the relative contribution of the CCR2 ligands, chemokines CCL2 and CCL7, in directing monocyte mobilization and leukocyte migration into the CNS is unclear. In this study, we demonstrate that, although both CCL2 and CCL7 are required for efficient monocytosis and monocyte accumulation in the CNS, only CCL7 deficiency resulted in increased viral burden in the brain and enhanced mortality. The enhanced susceptibility in the absence of CCL7 was associated with the delayed migration of neutrophils and CD8(+) T cells into the CNS compared with WT or Ccl2(-/-) mice. To determine whether CCL7 reconstitution could therapeutically alter the survival outcome of WNV infection, we administered exogenous CCL7 i.v. to WNV-infected Ccl7(-/-) mice and observed a significant increase in monocytes and neutrophils, but not CD8(+) T cells, within the CNS, as well as an enhancement in survival compared with Ccl7(-/-) mice treated with a linear CCL7 control peptide. Our experiments suggest that CCL7 is an important protective signal involved in leukocyte trafficking during WNV infection, and it may have therapeutic potential for the treatment of acute viral infections of the CNS.