RESUMEN
While the structure and composition of the scientific manuscript is well known within scientific communities, insider knowledge such as the tricks of the trade and editorial viewpoints of scientific publishing are often less known to early-career research scientists. This article focuses on the key aspects of scientific publishing, including tips for success geared towards senior postdocs and junior faculty. It also highlights important considerations for getting manuscripts published in an efficient and successful manner.
RESUMEN
Innate-like T cells, including invariant natural killer T cells, mucosal-associated invariant T cells, and γδ T cells, are present in various barrier tissues, including the lung, where they carry out protective responses during infections. Here, we investigate their roles during pulmonary pneumococcal infection. Following infection, innate-like T cells rapidly increase in lung tissue, in part through recruitment, but T cell antigen receptor activation and cytokine production occur mostly in interleukin-17-producing NKT17 and γδ T cells. NKT17 cells are preferentially located within lung tissue prior to infection, as are CD103+ dendritic cells, which are important both for antigen presentation to NKT17 cells and γδ T cell activation. Whereas interleukin-17-producing γδ T cells are numerous, granulocyte-macrophage colony-stimulating factor is exclusive to NKT17 cells and is required for optimal protection. These studies demonstrate how particular cellular interactions and responses of functional subsets of innate-like T cells contribute to protection from pathogenic lung infection.
Asunto(s)
Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Células T Asesinas Naturales/inmunología , Células T Asesinas Naturales/metabolismo , Animales , Línea Celular , Células Dendríticas/inmunología , Femenino , Humanos , Interferón gamma/inmunología , Interleucina-17/inmunología , Linfocitos Intraepiteliales/inmunología , Linfocitos Intraepiteliales/metabolismo , Pulmón/inmunología , Pulmón/microbiología , Activación de Linfocitos , Masculino , Ratones , Ratones Endogámicos C57BL , Infecciones Neumocócicas/inmunología , Receptores de Antígenos de Linfocitos T gamma-delta/inmunología , Streptococcus pneumoniae/inmunologíaRESUMEN
Streptococcus pneumoniae is an important bacterial pathogen that causes a range of noninvasive and invasive diseases. The mechanisms underlying variability in the ability of S. pneumoniae to transition from nasopharyngeal colonization to disease-causing pathogen are not well defined. Mucosal-associated invariant T (MAIT) cells are prevalent in mucosal tissues such as the airways and are believed to play an important role in the early response to infection with bacterial pathogens. The ability of MAIT cells to recognize and contain infection with S. pneumoniae is not known. In the present study, we analyzed MAIT-cell responses to infection with clinical isolates of S. pneumoniae serotype 19A, a serotype linked to invasive pneumococcal disease. We found that although MAIT cells were capable of responding to human dendritic and airway epithelial cells infected with S. pneumoniae, the magnitude of response to different serotype 19A isolates was determined by genetic differences in the expression of the riboflavin biosynthesis pathway. MAIT-cell release of cytokines correlated with differences in the ability of MAIT cells to respond to and control S. pneumoniae in vitro and in vivo in a mouse challenge model. Together, these results demonstrate first that there are genetic differences in riboflavin metabolism among clinical isolates of the same serotype and second that these likely determine MAIT-cell function in response to infection with S. pneumoniae. These differences are critical when considering the role that MAIT cells play in early responses to pneumococcal infection and determining whether invasive disease will develop.
Asunto(s)
Interacciones Huésped-Patógeno/fisiología , Mucosa Respiratoria/citología , Riboflavina/metabolismo , Streptococcus pneumoniae/metabolismo , Linfocitos T/microbiología , Animales , Citocinas/metabolismo , Células Dendríticas/microbiología , Regulación Bacteriana de la Expresión Génica , Humanos , Ratones Mutantes , Fagocitosis , Mucosa Respiratoria/microbiología , Riboflavina/genética , Streptococcus pneumoniae/aislamiento & purificación , Streptococcus pneumoniae/patogenicidadRESUMEN
During pneumococcal pneumonia, antibacterial defense requires the orchestrated expression of innate immunity mediators, initiated by alveolar macrophages and dependent on transcription driven by nuclear factor κB (NF-κB). Such immune pressure may select for pneumococci, which avoid or subvert macrophage NF-κB activation. Analyzing pneumococci collected from children in Massachusetts, we found that the activation of macrophage NF-κB by Streptococcus pneumoniae is highly diverse, with a preponderance of low NF-κB activators that associate particularly with complicated pneumonia. Low NF-κB activators cause more severe lung infections in mice, and they drive macrophages toward an alternate and detrimental cell fate of necroptosis. Both outcomes can be reversed by activation of macrophages with pneumococci that are high NF-κB activators. These results suggest that low NF-κB activation is a virulence property of pneumococci and that the appropriate activation of macrophages, including NF-κB, may hold promise as an adjunct therapeutic avenue for pneumococcal pneumonia.
Asunto(s)
Macrófagos Alveolares/metabolismo , FN-kappa B/metabolismo , Necrosis/inmunología , Neumonía Neumocócica/inmunología , Streptococcus pneumoniae , Animales , Modelos Animales de Enfermedad , Femenino , Inmunidad Innata , Macrófagos Alveolares/microbiología , Masculino , Ratones , Ratones Endogámicos C57BL , Necrosis/terapia , Neumonía Neumocócica/terapia , Células RAW 264.7 , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Zinc finger protein 36, C3H type-like 1 (ZFP36L1) is one of several Zinc Finger Protein 36 (Zfp36) family members, which bind AU rich elements within 3' untranslated regions (UTRs) to negatively regulate the post-transcriptional expression of targeted mRNAs. The prototypical member of the family, Tristetraprolin (TTP or ZFP36), has been well-studied in the context of inflammation and plays an important role in repressing pro-inflammatory transcripts such as TNF-α. Much less is known about the other family members, and none have been studied in the context of infection. Using macrophage cell lines and primary alveolar macrophages we demonstrated that, like ZFP36, ZFP36L1 is prominently induced by infection. To test our hypothesis that macrophage production of ZFP36L1 is necessary for regulation of the inflammatory response of the lung during pneumonia, we generated mice with a myeloid-specific deficiency of ZFP36L1. Surprisingly, we found that myeloid deficiency of ZFP36L1 did not result in alteration of lung cytokine production after infection, altered clearance of bacteria, or increased inflammatory lung injury. Although alveolar macrophages are critical components of the innate defense against respiratory pathogens, we concluded that myeloid ZFP36L1 is not essential for appropriate responses to bacteria in the lungs. Based on studies conducted with myeloid-deficient ZFP36 mice, our data indicate that, of the Zfp36 family, ZFP36 is the predominant negative regulator of cytokine expression in macrophages. In conclusion, these results imply that myeloid ZFP36 may fully compensate for loss of ZFP36L1 or that Zfp36l1-dependent mRNA expression does not play an integral role in the host defense against bacterial pneumonia.
Asunto(s)
Infecciones Bacterianas/metabolismo , Inflamación/metabolismo , Proteínas Nucleares/metabolismo , Neumonía Bacteriana/metabolismo , Proteínas de Unión al ARN/metabolismo , Animales , Infecciones Bacterianas/microbiología , Líquido del Lavado Bronquioalveolar/microbiología , Factor 1 de Respuesta al Butirato , Línea Celular , Citocinas/metabolismo , Humanos , Inflamación/microbiología , Pulmón/metabolismo , Pulmón/microbiología , Macrófagos/metabolismo , Macrófagos/microbiología , Ratones , Ratones Endogámicos C57BL , Células Mieloides/metabolismo , Células Mieloides/microbiología , Neumonía Bacteriana/microbiología , ARN Mensajero/metabolismoRESUMEN
Innate immunity is critical for clearing Pseudomonas aeruginosa from the lungs. In response to P. aeruginosa infection, a central transcriptional regulator of innate immunity-NF-kappaB-is translocated within 15 min to the nuclei of respiratory epithelial cells expressing wild-type (WT) cystic fibrosis (CF) transmembrane conductance regulator (CFTR). P. aeruginosa clearance from lungs is impaired in CF, and rapid NF-kappaB nuclear translocation is defective in cells with mutant or missing CFTR. We used WT and mutant P. aeruginosa and strains of transgenic mice lacking molecules involved in innate immunity to identify additional mediators required for P. aeruginosa-induced rapid NF-kappaB nuclear translocation in lung epithelia. We found neither Toll-like receptor 2 (TLR2) nor TLR4 nor TLR5 were required for this response. However, both MyD88-deficient mice and interleukin-1 receptor (IL-1R)-deficient mice failed to rapidly translocate NF-kappaB to the nuclei of respiratory epithelial cells in response to P. aeruginosa. Cultured human bronchial epithelial cells rapidly released IL-1beta in response to P. aeruginosa; this process was maximized by expression of WT-CFTR and dramatically muted in cells with DeltaF508-CFTR. The IL-1R antagonist blocked P. aeruginosa-induced NF-kappaB nuclear translocation. Oral inoculation via drinking water of IL-1R knockout mice resulted in higher rates of lung colonization and elevated P. aeruginosa-specific antibody titers in a manner analogous to that of CFTR-deficient mice. Overall, rapid IL-1 release and signaling through IL-1R represent key steps in the innate immune response to P. aeruginosa infection, and this process is deficient in cells lacking functional CFTR.
Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/fisiología , Interleucina-1beta/inmunología , Infecciones por Pseudomonas/inmunología , Pseudomonas aeruginosa/inmunología , Receptores de Interleucina-1/inmunología , Animales , Línea Celular , Núcleo Celular/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Dípteros , Humanos , Inmunidad Innata , Interleucina-1beta/metabolismo , Pulmón/inmunología , Pulmón/metabolismo , Ratones , Ratones Endogámicos , Ratones Noqueados , Microscopía Confocal , Factor 88 de Diferenciación Mieloide/genética , Factor 88 de Diferenciación Mieloide/inmunología , FN-kappa B/metabolismo , Infecciones por Pseudomonas/metabolismo , Receptores de Interleucina-1/genética , Receptor Toll-Like 2/inmunología , Receptor Toll-Like 4/inmunología , Receptor Toll-Like 5/inmunologíaRESUMEN
Two fully human mAbs specific for epitopes dependent on intact carboxylate groups on the C6 carbon of the mannuronic acid components of Pseudomonas aeruginosa alginate were found to promote phagocytic killing of both mucoid and nonmucoid strains as well as protection against both types of strains in a mouse model of acute pneumonia. The specificity of the mAbs for alginate was determined by ELISA and killing assays. Some strains of P. aeruginosa did not make detectable alginate in vitro, but in vivo protection against lethal pneumonia was obtained and shown to be due to rapid induction of expression of alginate in the murine lung. No protection against strains genetically unable to make alginate was achieved. These mAbs have potential to be passive therapeutic reagents for all strains of P. aeruginosa and the results document that alginate is a target for the proper type of protective Ab even when expressed at low levels on phenotypically nonmucoid strains.
Asunto(s)
Anticuerpos Antibacterianos/administración & dosificación , Anticuerpos Monoclonales/administración & dosificación , Ácido Glucurónico/inmunología , Ácidos Hexurónicos/inmunología , Infecciones por Pseudomonas/inmunología , Infecciones por Pseudomonas/prevención & control , Pseudomonas aeruginosa/inmunología , Enfermedad Aguda , Alginatos/metabolismo , Animales , Anticuerpos Antibacterianos/biosíntesis , Anticuerpos Antibacterianos/genética , Anticuerpos Antibacterianos/metabolismo , Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/metabolismo , Sitios de Unión de Anticuerpos , Modelos Animales de Enfermedad , Femenino , Ácido Glucurónico/metabolismo , Ácidos Hexurónicos/metabolismo , Humanos , Hibridomas , Región Variable de Inmunoglobulina/administración & dosificación , Región Variable de Inmunoglobulina/biosíntesis , Región Variable de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/metabolismo , Pulmón/inmunología , Pulmón/microbiología , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Fagocitosis/inmunología , Neumonía Bacteriana/inmunología , Neumonía Bacteriana/microbiología , Neumonía Bacteriana/prevención & control , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/patogenicidad , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/metabolismo , Especificidad de la EspecieRESUMEN
Acute pneumonias and corneal infections due to Pseudomonas aeruginosa are typically caused by lipopolysaccharide (LPS)-smooth strains. In cystic fibrosis patients, however, LPS-rough strains of P. aeruginosa, which lack O antigen, can survive in the lung and cause chronic infection. It is not clear whether an LPS-rough phenotype affects cytotoxicity related to the type III secretion system (TTSS). We previously reported that interruption of the galU gene in P. aeruginosa results in production of a rough LPS and truncated LPS core. Here we evaluated the role of the galU gene in the pathogenesis of murine lung and eye infections and in cytotoxicity due to the TTSS effector ExoU. We studied galU mutants of strain PAO1, of its cytotoxic variant expressing ExoU from a plasmid, and of the inherently cytotoxic strain PA103. The galU mutants were more serum sensitive than the parental strains but remained cytotoxic in vitro. In a corneal infection model, the galU mutants were significantly attenuated. In an acute pneumonia model, the 50% lethal doses of the galU mutants were higher than those of the corresponding wild-type strains, yet these mutants could cause mortality and severe pneumonia, as judged by histology, even with minimal systemic spread. These findings suggest that the galU gene is required for corneal infection and for efficient systemic spread following lung infection but is not required for infection confined to the lung. Host defenses in the lung appear to be insufficient to control infection with LPS-rough P. aeruginosa when local bacterial levels are high.
Asunto(s)
Córnea/microbiología , Infecciones Bacterianas del Ojo/enzimología , Pseudomonas aeruginosa/genética , UTP-Glucosa-1-Fosfato Uridililtransferasa/genética , Animales , Femenino , Pulmón/metabolismo , Pulmón/patología , Ratones , Mutación , Neumonía/metabolismo , Neumonía/patología , Pseudomonas aeruginosa/enzimología , Pseudomonas aeruginosa/inmunología , Pseudomonas aeruginosa/patogenicidad , VirulenciaRESUMEN
Deterioration of lung function in patients with cystic fibrosis (CF) is closely associated with chronic pulmonary infection with mucoid Pseudomonas aeruginosa. The mucoid exopolysaccharide (MEP) from P. aeruginosa has been shown to induce opsonic antibodies in mice that are protective against this chronic infection. MEP-specific opsonic antibodies are also commonly found in the sera of older CF patients lacking detectable P. aeruginosa infection. When used in a human vaccine trial, however, MEP only minimally induced opsonic antibodies. To evaluate whether conjugation of MEP to a carrier protein could improve its immunogenicity, we bound thiolated MEP to keyhole limpet hemocyanin (KLH) by using succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) as a linker. In contrast to the native MEP polymer, the MEP-KLH conjugate vaccine induced high titers of MEP-specific immunoglobulin G (IgG) in C3H-HeN mice and in a rabbit. Sera from mice immunized with MEP-KLH conjugate, but not from animals immunized with comparable doses of native MEP, demonstrated opsonic killing activity. Vaccination with MEP-KLH conjugate induced opsonic antibodies broadly cross-reactive to heterologous mucoid strains of P. aeruginosa. Preexisting nonopsonic antibodies to MEP are found in normal human sera, including young CF patients, and their presence impedes the induction of opsonic antibodies. Induction of nonopsonic antibodies by either intraperitoneal injection of MEP or injection or feeding of the cross-reactive antigen, seaweed alginate, reduced the level of overall IgG elicited by follow-up immunization with the MEP-KLH conjugate. However, the opsonic activity was lower only in the sera of MEP-KLH conjugate-immunized mice with preexisting antibodies induced by MEP but not with antibodies induced by seaweed alginate. Immunization with MEP-KLH elicited a significant proportion of antibodies specific to epitopes involving O-acetate residues, and this subpopulation of antibodies mediated opsonic killing of mucoid P. aeruginosa in vitro. These results indicate that conjugation of MEP to KLH significantly enhances its immunogenicity and the elicitation of opsonic antibodies in mice and rabbits, that the conjugate induces opsonic antibodies in the presence of preexisting nonopsonic antibodies, and that opsonic antibodies to MEP are directed at epitopes that include acetate residues on the uronic acid polymer.
Asunto(s)
Alginatos , Vacunas Bacterianas/inmunología , Polisacáridos Bacterianos/inmunología , Pseudomonas aeruginosa/inmunología , Alginatos/química , Animales , Anticuerpos Antibacterianos/sangre , Actividad Bactericida de la Sangre , Ensayo de Inmunoadsorción Enzimática , Epítopos , Hemocianinas/inmunología , Ratones , Ratones Endogámicos C3H , Conejos , Vacunas Conjugadas/inmunologíaRESUMEN
No transgenic cystic fibrosis (CF) mouse model developed to date mimics the major clinical phenotype found in humans with CF, chronic Pseudomonas aeruginosa lung infection. In a transgenic CF transmembrane conductance regulator (cftr) mouse colony, we found WT, heterozygous, and homozygous CF mice housed in the same cage became chronically colonized in the oropharynx with environmental P. aeruginosa when the bacterium was present in drinking water. Elimination of P. aeruginosa from drinking water resulted in clearance in most WT and CF heterozygous, but not homozygous mice. For experimental evaluation, a combination of specific animal husbandry techniques and an oral infection route showed cftr(-/-) mice but not WT mice can be chronically colonized by P. aeruginosa with subsequent lung translocation, yielding a pathologic picture indicative of chronic lung infection. In some instances, mucoid isolates of P. aeruginosa were recovered from lungs, indicating conditions were present for conversion to mucoidy. Overexpression of human CFTR in the lungs of WT mice markedly accelerated the clearance rate of P. aeruginosa, demonstrating that lung levels of CFTR play an important role in defense against infection. P. aeruginosa mutants unable to express the surface polysaccharide alginate or the global regulator GacA were deficient in their ability to colonize the mice. CF mice made potent immune responses to P. aeruginosa outer membrane antigens. Overall, we found that under the proper conditions, transgenic CF mice are hypersusceptible to P. aeruginosa colonization and infection and can be used for evaluations of lung pathophysiology, bacterial virulence, and development of therapies aimed at treating CF lung disease.
Asunto(s)
Fibrosis Quística/complicaciones , Boca/microbiología , Faringe/microbiología , Neumonía Bacteriana/complicaciones , Infecciones por Pseudomonas/complicaciones , Pseudomonas aeruginosa/aislamiento & purificación , Animales , Enfermedad Crónica , Fibrosis Quística/microbiología , Fibrosis Quística/patología , Ratones , Infecciones por Pseudomonas/microbiología , Infecciones por Pseudomonas/patología , Pseudomonas aeruginosa/patogenicidad , VirulenciaRESUMEN
Studies of immunity to Pseudomonas aeruginosa have indicated that a variety of potential immunogens can elicit protection in animal models, utilizing both antibody- and cell-mediated immune effectors for protection. To attempt to optimize delivery of multiple protective antigens and elicit a broad range of immune effectors, we produced an aroA deletion mutant of the P. aeruginosa serogroup O2/O5 strain PAO1, designated PAO1deltaaroA. Previously, we reported that this strain elicits high levels of opsonic antibody directed against many serogroup O2/O5 strains after nasal immunization of mice and rabbits. Here, we assessed the protective efficacy of immunization with PAO1deltaaroA against acute fatal pneumonia in mice. After active immunization, high levels of protection were achieved against an ExoU-expressing cytotoxic variant of the parental strain PAO1 at doses up to 1,000-fold greater than the 50% lethal dose. Significant protection against PAO1 and two of four other serogroup O2/O5 strains was also found, but there was no protection against serogroup-heterologous strains. The serogroup O2/O5 strains not protected against were killed in opsonophagocytic assays as efficiently as the strains with which protection was seen, indicating a lack of correlation of protection and opsonic killing within the serogroup. In passive immunization experiments using challenge with wild-type PAO1 or other noncytotoxic members of the O2/O5 serogroup, there was no protection despite the presence of high levels of opsonic antibody in the mouse sera. However, passive immunization did prevent mortality from pneumonia due to the cytotoxic PAO1 variant at low-challenge doses. These data suggest that a combination of humoral and cellular immunity is required for protection against P. aeruginosa lung infections, that such immunity can be elicited by using aroA deletion mutants, and that a multivalent P. aeruginosa vaccine composed of aroA deletion mutants of multiple serogroups holds significant promise.
Asunto(s)
Vacunas Bacterianas/inmunología , Antígenos O/fisiología , Neumonía Bacteriana/prevención & control , Infecciones por Pseudomonas/prevención & control , Administración Intranasal , Animales , Femenino , Inmunización , Dosificación Letal Mediana , Lipopolisacáridos/inmunología , Pulmón/patología , Ratones , Ratones Endogámicos , Antígenos O/genética , Conejos , Vacunas Atenuadas/inmunologíaRESUMEN
Antibodies to the lipopolysaccharide O antigen of Pseudomonas aeruginosa mediate high-level immunity, but protective epitopes have proven to be poorly immunogenic, while nonprotective or minimally protective O-antigen epitopes often elicit the best immune responses. With the goal of developing a broadly protective P. aeruginosa vaccine, we used a gene replacement system based on the Flp recombinase to construct an unmarked aroA deletion mutant of the P. aeruginosa serogroup O2/O5 strain PAO1. The resultant aroA deletion mutant of PAO1 is designated PAO1 Delta aroA. The aroA deletion was confirmed by both PCR and failure of the mutant to grow on minimal media lacking aromatic amino acids. When evaluated for safety and immunogenicity in mice, PAO1 Delta aroA could be applied either intranasally or intraperitoneally at doses up to 5 x 10(9) CFU per mouse without adverse effects. No dissemination of PAO1 Delta aroA to blood, liver, or spleen was detected after intranasal application, and histological evidence of pneumonia was minimal. Intranasal immunization of mice and rabbits elicited high titers of immunoglobulin G to whole bacterial cells and to heat-stable bacterial antigens of all seven prototypic P. aeruginosa serogroup O2/O5 strains. The mouse antisera mediated potent phagocytic killing of most of the prototypic serogroup O2/O5 strains, while the rabbit antisera mediated phagocytic killing of several serogroup-heterologous strains in addition to killing all O2/O5 strains. This live, attenuated P. aeruginosa strain PAO1 Delta aroA appears to be safe for potential use as an intranasal vaccine and elicits high titers of opsonic antibodies against multiple strains of the P. aeruginosa O2/O5 serogroup.