RESUMEN
The triterpene oil squalene is an essential component of nanoemulsion vaccine adjuvants. It is most notably in the MF59 adjuvant, a component in some seasonal influenza vaccines, in stockpiled, emulsion-based adjuvanted pandemic influenza vaccines, and with demonstrated efficacy for vaccines to other pandemic viruses, such as SARS-CoV-2. Squalene has historically been harvested from shark liver oil, which is undesirable for a variety of reasons. In this study, we have demonstrated the use of a Synthetic Biology (yeast) production platform to generate squalene and novel triterpene oils, all of which are equally as efficacious as vaccine adjuvants based on physiochemical properties and immunomodulating activities in a mouse model. These Synthetic Biology adjuvants also elicited similar IgG1, IgG2a, and total IgG levels compared to marine and commercial controls when formulated with common quadrivalent influenza antigens. Injection site morphology and serum cytokine levels did not suggest any reactogenic effects of the yeast-derived squalene or novel triterpenes, suggesting their safety in adjuvant formulations. These results support the advantages of yeast produced triterpene oils to include completely controlled growth conditions, just-in-time and scalable production, and the capacity to produce novel triterpenes beyond squalene.
Asunto(s)
Adyuvantes Inmunológicos/química , Vacunas contra la Influenza/inmunología , Triterpenos/química , Animales , Anticuerpos Antivirales/sangre , Betacoronavirus/aislamiento & purificación , COVID-19 , Infecciones por Coronavirus/prevención & control , Infecciones por Coronavirus/virología , Citocinas/sangre , Inmunoglobulina G/sangre , Vacunas contra la Influenza/química , Ratones , Ratones Endogámicos BALB C , Nanopartículas/química , Infecciones por Orthomyxoviridae/patología , Infecciones por Orthomyxoviridae/prevención & control , Infecciones por Orthomyxoviridae/virología , Pandemias/prevención & control , Neumonía Viral/prevención & control , Neumonía Viral/virología , SARS-CoV-2 , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Biología Sintética/métodosRESUMEN
While mammals tend to repair injuries, other adult vertebrates like salamanders and fish regenerate damaged tissue. One prominent hypothesis offered to explain an inability to regenerate complex tissue in mammals is a bias during healing toward strong adaptive immunity and inflammatory responses. Here we directly test this hypothesis by characterizing part of the immune response during regeneration in spiny mice (Acomys cahirinus and Acomys percivali) vs. fibrotic repair in Mus musculus. By directly quantifying cytokines during tissue healing, we found that fibrotic repair was associated with a greater release of pro-inflammatory cytokines (i.e., IL-6, CCL2, and CXCL1) during acute inflammation in the wound microenvironment. However, reducing inflammation via COX-2 inhibition was not sufficient to reduce fibrosis or induce a regenerative response, suggesting that inflammatory strength does not control how an injury heals. Although regeneration was associated with lower concentrations of many inflammatory markers, we measured a comparatively larger influx of T cells into regenerating ear tissue and detected a local increase in the T cell associated cytokines IL-12 and IL-17 during the proliferative phase of regeneration. Taken together, our data demonstrate that a strong adaptive immune response is not antagonistic to regeneration and that other mechanisms likely explain the distribution of regenerative ability in vertebrates.
Asunto(s)
Quimiotaxis de Leucocito , Citocinas/inmunología , Mediadores de Inflamación/inmunología , Piel/inmunología , Linfocitos T/inmunología , Cicatrización de Heridas , Heridas Penetrantes/inmunología , Inmunidad Adaptativa , Animales , Animales Salvajes , Proliferación Celular , Citocinas/sangre , Citocinas/metabolismo , Modelos Animales de Enfermedad , Femenino , Fibrosis , Mediadores de Inflamación/sangre , Mediadores de Inflamación/metabolismo , Activación de Linfocitos , Ratones , Transducción de Señal , Piel/lesiones , Piel/metabolismo , Piel/patología , Especificidad de la Especie , Linfocitos T/metabolismo , Factores de Tiempo , Heridas Penetrantes/metabolismo , Heridas Penetrantes/patologíaRESUMEN
Regeneration is rare in mammals, but spiny mice (Acomys spp.) naturally regenerate skin and ear holes. Inflammation is thought to inhibit regeneration during wound healing, but aspects of inflammation contribute to both regeneration and pathogen defense. We compared neutrophil traits among uninjured, regeneration-competent (Acomys: A. cahirinus, A. kempi, A. percivali) and -incompetent (Mus musculus: Swiss Webster, wild-caught strains) murids to test for constitutive differences in neutrophil quantity and function between these groups. Neutrophil quantity differed significantly among species. In blood, Acomys had lower percentages of circulating neutrophils than Mus; and in bone marrow, Acomys had higher percentages of band neutrophils and lower percentages of segmented neutrophils. Functionally, Acomys and Mus neutrophils did not differ in their ability to migrate or produce reactive oxygen species, but Acomys neutrophils phagocytosed more fungal zymosan. Despite this enhanced phagocytosis activity, Acomys neutrophils were not more effective than Mus neutrophils at killing Escherichia coli. Interestingly, whole blood bacteria killing was dominated by serum in Acomys versus neutrophils only or neutrophils and serum in Mus, suggesting that Acomys primarily rely on serum to kill bacteria whereas Mus do not. These subtle differences in neutrophil traits may allow regeneration-competent species to offset damaging effects of inflammation without compromising pathogen defense.
Asunto(s)
Ratones/sangre , Murinae/sangre , Neutrófilos/fisiología , Regeneración , Animales , Especificidad de la EspecieRESUMEN
How the immune system affects tissue regeneration is not well understood. In this study, we used an emerging mammalian model of epimorphic regeneration, the African spiny mouse, to examine cell-based inflammation and tested the hypothesis that macrophages are necessary for regeneration. By directly comparing inflammatory cell activation in a 4 mm ear injury during regeneration (Acomys cahirinus) and scarring (Mus musculus), we found that both species exhibited an acute inflammatory response, with scarring characterized by stronger myeloperoxidase activity. In contrast, ROS production was stronger and more persistent during regeneration. By depleting macrophages during injury, we demonstrate a functional requirement for these cells to stimulate regeneration. Importantly, the spatial distribution of activated macrophage subtypes was unique during regeneration with pro-inflammatory macrophages failing to infiltrate the regeneration blastema. Together, our results demonstrate an essential role for inflammatory cells to regulate a regenerative response.
Asunto(s)
Oído/lesiones , Activación de Macrófagos , Macrófagos/fisiología , Regeneración , Animales , Ratones , MurinaeRESUMEN
Why mammals have poor regenerative ability has remained a long-standing question in biology. In regenerating vertebrates, injury can induce a process known as epimorphic regeneration to replace damaged structures. Using a 4-mm ear punch assay across multiple mammalian species, here we show that several Acomys spp. (spiny mice) and Oryctolagus cuniculus completely regenerate tissue, whereas other rodents including MRL/MpJ 'healer' mice heal similar injuries by scarring. We demonstrate ear-hole closure is independent of ear size, and closure rate can be modelled with a cubic function. Cellular and genetic analyses reveal that injury induces blastema formation in Acomys cahirinus. Despite cell cycle re-entry in Mus musculus and A. cahirinus, efficient cell cycle progression and proliferation only occurs in spiny mice. Together, our data unite blastema-mediated regeneration in spiny mice with regeneration in other vertebrates such as salamanders, newts and zebrafish, where all healthy adults regenerate in response to injury.
Asunto(s)
Oído Externo/lesiones , Oído Externo/fisiopatología , Regeneración/fisiología , Cicatrización de Heridas/fisiología , Animales , Ciclo Celular/genética , Ciclo Celular/fisiología , Proliferación Celular/genética , Proliferación Celular/fisiología , Oído Externo/metabolismo , Femenino , Perfilación de la Expresión Génica/métodos , Masculino , Mamíferos/genética , Mamíferos/fisiología , Ratones , Murinae , Conejos , Regeneración/genética , Especificidad de la Especie , Cicatrización de Heridas/genéticaRESUMEN
African spiny mice (Acomys spp.) are unique precocial rodents that are found in Africa, the Middle East, and southern Asia. They exhibit several interesting life-history characteristics, including precocial development, communal breeding, and a suite of physiologic adaptations to desert life. In addition to these characteristics, African spiny mice are emerging as an important animal model for tissue regeneration research. Furthermore, their important phylogenetic position among murid rodents makes them an interesting model for evolution and development studies. Here we outline the necessary components for maintaining a successful captive breeding colony, including laboratory housing, husbandry, and health monitoring aspects. We also review past and present studies focused on spiny mouse behavior, reproduction, and disease. Last, we briefly summarize various current biomedical research directions using captive-bred spiny mice.
Asunto(s)
Crianza de Animales Domésticos , Ciencia de los Animales de Laboratorio/métodos , Murinae/genética , Murinae/fisiología , Animales , Cruzamiento , Filogenia , InvestigaciónRESUMEN
Progesterone is a steroid hormone that is necessary to maintain pregnancy in mammals. We recently found that mice with a conditional deletion of Becn1/Beclin 1 specifically in the progesterone-synthesizing cells of the corpus luteum, had reduced progesterone synthesis and these mice failed to maintain pregnancy.(1) Furthermore, we identified that lipid storage and feedback through PRLR (prolactin receptor) and LHCGR (luteinizing hormone/choriogonadotropin receptor) were negatively affected by Becn1 deletion. BECN1 is necessary for the interaction of the 2 catalytic subunits of the class III phosphatidylinositol 3-kinase complex, PIK3C3, and PIK3R4, which are responsible for the generation of phosphatidylinositol 3-phosphate that is required for nucleation of the phagophore. Work from Sun et al. and Itakura et al. demonstrated that this BECN1 complex is also necessary for the fusion of autophagosomes and endosomes with lysosomes. Therefore, we suspected that ablating Becn1 in luteal cells would inhibit macroautophagy, hereafter referred to as autophagy. In support, we provide evidence that autophagic flux is reduced in our model. Thus, this study provides evidence that Becn1 is necessary for steroid production in murine luteal cells.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Cuerpo Lúteo/metabolismo , Trabajo de Parto Prematuro/metabolismo , Autofagia , Beclina-1 , Femenino , Humanos , Trabajo de Parto Prematuro/patología , EmbarazoRESUMEN
Autophagy is an important cellular process that serves as a companion pathway to the ubiquitin-proteasome system to degrade long-lived proteins and organelles to maintain cell homeostasis. Although initially characterized in yeast, autophagy is being realized as an important regulator of development and disease in mammals. Beclin1 (Becn1) is a putative tumor suppressor gene that has been shown to undergo a loss of heterozygosity in 40-75% of human breast, ovarian, and prostate cancers. Because Becn1 is a key regulator of autophagy, we sought to investigate its role in female reproduction by using a conditional knockout approach in mice. We find that pregnant females lacking Becn1 in the ovarian granulosa cell population have a defect in progesterone production and a subsequent preterm labor phenotype. Luteal cells in this model exhibit defective autophagy and a failure to accumulate lipid droplets needed for steroidogenesis. Collectively, we show that Becn1 provides essential functions in the ovary that are essential for mammalian reproduction.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/deficiencia , Trabajo de Parto Prematuro/genética , Ovario/metabolismo , Progesterona/biosíntesis , Animales , Proteínas Reguladoras de la Apoptosis/genética , Autofagia , Beclina-1 , Vías Biosintéticas/genética , Endosomas/metabolismo , Endosomas/ultraestructura , Femenino , Expresión Génica , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Gotas Lipídicas/metabolismo , Gotas Lipídicas/ultraestructura , Células Lúteas/metabolismo , Masculino , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Microscopía Electrónica de Transmisión , Microscopía Fluorescente , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Fagosomas/metabolismo , Fagosomas/ultraestructura , EmbarazoRESUMEN
Autophagy is an evolutionarily conserved cellular process through which long-lived proteins and damaged organelles are recycled to maintain energy homeostasis. These proteins and organelles are sequestered into a double-membrane structure, or autophagosome, which subsequently fuses with a lysosome in order to degrade the cargo. Although originally classified as a type of programmed cell death, autophagy is more widely viewed as a basic cell survival mechanism to combat environmental stressors. Autophagy genes were initially identified in yeast and were found to be necessary to circumvent nutrient stress and starvation. Subsequent elucidation of mammalian gene counterparts has highlighted the importance of this process to normal development. This review provides an overview of autophagy, the types of autophagy, its regulation and its known impact on development gleaned primarily from murine models.
Asunto(s)
Autofagia , Crecimiento y Desarrollo , Animales , Apoptosis/genética , Autofagia/genética , Humanos , Modelos Biológicos , Fagosomas/metabolismo , Estrés Fisiológico/genéticaRESUMEN
It is estimated that infertility affects 15-20% of couples and can arise from female or male reproductive defects. Mouse models have ascribed roles to over 100 genes in the maintenance of female fertility. Although previous models have determined roles for apoptosis in male and female fertility, we find that compromised autophagy within the perinatal ovary, through the loss of Becn1 or Atg7, results in the premature loss of female germ cells. Becn1(+/-) ovaries have a 56% reduction of germ cells compared with control ovaries at post-natal day 1, whereas Atg7(-/-) ovaries lack discernable germ cells at this stage. Thus autophagy appears to be a cell survival mechanism to maintain the endowment of female germ cells prior to establishing primordial follicle pools in the ovary.