RESUMO
Helper T (Th) cell subsets direct immune responses by producing signature cytokines. Th2 cells produce IL-4, IL-5, and IL-13, which are important in humoral immunity and protection from helminth infection and are central to the pathogenesis of many allergic inflammatory diseases. Molecular analysis of Th2 cell differentiation and maintenance of function has led to recent discoveries that have refined our understanding of Th2 cell biology. Epigenetic regulation of Gata3 expression by chromatin remodeling complexes such as Polycomb and Trithorax is crucial for maintaining Th2 cell identity. In the context of allergic diseases, memory-type pathogenic Th2 cells have been identified in both mice and humans. To better understand these disease-driving cell populations, we have developed a model called the pathogenic Th population disease induction model. The concept of defined subsets of pathogenic Th cells may spur new, effective strategies for treating intractable chronic inflammatory disorders.
Assuntos
Helmintíase/imunologia , Hipersensibilidade/imunologia , Células Th2/imunologia , Animais , Diferenciação Celular , Modelos Animais de Doenças , Epigênese Genética , Fator de Transcrição GATA3/genética , Fator de Transcrição GATA3/metabolismo , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Imunidade Humoral , Memória Imunológica , Interleucina-13/metabolismo , Interleucina-4/metabolismo , Interleucina-5/metabolismo , Camundongos , Proteína de Leucina Linfoide-Mieloide/genética , Proteína de Leucina Linfoide-Mieloide/metabolismo , Proteínas do Grupo Polycomb/genética , Proteínas do Grupo Polycomb/metabolismoRESUMO
Retrospective lineage reconstruction of humans predicts that dramatic clonal imbalances in the body can be traced to the 2-cell stage embryo. However, whether and how such clonal asymmetries arise in the embryo is unclear. Here, we performed prospective lineage tracing of human embryos using live imaging, non-invasive cell labeling, and computational predictions to determine the contribution of each 2-cell stage blastomere to the epiblast (body), hypoblast (yolk sac), and trophectoderm (placenta). We show that the majority of epiblast cells originate from only one blastomere of the 2-cell stage embryo. We observe that only one to three cells become internalized at the 8-to-16-cell stage transition. Moreover, these internalized cells are more frequently derived from the first cell to divide at the 2-cell stage. We propose that cell division dynamics and a cell internalization bottleneck in the early embryo establish asymmetry in the clonal composition of the future human body.
Assuntos
Blastômeros , Linhagem da Célula , Embrião de Mamíferos , Feminino , Humanos , Blastômeros/citologia , Blastômeros/metabolismo , Divisão Celular , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário , Camadas Germinativas/citologia , Camadas Germinativas/metabolismo , Masculino , Animais , CamundongosRESUMO
Alveolar type 2 (AT2) cells are stem cells of the alveolar epithelia. Previous genetic lineage tracing studies reported multiple cellular origins for AT2 cells after injury. However, conventional lineage tracing based on Cre-loxP has the limitation of non-specific labeling. Here, we introduced a dual recombinase-mediated intersectional genetic lineage tracing approach, enabling precise investigation of AT2 cellular origins during lung homeostasis, injury, and repair. We found AT1 cells, being terminally differentiated, did not contribute to AT2 cells after lung injury and repair. Distinctive yet simultaneous labeling of club cells, bronchioalveolar stem cells (BASCs), and existing AT2 cells revealed the exact contribution of each to AT2 cells post-injury. Mechanistically, Notch signaling inhibition promotes BASCs but impairs club cells' ability to generate AT2 cells during lung repair. This intersectional genetic lineage tracing strategy with enhanced precision allowed us to elucidate the physiological role of various epithelial cell types in alveolar regeneration following injury.
Assuntos
Células Epiteliais Alveolares , Pulmão , Células-Tronco , Animais , Camundongos , Células Epiteliais Alveolares/metabolismo , Células Epiteliais Alveolares/citologia , Diferenciação Celular , Linhagem da Célula , Pulmão/citologia , Pulmão/metabolismo , Pulmão/fisiologia , Lesão Pulmonar/patologia , Camundongos Endogâmicos C57BL , Alvéolos Pulmonares/citologia , Alvéolos Pulmonares/metabolismo , Receptores Notch/metabolismo , Regeneração , Transdução de Sinais , Células-Tronco/metabolismo , Células-Tronco/citologiaRESUMO
Transposable elements represent nearly half of mammalian genomes and are generally described as parasites, or "junk DNA." The LINE1 retrotransposon is the most abundant class and is thought to be deleterious for cells, yet it is paradoxically highly expressed during early development. Here, we report that LINE1 plays essential roles in mouse embryonic stem cells (ESCs) and pre-implantation embryos. In ESCs, LINE1 acts as a nuclear RNA scaffold that recruits Nucleolin and Kap1/Trim28 to repress Dux, the master activator of a transcriptional program specific to the 2-cell embryo. In parallel, LINE1 RNA mediates binding of Nucleolin and Kap1 to rDNA, promoting rRNA synthesis and ESC self-renewal. In embryos, LINE1 RNA is required for Dux silencing, synthesis of rRNA, and exit from the 2-cell stage. The results reveal an essential partnership between LINE1 RNA, Nucleolin, Kap1, and peri-nucleolar chromatin in the regulation of transcription, developmental potency, and ESC self-renewal.
Assuntos
Fosfoproteínas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Diferenciação Celular , Linhagem Celular , Autorrenovação Celular , Imunoprecipitação da Cromatina , Retrovirus Endógenos/genética , Feminino , Proteínas de Homeodomínio/antagonistas & inibidores , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Hibridização in Situ Fluorescente , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Oligorribonucleotídeos Antissenso/metabolismo , Fosfoproteínas/antagonistas & inibidores , Fosfoproteínas/genética , Interferência de RNA , RNA Ribossômico/metabolismo , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/genética , Proteína 28 com Motivo Tripartido/antagonistas & inibidores , Proteína 28 com Motivo Tripartido/genética , Proteína 28 com Motivo Tripartido/metabolismo , Regulação para Cima , NucleolinaRESUMO
Tuft cells are a type of intestinal epithelial cells that exist in epithelial barriers and play a critical role in immunity against parasite infection. It remains insufficiently clear whether Tuft cells participate in bacterial eradication. Here, we identified Sh2d6 as a signature marker for CD45+ Tuft-2 cells. Depletion of Tuft-2 cells resulted in susceptibility to bacterial infection. Tuft-2 cells quickly expanded in response to bacterial infection and sensed the bacterial metabolite N-undecanoylglycine through vomeronasal receptor Vmn2r26. Mechanistically, Vmn2r26 engaged with N-undecanoylglycine activated G-protein-coupled receptor-phospholipase C gamma2 (GPCR-PLCγ2)-Ca2+ signaling axis, which initiated prostaglandin D2 (PGD2) production. PGD2 enhanced the mucus secretion of goblet cells and induced antibacterial immunity. Moreover, Vmn2r26 signaling also promoted SpiB transcription factor expression, which is responsible for Tuft-2 cell development and expansion in response to bacterial challenge. Our findings reveal an additional function of Tuft-2 cells in immunity against bacterial infection through Vmn2r26-mediated recognition of bacterial metabolites.
Assuntos
Anti-Infecciosos , Mucosa Intestinal , Antibacterianos , Anti-Infecciosos/metabolismo , Células Caliciformes , Prostaglandina D2/metabolismoRESUMO
Single-nucleotide polymorphisms in ETS1 are associated with systemic lupus erythematosus (SLE). Ets1-/- mice develop SLE-like symptoms, suggesting that dysregulation of this transcription factor is important to the onset or progression of SLE. We used conditional deletion approaches to examine the impact of Ets1 expression in different immune cell types. Ets1 deletion on CD4+ T cells, but not B cells or dendritic cells, resulted in the SLE autoimmunity, and this was associated with the spontaneous expansion of T follicular helper type 2 (Tfh2) cells. Ets1-/- Tfh2 cells exhibited increased expression of GATA-3 and interleukin-4 (IL-4), which induced IgE isotype switching in B cells. Neutralization of IL-4 reduced Tfh2 cell frequencies and ameliorated disease parameters. Mechanistically, Ets1 suppressed signature Tfh and Th2 cell genes, including Cxcr5, Bcl6, and Il4ra, thus curbing the terminal Tfh2 cell differentiation process. Tfh2 cell frequencies in SLE patients correlated with disease parameters, providing evidence for the relevance of these findings to human disease.
Assuntos
Diferenciação Celular/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Proteína Proto-Oncogênica c-ets-1/imunologia , Células Th2/imunologia , Animais , Autoimunidade/genética , Autoimunidade/imunologia , Linfócitos B/imunologia , Linfócitos B/metabolismo , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Diferenciação Celular/genética , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Expressão Gênica/imunologia , Perfilação da Expressão Gênica , Humanos , Lúpus Eritematoso Sistêmico/genética , Lúpus Eritematoso Sistêmico/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Proteína Proto-Oncogênica c-ets-1/genética , Proteína Proto-Oncogênica c-ets-1/metabolismo , Células Th2/metabolismoRESUMO
Asthma is a widespread airway disorder where GATA3-dependent Type-2 helper T (Th2) cells and group 2 innate lymphoid cells (ILC2s) play vital roles. Asthma-associated single nucleotide polymorphisms (SNPs) are enriched in a region located 926-970 kb downstream from GATA3 in the 10p14 (hG900). However, it is unknown how hG900 affects the pathogenesis of allergic airway inflammation. To investigate the roles of the asthma-associated GATA3 enhancer region in experimental allergic airway inflammation, we first examined the correlation between GATA3 expression and the activation of the hG900 region was analyzed by flow cytometry and ChIP-qPCR. We found that The activation of enhancers in the hG900 region was strongly correlated to the levels of GATA3 in human peripheral T cell subsets. We next generated mice lacking the mG900 region (mG900KO mice) were generated by the CRISPR-Cas9 system, and the development and function of helper T cells and ILCs in mG900KO mice were analyzed in steady-state conditions and allergic airway inflammation induced by papain or house dust mite (HDM). The deletion of the mG900 did not affect the development of lymphocytes in steady-state conditions or allergic airway inflammation induced by papain. However, mG900KO mice exhibited reduced allergic inflammation and Th2 differentiation in the HDM-induced allergic airway inflammation. The analysis of the chromatin conformation around Gata3 by circular chromosome conformation capture coupled to high-throughput sequencing (4C-seq) revealed that the mG900 region interacted with the transcription start site of Gata3 with an influencing chromatin conformation in Th2 cells. These findings indicate that the mG900 region plays a pivotal role in Th2 differentiation and thus enhances allergic airway inflammation.
Assuntos
Asma , Diferenciação Celular , Elementos Facilitadores Genéticos , Fator de Transcrição GATA3 , Células Th2 , Fator de Transcrição GATA3/metabolismo , Fator de Transcrição GATA3/genética , Animais , Células Th2/imunologia , Camundongos , Diferenciação Celular/imunologia , Asma/imunologia , Asma/genética , Asma/patologia , Humanos , Camundongos Knockout , Inflamação/imunologia , Inflamação/genética , Hipersensibilidade/imunologia , Hipersensibilidade/genética , Polimorfismo de Nucleotídeo Único , Camundongos Endogâmicos C57BLRESUMO
Upon encountering allergens, CD4+ T cells differentiate into IL-4-producing Th2 cells in lymph nodes, which later transform into polyfunctional Th2 cells producing IL-5 and IL-13 in inflamed tissues. However, the precise mechanism underlying their polyfunctionality remains elusive. In this study, we elucidate the pivotal role of NRF2 in polyfunctional Th2 cells in murine models of allergic asthma and in human Th2 cells. We found that an increase in reactive oxygen species (ROS) in immune cells infiltrating the lungs is necessary for the development of eosinophilic asthma and polyfunctional Th2 cells in vivo. Deletion of the ROS sensor NRF2 specifically in T cells, but not in dendritic cells, significantly abolished eosinophilia and polyfunctional Th2 cells in the airway. Mechanistically, NRF2 intrinsic to T cells is essential for inducing optimal oxidative phosphorylation and glycolysis capacity, thereby driving Th2 cell polyfunctionality independently of IL-33, partially by inducing PPARγ. Treatment with an NRF2 inhibitor leads to a substantial decrease in polyfunctional Th2 cells and subsequent eosinophilia in mice and a reduction in the production of Th2 cytokines from peripheral blood mononuclear cells in asthmatic patients. These findings highlight the critical role of Nrf2 as a spatial and temporal metabolic hub that is essential for polyfunctional Th2 cells, suggesting potential therapeutic implications for allergic diseases.
Assuntos
Asma , Fator 2 Relacionado a NF-E2 , Células Th2 , Animais , Feminino , Humanos , Camundongos , Asma/imunologia , Asma/metabolismo , Citocinas/metabolismo , Modelos Animais de Doenças , Eosinofilia/imunologia , Eosinofilia/metabolismo , Glicólise , Interleucina-33/metabolismo , Pulmão/imunologia , Pulmão/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 2 Relacionado a NF-E2/metabolismo , Fosforilação Oxidativa , PPAR gama/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Células Th2/imunologia , Células Th2/metabolismoRESUMO
Uncontrolled type 2 immunity by type 2 helper T (Th2) cells causes intractable allergic diseases; however, whether the interaction of CD4+ T cells shapes the pathophysiology of allergic diseases remains unclear. We identified a subset of Th2 cells that produced the serine proteases granzyme A and B early in differentiation. Granzymes cleave protease-activated receptor (Par)-1 and induce phosphorylation of p38 mitogen-activated protein kinase (MAPK), resulting in the enhanced production of IL-5 and IL-13 in both mouse and human Th2 cells. Ubiquitin-specific protease 7 (USP7) regulates IL-4-induced phosphorylation of STAT3, resulting in granzyme production during Th2 cell differentiation. Genetic deletion of Usp7 or Gzma and pharmacological blockade of granzyme B ameliorated allergic airway inflammation. Furthermore, PAR-1+ and granzyme+ Th2 cells were colocalized in nasal polyps from patients with eosinophilic chronic rhinosinusitis. Thus, the USP7-STAT3-granzymes-Par-1 pathway is a potential therapeutic target for intractable allergic diseases.
Assuntos
Hipersensibilidade , Células Th2 , Humanos , Animais , Camundongos , Granzimas/genética , Granzimas/metabolismo , Interleucina-5/metabolismo , Peptidase 7 Específica de Ubiquitina/metabolismo , Inflamação/metabolismo , Diferenciação Celular , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismoRESUMO
Totipotent stem cells are transiently occurring in vivo cells that can form all cell types of the embryo including placenta, with their in vitro counterparts being actively pursued. Subsequently, totipotent-like cells are established with variable robustness and biological relevance. Here, we summarize current progress on capturing these cells in culture.
Assuntos
Embrião de Mamíferos , Células-Tronco Totipotentes , Diferenciação Celular/genética , Células-Tronco Totipotentes/metabolismoRESUMO
Mouse embryonic stem cells (ESCs) display pluripotency features characteristic of the inner cell mass of the blastocyst. Mouse embryonic stem cell cultures are highly heterogeneous and include a rare population of cells, which recapitulate characteristics of the 2-cell embryo, referred to as 2-cell-like cells (2CLCs). Whether and how ESC and 2CLC respond to environmental cues has not been fully elucidated. Here, we investigate the impact of mechanical stress on the reprogramming of ESC to 2CLC. We show that hyperosmotic stress induces 2CLC and that this induction can occur even after a recovery time from hyperosmotic stress, suggesting a memory response. Hyperosmotic stress in ESCs leads to accumulation of reactive-oxygen species (ROS) and ATR checkpoint activation. Importantly, preventing either elevated ROS levels or ATR activation impairs hyperosmotic-mediated 2CLC induction. We further show that ROS generation and the ATR checkpoint act within the same molecular pathway in response to hyperosmotic stress to induce 2CLCs. Altogether, these results shed light on the response of ESC to mechanical stress and on our understanding of 2CLC reprogramming.
Assuntos
Células-Tronco Embrionárias , Transdução de Sinais , Animais , Camundongos , Espécies Reativas de Oxigênio/metabolismo , Células-Tronco Embrionárias/metabolismo , Células-Tronco Embrionárias Murinas/metabolismo , Blastocisto/metabolismo , Diferenciação CelularRESUMO
BACKGROUND: Immune dysregulation often presents as autoimmunity, inflammation, and/or lymphoproliferation. Several germline genetic defects have been associated with immune dysregulation; they include heterozygous gain-of-function (GOF) mutations in IKZF1, an essential transcription factor for hematopoiesis containing zinc finger domains (ZFs). However, in a large percentage of patients, the genetic origin of their immunedysregulation remains undetermined. OBJECTIVE: A family with 2 members presenting immune dysregulation signs was studied to identify the genetic cause of their disease. METHODS: Whole exome sequencing, analysis of immunologic parameters, and functional assays (including Western blotting, electrophoretic mobility shift assay during the cell cycle, and TH cell differentiation) were performed. RESULTS: The 2 patients carried a novel heterozygous mutation in IKZF1 (IKZF1T398M). IKZF1 heterozygous mutations have previously been shown to be responsible for several distinct human immunologic diseases by directly affecting the ability of ZFs to bind to DNA or to dimerize. Herein, we showed that the IKZF1T398M, which is outside the ZFs, caused impaired phosphorylation of IKZF1, resulting in enhanced DNA-binding ability at the S phase of the cell cycle, reduction of the G1-S phase transition, and decreased proliferation. Confirming these data, similar functional alterations were observed with IKZF1T398A, but not with IKZF1T398D, mimicking dephosphorylation and phosphorylation, respectively. In T lymphocytes, expression of IKZF1T398M led to TH cell differentiation skewed toward TH2 cells. Thus, our data indicate that IKZF1T398M behaves as a GOF variant underlying immune dysregulation. CONCLUSION: Disturbed IKZF1 phosphorylation represents a novel GOF mechanism (GOF by loss of phosphorylation (termed as GOF-LOP) associated with immune dysregulation, highlighting the regulatory role of IKZF1 during cell cycle progression through phosphorylation.
Assuntos
Mutação com Ganho de Função , Fator de Transcrição Ikaros , Humanos , Fator de Transcrição Ikaros/genética , Fosforilação , Feminino , Masculino , Linhagem , AdultoRESUMO
In a Japanese chemical factory, lung diseases such as pneumoconiosis have been reported among workers handling cross-linked water-soluble acrylic acid polymers (CWAAP). Our previous study reported that a single intratracheal administration of CWAAP induces acute inflammation and fibrosis. In this study, we investigated the effects of multiple intratracheal administrations of CWAAP on inflammatory responses and pulmonary fibrosis along with inducible bronchus-associated lymphoid tissues (iBALT) formation, which is involved in allergic inflammation. Male F344 rats (190-200 g) received single or multiple intratracheal administrations of phosphate-buffered saline (PBS) or CWAAP. To assess inflammatory responses and pulmonary fibrosis, immunohistochemical and histological staining was performed. CD68, CD163, CD169, TGF-ß, and collagen I positive cells/areas in the lungs of the CWAAP-group rats were significantly increased than those in the PBS group. Furthermore, the number of iBALT structures, CD4 + T cells, along with CD19, PAX5, IL-4, GATA-3, T-bet, and IgE-positive cells in the terminal bronchioles and blood vessels of the lungs were significantly increased in the CWAAP group. Moreover, pulmonary fibrosis, iBALT formation, and levels of specific IgG were significantly increased in rats who received multiple intratracheal administrations of CWAAP compared to those with single intratracheal administration. Multiple intratracheal administrations of CWAAP potentiated the classical fibrotic pathway (M2 macrophage-TGF-ß-collagen I) more potently than single intratracheal administration. Furthermore, it was possible that iBALT was formed around terminal bronchioles and blood vessels and the number of immune cells was increased, resulting in enhanced allergic inflammation and pulmonary fibrosis.
Assuntos
Acrilatos , Fibrose Pulmonar , Masculino , Ratos , Animais , Fibrose Pulmonar/patologia , Polímeros , Ratos Endogâmicos F344 , Tecido Linfoide , Brônquios/patologia , Pulmão/patologia , Inflamação/patologia , Fator de Crescimento Transformador beta , ColágenoRESUMO
COVID-19 syndrome is characterized by acute lung injury, hypoxemic respiratory failure, and high mortality. Alveolar type 2 (AT2) cells are essential for gas exchange, repair, and regeneration of distal lung epithelium. We have shown that the causative agent, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and other members of the ß-coronavirus genus induce an endoplasmic reticulum (ER) stress response in vitro; however, the consequences for host AT2 cell function in vivo are less understood. To study this, two murine models of coronavirus infection were used-mouse hepatitis virus-1 (MHV-1) in A/J mice and a mouse-adapted SARS-CoV-2 strain. MHV-1-infected mice exhibited dose-dependent weight loss with histological evidence of distal lung injury accompanied by elevated bronchoalveolar lavage fluid (BALF) cell counts and total protein. AT2 cells showed evidence of both viral infection and increased BIP/GRP78 expression, consistent with activation of the unfolded protein response (UPR). The AT2 UPR included increased inositol-requiring enzyme 1α (IRE1α) signaling and a biphasic response in PKR-like ER kinase (PERK) signaling accompanied by marked reductions in AT2 and BALF surfactant protein (SP-B and SP-C) content, increases in surfactant surface tension, and emergence of a reprogrammed epithelial cell population (Krt8+ and Cldn4+). The loss of a homeostatic AT2 cell state was attenuated by treatment with the IRE1α inhibitor OPK-711. As a proof-of-concept, C57BL6 mice infected with mouse-adapted SARS-CoV-2 demonstrated similar lung injury and evidence of disrupted surfactant homeostasis. We conclude that lung injury from ß-coronavirus infection results from an aberrant host response, activating multiple AT2 UPR stress pathways, altering surfactant metabolism/function, and changing AT2 cell state, offering a mechanistic link between SARS-CoV-2 infection, AT2 cell biology, and acute respiratory failure.NEW & NOTEWORTHY COVID-19 syndrome is characterized by hypoxemic respiratory failure and high mortality. In this report, we use two murine models to show that ß-coronavirus infection produces acute lung injury, which results from an aberrant host response, activating multiple epithelial endoplasmic reticular stress pathways, disrupting pulmonary surfactant metabolism and function, and forcing emergence of an aberrant epithelial transition state. Our results offer a mechanistic link between SARS-CoV-2 infection, AT2 cell biology, and respiratory failure.
Assuntos
COVID-19 , Estresse do Retículo Endoplasmático , Endorribonucleases , Homeostase , Vírus da Hepatite Murina , SARS-CoV-2 , Animais , Camundongos , COVID-19/metabolismo , COVID-19/patologia , COVID-19/virologia , COVID-19/complicações , Vírus da Hepatite Murina/patogenicidade , Endorribonucleases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Células Epiteliais Alveolares/metabolismo , Células Epiteliais Alveolares/virologia , Células Epiteliais Alveolares/patologia , Chaperona BiP do Retículo Endoplasmático , Infecções por Coronavirus/metabolismo , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Infecções por Coronavirus/complicações , Surfactantes Pulmonares/metabolismo , Resposta a Proteínas não Dobradas , Betacoronavirus , Insuficiência Respiratória/metabolismo , Insuficiência Respiratória/virologia , Insuficiência Respiratória/patologia , Modelos Animais de Doenças , eIF-2 Quinase/metabolismo , HumanosRESUMO
Many terrestrial plants produce large quantities of alkanes for use in epicuticular wax and the pollen coat. However, their carbon chains must be long to be useful as fuel or as a petrochemical feedstock. Here, we focus on Nymphaea odorata, which produces relatively short alkanes in its anthers. We identified orthologs of the Arabidopsis alkane biosynthesis genes AtCER1 and AtCER3 in N. odorata and designated them NoCER1A, NoCER3A and NoCER3B. Expression analysis of NoCER1A and NoCER3A/B in Arabidopsis cer mutants revealed that the N. odorata enzymes cooperated with the Arabidopsis enzymes and that the NoCER1A produced shorter alkanes than AtCER1, regardless of which CER3 protein it interacted with. These results indicate that AtCER1 frequently uses a C30 substrate, whereas NoCER1A, NoCER3A/B and AtCER3 react with a broad range of substrate chain lengths. The incorporation of shorter alkanes disturbed the formation of wax crystals required for water-repellent activity in stems, suggesting that chain-length specificity is important for surface cleaning. Moreover, cultured tobacco cells expressing NoCER1A and NoCER3A/B effectively produced C19-C23 alkanes, indicating that the introduction of the two enzymes is sufficient to produce alkanes. Taken together, our findings suggest that these N. odorata enzymes may be useful for the biological production of alkanes of specific lengths. 3D modeling revealed that CER1s and CER3s share a similar structure that consists of N- and C-terminal domains, in which their predicted active sites are respectively located. We predicted the complex structure of both enzymes and found a cavity that connects their active sites.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Nymphaea , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Nymphaea/metabolismo , Alcanos/metabolismo , Carbono-Carbono Liases/metabolismoRESUMO
Ingestion of Porphyromonas gingivalis, a periodontal pathogen, disrupts the intestinal barrier in mice. However, the involvement of outer membrane vesicles (OMVs) secreted from P. gingivalis in the destruction of the intestinal barrier remains unclear. In this study, we tested the hypothesis that OMVs carrying gingipains, the major cysteine proteases produced by P. gingivalis, affects the intestinal barrier function. OMVs increased the permeability of the Caco-2 cell monolayer, a human intestinal epithelial cell line, accompanied by degradation of the tight junction protein occludin. In contrast, OMVs prepared from mutant strains devoid of gingipains failed to induce intestinal barrier dysfunction or occludin degradation in Caco-2 cells. A close histological examination revealed the intracellular localization of gingipain-carrying OMVs. Gingipain activity was detected in the cytosolic fraction of Caco-2 cells after incubation with OMVs. These results suggest that gingipains were internalized into intestinal cells through OMVs and transported into the cytosol, where they then directly degraded occludin from the cytosolic side. Thus, P. gingivalis OMVs might destroy the intestinal barrier and induce systemic inflammation via OMV itself or intestinal substances leaked into blood vessels, causing various diseases.
Assuntos
Adesinas Bacterianas , Porphyromonas gingivalis , Animais , Camundongos , Humanos , Cisteína Endopeptidases Gingipaínas/metabolismo , Células CACO-2 , Porphyromonas gingivalis/fisiologia , Citosol/metabolismo , Ocludina/metabolismo , Adesinas Bacterianas/metabolismoRESUMO
Allergic asthma (AA) is closely associated with the polarization of T helper (Th)2 and Th17 cells. Interleukin (IL)-18 acts as an inducer of Th2 and Th17 cell responses. However, expressions of IL-18 and IL-18 receptor alpha (IL-18Rα) in blood Th2 and Th17 cells of patients with AA remain unclear. We therefore investigated their expressions in Th2 and Th17 cells using flow cytometric analysis, quantitative real-time PCR (qPCR), and murine AA model. We observed increased proportions of Th2, Th17, IL-18+, IL-18+ Th2, and IL-18+ Th17 cells in blood CD4+ T cells of patients with AA. Additionally, house dust mite seemed to upregulate further IL-18 expression in Th2 and Th17, and upregulate IL-18Rα expression in CD4+ T, Th2, and Th17 cells of AA patients. It was also found that the plasma levels of IL-4, IL-17A, and IL-18 in AA patients were elevated, and they were correlated between each other. In ovalbumin (OVA)-induced asthma mouse (AM), we observed that the percentages of blood CD4+ T, Th2, and Th17 cells were increased. Moreover, OVA-induced AM expressed higher level of IL-18Rα in blood Th2 cells, which was downregulated by IL-18. Increased IL-18Rα expression was also observed in blood Th2 cells of OVA-induced FcεRIα-/- mice. Collectively, our findings suggest the involvement of Th2 cells in AA by expressing excessive IL-18 and IL-18Rα in response to allergen, and that IL-18 and IL-18Rα expressing Th2 cells are likely to be the potential targets for AA therapy.
Assuntos
Alérgenos , Asma , Interleucina-18 , Células Th17 , Células Th2 , Humanos , Interleucina-18/imunologia , Interleucina-18/sangue , Asma/imunologia , Asma/sangue , Animais , Células Th2/imunologia , Camundongos , Feminino , Células Th17/imunologia , Masculino , Adulto , Alérgenos/imunologia , Pessoa de Meia-Idade , Regulação para Cima/imunologia , Subunidade alfa de Receptor de Interleucina-18/imunologia , Subunidade alfa de Receptor de Interleucina-18/genética , Ovalbumina/imunologia , Receptores de Interleucina-18/imunologia , Camundongos Endogâmicos BALB C , Modelos Animais de Doenças , Pyroglyphidae/imunologia , Adulto JovemRESUMO
BACKGROUND: Neoantigens have emerged as a promising area of focus in tumor immunotherapy, with several established strategies aiming to enhance their identification. Human leukocyte antigen class I molecules (HLA-I), which present intracellular immunopeptides to T cells, provide an ideal source for identifying neoantigens. However, solely relying on a mutation database generated through commonly used whole exome sequencing (WES) for the identification of HLA-I immunopeptides, may result in potential neoantigens being missed due to limitations in sequencing depth and sample quality. METHOD: In this study, we constructed and evaluated an extended database for neoantigen identification, based on COSMIC mutation database. This study utilized mass spectrometry-based proteogenomic profiling to identify the HLA-I immunopeptidome enriched from HepG2 cell. HepG2 WES-based and the COSMIC-based mutation database were generated and utilized to identify HepG2-specific mutant immunopeptides. RESULT: The results demonstrated that COSMIC-based database identified 5 immunopeptides compared to only 1 mutant peptide identified by HepG2 WES-based database, indicating its effectiveness in identifying mutant immunopeptides. Furthermore, HLA-I affinity of the mutant immunopeptides was evaluated through NetMHCpan and peptide-docking modeling to validate their binding to HLA-I molecules, demonstrating the potential of mutant peptides identified by the COSMIC-based database as neoantigens. CONCLUSION: Utilizing the COSMIC-based mutation database is a more efficient strategy for identifying mutant peptides from HLA-I immunopeptidome without significantly increasing the false positive rate. HepG2 specific WES-based database may exclude certain mutant peptides due to WES sequencing depth or sample heterogeneity. The COSMIC-based database can effectively uncover potential neoantigens within the HLA-I immunopeptidomes.
Assuntos
Antígenos de Neoplasias , Bases de Dados Genéticas , Antígenos de Histocompatibilidade Classe I , Linfócitos T , Humanos , Antígenos de Neoplasias/metabolismo , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/metabolismo , Mutação/genética , Peptídeos/químicaRESUMO
BACKGROUND: Wnt/ß-catenin signaling is critical for lung development and AT2 stem cell maintenance in adults, but excessive pathway activation has been associated with pulmonary fibrosis, both in animal models and human diseases such as idiopathic pulmonary fibrosis (IPF). IPF is a detrimental interstitial lung disease, and although two approved drugs limit functional decline, transplantation is the only treatment that extends survival, highlighting the need for regenerative therapies. METHODS: Using our antibody-based platform of Wnt/ß-catenin modulators, we investigated the ability of a pathway antagonist and pathway activators to reduce pulmonary fibrosis in the acute bleomycin model, and we tested the ability of a WNT mimetic to affect alveolar organoid cultures. RESULTS: A WNT mimetic agonist with broad FZD-binding specificity (FZD1,2,5,7,8) potently expanded alveolar organoids. Upon therapeutic dosing, a broad FZD-binding specific Wnt mimetic decreased pulmonary inflammation and fibrosis and increased lung function in the bleomycin model, and it impacted multiple lung cell types in vivo. CONCLUSIONS: Our results highlight the unexpected capacity of a WNT mimetic to effect tissue repair after lung damage and support the continued development of Wnt/ß-catenin pathway modulation for the treatment of pulmonary fibrosis.
Assuntos
Fibrose Pulmonar Idiopática , beta Catenina , Adulto , Animais , Humanos , beta Catenina/metabolismo , Pulmão/metabolismo , Fibrose Pulmonar Idiopática/induzido quimicamente , Fibrose Pulmonar Idiopática/tratamento farmacológico , Fibrose Pulmonar Idiopática/metabolismo , Via de Sinalização Wnt , Bleomicina/toxicidadeRESUMO
Compared with Li+, Na+ with a smaller stokes radius has faster de-solvation kinetics. An electrolyte with ultralow sodium salt (0.3â M NaPF6) is used to reduce the cell cost. However, the organic-dominated interface, mainly derived from decomposed solvents (SSIP solvation structure), is defective for the long cycling performance of sodium ion batteries. In this work, the simple application of dual additives, including sodium difluoro(oxalato)borate (NaDFOB) and tris(trimethylsilyl)borate (TMSB), is demonstrated to improve the cycling performance of the hard carbon/NaNi1/3Fe1/3Mn1/3O2 cell by constructing interface films on the anode and cathode. A significant improvement on cycling stability has been achieved by incorporating dual additives of NaDFOB and TMSB. Particularly, the capacity retention increased from 17 % (baseline) to 79 % (w/w, 2.0â wt % NaDFOB) and 83 % (w/w, 2.0â wt % NaDFOB and 1.0â wt % TMSB) after 200 cycles at room temperature. Insight into the mechanism of improved interfacial properties between electrodes and electrolyte in ultralow concentration electrolyte has been investigated through a combination of theoretical computation and experimental techniques.