RESUMEN
Influenza A virus (IAV) infection is a complicated process. After IAVs spread to the lung, extensive pro-inflammatory cytokines and chemokines are released, which largely determine the outcome of infection. Using a single-cell RNA sequencing (scRNA-seq) assay, we systematically and sequentially analyzed the transcriptome of more than 16,000 immune cells in the pulmonary tissue of infected mice, and demonstrated that two waves of pro-inflammatory factors were released. A group of IAV-infected PD-L1+ neutrophils were the major contributor to the first wave at an earlier stage (day 1-3 post infection). Notably, at a later stage (day 7 post infection) when IAV was hardly detected in the immune cells, a group of platelet factor 4-positive (Pf4+)-macrophages generated another wave of pro-inflammatory factors, which were probably the precursors of alveolar macrophages (AMs). Furthermore, single-cell signaling map identified inter-lineage crosstalk between different clusters and helped better understand the signature of PD-L1+ neutrophils and Pf4+-macrophages. Our data characteristically clarified the infiltrated immune cells and their production of pro-inflammatory factors during the immunopathogenesis development, and deciphered the important mechanisms underlying IAV-driven inflammatory reactions in the lung.
Asunto(s)
Virus de la Influenza A/inmunología , Pulmón/inmunología , Macrófagos Alveolares/inmunología , Animales , Plaquetas/inmunología , Quimiocinas/inmunología , Citocinas/inmunología , Femenino , Humanos , Inflamación/patología , Subtipo H1N1 del Virus de la Influenza A/inmunología , Virus de la Influenza A/metabolismo , Virus de la Influenza A/patogenicidad , Gripe Humana/virología , Macrófagos/inmunología , Macrófagos Alveolares/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Neutrófilos/inmunología , Infecciones por Orthomyxoviridae/inmunología , Análisis de Secuencia de ARN/métodos , Transducción de Señal/inmunología , Análisis de la Célula Individual/métodosRESUMEN
Activation-induced cytidine deaminase (AID) initiates class switch recombination and somatic hypermutation in Ig genes. The activity and protein levels of AID are tightly controlled by various mechanisms. In this study, we found that CUL7 E3 ubiquitin ligases specifically mediated AID ubiquitination. CUL7 overexpression or knockdown influenced the decay of AID, affecting AID protein levels and subsequently IgA class switching in CH12F3 cells, a mouse B lymphocyte cell line. Further analysis indicated that CUL7 mediated AID ubiquitination by forming a complex with FBXW11. In a CUL7 fl/fl CD19 cre+ mouse model, we demonstrated that CUL7 knockout significantly enhanced AID protein levels in B cells in the germinal center and increased both the IgG1 and IgA class switching. Collectively, our results reveal a subtle regulation mechanism for tightly controlling AID protein levels. The manipulation of this pathway may be useful for regulating AID abundance and efficiency of Ig class switching and is therefore a potential target for developing immunologic adjuvants for vaccines of various pathogens such as HIV-1 and influenza viruses.
Asunto(s)
Antígenos CD19/metabolismo , Linfocitos B/fisiología , Proteínas Cullin/metabolismo , Citidina Desaminasa/metabolismo , Centro Germinal/inmunología , Ubiquitina-Proteína Ligasas/metabolismo , Vacunas contra el SIDA , Adyuvantes Inmunológicos , Animales , Antígenos CD19/genética , Línea Celular , Proteínas Cullin/genética , Citidina Desaminasa/genética , Inmunidad Humoral , Inmunoglobulina A/genética , Cambio de Clase de Inmunoglobulina , Inmunoglobulina G/genética , Vacunas contra la Influenza , Activación de Linfocitos , Ratones , Ratones Noqueados , Transducción de Señal , Ubiquitina-Proteína Ligasas/genética , UbiquitinaciónRESUMEN
OBJECTIVE: Quanzhen Yiqi decoction (QZYQ) is a traditional Chinese medicine for treating chronic obstructive pulmonary disease. METHODS: Mice were exposed to cigarette smoke (CS) 6 days/week (40 cigarettes/day) for 24 weeks and then intragastrically administered QZYQ (4.72, 9.45, or 18.89 g/kg) or dexamethasone (DEX, 0.6 mg/kg) for 6 weeks. We examined the lung function and collected bronchoalveolar lavage fluid for inflammatory cell and cytokine quantification. The pathological lung changes, ROS and oxidative biomarkers were measured. We used immunohistochemistry and western blotting to evaluate the levels of Nrf2/HO-1, NLRP3/ASC/Caspase1/IL-1ß/IL-18. RESULTS: The CS group showed significant increases in the forced vital capacity, lung resistance, and chord compliance and a lower FEV50/FVC compared with the control, and QZYQ improved these changes. In addition, QZYQ effectively reduced emphysema, immune cell infiltration, and airway remodeling. QZYQ stimulated HO-1 expression and reduced oxidative stress through the Nrf2 pathway. QZYQ inhibited the production of NLRP3/ASC/Caspase-1 to inhibit IL-1ß and IL-18. CONCLUSION: Our study suggested that QZYQ can improve the function and histology of the lungs and reduce inflammatory cell recruitment. QZYQ inhibits ROS production and NLRP3 inflammasome activation by upregulating Nrf2 to reduce lung injury. The anti-inflammatory effects of QZYQ are similar to those of DEX.
RESUMEN
Acute myeloid leukemia (AML) is caused by clonal disorders of hematopoietic stem cells. Differentiation therapy is emerging as an important treatment modality for leukemia, given its less toxicity and wider applicable population, but the arsenal of differentiation-inducing agents is still very limited. In this study, we adapted a competitive peptide phage display platform to search for candidate peptides that could functionally induce human leukemia cell differentiation. A monoclonal phage (P6) and the corresponding peptide (pep-P6) were identified. Both L- and D-chirality of pep-P6 showed potent efficiency in inducing AML cell line differentiation, driving their morphologic maturation and upregulating the expression of macrophage markers and cytokines, including CD11b, CD14, IL-6, IL-1ß, and TNF-α. In the THP-1 xenograft animal model, administration of D-pep-P6 was effective in inhibiting disease progression. Importantly, exposure to D-pep-P6 induced the differentiation of primary human leukemia cells isolated AML patients in a similar manner to the AML cell lines. Further mechanism study suggested that D-pep-P6 induced human leukemia cell differentiation by directly activating a TLR-2 signaling pathway. These findings identify a novel D-peptide that may promote leukemia differentiation therapy.
RESUMEN
Chronic obstructive pulmonary disease (COPD) is a serious chronic lung disease. Schisandrin A (SchA) is one of the most important active ingredients in Schisandra chinensis and has been used to treat various lung diseases in several countries. Here, we studied the pharmacological effect of SchA on airway inflammation induced by cigarette smoke (CS) and explored the therapeutic mechanism of SchA in COPD model mice. Our results showed that SchA treatment significantly improved the lung function of CS-induced COPD model mice and reduced the recruitment of leukocytes and hypersecretion of interleukin-6 (IL-6), interleukin-1ß (IL-1ß) and tumor necrosis factor α (TNF-α) in bronchoalveolar lavage fluid (BALF). H&E staining showed that SchA treatment could effectively reduce emphysema, immune cell infiltration and airway wall destruction. In addition, we found that SchA treatment can stimulate the expression of heme oxygenase-1 (HO-1) through the nuclear factor-erythroid 2-related factor (Nrf2) pathway, significantly reduce oxidative stress, increase catalase (CAT) and superoxide dismutase (SOD) levels, and suppress the level of malondialdehyde (MDA) in COPD model mice. Moreover, SchA treatment suppressed the generation of the NLRP3/ASC/Caspase1 inflammasome complex to inhibit the inflammatory response caused by IL-1ß and IL-18 and pyroptosis caused by GSDMD. In conclusion, our study shows that SchA treatment can inhibit the production of ROS and the activation of the NLRP3 inflammasome by upregulating Nrf-2, thereby producing anti-inflammatory effects and reducing lung injury in COPD model mice. More importantly, SchA exhibited similar anti-inflammatory effects to dexamethasone in COPD model mice, and we did not observe substantial side effects of SchA treatment. The high safety of SchA makes it a potential candidate drug for the treatment of COPD.
Asunto(s)
Inflamasomas , Enfermedad Pulmonar Obstructiva Crónica , Animales , Ratones , Antiinflamatorios/farmacología , Antiinflamatorios/uso terapéutico , Inflamasomas/metabolismo , Factor 2 Relacionado con NF-E2 , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Enfermedad Pulmonar Obstructiva Crónica/tratamiento farmacológico , Piroptosis , Transducción de SeñalRESUMEN
Activation-induced cytidine deaminase (AID) initiates class-switch recombination and somatic hypermutation (SHM) in antibody genes. Protein expression and activity are tightly controlled by various mechanisms. However, it remains unknown whether a signal from the extracellular environment directly affects the AID activity in the nucleus where it works. Here, we demonstrated that a deubiquitinase USP10, which specifically stabilizes nuclear AID protein, can translocate into the nucleus after AKT-mediated phosphorylation at its T674 within the NLS domain. Interestingly, the signals from BCR and TLR1/2 synergistically promoted this phosphorylation. The deficiency of USP10 in B cells significantly decreased AID protein levels, subsequently reducing neutralizing antibody production after immunization with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or human immunodeficiency virus type 1 (HIV-1) nanoparticle vaccines. Collectively, we demonstrated that USP10 functions as an integrator for both BCR and TLR signals and directly regulates nuclear AID activity. Its manipulation could be used for the development of vaccines and adjuvants.
Asunto(s)
Vacunas contra el SIDA/inmunología , Factor Activador de Células B/inmunología , Vacunas contra la COVID-19/inmunología , Citidina Desaminasa/inmunología , VIH-1/inmunología , Nanopartículas , SARS-CoV-2/inmunología , Transducción de Señal/inmunología , Ubiquitina Tiolesterasa/inmunología , Ubiquitinación/inmunología , Vacunas contra el SIDA/genética , Animales , Factor Activador de Células B/genética , Vacunas contra la COVID-19/genética , Citidina Desaminasa/genética , Células HEK293 , VIH-1/genética , Humanos , Ratones , Ratones Noqueados , SARS-CoV-2/genética , Transducción de Señal/genética , Ubiquitina Tiolesterasa/genéticaRESUMEN
Kelch superfamily involves a variety of proteins containing multiple kelch motif and is well characterized as substrate adaptors for CUL3 E3 ligases, which play critical roles in carcinogenesis. However, the role of kelch proteins in lung cancer remains largely unknown. In this study, the non-small cell lung cancer (NSCLC) patients with higher expression of a kelch protein, kelch domain containing 3 (KLHDC3), showed worse overall survival. KLHDC3 deficiency affected NSCLC cell lines proliferation in vitro and in vivo. Further study indicated that KLHDC3 mediated CUL2 E3 ligase and tumor suppressor p14ARF interaction, facilitating the N-terminal ubiquitylation and subsequent degradation of p14ARF. Interestingly, Gefitinib-resistant NSCLC cell lines displayed higher KLHDC3 protein levels. Gefitinib and Osimertinib medications were capable of upregulating KLHDC3 expression to promote p14ARF degradation in the NSCLC cell lines. KLHDC3 shortage significantly increased the sensitivity of lung cancer cells to epidermal growth factor receptor (EGFR)-targeted drugs, providing an alternative explanation for the development of Gefitinib and Osimertinib resistance in NSCLC therapy. Our works suggest that CRL2KLHDC3 could be a valuable target to regulate the abundance of p14ARF and postpone the occurrence of EGFR-targeted drugs resistance.
Asunto(s)
Antineoplásicos , Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Línea Celular Tumoral , Proliferación Celular , Resistencia a Antineoplásicos/genética , Receptores ErbB/genética , Receptores ErbB/metabolismo , Gefitinib/farmacología , Gefitinib/uso terapéutico , Humanos , Secuencia Kelch , Neoplasias Pulmonares/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Proteína p14ARF Supresora de Tumor/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , UbiquitinaciónRESUMEN
Butenyl-spinosyn is a highly effective and broad-spectrum biopesticide produced by Saccharopolyspora pogona. However, the yield of this compound is difficult to increase because the regulatory mechanism of secondary metabolism is still unknown. Here, the transcriptional regulator Sp13016 was discovered to be highly associated with butenyl-spinosyn synthesis and bacterial growth. Overexpression of sp13016 improved butenyl-spinosyn production to a level that was 2.84-fold that of the original strain, while deletion of sp13016 resulted in a significant decrease in yield and growth inhibition. Comparative proteomics revealed that these phenotypic changes were attributed to the influence of Sp13016 on the central carbon metabolism pathway to regulate the supply of precursors. Our research helps to reveal the regulatory mechanism of butenyl-spinosyn biosynthesis and provides a reference for increasing the yield of natural products of Actinomycetes.
Asunto(s)
Proteómica , Saccharopolyspora , Proteínas Bacterianas/genética , Macrólidos , Saccharopolyspora/genéticaRESUMEN
The etiologic agent of COVID-19 is highly contagious and has caused a severe global pandemic. Until now, there has been no simple and reliable system available in a lower-biosafety-grade laboratory for SARS-CoV-2 virologic research and inhibitor screening. In this study, we reported a replicon system which consists of four plasmids expressing the required segments of SARS-CoV-2. Our study revealed that the features for viral RNA synthesis and responses to antivirus drugs of the replicon are similar to those of wild-type viruses. Further analysis indicated that ORF6 provided potent in trans stimulation of the viral replication. Some viral variations, such as 5'UTR-C241T and ORF8-(T28144C) L84S mutation, also exhibit their different impact upon viral replication. Besides, the screening of clinically used drugs identified that several tyrosine kinase inhibitors and DNA-Top II inhibitors potently inhibit the replicon, as well as authentic SARS-CoV-2 viruses. Collectively, this replicon system provides a biosafety-worry-free platform for studying SARS-CoV-2 virology, monitoring the functional impact of viral mutations, and developing viral inhibitors.IMPORTANCE COVID-19 has caused a severe global pandemic. Until now, there has been no simple and reliable system available in a lower-biosafety-grade laboratory for SARS-CoV-2 virologic research and inhibitor screening. We reported a replicon system which consists of four ordinary plasmids expressing the required segments of SARS-CoV-2. Using the replicon system, we developed three application scenarios: (i) to identify the effects of viral proteins on virus replication, (ii) to identify the effects of mutations on viral replication during viral epidemics, and (iii) to perform high-throughput screening of antiviral drugs. Collectively, this replicon system would be useful for virologists to study SARS-CoV-2 virology, for epidemiologists to monitor virus mutations, and for industry to develop antiviral drugs.
Asunto(s)
Antivirales/farmacología , COVID-19/virología , ARN Viral/biosíntesis , Replicón/efectos de los fármacos , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/genética , Evaluación Preclínica de Medicamentos/métodos , Femenino , Ingeniería Genética , Células HEK293 , Ensayos Analíticos de Alto Rendimiento , Humanos , Concentración 50 Inhibidora , Mutación , Pandemias , ARN Viral/genética , Replicón/genética , SARS-CoV-2/metabolismo , Replicación Viral/efectos de los fármacosRESUMEN
Butenyl-spinosyn, a promising biopesticide produced by Saccharopolyspora pogona, exhibits stronger insecticidal activity and a broader pesticidal spectrum. However, its titre in the wild-type S. pogona strain is too low to meet the industrial production requirements. Deletion of non-target natural product biosynthetic gene clusters resident in the genome of S. pogona could reduce the consumption of synthetic precursors, thereby promoting the biosynthesis of butenyl-spinosyn. However, it has always been a challenge for scientists to genetically engineer S. pogona. In this study, the Latour gene knockout system (linear DNA fragment recombineering system) was established in S. pogona. Using the Latour system, a hybrid NRPS-T1PKS cluster (Ë20 kb) which was responsible for phthoxazolin biosynthesis was efficiently deleted in S. pogona. The resultant mutant S. pogona-Δura4-Δc14 exhibited an extended logarithmic phase, increased biomass and a lower glucose consumption rate. Importantly, the production of butenyl-spinosyn in S. pogona-Δura4-Δc14 was increased by 4.72-fold compared with that in the wild-type strain. qRT-PCR analysis revealed that phthoxazolin biosynthetic gene cluster deletion could promote the expression of the butenyl-spinosyn biosynthetic gene cluster. Furthermore, a TetR family transcriptional regulatory gene that could regulate the butenyl-spinosyn biosynthesis has been identified from the phthoxazolin biosynthetic gene cluster. Because dozens of natural product biosynthetic gene clusters exist in the genome of S. pogona, the strategy reported here will be used to further promote the production of butenyl-spinosyn by deleting other secondary metabolite synthetic gene clusters.
Asunto(s)
Macrólidos , Saccharopolyspora , Proteínas Bacterianas/genética , Técnicas de Inactivación de Genes , Familia de Multigenes , Saccharopolyspora/genéticaRESUMEN
Spinosyns, the secondary metabolites produced by Saccharopolyspora spinosa, are the active ingredients in a family of novel biological insecticides. Although the complete genome sequence of S. spinosa has been published, the transcriptome of S. spinosa remains poorly characterized. In this study, high-throughput RNA sequencing (RNA-seq) technology was applied to dissect the transcriptome of S. spinosa. Through transcriptomic analysis of different periods of S. spinosa growth, we found large numbers of differentially expressed genes and classified them according to their different functions. Based on the RNA-seq data, the CRISPR-Cas9 method was used to knock out the PEP phosphonomutase gene (orf 06952-4171). The yield of spinosyns A and D in S. spinosa-ΔPEP was 178.91 mg/L and 42.72 mg/L, which was 2.14-fold and 1.76-fold higher than that in the wild type (83.51 and 24.34 mg/L), respectively. The analysis of the mutant strains also verified the validity of the transcriptome data. The deletion of the PEP phosphonomutase gene leads to an increase in pyruvate content and affects the biosynthesis of spinosad. The replenishment of phosphoenol pyruvate in S. spinosa provides the substrate for the production of spinosad. We envision that these transcriptomic analysis results will contribute to the further study of secondary metabolites in actinomycetes.
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Proteínas Bacterianas/metabolismo , Saccharopolyspora/enzimología , Proteínas Bacterianas/genética , Perfilación de la Expresión Génica , Macrólidos/metabolismo , Mutación , Ácido Pirúvico/metabolismo , RNA-Seq , Saccharopolyspora/genética , Saccharopolyspora/metabolismo , TranscriptomaRESUMEN
Immunotherapy has been demonstrated as a promising strategy in controlling head and neck squamous cell carcinoma (HNSC). The AID/APOBEC family is well characterized as DNA mutator and considered to play critical roles in immune responses in HNSC. However, the expression pattern and deamination-dependent demethylation roles of AID/APOBECs in HNSC are unclear. In this study, the RNA-seq and DNA methylation profiles of HNSC from TCGA database and cell-based experiments were applied to analyze the relationships between AID/APOBEC expression levels, patients' clinical outcomes, methylation alterations, and immune responses. Here, we found that APOBEC3H was abnormally upregulated in HNSC patients. HPV+ patients tended to have higher APOBEC3H levels than HPV- patients. Remarkably, patients with high APOBEC3H levels showed a favorable overall survival. Furthermore, tumors with high APOBEC3H levels exhibited a genome-wide DNA hypomethylation pattern. APOBEC3H was identified to demethylate and upregulate CXCL10 and improve CD8+ T cell tumor infiltration in the tumor microenvironment. Collectively, APOBEC3H plays critical roles in CD8+ T cell immune infiltration and activation in HNSC, which may be a potential biomarker for oncoimmunotherapy in HNSC.
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Aminohidrolasas/inmunología , Biomarcadores de Tumor/inmunología , Bases de Datos de Ácidos Nucleicos , Neoplasias de Cabeza y Cuello/inmunología , Proteínas de Neoplasias/inmunología , Carcinoma de Células Escamosas de Cabeza y Cuello/inmunología , Femenino , Neoplasias de Cabeza y Cuello/mortalidad , Neoplasias de Cabeza y Cuello/terapia , Humanos , Inmunoterapia , Masculino , Metilación , Carcinoma de Células Escamosas de Cabeza y Cuello/mortalidad , Carcinoma de Células Escamosas de Cabeza y Cuello/terapiaRESUMEN
The antiviral activity of host factor apolipoprotein B mRNA editing enzyme catalytic polypeptide-like 3G (APOBEC3G, A3G) and its degradation mediated by human immunodeficiency virus type 1 (HIV-1) Vif protein are important topics. Although accumulating evidence indicates the importance of deubiquitination enzymes (DUBs) in innate immunity, it is unknown if they participate in A3G stability. Here, we found that USP49 directly interacts with A3G and efficiently removes ubiquitin, consequently increasing A3G protein expression and significantly enhancing its anti-HIV-1 activity. Unexpectedly, A3G degradation was also mediated by a Vif- and cullin-ring-independent pathway, which was effectively counteracted by USP49. Furthermore, clinical data suggested that USP49 is correlated with A3G protein expression and hypermutations in Vif-positive proviruses, and inversely with the intact provirus ratio in the HIV-1 latent reservoir. Our studies demonstrated a mechanism to effectively stabilize A3G expression, which could comprise a target to control HIV-1 infection and eradicate the latent reservoir.
Asunto(s)
Desaminasa APOBEC-3G/metabolismo , VIH-1/crecimiento & desarrollo , VIH-1/inmunología , Factores Inmunológicos/metabolismo , Ubiquitina Tiolesterasa/metabolismo , Ubiquitina/metabolismo , Replicación Viral , Células HEK293 , Células HeLa , Humanos , Inmunidad InnataRESUMEN
Comprehensively elucidating the molecular mechanisms of human immunodeficiency virus type 1 (HIV-1) latency is a priority to achieve a functional cure. As current 'shock' agents failed to efficiently reactivate the latent reservoir, it is important to discover new targets for developing more efficient latency-reversing agents (LRAs). Here, we found that TRIM28 potently suppresses HIV-1 expression by utilizing both SUMO E3 ligase activity and epigenetic adaptor function. Through global site-specific SUMO-MS study and serial SUMOylation assays, we identified that P-TEFb catalytic subunit CDK9 is significantly SUMOylated by TRIM28 with SUMO4. The Lys44, Lys56 and Lys68 residues on CDK9 are SUMOylated by TRIM28, which inhibits CDK9 kinase activity or prevents P-TEFb assembly by directly blocking the interaction between CDK9 and Cyclin T1, subsequently inhibits viral transcription and contributes to HIV-1 latency. The manipulation of TRIM28 and its consequent SUMOylation pathway could be the target for developing LRAs.