Involvement of Hdac3-mediated inhibition of microRNA cluster 17-92 in bronchopulmonary dysplasia development

Abstract Background The incidence of bronchopulmonary dysplasia (BPD), a chronic lung disease of newborns, has been paradoxically rising despite medical advances. Histone deacetylase 3 (Hdac3) has been reported to be a crucial regulator in alveologenesis. Hence, this study aims to investigate the mechanism of Hdac3 in the abnormal pulmonary angiogenesis and alveolarization of BPD. Methods A hyperoxia-induced BPD model of was developed in newborn mice, and primary lung fibroblasts were isolated from adult mice. Hdac3 was knocked out in vivo and knocked down in vitro, while microRNA (miR)-17 was downregulated in vivo and in vitro to clarify their roles in abnormal pulmonary angiogenesis and alveolarization. Mechanistic investigations were performed on the interplay of Hdac3, miR-17-92 cluster, enhancer of zeste homolog 1 (EZH1), p65 and placental growth factor (Pgf). Results Hdac3 was involved in abnormal alveolarization and angiogenesis in BPD mice. Further, the expression of the miR-17-92 cluster in BPD mice was downregulated by Hdac3. miR-17 was found to target EZH1, and Hdac3 rescued the inhibited EZH1 expression by miR-17 in lung fibroblasts. Additionally, EZH1 augmented Pgf expression by recruiting p65 thus enhancing the progression of BPD. Hdac3 augmented the recruitment of p65 in the Pgf promoter region through the miR-17/EZH1 axis, thus enhancing the transcription and expression of Pgf, which elicited abnormal angiogenesis and alveolarization of BPD mice. Conclusions Altogether, the present study revealed that Hdac3 activated the EZH1-p65-Pgf axis through inhibiting miR-17 in the miR-17-92 cluster, leading to accelerated abnormal pulmonary angiogenesis and alveolarization of BPD mice.

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Spleen cells from adult mice given total lymphoid irradiation or from newborn mice have similar regulatory effects in the mixed leukocyte reaction. I. Generation of antigen-specific suppressor cells in the mixed leukocyte reaction after the addition of spleen cells from adult mice given total lymphoid irradiation.

Journal of Experimental Medicine ◽

10.1084/jem.156.2.522 ◽

1982 ◽

Vol 156 (2) ◽

pp. 522-538 ◽

Cited By ~ 22

Author(s):

S Okada ◽

S Strober

Keyword(s):

Suppressor Cells ◽

Allogeneic Bone ◽

Mixed Leukocyte Reaction ◽

Spleen Cells ◽

Newborn Mice ◽

Total Lymphoid Irradiation ◽

Adult Mice ◽

Regulatory Effects

We added spleen cells from adult BALB/c mice treated with total lymphoid irradiation (TLI) to the mixed leukocyte reaction (MLR) using a variety of responder and stimulator cells. The spleen cells nonspecifically suppressed the uptake of [3H]-thymidine and the generation of cytolytic cells regardless of the responder-stimulator combination used. We also examined the effect of the spleen cells on the generation of antigen-nonspecific and antigen-specific suppressor cells in the MLR. The experimental results suggest that the spleen cells from TLI-treated mice inhibit the generation of nonspecific suppressor cells, but do not inhibit the generation of antigen-specific suppressor cells. Thus, alloantigenic stimulation of normal responder cells in vitro in the presence of spleen cells from TLI-treated mice generates large numbers of antigen-specific suppressor cells, but few cytolytic cells or nonspecific suppressor cells. Similar nonspecific inhibition of the MLR was observed with neonatal spleen cells. This in vitro system provides a regulatory model for the induction and maintenance of tolerance in vivo, in which adult mice given TLI or neonatal mice accept allogeneic bone marrow transplants without graft-vs.-host disease.

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The involvement of HDAC3-mediated inhibition of microRNA cluster 17-92 in hyperoxia-mediated impairment of lung development in neonatal rats

10.1101/2020.03.01.971663 ◽

2020 ◽

Author(s):

Di Wang ◽

Hui Hong ◽

Xiao-Xia Li ◽

Jing Li ◽

Zhi-Qun Zhang

Keyword(s):

Promoter Region ◽

Immunofluorescence Assay ◽

Lung Fibroblasts ◽

Luciferase Reporter ◽

Luciferase Reporter Gene ◽

Alveolar Volume ◽

Histone Deacetylase 3 ◽

Gene Assay ◽

Medical Advances

AbstractBackgroundThe incidence of bronchopulmonary dysplasia (BPD), a chronic lung disease of newborns, has been paradoxically rising despite medical advances. Histone deacetylase 3 (HDAC3) has been reported to be a crucial regulator in alveologenesis. Hence, this study aims to investigate the mechanism of HDAC3 in the pulmonary angiogenesis and alveolarization of BPD.MethodsA hyperoxia-induced mouse model of BPD was constructed. The mean liner intercept (MLI) and alveolar volume were measured to evaluate the alveolarization in BPD mice. Immunofluorescence assay was performed to detect the microvessel density (MVD) of lung tissues. Next, the expression of HDAC3 and its enrichment in the promoter region of microRNA (miR)-17-92 cluster, as well as the enrichment of p65 in the placental growth factor (Pgf) promoter region were detected by Western blot analysis and chromatin immunoprecipitation (ChIP) assay. The effect of HDAC3 and p65 on the activity of miR-17-92 promoter and Pgf promoter were examined by dual-luciferase reporter gene assay, respectively. Finally, the role of HDAC3 in angiogenesis and alveolarization through miR-17 regulated EZH1-p65-Pgf axis was validated in BPD mouse models.ResultsHDAC3 was involved in the regulation of alveolarization and angiogenesis in BPD. Results demonstrated that the expression of the miR-17-92 cluster in BPD was regulated by HDAC3. miR-17 was related to the regulatory role of HDAC3 in regulating EZH1 expression and in lung fibroblasts of BPD. Besides, results showed that EZH1 could promote Pgf expression by recruiting p65 to regulate BPD. HDAC3 regulated the expression of EZH1 through miR-17 to promote the recruitment of p65 in the Pgf promoter region, thus enhancing the transcription and expression of Pgf. HDAC3 was demonstrated to regulate Pgf through the miR-17-EZH1-p65 axis to mediate angiogenesis and alveolarization of BPD mice.ConclusionAltogether, the present study revealed that HDAC3 could regulate the EZH1-p65-Pgf axis through miR-17 in the miR-17-92 cluster in the pulmonary angiogenesis and alveolarization of BPD mice.

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Development of DNA Vaccines against Hemolytic-Uremic Syndrome in a Murine Model

Infection and Immunity ◽

10.1128/iai.71.7.3971-3978.2003 ◽

2003 ◽

Vol 71 (7) ◽

pp. 3971-3978 ◽

Cited By ~ 20

Author(s):

Alejandra V. E. Capozzo ◽

Virginia Pistone Creydt ◽

Graciela Dran ◽

Gabriela Fernández ◽

Sonia Gómez ◽

...

Keyword(s):

Hemolytic Uremic Syndrome ◽

Humoral Response ◽

Host Cells ◽

Newborn Mice ◽

B Subunit ◽

Adult Mice ◽

Uremic Syndrome ◽

A Subunit

ABSTRACT Shiga toxin type 2 (Stx2) produced by Escherichia coli O:157H7 can cause hemolytic-uremic syndrome in children, a disease for which there is neither a vaccine nor an effective treatment. This toxin consists of an enzymatically active A subunit and a pentameric B subunit responsible for the toxin binding to host cells, and also found to be immunogenic in rabbits. In this study we developed eukaryotic plasmids expressing the B subunit gene of Stx2 (pStx2B) and the B subunit plus the gene coding for the A subunit with an active-site deletion (pStx2ΔA). Transfection of eukaryotic cells with these plasmids produced proteins of the expected molecular weight which reacted with specific monoclonal antibodies. Newborn and adult BALB/c mice immunized with two intramuscular injections of each plasmid, either alone or together with the same vector expressing the granulocyte and monocyte colony-stimulating factor (pGM-CSF), elicited a specific Th1-biased humoral response. The effect of pGM-CSF as an adjuvant plasmid was particularly notable in newborn mice and in pStx2B-vaccinated adult mice. Stx2-neutralizing activity, evaluated in vitro on VERO cell monolayers, correlated with in vivo protection. This is the first report using plasmids to induce a neutralizing humoral immune response against the Stx2.

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Lactate secreted by PKM2 upregulation promotes Galectin-9-mediated immunosuppression via inhibiting NF-κB pathway in HNSCC

Cell Death and Disease ◽

10.1038/s41419-021-03990-4 ◽

2021 ◽

Vol 12 (8) ◽

Author(s):

Hanyue Chang ◽

Qiaoshi Xu ◽

Jiayi Li ◽

Mingyu Li ◽

Zhiyuan Zhang ◽

...

Keyword(s):

Critical Role ◽

Lactate Production ◽

Metabolic Reprogramming ◽

Migration And Invasion ◽

Histone Deacetylase 3 ◽

Proliferation And Metastasis ◽

Immunosuppressive Microenvironment ◽

Transcriptional Complex

AbstractPyruvate kinase M2 as a key rate-limiting enzyme in glycolysis, it plays a critical role in metabolic reprogramming and carcinogenesis. However, whether PKM2 can promote immunosuppressive microenvironment formation remains unknown in head and neck squamous cell carcinoma (HNSCC). PKM2 expression was detected using immunohistochemical staining. The biological functions of PKM2 were investigated in vitro and in vivo. Lactate production and the expression of Galectin-9, a critical immunosuppression molecule, were detected after PKM2 knockdown and overexpression in HNSCC cells. The mechanism of lactate regulating Galectin-9 expression through NF-κB signaling was explored in vitro. Overexpression of PKM2 correlates with poor prognosis in HNSCC patients. Silencing PKM2 markedly inhibits proliferation and metastasis capacity in vivo and in vitro, and vice versa. The glycolysis and glycolytic capacity are significantly decreased after PKM2 silencing. Lactate secretion induced by PKM2 significantly promotes migration and invasion capacity. Furthermore, a positive correlation between PKM2 and Galectin-9 expression is observed in HNSCC tissues. The induction of Galectin-9 expression by PKM2 can be affected by a lactate transporter inhibitor. Mechanically, lactate impeded the suppressive transcriptional complex formation of NF-κB and histone deacetylase 3 (HDAC3), which released the transcription of Galectin-9 mediated by NF-κB signaling. Our findings demonstrate that lactate produced by PKM2 upregulation promotes tumor progression and Galectin-9-mediated immunosuppression via NF-κB signaling inhibition in HNSCC, which bridges metabolism and immunosuppression. The novel PKM2-lactate-Galectin-9 axis might be a potential therapeutic target in HNSCC.

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Rotavirus Infection Is Not Associated with Small Intestinal Fluid Secretion in the Adult Mouse

Journal of Virology ◽

10.1128/jvi.00152-06 ◽

2006 ◽

Vol 80 (22) ◽

pp. 11355-11361 ◽

Cited By ~ 17

Author(s):

Shirin Kordasti ◽

Claudia Istrate ◽

Mahanez Banasaz ◽

Martin Rottenberg ◽

Henrik Sjövall ◽

...

Keyword(s):

Fluid Secretion ◽

Chloride Secretion ◽

Potential Difference ◽

Pathophysiological Mechanism ◽

Rotavirus Infection ◽

In Vivo Experiments ◽

Adult Mice ◽

Small Intestines

ABSTRACT In contrast to humans, adult but not infant small animals are resistant to rotavirus diarrhea. The pathophysiological mechanism behind this age-restricted diarrhea is currently unresolved, and this question was investigated by studying the secretory state of the small intestines of adult mice infected with rotavirus. Immunohistochemistry and histological examinations revealed that rotavirus (strain EDIM) infects all parts of the small intestines of adult mice, with significant numbers of infected cells in the ilea at 2 and 4 days postinfection. Furthermore, quantitative PCR revealed that 100-fold more viral RNA was produced in the ilea than in the jejuna or duodena of adult mice. In vitro perfusion experiments of the small intestine did not reveal any significant changes in net fluid secretion among mice infected for 3 days or 4 days or in those that were noninfected (37 ± 9 μl · h−1 · cm−1, 22 ± 13 μl · h−1 · cm−1, and 33 ± 6 μl · h−1 · cm−1, respectively) or in transmucosal potential difference (4.0 ± 0.3 mV versus 3.9 ± 0.4 mV), a marker for active chloride secretion, between control and rotavirus-infected mice. In vivo experiments also did not show any differences in potential difference between uninfected and infected small intestines. Furthermore, no significant differences in weight between infected and uninfected small intestines were found, nor were any differences in fecal output observed between infected and control mice. Altogether, these data suggest that rotavirus infection is not sufficient to stimulate chloride and water secretion from the small intestines of adult mice.

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A Tissue-Engineered Tracheobronchial In Vitro Co-Culture Model for Determining Epithelial Toxicological and Inflammatory Responses

Biomedicines ◽

10.3390/biomedicines9060631 ◽

2021 ◽

Vol 9 (6) ◽

pp. 631

Author(s):

Luis Soriano ◽

Tehreem Khalid ◽

Fergal J. O'Brien ◽

Cian O'Leary ◽

Sally-Ann Cryan

Keyword(s):

Epithelial Cells ◽

Inflammatory Responses ◽

Bronchial Epithelial Cells ◽

Lung Fibroblasts ◽

Drug Induced ◽

Bronchial Epithelial ◽

Culture Model ◽

Epithelial Cultures

Translation of novel inhalable therapies for respiratory diseases is hampered due to the lack of in vitro cell models that reflect the complexity of native tissue, resulting in many novel drugs and formulations failing to progress beyond preclinical assessments. The development of physiologically-representative tracheobronchial tissue analogues has the potential to improve the translation of new treatments by more accurately reflecting in vivo respiratory pharmacological and toxicological responses. Herein, advanced tissue-engineered collagen hyaluronic acid bilayered scaffolds (CHyA-B) previously developed within our group were used to evaluate bacterial and drug-induced toxicity and inflammation for the first time. Calu-3 bronchial epithelial cells and Wi38 lung fibroblasts were grown on either CHyA-B scaffolds (3D) or Transwell® inserts (2D) under air liquid interface (ALI) conditions. Toxicological and inflammatory responses from epithelial monocultures and co-cultures grown in 2D or 3D were compared, using lipopolysaccharide (LPS) and bleomycin challenges to induce bacterial and drug responses in vitro. The 3D in vitro model exhibited significant epithelial barrier formation that was maintained upon introduction of co-culture conditions. Barrier integrity showed differential recovery in CHyA-B and Transwell® epithelial cultures. Basolateral secretion of pro-inflammatory cytokines to bacterial challenge was found to be higher from cells grown in 3D compared to 2D. In addition, higher cytotoxicity and increased basolateral levels of cytokines were detected when epithelial cultures grown in 3D were challenged with bleomycin. CHyA-B scaffolds support the growth and differentiation of bronchial epithelial cells in a 3D co-culture model with different transepithelial resistance in comparison to the same co-cultures grown on Transwell® inserts. Epithelial cultures in an extracellular matrix like environment show distinct responses in cytokine release and metabolic activity compared to 2D polarised models, which better mimic in vivo response to toxic and inflammatory stimuli offering an innovative in vitro platform for respiratory drug development.

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Human Cytomegalovirus Replication and Infection-Induced Syncytia Formation in Labial, Foreskin, and Fetal Lung Fibroblasts

Viruses ◽

10.3390/v13122355 ◽

2021 ◽

Vol 13 (12) ◽

pp. 2355

Author(s):

Alexis Aguiar ◽

Melissa Galinato ◽

Maite’ Bradley Silva ◽

Bryant Toth ◽

Michael A. McVoy ◽

...

Keyword(s):

Endothelial Cells ◽

Human Cytomegalovirus ◽

Fetal Lung ◽

Lung Fibroblasts ◽

Anatomical Site ◽

Cmv Infection ◽

Viral Spread ◽

Syncytia Formation

Only a handful of cell types, including fibroblasts, epithelial, and endothelial cells, can support human cytomegalovirus (CMV) replication in vitro, in striking contrast to the situation in vivo. While the susceptibility of epithelial and endothelial cells to CMV infection is strongly modulated by their anatomical site of origin, multiple CMV strains have been successfully isolated and propagated on fibroblasts derived from different organs. As oral mucosal cells are likely involved in CMV acquisition, we sought to evaluate the ability of infant labial fibroblasts to support CMV replication, compared to that of commonly used foreskin and fetal lung fibroblasts. No differences were found in the proportion of cells initiating infection, or in the amounts of viral progeny produced after exposure to the fibroblast-adapted CMV strain AD169 or to the endothelial cell-adapted strain TB40/E. Syncytia formation was, however, significantly enhanced in infected labial and lung fibroblasts compared to foreskin-derived cells, and did not occur after infection with AD169. Together, these data indicate that fibroblast populations derived from different tissues are uniformly permissive to CMV infection but retain phenotypic differences of potential importance for infection-induced cell–cell fusion, and ensuing viral spread and pathogenesis in different organs.

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Single amino acid mutations effect Zika virus replication in vitro and virulence in vivo

10.1101/2020.08.06.239392 ◽

2020 ◽

Author(s):

Nicole M. Collette ◽

Victoria H.I. Lao ◽

Dina R. Weilhammer ◽

Barbara Zingg ◽

Shoshana D. Cohen ◽

...

Keyword(s):

Amino Acid ◽

Zika Virus ◽

Model Systems ◽

Single Amino Acid ◽

Replication Rate ◽

Adult Mice ◽

Naturally Occurring ◽

Viral Virulence

AbstractThe 2014-2016 Zika virus (ZIKV) epidemic in the Americas resulted in large deposits of next-generation sequencing data from clinical samples. This resource was mined to identify emerging mutations and trends in mutations as the outbreak progressed over time. Information on transmission dynamics, prevalence and persistence of intra-host mutants, and the position of a mutation on a protein were then used to prioritize 544 reported mutations based on their ability to impact ZIKV phenotype. Using this criteria, six mutants (representing naturally occurring mutations) were generated as synthetic infectious clones using a 2015 Puerto Rican epidemic strain PRVABC59 as the parental backbone. The phenotypes of these naturally occurring variants were examined using both cell culture and murine model systems. Mutants had distinct phenotypes, including changes in replication rate, embryo death, and decreased head size. In particular, a NS2B mutant previously detected during in vivo studies in rhesus macaques was found to cause lethal infections in adult mice, abortions in pregnant females, and increased viral genome copies in both brain tissue and blood of female mice. Additionally, mutants with changes in the region of NS3 that interfaces with NS5 during replication displayed reduced replication in the blood of adult mice. This analytical pathway, integrating both bioinformatic and wet lab experiments, provides a foundation for understanding how naturally occurring single mutations affect disease outcome and can be used to predict the of severity of future ZIKV outbreaks.Author summaryTo determine if naturally occurring individual mutations in the Zika virus epidemic genotype effect viral virulence or replication rate in vitro or in vivo, we generated an infectious clone representing the epidemic genotype of stain Puerto Rico, 2015. Using this clone, six mutants were created by changing nucleotides in the genome to cause one to two amino acid substitutions in the encoded proteins. The six mutants we generated represent mutations that differentiated the early epidemic genotype from genotypes that were either ancestral or that occurred later in the epidemic. We assayed each mutant for changes in growth rate, and for virulence in adult mice and pregnant mice. Three of the mutants caused catastrophic embryo effects including increased embryonic death or significant decrease in head diameter. Three other mutants that had mutations in a genome region associated with replication resulted in changes in in vitro and in vivo replication rates. These results illustrate the potential impact of individual mutations in viral phenotype.

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Characterization of a Chlamydia psittaciDNA Binding Protein (EUO) Synthesized during the Early and Middle Phases of the Developmental Cycle

Infection and Immunity ◽

10.1128/iai.66.3.1167-1173.1998 ◽

1998 ◽

Vol 66 (3) ◽

pp. 1167-1173 ◽

Cited By ~ 31

Author(s):

Li Zhang ◽

Annemarie L. Douglas ◽

Thomas P. Hatch

Keyword(s):

Promoter Region ◽

Chlamydia Psittaci ◽

Host Cells ◽

Upstream Open Reading Frame ◽

Developmental Cycle ◽

Binding Property ◽

Logarithmic Growth Phase ◽

Reading Frame

ABSTRACT The EUO gene (for early upstream open reading frame) ofChlamydia psittaci was previously found to be transcribed better at 1 than at 24 h postinfection. We found that the EUO gene encodes a minor protein that is expressed within 1 h of infection of host cells with C. psittaci 6BC but that protein quantity peaks during the logarithmic growth phase of reticulate bodies (RBs), declines late in the infection (after 20 h) when RBs reorganize into elementary bodies (EBs), and is absent in infectious EBs. EUO protein lacks homology to known proteins but does contain a putative helix-turn-helix motif. We found that recombinant EUO binds to DNA in vitro with a relatively broad specificity. Using the bp −200 to +67 promoter region of the cysteine-rich envelope protein (crp) operon as a model, we show that EUO protein preferentially binds to AT-rich sequences and protects crpDNA from DNase I from approximately bp −60 to −9. We also found that native EUO protein in extracts of RBs binds to the promoter region of the crp operon, demonstrating that the DNA binding property of EUO protein is not an artifact of recombinant methods. Although EUO protein appears to bind to the crp operon with high affinity in vitro (Kd of about 15 nM), it is not known whether the protein binds the crp DNA in vivo.

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lncRNA MIR22HG-Derived miR-22-5p Enhances the Radiosensitivity of Hepatocellular Carcinoma by Increasing Histone Acetylation Through the Inhibition of HDAC2 Activity

Frontiers in Oncology ◽

10.3389/fonc.2021.572585 ◽

2021 ◽

Vol 11 ◽

Author(s):

Qiao Jin ◽

Hao Hu ◽

Siqi Yan ◽

Long Jin ◽

Yuliang Pan ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Histone Acetylation ◽

Chromatin Immunoprecipitation ◽

Promoter Region ◽

Animal Experiments ◽

Primary Hepatocellular Carcinoma ◽

Acetylation Level ◽

Expression Levels

BackgroundWith the development of radiotherapy technology, radiotherapy has been increasingly used to treat primary hepatocellular carcinoma (HCC). However, due to radioresistance and the intolerance of the adjacent organs to radiation, the effects of radiotherapy are often unsatisfactory. Therefore, it is necessary to study radiosensitization in HCC.MethodA microarray was used to analyze the genes that were significantly associated with radiosensitivity. HCC cells, HepG2 and MHCC97H, were subjected to radiation in vitro. Real-time PCR was performed to determine MIR22HG (microRNA22 host gene) and miR-22-5p expression levels. Western blotting was performed to determine histone expression levels. A histone deacetylase (HDAC) whole cell assay was used to determine the activity of HDAC2. MTT, colony formation, 5-ethynyl-2′-deoxyuridine, and wound healing assays were performed to examine the function of MIR22HG and miR-22-5p in cellular radiosensitivity. Chromatin immunoprecipitation-PCR was used to confirm that HDAC2 affects the acetylation level of the MIR22HG promoter region. Finally, animal experiments were performed to demonstrate the in vivo effect of MIR22HG on the radiosensitivity of hepatoma.ResultsIrradiation can up-regulate MIR22HG expression and down-regulate HDAC2 expression. Inhibition of HDAC2 expression promotes histone acetylation in the MIR22HG promoter region and up-regulates MIR22HG expression. MIR22HG can increase radiosensitivity via miR-22-5p in HCC.ConclusionInhibition of HDAC2 expression promotes histone acetylation in the MIR22HG promoter region, thereby up-regulating the expression of MIR22HG and promoting the production of miR-22-5p, and ultimately increasing the sensitivity of liver cancer radiotherapy.

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