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1.
mBio ; 12(5): e0212721, 2021 Oct 26.
Artigo em Inglês | MEDLINE | ID: mdl-34488445

RESUMO

Interferon (IFN) signaling is key to mucosal immunity in the gastrointestinal tract, but cellular regulatory elements that determine interferon gamma (IFN-γ)-mediated antimicrobial defense in intestinal epithelial cells are not fully understood. We report here that a long noncoding RNA (lncRNA), GenBank accession no. XR_001779380, was increased in abundance in murine intestinal epithelial cells following infection by Cryptosporidium, an important opportunistic pathogen in AIDS patients and a common cause of diarrhea in young children. Expression of XR_001779380 in infected intestinal epithelial cells was triggered by TLR4/NF-κB/Cdc42 signaling and epithelial-specific transcription factor Elf3. XR_001779380 primed epithelial cells for IFN-γ-mediated gene transcription through facilitating Stat1/Swi/Snf-associated chromatin remodeling. Interactions between XR_001779380 and Prdm1, which is expressed in neonatal but not adult intestinal epithelium, attenuated Stat1/Swi/Snf-associated chromatin remodeling induced by IFN-γ, contributing to suppression of IFN-γ-mediated epithelial defense in neonatal intestine. Our data demonstrate that XR_001779380 is an important regulator in IFN-γ-mediated gene transcription and age-associated intestinal epithelial antimicrobial defense. IMPORTANCE Epithelial cells along the mucosal surface provide the front line of defense against luminal pathogen infection in the gastrointestinal tract. These epithelial cells represent an integral component of a highly regulated communication network that can transmit essential signals to cells in the underlying intestinal mucosa that, in turn, serve as targets of mucosal immune mediators. LncRNAs are recently identified long noncoding transcripts that can regulate gene transcription through their interactions with other effect molecules. In this study, we demonstrated that lncRNA XR_001779380 was upregulated in murine intestinal epithelial cells following infection by a mucosal protozoan parasite Cryptosporidium. Expression of XR_001779380 in infected cells primed host epithelial cells for IFN-γ-mediated gene transcription, relevant to age-dependent intestinal antimicrobial defense. Our data provide new mechanistic insights into how intestinal epithelial cells orchestrate intestinal mucosal defense against microbial infection.

2.
Front Immunol ; 12: 705232, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34295340

RESUMO

Increasing evidence supports that N6-methyladenosine (m6A) mRNA modification may play an important role in regulating immune responses. Intestinal epithelial cells orchestrate gastrointestinal mucosal innate defense to microbial infection, but underlying mechanisms are still not fully understood. In this study, we present data demonstrating significant alterations in the topology of host m6A mRNA methylome in intestinal epithelial cells following infection by Cryptosporidium parvum, a coccidian parasite that infects the gastrointestinal epithelium and causes a self-limited disease in immunocompetent individuals but a life-threatening diarrheal disease in AIDS patients. Altered m6A methylation in mRNAs in intestinal epithelial cells following C. parvum infection is associated with downregulation of alpha-ketoglutarate-dependent dioxygenase alkB homolog 5 and the fat mass and obesity-associated protein with the involvement of NF-кB signaling. Functionally, m6A methylation statuses influence intestinal epithelial innate defense against C. parvum infection. Specifically, expression levels of immune-related genes, such as the immunity-related GTPase family M member 2 and interferon gamma induced GTPase, are increased in infected cells with a decreased m6A mRNA methylation. Our data support that intestinal epithelial cells display significant alterations in the topology of their m6A mRNA methylome in response to C. parvum infection with the involvement of activation of the NF-кB signaling pathway, a process that modulates expression of specific immune-related genes and contributes to fine regulation of epithelial antimicrobial defense.

3.
Medicine (Baltimore) ; 100(12): e24765, 2021 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-33761638

RESUMO

ABSTRACT: MicroRNA (miR)-26a-5p is an oncogene significantly associated with osteosarcoma. We try to evaluate expression of circulating miR-26a-5p in osteosarcoma patients and evaluate its significance.A total of 243 consecutive osteosarcoma patients and 96 healthy participates were enrolled. Circulating miR-26a-5p levels were evaluated by using real-time quantitative reverse transcription polymerase chain reactions (RT-PCR). The association between circulating miR-26a-5p level and survival outcomes was evaluated by univariate and multivariate analysis.Circulating miR-26a-5p levels in osteosarcoma patients was significantly higher than that of healthy volunteers (P < .05). Upregulated miR-26a-5p was significantly related to advanced cancer and metastasis (both P < .05). Moreover, patients with a high serum miR-26a-5p had a poorer overall survival than those with a low serum miR-26a-5p levels (P < .05). Circulating miR-26a-5p level also been showed as independent risk factor for osteosarcoma in multivariate analysis (hazard ratio [HR], 0.38; 95% confidence interval [CI]: 0.11-0.98; P < .01).Circulating miR-26a-5p was significantly upregulated in osteosarcoma patients and remarkably associated with poor prognosis, indicating that circulating miR-26a-5p might serve as a useful diagnostic and prognostic biomarker for osteosarcoma.


Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias Ósseas/mortalidade , MicroRNA Circulante/metabolismo , MicroRNAs/metabolismo , Recidiva Local de Neoplasia/epidemiologia , Osteossarcoma/mortalidade , Adulto , Biomarcadores Tumorais/sangue , Neoplasias Ósseas/sangue , Neoplasias Ósseas/genética , Neoplasias Ósseas/cirurgia , MicroRNA Circulante/sangue , Intervalo Livre de Doença , Feminino , Seguimentos , Regulação Neoplásica da Expressão Gênica , Humanos , Estimativa de Kaplan-Meier , Biópsia Líquida , Masculino , MicroRNAs/sangue , Recidiva Local de Neoplasia/genética , Osteossarcoma/sangue , Osteossarcoma/genética , Osteossarcoma/cirurgia , Estudos Retrospectivos , Medição de Risco/métodos , Regulação para Cima , Adulto Jovem
4.
Mol Biol Rep ; 48(1): 563-584, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33387198

RESUMO

Emerging evidence suggests that microglia can support neurogenesis. Little is known about the mechanisms by which microglia regulate the cortical environment and stimulate cortical neurogenesis. We used an in vitro co-culture model system to investigate the hypothesis that microglia respond to soluble signals from cortical cells, particularly following mechanical injury, to alter the cortical environment and promote cortical cell proliferation, differentiation, and survival. Analyses of cortical cell proliferation, cell death, neurogenic protein expression, and intracellular signaling were performed on uninjured and injured cortical cells in co-culture with microglial cell lines. Microglia soluble cues enhanced cortical cell viability and proliferation cortical cells. Co-culture of injured cortical cells with microglia significantly reduced cell death of cortical cells. Microglial co-culture significantly increased Nestin + and α-internexin + cortical cells. Multiplex ELISA and RT-PCR showed decreased pro-inflammatory cytokine production by microglia co-cultured with injured cortical cells. Inhibition of AKT phosphorylation in cortical cells blocked microglial-enhanced cortical cell viability and expression of neurogenic markers in vitro. This in vitro model system allows for assessment of the effect of microglial-derived soluble signals on cortical cell viability, proliferation, and stages of differentiation during homeostasis or following mechanical injury. These data suggest that microglia cells can downregulate inflammatory cytokine production following activation by mechanical injury to enhance proliferation of new cells capable of neurogenesis via activation of AKT intracellular signaling. Increasing our understanding of the mechanisms that drive microglial-enhanced cortical neurogenesis during homeostasis and following injury in vitro will provide useful information for future primary cell and in vivo studies.


Assuntos
Córtex Cerebral/crescimento & desenvolvimento , Microglia/citologia , Neurogênese/genética , Fosfatidilinositol 3-Quinases/genética , Proteínas Proto-Oncogênicas c-akt/genética , Animais , Diferenciação Celular/genética , Proliferação de Células/genética , Sobrevivência Celular/genética , Células Cultivadas , Córtex Cerebral/metabolismo , Técnicas de Cocultura , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteínas de Filamentos Intermediários/genética , Microglia/metabolismo , Nestina/genética , Neurônios/metabolismo , Cultura Primária de Células , Ratos , Transdução de Sinais/genética
5.
PLoS Pathog ; 17(1): e1009241, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33481946

RESUMO

The gastrointestinal epithelium guides the immune system to differentiate between commensal and pathogenic microbiota, which relies on intimate links with the type I IFN signal pathway. Epithelial cells along the epithelium provide the front line of host defense against pathogen infection in the gastrointestinal tract. Increasing evidence supports the regulatory potential of long noncoding RNAs (lncRNAs) in immune defense but their role in regulating intestinal epithelial antimicrobial responses is still unclear. Cryptosporidium, a protozoan parasite that infects intestinal epithelial cells, is an important opportunistic pathogen in AIDS patients and a common cause of diarrhea in young children in developing countries. Recent advances in Cryptosporidium research have revealed a strong type I IFN response in infected intestinal epithelial cells. We previously identified a panel of host cell lncRNAs that are upregulated in murine intestinal epithelial cells following microbial challenge. One of these lncRNAs, NR_033736, is upregulated in intestinal epithelial cells following Cryptosporidium infection and displays a significant suppressive effect on type I IFN-controlled gene transcription in infected host cells. NR_033736 can be assembled into the ISGF3 complex and suppresses type I IFN-mediated gene transcription. Interestingly, upregulation of NR_033736 itself is triggered by the type I IFN signaling. Moreover, NR_033736 modulates epithelial anti-Cryptosporidium defense. Our data suggest that upregulation of NR_033736 provides negative feedback regulation of type I IFN signaling through suppression of type I IFN-controlled gene transcription, and consequently, contributing to fine-tuning of epithelial innate defense against microbial infection.


Assuntos
Criptosporidiose/imunologia , Cryptosporidium/imunologia , Interferon Tipo I/metabolismo , RNA Longo não Codificante/genética , Transdução de Sinais , Animais , Animais Recém-Nascidos , Criptosporidiose/parasitologia , Diarreia/imunologia , Diarreia/parasitologia , Células Epiteliais/parasitologia , Trato Gastrointestinal/imunologia , Trato Gastrointestinal/parasitologia , Humanos , Mucosa Intestinal/imunologia , Mucosa Intestinal/parasitologia , Intestinos/parasitologia , Camundongos , Transcrição Genética , Regulação para Cima
6.
J Neuroinflammation ; 18(1): 16, 2021 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-33407594

RESUMO

BACKGROUND: Microglia are resident immunocompetent and phagocytic cells in the CNS. Pro-inflammatory microglia, stimulated by microbial signals such as bacterial lipopolysaccharide (LPS), viral RNAs, or inflammatory cytokines, are neurotoxic and associated with pathogenesis of several neurodegenerative diseases. Long non-coding RNAs (lncRNA) are emerging as important tissue-specific regulatory molecules directing cell differentiation and functional states and may help direct proinflammatory responses of microglia. Characterization of lncRNAs upregulated in proinflammatory microglia, such as NR_126553 or 2500002B13Rik, now termed Nostrill (iNOS Transcriptional Regulatory Intergenic LncRNA Locus) increases our understanding of molecular mechanisms in CNS innate immunity. METHODS: Microglial gene expression array analyses and qRT-PCR were used to identify a novel long intergenic non-coding RNA, Nostrill, upregulated in LPS-stimulated microglial cell lines, LPS-stimulated primary microglia, and LPS-injected mouse cortical tissue. Silencing and overexpression studies, RNA immunoprecipitation, chromatin immunoprecipitation, chromatin isolation by RNA purification assays, and qRT-PCR were used to study the function of this long non-coding RNA in microglia. In vitro assays were used to examine the effects of silencing the novel long non-coding RNA in LPS-stimulated microglia on neurotoxicity. RESULTS: We report here characterization of intergenic lncRNA, NR_126553, or 2500002B13Rik now termed Nostrill (iNOS Transcriptional Regulatory Intergenic LncRNA Locus). Nostrill is induced by LPS stimulation in BV2 cells, primary murine microglia, and in cortical tissue of LPS-injected mice. Induction of Nostrill is NF-κB dependent and silencing of Nostrill decreased inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production in BV2 and primary microglial cells. Overexpression of Nostrill increased iNOS expression and NO production. RNA immunoprecipitation assays demonstrated that Nostrill is physically associated with NF-κB subunit p65 following LPS stimulation. Silencing of Nostrill significantly reduced NF-κB p65 and RNA polymerase II recruitment to the iNOS promoter and decreased H3K4me3 activating histone modifications at iNOS gene loci. In vitro studies demonstrated that silencing of Nostrill in microglia reduced LPS-stimulated microglial neurotoxicity. CONCLUSIONS: Our data indicate a new regulatory role of the NF-κB-induced Nostrill and suggest that Nostrill acts as a co-activator of transcription of iNOS resulting in the production of nitric oxide by microglia through modulation of epigenetic chromatin remodeling. Nostrill may be a target for reducing the neurotoxicity associated with iNOS-mediated inflammatory processes in microglia during neurodegeneration.


Assuntos
Microglia/metabolismo , Óxido Nítrico Sintase Tipo II/biossíntese , RNA Longo não Codificante/biossíntese , Transcrição Genética/fisiologia , Animais , Linhagem Celular , Células Cultivadas , Feminino , Lipopolissacarídeos/toxicidade , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microglia/efeitos dos fármacos , Microglia/patologia , Síndromes Neurotóxicas/genética , Síndromes Neurotóxicas/metabolismo , Síndromes Neurotóxicas/patologia , Óxido Nítrico Sintase Tipo II/genética , RNA Longo não Codificante/genética , Transcrição Genética/efeitos dos fármacos
7.
Microorganisms ; 9(1)2021 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-33445463

RESUMO

Cryptosporidium is a genus of protozoan parasites that infect the gastrointestinal epithelium of a variety of vertebrate hosts. Intestinal epithelial cells are the first line of defense and play a critical role in orchestrating host immunity against Cryptosporidium infection. To counteract host defense response, Cryptosporidium has developed strategies of immune evasion to promote parasitic replication and survival within epithelial cells, but the underlying mechanisms are largely unclear. Using various models of intestinal cryptosporidiosis, we found that Cryptosporidium infection caused suppression of mitogen-activated protein kinase (MAPK) signaling in infected murine intestinal epithelial cells. Whereas expression levels of most genes encoding the key components of the MAPK signaling pathway were not changed in infected intestinal epithelial cells, we detected a significant downregulation of p38/Mapk, MAP kinase-activated protein kinase 2 (Mk2), and Mk3 genes in infected host cells. Suppression of MAPK signaling was associated with an impaired intestinal epithelial defense against C. parvum infection. Our data suggest that cryptosporidial infection may suppress intestinal epithelial cell MAPK signaling associated with the evasion of host antimicrobial defense.

8.
Oncol Lett ; 20(3): 2191-2198, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32782536

RESUMO

N6-methyladenosine (m6A) RNA modification regulates multiple biological functions. Methyltransferase like 3 (METTL3), one of the major N6-methyltransferases, is highly expressed in gastric cancer, but its potential role in disease is unclear. The current study knocked out METTL3 (METTL3-KO) in human gastric cancer AGS cells using CRISPR/Cas9. METTL3-KO AGS cells exhibited decreased m6A methylation levels. A significant inhibition of cell proliferation was observed in METTL3-KO AGS cells. Silencing METTL3 in AGS cells altered the expression profile of many effector molecules that were previously demonstrated to serve key roles in AGS cell proliferation, including the suppressor of cytokine signaling (SOCS) family of proteins. The results further demonstrated that SOCS2 upregulation in METTL3-KO AGS cells was associated with a decreased RNA decay rate. Furthermore, SOCS2 KO or SOCS2 overexpression caused a significant increase and decrease in AGS cell proliferation, respectively. The current data suggested that METTL3-KO in gastric cancer cells resulted in the suppression of cell proliferation by inducing SOCS2, suggesting a potential role of elevated METTL3 expression in gastric cancer progression.

9.
Methods Mol Biol ; 2052: 205-218, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31452164

RESUMO

MicroRNAs (miRNAs) represent a subclass of endogenous small noncoding RNAs that have been identified in both mammalian and nonmammalian cells. miRNAs are an essential part of the complex regulatory networks that control numerous biological processes and may play an important role in host defense and/or microbial offense during host-parasite interactions. Here, several methodologies to explore the role for miRNAs in host-parasite interactions are briefly summarized, including the detection, quantification, and intracellular localization of miRNAs, identification and validation of miRNA targets, and functional manipulation of specific miRNAs.


Assuntos
Cryptosporidium/genética , Interações Hospedeiro-Parasita/genética , MicroRNAs/genética , Northern Blotting/métodos , Western Blotting/métodos , Linhagem Celular , Cryptosporidium/patogenicidade , Bases de Dados Genéticas , Células Epiteliais/metabolismo , Células Epiteliais/parasitologia , Genes Reporter , Humanos , Hibridização In Situ/métodos , Luciferases/genética , Luciferases/metabolismo , MicroRNAs/isolamento & purificação , MicroRNAs/metabolismo , Interferência de RNA , Reação em Cadeia da Polimerase em Tempo Real/métodos , Fluxo de Trabalho
10.
Eur Arch Otorhinolaryngol ; 276(8): 2283-2287, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31177327

RESUMO

PURPOSE: To assess autonomic nerve function in patients with laryngopharyngeal reflux disease (LPRD) and determine the correlation between LPRD and autonomic nerve dysfunction. METHODS: Patients with suspected LPRD who visited our outpatient department were assessed using the reflux symptom index (RSI) and reflux finding score (RFS) scales. Eighty-one suspected LPRD patients with RSI > 13 and RFS > 7 were examined using 5-min short-range heart rate variability, and all were given proton pump inhibitor diagnostic treatment. RESULTS: The root mean square of successive R-R intervals, high-frequency (HF) power, standardized HF, and HF % were significantly lower in the case group than in the control group (p < 0.05); however, the low frequency (LF)/HF ratio was significantly higher in the case group (p < 0.05). There were no significant differences in the standard deviation of the average normal-to-normal interval, total power, LF power, and LF % between the two groups (p > 0.05). RSI, RFS, and disease duration were negatively correlated with HF power (r = - 0.89, -0.77, and -0.315, respectively; p < 0.05). The LF/HF ratio and disease duration were positively correlated (r = 0.315, p < 0.05). CONCLUSIONS: Autonomic nerve dysfunction was observed in our patients with LPRD. LPRD severity was significantly correlated with autonomic nerve dysfunction and negatively correlated with vagal nerve function.


Assuntos
Doenças do Sistema Nervoso Autônomo/diagnóstico , Doenças do Sistema Nervoso Autônomo/etiologia , Vias Autônomas/fisiopatologia , Refluxo Laringofaríngeo/complicações , Adulto , Idoso , Feminino , Frequência Cardíaca , Humanos , Refluxo Laringofaríngeo/diagnóstico , Refluxo Laringofaríngeo/tratamento farmacológico , Masculino , Pessoa de Meia-Idade , Inibidores da Bomba de Prótons/uso terapêutico
11.
Infect Immun ; 87(4)2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30642905

RESUMO

Cryptosporidium, a protozoan parasite that infects the gastrointestinal epithelium and other mucosal surfaces in humans and animals, is an important opportunistic pathogen in AIDS patients and one of the most common enteric pathogens affecting young children in developing regions. This parasite is referred to as a "minimally invasive" mucosal pathogen, and epithelial cells play a central role in activating and orchestrating host immune responses. We previously demonstrated that Cryptosporidium parvum infection stimulates host epithelial cells to release exosomes, and these released exosomes shuttle several antimicrobial peptides to carry out anti-C. parvum activity. In this study, we detected the upregulation of inflammatory genes in the liver and spleen following C. parvum intestinal infection in neonatal mice. Interestingly, exosomes released from intestinal epithelial cells following C. parvum infection could activate the nuclear factor kappa B signaling pathway and trigger inflammatory gene transcription in isolated primary splenocytes. Several epithelial cell-derived proteins and a subset of parasite RNAs were detected in the exosomes released from C. parvum-infected intestinal epithelial cells. Shuttling of these effector molecules, including the high mobility group box 1 protein, was involved in the induction of inflammatory responses in splenocytes induced by the exosomes released from infected cells. Our data indicate that exosomes released from intestinal epithelial cells upon C. parvum infection can activate immune cells by shuttling various effector molecules, a process that may be relevant to host systemic responses to Cryptosporidium infection.


Assuntos
Criptosporidiose/imunologia , Criptosporidiose/parasitologia , Cryptosporidium parvum/fisiologia , Células Epiteliais/imunologia , Exossomos/imunologia , Intestinos/imunologia , Baço/citologia , Animais , Criptosporidiose/genética , Células Epiteliais/parasitologia , Exossomos/genética , Feminino , Proteínas de Grupo de Alta Mobilidade/genética , Proteínas de Grupo de Alta Mobilidade/imunologia , Humanos , Intestinos/parasitologia , Fígado/imunologia , Fígado/parasitologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/genética , NF-kappa B/imunologia , Baço/imunologia , Baço/parasitologia
12.
J Immunol ; 201(12): 3630-3640, 2018 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-30446564

RESUMO

Cryptosporidium is an important opportunistic intestinal pathogen for immunocompromised individuals and a common cause of diarrhea in young children in developing countries. Gastrointestinal epithelial cells play a central role in activating and orchestrating host immune responses against Cryptosporidium infection, but underlying molecular mechanisms are not fully understood. We report in this paper that C. parvum infection causes significant alterations in long noncoding RNA (lncRNA) expression profiles in murine intestinal epithelial cells. Transcription of a panel of lncRNA genes, including NR_045064, in infected cells is controlled by the NF-κB signaling. Functionally, inhibition of NR_045064 induction increases parasite burden in intestinal epithelial cells. Induction of NR_045064 enhances the transcription of selected defense genes in host cells following C. parvum infection. Epigenetic histone modifications are involved in NR_045064-mediated transcription of associated defense genes in infected host cells. Moreover, the p300/MLL-associated chromatin remodeling is involved in NR_045064-mediated transcription of associated defense genes in intestinal epithelial cells following C. parvum infection. Expression of NR_045064 and associated genes is also identified in intestinal epithelium in C57BL/6J mice following phosphorothioate oligodeoxynucleotide or LPS stimulation. Our data demonstrate that lncRNAs, such as NR_045064, play a role in regulating epithelial defense against microbial infection.


Assuntos
Criptosporidiose/genética , Cryptosporidium parvum/fisiologia , Mucosa Intestinal/fisiologia , RNA Longo não Codificante/genética , Animais , Anti-Infecciosos , Linhagem Celular , Criptosporidiose/imunologia , Modelos Animais de Doenças , Regulação da Expressão Gênica , Humanos , Imunidade/genética , Lipopolissacarídeos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo
13.
Mol Ther Nucleic Acids ; 13: 450-463, 2018 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-30388619

RESUMO

Impairment of microglial functions, such as phagocytosis and/or dysregulation of immune responses, has been implicated as an underlying factor involved in the pathogenesis of various neurodegenerative disorders. Our previous studies have demonstrated that long intergenic noncoding RNA (lincRNA)-Cox2 expression is influenced by nuclear factor κB (NF-κB) signaling and serves as a coactivator of transcriptional factors to regulate the expression of a vast array of immune-related genes in microglia. Extracellular vesicles (EVs) have been recognized as primary facilitators of cell-to-cell communication and cellular regulation. Herein, we show that EVs derived from astrocytes exposed to morphine can be taken up by microglial endosomes, leading, in turn, to activation of Toll-like receptor 7 (TLR7) with a subsequent upregulation of lincRNA-Cox2 expression, ultimately resulting in impaired microglial phagocytosis. This was further validated in vivo, wherein inhibition of microglial phagocytic activity was also observed in brain slices isolated from morphine-administrated mice compared with control mice. Additionally, we also showed that intranasal delivery of EVs containing lincRNA-Cox2 siRNA (small interfering RNA) was able to restore microglial phagocytic activity in mice administered morphine. These findings have ramifications for the development of EV-loaded RNA-based therapeutics for the treatment of various disorders involving functional impairment of microglia.

14.
J Infect Dis ; 218(8): 1336-1347, 2018 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-30052999

RESUMO

Intestinal infection by Cryptosporidium is known to cause epithelial cell migration disorder but the underlying mechanisms are unclear. Previous studies demonstrated that a panel of parasite RNA transcripts of low protein-coding potential are delivered into infected epithelial cells. Using multiple models of intestinal cryptosporidiosis, we report here that C. parvum infection induces expression and release of the dickkopf protein 1 (Dkk1) from intestinal epithelial cells. Delivery of parasite Cdg7_FLc_1030 RNA to intestinal epithelial cells triggers transactivation of host Dkk1 gene during C. parvum infection. Release of Dkk1 is involved in C. parvum-induced inhibition of cell migration of epithelial cells, including noninfected bystander cells. Moreover, Dkk1-mediated suppression of host cell migration during C. parvum infection involves inhibition of Cdc42/Par6 signaling. Our data support the hypothesis that attenuation of intestinal epithelial cell migration during Cryptosporidium infection involves parasite Cdg7_FLc_1030 RNA-mediated induction and release of Dkk1 from infected cells.


Assuntos
Cryptosporidium parvum/metabolismo , Células Epiteliais/parasitologia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Mucosa Intestinal/citologia , RNA de Protozoário/farmacologia , Animais , Linhagem Celular , Cryptosporidium parvum/genética , Células Epiteliais/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/imunologia , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Camundongos , Ativação Transcricional
15.
Cytokine ; 110: 29-38, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-29698843

RESUMO

Creatinine is the breakdown product of creatine, a key participant in the generation of ATP and is traditionally considered to be a biologically inert waste product. Based on our earlier work, we analyzed the effects of creatinine hydrochloride on the expression of tumor necrosis factor-alpha (TNF-α), a pro-inflammatory cytokine, in a human T cell line, as well as human and mouse macrophage cell lines. Exposing cells to creatinine hydrochloride significantly reduced TNF-α mRNA and protein levels compared to control-treated cultures in all cell lines tested. Lipopolysaccharide (LPS), a potent inducer of inflammation, was employed with in mouse macrophage cell lines to induce high levels of TNF-α in order to determine whether creatinine hydrochloride could reduce preexisting inflammation. Cells treated with LPS and creatinine hydrochloride had significantly reduced TNF-α levels compared to cells treated with LPS alone. As the NF-κB signaling pathway represents a major mechanism of TNF-α generation, nuclear extracts were examined for NF-κB pathway activation. Cells exposed to CRN had significantly lower levels of NF-κB in the nucleus compared to control-treated cells. Together, these results support the hypothesis that CRN can alter anti-inflammatory responses by interfering with the activation of the NF-κB pathway.


Assuntos
Creatinina/metabolismo , Regulação para Baixo/fisiologia , Macrófagos/metabolismo , Linfócitos T/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Linhagem Celular , Linhagem Celular Tumoral , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Citocinas/metabolismo , Regulação para Baixo/efeitos dos fármacos , Humanos , Inflamação/induzido quimicamente , Inflamação/metabolismo , Células Jurkat , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Camundongos , NF-kappa B/metabolismo , Células RAW 264.7 , RNA Mensageiro/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Linfócitos T/efeitos dos fármacos , Células THP-1/efeitos dos fármacos , Células THP-1/metabolismo
16.
Vet Parasitol ; 251: 27-33, 2018 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-29426472

RESUMO

Intestinal infection by the zoonotic protozoan, Cryptosporidium parvum, causes significant alterations in the gene expression profile in host epithelial cells. The molecular mechanisms of how C. parvum may modulate host cell gene transcription and the pathological significance of such alterations are largely unclear. Previous studies demonstrate that a panel of parasite RNA transcripts are delivered into infected host cells and may modulate host gene transcription. Using in vitro models of intestinal cryptosporidiosis, in this study, we analyzed the impact of host delivery of C. parvum Cdg2_FLc_0220 RNA transcript on host gene expression profile. We found that alterations in host gene expression profile following C. parvum infection were partially associated with the nuclear delivery of Cdg2_FLc_0220. Specifically, we identified a total of 46 overlapping upregulated genes and 8 overlapping downregulated genes in infected cells and cells transfected with Full-Cdg2_FLc_0220. Trans-suppression of the DAZ interacting zinc finger protein 1 like (DZIP1L) gene, the top overlapping downregulated gene in host cells following C. parvum infection and cells transfected with Full-Cdg2_FLc_0220, was mediated by G9a, independent of PRDM1. Cdg2_FLc_0220-mediated trans-suppression of the DZIP1L gene was independent of H3K9 and H3K27 methylation. Data from this study provide additional evidence that delivery of C. parvum Cdg2_FLc_0220 RNA transcript in infected epithelial cells modulates the transcription of host genes, contributing to the alterations in the gene expression profile in host epithelial cells during C. parvum infection.


Assuntos
Cryptosporidium parvum/genética , Interações Hospedeiro-Patógeno/genética , Mucosa Intestinal/parasitologia , RNA de Protozoário/genética , Transcrição Genética , Animais , Linhagem Celular Tumoral , Criptosporidiose/parasitologia , Cryptosporidium parvum/fisiologia , Regulação da Expressão Gênica , Humanos , Fator 1 de Ligação ao Domínio I Regulador Positivo/genética , Transcriptoma , Regulação para Cima
17.
Int J Parasitol ; 48(6): 423-431, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29438669

RESUMO

Intestinal infection by Cryptosporidium parvum causes significant alterations in the gene expression profile in host epithelial cells. Previous studies demonstrate that a panel of parasite RNA transcripts of low protein-coding potential are delivered into infected host cells and may modulate host gene transcription. Using in vitro models of human intestinal cryptosporidiosis, we report here that trans-suppression of the cadherin 3 (CDH3) and lysyl oxidase like 4 (LOXL4) genes in human intestinal epithelial cells following C. parvum infection involves host delivery of the Cdg7_FLc_1000 RNA, a C. parvum RNA that has been previously demonstrated to be delivered into the nuclei of infected host cells. Downregulation of CDH3 and LOXL4 genes was detected in host epithelial cells following C. parvum infection or in cells expressing the parasite Cdg7_FLc_1000 RNA. Knockdown of Cdg7_FLc_1000 attenuated the trans-suppression of CDH3 and LOXL4 genes in host cells induced by infection. Interestingly, Cdg7_FLc_1000 was detected to be recruited to the promoter regions of both CDH3 and LOXL4 gene loci in host cells following C. parvum infection. Host delivery of Cdg7_FLc_1000 promoted the PH domain zinc finger protein 1 (PRDM1)-mediated H3K9 methylation associated with trans-suppression in the CDH3 gene locus, but not the LOXL4 gene. Therefore, our data suggest that host delivery of Cdg7_FLc_1000 causes CDH3 trans-suppression in human intestinal epithelial cells following C. parvum infection through PRDM1-mediated H3K9 methylation in the CDH3 gene locus, whereas Cdg7_FLc_1000 induces trans-suppression of the host LOXL4 gene through H3K9/H3K27 methylation-independent mechanisms.


Assuntos
Transporte Ativo do Núcleo Celular/fisiologia , Aminoácido Oxirredutases/metabolismo , Caderinas/metabolismo , Cryptosporidium parvum/fisiologia , Proteínas de Protozoários/farmacologia , RNA de Protozoário/metabolismo , Aminoácido Oxirredutases/genética , Caderinas/genética , Linhagem Celular , Cryptosporidium parvum/genética , Regulação para Baixo , Células Epiteliais/metabolismo , Células Epiteliais/parasitologia , Técnicas de Silenciamento de Genes , Inativação Gênica , Humanos , Regiões Promotoras Genéticas , Processamento de Proteína Pós-Traducional , Proteína-Lisina 6-Oxidase , Proteínas de Protozoários/metabolismo
18.
Parasitol Res ; 117(3): 831-840, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29374323

RESUMO

To counteract host immunity, Cryptosporidium parvum has evolved multiple strategies to suppress host antimicrobial defense. One such strategy is to reduce the production of the antimicrobial peptide beta-defensin 1 (DEFB1) by host epithelial cells but the underlying mechanisms remain unclear. Recent studies demonstrate that a panel of parasite RNA transcripts of low protein-coding potential are delivered into infected host cells and may modulate host gene transcription. Using in vitro models of intestinal cryptosporidiosis, in this study, we analyzed the expression profile of host beta-defensin genes in host cells following infection. We found that C. parvum infection caused a significant downregulation of the DEFB1 gene. Interestingly, downregulation of DEFB1 gene was associated with host delivery of Cdg7_FLc_1000 RNA transcript, a C. parvum RNA that has previously demonstrated to be delivered into the nuclei of infected host cells. Knockdown of Cdg7_FLc_1000 in host cells could attenuate the trans-suppression of host DEFB1 gene and decreased the parasite burden. Therefore, our data suggest that trans-suppression of DEFB1 gene in intestinal epithelial cells following C. parvum infection involves host delivery of parasite Cdg7_FLc_1000 RNA, a process that may be relevant to the epithelial defense evasion by C. parvum at the early stage of infection.


Assuntos
Criptosporidiose/genética , Cryptosporidium parvum , Mucosa Intestinal/parasitologia , RNA de Protozoário/fisiologia , beta-Defensinas/genética , Animais , Linhagem Celular , Criptosporidiose/imunologia , Criptosporidiose/parasitologia , Cryptosporidium parvum/genética , Regulação da Expressão Gênica , Humanos , Mucosa Intestinal/metabolismo , Camundongos
19.
J Immunol ; 199(10): 3571-3582, 2017 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-28993514

RESUMO

Long noncoding RNAs, a newly identified class of noncoding RNAs, are important regulators of gene expression in innate immunity. We report in this study that the transcription of FIRRE, a conserved long noncoding RNA between humans and mice, is controlled by NF-κB signaling in macrophages and intestinal epithelial cells. Functionally, FIRRE appears to positively regulate the expression of several inflammatory genes in macrophages or intestinal epithelial cells in response to LPS stimulation via posttranscriptional mechanisms. Specifically, FIRRE physically interacts with heterogeneous nuclear ribonucleoproteins U, regulating the stability of mRNAs of selected inflammatory genes through targeting the AU-rich elements of their mRNAs in cells following LPS stimulation. Therefore, our data indicate a new regulatory role for NF-κB-responsive FIRRE in the posttranscriptional regulation of inflammatory genes in the innate immune system.


Assuntos
Sequência Conservada/genética , Inflamação/genética , Mucosa Intestinal/imunologia , Macrófagos/imunologia , NF-kappa B/metabolismo , RNA Longo não Codificante/genética , Elementos de Resposta/genética , Animais , Regulação da Expressão Gênica , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Humanos , Imunidade Inata , Camundongos , RNA Longo não Codificante/metabolismo , RNA Interferente Pequeno/genética , Células U937
20.
J Infect Dis ; 217(1): 122-133, 2017 12 27.
Artigo em Inglês | MEDLINE | ID: mdl-28961856

RESUMO

Intestinal infection by Cryptosporidium parvum causes inhibition of epithelial turnover, but underlying mechanisms are unclear. Previous studies demonstrate that a panel of parasite RNA transcripts of low protein-coding potential are delivered into infected epithelial cells. Using in vitro and in vivo models of intestinal cryptosporidiosis, we report here that host delivery of parasite Cdg7_FLc_1000 RNA results in inhibition of epithelial cell migration through suppression of the gene encoding sphingomyelinase 3 (SMPD3). Delivery of Cdg7_FLc_1000 into infected cells promotes the histone methyltransferase G9a-mediated H3K9 methylation in the SMPD3 locus. The DNA-binding transcriptional repressor, PR domain zinc finger protein 1, is required for the assembly of Cdg7_FLc_1000 into the G9a complex and associated with the enrichment of H3K9 methylation at the gene locus. Pathologically, nuclear transfer of Cryptosporidium parvum Cdg7_FLc_1000 RNA is involved in the attenuation of intestinal epithelial cell migration via trans-suppression of host cell SMPD3.


Assuntos
Movimento Celular , Criptosporidiose/patologia , Cryptosporidium parvum/patogenicidade , Regulação para Baixo , Células Epiteliais/fisiologia , RNA de Protozoário/metabolismo , Esfingomielina Fosfodiesterase/biossíntese , Animais , Linhagem Celular , Modelos Animais de Doenças , Epigênese Genética , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Humanos , Enteropatias/patologia , Metilação , Camundongos , Processamento de Proteína Pós-Traducional
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