RESUMO
BACKGROUND: To investigate the involvement of LINC02605 in the progression of paediatric Mycoplasma pneumoniae pneumonia (MPP). METHODS: One hundred and thirty-two children with MPP (90 simple MPP and 42 MPP + diarrhoea) were enrolled, and their plasma was collected for detection of LINC026505 expression. CCK-8 kit and commercial apoptosis kit were introduced to determine cell growth and apoptosis. In silico prediction analyses were conducted to predict the downstream miRNA for LINC02605, following verification by dual luciferase reporter assay. The lipid-associated membrane proteins (LAMPs) were used to treat A549 and Coca-2 cells. RESULTS: LIN02605 was highly expressed in the MPP, especially in MPP complicated with diarrhoea. LINC02605 downregulation in A549 cells correlated with significant suppression of cell apoptosis rate and growth inhibition rate in vitro. Introduction of miR-539-5p inhibited luciferase activity in a reporter system containing the wild-type LINC02605 and CXCL1. After stimulation with LAMPs, overexpression of LINC02605 and CXCL1 and inhibition of miR-539-5p were found. miR-539-5p and CXCL1 knockdown resulted in a rescue effect on the LINC02605-inhibited cell apoptosis. LAMPs induced IL-1ß in intestinal epithelial cells and IL-1ß induced LINC02605 expression in A549 cells. CONCLUSIONS: LINC02605 was upregulated in MPP and miR-539-5p was a target for LINC02605. LINC02605 may be involved in the crosstalk between the gastrointestinal tract and the respiratory tract.
Assuntos
Apoptose , Quimiocina CXCL1 , Diarreia , MicroRNAs , Pneumonia por Mycoplasma , RNA Longo não Codificante , Humanos , MicroRNAs/metabolismo , MicroRNAs/genética , Pneumonia por Mycoplasma/genética , Apoptose/genética , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Masculino , Diarreia/genética , Feminino , Quimiocina CXCL1/metabolismo , Quimiocina CXCL1/genética , Células A549 , Pré-Escolar , Criança , Regulação para BaixoRESUMO
Mycoplasma pneumoniae pneumonia (MPP) represents a common respiratory disease in children patients. Kukoamine A (KuA) is a spermine alkaloid found in the Chinese herb Cortex Lycii radices, which has a variety of pharmacological properties. However, no study has been reported on the role of KuA in MPP. Exosomes, a type of lipid bilayer-enclosed extracellular vesicles, can be delivered to the target cells, where they regulate function and physiology. With the use of human alveolar basal epithelial cells (HABECs) as an in vitro model, in this study, we sought to characterize the changes in levels of superoxide dismutase 2 (SOD2) and proinflammatory cytokines including IL-6 and TNF-α in HABECs in response to exosomes, which were isolated from peripheral blood serum of MPP patients. We found that, compared to normal, MPP patients exhibited a significant up-regulated miR-222-3p. Further, exosomal miR-222-3p downregulated SOD2 activity but promoted nuclear NF-κB activity and expression of IL-6 and TNF-α in HABECs, ultimately leading to an oxidative stress condition. Interestingly, such stimulating effects were attenuated by the pretreatment of KuA. This study suggests a critical role possessed by KuA in MPP by regulating the miR-222-3p/SOD2 axis, which represents a promising strategy for the treatment of MPP.
Assuntos
MicroRNAs , Mycoplasma pneumoniae , Pneumonia por Mycoplasma , Espermina , Criança , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Mycoplasma pneumoniae/genética , Pneumonia por Mycoplasma/tratamento farmacológico , Pneumonia por Mycoplasma/genética , Pneumonia por Mycoplasma/metabolismo , Espermina/análogos & derivados , Espermina/farmacologia , Superóxido Dismutase , Fator de Necrose Tumoral alfa/metabolismoRESUMO
OBJECTIVE: This study aimed to investigate the role of p50-associated cyclooxygenase- (COX-2) extragenic RNA (PACER) on the inflammation of airway epithelium caused by Mycoplasma pneumoniae (MP) infection. METHODS: A549 cells and MP strain were cultured respectively. The expressions of PACER, IL-8, TNF-α and COX-2 in MP-infected cells were detected by qRT-PCR, the concentration of IL-8 and TNF-α in the supernatant of the cells were detected by ELISA, and the expression of COX-2 protein in the cells was detected by western-blot. After knockdown of PACER, the expression of IL-8, TNF-α and COX-2 in MP infected cells were observed. The activity of NF-κB in cells was detected by fluorescence reporter assay, and the interaction between PACER and NF-κB was verified by RNA immunoprecipitation. RESULTS: First, we observed that PACER was upregulated in MP infected A549 cells. Knockdown of PACER suppressed the production of inflammatory cytokines as well as the expression of COX-2 in A549 cells after MP infection. By performing luciferase reporter assay, we found PACER knockdown inhibited NF-κB activation induced by MP. Furthermore, RNA immunoprecipitation showed that PACER could physically bind to NF-κB p50 in MP-treated A549 cells. CONCLUSION: Collectively, our data demonstrated that attenuation of PACER reduces the inflammatory response of MP-infected epithelial cells via regulating NF-κB.
Assuntos
Mycoplasma pneumoniae , NF-kappa B , Pneumonia por Mycoplasma , RNA Longo não Codificante , Células A549 , Humanos , Inflamação/genética , Inflamação/metabolismo , Inflamação/microbiologia , NF-kappa B/metabolismo , Pneumonia por Mycoplasma/genética , Pneumonia por Mycoplasma/metabolismo , Pneumonia por Mycoplasma/microbiologia , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Pneumonia caused by Mycoplasma pneumoniae (M. pneumoniae) is a major cause of communityacquired pneumonia in children. In some cases, M. pneumoniae pneumonia (MPP) can develop into refractory MPP (RMPP), which shows no clinical or radiological response to macrolides, and can progress to severe and complicated pneumonia. However, the pathogenesis of RMPP remains poorly understood. The present study aimed to identify target genes that could be used as biomarkers for the clinical diagnosis of earlystage RMPP through highthroughput sequencing technology. The differences in long noncoding (lnc)RNAs, mRNAs and circular (circ)RNAs were examined between wholeblood samples from two patients with nonrefractory MPP (NRMPP), two patients with RMPP and three healthy children using ribosomal (r)RNAdepleted RNAsequencing techniques and an integrated mRNA/circRNA analysis. A total of 17 lncRNAs (four upregulated and 13 downregulated), 18 mRNAs (six upregulated and 12 downregulated) and 24 circRNAs (12 upregulated and 12 downregulated) were the most significantly differentially expressed (P<0.05) between the NRMPP and RMPP groups. Upon functional analysis, the significantly differentially expressed genes encoded by the targeting mRNAs (prostaglandinendoperoxide synthase 2, IL8 and foslike antigen 1) were screened and identified to be enriched in the 'IL17 signaling pathway'. Furthermore, the key circRNAs in the NRMPP and RMPP comparative groups were primarily enriched in 'herpes simplex virus 1 infection', 'viral carcinogenesis' and 'RNA transport'. In the present study, a comprehensive analysis of the differences between the NRMPP and RMPP cases was performed based on rRNAdepleted RNAsequencing techniques, and the selected genes and circRNAs may be closely associated with the complex pathogenesis of RMPP.
Assuntos
Antibacterianos/farmacologia , Infecções Comunitárias Adquiridas/tratamento farmacológico , Farmacorresistência Bacteriana/genética , Mycoplasma pneumoniae/patogenicidade , Pneumonia por Mycoplasma/tratamento farmacológico , Antibacterianos/uso terapêutico , Biomarcadores/análise , Criança , Pré-Escolar , Infecções Comunitárias Adquiridas/genética , Infecções Comunitárias Adquiridas/imunologia , Infecções Comunitárias Adquiridas/microbiologia , Feminino , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Humanos , Lactente , Macrolídeos/farmacologia , Macrolídeos/uso terapêutico , Masculino , Pneumonia por Mycoplasma/genética , Pneumonia por Mycoplasma/imunologia , Pneumonia por Mycoplasma/microbiologia , RNA Circular/análise , RNA Longo não Codificante/análise , RNA Mensageiro/análise , RNA Ribossômico , Análise de Sequência de RNA/métodosRESUMO
The secretory function of airway epithelial cells is important in the pathogenesis of Mycoplasma pneumoniae pneumonia (MPP). To investigate the regulatory function of NKILA (nuclear factor-κB (NF-κB) interacting long noncoding RNA (lncRNA)) in MPP, we first detected NKILA as well as the concentration of interleukin 8 (IL-8) and tumor necrosis factor-α (TNF-α) in bronchoalveolar lavage fluid of children with MPP. Then, NKILA was knocked down in epithelial cells to investigate its effect on their secretory function. The results suggested that NKILA was downregulated in children with MPP, while IL-8 and TNF-α levels increased. Knockdown of NKILA in vitro promoted the inflammatory effects of Mycoplasma pneumoniae (MP) in epithelial A549 and BEAS-2B cells. Knockdown of NKILA promoted inhibitor of κBα (IκBα) phosphorylation and degradation, and NF-κB p65 nuclear translocation. Furthermore, RNA immunoprecipitation showed that NKILA could physically bind to IκBα in MP-treated A549 cells. Collectively, our data demonstrated that attenuation of NKILA enhances the effects of MP-stimulated secretory functions of epithelial cells via regulation of NF-κB signaling.
Assuntos
Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Pulmão/patologia , Mycoplasma pneumoniae/fisiologia , NF-kappa B/metabolismo , RNA Longo não Codificante/metabolismo , Células A549 , Líquido da Lavagem Broncoalveolar , Pré-Escolar , Citocinas/genética , Citocinas/metabolismo , Regulação para Baixo/genética , Células Epiteliais/patologia , Feminino , Humanos , Mediadores da Inflamação/metabolismo , Masculino , Inibidor de NF-kappaB alfa/metabolismo , Fosforilação , Pneumonia por Mycoplasma/genética , Pneumonia por Mycoplasma/microbiologia , Ligação Proteica , Transporte Proteico , RNA Longo não Codificante/genética , Fator de Transcrição RelA/metabolismoRESUMO
Mycoplasma pneumoniae pneumonia (MPP) is a type of pneumonia induced by M. pneumoniae (MP) infection. The present study investigated the effect of long noncoding RNA growth arrestspecific 5 (GAS5) in MPP and the underlying molecular mechanism of this. The expression of GAS5, microRNA2223p, (miR2223p) and tissue inhibitor of metalloproteinases3 (TIMP3) in MPP was investigated using reverse transcriptionquantitative PCR. Lipidassociated membrane protein (LAMP)induced THP1 cells were used to model MPP. The viability of LAMPinduced THP1 cells was analyzed using an MTT assay. Expression levels of interleukin (IL)1ß, IL6 and tumor necrosis factorα (TNFα) proinflammatory cytokines, and the antiinflammatory cytokine heme oxygenase1 (HO1) in LAMPinduced THP1 cells were measured by ELISA. A dualluciferase reporter assay assessed the associations among GAS5, miR2223p and TIMP3. The expression of GAS5 and TIMP3 was downregulated in MPP. Expression of miR2223p was upregulated. GAS5overexpression increased the viability of LAMPinduced THP1 cells. GAS5 upregulation decreased the levels of IL1ß, IL6, TNFα and HO1 levels in LAMPinduced THP1 cells. GAS5 directly interacted with miR2223p. TIMP3 was a target of miR2223p. miR2223p upregulation or TIMP3knockdown reversed the promotion effect on cell viability as well as the inhibitory effect on inflammation caused by GAS5overexpression in LAMPinduced THP1 cells. GAS5overexpression increased the viability and decreased the inflammation of LAMPinduced THP1 cells by regulating the miR2223p/TIMP3 axis. These results demonstrated a potential therapeutic target for MPP treatment.
Assuntos
Inflamação/genética , MicroRNAs/genética , Pneumonia por Mycoplasma/genética , RNA Longo não Codificante/genética , Inibidor Tecidual de Metaloproteinase-3/genética , Apoptose/genética , Citocinas/genética , Regulação da Expressão Gênica/genética , Humanos , Inflamação/microbiologia , Inflamação/patologia , Mycoplasma pneumoniae/genética , Mycoplasma pneumoniae/patogenicidade , Pneumonia por Mycoplasma/microbiologia , Pneumonia por Mycoplasma/patologiaRESUMO
Herein, we found that serum concentration of superoxide dismutase 3 (SOD3) was significantly reduced in children with mycoplasma pneumonia (MP) infection. To study the roles of SOD3 in inflammatory regulation of MP infection, human A549 type II alveolar epithelial cells were stimulated with 107 CCU/ml of MP to build MP infection in vitro. Secretion of pro-inflammatory cytokine interleukin (IL)-8 and tumor necrosis factor (TNF)-α were measured via enzyme-linked immunosorbent assay (ELISA) to assess the inflammatory response of A549 cells. Levofloxacin (LVFX) was used as an anti-inflammatory drug while recombinant TNF-α was used as an inflammatory promotor in MP-infected cells. Transcriptional activity of nuclear factor (NF)-κB was assessed by detecting protein levels of nuclear NF-κB and cytoplasm NF-κB using Western blot analysis. Our data suggested that the expression of SOD3 mRNA and protein, as well as content of SOD3 in cultured supernatant, were time-dependently inhibited in MP-infected A549 cells. However, lentiviruses-mediated SOD3 overexpression alleviated inflammatory response of MP-infected A549 cells, and prevented the unclear translocation of NF-κB, as evidenced by obviously reducing the production of IL-8 and TNF-α in cell cultured supernatant, as well as decreasing nuclear NF-κB while increasing cytoplasm NF-κB. Inspiringly, SOD3 overexpression induced anti-inflammatory effect and the inactivation of NF-κB was similar to that of 2 lg/ml of LVFX, but reversed by additional TNF-α treatment. Therefore, we can conclude that transcriptional activity of NF-jB was the underlying mechanism, by which SOD3 regulated inflammatory response in MP infection in vitro.
Assuntos
Inflamação/genética , Interleucina-8/genética , Pneumonia por Mycoplasma/genética , Superóxido Dismutase/genética , Fator de Necrose Tumoral alfa/genética , Células A549 , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/genética , Criança , Humanos , Inflamação/tratamento farmacológico , Inflamação/microbiologia , Levofloxacino/farmacologia , Lipopolissacarídeos/farmacologia , Mycoplasma pneumoniae/patogenicidade , Pneumonia por Mycoplasma/microbiologia , Pneumonia por Mycoplasma/patologia , RNA Mensageiro/genéticaRESUMO
Human surfactant protein-A2 (hSP-A2) is a component of pulmonary surfactant that plays an important role in the lung's immune system by interacting with viruses, bacteria, and fungi to facilitate pathogen clearance and by downregulating inflammatory responses after an allergic challenge. Genetic variation in SP-A2 at position Gln223Lys is present in up to â¼30% of the population and has been associated with several lung diseases, such as asthma, pulmonary fibrosis, and lung cancer (M. M. Pettigrew, J. F. Gent, Y. Zhu, E. W. Triche, et al., BMC Med Genet 8:15, 2007, https://bmcmedgenet.biomedcentral.com/articles/10.1186/1471-2350-8-15; Y. Wang, P. J. Kuan, C. Zing, J. T. Cronkhite, et al., Am J Hum Genet 84:52-59, 2009, https://www.cell.com/ajhg/fulltext/S0002-9297(08)00595-8). Previous work performed by our group showed differences in levels of SP-A binding to non-live mycoplasma membrane fractions that were dependent on the presence of a lysine (K) or a glutamine (Q) at amino acid position 223 in the carbohydrate region of SP-A2. On the basis of these differences, we have derived 20-amino-acid peptides flanking this region of interest in order to test the ability of each to regulate various immune responses to live Mycoplasma pneumoniae in SP-A knockout mice and RAW 264.7 cells. In both models, the 20-mer containing 223Q significantly decreased both tumor necrosis factor alpha (TNF-α) mRNA levels and protein levels in comparison to the 20-mer containing 223K during M. pneumoniae infection. While neither of the 20-mer peptides (223Q and 223K) had an effect on p38 phosphorylation during M. pneumoniae infection, the 223Q-20mer peptide significantly reduced NF-κB p65 phosphorylation in both models. Taken together, our data suggest that small peptides derived from the lectin domain of SP-A2 that contain the major allelic variant (223Q) maintain activity in reducing TNF-α induction during M. pneumoniae infection.
Assuntos
Anti-Inflamatórios/farmacologia , Interações entre Hospedeiro e Microrganismos/imunologia , Mycoplasma pneumoniae/imunologia , Peptídeos/farmacologia , Pneumonia por Mycoplasma/tratamento farmacológico , Proteína A Associada a Surfactante Pulmonar/imunologia , Fator de Necrose Tumoral alfa/imunologia , Adesinas Bacterianas/genética , Adesinas Bacterianas/imunologia , Animais , Anti-Inflamatórios/síntese química , Modelos Animais de Doenças , Regulação da Expressão Gênica , Interações entre Hospedeiro e Microrganismos/genética , Humanos , Pulmão/efeitos dos fármacos , Pulmão/imunologia , Pulmão/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mycoplasma pneumoniae/efeitos dos fármacos , Mycoplasma pneumoniae/patogenicidade , Neutrófilos/efeitos dos fármacos , Neutrófilos/imunologia , Neutrófilos/microbiologia , Peptídeos/síntese química , Pneumonia por Mycoplasma/genética , Pneumonia por Mycoplasma/imunologia , Pneumonia por Mycoplasma/microbiologia , Domínios Proteicos , Proteína A Associada a Surfactante Pulmonar/química , Proteína A Associada a Surfactante Pulmonar/deficiência , Proteína A Associada a Surfactante Pulmonar/genética , Células RAW 264.7 , RNA Mensageiro/genética , RNA Mensageiro/imunologia , Transdução de Sinais , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/imunologia , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/genética , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/imunologiaRESUMO
Interleukin 1 receptor-like 1 (IL1RL1), also known as suppression of tumorigenicity 2 (ST2), is the receptor for interleukin 33 (IL-33) and has been increasingly studied in type 2 inflammation. An increase in airway IL-33/ST2 signaling in asthma has been associated with eosinophilic inflammation, but little is known about the role of ST2 in neutrophilic inflammation. Airway Mycoplasma pneumoniae and human rhinovirus (HRV) infections are linked to neutrophilic inflammation during acute exacerbations of asthma. However, whether ST2 contributes to M. pneumoniae- and HRV-mediated airway inflammation is poorly understood. The current study sought to determine the functions of ST2 during airway M. pneumoniae or HRV infection. In cultured normal human primary airway epithelial cells, ST2 overexpression (OE) increased the production of neutrophilic chemoattractant IL-8 in the absence or presence of M. pneumoniae or HRV1B infection. ST2 OE also enhanced HRV1B-induced IP-10, a chemokine involved in asthma exacerbations. In the M. pneumoniae-infected mouse model, ST2 deficiency, in contrast to sufficiency, significantly reduced the levels of neutrophils following acute (≤24 h) infection, while in the HRV1B-infected mouse model, ST2 deficiency significantly reduced the levels of proinflammatory cytokines KC, IP-10, and IL-33 in bronchoalveolar lavage (BAL) fluid. Overall, ST2 overexpression in human epithelial cells and ST2 sufficiency in mice increased the M. pneumoniae and HRV loads in cell supernatants and BAL fluid. After pathogen infection, ST2-deficient mice showed a higher level of the host defense protein lactotransferrin in BAL fluid. Our data suggest that ST2 promotes proinflammatory responses (e.g., neutrophils) to airway bacterial and viral infection and that blocking ST2 signaling may broadly attenuate airway infection and inflammation.
Assuntos
Infecções por Enterovirus/imunologia , Enterovirus/fisiologia , Proteína 1 Semelhante a Receptor de Interleucina-1/imunologia , Mycoplasma pneumoniae/fisiologia , Pneumonia por Mycoplasma/microbiologia , Sistema Respiratório/imunologia , Animais , Líquido da Lavagem Broncoalveolar/imunologia , Líquido da Lavagem Broncoalveolar/microbiologia , Líquido da Lavagem Broncoalveolar/virologia , Enterovirus/genética , Infecções por Enterovirus/genética , Infecções por Enterovirus/virologia , Células Epiteliais/imunologia , Células Epiteliais/microbiologia , Células Epiteliais/virologia , Humanos , Proteína 1 Semelhante a Receptor de Interleucina-1/genética , Interleucina-33/genética , Interleucina-33/imunologia , Interleucina-8/genética , Interleucina-8/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Mycoplasma pneumoniae/genética , Pneumonia por Mycoplasma/genética , Pneumonia por Mycoplasma/imunologia , Sistema Respiratório/microbiologia , Sistema Respiratório/virologiaRESUMO
Mycoplasma pneumoniae is one of the most common pathogenic causes of community-acquired pneumonia. Hydrogen sulfide, alanine, and pyruvate producing enzyme (HapE) is a recently discovered M. pneumoniae virulence factor that can produce H2S to promote erythrocyte lysis. However, other cytotoxic effects of HapE have not been explored. The present study examined the effects of this enzyme on normal human bronchial epithelial (NHBE) cells, in an attempt to identify additional mechanisms of M. pneumoniae pathogenesis. Recombinant HapE was purified for use in downstream assays. MTT and colony formation assays were conducted to determine the effects of HapE on cell viability and growth, while flow cytometry was used to examine changes in cell proliferation and cell cycle function. ELISA was performed to examine changes in the cytokine profile of HapE-treated cells. HapE treatment arrested NHBE cells in S phase and inhibited cell proliferation in a concentration-dependent manner. The anti-inflammatory factors interleukin (IL)-4 and IL-6 were significantly enhanced following HapE treatment. Increased secretion of pro-inflammatory factors was not observed. The effects of HapE on the respiratory epithelium may have an impact on the efficiency of host immune surveillance and pathogen elimination, and contribute to the pathogenesis of M. pneumoniae.
Assuntos
Proteínas de Bactérias/genética , Brônquios/microbiologia , Mycoplasma pneumoniae/enzimologia , Pneumonia por Mycoplasma/enzimologia , Fatores de Virulência/genética , Proteínas de Bactérias/farmacologia , Brônquios/enzimologia , Proliferação de Células/genética , Citocinas/genética , Citocinas/farmacologia , Células Epiteliais/enzimologia , Células Epiteliais/microbiologia , Humanos , Mycoplasma pneumoniae/genética , Mycoplasma pneumoniae/patogenicidade , Pneumonia por Mycoplasma/genética , Pneumonia por Mycoplasma/microbiologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Fatores de Virulência/farmacologiaRESUMO
Herein, we found that serum content of the triggering receptor expressed on myeloid cells-1 (TREM1) was increased, and positively correlated with Mycoplasma pneumoniae (MP)-DNA in children with MP infection. In this study, A549 cells, known as human lung epithelial cells, were co-cultured with 107 CCU/ml of MP to established in vitro model of MP infection. We studied the roles of TREM1 in inflammatory response of A549 cell by regulating the secretions of cytokine interleukin (IL)-8 and tumor necrosis factor (TNF)-α in cell culture supernatants. Moreover, transcriptional activity of nuclear factor kappa B (NF-кB) was assessed by measuring protein levels of NF-кB in the cytoplasm and nuclear. Our data suggested that sanguinarine chloride significantly decreased TREM1 expression, and alleviated inflammatory response of MP-infected A549 cells via preventing NF-кB nuclear translocation. To study the roles of TREM1 in inflammatory regulation in MP-infected A549 cells and the underlying mechanisms, we established TREM1 overexpression transfected A549 cells. PDTC was used for inhibiting NF-кB nuclear translocation. We found that TREM1 overexpression induced server inflammatory response of A549 cells. Besides, TREM1 overexpression attenuated anti-inflammatory effects of sanguinarine chloride in MP-infected cells. More importantly, pro-inflammatory effects of TREM1 overexpression was significantly reversed with additional PDTC treatment in MP-infected cells treated with sanguinarine chloride, suggesting that TREM1 was a pro-inflammatory factor via regulating NF-кB nuclear translocation in MP-infected A549 cells.
Assuntos
NF-kappa B/metabolismo , Pneumonia por Mycoplasma/patologia , Receptor Gatilho 1 Expresso em Células Mieloides/sangue , Receptor Gatilho 1 Expresso em Células Mieloides/genética , Células A549 , Transporte Ativo do Núcleo Celular , Benzofenantridinas/farmacologia , Células Epiteliais/patologia , Regulação da Expressão Gênica , Humanos , Inflamação/genética , Inflamação/microbiologia , Inflamação/patologia , Interleucina-8/metabolismo , Isoquinolinas/farmacologia , Pulmão/citologia , Pulmão/microbiologia , Pulmão/patologia , Mycoplasma pneumoniae/isolamento & purificação , Pneumonia por Mycoplasma/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Excessive immune responses played an important role in pathophysiology of mycoplasma pneumonia (MP) infection. Tumor necrosis factor-α-induced protein 8-like 2 (TIPE2) is a negative regulator of immune response. This study investigated the expression change of TIPE2 and its role in immune defense against MP infection, as well as the underlying mechanisms. Expressions of TIPE2 both in patients and in macrophages in vitro after MP infection were measured. We further studied cytokine production and mitogen-activated protein kinase (MAPK) signaling function in macrophages with interfered expression of TIPE2 upon MP infection. A significant decrease of TIPE2 mRNA expression was observed in peripheral blood mononuclear cells (PBMCs) from MP patients, which was correlated with the severity of infection. Accordingly we found down-regulation of TIPE2 expression in macrophages after MP infection. In vitro study further suggested that TIPE2 jeopardized inflammatory cytokine production trigged by MP infection via inhibiting MAPK signaling pathway. These findings provided evidences of the novel function of TIPE2 in anti-MP immunity and its possible clinical utility related clinical significance.
Assuntos
Interações Hospedeiro-Patógeno/imunologia , Imunidade , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Sistema de Sinalização das MAP Quinases , Mycoplasma ovipneumoniae/imunologia , Pneumonia por Mycoplasma/imunologia , Pneumonia por Mycoplasma/metabolismo , Animais , Biomarcadores , Estudos de Casos e Controles , Linhagem Celular , Citocinas/metabolismo , Expressão Gênica , Humanos , Mediadores da Inflamação/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Pneumonia por Mycoplasma/genética , Pneumonia por Mycoplasma/microbiologiaRESUMO
Mycoplasma pneumoniae (M. pneumoniae) infection is closely associated with pneumonia in children. Apoptosis of alveolar epithelial cells is involved in the development of pneumonia in children. The present study aimed to examine how caspase3 influences apoptosis rates in M. pneumoniaeinfected alveolar epithelial cells. A549 alveolar epithelial cells were treated with M. pneumoniae, and cells and culture supernatant were collected at different time points. Alterations in apoptosis rates and caspase3 mRNA and protein levels were measured for each treatment group. Cell apoptosis was detected using flow cytometry and TUNEL assay, and cell proliferation was detected using Cell Counting Kit8 assay. Caspase3 expression in A549 cells was inhibited via small interfering RNA (siRNA) knockdown and relative alterations in mRNA and protein levels and apoptosis rates were measured. Cytokine levels were measured using ELISA assay. Apoptosis rates of alveolar epithelial cells increased with prolonged exposure to M. pneumoniae (P<0.05). M. pneumoniae infection increased interleukin (IL)4, IL6 and IL13 levels and reduced IL10 levels. Caspase3 was upregulated, whereas B cell lymphoma (Bcl)2 was downregulated upon M. pneumoniae exposure for 24 h (P<0.05). Following 12 and 24 h of treatment, caspase3 levels in the siRNAtreated cells were decreased compared with control group (P<0.05). M. pneumoniae also significantly altered caspase3 and Bcl2 protein expression. M. pneumoniae promoted apoptosis in alveolar epithelial cells via activation of the external death receptor pathway. Therefore, M. pneumoniae infection may affect the development of pneumonia in children by regulating caspase3 expression and promoting apoptosis.
Assuntos
Células Epiteliais Alveolares/metabolismo , Apoptose , Caspase 3/metabolismo , Mycoplasma pneumoniae/fisiologia , Linhagem Celular Tumoral , Proliferação de Células , Citocinas/metabolismo , Expressão Gênica , Humanos , Pneumonia por Mycoplasma/genética , Pneumonia por Mycoplasma/metabolismo , Pneumonia por Mycoplasma/microbiologiaRESUMO
Mycoplasma pneumoniae is a common cause of community-acquired pneumonia (CAP) and the clinical presentation of mycoplasma pneumoniae pneumonia (MPP) varies widely. Genetic variability affecting the host response may also influence the susceptibility to MPP. Several studies have investigated the association between single nucleotide polymorphism (SNP) of some genes and the risks of CAP; however, the results were inconsistent. Here, we investigated the association of 5 functional genes and the risks of MPP, including ACE (rs4340), GSTM1 (Ins/del), IL-6 (rs1800795), NOS3 (rs1799983), and CYP1A1 (rs2606345) in a total of 715 subjects (415 cases, 300 controls) by using tetra-primer allele-specific polymerase chain reaction (PCR) and Sanger sequencing. The gene-gene interactions were analyzed using the Multifactor Dimensionality Reduction and cumulative genetic risk score approaches. Our results showed that 3 SNPs of ACE rs4340, IL-6 rs1800795, and NOS3 rs1799983 were significantly associated with the risks of MPP, while no differences were observed in genotype frequencies of GSTM1 (Ins/del) and CYP1A1 rs2606345 between both groups. The combinations of ACE rs4340D/NOS3 rs1799983T/CYP1A1 rs2606345G and ACE rs4340D/NOS3 rs1799983T contribute to the genetic susceptibility of MPP in Chinese children.
Assuntos
Citocromo P-450 CYP1A1/genética , Glutationa Transferase/genética , Interleucina-6/genética , Óxido Nítrico Sintase Tipo III/genética , Peptidil Dipeptidase A/genética , Pneumonia por Mycoplasma/genética , Alelos , Povo Asiático/genética , Estudos de Casos e Controles , Criança , Pré-Escolar , China , Feminino , Frequência do Gene , Predisposição Genética para Doença , Humanos , Masculino , Mycoplasma pneumoniae , Pneumonia por Mycoplasma/microbiologia , Reação em Cadeia da Polimerase , Polimorfismo de Nucleotídeo Único , Fatores de RiscoRESUMO
OBJECTIVES: Activating transcription factor-3 (ATF3) is a key regulator of inflammatory responses. We aimed to investigate the effects and mechanisms of ATF3 on the inflammatory cytokines are induced by Mycoplasma pneumonia (MP). STUDY DESIGN: RAW264.7 and mouse peritoneal macrophages were exposed to various time with or without MP infection (3, 6, 12, 24, and 48 h), and detect the expression of ATF3. Adenovirus-expression of ATF3 (Ad/ATF3) or Ad/ßgal was transfected into cells which were exposed to MP for 48 h, RT-PCR and ELISA was used to evaluate the expression and secretion of TNF-α, IL-1ß, IL-6, and IL-18. In addition, intravenous administration Ad/ATF3 or Ad/ßgal into the mice, the secretion of inflammatory cytokines were detected using ELISA. ChIP assay was used to determine whether ATF3 can bind to the promoter of Early growth response protein 1 (Egr-1). Western blot was used to detect the expression of Egr-1 and Fyn. RESULTS: ATF3 was increased at 3, 6, 12, and 24 h and the highest expression levels occurs in 6 h, there is no significant differences at 24 and 48 h compared with 0 h or CON group in RAW 264.7. Similar results were seen in mouse peritoneal macrophages. Overexpression of ATF3 resulted in the reduction of inflammatory cytokines. ChIP assay revealed that ATF3 can bind to the promoter of Egr-1. Overexpression of ATF3 inhibited the protein expression of Egr-1 and Fyn; conversely, ATF3-deficiency promoted the expression of Egr-1 and Fyn. Overexpression of Egr-1 reduced the anti-inflammatory action of ATF3. CONCLUSIONS: ATF3 inhibit the expression and release of TNF-α, IL-1ß, IL-6, and IL-18 induced by MP in vitro and in vivo, which is associated with its negative regulation of Egr-1/Fyn signaling pathway.
Assuntos
Fator 3 Ativador da Transcrição/metabolismo , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Inflamação/metabolismo , Pneumonia por Mycoplasma/metabolismo , Proteínas Proto-Oncogênicas c-fyn/metabolismo , Fator 3 Ativador da Transcrição/genética , Adenoviridae/genética , Animais , Células Cultivadas , Citocinas/metabolismo , Proteína 1 de Resposta de Crescimento Precoce/genética , Feminino , Inflamação/genética , Macrófagos Peritoneais/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Pneumonia por Mycoplasma/genética , Regiões Promotoras Genéticas , Células RAW 264.7 , Transdução de SinaisRESUMO
Hepatocyte growth factor (HGF) promotes lung epithelial repair after injury. Because prior studies established that human neutrophil proteases inactivate HGF in vitro, we predicted that HGF levels decrease in lungs infiltrated with neutrophils and that injury is less severe in lungs lacking HGF-inactivating proteases. After establishing that mouse neutrophil elastase cleaves mouse HGF in vitro, we tested our predictions in vivo by examining lung pathology and HGF in mice infected with Mycoplasma pulmonis, which causes neutrophilic tracheobronchitis and pneumonia. Unexpectedly, pneumonia severity was similar in wild type and dipeptidylpeptidase I-deficient (Dppi-/-) mice lacking neutrophil serine protease activity. To assess how this finding related to our prediction that Dppi-activated proteases regulate HGF levels, we measured HGF in serum, bronchoalveolar lavage fluid, and lung tissue from Dppi(+/+) and Dppi(-/-) mice. Contrary to prediction, HGF levels were higher in lavage fluid from infected mice. However, serum and tissue concentrations were not different in infected and uninfected mice, and HGF lung transcript levels did not change. Increased HGF correlated with increased albumin in lavage fluid from infected mice, and immunostaining failed to detect increased lung tissue expression of HGF in infected mice. These findings are consistent with trans-alveolar flux rather than local production as the source of increased HGF in lavage fluid. However, levels of intact HGF from infected mice, normalized for albumin concentration, were two-fold higher in Dppi(-/-) versus Dppi(+/+) lavage fluid, suggesting regulation by Dppi-activated proteases. Consistent with the presence of active HGF, increased expression of activated receptor c-Met was observed in infected tissues. These data suggest that HGF entering alveoli from the bloodstream during pneumonia compensates for destruction by Dppi-activated inflammatory proteases to allow HGF to contribute to epithelial repair.
Assuntos
Fator de Crescimento de Hepatócito/metabolismo , Peptídeo Hidrolases/metabolismo , Pneumonia/metabolismo , Alvéolos Pulmonares/metabolismo , Animais , Líquido da Lavagem Broncoalveolar , Catepsina C/genética , Modelos Animais de Doenças , Expressão Gênica , Fator de Crescimento de Hepatócito/química , Fator de Crescimento de Hepatócito/genética , Elastase de Leucócito/metabolismo , Pulmão/metabolismo , Pulmão/microbiologia , Pulmão/patologia , Camundongos , Camundongos Knockout , Modelos Biológicos , Mycoplasma pulmonis/enzimologia , Especificidade de Órgãos/genética , Pneumonia/genética , Pneumonia/microbiologia , Pneumonia/patologia , Pneumonia por Mycoplasma/genética , Pneumonia por Mycoplasma/metabolismo , Pneumonia por Mycoplasma/microbiologia , Pneumonia por Mycoplasma/patologia , Domínios e Motivos de Interação entre Proteínas , Proteólise , Proteínas Proto-Oncogênicas c-met/genética , Proteínas Proto-Oncogênicas c-met/metabolismo , Serina Endopeptidases/metabolismoRESUMO
Mycoplasma pneumoniae is an extracellular pathogen that colonizes mucosal surfaces of the respiratory tract and is associated with asthma exacerbations. Previous reports demonstrate that surfactant protein-A (SP-A) binds live M. pneumoniae and mycoplasma membrane fractions (MMF) with high affinity. Humans express a repertoire of single-amino acid genetic variants of SP-A that may be associated with lung disease, and our findings demonstrate that allelic differences in SP-A2 (Gln223Lys) affect the binding to MMF. We show that SP-A(-/-) mice are more susceptible to MMF exposure and have significant increases in mucin production and neutrophil recruitment. Novel humanized SP-A2-transgenic mice harboring the hSP-A2 223K allele exhibit reduced neutrophil influx and mucin production in the lungs when challenged with MMF compared with SP-A(-/-) mice. Conversely, mice expressing hSP-A2 223Q have increased neutrophil influx and mucin production that are similar to SP-A(-/-) mice. Using tracheal epithelial cell cultures, we show that enhanced mucin production to MMF occurs in the absence of SP-A and is not dependent upon neutrophil recruitment. Increased phosphorylation of the epidermal growth factor receptor (EGFR) was evident in the lungs of MMF-challenged mice when SP-A was absent. Pharmacologic inhibition of EGFR prior to MMF challenge dramatically reduced mucin production in SP-A(-/-) mice. These findings suggest a protective role for SP-A in limiting MMF-stimulated mucin production that occurs through interference with EGFR-mediated signaling. SP-A interaction with the EGFR signaling pathway appears to occur in an allele-specific manner that may have important implications for SP-A polymorphisms in human diseases.
Assuntos
Variação Genética , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Mycoplasma pneumoniae/imunologia , Mycoplasma pneumoniae/metabolismo , Pneumonia por Mycoplasma/genética , Pneumonia por Mycoplasma/imunologia , Proteína A Associada a Surfactante Pulmonar/genética , Animais , Membrana Celular/imunologia , Membrana Celular/metabolismo , Quimiotaxia/imunologia , Receptores ErbB/metabolismo , Feminino , Expressão Gênica , Ordem dos Genes , Marcação de Genes , Vetores Genéticos/genética , Humanos , Pulmão/imunologia , Pulmão/metabolismo , Pulmão/microbiologia , Pulmão/patologia , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Mucina-5AC/genética , Mucina-5AC/metabolismo , Muco/metabolismo , Neutrófilos/imunologia , Neutrófilos/metabolismo , Fosforilação , Pneumonia por Mycoplasma/metabolismo , Ligação Proteica , Proteína A Associada a Surfactante Pulmonar/metabolismoRESUMO
The aim of this study was to investigate the pathogenesis of Mycoplasma pneumoniae (MP) infection and its association with cardiac and hepatic damage. Between March 2013 and March 2014, 59 children with MP pneumonia (MPP) and 30 healthy children were enrolled. Serum titers of TLR4, T cell immunoglobulin and mucin-domain (TIM) 3, IL-10, TNF-α, and IFN-γ were measured both in children with MPP and healthy children. Additionally, MP-specific antibody titer and creatine kinase-MB (CK-MB), and alanine transaminase (ALT) titers were measured in patients with MPP. There were significant differences between the MPP patients and healthy controls in titers of TIM1 (P < 0.01), TLR2 (P = 0.028), TLR4 (P = 0.019), IL-10 (P < 0.01), TNF-α (P < 0.01) and IFN-γ (P < 0.01); however, no significant difference was found in TIM3 titers (P = 0.8181). TIM1 was correlated with CK-MB (P = 0.025), whereas both TIM1 and TLR2 titers were correlated with MP-specific antibody titers (P < 0.001; P = 0.003, respectively). Additionally, there were correlations between ALT, TIM3, and TLR2 titers (P = 0.025; P = 0.037, respectively). The titers of TIM1 were significantly higher in patients with cardiac damage (P = 0.007) than in those without it, whereas the titers of TLR2 were significantly higher in patients with hepatic damage (P = 0.026) than in those without it. TLR2, TLR4 and TIM1 may be involved in the process of MP infection. Additionally, TLR2, TLR4, TIM1 and TIM3 may play particular roles in the pathogenesis of MPP-associated cardiac and hepatic damage.
Assuntos
Fígado/imunologia , Mycoplasma pneumoniae/fisiologia , Miocárdio/imunologia , Pneumonia por Mycoplasma/imunologia , Anticorpos Antibacterianos/sangue , Criança , Pré-Escolar , Feminino , Receptor Celular 2 do Vírus da Hepatite A , Humanos , Lactente , Interleucina-10/sangue , Masculino , Proteínas de Membrana/sangue , Pneumonia por Mycoplasma/sangue , Pneumonia por Mycoplasma/genética , Pneumonia por Mycoplasma/microbiologia , Fator de Necrose Tumoral alfa/sangueRESUMO
The DNA recombination and repair machineries of Mycoplasma genitalium and Mycoplasma pneumoniae differ considerably from those of gram-positive and gram-negative bacteria. Most notably, M. pneumoniae is unable to express a functional RecU Holliday junction (HJ) resolvase. In addition, the RuvB homologues from both M. pneumoniae and M. genitalium only exhibit DNA helicase activity but not HJ branch migration activity in vitro. To identify a putative role of the RuvA homologues of these mycoplasmas in DNA recombination, both proteins (RuvA(Mpn) and RuvA(Mge), respectively) were studied for their ability to bind DNA and to interact with RuvB and RecU. In spite of a high level of sequence conservation between RuvA(Mpn) and RuvA(Mge) (68.8% identity), substantial differences were found between these proteins in their activities. First, RuvA(Mge) was found to preferentially bind to HJs, whereas RuvA(Mpn) displayed similar affinities for both HJs and single-stranded DNA. Second, while RuvA(Mpn) is able to form two distinct complexes with HJs, RuvA(Mge) only produced a single HJ complex. Third, RuvA(Mge) stimulated the DNA helicase and ATPase activities of RuvB(Mge), whereas RuvA(Mpn) did not augment RuvB activity. Finally, while both RuvA(Mge) and RecU(Mge) efficiently bind to HJs, they did not compete with each other for HJ binding, but formed stable complexes with HJs over a wide protein concentration range. This interaction, however, resulted in inhibition of the HJ resolution activity of RecU(Mge).
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Mycoplasma genitalium/metabolismo , Pneumonia por Mycoplasma/metabolismo , Homologia de Sequência de Aminoácidos , Adenosina Trifosfatases/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , DNA Cruciforme/genética , DNA Cruciforme/metabolismo , Dados de Sequência Molecular , Mycoplasma genitalium/genética , Oligodesoxirribonucleotídeos/metabolismo , Fases de Leitura Aberta/genética , Pneumonia por Mycoplasma/genética , Ligação Proteica , Recombinação GenéticaRESUMO
In mycoplasmal pneumonia, the bronchi are histopathologically filled with polymorphonuclear leukocytes. The EGFR pathway is involved in IL-8 production. We investigated the contribution of the EGFR pathway to IL-8 production by bronchial epithelial cells (A549) stimulated with Mp-Ag. The IL-8 production by A549 cells stimulated with Mp-Ag was decreased by the addition of an EGFR kinase inhibitor or transfection with small interfering RNA against EGFR. The levels of epiregulin mRNA in A549 cells were increased by stimulation with Mp-Ag. In conclusion, the EFGR pathway participates in IL-8 production by bronchial epithelial cells stimulated with Mp-Ag.