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Co-Transcriptomes of Initial Interactions In Vitro between Streptococcus Pneumoniae and Human Pleural Mesothelial Cells.

Heath CJ, del Mar Cendra M, Watson A, Auger JP, Pandey A, Tighe P, Christodoulides M - PLoS ONE (2015)

Bottom Line: After Spn initial contact, 870 human genes were differentially regulated and the largest numbers of significant gene expression changes were found in canonical pathways for eukaryotic initiation factor 2 signaling (60 genes out of 171), oxidative phosphorylation (32/103), mitochondrial dysfunction (37/164), eIF4 and p70S6K signaling (28/142), mTOR signaling (27/182), NRF2-mediated oxidative stress response (20/177), epithelial adherens junction remodeling (11/66) and ubiquitination (22/254).Spn did not activate NF-kB or phosphorylate p38 MAPK or induce cytokine production from PMC.Moreover, Spn infection of TNF-α pre-stimulated PMC inhibited production of IL-6 and IL-8 secretion by >50% (p<0.01).

View Article: PubMed Central - PubMed

Affiliation: Neisseria Research Group, Molecular Microbiology, Clinical and Experimental Sciences, Sir Henry Wellcome laboratories, University of Southampton Faculty of Medicine, Southampton General Hospital, Southampton, United Kingdom.

ABSTRACT
Streptococcus pneumoniae (Spn) is a major causative organism of empyema, an inflammatory condition occurring in the pleural sac. In this study, we used human and Spn cDNA microarrays to characterize the transcriptional responses occurring during initial contact between Spn and a human pleural mesothelial cell line (PMC) in vitro. Using stringent filtering criteria, 42 and 23 Spn genes were up-and down-regulated respectively. In particular, genes encoding factors potentially involved in metabolic processes and Spn adherence to eukaryotic cells were up-regulated e.g. glnQ, glnA, aliA, psaB, lytB and nox. After Spn initial contact, 870 human genes were differentially regulated and the largest numbers of significant gene expression changes were found in canonical pathways for eukaryotic initiation factor 2 signaling (60 genes out of 171), oxidative phosphorylation (32/103), mitochondrial dysfunction (37/164), eIF4 and p70S6K signaling (28/142), mTOR signaling (27/182), NRF2-mediated oxidative stress response (20/177), epithelial adherens junction remodeling (11/66) and ubiquitination (22/254). The cellular response appeared to be directed towards host cell survival and defense. Spn did not activate NF-kB or phosphorylate p38 MAPK or induce cytokine production from PMC. Moreover, Spn infection of TNF-α pre-stimulated PMC inhibited production of IL-6 and IL-8 secretion by >50% (p<0.01). In summary, this descriptive study provides datasets and a platform for examining further the molecular mechanisms underlying the pathogenesis of empyema.

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Related in: MedlinePlus

Inflammatory cytokine responses of Met-5A PMC.A) Met-5A cell monolayers (n = 3 wells) were infected in triplicate wells with live D39 (MOI 1–200), stimulated with pure TNF-α (100ng/ml) or Nm-OM (100ng/ml) and also pre-stimulated with TNF-α (100ng/ml) for 4h and then infected with various MOI (1–200) of live D39. Control cells were left with medium alone. Cells were also treated with live D39 without any other stimulation (no cytokine production at any of the concentrations tested between MOI 1–200). After 24h, supernatants were removed and production of IL-6 and IL-8 quantified by ELISA. The bars represent mean cytokine levels and the errors bars the standard deviation from triplicate wells from a representative experiment (n = 2). * denotes statistically significant reduction in IL-6 or IL-8 compared to cells treated with TNFα only (p<0.05). B) Met-5A cells were challenged in triplicate wells with live D39 bacteria (1–100 MOI), TNFα (30 ng/monolayer) or Nm-OM (30ng/monolayer). At intervals up to 2h, cells were processed and NFkB p65 protein measured by ELISA. The columns and symbols denote the mean absorbance readings from n = 3 experiments and the error bars the standard errors of the mean. * denotes statistically significant increase compared to control cells (p<0.05). C) Met-5A cells were challenged in triplicate wells with live D39 bacteria (1–100 MOI), TNFα (30 ng/monolayer) or Nm-OM (30ng/monolayer). At intervals up to 2h, cells were processed and p38 MAP Kinase protein measured by ELISA. The columns and symbols denote the mean absorbance readings from n = 3 experiments and the error bars the standard errors of the mean. * denotes statistically significant increase compared to control cells (p<0.05).
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pone.0142773.g004: Inflammatory cytokine responses of Met-5A PMC.A) Met-5A cell monolayers (n = 3 wells) were infected in triplicate wells with live D39 (MOI 1–200), stimulated with pure TNF-α (100ng/ml) or Nm-OM (100ng/ml) and also pre-stimulated with TNF-α (100ng/ml) for 4h and then infected with various MOI (1–200) of live D39. Control cells were left with medium alone. Cells were also treated with live D39 without any other stimulation (no cytokine production at any of the concentrations tested between MOI 1–200). After 24h, supernatants were removed and production of IL-6 and IL-8 quantified by ELISA. The bars represent mean cytokine levels and the errors bars the standard deviation from triplicate wells from a representative experiment (n = 2). * denotes statistically significant reduction in IL-6 or IL-8 compared to cells treated with TNFα only (p<0.05). B) Met-5A cells were challenged in triplicate wells with live D39 bacteria (1–100 MOI), TNFα (30 ng/monolayer) or Nm-OM (30ng/monolayer). At intervals up to 2h, cells were processed and NFkB p65 protein measured by ELISA. The columns and symbols denote the mean absorbance readings from n = 3 experiments and the error bars the standard errors of the mean. * denotes statistically significant increase compared to control cells (p<0.05). C) Met-5A cells were challenged in triplicate wells with live D39 bacteria (1–100 MOI), TNFα (30 ng/monolayer) or Nm-OM (30ng/monolayer). At intervals up to 2h, cells were processed and p38 MAP Kinase protein measured by ELISA. The columns and symbols denote the mean absorbance readings from n = 3 experiments and the error bars the standard errors of the mean. * denotes statistically significant increase compared to control cells (p<0.05).

Mentions: In the current study, microarray gene expression for inflammatory cytokines was not observed, which was confirmed by the absence of cytokine proteins in culture supernatants from PMC infected for 24h with live D39 (MOI ranging from 2.5× 10−5 to 250). There was no significant production of IL-6, TNF-α, IL-1β, IL-8, IL-10 or TGF-β, compared to control, uninfected cells (P>0.05). Furthermore, treatment of Met-5A cells with a whole crude lysate from mechanically lysed D39 and a sterile filtrate, which both contained active pneumolysin as demonstrated by erythrocyte haemolysis, did not induce significant cytokine production, compared to untreated cells (p>0.05). PMC were capable of producing cytokines, but only a high concentration of heat-inactivated bacteria (100μg/monolayer equivalent to a MOI of ~450) was capable of inducing significant IL-6 (~500 pg/ml) and IL-8 (~565 pg/ml) secretion (p<0.05) by Met-5A cells. PMC responded to Nm-OM and pure TNF-α alone with similarly detectable levels of IL-6 and IL-8 secretion (p<0.05) (Fig 4A).


Co-Transcriptomes of Initial Interactions In Vitro between Streptococcus Pneumoniae and Human Pleural Mesothelial Cells.

Heath CJ, del Mar Cendra M, Watson A, Auger JP, Pandey A, Tighe P, Christodoulides M - PLoS ONE (2015)

Inflammatory cytokine responses of Met-5A PMC.A) Met-5A cell monolayers (n = 3 wells) were infected in triplicate wells with live D39 (MOI 1–200), stimulated with pure TNF-α (100ng/ml) or Nm-OM (100ng/ml) and also pre-stimulated with TNF-α (100ng/ml) for 4h and then infected with various MOI (1–200) of live D39. Control cells were left with medium alone. Cells were also treated with live D39 without any other stimulation (no cytokine production at any of the concentrations tested between MOI 1–200). After 24h, supernatants were removed and production of IL-6 and IL-8 quantified by ELISA. The bars represent mean cytokine levels and the errors bars the standard deviation from triplicate wells from a representative experiment (n = 2). * denotes statistically significant reduction in IL-6 or IL-8 compared to cells treated with TNFα only (p<0.05). B) Met-5A cells were challenged in triplicate wells with live D39 bacteria (1–100 MOI), TNFα (30 ng/monolayer) or Nm-OM (30ng/monolayer). At intervals up to 2h, cells were processed and NFkB p65 protein measured by ELISA. The columns and symbols denote the mean absorbance readings from n = 3 experiments and the error bars the standard errors of the mean. * denotes statistically significant increase compared to control cells (p<0.05). C) Met-5A cells were challenged in triplicate wells with live D39 bacteria (1–100 MOI), TNFα (30 ng/monolayer) or Nm-OM (30ng/monolayer). At intervals up to 2h, cells were processed and p38 MAP Kinase protein measured by ELISA. The columns and symbols denote the mean absorbance readings from n = 3 experiments and the error bars the standard errors of the mean. * denotes statistically significant increase compared to control cells (p<0.05).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4643877&req=5

pone.0142773.g004: Inflammatory cytokine responses of Met-5A PMC.A) Met-5A cell monolayers (n = 3 wells) were infected in triplicate wells with live D39 (MOI 1–200), stimulated with pure TNF-α (100ng/ml) or Nm-OM (100ng/ml) and also pre-stimulated with TNF-α (100ng/ml) for 4h and then infected with various MOI (1–200) of live D39. Control cells were left with medium alone. Cells were also treated with live D39 without any other stimulation (no cytokine production at any of the concentrations tested between MOI 1–200). After 24h, supernatants were removed and production of IL-6 and IL-8 quantified by ELISA. The bars represent mean cytokine levels and the errors bars the standard deviation from triplicate wells from a representative experiment (n = 2). * denotes statistically significant reduction in IL-6 or IL-8 compared to cells treated with TNFα only (p<0.05). B) Met-5A cells were challenged in triplicate wells with live D39 bacteria (1–100 MOI), TNFα (30 ng/monolayer) or Nm-OM (30ng/monolayer). At intervals up to 2h, cells were processed and NFkB p65 protein measured by ELISA. The columns and symbols denote the mean absorbance readings from n = 3 experiments and the error bars the standard errors of the mean. * denotes statistically significant increase compared to control cells (p<0.05). C) Met-5A cells were challenged in triplicate wells with live D39 bacteria (1–100 MOI), TNFα (30 ng/monolayer) or Nm-OM (30ng/monolayer). At intervals up to 2h, cells were processed and p38 MAP Kinase protein measured by ELISA. The columns and symbols denote the mean absorbance readings from n = 3 experiments and the error bars the standard errors of the mean. * denotes statistically significant increase compared to control cells (p<0.05).
Mentions: In the current study, microarray gene expression for inflammatory cytokines was not observed, which was confirmed by the absence of cytokine proteins in culture supernatants from PMC infected for 24h with live D39 (MOI ranging from 2.5× 10−5 to 250). There was no significant production of IL-6, TNF-α, IL-1β, IL-8, IL-10 or TGF-β, compared to control, uninfected cells (P>0.05). Furthermore, treatment of Met-5A cells with a whole crude lysate from mechanically lysed D39 and a sterile filtrate, which both contained active pneumolysin as demonstrated by erythrocyte haemolysis, did not induce significant cytokine production, compared to untreated cells (p>0.05). PMC were capable of producing cytokines, but only a high concentration of heat-inactivated bacteria (100μg/monolayer equivalent to a MOI of ~450) was capable of inducing significant IL-6 (~500 pg/ml) and IL-8 (~565 pg/ml) secretion (p<0.05) by Met-5A cells. PMC responded to Nm-OM and pure TNF-α alone with similarly detectable levels of IL-6 and IL-8 secretion (p<0.05) (Fig 4A).

Bottom Line: After Spn initial contact, 870 human genes were differentially regulated and the largest numbers of significant gene expression changes were found in canonical pathways for eukaryotic initiation factor 2 signaling (60 genes out of 171), oxidative phosphorylation (32/103), mitochondrial dysfunction (37/164), eIF4 and p70S6K signaling (28/142), mTOR signaling (27/182), NRF2-mediated oxidative stress response (20/177), epithelial adherens junction remodeling (11/66) and ubiquitination (22/254).Spn did not activate NF-kB or phosphorylate p38 MAPK or induce cytokine production from PMC.Moreover, Spn infection of TNF-α pre-stimulated PMC inhibited production of IL-6 and IL-8 secretion by >50% (p<0.01).

View Article: PubMed Central - PubMed

Affiliation: Neisseria Research Group, Molecular Microbiology, Clinical and Experimental Sciences, Sir Henry Wellcome laboratories, University of Southampton Faculty of Medicine, Southampton General Hospital, Southampton, United Kingdom.

ABSTRACT
Streptococcus pneumoniae (Spn) is a major causative organism of empyema, an inflammatory condition occurring in the pleural sac. In this study, we used human and Spn cDNA microarrays to characterize the transcriptional responses occurring during initial contact between Spn and a human pleural mesothelial cell line (PMC) in vitro. Using stringent filtering criteria, 42 and 23 Spn genes were up-and down-regulated respectively. In particular, genes encoding factors potentially involved in metabolic processes and Spn adherence to eukaryotic cells were up-regulated e.g. glnQ, glnA, aliA, psaB, lytB and nox. After Spn initial contact, 870 human genes were differentially regulated and the largest numbers of significant gene expression changes were found in canonical pathways for eukaryotic initiation factor 2 signaling (60 genes out of 171), oxidative phosphorylation (32/103), mitochondrial dysfunction (37/164), eIF4 and p70S6K signaling (28/142), mTOR signaling (27/182), NRF2-mediated oxidative stress response (20/177), epithelial adherens junction remodeling (11/66) and ubiquitination (22/254). The cellular response appeared to be directed towards host cell survival and defense. Spn did not activate NF-kB or phosphorylate p38 MAPK or induce cytokine production from PMC. Moreover, Spn infection of TNF-α pre-stimulated PMC inhibited production of IL-6 and IL-8 secretion by >50% (p<0.01). In summary, this descriptive study provides datasets and a platform for examining further the molecular mechanisms underlying the pathogenesis of empyema.

Show MeSH
Related in: MedlinePlus