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Streptococcus pneumoniae stabilizes tumor necrosis factor alpha mRNA through a pathway dependent on p38 MAPK but independent of Toll-like receptors.

Mogensen TH, Berg RS, Ostergaard L, Paludan SR - BMC Immunol. (2008)

Bottom Line: The ability of S. pneumoniae to stabilize TNF-alpha mRNA was dependent on the mitogen-activated protein kinase (MAPK) p38 whereas inhibition of Toll-like receptor signaling via MyD88 did not affect S. pneumoniae-induced mRNA stabilization.P38 was activated through a pathway involving the upstream kinase transforming growth factor-activated kinase 1 and MAPK kinase 3.Production of TNF-alpha may contribute significantly to the inflammatory response raised during pneumococcal infection.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Infectious Diseases, Skejby Hospital - Aarhus University Hospital, DK-8200, Aarhus N, Denmark. trine.mogensen@dadlnet.dk

ABSTRACT

Background: Streptococcus pneumoniae is a human pathogenic bacteria and a major cause of severe invasive diseases, including pneumonia, bacteremia, and meningitis. Infections with S. pneumoniae evoke a strong inflammatory response, which plays a major role in the pathogenesis of pneumococcal disease.

Results: In this study, we have examined how S. pneumoniae affects expression of the inflammatory cytokine tumor necrosis factor (TNF) alpha, and the molecular mechanisms involved. Secretion of TNF-alpha was strongly induced by S. pneumoniae, which was able to stabilize TNF-alpha mRNA through a mechanism dependent on the viability of the bacteria as well as the adenylate uridylate-rich elements in the 3'untranslated region of TNF-alpha mRNA. The ability of S. pneumoniae to stabilize TNF-alpha mRNA was dependent on the mitogen-activated protein kinase (MAPK) p38 whereas inhibition of Toll-like receptor signaling via MyD88 did not affect S. pneumoniae-induced mRNA stabilization. P38 was activated through a pathway involving the upstream kinase transforming growth factor-activated kinase 1 and MAPK kinase 3.

Conclusion: Thus, S. pneumoniae stabilizes TNF-alpha mRNA through a pathway dependent on p38 but independent of Toll-like receptors. Production of TNF-alpha may contribute significantly to the inflammatory response raised during pneumococcal infection.

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S. pneumoniae mediates TNF-α mRNA stability independent of TLRs. (A) RAW-TNF-AU+ cells were treated with vehicle, S. pneumoniae (5 × 107 bacteria/ml), LPS (100 ng/ml), Pam3Csk4 (200 ng/ml), or ODN1826 (1 μM). Total cell lysates were harvested 20 h later and CAT was measured by ELISA. (B) RAW-TNF-AU+ cells were treated with the TLR9 antagonist ODN2088 (3 μM) 15 min prior to addition of vehicle or S. pneumoniae (5 × 107 bacteria/ml) to the cell cultures. Total cell lysates were harvested 20 h later and CAT was measured by ELISA. (C and D) RAW-TNF-AU+ cells were treated with a MyD88 inhibitor peptide or a control peptide for 24 h before addition of (C) vehicle or S. pneumoniae (5 × 107 bacteria/ml) or (D) LPS (100 ng/ml) or TNF-α (25 ng/ml) plus IFN-γ (10 ng/ml). (C) Total cell lysates were harvested 20 h later and CAT was measured by ELISA. (D) Supernatants were harvested 36 h post stimulation, and nitrite was measured by Griess assay. Similar results were obtained in 2–3 independent experiments. The data are shown as means +/- SEM.
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Figure 3: S. pneumoniae mediates TNF-α mRNA stability independent of TLRs. (A) RAW-TNF-AU+ cells were treated with vehicle, S. pneumoniae (5 × 107 bacteria/ml), LPS (100 ng/ml), Pam3Csk4 (200 ng/ml), or ODN1826 (1 μM). Total cell lysates were harvested 20 h later and CAT was measured by ELISA. (B) RAW-TNF-AU+ cells were treated with the TLR9 antagonist ODN2088 (3 μM) 15 min prior to addition of vehicle or S. pneumoniae (5 × 107 bacteria/ml) to the cell cultures. Total cell lysates were harvested 20 h later and CAT was measured by ELISA. (C and D) RAW-TNF-AU+ cells were treated with a MyD88 inhibitor peptide or a control peptide for 24 h before addition of (C) vehicle or S. pneumoniae (5 × 107 bacteria/ml) or (D) LPS (100 ng/ml) or TNF-α (25 ng/ml) plus IFN-γ (10 ng/ml). (C) Total cell lysates were harvested 20 h later and CAT was measured by ELISA. (D) Supernatants were harvested 36 h post stimulation, and nitrite was measured by Griess assay. Similar results were obtained in 2–3 independent experiments. The data are shown as means +/- SEM.

Mentions: The S. pneumoniae SK1013 strain used in this study has previously been demonstrated to be recognized by TLR2 and 9 [16], with the former recognizing bacterial peptidoglycan and lipoteichoic acid and the latter recognizing bacterial DNA [16-18,26]. We have previously demonstrated that TLR9 recognizes live but not heat-killed S. pneumoniae [16] wherefore the data shown in Fig. 2 prompted us to examine the role of TLR9 in stabilization of TNF-α mRNA. We first examined how pure TLR agonists affected CAT expression in the RAW-TNF-α 3'-UTR AU+ cell line and compared this with the response evoked by live S. pneumoniae. The agonists for TLR2, 4, and 9 all enhanced CAT expression but to a rather moderate extent compared to what was observed in cells receiving live S. pneumoniae (Fig. 3A). To directly assess the role of TLR9, we treated cells with the TLR9 antagonist ODN2088 prior to addition of S. pneumoniae and measured CAT expression in lysates at later time points. Although ODN2088 inhibited induction of TNF-α by the TLR9 agonist ODN1826 by more than 95% (data not shown), the presence of the TLR9 antagonist had no effect on CAT expression induced by S. pneumoniae (Fig. 3B), thus suggesting that mRNA stabilization by S. pneumoniae is independent of TLR9.


Streptococcus pneumoniae stabilizes tumor necrosis factor alpha mRNA through a pathway dependent on p38 MAPK but independent of Toll-like receptors.

Mogensen TH, Berg RS, Ostergaard L, Paludan SR - BMC Immunol. (2008)

S. pneumoniae mediates TNF-α mRNA stability independent of TLRs. (A) RAW-TNF-AU+ cells were treated with vehicle, S. pneumoniae (5 × 107 bacteria/ml), LPS (100 ng/ml), Pam3Csk4 (200 ng/ml), or ODN1826 (1 μM). Total cell lysates were harvested 20 h later and CAT was measured by ELISA. (B) RAW-TNF-AU+ cells were treated with the TLR9 antagonist ODN2088 (3 μM) 15 min prior to addition of vehicle or S. pneumoniae (5 × 107 bacteria/ml) to the cell cultures. Total cell lysates were harvested 20 h later and CAT was measured by ELISA. (C and D) RAW-TNF-AU+ cells were treated with a MyD88 inhibitor peptide or a control peptide for 24 h before addition of (C) vehicle or S. pneumoniae (5 × 107 bacteria/ml) or (D) LPS (100 ng/ml) or TNF-α (25 ng/ml) plus IFN-γ (10 ng/ml). (C) Total cell lysates were harvested 20 h later and CAT was measured by ELISA. (D) Supernatants were harvested 36 h post stimulation, and nitrite was measured by Griess assay. Similar results were obtained in 2–3 independent experiments. The data are shown as means +/- SEM.
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Figure 3: S. pneumoniae mediates TNF-α mRNA stability independent of TLRs. (A) RAW-TNF-AU+ cells were treated with vehicle, S. pneumoniae (5 × 107 bacteria/ml), LPS (100 ng/ml), Pam3Csk4 (200 ng/ml), or ODN1826 (1 μM). Total cell lysates were harvested 20 h later and CAT was measured by ELISA. (B) RAW-TNF-AU+ cells were treated with the TLR9 antagonist ODN2088 (3 μM) 15 min prior to addition of vehicle or S. pneumoniae (5 × 107 bacteria/ml) to the cell cultures. Total cell lysates were harvested 20 h later and CAT was measured by ELISA. (C and D) RAW-TNF-AU+ cells were treated with a MyD88 inhibitor peptide or a control peptide for 24 h before addition of (C) vehicle or S. pneumoniae (5 × 107 bacteria/ml) or (D) LPS (100 ng/ml) or TNF-α (25 ng/ml) plus IFN-γ (10 ng/ml). (C) Total cell lysates were harvested 20 h later and CAT was measured by ELISA. (D) Supernatants were harvested 36 h post stimulation, and nitrite was measured by Griess assay. Similar results were obtained in 2–3 independent experiments. The data are shown as means +/- SEM.
Mentions: The S. pneumoniae SK1013 strain used in this study has previously been demonstrated to be recognized by TLR2 and 9 [16], with the former recognizing bacterial peptidoglycan and lipoteichoic acid and the latter recognizing bacterial DNA [16-18,26]. We have previously demonstrated that TLR9 recognizes live but not heat-killed S. pneumoniae [16] wherefore the data shown in Fig. 2 prompted us to examine the role of TLR9 in stabilization of TNF-α mRNA. We first examined how pure TLR agonists affected CAT expression in the RAW-TNF-α 3'-UTR AU+ cell line and compared this with the response evoked by live S. pneumoniae. The agonists for TLR2, 4, and 9 all enhanced CAT expression but to a rather moderate extent compared to what was observed in cells receiving live S. pneumoniae (Fig. 3A). To directly assess the role of TLR9, we treated cells with the TLR9 antagonist ODN2088 prior to addition of S. pneumoniae and measured CAT expression in lysates at later time points. Although ODN2088 inhibited induction of TNF-α by the TLR9 agonist ODN1826 by more than 95% (data not shown), the presence of the TLR9 antagonist had no effect on CAT expression induced by S. pneumoniae (Fig. 3B), thus suggesting that mRNA stabilization by S. pneumoniae is independent of TLR9.

Bottom Line: The ability of S. pneumoniae to stabilize TNF-alpha mRNA was dependent on the mitogen-activated protein kinase (MAPK) p38 whereas inhibition of Toll-like receptor signaling via MyD88 did not affect S. pneumoniae-induced mRNA stabilization.P38 was activated through a pathway involving the upstream kinase transforming growth factor-activated kinase 1 and MAPK kinase 3.Production of TNF-alpha may contribute significantly to the inflammatory response raised during pneumococcal infection.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Infectious Diseases, Skejby Hospital - Aarhus University Hospital, DK-8200, Aarhus N, Denmark. trine.mogensen@dadlnet.dk

ABSTRACT

Background: Streptococcus pneumoniae is a human pathogenic bacteria and a major cause of severe invasive diseases, including pneumonia, bacteremia, and meningitis. Infections with S. pneumoniae evoke a strong inflammatory response, which plays a major role in the pathogenesis of pneumococcal disease.

Results: In this study, we have examined how S. pneumoniae affects expression of the inflammatory cytokine tumor necrosis factor (TNF) alpha, and the molecular mechanisms involved. Secretion of TNF-alpha was strongly induced by S. pneumoniae, which was able to stabilize TNF-alpha mRNA through a mechanism dependent on the viability of the bacteria as well as the adenylate uridylate-rich elements in the 3'untranslated region of TNF-alpha mRNA. The ability of S. pneumoniae to stabilize TNF-alpha mRNA was dependent on the mitogen-activated protein kinase (MAPK) p38 whereas inhibition of Toll-like receptor signaling via MyD88 did not affect S. pneumoniae-induced mRNA stabilization. P38 was activated through a pathway involving the upstream kinase transforming growth factor-activated kinase 1 and MAPK kinase 3.

Conclusion: Thus, S. pneumoniae stabilizes TNF-alpha mRNA through a pathway dependent on p38 but independent of Toll-like receptors. Production of TNF-alpha may contribute significantly to the inflammatory response raised during pneumococcal infection.

Show MeSH
Related in: MedlinePlus