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MiR-155 Induction by Microbes/Microbial Ligands Requires NF-κB-Dependent de novo Protein Synthesis

Cremer, Thomas J., Fatehchand, Kavin, Shah, Prexy, Gillette, Devyn, Patel, Hemal, Marsh, Rachel L., Besecker, Beth Y., Rajaram, Murugesan V. S., Cormet-Boyaka, Estelle, Kanneganti, Thirumala-Devi, Schlesinger, Larry S., Butchar, Jonathan P., Tridandapani, Susheela·2012·Frontiers in Cellular and Infection Microbiology

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Figure 1 MiR-155 induction requires de novo host-protein synthesis. (A) PBM were infected with F. novicida (FN) at an MOI of 50 for 6 h in the presence or absence of 2 μM cycloheximide (CHX), and then RNA was isolated. “R” stands for resting/uninfected cells. Mature miR-155 expression was assayed by qRT-PCR and data expressed as relative expression from triplicate samples. (B) TNFα mRNA was assayed from the same samples as in (A). (C) Secreted TNFα was measured from the media of the cells in (A) by ELISA. *Designates a p value < 0.05 by Students t-test, while N.S. designates a p value > 0.05. These data are representative of three independent experiments.
Figure 1 MiR-155 induction requires de novo host-protein synthesis. (A) PBM were infected with F. novicida (FN) at an MOI of 50 for 6 h in the presence or absence of 2 μM cycloheximide (CHX), and then RNA was isolated. “R” stands for resting/uninfected cells. Mature miR-155 expression was assayed by qRT-PCR and data expressed as relative expression from triplicate samples. (B) TNFα mRNA was assayed from the same samples as in (A). (C) Secreted TNFα was measured from the media of the cells in (A) by ELISA. *Designates a p value < 0.05 by Students t-test, while N.S. designates a p value > 0.05. These data are representative of three independent experiments.CC BY-NC · PubMed Central
Figure 2 MiR-155 induction can be mediated indirectly though secreted factors. (A) PBM were infected with FN at an MOI of 50 for 24 h. Media from infected cells was sterile filtered to remove bacteria and RNA was isolated from the cells (R and Fn). New PBM were cultured in the conditioned media for 24 h (CM R and CM FN) followed by RNA isolation. Mature miR-155 expression was assayed by qRT-PCR. (B) PBM were infected for 24 h, then the media from the infected cells was sterile filtered to generate conditioned media. New PBM were infected with F.n. at an MOI of 50 for 6 h or treated with conditioned media for 6 h in the presence of BAY-7085-11 (5 μM) or DMSO control. MiR-155 expression was assayed by qRT-PCR. These data are representative of at least three independent experiments. *Designates a p value < 0.05.
Figure 2 MiR-155 induction can be mediated indirectly though secreted factors. (A) PBM were infected with FN at an MOI of 50 for 24 h. Media from infected cells was sterile filtered to remove bacteria and RNA was isolated from the cells (R and Fn). New PBM were cultured in the conditioned media for 24 h (CM R and CM FN) followed by RNA isolation. Mature miR-155 expression was assayed by qRT-PCR. (B) PBM were infected for 24 h, then the media from the infected cells was sterile filtered to generate conditioned media. New PBM were infected with F.n. at an MOI of 50 for 6 h or treated with conditioned media for 6 h in the presence of BAY-7085-11 (5 μM) or DMSO control. MiR-155 expression was assayed by qRT-PCR. These data are representative of at least three independent experiments. *Designates a p value < 0.05.CC BY-NC · PubMed Central
Figure 3 F. novicida induction miR-155 expression is not mediated by TNFα or IFNβ. (A) PBM were treated with TNFα neutralizing antibody or isotype control antibody, then infected with F.n. at an MOI of 50 for 6 h. RNA was collected and assayed for miR-155 expression. (B) The media from the samples in (A) was assayed for human TNFα by ELISA. (C) Conditioned media (CM) was obtained as in Figures 2B. Conditioned media from monocytes uninfected (CM:R) or infected (CM:Fn) with F.n. at an MOI of 50 was sterile filtered and incubated with neutralizing antibody or isotype control antibody at 37°C for 1 h. New PBM were then cultured in the conditioned media conditions, uninfected (R), or direct infection (FN) for 6 h. RNA was collected and miR-155 expression was assayed by qRT-PCR. (D) Effective neutralization of IFNβ was demonstrated by pre-treating media with recombinant IFNβ (500 U/ml) with or without neutralizing antibody or isotype control antibody. Then PBM were cultured in media alone, IFNβ, IFNβ plus neutralizing antibody, or IFNβ plus isotype control for 15 min. Cells were lysed and subject to western blot for phosphorylated STAT1, followed by Actin re-probe.
Figure 3 F. novicida induction miR-155 expression is not mediated by TNFα or IFNβ. (A) PBM were treated with TNFα neutralizing antibody or isotype control antibody, then infected with F.n. at an MOI of 50 for 6 h. RNA was collected and assayed for miR-155 expression. (B) The media from the samples in (A) was assayed for human TNFα by ELISA. (C) Conditioned media (CM) was obtained as in Figures 2B. Conditioned media from monocytes uninfected (CM:R) or infected (CM:Fn) with F.n. at an MOI of 50 was sterile filtered and incubated with neutralizing antibody or isotype control antibody at 37°C for 1 h. New PBM were then cultured in the conditioned media conditions, uninfected (R), or direct infection (FN) for 6 h. RNA was collected and miR-155 expression was assayed by qRT-PCR. (D) Effective neutralization of IFNβ was demonstrated by pre-treating media with recombinant IFNβ (500 U/ml) with or without neutralizing antibody or isotype control antibody. Then PBM were cultured in media alone, IFNβ, IFNβ plus neutralizing antibody, or IFNβ plus isotype control for 15 min. Cells were lysed and subject to western blot for phosphorylated STAT1, followed by Actin re-probe.CC BY-NC · PubMed Central
Figure 4 MiR-155 induction is not mediated through host cytokines, but rather by soluble bacterial factors. (A) PBM were infected with F.n. at an MOI of 50 for 16 h in the presence of Brefeldin A (BFA) or DMSO vehicle control. RNA was isolated and mature miR-155 expression was assayed by qRT-PCR. (B) Extracellular TNFα levels were measured by ELISA. (C) Intracellular TNFα levels were measured by ELISA from the cell lysate. (D) WT and MyD88−/− BMMs were infected for 24 h at an MOI of 50 and then sterile filtered to remove bacteria to generate conditioned media (CM). RNA was collected from the infected cells for qRT-PCR (Bars 1, 2, 7, 8). The conditioned media was then placed on new wild-type BMM for 24 h and RNA was subsequently collected for qRT-PCR (Bars 3, 4, 5, 6). These data are representative of three independent experiments. *Designates a p value < 0.05. (E) F.n. was suspended in RPMI-1640 with 10% heat-inactivated fetal bovine serum at a concentration of 3.5 × 108 bacteria/ml (comparable to 5 × 106 PBM infected at MOI 50 in 1 ml) and incubated at 37°C for 24 h. The media was sterile filtered to remove bacteria. PBM were directly infected with F.n. at an MOI of 50 or treated with the filtered media that previously contained F.n. for 16 h. RNA was isolated and miR-155 expression assayed by qRT-PCR. These data are representative of three independent experiments. *Designates a p value < 0.05.
Figure 4 MiR-155 induction is not mediated through host cytokines, but rather by soluble bacterial factors. (A) PBM were infected with F.n. at an MOI of 50 for 16 h in the presence of Brefeldin A (BFA) or DMSO vehicle control. RNA was isolated and mature miR-155 expression was assayed by qRT-PCR. (B) Extracellular TNFα levels were measured by ELISA. (C) Intracellular TNFα levels were measured by ELISA from the cell lysate. (D) WT and MyD88−/− BMMs were infected for 24 h at an MOI of 50 and then sterile filtered to remove bacteria to generate conditioned media (CM). RNA was collected from the infected cells for qRT-PCR (Bars 1, 2, 7, 8). The conditioned media was then placed on new wild-type BMM for 24 h and RNA was subsequently collected for qRT-PCR (Bars 3, 4, 5, 6). These data are representative of three independent experiments. *Designates a p value < 0.05. (E) F.n. was suspended in RPMI-1640 with 10% heat-inactivated fetal bovine serum at a concentration of 3.5 × 108 bacteria/ml (comparable to 5 × 106 PBM infected at MOI 50 in 1 ml) and incubated at 37°C for 24 h. The media was sterile filtered to remove bacteria. PBM were directly infected with F.n. at an MOI of 50 or treated with the filtered media that previously contained F.n. for 16 h. RNA was isolated and miR-155 expression assayed by qRT-PCR. These data are representative of three independent experiments. *Designates a p value < 0.05.CC BY-NC · PubMed Central
Figure 5 Fos and Jun expression is highly up-regulated by infection in an NF-κB-dependent mechanism to control AP-1 activity. (A) PBM were pre-treated with DMSO or BAY-11-7085 (5 μM; designated as BAY in the figure) for 30 min then infected with F.n. at an MOI of 50 for 4 h. Cells were lysed and subject to western blotting for c-Fos, followed by actin re-probe. (B) Protein-matched lysates were probed for c-Jun and re-probed with actin antibody. (C) RAW264.7 macrophages were transfected with an AP-1 luciferase reporter. 14 h post-transfection cells were uninfected or infected with F.n. at an MOI of 50 for 1, 4, 8, or 24 h. Cells were lysed and luciferase activity was measured by a luminometer in triplicate. Data are represented as % increase over uninfected control. (D) RAW264.7 macrophages were transfected with the AP-1 reporter as in (C). Fourteen hours post-transfection cells were pre-treated with DMSO vehicle control, BAY-11-7085, or U0126 then infected for 8 h. Data are represented as % increase over uninfected samples. These data are representative of three independent experiments. *Designates a p value < 0.05.
Figure 5 Fos and Jun expression is highly up-regulated by infection in an NF-κB-dependent mechanism to control AP-1 activity. (A) PBM were pre-treated with DMSO or BAY-11-7085 (5 μM; designated as BAY in the figure) for 30 min then infected with F.n. at an MOI of 50 for 4 h. Cells were lysed and subject to western blotting for c-Fos, followed by actin re-probe. (B) Protein-matched lysates were probed for c-Jun and re-probed with actin antibody. (C) RAW264.7 macrophages were transfected with an AP-1 luciferase reporter. 14 h post-transfection cells were uninfected or infected with F.n. at an MOI of 50 for 1, 4, 8, or 24 h. Cells were lysed and luciferase activity was measured by a luminometer in triplicate. Data are represented as % increase over uninfected control. (D) RAW264.7 macrophages were transfected with the AP-1 reporter as in (C). Fourteen hours post-transfection cells were pre-treated with DMSO vehicle control, BAY-11-7085, or U0126 then infected for 8 h. Data are represented as % increase over uninfected samples. These data are representative of three independent experiments. *Designates a p value < 0.05.CC BY-NC · PubMed Central
Figure 6 MiR-155 induction is dependent upon c-Fos. (A) RAW 264.7 macrophages were transfected with 4.0 μM siRNA control or siRNA against c-Fos. Thirty minutes post-transfection cells were left uninfected or infected with F.n. at an MOI of 50 for 4 h. Cells were then lysed and subject to western blotting for c-Fos. An antibody against Actin was used to re-probe the blots to ensure equal loading. (B) RAW 264.7 macrophages were transfected as done in part A and infected for 16 h. RNA was collected and miR-155 expression was assayed by qRT-PCR. These data are representative of three independent experiments. *Designates a p value < 0.05.
Figure 6 MiR-155 induction is dependent upon c-Fos. (A) RAW 264.7 macrophages were transfected with 4.0 μM siRNA control or siRNA against c-Fos. Thirty minutes post-transfection cells were left uninfected or infected with F.n. at an MOI of 50 for 4 h. Cells were then lysed and subject to western blotting for c-Fos. An antibody against Actin was used to re-probe the blots to ensure equal loading. (B) RAW 264.7 macrophages were transfected as done in part A and infected for 16 h. RNA was collected and miR-155 expression was assayed by qRT-PCR. These data are representative of three independent experiments. *Designates a p value < 0.05.CC BY-NC · PubMed Central
Figure 7 MiR-155 is induced by microbes/microbial ligands with a concomitant decrease in SHIP expression. Human PBM were treated with LPS (500 ng/ml), R848 (1 uM), or infected with B. cenocepacia (designated as Bc in the figure) at MOI 5, or M. smegmatis (designated as Ms in the figure) at MOI 5. mRNA was isolated from the cells 16 h later and assayed by qRT-PCR for (A) miR-155 and (B) SHIP expression. These data are representative of six independent experiments. *Designates a p value < 0.05.
Figure 7 MiR-155 is induced by microbes/microbial ligands with a concomitant decrease in SHIP expression. Human PBM were treated with LPS (500 ng/ml), R848 (1 uM), or infected with B. cenocepacia (designated as Bc in the figure) at MOI 5, or M. smegmatis (designated as Ms in the figure) at MOI 5. mRNA was isolated from the cells 16 h later and assayed by qRT-PCR for (A) miR-155 and (B) SHIP expression. These data are representative of six independent experiments. *Designates a p value < 0.05.CC BY-NC · PubMed Central
Figure 8 MiR-155 induction by microbes/microbial ligands is NF-κB-dependent. (A–D) Human PBM were pre-treated with or without the NF-κB inhibitor (BAY-11-7085, designated as BAY in the figure), and subsequently treated with (A) LPS (500 ng/ml), (B) R848 (1 uM), or infected with (C) B. cenocepacia (Bc) at MOI 5, or (D) M. smegmatis (Ms) at MOI 5. mRNA was isolated from the cells 16 h later and assayed for miR-155 by qRT-PCR. These data are representative of six independent experiments. *Designates a p value < 0.05.
Figure 8 MiR-155 induction by microbes/microbial ligands is NF-κB-dependent. (A–D) Human PBM were pre-treated with or without the NF-κB inhibitor (BAY-11-7085, designated as BAY in the figure), and subsequently treated with (A) LPS (500 ng/ml), (B) R848 (1 uM), or infected with (C) B. cenocepacia (Bc) at MOI 5, or (D) M. smegmatis (Ms) at MOI 5. mRNA was isolated from the cells 16 h later and assayed for miR-155 by qRT-PCR. These data are representative of six independent experiments. *Designates a p value < 0.05.CC BY-NC · PubMed Central
Figure 9 c-Jun and c-Fos are induced by microbes/microbial ligands in an NF-κB-dependent manner. (A–D) Human PBM were pre-treated with or without the NF-κB inhibitor (BAY-11-7085, designated as BAY in the figure), and subsequently treated with (A) LPS (500 ng/ml), (B) R848 (1 uM), or infected with (C) B. cenocepacia (Bc) at MOI 5, or (D) M. smegmatis (Ms) at MOI 5. Cell lysates were collected 16 h later. Protein-matched lysates were analyzed by Western blotting with c-Jun or c-Fos antibodies. All membranes were re-probed with Actin antibody to ensure equal loading of protein in all lanes. These data are representative of four independent experiments.
Figure 9 c-Jun and c-Fos are induced by microbes/microbial ligands in an NF-κB-dependent manner. (A–D) Human PBM were pre-treated with or without the NF-κB inhibitor (BAY-11-7085, designated as BAY in the figure), and subsequently treated with (A) LPS (500 ng/ml), (B) R848 (1 uM), or infected with (C) B. cenocepacia (Bc) at MOI 5, or (D) M. smegmatis (Ms) at MOI 5. Cell lysates were collected 16 h later. Protein-matched lysates were analyzed by Western blotting with c-Jun or c-Fos antibodies. All membranes were re-probed with Actin antibody to ensure equal loading of protein in all lanes. These data are representative of four independent experiments.CC BY-NC · PubMed Central
Figure 10 MiR-155 induction by microbes/microbial ligands requires de novo protein synthesis. (A–D) Human PBM were pre-treated with or without the protein synthesis inhibitor (CHX), and subsequently treated with (A) LPS (500 ng/ml), (B) R848 (1 uM), or infected with (C) B. cenocepacia (Bc) at MOI 5, or (D) M. smegmatis (Ms) at MOI 5. mRNA was isolated from the cells 16 h later and assayed for miR-155 by qRT-PCR. These data are representative of six independent experiments. *Designates a p value < 0.05.
Figure 10 MiR-155 induction by microbes/microbial ligands requires de novo protein synthesis. (A–D) Human PBM were pre-treated with or without the protein synthesis inhibitor (CHX), and subsequently treated with (A) LPS (500 ng/ml), (B) R848 (1 uM), or infected with (C) B. cenocepacia (Bc) at MOI 5, or (D) M. smegmatis (Ms) at MOI 5. mRNA was isolated from the cells 16 h later and assayed for miR-155 by qRT-PCR. These data are representative of six independent experiments. *Designates a p value < 0.05.CC BY-NC · PubMed Central

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Metadata

Authors
Cremer, T. J., Fatehchand, K., Shah, P., Gillette, D., Patel, H., Marsh, R. L., Besecker, B. Y., Rajaram, M. V. S., Cormet-Boyaka, E., Kanneganti, T.-D., Schlesinger, L. S., Butchar, J. P., & Tridandapani, S.
Venue
Frontiers in Cellular and Infection Microbiology
Published
2012
Volume
2
ISSN
2235-2988
Cited by
29
References
2

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Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

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