Fig 1.
M.tb induces CREB activation independent of cAMP in human macrophages.
MDMs were infected with M.tb H37Rv by synchronized phagocytosis at the indicated MOI. A) Western blot was performed to detect levels of phosphorylated and total CREB protein at the indicated time points. WB is representative of n = 4 donors. B) Densitometry analysis was performed and ratios of pCREB/total CREB were determined. Data are cumulative ± SEM of n = 4 donors. One-way ANOVA with Tukey’s post-test. C) MDMs were stimulated with cAMP agonists PGE2 or forskolin ± PDE inhibitor IBMX for 30 min. Data are representative ± SD of n = 4 donors. One-way ANOVA with Tukey’s post-test. D) cAMP levels in M.tb-infected (MOI 5) MDM lysates were determined. Graph is representative ± SD of n = 11 donors. One-way ANOVA with Tukey’s post-test. E) MDMs were infected with different strains of mycobacteria (MOI 5) or forskolin + IBMX as a control for cAMP production. Lysates were collected and analyzed for cAMP production. Dotted line indicates minimum level of detection for the assay. Data are representative ± SD of n = 2 donors; *p < 0.05, ***p < 0.001, ****p < 0.0001.
Fig 2.
M.tb-induced CREB activation is p38-dependent and ERK-independent.
A) MDMs were infected with M.tb H37Rv by synchronized phagocytosis at the indicated MOI. WB was performed to detect levels of phosphorylated and total p38 and ERK1/2 protein at the time points specified. Data are representative of n = 4 donors. B,C) Densitometry of phosphorylated protein normalized to total protein. Data are cumulative ± SEM of n = 4 donors. One-way ANOVA with Tukey’s post-test. D-E) MDMs were pretreated with SB203580 or UO126 for 60 min prior to infection with M.tb H37Rv at MOI 10. D) WB was performed to detect levels of phosphorylated and total levels of CREB, p38, ERK1/2, and MK2 protein at the specified time points. Data are representative of n = 4 donors. E) Densitometry of phosphorylated protein normalized to total protein. Data are cumulative ± SEM of n = 4 donors. One-way ANOVA with Tukey’s post-test; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
Fig 3.
M.tb-induced CREB activation is important for induction of immediate early genes in human macrophages.
MDMs were pretreated with DMSO or 666–15 for 60 min and subsequently infected with M.tb H37Rv at the MOI 10 (A-F,H) by synchronized phagocytosis or MOI 5(E). RNA or protein lysates were collected at the indicated time points. A-D) Gene expression of COX2, MCL-1, CCL8 and c-FOS was determined by qRT-PCR and data are shown as fold change ± SEM compared to uninfected MDMs. Data are cumulative of n = 3–5 donors. One-way ANOVA with Tukey’s post-test. E) Cell lysates were probed by WB for COX2, MCL-1 and β-actin. A representative experiment is shown of n = 3–4 donors. F,H) Densitometry at 3h post infection compared to M.tb-infected MDMs. Data are cumulative ± SEM of n = 3–4 donors. Unpaired t test. G) PGE2 production was measured by competitive binding ELISA at 6h post infection. Data are cumulative ± SEM of n = 4 donors. Unpaired t test; *p < 0.05, **p < 0.01, ****p < 0.0001.
Fig 4.
Inhibition of M.tb-induced CREB activation allows for prolonged nuclear colocalization of NF-kB p65 in human macrophages.
MDMs were pretreated with DMSO or the CREB inhibitor, 666–15, for 60 min then infected with M.tb H37Rv at MOI 10. A) At 1h and 3h post-infection, cells were fixed, permeabilized, and stained for NF-kB p65 (red) and DAPI in the nucleus (blue). MFI of NF-kB p65 signal that colocalized with DAPI (magenta) was calculated using ImageJ software, normalized to total number of cells per field and graphed as fold change ± SEM compared to infected cells. Data are cumulative of n = 3–6 donors. One-way ANOVA with Tukey’s post-test. B) Images were taken at 3h post-infection at 20x magnification. Images shown are representative of n = 3–6 donors; *p < 0.05, **p < 0.01, ****p < 0.0001.
Fig 5.
M.tb-induced CREB activation is important for bacterial growth in human macrophages.
A) MDMs were pretreated for 60 min with CREB inhibitor 666–15 or DMSO, then infected with M.tb H37Rv MOI 2 and CFUs were determined at the indicated time points. Data are cumulative ± SEM of n = 6 donors. Unpaired t test. B) MDMs were transfected with CREB targeting siRNA or scrambled control for 72h prior to infection with M.tb H37Rv MOI 2. A representative WB is shown and graphed data are cumulative ± SEM of n = 2 donors. C) CFUs were determined at the indicated time points. Data are representative ± SD of n = 2 donors. Unpaired t test; *p < 0.05, **p < 0.01, ***p < 0.001.
Fig 6.
CREB inhibition induces activation of the necroptotic signaling pathway in M.tb-infected macrophages, but does not affect cell viability.
MDMs were pretreated with 666–15 or DMSO control for 60 min and subsequently infected with M.tb H37Rv at MOI 2 (A,D) or MOI 10 (B,C). A) Bright field images of the cells were taken at 40x; Data shown are representative of n = 10 donors. B,C) Cell lysates were collected and probed by WB blot for the indicated phosphorylated and total proteins. Densitometry was determined and graphed as fold change ± SEM of phosphorylated protein to total protein; A representative experiment is shown and graphed data are cumulative of n = 4 donors. One-way ANOVA with Tukey’s post-test. D) Membrane integrity and cell viability was determined by Cytotox Glo assay. Data are cumulative ± SEM of n = 3 donors. Two-way ANOVA with Tukey’s post-test; *p < 0.05, **p < 0.01, ***p < 0.001.
Fig 7.
pMLKL induced by CREB inhibition in human macrophages infected with M.tb is dependent on RIPK1/RIPK3.
A) MDMs were pretreated with for 60 min with DMSO or CREB inhibitor 666–15 +/- Nec-1 or GSK’872 then infected or not with M.tb H37Rv MOI 10. Cell lysates were collected and probed by WB for phosphorylated and total MLKL at the indicated time points. A representative experiment is shown of n = 5, 4 donors. B) Densitometry was determined and graphed as fold change ± SEM of phosphorylated protein to total protein. n = 5, 4 donors. One-way ANOVA with Tukey’s post-test; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
Fig 8.
CREB signaling in human macrophages inhibits phagolysosomal fusion.
A) MDMs were plated on glass coverslips and pretreated with DMSO or CREB inhibitor 666–15 for 60 min then infected with mCherry expressing M.tb H37Rv (red) MOI 10. MDMs were fixed, permeabilized, and stained for LAMP-1 (green) and DAPI (blue). A representative experiment is shown of n = 3 donors. B) At the indicated time points, the percent of M.tb colocalizing with LAMP-1 was calculated following manual counting. White arrows indicate colocalization. Data are cumulative ± SEM of n = 3 donors. Unpaired t test; *p < 0.05.
Fig 9.
Increased phagolysosomal fusion during CREB inhibition requires RIPK3 activity.
A) MDMs were plated on glass coverslips and pretreated for 60 min with DMSO or CREB inhibitor 666–15 +/- Nec-1, GSK’872, or NSA, then infected with mCherry M.tb H37Rv (red) MOI 10. MDMs were fixed, permeabilized, and stained for LAMP-1 (green) and DAPI (blue). A representative experiment is shown of n = 4–5 donors. B) At the indicated time points, the percent of M.tb colocalizing with LAMP-1 was calculated following manual counting. White arrows indicate colocalization. Data are cumulative ± SEM of n = 4–5 donors. One-way ANOVA with Tukey’s post-test; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
Fig 10.
MLKL is essential for phagolysosomal fusion in the absence of CREB signaling in M.tb-infected human macrophages.
MDMs were plated on glass coverslips and transfected with siRNA targeting MLKL or scrambled control siRNA for 48h. MDMs were then pretreated for 60 min with CREB inhibitor 666–15 and infected with mCherry M.tb H37Rv (red) MOI 10. A) Knockdown of MLKL was verified by WB and percent signal quantified relative to scrambled control. A representative experiment is shown and graphed data are cumulative of n = 3 donors. Unpaired t test. B) MDMs were fixed, permeabilized, and stained for LAMP-1 (green) and DAPI (blue). A representative experiment is shown of n = 3 donors. C) At the indicated time points, the percent of M.tb colocalizing with LAMP-1 was calculated following manual counting. Data are shown as fold change of percent colocalization compared to scrambled control for each time point. White arrows indicate colocalization. Data are cumulative ± SEM of n = 3 donors. Unpaired t test; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
Fig 11.
In human macrophages, M.tb infection induces phosphorylation of p38 MAPK resulting in downstream phosphorylation of the transcription factor CREB. A) Phosphorylated CREB enters the nucleus and forms a transcriptional complex binding coactivator CREB binding protein (CBP), corresponding with decreased nuclear colocalization of NF-kB, which also requires CBP [10]. Activated CREB transcribes M.tb-induced genes including early expression of COX2, MCL-1, CCL8 and c-FOS. CREB also prevents phosphorylation of RIPK3 and MLKL (red blocking arrow) limiting pMLKL-dependent lysosomal fusion with the M.tb-containing phagosome, all things that contribute to intramacrophage bacterial growth. B) When CREB binding to CBP is inhibited with 666–15 constraining CREB signaling (red blocking arrow), less CREB is localized to the nucleus. In contrast, NF-kB nuclear localization is increased during CREB inhibition. It is possible that CBP is now available to bind NF-kB, likely leading to increased transcription of NF-kB regulated gene expression. CREB inhibition also leads to increased phosphorylation of RIPK3 and MLKL, resulting in increased phagolysosomal fusion (P-L fusion) corresponding with decreased bacterial growth in human macrophages. Phosphorylated MLKL can be expelled in extracellular vesicles or tagged for proteosomal or lysosomal degradation (pMLKL in red endosome), preventing necroptosis and macrophage death [38,43].