CREB inhibition induces activation of the necroptotic signaling pathway in <i>M</i>.<i>tb</i>-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.</p

Increased phagolysosomal fusion during CREB inhibition requires RIPK3 activity.

A) MDMs were plated on glass coverslips and pretreated with 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.</p

Treatment with 666–15 inhibits CREB nuclear colocalization.

MDMs were plated on glass coverslips and pretreated with DMSO or the CREB inhibitor, 666–15, for 60 min then infected with M.tb H37Rv at MOI 10. At 1h post-infection, cells were fixed, permeabilized, and stained for pCREB (red) and DAPI in the nucleus (blue). A) MDMs were imaged at 20x and a representative experiment is shown of n = 5 donors. B) MFI of pCREB signal that colocalized with DAPI (magenta) was calculated using ImageJ Fiji software, normalized to total number of cells per field and graphed as fold change ± SEM compared to infected, cells. Data are cumulative of n = 5 donors. C) MDMs were pretreated as described and infected with mCherry M.tb H37Rv. Bacteria associated with MDMs were counted and normalized to total cell number in each field and graphed as fold change ± SEM compared to M.tb infected cells. Data are cumulative of n = 4 donors; p (TIF)</p

Phosphorylation of CREB in human macrophages based on <i>M</i>.<i>tb</i> MOI.

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 1h post-infection. WB is representative of n = 2 donors. B) Densitometry analysis was performed and ratios of pCREB/total CREB were determined. Data are cumulative ± SEM of n = 2 donors. One-way ANOVA with Tukey’s post-test; *p (TIF)</p

<i>M</i>.<i>tb</i> 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.</p

Inhibition of the necroptotic signaling pathway is not sufficient to affect phagolysosomal fusion in <i>M</i>.<i>tb</i>-infected human macrophages.

A) MDMs were plated on glass coverslips and pretreated with 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 = 2–3 donors. B) At 2h post-infection, the percent of M.tb colocalizing with LAMP-1 was calculated following manual counting. White arrows indicate colocalization. Data are representative ± SD of n = 2–3 donors. One-way ANOVA with Tukey’s post-test; *p (TIF)</p

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, ***p < 0.01.</p

MLKL is essential for phagolysosomal fusion in the absence of CREB signaling in <i>M</i>.<i>tb</i>-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.</p

Effect of <i>M</i>.<i>tb</i> infection and CREB inhibition on expression of select genes and c-FOS protein.

MDMs were pretreated with DMSO or 666–15 for 60 min and subsequently infected with M.tb H37Rv at the MOI 10 by synchronized phagocytosis. A) RNA was collected at the indicated time points and gene expression of the indicated genes was determined by qRT-PCR. 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. B) Cell lysates were probed by WB for c-FOS and β-actin. Shown is a representative experiment of n = 3. C) Densitometry at 3h post infection compared to M.tb-infected MDMs. Data are cumulative ± SEM of n = 3 donors. Unpaired t test; *p (TIF)</p

pMLKL induced by CREB inhibition in human macrophages infected with <i>M</i>.<i>tb</i> 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.</p