Figures and data
Susceptible B6.Sst1S macrophages undergo a persistent pathological activation state (pPAS) during TNF stimulation.
A. Schematic of the experimental workflow. BMDMs from B6 and B6.Sst1S mice were stimulated with 10 ng/mL TNF for 24 h. Cells were either harvested immediately or cultured for an additional 16 h in fresh medium containing either TNF (TNF restimulation) or no TNF (TNF withdrawal) before harvest at 40 h after the initial stimulation. B. Persistent accumulation of ROS and 4-HNE adducts in B6.Sst1S BMDMs. BMDMs from B6 and B6.Sst1S mice were treated as described in panel A. Intracellular ROS levels (left panel) and accumulation of 4-HNE adducts (right panel) were subsequently assessed. ROS accumulation was detected using CellROX Green staining, and fluorescence images were acquired by fluorescence microscopy. Accumulation of the lipid peroxidation product 4-HNE was detected by confocal microscopy using a 4-HNE-specific antibody. Scale bar: 20 μm. C. IFN-I signaling remains persistently super-induced in B6.Sst1S BMDMs following TNF withdrawal. BMDMs from B6 and B6.Sst1S mice were treated as described in panel A. The mRNA expression levels of Ifnb1 were quantified by qRT-PCR at 24 and 40 h post stimulation. Fold change was calculated relative to the unstimulated B6 control using the ΔΔCt method with 18S as an internal control. D. Lipid biosynthesis genes remain downregulated in B6.Sst1S BMDMs following TNF withdrawal. BMDMs from B6 and B6.Sst1S mice were treated as described in panel A. The mRNA expression levels of Srebf2, Scd2, and Dhcr24 were quantified by qRT-PCR at 40 h post TNF stimulation. Fold change was calculated relative to the unstimulated B6 control using the ΔΔCt method with 18S as an internal control. E. B6.Sst1S BMDMs fail to respond to IFNγ during the pPAS state. B6.Sst1S BMDMs were stimulated with TNF for 18 h. After 18 h of TNF stimulation, IFNγ ( 5 U/mL) was added to the culture media, and cells were harvested at 40 h. The mRNA expression levels of Ciita was quantified by qRT-PCR. Fold change was calculated relative to the unstimulated control using the ΔΔCt method with 18S as an internal control. F. TNF restimulation during pPAS further enhances IFN-I signaling in B6.Sst1S BMDMs. BMDMs derived from B6 and B6.Sst1S mice were stimulated with TNF as described in panel A, including TNF restimulation at 24 h. Cells were harvested at 40 h, and Ifnb1 mRNA expression levels were quantified by qRT-PCR. Fold change was calculated relative to the unstimulated B6 control using the ΔΔCt method with 18S as an internal control. G. TNF restimulation sustains elevated ROS levels and further increases 4-HNE adduct accumulation in B6.Sst1S BMDMs. BMDMs derived from B6 and B6.Sst1S mice were treated as described in panel A, including TNF restimulation at 24 h. Intracellular ROS levels were assessed using CellROX Green staining, and fluorescence images were acquired by fluorescence microscopy (left panel). Accumulation of 4-HNE adducts was detected by confocal microscopy using a 4-HNE-specific antibody (right panel). Scale bar: 20 μm. H. Lipid biosynthesis genes remain suppressed in B6.Sst1S BMDMs during TNF restimulation. BMDMs derived from B6 and B6.Sst1S mice were treated as described in panel A, including TNF restimulation at 24 h. The mRNA expression levels of Srebf2 were quantified by qRT-PCR at 40 h. Fold change was calculated relative to the unstimulated B6 control using the ΔΔCt method with 18S as an internal control. The data are presented as mean ± standard deviation (SD) from three-five samples per experiment, representative of three independent experiments. The statistical analysis was performed by two-way ANOVA with Tukey’s multiple comparisons test (Panel D and E) and Sidak’s multiple comparison test (Panel F and H)), and one-way ANOVA with Tukey’s multiple comparisons test (Panel C) Significant differences are indicated with asterisks (ns, non significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001).
IFN-I autocrine signaling sustains pathological macrophage activation in TNF-independent manner.
A. Schematic of the experimental workflow. BMDMs from B6 and B6.Sst1S mice were stimulated with 10 ng/mL TNF for 24 h. Cells were either harvested immediately or cultured for an additional 16 h following replacement with fresh medium containing either TNF (TNF restimulation) or no TNF (TNF withdrawal). Where indicated, cells additionally received isotype control antibodies or anti-IFNAR antibodies (5 μg/mL) at the time of media replacement. Cells were harvested at 40 h after the initial TNF stimulation. B. IFN-I signaling suppresses IFNã responsiveness in B6.Sst1S BMDMs during the pPAS state. B6.Sst1S BMDMs were stimulated with TNF for 18 h, followed by addition of IFNã (5 U/mL) in the presence of either isotype control antibodies or anti-IFNAR antibodies, where indicated. Cells were harvested at 40 h after the initial TNF stimulation, and Ciita mRNA expression was quantified by qRT-PCR. Gene expression was normalized to 18S and expressed relative to unstimulated controls using the ΔΔCt method. C. IFN-I sustains the 4-HNE adducts accumulation in B6.Sst1S BMDMs during pPAS. BMDMs from B6.Sst1S mice were treated as described in A. The accumulation of lipid peroxidation product, 4-HNE was detected by confocal microscopy using 4-HNE specific antibody. Scale bar-20 μm. D. IFN-I signaling initiates and sustains lipid peroxidation product synthesis in B6.Sst1S BMDMs during TNF stimulation. B6.Sst1S BMDMs were stimulated with TNF, and either isotype control antibodies or anti-IFNAR antibodies were added at 2 h or 12 h after TNF stimulation. Cells receiving antibody treatment at 2 h were harvested at 12 h following TNF stimulation (top panel), whereas cells receiving antibody treatment at 12 h were harvested at 24 h following TNF stimulation (bottom panel). Lipid peroxidation product synthesis was assessed by LA alkyne staining. Representative fluorescence images are shown. Scale bar, 20 μm. E. IFNâ induces lipid peroxidation product synthesis independently of the sst1 locus. BMDMs from B6 and B6.Sst1S mice were treated with IFNβ (300 U/mL) for 12 h, followed by assessment of lipid peroxidation product synthesis by LA alkyne staining. Representative fluorescence images are shown. Scale bar, 20 μm. F. IFNâ induces 4-HNE adduct accumulation independently of the sst1 locus. BMDMs from B6 and B6.Sst1S mice were treated with IFNβ (300 U/mL) for 24 h, followed by immunofluorescence staining for 4-HNE adducts using 4-HNE specific antibodies. Representative confocal images are shown. Scale bar, 20 μm. G. Direct 4-octyl itaconate treatment induces 4-HNE adduct accumulation in B6.Sst1S BMDMs. B6.Sst1S BMDMs were treated with 4-octyl itaconate (4-OI) (5 μM) for 24 h, followed by immunofluorescence staining for 4-HNE adducts using 4-HNE specific antibodies. Representative confocal images are shown. Scale bar, 20 μm. H. IFNâ does not alter the labile iron pool in B6.Sst1S BMDMs. B6.Sst1S BMDMs were treated with TNF (10 ng/mL) or IFNβ (300 U/mL) for 24 h and the cells were stained with FerroOrange to assess intracellular labile iron levels. Representative fluorescence images are shown. Fluorescence intensity was quantified and expressed as fold change relative to untreated controls. Scale bar, 20 μm. I. IFN-I signaling does not regulate iron metabolism during TNF stimulation in B6.Sst1S BMDMs. B6.Sst1S BMDMs were stimulated with TNF (10 ng/mL), followed by addition of either isotype control antibodies or anti-IFNAR antibodies at 2 h post stimulation. Cells were stained with FerroOrange at 24 h to assess intracellular labile iron levels. Fluorescence intensity was quantified and expressed as fold change relative to isotype control antibody-treated samples. J. IFN-I signaling suppresses lipid biosynthesis gene expression during the pPAS state. B6.Sst1S BMDMs were treated as described in panel A under the TNF withdrawal condition. Cells were harvested at 40 h after the initial TNF stimulation, and expression of Srebf2, Scd2, and Dhcr24 was quantified by qRT-PCR. Gene expression was normalized to 18S and expressed relative to unstimulated controls using the ΔΔCt method. The data are presented as mean ± SD from three-five samples per experiment, representative of three independent experiments. The statistical analysis was performed by two-way ANOVA with Tukey’s multiple comparisons test (Panel J), and one-way ANOVA with Tukey’s multiple comparisons test (Panel B, H, and I). Significant differences are indicated with asterisks (ns, non significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001).
IFNγ priming prevents pPAS.
A. IFNã controls intracellular Mtb growth in both B6 and B6.Sst1S BMDMs following TNF stimulation. BMDMs were stimulated with TNF (10 ng/mL) in the presence or absence of IFNγ (5 U/mL). The following day, cells were infected with Mtb Erdman reporter strain (SSB-GFP, smyc’::mCherry) at MOI = 1 and cultured for 5 days. On day 5 post-infection, intracellular Mtb load was quantified by qRT-PCR-based method. B. IFNã priming attenuates TNF-induced oxidative stress and lipid peroxidation in B6.Sst1S BMDMs. B6.Sst1S BMDMs were primed with IFNã (5 U/mL) for 16 h, followed by TNF (10 ng/mL) stimulation for 36 h (24 h for LA alkyne). ROS levels were assessed using CellROX Green staining and quantified by fluorescence microscopy (top panel). Accumulation of 4-HNE adducts was evaluated by confocal microscopy using a 4-HNE-specific antibody (middle panel). Lipid peroxidation product synthesis was assessed by LA alkyne staining, with images acquired by confocal microscopy (bottom panel). Representative images are shown. Scale bar, 20 μm. C-D. IFNγ priming prevents IFN-I super-induction and integrated stress response during TNF stimulation. B6.Sst1S BMDMs were primed with IFNγ (5 U/mL) for 16 h, followed by TNF (10 ng/mL) stimulation for 24 h. The mRNA levels of Ifnb1 and Rsad2 (Panel C) and Trib3 and Chac1 (Panel D) were quantified by qRT-PCR. The fold change was calculated normalizing with the untreated control using ΔΔCt method using 18S as an internal control. E. IFN-I maintains Acod1 overexpression during TNF stimulation in B6.Sst1S BMDMs. B6.Sst1S BMDMS were treated with TNF (10 ng/mL) for 40 h and the mRNA levels of Acod1 was quantified by qRT-PCR. The fold change was calculated normalizing with the untreated control using ΔΔCt method using 18S as an internal control. Isotype control or anti-IFNAR antibodies were added to the culture at 2 h after TNF stimulation. F. IFNã priming prevents Acod1 overexpression during TNF stimulation. B6.Sst1S BMDMs were either primed with IFNγ (5 U/mL) for 16 h or left unprimed, followed by treatment with TNF (10 ng/mL) for an additional 24 h. The Acod1 mRNA expression was quantified by qRT-PCR and presented as fold change. Gene expression was normalized to 18S and expressed compared to unstimulated controls using the ΔΔCt method. G. IFNγ priming prevents 4-OI induced 4-HNE adducts accumulation. B6.Sst1S BMDMs were either primed with IFNγ (5 U/mL) for 16 h or left unprimed, followed by treatment with 4-octyl itaconate (4-OI; 5 μM) for an additional 36 h. 4-HNE adducts accumulation was observed by confocal microscopy using 4-HNE specific antibody. Scale bar-20 μm. H. IFNγ priming do not restore lipid biosynthesis gene expression during TNF stimulation. B6.Sst1S BMDMs were primed with IFNγ (5 U/mL) for 16 h, followed by TNF (10 ng/mL) stimulation for an additional 24 h. The mRNA levels of Srebf2 and Scd2 were quantified by qRT-PCR and presented as fold change. Gene expression was normalized to 18S and expressed compared to unstimulated controls using the ΔΔCt method. I. The PCA plot showing the combination of IFNγ and TNF leads to a distinct transcriptomic profile compared to the individual counterparts with least variation between B6 and B6.Sst1S macrophages. B6 and B6.Sst1S BMDMs were treated individually with IFNγ (5 U/mL) or TNF (10 ng/mL) or in combination for 12 h. Cells were harvested, and transcriptome was analyzed by RNA-seq. J. The combination of IFNγ and TNF causes similar transcriptomic profile in B6 and B6.Sst1S macrophages. The differential gene expression analysis showing the significantly upregulated and downregulated genes were common in B6 and B6.Sst1S macrophages when treated in combination of IFNγ and TNF compared to TNF alone. The differentially expressed genes with adjusted p-value < 0.05 and absolute fold change >1.5 were considered. K. The type II interferon response, glucose catabolic processes were upregulated, and cell cycle related processes were downregulated in both B6 and B6.Sst1S macrophages upon stimulation in combination of IFNγ and TNF compared to TNF alone. The gene set enrichment analysis based on biological processes comparing the IFNγ and TNF combination treatment to TNF alone in B6 and B6.Sst1S macrophages. The significant enriched pathways were defined with adjusted p-value < 0.05. L. The single sample gene set enrichment analysis showing downregulation of genes involved in pro-ferroptosis, cholesterol biosynthesis, lipid metabolism, cell cycle and upregulation of genes involved in Hif1a pathway, glycolysis when treated with both IFNγ and TNF in B6 and B6.Sst1S macrophages independent of sst1. M. IFNγ priming enhances ferritins heavy chain and ferritin light chain expression independent of sst1. B6.Sst1S BMDMs were primed with IFNγ (5 U/mL) for 16 h, followed by TNF (10 ng/mL) stimulation for 24 h. and Fth1 and Ftl1 protein levels were quantified by Western immunoblotting. β-tubulin was used as loading control. Average densitometric values from two independent experiments were included above the blot. N. IFNγ priming suppresses labile iron pool during TNF stimulation. B6.Sst1S BMDMs were primed with IFNγ (5 U/mL) for 16 h, followed by TNF (10 ng/mL) stimulation for 24. After 24 h the cells were stained with FerroOrange and fluorescence intensities were quantified. Bar graphs showing the fold change in fluorescence intensities compared to the untreated samples. (n=4). O. IFNγ priming enhances Hif1a protein levels in B6.Sst1S BMDMs. B6.Sst1S BMDMs were primed with IFNγ (5 U/mL) for 16 h, followed by TNF (10 ng/mL) stimulation for 24 h and HIF1α protein levels were quantified by Western immunoblotting. β-tubulin was used as loading control. Average densitometric values from two independent experiments were included above the blot. P. The iron metabolism regulated by IFNγ priming enhances Hif-1α protein levels during TNF stimulation in B6.Sst1S BMDMs. B6.Sst1S BMDMs were primed with IFNγ (5 U/mL) for 16 h, followed by stimulation with TNF (10 ng/mL) alone or in presence of either FeSO4 or deferiprone (DFP) for 24 h. Hif1a protein levels were quantified by Western immunoblotting. β-tubulin was used as loading control. Average densitometric values from two independent experiments were included above the blot. The data are presented as mean ± SD from three-five samples per experiment, representative of three independent experiments. The statistical analysis was performed by two-way ANOVA with Tukey’s multiple comparisons test (Panel A), and one-way ANOVA with Tukey’s multiple comparisons test (Panel C, D, E, F,H, and N). Significant differences are indicated with asterisks (ns, non significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001).
IFNγ mimetics prevents pPAS.
A. The PCA plot illustrating qualitative separation between TB-resistant and TB-sensitive states. Each dots shows average of 3 biological replicates, presented individually at Figure 2I. B. Comparison of DPD_TB scores of macrophage resistance towards Mtb (DPD_TB) calculated for B6.Sst1S (red) and B6 (blue) macrophages treated with TNF (10 ng/mL), IFNγ (5 U/mL) and their combination for 12 hours. The DPD scores were computed using the changes in macrophage transcriptomics patterns. Higher DPD_TB scores indicate the greater the Mtb resistance. C. cSTAR network predicted responses of the DPD_TB phenotypic score to activation of core network modules. The responses of the DPD_TB score were calculated using the inferred influences of the core network pathways depicted in (C). Negative values represent decreases in macrophage resistance to Mtb, positive values indicate enhanced Mtb resistance following an increase in each core pathway activity. D. Schematic of the experimental workflow. B6.Sst1S BMDMs were pretreated with CDK4/6 inhibitor, trilaciclib (3 μM) for 12 h followed by TNF (10 ng/mL) stimulation for 24 h or 36 h. E. Trilaciclib pretreatment prevents IFN-I super-induction during TNF stimulation. B6.Sst1S BMDMs were pretreated with trilaciclib (3 μM) for 12 h followed by TNF stimulation for additional 24 h. The mRNA levels of Ifnb1 and Rsad2 were quantified by qRT-PCR. The fold change was calculated by normalizing with the untreated control using ΔΔCt method. 18S was used an an internal control. F. Trilaciclib pretreatment reduces ROS accumulation and lipid peroxidation during TNF stimulation. B6.Sst1S BMDMs were pretreated with trilaciclib (3 μM) for 12 h, followed by TNF (10 ng/ml) stimulation for an additional 36 h (24 h for LA alkyne staining). Separate control groups were treated with either TNF alone or trilaciclib alone. Intracellular ROS levels were assessed using CellROX Green staining and quantified by fluorescence microscopy (top panel). Accumulation of 4-HNE adducts was evaluated by confocal microscopy using a 4-HNE-specific antibody (middle panel). Lipid peroxidation product synthesis was assessed by LA alkyne staining, and images were acquired by confocal microscopy (bottom panel). Representative images are shown. Scale bar: 20 μm. Bar graphs show the fold change in fluorescence intensity relative to untreated controls. Data represent mean ± SEM from three independent experiments (n=3). G. Trilaciclib pretreatment enhances ferritins heavy chain and ferritin light chain expression during TNF stimulation. B6.Sst1S BMDMs were pretreated with trilaciclib for 12 h followed by TNF stimulation for additional 24 h. Fth1 and Ftl1 protein levels were measured by western immunoblotting. Average densitometric values from two independent experiments were included above the blot. β-tubulin was used as loading control. H. Trilaciclib pretreatment suppresses labile iron pool during TNF stimulation. B6.Sst1S BMDMs were pretreated with trilaciclib (3 μM) for 12 h followed by TNF (10 ng/mL) stimulation for additional 24 h. Cells were stained with FerroOrange, and fluorescence intensities were quantified by florescence microscopy. Bar graphs showing the fold change in fluorescence intensities compared to the untreated samples. (n=5). I. Trilaciclib pretreatment enhances Hif1a protein levels in B6.Sst1S BMDMs. B6.Sst1S BMDMs were pretreated with trilaciclib (3 μM) for 12 h followed by TNF stimulation for additional 24 h. Hif1a protein level was measured by Western immunoblotting. Average densitometric values from two independent experiments were included above the blot. β-tubulin was used as loading control. J. Trilaciclib pretreatment enhances lipid biosynthesis gene expression during TNF stimulation. B6.Sst1S BMDMs were pretreated with trilaciclib (3 μM) for 12 h followed by TNF stimulation for additional 24 h. The mRNA levels of Srebf2, Scd2, and Dhcr24 were quantified by qRT-PCR. The fold change was calculated normalizing with the untreated control using ΔΔCt method. 18S was used as an internal control. K. Schematic of the experimental workflow. B6.Sst1S BMDMs were pretreated with All trans Retinoic acid (ATRA) (2 μM) for 12 h followed by TNF (10 ng/mL) stimulation for 24 h or 36 h. L. ATRA pretreatment show minimal effect ROS levels during TNF stimulation. B6.Sst1S BMDMs were pretreated with ATRA (2 μM) for 12 h, followed by TNF (10 ng/ml) stimulation for an additional 36 h. Separate control groups were treated with either TNF alone or ATRA alone. Intracellular ROS levels were assessed using CellROX Green staining and quantified by fluorescence microscopy (top panel). Accumulation of 4-HNE adducts was evaluated by confocal microscopy using a 4-HNE-specific antibody (bottom panel). Scale bar: 20 μm. Bar graphs show the fold change in fluorescence intensity relative to untreated controls. Data represent mean ± SEM from three independent experiments (n=3). M. ATRA pretreatment controls IFN-I super-induction during TNF stimulation. B6.Sst1S BMDMs were pretreated with ATRA (2 μM) for 12 h followed by TNF stimulation for additional 24 h. The mRNA levels of Ifnb1 and Rsad2 were quantified by qRT-PCR. The fold change was calculated by normalizing with the untreated control using ΔΔCt method. 18S was used an an internal control. N. ATRA pretreatment reduces labile iron pool during TNF stimulation. B6.Sst1S BMDMs were pretreated with ATRA (2 μM) for 12 h followed by TNF (10 ng/mL) stimulation for additional 24 h. Cells were stained with FerroOrange, and fluorescence intensities were quantified by florescence microscopy. Bar graphs showing the fold change in fluorescence intensities compared to the untreated samples. (n=5). O. ATRA pretreatment enhances ferritins heavy chain and ferritin light chain expression during TNF stimulation. B6.Sst1S BMDMs were pretreated with ATRA (2 μM) for 12 h followed by TNF stimulation for additional 24 h. Fth1 and Ftl1 protein levels were measured by western immunoblotting. Average densitometric values from two independent experiments were included above the blot. β-tubulin was used as loading control. P. ATRA pretreatment enhances GPX4 protein expression in B6.Sst1S BMDMs. B6.Sst1S BMDMs were pretreated with ATRA (2 μM) for 12 h followed by TNF stimulation for additional 24 h. GPX4 protein levels was measured by Western immunoblotting. Average densitometric values from two independent experiments were included above the blot. β-tubulin was used as loading control. Q. ATRA pretreatment enhances lipid biosynthesis gene expression during TNF stimulation. B6.Sst1S BMDMs were pretreated with ATRA (2 μM) for 12 h followed by TNF stimulation for additional 24 h. The mRNA levels of Srebf2, Scd2, and Dhcr24 were quantified by qRT-PCR. The fold change was calculated normalizing with the untreated control using ΔΔCt method. 18S was used as an internal control. The data are presented as mean ± SD from three-five samples per experiment, representative of three independent experiments. The statistical analysis was performed one-way ANOVA with Tukey’s multiple comparisons test (Panel E, F, H, J, L, M, N, and Q). Significant differences are indicated with asterisks (ns, non significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001).
The combination of CDK4/6 inhibition and RAR activation allows dose reduction while preventing pPAS and controlling intracellular Mtb growth.
A. cSTAR-inferred network that controls resistance priming of macrophages. Black arrows indicate activation, while red arrows indicate inhibition. The intensity of the color is proportional to the strength of connection. Perturbation data for THP1 cell line were used for network inference. B. Hierarchical clustering of the cSTAR-predicted global response matrix. Each column of the heatmap shows responses of all nodes to activation of the node in question, while each row of the heatmap shows responses of the node in question to activation of all other nodes. Correlation distance was used for the clustering algorithm. C. The combination of trilaciclib and ATRA enables dose reduction effectively suppressing 4-HNE adducts accumulation during TNF stimulation. B6.Sst1S BMDMs were pretreated with Trilaciclib and ATRA at different concentration combinations for 12 h followed by TNF (10 ng/mL) for 36 h. Accumulation of 4-HNE adducts was evaluated by confocal microscopy using a 4-HNE-specific antibody. Scale bar-20 μm. D. Heat map showing the average fold change in 4-HNE adducts fluorescence upon Trilaciclib and ATRA combinations compared to the untreated samples. (n=3). E-G. The combination of 30 nM of trilaciclib and 30 nM ATRA did not cause B6.Sst1S BMDMs cell death during Mtb infection. (n=4). The combination of trilaciclib and ATRA controls the intracellular Mtb growth. B6.Sst1S BMDMs were pretreated with trilaciclib (30 nM) and ATRA (30 nM or 60 nM) alone or in combination for 12 h followed by TNF (10 ng/mL) for 24 h. Following day, cells were infected with Mtb Erdman reporter strain (SSB-GFP, smyc’::mCherry) at MOI=1 for five days. Day 1 and 5 post infection, total cell numbers (E) were quantified by automated microscopy and intracellular Mtb load was quantified by qPCR based assay (G). Images of infected BMDMs were acquired by confocal microscopy 5 days post infection (F). Scale bar: 20 μm. (n=4). The data are presented as mean ± standard deviation (SD) from three-five samples per experiment, representative of three independent experiments. The statistical analysis was performed by one-way ANOVA with Tukey’s multiple comparisons test (Panel E and G). Significant differences are indicated with asterisks (ns, non significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001).
Differential regulation of pPAS features by interferons
IFNγ mimicking effect of trilaciclib and ATRA on pPAS features during TNF stimulation
A. ROS levels remain elevated in B6.Sst1S BMDMs following TNF withdrawal. BMDMs derived from B6 and B6.Sst1S mice were treated as described in Figure 1A. Accumulation of ROS was detected using CellROX Green staining, and fluorescence images were acquired by fluorescence microscopy. Bar graphs show the fold change in fluorescence intensity relative to unstimulated B6 controls. B. Accumulation of 4-HNE adducts persists in B6.Sst1S BMDMs following TNF withdrawal. BMDMs from B6 and B6.Sst1S mice were treated as described in Figure 1A. The accumulation of lipid peroxidation product, 4-HNE was detected by confocal microscopy using 4-HNE specific antibody. The 4-HNE accumulation was quantified using ImageJ and plotted as fold accumulation compared to untreated B6 group. C. IFN-I signaling remains persistently super-induced in B6.Sst1S BMDMs following TNF withdrawal. BMDMs from B6 and B6.Sst1S mice were treated as described in Figure 1A. The mRNA expression levels of Rsad2 were quantified by qRT-PCR at 24 and 40 h post TNF stimulation. Fold change was calculated relative to the unstimulated B6 control using the ΔΔCt method with 18S as an internal control. D. Lipid biosynthesis genes are downregulated in B6.Sst1S BMDMs during TNF stimulation. Heatmap showing the average fold change in FPKM values from RNA-seq analysis of B6 and B6.Sst1S BMDMs following 12 h TNF stimulation. Fold change was calculated relative to unstimulated controls. E. Lipid biosynthesis genes remain downregulated in B6.Sst1S BMDMs following TNF stimulation. BMDMs from B6 and B6.Sst1S mice were treated with 10 ng/mL TNF for 24 h. The mRNA expression levels of Srebf2, Scd2, and Dhcr24 were quantified by qRT-PCR. Fold change was calculated relative to the unstimulated B6 control using the ΔΔCt method with 18S rRNA as an internal control. F. TNF restimulation further enhances IFN-I signaling in B6.Sst1S BMDMs. BMDMs from B6 and B6.Sst1S mice were treated as described in Figure 1A, including TNF restimulation at 24 h. Cells were harvested at 40 h, and Rsad2 mRNA expression levels were quantified by qRT-PCR. Fold change was calculated relative to the unstimulated B6 control using the ΔΔCt method with 18S as an internal control. G. TNF restimulation during pPAS sustains elevated ROS levels in B6.Sst1S BMDMs. BMDMs from B6 and B6.Sst1S mice were treated as described in Figure 1A, including TNF restimulation at 24 h for additional 16 h. Accumulation of ROS was detected using CellROX Green staining, and fluorescence images were acquired by fluorescence microscopy. Bar graphs show the fold change in fluorescence intensity relative to unstimulated B6 controls. H. TNF restimulation during pPAS further increases 4-HNE adduct accumulation in B6.Sst1S BMDMs. BMDMs derived from B6 and B6.Sst1S mice were treated as described in Figure 1A, including TNF restimulation at 24 h for additional 16 h. The accumulation of lipid peroxidation product, 4-HNE was detected by confocal microscopy using 4-HNE specific antibody. The 4-HNE accumulation was quantified using ImageJ and plotted as fold accumulation compared to untreated B6 group. I. Lipid biosynthesis genes remain suppressed in B6.Sst1S BMDMs during TNF restimulation. BMDMs derived from B6 and B6.Sst1S mice were treated as described in Figure 1A, including TNF restimulation at 24 h for additional 16 h. Cells were harvested at 40 h, and the mRNA expression levels of Scd2 and Dhcr24 were quantified by qRT-PCR. Fold change was calculated relative to the unstimulated B6 control using the ΔΔCt method with 18S as an internal control. J. Sustained lipid peroxidation in B6.Sst1S BMDMs is independent of continued TNF signaling. B6.Sst1S BMDMs were stimulated with 10 ng/mL TNF for 24 h. Following the 24 h TNF stimulation, cells were treated with either isotype control antibodies or anti-TNFR antibodies and harvested at 36 h. The accumulation of lipid peroxidation product, 4-HNE was detected by confocal microscopy using 4-HNE specific antibody. The 4-HNE accumulation was quantified using ImageJ and plotted as fold accumulation compared to untreated group. Scale bar: 20 μm. The data are presented as mean ± SD from three-five samples per experiment, representative of three independent experiments. The statistical analysis was performed by two-way ANOVA with Tukey’s multiple comparisons test (Panel A, B, C, and E), Sidak’s multiple comparison test (Panel F, G, H, and I), and one-way ANOVA with Tukey’s multiple comparisons test (Panel J). Significant differences are indicated with asterisks (ns, non significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001).
A. IFN-I sustains the 4-HNE adducts accumulation in B6.Sst1S BMDMs during pPAS. Bar graphs showing the fold change in fluorescence compared to the unstimulated samples. N=3. B and C. IFN-I initiates and maintains lipid peroxidation product synthesis in B6.Sst1S BMDMs during TNF stimulation. B6.Sst1S BMDMs were stimulated with TNF. Either at 2 h or 12 h, isotype control or anti-IFNAR antibodies were added to the culture, and the LA alkyne staining was carried out at either 12 h (B) or 24 h (C), respectively. Bar graphs showing the fold change in fluorescence compared to the respective untreated samples. N=3. D. Direct 4-Octyl itaconate treatment leads to 4-HNE adducts accumulation. B6.Sst1S BMDMs were treated with 4-OI for 24 h and the cells were immuno-stained for 4-HNE adducts. Bar graphs showing the fold change in fluorescence compared to the untreated samples. n=3. E. IFN-I downregulates the lipid biosynthesis gene expression during pPAS. Cells were harvested at 40 h for TNF restimulation condition and the mRNA levels of Srebf2, Scd2, and Dhcr24 were quantified by RT-PCR. The fold change was calculated normalizing with the untreated control using ΔΔCt method using 18S as endogenous control. F and G. IFN-I do not regulate ROS during the pPAS. Confocal images showing the ROS during TNF withdrawal (F) or TNF restimulation (G) upon blocking IFN-I signaling. Scale bar-20 μm. Bar graphs showing the fold change in fluorescence compared to the unstimulated samples. n=3. The data are presented as mean ± SD from three-five samples per experiment, representative of three independent experiments. The statistical analysis was performed by two-way ANOVA with Tukey’s multiple comparisons test (Panel A, E, F, and G), and one-way ANOVA with Tukey’s multiple comparisons test (Panel B and C). Significant differences are indicated with asterisks (ns, non significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001).
A. Experimental workflow. B6.Sst1S BMDMs were primed with IFNγ 12 h prior TNF stimulation. Cells were harvested either at 24 h or 36 h to understand the role of IFNγ on the IFN-I maintained pathological activation state. B. IFNγ priming reduces ROS levels during TNF stimulation. Upon IFNγ priming followed by TNF stimulation, B6.Sst1S BMDMs were stained with CellROX Green at 36 h. Bar graphs showing the fold change in fluorescence compared to the untreated samples. N=3. IFNγ priming prevents 4-HNE adducts accumulation during TNF stimulation. Upon IFNγ priming followed by TNF stimulation, B6.Sst1S BMDMs were immuno-stained for 4-HNE adducts at 36 h. Bar graphs showing the fold change in fluorescence compared to the untreated samples. N=3. IFNγ priming prevents lipid peroxidation product synthesis during TNF stimulation. Upon IFNγ priming followed by TNF stimulation, B6.Sst1S BMDMs were stained for LA alkyne at 24 h. Bar graphs showing the fold change in fluorescence compared to the untreated samples. n=3. C. IFNγ priming prevents 4-OI induced 4-HNE adducts accumulation. B6.Sst1S BMDMs were treated with 4-OI with or without IFNγ priming. Cells were immuno-stained for 4-HNE adducts at 36 h. Bar graphs showing the fold change in fluorescence compared to the untreated samples. n=3. D. IFNγ priming do not restore lipid biosynthesis gene expression during TNF stimulation. Upon IFNγ priming followed by TNF stimulation, B6.Sst1S BMDMs were harvested at 24 h and the mRNA levels of Dhcr24 were quantified by RT-PCR. The fold change was calculated normalizing with the untreated control using ΔΔCt method using 18S as endogenous control. E. IFNγ priming do not enhance GPX4 expression. Upon IFNγ priming followed by TNF stimulation, B6.Sst1S BMDMs were harvested at 24 h GPX4 protein levels were quantified. Western blot images showing GPX4 protein expression considering β-tubulin as loading control. The average fold change in expression was mentioned compared to the untreated sample, normalized with β-tubulin levels. The data are presented as mean ± SD from three-five samples per experiment, representative of three independent experiments. The statistical analysis was performed by one-way ANOVA with Tukey’s multiple comparisons test (Panel B, C, and D). Significant differences are indicated with asterisks (ns, non significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001).
A. Palbociclib pretreatment partially prevents IFN-I super-induction during TNF stimulation. B6.Sst1S BMDMs were pretreated with palbociclib (3 μM) for 12 h then with TNF. After 24 h of TNF stimulation, cells were harvested and the mRNA levels of Ifnb1 and Rsad2 were quantified by RT-PCR. The fold change was calculated normalizing with the untreated control using ΔΔCt method using 18S as endogenous control. B. Trilaciclib pretreatment enhances Hi1f1a during TNF stimulation. B6.Sst1S BMDMs were pretreated with trilaciclib (3 μM) for 12 h then with TNF. After 24 h of TNF stimulation, cells were harvested and the mRNA levels of Hif1a were quantified by RT-PCR. The fold change was calculated normalizing with the untreated control using ΔΔCt method using 18S as endogenous control. C. Trilaciclib pretreatment do not enhance GPX4 expression. Upon Trilaciclib (3 μM) pretreatment followed by TNF stimulation, B6.Sst1S BMDMs were harvested at 24 h GPX4 protein levels were quantified. Western blot images showing GPX4 protein expression considering β-tubulin as loading control. The average fold change in expression was mentioned compared to the untreated sample, normalized with β-tubulin levels. The data are presented as mean ± SD from three-five samples per experiment, representative of three independent experiments. The statistical analysis was performed by one-way ANOVA with Tukey’s multiple comparisons test (Panel A and B). Significant differences are indicated with asterisks (ns, non significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001).
A-B. Higher concentration of Trilaciclib (1.5 μM) and ATRA (2 μM) induces cell loss in B6.Sst1S BMDMs under TNF stimulated and Mtb infected condition. B6.Sst1S BMDMs were pretreated with Trilaciclib (1.5 μM) and ATRA (2 μM) for 12 h followed by TNF stimulation for additional 24 h. Following day cells were infected with Mtb (MOI=1). Percent cell number was observed 5 days post infection using automated microscopy. (n=4). C – F. B6.Sst1S BMDMs were pretreated with trilaciclib (30 nM) and ATRA (30 nM or 60 nM), either alone or in combination, for 12 h followed by TNF stimulation (10 ng/mL) for an additional 24 h. Cells were subsequently infected with the Mtb Erdman reporter strain (SSB-GFP, smyc’::mCherry) at an MOI of 1 for 5 days. Total cell numbers were quantified by automated microscopy on days 1 and 5 post-infection (C). Combination treatment with 30 nM trilaciclib and either 30 nM or 60 nM ATRA reduced intracellular Mtb growth in B6.Sst1S BMDMs, as determined by qPCR-based assay on day 5 post-infection (D – F). Bar graphs show the fold change in Mtb burden from day 1 to day 5 post-infection following combination treatment. Data represent mean ± SEM from four independent experiments (n=4). The data are presented as mean ± standard deviation (SD) from three-five samples per experiment, representative of three independent experiments. The statistical analysis was one-way ANOVA with Tukey’s multiple comparisons test. Significant differences are indicated with asterisks (ns, non significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001).
