TOLLIP inhibits lipid accumulation and the integrated stress response in alveolar macrophages to control Mycobacterium tuberculosis infection

Abstract

A polymorphism causing deficiencies in Toll-interacting protein (TOLLIP), an inhibitory adaptor protein affecting endosomal trafficking, is associated with increased tuberculosis (TB) risk. It is, however, unclear how TOLLIP affects TB pathogenesis. Here we show that TB severity is increased in Tollip^-/- mice, characterized by macrophage- and T cell-driven inflammation, foam cell formation and lipid accumulation. Tollip^-/- alveolar macrophages (AM) specifically accumulated lipid and underwent necrosis. Transcriptional and protein analyses of Mycobacterium tuberculosis (Mtb)-infected, Tollip^-/- AM revealed increased EIF2 signalling and downstream upregulation of the integrated stress response (ISR). These phenotypes were linked, as incubation of the Mtb lipid mycolic acid with Mtb-infected Tollip^-/- AM activated the ISR and increased Mtb replication. Correspondingly, the ISR inhibitor, ISRIB, reduced Mtb numbers in AM and improved Mtb control, overcoming the inflammatory phenotype. In conclusion, targeting the ISR offers a promising target for host-directed anti-TB therapy towards improved Mtb control and reduced immunopathology.

Extended Data 1.. Tollip^−/− macrophages are hyperinflammatory.

A-B) Peritoneal extract macrophages (PEM) were isolated from Tollip^−/− and B6 mice, plated, stimulated with control (Uns), LPS (10ng/ml), PAM3 (250ng/ml), or Mtb whole cell lysate (1μg/ml) overnight and A) TNF and B) IL-10 were measured from cellular supernatants by ELISA.C-E) PEM were infected with Mtb H37Rv strain (MOI 2.5) overnight ex vivo, then C) TNF, D) IL-1β, and E) IL-10 were measured from cellular supernatants by ELISA. F-H) Baseline immune cell number in Tollip^−/− mice. Total number of F) splenocytes, G) CD3+, C4+ CD8+, F4/80+, and NK1.1+ splenocytes, and the H) proportion of alveolar macrophages (AM) in the lungs of healthy 8-week-old mice, measured by flow cytometry. N = 3 measurements per experiment; each experiment was performed twice. * p<0.05, student’s two sided t-test.

Extended Data 2.. Related to Figure 1. Characteristics of Mtb-infected Tollip^−/− lungs.

A-G) Gating strategy used to identify Mtb-infected lung-resident myeloid cell subsets. A) Live/Dead Fixability dye was used to exclude dead cells, B) AM were identified by coexpression of SiglecF and CD11c. C) SiglecF− cells were gated; CD11b+Ly6G+ cells were classified as neutrophils, and CD11b+Ly6G− cells were considered macrophages. D) MHC-II+CD11c+ macrophages were subclassified as monocyte-derived macrophage cells (MDM) and MHC-II+CD11c− as interstitial macrophages (IM). E) CD11b-Ly6G− cells were gated and MHC-II+CD11c+ cells were classified as conventional DC (cDC) or F) CD103+ cDC) was measured. G) Representative flow cytometry images of the proportion of Mtb-infected myeloid subsets 28 and 56 days post infection (dpi). H-I) Proportion of total alveolar macrophages (AM), monocyte-derived macrophages (MDM), interstitial macrophages (IM), neutrophils (PMN), and dendritic cells (DC) in Mtb-infected mice H) 28 and I) 56 days after infection; n= 10; two-sided t-test. J) Representative flow cytometry images of Mtb-mCherry expression in AM and MDM from Rag1^−/− and Tollip^−/−Rag1^−/− mice 28 days after infection.

Extended Data 3.. Related to Figure 2. Gating strategy used to identify lung-resident myeloid cells in mixed bone marrow chimeric mice.

A-D) Mixed bone marrow chimeric mice were infected with Mtb H37Rv expressing mCherry reporter plasmid (Mtb-mCherry) (50–100 CFU) via aerosol. At selected time points flow analysis was performed to identify populations infected with Mtb. Myeloid populations were identified as in Extended Data 2. A) Alveolar macrophages (AM), neutrophils (PMN), monocyte-derived macrophages (MDM), andinterstitial macrophages (IM) were subclassified as B6 or Tollip^−/− based on CD45.1/CD45.2 or CD45.2 expression, respectively, and the proportion of mCherry-Mtb cells measured. Representative images from 28 days post infection are shown. E) Representative image of CD45.2+ Tollip^−/− AM adoptively transferred into CD45.1+ mice 56 days after AM depletion.

Extended Data 4.. Related to Figure 3. Gating strategy used for sorting Mtb-infected AM.

Mixed bone marrow mice were infected with Mtb H37Rv (50–100 CFU) via aerosol and AM were sorted for RNA seq analysis 28 days post infection, from left to right. A) Lymphocyte gating, followed by B) Singlet identification. C) Live/Dead Fixability dye was used to exclude dead/dying cells, and D) SiglecF+ CD11c+ cells were classified as AM. E) Uninfected and Mtb-infected AM were identified by mCherry expression F) Uninfected AM and G) Mtb-infected AM genetic lineage was defined based on CD45.1/CD45.2 (F1 B6) or CD45.2 (Tollip^−/−) expression, respectively.

Extended Data 5.. Related to Figure 4. Tollip is dispensable for bulk autophagy in macrophages.

Bone marrow from B6 (white bars) or Tollip^−/− (black bars) were differentiated ex vivo to macrophages using 40ng/mL M-CSF for 7–9 days. Following differentiation, BMDMs were treated for 6hr with or without 250nM Bafilomycin A (BafA). A) Representative western blot results showing protein levels of LC3I/II and p62. B) Quantification of protein levels (by densitometry) of LC3II using total LC3 (I+II) as a loading control. ***p<0.001 2-way ANOVA for an effect of BafA. C) Quantification of p62 levels (by densitometry) using vinculin as a loading control. p=0.056 2-way ANOVA effect of BafA. D-F) TNF concentrations in the supernatants of TOLLIP-deficient D) BMDM, E) alveolar macrophages (p=0.02 between genotypes in Mtb and p=0.03 in MA+Mtb groups; AM), and F) THP-1 cells 24 hours after Mtb infection (MOI 5) and mycolic acid (MA; 10μg/ml) treatment by ELISA (p=0.0002 between genotypes in Mtb and p<0.0001 in MA+Mtb groups; AM)* p < 0.05, ** p<0.01, *** p<0.001, two-sided t-test. Experiment was performed in BMDM twice and all other cell types three times. G) Expression of EIF2AK1, EIF2AK2, EIF2AK4, and TOLLIP in human whole blood in heathy controls (control) or patients with latent tuberculosis infection (LTBI), active symptomatic pulmonary TB disease (Mtb). EIF2AK3 was not detected in this dataset. Data are shown as violin plots with lines indicating 25^th, 50^th, and 75^th percentile, extending to minimum and maximum value. Obtained from GSE 19491.

Extended Data 6.. Related to Figure 6. Evaluation of integrated stress responses in Tollip^−/− mice and macrophages

A-C) B6 and Tollip^−/− peritoneal extract macrophages (PEM) were incubated with media or mycolic acid (MA; 10μg/ml) for 72 hours, then infected with Mtb (MOI 1) overnight. mRNA transcripts of key regulatory genes of the cellular stress response were measured before and after Mtb infection. A) Ern1 (IRE1a; p = 0.001 for media, p= 0.049 for MA), B) Eif2ak3 (PERK; p = 0.002 for media, p= 0.007 for MA), and C) Atf6 (ATF6; p = 0.02 for media) were measured and displayed as their fold change from baseline after Mtb infection. FC = (Normalized mRNA expression after Mtb infection) / (Normalized mRNA expression after media control stimulation). N=2/group and are representative of at least two independent experiments. D) Western blot of PEM incubated as above measuring pEIF2 and tubulin expression. E) Optical density at 600nm (OD600) of Mtb H37Rv in 7H9 broth culture in the presence of raphin-1 (10 μM), ISRIB (250 nM), or vehicle control over time. N = 2 over two independent experiments; error bars – SEM. F-H) TNF concentrations in cellular supernatants from TOLLIP-deficient F) bone marrow-derived macrophages (BMDM), G) alveolar macrophages (AM; p = 0.04 for Mtb+MA, p=0.003 for Mtb+ISRIB) and H) THP-1 cells (p = 0.002 for Mtb+MA, p=0.005 for Mtb+ISRIB, p=0.004 for Mtb+MA+ISRIB), after 24 hours of Mtb infection (MOI 5), mycolic acid (MA, 10μg/ml), and ISRIB (250nM), measured by ELISA. This experiment was performed twice, each with three technical replicates. * p < 0.05, ** p <0.01, two-sided t-test.

Extended Data 7.. Images related to Figure 6 histocytometry studies.

A) Representative confocal microscopy image of the lungs of a B6 mouse infected with Mtb for 56 days. Yellow – SiglecF; green – CD11b; red – pEIF2; blue – LipidTox; white --- PPD. B) Histocytometry positional mapping of AM and MDM within the Mtb-infected lung. C) Proportion of pEIF2+ PPD+ AM (p = 0.017 for overall ANOVA effect) and MDM (p = 0.016 for overall ANOVA effect) in B6 and Tollip^−/− mice at baseline and after ISRIB treatment. p = 0.02 for overall ANOVA effect. * p<0.05, one-sided ANOVA. N = 4 B6 control mice, N = 5 B6 ISRIB mice, N = 6 Tollip^−/− control, and N = 6 Tollip^−/− ISRIB mice. D) Representative images staining from the lungs of Mtb-infected B6 and Tollip^−/− mice with and without ISRIB treatment. Red -- pEIF2; green -- CD68; blue – SiglecF; white -- PPD. E) Spatial correlation analysis of cell types within 20-mm-radius neighborhoods in B6 and Tollip^−/− mice. Red shades indicate positive correlation, and blue shades indicate negative correlation.

Extended Data 8.. Overall experimental model

. A) Basal homeostasis. During chronic Mtb infection, lipid products are released in Mtb-infected AM. TOLLIP prevents lipid accumulation and controls inflammation, which maintains EIF2 signaling at a basal level. B) Tollip^−/− AM. Tollip^−/− mice develop excess TNF and IFNα responses from macrophages and T cells. Mtb-infected Tollip^−/− AM undergo lipid accumulation, which increases EIF2 phosphorylation. Excess pEIF2 induces sensitivity to inflammation in AM. C) Chronic infection in Tollip^−/− AM. During prolonged infection, increased and prolonged EIF2 phosphorylation from lipids and inflammation leads to cellular necrosis, decreasing the Mtb burden within individual AM and releasing extracellular Mtb. D) ISRIB treatment. ISRIB improves AM host defense, which improves Mtb control in both B6 and Tollip^−/− mouse models, making it an effective therapeutic across genetic backgrounds.

Figure 1.. TOLLIP is required for Mtb control in mice

A) Experimental timeline. B-C) Bacterial burden in B6 (clear circle) and Tollip^−/− (black square) mice in B) lung (p = 0.0016) and C) spleen (p = 0.0092). ** p < 0.01 *** p < 0.001, two-sided mixed effects model. n = 50. Error bars – SEM. D) Percentage of initial body weight. Circle – B6 mice; square - Tollip^−/− mice. N = 20 *** p < 0.001, P = 0.0005, two-way ANOVA accounting for time and genotype. Error bars – SEM. E) Survival curve analysis of B6 (clear circle) and Tollip^−/− (black square) mice after aerosol infection. N = 20. *** p < 0.001, two-sided Mantel-Cox test. F) Hematoxylin and eosin stained lung tissue 28 and 56 days after Mtb infection. Low magnification scale bar - 250μm. High magnification scale bar - 100μm. G-H) Fluorescence lipid staining in Mtb-infected lungs 56 days after infection. Lipid (green) and DAPI (blue); scale bar – 10μm. H) Integrated fluorescence density of lipid staining (area of excess fluorescence above background x fluorescence intensity). N = 20 fields / genotype of four independent experiments. **** p < 0.0001, two-sided Mann-Whitney test. I-J) The percentage of Mtb-infected (mCherry+) alveolar macrophages (AM), monocyte-derived macrophages (MDM), interstitial macrophages (IM), and neutrophils (PMN) G) 28 and H) 56 days after infection. n = 5/group; * p < 0.05, two-sided t-test. K-L) The proportion of lung-resident K) ESAT6(4–17)+CD44+CD4+ T cells after Mtb infection. L) Representative image. N = 5 / group 28 (p=0.002) and 56 days after infection, N = 4 180 days after infection. * p<0.05, two-sided t-test. Error bars – SEM. M-N) The proportion of M) PD1-KLRG1+ESAT6+CD4+CD44+ T cells and N) representative image after Mtb infection. N = 9 mice / genotype at days 28 and 56 (p=0.016), n = 4 mice/genotype at day 180 (p=0.007) after infection, error bars represent SEM. * p<0.05, ** p<0.01, two-sided t-test.

Figure 2.. Tollip^−/− AM exhibit diminished Mtb intracellular carriage in a cell-autonomous manner

A) Experimental outline. B) Lung bacterial burden in chimeric mice 56 days after Mtb infection (dpi). N = 20. * p < 0.05, ** p < 0.01. Two-sided ANOVA with Tukey’s test. P = 0.0023 for B6B6 vs Tollip−/−B6, p= 0.01 for B6B6 vs Tollip−/−Tollip−/−, p =0.043 for Tollip−/−Tollip−/− vs B6Tollip-/−. C) Bacterial burden in T cell chimeric mice lungs 56 dpi. N = 11. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p<0.0001, two-sided t-test. D) Bacterial burden in the lungs of B6 (n = 5), Tollip^−/− (n = 5), Rag1^−/− (n = 8), and Rag1^−/−Tollip^−/− (n = 9) mice 28 dpi. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p<0.0001, two-sided t-test. E-F) Proportion of Mtb-infected (mCherry+) alveolar macrophages (AM), monocyte-derived macrophages (MDM), and neutrophils (PMN) from Rag1^−/− (clear circle) and Tollip^−/− Rag1^−/− (black square) mice E) 28 (n = 17) and F) 44 (n = 22) dpi. P = 0.04 for 2F. * p < 0.05, ** p <0.01, *** p < 0.001, **** p < 0.0001; two-sided t-test. G) Mixed bone marrow chimera experimental outline. H) Distribution of CD45 expression in naïve mixed bone marrow chimeric mice. N = 8. I) Composition of mCherry+ cells among measured lung myeloid cell subpopulations after Mtb infection. N = 5, Bars - mean; error bar - SD. J-K) The proportion of J) Mtb-infected lung-specific myeloid cells (P=0.015 for AM and p=0.023 for PMN) and K) MFI of mCherry in Mtb+ AM 28 dpi (p = 0.0076) in mixed bone marrow chimeric mice. Lines connect genotypes from paired samples. * p < 0.05, ** p < 0.01, paired two-sided t-test. N = 5/group. L-M) The proportion of Mtb-infected and Mtb CFU / 1000 from sorted L) AM (p=0.0004 and p=0.08, respectively) or M) MDM from mixed bone marrow chimeric mice 56 dpi. Lines connect B6 (white circle) and Tollip^−/− (black square) genotypes. *** p < 0.001, paired two-sided t-test. N = 7/group. N) Representative images from M.

Figure 3.. Tollip^−/− AM undergo increased necrosis in a cell-autonomous manner during prolonged Mtb infection

A) Proportion of apoptotic (Annexin V+) and necrotic (amine reactive dye+) AM from mixed bone marrow chimeric mice 28 days after infection, stratified by genotype. Lines connect B6 (white circle) and Tollip^−/− (black square) genotypes from paired samples. P = 0.005. ** p < 0.01, paired two-sided t-test. N = 5. Representative images from B6 and Tollip^−/− genotype with Annexin V flow minus one (FMO) control are shown, demonstrating proportions of amine-reactive dye+Annexin V- (necrotic) cells on the top line, and total proportion of Annexin V+ cells (apoptotic) on the bottom line. B) Proportion of necrotic AM (amine reactive dye+) in mixed bone marrow chimeric mice 56 days after Mtb infection with representative images. Lines connect B6 (white circle) and Tollip^−/− (black square) genotypes from paired samples. P=0.021. * p < 0.05, paired two-sided t-test. N = 5. C) Experimental outline of AM adoptive transfer. D) Proportion of apoptotic (Annexin V+; p = 0.04) and necrotic (amine reactive dye+; p=0.02) Tollip^−/− (black square) donor AM transferred into CD45.1 mice compared with CD45.1+ recipient AM (white circle). Lines connect AM from the same animal. * p < 0.05, paired two-sided t-test. N = 5. Representative images from B6 and Tollip^−/− genotype with Annexin V flow minus one (FMO) control are shown. The top number represents the proportion of amine-reactive dye+Annexin V- (necrotic) cells, and the bottom number represents the proportion of Annexin V+ cells (apoptotic). E) The proportion of apoptotic and necrotic AM in B6 donor (white circle) and CD45.1 recipient (black circle) AM 56 days after aerosol Mtb infection. Paired two-sided t-test. N = 5/group. Representative images from B6 and Tollip^−/− genotype with flow minus one (FMO) control are shown. All experiments were performed at least three times for reproducibility.

Figure 4.. Tollip^−/− AM autonomously develop increased EIF2 phosphorylation after prolonged Mtb infection

A) Volcano plot of gene expression (log2 fold change) and significance (-log10(FDR)) for genes between Tollip^−/− (CD45.2) and B6 (CD45.1+CD45.2+) in Mtb-uninfected and Mtb-infected alveolar macrophages (AMs) sorted from mixed bone marrow chimeric mice 28 days after infection (dpi). Horizontal lines indicate differentially expressed genes (DEGs; FDR q < 0.05) and genes of interest (FDR q < 0.3). B) Ingenuity Causal Network Analysis on DEGs between B6 and Tollip^−/− Mtb-infected AM (FDR q < 0.05). P value cutoff = 5×10⁻³, calculated using two-sided Fisher’s exact test. C) Gene set enrichment analysis of DEGs (FDR q<0.05) for Reactome pathways. FDR cutoff <10⁻⁴ to account for multiple comparisons. Reactome pathway cut off with ellipsis is “Response of EIF2AK4 (GCN2) to Amino Acid Starvation.” D) Heatmap of gene expression (log2 fold change over average of B6 AM) for DEGs between Mtb-infected AM. Columns are independent experiments of pooled mice, and rows are genes. Called genes are stress response genes of interest. E-F) pEIF2 MFI in AM and monocyte-derived macrophages (MDM) from mixed bone marrow chimeric mice D) 28 days (p=0.003) and E) 56 (p=0.002) dpi. B6 – clear circle, Tollip^−/− -- black square; lines connect genotypes from paired samples. * p < 0.05, ** p < 0.01, paired two-sided t-test. N = 6/group. Representative histograms B6 – blue, Tollip^−/− -- red, IgG control – gray. G) pEIF2 MFI in adoptively transferred Tollip^−/− AM (black square) compared to CD45.1+ recipient AM (CD45.1+, white circle). N = 4, p=0.001. ** p < 0.01, paired two-sided t-test. H) Representative pEIF2 and EIF2A stains of lung tissue 56 dpi. I-J) Semiquantitative analysis of H) pEIF2 (p=0.002) and I) EIF2A protein expression. Magnification - 20x; scale bar - 100μm. * p < 0.05, ** p < 0.01, two-sided t-test. N = 5 mice/group.100 high-powered fields were sampled for semiquantitative analysis.

Figure 5.. Mycolic acid-treated Tollip^−/− macrophages accumulate lipids and permit increased Mtb replication

A-B) LipidTox neutral lipid stain MFI in alveolar macrophages (AM; p=0.03), monocyte-derived macrophages (MDM), and neutrophils (PMN) from mixed bone marrow chimeric mice 28 days after Mtb infection (dpi). B) Representative histogram of AM and MDM lipid staining (B6 – blue; Tollip^−/− – red). Lines connect B6 and Tollip^−/− AM from the same mouse. * p < 0.05, paired two-sided t-test; N = 5. Experiment was performed five times. C) Ifnb1 mRNA expression, normalized to Gapdh, in the lungs of Mtb-infected mice 56 dpi. p=0.009, N = 6 mice over two experiments. B6 (white circle) and Tollip^−/− (black square) genotype. * p<0.05, **p<0.01, ***p<0.001, two-sided t-test. D) Relative Ifnb1 mRNA expression in B6 (clear circle) and Tollip^−/− (black square) AM, normalized to Gapdh, after TNF instillation (10 ng/ml). p = 0.01 at 20 hours and p = 0.03 at 24 hours N = 6 replicates in each group over two independent experiments. * p<0.05, **p<0.01, ***p<0.001, two-sided t-test, unadjusted for comparisons. Error bars – SEM. E-G) Representative lipid staining of PEM incubated with mycolic acid (MA) for 72hrs. LipidTOX Red stain (red) and DAPI (blue). Magnification 100x; scale bar 16μm. F) Percentage of PEM with lipid droplets (LD) and G) total number of LD per cell (p=0.002). 100 cells imaged. Bar – mean value. H) LipidTox neutral lipid stain MFI in alveolar macrophages (AM; p=0.03), MDM, and PMN 5 days after intratracheal MA instillation. *p<0.05, two-sided t-test I-L) Relative luminescence (RLU) of B6 and Tollip^−/− I) PEM (p=0.03), J) AM (p=0.05), K) TOLLIP-deficient THP-1 (TOLLIP-KO; p=0.001), and L) BMDM after Mtb H37Rv-lux infection, normalized to initial luminescence.; N = 6 over three independent experiments. * p < 0.05; 3-way ANOVA for an effect of MA on Tollip^−/− macrophages. Error bars – SEM. M) EIF2AK1, EIF2AK2, EIF2AK3, EIF2AK4, TOLLIP, and EIF2S1 expression in human caseous granuloma tissue compared with healthy-appearing lung tissue from different lobes in the same individuals, normalized to median value. Data are from GSE20050. FDR is indicated above each data point.

Figure 6.. ISRIB treatment restores immune control in Tollip^−/− mice

A) Relative luminescence (RLU) in B6 peritoneal macrophages (PEM) treated with raphin-1 (black and white circles; 10μM) over time. Error bars -- SEM. P = 0.02; * p < 0.05; 2-way ANOVA for an effect of raphin-1 controlling for time. N = 6, experiment was performed three times. B-D) Normalized RLU was measured 24–72 hours after after Mtb-lux infection in Tollip^−/− B) PEM (p=0.008), C) alveolar macrophages (AM; p=0.03), and D) THP-1 lacking TOLLIP (TOLLIP-KO; p=0.001). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p< 0.0001, 2-way ANOVA accounting for time and drug treatment. N = 6, experiment was performed three times. Error bars – SEM. E) Experimental design. B6 and Tollip^−/− mice were infected with 50–100 colony forming units (CFU) of Mtb and 15 days after infection, vehicle control, ISRIB (1 mg/kg/day), or raphin-1 (1 mg/kg/day) were instilled intraperitoneally. Eight weeks after infection, mice were euthanized, bacterial CFU was measured, and tissue was collected for pathology analysis. N = 22. F-G) The percentage of purified protein-derivative-expressing (PPD+) F) AM (SiglecF+) and G) monocyte-derived macrophages (MDM; CD11b+) in Mtb-infected lung tissue, measured by multiparameter confocal microscopy. H-I) Mtb CFU in H) lungs and I) spleen from mice treated with ISRIB. J-K) Mtb CFU in J) lungs and K) spleen from mice treated with raphin-1. L) Representative images of lung tissue from B6 and Tollip^−/− mice, stained with hematoxylin and eosin. Magnification 5x; scale bar – 500μm. M) Number of nucleated cells per 20x high-powered field in Mtb-infected mice after ISRIB and raphin-1 treatment. B6 vs. Tollip^−/− p = 0.0001, Tollip^−/− vs Tollip^−/− with ISRIB p <0.0001, Tollip^−/− vs Tollip^−/− with raphin-1 p =0.0013. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p< 0.0001, 2-way ANOVA accounting for genotype and drug treatment. N = 11 total. N) Lung inflammation score, from a blinded veterinary pathologist, after 56 days of Mtb infection. N = 30 mice total. B6 vs. Tollip^−/− p = 0.02, Tollip^−/− vs Tollip^−/− with ISRIB p = 0.001. * p < 0.05, ** p<0.01, 2-sided t-test.