Fig 1.
Transcriptional change induced by acidosis and M.tb infection in macrophages.
RNA-seq was performed on control and M.tb infected MDMs incubated at pH 7.4 or acidic pH 7.0. (A) Principal Component Analysis (PCA) showing the first and second principal component on the x and y axis respectively. Total variance attributable to that PC indicated. (B) Clustered expression intensities standardised to z-score are shown for the 50 most differentially expressed genes (by p value) across all samples. Each sample is labelled with condition, pH and replicate number (C) Schematic of the conditions compared in differential expression analysis. (D) Venn diagram showing overlap of differentially expressed genes between the effect of infection at pH 7.4 and infection at pH 7.0 (E) Magnitude and number of differential gene expression in each analysis. Light bars <2 fold, medium shading 2–4 fold, dark bars >4 fold.
Fig 2.
Functional analysis of acidosis regulated genes in uninfected macrophages.
Gene Set Enrichment Analysis (GSEA) was performed on gene lists in the Acidosis (Uninfected) dataset ranked by fold change or significance (p value). Gene sets were considered enriched if FDR q-value is <0.05 in both fold change and significance ranking. Plotted FDR-q value is significance ranking value. No gene sets were enriched amongst upregulated genes. (A) The 219 Reactome gene sets downregulated by acidosis (pH 7.0) in uninfected cells. Gene sets are categorised by the uppermost level of the Reactome pathway event hierarchy. (B) Specific Immune System gene sets downregulated by acidosis. (C) Selected acidosis downregulated gene sets of importance to macrophage M.tb responses.
Fig 3.
Acidosis upregulates proinflammatory genes in M.tb infected macrophages.
Gene expression heatmaps of selected Cytokine, Chemokine, MMP and TIMP genes. Box shading indicates the the Log2 Fold Change in the Acidosis (uninfected), Infected (pH 7.4) and Infected (pH 7.0) analyses. Boxes unshaded (white) if gene expression not above threshold of detection. Black Circle indicates genes with individual p value <0.01.
Fig 4.
Extracellular Matrix degradation pathways are enhanced by acidosis in M.tb infection.
GSEA of gene lists comparing M.tb infection of MDMs at pH 7.0 versus pH 7.4 (Acidosis (Infected)) shows upregulation of ECM degradation pathways due to increased MMP expression. (A) Broad classification of the 22 enriched Reactome pathways in the Acidosis (Infected) analysis (top panel) and the individual gene sets ordered by FDR q-value (bottom panel). (B) GO terms upregulated in Acidosis (Infected) analysis plotted using REViGO. Magnitude of bubble red shading indicates degree of significance and edges link related terms. (C) Broad classification of the 168 Reactome downregulated gene sets. (D) Heatmap of gene expression of the individual genes included in the 5 upregulated Extracellular Matrix Organisation gene sets. Gene Log2 fold changes in the Acidosis (Uninfected), Infection (pH7.4) and Infection (pH 7.0) analysis (left to right) plotted. Only genes in the gene set with expression in one or more conditions are shown.
Fig 5.
Effect of Acidosis on secretion of MMPs, TIMPs and Cytokines from M.tb infected MDMs.
MDMs were infected with M.tb at pH 7.4 or pH 7.0 for 24 hours and supernatant protein levels measured at 24 hours. (A) MMP-1.(B) MMP-3. (C) MMP-10. (D) MMP-1 gene expression. (E) TIMP-1. (F) TIMP-2. (G) IL-1β. (H) TNF-α. (I) IFN-γ. (J) IL-6. (K) IL-8. (L). IL-10. Graphs are representative of n = 4 donors. Bars are means +/- SD. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. NS–not significant.
Fig 6.
Expression of TDAG-8 and OGR-1 and role of receptor signaling in mediating MDM acidosis responses.
(Α) Lung sections of mice infected with M.tb by the intranasal route were stained for either (i) isotype control antibody (ii) TDAG-8 or (iii) OGR-1. Images representative from n = 3 animals. Gene expression of acidosis GPRCRs in primary human MDMs from healthy donors (n = 5) was measured by qPCR. (B) Representative relative expression of acidosis GPCRs from one human donor normalised to 18S. (C) Average expression across n = 5 donors normalised to TDAG-8 expression. Changes in expression of (D) TDAG-8 and (E) OGR-1 with M.Tb and acidosis. Effect of TDAG-8 agonist BTB09089 on (F) MMP-1 and (G) TNF-α secretion. Effect of OGR-1 agonists (E) ISX-9 on MMP-1 secretion and (F) Ogerin on TNF-α secretion. (J) Effect of gene knockdown of acidosis GPCRs on TNF-α secretion. B-J representative of n = 3 donors.
Fig 7.
TDAG-8 and OGR-1 expression in patients with TB Lymphadenitis.
Immunohistochemistry of lymph node sections from patients with TB lymphadenitis (n = 5). (A) Haemotoxylin/Eosin stain showing necrotising granulomatous inflammation with multinucleate giant cell (arrowed) characteristic of TB. (B) Isotype control staining. (C) TDAG-8 staining with increased staining in the macrophage containing granuloma center. (D) Infiltrating monocytes staining strongly for TDAG-8 (arrowed). (E) Multinucleate Giant Cells stain strongly for TDAG-8. (F) Staining for OGR-1. Staining in the center of the granuloma is marked with an arrow. Multinucleate Giant cell marked MGC in the figure. Images are representative of n = 5 donors. Scale bars 100μM.
Fig 8.
Effect of Lactate on Macrophage MMP and Cytokine Secretion.
The effect of extracellular lactate at pH 7.4 on MDM secretion of (A) TNF-α and (B) MMP-1. (C) Gene expression of MMP-1with lactate (25mM). (D) Intracellular and cell free M.tb growth is unaffected by extracellular pH or lactate (25mM) at 24 hours. Gene expression of (E) SLC16A1 (MCT-1) and (F) SLC16A3 (MCT-4) with lactate and M.tb infection. (G) Western blots for MDM MCT-1 and -4. (H) Effect of an MCT-1 inhibitor on MMP-1 secretion. All experiments representative of n = 3 donors and performed with MDMs incubated for 24 hours. Bars are means +/- SD. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
Fig 9.
Summary of the effects of microenvironment acidosis and lactate on M.tb infected MDMs.
Review of the main changes in protein secretion and gene transcription in M.tb infected MDMs exposed to extracellular acidosis and lactate.