Figure 1. Tuberculosis-associated microenvironments induce Siglec-1 expression in macrophages.

(A–D) For 3 days, human monocytes were differentiated into macrophages with cmCTR (white) or cmMTB (black) supernatants. (A) Heatmap from a transcriptomic analysis (GEO submission GSE139511) illustrating the top 60 differentially expressed genes (DEGs) between cmCTR- or cmMTB-cells. Selection of the top DEGs was performed using an adjusted p-value ≤ 0.05, a fold change of at least 2, and a minimal expression of 6 in a log₂ scale. Hierarchical clustering was performed using the complete linkage method and the Pearson correlation metric with Morpheus (Broad Institute). Interferon-stimulated genes (ISG) are labelled with an asterisk and Siglec-1 is indicated in red. (B–D) Validation of Siglec-1 expression in cmMTB-treated macrophages. Vertical scatter plots showing the relative abundance to mRNA (B), median fluorescent intensity (MFI) (C), and mean number of Siglec-1 antibody binding sites per cell (D). Each circle represents a single donor and histograms median values. (E) Representative immunohistochemical images of Siglec-1 staining (brown) in lung biopsies of healthy, SIV infected (SIV), active TB (ATB), and co-infected (ATB-SIV) non-human primates (NHP). Scale bar, 100 µm. Insets are 2x zoom. (F) Vertical Box and Whisker plot indicating the distribution of the percentage of Siglec-1⁺ alveolar macrophages in lung biopsies from the indicated NHP groups. Quantification analysis from n = 800 alveolar macrophages grouped from three independent animals per NHP group. (G) Vertical scatter plots displaying the number of cells that are positive for Siglec-1 per mm² of lung biopsies from the indicated NHP groups. Each symbol represents a single animal per NHP group. (H) Correlation between Siglec-1⁺ cells per mm² of lung tissue and the pathological score for healthy (white circle), SIV⁺ (white triangles), latent (black circle) or active (black square) TB, and SIV⁺ with latent (grey circle) or active (grey square) TB. Each symbol represents a single animal per NHP group. Mean value is represented as a black line. Statistical analyses: Two-tailed, Wilcoxon signed-rank test (B–D), Mann-Whitney unpaired test (F–G), Spearman correlation (H). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. ns: not significant. See Figure 1—source data 1.

Figure 1—figure supplement 1. Tuberculosis-associated microenvironments increase Siglec-1 expression in human macrophages.

(A–D) For 3 days, human monocytes were differentiated into macrophages with cmCTR (white) or cmMTB (black) supernatants. (A) (Left) Gene set enrichment plot of the interferon alpha (IFNα) response (hallmark collection of MSigDB). This plot shows the distribution of the barcode between macrophages exposed to cmCTR (red) versus cmMTB (blue) supernatants. Each bar of the barcode corresponds to a signature gene of the gene set. The skewing to the right indicates enrichment in macrophages exposed to cmMTB versus cmCTR supernatant of genes up-regulated in response to IFNα. (Right) Gene set enrichment plot of the IFNγ response (hallmark collection of MSigDB). (B) Flow cytometry gating strategy to assess Siglec-1 cell-surface expression in human macrophages exposed to cmCTR (white) and cmMTB (black). (Left) Based on size (FSC-A) and granularity (SSC-A), a gate was created to separate human macrophages from cell debris and dying cells. Macrophages were then subjected through a second gate based FSC Area and Height Scaling (FSC-A and FSC-H) to separate singlets from doublets. (Right) Based on the singlet gate, the histogram plot illustrates Siglec-1 expression that is higher in cmMTB- (black) than in cmCTR-treated (white) macrophages. (C) Representative immunofluorescence of Siglec-1 intracellular staining (green), actin (red) and nuclei (blue) after 3 days of monocyte conditioning with cmMTB. Scale bar, 10 μm. (D) Vertical scatter plot showing the quantification of Siglec-1 intracellular staining in cmCTR- or cmMTB-treated cells in the presence of an IFNAR-2 blocking (α-IFNAR) or control (α-IgG) antibodies during cell conditioning. Each circle represents a single donor and histograms median value. (E) Median fluorescence intensity (MFI) of Siglec-1 cell-surface expression in human macrophages exposed to cmCTR and infected with HIV-1 assessed by flow cytometry. The histogram plot illustrates Siglec-1 expression that increases within days post-HIV-1 infection of cmCTR-macrophages. Statistical analyses: Two-tailed, Wilcoxon signed-rank test (D). *p<0.05, **p<0.01, ns: not significant.

Figure 1—figure supplement 2. Tuberculosis-associated microenvironments increase Siglec-1 expression in non-human primate alveolar macrophages.

(A) Accumulation of Siglec-1⁺ alveolar macrophages in the lung of co-infected non-human primates (NHP). Representative immunohistochemical images of Siglec-1 staining (brown) in lung biopsies of healthy, SIV-infected (SIV), active tuberculosis (ATB) and co-infected with SIV (ATB-SIV) NHP. Scale bars from top to bottom: 2 mm, 500 µm and 50 µm. (B–C) Siglec-1⁺ cells display the alveolar macrophage morphology. (B) Representative immunohistochemistry image from lung biopsy of an ATB-SIV NHP stained for Siglec-1 (brown). Siglec-1⁺ cells display a cell morphology with a single nucleus and large cytoplasm reminiscent of macrophage (black arrowhead); Siglec-1^- cells display a different nucleus morphology and small cytoplasm reminiscent of neutrophils (red arrowhead). Scale bar, 20 µm. (C) Representative immunofluorescence images of alveolar macrophages found in lung biopsy of a representative ATB-SIV NHP stained for Siglec-1 (red), CD163 (green) and nuclei (DAPI, blue). Scale bar, 20 µm.