Immune microenvironment analysis pre- and postmycobacterial granuloma formation. (A) UMAP plot of all cells from the four samples, with 15 generic cell types identified by different colors, as indicated on the Right side of the plot. (B) Proportions of different cell populations in the four samples, with colors of the cell groups corresponding to the annotations on the Right side. (C) Following the intraperitoneal injection of M.m into Tg(mpeg1-LRLG (mpeg1:loxP-DsRed2-loxP-GFP), macrophages labeled with red fluorescence) zebrafish, samples were obtained at 14 dpi. Flow cytometry (FACS) was used to analyze the proportion of DsRed2+ macrophages in the four samples. (D) Statistics of the proportions of macrophages in the four samples shown in panel (C). (D) analyzed with one-way ANOVA, *P < 0.05; **P < 0.01.

Analysis of macrophage subpopulations during granuloma formation. (A) UMAP plots showing Macrophage_1/2/3/4. (B) Proportions of Macrophage_1/2/3/4 in samples K1, K2, G1, and G2. The colors of the four macrophage groups correspond to those shown in panel (A). (C) Heatmap of the top 20 DEGs for Macrophage_1/2/3/4. Each row corresponds to a gene’s expression across different macrophages, with gene names annotated on the Left and expression levels on the Right. (D) Distribution of macrophages in zebrafish granulomas. Following the intraperitoneal injection of M.m into Tg(mpeg1-LRLG; grna.2-GFP-NTR, grna.2+ macrophages labeled with green fluorescence) zebrafish, samples were obtained at 14 dpi. Frozen sections were stained with antibodies against DsRed2 and GFP. White dashed lines indicate granulomas core. (E) Flow cytometry analysis of GFP+ grna.2+ macrophages from the kidney of M.m-infected Tg(mpeg1-LRLG; grna.2-GFP-NTR) zebrafish. [Scale bar: 50 μm (D).]

Functional assay suggesting grna.2+ macrophages are protective during mycobacterial infection. (A) Survival curves of zebrafish in the Ctr, NTR, Ctr + M.m, and NTR + M.m groups. Each group contains 10 fish. (B) Survival curves of zebrafish in the Ctr + M.m + MTZ and NTR + M.m + MTZ groups. The data shown combine three independent biological replicates. (C) Differences in bacterial load in zebrafish from the Ctr + M.m + MTZ and NTR + M.m + MTZ groups. Each point represents the bacterial load in a single zebrafish from one biological replicate. Different colors indicate independent biological replicates. (D–H) HE staining of paraffin sections of zebrafish from the Ctr + M.m + MTZ and NTR + M.m + MTZ groups. Blue arrows indicate granulomas. The Top Right panel shows an enlarged view of the region within the black box. Blue dashed lines indicate granulomas. Panel (G–I) show representative images of early immune cell infiltration, nonnecrotic granulomas, and necrotic granulomas, respectively. Blue dashed lines indicate granulomas, and yellow dashed lines indicate necrotic cores. (I) Quantification of granulomas in zebrafish from the Ctr + M.m + MTZ and NTR + M.m + MTZ groups. Each point represents the total number of granulomas in one zebrafish. (J) Quantification of granulomas at different developmental stages from panel (J). (K) Number of immune cells around granulomas of zebrafish from the Ctr + M.m + MTZ and NTR + M.m + MTZ groups. (L) Area of immune infiltration of the Ctr + M.m + MTZ and NTR + M.m + MTZ groups. (A and B) analyzed with simple survival analysis (Kaplan–Meier), and (C, I, J, K, and L) analyzed with the unpaired two-tailed t test, **P < 0.01, ***P < 0.001, and ns = nonsignificant. [Scale bar: 500 μm (D and E), 50 μm (D and E (Top Right panel)), 25 μm (F, G, and H).]

Investigation of the origin and the role of grna.2+ macrophages in host defense ex vivo. (A) Fluorescence changes of grna.2+ macrophages (green) and M.m (red) in granulomas at different time points in the control and 2 mM MTZ treatment groups. Time-lapse intervals were 10 min. (B) Fluorescence changes of grna.2+ macrophages (green) and M.m (red) in granulomas at different time points after removal of MTZ. Time-lapse intervals were 10 min. The Bottom Left panel shows an enlarged view of the region within the blue box. Red arrows indicate reappear grna.2+ macrophages. [Scale bar: 50 μm (A and B).]

Depletion of grna.2+ macrophages altered the distribution of immune cells in granulomas. (A) Distribution of macrophages in granulomas of zebrafish from the Ctr + M.m + MTZ and NTR + M.m + MTZ groups. Following intraperitoneal injection of M.m into Tg(mpeg1-LRLG; grna.2-GFP-NTR) zebrafish, samples were obtained at 14 dpi. Frozen sections were stained with DAPI and antibodies against DsRed2. (B) Density value distribution of macrophages in panel (A). Each point represents the relative coordinates of a single macrophage. The R script was used to calculate the density value of each point relative to all other points. (C) Proportion of HDV macrophages in panel (B). Each point represents the statistical value for a single granuloma, with different colors indicating three independent biological replicates, with data from three zebrafish per experiment (each zebrafish having 2 to 3 granulomas). (D) Representative images of lck+ in situ hybridization signals in granulomas of zebrafish from the Ctr + M.m + MTZ and NTR + M.m + MTZ groups. Red dashed lines outline the granulomas, yellow dashed lines indicate necrotic cores, and red arrows point to lck+ in situ hybridization signals. (E) Quantification of the number of lck+ in situ hybridization signals in different types of granulomas from the Ctr + M.m + MTZ and NTR + M.m + MTZ groups. Each point represents the number of lck+ in situ hybridization signals in a single granuloma. (F) Relative mRNA expression levels of TCR-related genes trac and cd3 in granulomas from the Ctr + M.m + MTZ and NTR + M.m + MTZ groups. Each group consists of three granuloma samples, with each sample derived from three zebrafish. (C) analyzed with the unpaired two-tailed t test, and (E and F) analyzed with one-way ANOVA, *P < 0.05, **P < 0.01, ***P < 0.001, and ns = nonsignificant. [Scale bar: 50 μm (A and D).]

Depletion of grna.2+ macrophages led to a hyperinflammatory granuloma microenvironment and promoted macrophage lytic cell death and tissue damage. (A) The bacterial load in the early stages of infection. Each point represents the bacterial load in an individual fish. (B) The number of granulomas in the early stages of infection. Each group consists of 5 zebrafish. (C) Representative morphology of granulomas shown in panel (B). Red dashed lines indicate granulomas, yellow dashed lines indicate necrotic cores. (D) Relative mRNA levels of proinflammatory cytokines il1β, tnfα, and il6 mRNA from the Ctr + M.m + MTZ and NTR + M.m + MTZ groups in the early stages of infection. Each group consists of three kidney samples, each sample derived from three zebrafish. (E) Schematic diagram of experimental design for RNA-seq analysis of kidney samples from Ctr + M.m + MTZ and NTR + M.m + MTZ groups of zebrafish. (F) GO enrichment analysis of the top 10 upregulated pathways in the NTR + M.m + MTZ group. The chart displays the top 10 most significant BP terms. (G) Proportion of dead macrophages (7-AAD+) in the Ctr + M.m + MTZ and NTR + M.m + MTZ groups. Following the intraperitoneal injection of M.m into Tg(mpeg1-LRLG; grna.2-GFP-NTR) zebrafish, samples were obtained at 14 dpi. (H) Statistical analysis of the proportion of dead macrophages in panel (G). Each point represents the proportion of dead cells in an individual zebrafish. (I) Schematic diagram of experimental design for sorting of kidney samples from NTR + M.m + MTZ and NTR + M.m groups of zebrafish. (J–L) Following the intraperitoneal injection of Katushka-labeled M.m into Tg(mpeg1-LRLG; grna.2-GFP-NTR) zebrafish, samples were obtained at 7 dpi. FACS was used to analyze the proportion of Katushka+ macrophages in grna.2− macrophages from NTR + M.m + MTZ group, grna.2+ and grna.2− macrophages from NTR + M.m group. (M) Statistical analysis of the MFI of Katushka+ macrophages in three samples shown in panel (J–L). (A, B, D, and H) analyzed with the unpaired two-tailed t test, and (M) analyzed with one-way ANOVA, *P < 0.05, **P < 0.01, ***P < 0.001, and ns = nonsignificant. [Scale bar: 25 μm (C).]

GRN exerted an anti-inflammation function in human mycobacterial granulomas. (A) Representative images of GRN expression in lung granulomas from a tuberculosis patient. The Left panel shows the overall granuloma structure, while the Right panel provides an enlarged view of the red boxed region. Brown spots indicate GRN positive signals, and blue spots indicate the nucleus. GRN-positive signals are predominantly located at the periphery of the granuloma. (B) Schematic diagram of TB patients with PET-CT and scRNA-seq of granulomas (82). (C) Expression level of GRN mRNA in the lung of tuberculosis patients with high 18F-FDG-avidity and low 18F-FDG-avidity. Data are sourced from ref. 82. PKM, Per Kilobase of transcript per Million mapped reads. (D) UMAP plot of scRNA-seq data of all macrophages from publicly available scRNA-seq data about human tuberculosis granulomas. (E) Expression map of GRN in macrophages. The red dashed line indicates the area of macrophages with high expression of GRN. (F) UMAP plot of scRNA-seq data of all macrophages in PET-CT_high and PET-CT_low groups. The black dashed line indicates the area of macrophages with high expression of GRN. (G) The GRN high macrophage ratios in PET-CT_high (n = 5) and PET-CT_low groups (n = 6). (C) analyzed with the unpaired two-tailed t test, ***P < 0.001. [Scale bar: 50 μm (A).]