In vivo inhibition of tryptophan catabolism reorganizes the tuberculoma and augments immune-mediated control of Mycobacterium tuberculosis

Abstract

Mycobacterium tuberculosis continues to cause devastating levels of mortality due to tuberculosis (TB). The failure to control TB stems from an incomplete understanding of the highly specialized strategies that M. tuberculosis utilizes to modulate host immunity and thereby persist in host lungs. Here, we show that M. tuberculosis induced the expression of indoleamine 2,3-dioxygenase (IDO), an enzyme involved in tryptophan catabolism, in macrophages and in the lungs of animals (mice and macaque) with active disease. In a macaque model of inhalation TB, suppression of IDO activity reduced bacterial burden, pathology, and clinical signs of TB disease, leading to increased host survival. This increased protection was accompanied by increased lung T cell proliferation, induction of inducible bronchus-associated lymphoid tissue and correlates of bacterial killing, reduced checkpoint signaling, and the relocation of effector T cells to the center of the granulomata. The enhanced killing of M. tuberculosis in macrophages in vivo by CD4⁺ T cells was also replicated in vitro, in cocultures of macaque macrophages and CD4⁺ T cells. Collectively, these results suggest that there exists a potential for using IDO inhibition as an effective and clinically relevant host-directed therapy for TB.

Keywords: IDO; T cell; granuloma; macaque; tuberculosis.

Fig. 1.

Expression of IDO1 in in vitro as well as in vivo experimental models of M. tuberculosis infection. IDO levels detected by qRT-PCR at 0 (gray) and 24 h (red) after M. tuberculosis infection relative to uninfected BMDMs from three biological replicates (A) C3HeB/FeJ mice (70) and (B) rhesus macaques (71). The average relative expression (2^−∆∆Ct) of IDO in C3HeB/FeJ mice lungs at weeks 8, 12–20, and 24 in respect to postday 1 (base line) M. tuberculosis-infected lungs by qRT PCR (C) and microarray (D) (70). Linear regression (r² = 0.68, P < 0.05) plot of IDO1 expression correlates to bacillary load in C3HeB/FeJ mice lungs at weeks 8 and 12 obtained is from C (E). IDO expression in the lung of rhesus macaques with ATB (dark blue bars), LTBI (intermediate blue bars), chemotherapeutic treatment of ATB (light blue bars), or nonpathogenic M. tuberculosis infection (dark and light purple bars) by qRT-PCR (F) and microarray (G). IDO expression in M. tuberculosis-infected (dark blue bars), LTBI-coinfected SIV lungs either exhibited a reactivation (dark brown bars) or nonreactivation phenotype (light brown bars) (10). IDO expression in lungs of rhesus macaques infected with nonpathogenic Mtb∆dosR (dark purple bars) or Mtb∆sigH (light purple bars) (11, 13). Linear regression (r² = 0.48, P < 0.0001) plot of IDO expression correlates to bacterial burdens in the lungs of rhesus macaques with ATB (24–37 d postinfection) and LTBI (166–180 d postinfection) (H). Expression of Trp biosynthetic pathway genes in ATB (dark blue) and LTBI (light blue) animals (I) and their linear regression with IDO expression levels in ATB and LTBI animals (r² = 0.41, P < 0.005) (J). Data are means ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.001 using (A, B, and I) Student’s t test, (E, H, and J) linear regression analysis, or (C, D, F, and G) one-way ANOVA.

Fig. 2.

Clinical, microbiology and pulmonary pathology measures of infection and disease in control and D-1MT–treated macaques. Linear regression of serum CRP (μg/mL) between two groups (A). Linear regression of weight change over the course of time (weeks postinfection) between two groups: D-1MT–treated (orange circle), control (gray circle) (B). (C–E) Bacterial burdens detected by CFU assay in BAL (C), per gram of lung tissues (D), and per gram tissue for each BLN, spleen, liver, and kidney (E). Subgross H&E staining of lung sections from D-1MT (F) and control animals (G). (Scale bars, 250 µm.) Morphometric measure of TB-related total lung pathology (H). At least three systematic random microscopic fields from each lung, representing all lung lobes, from at least two of the animals in every group, were used for the morphometric analysis of histopathology. Data are means ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001 using (C) one-way ANOVA with Bonferroni post correction or (D, E, and H) Student’s t test; ns, not significant.

Fig. 3.

Assessing the IDO enzymatic activity in M. tuberculosis-infected macaques in vivo and its impacts on T and B cell phenotypes. BAL staining 3 wk after M. tuberculosis-infection; red, Kyn; blue, nuclei; gray, DIC marked with white arrowheads pointing toward the lining that appears a cell membrane indicates Kyn deposition within a cell. [Scale bars, 100 μm (Upper); 20 μm (Lower).] (A) Kyn quantification (B) and Kyn/Trp ratio by ELISA (C). In A, images are shown at different scale with more number of cells in a field from D-1MT–treated animals. Phenotype of memory T cells in BAL and lung samples at necropsy with respect to proliferation as measured by K_i67 positivity in D-1MT–treated (orange) and controlanimals (gray) (D–H). A representative flow-density plot from lungs of memory T cells expressing K_i67 (H). Costaining with CD20 and CD3 exhibits iBALT in D-1MT–treated (I), control animals (J): red, CD20⁺ B cells; green, CD3⁺ T cells; blue, macrophages. [Scale bars, 20 μm (I, Left and Right, and J, Right); 40 μm (I, Middle and J, Left).] White box indicates CD3+ T cells (I) and CD20⁺ B cells (J) found in iBALT follicle. Quantification of B cells in the multiple lesions from both groups: D-1MT–treated (orange circle) and control (gray circle) (K) (means ± SEM). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.001 using a (B and K) Student’s t test or (C) two-way ANOVA or (D–G) repeated-measures t test.

Fig. 4.

Impact of inhibiting IDO on lung gene expression. Selected modules derived from significantly enriched pathways based on the method described elsewhere (70) in BAL microarray datasets are shown. The horizontal colored bar on top of each module for a category represents a range in gene-expression magnitude in logarithmic base2. Heat-map clusters: “black” to “yellow” to “red,” lower (fold-change ∼1.5 cut-off) to higher expression (A). The change in gene expression (2^−∆∆Ct) by RT-PCR (B), cytokine assay (C) in lung homogenates of D-1MT–treated (orange) and control animals (gray) relative to uninfected lung samples as base line. GAPDH was used as an internal reference. Bars with no statistics shown are nonsignificant between two groups (e.g., IFN-γ) (P = 0.3381) (C). Immunofluorescence-based detection of T cell apoptosis by TUNEL assay. The arrowheads (white) in each magnified image indicate apoptotic cells [scale bars, 20 μm (Right); magnification, 20× (Left)] (D). Data obtained by counting multiple fields with T cells positive for TUNEL staining using a Leica confocal microscope (Leica Microsystems) (E). The data (means ± SEM) from animals from both groups were used for analysis; *P < 0.05, ****P < 0.001 using a Student’s t test.

Fig. 5.

Extensive relocalization of T cells to the internal regions of the macaque granulomata after inhibition of IDO-signaling. H&E staining of a representative lesion from D-1MT–treated (Left) and control (Right) groups is shown (A). A schematic representation of the granulomata shown in A is drawn in B, distinctly differentiating the lymphocytic and macrophage layers from the necrotic center. The expression of IDO was measured as a function of its presence in either of the three intragranulomatous compartments (necrotic, macrophage, or lymphocytic layer) by immunostaining; IDO (green), CD3 (red), and nuclei (blue) (C). A magnification of the white square area in C is shown in D, with white arrowheads pointing to CD3⁺ cells in red. The number of CD3⁺ cells (E, Upper) as well as total nuclei (E, Lower) in multiple granulomata in D-1MT–treated (orange with circular data points) and control (gray with triangle data points) animals enumerated in the lymphocytic, macrophage, and necrotic center compartments are shown. For quantification, 10 fields from each compartment were counted under a fixed magnification (corresponding to an area of 0.05 mm²) using a multispectral imaging camera (CRi Nuance). The data are means ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001 using a Student’s t test. [Scale bars, 20 μm (C, also applies to A and B); 5 μm (D).]

Fig. 6.

Granuloma performance in D-1MT–treated and control animals. Immunohistochemistry staining of lung sections for CD3⁺ T cells; (A) K_i67 (red), (B) granzyme B (red) with CD3 in green and nuclei (TOPRO3) stained in blue (A and B) and M. tuberculosis (red) staining (C). Multiple granzyme B⁺ cells can be observed in the necrotic center of granuloma from D-1MT–treated animals and are marked with white arrowheads. The far-right images in each panel are close-ups of the boxed region. (Scale bars, 150 µm.) The total number of cells positive for both CD3 and K_i67 as well as CD3 and granzyme B in A and B, respectively, as well as total nuclei in each panel were counted in multiple granulomata for each group and plotted. The graphs (far right in each panel) show percentages of cells positive for K_i67 (A) and granzyme B (B). The H&E staining of a representative lung lesion from D-1MT–treated (Upper) and control animals (Lower). [Scale bars, 20 µm (C, H&E); 150 µm (C, immunostaining).] A schematic representation of the granulomata and demarcation (L, lymphocytic layer; M, macrophage layer; N, necrotic center) in C is shown as described and is also applicable to A and B. The far-right image in C is the close-up of the boxed region and the graph shows the enumeration of bacilli in both groups (C). ***P < 0.001, ****P < 0.0001 using a Student’s t test. Data are means ± SEM, D-1MT treated (orange circle), controls (gray circle).