Macrophage arginase-1 controls bacterial growth and pathology in hypoxic tuberculosis granulomas

Abstract

Lung granulomas develop upon Mycobacterium tuberculosis (Mtb) infection as a hallmark of human tuberculosis (TB). They are structured aggregates consisting mainly of Mtb-infected and -uninfected macrophages and Mtb-specific T cells. The production of NO by granuloma macrophages expressing nitric oxide synthase-2 (NOS2) via l-arginine and oxygen is a key protective mechanism against mycobacteria. Despite this protection, TB granulomas are often hypoxic, and bacterial killing via NOS2 in these conditions is likely suboptimal. Arginase-1 (Arg1) also metabolizes l-arginine but does not require oxygen as a substrate and has been shown to regulate NOS2 via substrate competition. However, in other infectious diseases in which granulomas occur, such as leishmaniasis and schistosomiasis, Arg1 plays additional roles such as T-cell regulation and tissue repair that are independent of NOS2 suppression. To address whether Arg1 could perform similar functions in hypoxic regions of TB granulomas, we used a TB murine granuloma model in which NOS2 is absent. Abrogation of Arg1 expression in macrophages in this setting resulted in exacerbated lung granuloma pathology and bacterial burden. Arg1 expression in hypoxic granuloma regions correlated with decreased T-cell proliferation, suggesting that Arg1 regulation of T-cell immunity is involved in disease control. Our data argue that Arg1 plays a central role in the control of TB when NOS2 is rendered ineffective by hypoxia.

Fig. 1.

Arg1 expression in lung granulomas of Mtb-infected Nos2^−/− mice and association with necrosis. (A) Lung cfus from infected WT, Nos2^−/−, and IFN-γ–blocked Nos2^−/− mice at day 56 p.i. Data are shown as median and interquartile range; n = 6 mice; **P = 0.0022 (Mann–Whitney test). ND, not detectable. (B and C) Number of total granulomas (B) and number of necrotic granulomas (C) per lung section in infected WT (n = 72), Nos2^−/− (n = 66), and IFN-γ–blocked Nos2^−/− (n = 62) mice at day 56 p.i. Data are shown as median and interquartile range; P < 0.0001 (Gaussian approximation; Kruskal–Wallis test and Dunn’s post test). (D) Arg1 expression in lung homogenates from infected WT, Nos2^−/−, and IFN-γ–blocked Nos2^−/− mice at day 56 p.i. Protein (20 μg) was immunoblotted with anti-Arg1 and anti–β-actin. (E) IHC staining of lung tissue from infected WT and Nos2^−/− mice with anti-Arg1 and isotype at day 56 p.i. (F) Number of Arg1⁺ granulomas per lung section in infected WT (n = 72), Nos2^−/− ( n = 64), and IFN-γ–blocked Nos2^−/− (n = 62) mice at day 56 p.i. Data are shown as median and interquartile range. P < 0.0001 (Gaussian approximation; Kruskal–Wallis test and. Dunn’s post test). (G) Representative Arg1 staining of a lung section from an IFN-γ–blocked Nos2^−/− mouse at day 56 p.i. (H) Correlation between the number of necrotic and Arg1⁺ granulomas in the lungs of infected IFN-γ–blocked Nos2^−/− mice. Spearman correlation; P = 0.0001. Data are representative of two independent experiments. **P < 0.005; ***P < 0.0005.

Fig. 2.

Macrophages are the main cell type expressing Arg1 in lungs from Mtb-infected Nos2^−/− mice. (A) Single-cell lung suspensions prepared at day 56 p.i. were analyzed by flow cytometry to detect the frequency of the indicated immune cell populations in the lungs of infected Nos2^−/− and IFN-γ–blocked Nos2^−/− mice. (B) Percentages of Arg1⁺ cells in neutrophils, inflammatory macrophages, and monocytes in lungs of infected Nos2^−/− and IFN-γ–blocked Nos2^−/− mice at day 56 p.i. Histograms show Arg1 expression in neutrophils, inflammatory macrophages, and monocytes. The black line depicts Nos2^−/− mice (n = 12), and the blue line depicts IFN-γ–blocked Nos2^−/− mice (n = 14). Data are shown as median and interquartile range. Mann–Whitney test. Data are representative of two independent experiments. *P < 0.05; **P < 0.01.

Fig. 3.

Arg1 metabolizes l-arginine, producing l-ornithine and polyamines in lungs of Mtb-infected Nos2^−/− mice. (A) Arginase activity measured by the conversion of l-arginine into l-ornithine. (B) Amounts of l-arginine, l-ornithine, putrescine, and spermidine detected by HPLC in lung homogenates of infected WT (n = 7), Nos2^−/− (n = 6), and IFN-γ–blocked Nos2^−/− (n = 6) mice at day 56 p.i. Data are shown as median and interquartile range. Kruskal–Wallis test and Dunn’s post test. Data are representative of two independent experiments. *P < 0.05; **P < 0.005; ***P < 0.0005.

Fig. 4.

Abrogation of Arg1 exacerbates Mtb growth and pathology in TB lung granulomas. (A) H&E staining of lung tissue from infected Nos2^−/− and Arg1-Nos2 DKO mice at day 56 p.i. (B) Number of total and necrotic granulomas per lung section in infected Nos2^−/− (n = 259) and Arg1-Nos2 DKO (n = 196) mice on day 56 p.i. Data are shown as median and interquartile range. ****P < 0.0001 (Gaussian approximation; Mann–Whitney test). (C) Lung cfus from infected Nos2^−/− (n = 6) and Arg1-Nos2 DKO (n = 7) mice at day 56 p.i. Data are shown as median and interquartile range. Mann–Whitney test. Data are representative of three independent experiments.

Fig. 5.

Proximity of Arg1⁺ cells affects T-cell proliferation in microenvironments within granulomas. (A) Representative nonnecrotic and necrotic granulomas from infected Nos2^−/− and Arg1-Nos2 DKO mice at day 56 p.i. stained with anti-Arg1 (brown staining, red arrow) and anti-Ki67 (fuchsia staining, green arrow). White arrow and numeration on pictures taken at 50× and 100× magnification show the position where pictures at 400× magnification were acquired starting from the center (1) and moving toward the outer limits of the granuloma (2–4). (B) Correlation between area (in square pixels) of Arg1⁺ staining and the number of Ki-67⁺ cells quantified in granulomas from infected Nos2^−/− and IFN-γ–blocked Nos2^−/− mice at day 56 p.i. The red lines show the log-linear regression fit for data points with positive value; the pink area denotes the 95% confidence band. The fitted regression coefficient is significant with P < 0.0001 (r² = 0.2; Spearman correlation) (n = 134 pictures from regions of 50 granulomas). P < 0.00001 (r = −0.49). (C) Linear regression of gradient of numbers of Ki-67⁺ cells in necrotic granulomas from infected Nos2^−/− and IFN-γ–blocked Nos2^−/− mice (blue line) and Arg1-Nos2 DKO mice (red lines) at day 56 p.i. Lines correspond to linear fits of centered Ki-67⁺ cells’ values on a separate granuloma region for each of the two subgroups. Shaded areas show corresponding 95% confidence bands. The interaction between mouse strain and granuloma region is significant (P < 0.02) as tested by a separate complete mixed-effects model.