Ferritin H Deficiency in Myeloid Compartments Dysregulates Host Energy Metabolism and Increases Susceptibility to Mycobacterium tuberculosis Infection

Abstract

Iron is an essential factor for the growth and virulence of Mycobacterium tuberculosis (Mtb). However, little is known about the mechanisms by which the host controls iron availability during infection. Since ferritin heavy chain (FtH) is a major intracellular source of reserve iron in the host, we hypothesized that the lack of FtH would cause dysregulated iron homeostasis to exacerbate TB disease. Therefore, we used knockout mice lacking FtH in myeloid-derived cell populations to study Mtb disease progression. We found that FtH plays a critical role in protecting mice against Mtb, as evidenced by increased organ burden, extrapulmonary dissemination, and decreased survival in Fth^-/- mice. Flow cytometry analysis showed that reduced levels of FtH contribute to an excessive inflammatory response to exacerbate disease. Extracellular flux analysis showed that FtH is essential for maintaining bioenergetic homeostasis through oxidative phosphorylation. In support of these findings, RNAseq and mass spectrometry analyses demonstrated an essential role for FtH in mitochondrial function and maintenance of central intermediary metabolism in vivo. Further, we show that FtH deficiency leads to iron dysregulation through the hepcidin-ferroportin axis during infection. To assess the clinical significance of our animal studies, we performed a clinicopathological analysis of iron distribution within human TB lung tissue and showed that Mtb severely disrupts iron homeostasis in distinct microanatomic locations of the human lung. We identified hemorrhage as a major source of metabolically inert iron deposition. Importantly, we observed increased iron levels in human TB lung tissue compared to healthy tissue. Overall, these findings advance our understanding of the link between iron-dependent energy metabolism and immunity and provide new insight into iron distribution within the spectrum of human pulmonary TB. These metabolic mechanisms could serve as the foundation for novel host-directed strategies.

Keywords: bioenergetics; energy metabolism; ferritin H chain; immunometabolism; iron; tuberculosis.

Figure 1

Survival, bacillary burden, and pathology of mice following Mtb infection. (A) Kaplan–Meier survival curve showing the death of Fth^−/− mice following high dose and medium dose of Mtb infection (n = 11). (B,C) Bacillary burden in the lungs and spleens of mice at 4 and 9 weeks postinfection (n = 7). (D) Representative photograph of mouse eyes at 9 weeks postinfection (n = 5). (E) Representative H&E staining of eyes showing more vasculitis in Fth^−/− mice compared to Fth^+/+ mice (n = 5). (F,G) Bacillary burden in the eyes and brains of mice at 4 and 9 weeks postinfection (n = 7). (H) Representative photograph of brains from Mtb-infected mice 30 min after i.v. injection of Evans blue dye (n = 5). (I) Quantitation of Evans blue dye extracted from the brains of infected mice (n = 5). (J,K) Gross pathology of lungs and spleen of mice at 4 weeks postinfection (n = 5). (L) Representative micrographs of H&E staining of lungs from mice at 4 and 9 weeks postinfection (n = 7). Peribronchial and perivascular aggregation of histiocytes and the position of fibrous tissues in tuberculosis lesions in wild-type and knockout mice at 4 and 9 weeks postinfection. Scale bar; 500 µm. Statistical testing was performed using the unpaired Student’s t-test. Data are represented as mean ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001).

Figure 2

Quantification of iron in mouse tissues following Mtb infection. (A–C) Iron levels in lungs, spleens, and sera of Fth^+/+ and Fth^−/− mice by inductively coupled plasma mass spectrometry (n = 7). (D) Hepcidin levels in the sera of mice at 4 and 9 weeks postinfection (n = 7). (E) Iron levels in urine of mice at 4 and 9 weeks postinfection (n = 7). Iron levels in urine were normalized to creatinine. (F) Iron levels in feces of mice at 4 and 9 weeks postinfection (n = 7). (G) Iron Von Giesen stain demonstrating minimal hemosiderin deposition in an Fth^−/− mouse lung, representative of (n = 6). (H) Conspicuous hemosiderin deposition in an Fth^+/+ mouse lung, representative of (n = 6). Inset: high-magnification iron Von Giesen stain demonstrating blue-green siderophages (arrows) and foci of hemorrhage (arrowheads) in Fth^+/+ mouse. (I) Western blot showing ferroportin (FPN) in the duodenum of three mice at 4 weeks postinfection. (J) Densitometry analysis of relative FPN expression in duodenum. Expression of iron related proteins in lungs following Mtb infection. Statistical testing was performed using the unpaired Student’s t-test. Data are represented as mean ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Figure 3

Expression of iron related proteins in mouse lungs following Mtb infection. (A) Western blot showing expression of FtH, FtL, ferroportin (FPN), and heme oxygenase-1 (HO-1) in the lungs of Fth^+/+ and Fth^−/− mice in uninfected animals and at 4 and 9 weeks postinfection (pooled samples, n = 7). (B) Densitometric quantitation of western blot bands in panel (A) shown as relative protein expression normalized to GAPDH band intensity. (C) Representative image of immunohistochemical analysis of FtH and FtL expression in the lungs of mice at 9 weeks postinfection (n = 7). Scale bar; 100 µm.

Figure 4

Fth^−/− mice elicited strong Th-1 response upon Mtb infection. (A–H) Levels of TNF-α, IFN-γ, GM-CSF, IL-2, IL-4, IL-5, IL-8, and IL-10 in bronchoalveolar lavage fluid obtained from uninfected Fth^+/+ and Fth^−/− mice and at 4 and 9 weeks postinfection (n = 7). (I) Representative contour plots showing the percent of CD11b⁺/Gr-1(1A8)^bright neutrophils (box) isolated from lungs 4 weeks postinfection. (J) Percentage of neutrophils in the lungs of mice at 4 weeks postinfection (n = 4). (K) Percentage of CD45⁺ cells in the lungs of mice at 4 weeks postinfection (n = 4). (L) Representative contour plots showing the percent of CD11b⁺/Gr-1(1A8)^bright neutrophils (box) isolated from spleens 4 weeks postinfection. (M) Percentage of neutrophils in spleens of mice at 4 weeks postinfection (n = 4). (N) Percentage of CD11b⁺/ly6C⁺ macrophages in the spleen of mice at 4 weeks postinfection (n = 4). (O) Representative contour plots showing the percent of CD4⁺ cells (box) from the lungs of mice at 4 weeks postinfection. (P) Percentage of CD4⁺ cells in the lungs of mice at 4 weeks postinfection (n = 4). (Q) Relative expression of CD4 mRNA levels in the lungs of Fth^+/+ and Fth^−/− mice at 4 weeks postinfection (n = 4). Expression levels were normalized to β-2 microglobulin. (R) Representative contour plots showing percentage of CD4⁺ cells (box) from the spleens of mice at 4 weeks postinfection. (S) Percentage of CD4⁺ cells in the spleens of mice at 4 weeks postinfection (n = 4). (T) Relative expression of CD8 mRNA levels in the lungs of Fth^+/+ and Fth^−/− mice at 4 weeks postinfection (n = 4). Expression levels were normalized to β-2 microglobulin. Statistical testing was performed using the unpaired Student’s t-test. Data are represented as mean ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Figure 5

Gene expression analysis of lungs from Mtb-infected mice. (A) Heat map showing differentially regulated genes in the lungs of Fth^−/− mice compared to wild-type mice at 9 weeks postinfection (n = 3). (B) Ingenuity pathway analysis (IPA) showing how upregulation of immunomodulators in Fth^−/− mice affect immune functions. (C) IPA shows that pathways involved in oxidative phosphorylation, mitochondrial dysregulation, and the IL-17 family are altered in Fth^−/− mice at 9 weeks postinfection. (D) IPA shows that pathways involved in the electron transport chain are altered in Fth^−/− mice at 9 weeks postinfection. Bioenergetic analysis of peritoneal macrophages from Fth^+/+ and Fth^−/− mice infected with Mtb. (E) Oxygen consumption rate (OCR) of uninfected peritoneal macrophages from Fth^+/+ and Fth^−/− mice. (F–H) Basal respiration, maximal respiration (MR), and spare respiratory capacity (SRC) of uninfected macrophages. (I) Extracellular acidification rate (ECAR) of uninfected peritoneal macrophages from Fth^+/+ and Fth^−/− mice. (J) OCR of Mtb-infected peritoneal macrophages at 24 h postinfection. (K–M) Basal respiration, MR, and SRC of Mtb-infected macrophages. (N) ECAR of Mtb-infected peritoneal macrophages at 24 h postinfection. (O) ECAR of uninfected macrophages using glycolytic stress test. (P) ECAR of Mtb-infected macrophages using glycolytic stress test. (Q) Phenogram (OCR Vs ECAR) of uninfected macrophages. (R) Phenogram (OCR Vs ECAR) of Mtb-infected macrophages. Data in panel (A) represents the mean fold change of three mice with q < 0.05 and cutoff ±1.5-fold. Statistical testing was performed using the unpaired Student’s t-test. Data are represented as mean ± SEM of three independent experiments (*p < 0.05, **p < 0.01, ***p < 0.001).

Figure 6

Metabolite clustering and MetPa Analysis of metabolites isolated from lungs of Mtb-infected mice. (A) Two-way hierarchical clustering analysis identified compounds measured by CE-MS and GC-MS of lung samples of Fth^+/+ and Fth^−/− mice at 9-week postinfection. (B,C) MetPa analysis of metabolic pathways significantly altered in Fth^−/− mice infected with Mtb. Statistical testing was performed using the unpaired Student’s t-test.

Figure 7

Metabolomic fingerprint in the lungs of Mtb-infected mice. (A) Heat map showing the percentage change in the levels of metabolites in the lungs of Fth^−/− mice compared to Fth^+/+ mice at 4 and 9 weeks postinfection (n = 4). (B) Levels of essential amino acids in the lungs of mice at 4 weeks postinfection. (C) Levels of polyamines in the lungs of Mtb-infected mice at 4 weeks postinfection. (D) Amino acids feeding into the TCA cycle. Arg and polyamine biosynthesis pathway at 9 weeks postinfection in lungs of Fth^−/− mice compared to Fth^+/+ mice. ODC, Ornithine decarboxylase; SPDS, spermidine synthase; SPS, spermine synthase; SSAT, spermidine and spermine acetyl transferase; DHS, deoxyhypusine synthase; DOHH, deoxyhypusine hydroxylase. Green text indicates increased levels of metabolite and red indicates reduced levels in the lungs of Fth^−/− mice at 9 weeks postinfection. Statistical testing was performed using the unpaired Student’s t-test. Data are represented as mean ± SEM of four mice (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Figure 8

Spatial distribution of iron in the human lung tubercle. (A) Low power depiction of Iron van Gieson (FeVG stains hemosiderin a sea-green, and collagen a red color) stained lung section highlighting tubercles (T). Inset (I): high power demonstration of scattered sea-green siderophages (arrows) in central necrotic caseative debris. Inset (II): abundant hemosiderin in adjacent lung tissue within patent (arrowheads) and collapsed (asterisks) alveolar spaces. (B) High power depiction of hemosiderin distribution in fibro-inflammatory response in rectangle (A) with pale hemosiderin in focal histiocytes and giant cells [arrow and inset (III)] within granulomas (Gr). Conspicuous iron in inflamed granulation tissue (IGT). Less, but noticeable iron in outer lamellar fibrosis (LF) (AL, adjacent lung; ND, necrotic debris in caseative focus). (C). Iron levels in the lungs of healthy and TB patients (**p = 0.0034).

Figure 9

Microscopic iron distribution in the human tuberculous lung. FeVG-stained section of non-necrotizing granulomatous inflammation (NNGI) showing confluent, NNGI with stippled pale staining hemosiderin in organized granulomas (G) and in Langhans giant cells (arrows). Conspicuous sea-green hemosiderin deposits in intervening and surrounding, variably fibrotic, lung (L) parenchyma.

Figure 10

Proposed mouse model. Following exposure, Mtb resides inside the lung macrophages and replicate by using host iron to cause infection. Fth^−/− mice tries to reduce the availability of iron in the lung cells by upregulating FPN, an iron exporter. FtH is essential for maintaining mitochondrial function through oxidative phosphorylation (OXPHOS) and demonstrated an essential role for FtH in maintenance of central intermediary metabolism.