Increased expression of host iron-binding proteins precedes iron accumulation and calcification of primary lung lesions in experimental tuberculosis in the guinea pig

Abstract

The growth and virulence of Mycobacterium tuberculosis depends on its ability to scavenge host iron, an essential and limited micronutrient in vivo. In this study, we show that ferric iron accumulates both intra- and extra-cellularly in the primary lung lesions of guinea pigs aerosol-infected with the H37Rv strain of M. tuberculosis. Iron accumulated within macrophages at the periphery of the primary granulomatous lesions while extra-cellular ferric iron was concentrated in areas of lesion necrosis. Accumulation of iron within primary lesions was preceded by an increase in expression of heavy chain (H) ferritin, lactoferrin and receptors for transferrin, primarily by macrophages and granulocytes. The increased expression of intra-cellular H ferritin and extra-cellular lactoferrin, more so than transferrin receptor, paralleled the development of necrosis within primary lesions. The deposition of extra-cellular ferric iron within necrotic foci coincided with the accumulation of calcium and phosphorus and other cations in the form of dystrophic calcification. Primary lung lesions from guinea pigs vaccinated with Mycobactrium bovis BCG prior to experimental infection, had reduced iron accumulation as well as H ferritin, lactoferrin and transferrin receptor expression. The amelioration of primary lesion necrosis and dystrophic calcification by BCG vaccination was coincident with the lack of extra-cellular ferric iron and lactoferrin accumulation. These data demonstrate that BCG vaccination ameliorates primary lesion necrosis, dystrophic mineralization and iron accumulation, in part by down-regulating the expression of macrophage H ferritin, lactoferrin and transferrin receptors, in vivo.

FIG. 1

The pattern of ferric iron accumulation and calcification within the primary lung lesions in guinea pigs sham vaccinated with saline prior to infection by low-dose aerosol with the H37Rv strain of M. tuberculosis differs depending on progression of primary lesions. (A) In less advanced lesions 60 days post-infection, extra-cellular (inset) aggregates of ferric iron (arrows) are scattered amongst the necrotic cellular debris within the center of primary lesions. (B) In the same lesion, the pattern of extra-cellular calcium deposition (arrows) is similar to that of ferric iron, being concentrated within the areas of central necrosis. (C) In more advanced lesions 60 days post-infection, extra-cellular ferric iron is at the periphery (black arrow) of the area of calcified necrotic debris, while the intra-cellular (inset) ferric iron is within macrophages at the periphery granuloma (white arrow). (D) In the more advanced lesions 60 days post-infection, the intra- and extra-cellular calcification coalesce to form a well delineated center of calcified necrotic debris (arrow). Primary lung lesions in guinea pigs BCG-vaccinated prior to infection by low-dose aerosol with the H37Rv strain of M. tuberculosis fail to progress to necrosis at 30 days (E) and , calcification and extra-cellular ferric iron accumulation by day 60 (F) with Von Kossa's stain for calcium. Pearl's Prussian blue for ferric iron (A, C, E), Von Kossa's for calcium (B, D, F). A, B, D, Bars = 130 μm, 100× magnification. C, E, F Bars = 55.0 μm, 200× magnification.

FIG. 2

Trace mineral analysis of isolated of primary lung lesions of guinea pigs challenged with a low-dose aerosol of the H37Rv strain of M. tuberculosis or sterile water, without or with prior BCG-vaccination. The marked increase in calcium (A), phosphorus (B) and total iron (C) content of primary lesions at day 60 post-infection was ameliorated by BCG vaccination which reflects the lack of dystrophic calcification and ferric iron deposition seen microscopically. Data represents the mean ± standard deviation parts per million (PPM) from 2 separate experiments. Control n = 10, BCG- and sham-vaccinated n = 8. Asterisks denote values significantly different between BCG- and sham-vaccinated controls. * P≤ 0.05, ** P≤ 0.01.

FIG. 3

Expression of ferritin heavy chain, (H ferritin) as demonstrated by immunohistochemistry, by macrophages within primary lung lesions of guinea pigs challenged with a low-dose aerosol of the H37Rv strain of M. tuberculosis, without (A, C) or with prior BCG-vaccination (B,D). Lesions from sham-vaccinated animals had few cells in surrounding foci of lesion necrosis (arrows) that showed weak immunoreactivity at day 30 (inset) (A) that intensified by day 60 (C). Immunoreactivity was less in lesions from BCG-vaccinated animals at both day 30 (B) and day 60 (D) but differences were not statically different at day 60 (E). Expression of ferritin heavy chain was not significantly increased compared to non-infected controls, until 20 days post-infection where it was first seen in animals sham-vaccinated with saline. Expression was significantly increased over BCG-vaccinated animals at days 20 and 30 but was not different at day 60 post-infection. Expression of ferritin was not seen in BCG-vaccinated animals until 30 days post-infection. Ferritin showed no significant expression in non-infected animals (water aerosolized). Data represents the mean ± standard deviation from 1 representative experiment. n = 3. Asterisks denote values significantly different between BCG- and sham-vaccinated controls. * P≤ 0.05, ** P≤ 0.01. Bars = 340.0 μm, 40× magnification, insets 200× magnification.

FIG. 4

Expression of transferrin receptor (CD71) as demonstrated by immunohistochemistry, by macrophages within primary lung lesions of guinea pigs challenged with a low-dose aerosol of the H37Rv strain of M. tuberculosis, without (A, C) or with prior BCG-vaccination (B,D). Transferrin receptor expression in primary lesions in both BCG- and sham-vaccinated animals increased significantly from day 5 to 60 post-infection. Expression was highest in BCG-vaccinated animals at days 30 and 60 but based on scores, differences were not statistically different from sham-vaccinated animals (E). Data represents the mean ± standard deviation from 1 representative experiment. n = 3. Asterisks denote values significantly different between BCG- and sham-vaccinated controls. * P≤ 0.05, ** P≤ 0.01. Bars = 340 μm, 40× magnification, insets 200× magnification.

FIG. 5

Expression of lactoferrin, as demonstrated by immunohistochemistry, by cells within the primary lesions of guinea pigs at day 60 following challenge with a low-dose aerosol of the H37Rv strain of M. tuberculosis, without (A) or with prior BCG-vaccination (B). In the non-vaccinated animals labeling was particularly prominent in the necrotic centers (arrow) and appeared to be both intracellular, in degenerating cells, and extra-cellular in the necrotic debris (inset) (A). The surrounding macrophages expressed no or only low levels of lactoferrin. In contrast, in BCG-vaccinated animals the macrophages (arrow) in primary granulomas expressed moderate to high levels of lactoferrin (B). Lactoferrin expression increased progressively in non-vaccinated animals from 20 to 60 days but was only significantly different from BCG vaccinated animals at day 30 (C). Data represents the mean ± standard deviation from 1 representative experiment. n = 3. Asterisks denote values significantly different between BCG- and sham-vaccinated controls. * P≤ 0.05, ** P≤ 0.01. Bar = 130 μm, 100× magnification, inset 200× magnification.