Effect of Iron Supplementation on the Outcome of Non-Progressive Pulmonary Mycobacterium tuberculosis Infection

Abstract

The human response to Mycobacterium tuberculosis (Mtb) infection is affected by the availability of iron (Fe), which is necessary for proper immune cell function and is essential for the growth and virulence of bacteria. Increase in host Fe levels promotes Mtb growth and tuberculosis (TB) pathogenesis, while Fe-supplementation to latently infected, asymptomatic individuals is a significant risk factor for disease reactivation. However, the effect of Fe-supplementation on the host immunity during latent Mtb infection remains unclear, due partly to the paucity in availability of animal models that recapitulate key pathophysiological features seen in humans. We have demonstrated that rabbits can develop non-progressive latency similar to infected humans. In this study, using this model we have evaluated the effect of Fe-supplementation on the bacterial growth, disease pathology, and immune response. Systemic and lung Fe parameters, gene expression profile, lung bacterial burden, and disease pathology were determined in the Mtb-infected/Fe- or placebo-supplemented rabbits. Results show that Fe-supplementation to Mtb-infected rabbits did not significantly change the hematocrit and Hb levels, although it elevated total Fe in the lungs. Expression of selected host iron- and immune-response genes in the blood and lungs was perturbed in Mtb-infected/Fe-supplemented rabbits. Iron-supplementation during acute or chronic stages of Mtb infection did not significantly affect the bacterial burden or disease pathology in the lungs. Data presented in this study is of significant relevance for current public health policies on Fe-supplementation therapy given to anemic patients with latent Mtb infection.

Keywords: Mycobacterium tuberculosis; Perls’ stain; gene expression; immune response; iron supplementation; latent infection; pathology; pulmonary; rabbit; tuberculosis.

Figure 1

Effect of Fe-supplementation on plasma iron parameters of rabbits at 8 weeks (A,C,E) or 16 weeks (B,D,F) post-Mtb infection. Total iron binding capacity (TIBC) (top), total iron (middle), and percent transferrin saturation (bottom) were determined in plasma samples of Mtb-infected and placebo- or Fe-treated rabbits at the conclusion of treatment (i.e., 8 weeks or 16 weeks post-infection). Data was analyzed by one-way Anova with Tukey’s multiple comparison test. Values plotted are mean +/– sd with n = 4 per group per time point. * p < 0.05; ** p < 0.01.

Figure 2

Effect of Fe-supplementation on the lung iron parameters of rabbits at 8 and 16 weeks post infection. TIBC (top; A), total iron (middle; B) and percent transferrin saturation (bottom; C) were determined in the homogenates of Mtb-infected and placebo (-) or Fe (+) treated rabbits. Data was analyzed by one-way Anova with Tukey’s multiple comparison test. Values plotted are mean +/– sd with n = 4 per group per time point. * p < 0.05.

Figure 3

Expression of iron-responsive genes in Mycobacterium tuberculosis (Mtb)-infected rabbits at 4- and 12-weeks post infection. Data shown are expression of target genes in the blood (A,B) or lung (C,D) during acute (8 weeks; A,C) or chronic (16 weeks; B,D) stages of infection. The gene expression levels in Mtb-infected animals was calibrated with the corresponding levels in uninfected rabbits. Host house-keeping gene (GAPDH) expression was used to normalize the level of target gene expression. Data was analyzed by one-way Anova with Tukey’s multiple comparison test. Values plotted are mean +/– sd with n = 4 per group per time point. All tested genes were statistically significant in Figure 3A–D.

Figure 4

Effect of Fe-supplementation on the expression of host iron-responsive genes in Mtb-infected rabbits. Heat map of selected immune response gene expression in the blood (A–D) or lung (E–H) during acute (4 and 8 weeks; A,B,E,F) or chronic (12 and 16 weeks; C,D,G,H) stages of infection in rabbits treated with Fe. The data from Fe-supplemented animals was calibrated with the corresponding levels in placebo-treated animals. Host house-keeping gene (GAPDH) expression was used to normalize target gene expression. Red color indicates up-regulation and blue color indicates down-regulation. The gradient in color indicates the trend towards up- or down-regulation.

Figure 5

Expression of host pro- and anti-inflammatory response genes in Mtb-infected rabbits at 8 and 16 weeks post infection. Data shown are expression of target genes in the blood (A,B) or lung (C,D) during acute (8 weeks; A,C) or chronic (16 weeks; B,D) stages of infection. The gene expression levels in Mtb-infected animals was calibrated with the corresponding levels in uninfected rabbits. Host house-keeping gene (GAPDH) expression was used to normalize the level of target gene expression. Data was analyzed by one-way Anova with Tukey’s multiple comparison test. Values plotted are mean +/– sd with n = 4 per group per time point. All tested genes were statistically significant (p < 0.05) in Figure 5A–D.

Figure 6

Effect of Fe-supplementation on the expression of host immune response genes in Mtb-infected rabbits. Heat map of selected immune response gene expression in the blood (A–D) or lung (E–H) during acute (4 and 8 weeks; A,B,E,F) or chronic (12 and 16 weeks; C,D,G,H) stages of infection in rabbits treated with Fe. The data from Fe-supplemented animals was calibrated with the corresponding levels in placebo-treated animals. Host house-keeping gene (GAPDH) expression was used to normalize target gene expression. Red color indicates up-regulation and blue color indicates down-regulation. The gradient in color indicates the trend towards up- or down-regulation

Figure 7

Effect of Fe-supplementation on the lung bacillary load of Mtb-infected rabbits. Number of bacterial colony-forming units (CFU) was determined in the lungs of rabbits supplemented with Fe or placebo during acute (A) or chronic (B) stages of Mtb infection. Data was analyzed by one-way Anova with Tukey’s multiple comparison test. Values plotted are mean +/– sd with n = 5 per group per time point. Rx denotes treatment start time (day-1 or 8 weeks post-infection). The treatment was continued for 8 weeks in both acute and chronic infection groups.

Figure 8

Effect of Fe-supplementation on rabbit lung pathology at 8 weeks post Mtb infection. Histopathology of rabbit lungs infected with Mtb CDC1551 at 8 weeks post infection with (A–C) or without (D–F) Fe-supplementation showing disease pathology (H&E stain; A,D), iron deposition (Perls’ iron stain; B,E) and Mtb (by immunohistochemistry; C,F). Dark arrows in (E) show cellular iron deposition (blue color). White arrows in (C,F) show Mtb (purple color). The scale bar for all the images is 50 µm. Sections were photographed at 400× (A,B,D,E) or 600× (C,F) of original magnification.

Figure 9

Effect of Fe-supplementation on rabbit lung pathology at 16-weeks post Mtb infection. Histopathology of rabbit lungs infected with Mtb CDC1551 at 16 weeks post infection with (A–C) or without (D–F) Fe-supplementation showing disease pathology (H&E stain; A,D), iron deposition (Perls’ iron stain; B,E) and Mtb (by immunohistochemistry; C,F). Dark arrows in (E) show cellular iron deposition (blue color). White arrows in (C,F) show Mtb (purple color). The scale bar for all the images is 50 µm. Sections were photographed at 400× (A,B,D,E) or 600× (C,F) of original magnification.