The influence of haemoglobin and iron on in vitro mycobacterial growth inhibition assays.

The current vaccine against tuberculosis, live attenuated Mycobacterium bovis BCG, has variable efficacy, but development of an effective alternative is severely hampered by the lack of an immune correlate of protection. There has been a recent resurgence of interest in functional in vitro mycobacterial growth inhibition assays (MGIAs), which provide a measure of a range of different immune mechanisms and their interactions. We identified a positive correlation between mean corpuscular haemoglobin and in vitro growth of BCG in whole blood from healthy UK human volunteers. Mycobacterial growth in peripheral blood mononuclear cells (PBMC) from both humans and macaques was increased following the experimental addition of haemoglobin (Hb) or ferric iron, and reduced following addition of the iron chelator deferoxamine (DFO). Expression of Hb genes correlated positively with mycobacterial growth in whole blood from UK/Asian adults and, to a lesser extent, in PBMC from South African infants. Taken together our data indicate an association between Hb/iron levels and BCG growth in vitro, which may in part explain differences in findings between whole blood and PBMC MGIAs and should be considered when using such assays

Hepcidin deficiency and iron deficiency do not alter tuberculosis susceptibility in a murine M.tb infection model.

Tuberculosis (TB), caused by the macrophage-tropic pathogen Mycobacterium tuberculosis (M.tb) is a highly prevalent infectious disease. Since an immune correlate of protection or effective vaccine have yet to be found, continued research into host-pathogen interactions is important. Previous literature reports links between host iron status and disease outcome for many infections, including TB. For some extracellular bacteria, the iron regulatory hormone hepcidin is essential for protection against infection. Here, we investigated hepcidin (encoded by Hamp1) in the context of murine M.tb infection. Female C57BL/6 mice were infected with M.tb Erdman via aerosol. Hepatic expression of iron-responsive genes was measured by qRT-PCR and bacterial burden determined in organ homogenates. We found that hepatic Hamp1 mRNA levels decreased post-infection, and correlated with a marker of BMP/SMAD signalling pathways. Next, we tested the effect of Hamp1 deletion, and low iron diets, on M.tb infection. Hamp1 knockout mice did not have a significantly altered M.tb mycobacterial load in either the lungs or spleen. Up to 10 weeks of dietary iron restriction did not robustly affect disease outcome despite causing iron deficiency anaemia. Taken together, our data indicate that unlike with many other infections, hepcidin is decreased following M.tb infection, and show that hepcidin ablation does not influence M.tb growth in vivo. Furthermore, because even severe iron deficiency did not affect M.tb mycobacterial load, we suggest that the mechanisms M.tb uses to scavenge iron from the host must be extremely efficient, and may therefore represent potential targets for drugs and vaccines

Cytomegalovirus infection is a risk factor for tuberculosis disease in infants.

Immune activation is associated with increased risk of tuberculosis (TB) disease in infants. We performed a case-control analysis to identify drivers of immune activation and disease risk. Among 49 infants who developed TB disease over the first 2 years of life, and 129 healthy matched controls, we found the cytomegalovirus-stimulated (CMV-stimulated) IFN-γ response to be associated with CD8+ T cell activation (Spearman's rho, P = 6 × 10-8). A CMV-specific IFN-γ response was also associated with increased risk of developing TB disease (conditional logistic regression; P = 0.043; OR, 2.2; 95% CI, 1.02-4.83) and shorter time to TB diagnosis (Log Rank Mantel-Cox, P = 0.037). CMV+ infants who developed TB disease had lower expression of NK cell-associated gene signatures and a lower frequency of CD3-CD4-CD8- lymphocytes. We identified transcriptional signatures predictive of TB disease risk among CMV ELISpot-positive (area under the receiver operating characteristic [AUROC], 0.98, accuracy, 92.57%) and -negative (AUROC, 0.9; accuracy, 79.3%) infants; the CMV- signature was validated in an independent infant study (AUROC, 0.71; accuracy, 63.9%). A 16-gene signature that previously identified adolescents at risk of developing TB disease did not accurately classify case and control infants in this study. Understanding the microbial drivers of T cell activation, such as CMV, could guide new strategies for prevention of TB disease in infants

The effect of more severe iron deficiency on susceptibility to <i>M</i>.<i>tb</i>.

<p>Female 4–5 week old C57BL/6 mice were fed an iron deficient (2–6ppm) or control (200ppm) diet for 6 weeks prior to infection with 50–100 CFU of aerosolised <i>M</i>.<i>tb</i> Erdman (A). Mice remained on the respective diet until 4 weeks post-infection, when animals were sacrificed (a total of 10 weeks on their respective diets). Lungs and spleen were harvested for enumeration of CFU (B+C), and livers for gene expression analyses for <i>Hamp1</i> (D), <i>Fpn1</i> (E), <i>Id1</i> (F), <i>Bmp6</i> (G) and <i>Tfrc</i> (H). Mann-Whitney tests were performed to compare groups where *, **, *** and **** indicate p = <0.05, p = <0.01, p = <0.001 and p = <0.0001, respectively. N = 8 animals in infected groups and n = 6 in uninfected groups. Black symbols represent uninfected animals, red points infected animals except for CFU graphs where all animals are infected. Closed circles represent control animals and open circles, iron deficient animals.</p

The effect of <i>M</i>.<i>tb</i> infection on the expression of genes involved in the regulation of iron homeostasis.

<p>Female 6–10 week old C57BL/6 mice were infected with 100–200 CFU of <i>M</i>.<i>tb</i> Erdman via aerosol. Animals were sacrificed at day (D) 0 (baseline), D1, D7, D14, D28 and D56 post-infection (outlined in <b>A</b>). Hepatic gene expression analyses over the time course are shown for <i>Hamp1</i> (<b>B</b>), <i>Fpn1</i> (<b>D</b>), <i>Fga</i> (<b>F</b>) and <i>Id1</i> (<b>H</b>). Comparisons of <i>Hamp1</i> and <i>Fpn1</i> gene expression respective to uninfected controls are depicted in <b>C</b> and <b>E</b> respectively. Correlations between <i>Hamp1</i> and <i>Id1</i>, and <i>Hamp1</i> and <i>Fga</i> are shown in <b>G</b> and <b>I</b>, respectively. Expression of immune genes <i>Ifng</i> and <i>Tnfa</i> are shown in figures <b>J</b> and <b>K,</b> respectively. Kruskal-Wallis tests with Dunn’s post-tests for multiple comparisons were done for time course studies, and Mann-Whitney tests for comparisons to uninfected controls. Correlations were Spearman’s rank correlations. In all cases *, **, *** and **** indicate p = <0.05, p = <0.01, p = <0.001 and p = <0.0001, respectively. Baseline values (D0) were not included in correlations. In panels C and E, black symbols represent uninfected animals and red symbols infected animals. In all other panels, animals are infected. N = 5 per group.</p

Characterisation of more severe iron deficiency in younger mice.

<p>Female 4–5 week old mice were fed an iron deficient (2–6ppm iron) or control (200ppm iron) diet for a total of 6 or 10 weeks. Animals were bled via cardiac puncture under terminal anaesthesia and liver and spleen were removed for quantification of tissue iron. Haematological parameters are shown in panels A-F, and liver and spleen non-heme iron content shown in G-H, respectively. Mann-Whitney tests were performed to compare groups where *, **, *** and **** indicate p = <0.05, p = <0.01, p = <0.001 and p = <0.0001, respectively. N = 6 per group.</p

The effect of iron deficiency on susceptibility to <i>M</i>.<i>tb</i>.

<p>Female 6–10 week old C57BL/6 mice were fed an iron deficient (2–6ppm) or control (200ppm) diet for 2 weeks prior to infection with 50–100 CFU of aerosolised <i>M</i>.<i>tb</i> Erdman (<b>A</b>). Mice remained on the respective diet until 4 weeks or 8 weeks post-infection, when animals were sacrificed. Lungs and spleen were harvested for enumeration of CFU (<b>B-E</b>) and livers (from 4-week infected animals) for gene expression analyses. Gene expression data is shown for <i>Hamp1</i> (<b>F</b>), <i>Fpn1</i> (<b>G</b>), <i>Id1</i> (<b>H</b>), <i>Bmp6</i> (<b>I</b>) and <i>Tfrc</i> (<b>J</b>). Mann-Whitney tests were performed to compare groups where *, **, *** and **** indicate p = <0.05, p = <0.01, p = <0.001 and p = <0.0001, respectively. N = 8 per group for <b>B</b> and <b>C</b>, n = 10 per group for <b>D</b> and <b>E</b>. Gene expression data is representative of the two experiments where n = 10 for infected groups and 6 for uninfected controls. In Panels F-J, black symbols represent uninfected animals, red symbols infected animals except for CFU graphs where all animals are infected. Closed circles represent control animals and open circles represent iron deficient animals in all panels.</p

Characterisation of the iron deficient mouse model.

<p>Female 6–10 week old C57BL/6 mice were fed an iron deficient (2–6ppm) or control (200ppm) diet for a total of 2 weeks. Animals were sacrificed and hepatic gene expression analyses are shown for <i>Hamp1</i> (A), <i>Fpn</i> (B), <i>Id1</i> (C), and <i>Bmp6</i> (D). Mann-Whitney tests were performed to compare groups where *, **, *** and **** indicate p = <0.05, p = <0.01, p = <0.001 and p = <0.0001 respectively. N = 6 animals per group. Closed circles represent control animals and open circles represent iron deficient animals.</p

The effect of <i>Hamp1</i> deletion in murine <i>M</i>.<i>tb</i> infection and iron-related hepatic gene expression.

<p>Female 16–20 week old <i>Hamp1</i>^<i>-/-</i> mice and wild type controls were infected with 50–100 CFU of <i>M</i>.<i>tb</i> Erdman strain via aerosol (A). Animals were sacrificed 4 weeks later and CFU enumerated in organ homogenates (B-C). Hepatic gene expression is shown for <i>Fpn1</i> (D), <i>Id1</i> (E), <i>Bmp6</i> (F), <i>Tfrc</i> (G), <i>Ftl</i> (H) and <i>Fth</i> (I). Mann-Whitney tests were performed to compare groups where *, **, ***, and **** indicate p = <0.05, p = <0.01, p = <0.001 and p = <0.0001, respectively. N = 10 animals per group. Closed circles represent wild type animals and open circles represent <i>Hamp1</i>^<i>-/-</i> animals.</p