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

Rachel Tanner,Hal Drakesmith,Matthew K O’Shea,Magali Matsumiya,Julius Müller,Elena Stylianou,Helen A Fletcher,Paulo Bettencourt,Sally Sharpe,Stephanie Harris,Mike J Dennis,Vivek Naranbhai,Eneida A Parizotto,Rachel Harrington-Kandt,Andrew D White,Helen Mcshane

doi:10.1038/srep43478

Abstract

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.

Highlights

Transferrin and lactoferrin and transport it to mycobactins in the cell wall or the iron transport system[19]
Our data indicate an association between Hb/iron levels and BCG growth in vitro, which may be a confounding factor and contribute to variability when using whole blood, and to a lesser extent, peripheral blood mononuclear cells (PBMC) mycobacterial growth inhibition assays in vaccine and mycobacterial immunological studies
Where data was available from mycobacterial growth indicator tube (MGIT) assays performed using PBMC and separately whole blood from the same individuals from the previously reported BCG vaccination study in UK adults[8] (n = 10), correlations were performed between outcomes of the two assays

Summary

Introduction

Transferrin and lactoferrin and transport it to mycobactins in the cell wall or the iron transport system[19]. The majority of dietary iron in the host (~70–80%) is stored in the form of heme: primarily as haemoglobin[21]. Addition of exogenous heme to an M.tb mutant with an interrupted heme biosynthetic pathway restores growth[26], and addition of haemoglobin increases mycobacterial growth in vitro[12]. We have evaluated the effect of Hb and iron on mycobacterial growth in vitro using the previously-described MGIT assay[7,8,9,10]. This was investigated across different species commonly used in TB vaccine testing; human, mouse and non-human primates (NHPs). Our data indicate an association between Hb/iron levels and BCG growth in vitro, which may be a confounding factor and contribute to variability when using whole blood, and to a lesser extent, PBMC mycobacterial growth inhibition assays in vaccine and mycobacterial immunological studies

Methods

Results

Conclusion