Macrophage susceptibility to infection by Ghanaian Mycobacterium tuberculosis complex lineages 4 and 5 varies with self-reported ethnicity

Abstract

Background: The epidemiology of Mycobacterium tuberculosis complex (MTBC) lineage 5 (L5) infections in Ghana revealed a significantly increased prevalence in Ewes compared to other self-reported ethnic groups. In that context, we sought to investigate the early phase of tuberculosis (TB) infection using ex vivo infection of macrophages derived from the blood of Ewe and Akan ethnic group volunteers with MTBC L4 and L5 strains.

Methods: The study participants consisted of 16 controls, among which self-reported Akan and Ewe ethnicity was equally represented, as well as 20 cured TB cases consisting of 11 Akans and 9 Ewes. Peripheral blood mononuclear cells were isolated from both healthy controls and cured TB cases. CD14⁺ monocytes were isolated and differentiated into monocyte-derived macrophages (MDMs) before infection with L4 or L5 endemic strains. The bacterial load was assessed after 2 hours (uptake) as well as 3 and 7 days post-infection.

Results: We observed a higher capacity of MDMs from Ewes to phagocytose L4 strains (p < 0.001), translating into a higher bacillary load on day 7 (p < 0.001) compared to L5, despite the higher replication rate of L5 in Ewe MDMs (fold change: 1.4 vs. 1.2, p = 0.03) among the controls. On the contrary, within macrophages from Akans, we observed a significantly higher phagocytic uptake of L5 (p < 0.001) compared to L4, also translating into a higher load on day 7 (p = 0.04). However, the replication rate of L4 in Akan MDMs was higher than that of L5 (fold change: L4 = 1.2, L4 = 1.1, p = 0.04). Although there was no significant difference in the uptake of L4 and L5 among cured TB cases, there was a higher bacterial load of both L4 (p = 0.02) and L5 (p = 0.02) on day 7 in Ewe MDMs.

Conclusion: Our results suggest that host ethnicity (driven by host genetic diversity), MTBC genetic diversity, and individual TB infection history are all acting together to modulate the outcome of macrophage infections by MTBC.

Keywords: Akan; Ewe; Ghana; Mycobacterium tuberculosis complex; West Africa; ethnicity; macrophage infection; tuberculosis.

Figure 1

Phylogenetic analysis of mycobacterial isolates used for the study. Phylogenetic tree based on whole-genome sequencing data from L4 (red) and L5 (brown) mycobacterial isolates (TBNM) used for the infection assays as well as other West African sequence data (Genome IDs) and all four other major MTBC lineages (L6 in green, L1 in pink, L2 in blue, and L3 in purple). MTBC, Mycobacterium tuberculosis complex.

Figure 2

Flow cytometry gating strategy. (A) (i) Morphological cell gating on lymphocyte and monocyte based on forward versus side scatter signals was used to determine the representation of (ii) CD3⁺CD4⁺ T cells or (iii) CD14⁺CD16⁺ monocytic cells and (iv) CD14⁺HLA-DR⁺ monocytes within PBMCs. (B) Isolated monocytes were analyzed first following morphological gating (i) and then based on their propensity to express (ii) CD14 and/or CD16 and (iii) CD16 and/or HLA-DR. (C) Monocyte-derived macrophages were also first analyzed based on their morphology (i) and subsequent fluorescence propensity to express (ii) CD206 and CD40 or (iii) CD14 and CD206. MTBC, Mycobacterium tuberculosis complex; PBMCs, peripheral blood mononuclear cells.

Figure 3

Comparison of the cellular composition of PBMCs from participants. Phenotypic and quantitative characterization of cells in PBMCs of (A) healthy controls [Akan, n = 10; Ewe, n = 10] and (B) cured TB cases [Akan, n = 18; Ewe, n = 15] of the two ethnic groups. Significant difference was observed only in the proportions (p = 0.02) and counts of CD3⁺CD4⁺ cells (p = 0.02) among the two ethnic groups using Mann–Whitney test. PBMCs, peripheral blood mononuclear cells; TB, tuberculosis. *p-value ≤ 0.05; ns, not significant (statistically), that is, p-value > 0.05.

Figure 4

Phenotypic profiling of sorted monocytes and derived macrophages. (A.i) Comparison of the proportions of sorted monocytes (CD14⁺CD16⁺, CD14⁺CD16⁻, CD14⁺HLA-DR⁺, and median fluorescent intensity (MFI) of HLA-DR) for controls [Akan, n = 8; Ewe, n = 8]. (A.ii) Comparison of derived macrophages (CD206⁺CD40⁺ and MFI of CD40 and CD206) for controls. (B.i) Comparison of the proportions of sorted monocytes (CD14⁺CD16⁺, CD14⁺CD16⁻, CD14⁺HLA-DR⁺, and MFI of HLA-DR) for cured TB cases [Akan, n = 11; Ewe, n = 9] across ethnicity. (B.ii) Comparison of the proportions of macrophages (CD206⁺CD40⁺ and MFI of CD40 and CD206) among the ethnic groups for cured TB cases. A significant difference was seen for MFI of HLA-DR of the controls (p = 0.03) using Mann–Whitney test. TB, tuberculosis. *p-value ≤ 0.05; ns, not significant (statistically), that is, p-value > 0.05.

Figure 5

Growth assessment of L4 and L5 in control macrophages (A) [Akan, n = 8; Ewe, n = 8] and cured TB cases (B) [Akan, n = 11; Ewe, n = 9]. (i) Comparison of growth of L4 and L5 at uptake, day 3 (D3), and day 7 (D7) in Akan macrophages. (ii) Comparison of the growth rate of L4 and L5 in Akan macrophages between uptake and D7. (iii) Comparison of the growth of L4 and L5 in Ewe macrophages (iv) using multiple Mann–Whitney test. TB, tuberculosis. *p-value ≤ 0.05; ***p-value ≤ 0.001; ns, not significant (statistically), that is, p-value > 0.05.

Figure 6

Comparison of Akan and Ewe macrophages from control (A) [Akan, n = 8; Ewe, n = 8] and cured TB cases (B) [Akan, n = 11; Ewe, n = 9] in support of MTBC growth. (i) Comparison of the growth of L4 at uptake, D3, and D7 in Akan and Ewe macrophages. (ii) Comparison of the growth rate of L4 in Akan and Ewe macrophages between uptake and D7. (iii) Comparison of the growth of L5 in Akan and Ewe macrophages. (iv) Growth rate comparison of L5 in Akan and Ewe macrophage between uptake and D7 using multiple Mann–Whitney test. TB, tuberculosis; MTBC, Mycobacterium tuberculosis complex. *p-value ≤ 0.05; **p-value ≤ 0.01; ***p-value ≤ 0.001; ns, not significant (statistically), that is, p-value > 0.05.