Peripheral blood mononuclear cells (PBMCs) were isolated from tuberculosis (TB) patients and stimulated with 10 μg/ml of either Mycobacterium
tuberculosis lab-adapted strain H37Rv or clinical strain HN878 cell wall extract. Levels of (A) IL-1β (n = 19 patients) and (B) IL-17 (n = 20 patents) in culture supernatants were measured by ELISA. (C) Expression of HIF1A mRNA following stimulation with 10 μg/ml HN878 cell wall extract was determined by RT-PCR in PBMCs from TB patients (n = 30), latently infected TB (LTBI) patients (n = 30), or household controls (n = 55). (D) Formalin-fixed, paraffin-embedded lung sections from TB patients were stained for CD68 (green) and HIF1α (red) by immunofluorescence. Representative images shown (×200 magnification). (E) TB patients or household controls (HC) were genotyped for G/A, G/G, or A/A alleles of the rs2275913 SNP using the Sequenom assay as described in the Methods (nTotal = 263 patients; n = 94 HC with G/G, n = 54 HC with G/A or A/A, n = 87 TB patients with G/G, and n = 28 TB patients with G/A or A/A). (F) IL-17 production in culture supernatants of HN878 cell wall extract–stimulated (10 μg/ml) PBMCs isolated from either G/A, A/A, or G/G genotype TB patients was measured by multiplex assay (n = 12 patients for G/G allele, n = 4 patients for untreated G/A or A/A alleles, and n = 5 patients for HN878-treated G/A or A/A alleles). *P < 0.05, **P < 0.01, ***P < 0.001 by 2-way ANOVA (A and B), 1-way ANOVA (C), or repeated-measures ANOVA (F).