Epiregulin (EREG) variation is associated with susceptibility to tuberculosis

Abstract

Although host genetics influences susceptibility to Mycobacterium tuberculosis, the human genes regulating pathogenesis remain largely unknown. We used M. tuberculosis-stimulated macrophage gene expression profiling in conjunction with a case-control genetic association study to discover epiregulin (EREG), as a novel candidate tuberculosis (TB) susceptibility gene. Using a genome-wide association study dataset, we found that among the 21 genes with greater than 50-fold induction, EREG had the most polymorphisms associated with TB. We genotyped haplotype-tagging polymorphisms in discovery (N = 337 cases, N = 380 controls) and validation (N = 332 cases) datasets and an EREG polymorphism (rs7675690) was associated with susceptibility to TB (genotypic comparison; corrected P = 0.00007). rs7675690 was also associated more strongly with infections caused by the Beijing lineage of M. tuberculosis when compared with non-Beijing strains (controls vs Beijing, OR 7.81, P = 8.7 × 10(-5); non-Beijing, OR 3.13, P = 0.074). Furthermore, EREG expression was induced in monocytes and peripheral blood mononuclear cells stimulated with M. tuberculosis as well as TLR4 and TLR2/1/6 ligands. In murine macrophages, EREG expression induced by M. tuberculosis was MYD88- and TLR2-dependent. Together, these data provide the first evidence for an important role for EREG as a susceptibility gene for human TB.

Figure 1

Regulation of EREG in mice and human cells. EREG mRNA expression in human cells stimulated with Mtb and TLR ligands; (a) in human epithelial cell lines Hela (open columns) and A549 (solid columns); (b) in human peripheral blood mononuclear cell (PBMC) (gray columns), THP1 (open columns) and U937 (solid columns). (c) EREG mRNA expression in PM (open columns) and bone marrow macrophages (BMM; solid columns) from wild-type C57Bl/six mice, which were stimulated with Mtb and TLR ligands. Cells were stimulated with LPS (100 ng ml⁻¹), whole cell lysates of H37Rv Mtb (50 μg ml⁻¹) and lipopeptides PAM2 or PAM3 (250 ng ml⁻¹). After stimulation for 4 h, mRNA was extracted and measured by real-time PCR. (d) EREG expression in PM from wild-type, Myd88−/−, TLR2−/− and TLR4−/− knockout mice. Peritoneal macrophages were stimulated with LPS (100 ng ml⁻¹), Mtb (100 μg ml⁻¹), PAM2 (250 ng ml⁻¹) or PAM3 (250 ng ml⁻¹) for 4 h. EREG expression (performed in triplicate, mean±s.d.) was determined by real-time PCR. Student’s t-test for comparisons of EREG expression between WT and KO mice stimulated with either media (non-stimulated), LPS, Mtb, PAM2 or PAM3 have P<0.001 (*), <0.0001 (^&) and <0.00001 (^#).