Using a genome-wide expression study with Mtb-stimulated macrophages, we identified EREG as a highly induced TLR-dependent gene with a human polymorphism associated with susceptibility to TB. In addition, the risk of TB was further increased in individuals who had EREG polymorphism rs7675690 and were infected with the Beijing strain of Mtb.
As a member of the epidermal growth factor (EGF) family, EREG is better known for its role in cell growth and homeostasis. However, our study supports a broader biological role for EREG with evidence that it is involved in the macrophage response to Mtb
infection. Unlike other EGF family members, EREG expression is not ubiquitous in normal human tissue at the mRNA level.23
Interestingly, we found that EREG induction was selective for myeloid cells after TLR or Mtb
stimulation. Previous studies have also found that EREG is induced in murine tissue and PM by the TLR agonists LPS and peptidoglycan. The molecular mechanism of how EREG modulates macrophage function is only partially understood. Shirawasa et al
found that IL6 and TNF-α were reduced in PM from EREG knockout mice in response to LPS and PGN, compared with cytokine levels in wild-type mice. Along with our data, this suggests that EREG expression is dependent upon TLR activation and may also modulate TLR-mediated signaling in macrophages. EREG has both a membrane-bound and a soluble form, which can act as an autocrine growth factor for keratinocytes in vitro
The membrane-anchored form, but not the soluble form, regulates cytokine production in macrophages.16,25
on the basis of the literature and our data,16,25,26
we propose a multi-step model of EREG-dependent function in Mtb
-infected macrophages. First, Mtb
stimulates EREG expression and other inflammatory molecules in a TLR2-dependent manner. EREG subsequently acts in a membrane or soluble form to stimulate cells through EGF receptor and further amplify cytokine production in an autocrine or paracrine manner. Interestingly, EREG modulation of cytokine production may be dependent on the specific TLR which is stimulated (for example, regulation of EREG when TLR2 is stimulated with peptidoglycan, but not when TLR9 is stimulated with CpG DNA).16,17
In addition, previous studies suggest that soluble EREG may modulate inflammatory pathways in a selective manner in some cell types by downregulating IL-18 expression.16
How EREG modulates inflammatory pathways in Mtb
-stimulated macrophages is incompletely understood and will require further study.
The mechanistic role of EREG in TB pathogenesis is not yet known. EREG expression was not only induced in Mtb
stimulated macrophages, but displayed higher expression in macrophages from patients with TBM compared with PTB patients and LTB subjects. A recent study demonstrated that EREG expression is induced in murine interstitial lung macrophages quite some time after low dose aerosol infection of Mtb
[(after 360 days, but not after 56 or 180 days)18
]. Other studies have shown that a group of genes including EREG
are expressed in human breast cancer cells. These genes are required to breach the lung vasculature and enable extravasation of cancer cells during their dissemination from mammary tumors to the lungs.27,28
-infected people, after bacilli invade the pulmonary alveolar macrophage within the lung, they replicate and disseminate to the regional lymph nodes. In the case of extra-pulmonary disease, bacteria spread haematogenously to other organs in the body. TBM subjects with elevated EREG expression may have a higher risk of dissemination due to an effect on one of these steps in pathogenesis.
The polymorphism associated with TB is located within an intronic region of the EREG gene and is likely a genetic marker in linkage disequilibrium with the functional and disease causative allele. The association of this polymorphism with TB was identified in our discovery cohort and was ‘validated’ in a second cohort with independent cases. This technically does not constitute a ‘complete validation’ cohort as the same controls were used for both cohorts. It is possible that random variation in this one control group could be responsible for the observed associations. However this is unlikely, and the combination of phenotype-genotype association and expression-genotype association provides a useful validation, especially as it suggests actual biological consequences with regard to altering gene expression. Although we cannot exclude that the causative variant is within a neighboring gene (EPGN encoding Epigen and AREG encoding Amphiregulin, both EGF family ligands for ERBB family receptors), it is unlikely because of low linkage disequilibrium between the associated EREG SNPs and the haplotype block defined by SNPs in EPGN and AREG (r2<0.50). To our knowledge this is the first association between EREG SNPs and any disease.
There is a evidence to support the hypothesis that some strains of Mtb
are more virulent than others,29
however, it remains unclear whether bacterial lineage influences the development of human TB disease. We previously found that Euro–American lineage strains of Mtb
were significantly less likely to cause disseminated TB with meningitis than the East/Asian Beijing and Indo-Oceanic lineage strains.20
In addition the combination of a particular host genotype (the C allele of TLR-2 T597C) with TB caused by the East-Asian/Beijing lineage increased the risk of TBM. Here, the association of the EREG SNP rs7675690 was the strongest in TBM patients that had been infected with Mtb
of the Beijing lineage. This suggests that the causative variant of EREG may be associated with an impaired immune response to Mtb
leading to more aggressive disease, prolonged bacteraemia and an increased chance of seeding to the meninges. This demonstrates a further significant interaction between host and bacterial genotypes and the development of TB.
Together, these data provide the first evidence to suggest an important role for EREG in the human immune response to TB. By combining macrophage transcriptional profiling with human genetic studies, we identified a potential human susceptibility gene that was not previously thought to be involved with TB pathogenesis.