Understanding the expression of Toll-like receptors in Asian Indians predisposed to coronary artery disease The Bruneck study provided the first epide- miological evidence that circulating pro-inflam- matory mediators such as bacterial lipopolysac- charides (LPS) can constitute a strong risk factor for atherosclerosis development [30]. Subsequently, TLR-1, TLR-2 and TLR-4 were shown to be markedly elevated in human atherosclerotic plaque specimens [10] and in the medium and large arterial trees [31]. The above studies highlight the prominent role of TLRs as an emerging biomarker of atherosclerosis and a potential therapeutic target against CAD. Given this topical interest in TLRs, the present study was an exploratory study to understand the role of circulating TLR expression in CAD patients with strong familial predisposition to CAD. Our initial findings reveal that there is a differential pattern of gene expression in CAD patients as compared to the controls. In our study, the expression of TLR-2, was found to be significantly elevated while TLR-9 was lower in CAD patients as compared to controls. On the other hand, there was no difference in the levels of TLR-4 expression between the two groups. High levels of TLR-2 expression were reported on the surface of circulating monocytes, independent of established risk factors [32]. Genetic variations in the TLRs can attenuate (Asp299Gly in TLR-4) [33] or enhance the inflammatory and immune response to pathogenic challenge and thereby influence the pathogenesis or outcome of disease [34, 35]. Novel mutations have been identified in TLR-1 (Ser150Gly and Val220Met) and TLR-2 (Phe670Leu) that alter the ability of TLRs to recognize specific PAMPs associated with infection by Mycobacterium avium subsp. paratuberculosis (MAP) [36]. In the IARS, we have measured the antibody titres to various common pathogens such as Cytomegalovirus, Chlamydia pneumoniae, Helico- bacter pylori and Herpes simplex virus 1. Sero- positivity to the first three pathogens was found to be significantly higher in CAD patients (p < 0.05) as compared to their unaffected family members; also, the number of CAD patients was higher in the group with multiple rather than single infections (data unpublished), supporting the hypothesis that total pathogen burden rather than single infections are an important aetiopathological factor for CAD [37, 38]. Given this background knowledge, it is not surprising that we observed enhanced expression for TLR-2 in our patients selected from the IARS cohort. However, these preliminary findings have to be extended to a larger cohort to understand the true implications of the fold changes amongst the various TLRs and their downstream modulation of proinflammatory cytokines. In a recent report on the analysis of vessel-specific profiling of various TLRs in the medium and large arteries, it has been shown that while TLR-2 and TLR-4 are ubiquitously expressed, TLR-7 and TLR-9 may be infrequent and the other TLRs may show selective expression [31]. This again has to be further validated in the present context. In a recent study, a decrease in the expression of TLR-4 was shown to be associated with progression of cervical neoplasia in cases of infections with human papillomavirus (HPV) [39]. Yet another interesting study on Sprague-Dawley rats has shown that the underlying mechanism of ischemic preconditioning during repeated ischemic reperfusion might be related to decreased levels of TLR-4 expression and reduced levels of proin- flammatory cytokines, TNF-α and IL-1β [40]. TLR signalling has been shown to increase plaque vulnerability through myeloid differentiation primary response gene 88 (MyD88), a signalling adaptor shared by most TLR genes [17, 40]. In our study over 41% of CAD patients had experienced an MI event, suggesting the possible association of enhanced TLR expression with adverse cardio- vascular outcome. Statin treatment, primarily advocated for lowering of cholesterol levels, has been shown to decrease TLR-4 signalling through attenuation of the LPS responsiveness [41]. In contrast, such signalling has also been shown to enhance TLR- mediated cytokine expression in astrocytes through negative feedback regulation of the Rho proteins [42]. However, in the present study, statins did not appear to influence the pattern of TLR expression in CAD patients. Although our pilot study reveals alterations in expression levels of TLR-2 and TLR-9 genes in the present study, there are certain limitations such as the small sample size, increased prevalence of co- morbidities such as diabetes and hypertension and use of statins in the patient group as compared to the controls. These are being addressed in a further study involving well-matched control groups. Furthermore, we need to evaluate the influence of altered TLR expression on downstream cytokines that might provide stronger evidence on the mode of TLR regulation in atherosclerosis. An interesting association has been reported on the enhanced expression of IL-10-dependent elevated expression of TLR-9 by Treg cells through induction by CD3+ T cells in the presence of vitamin D, which has significant implications for vaccine design [43]. Developing a vaccine against atherosclerosis is one of our primary objectives, and the report by Urry et al. provides critical insights into mechanism of TLR regulation and the balance achieved by the T regulatory (Treg) cells in coordinating an effective immune response. We have observed that over 97% of the CAD patients have chronic vitamin D defi- ciency in a pilot study in the IARS (Insert reference # 44 – PMID: 216110492). The reported association of vitamin D mediated immune response of TLRs opens up exciting avenues for detailed inves- tigations. Arch Med Sci 5, October / 2011 785