Analysis of Chlamydia and Mycoplasma Expression in Atherosclerotic plaques and its Relation with P2X7 and TLR Expression

Introduction: We analysed the expression two infectious agents (C. pneumoniae and Mycoplasma spp.) and their possible relation with danger signal receptors (P2X7, TLR2 and 4) in atherosclerotic lesions. Danger signals are intracellular molecules released from damaged cells at inflammatory sites that include ATP (recognized by P2X7 receptors) and heat shock proteins (recognized by TLR receptors). Once activated, P2X7 receptor mediates IL-1 beta secretion and it is also involved in selective death of mycobacteria and Chlamydia infected macrophages. Besides being danger signal sensors, TLR are also well known for their interaction with bacterial ligands such as LPS, activating the secretion of inflammatory cytokines. Methods: We investigated specimen from 10 patients with severe stenosis of the internal carotid artery. Each sample was divided in three parts: proximal tract to heart, healthy, without stenosis, medial tract, corresponding to the atheromatous plaque and a distal tract, above the plaque. Total RNA was extracted and tested by a reverse transcriptase (RT)-PCR from each tissue sample. To detect the presence of C. pneumoniae and Mycoplasma spp. in atheromatous carotid plaque (APC), different genes were investigated (16s rRNA, Hsp60 and 16s rRNA, respectively). Moreover, expression levels of immune receptors (P2X7, TLR2 and 4) were evaluated by semiquantitative RT-PCR. Results: Among the samples studied, two resulted positive for Chlamydia expression both in the proximal tract. The one positive for the hsp60 gene of Chlamydia was from a patient with hemorrhagic evolution of the APC, while the one positive for 16s rRNA was from a patient with milder condition. In both these plaques, P2X7 receptor expression was down regulated if compared with the only plaque found completely negative for infectious agents. On the other hand, TLR2 and 4 were expressed just by the Hsp60 positive patient. The remaining seven plaques all showed positivity for a generic mycoplasma infection but only three of them also expressed 16s rRNA of M. hominis in whole three portions. No significant association of P2X7 expression levels was revealed for these patients, while whole plaques positive for M. hominis were also TLR2 positive. Conclusions: These preliminary data suggest a possible association among chlamydial infection and severity of the atherosclerotic lesion, that would be also related to a down modulation of P2X7 receptor. This could be explained by the ability of P2X7 of restricting Chlamydia widespread. Moreover, TLR2 expression seems to be upregulated by Mycoplasma infection. To further verify the significance of these associations, an analysis of a wider sample size will be required. Moreover, it could be of interest to study, in vitro, the modulation of danger signals receptors in foamy macrophages obtained in the presence of Chlamydia and Mycoplama.