Toll-like receptor 9 mediated responses in cardiac fibroblasts.

Altered cardiac Toll-like receptor 9 (TLR9) signaling is important in several experimental cardiovascular disorders. These studies have predominantly focused on cardiac myocytes or the heart as a whole. Cardiac fibroblasts have recently been attributed increasing significance in mediating inflammatory signaling. However, putative TLR9-signaling through cardiac fibroblasts remains non-investigated. Thus, our aim was to explore TLR9-signaling in cardiac fibroblasts and investigate the consequence of such receptor activity on classical cardiac fibroblast cellular functions. Cultivated murine cardiac fibroblasts were stimulated with different TLR9 agonists (CpG A, B and C) and assayed for the secretion of inflammatory cytokines (tumor necrosis factor α [TNFα], CXCL2 and interferon α/ß). Expression of functional cardiac fibroblast TLR9 was proven as stimulation with CpG B and -C caused significant CXCL2 and TNFα-release. These responses were TLR9-specific as complete inhibition of receptor-stimulated responses was achieved by co-treatment with a TLR9-antagonist (ODN 2088) or chloroquine diphosphate. TLR9-stimulated responses were also found more potent in cardiac fibroblasts when compared with classical innate immune cells. Stimulation of cardiac fibroblasts TLR9 was also found to attenuate migration and proliferation, but did not influence myofibroblast differentiation in vitro. Finally, results from in vivo TLR9-stimulation with subsequent fractionation of specific cardiac cell-types (cardiac myocytes, CD45+ cells, CD31+ cells and cardiac fibroblast-enriched cell-fractions) corroborated our in vitro data and provided evidence of differentiated cell-specific cardiac responses. Thus, we conclude that cardiac fibroblast may constitute a significant TLR9 responder cell within the myocardium and, further, that such receptor activity may impact important cardiac fibroblast cellular functions.

Toll-like receptor 9-activation during onset of myocardial ischemia does not influence infarct extension.

AIM: Myocardial infarction (MI) remains a major cause of death and disability worldwide, despite available reperfusion therapies. Inflammatory signaling is considered nodal in defining final infarct size. Activation of the innate immune receptor toll-like receptors (TLR) 9 prior to ischemia and reperfusion (I/R) reduces infarct size, but the consequence of TLR9 activation timed to the onset of ischemia is not known. METHODS AND RESULTS: The TLR9-agonist; CpG B was injected i.p. in C57BL/6 mice immediately after induction of ischemia (30 minutes). Final infarct size, as well as area-at-risk, was measured after 24 hours of reperfusion. CpG B injection resulted in a significant increase in circulating granulocytes and monocytes both in sham and I/R mice. Paradoxically, clear evidence of reduced cardiac infiltration of both monocytes and granulocytes could be demonstrated in I/R mice treated with CpG B (immunocytochemistry, myeloperoxidase activity and mRNA expression patterns). In addition, systemic TLR9 activation elicited significant alterations of cardiac inflammatory genes. Despite these biochemical and cellular changes, there was no difference in infarct size between vehicle and CpG B treated I/R mice. CONCLUSION: Systemic TLR9-stimulation upon onset of ischemia and subsequent reperfusion does not alter final infarct size despite causing clear alterations of both systemic and cardiac inflammatory parameters. Our results question the clinical usefulness of TLR9 activation during cardiac I/R.