Distinct transcriptional, cytokine, and γ-protocadherin responses to TLR1/2, TLR3, and TLR7/8 agonists in human cardiac endothelial cells

Toll-like receptors (TLRs) are key mediators of innate immune signaling and have been implicated in cardiovascular disease. TLR1, TLR3, and TLR7 are upregulated in diseased myocardium, but responses to ligands engaging these receptors in cardiac endothelial cells (ECs) remain poorly characterized. In the present study, primary human cardiac microvascular ECs were stimulated for 24 hours with Pam3CSK4, a TLR1/TLR2 agonist, Poly I:C, a TLR3 agonist, or R848, a TLR7/TLR8 agonist. Basal and stimulus-induced expression of TLR1, TLR2, TLR3, TLR7, and TLR8 was assessed by RT-qPCR. In addition, expression of a predefined panel of 84 NF-κB pathway-associated genes was profiled, secreted cytokine concentrations were quantified, and γ-protocadherin (PCDHG) family transcript levels were determined. TLR1, TLR2, and TLR3 were readily detectable in untreated ECs, whereas TLR7 and TLR8 transcripts were undetectable. Poly I:C markedly increased expression of TLR2, TLR3, and TLR7, while Pam3CSK4 and R848 did not significantly alter expression of the TLRs examined. The three stimuli produced distinct transcriptional profiles within the focused gene panel. Poly I:C induced extensive transcriptional changes and the strongest cytokine response while reducing all detectable PCDHG transcripts. Pam3CSK4 also induced extensive transcriptional changes and cytokine secretion but generated a different expression profile and generally increased PCDHG transcript levels. R848 produced limited transcriptional changes and did not significantly alter cytokine secretion or PCDHG expression at the 24-hour time point. These findings demonstrate stimulus-dependent differences in inflammatory and PCDHG responses in cardiac ECs and show that the endothelial TLR repertoire itself is differentially regulated by innate immune stimulation. Under the conditions tested, Poly I:C elicited the strongest inflammatory response and the most pronounced reduction in PCDHG expression, whereas R848 produced comparatively limited effects. Differences from previously observed responses in cardiac fibroblasts further highlight the cell-type-specific nature of innate immune responses in the human heart.