Potentiation and tolerance of toll-like receptor priming in human endothelial cells.

ABSTRACT

Repeated challenge of lipopolysaccharide (LPS) alters the response to subsequent LPS exposures via modulation of toll-like receptor 4 (TLR4). Whether activation of other TLRs can modulate TLR4 responses, and vice versa, remains unclear. Specifically with regards to endothelial cells, a key component of innate immunity, the impact of TLR cross-modulation is unknown. We postulated that TLR2 priming (via Pam3Csk4) would inhibit TLR4-mediated responses while TLR3 priming (via Poly IC) would enhance subsequent TLR4-inflammatory signaling. We studied human umbilical vein endothelial cells (HUVECs) and neonatal human dermal microvascular endothelial cells (HMVECs). Cells were primed with a combination of Poly IC (10 µg/ml), Pam3Csk4 (10 µg/ml), or LPS (100 ng/ml), then washed and allowed to rest. They were then rechallenged with either Poly IC, Pam3Csk4 or LPS. Endothelial cells showed significant tolerance to repeated LPS challenge. Priming with Pam3Csk4 also reduced the response to secondary LPS challenge in both cell types, despite a reduced proinflammatory response to Pam3Csk4 in HMVECs compared to HUVECs. Poly IC priming enhanced inflammatory and interferon producing signals upon Poly IC or LPS rechallenge, respectively. Poly IC priming induced interferon regulatory factor 7, leading to enhancement of interferon production. Finally, both Poly IC and LPS priming induced significant changes in receptor-interacting serine/threonine-protein kinase 1 activity. Pharmacological inhibition of receptor-interacting serine/threonine-protein kinase 1 or interferon regulatory factor 7 reduced the potentiated phenotype of TLR3 priming on TLR4 rechallenge. These results demonstrate that in human endothelial cells, prior activation of TLRs can have a significant impact on subsequent exposures and may contribute to the severity of the host response.