Endothelial cell tolerance to lipopolysaccharide challenge is induced by monophosphoryl lipid A

Abstract

Prior exposure to lipopolysaccharide (LPS) produces a reduced or “tolerant” inflammatory response to subsequent challenges with LPS, however the potent pro-inflammatory effects of LPS limit its clinical benefit. The adjuvant Monophosphoryl lipid A (MPLA) is a weak toll-like receptor 4 (TLR4) agonist that induces negligible inflammation but retains potent immunomodulatory properties. We postulated that pre-treatment with MPLA would inhibit the inflammatory response of endothelial cells to secondary LPS challenge. Human umbilical vein endothelial cells (HUVECs), were exposed to MPLA (10 µg/ml), LPS (100 ng/ml) or vehicle control. HUVECs were then washed and maintained in culture for 24 hours before being challenged with LPS (100 ng/ml). Supernatants were collected and examined for cytokine production in the presence or absence of siRNA inhibitors of critical TLR4 signaling proteins. Pretreatment with MPLA attenuated IL-6 production to secondary LPS challenge to a similar degree as LPS. The application of MyD88 siRNA dramatically reduced MPLA-induced tolerance while TRIF siRNA had no effect. The tolerant phenotype in endothelial cells was associated with reduced IKK, p38 and JNK phosphorylation and enhanced IRAK-M expression for LPS primed HUVECs, but less so in MPLA primed cells. Instead, MPLA-primed HUVECs demonstrated enhanced ERK phosphorylation. In contrast to leukocytes in which tolerance is largely TRIF-dependent, MyD88 signaling mediated endotoxin tolerance in endothelial cells. Most importantly, MPLA, a vaccine adjuvant with a wide therapeutic window, induced tolerance to LPS in endothelial cells.

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

“…Based on previous studies, LPS causes limited production of IP-10, owing to poor induction of the TRIF pathway after TLR4 stimulation in endothelial cells. 19 However, as shown in Fig 3 B (and Fig S1 C ), when both types of endothelial cells were primed with Poly I:C, there was a significant induction of IP-10 with a greater than 17-fold increased level compared to unprimed cells in HMVECs and greater than 7-fold in HUVECs. Moreover, priming by Pam3Csk4 reduced IP-10 production by LPS rechallenge compared to unprimed cells in HMVECs, though this effect was not as significant as LPS priming.…”

“…18 Further confounding these results, we have demonstrated that the mechanisms of TLR4 signaling tolerance in endothelial cells are differentially regulated compared to those of leukocytes, suggesting that modulation of TLR signals in human endothelial cells may not be conserved to that reported in mouse macrophages. 19 Understanding this difference in the human endothelium is emphasized by the role it plays in vascular homeostasis and innate immunity activation, making its modulation a key driver of the host response to infection. 20 …”

“…16 However, we have demonstrated that in endothelial cells, TLR4 binding to LPS results primarily in MyD88 activation with little TRIF activation, making the role of MyD88 and TRIF in tolerance less clear. 19 Given the fundamental difference between endothelial cells and leukocytes, we sought to explore how the differential regulation of TLR-mediated MyD88 and TRIF pathway activation altered the response of endothelial cells to multiple TLR ligands compared to that published in leukocytes. In addition, based on clinical data that coinfections lead to worse outcomes and previous work on TLR signaling, we hypothesized that TLRs that used the same signaling pathway (eg, MyD88 only) would induce cross-tolerance while TLRs that signal via different pathways (TRIF vs MyD88) would lead to potentiation.…”

Interactions of TLR4 and PPARγ, Dependent on AMPK Signalling Pathway Contribute to Anti-Inflammatory Effects of Vaccariae Hypaphorine in Endothelial Cells

“…Toll-like receptors complex (TLRs) signalling pathways, particularly TLR4, are activated by LPS, which induces excessive expressions of inflammatory cytokines [26]. We next determined whether TLR4 was involved in the anti-inflammatory roles of VH on EA.hy926 cells.…”

Stroma: the forgotten cells of innate immune memory

“…The role of EC as inflammatory contributors to atherosclerosis is well established [44,67], and the role of myeloid trained immunity in the disease is also gaining attention (reviewed in [68,69]. These publications and others [34,37,40,48,70] acknowledge the dangers of innate over-aggressive inflammatory responses based on priming by endogenous mediators. DAMPs and other host products inducing this high alert state may lead to chronic inflammatory conditions because they will tend to favour sustained inflammatory responses rather than resolution of inflammation.…”