The effect of systemic iNOS inhibition during human endotoxemia on the development of TLR-tolerance in vitro

Introduction

The phenomenon of repeated exposure to endotoxin resulting in diminished release of proinflammatory cytokines is called endotoxin tolerance. After receiving a low-dose pretreatment with endotoxin, tolerant animals survive a challenge with a 'lethal dose' of endotoxin. Recent studies suggests a pivotal role for NO arising from iNOS in the development of tolerance. Toll-like receptors (TLRs) are receptors that recognize specific pathogen-associated molecular patterns. Ten members of the so-called TLR family have been identified in humans, and several of them appear to recognize specific microbial products, including LPS (TLR-4), peptidoglycan (TLR-2) and flagelline (TLR-5). Recognition leads to activation of intracellular signaling pathways, which upregulate a wide array of inflammatory modulators that contribute to the early host cell response. The aim of this study was to test whether administration of the selective iNOS inhibitor aminoguanidine during human experimental endotoxemia influences the development of tolerance to several TLR-agonists in vitro.

Methods

Seventeen healthy volunteers (age 22.2 ± 1.8 years) were treated with an i.v. bolus injection of 2 ng/kg Escherichia coli LPS in the absence (n = 10) or presence (n = 7) of the selective iNOS inhibitor aminoguanidine (bolus of 370 mg and continuous infusion 1 mg/min 1 hour after administration of LPS). Whole blood was stimulated in vitro with different TLR agonists before, and 2 and 4 hours after LPS treatment. The TLR agonists used were Pam 3 Cys, 1 μg/ml (TLR-2); Poly I:C, 50 μg/ml (TLR-3); E. coli LPS, 1 ng/ml (TLR-4); Flagelline, 10 ng/ml (TLR-5); and Loxoribine, 50 μg/ml (TLR-7). The samples were incubated at 37°C for 24 hours, after which cell – free super natant was obtained ( centrifugation at 2000 × g at 4°C for 15 min) and stored at -80°C until ELISAs were performed.

Results

In-vitro incubation with the different TLR-agonists resulted in release of cytokines in blood samples taken prior to the LPS administration in vivo. The cytokine release was attenuated 2 and 4 hours after LPS administration in vivo (see Fig. 1a). This development of tolerance occurred in TLR-3, TLR-4, TLR-5 stimulated whole blood. In-vivo administration of the iNOS inhibitor amino-guanidine had no significant effect on the development of tolerance in TLR-3 and TLR-4 stimulated whole blood, but TLR-5 agonist stimulated whole blood showed that tolerance was inhibited (P < 0.05; see Fig. 1b).

Figure 1

TNF response to (a) TLR-4 and (b) TLR-5 agonist stimulation in vitro in the absence (straight line) and presence (dotted line) of in-vivo treatment with iNOS inhibitor aminoguanidine.

Conclusion

LPS administration in vivo attenuates the in-vitro cytokine response on various TLR-agonists. Inhibition of iNOS in vivo had no effect on the development of tolerance in TLR-3 and TLR-4 stimulated whole blood, but tolerance diminished in TLR-5 agonist stimulated whole blood. This suggests that NO is involved in the development of tolerance to flagelline.