Figure 4. In vitro endotoxin (LPS) ‘tolerance’ and TLR ‘priming’ of cytokine responses by single-stranded RNA.

Data for triplicate experiments in PBMCs from two HIV uninfected subjects are shown in (A and B) using 0.1 ug/ml of LPS, 1 ug/ml ssRNA40, or media alone for initial priming and subsequent stimulation steps (e.g. ‘media-LPS’). In the first subject, pre-incubating PBMCs with LPS dramatically reduced subsequent LPS-induced TNF-α responses (P<0.0001). However, pre-incubating with HIV ssRNA (RNA40) increased subsequent LPS-induced TNF-α responses (P = 0.0011) when compared with pre-incubating with culture media alone. Conversely, LPS pre-incubation resulted in higher endotoxin-induced IL-10 responses than either media or HIV ssRNA-primed endotoxin stimulations (P = 0.0039, P = 0.025, and P = 0.098). In a second subject (B), with lower overall cytokine production from PBMC, LPS priming also reduced subsequent LPS induced TNF-α production (P = 0.0015), but priming with ssRNA did not result in significantly altered TNF-α responses (P = 0.39). Also in the second subject, ssRNA primed PBMCs induced lower IL-10 to subsequent LPS stimulation than media primed PBMCs at borderline statistical significance (P = 0.056). (C) The same experiment design but with reduced LPS levels to 0.01 ug/ml include one uninfected and one HIV infected subject. Lowering the LPS dose to 0.01 ug/ml resulted a trend toward LPS tolerance in the uninfected subjects (P = 0.097), but no significant tolerance in the HIV infected subjects (P = 0.325). However, in both cases, pre-stimulating PBMC with ssRNA40 dramatically enhanced subsequent LPS induced TNF-α (P<0.0001 for both). Between priming and the subsequent LPS stimulations, PBMCs were washed to remove solublized cytokines; and carry-over cytokines tested were negligible. All experiments were run in triplicate. Unpaired two tailed t tests were used. Level of significance ns = not significant, t = trend(P<0.1), *P≤0.05, **P≤0.01, and ***P≤0.001.