Toll-like receptor agonists abrogate viremia.

<p>A: Left panel, a peak of viremia was detected at day 4 after DENV infection in two of three animals (44Z and AM28). However, this peak was completely absent in the four animals of the DENV/TLR group at 48 hours after the TLR3 and 7/8 agonists [poly (I:C) and CL097M-012, respectively] were administered (right panel). Results are expressed in milliequivalent genomes per milliliter. B: Plasma levels of DENV protein NS1 were not significantly different between the DENV group and the treated group (DENV/TLR). However, NS1 levels were significantly higher in the DENV group compared to the Control group (p<0.018), whereas they were not significantly different between the DENV/LTR (despite the Dengue infection) and the Control groups. This confirmatory assay corroborates the role of TLRs in controlling DENV replication. The results reflect the ratio of the sample O.D./Control O.D. according to the manufacturer's instructions.</p

Dengue virus inhibits TLR-induced activation of mDC.

<p>A: Left panel, 48 hours after the first TLR stimulation, mDC were activated in the groups receiving TLRs but not in the group receiving only the virus. Coincident with the peak of viremia at day 4, an inhibitory effect of DENV on the mDC subset was evidenced by the frequency of activated cells in the DENV group, which was significantly lower compared to the DENV/TLR and TLR groups. Right panel, 10 days after the infection (72 hours after second TLR stimulation) the mDC subset was still significantly activated in both groups that received TLRs when compared to the Control group. However, the inhibitory effect of DENV was absent as there were no significant differences between the DENV and DENV/TLR groups, supporting the role of DENV replication in the inhibitory effect on mDC activation on day 4. B: Ten days after infection and 72 hours after the secondary TLR stimulation the frequency of activated pDC was significantly higher in the DENV/TLR and TLR groups compared to the DENV and Control groups. However, an inhibitory effect of DENV on this subset of DC was not observed. Asterisks denote significant differences, (*) p <0.05, (**) p <0.01, and (***) p<0.00.</p

TLR3-7/8 stimulations in synergism with DENV induce quantitative and qualitative modifications of the humoral response.

<p>A: Ten days after the infection, the frequency of activated B cells was significantly higher in DENV/TLR group when compared to all other groups, showing a synergistic effect of DENV with the TLR agonists in this activation. DENV alone was able to induce activation of B cells when compared to the TLR and Control groups. However, in our model, TLR agonists alone were not sufficient to induce B-cell activation. B: The activation profile of B cells correlates with the levels of anti-DENV antibodies. Antibody levels were significantly higher in the DENV/TLR group compared to the DENV group. C: As early as 10 days after infection and coincident with activation of B cells, the switch to IgG1 was significantly diminished in the DENV/TLR group. This difference had disappeared by 15 days after the infection, but it was re-established on day 30. D: Although antibody O.D. values were limited, the difference in the IgG1 and IgG2 levels translated into a significant increase of the IgG2/IgG1 ratio (>11 and 6 times on days 10 and 30 after the infection, respectively). Asterisks denote significant differences, (*) p<0.05, (**) p<0.01, and (***) p<0.001.</p

Serum levels of CXCL-10 and IL-1Ra are increased significantly after TLR stimulation and are inhibited by DENV.

<p>A: Coincident with the higher level of mDC activation, serum levels of CXCL-10 increased significantly only in the TLR group when compared to the Control group. Higher levels, albeit not significant, were also found in the DENV/TLR group. The lowest levels among the experimental groups were found in animals of the DENV group. This trend in the serum levels of CXCL-10 among different groups confirms a stimulatory effect of the TLR agonist but at the same time suggests an inhibitory role of Dengue virus in the secretion of this cytokine. B: Serum levels of IL-1Ra peaked 48 hours after the first TLR stimulation. An inhibitory effect of DENV at this time point (also coincident with the viremia peak) is supported by significantly higher values of this cytokine in the TLR group when compared to the DENV and DENV/TLR groups. Both groups, DENV/TLR and TLR, showed significantly higher serum levels of this cytokine when compared to the DENV and Control groups. Ten days after the infection (72 hours after second TLR stimulation), IL1-Ra values were still increased in both groups receiving TLR agonists. Significantly higher levels were present up to 15 days after the infection (8 days after the last TLR stimulation). However, at the later time point, there was no significant difference between the DENV/TLR and TLR groups. Asterisks denote significant differences, (*) p <0.05, (**) p<0.01, and (***) p<0.001.</p