HCMV induced effects of recombinant TLR4/MD2 soluble receptors on THP-1 cells.

<p>CD14 mRNA (A) increases at 6 hours were blunted in the presence of TLR4/MD2 soluble receptors but not in media absent the exogenous soluble receptors, consistent with the results seen for neutralizing TLR4 antibody in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044500#pone-0044500-g003" target="_blank">Figure 3</a>. Similar effects were seen on TIRAP (B), IL-6 (E) and IL-8 (F), suggesting that TLR4/MD2 complex signaling was essential for IL-6 and IL-8 induction. However, the soluble receptors had a minimal effect on TRAM (C) and TRAF6 (D). (means±S.D. n = 4). IL-6 (G) and IL-8 (H) secretion measured by ELISA showed similar findings. All data are means<u>+</u>S.D. of experiments performed in triplicate unless otherwise noted. *<i>p</i><0.05; ** p<0.01 by student T-test.</p

Human Cytomegalovirus Induces TLR4 Signaling Components in Monocytes Altering TIRAP, TRAM and Downstream Interferon-Beta and TNF-Alpha Expression

<div><p>Using TLR pathways, primary human cytomegalovirus (HCMV) induces innate responses including the production of inflammatory cytokines. Mounting evidence suggests that LPS recognition by TLR4/MD2/CD14 results in differential utilization of TIRAP-TRAF6 and TRAM-TRIF signaling, thereby leading to transcriptional activation of various cytokine genes. However, relative roles of the TLR4/MD2/CD14 complex and its adaptor proteins TIRAP and TRAM involved in regulating monocyte responses to HCMV are incomplete. Here, we provided evidence supporting the notion that the TLR4/MD2/CD14 complex contributes notably to HCMV-induced signaling and subsequent cytokine production in monocytes. In particular, induction of both IL-6 and IL-8 is associated with elevated TIRAP and reduced TRAM mRNA expression. The latter may serve in a compensatory pathway that yields a robust IFN response when TIRAP signaling is blocked in monocytes incubated with Toledo strain HCMV. Inhibitory studies using antisense oligonucleotides or neutralizing antibodies indicate that IL-6 induction by TLR4/MD2 complex is important for the activation of endogenous CD14 which later acts in concert or synergy with TLR4/MD2 as a factor resulting in IL-8 gene expression. We further show that exogenous recombinant CD14 can potentiate innate immune response via TLR4-dependent and possibly via TLR9-dependent pathways to promote enhanced expression/production of IL-8 and IFN-β, respectively.</p> </div

Role for CD14 in mediating CMV-induced cytokine expression.

<p>TLR4 (A) and TIRAP (C) mRNA elevations at 6 hours of HCMV co-incubation were suppressed in the CD14 antisense-treated THP-1 cells, but little effect was seen on TRAM (D) and MD2 (B). However, IL-8 (F) and IFN-β (H) were abolished by the CD14 antisense whereas IL-6 (E) and TRIF (G) were unaffected, suggesting that CD14 is the responsible receptor for inducing IL-8 and IFN-β transcription via the TLR4/TIRAP-dependent pathway and possibly the TLR9/MyD88-dependent pathway, respectively. (means±S.D. n = 4) (*<i>p</i><0.05, **p<0.01 by Student T-test compared with control at 1 hour and 6 hours) open squares = missense, closed squares = CD14 antisense. ELISA analysis showed that the CD14 blockade reduced the in vitro production of IL-6 (I), IL-8 (J) and sCD14 (K). Western blot for CD14 confirmed sequence-specific effect of the antisense (L). (n = 3). All data are means of experiments performed in triplicate except where noted.</p

Quantitative analysis of TLR4-mediated signaling molecules and cytokine transcriptions in THP-1 cells induced by HCMV.

<p>All data are means±S.D. of experiments performed in triplicate. A: TIRAP levels were seen to increase even at the earliest times after incubation with HCMV. B: TRAM conversely showed a marked reduction of mRNA levels in response to HCMV, particularly at 6 hours. C: TRAF6, which potentially serves as a rate-limiting factor, was not found to be significantly altered at any time point. D and E: TLR4 regulating cytokine IL-6 and IL-8 expressions were elevated at 1 hour and 6 hours, respectively. F and G: The production of IL-6 and IL-8 measured by ELISA correlated well with the results of D and E. *<i>p</i><0.05; ** p<0.01 by student T-test.</p

Role of TIRAP in TLR4-mediated regulation of cytokine gene expression in THP-1 cells induced by HCMV.

<p>A: TIRAP antisense treatment dramatically enhances levels of TRAM mRNA. B–D: Unlike previous elevations of TRAF6 mRNA (B), it was greatly inhibited by the TIRAP antisense. TIRAP-TRAF6-induced IL-6 (C) and IL-8 (D) mRNA elevation was also accompanied by a marked suppression. Interestingly, TRIF-induced (E) activation of IFN-beta (F) was markedly elevated by the TIRAP antisense, suggesting that a reversal exchange took place through the action of an alternative mechanism to activate IFN-inducible genes via the TRAM-TRIF pathway. (means±S.D. n = 4) (*<i>p</i><0.05, **p<0.01 by student T-test compared with control at 1 hour and 6 hours) open squares = missense, closed squares = TIRAP antisense. The ELISA results showed an inhibition of both IL-6 (G) and IL-8 (H) releases whereas secretion of IFN-β was significantly enhanced. All data are means of experiments performed in triplicate except where noted. J: Western blotting confirms the inhibitory effects of the antisense on TIRAP levels; (n = 3). β-actin served as control for equal loading.</p

Role of IL-6 in THP-1 cells incubated with HCMV.

<p>No change was seen for TLR4 (A) and TRAM (D) in the presence of IL-6 antibody. However, the IL-6 antibody had an inhibitory effect on CD14 (B), TIRAP (C), TRAF6 (E) and IL-8 (F). (means±S.D. n = 4) (*<i>p</i><0.05, **p<0.01 by student T-test compared with control at 6 hours). The secretion of cytokines IL-8 (G) and sCD14 (H) from IL-6-antibody-treated THP-1 cells was analyzed by ELISA and the results were similar to those observed by real-time PCR. Elisa data are means of experiments performed in triplicate.</p

Rescue effects of exogenous recombinant CD14 in THP-1 cells incubated with HCMV and treated with IL-6 antisense.

<p>TIRAP (A), TRAF6 (C), and IL-8 (D) levels at 6 hours were up-regulated by the addition of 20 ug/ml CD14. Rescue of IFN-β (F) levels at 6 hours was also seen. However, rescued cells showed no change in TRAM (B) and TRIF (E) levels. (means±S.D. n = 4) (*<i>p</i><0.05, **p<0.01 by student T-test compared with control at 6 hours). ELISA revealed a dramatic increase in IL-8 (G) and IFN-β (H) production. Elisa data are means of experiments performed in triplicate.</p

Effects of neutralizing TLR4 antibody compared to goat IgG control antibody on TLR4-induced gene transcription and cytokine activation.

<p>A: The increase in CD14 mRNA levels upon incubation with HCMV for 6 hours was significantly reduced by the TLR4 neutralizing antibody. B–F: Similar results were obtained for MD2 (B), TIRAP (C), IL-6 (E) and IL-8 (F). However, no effect on TRAM (D) was seen. (means±S.D. n = 4) (*<i>p</i><0.05, **p<0.01 by students T test compared with control at 1 hour and 6 hours). G and H: ELISA analysis confirmed expected decreased levels of IL-6 (G) and IL-8 (H). All data are means of experiments performed in triplicate except where noted.</p

PCR Primer Sequences.

<p>(FP = Forward Primer; RP = Reverse Primer; bp = basepair).</p

HCMV activates THP-1 cells via the TLR4/MD2/CD14 receptor complex.

<p>A and B: Immunostaining of THP-1 cells with and without HCMV stimulation for TLR4 showed marked upregulation of TLR4-positive staining. C–E: Quantitative gene expression analysis using real-time qPCR in TLR4, MD2, and CD14 reflected similar upregulation seen in A. All mRNAs were analyzed from the same preparation. All data are means±S.D. of experiments performed in triplicate. Reaction mixtures without reverse transcriptase served as controls for genomic DNA contamination in all cases (Data not shown).</p