HCMV-induced effects of TRAM in TLR4-mediated regulation of cytokine gene expression in THP-1 cells.

<p>A–F: The mRNA levels of all of the key molecules remained stable in the presence of the TRAM antisense, which is the opposite result of what is seen with the TIRAP antisense, suggesting that TRAM may not be a critical regulator in signaling triggered by HCMV in THP-1 cells. Open squares = missense, closed squares = TRAM antisense. G: Western blot confirmed sequence-specific decreases in target TRAM by antisense. All data are means±S.D. of experiments performed in triplicate.</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

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

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

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

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

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