Subversion of innate defenses by the interplay between DENV and pre-existing enhancing antibodies: TLRs signaling collapse

Abstract

Background: The phenomenon of antibody dependent enhancement as a major determinant that exacerbates disease severity in DENV infections is well accepted. While the detailed mechanism of antibody enhanced disease severity is unclear, evidence suggests that it is associated with both increased DENV infectivity and suppression of the type I IFN and pro-inflammatory cytokine responses. Therefore, it is imperative for us to understand the intracellular mechanisms altered during ADE infection to decipher the mechanism of severe pathogenesis.

Methodology/principal findings: In this present work, qRT-PCR, immunoblotting and gene array analysis were conducted to determine whether DENV-antibody complex infection exerts a suppressive effect on the expression and/or function of the pathogen recognition patterns, focusing on the TLR-signaling pathway. We show here that FcγRI and FcγRIIa synergistically facilitated entry of DENV-antibody complexes into monocytic THP-1 cells. Ligation between DENV-antibody complexes and FcR not only down regulated TLRs gene expression but also up regulated SARM, TANK, and negative regulators of the NF-κB pathway, resulting in suppression of innate responses but increased viral production. These results were confirmed by blocking with anti-FcγRI or anti-FcγRIIa antibodies which reduced viral production, up-regulated IFN-β synthesis, and increased gene expression in the TLR-dependent signaling pathway. The negative impact of DENV-ADE infection on the TLR-dependent pathway was strongly supported by gene array screening which revealed that both MyD88-dependent and -independent signaling molecules were down regulated during DENV-ADE infection. Importantly, the same phenomenon was seen in PBMC of secondary DHF/DSS patients but not in PBMC of DF patients.

Conclusions/significance: Our present work demonstrates the mechanism by which DENV uses pre-existing immune mediators to defeat the principal activating pathway of innate defense resulting in suppression of an array of innate immune responses. Interestingly, this phenomenon specifically occurred during the severe form of DENV infection but not in the mild form of disease.

Figure 1. Kinetics of DENV-replication and IFN-β production in THP-1 cells infected with DEN-2 or DENV-Ab complexes.

THP-1 cells were pretreated with anti-FcγRI or/and FcγRIIa antibody before being infected with DENV-2 or DENV-enhancing antibody complexes. Supernatants were harvested every 24 h. post inoculation and were subjected to DENV RNA synthesis, infectious virion production and IFN-β production using qRT-PCR, plaque assay and ELISA, respectively. (A and B) Levels of viral RNA synthesis and infectious virus production. Three independent experiments were performed and results are expressed as mean ± SD. Asterisk indicates significant differences between DENV-ADE infected cultures with or without anti-FcR antibody pretreatment at P≤0.05. Triangle indicates the significant differences between DENV-ADE infected cultures pretreated with anti-FcγRI or with anti-FcγRIIa at P≤0.05. The significant differences were tested using ANOVA analysis, SPSS program. (C) Quantitation of IFN- β production by ELISA. Supernatant fluids harvested at 24 hr. of infection were quantitated for IFN-β production. Asterisk indicates signicficant differences in IFN-β production from DENV-ADE infected cells pretreated with anti-FcR antibodies or mock pretreatment. The P values were obtained from ANOVA analysis.

Figure 2. Expression levels of TLRs and TLR-signaling molecules during DENV or DENV-Ab complexes infection.

THP-1 cells were pre-treated with either anti-FcγRI antibody or anti-FcγRIIa antibody or both before being infected with DENV or DENV antibody complex. RNA was extracted from harvested cells at the indicated times and RNA copy numbers of (A) TLR-4, (B) TLR-7, (C) TLR-3, (D) TRAF-6 and (E) TRIF were quantified using qRT-PCR. Three independent experiments were performed and results are expressed as mean ± SD. Asterisk indicates significant differences between DENV-ADE infection, DENV infection, DENV-ADE infected cells pretreated with anti-FcγRI antibody or anti-FcγRIIa antibody or pretreated with both antibodies, at P≤0.05 using SPSS, ANOVA analysis. Significant differences between DENV-ADE infected cells pretreated with anti- FcγRI and anti-FcγRIIa antibody at the indicated P values were tested using Student's t-test. Figure 2 (F–I) are TRAF6, IRAK4 and TLR7 protein production detected by immunblotting using specific monoclonal antibody.

Figure 3. Activation of TANK and SARM during DENV and DENV-Ab infection.

THP-1 cells were infected with DENV alone or with DENV enhancing antibody complexes. The expression levels of (A) TANK and (B) SARM were quantified by qRT-PCR at various time points. Three independent experiments were performed and results are expressed as mean ± SD. Asterisk indicates the significant difference between DENV-ADE infection and DENV infection at P≤0.05. (C–D) detection of SARM at the protein level using immunoblotting.

Figure 4. Suppression of the TLRs-dependent signaling pathway during natural DENV infection.

PBMCs obtained from DF and secondary DHF patients (n = 15 for each group) on fever and convalescence days were subjected to monitoring the mRNA copy numbers of (A) TLR-3, (B) TLR-4, (C) TLR-7 and (D) TRAF-6 by qRT-PCR. The expression levels of these genes were expressed as mean ± SD of three independent experiments. Asterisk indicates significant differences at P≤0.05 using ANOVA analysis.

Figure 5. Validation of cDNA array expression data by qRT-PCR and immunoblotting.

RT-PCR was carried out using total RNA from THP-1 cells infected with either DENV or DENV-enhancing antibody complexes. The mRNA copy numbers of (A) TICAM2, (B) TIRAP and (C) IRAK-4 were monitored by qRT-PCR at indicated time points. The level of (D) IκB-α and (E) NF-κB productions were semi-quantified using immunoblotting. Three independent experiments were performed and results are expressed as mean ± SD. Asterisk indicates significant difference at P≤0.05, using ANOVA analysis. (F) is the representative of IκB-α and NF-κB western blotting.

Figure 6. Model illustrating TLR and TLR-signaling pathway collapse during DENV-Ab complex infection.

DENV-ADE infection down-regulates TLR-4 and endosomal TLRs synthesis while strongly stimulates SARM and TANK, negative regulators of TRIF and TRAF-6, respectively. These events result in suppression of innate responses mediated through the TLR-signaling pathway.