Scavenger receptor A1 is required for sensing HCMV by endosomal TLR-3/-9 in monocytic THP-1 cells

Abstract

Monocytes provide initial surveillance for pathogenic glycopeptides via scavenger receptors (SRs) and for viruses via Toll-like receptors (TLRs) which trigger pro-inflammatory response. However, specific interactions between SR-A1 and TLRs have not yet been assessed in human cytomegalovirus (HCMV)-exposed monocytes. Our results showed two patterns of gene expression upon HCMV exposure: genes that were induced within 10 min include SR-A1, Lyn, TLR-2, and IL-12p35, whereas those induced at 1 h are TLR-3, TLR-9, TRIF, IRF-3, and IFN-β. NF-κB p65 and TNF-α were elevated at both 10 min and 1 h post exposure. Further, inhibitory studies using neutralizing antibodies and morpholino antisense oligonucleotides suggested that within 10 min of HCMV exposure, transcription of TNF-α and IL-12 genes is TLR-2-dependent fashion. However, induction of both TLR-3-mediated IFN-β and TLR-9-mediated TNF-α at 1 h was dependent on SR-A1. These findings reveal a novel mechanistic insight into an interrelationship between SR-A1 and TLR-3/-9 signaling in HCMV-exposed monocytes.

Introduction

Human cytomegalovirus (HCMV) is the leading cause of human viral congenital infection and its sequelae can include sensorineural hearing loss and mental retardation (de la Hoz et al., 2002, Landolfo et al., 2003, Kumazaki et al., 2002). The majority of the congenitally infected infants are asymptomatic at birth but some present with cytomegalic inclusion disease, comprising microcephaly, organomegaly, hepatitis and thrombocytopenia with ∼10% mortality and debilitating sequelae in most survivors (Kumazaki et al., 2002, Azam et al., 2001, Lazzarotto et al., 2000). HCMV also can lead to retinitis and blindness in HIV-patients not receiving antiretrovirals. Further, concern for HCMV requires prophylaxis to reduce the risk of disseminated disease or organ rejection in transplant recipients (Myerson et al., 1984, Sano and Izumi, 1991) as well as stenosis of coronary arteries in cardiac transplants. A pathogenic advantage to HCMV is the ability to interact with and modify a broad spectrum of target cell types in vivo, such as monocytes/macrophages (Ibanez et al., 1991, Maciejewski et al., 1993), fibroblasts (Maciejewski et al., 1993), endothelial cells (Fish et al., 1998), coronary smooth muscle cells (Speir et al., 1998), hematopoietic progenitor cells (Maciejewski and St Jeor, 1999), neuronal cells (Tsutsui et al., 2005) as well as human cytotrophoblasts (LaMarca et al., 2004).

During acute HCMV infection, monocytes and their derivatives are key innate immune effector cells and are thought to participate in initial immune responses aimed at restricting viral dissemination. HCMV may enter monocytes to establish non-productive or at times productive infection (Smith et al., 2004), leading potentially to latent infection in vivo (Taylor-Wiedeman et al., 1991, Gredmark et al., 2004, Jin et al., 2007). Further, monocytes carrying HCMV genome are potential factors in the spread of HCMV to privileged sites such as the placenta and brain. Information on the nature and precise mechanism of HCMV-mediated pro-inflammatory cytokine induction at the mRNA level in monocytes shortly after exposure to HCMV is evolving. We sought to add to existing data by further delineating the initial aspects of HCMV-induced signaling that results in the induction of pro-inflammatory cytokines in monocytes. We investigated HCMV-exposed human monocytic THP-1 cells as an in vitro model of the intracellular signaling and the subsequent pro-inflammatory cytokine gene expression initiated by HCMV and triggered by engagement of pattern recognition receptor (PRR) motifs.

Among PRR receptor families are Toll-like receptors (TLRs) and scavenger receptors (SRs) (Barton and Medzhitov, 2002, Pasare and Medzhitov, 2005, Rice et al., 2002). TLRs and SRs are cell membrane proteins involved in both sensing of foreign, damaged or viral cell components (Tabeta et al., 2004, Compton et al., 2003, Carlquist et al., 2004) and in the induction of pro-inflammatory cytokines (Tabeta et al., 2004, Compton et al., 2003). TLRs consist of cell surface membrane receptors (TLR-1, TLR-2, TLR-4, TLR-5, TLR-6, and TLR-10) and endosomal membrane receptors (TLR-3, TLR-7, TLR-8 and TLR-9). All TLRs mediate signaling through their cytoplasmic TIR domains linked to adaptor proteins, such as MYD-88 and TRIF which induce the activation of NF-κB and IRF-3, respectively. Subsequently, these complexes translocate into the nucleus where they regulate transcription of target genes. For instance, TLR-3 signaling activates TRIF and IRF-3 which then mediate IFN-β promoter activation as an innate response to infectious agents (Oshiumi et al., 2003). In contrast, TLR-2 and TLR-9 engagement triggers MYD-88 and NF-κB p65 activation which regulates gene expression of multiple cytokines, e.g. TNF-α (Ryu et al., 2007, Tran et al., 2007) and IL-12 (Ryu et al., 2007). Of the known scavenger receptors, SR-A1 (Class A SRs, CD204) has been most studied primarily as a factor in atherosclerosis because it binds and mediates the endocytosis of modified low density lipoproteins (LDLs). However, other studies have shown SR-A1 functioning as a PRR by engaging components of several bacteria (Dunne et al., 1994) and viruses (Emile Gras et al., 2006), indicating a role in innate immunity and host defense.

Monocyte activation by viral/fungal/bacterial components via TLRs or by glycopeptides via SRs (Rice et al., 2002, Tabeta et al., 2004, Compton et al., 2003, Carlquist et al., 2004) plays a pivotal role in initial innate immune responses. In spite of extensive research of SRs and TLRs on the mice or fibroblast model, the signaling mechanisms involved in the pro-inflammatory cytokine induction by SR-A1 and TLR-2, -3, and -9 in HCMV-exposed human monocytes are not completely understood, and the possible role of interplay between these two receptor families has not yet to be addressed. Our goal is to evaluate CMV-induced triggering of TLR-2, -3, and -9 and whether surface receptor, e.g. SR-A1 may facilitate signaling of the endosomal TLR3 and/or TLR9. We hypothesized a possible interaction between SR-A1 and selected TLRs (TLR-2, -3, and -9) known to be involved in CMV responses in mouse NK cells (Tabeta et al., 2004) and human PBMC (Compton et al., 2003). This seemed plausible because HCMV expresses multiple envelope glycoproteins that might engage SR-A1 in addition to previously described TLR engagement. We first found that changes in the mRNA level of SR-A1 as well as TLR-2 occurred in THP-1 cells within 10 min, whereas TLR-3 and TLR-9 occurred later but within the first hour after exposure to HCMV. In a series of inhibitory experiments, we also showed that while both SR-A1 and TLRs are involved in HCMV sensing, SR-A1 is a required factor for TLR-3 and TLR-9 signaling. In contrast, TLR-2 acts independently of SR-A1 in regulating the induction of pro-inflammatory cytokines. Further, we report that the regulation of IL-12p35 and TNF-α expression is mediated via a TLR-2-dependent mechanism. This contrasts with the fact that both the induction of TLR-3-mediated IFN-β and a later TLR-9-mediated 15-fold increase in TNF-α mRNA is dependent on activation of SR-A1. Here, we present the first evidence of a distinct interrelationship between SR-A1 and the endosomal TLR-3 and -9, but a lack of interdependence between SR-A1 and TLR-2 that is essential for potent immunoregulation of pro-inflammatory cytokines following HCMV exposure of human monocytic THP-1 cells.