Respiratory Syncytial Virus Fusion Protein-Induced Toll-Like Receptor 4 (TLR4) Signaling Is Inhibited by the TLR4 Antagonists Rhodobacter sphaeroides Lipopolysaccharide and Eritoran (E5564) and Requires Direct Interaction with MD-2

Abstract

Respiratory syncytial virus (RSV) is a leading cause of infant mortality worldwide. Toll-like receptor 4 (TLR4), a signaling receptor for structurally diverse microbe-associated molecular patterns, is activated by the RSV fusion (F) protein and by bacterial lipopolysaccharide (LPS) in a CD14-dependent manner. TLR4 signaling by LPS also requires the presence of an additional protein, MD-2. Thus, it is possible that F protein-mediated TLR4 activation relies on MD-2 as well, although this hypothesis has not been formally tested. LPS-free RSV F protein was found to activate NF-κB in HEK293T transfectants that express wild-type (WT) TLR4 and CD14, but only when MD-2 was coexpressed. These findings were confirmed by measuring F-protein-induced interleukin 1β (IL-1β) mRNA in WT versus MD-2−/− macrophages, where MD-2−/− macrophages failed to show IL-1β expression upon F-protein treatment, in contrast to the WT. Both Rhodobacter sphaeroides LPS and synthetic E5564 (eritoran), LPS antagonists that inhibit TLR4 signaling by binding a hydrophobic pocket in MD-2, significantly reduced RSV F-protein-mediated TLR4 activity in HEK293T-TLR4–CD14–MD-2 transfectants in a dose-dependent manner, while TLR4-independent NF-κB activation by tumor necrosis factor alpha (TNF-α) was unaffected. In vitro coimmunoprecipitation studies confirmed a physical interaction between native RSV F protein and MD-2. Further, we demonstrated that the N-terminal domain of the F1 segment of RSV F protein interacts with MD-2. These data provide new insights into the importance of MD-2 in RSV F-protein-mediated TLR4 activation. Thus, targeting the interaction between MD-2 and RSV F protein may potentially lead to novel therapeutic approaches to help control RSV-induced inflammation and pathology.

Eritoran Suppresses Colon Cancer by Altering a Functional Balance in Toll-like Receptors That Bind Lipopolysaccharide

“…Other studies in human colorectal and esophageal cancer cell lines have indicated that eritoran blocks LPS-induced TLR4 signaling for b1 integrin-and selectin-dependent cell adhesion and that it inhibits their migratory capability (54,55). Furthermore, eritoran has previously been shown to inhibit monocytic inflammatory responses in endotoxemic rats and virus-infected mice (22,29,30). The possibility that the inhibitory effect of eritoran on inflammatory cytokine production may help suppress tumor malignancy cannot be ruled out.…”

“…However, eritoran (with only 4 acyl chains) does not induce TLR4/MD2 complex dimerization or cytoplasmic signaling (27). Animal studies using eritoran have demonstrated an inhibition of proinflammatory TLR4 signaling, improvement of intestinal microcirculation, and higher survival rates in endotoxemic rodents (22,28) and in influenza-and syncytial virusinfected mice (29,30). However, whether eritoran binds to CD14 and triggers CD14-mediated apoptosis in colon cancer cells remains unclear.…”

Human Coagulation Factor X-Adenovirus Type 5 Complexes Poorly Stimulate an Innate Immune Response in Human Mononuclear Phagocytes

“…TLR4 forms a complex with MD-2 for efficient binding on the cell surface (43) and with CD14 for the internalization of TLR4 (35). MD-2 acts as a coreceptor for recognition of both exogenous ligands (43,44) and endogenous ligands (45,46). In addition, TLR4 can bind bacterial and viral PAMPs and, under inflammatory conditions, also endogenous damage-associated molecular pattern molecules (9).…”

Type III Secretion Needle Proteins Induce Cell Signaling and Cytokine Secretion via Toll-Like Receptors

“…TLR4's role in recognition of needle proteins was less expected than that of the more promiscuous TLR2. However, there are several documented cases of TLR4 interaction with pathogen-associated substrates other than LPS, including respiratory syncytial virus (RSV) fusion protein (30), chlamydial Hsp60 (31), pneumolysin (32), Francisella tularensis DnaK (33), Ebola virus glycoprotein (34), and cell wall components from Pseudallescheria boydii (35). The interaction of these PAMPs and TLR4 are well known, and future research will hopefully elucidate how needle proteins interact with TLR4, including whether MD-2 or CD-14 is required for this interaction.…”