Inhibitors of TLR8 Reduce TNF Production from Human Rheumatoid Synovial Membrane Cultures

Abstract

The advent of anti-TNF biologicals has been a seminal advance in the treatment of rheumatoid arthritis (RA) and has confirmed the important role of TNF in disease pathogenesis. However, it is unknown what sustains the chronic production of TNF. In this study, we have investigated the anti-inflammatory properties of mianserin, a serotonin receptor antagonist. We discovered mianserin was able to inhibit the endosomal TLRs 3, 7, 8, and 9 in primary human cells and inhibited the spontaneous release of TNF and IL-6 from RA synovial membrane cultures. This suggested a role for these TLRs in production of TNF and IL-6 from RA which was supported by data from chloroquine, an inhibitor of endosomal acidification (a prerequisite for TLRs 3, 7, 8, and 9 activation) which also inhibited production of these cytokines from RA synovial cultures. Only stimulation of TLR 3 or 8 induced TNF from these cultures, indicating that TLR7 and TLR9 were of less consequence in this model. The key observation that indicated the importance of TLR8 was the inhibition of spontaneous TNF production by imiquimod, which we discovered to be an inhibitor of TLR8. Together, these data suggest that TLR8 may play a role in driving TNF production in RA. Because this receptor can be inhibited by small m.w. molecules, it may prove to be an important therapeutic target.

Targeting DAMP Activation of Toll-Like Receptors: Novel Pathways to Treat Rheumatoid Arthritis?

“…While the same workers found that TLRs 7 and 9 were not responsive to their respective ligands in RA cultures, stimulation of TLRs 3 and 8 did increase cytokine production. The contribution of endosomal TLRs to cytokine synthesis in RA is also supported by other studies; chloroquine, which prevents intracellular TLR function by inhibiting endosomal acidification, reduces cytokine release in synovial cells (Sacre et al, 2008). The selective serotonin reuptake inhibitors, antidepressant drugs fluoxetine and citalopram and the antidepressant small molecule mianserin are also efficacious in inhibiting synovial cell cytokine release (Sacre et al, 2010).…”

“…Inhibition of TLR4 by the naturally occurring antagonist LPS isolated from Bartonella Quintana, also partially inhibited cytokine release in RA synovial biopsies . Stimulation of TLRs 2, and 4 has also been shown to induce cytokine synthesis in cell cultures isolated from RA synovia (Sacre et al, 2008). While the same workers found that TLRs 7 and 9 were not responsive to their respective ligands in RA cultures, stimulation of TLRs 3 and 8 did increase cytokine production.…”

C/EBPδ and STAT-1 Are Required for TLR8 Transcriptional Activity

“…Recent data reported HIV ssRNA sequences that are specific for TLR8 (26) and epidemiological studies showed the association of a functional TLR8 variant with HIV progression disease (27) and with pulmonary tuberculosis susceptibility (28). TLR8 activity is an important source of TNF-␣ in the sinovial membrane cultures in rheumatoid arthritis (29). Based on these findings, it is thus important to understand how this receptor is regulated prior to and during an immune response.…”

Simvastatin Inhibits Toll-like Receptor 8 (TLR8) Signaling in Primary Human Monocytes and Spontaneous Tumor Necrosis Factor Production from Rheumatoid Synovial Membrane Cultures

“…In previous studies, we had identified a role for TLRs and, in particular, TLR8 in the production of spontaneous TNF in human RA synovial membrane cultures (32,42).…”

“…Simvastatin had no effect on TNF secretion in response to stimulation of TLR2, -4 or -5 but did result in a decrease in TNF produced in response to R-848 ( Figure 1A), with no effect on cell viability as shown by MTT assay ( Figure 1B). This effect was assumed to be due to inhibition of TLR8, rather than TLR7, signaling, since it has previously been demonstrated that monocytes do not produce TNF in response to TLR7 ligands (32). To investigate the mechanism by which simvastatin inhibits R-848-induced TNF secretion, a downstream product of the cholesterol pathway (GGPP) was added to the cultures together with simvastatin in an attempt to reverse this effect.…”