Human Toll-like Receptor 8 (TLR8) Is an Important Sensor of Pyogenic Bacteria, and Is Attenuated by Cell Surface TLR Signaling

Abstract

TLR8 is an endosomal sensor of RNA degradation products in human phagocytes, and is involved in the recognition of viral and bacterial pathogens. We previously showed that in human primary monocytes and monocyte derived macrophages, TLR8 senses entire Staphylococcus aureus and Streptococcus agalactiae (group B streptococcus , GBS), resulting in the activation of IRF5 and production of IFNβ, IL-12p70, and TNF. However, the quantitative and qualitative impact of TLR8 for the sensing of bacteria have remained unclear because selective inhibitors have been unavailable. Moreover, while we have shown that TLR2 activation attenuates TLR8-IRF5 signaling, the molecular mechanism of this crosstalk is unknown. We here used a recently developed chemical antagonist of TLR8 to determine its role in human primary monocytes challenged with S. aureus , GBS, Streptococcus pneumonia, Pseudomonas aeruginosa , and E. coli . The inhibitor completely blocked cytokine production in monocytes stimulated with TLR8-agonists, but not TLR2-, and TLR4-agonists. Upon challenge with S. aureus , GBS, and S. pneumonia , the TLR8 inhibitor almost eliminated the production of IL-1β and IL-12p70, and it strongly reduced the release of IL-6, TNF, and IL-10. With P. aeruginosa infection, the TLR8 inhibitor impaired the production of IL-12p70 and IL-1β, while with E. coli infection the inhibitor had less effect that varied depending on the strain and conditions. Signaling via TLR2, TLR4, or TLR5, but not TLR8, rapidly eliminated IRAK-1 detection by immunoblotting due to IRAK-1 modifications during activation. Silencing of IRAK-1 reduced the induction of IFNβ and TNF by TLR8 activation, suggesting that IRAK-1 is required for TLR8-IRF5 signaling. The TLR-induced modifications of IRAK-1 also correlated closely with attenuation of TLR8-IRF5 activation, suggesting that sequestration and/or modification of Myddosome components by cell surface TLRs limit the function of TLR8. Accordingly, inhibition of CD14- and TLR4-activation during E. coli challenge increased the activation of IRF5 and the production of IL-1β and IL-12p70. We conclude that TLR8 is a dominating sensor of several species of pyogenic bacteria in human monocytes, while some bacteria attenuate TLR8-signaling via cell surface TLR- activation. Taken together, TLR8 appears as a more important sensor in the antibacterial defense system than previously known.

Nucleic Acid-Sensing Toll-Like Receptors Play a Dominant Role in Innate Immune Recognition of Pneumococci

“…However, further studies using various bacterial species are required to better analyze the role of endosomal TLRs in the recognition of Gram-negative pathogens. Our data using mouse macrophages parallel recent findings using human cells on the importance of RNA-sensing receptors in antipneumococcal responses, specifically TLR8 in primary monocyte-derived macrophages (49) and TLR3 in dendritic cells (35). Clearly, RNA-sensing TLRs and their cognate target sequences differ in humans and mice, since murine TLR8 is largely nonfunctional (50), while TLR13 is absent in humans.…”

“…However, the general strategy of focusing on prokaryotic-type RNA for bacterial sensing seems evolutionarily conserved. For example, in human monocytederived macrophages, TLR8 is generally responsible for the recognition of bacterial species that stimulate TLR13 in mouse cells (45,49,(51)(52)(53). However, it is possible that other RNA-sensing receptors (such as TLR7 and TLR3) have a prominent role in other types of human cells, and studies are under way to clarify this point.…”

Staphylococcus aureus secretes immunomodulatory RNA and DNA via membrane vesicles

“…A conserved sequence embedded within 23S ribosomal RNA has been identified as the minimal S. aureus RNA sequence required for TLR13 activation in mouse immune cells 7 . In human monocytes, TLR8 can sense S. aureus ssRNA degradation products containing a UR/URR RNA consensus motif, leading to IFN-ß production 8 , 9 . S. aureus tRNA was previously reported to induce Type I IFN production in peripheral blood mononuclear cells by activating TLR7 10 .…”

Staphylococcus aureussecretes immunomodulatory RNA and DNA via membrane vesicles

“…A conserved sequence embedded within 23S ribosomal RNA has been identified as the minimal S. aureus RNA sequence required for TLR13 activation in mouse immune cells 7 . In human monocytes, TLR8 can sense S. aureus ssRNA degradation products containing a UR/URR RNA consensus motif, leading to IFN-ß production 8,9 . S. aureus tRNA was previously reported to induce Type I IFN production in peripheral blood mononuclear cells by activating TLR7 10 .…”

“…A subset of immunomodulatory MV-associated nucleic acids are protected from nuclease degradation Transfection reagents are known to facilitate RNA uptake by mammalian cells while protecting the RNA from nuclease degradation. Transfection of S. aureus RNA into innate immune cells was previously shown to trigger the production of Type I IFNs and pro-inflammatory cytokines [6][7][8][9][10] . We verified that RNA extracted from S. aureus whole-cells induce IFN-β mRNA expression in RAW 264.7 cells after 3h of transfection with lipofectamine ( Fig.…”