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A2247 - Toll-Like Receptor 8 (TLR8) Protein Degradation Limits Inflammatory Signaling
Author Block: J. Evankovich1, T. Lear1, R. K. Mallampalli2, B. Chen3; 1ALI Center of Excellence, Division of Pulmonary, Allergy, and Critical Care Medicine, University of Pittsburgh, Pittsburgh, PA, United States, 2ALI Center of Excellence, Division of Pulmonary, Allergy, and Critical Care Medicine, VAMC, University of Pittsburgh, VAMC, Pittsburgh, PA, United States, 3Pulmonary, Allergy, and Critical Care Medicine, University of Pittsburgh, Pittsburgh, PA, United States.
RATIONALE: Toll-Like Receptor 8 (TLR8) is a pattern recognition receptor that senses RNA in endosomes, initiating innate immune signaling through NF-κB. In monocytes TLR8 is activated in response to bacterial RNA, which is present in bacterial infection, a well-established risk factor for ARDS. Thus, TLR8 signaling may be a previously unrecognized contributor to innate immune activation in severe infection and ARDS. Mechanisms regulating TLR8 protein abundance and activation are not completely understood. Protein degradation is a cellular mechanism controlling protein abundance, accomplished through ubiquitin transfer by E3-Ligase proteins. The E3-Ligase protein Triad3A ubiquitinates and coordinates the disposal of similar TLRs in different cell types, but its relationship to TLR8 has not been examined. We hypothesized that TLR8 protein degradation, coordinated by the E3-Ligase Triad3A, would reduce TLR8-dependent NF-κB activation in the presence of activating signals.
METHODS: THP-1 cells were treated with the protein synthesis inhibitor cycloheximide (CHX, 40μg/mL), a proteasome inhibitor MG-132 (40 mMol), a lysosomal inhibitor Leupeptin (40 mMol), or their combination. Proteins levels were determined by immunoblotting. Experiments were repeated after treatment with the TLR8 agonist R848 (1 μg/mL). TLR8 ubiquitination was examined by agarose conjugated Tandem Ubiquitin Binding Entities (TUBEs) reagent (LifeSensors, Inc), and TLR8/Triad3A assocation was examined by co-immunoprecipitation. TLR8 activation was examined by using HEK-Blue TLR8 cells (Invivogen). HEK-Blue TLR8 cells were transfected with an empty vector or a plasmid containing Triad3A and stimulated with the TLR8 agonist R848.
RESULTS: TLR8 half-life was 60 minutes in THP-1 cells, and pretreatment with both the proteasomal inhibitor MG-132 and the lysosomal inhibitor Leupeptin stabilized TLR8 protein levels in CHX chase experiments. Ubiquitinated TLR8 was detected by TUBEs pull-down, and TLR8/Triad3A association was detected by co-immunoprecipitation. Treatment with the TLR8 agonist R848 decreased TLR8 protein levels at 4 h, an effect that was similarly prevented with MG-132 or Leupeptin pretreatment. In HEK-Blue TLR8 cells, TLR8 agonist R848 increased TLR8-dependent NF-κB activation. This effect was exacerbated by MG-132 and Leupeptin pretreatment. Lastly, TLR8-dependent NF-κB activation was abrogated by Triad3A over-expression.
CONCLUSIONS: TLR8 undergoes protein degradation and has a short half-life of 60 minutes. Subcellularly, both the proteasome and the lysosome may participate in TLR8 disposal. TLR8-dependent NF-κB signaling is enhanced by preventing TLR8 degradation. Thus, TLR8 degradation, coordinated by Triad3A, may be a mechanism to limit excessive inflammatory signaling response to TLR8 agonists. Future studies will examine if these mechanisms are operant in humans with severe infection and ARDS.