Negatively Regulating TLR-4/NF-κB Signaling Pathway Via Fenofibrate In Ocuolar Inflammation

PPARα is an important transcription factor known to regulate lipid metabolism,although its role in regulating TLR4signaling in inflammatory and autoimmunediseases has not been investigated. The results presented here are the first todemonstrate that PPARα activator, fenofibrate, down-regulated TLR4expression viasuppression of TLR4promoter transcriptional activity. The present study also showedthat PPARα agonists significantly prevented LPS-induced uveitis and inhibitedTLR/NF-κB signaling during inflammation in EIU rats. Moreover, PPARα activationdecreased TLR4levels and inhibited the NF-κB signaling pathway induced by LPS inRPE cells and NF-κB reporter cells. Furthermore, down-regulation or deletion ofPPARα resulted in increased TLR4levels and the activation of NF-κB signaling inRPE cells and rendered fenofibrate unable to deliver its anti-inflammatory effects.These observations indicate that PPARα plays an important role in regulating TLR4expression and downstream signaling and might therefore represent a new therapeutictarget for the treatment of inflammatory and autoimmune complications.Our present study evaluated the hypothesis that PPARα activation wouldreduce TLR4promoter transcriptional activity and suppress TLR4signaling, thusinhibiting the subsequent induction of IKK activity and inhibition of the NF-κBpathway. Our results indicate the importance of PPARα in TLR4signaling, as thedeletion or down-regulation of PPARα resulted in a significant increase in TLR4levels in primary cultured cells from PPARα-knockout mice. The data presented inthis study demonstrate that the ability of PPARα to inhibit LPS/TLR4signaling playsa central role in the prevention LPS-induced uveitis. Exposure of rats to LPS resultedin the breakdown of the blood-aqueous barrier, resulting in plasma proteinextravasation into the AqH and cellular infiltration into the anterior chamber, andthese increases were significantly reduced by oral fenofibrate treatment. To the best ofour knowledge, this is the first evidence demonstrating the therapeutic potential offenofibrate to act on the inflammatory processes of the anterior chamber of the eye. Inaddition to LPS-induced ocular inflammation, previous clinical trials and animalstudies have shown the dramatic therapeutic impacts of fenofibrate on reducing inflammatory cytokine production during diabetic retinopathy in type2diabeticpatients. Furthermore, genetic studies have shown that mutant TLR4silences theoccurrence of diabetic retinopathy in type2diabetic patients. Our unpublished datahave also revealed that low levels of PPARα in type2diabetic patients can be used asa predictor of diabetic retinopathy. Together, these results indicate a novel role forPPARα in TLR4-mediated inflammation, and they highlight PPARα as a noveltherapeutic target for inflammatory disease, which could herald the development ofnew classes of medicine for inflammatory autoimmune diseases. This study providesthe first evidence that the application of PPARα agonists could exert therapeuticeffects on the inflammatory processes of the anterior chamber of the eye. Moreover,because fenofibrate is a widely used clinical drug due to its low cost, low toxicity, andbetter clinical tolerance, oral fenofibrate represents a safe and useful approach for theprevention and treatment of systemic inflammation.As a transcription factor, PPARα regulates and interacts with a diverse groupof molecules. The effects of PPARα activation on crucial elements of the LPS-inducedsignal transduction pathway have previously been studied. For example, PPARαactivation was shown to decrease the expression of the inflammatory factors COX2,IL1, and TNF-α by inhibiting the NF-κB pathway. Moreover, activation of NF-κBresulted in the increased transcription of a number of genes involved in immuneresponses, including those encoding TNF-α, IL-1β, ICAM-1, and VEGF. PPARαinhibition of the NF-κB pathway was also shown through ligand-dependenttrans-repression or through increased expression of Iκa; however, it remains unclearwhether PPARα increases Iκa expression through the TLR pathway.In addition to the aforementioned anti-inflammatory functions, the moststudied role of PPARα is regulating a number of genes involved in free fatty acids(FFAs) metabolism and insulin resistance. PPARα is a ligand-activated transcriptionalfactor regulating a number of genes involved in FFA metabolism and insulinresistance. The effects of PPARα activity include the up-regulated expression of genesinvolved in the transportation and oxidation of FFAs, which reduce de novo FFAsynthesis. Recently, several plausible studies have revealed the ability of FFAs tostimulate TLR4-dependent inflammatory pathways. For example, FFAs act as directligands for TLR4, and they indirectly activate the TLR4pathway through binding to the adaptor protein Fetuin-A. In addition to these actions, FFAs have also been shownto promote the accumulation and homodimerization of TLR4in the cell membrane,leading to activation of the TLR4pathway. Therefore, our present findings concerningPPARα are relevant because PPARα activation inhibits TLR4signaling through thedown-regulation of TLR4transcription, and PPARα activation might also reduce thesynthesis or increase the clearance of FFAs.The inhibitory effect of LPS on PPARα reported here was previously unknown.In a cell-based reporter assay, we found that LPS dose-dependently inhibited severalPPARα-specific agonists. This result indicates that LPS is a PPARα antagonist. Thebinding of LPS to PPARα reduced PPARα activity, which contributed, at leastpartially, to the effects of LPS-induced TLR4signaling. This finding also verified thatPPARα negatively regulates the TLR4signaling, although the precise mechanism bywhich LPS inhibits the PPARα activation remains unclear. LPS mainly consists of apolysaccharide region and an endotoxin, a specific carbohydrate lipid moiety termedlipid A, which is responsible for the immunostimulatory activity of LPS. PPARαconsists of a DNA-binding domain, a flexible hinge region, and a ligand bindingdomain (LBD). Crystal structure analysis of PPARα agonists and antagonists revealedthat a hydrogen bonded interaction between the carboxylic acid group of a PPARαagonist and Y464on the C-terminal AF-2helix of PPARα stabilized the receptor andthe active confirmation change. Normally, nuclear transcription repression of genetranscription is mediated through interactions with co-repressor proteins such asSMRT and N-CoR, which subsequently recruitment histone deacetylases to thechromatin. One example is the PPARα antagonist GW6471, as a PPARα LBD boundto GW6471at a SMRT co-repressor motif led to SMRT motif structural changes andprevented activation of functional conformation. However, whether this mechanismapplies to the interaction between LPS and PPARα remains unclear, and biochemicalanalyses and structure-based mutagenesis of LPS could provide additionalinformation. Moreover, whether the presence of a PPARα agonist interrupts thebinding of LPS to TLR4requires further study.Overall, our study that PPARα activation has the novel function of negativelyregulating TLR4signaling during inflammatory and autoimmune conditions. Thus,the up-regulation or activation of PPARα could represent a new therapeutic strategy for the treatment of inflammatory diseases.