Prevention of fatty acid-induced inflammasome activation by a bioactive lipokine

Abstract

Exposure to excess lipids such as fatty acids and cholesterol leads to cellular stress, release of reactive oxygen species (ROS), inflammation dysfunction and death. These responses have important role in the pathogenesis of chronic metabolic and inflammatory diseases such asobesity, diabetes and atherosclerosis. Recent studies show that fatty acids also cause the formation of inflammatoryprotein complexes that are called inflammasome. Inflammasome promotesthe activation of caspase-1 protein and cleavage of inactive interleukin-1 beta(IL-1β) and interleukin-18 (IL-18) into their active, secreted forms.Two signals are required for the activation of inflammasome. The first signal (also known as priming step) is needed for the inducing the expression ofthe proinflammatory factors pro-IL-1β and pro-IL-18 andthe nucleotide-binding oligomerization domain receptor (NOD-like receptor) family, pyrin domain containing-3 (NLRP3) proteins through the activation of nuclear factor kappa beta (NF-κB),a transcription factor. The second signal is needed for the activation of caspase-1 and formation of the inflammasome complex. Saturated free fatty acids such as palmitate are known to cause the activation of inflammasome through generation of mitochondrial ROS as a second signal for inflammasome complex formation. In this study, I investigated the effect of palmitoleate, a bioactive monounsaturated fatty acidpreviously shown to counteract lipid-induced ER stress and enhance insulin sensitivity, on palmitate-induced activation of inflammasome complex. I observed that palmitoleate lead to a significant reduction in palmitate-stimulatedIL-1β transcription. Moreover, it reduced the expression of palmitate-stimulated secondary proinflammatory factors such as tumor necrosis factor alpha(TNF-a).Palmitoleate also diminished the palmitate induced activation of caspase-1, maturation and secretion of IL-1β in macrophages. To further understand the mechanism of the protective role of palmitoleate on palmitate induced inflammasome activation,I analyzed the effect of palmitoleate on palmitate-induced mtROS in macrophages cells were analyzed. These studies showed palmitoleate decreased palmitate induced mitochondrial oxygen species in macrophages. Moreover, I investigated the effect of palmitoleate on palmitate-induced inactivation of 5' AMP-activated protein kinase (AMPK)controls autopaghy and clearance of dysfunctional mitochondria, thereby reducing mtROS formation. Palmitoleate blocked the suppression of AMPK phosphorylation that was suppressed by palmitate, suggesting PAO's impact on mtROS secretion and inflammasome activation occurs by this upstream mechanism. Saturated fatty acids like palmitate are also known to cause endoplasmic reticulum (ER) stress, whichbe counteracted by palmitolate treatment. When ER stress happens, cells try to solve stress by activating the unfolded protein response (UPR). There are three arms of the UPR are Protein Kinase R-resemble like protein kinase (PERK), inositol-requiring enzyme-1 (IRE1) and activating factor 6 (ATF6). In this thesis, contribution of PERK and IRE1 on palmitate-induced activation of inflammasome was investigated. First, the relation between PERK and IRE1 with palmitate-stimulated mtROS was analyzedusing PERK- and IRE1- deficient mouse embryonic fibroblast cells (MEFs).The results of this study show PERK leads to a marked reduction in the formation of mtROS while IRE1 enhanced palmitate induced mtROS.Furthermore, palmitoleate suppressed palmitate-induced autophosphorylation of IRE1 while there was no effect of palmitoleate on palmitate-induced phosphorylation of PERK. In summary, palmitate-induced inflammasome activation can be inhibited by palmitoleate in boththe priming step and the second step.Palmitoleate blocks palmitate-induced suppression of phosphorylation of AMPK, leading to its reactivation and subsequent reduction in mtROS formation. Palmitate is a well-known inducer of ER stress, which can be counteracted by palmitoleic acid. Moreover, the IRE1 branch of the UPR has been shown to control inflammasome activationby regulating of mtROS production under lipotoxicity. The outcome of my studies show that the UPR branches initiated by PERK and IRE1 regulate mtROS production and therefore, palmitoleate may also block inflammasome activation induced by palmitate through this alternative mechanism.These findings imply that palmitoleate may have therapeutic applications inthe management of diseases where inflammasome activation has been shown to play a causal role such as obesity, diabetes and atherosclerosis.