Investigation of the effect of valproic acid, an hdac inhibitor, on the relationship between oxidative stress and autophagy in human eosinophil

Abstract

Epigenetics bridges the gaps between phenotype and genotype. Any process that modulates the gene activity without causing any change in DNA sequence is called epigenetic modification. Epigenetic modifications are divided into three main groups: histone modifications, DNA methylation, and non-coding RNAs. These changes are reversible because they do not alter DNA sequence. However, dysregulation in modifications can lead to several diseases such as cancer, metabolic, and neurological disorders. Inhibitors that suppress modifications such as DNA methylation, histone deacetylase, histone acetyl transferase, and protein methyltransferase have been developed to reverse the epigenetic processes that drive the pathogenesis of diseases and are used as epigenetic drugs for treatments. One of the most widely used inhibitors in therapy is histone deacetylation inhibitors (HDACi). Valproic acid (VPA), a branched short-chain fatty acid obtained from valeric acid, is one of the drugs used as HDACi. VPA is generally used as an anticonvulsant drug for the effective treatment of different types of diseases such as epileptic seizures, migraine headaches, bipolar disorder. In addition, VPA has the promising potential to be effective in cancer therapy and controlling allergic responses because it can mediate the expression of important genes involved in anti-tumor immunity and activation of cells. Nevertheless, even when VPA is used therapeutically, studies have revealed that VPA induces oxidative stress, and can activates autophagy pathway, inflammasome response in different cells as a side effect. In this thesis, our aim is primarily to investigate the effects of valproic acid-induced stress on the activation of the antioxidant pathway, inflammasome complexes, and autophagy pathways that maintain cell balance in Eol-1 cells. Eol-1 human eosinophilic cells were used in this study since eosinophils have a key effector role in the response to allergic reactions. When the balance of reactive oxygen species (ROS) production in the cell is disrupted, oxidative stress arises in cells. ROS are highly reactive molecules and, elevated levels of ROS can damage cell compounds. Therefore, regulation of reactive oxygen species (ROS) generation is crucial for the proper functioning of cells. To maintain homeostasis, cells develop a series of antioxidant responses to reduce the toxicity caused by oxidative stress. In this case, the main regulator in the cell is the antioxidant transcription factor nuclear factor erythroid 2-associated factor 2 (Nrf2). Under normal conditions, Nrf2 interacts with Kelch-like ECH-associated protein 1 (Keap1) in the cytoplasm and leads to degradation of Nrf2 by the 26s proteasome, thus it is a negative regulator of Nrf2. Oxidative stress causes a conformational change in Keap1, leading to the dissociation of Nrf2 and Keap1. This enhances the acetylation and nuclear translocation of Nrf2. Proteins which transcribed by Nrf2 are involved in the activation of both anti-inflammatory and pro-inflammatory pathways in the cell to inhibit excessive immune responses caused by oxidative stress. Our results showed that VPA stimulation increased the cellular ROS formation in a dose-dependent manner at 24 h post-stimulation without any change in viability of Eol-1 cells. Moreover, the activation marker CD69 was upregulated via VPA treatment in dose-dependent manner. At lower concentrations, VPA augmented the protein level of Nrf2 and acetylated Nrf2, while attenuating the protein level of Keap1 in a dose-dependent manner. The immune system represents cells, tissues, organs, and components that come together to form a defense network to fight various pathogens or diseases by the excessive reaction and invasion of the microorganisms and to maintain the homeostasis of the host. The immune system consists of two arms: innate immunity and adaptive immunity. The first defense against pathogens is provided by innate immunity and white blood cells such as granulocytes (basophils, mast cells, eosinophils, neutrophils), monocytes, and Langerhans cells which are the main effector cells in innate immunity. The innate immune response is controlled by membrane/cytoplasmic receptors, inflammatory proteins, secreted cytokines, and chemokines. Pattern recognition receptors (PRRs) are mainly categorized as Toll-like receptors (TLR), NOD-like receptors (NLR), C-type Lectin (CLR) and RIG-I-like receptors (RLR). They can recognize different pathogen associated molecular patterns (PAMPs) and danger associated molecular patterns (DAMPs). Ligand-receptor interaction initiates inflammatory responses. The major canonical inflammatory responses are driven by NLRP3, NLRC4 inflammasome complexes against microbial structures and danger singals such as bacteria, mitochondrial ROS, and ATP. The NLRP3 and NLRC4 proteins combine with ASC and caspase 1 separately to form complexes. Activated caspase-1 cleaves pro forms of IL-1B, IL-18, and gastermin D (GSDMD) into mature forms. Firstly, GSDMD oligomerizes, then migrates to the membrane to form pores, so mature cytokines can be released from the pores. Cell membrane integrity is disrupted, and cells eventually die; this process is called pyroptosis. The release of cytokines and chemokines induces the migration and activation of many inflammatory cells, which is important for the proper immune response in microbial defense. According to our findings VPA treatment reduced the protein level of NLRC4 in a dose dependent fashion, but interestingly NLRP3 and caspase-1 cleavage, cleaved IL1β protein levels were elevated at lower doses in Eol-1 cells. IL-1B cleavage increased in parallel to the activity of caspase-1 and the inflammasome complex. Although cleaved IL1B was increased intracellularly, IL-1B and IL-10 secretion via VPA stimulation did not significantly change as compared to non- treated group. Autophagy is a mechanism responsible for maintaining homeostasis in the cell under various stress conditions. Cells remove damaged and unnecessary cell components through lysosomal degradation in response to cellular stresses such as nutrient deficiency or high levels of reactive oxygen species (ROS). As the most studied autophagy type, in macroautophagy, cytoplasmic components merge with the lysosome with the help of vesicles called autophagosome, forming autolysosome structures and the components inside the autophagosome are degraded. Various genes/proteins and pathways are involved in the maintenance of autophagy. The MAPK (mitogen-activated protein kinase), Akt (alpha serine/threonine-protein kinase) and mTOR (mammalian target of rapamycin) pathways are in the upstream of the autophagy pathway and are responsible for the regulation of cell growth, cellular metabolism, cell survival, and proliferation of cells. ERK1/2 is phosphorylated in the presence of autophagosome and thus it can be used as a positive marker in the autophagy pathway. Conversely, Akt and mTOR pathways negatively regulate the autophagy pathway. Phosphorylation of the Akt signaling pathway affects activation of the mTOR pathway as mTOR is downstream of Akt. In addition to these, microtubule‑associated protein light chain 3 (LC3) is the main marker in the autophagy flux. The cytosolic form LC3-I is converted to the LC3-II form through autophagy activation. LC3-II binds to the inner and outer membrane of the autophagosomes and preautophagosomal structure (PAS). Also, Beclin 1 is used as a marker for autophagy activation because it is crucial for the autophagosome structure and suppresses mTOR activity while inducing ERK activity. Our findings indicated that following VPA treatment, p44/42 MAPK (ERK1/2) protein increased at ascending doses, and phosphorylation of p44 MAPK was upregulated at lower doses. On the other hand, while the protein level of Akt didn't change after VPA, phosphorylation of Akt on both serine 473 and threonine 308 was downregulated via VPA stimulation. Moreover, the mTOR protein levels and the phosphorylation of mTOR decreased at high VPA doses, while protein levels of LC3B II was increased by VPA stimulation. However, increasing concentrations of VPA decreased the Beclin-1 protein level. Our results suggest that VPA stimulation induced the autophagy pathway independently of Beclin1in Eol-1 cells. Based on our current data, VPA led to oxidative stress by increasing ROS production levels and activated the antioxidant transcription factor NRF2 in Eol-1 cells. Also, VPA activated Eol-1 cells. VPA induced inflammasome complex formation without any change in the secretion of proinflammatory cytokines. Finally, VPA activated the autophagy pathway independently of Beclin1 in Eol-1 cells. Our results demonstrated the side effects that should be considered in the therapeutic use of VPA. In addition, the fact that VPA increases the activation of Eol-1 cells raises the question of whether VPA can not be used as a drug in allergic responses since it is not desired to increase the severity of the reaction in allergic responses.