Student Theses, Dissertations and Capstones
Document Type
Dissertation - NSU Access Only
Degree Name
Doctor of Philosophy (PhD) in Pharmacy
First Advisor
Richard Deth
Second Advisor
Ana Castejon
Defense Date
1-1-2024
NSUWorks Citation
Karin Hsiao. 2024. REDOX REGULATION OF THE NLRP3 INFLAMMASOME: IMPLICATIONS FOR IMMUNE SENESCENCE, INFLAMMAGING, AND NEURODEGENERATIVE DISORDERS. Doctoral dissertation. Nova Southeastern University. Retrieved from NSUWorks, . (19)
https://nsuworks.nova.edu/hpd_corx_stuetd/19.
Abstract
Background: The aging population is increasing, and the number of age-related diseases, such as neurodegenerative disorders, is projected to increase dramatically. Recent studies on neuroinflammation and the role of the nucleotide-binding domain (NOD)-like receptor protein 3 (NLRP3) inflammasome have gained considerable attention, largely due to its activity promoting inflammatory cytokine in COVID-19 cases. Moreover, NLRP3 activation has been associated with aging-related neurological diseases, including Alzheimer’s disease and Parkinson’s disease. Considering that NLRP3 activity is promoted by oxidative stress, we hypothesized that changes in redox status might play a crucial role in the low-grade inflammatory disease associated with aging, collectively known as "inflammaging.” Study: This study investigated changes in antioxidant-related metabolic pathways during NLRP3 inflammasome activation and thiol changes during transsulfuration pathways due to aging. Immune cells (monocytic leukemia cell line THP-1) treated with the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA), which differentiates the monocytes into macrophage-like cells, served as a study model for NLRP3-mediated induction of IL-1β secretion in conjunction with a secondary inflammatory stimulus such as lipopolysaccharide (LPS, endotoxin), ATP, and casein-derived opioid β-casomorphin- 7 (BCM7), which has previously shown to inhibit cysteine transport. In this study, we also treated THP-1 cells with hydroxocobinamide, a precursor in the synthesis of vitamin B12, and suramin, an inhibitor of the P2X7 purinergic receptor, whose activation increases NLRP3 activity. Thiol metabolites (methionine, homocysteine, cysteine, cytine, reduced glutathione (GSH), oxidized glutathione (GSSG), homocysteine, cystathionine, S-adenosylmethionine (SAM), and S- adenosylhomocysteine (SAH) and gene expression of postmortem human and mouse brain tissues were also analyzed to investigate the role of oxidative conditions in promoting NLRP3 inflammasome activity via ELISA-based assessment of IL-1β formation and LC/MS measurement of redox/methylation metabolites. Gene expression of pro-inflammatory cytokines (IL-1β, IL-18), transsulfuration enzymes (CBS, CSE), and other neurodegeneration-associated markers (TGF-β1, P2X7, Nrf2, and NLRP3) were analyzed for changes in young, old and AD postmortem human brain samples. Western blots were also conducted in THP-1 cells and postmortem brain tissues to evaluate NLRP3 status further. Results: PMA action was associated with significant (p <0.05) changes in thiol metabolites. Thiol levels increased uniformly with PMA stimulation, except for homocysteine and cystathionine. Subsequent LPS treatment significantly (p <0.05) increased levels of IL-1β, indicative of M1 polarization state, which was associated with significant decreases in multiple thiols, including cysteine and glutathione. However, self-limiting feedback of IL-1β and thiol levels was observed with a higher LPS concentration. A time course study with LPS revealed a slow onset of IL-1β production starting after 1 hr. and reaching a higher level at 4 and 8 hrs., stabilizing at 24 hrs. When cells were treated with both LPS and ATP, significantly (p <0.05) accelerated production of IL-1β was observed, indicating a promotional role for ATP-activated P2X7 receptors, and treatment with suramin blocked the ability of ATP to accelerate LPS-induced IL-1β formation. BCM7 treatment caused a modest augmentation of IL-1β formation at low concentration of LPS, consistent with inhibition of cysteine uptake. Hydroxocobinamide significantly (p <0.05) decreased LPS-induced IL-1β formation, greater than methylcobalamin. IL-1β formation was confirmed by western blot, and RT-qPCR studies revealed significant (p <0.05) LPS-induced changes in the expression of inflammasome-related and redox pathway-related genes. Postmortem brain studies in the human cortex and hippocampus revealed higher GSH levels and higher oxidized GSSG in older vs. younger subjects, but there was no difference (p >0.05) between older age and Alzheimer's disease subjects for any of the thiols. Cystathionine and lanthionine increased with age, which may indicate age-dependent changes in transsulfuration pathway activity. RT-qPCR in postmortem tissues confirmed significant (p <0.05) changes in gene expression between young and old subjects but not in Alzheimer’s disease. Changes in P2X7 paralleled those for the transsulfuration enzyme CBS results, and Nrf2 expression significantly (p <0.05) increased in older age control and AD groups, indicating its response to oxidative stress with aging. Conclusion: My studies demonstrate that inflammatory stimulation with LPS is exacerbated with ATP treatment, while hydroxocobinamide and suramin treatment inhibit ATP activity and decrease inflammation. mRNA expression levels of NLRP3 and IL-1β genes are abnormal in postmortem AD brains compared to elderly control subjects, suggesting that aging-related changes contribute to NLRP3 inflammasome activation in AD. However, further studies are required to comprehend the relationship between NLRP3 and oxidative stress in AD. This project suggests that inflammaging, exacerbated by oxidative stress, may lead to aging-related diseases that involve neurodegeneration. Importantly, these diseases could potentially be modulated with thiol metabolites and P2X7 inhibitor drugs and/or cobinamide to aid current drug therapy for aging-related diseases; however, there is a need for more research related to aging diseases to help with the expected increase in the aging population worldwide.
Subject Categories
Pharmacy and Pharmaceutical Sciences
Keywords
aging; Alzheimer’s disease; inflammaging; Neurodegenerative disease; NLRP3; P2X7