Analysis of the inflammatory response of dTHP-1 cells treated with urate hydroperoxide.

• Grant number: 24/08974-9
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: September 01, 2024
• End date: August 31, 2025
• Field of knowledge: Biological Sciences - Biochemistry
• Principal Investigator: Flavia Carla Meotti
• Grantee: Antônio Paulo Siqueira Pratti
• Host Institution: Instituto de Química (IQ). Universidade de São Paulo (USP). São Paulo , SP, Brazil
• Associated research grant: 18/14898-2 - Investigations of the redox processes in inflammatory response and associated pathologies, AP.JP2

Abstract

Being derived from the degradation product of purines, uric acid has been associated with diseases of chronic inflammatory profile, such as atherosclerosis, with macrophages also being a fundamental cell in the genesis of this disease. Macrophages are known as cells of the innate immune system, which respond to oxidative stimuli by activating two main inflammatory pathways: the NF-kB pathway and the Nrf2 pathway, with the initial step of both pathways being the translocation of the NF-kB and Nrf2 transcription factors to the nucleus. However, little is known about the effects caused in these pathways by urate hydroperoxide (HOOU), an oxidant synthesized in the inflammatory oxidative burst by the enzyme myeloperoxidase, from uric acid. Thus, the present study aims to evaluate the signaling response of differentiated Human Monocytic Leukemic Cells (dTHP-1) to treatment with HOOU, comparing the levels of expression of NF-kB and Nrf2 in the cytosolic and nuclear fractions, thus allowing a better understanding of the biological effect of HOOU in an inflammatory process. Treatments with LPS, TNF-±, tBHQ, HOOU, or mobile phase (MP) will be performed to obtain two fractions, one enriched with cytoplasmic content and the other nuclear. This enrichment will be achieved through initial cytoplasmic lysis, followed by nucleus precipitation, supernatant collection, and finally, lysis of the precipitated nucleus. The expression of NF-kB and Nrf2 will be measured in both fractions via Western Blot, and the efficacy of fraction separation will be determined by the presence of ²-actin exclusively in the cytosolic fraction and lamin B1 only in the nuclear fraction, as these proteins are exclusive to these compartments. In addition, following the same treatments but without fraction separation, the effect of HOOU on the production of IL-1² by ELISA and on the expression of mRNA HO-1 and mRNA IL-1² by RT-PCR will be determined. Standardization of a co-culture of dTHP-1 and differentiated human myeloid leukemia cells (dHL-60) will also be performed, establishing the proportion of both cells and the incubation time to obtain a response similar to immune system activation in a biological medium, using control groups without treatment, 1 ¼g/mL LPS, 5 ng/mL TNF-±, and 20 ¼M tBHQ to determine the ideal conditions. The response will be determined by the activation of the promoter present in the NF-kB firefly plasmid in previously transfected dTHP-1 cells. After standardization, cells will be incubated with 200 ¼M uric acid to induce HOOU formation, and experiments will be repeated.