Evaluation of gene expression profile and pro-inflammatory molecules production in microglial cells after TLR2 stimulation

MANZONE-RODRÍGUEZ, C; RODRIGUEZ, CM; IRIBARREN, P

San Luis

Congreso; LXXI REUNIÓN CIENTÍFICA ANUAL DE LA SOCIEDAD ARGENTINA DE INMUNOLOGÍA (SAI); 2023

Objective: Toll-like receptors (TLRs) are a family of pattern-recognition receptors in the innate immune system. Exogenous and endogenous TLR ligands activate microglia that trigger inflammatory reactions in CNS.Studies using a mouse experimental brain abscess model have revealed a complex role for TLRs in the disease pathogenesis. These studies showed that TLR2 participates in the innate immune response, during the acute stage of brain abscess formation induced by Staphylococcus aureus influencing the adaptive immune response. Therefore, identification of potentially neurotoxic molecules released by TLR stimulation, may provide clues about neuropathological mechanisms involved in these diseases. Thus, here we evaluated gene expression profiles and proinflammatory molecules production in murine microglial cells after TLR2 stimulation. Methods: RNA-seq public datasets from mouse microglial cells and/or macrophages stimulated with TLR2 ligands, were obtained from NCBI Gene Expression Omnibus (GEO) repository and analyzed using integrated Differential Expression and Pathway (iDEP) analysis tool. Murine BV2 microglial cells were cultured in the presence or the absence of autophagy inhibitors (3-MA) or a general phosphatidyl-inositol-3 kinase (PI3K) inhibitor (LY294002) and then stimulated with Pam3CSK4 or LPS (control) at different time points. After treatment, the cells were processed to evaluate: (1) cytokine production by ELISA and (2) nitric oxide (NO) production by Griess reaction. All experiments were performed 3 times and p < 0.05 was considered statistically significant.Results: Preliminary exploration of the RNA-seq data showed enhanced pro-inflammatory gene expression in microglial cells stimulated with TLR2 agonists, compared to the control groups.On the other hand, we confirmed that activation of microglial cells with Pam3CSK4 induced increased production of TNFα and NO (p < 0.001).Interestingly, treatment of microglial cells with autophagy or a general PI3K inhibitors, prevented the increased production of NO (p < 0.001). Additional experiments are currently performed to evaluate the effects of these inhibitors on TNFα production. Conclusions: Both, RNA-seq datasets analysis and cell culture experiments suggest that TLR2 stimulation of microglial cells modulates pro-inflammatory gene expression and cell responses that may involve the participation of PI3K activation.