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HSP60 plays a regulatory role in IL-1β-induced microglial inflammation via TLR4-p38 MAPK axis
Shalini Swaroop1, Nabonita Sengupta1, Amol Ratnakar Suryawanshi2
1National Brain Research Centre, Manesar, Haryana, 122051, India.
Background:
IL-1β, also known as "the master regulator of inflammation", is a potent pro-inflammatory cytokine secreted by activated microglia in response to pathogenic invasions or neurodegeneration. It initiates a vicious cycle of inflammation and orchestrates various molecular mechanisms involved in neuroinflammation. The role of IL-1β has been extensively studied in neurodegenerative disorders; however, molecular mechanisms underlying inflammation induced by IL-1β are still poorly understood. The objective of our study is the comprehensive identification of molecular circuitry involved in IL-1β-induced inflammation in microglia through protein profiling.
Methods:
To achieve our aim, we performed the proteomic analysis of N9 microglial cells with and without IL-1β treatment at different time points. Expression of HSP60 in response to IL-1β administration was checked by quantitative real-time PCR, immunoblotting, and immunofluorescence. Interaction of HSP60 with TLR4 was determined by co-immunoprecipitation. Inhibition of TLR4 was done using TLR4 inhibitor to reveal its effect on IL-1β-induced inflammation. Further, effect of HSP60 knockdown and overexpression were assessed on the inflammation in microglia. Specific MAPK inhibitors were used to reveal the downstream MAPK exclusively involved in HSP60-induced inflammation in microglia.
Results:
Total 21 proteins were found to be differentially expressed in response to IL-1β treatment in N9 microglial cells. In silico analysis of these proteins revealed unfolded protein response as one of the most significant molecular functions, and HSP60 turned out to be a key hub molecule. IL-1β induced the expression as well as secretion of HSP60 in extracellular milieu during inflammation of N9 cells. Secreted HSP60 binds to TLR4 and inhibition of TLR4 suppressed IL-1β-induced inflammation to a significant extent. Our knockdown and overexpression studies demonstrated that HSP60 increases the phosphorylation of ERK, JNK, and p38 MAPKs in N9 cells during inflammation. Specific inhibition of p38 by inhibitors suppressed HSP60-induced inflammation, thus pointed towards the major role of p38 MAPK rather than ERK1/2 and JNK in HSP60-induced inflammation. Furthermore, silencing of upstream modulator of p38, i.e., MEK3/6 also reduced HSP60-induced inflammation.
Conclusions:
IL-1β induces expression of HSP60 in N9 microglial cells that further augments inflammation via TLR4-p38 MAPK axis.
Insights
Interleukin-1 beta (IL-1β) triggers heat shock protein 60 (HSP60) in microglia, which amplifies neuroinflammation through the TLR4-p38 MAPK pathway. This study identifies HSP60 as a key mediator in IL-1β-induced inflammatory responses.
Area of Science:
- Neuroscience
- Immunology
- Cell Biology
Background:
- Interleukin-1 beta (IL-1β) is a key pro-inflammatory cytokine secreted by microglia, central to neuroinflammation in neurodegenerative diseases.
- While IL-1β's role is established, the precise molecular mechanisms driving IL-1β-induced inflammation remain incompletely understood.
- This study aims to comprehensively identify the molecular circuitry mediating IL-1β-induced inflammation in microglia using proteomic profiling.
Purpose of the Study:
- To identify key proteins and molecular pathways involved in IL-1β-induced microglial inflammation.
- To elucidate the role of Heat Shock Protein 60 (HSP60) in IL-1β-mediated inflammatory signaling.
- To investigate the involvement of Toll-Like Receptor 4 (TLR4) and Mitogen-Activated Protein Kinase (MAPK) pathways in HSP60-induced inflammation.
Main Methods:
- Proteomic analysis of N9 microglial cells treated with IL-1β.
- Validation of Heat Shock Protein 60 (HSP60) expression and secretion using qPCR, immunoblotting, and immunofluorescence.
- Co-immunoprecipitation to confirm HSP60-TLR4 interaction; use of TLR4 inhibitors, HSP60 knockdown/overexpression, and specific MAPK inhibitors (including MEK3/6 inhibitors).
Main Results:
- Proteomic analysis identified 21 differentially expressed proteins, highlighting unfolded protein response and identifying HSP60 as a central molecule.
- IL-1β significantly induced HSP60 expression and secretion, which subsequently bound to TLR4, suppressing inflammation upon TLR4 inhibition.
- HSP60 overexpression increased ERK, JNK, and p38 MAPK phosphorylation; p38 MAPK inhibition, and MEK3/6 silencing, significantly reduced HSP60-induced inflammation.
Conclusions:
- IL-1β induces HSP60 expression in microglia, which amplifies inflammation.
- The TLR4-p38 MAPK axis is a critical pathway mediating HSP60-induced microglial inflammation.
- Targeting the HSP60-TLR4-p38 MAPK pathway may offer therapeutic strategies for neuroinflammatory conditions.
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