Abstract
Microglia are resident macrophages in the central nervous system that are involved in immune responses driven by Toll-like receptors (TLRs). Microglia-mediated inflammation can lead to central nervous system disorders, and more than one TLR might be involved in these pathological processes. The cysteine peptidase cathepsin X has been recognized as a pathogenic factor for inflammation-induced neurodegeneration. Here, we hypothesized that simultaneous TLR3 and TLR4 activation induces synergized microglia responses and that these phenotype changes affect cathepsin X expression and activity. Murine microglia BV2 cells and primary murine microglia were exposed to the TLR3 ligand polyinosinic-polycytidylic acid (poly(I:C)) and the TLR4 ligand lipopolysaccharide (LPS), individually and simultaneously. TLR3 and TLR4 co-activation resulted in increased inflammatory responses compared to individual TLR activation, where poly(I:C) and LPS induced distinct patterns of proinflammatory factors together with different patterns of cathepsin X expression and activity. TLR co-activation decreased intracellular cathepsin X activity and increased cathepsin X localization at the plasma membrane with concomitant increased extracellular cathepsin X protein levels and activity. Inhibition of cathepsin X in BV2 cells by AMS36, cathepsin X inhibitor, significantly reduced the poly(I:C)- and LPS-induced production of proinflammatory cytokines as well as apoptosis. Additionally, inhibiting the TLR3 and TLR4 common signaling pathway, PI3K, with LY294002 reduced the inflammatory responses of the poly(I:C)- and LPS-activated microglia and recovered cathepsin X activity. We here provide evidence that microglial cathepsin X strengthens microglia activation and leads to subsequent inflammation-induced neurodegeneration. As such, cathepsin X represents a therapeutic target for treating neurodegenerative diseases related to excess inflammation.
Highlights
Innate immune pathways are early responses that are important for pathogen control
The proinflammatory activity of microglia is characterized by the excessive release of nitric oxide (NO) and proinflammatory cytokines [50]; where the amounts of the released factors depend on the type and intensity of the inflammatory stimulus [3, 51]
The phosphorylated Akt levels were significantly increased after 30 min of LPS/ poly(I:C) co-stimulation; this phosphorylation of Akt was completely blocked by pre-treatment with AMS36, i.e., by cathepsin X inhibition (Fig. 7E). These results indicate that cathepsin X is involved in regulating the phosphoinositide 3 kinase (PI3K)/ Akt signaling pathway that is activated by TLR4 and TLR3 ligands, which trigger microglia activation and inflammatory responses
Summary
Innate immune pathways are early responses that are important for pathogen control They can be activated by specific pattern recognition receptors that recognize pathogen-associated molecule patterns (PAMPs) or danger-associated molecule patterns [1,2,3]. Homeostatic microglia are transformed into their activated phenotype, which can lead to neuronal tissue damage and the expression of genes related to neuroinflammation [5,6,7] This phenotype includes the expression of the cell surface receptors, which make these cells highly reactive to a variety of stimuli [2, 8]. Poly(I:C) is a synthetic analog of double-stranded RNA and can be generated during the replication of RNA and DNA viruses [18] This PAMP is mainly recognized by the TLR3 receptor [19, 20]. This, in turn, induces the production of proinflammatory cytokines, such as tumor necrosis factor (TNF)-α and interleukin (IL)-6 [3, 22]
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