Crucial Role of the Mammalian Target of Rapamycin (mTOR) in Activation of Human Microglia, Mast Cells and Keratinocytes, Inhibitable by Methoxyluteolin.
Abstract: Activation of microglia, the resident immune cells of the brain and increased levels of pro-inflammatory cytokines and chemokines have been reported in patients with Autism Spectrum Disorders (ASD); however the stimulus is unknown. Inflammation of the brain in ASD could result from cross-talk between microglia and mast cells (MC), the unique immune cells responsible for allergic and infl... read moreammatory processes. ASD risk is associated with gene mutations of regulatory proteins increasing activation of the phosphatidylinositol-3-kinase (PI3K)-dependent mammalian target of rapamycin (mTOR) signaling, and those lowering moesin, an ezrin/radixin/moesin (ERM) cytoskeletal protein. Increased mTOR activity is also evident in MC and in keratinocyte proliferation, resulting in allergies, asthma, mastocytosis or psoriasis, common in mothers having children with ASD. We had reported the elevated serum levels of the peptide neurotensin (NT) in patients with ASD and psoriasis. We hypothesized that aberrant activation of human microglia, MC and keratinocytes in response to peptides or cytokines will depend on PI3K/mTOR signaling, and will be inhibited by the novel flavonoid methoxyluteolin. We investigated the involvement of PI3K/mTOR activation in neuropeptide or cytokine stimulated cells using specific inhibitors and the natural flavonoids luteolin and methoxyluteolin. The gene expression and release of the pro-inflammatory mediators were increased for: (a) IL-1β, CXCL8, CCL2 and CCL5 from microglia stimulated by NT or LPS, (b) TNF, CXCL8 and VEGF from MC stimulated by NT or SP and (c) IL-6, CXCL8 and VEGF from human keratinocytes stimulated by TNF. Methoxyluteolin attenuated mTOR activation, and pro-inflammatory mediator expression and release from stimulated cells. We show that ERM regulate human MC mediator release, and speculate that kinases downstream of mTOR promote moesin phosphorylation, which is also inhibited by methoxyluteolin. NT-induced human microglia stimulation and which included cell proliferation were mediated via its receptor NTR3/sortilin, which is secreted extracellularly and is uniquely elevated in the serum of ASD patients. Our findings reveal NT as the potential stimulus for activation of human microglia contributing to inflammation of the brain in ASD. Thus, targeting NTR3/sortilin and/or using methoxyluteolin may provide important novel therapeutic options for ASD, and other inflammatory disorders involving abnormal activation of MC and keratinocytes.
Thesis (Ph.D.)--Tufts University, 2016.
Submitted to the Dept. of Cell, Molecular & Developmental Biology.
Advisor: Theoharis Theoharides.
Committee: Alexei Degterev, Alex Bohm, John Castellot, and Margaret Bauman.
Keywords: Biochemistry, Cellular biology, and Pharmacology.read less