Arti B. Patel

Focal brain inflammation and autism.

Increasing evidence indicates that brain inflammation is involved in the pathogenesis of neuropsychiatric diseases. Autism spectrum disorders (ASD) are characterized by social and learning disabilities that affect as many as 1/80 children in the USA. There is still no definitive pathogenesis or reliable biomarkers for ASD, thus significantly curtailing the development of effective therapies. Many children with ASD regress at about age 3 years, often after a specific event such as reaction to vaccination, infection, stress or trauma implying some epigenetic triggers, and may constitute a distinct phenotype. ASD children respond disproportionally to stress and are also affected by food and skin allergies. Corticotropin-releasing hormone (CRH) is secreted under stress and together with neurotensin (NT) stimulates mast cells and microglia resulting in focal brain inflammation and neurotoxicity. NT is significantly increased in serum of ASD children along with mitochondrial DNA (mtDNA). NT stimulates mast cell secretion of mtDNA that is misconstrued as an innate pathogen triggering an auto-inflammatory response. The phosphatase and tensin homolog (PTEN) gene mutation, associated with the higher risk of ASD, which leads to hyper-active mammalian target of rapamycin (mTOR) signalling that is crucial for cellular homeostasis. CRH, NT and environmental triggers could hyperstimulate the already activated mTOR, as well as stimulate mast cell and microglia activation and proliferation. The natural flavonoid luteolin inhibits mTOR, mast cells and microglia and could have a significant benefit in ASD.

Atopic diseases and inflammation of the brain in the pathogenesis of autism spectrum disorders

Autism spectrum disorders (ASDs) affect as many as 1 in 45 children and are characterized by deficits in sociability and communication, as well as stereotypic movements. Many children also show severe anxiety. The lack of distinct pathogenesis and reliable biomarkers hampers the development of effective treatments. As a result, most children with ASD are prescribed psychopharmacologic agents that do not address the core symptoms of ASD. Autoantibodies against brain epitopes in mothers of children with ASD and many such children strongly correlate with allergic symptoms and indicate an aberrant immune response, as well as disruption of the blood–brain barrier (BBB). Recent epidemiological studies have shown a strong statistical correlation between risk for ASD and either maternal or infantile atopic diseases, such as asthma, eczema, food allergies and food intolerance, all of which involve activation of mast cells (MCs). These unique tissue immune cells are located perivascularly in all tissues, including the thalamus and hypothalamus, which regulate emotions. MC-derived inflammatory and vasoactive mediators increase BBB permeability. Expression of the inflammatory molecules interleukin (IL-1β), IL-6, 1 L-17 and tumor necrosis factor (TNF) is increased in the brain, cerebrospinal fluid and serum of some patients with ASD, while NF-kB is activated in brain samples and stimulated peripheral blood immune cells of other patients; however, these molecules are not specific. Instead the peptide neurotensin is uniquely elevated in the serum of children with ASD, as is corticotropin-releasing hormone, secreted from the hypothalamus under stress. Both peptides trigger MC to release IL-6 and TNF, which in turn, stimulate microglia proliferation and activation, leading to disruption of neuronal connectivity. MC-derived IL-6 and TGFβ induce maturation of Th17 cells and MCs also secrete IL-17, which is increased in ASD. Serum IL-6 and TNF may define an ASD subgroup that benefits most from treatment with the natural flavonoid luteolin. Atopic diseases may create a phenotype susceptible to ASD and formulations targeting focal inflammation of the brain could have great promise in the treatment of ASD.

Luteolin Inhibits Human Keratinocyte Activation and Decreases NF-κB Induction That Is Increased in Psoriatic Skin

Psoriasis (Ps) is an autoimmune disease characterized by keratinocyte hyperproliferation and chronic inflammation, with increased expression of tumor necrosis factor (TNF) and vascular endothelial growth factor (VEGF). Anti-TNF biologic agents are effective in treating Ps, but are associated with increased risk of infections and blood malignancies. Moreover, keratinocyte hyperproliferation and activation have yet to be addressed. Flavonoids, such as luteolin, are natural compounds with potent anti-inflammatory properties, but their actions on keratinocytes remain unknown. We show that TNF (50 ng/mL) triggers significant production of inflammatory mediators interleukin-6, interleukin-8 and VEGF from both human HaCaT and primary keratinocytes. Pretreatment with the flavonoid luteolin (10–100 µM) significantly inhibits mRNA expression and release of all three mediators in a concentration-dependent manner. More importantly, luteolin decreases TNF-induced phosphorylation, nuclear translocation and DNA binding of the nuclear factor-kappa B (NF-κB) typically involved in inflammatory mediator transcription. We also report that luteolin reduces TNF-induced mRNA expression of two genes (NFKB1 and RELA) encoding two NF-κB subunits (NF-κB p50 and NF-κB p65, respectively). Interestingly, we show that gene expression of RELA is increased in human psoriatic skin. Keratinocyte proliferation, which is a characteristic feature of psoriatic skin, is effectively reduced by luteolin in HaCaT cells, but not in primary keratinocytes. Finally, luteolin does not affect intracellular ATP production or viability. Appropriate formulations of luteolin and related flavones may be promising candidates to be developed into local and systemic treatments for Ps and other inflammatory skin diseases.

Neurotensin stimulates sortilin and mTOR in human microglia inhibitable by methoxyluteolin, a potential therapeutic target for autism.

Significance Human microglia, the resident immune cells of the brain, express only the neurotensin (NT) receptor-3/sortilin. NT significantly increases microglia synthesis and release of proinflammatory cytokine IL-1β and chemokine (C-X-C motif) ligand 8 (CXCL8), chemokine (C-C motif) ligand 2 (CCL2), and CCL5 via NTR3/sortilin. A soluble form of this receptor is secreted from stimulated microglia and is increased in the serum of children with autism spectrum disorders (ASD). These responses and the NT-stimulated increases in microglia numbers are mediated via mammalian target of rapamycin (mTOR) activation and are inhibitable by the natural flavonoids luteolin and methoxyluteolin. We had reported elevated serum levels of the peptide neurotensin (NT) in children with autism spectrum disorders (ASD). Here, we show that NT stimulates primary human microglia, the resident immune cells of the brain, and the immortalized cell line of human microglia-SV40. NT (10 nM) increases the gene expression and release (P < 0.001) of the proinflammatory cytokine IL-1β and chemokine (C-X-C motif) ligand 8 (CXCL8), chemokine (C-C motif) ligand 2 (CCL2), and CCL5 from human microglia. NT also stimulates proliferation (P < 0.05) of microglia-SV40. Microglia express only the receptor 3 (NTR3)/sortilin and not the NTR1 or NTR2. The use of siRNA to target sortilin reduces (P < 0.001) the NT-stimulated cytokine and chemokine gene expression and release from human microglia. Stimulation with NT (10 nM) increases the gene expression of sortilin (P < 0.0001) and causes the receptor to be translocated from the cytoplasm to the cell surface, and to be secreted extracellularly. Our findings also show increased levels of sortilin (P < 0.0001) in the serum from children with ASD (n = 36), compared with healthy controls (n = 20). NT stimulation of microglia-SV40 causes activation of the mammalian target of rapamycin (mTOR) signaling kinase, as shown by phosphorylation of its substrates and inhibition of these responses by drugs that prevent mTOR activation. NT-stimulated responses are inhibited by the flavonoid methoxyluteolin (0.1–1 μM). The data provide a link between sortilin and the pathological findings of microglia and inflammation of the brain in ASD. Thus, inhibition of this pathway using methoxyluteolin could provide an effective treatment of ASD.

Mast Cells Regulate Wound Healing in Diabetes

Diabetic foot ulceration is a severe complication of diabetes that lacks effective treatment. Mast cells (MCs) contribute to wound healing, but their role in diabetes skin complications is poorly understood. Here we show that the number of degranulated MCs is increased in unwounded forearm and foot skin of patients with diabetes and in unwounded dorsal skin of diabetic mice (P < 0.05). Conversely, postwounding MC degranulation increases in nondiabetic mice, but not in diabetic mice. Pretreatment with the MC degranulation inhibitor disodium cromoglycate rescues diabetes-associated wound-healing impairment in mice and shifts macrophages to the regenerative M2 phenotype (P < 0.05). Nevertheless, nondiabetic and diabetic mice deficient in MCs have delayed wound healing compared with their wild-type (WT) controls, implying that some MC mediator is needed for proper healing. MCs are a major source of vascular endothelial growth factor (VEGF) in mouse skin, but the level of VEGF is reduced in diabetic mouse skin, and its release from human MCs is reduced in hyperglycemic conditions. Topical treatment with the MC trigger substance P does not affect wound healing in MC-deficient mice, but improves it in WT mice. In conclusion, the presence of nondegranulated MCs in unwounded skin is required for proper wound healing, and therapies inhibiting MC degranulation could improve wound healing in diabetes.

Methoxyluteolin Inhibits Neuropeptide-stimulated Proinflammatory Mediator Release via mTOR Activation from Human Mast Cells

Mast cells (MCs) are critical for allergic reactions but are also important in inflammatory processes. Stimulation by neuropeptides, such as substance P (SP) and neurotensin (NT), leads to release of preformed molecules stored in numerous MC secretory granules and newly synthesized proinflammatory mediators, including tumor necrosis factor, C-X-C motif chemokine ligand 8, and vascular endothelial growth factor. Here, we investigate the role of mammalian target of rapamycin (mTOR) signaling in the stimulation of cultured human LAD2 MCs by NT or SP, as well as the inhibitory effect of the natural flavonoids 3′,4′,5,7-tetrahydroxyflavone (luteolin) and its novel structural analog 3′,4′,5,7-tetramethoxyflavone (methoxyluteolin). Stimulation by NT (10 μM) or SP (1 μM) increases (P < 0.0001) gene expression (after 6 hours) and release (after 24 hours) of tumor necrosis factor, C-X-C motif chemokine ligand 8, and vascular endothelial growth factor. This occurs via activation of both mTOR complexes, as denoted by the increased phosphorylated (p) protein levels (P < 0.0001) of the downstream mTORC1 substrate pp70S6KThr389 and mTORC2 component pmTORSer2448. Pretreatment of human MCs using the mTORC1 inhibitor rapamycin, the mTORC1/mTORC2 inhibitor Torin1, or the two flavonoids decreases both gene expression and release (P < 0.0001) of all three mediators. Methoxyluteolin is a more potent human MC inhibitor than luteolin or Torin1, implicating other MC protein targets in addition to the mTOR complex. These findings indicate that mTOR is partially involved in the neuropeptide stimulation of MCs, but the novel flavonoid methoxyluteolin inhibits the response entirely, suggesting that it may be developed for treatment of allergic and inflammatory diseases.

TNF stimulates IL‐6, CXCL8 and VEGF secretion from human keratinocytes via activation of mTOR, inhibited by tetramethoxyluteolin

Psoriasis is an autoimmune skin disease characterized by keratinocyte hyperproliferation and chronic inflammation. The pathogenesis of psoriasis involves proinflammatory cytokines, such as tumor necrosis factor (TNF), but the mechanism of keratinocyte activation is not well understood. Here, we show that TNF (10 or 50 ng/mL) stimulates a significant (P < .0001) gene expression and secretion of proinflammatory IL‐6, CXCL8 and VEGF from both cultured human HaCaT and normal epidermal human keratinocytes (NHEKs). This effect occurs via activation of the mammalian target of rapamycin (mTOR) signalling complex as shown by Western blot analysis and phospho‐ELISAs. Pretreatment with the novel natural flavonoid tetramethoxyluteolin (10‐100 μmol L−1) significantly (P < .0001) inhibits gene expression and secretion (P < .0001) of all 3 mediators in a concentration‐dependent manner. Moreover, tetramethoxyluteolin (50 μmol L−1) appears to be a potent inhibitor of the phosphorylated mTOR substrates (pmTORSer2448, pp70S6KThr389 and p4EBP1Thr37/46) as compared to known mTOR inhibitors in keratinocytes. The present findings indicate that TNF stimulates skin inflammation via mTOR signalling. Inhibition by tetramethoxyluteolin may be used in the treatment for psoriasis.