Polyunsaturated Fatty Acids Mend Macrophage Transcriptome, Glycome, and Phenotype in the Patients with Neurodegenerative Diseases, Including Alzheimer's Disease.

BACKGROUND: Macrophages of healthy subjects have a pro-resolution phenotype, upload amyloid-ß (Aß) into endosomes, and degrade Aß, whereas macrophages of patients with Alzheimer's disease (AD) generally have a pro-inflammatory phenotype and lack energy for brain clearance of Aß. OBJECTIVE: To clarify the pathogenesis of sporadic AD and therapeutic effects of polyunsaturated fatty acids (PUFA) with vitamins B and D and antioxidants on monocyte/macrophage (MM) migration in the AD brain, MM transcripts in energy and Aß degradation, MM glycome, and macrophage clearance of Aß. METHODS: We followed for 31.3 months (mean) ten PUFA-supplemented neurodegenerative patients: 3 with subjective cognitive impairment (SCI), 2 with mild cognitive impairment (MCI), 3 MCI/vascular cognitive impairment, 2 with dementia with Lewy bodies, and 7 non-supplemented caregivers. We examined: monocyte migration in the brain and a blood-brain barrier model by immunochemistry and electron microscopy; macrophage transcriptome by RNAseq; macrophage glycome by N-glycan profiling and LTQ-Orbitrap mass spectrometry; and macrophage phenotype and phagocytosis by immunofluorescence. RESULTS: MM invade Aß plaques, upload but do not degrade Aß, and release Aß into vessels, which develop cerebrovascular amyloid angiopathy (CAA); PUFA upregulate energy and Aß degradation enzyme transcripts in macrophages; PUFA enhance sialylated N-glycans in macrophages; PUFA reduce oxidative stress and increase pro-resolution MM phenotype, mitochondrial membrane potential, and Aß phagocytosis (p < 0.001). CONCLUSION: Macrophages of SCI, MCI, and AD patients have interrelated defects in the transcriptome, glycome, Aß phagocytosis, and Aß degradation. PUFA mend macrophage transcriptome, enrich glycome, enhance Aß clearance, and benefit the cognition of early-stage AD patients.

Omega-3 fatty acids increase OXPHOS energy for immune therapy of Alzheimer disease patients.

Sporadic late-onset Alzheimer disease (LOAD) preceded by mild cognitive impairment (MCI) is the most common type of dementia. Long-term studies of immunity to pathogenic amyloid-ß (Aß) in LOAD are lacking. Innate immunity of LOAD patients is malfunctioning in phagocytosis and degradation of Aß and LOAD patients' macrophage transcriptome and metabolome are deregulated. We previously showed omega-3 fatty acid (ω-3)-mediated repair of unfolded protein response and here we show much broader transcriptomic effects. ω-3 treatment in vitro and ω-3 supplementation by the drink Smartfish (SMF) in vivo increased the transcripts of the genes and pathways of immunity, glycolysis, tricarboxylic acid cycle, OX-PHOS, nicotinamide dinucleotide (NAD+ ) synthesis, and reversed the defects in Aß phagocytosis. In both peripheral blood mononuclear cells (PBMC) and macrophages, ω-3 increased ATP-linked oxygen consumption rate (OCR) and ω-3 with carnitine was superior to ω-3. ω-3 treatment in vitro and supplementation by the ω-3 drink SMF in vivo rescued macrophage phagocytosis when glycolysis or glycosylation were blocked. ω-3 provide flexible energy for immune clearance of the brain throughout the diurnal cycle, even in hypo- or hyper-glycemia. In certain LOAD patients, ω-3 may delay progression to dementia.

Omega-3 Fatty Acids Increase Amyloid-ß Immunity, Energy, and Circadian Rhythm for Cognitive Protection of Alzheimer's Disease Patients Beyond Cholinesterase Inhibitors.

BACKGROUND: The cholinesterase inhibitor therapeutics (CI) approved for use in Alzheimer's disease (AD) are palliative for a limited time. OBJECTIVE: To examine the outcome of AD patients with add-on therapy of the omega-3 fatty acid drink Smartfish. METHODS: We performed a prospective study using Mini-Mental State Examination, amyloid-ß (Aß) phagocytosis blood assay, and RNA-seq of peripheral blood mononuclear cells in 28 neurodegenerative patients who had failed their therapies, including 8 subjective cognitive impairment (SCI), 8 mild cognitive impairment (MCI), 2 AD dementia, 1 frontotemporal dementia (FTD), 2 vascular cognitive impairment, and 3 dementia with Lewy bodies (DLB) patients. RESULTS: MCI, FTD, and DLB patients patients volunteered for the addition of a ω-3 fatty acid drink Smartfish protected by anti-oxidants to failing CI therapy. On this therapy, all MCI patients improved in the first year energy transcripts, Aß phagocytosis, cognition, and activities of daily living; in the long term, they remained in MCI status two to 4.5 years. All FTD and DLB patients rapidly progressed to dementia. On in vivo or in vitroω-3 treatments, peripheral blood mononuclear cells of MCI patients upregulated energy enzymes for glycolysis and citric acid cycle, as well as the anti-inflammatory circadian genes CLOCK and ARNTL2. CONCLUSION: Add-on ω-3 therapy to CI may delay dementia in certain patients who had failed single CI therapy.

Immunotherapy of Mild Cognitive Impairment by ω-3 Supplementation: Why Are Amyloid-ß Antibodies and ω-3 Not Working in Clinical Trials?

This article reviews the basic tenets of a clinical approach to effective immunotherapy of Alzheimer's disease (AD) in patients with mild cognitive impairment (MCI). Although one randomized controlled study in early MCI patients by fish-derived omega-3 fatty acids (ω-3) showed slowing of disease progression, large clinical trials with different products have failed to show cognitive effects. Macrophages of healthy subjects phagocytize and degrade amyloid-ß1 - 42 (Aß) in the brain tissues, whereas macrophages of patients with AD and MCI are functionally defective. ω-3 and ω-3-derived specialized proresolving mediators (SPMs), such as resolvin D1, have powerful biochemical and immunological effects, which may repair the functions of MCI patients' macrophages in the brain's clearance of Aß. Unfortunately, ω-3 products on the market have a variable quality. Nutritional supplementation with a combination drink called Smartfish with an emulsion of ω-3 and other fatty acids, antioxidants, 1,25-dihydroxy vitamin D3, and resveratrol improved the innate immune system of MCI patients by modulation of macrophage type to the pro-phagocytic M1-M2 type with an effective unfolded protein response against endoplasmic reticulum stress. Some MCI patients maintained their initial cognitive status for three years on Smartfish supplementation. Future randomized clinical trials should investigate the immune effects of ω-3, 1,25-dihydroxy vitamin D3, and SPMs on macrophage type, function, and biochemistry in parallel with cognitive effects.

Omega-3 fatty acids increase the unfolded protein response and improve amyloid-ß phagocytosis by macrophages of patients with mild cognitive impairment.

Macrophages (Mϕs) of patients with Alzheimer's disease and mild cognitive impairment (MCI) are defective in amyloid-ß1-42 (Aß) phagocytosis and have low resistance to apoptosis by Aß. Omega-3 fatty acids (ω-3s) in vitro and in vivo and the ω-3 mediator, resolvin D1, in vitro increase Aß phagocytosis by Mϕs of patients with MCI. We have investigated the unfolded protein response (UPR) to endoplasmic reticulum (ER) stress by Mϕs in a longitudinal study of fish-derived, ω-3-supplemented patients with MCI. Patients in the apolipoprotein E (ApoE)e3/e3 subgroup over time exhibited an increase of protein kinase RNA-like ER kinase (PERK) expression, Aß phagocytosis, intermediate M1-M2 Mϕ type, and a Mini-Mental State Examination (MMSE) rate of change of +1.8 points per year, whereas patients in the ApoEe3/e4 subgroup showed individually divergent results with an MMSE rate of change of -3.2 points per year. In vitro treatment of Mϕs by fish-derived ω-3 emulsion increased Aß phagocytosis, PERK expression, and UPR RNA signature, and decreased ER stress signature. Augmented genes in the UPR signature included chaperones, lectins, foldases, and N-linked glycosylation enzymes. In summary, fish-derived ω-3s increase cytoprotective genes and decrease proapoptotic genes, improve immune clearance of Aß, and are associated with an improved MMSE rate of change in ApoEe3/e3 vs. ApoEe3/e4 patients.-Olivera-Perez, H. M., Lam, L., Dang, J., Jiang, W., Rodriguez, F., Rigali, E., Weitzman, S., Porter, V., Rubbi, L., Morselli, M., Pellegrini, M., Fiala, M. Omega-3 fatty acids increase the unfolded protein response and improve amyloid-ß phagocytosis by macrophages of patients with mild cognitive impairment.

Modulation of innate immunity of patients with Alzheimer's disease by omega-3 fatty acids.

The innate immune system of patients with Alzheimer's disease and mild cognitive impairment (MCI) is deregulated with highly increased or decreased transcription of inflammatory genes and consistently depressed phagocytosis of amyloid-ß1-42 (Aß) by monocytes and macrophages. Current immune therapies target single mechanisms in the adaptive immune system but not innate immunity. Here, we summarize recent advances in therapy by ω-3, ω-6, and epoxy fatty acids; specialized proresolving mediators; and vitamin D3 that have proven immune effects and emerging cognitive effects in patients with MCI. The hypothesis of this approach is that macrophages of normal participants, but not those of patients with Alzheimer's disease and MCI, possess effective phagocytosis for Aß and protect homeostasis of the brain and, furthermore, that defective MCI macrophages recover phagocytic function via ω-3. Recent studies of fish-derived ω-3 supplementation in patients with MCI have shown polarization of Apoε3/ε3 patients' macrophages to an intermediate M1-M2 phenotype that is optimal for Aß phagocytosis and the stabilization of cognitive decline. Therefore, accumulating preclinical and preliminary clinical evidence indicates that ω-3 supplementation should be tested in a randomized controlled clinical trial and that the analysis should involve the apolipoprotein E genotype and intervening conditions during trial.-Fiala, M., Kooij, G., Wagner, K., Hammock, B., Pellegrini, M. Modulation of innate immunity of patients with Alzheimer's disease by omega-3 fatty acids.

Increased intermediate M1-M2 macrophage polarization and improved cognition in mild cognitive impairment patients on ω-3 supplementation.

Monocyte/macrophages of patients with mild cognitive impairment (MCI) and Alzheimer disease (AD) are defective in phagocytosis and degradation amyloid ß1-42 (Aß1-42), but are improved by ω-3 fatty acids (ω-3s). The hypothesis of this study was that active Aß1-42 phagocytosis by macrophages prevents brain amyloidosis and thus maintains cognition. We studied the effects of self-supplementation with a drink with ω-3s, antioxidants, and resveratrol on Mini-Mental State Examination (MMSE) scores, macrophage M1M2 phenotype [the ratio of inflammatory cluster of differentiation (CD)54+CD80 and proresolution markers CD163+CD206], and Aß1-42 phagocytosis in patients initially diagnosed as having MCI or subjective cognitive impairment (SCI). At baseline, the median MMSE score in patients in both the apolipoprotein E (ApoE) ε3/ε3 and ApoE ε3/ε4 groups was 26.0 and macrophage Aß1-42 phagocytosis was defective. The MMSE rate of change increased in the ApoE ε3/ε3 group a median 2.2 points per year (P = 0.015 compared to 0) but did not change in the ApoE ε3/ε4 group (P = 0.014 between groups). In the ApoE ε3/ε3 group, all patients remained cognitively stable or improved; in the ApoE ε3/ε4 group, 1 recovered from dementia, but 3 lapsed into dementia. The macrophage phenotype polarized in patients bearing ApoE ε3/ε3 to an intermediate (green zone) M1-M2 type at the rate of 0.226 U/yr, whereas in patients bearing ApoE ε3/ε4, polarization was negative (P = 0.08 between groups). The baseline M1M2 type in the extreme M1 (red zone) or M2 (white zone) was unfavorable for cognitive outcome. Aß1-42 phagocytosis increased in both ApoE groups (P = 0.03 in each groups). In vitro, the lipidic mediator resolvin D1 (RvD1) down regulated the M1 type in patients with ApoE ε3/ε3 but in some patients with ε3/ε4, paradoxically up-regulated the M1 type. Antioxidant/ω-3/resveratrol supplementation was associated with favorable immune and cognitive responses in ApoE ε3/ε3 and individual patients bearing ApoE ε3/ε4, and brings into personalized clinical practice the immune benefits expected from ω-3 mediators called resolvins. The validity of this study is limited by its small size and uncontrolled design.-Famenini, S., Rigali, E. A., Olivera-Perez, H. M., Dang, J., Chang, M T., Halder, R., Rao, R. V., Pellegrini, M., Porter, V., Bredesen, D., Fiala, M. Increased intermediate M1-M2 macrophage polarization and improved cognition in mild cognitive impairment patients on ω-3 supplementation.

Specialized Pro-Resolving Mediators from Omega-3 Fatty Acids Improve Amyloid-ß Phagocytosis and Regulate Inflammation in Patients with Minor Cognitive Impairment.

In this review we discuss the immunopathology of Alzheimer's disease (AD) and recent advances in the prevention of minor cognitive impairment (MCI) by nutritional supplementation with omega-3 fatty acids. Defective phagocytosis of amyloid-ß (Aß) and abnormal inflammatory activation of peripheral blood mononuclear cells (PBMCs) are the two key immune pathologies of MCI and AD patients. The phagocytosis of Aß by PBMCs of MCI and AD patients is universally defective and the inflammatory gene transcription is heterogeneously deregulated in comparison to normal subjects. Recent studies have discovered a cornucopia of beneficial anti-inflammatory and pro-resolving effects of the specialized proresolving mediators (SPMs) resolvins, protectins, maresins, and their metabolic precursors. Resolvin D1 and other mediators switch macrophages from an inflammatory to a tissue protective/pro-resolving phenotype and increase phagocytosis of Aß. In a recent study of AD and MCI patients, nutritional supplementation by omega-3 fatty acids individually increased resolvin D1, improved Aß phagocytosis, and regulated inflammatory genes toward a physiological state, but only in MCI patients. Our studies are beginning to dissect positive factors (adherence to Mediterranean diet with omega-3 and exercise) and negative factors (high fat diet, infections, cancer, and surgeries) in each patient. The in vitro and in vivo effects of omega-3 fatty acids and SPMs suggest that defective phagocytosis and chronic inflammation are related to defective production and/or defective signaling by SPMs in immune cells.

ω-3 Supplementation increases amyloid-ß phagocytosis and resolvin D1 in patients with minor cognitive impairment.

We investigated the effects of 4-17 month supplementation with ω-3 fatty acids and antioxidants (Smartfish drink; Smartfish AS, Oslo, Norway) in 12 patients with minor cognitive impairment (MCI) [minimental state examination (MMSE) ≥19], 2 patients with pre-MCI (normal MMSE), and 7 patients with Alzheimer disease (AD) (MMSE <19). We measured the phagocytosis of amyloid-ß 1-42 (Aß) by flow cytometry and microscopy, the transcription of inflammatory genes by RT-PCR, the production of resolvin D1 (RvD1) by enzyme immunoassay, and the cognitive status by MMSE. In patients with MCI and pre-MCI, phagocytosis of Aß by monocytes increased from 530 to 1306 mean fluorescence intensity units (P = 0.016). The increase in patients with AD was not significant (N.S.). The lipidic mediator RvD1, which stimulates Aß phagocytosis in vitro, increased in macrophages in 80% of patients with MCI and pre-MCI (mean increase 9.95 pg/ml) (N.S.). Transcription of inflammatory genes' mRNAs was increased in a subgroup of patients with low transcription at baseline, whereas it was not significantly changed in patients with high transcription at baseline. The mean MMSE score of patients with MCI and pre-MCI was 25.9 at baseline and 25.7 after 4-17 months (N.S.). Our study is the first to show significant immune and biochemical effects of ω-3 fatty acids with antioxidants in patients with MCI. Cognitive benefits of ω-3 supplementation in patients with MCI should be tested in a clinical trial.

Curcumin and omega-3 fatty acids enhance NK cell-induced apoptosis of pancreatic cancer cells but curcumin inhibits interferon-γ production: benefits of omega-3 with curcumin against cancer.

STAT-3 and STAT-1 signaling have opposite effects in oncogenesis with STAT-3 acting as an oncogene and STAT-1 exerting anti-oncogenic activities through interferon-γ and interferon-α. The cytokine IL-6 promotes oncogenesis by stimulation of NFκB and STAT-3 signaling. Curcuminoids have bi-functional effects by blocking NFκB anti-apoptotic signaling but also blocking anti-oncogenic STAT-1 signaling and interferon-γ production. In our recent study (unpublished work [1]) in pancreatic cancer cell cultures, curcuminoids enhanced cancer cell apoptosis both directly and by potentiating natural killer (NK) cell cytotoxic function. The cytotoxic effects of curcuminoids were increased by incubation of cancer cells and NK cells in an emulsion with omega-3 fatty acids and antioxidants (Smartfish), which enhanced cancer cell apoptosis and protected NK cells against degradation. However, as also shown by others, curcuminoids blocked interferon-γ production by NK cells. The combined use of curcuminoids and omega-3 in cancer immunotherapy will require deeper understanding of their in vivo interactions with the immune system.

MGAT3 mRNA: a biomarker for prognosis and therapy of Alzheimer's disease by vitamin D and curcuminoids.

Practical biomarkers of Alzheimer's disease (AD) prognosis are lacking. Correspondingly, no drugs are known to decrease disease progression, although vitamin D3 has positive effects on cognition in vivo and 1α, 25-dihydroxyvitamin D3 (1,25 D3) on amyloid-ß 1-42 (Aß) phagocytosis in vitro. We have examined in a pilot study a new biomarker in peripheral blood mononuclear cells, the transcription of mRNA of ß-1,4-mannosyl-glycoprotein 4-ß-N-acetylglucosaminyltransferase (MGAT3), the essential gene for Aß phagocytosis. The transcription of MGAT3 stimulated by Aß distinguishes macrophages into Type 0 (very low MGAT3 transcription), Type I (low MGAT3 transcription up regulated by bisdemethoxycurcumin), and Type II (high MGAT3 transcription down regulated by bisdemethoxycurcumin). In this pilot study of 20 AD patients and 20 control subjects, 45% patients, but only 10% control subjects, were Type 0 (p-value = 0.009). Type 0 AD patients had worse 2-year prognosis regarding loss of independence than Type I and Type II patients (p-value = 0.013). Phagocytosis of Aß in Type I and II patients was shown to be dependent on 1,25 D3 using a specific inhibitor of the 1,25 D3-VDR activated nuclear receptor transcription factor. In a Type II patient, recovery from cognitive dysfunction related to surgical anesthesia was preceded by an improvement in phagocytosis of Aß. The results of this pilot study suggest that the MGAT3 Type biomarker may characterize subgroups of AD patients with different disease progression. In vitro results suggest that vitamin D3 supplementation might be beneficial in both Type I and II patients, whereas curcuminoids only in Type I. These results must be investigated in a large prospective study.

Re-balancing of inflammation and abeta immunity as a therapeutic for Alzheimer's disease-view from the bedside.

Morbidities of aging and Alzheimer's disease (AD) have been related to defective functions of both T cells and macrophages leading to brain amyloidosis and inflammation. In AD patients, "inflammaging" may be associated with an increase of incompetent memory T cells and inflammatory cytokines produced by macrophages, whereas defective clearance of amyloid-beta 1-42 (Abeta) may be related to defective transcription of immune genes necessary for Abeta phagocytosis, beta-1,4-mannosyl-glycoprotein 4-beta-N-acetylglucosaminyltransferase and Toll-like receptors. However, AD shows considerable heterogeneity of disease manifestations and mechanisms. The approaches to re-balancing Abeta immunity and inflammation are being pursued in transgenic animal models and peripheral blood mononuclear cells of patients. The regulatory signaling pathways of microglial phagocytosis and inflammation involving co-receptors and transforming growth factor-beta have been considerably clarified in animal studies. Natural immunostimulating therapies using vitamin D3 and curcuminoids have been developed in macrophages of AD patients. AD patients possess two types of macrophages: a majority has "Type I", which are improved by curcuminoids and vitamin D3; whereas a minority has "Type II" responding positively to vitamin D3 but not to curcuminoids. Other nutritional substances, such as plant polyphenols and omega-3 fatty acids, may inhibit inflammation and stimulate immunity. More invasive immune approaches involve Abeta vaccine and cytokine antagonists. Increased inflammation may represent the "first hit", and defective transcription of immune genes the "second hit" in the pathogenesis of AD.