Clinical and neurological examinations of AD patients were conducted at the IRCCS Mondino Foundation (Pavia, Italy). Healthy volunteers were recruited to the Immunohematological and Transfusional Service and Centre of Transplantation Immunology (IRCCS Foundation “San Matteo,” Pavia, Italy). Peripheral blood samples were collected from 30 AD patients with no family history of dementia and 28 healthy volunteers over 65 years of age after obtaining written informed consent (Table 1). Before being enrolled, the subjects that participated in the study signed an informed consent form (Protocol n°375/04 – version 07/01/2004). AD diagnosis was made according to the National Institute of Neurological and Communicative Disorders and Stroke and the AD and Related Disorders Association (NINCDS-ADRDA) criteria (Aggarwal and Harikumar, 2009). Quantification of neuropsychological testing, a detailed interview and clinical examinations were conducted with each subject. The severity of dementia in the AD patients was evaluated by the Mini Mental State Examination (MMSE) and carried out in accordance with The Code of Ethics of the World Medical Association (Declaration of Helsinki).

Peripheral blood mononuclear cells were prepared by layering peripheral blood on Ficoll-Histopaque (density = 1.077) and centrifugation at 950 ×g for 30 min. After isolation on a Ficoll-Histopaque layer (Sigma, Italy), cell viability was assayed by a trypan blue exclusion test and by cytometric analysis (Strober, 2001). Viable cells were used for in vitro studies with curcumins. PBMCs (5 × 10⁶ cells with viability ≥80%) were independently treated for 24 h with five different curcumins (0.1 μM) obtained from the Human BioMolecular Research Institute in San Diego, CA, United States. The curcumins were placed in DMSO and administered to cells in media. The five curcuminoids with the greatest potency for inducing expression of genes relevant to Aβ phagocytosis were selected based on results from a previous report (Gagliardi et al., 2012). The chemical structures of curcumins used in the studies are shown in Figure 1. About 5 × 10⁶ PBMCs were treated for 24 h with 0.1 μM of each curcuminoid and then with Aβ 1–42 (1 μM) (Sigma-Aldrich) for an additional 24 h. The conditions for tests included (Figure 2): untreated, treated only with curcuminoids, treated only with Aβ and, finally, treated with both curcuminoids and Aβ.

Trypan blue exclusion tests were conducted to assay cell viability after treatments. Curcuminoids and Aβ at 0.001–10 μM were used to treat PBMCs from AD patients and healthy volunteers for 24 h. The concentration range was determined based on data from similar studies carried out in different experimental models (Gagliardi et al., 2012).

To confirm the presence/absence of intact curcuminoids in cell culture supernatants and cell pellets, an HPLC method with ultraviolet (UV) detection for the determination of the selected compounds was used. A 1000 μL aliquot of cell culture supernatant was introduced onto an EMPORE Extraction Disk Cartridge (Sigma-Aldrich) under vacuum at 15 in Hg. Cartridges were conditioned sequentially with 1000 μL of methanol and 1000 μL of water. Interfering substances were washed out with water (1000 μL). Analytes were eluted with acetonitrile (80 μL) and water (120 μL). The eluate was then centrifuged at 1500 ×g for 5 min at 4°C and 50 μL was injected onto the HPLC system. HPLC analyses were carried out using a Shimadzu LC-10Av chromatograph (Shimadzu Scientific Instrument, Inc., Columbia, MD, United States) equipped with a System Controller SCL-10Avp, an LP-10ADvp pump, an on-line Degasser DGU-14A, a SIL-10ADvp auto injector and a model CTOXX temperature controller. The system was connected to a LaChrom L-7400 variable wavelength detector (Merck, Darmstadt, Germany). Separation of selected compounds was achieved on a Chromolith Performance Column (100 mm × 4.6 mm i.d., RP-18e, Merck, Darmstadt, Germany), protected by a guard column of the same composition (5-4.6 mm i.d., RP-18e, Merck, Darmstadt, Germany). The mobile phase was prepared by adding 1 mL of 85% phosphoric acid to 1 L of a mixture of water/acetonitrile (60:40 vol/vol) and thoroughly mixed. The column temperature was maintained at 50°C and the flow rate was 1.5 mL/min. Curcuminoids were detected at 420 nm. Data were acquired and analyzed using the Shimadzu LabSolution Lite software.

RNA was extracted using the Maxwell 16, Blood kit (Promega and automated purification system Maxwell 16). RNA was analyzed by a Bioanalyzer spectrophotometer to verify the quality and the concentration and stored at -80°C. RNA libraries were prepared by TruSeq Targeted RNA Expression NF-κB Panel (Illumina, United States) according to the manufacturers’ instructions. The panel was integrated by BACE1 as an AD-linked gene, and VDR and MGAT3 to confirm the involvement of these two genes along the lines reported previously (Gagliardi et al., 2012). RNA processing and RNA-seq analysis was carried out using Illumina MiSeq sequencer. RNA sequencing data is available in GEO repository (GSE122438).

Differential gene expression analysis was conducted on data obtained with TruSeq Targeted RNA Expression Kit with a MiSeq sequencer. Cells from AD patients and healthy volunteers were treated with different curcuminoids, both separately and in combination and were compared to identify differentially expressed genes.

The tables of counts obtained via MiSeq reporter analysis was imported in R¹ and differentially expressed genes were evaluated using a negative binomial GLM approach (EdgeR Bioconductor). Batch effects were removed with the RemoveBatchEffects function. Differentially expressed genes had | log2FC | > 1 and p-values <0.1.

To validate the expression profiles obtained by RNA-Seq, real-time PCR was conducted on MAGT3, VDR, NF-κB, and BACE1 genes selected for high or low expression levels.

1 × 10⁵ cells were placed on a poly-L-lysine slide (Thermo Fisher Scientific) and incubated at 37°C to allow cell attachment to the slide. Cells were rinsed with 1× PBS and fixed in 4% paraformaldehyde in 1× PBS. Fixed cells were washed with 1× PBS and blocked with 5% normal goat serum in 0.1% Tween-PBS for 1 h. Incubation with the primary mouse anti-beta amyloid antibody (1:100 dilution; Abcam) was conducted in blocking solution overnight at 4°C. The fluorescently tagged secondary antibody CFTM 488A goat anti-rabbit (1:700 dilution; Sigma-Aldrich) was used for detection. Slides were mounted with Prolong^® Gold anti-fade reagent with 4′6-diamidino-2-phenylindole (DAPI) (Invitrogen) and images were acquired by confocal microscopy (Leica DM IRBE, Leica Microsystems Srl, Italy).

Immediately after PBMCs were treated with curcumins, they were centrifuged at 16,000 rpm for 8 min at room temperature and the cell pellet was incubated with lysis buffer supplemented with protease inhibitors at 4°C for 10 min and then centrifuged at 18,000 ×g for 15 min at 4°C. The supernatant was used for measuring Aβ 1–42. The Aβ 1–42 content was determined using the AlphaLISA technique with the AlphaLISA human amyloid beta 1–42 (high specificity) kit (PerkinElmer) according to the manufacturers’ instructions. In the assay, biotinylated antibody against Aβ 1–42 and Acceptor beads (PerkinElmer) conjugated to a 2nd antibody against Aβ 1–42 was added to samples in 1× HiBlock AlphaLISA buffer on the assay plate. One antibody was specific to the β-secretase cleavage site at the N-terminus (mouse monoclonal antibody, clone number 82E1), the second antibody was specific to the C-terminus (mouse monoclonal antibody, clone number 12F4). The plate was sealed and incubated for 1 h at room temperature. Streptavidin-coated Donor beads were then added under subdued lighting, followed by 1 h incubation in the dark to bind the biotinylated antibody. When both antibodies bound Aβ 1–42 fragments, they were in close enough proximity and the excitation at 680 nm resulted in the production of ¹O₂ from the Donor beads. Energy is transferred from the singlet oxygen to the Acceptor beads, subsequently culminating in light production at 615 nm that was detected using an EnVision plate reader (PerkinElmer). For each group of cells, Aβ 1–42 content was determined by interpolating the standard curve using 4PL non-linear regression. As a control the same procedure was conducted with PBMCs treated only with Aβ in the absence of BDC.

Cells treated with 10 μM curcumin and 10 μM Aβ showed a greater decrease in viability compared with lower concentrations examined. Therefore, curcumins at 0.1 μM and Aβ at 1 μM were used for experiments (Figures 3A,B). Treatments with both molecules at the chosen concentrations did not show a significant alteration of cells viability (Figure 3C).

Although 10 μM curcuminoid was the concentration with the greatest impact on cells viability, the percentage of apoptosis was considered acceptable (20%) so it was used for HPLC analysis due to the sensitivity of the method. Under the chromatographic conditions the duration of each chromatographic run was 8 min and COMPOUND 1 eluted at 7 min. HPLC analysis conducted on PBMC samples from AD patients and healthy volunteers treated with curcuminoids (i.e., 10 μM for 24 h) showed the presence of compounds in the cellular pellet. Curcumins were absent in the supernatant of cells from both AD patients and healthy subjects treated with curcumins (Figures 4A,C). The presence of curcuminoids in the cellular pellet was reasonable based on the lipophilicity and protein binding properties of the compounds and confirmed that they partitioned into cells (Figures 4B,D).

Compared to untreated PBMC cells of AD patients, mRNA expression of 12 selected genes in PBMCs from healthy controls showed a significant difference (Table 2). Among the 12 genes examined, 6 genes (i.e., NF-κB, BACE1, VDR, MGAT3, TLR7, and TLR8) were previously reported to be targets of curcumin action. Alterations of NF-κB, BACE1, MGAT3, and VDR gene expression were validated with Real-Time PCR. The results confirmed the next generation sequencing (NGS) data that showed an increase of NF-κB and BACE1 in cells from AD patients compared to cells from healthy volunteers and a decrease of MGAT3 and VDR in cells from AD patients compared to cells from healthy controls (Table 2).

Peripheral blood mononuclear cells from AD patients and healthy volunteers treated with curcuminoids (i.e., 0.1 μM, 24 h) showed a significant alteration in gene expression in cells from AD patients (Supplementary Table S1). Compared to cells from healthy volunteers, COMPOUND 1 was the most potent compound in regulating genes expression and caused a twofold increase after treatment in cells from AD patients Compared to PBMCs from healthy controls, down-regulation of MGAT3 was observed (log2FC = 18) in untreated PBMCs of AD patients. In contrast, over-expression of MGAT3 was observed in PBMCs of AD patients treated with curcuminoids (i.e., 7- and 18-fold increase with COMPOUND 1 and 3, respectively). This showed that curcuminoids were able to induce transcription of MGAT3 in PBMCs from AD patients even in the absence of Aβ.

Treatment of PBMCs from AD patients with Aβ 1–42 (1 μM) caused significant alterations in genes expression but only in cells from AD patients (Supplementary Table S2). Compared to untreated PBMC cells from healthy volunteers, results showed that treatment of PBMCs from AD patients afforded a greater number of altered genes. Compared to PBMCs from healthy volunteers, 20 genes were up-regulated in PBMCs from AD patients after treatment with Aβ (0.1 μM, 24 h). For example, MGAT3 and VDR were increased log2FC, five and twofold, respectively. TLR4, 5, 7, and 8 were up-regulated log2FC, X-, X-, etc-fold, respectively. Compared to PBMCs from healthy volunteers, 15 genes were down-regulated in PBMCs from AD patients treated with 0.1 μM for 24 h. For example, CCR5, CD27, and CD83 genes involved in inflammatory pathway were down-regulated four-, three-, and twofold log2FC, respectively.

Individual PBMCs from 30 AD patients and 28 healthy volunteers were treated with COMPOUND 1 (i.e., 0.1 μM, 24 h), and then Aβ 1 μM was added to the cell culture for the next 24 h and Aβ and gene expression was quantified.

Peripheral blood mononuclear cells from 30 AD patients and 28 healthy subjects were treated with COMPOUND 2 (i.e., 0.1 μM for 24 h) and then Aβ (i.e., 1 μM) was added to the cells for the next 24 h.

Peripheral blood mononuclear cells from 30 AD patients and 28 healthy volunteers were treated with COMPOUND 3 (i.e., 0.1 μM for 24 h) and then Aβ (i.e., 1 μM) was added to cells for the next 24 h.