Patients and controls
Ninety elderly Italian individuals were enrolled in the study by the Neurology Department of the IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Italy. Thirthy patients had a diagnosis of AD, 30 patients had a diagnosis of mild cognitive impairment (MCI) and 30 individuals were age and sex matched healthy controls (HC). The clinical diagnosis of AD was performed according to NINCDS-ADRDA work group criteria [28] and further revisions [37], the clinical diagnosis of MCI fulfilled Petersen’s operational criteria [29]. Neuropsychological evaluation was performed with a Mini-Mental State Examination (MMSE) [38] and Clinical Dementia Rating Scale (CDR) [39].
All patients underwent a clinical interview, neurological and neuropsychological examination, routine blood tests, brain MRI, and lumbar puncture (LP). The mean age of AD patients (13 males and 17 females) was 74.3 years (age range 54-88 years) and that of MCI patients (8 males and 22 females) was 75.3 years (age range 63-84 years). All the AD patients were enrolled in a Multidimensional Stimulation Rehabilitation program designed for this pathology. Finally, the thirty age- and sex-matched elderly subjects (HC) who were included in the study were selected according to the SENIEUR protocol for immuno-gerontological studies of European Community’s Control Action Programme on Aging [40]; their MMSE score was > 28.
The study was approved by the Ethics Committee of Don Gnocchi Foundation and informed written consent was obtained from all the included subjects before study initiation.
Serum and CSF
Serum was collected in vacutainer tubes containing serum separator (Becton Dickinson and Co., Rutherford, NJ, USA) and were centrifuged at 3,000 rpm for 10 min to separate sera; CSF samples were collected by lumbar puncture. Serum and CSF were used immediately or stored at -80°C.
Blood sample collection and cell separation
Whole blood of a subset of individuals (5 AD, 5 MCI and 5 HC) was collected in vacutainer tubes containing ethylenediaminetetraacetic acid (EDTA) (Becton Dickinson). Peripheral blood mononuclear cells (PBMC) were separated on lympholyte separation medium (Cedarlane, Hornby, Ontario, CA) and washed twice in PBS at 1500 rpm for 10 min; viable leukocytes were determined using a TC20 Automated Cell Counter (Biorad, Hercules, CA, USA).
Cell cultures
PBMC (1x106/ml) were cultured in RPMI 1640 supplemented with 10% human serum, 2mM L-glutamine, and 1% penicillin (Invitrogen Ltd, Paisley, UK) and incubated at 37°C in a humidified 5% CO2 atmosphere for 2 hours in a 12 wells plate for monocyte adhesion. After 2 hours, non- adhering PBMC were harvested and discarded and monocytes grown on plate were either culture in medium alone (unstimulated) or were or primed with 2mg/ml Lipopolysaccharide (LPS) for 2 hours (Sigma-Aldrich, St. Louis, MO, USA) before stimulation with 10mg/ml of 1-42 amyloid-beta peptide (Ab42) (Sigma-Aldrich) in the absence/presence of 10ng/ml of Human Recombinant IL-33 (Biolegend, San Diego, CA, USA) for 24h at 37°C in a humidified 5% CO2 atmosphere. After 24 hour, supernatants were collected and stored at -20°C; adhering cells (monocytes) were collected and prepared for FlowSight analysis.
Enzyme-linked immunosorbent assay (ELISA)
IL-33 (catalog number D3300B, sensitivity: 1.51 pg/mL, assay range: 3.1–200 pg/ml, minimum detectable dose (MDD): ranged from 0.069-1.51 pg/mL), sST2 (catalog number DST200, sensitivity 13.5 pg/mL, assay range: 31.3-2000 pg/mL, MDD: ranged from 2.45-13.5 pg/mL), IL-1b (catalog number DLB50, sensitivity: 1 pg/mL, assay range: 3.9-250 pg/ml MDD: less than 1 pg/mL), IL-6 (catalog number D6050, sensitivity:0.70 pg/mL, assay range: 3.1–300 pg/ml MDD: less than 0.70 pg/mL) and IL-10 (catalog number D1000B, sensitivity: 3.90 pg/mL, assay range: 7.8–500 pg/ml, MDD: less than 3.90 pg/mL) concentration was analyzed in serum and CSF by commercially-available ELISA according to the manufacturer’s recommendations (Quantikine Immunoassay; R&D Systems, Minneapolis, MN, USA or Thermo Fisher Scientific, Waltham, MA, USA). A plate reader (Sunrise, Tecan, Mannedorf, Switzerland) was used and optical densities (OD) were determined at 450/620 nm. All samples were performed in duplicates. The same methods were used to measure IL-1b, IL-6 and IL-10 in unstimulated and in LPS-primed and Ab42- stimulated-PBMC supernatants in the presence/absence of recombinant IL-33 (see above).
Western blot
A Qubit Protein Assay Kit was used for protein extraction; total protein amount was measured
using a Qubit 3.0 Fluorometer (Thermo Fisher Scientific). Equal amounts of protein (15 µg) from each sample, or 2 ng of artificial truncated IL-33 (amino acids 112-270) (20 KD) (Recombinant Human IL33 carrier-free, Biolegend, San Diego, CA, USA) used as positive control to confirm that the bands correspond to IL-33 full length (amino acids 1-270) (34-30 KD), to the cleaved inactive forms (amino acids 1-178)(22-20 KD) and (amino acids 179-270) (13- 12KD) [2-3], or, finally, to the cleaved active forms (amino acids 95-270)(amino acids 99-270) and (amino acids 109-270) (19-15 KD) [4], were separated by 10% SDS-PAGE and transferred to PVDF membranes (Genscript). A pre-stained marker, broad range 11-190 KDa (Cell signaling, Danvers, MA, USA), was also used, PVDF membrane were treated by ONE-HOUR western detection kit (Genscript) and proteins were visualized using a Chromosensor TMB substrate (Genscript). After protein transfer, PVDF membranes were incubated with Pretreatment Solution for 5 min RT and then with the WB solution and an a-human IL-33 Ab (Nessy-1)(Abcam, Cambridge, UK) for 40 min. After three washes membranes were developed with a TMB substrate.
Cell culture and NFkB /7AAD intracellular staining
Monocytes that were either unstimulated or LPS-primed and Ab42- stimulated in the presence/absence of 10ng/ml of recombinant IL-33 were analyzed to evaluate nuclear translocation of NF-kB; the Amnis® NF-kB Translocation Kit was used according to the manufacturer’s recommendations (Merck KGaA, Darmstadt, Germany). Briefly, cells were fixed, permeabilized and stained with Anti-Hu NF-κB(p50) Alexa Fluor® 488 for 30 min RT. After incubation, monocyte were washed, fixed and 10ml of 7AAD were added.
FlowSight Analysis
The FlowSight (Amnis Corporation, Seattle, WA, USA) is equipped with two lasers operating at 488 and 642 nm, two camera and twelve standard detection channels. It simultaneously produce side scatter (darkfield) images, one or two transmitted light (brightfield) images, and up to ten channels of fluorescence imagery of every cell. FlowSight acquires 2000 cells/second and operates with a 1mm pixel size (~20X magnification) allowing visualization of fluorescence from the membrane, cytoplasm, or nucleus. The IDEAS image analysis software allows quantification of cellular morphology and fluorescence at different cellular localizations by defining specific cellular regions (masks) and mathematical expressions that uses image pixel data or masks (feature) by different wizards. Analysis of NF-kB translocation was performed by Nuclear Localization Wizard using Similarity Feature. Briefly, nuclear translocation has occurred if the NF-κB and nuclear fluorescence signals overlap with similar shapes. The Bright Detail Similarity is designed to specifically to compare the small bright image detail of two images and can be used to quantify the co-localization of two probes (NF-kB and 7AAD) in a defined region. The Similarity score is the log transformed Pearson’s Correlation Coefficient and it is a measure of the degree to which two images are linearly correlated within a masked region and is calculated on double positive region (NF-kB+7AAD+).
Statistical analysis
Quantitative data were not normally distributed (Shapiro–Wilk test) and were summarized as median and Interquartile Range (IQR) (25° and 75° percentile). Comparisons between groups were performed used a Kruskal-Wallis ANOVA for each variable. Comparisons among the different groups were made using a 2-tailed Mann-Whitney U test performed for independent samples. Data analysis was performed using the MedCalc statistical package (MedCalc Software bvba, Mariakerke, Belgium).