The in vitro GcMAF effects on endocannabinoid system transcriptionomics, receptor formation, and cell activity of autism-derived macrophages

We investigated 22 children with autism, and compared them to 20 age and sex matched healthy children used as a control group (age ranging 3 to 11 years; mean age: 7.06 ± 1.52 versus 7.38 ± 2.33 in autistic and healthy individuals, respectively). The subjects with autism were recruited into the study from the outpatient Biomedical Center for Autism Research and Treatment, Bari, Italy. Before entering the study, all of the children were administered the Autism Diagnostic Interview-Revised version [23], the Childhood Autism Rating Scales (CARS) [24], and the Autism Diagnostic Observation Schedule-Generic [25] to document the diagnosis of autism. All included patients met the Diagnostic and Statistical Manual of Mental Disorders-IV criteria for autism (DSM-IV-TR) [1]. In addition to meeting the criteria for autistic disorder, subject children were required to score at least 30 points on the CARS scale. Twenty healthy children were recruited among staff family members. Potential subjects were excluded if they had any of the following: a neurological or comorbid psychiatric disorder, epilepsy, history of liver, renal or endocrine disorders, current infection of any origin. Mental retardation or behavioral disorders, including Pervasive Developmental Disorder - Not Otherwise Specified (PDD-NOS), and inclusion criteria for attention deficit-hyperactivity disorder, were all considered exclusion criteria for control children. Children diagnosed with Asperger’s syndrome, fragile X syndrome and tuberous sclerosis were also excluded from the study. IQ test was not performed. Neither autistic subjects nor controls were receiving pharmacological interventions. Other exclusion criteria were celiac disease and/or other major diseases of the intestinal tract, such as inflammatory bowel disease or hepatic disorders.

Informed consent was obtained from the parents of all children enrolled in this study and assent was obtained from the healthy controls in compliance with Italian legislation and the Code of Ethical Principles for Medical Research Involving Human Subjects of the World Medical Association (Declaration of Helsinki).

Mononuclear cells were extracted as previously described [9, 19]. Briefly, less than 10 ml of fresh peripheral blood samples from autistic subjects and control donors were drawn and collected in sterile EDTA tubes (Becton Dickinson, Franklin Lakes, NJ, USA). Peripheral blood mononuclear cells (PMBCs) were isolated by centrifugation over Histopaque 1077 density gradient (Sigma Chemical, St Louis, MO, USA). Briefly, blood was diluted 1:1 in PBS (Sigma, St. Louis, MO, USA), overlaid onto lymphocyte separation media (Lymphocyte Separation Medium -Lonza, Walkersville, MD, USA), centrifuged at 2,200 rpm for 30 minutes at room temperature and plasma was removed. Mononuclear cell fraction was harvested and washed twice in PBS. The final pellet was re-suspended in RPMI 1640 complete medium (Lonza, Verviers, Belgium) containing 10% FBS (EuroClone-Celbio, Milan, Italy), 2 mM L-glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin (all Lonza, Verviers, Belgium) and incubated at 37°C with 5% CO₂. Lymphocytes (non-adherent cells) were removed.

In order to obtain fully differentiated human blood monocyte-derived macrophages (BMDMs), PBMCs were then cultured for about ten days in the presence of 25 ng/mL recombinant human macrophage colony-stimulating factor (Peprotech, London, UK) [26, 27] (Figure 1).

GcMAF was added at 0.1 ng/ml final concentration to BMDMs from healthy control and autistic patients. The dose of GcMAF was chosen on the basis of a previous work demonstrating that maximal stimulation of PBMCs was achieved with 0.1 ng/ml [28]. GcMAF was kindly provided by Immuno Biotech (St. Peter Port, Guernsey, UK). Following incubation of the cells for 24 hours in the presence of GcMAF, some cells were lysed for the extraction and analysis of RNA (Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR)), and proteins via Western blot analysis; alternatively, other cells were fixed for fluorescence-based immunocytochemistry analysis. Comparisons between pre-post treatment levels of RNA and protein expression were made for both autistic individuals and healthy controls.

The RNA was extracted from BMDMs using a RNA Tri-Reagent (Molecular Research Center Inc., Cincinnati, OH, USA) according to the manufacturer’s protocol. The total RNA concentration and integrity were determined by Nanodrop® ND-1000 UV spectrophotometer (Nano-Drop® Technologies, Thermo Scientific, Wilmington, DE, USA). The mRNA levels of the EC genes under analysis were measured by RT-PCR amplification, as previously reported [19]. Reverse Transcriptase from Avian Myeloblastosis Virus (AMV-RT; Promega, Madison, WI, USA) was used. For first-strand cDNA synthesis, 200 ng total RNA, random hexamers, dNTPs (Promega, Madison, WI, USA), AMV buffer, AMV-RT and recombinant RNasinribonuclease inhibitor (Promega, Madison, WI, USA) were assembled in diethyl-pyrocarbonate-treated water to a 20 μl final volume and incubated for ten minutes at 65°C and one hour at 42°C. RT minus controls were carried out to check potential genomic DNA contamination. These RT minus controls were performed without using the reverse transcriptase enzyme in the reaction mix. Aliquots of 2 μl cDNA were transferred into a 25 μl PCR reaction mixture containing dNTPs, MgCl₂, reaction buffer, specific primers and GoTaq Flexi DNA polymerase (Promega, Madison, WI, USA), and amplification reactions using specific primers and conditions for human genes under analysis were carried out. Sequences for the human mRNAs from GeneBank (DNASTAR Inc., Madison, WI, USA) were used to design specific primer pairs for RT-PCRs (OLIGO 4.05 software, National Biosciences Inc., Plymouth, MN, USA) (Table 1) [19]. Each RT-PCR was repeated at least three times to achieve the best reproducibility data. The levels of mRNA measured were normalized with respect to glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which was chosen as the housekeeping gene. Indeed GAPDH is one of the most stably expressed genes in human peripheral blood [29]. To our knowledge, there is no molecular evidence of variation in GAPDH mRNAlevels in autism disorders [19]. The gene expression values were expressed as arbitrary units ± SEM. Amplification of the genes of interest and GAPDH was performed simultaneously. PCR products were resolved into 2% agarose gel. A semi-quantitative analysis of mRNA levels was carried out by the Gel Doc EZ UV System (Bio-Rad, Hercules, CA, USA).

For protein extraction, BMDMs were suspended in protein lysis buffer (HEPES 25 mM; EDTA 5 mM; SDS 1%; Triton X-100 1%; PMSF 1 mM; MgCl₂ 5 mM; Protease Inhibitor Cocktail (Roche, Mannheim, Germany); Phosphatase Inhibitor Cocktail (Roche, Mannheim, Germany)). Protein concentration was determined using the method described by Bradford [30]. For CB2R detection, each sample was loaded, electrophoresed in a 15% SDS-polyacrylamide gel and electroblotted onto a nitrocellulose membrane. The membrane was blocked in 5% milk, 1X Tris-buffered saline and 0.05% Tween-20. Primary antibodies to detect CB2R (Calbiochem-Merck, Darmstadt, Germany) were used according to the manufacturer’s instructions at 1:250 dilutions [19]. The rabbit anti-CB2R antibody detects endogenous levels of the human 45 kDa fragment of CB2R protein. The antibody does not cross-react with the CB1 receptor protein and, according to the manufacturer, was validated with a recombinant protein consisting of the first 33 amino acids of human CB2R used as a positive control. For mannose receptor detection, each sample was loaded, electrophoresed in a precast gradient 4 to 12% SDS-polyacrylamide gel using Bolt® system (Life Technologies, Monza, Italy) and electroblotted onto a nitrocellulose membrane. The membrane was blocked in 5% milk, 1X Tris-buffered saline and 0.05% Tween-20. Primary antibodies to detect mannose receptor (ab64693 Abcam, Cambridge, UK) were used according to the manufacturer’s instructions at 1:1,000 dilutions. Immunoreactive signals were detected with a horseradish peroxidase-conjugated secondary antibody and reacted with an ECL system (Amersham Pharmacia, Uppsala, Sweden). To assess equal loading, protein levels were normalized with respect to the signal obtained with Coomassie Blue staining, as previously reported [31]. We used Coomassie Blue staining as equal loading control as this method overcomes the possibility that housekeeping proteins could vary in this pathology or be saturated at the levels of loading [19]. However, we confirmed the signals obtained by Coomassie Blue staining with respect to the signal obtained with anti-β-tubulin monoclonal antibodies (A2066 Sigma Chemical, St Louis, MO, USA; 1:1,000 dilution). The semi-quantitative analysis of protein levels was carried out by the ChemiDoc-It 5000, using VisionWorks Life Science Image Acquisition and Analysis software (UVP, Upland, CA, USA).

For immunocytochemical analysis, BMDMs were re-suspended at 1x10⁶ cell/mL in RPMI 1640 complete medium (Lonza, Verviers, Belgium) containing 10% FBS (EuroClone-Celbio, Milan, Italy), 2 mM L-glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin (all Lonza, Verviers, Belgium), were plated on slides with a 12-well plate and incubated at 37°C with 5% CO₂. Cells were then fixed with 4% paraformaldehyde fixative. After washing in PBS, non-specific antibody binding was inhibited by incubation for 30 minutes in blocking solution (1% BSA in PBS). Primary antibodies were diluted in PBS blocking buffer and slides were incubated overnight at 4°C in primary antibodies to human Notch (1:100; Santa Cruz Biotechnology, Santa Cruz, CA, USA) or to human Ki67 proliferation marker (1:200; Santa Cruz Biotechnology, Santa Cruz, CA, USA). Fluorescent-labeled secondary antibodies (1:1,000; Alexa Fluor 488, Molecular Probe; Invitrogen, Carlsbad, CA, USA) specific to the IgG species used as a primary antibody were used to locate the specific antigens in each slide. Cells were counterstained with bisbenzimide (Hoechst 33258; Hoechst, Frankfurt, Germany) and mounted with mounting medium (90% glycerol in PBS). Fluorescently-labeled slides were viewed with a fluorescence microscope (Leica, Wetzlar, Germany) and with a fluorescence confocal microscope (LSM 710, Zeiss, Oberkochen, Germany). Immunofluorescence images were analyzed with Leica FW4000 software (Leica, Wetzlar, Germany) and with Zen Zeiss software (Zeiss, Oberkochen, Germany). Quantification of Ki67-ir profiles was performed by an observer blind to the treatment. Cell positive profile quantification was performed on each digitized image, and the reported data are the intensity means ± SE on counterstained cells per group. Only bisbenzimide counterstained cells were considered as positive profiles so as to avoid overcounting cells.

Biomolecular data are expressed as means ± SEM. ANOVA, followed by Student-Neuman-Keuls post hoc test, was used to determine the statistical significance among groups. P < 0.05 was considered statistically significant.

As we already demonstrated [19], studying EC system gene expression mainly by RT-PCR is far more sensitive for the detection of gene expression than immunocytochemistry [9, 32]. We evaluated the GcMAF effects on NAPE-PLD (N-acyl phosphatidylethanolamine phospholipase D), a protein-coding gene [GC07M102742]. The gene codes for the enzyme which hydrolyzes N-acyl-phosphatidylethanolamines (NAPEs) to produce N-acylethanolamines (NAEs) and phosphatidic acid and, specifically, the generation of anandamide (N-arachidonoylethanolamine), the ligand of cannabinoid receptors. When compared to healthy controls, the semiquantitative analysis of BMDM-extracted mRNA levels, measured by RT-PCR amplification, showed an increase in the NAPE-PLD gene in BMDMs of autistic patients (mean ± SE of arbitrary units: 1.20 ± 0.34 versus 0.71 ± 0.11, P < 0.05, in BMDMs from autistic children and healthy subjects, respectively).

We also evaluated the gene for fatty acid amide hydrolase, (FAAH) [GC01P046860]. FAAH is a membrane associated enzyme which hydrolyzes bioactive amides, including the EC, anandamide. We observed that the mRNA levels of the FAAH enzyme gene were decreased (mean ± SE of arbitrary units: 0.40 ± 0.08 versus 1.60 ± 0.06, P < 0.05, in BMDMs from autistic children and healthy subjects, respectively); the NAPE-PLD/FAAH ratio was significantly increased (mean ± SE of arbitrary units: 3.00 ± 0.84 versus 0.44 ± 0.07 in BMDMs from autistic children compared to healthy subjects, respectively); mRNA levels of CB2R gene (mean ± SE of arbitrary units: 0.40 ± 0.01 versus 0.43 ± 0.02, P > 0.05, in BMDMs from autistic children and healthy subjects, respectively) were not changed (Figure 2).

In BMDMs of autistic children, GcMAF treatment was able to significantly increase gene expressions both NAPE-PLD (mean ± SE of arbitrary units: 1.20 ± 0.34 and 1.74 ± 0.18, P < 0.05, before and after GcMAF treatment in autistic BMDMs, respectively) and FAAH (mean ± SE of arbitrary units: 0.40 ± 0.08 versus 0.95 ± 0.04, P < 0.05, before and after GcMAF treatment in autistic BMDMs, respectively), whereas the NAPE-PLD/FAAH ratio was significantly reduced (mean ± SE of arbitrary units: 3.00 ± 0.84 versus 1.83 ± 0.19, before and after GcMAF treatment in autistic BMDMs, respectively). The mRNA levels of CB2R gene were not affected by GcMAF treatment (mean ± SE of arbitrary units: 0.40 ± 0.01 versus 0.54 ± 0.01, P > 0.05, before and after GcMAF treatment in autistic BMDMs, respectively). No changes were observed in GcMAF treated BMDMs of healthy control children with respect to untreated BMDMs, except for a slight decrease in NAPE-PLD gene expression (not affecting NAPE-PLD/FAAH ratio) (Figure 2).

GcMAF was also able to reduce the protein levels for CB2R in BMDMs from autistic children with respect to treated BMDMs from healthy controls. Since CB2Rs are G protein-coupled receptors, they show post-translational regulation [33]. We therefore determined the protein levels of CB2R by Western blot analysis.

Western blot analysis showed a strong decrease in CB2R protein levels in GcMAF treated BMDMs from autistic children as compared to untreated macrophages (mean ± SE of arbitrary units: 3.24 ± 0.54 versus 1.66 ± 0.39, P < 0.05, before and after GcMAF treatment in autistic BMDMs, respectively). Interestingly, GcMAF was also able to decrease CB2R protein levels in GcMAF treated BMDMs from healthy controls as compared to untreated BMDMs (Figure 3) (mean ± SE of arbitrary units: 5.97 ± 0.34 versus 2.39 ± 0.43, P < 0.05, before and after GcMAF treatment in healthy control BMDMs, respectively).

The difference between the unchanged CB2R mRNA levels and the decrease in CB2R protein levels in autistic GcMAF treated cells is not surprising, as we have already shown [19]. Indeed, protein levels and functions are affected by post-translational control. The levels of CB2R in the cell are strictly regulated in a multilevel system of regulation [34], and there is not a direct correlation between mRNA transcripts and protein levels [19].

In order to check cellular activation, fluorescence-based immunocytochemical analysis on macrophage cell culture was performed. In detail, Notch staining was early carried out. Notch is a protein mainly involved in stem cell maintenance, cell differentiation and cellular homeostasis regulation [35]; however, Notch signaling pathway was reported in activated pro-inflammatory macrophages and it is involved in regulating the expression of il12p40 [36]. Recently, it has been proposed a putative role of Notch signaling in autism [37]. We did not find any changes in Notch immunostaining profiles in GcMAF treated blood monocyte-derived macrophages from autistic children as compared to untreated macrophage cells, as analyzed by fluorescence microscopy (Figure 4).

As macrophages possess proliferation capacity [38], we investigated the effect of GcMAF on this cellular activity through Ki67 proliferation marker immunostaining. Interestingly, GcMAF treatment showed reduction in the immunostaining profiles of the proliferation marker Ki67 in GcMAF treated monocyte-derived macrophages from autistic children, as compared to untreated macrophage cells (Figure 4). We quantified this reduction through confocal fluorescence microscopy, showing a decrease of 23% in GcMAF treated monocyte-derived macrophages from autistic children as compared to untreated macrophage cells (mean ± SE of arbitrary units: 0.32 ± 0.04 versus 0.24 ± 0.06, P < 0.05, before and after GcMAF treatment in autistic BMDMs, respectively) (Figure 5). GcMAF was also able to reduce Ki67 immunostaining in BMDMs from healthy controls, as compared to untreated macrophage cells (mean ± SE of arbitrary units: 0.17 ± 0.01 versus 0.012 ± 0.003, P < 0.05, before and after GcMAF treatment in healthy control BMDMs, respectively) (picture not shown). It is noteworthy to consider that BMDMs from autistic children were more activated than BMDMs from healthy controls (mean ± SE of arbitrary units: 0.32 ± 0.04 versus 0.17 ± 0.01, P < 0.05, in BMDMs from autistic children and healthy subjects, respectively).

To further investigate the GcMAF effect in specific cellular activation in macrophages, we quantified the protein levels of mannose receptor by Western blot analysis. The macrophage mannose receptor (alternative name CD206) mediates the endocytosis of glycoproteins by macrophages and it is considered a specific marker for alternative activated phenotype M2 macrophages [39, 40]. Western blot analysis showed a decrease in mannose receptor protein levels in GcMAF treated BMDMs from autistic children as compared to untreated macrophages (mean ± SE of arbitrary units: 28.1 ± 0.41 versus 16.7 ± 0.94, P < 0.05, before and after Gc-MAF treatment in autistic BMDMs, respectively) (Figure 6). GcMAF was also able to decrease mannose receptor protein levels in GcMAF treated BMDMs from healthy controls as compared to untreated BMDMs (mean ± SE of arbitrary units: 26.8 ± 0.21 versus 18.9 ± 0.47, P < 0.05, before and after GcMAF treatment in healthy control BMDMs, respectively) (Figure 6).