Participants of the study were 62 apparent healthy older adult volunteers (29 female, age range 65–74 years of age), recruited with the use of posters, pamphlets and word of mouth at recreational clubs. Thirty-one apparent healthy young adults (16 female, age 19–28 years of age), were also recruited by word of mouth and with pamphlets at University G. d’Annunzio, CH-PE, Italy. The exclusion criteria regarding all participants were summarized in Table 1.

Briefly, subjects with the BMI < 20 and >33 k/m² were excluded (Travison et al., 2007; Mangnus et al., 2016). Subjects with unusual dietary habits (e.g., vegetarians) were also excluded (Sutliffe et al., 2015). Hematological and biochemical tests needed to be within the normal range. Negative serologies for the HIV and Hepatitis C viruses were verified. To assess the inflammatory status, all recruited subjects underwent the same laboratory blood tests: Erythrocyte Sedimentation Rate (ESR) and C-Reactive Protein (CRP) were measured as non-specific markers for inflammation and were utilized as exclusion criteria. The upper limit of the ESR is 20 mm/hr, for males, and 30 mm/hr for females (Sox and Liang, 1986; Caswell, 1993). To avoid the inclusion of subjects in an acute phase of inflammation (e.g., infection, trauma and tissue necrosis, malignancies, and autoimmune disorders), the subjects with CRP levels > 5 mg/L were also excluded (Gabay and Kashner, 1999; Ablij and Meinders, 2002; Biasucci, 2004).

Habitual smokers were excluded because this factor has already been significantly marked as a modulator of C level and strong pro-inflammatory (Mendelson et al., 2008; Messner and Bernhard, 2014). The participants were invited not to consume alcohol starting 1 week before the sample collection, in order to avoid any effects on C and the systemic inflammation levels (Sher et al., 2007; Morris et al., 2015).

Subjects who had current infections, allergies, or a present and past history of autoimmune disorders, and those on current medication (including herbal remedies or vitamins) such as anti-inflammatory, antiviral agents or immunosuppressive medication that could directly or indirectly affect the systemic inflammatory state were excluded.

Older adults reaching the “exclusion criteria” adapted from the SENIEUR protocol for demographic suitability was excluded from the study (Ligthart et al., 1984). The exclusion criteria for older adults included factors thought to influence the relationship between the cognitive function and chronic inflammation such as the presence of dementia, and depression. Volunteers were invited to a preliminary screening session based on a full medical history and examination including, anthropometric measurements, the assessment of dietary habits, tobacco and alcohol consumption, and screening for cognitive impairment using the Mini-Mental State Examination (MMSE, Folstein et al., 1975), and the Geriatric Depression Scale (GDS), that provides a screening measure of depressive symptoms in adults over 55 years (Yesavage et al., 1982). All participants had their cognitive functions assessed using a neurocognitive battery of tests as described to follow. The neurocognitive battery was designed in order to evaluate the memory and attention which are frequently affected in older persons. The subjects with depression (score ≥ 11 on the GDS) and subjects with dementia (score ≤ 24 on the MMSE) were excluded.

A total of 94 consecutive older adults (39 female) were invited to participate in the study. Ten subjects (4 female) were excluded as they were smokers; 3 male subjects were excluded because they had elevated ESR values, one female subject for an elevated CRP value. Eight male and 10 female were excluded as they were on medication. In the end, 62 consecutive subjects (of which 66% subjects contacted) were included in the study. After having signed the consent, 34 subjects were randomly assigned to an Experimental Group (EG) (16 female) and 28 to a Control Group (CG) (13 female) before the cognitive screening assessment.

A total of 43 consecutive young adults (20 female) were invited to participate in the study. Eight subjects (2 female) were excluded as they were smokers, four (2 female) were excluded as they were on medication. In the end, 31 consecutive subjects (of which 74% of subjects contacted) were included in the study.

The study procedures were described in detail to all participants, a minimum reflection time of 24 h was given before written informed consent was obtained. Participants not having the capacity to consent to research participation and non-Italian-speaking participants (because the inclusion of these participants would have meant not to be able to use standardized assessment techniques) were also excluded from the study.

The study was conducted according to the principles expressed in the Declaration of Helsinki and subsequent revisions and was approved by the Ethics Committee of G. d’Annunzio University, Chieti, Italy.

Demographic data of the subjects studied are listed in Table 2, together with BMI value and plasmatic level of CRP.

The CG included 28 subjects (13 women; age, mean ± SD, 69.0 ± 8.0 years). The EG included 34 subjects (16 women; age, mean ± SD, 70.6 ± 7.0 years).

The two groups were not significantly different at the baseline (T0) in terms of age (p = 0.428), male/female ratio (p = 0.730), BMI (p = 0.841), education (p = 0.681), and plasma CRP (p = 0.530). They did not significantly differ for any neuropsychological scores obtained at the beginning in which all scores were within the normal range. At this period all participants were considered healthy and defined as not suffering from any recent episode that could condition the basal inflammatory level and cognitive performance (i.e., infection and malignancy).

Cognitive functioning in CG and EG was measured using a comprehensive neuropsychological test battery at baseline (T0) and after 6 months (T1), 1 day before blood sample collection. For all subjects and controls, the same test administration order was used. The battery was comprised of tests with strong psychometric properties and assessed the following cognitive domains:

Global Assessment of Cognitive Function. MMSE (Folstein et al., 1975) is the use a psychometric test used to quantify the global cognitive functioning and the cognitive change in population-based longitudinal studies. MMSE consists of a series of questions with the aim of quantifying the global cognitive functioning on a 0–30 scale.

The following instruments were administered (in this fixed order), by a psychologist for the overall assessment of each participant, of which had a duration of approximately 90 min, with breaks provided, as requested by participants:

The participants of EG were submitted to MT from T0 to T1. The MT is based on the concept that repeated practice within a specific domain results in gains in both cognitive and behavioral efficiency of the targeted domain, as well as subsequently transferring improvements to untrained domains.

The intervention was conducted in several laboratories. All sessions lasted 1 h and were held twice a week, with a total of 48 laboratories (Figure 1).

During MT, two different mnemonics were used. To assess the EG visual mnemonic (Loci) and general strategies (Strategic) were used (Cavallini et al., 2003). The Loci mnemonics is based on participants that generate sequences of visuo-spatial images of familiar locations, and further produce an image that is more complex, with the initial scene and the name/object to remember. The second training was characterized by the use of disparate strategies in different situations and each of them required a special strategy (generating mental images, association or categorization of items).

Older adults are particularly sensitive to the setting and emotional conditions. For this reason, we used ecological tasks to reproduce daily activities (Baltes and Baltes, 1990).

Story recall. All participants had 5 min to study a short story composed of 22 units. They then had to write what they remembered. Performance was assessed according to the correct number of units they could remember.

Shopping list recall. A shopping list was read to all the participants and they had 5 min to remember and subsequently put it in writing. Performance was evaluated according to the correct number of products which were remembered.

Memory for faces/names. Twelve photographs, each coupled with a name, on a computer screen was presented to all participants for 30 s. The same faces, were then shown without names and participants had to remember the names. Performance was assessed according to the correct face/names associations made.

Memory for places. A map of an Italian city with the name and the position of 10 monuments was presented to all participants for 5 min. They then had to remember and to write the name and the position of the monuments on a blank map. Performance was evaluated according to the correct names and positions which were remembered (Cavallini et al., 2003).

Blood samples were collected at T0 and T1. Veni-puncture was performed in the morning between 08.00 and 10.00 am. in order to avoid the effect of diurnal variation, and peripheral blood samples were collected in 4 ml endotoxin-free Heparin tubes (Vacutainer, Becton Dickinson, NJ, United States). Tubes were kept at room temperature and transported to the laboratory for processing within 1 h of collection. The plasma was obtained by blood centrifugation as described previously and was kept frozen at -20°C (Pesce et al., 2014).

The PBMCs were isolated as described previously and used freshly for the evaluation of the WIP-1 mRNA and protein expressions and for the study of NF-kB signaling (Speranza et al., 2013). Briefly, the PBMCs were isolated by density-gradient centrifugation through Ficoll-Hypaque (Pharmacia), suspended (10⁶/ml) in RPMI 1640 medium (Sigma-Aldrich, St Louis, MO, United States), containing L-glutamine 1% and antibiotics (penicillin 100 U/ml-streptomycin 100 ug/ml) with 10% heat-inactivated fetal calf serum (Sigma, CA, United States), seeded in polypropylene tubes (Falcon, BD, Lincoln Park, NJ, United States) and incubated at 37°C in a 95% humidified 5% CO₂ cell culture incubator. Cell viability in each culture was assessed by Trypan blue die exclusion. All solutions were prepared using pyrogen-free water and sterile polypropylene plastic-ware and were free of detectable LPS (<0.1 EU/ml), as determined by the Limulus amoebocyte lysate assay (sensitivity limit 12 pg/ml; Associates of Cape Cod, Falmouth, MA, United States). All reagents used were tested before use for Mycoplasma contamination (minimum detection level 0.1 μg/ml) (Whittaker Bioproducts, Walkersville, MD, United States) and found negative. The same batches of serum and medium were used in all experiments. After 24 h incubation, samples were centrifuged at 400 × g for 10 min at room temperature, supernatants were collected and stored at -80°C pending assay (Pesce et al., 2013). The PBMCs yield per mL of blood was approximately 1 × 10⁶ cells.

Total RNA was extracted from PBMCs of all subjects using the TRIzol reagent (Invitrogen, Life Technologies, Paisley, United Kingdom) according to the manufacturer’s protocol. The RNA concentration and purity were determined by measuring absorbencies at 260 and 280 nm; a 260:280 ratio of 1.9 being considered acceptable for analysis. The RNA concentration was estimated by measuring the absorbance at 260 nm using a Bio-Photometer (Eppendorf AG, Hamburg, Germany), and RNA samples were kept frozen at –80°C until use. Purified RNA was electrophoresed on a 1% agarose gel to assess the integrity of the purified RNA. The RNA yield was 8.2 ± 2.7 μg/10⁶ cells (mean ± SD).

Before Reverse Transcription, residual genomic DNA was removed from Total RNA using DNaseI (Thermo Scientific). The reaction mix was as follows: 1 μg of Total RNA, 1 μl of 10x Reaction Buffer, 1 U of DNaseI and water up to 10 μl. The mix was incubated at 37°C for 30 min and stopped by adding 1 μl of EDTA 50 mM and incubating at 65°C for 10 min. A High Capacity cDNA Reverse Transcription kit (Applied Biosystem) was used for reverse transcription of mRNAs. The reaction mix was prepared as follows: 2 μg of total RNA, 2 μl of 10x RT Buffer, 0.8 μl of dNTP, 2 μl of 10x Random Hexamer, 50 U of MultiScribe Reverse Transcriptase, 1 μl of RNase Inhibitor and water up to 20 μl. cDNA synthesis was performed in a thermal block cycler (Bio-rad) following these steps: priming at 25°C for 10 min, transcription at 37°C for 120 min and enzyme inactivation at 85°C for 5 min. All cDNA samples were stored at -20°C pending qPCR analysis.

A qPCR assay was carried out in an Eppendorf Mastercycler EP Realplex (Eppendorf AG). Briefly, preliminary PCR reactions were run to optimize the concentration and ratio of each primer set. For all the cDNA templates 2 μL was used in a 20 μL qPCR amplification system of the SYBR Green Real Master Mix Kit. Primers for human WIP-1 gene and GAPDH as reference gene were designed using GeneWorks software (IntelliGenetix, Inc., Mountain View, CA, United States).

The primer pairs used were as follows: WIP-1 (NM003620) forward-5′-TTTTTATATTGTTTTTAGGTTATT-3′ and reverse-5′-ATCTATATAAACTTTTAACTCAATC-3′; GAPDH (NM002046) forward-5′-ACCACCATGGAGAAGGC-3′ and reverse-5′-GGCATGGACTGTGGTCATGA-3′. The amplification procedures and data computation followed were similar to those described above (Patruno et al., 2012). In this study stability of GAPDH did not vary in the cells whatever the conditions. Thus, the relative expression of WIP-1 was normalized to GAPDH using the ΔC_q method [relative expression = 2^-ΔCq, where ΔC_q = C_q(WIP1) – C_q(GAPDH)].

Peripheral Blood Mononuclear Cells were washed once in cold phosphate-buffered saline (PBS; 0.5 mol/L sodium phosphate, pH 7.5), harvested by gentle scraping, and used to prepare total or nuclear protein extracts. Total protein extracts were prepared as described previously by treating cells with lysis buffer for 30 min at 4°C (Felaco et al., 2000). Nuclear extracts were prepared as previously described (Speranza et al., 2009). The protein concentrations of the extracts were determined using the Bradford method (Bio-Rad protein assay, Hercules, CA, United States).

For Western blotting (WB) analysis, 50 μg of protein per lane was separated on a 4-12% NuPAGE gradient gel (Gibco Invitrogen), electrotransferred onto a nitrocellulose membrane and blocked with 10% skimmed milk in PBS containing 0.1% Tween-20 (Franceschelli et al., 2011). Blots were probed and incubated overnight at 4°C with polyclonal rabbit IgG anti-WIP1, and pNF-κB (p-p65^Ser536) (Santa Cruz Biotechnology, Santa Cruz, CA, United States), all at 0.2 μg mL^-1 in Tris-buffered saline (TBS)/0.1% Tween-20. A mouse antihuman monoclonal antibody recognizing human β-actin (A5441; Sigma–Aldrich) was used as the control in all experiments. A rabbit antihuman antibody for β-tubulin was used as the control in experiments with nuclear extract proteins (Santa Cruz Biotechnology, Santa Cruz, CA, United States). Three microgram of human recombinant the WIP-1 protein was used as positive control (Lifespan Biosciences, Seattle, WA, United States). Fifty microgram of nuclear extract proteins from PBMCs previously treated with the LPS (10 μg/mL) and INFγ (20 ng/mL) was used as positive control for the detection of p-p65^Ser536 (Pesce et al., 2014).

Blots were then washed and incubated for 1 h with goat antirabbit-horseradish peroxidase (Pierce Biotechnology, Rockford, IL, United States) diluted 1:10000 in TBS/0.1% Tween-20. Immunoblot signals were developed using Super Signal Ultra chemiluminescence detection reagents (Pierce Biotechnology). The blot images were analyzed by a gel analysis software package (Gel Doc 1000; Bio-Rad, Milan, Italy). Data are expressed as the mean ± SD intensity of optical density.

In order to detect the role of the WIP-1 in regulating NF-κB signaling, the PBMCs were isolated and cultured during the study, as above described. The cultured cells were pre-treated or not treated with the WIP-1 inhibitor GSK2830371 for 1 h, and later the cells were stimulated with the LPS and INFγ. The LPS was used at 10 μg/mL, IFNγ at 20 ng/mL, and GSK2830371 at 10 μM (Sigma–Aldrich, St Louis, MO, United States) (Pesce et al., 2014).

Plasma was obtained with blood centrifugation at 1500 × g for 7 min and kept frozen at -20°C from all participants. The amount of plasma circulating IL-1β, IL-18, IL-6, C, and the CRP was assayed with the use of specific ELISA development systems (Pierce, Rockford, IL, United States) in all individuals. The amount of IL-1β, IL-18, IL-6 from the culture supernatants of the PBMCs was assayed with the use of specific ELISA development systems (Pierce, Rockford, IL, United States) in older adults after 6 months of being on a MT program. The experiments were performed according to the manufacturer’s instructions.

Plates were scanned using a specialized Charge Coupled Device cooled tool. The integrated density values of the spots of known standards were used to generate a standard curve. Density values for unknown samples were determined by using the standard curve for each subject in order to calculate the real values in pg/mL. All steps were performed in duplicate and at room temperature. The IL-1β assay sensitivity was ≤1.0 pg/ml, for IL-18 it was ≤12.5 pg/mL, for IL-6 it was ≤1 pg/ml, for C it was ≤56.7 pg/mL, and for CRP ≤ 10.0 pg/ml. The values below the assay sensitivity have been excluded. The intra- and inter-assay reproducibility was >90%. Duplicate values that differed from the mean by more than 10% were considered suspect and were repeated.

The normal distributions of data were tested by the Skewness and Kurtosis measurement. All the variables considered, resulted distributed in a normal manner. The results were reported separately for each group: young adults, total older adults, older adults Control Group (CG) and older adults Experimental Group (EG). The Unpaired Student t-test was used to assess significant differences between the young group and total older adults. The unpaired Student t-test and Chi-squared test were applied to evaluate significant differences between the CG and the EG. The same statistical test was applied to evaluate the differences for biological variables. Pearson’s (r) co-efficient correlation was applied to assess the strength of the relationship between the basal levels of age, BMI, WIP-1, C, IL-1β, IL-18, IL-6, CRP and of the MMSE scores in older adult subjects. In order to assess the multi-collinearity of the variables, the diagnostic factors, Tolerance and Variance Inflation Factor (VIF) were considered. Accordingly, the MMSE scores were used as an dependent variable in a multiple regression analysis, whereas Age, as well as C, WIP-1, IL-1β, IL-18, and IL-6 were used as independent ones. The differences pre- and post-cognitive training were analyzed by the Paired samples t-test. Statistical analyses were performed using the SPSS 19.0 statistic (SPSS Inc., Chicago, IL, United States) for Windows (IBM). Results are described as means ± SD for each assessment performed in triplicate. All statistical tests were two-tailed and were evaluated at an alpha level of 0.05.

Table 3 shows the bivariate correlations at T0 between Age, BMI, the plasmatic level of C, CRP, the pro-inflammatory cytokines IL-1β, IL-18, IL-6, the mRNA expression of the WIP-1 from ex vivo PBMCs, and MMSE score of all older adults (n = 62).

The Age parameter correlated significantly as well as negatively with the WIP-1 mRNA expression (r = -0.368, p < 0.01), whereas it correlated positively with the IL-1β (r = 0.320, p < 0.05), the IL-18 (r = 0.371, p < 0.01), and the IL-6 (r = 0.327, p < 0.05). The BMI did not correlate with any variable considered, except for the IL-6 (r = 0.254, p < 0.05).

The MMSE score correlated positively to the WIP-1 (r = 0.322; p < 0.01), and negatively to C (r = 0.340; p < 0.01) and to all the pro-inflammatory cytokines analyzed. Once again, the plasmatic level of C at baseline was significantly as well as negatively associated to the WIP-1 mRNA expression, and positively associated to IL-1β, IL-18, and IL-6. The cytokines were strongly and positively associated with each other. None of the variables considered, correlated significantly with the CRP.

The correlation values obtained at T0 were not confirmed at T1 for Age and the MMSE score, Age and C, and Age as well as immune markers evaluated. The same significant loss also occurred when the correlation were analyzed for the EG at T1.

Table 4 presents the multiple linear regression analysis results. The MMSE score was considered as a dependent variable. The variables which show significant correlation vs. MMSE score were considered as potential independent variables for the analysis. In order to assess multi-collinearity, the diagnostic factors, Tolerance and VIF were evaluated. As a consequence, none of the variables were excluded. The analysis was carried out using Age, C, WIP-1, IL-1β, IL-18, and IL-6 as independent variables that were added stepwise. The model including Age, C, and amount of the WIP-1 mRNA in ex vivo PBMCs resulted as the most significant, accounting for 13.4% of the MMSE variance [F_(1,60)= 4.105; p = 0.010] (Supplementary Table 1).

Cognitive functioning was directly assessed using a neuropsychological test battery. In 62 apparent older adults, the global cognitive function was measured by the MMSE at T0, and after 6 months of the MT program (T1). At the same time, verbal memory was assessed to measure the ability to retain (Short Term Memory, STM) and recall (Long Term Memory, LTM) verbal information using the RAVL Test. Visual memory was evaluated through the ROCF Test. Attention was assessed by the TMT A, the TMT B and the test of Attentive Matrices.

The results reported in Table 4 show that the MMSE score significantly increased at T1 in EG when compared to their baseline score (28.3 ± 1.6 vs. 26.1 ± 1.7, p < 0.01). We also observed that the MMSE score of EG at T1 was significantly higher than those of CG (28.3 ± 1.6 vs. 26.4 ± 1.9, p < 0.01).

With close attention, we observed significant differences pre- and post- the MT program for attentive test targets. The medium number of targets attempted was augmented in the EG over time (48.8 ± 6.2 vs. 55.7 ± 5.1, p < 0.05), and between the EG and the CG at T1 (55.7 ± 5.1 vs. 50.2 ± 5.0, p < 0.05). The two groups did not differ significantly in their TMT A (p = 0.611) or TMT B (p = 0.684) performance concerning completion time at T0. However, the completion time was different between the two groups at the T1, in which it was lower in the EG both for the TMT A (35.1 ± 8.1 vs. 42.2 ± 11.5, p > 0.05), that the TMT B (66.2 ± 20.7 vs. 78.9 ± 27.7, p > 0.05), but it did not reach the significance. The EG did not show significant variation of scores when compared to themselves at T0 for the TMT A (35.1 ± 8.1 vs. 41.8 ± 10.5, p > 0.05), and B (66.2 ± 20.7 vs. 79.3 ± 28.4, p > 0.05). We again found that the RAVLT score followed the same trend over time observed in the EG for global cognitive assessment. In detail, the STM score significantly increased from 2.5 ± 1.2 to 3.4 ± 1.0 (p < 0.01), and the LTM significantly improved from 2.3 ± 1.2 to 3.3 ± 1.2 (p < 0.01). We did not observe any variation over time in the CG. Nonetheless, the EG showed a significantly higher RAVLT score for the STM at T1 when compared to CG, whereas the groups did not differ significantly for STM and for any neuropsychological measure at the baseline (Table 5).

The data obtained for Visual memory through the ROCF Test, suggested that the MT program also significantly improved this function in subjects of the EG over time. We did not observe any difference between groups at the T1 (Table 5).

The expression of WIP-1 has been shown to decrease during aging in murine model (Wong et al., 2009). In order to confirm on human the differentiated expression of the WIP-1 during age, qPCR, and WB experiments were performed to detect the mRNA and protein level, respectively, on freshly isolated PBMCs from healthy young and older adults. The results showed in Figure 2, suggest that older adults were characterized by significantly lower levels of the WIP-1 expression.

We determined the expression of the WIP-1 mRNA transcripts in freshly isolated PBMCs from selected healthy older adults by the qPCR at the baseline and after 6 months of the MT program in the CG and in the EG. Figure 3A shows the results from subjects of both groups. Compared with the CG, the PBMCs obtained from the EG were found to contain much higher levels of the mRNA for the WIP-1 at T1, determined as its expression relative to the reference gene GAPDH obtained from all subjects at each time. We did not observe any difference between groups at T0. Nevertheless, the WIP-1 mRNA expression was significantly higher in the PBMCs of the EG at T1 when compared to their value at the baseline. In detail, the detected amount of the WIP-1 mRNA in PBMCs from subjects of the EG at T1, is increased by more than 40% when compared to the EG at T0, and the CG at T1.

The levels of the WIP-1 protein in PBMCs were measured by the WB. In accordance with the mRNA levels, the PBMCs from the CG had significantly lower levels of the WIP-1 protein versus the EG after the MT program. The expression of the WIP-1 protein was also significantly increased within the EG over time. According to those reported for the mRNA, the protein level did not significantly differ between groups at T0 (Figure 3B).

In order to highlight the effects of the MT on the C level and the low-grade of inflammation which characterizes healthy older adults, the ELISA test was used to detect the plasmatic level of C as well as determining the non-specific marker for inflammation CRP in both groups at baseline and after the MT. Significant variation for CRP was not observed. As reported in Table 6, our data suggest that the MT significantly modulates the C plasma level. That level had significantly decreased in the EG at T1 when compared vs. CG at T1 and vs. EG at T0. In order to investigate any involvement of the MT on cytokine production in healthy older adults, the plasma levels of the IL-1β, the IL-18, and the IL-6 were evaluated. Table 5 also shows the concentrations of cytokines detected in plasma of both groups at T0 and T1. The levels of the IL-1β, the IL-18 and the IL-6 detected at T0 did not significantly differ between the CG and the EG. On the other hand, the submission to the MT program significantly decreased the plasmatic level of all cytokines analyzed at T1 in the EG. Consequently, the subjects of this group showed a significant decrease over time.

The role of the NF-κB in the expression of pro-inflammatory cytokines has been broadly reviewed (Chung et al., 2011). The post-translational modification of the NF-κB (p65) affects its transcriptional activity; in particular, several reports have documented negative regulation of the NF-κB by the WIP-1, also through the direct dephosphorylation of Ser-536 of p65 (Chew et al., 2009). We next further performed WB studies to determine the phosphorylation of p65 at Ser-536, using specific phospho-p65 antibody on nuclear protein extracts. In the EG, we observed a down-regulation of nuclear translocation of the p-p65^Ser536 as compared to the CG group at T1. As a result, the phosphorylated p65 in the EG showed low levels at T1 when compared to their own levels at T0 (Figure 4A).

In order to better investigate the role of this phosphatase in regulating the NF-κB nuclear translocation, the PBMCs from the CG and the EG at T0 and T1 were cultured and treated with a selective inhibitor of the WIP-1 (GSK 2830371) and/or LPS + INFγ, while nuclear protein extracts were detected by an antibody specific for the p-p65. As shown in Figure 4B, treatment with the LPS + INFγ increased the p-p65^Ser536 expression in nuclei. This response was significantly elevated in the PBMCs from the CG as compared to the EG at T1. It should also be noted that the WIP-1 inhibition resulted in a significant increase in the constitutive and the LPS + INFγ-induced p65 phosphorylation and nuclear translocation in both groups. These findings suggested that the WIP-1 expression is significantly involved in the negative regulation of the NF-κB activity.

Furthermore, the inhibition of the activity of the WIP-1 resulted in a significantly higher constitutive and induced the release of the IL-1β, of the IL-18 and of the IL-6 in the medium of cultured cells (Table 7). We did not observe any significant differences between cells treated with an inhibitor and a medium alone, either in the p-p65^Ser536 nuclear levels (Figure 4B) or in cytokines release (Table 7).

Altogether, these findings suggest a role for the WIP-1 activity in mediating the NF-κB nuclear levels and pro-inflammatory cytokines released in older adults. Furthermore, the submission to 6 months of the MT significantly restored the WIP-1 inhibitory function.