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Nicotinic receptor activation negatively modulates pro-inflammatory cytokine production in multiple sclerosis patients

International Immunopharmacology, In Press, Corrected Proof, Available online 23 July 2015, January 1970

Abstract

Acetylcholine (ACh) and its receptors of muscarinic and nicotinic types are involved in the modulation of immune and inflammatory responses. In present work we have characterized the nicotinic receptors expression in PBMC of RR-MS patients and healthy donors (HD) and their ability to modulate pro-inflammatory cytokines. Here we report that the IL-1β e IL-17 levels are significantly increased in serum of RR-MS patients in respect to HD and that the PBMC stimulation with PHA caused a significant increase in pro-inflammatory cytokine levels both in RR-MS and HD subjects, with higher increase of protein release in RR-MS patients than in HD. The PBMC treatment with PHA plus nicotine produced a significant decrease of IL-1β e IL-17 both as transcript and as protein, confirming that the PBMC of the patients respond to the cholinergic stimulation more than PBMC of HD. By real time PCR and western blot analysis we have also demonstrated that in particular α7 receptor subtype appeared expressed at comparable levels both in RR-MS patients and HD. The PHA stimulation results to inhibit the α7 subunit expression while the nicotine causes a significant increase in α7 transcripts but only in MS patients. The data obtained highlight the role of α7 receptor subtype in the modulation of anti-inflammatory cytokines also in MS. Moreover the ability of nicotine to up-regulate the expression of α7 receptor subtype in RR-MS patients, indicates that nicotinic receptor stimulation may contribute to down-modulate the inflammation occurred in MS by a positive feedback control of its expression.

Highlights

 

  • Characterization of levels of pro-inflammatory cytokines in RR-MS patients
  • Characterization of expression of nicotinic receptors in PBMC (monocytes and lymphocytes) in MS patients compared with HD
  • Ability of nicotinic stimulation to decrease the level of pro-inflammatory cytokines in MS patients

Keywords: Nicotinic receptors, Acetylcholine, Pro-inflammatory cytokines, Multiple sclerosis.

1. Introduction

The role of the cholinergic system in the pathogenesis of human diseases and its involvement in the modulation of the inflammatory states in cancers and in several autoimmune pathologies is largely emerging [1] . Different brain degenerative disorders are characterized by cholinergic deficiency that may be responsible of the cognitive disabilities and in the same time contribute to the dis-regulation of the inflammatory processes in the brain [2] and [3].

At least the status of the knowledge of cholinergic system activity in multiple sclerosis (MS) is very poor. Several observations, obtained in autoimmune encephalomyelitis (EAE) animal models, have demonstrated that acetylcholine (ACh) reduces the inflammatory state, as indicated by a consistent reduction of CNS lymphocyte infiltrates in brain. Moreover, chronic administration of acetylcholinesterase (AChE) inhibitors ameliorates clinical symptoms [4] as well as nicotine exposure significantly delays and attenuates inflammatory and autoimmune responses. Considering these evidences, the ACh presents in inflammatory sites, probably synthesized by immune cells, might play a relevant role in modulating inflammatory states also in MS. Although the data obtained in EAE model, currently there is not any evidence on the cholinergic system activity in MS patients. Recently, our group have demonstrated that the ACh levels appear significantly decreased both in serum and in cerebrospinal fluid of Relapse Remitting-Multiple Sclerosis (RR-MS) patients compared with subjects affected by neurological non-inflammatory diseases (OND), suggesting that a dis-regulation of acetylcholine levels may influence the inflammatory state in RR-MS patients [5] .

Cholinergic receptors are expressed in immune cells and their stimulation by ACh directly produced by immune system cells, may contribute to modulate immune response in autocrine/paracrine manner [6] and [7].

The cholinergic anti-inflammatory effects are in general mediated by nicotinic receptors. In fact it has been demonstrated that nicotinic receptors inhibit, in the periphery, the proliferation of the auto-reactive T cells and alters the cytokine profile of T helper cells [8] and [9]. In CNS the nicotine exposure reduces number of dendritic cells, infiltrating monocytes and resident microglial cells and down-regulates the expression of MHC class II [10] and [11]. Moreover, in particular α7 nicotinic receptors are expressed also in microglial cells where their activation plays a role in the modulation of inflammation also in the brain, by affecting the release of pro-inflammatory agents [12] .

In RR-MS patients the decreased levels of ACh are correlated to higher levels of pro-inflammatory cytokines [5] . In order to evaluate if the effects of the cholinergic system dis-regulation on pro-inflammatory cytokine production may be counteracted by nicotinic receptor stimulation, in the present work we have evaluated the ability of the nicotinic agonist nicotine, to modulate the synthesis and production of IL-1β and IL-17 in peripheral blood mononuclear cells (PBMC) of RR-MS patients and in healthy donors (HD).

Considering the relevant role of α7 receptor subtypes in the anti-inflammatory responses [8] , we have also evaluated the expression of this nicotinic receptor subunit in RR-MS patients and HD and the ability of nicotine stimulation to modulate the expression of this receptor subtype.

The data obtained may contribute to increase the knowledge on role of nicotinic receptors in the modulation of the inflammatory state in MS patients.

2. Material and methods

2.1. Subjects

RR-MS patients followed at U.O. of Neurology Villa Serena Hospital (Citta Sant' Angelo, Pescara, Italy) were enrolled. Definition of RR-MS course was established considering clinical [13], [14], and [15] and a Kurtzke's EDSS score ≤ 5.5 [16] . The diagnosis of RR-MS was confirmed by brain magnetic resonance imaging (MRI) with gadolinium. Pregnant or lactating patients as well as patients treated with immunosuppressive drugs were excluded. Patients with known sensitivity to gadolinium chelates or for inability to undergo MRI were not considered. Moreover patients with recent vaccination were also excluded. Before inclusion in the study, all patients were screened for infectious conditions. Healthy donors (HD) from the Transfusion Blood Bank Services of Chieti, Italy, frequency matched for age were enrolled. Mean age, mean disease duration, mean EDSS are shown in Table 1 . None of the participants were smokers or vegetarians, none were known to be taking vitamin B12 or folates (including multivitamins) or prescribed drugs known to affect circulating homocysteine or nitric oxide concentrations. The study was approved by the Ethics Committee of Chieti e Pescara (Italy) and all patients and controls included, signed an informed consent.

Table 1 Characteristics of the subjects involved in this study.

Variable Control group (n = 15) RR-MS group (n = 15) p-value
Gender, n (%)     0.908 a
 Male 2 (13.3) 3 (20.0)  
 Female 13 (86.7) 12 (80.0)  
Age (years), median (range) 40 (19–76) 38 (18–59) 0.680 b
BBB impairment, median (range) 5.5 (2.8–15.2)  
Duration of disease (years), median (range) 5 (3–6)  
EDSS, median (range) 2.5 (0.0–6.0)  

a Fisher's exact test.

b Mann–Whitney U test.

BBB: Blood–brain Barrier; EDSS: Expanded Disability Status Scale.

2.2. Cell culture

Venipuncture was performed in the morning between 08:00 and 10:00. Serum was immediately stored at − 20 °C after separation and a consecutive code number was assigned to each sample to ensure that all assays were performed in a blinded condition. Peripheral blood samples were collected into endotoxin-free EDTA tubes (Vacutainer, Becton Dickinson, NJ, USA) and transported to the laboratory for processing within 1 h of collection. PBMC were isolated by Ficoll–Hypaque density gradient centrifugation and re-suspended in complete culture media consisting of RPMI 1640 medium supplemented with 10% fetal calf serum, 4 mM l-glutamine, 25 mM Hepes buffer, 50 U/ml penicillin, and 50 mg/L streptomycin. PBMC (2 × 106) were immediately placed in polypropylene culture tubes (Bibby Sterilin Italia, Italy) in a volume of 1 ml of complete culture medium and incubated at 37 °C in 95% air and 5% CO2 cell culture incubator for 24 h. Nicotine and phytohemagglutinin (PHA) were dissolved in culture medium and added to cell cultures at the final concentration of 10 μM and 20 μg/ml respectively. An equal volume of media was added to control samples to make normalized volumes. Supernatants were collected at the end of incubation and frozen at − 80 °C until assay. Cell pellets were also kept at − 80 °C until analysis. Cell viability in each culture was assessed by trypan blue dye exclusion. All reagents used were tested before use for endotoxin (< 10 pg/ml; Associates of Cape Cod, Inc., Woods Hole, MA, USA) and mycoplasma contamination (General-probe II; General-probe Inc., San Diego, CA, USA) and found negative. The same batch of serum and medium were used in all experiments. All media and reagents were purchased from Sigma (Mi, Italy).

2.3. IL-1β and IL-17 ELISA assay

Supernatants from the PBMC cultures were collected and frozen at − 80 °C until the ELISA assay was performed at a single time, so as to avoid a “batching” effect, and preliminary analyses have showed that storage of supernatants for ≈ 8 months had no effect on cytokine levels. IL-1β and IL-17 release were evaluated by commercial ELISA kits (Thermo Fisher, Rockford, IL, USA) following the manufacturer's instructions. A standard curve was generated using known amounts of recombinant cytokine and IL-1β and IL-17 levels were then calculated plotting the optical density (O.D.) of each sample against the standard curve. All samples were analyzed in duplicate. The detection limit of the assay was ≤ 5 pg/ml for IL-17 and ≤ 1 pg/ml for IL-1β. The range of analysis was between 31.25 to 2000 pg/mL for IL-17 and 10.2 to 400 pg/mL for IL-1β. The intra- and inter-assay reproducibility was > 90%. Duplicate values that differed from the mean by greater than 10% were not considered.

2.4. RNA extraction and mRNA expression analysis

Total RNA was extracted from PBMC using TRIzol reagent (Invitrogen, Life Technologies, Paisley, U.K.) and then digested with DNAse I (Ambion-Life Technologies Italia, Monza, Italy). The RNA concentration was estimated by measuring the absorbance at 260 nm (λ) using a Bio-Photometer (Eppendorf AG, Hamburg, Germany). 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. One microgram of RNA was reverse transcribed into cDNA using a High Fidelity Superscript reverse transcriptase commercially available kit (Applied Biosystems, Foster City, CA, USA), in accord with the manufacturer's instructions. PCR was performed using specific primer pairs, following reported:

  • IL1 β: forward, 5′ TGAGGATGACTTGTTCTTTGAAG-3′;
  • reverse, 5′-GTGGTGGTCGGAGATTCG-3′
  • IL17 α: forward, 5′-CAACGATGACTCCTGGGAAG-3′;
  • reverse, 5′-GGGATTGGTATTGGTATTCCGG-3′
  • 18S: forward, 5′ CTTTGCCATCACTGCCATTAAG-3′;
  • reverse, 5′-TCCATCCTTTACATCCTTCTGTC-3′

All polymerase chain reactions (PCRs) were performed in PCR-express cyclers (Hybaid, Heidelberg, Germany). To be within the exponential phase of the semi-quantitative PCR reaction, the appropriate number of cycles was pre-established for every set of samples. PCR products were separated by gel electrophoresis on 2% agarose gels and visualized by ethidium bromide staining. All gels were scanned and the normalized intensities of all reverse transcription (RT)-PCR products were determined by the BioRad gel documentation system (BioRad, Hercules, CA, USA). Mean and standard deviation were calculated for all RT-PCR experiments.

Quantitative real-time PCR was performed with GoTaq qPCR Master mix (Promega Italia, Mi, Italy) using Cycler IQTM Multicolor Real Time Detection System (Biorad, Hercules, CA, USA). A quantity of 50 ng of cDNA was used as template in each tube. Gene expression analysis was performed using the comparative cycle threshold with 18S used as housekeeping gene.

The sequences of the primers used in real-time PCR were:

α7 nicotinic receptor: Forward, 5′-TTCACCATCATCTGCACCAT-3′;
  Reverse, 5′-AAGAAACCGTAAGCAACACGA-3′
α4 nicotinic receptor: Forward, 5′-TATTCAGGACCCCATCTGCT-3′;
  Reverse, 5′-AGCTGGGTCCACTTATGCAA-3′
18S: Forward, 5′-CCAGTAAGTGCGGGTCATAAGC-3′;
  Reverse, 5′-AACGATCCAATCGGTAGTAGCG-3′

2.5. Western blotting analysis

Total protein content from PBMCs was obtained using All-in-one DNA/RNA/protein Mini-preps kit (BioBasic, Canada). The samples were resolved by SDS-PAGE, transferred onto PDVF membrane and immunostained with anti-α7 nAChR antibody (1 μg/ml; Abcam, Cambridge, UK) overnight at 4 °C. Blots were washed and then incubated for 1 h with anti-rabbit secondary antibody horseradish-peroxidase-conjugated (Promega Italia). The reaction was obtained by ECL chemiluminescence reagent (Euroclone, Milan, Italy). The bands were revealed by exposition to Molecular Imager Chemidoc XRS (Bio-Rad, CA, USA) and the optical density of the bands was quantified by ImageJ software (National Institutes of Health). GAPDH was used to normalize the intensity of the bands.

2.6. Statistical analysis

The quantitative variables were summarized as mean and standard error (SEM), qualitative variables as frequency and percentage. The results are reported separately for each of two groups (HD and RR-MS). Statistical analysis was performed using non-parametric tests when the distribution of the variables was not normal, as assessed by the Shapiro–Wilk test. Mann–Whitney U test was applied for assessing the comparison of the quantitative variables between two groups while the Wilcoxon signed-rank test was applied for comparison within groups. Fisher's exact test was applied for comparison of the qualitative variables. Two-way analysis of variance (ANOVA) for repeated measures (group × treatment conditions) was used to compare the IL-1β and IL-17 level in different treatment conditions. Contrast analysis, a priori specified, was also used to evaluate the difference between groups at each treatment conditions.

The correlations between serum IL-1β, IL-17, α7 nicotinic receptors were evaluated by non-parametric Spearman rho correlation coefficient (ρ). All statistical tests were evaluated at an alpha level of 0.05. Statistical analysis was performed using SPSS® Advanced Statistical 11.0 software (SPSS Inc., Chicago, Illinois, USA).

3. Results

3.1. IL-1β and IL-17 levels in PBMCs of HD and RR-MS patients

First of all we evaluated the circulating levels of IL-1β and IL-17 in HD (n = 15) and RR-MS patients (n = 15) (see Table 1 ). Levels of IL-1β and IL-17 in serum were significantly higher in RR-MS patients than in HD subjects ( Fig. 1 ). These data, as also previously reported [5] , are in agreement to an active pro-inflammatory state characterizing the RR-MS patients. No statistical significant correlation was observed in two groups between IL-1β and IL-17 serum levels.

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Fig. 1 Serum levels of IL-1β and IL-17 (pg/ml) in HD (n = 15) and RR-MS patients (n = 15). (p-Value reported in figure are relative to comparison between groups).

In order to investigate whether IL-1β and IL-17 were differently produced by PBMC from RR-MS and HD subjects, we compare the production of these pro-inflammatory cytokines in unstimulated and PHA-stimulated PBMC. Elisa assay of IL-1β in cell free-supernatants revealed that its spontaneous level production was higher in RR-MS patients than in HD subjects (56.3 ± 13.3 vs 12.4 ± 4.4 respectively; p = 0.011), such as IL-17 levels (24.13.3 vs 21.9 ± 4.8 respectively; p = 0.011), in agreement with levels detected in serum. Upon PHA (20 μg/ml) stimulation, release of IL-1β and IL-17 increased in HD and in RR-MS ( Fig. 2 , panel A) (p < 0.001 for both cytokines), and statistical analysis revealed higher increase of IL-1β and IL-17 in RR-MS patients compared to HD (p = 0.050 and p = 0.001, respectively). To examine the effect of nicotine on the production of IL-1β and IL-17 from PBMC of RR-MS and HD, cells were stimulated with PHA and incubate in presence of 10 μM nicotine for 24 h. In both groups nicotine decrease the levels of IL-1β (p < 0.05) and IL-17 released (p < 0.05) respect to PHA stimulated cells. The higher decrease was observed in RR-MS group for both cytokines (p = 0.050 and p = 0.017, respectively), although the IL-17 levels were more marked than IL-1β ( Fig. 2 , panel B). In order to evaluate whether nicotine may control the pro-inflammatory cytokines production only by post-transcriptional mechanism, we analyzed the expression of the cytokines transcripts in PBMC after nicotine treatment. By RT-PCR we evaluate that the spontaneous IL-17 mRNA expression was significantly higher in RR-MS patients than in HD (p = 0.005), while not statistically significant differences were observed for IL-1β expression between RR-MS and HD subjects (data not shown). When PBMC were stimulated with PHA (20 μg/ml) for 24 h, the expression of both cytokine transcripts increased in both group, with higher increases of IL-17 in HD (p < 0.001) ( Fig. 2 , panel C). Incubation of PBMC with PHA plus nicotine significantly reduced the expression of IL-1β in both RR-MS and HD (p < 0.05), with higher reduction in RR-MS patients (p = 0.068). Although the expression of IL-17 was higher in RR-MS than HD, down-regulation of IL-17 transcript by nicotine was higher in HD than in RR-MS (p = 0.048) ( Fig. 2 , panel D).

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Fig. 2 A) Percentage increase (PHA vs basal) of IL-1β and IL-17 levels detected in cell free supernatants of PBMC of HD subjects and RR-MS patients. B) Percentage decrease (PHA + Nic vs PHA) of IL-1β and IL-17 levels detected in cell free supernatants of PBMC of HD subjects and RR-MS patients. C) Percentage increase (PHA vs basal) of IL-1β and IL-17 gene expression in PBMC of HD subjects and RR-MS patients. D) Percentage decrease (PHA + Nic vs PHA) of IL-1β and IL-17 gene expression in PBMC of HD subjects and RR-MS patients. Error bars represent the standard error. (p-Value reported in figure are relative to comparison between groups).

3.2. Nicotinic receptor expression

In order to evaluate the expression of nicotinic receptors in PBMC, we have firstly performed an analysis of the transcript of α7 and α4 nicotinic receptor subunits by quantitative RT-PCR. We have decided to work with PBMC for two reasons: 1) Stimulation of PBMC has the advantage of reflecting the responses of the immune cell network in comparable manner to the in vivo condition; 2. the previous characterization of the α7 and α4 nicotinic receptor subunit in HD subjects has indicated that both monocytes and lymphocytes expressed the two nicotinic receptor subunits ( Fig. 3 ). Taking into account these considerations, we have evaluated the expression of nicotinic receptors in PBMC of the RR-MS patient group (n = 15) and HD subjects (n = 15) in which pro-inflammatory cytokine production was analyzed. As reported in Fig. 4 A and B, the α7 and α4 nicotinic receptor subunits are expressed both in RR-MS patients and in HD. Any statistically significant differences for nicotinic receptor subunits mRNA expression were observed between RR-MS patients and HD. Since α7 nicotinic receptors are the nicotinic subtypes mainly involved in the modulation of immune and inflammatory responses, we have analyzed the expression of α7 receptor protein by western blot analysis. Preliminary results, have confirmed the presence of the α7 subunit protein in both RR-MS and HD subjects ( Fig. 4 C), indicating a relationship between the α7 transcript and protein; in fact the patients presenting more transcript present also more protein.

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Fig. 3 (A) Expression of α7 and α4 nicotinic receptor transcript (RT-PCR) in monocytes and lymphocytes from HD (n = 2). (B) Western blot analysis for α7 nicotinic receptors in HD.

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Fig. 4 mRNA expression of α7 (A) and α4 (B) nicotinic receptor subunits was measured by real-time RT-PCR. 18 s was used as housekeeping gene. HD (n = 15), RR-MS patients (n = 15); (p-value reported in figure are relative to comparison between groups). C) Western blotting analysis of α7 receptor protein performed in PBMC from HD (n = 3) and RR-MS patients (n = 3). GAPDH was used as loading control. The graph below represents the mean value of the densitometric analysis of the α7/GAPDH bands in the two groups (HD and RR-MS). D) PBMCs from HD and RR-MS subjects were stimulated with PHA or PHA + nicotine. mRNA levels of α7 nAChR were measured by real-time PCR. (*p < 0.05; **p < 0.01).

To evaluate whether the α7 nicotinic receptor transcript was modulated by PHA stimulation, we have analyzed by quantitative RT-PCR the expression of α7 nicotinic receptor mRNA in PBMC maintained in culture for 24 h in presence or absence of 20 μg/ml PHA. We have observed that PHA stimulation significantly decrease mRNA levels for α7 receptor subunit both in RR-MS patients and in HD subjects (p < 0.05). Interestingly the co-treatment of PBMC with PHA and 10 μM nicotine does not modify the expression of α7 subunit mRNA in HD (PHA vs PHA + nic; p = 0.562), while induces a significant increase of α7 transcript levels in RR-MS patients (p < 0.05) ( Fig. 4 D).

4. Discussion

ACh involvement in the modulation of inflammatory states, as well as the expression of cholinergic markers in immune system cells has been reported [4], [6], and [11]. Lymphocytes and macrophages can synthesize and degrade ACh respectively by choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) enzymes [7] . Activation of the immune cells increases their ability to synthesize and to secrete ACh as indicated by the augmented ChAT expression and ACh levels [17] . Moreover T-cells and macrophages express both muscarinic (mAChRs) and nicotinic (nAChRs) receptors. Their activation produces differential effects since muscarinic receptors enhance pro-inflammatory mediators, while nicotinic receptor activation exerts anti-inflammatory responses [7] and [18]. Expression of nicotinic and muscarinic receptors changes during T-cell differentiation, suggesting that T-cell cholinergic receptor repertoire is dynamic and influenced by T-cell activation state and Th commitment [19] and [20]. In particular nicotinic receptors are expressed both in monocytes and lymphocytes, suggesting that nicotinic stimulation can modulate the immune/inflammatory responses activating different cell types ( Fig. 3 ).

The role of the cholinergic system in the pathogenesis of human diseases is underscored by the progress in our knowledge of its involvement in cancer and various autoimmune pathologies [1] and [21]. Moreover different neurodegenerative diseases are characterized by cholinergic deficiency that may be responsible for cognitive disabilities and at the same time contributes to dis-regulation of the inflammatory processes in the brain [2] and [3].

The knowledge of cholinergic system activity in MS is limited. Recently we demonstrated that ACh levels both in serum and cerebrospinal fluid (CSF) of RR-MS patients appeared significantly lower, as compared with subjects affected by other non-neuronal diseases [5] . These results may suggest that the decreased ACh levels may contribute to up-regulation of inflammation in MS. Considering the anti-inflammatory role of nicotinic receptors, in the present work we have evaluated the presence of nicotinic receptors in PBMC of RR-MS patients. Moreover the ability of nicotine to modulate the pro-inflammatory cytokines in PBMC of RR-MS patients was also evaluated.

First of all we have measured the levels of IL-1β and IL-17 in serum of RR-MS patients and HD subjects. As expected and as demonstrated in our previous study on other different RR-MS patients [5] , the levels of both pro-inflammatory cytokines resulted significantly higher in RR-MS patients than in HD. PHA stimulates lymphocytes proliferation and is also able to activate cholinergic system in immune cells [17] , for this reason we have treated the PBMC of RR-MS patients and HD with PHA and evaluated the levels of IL-1β and IL-17 released in the culture medium. The results indicate that the release of pro-inflammatory cytokines increased significantly both in RR-MS patients and HD subjects, although the increase was higher in the RR-MS patients, confirming the inflammatory state of MS patients. The analyses of the transcripts for these cytokines have also indicated an increase of mRNA levels for both cytokines.

Considering the decreased levels of ACh in RR-MS patients [5] and the ability of nicotine to negatively modulate anti-inflammatory cytokines [20] , we have also evaluated whether the nicotine stimulation was able to counteract the PHA-induced pro-inflammatory cytokines production. As shown in Fig. 2 B and D, 10 μM nicotine caused a significant decrease of the protein and transcript levels of IL-1β and IL-17 both in RR-MS and in HD, although the decrease of IL-17 mRNA in RR-MS patients was not comparable to the decrease observed for the protein. These results suggest that nicotine modulates IL-1β expression and protein production, while nicotine may control IL-17 production mainly at post-transcriptional levels. In fact it is not possible to exclude that nicotine stimulation may influence Th 17 differentiation modulating specific pool of miRNAs [21] and [22].

To understand the mechanism of PBMC from RR-MS patients of the response to nicotine stimulation, we have evaluated the expression of some nicotinic receptors subtype in RR-MS and HD subjects, demonstrating that the expression of α4 and α7 subunits mRNA were expressed at comparable levels in RR-MS patients and HD. Considering the relevant role of α7 receptors in modulation of inflammation and immune response both in periphery and in central nervous system [9] and [20], we have better characterized the levels of expression of α7 nicotinic receptor in RR-MS patients. The western blot analysis has confirmed the real time PCR results, demonstrating that also α7 protein results expressed in comparable manner in RR-MS patients and HD. However when we treated the PBMC with PHA and then with PHA + nicotine we observed that the expression of α7 transcripts appears differently modulated. In fact in both basal and PHA-stimulated condition, the α7 mRNA appeared expressed at the same levels in RR-MS and HD, with a significant decrease of α7 expression after PHA stimulation, probably as consequence of increased PBMC proliferation and pro-inflammatory cytokine production. Interestingly, PHA plus nicotine induced a strong increase of α7 expression in PBMC of RR-MS patients, while in PBMC of HD not significant difference between two experimental conditions was observed. Moreover a negative correlation between the variation of α7 subunit and the variation of pro-inflammatory cytokines after PHA + nicotine was observed in two groups, demonstrating that the decrease of the pro-inflammatory cytokines in RR-MS patients is directly correlated with the α7 receptor increase. This different response may be probably dependent on the higher levels of pro-inflammatory cytokines in RR-MS patients; in fact the increased inflammatory state in RR-MS patients may require an up-regulation of factors that contribute to balance the inflammatory responses. On the other hand it is not possible to exclude that different mechanism of regulation of α7 nicotinic receptors may occur in patients with a pro-inflammatory state such as RR-MS patients. However, interestingly, evaluating in a limited number of subjects, the ability of unselective cholinergic agonist carbachol to modulate the IL-1β production after PHA stimulation, we observed that the simultaneous stimulation of muscarinic and nicotinic receptors was not able to decrease the IL-1β release (suppl. Fig. 1 ). Although this result should be further confirmed in a large number of patients, it reinforces that idea that the selective activation of nicotinic receptors may be of strategic relevance for the treatment of inflammation.

In conclusion the present data clearly demonstrate that the nicotinic receptors are expressed in PBMC of RR-MS patients and their stimulation can negatively modulate pro-inflammatory cytokines. Nevertheless, the inability of RR-MS patients to promptly counteract the inflammation and altered immune response should not dependent on the poor activity or expression of nicotinic receptors, but by decreased levels of ACh, as previously demonstrated, [5] . Although the results obtained require further investigations, they highlight the relevant role of nAChR activation in the modulation of inflammatory responses in RR-MS and how their selective activation may contribute to balance the cholinergic dysfunction occurred in RR-MS patients [5] and [23].

The following is the supplementary data related to this article.

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Suppl. Fig. 1 Serum levels of IL-1β in RR-MS (n = 4) and HD (n = 4) in control condition, after PHA stimulation and after PHA + Charbacol (Pha + C). (*p < 0.05 PHA and PHA + C vs respective control).

Conflict of interest

The authors declare there are no conflicts of interest.

Acknowledgments

This work was supported by FISM – Fondazione Italiana Sclerosi Multipla – Cod. 2013/R/25. MDB was supported by fellowship on FISM project 2013/R/25.

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Footnotes

a Department of Medical, Oral and Biotechnological Science, University “G. d'Annunzio” Chieti-Pescara, Chieti, Italy

b Department of Biology and Biotechnologies C. Darwin, Research Center of Neurobiology Daniel Bovet, “Sapienza” University of Rome, Italy

c Villa Serena Hospital, Città Sant'Angelo, Pescara, Italy

Corresponding author at: Dipartimento di Biologia e Biotecnologie Charles Darwin, Centro di ricerca in Neuroscienze Daniel Bovet, Sapienza, Università di Roma, P.le Aldo Moro, 5, 00185 Roma, Italy.

1 These authors have equally contributed to this work.


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    Timothy Vartanian, Professor at the Brain and Mind Research Institute and the Department of Neurology, Weill Cornell Medical College, Cornell...
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    Claire S. Riley, MD is an assistant attending neurologist and assistant professor of neurology in the Neurological Institute, Columbia University,...
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    Rebecca Farber, MD is an attending neurologist and assistant professor of neurology at the Neurological Institute, Columbia University, in New...

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