Journal of Neuroimmunology 277 (2014) 186–188

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Journal of Neuroimmunology

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Short communication

Gene expression analysis of histamine receptors in peripheral bloodmononuclear cells from individuals with clinically-isolated syndromeand different stages of multiple sclerosis

Massimo Costanza a, Marco Di Dario a,b, Lawrence Steinman c, Cinthia Farina a,b, Rosetta Pedotti a,⁎,1

a Neuroimmunology and Neuromuscular Disorder Unit, Neurological Institute Foundation IRCCS C. Besta, Via Amadeo 42, 20133 Milan, Italyb Institute of Experimental Neurology (INSpe), San Raffaele Scientific Institute, Via Olgettina 60, 20132 Milan, Italyc Departments of Pediatrics, Neurology and Neurological Sciences, Stanford University, Stanford, CA, USA

Abbreviations:CIS, clinically isolated syndrome; CNS, cperimental autoimmune encephalomyelitis;HC, healthy co1; H2R, histamine receptor 2; H3R, histamine receptor 3;multiple sclerosis; PBMC, peripheral blood mononuprogressive multiple sclerosis; RR-MS, relapsing-remitsecondary-progressivemultiple sclerosis; Th, T helper; Tre⁎ Corresponding author. Tel.: +39 02 23944654; fax: +

E-mail address: rpedotti@istituto-besta.it (R. Pedotti).1 Present address: Departments of Neurology and N

University, Stanford, CA, USA.

http://dx.doi.org/10.1016/j.jneuroim.2014.09.0180165-5728/© 2014 Elsevier B.V. All rights reserved.

a b s t r a c t

a r t i c l e i n f o

Article history:Received 26 June 2014Received in revised form 18 September 2014Accepted 22 September 2014

Keywords:Multiple sclerosisHistamine receptorsPeripheral blood mononuclear cells

Along with their established role in allergic reactions, histamine and its receptors have been implicated in thepathology of multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis. Inthis study we analyzed the gene expression of histamine receptor 1 (HRH1), HRH2 and HRH4 in peripheralblood mononuclear cells derived from patients with clinically isolated syndrome (CIS), relapsing-remitting(RR) MS, secondary-progressive (SP) MS, primary-progressive (PP) MS, and healthy controls (HC). We foundthat HRH1 transcript was significantly down-modulated in SP-MS compared with HC, and HRH4was increasedin this group compared to HC, CIS and RR-MS. No other differences in the expression of histamine receptorswere observed between HC, CIS and other clinical forms of definite MS.

© 2014 Elsevier B.V. All rights reserved.

1. Introduction

Multiple sclerosis (MS) is an immune-mediated demyelinatingdisorder of the central nervous system (CNS), affecting more than 2.5million peopleworldwide (Steinman, 2014).MS is generally considereda CNS autoimmune disorder in which myelin-specific T helper (Th) 1and Th17 cells direct an inflammatory response against the myelinsheath, leading to myelin damage and axonal injury (Weiner, 2009).Classically envisioned as a key mediator in allergic reactions, histaminehas been suggested to play a role in CNS autoimmune responses occur-ring in MS and experimental autoimmune encephalomyelitis (EAE),animal model for this disease (Ma et al., 2002; Pedotti et al., 2003).Histamine acts through four G-protein-coupled receptors (Thurmondet al., 2008), histamine receptor 1 (H1R), H2R, H3R, and H4R. HRH1transcript was found overexpressed in chronic plaques of MS patients(Lock et al., 2002). Mononuclear cells infiltrating the CNS in EAE mice

entral nervous system; EAE, ex-ntrols;H1R, histamine receptorH4R, histamine receptor 4; MS,clear cells; PP-MS, primary-ting multiple sclerosis; SP-MS,gs, regulatory T cells.39 02 23944708.

eurological Sciences, Stanford

express H1R and H2R (Pedotti et al., 2003), and mRNA levels for thesereceptors are significantly modulated in peripheral T cells in differentstages of EAE (Lapilla et al., 2011). Studies performedwith histamine re-ceptors or histidine-decarboxylase knockout mice have uncovered along unappreciated role for the histaminergic network in the regulationof EAE development, and suggest that histamine can play pathogenic orprotective roles in EAE depending on the receptors engaged (reviewedin Panula and Nuutinen, 2013). In order to determine whether thesefindings could be relevant for the human disease, in the present studywe sought to investigate the gene expression of histamine receptors inperipheral immune cells of subjects with clinically isolated syndrome(CIS) and different forms of MS.

2. Material and methods

2.1. Inclusion criteria for patients and healthy controls

The study was approved by the Institutional Ethical Committee andall individuals gave signed informed consent. All MS patients fulfilledthe McDonald criteria (Polman et al., 2011) and consisted ofrelapsing-remitting (RR-MS; n = 15), secondary-progressive (SP-MS;n = 9) and primary-progressive (PP-MS; n = 9) subjects. Patientswith a diagnosis of CIS (n = 16) were also enrolled in the study andblood sampling was performed between 30 and 90 days after the firstclinical attack. All patients were clinically stable, had not started anyimmunomodulatory therapy until blood sampling and had no otherchronic or acute inflammatory disorders. Healthy subjects, who had

HCCIS

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Fig. 1. PBMCwere isolated from HC (n= 17), CIS (n= 16), RR-MS (n= 15), SP-MS (n=9) and PP-MS (n = 9) subjects. To reduce variability, all patients had never received im-mune-modulating therapies, and were free from clinical attacks or drug treatment for atleast 4 weeks before sampling. Transcripts for HRH1 (A), HRH2 (B) and HRH4 (C) werequantified by real-time PCR as described in Materials and methods. Data are representedas median (range). *, p b 0.05; **, p = 0.01 by the Mann–Whitney U test.

187M. Costanza et al. / Journal of Neuroimmunology 277 (2014) 186–188

no acute or chronic inflammatory diseases or autoimmune disorders,were included as controls (HC; n = 17). An overview on the clinicaland demographic features of the subjects enrolled in this study issummarized in Table 1.

2.2. PBMC isolation

Peripheral blood mononuclear cells (PBMC) were isolated fromanticoagulated whole blood as previously described (Menon et al.,2012).

2.3. Gene expression analysis

Total RNA was isolated from PBMC with TriReagent (Ambion)according to the manufacturer's guidelines. Total RNA was treatedwith DNase (DNA-free kit, Ambion) and reverse transcribed to cDNAby using random hexamers and Superscript III® Reverse Transcriptasekit (Invitrogen), following the manufacturer's instructions. Real TimePCR was performed on 7500 Fast Real-time PCR system (AppliedBiosystems) by using Taqman® Fast Universal PCR Master Mix andTaqMan® gene expression assays for HRH1 (Hs00185542_m1), HRH2(Hs00254569_s1) and HRH4 (Hs00222094_m1), normalized oncyclophilin A (PPIA) (Hs99999904_m1) according to themanufacturer'sprotocol (Applied Biosystems). Transcript levels of target genes werequantified by the comparative threshold cycle method and areexpressed as the percentage of the housekeeping gene PPIA ± SEM.

2.4. Statistical analysis

The Mann–Whitney U test was used to compare results betweengroups. In all tests, *p b 0.05 was considered statistically significant.

3. Results

We performed a quantitative PCR study of HRH1, HRH2 and HRH4genes, known to be expressed in immune cells (Thurmond et al.,2008). HRH3 has not been included in our analysis, as its expression ismostly restricted to the CNS, where it regulates the release of neuro-transmitters (Panula and Nuutinen, 2013). We evaluated transcriptlevels of HRH1, HRH2 and HRH4 in PBMC isolated from HC (n = 17),CIS (n= 16), RR-MS (n= 15), SP-MS (n= 9), and PP-MS (n= 9) sub-jects. H1R, H2R and H4R were all expressed at the mRNA level in PBMCof all groups, and HRH2 transcripts were always more abundant (meanrelative expression was 5.018 ± 0.675 in HC) than those for HRH4(mean relative expression was 0.237 ± 0.036 in HC) and HRH1 (meanrelative expression was 0.022 ± 0.004 in HC) (Fig. 1). Gene expressionanalysis of HRH1 revealed a significantly lower abundance of thistranscript in SP-MS patients in comparison to HC (mean relativeexpression of HRH1 was 0.022 ± 0.004 in HC vs. 0.009 ± 0.003 inSP-MS; p = 0.04) (Fig. 1A). Moreover, HRH1 was undetectable in 44%(4/9) of SP-MS versus 12% of HC (2/17), 25% (4/16) of CIS, 20% (3/15)of RR-MS, and 11% (1/9) of PP-MS patients.We did notfind a substantial

Table 1Clinical and demographic features of the subjects enrolled in the study.

N Age(years)a

EDSSa Disease duration(years)a

Relapseratea

HC 17 34.4 ± 9.96 – – –

CIS 16 36.9 ± 10.53 1.0 ± 0.80 – –

RR-MS 15 38.3 ± 9.25 2.0 ± 1.42 6 ± 4.9 1.0 ± 1.98SP-MS 9 55.8 ± 10.24 6.0 ± 2.56 24 ± 4.5 –

PP-MS 9 55.6 ± 8.16 5.0 ± 2.50 9 ± 7.3 –

a Data are shown as mean ± SD. EDSS, Expanded Disability Status Scale; HC, healthycontrols; CIS, clinically isolated syndrome; RR-MS, relapsing-remitting multiple sclerosis;SP-MS, secondary-progressive multiple sclerosis; PP-MS, primary-progressive multiplesclerosis.

difference in HRH1 expression between HC, CIS (p = 0.46), RR-MS(p = 0.46) and PP-MS (p = 0.37) subjects (Fig. 1A). Similar amountsof HRH2 transcripts were detected in all groups (CIS, p = 0.60; RR-MS, p =0.62; SP-MS, p = 0.46; PP-MS, p = 0.40 compared with HC)(Fig. 1B). HRH4 expression was mildly but significantly increased inSP-MS patients in comparison to HC (mean relative expression ofHRH4was 0.237 ± 0.036 in HC vs. 0.334 ± 0.041 in SPMS; p = 0.03).SP-MS patients displayed significantly higher levels of HRH4 mRNAalso if compared to CIS (mean relative expression was 0.191 ± 0.027in CIS; p = 0.01) and RR-MS (mean relative expression was0.214 ± 0.034 in RR-MS; p = 0.048). No substantial changes inHRH4 expression were detected between HC and CIS (p = 0.49),RR-MS (p = 0.62) or PP-MS (p = 0.20) subjects (Fig. 1C), oramong any of these disease groups.

188 M. Costanza et al. / Journal of Neuroimmunology 277 (2014) 186–188

4. Discussion

Even though histamine is classically associated with Th2-driven im-mune responses and allergy, numerous studies have pointed out thatthis mediator is endowed with a broad spectrum of immune-modulating functions. Histamine has been shown to increase humanTh1 responses by activating H1R and to decrease both Th1 and Th2responses through H2R (Jutel et al., 2001). In MS, HRH1 mRNA wasupregulated in brain lesions (Lock et al., 2002), and histamine levelswere found increased in the cerebrospinal fluid of patients (Tuomistoet al., 1983; Kallweit et al., 2013). In this study we evaluated transcriptlevels of histamine receptors in the peripheral immune cell compart-ment extending the analysis from subjects suffering a first clinical CNSdemyelinating episode (CIS) to patients affected by several forms ofdefinite MS. In PBMC, T cells, B cells, monocytes, dendritic cells and NKcells have all been reported to express H1R, H2R and H4R (Damajet al., 2007; Jutel et al., 2009). To limit variations in the gene expression,all patients included in thisworkwere clinically stable and none of themhad started any immunomodulatory therapy until sampling. Our studyshows that, among HC, CIS and the different forms of MS, only SP-MSpatients exhibit a differential expression of histamine receptors genes,displaying significantly reduced amount of HRH1 transcripts comparedto HC and increased HRH4 transcripts. No other difference in histaminereceptor expression was observed between HC, CIS, RR-MS and PP-MS.HRH1 gene is upregulated in human Th1-polarized cells in comparisonwith Th2 cells, which conversely express elevated levels of HRH2mRNA (Jutel et al., 2001). In line with these findings, higher expressionof Hrh1 has been found in murine Th1-polarized cell lines activatedagainst myelin compared with Th2 cell lines, which have instead in-creased expression of Hrh2 (Pedotti et al., 2003). Moreover, T cells iso-lated from H1R-deficient mice display a Th2-biased phenotype,characterized by reduced production of IFN-γ and enhanced secretionof IL-4 and IL-13 (Jutel et al., 2001). HRH4 transcript is upregulated inhuman Th2-polarized cells in comparison to naïve or Th1-cells(Gutzmer et al., 2009). Also, regulatory T cells (Tregs) in mice havebeen reported to express higher levels of H4R than effector T cells, andEAE studies conducted on H4R-deficient mice suggest that this receptorcontrols frequency and suppressive function of Tregs (Del Rio et al.,2012). The concomitant reduction of HRH1 gene expression and theincrease of HRH4 in SP-MS patients might reflect a shift toward a Th2phenotype in this advanced stage of disease, when acute inflammatoryevents are less frequent and innate rather than adaptive immunity isbelieved to perpetuate the immune-mediated injury into the CNS(Weiner, 2009). In line with this possible interpretation, we observeda higher abundance of HRH4 transcripts in SP-MS not only comparedto HC, but also compared with stages of the disease characterized byprominent inflammation, such as CIS and RR-MS. In addition, becausethe effects of histamine on immune cells depend on the receptor/sengaged, these results would also suggest that in PBMC of SP-MSpatients histamine may promote a Th2 shift and modulate Tregfunctions.

One limitation of this study is the rather small size of the samplegroup. This study should be considered exploratory. However, underthese conditions we were able to find in SP-MS patients reducedexpression of HRH1 in comparison to HC and increased HRH4 expres-sion. An extension of this analysis on larger groups of MS patients, aswell as a longitudinal study on patients progressing from CIS and RR-MS to SP-MS, might help clarify whether the expression of HRH1and HRH4 can be used as biomarker associated with secondary-progressive MS.

Statement of conflicts of interest

The authors declare no conflicts of interest.

Acknowledgments

We thank all the patients and healthy volunteerswho donated bloodfor this study, andDr. Loredana LaMantia, Dr. ClaraMilanese and theMScenter of the Besta Neurological Institute for referring the patients. Thisinvestigationwas supported by grants from Fondazione Italiana SclerosiMultipla (FISM-AISM) (FISM cod. 2012/R/13 to R.P.), and ItalianMinistry of Health (GR-2009-1607206 to R.P. and C.F.).

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