IFN-β therapy modulates B-cell and monocyte crosstalk via TLR7 in multiple sclerosis patients

Eur. J. Immunol. 2013. 43: 1963–1972 Clinical immunologyDOI: 10.1002/eji.201243212 1963

IFN-β therapy modulates B-cell and monocyte crosstalkvia TLR7 in multiple sclerosis patients

Elena Giacomini∗1, Martina Severa∗1, Fabiana Rizzo1, Rosella Mechelli2,Viviana Annibali2, Giovanni Ristori2, Valeria Riccieri3, Marco Salvetti2

and Eliana Marina Coccia1

1 Department of Infectious, Parasitic and Immune-mediated Diseases, Istituto Superiore diSanita, Rome, Italy

2 Centre for Experimental Neurological Therapies (CENTERS), S. Andrea Hospital-site,Sapienza University, Rome, Italy

3 Internal Medicine and Medical Specialities Department, Sapienza University, Rome, Italy

The implication of B lymphocytes in the immunopathology of multiple sclerosis (MS) isincreasingly recognized. Here we investigated the response of B cells to IFN-β, a first-linetherapy for relapsing-remitting MS patients, upon stimulation with TLR. IFN-β restoredthe frequency of TLR7-induced IgM and IgG-secreting cells in MS patients to the levelsfound in healthy donors, showing a specific deficiency in the TLR7 pathway. How-ever, no difference was observed in the TLR9 response. Furthermore, in MS-derivedPBMCs, TLR7-mediated production of IL-6 and the ex vivo expression of B-cell-activatingfactor of the TNF family, two crucial cytokines for B-cell differentiation and survival, wereinduced by IFN-β. Depletion of monocytes, which are key producers of both IL-6 and B-cell-activating factor of the TNF family, showed that TLR7-mediated B-cell differentiation intoIg-secreting cells is strongly dependent on the cross-talk between B cells and monocytes.Accordingly, impaired expression of TLR7 mRNA was observed in PBMCs and mono-cytes isolated from MS-affected individuals as compared with those from healthy donors,which was rescued by IFN-β therapy. Collectively, our data unveil a novel TLR7-regulatedmechanism in in vivo IFN-β-stimulated whole leukocytes that could be exploited to definenew TLR7-based strategies for the treatment of MS.

Keywords: B cell � IFN-β � Monocyte � Multiple sclerosis � TLR

� Additional supporting information may be found in the online version of this article at thepublisher’s web-site

Introduction

Growing evidence is accumulating on the central role that Blymphocytes play in the immunopathology of multiple sclerosis(MS) [1, 2]. For example, oligoclonal IgG bands, found in thecerebrospinal fluid of more than 90% of patients with MS, indi-cate an intrathecal Ab production [3]. The presence of clonally

Correspondence: Dr. Eliana Marina Cocciae-mail: [email protected]

expanded B cells, plasma cells, complement and myelin-specificAbs in chronic MS lesions also suggested an intrathecal, Ag-driven humoral immune response in the central nervous system ofMS sufferers [4–6]. In addition, B-cell follicle-like structures aredetectable in the meninges of MS patients [7, 8]. More recently,B-cell depleting therapies, such as Rituximab (that targets theB lymphocyte surface antigen CD20 [9–11]), together with

∗These authors contributed equally to this work.

C© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu

1964 Elena Giacomini et al. Eur. J. Immunol. 2013. 43: 1963–1972

Ocrelizumab and Ofatumumab (two other humanized anti-CD20monoclonal Abs), are proving their efficacy at various stages ofclinical development [12]. All together these findings contributeto the compelling evidence that B cells and the humoral immuneresponse are implicated in the pathogenesis of MS and suggest thetherapeutic implications that all this may have for the treatmentof this disease.

B lymphocytes play an essential role in bridging innate andadaptive immunity. To differentiate into specialized cells capableof communicating with helper T cells and undergo Ab diversifica-tion, clonal expansion, and Ig secretion, B lymphocytes need thesupport of a coordinated network of cytokines, growth factors,adhesion, and ligand-receptor signals [13]. Among B-cell recep-tors, the TLRs and their natural agonists have raised interest sincethey elicit direct effects on human B cells [14]. TLRs are germ-line encoded pattern recognition receptors that can detect con-served molecular patterns either expressed on microorganisms orof self-origin. Targeting them or modulating their functions mayhave therapeutic potential in autoimmune diseases, including MS[15]. B lymphocytes selectively express TLR7 and TLR9 and areactivated by their specific ligands [16,17]. At variance with otherTLRs, TLR7 and TLR9 share relevant properties. Indeed, they bothrecognize microbial and endogenous nucleic acids; in particular,TLR7 specifically binds guanosine- and uridine-rich ssRNAs whileTLR9 senses hypomethylated CpG-rich dsDNAs. Furthermore, theyboth reside in the endosomal compartments, unlike the other TLRspresent on the cell surface.

In this study, we characterized the TLR-induced modulationof B-cell responses and differentiation in MS patients undergoingIFN-β therapy, a first-line drug used in the treatment of relaps-ing remitting MS (RRMS), whose mechanism of action remainspoorly understood. IFN-β-mediated immunomodulatory functionsmay differentially operate depending on the responding cell subsetacting on T- or B-cell proliferation, modulation of cytokine produc-tion, and regulation of adhesion molecules involved in lymphocytemigration across the blood-brain barrier [18]. For these reasons,investigating the action of IFN-β therapy on B cells might be ofgreat relevance to understand their pathogenic role in the devel-opment and regulation of autoimmune inflammatory response inMS.

Results

IFN-β therapy selectively promotes TLR7-mediatedB-cell maturation and Ig production

There is increasing recognition that TLRs and TLR-drivenresponses can play a key role in the pathogenesis of severalautoimmune diseases, including MS. TLR7 and TLR9 are selec-tively expressed by B cells, and when activated by specific ligands,lead to their proliferation and differentiation into Ig-secretingcells. Given the key importance of B lymphocytes in MS disease,we investigated whether IFN-β therapy would modulate Ig synthe-sis in MS patients by performing a longitudinal study conducted

Figure 1. TLR7-mediated Ig production is selectively promoted by IFN-βtherapy. PBMCs from 10 HDs or 15 MS patients before (T0) and1 month (T1) after IFN-β therapy were treated with TLR7 (3M001) or TLR9(CpG) agonist for 7 days. (A) IgM- or (B) IgG-secreting cells were detectedand enumerated by isotype-specific ELISPOT. The values shown repre-sent the means +SEM of results pooled from the indicated number ofdonors. p-values were calculated by two-tailed Student’s t-test. For IgM:*p = 0.0353, **p = 0.0138. For IgG: *p = 0.0332, **p = 0.0169.

with unseparated PBMCs isolated from 15 MS patients before (T0)and 1 month after (T1) the beginning of IFN-β therapy. Moreover,PBMCs isolated from 10 healthy donors (HDs) were also includedin this study as comparative control.

To this end, PBMCs were cultured in vitro with either aspecific TLR7 (the synthetic small molecule 3M001) or TLR9(a type B CpG, 2006) agonist for 7 days and then IgM (Fig. 1A) andIgG production were evaluated by Elispot (Fig. 1B) and Elisa assay(Supporting Information Fig. 1). The TLR9-mediated B-cell stim-ulation led to a similar frequency of IgM- and IgG-secreting cellsin both HD- and MS-affected individuals and this Ab release wasnot modified in response to IFN-β treatment. On the other hand,it was very interesting to find that the basal level of TLR7-inducedIg production was significantly lower in MS patients as comparedwith that in HD, showing a specific defect in TLR7 responses inB cells from MS sufferers.

Surprisingly, 1 month of IFN-β therapy was able to partiallyrestore this deficiency and selectively increase the production ofIgM and IgG upon TLR7 triggering, re-establishing the level ofAb release found in HDs. The analysis of Ig content by Elisaconfirmed the results obtained by Elispot assay (SupportingInformation Fig. 1). IFN-β-mediated effect was long-lasting sinceit was still observed after 6 months of IFN-β treatment (data not


Eur. J. Immunol. 2013. 43: 1963–1972 Clinical immunology 1965

shown). However, IFN-β did not enhance auto-Ab production asdemonstrated by measurement of both homogeneous and speck-led patterns of anti-ANA Abs on sera of MS patients before andafter therapy (data not shown) [19].

In accordance with the Ig production, flow cytometry analysisof the co–stimulatory marker CD86 also highlighted an increasedmaturation status in in vivo IFN-β-treated CD19+ B cells fromMS patients stimulated with TLR7, whereas no major differ-ences were found upon TLR9 stimulation (Supporting InformationFig. 2A). In this experimental setting, we also observed a signif-icant increase in the expression of the activation marker CD38on B-cell surface after IFN-β treatment (Supporting InformationFig. 2B). Given that this protein is notoriously type I IFN inducible[20], this result clearly shows that B lymphocytes are target of theIFN-β therapy confirming previous study by Zula et al. [21] whodescribed a rapid activation of IFN signal transduction pathwaysin B cells present in unseparated blood from RRMS patients soonafter IFN-β injection.

TLR7 expression is lower in PBMCs and monocytes ofMS patients compared with HDs

In the past, we dissected the regulation of TLR7 in maturingmonocyte-derived DCs and observed that its transcription wasdependent on the endogenous IFN-β release [22]. Thus, to eval-uate whether IFN-β therapy would modulate TLR7 expression inMS patients, we first monitored by real-time RT-PCR TLR7 levelof transcription, together with that of TLR9, in MS patients versusHDs. It was of great interest to find that PBMCs obtained from MSpatients display a clear defect, as compared with those of HDs, inTLR7 expression that was statistically significant (25 HDs and45 MS patients analyzed) (Fig. 2A). This difference was notobserved in the transcription of TLR9 gene (Fig. 2B), demonstrat-ing that in MS patients, the defective TLR7 expression is specific.Furthermore, we observed that in PBMCs isolated from the sameMS patients following 1 month of IFN-β therapy, the level of TLR7mRNA was restored to the level observed in HDs, while that ofTLR9 was not modulated (Fig. 2A and B).

In the attempt to investigate which TLR7-expressing cell typesin the peripheral blood might be responsible for this defect inMS patients, B cells and monocytes were purified from both HDsand MS patients at baseline and 1 month after the beginning ofIFN-β therapy, since these two leukocyte populations expressTLR7. Data on TLR7 expression in B cells isolated from HDs or MS(7 and 13 individuals, respectively) did not mirror the impairmentobserved in the context of the mixed cell population of PBMCs(Fig. 2C and D), although a slightly enhanced level of TLR7 tran-scription in response to IFN-β occurred also in this experimen-tal setting. As observed in unseparated PBMCs, TLR9 levels of Bcells did not differ in HDs and MS patients irrespective of IFN-βtreatment. Interestingly, when the expression of TLR7 was ana-lyzed in monocytes of MS patients (13 individuals), a differentpicture appeared. Indeed, a lower TLR7 mRNA level was high-lighted in monocytes from MS patients than that obtained from HD

Figure 2. PBMCs and monocytes derived from MS patients display alow level of TLR7 gene expression. Cells were isolated from freshlydrawn blood of HDs and MS patients before (T0) and after 1 month (T1)of IFN-β therapy. (A, B) Total RNA was prepared and (A) TLR7 or (B)TLR9 gene expression was evaluated by quantitative real-time RT-PCR.Data obtained from all enrolled HDs and MS individuals are shown asmean ± SEM. (C–E) Twenty-five HDs and 45 MS were enrolled for PBMCisolation; (C, D) 7 HDs and 13 MS for B cells purification; (E) 8 HDs,8 MS patients without treatment (T0); and additional 5 MS patientsbefore (T0) and after 1 month (T1) of IFN-β therapy for monocyte iso-lation. The values shown represent the means +SEM of results pooledfrom the indicated number of donors. *p = 0.03965 (two-tailed Student’st-test for unpaired data); **p = 0.029 (two-tailed Student’s t-test forpaired data); ***p = 0.0466 (two-tailed Student’s t-test for unpaired data);****p = 0.0014 (two-tailed Student’s t-test for paired data).

(8 individuals) and, moreover, also a robust induction wasobserved in response to IFN-β therapy (longitudinal analysis of5 patients at baseline and 1 month after IFN-β treatment) (Fig. 2E).TLR9 expression was absent in monocytes (data not shown). Thesedata for the first time indicated a defect in TLR7 signaling in mono-cytes of MS patients.

IFN-β enhances the expression of soluble factorsinvolved in B-cell differentiation

To mature and differentiate into Ig-secreting cells, B lympho-cytes need the support of a coordinated action of many cytokines,



Figure 3. IL-6 and BAFF expression is strongly induced in MS patientsundergoing IFN-β therapy. Whole PBMCs obtained from 10 HDs and15 MS were cultured for 24 h with a TLR7 (3M001) or a TLR9 (CpG)ligand, respectively 3M001 and CpG. (A) IL-6 was measured in culturesupernatants by ELISA. Data are shown as mean +SEM of results pooledfrom 10 HDs and 15 MS individuals before (T0) and after 1 month (T1)of IFN-β therapy. (B, C) BAFF expression was evaluated in HDs or MSpatients before and after IFN-β therapy either (B) at the mRNA levelby quantitative real-time PCR in freshly isolated PBMCs (5 HDs and7 MS patients) or (C) at protein level by BAFF ELISA in sera (6 HDs or12 MS patients). The values shown represent the means +SEM of resultspooled from the indicated number of donors. For IL-6 *p = 0.005 (two-tailed Student’s t-test for paired data).

growth factors, and adhesion-dependent signals. Among them,IL-6 plays a pivotal and not redundant role acting on the survivaland on the Ig secretion capacity of B cells [23].

Thus, to characterize the mechanisms underlying the differ-ential responsiveness of B cells from MS patients to TLR7 andTLR9 stimulation, we measured by ELISA the production of IL-6in PBMCs isolated from 10 HDs and from 15 MS patients beforeand after IFN-β therapy (Fig. 3A). PBMCs were treated for 24 hwith the TLR7 and TLR9 ligands, 3M001 and CpG, respec-tively, and cytokine release was quantified. In line with the dataobtained for Ig production, HD PBMCs secreted a robust level ofIL-6 following TLR7 triggering that was significantly higher thanthe production seen in PBMCs of therapy-free MS patients. Thelower cytokine release observed in MS patients was almost com-

pletely restored following IFN-β administration. TLR9 stimulationinduced low amounts of this cytokine in HDs and the level of pro-duction was not differently modulated in MS individuals beforeand after IFN-β therapy.

In the same way, another key component of the milieuresponsible for B-cell proliferation and differentiation into plasmacells is the B-cell-activating factor of the TNF family (BAFF),whose mRNA expression was found to be comparable betweenPBMCs of HDs and untreated MS patients but strongly inducedupon IFN-β therapy (Fig. 3B). Accordingly, similar levels ofBAFF were present in the sera of HDs and MS patients andwere induced in response to IFN-β therapy (Fig. 3C), confirm-ing previous data from Krumbholz et al. [24]. All togetherthese results show that in MS patients, the lower humoralimmune response upon TLR7 triggering is replenished by IFN-βtreatment likely through the release of factors, such as IL-6and BAFF, that mediate B-cell differentiation into Ig-secretingcells.

Monocytes have a key role in TLR7-stimulated B-celldifferentiation and Ig production in MS patients

Having found that in MS patients monocytes display an impairedexpression of the TLR7 gene (Fig. 2E), we hypothesized that thiscell type might have a role in the defective TLR7-induced Abresponse of MS patients through the release of cytokines involvedin B-cell differentiation and activation, such as IL-6 and BAFF[25–27].

To test our hypothesis, we depleted PBMCs from 4 IFN-β-treated MS patients of monocytes and cultured total or monocyte-depleted PBMCs with the TLR7- or TLR9-specific ligands. Whilethe poor induction of IL-6 was not affected by the depletion ofmonocytes upon TLR9 stimulation, a strong dependence on mono-cytes was observed for IL-6 release in response to TLR7 (Fig. 4A).In a similar manner, BAFF mRNA, strongly expressed in freshlydrawn total PBMCs upon IFN-β therapy, displayed a clear reduc-tion in the level of expression in IFN-β-treated monocyte-depletedPBMCs (Fig. 4B).

In this scenario, it was not surprising to find that the TLR7-driven IgM and IgG synthesis, observed in PBMCs from IFN-β-treated MS patients, was dramatically decreased when monocyteswere depleted (Fig. 5A and B). Similarly, when BAFF activity wasprevented by the addition of a specific BAFF neutralizing Ab toPBMC cultures, a reduction in the TLR7-stimulated IgM and IgGproduction was obtained (Supporting Information Fig. 3). A dif-ferent picture was found when Ig release was measured uponTLR9 triggering in either monocyte-depleted PBMCs or wholePBMCs treated with anti-BAFF Ab. Indeed, an enhanced releaseof both IgM and IgG was observed in response to TLR9 stimu-lation in the absence of monocytes while the neutralization ofBAFF poorly affected Ig production (Fig. 5A and B and SupportingInformation Fig. 3, respectively). This result was not obvious and,at this stage, it is difficult to explain but it suggests that mono-cytes could be associated to a negative feedback loop on TLR9-driven B-cell differentiation while they positively act on the TLR7



Figure 4. Monocyte depletion completely abrogates IFN-β-induced IL-6and BAFF expression in PBMCs from MS patients. (A) Supernatants wereharvested from cultures of TLR7- or TLR9-stimulated total or monocyte-depleted PBMCs obtained from 4 IFN-β-treated MS patients. IL-6 levelswere measured by ELISA. Data are shown as mean +SEM of resultsobtained from the four IFN-β-treated MS patients analyzed. (B) RNAwas isolated from total or monocyte-depleted PBMCs obtained from4 IFN-β-treated MS patients and BAFF expression calculated by real-time RT-PCR as specified above. The values shown represent the means+SEM of results pooled from the indicated number of donors.

responsiveness of Ig-producing B cells. Thus, we can envisage thatchanges in the basal and/or TLR-induced cytokine milieu of in vivoIFN-β-conditioned PBMCs could profoundly impact on Ig produc-tion from B cells in response to TLR7 or TLR9 stimulation.

Collectively, these findings demonstrate that the cross-talkbetween monocytes and B cells is essential for the release of aneffective humoral immune response in the context of TLR7 stim-ulation affecting the maturation and differentiation status of Blymphocytes into Ig-secreting cells.

Discussion

Over the past decade, there has been growing understanding andacceptance of the pathological involvement of B cells and humoralresponse in MS [1, 2]. The demonstration that peripheral B-celldepletion leads to a rapid decline in disease activity in MS isthe strongest evidence of the central role of these cells in MSautoimmunity [9,11]. However, the key question that still remains

Figure 5. In the absence of monocytes TLR7-treated B cells do not dif-ferentiate into Ig-secreting cells. Whole or monocyte-depleted PBMCsobtained from IFN-β-treated MS patients were treated with TLR7(3M001) or TLR9 (CpG) agonists for 7 days. (A) IgM- or (B) IgG-secretingcells were detected and enumerated by isotype-specific ELISPOT. Thevalues shown represent the means +SEM of results pooled from 4 MSpatients. For IgM *p = 0.0325 and for IgG **p = 0.039 (two-tailed Student’st-test for paired data).

unsolved is when and how in the life of an individual B cell doesprovide immunopathogenic support or arise as a disease-relevantcell type in MS.

In this study, we investigated whether IFN-β targets B lym-phocytes and modulates their functions contributing to the pro-tective effects of this treatment. Only a few studies have thusfar addressed this point and most have investigated the abilityof highly purified B cells from MS patients to present antigensand subsequently regulate T-cell responses [28, 29]. In contrast,we studied whether IFN-β therapy would regulate the maturationand differentiation of B cells into Ig-secreting cells in response toTLR7 or TLR9 stimulation. Indeed, it has been shown that TLRtriggering is necessary for extensive human naıve B-cell prolifera-tion, isotypic switching, and production of Abs providing the thirdsignal upon BCR cross-linking by antigen and interaction withT helper cells [30]. However, compared with naıve B cells, mem-ory B cells have less stringent triggering requirements and can bereadily activated also in the absence of BCR stimulation by poly-clonal stimuli such as bystander T-cell help and TLR triggering,



which modifies also the cytokine milieu, accelerating Ab produc-tion in secondary responses and maintaining the serological mem-ory [31, 32]. Thus, in our experimental setting, the simultaneouspresence of different immune populations in total PBMCs assuredthe presence of all the required signals for B-cell differentiationand offered a faithful representation of what is actually happeningin vivo in the peripheral blood of MS patients.

Our results demonstrate a fundamental difference in the out-come of either TLR7 or TLR9 stimulation of B cells in the con-text of PBMCs isolated from HDs or MS patients. Indeed, whilethe treatment with a TLR9 ligand induced a comparable pro-duction of both IgG and IgM in control or MS-affected indi-viduals, we highlighted for the first time a clear deficiency inTLR7-mediated B-cell differentiation into Ig-secreting cells in MSpatients.

In vivo administered IFN-β is able to replenish in MS patientsthe low TLR7-induced Ig production to the level observed in HDs.In line with this evidence and consistent with previous findings[33], TLR7 expression was also upregulated by IFN-β both inwhole PBMCs, purified B cells, and monocytes. Furthermore, threestudies reported with different experimental approaches howIFN-α, another subtype of the type I IFN family to which IFN-βbelongs, exogenously provided or in situ produced by plasma-cytoid DC, enhances B-cell differentiation into IgM- and IgG-producing cells only in response to TLR7, but not TLR9, triggering[34–36]. We believe that in our settings in vivo IFN-β therapymight have similar activity to what is described in vitro for IFN-α.

IFN-β treatment enhances TLR7-induced B-cell responses inMS patients acting at different steps: not only on the regulationof TLR7 gene expression but also on the secretion of soluble fac-tors of key importance for B-cell differentiation, namely IL-6 andBAFF. IL-6 promotes terminal differentiation of B cells to plasmacells [23, 37] and exerts also a pronounced effect on the survivaland/or Ig secretion [38]. BAFF regulates, in tandem with APRIL(a proliferation-inducing ligand), B-cell survival, differentiationand class switching, determines the size of the peripheral B-cellpool and is essential for maintenance of the peripheral B-cellrepertoire and initiation of T-cell independent B-cell responses[39]. BAFF has been implicated in the development of autoimmu-nity in experimental settings and in several human B-cell-relatedautoimmune diseases, including MS [39]. Interestingly, Serafiniand Aloisi in collaboration with our team also found that BAFF isexpressed in EBV-infected B cells in acute MS lesions and ectopicB-cell follicles [40], highlighting the key role of this factor in B-cellactivation also in the MS brain.

In our experimental setting, we observed that IL-6 was stronglyinduced in TLR7-treated PBMCs derived from IFN-β-treated MSpatients and this result likely correlates with the replenishment ofTLR7 expression and responsiveness in in vivo IFN-β-conditionedmonocytes. Indeed, when PBMCs derived from IFN-β-treatedpatients were depleted of monocytes, the strong induction ofIL-6 observed in total PBMCs was completely lost. In addition,a strong reduction of BAFF expression was observed in in vivoIFN-β-conditioned PBMCs after the depletion of monocytes. In asimilar fashion, in the absence of monocytes, there was no induc-

tion of TLR7-driven IgM and IgG production, indicating that IFN-βtreatment could exert its therapeutic effects by fine-tuning mono-cyte functions, in the context of TLR7 stimulation, that act throughbystander mechanisms on the differentiation of B lymphocytes.Taking into account that TLR7 is crucial for type I IFN releasefrom pDC [41] and is, at the same time, an IFN-inducible gene[22], we can envisage the existence of a tight relation betweenIFN-β response and TLR7 responsiveness of MS monocytes, whosefull comprehension deserves further investigation.

In line with this view, recent data obtained by Molnarfi andcollaborators showed that monocytes from RRMS patients exhib-ited a reduced ability to produce HGF, a neuroprotective andneuroinflammation-suppressive mediator, when compared withHD [42]. Treatment with IFN-β significantly enhanced HGF syn-thesis and secretion by blood monocytes, contributing to the clin-ical benefit of IFN-β in RRMS via the combined HGF-mediatedneuroprotective and anti-inflammatory mechanisms.

In this context, it is also important to remind that monocytesare abundant in inflammatory MS brain lesions and displayedalso altered functions and an activated innate immune signaturein MS patients with clinically more severe course [43]. In par-ticular, the type I IFN pathway is dysregulated in these mono-cytes, which may contribute to more active disease. In additionto that, conditional genetic knockout of IFNAR1 in monocytes,but not in T cells, B cells, or central nervous system cells, leadsto enhanced disease severity in the animal model of MS [44].All these evidences indicate that perturbations of the type I IFNsignaling pathway and response in monocytes could representcrucial events in MS immunopathology and, at the same time,a key target of IFN-β therapy. On the other hand, we cannotexclude that the replenished TLR7 responsiveness in PBMCs andmonocytes of IFN-β-treated MS patients could be related to therescue or prevention of TLR7 tolerance, that is generally inducedby specific ligands of this receptor and leads to a reduced cytokineand Ig production [45]. Indeed, Poovassery and Bishop [45]recently demonstrated that IFN-β controls TLR7 tolerance andactivation through the PI3K/Akt/mammalian target of rapamycinsignaling pathway but also enhancing TLR7 expression in humanB cells. These data allow us to envisage that a chronic stimulationof TLR7 by a not-yet identified microbial or self-derived agonistcould lead to a reduced sensitivity to TLR7 stimulation in MSpatients, which might result in altered B-cell functions.

To date, the enhancement of Ab synthesis mediated by IFN-βtreatment is not resulting in an excessive Ig production or in aninduction of auto-Abs (data not shown and [46]). Rather, thistherapy restores via monocyte-mediated bystander mechanismsthe correct TLR7 responsiveness of MS-derived B cells, which inthis way fully acquire the capacity to mature into Ig-producingcells, similar to HDs. In this scenario, the study from Warring-ton et al. [47] is of great interest that demonstrates how nat-urally occurring polyclonal human Abs (in particular IgM) canstrongly promote remyelination inducing a transient Ca2+ influx inmyelin-forming cells. Thus, the ability of IFN-β therapy to inducepolyclonal Abs (and in particular IgM) with potential remyeli-nating activity reveals another mechanism of protection possibly



mediated by this drug, that could lead to amelioration of neuro-logical symptoms in MS patients.

An additional aspect to take into account from our find-ings is that the deficient TLR7-induced IgM and IgG produc-tion observed in MS patients might correlate with worsening ofdisease or impaired immune responses against infections withTLR7-recognized RNA viruses, such as influenza, or upon vac-cination. Many studies have been conducted in this regard. Dif-ferent groups have reported that the risk of relapse is increasedin individuals with MS bacterial or viral infections [48, 49]. Inthe case of influenza, it was shown that the reduction of infec-tion episodes leads to a lower number of exacerbations in MSsufferers. In a study with 180 RRMS patients, 33% of individ-uals, who became infected with this virus, developed an acuterelapse within 6 weeks [50]. However, randomized, double-blind,placebo-controlled studies during the past decade have shown thatinfluenza vaccination of MS patients neither increases the relapserate nor worsens the course of disease [51]. Indeed, the admin-istration of standard vaccines in MS patients is considered safeworldwide, it follows the same recommendations as in healthyadults and actually should be recommended to MS patients inorder to avoid attacks of the disease [52]. Having all this in mind,it cannot be excluded that our data on the reduced level of secretedAbs in response to TLR7 stimulation can have a role in the exac-erbation of relapses observed in MS-affected individuals alongepisodes of influenza infection. The increasing recognition thatviruses, and in particular EBV, can be etiological factors driving thedevelopment of MS or other autoimmune diseases in geneticallysusceptible individuals further strengthens the potential of admin-istering anti-viral therapies to people affected by these disorders[12]. In line with this view, the increased TLR7 gene expressionobserved upon IFN-β might be part of a specific antiviral pro-gram induced by this cytokine that could counteract dysregulatedresponses to viral infection in MS patients.

Targeting TLRs and modulating their functions are increasinglyrecognized as potential therapeutics for different autoimmunediseases, including rheumatoid arthritis, systemic lupus erythe-matosus, and MS [15, 53]. Indeed, a common trait of most auto-immune disorders is a chronic inflammation occurring at specificsites within the body or as a systemic complication suggested tobe sustained also by TLR activation. In our study, we highlighteda defect in TLR7 gene expression in PBMCs of MS patients ascompared with HDs. TLR7 is a member of the TLR family thathas been implicated at different levels in autoimmunity. Polymor-phisms in the TLR7 gene were shown to have a role in time todisease progression in individuals affected by MS [54] but also inpredisposition for systemic lupus erythematosus in Asian popula-tion [55].

All together, the above evidence suggests how a tight reg-ulation of both TLR expression and TLR-induced responses, inparticular those driven by TLR7 triggering, is necessary to main-tain a healthy and tolerant immune environment. Having foundthat TLR7 responsiveness was clearly rescued by IFN-β treatment,we can envisage that IFN-β therapy creates a new microenviron-ment in PBMCs and, likely, in other anatomical sites, where novel

interactions among leukocyte subsets are established and mightinfluence the outcome of the immune process. These new insightsin MS immunopathology and in the therapeutic effects of IFN-βcould help to improve existing therapies or define new therapeu-tic strategies for MS targeting TLR expression or TLR-inducedresponses.

Materials and methods

Patients

Sixty patients with definite RRMS according to McDonald’s criteria[56] (age, 36.8 ± 7.4 years (mean ± SD)) and 35 age- and sex-matched healthy subjects (40 ± 6.3 years) were enrolled at the S.Andrea Hospital MS Center. Patients were longitudinally studiedright before (T0) and 1 month (T1) after the beginning of IFN-βtreatment (recombinant IFN-β1b in the formulation of Betaferon,Bayer, 250 μg subcutaneously, every other day). Mean ExpandedDisability Status Scale was 1.5 (range 0–6), disease duration wasfrom 1 to 26 years. Patients had neither taken steroids during the3 months preceding enrollment, nor had received other diseasemodifying therapies before. The study was approved by the EthicsCommittee of S. Andrea Hospital and all the subjects involved inthe study gave written informed consent.

Cell isolation and stimulation

Peripheral blood (20–50 mL) was collected from MS patientsand HDs and PBMCs isolated by density gradient centrifugationusing Lympholyte-H (Cedarlane Laboratories, Hornby, Ontario,Canada). B cells and monocytes were obtained by positive sort-ing by using anti-CD19 and anti-CD14 conjugated magneticmicrobeads (Miltenyi Biotec, Bergish Gladbach, Germany), respec-tively. The recovered cells were >90–95% pure as determined byflow cytometry using anti-CD19 and anti-CD14 Ab (BD Pharmin-gen, San Diego, CA, USA).

PBMCs (1 × 106/mL) were cultured in RPMI 1640 (BioWhit-taker Europe, Verviers, Belgium) supplemented with 2 mML-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin(Gibco, Grand Island, NY, USA), and 10% FBS (BioWhittakerEurope).

PBMCs were subjected to positive sorting using anti-CD14 con-jugated magnetic microbeads (Miltenyi Biotec) to remove mono-cytes from whole PBMCs.

Whole or monocyte-depleted PBMCs were stimulated withoptimal doses of TLR7 and TLR9 agonists: 3M001 (25 μM, akind gift of Dr. Mark Tomai, 3M pharmaceuticals) and typeB phosphorothioate-CpG 2006 oligodeoxynucleotides (3 μg/mL,synthetized by Eurofins MWG Operon), respectively.

Monoclonal anti-human BAFF Ab (20 μg/mL; R&D Systems,Minneapolis, MN, USA) was used to block BAFF biological activity,where indicated.



Flow cytometry analysis

Monoclonal Abs for CD19, CD38, CD86 as well as IgG1, IgG2acontrol Abs (BD Pharmingen), conjugated with FITC, PE, or PERcPas needed, were used for flow cytometry analysis. Briefly, cells(1 × 105) were collected and washed once in PBS containing 2%FBS, then incubated with Abs at 4◦C for 30 min. After staining,cells were fixed with 2% paraformaldehyde before analysis onan FACSCan (BD Pharmingen). CD38 and CD86 expression wasevaluated in the CD19+/SSC gate.

Elispot assay

PBMCs from HD or MS patients before and after IFN-β ther-apy were treated with the TLR7 or TLR9 agonist for 7 days asspecified. For Elispot assay, cells were then recovered and incu-bated for 3 h at 37◦C in IgM- or IgG-coated 96-well flat-bottomedmicrotiter plates. Wells were subsequently washed and then incu-bated overnight at 4◦C with alkaline phosphatase-conjugated goatanti-human IgM or IgG (Sigma). After extensive washings withPBS-Tween, the alkaline phosphatase substrate 5-bromo-4-chloro-3-indolyl phosphate (BCIP; Sigma) was added to each well. Afterrinsing and drying, the spots were enumerated under a stereomi-croscope with 40-fold magnification. The ratio between the num-ber of Ig-secreting cells and the number of CD19+ cells present ineach culture was evaluated in 10 HDs and 15 MS patients ana-lyzed before and after IFN-β therapy. The values represent themeans ± SEM.

ELISA

Supernatants from PBMC cultures were prepared as described inthe text, harvested, and stored at −80◦C. ELISA kit for IL-6 waspurchased from Bender MedSystems (Burlingame, CA, USA). Thevalues shown represent the means ± SEM of the cytokine concen-trations detected in the supernatants of cultures collected fromindependent experiments.

IgM and IgG content present in the supernatants of PBMCsobtained from 6 MS patients and 5 HDs was evaluated byElisa kit (Bethyl Laboratories, Inc.). The values represent themeans ± SEM of Ig concentration.

Sera from 6 HDs and 12 MS patients were also collected andBAFF level was evaluated by Quantikine BAFF immunoassay (R&DSystems) according to the manufacturers’ instruction.

RNA purification and real-time RT-PCR

DNase-I-treated total RNA was purified from MS patient- or HD-derived PBMCs using the RNeasy Mini Kit (Qiagen, Valencia, CA,USA) or B cells and monocytes using the high pure RNA isola-tion kit (Roche Diagnostic GmbH, Mannheim, Germany). Reversetranscription was primed with oligo (dT) and random hexamers by

using the Murine Leukaemia Virus Reverse Transcriptase (Invit-rogen Life Technologies, Carlsbad, CA, USA). Quantitative PCRassays for GAPDH, TLR7, TLR9, and BAFF were done at least induplicates by using the Light Cycler Fast Start DNA SYBR GreenI Master Mix in the presence of 3 mM MgCl2 on a LightCyclerInstrument (Roche Diagnostics) as previously described [22].

Sample values were normalized by calculating the relativequantity of each mRNA to that of GAPDH using the formula 2−�Ct

and expressed as mean ± SD.Primer pairs for GAPDH and TLR7 was as previously described

[22]. TLR9 and BAFF primers used in this study were as follows:

TLR9_forward: 5′-TGAAGACTTCAGGCCCAACTG-3′

TLR9_reverse: 5′-TGCACGGTCACCAGGTTGT-3′

BAFF_forward: 5′-TGAAACACCAACTATACAAAAG-3′

BAFF_reverse: 5′-TCAATTCATCCCCAAAGACAT-3′

Statistical analysis

Statistical significance of differences was determined by Student’st-test for paired or unpaired data (p < 0.05 was considered signif-icant) from JAVA Applets & Servlets for Biostatistics software.

Acknowledgements: This work was supported by the Ital-ian Multiple Sclerosis Foundation # 2009/R/7 (to E.M.C.). Wethank Dr. Mark Tomai (3M pharmaceuticals) and Francesca Aloisi(Department of Cell Biology and Neurosciences, Istituto Superioredi Sanita, Rome, Italy) for their helpful discussion. We acknowl-edge Dr. Silvia Romano, Dr. Giulia Coarelli, and Dr. AriannaFornasiero, who took care of patients and helped with sampling.Furthermore, we thank Eugenio Morassi (Division Service for DataManagement, Documentation, Library and Publishing Activities,Istituto Superiore di Sanita, Rome, Italy) for preparing drawings.

Conflict of interest: Marco Salvetti received lecture feesfrom Biogen-Dompe and received research support from Bayer-Schering, Biogen-Dompe, Merck-Serono, and Sanofi-Aventis.

References

1 Franciotta, D., Salvetti, M., Lolli, F., Serafini, B. and Aloisi, F., B cells and

multiple sclerosis. Lancet Neurol. 2008. 7: 852–858.

2 Krumbholz, M., Derfuss, T., Hohlfeld, R. and Meinl, E., B cells and anti-

bodies in multiple sclerosis pathogenesis and therapy. Nat. Rev. Neurol.

2012. 8: 613–623.

3 Walsh, M. J., Tourtellotte, W. W., Roman, J. and Dreyer, W., Immunoglob-

ulin G, A, and M–clonal restriction in multiple sclerosis cerebrospinal

fluid and serum–analysis by two-dimensional electrophoresis. Clin.

Immunol. Immunopathol. 1985. 35: 313–327.



4 Genain, C. P., Cannella, B., Hauser, S. L. and Raine, C. S., Identification

of autoantibodies associated with myelin damage in multiple sclerosis.

Nat. Med. 1999. 5: 170–175.

5 O’Connor, K. C., Appel, H., Bregoli, L., Call, M. E., Catz, I., Chan, J. A.,

Moore, N. H. et al., Antibodies from inflamed central nervous system

tissue recognize myelin oligodendrocyte glycoprotein. J. Immunol. 2005.

175: 1974–1982.

6 Qin, Y., Duquette, P., Zhang, Y., Olek, M., Da, R. R., Richardson, J.,

Antel, J. P. et al., Intrathecal B-cell clonal expansion, an early sign of

humoral immunity, in the cerebrospinal fluid of patients with clinically

isolated syndrome suggestive of multiple sclerosis. Lab. Invest. 2003. 83:

1081–1088.

7 Magliozzi, R., Howell, O., Vora, A., Serafini, B., Nicholas, R., Puopolo,

M., Reynolds, R. et al., Meningeal B-cell follicles in secondary progres-

sive multiple sclerosis associate with early onset of disease and severe

cortical pathology. Brain 2007. 130: 1089–1104.

8 Serafini, B., Rosicarelli, B., Magliozzi, R., Stigliano, E. and Aloisi, F., Detec-

tion of ectopic B-cell follicles with germinal centers in the meninges of

patients with secondary progressive multiple sclerosis. Brain Pathol. 2004.

14: 164–174.

9 Hauser, S. L., Waubant, E., Arnold, D. L., Vollmer, T., Antel, J., Fox, R. J.,

Bar-Or, A. et al., B-cell depletion with rituximab in relapsing-remitting

multiple sclerosis. N. Engl. J. Med. 2008. 358: 676–688.

10 Bar-Or, A., Calabresi, P. A., Arnold, D., Markowitz, C., Shafer, S., Kasper,

L. H., Waubant, E. et al., Rituximab in relapsing-remitting multiple

sclerosis: a 72-week, open-label, phase I trial. Ann. Neurol. 2008. 63:

395–400.

11 Stuve, O., Leussink, V. I., Frohlich, R., Hemmer, B., Hartung, H. P., Menge,

T. and Kieseier, B. C., Long-term B-lymphocyte depletion with rituximab

in patients with relapsing-remitting multiple sclerosis. Arch. Neurol. 2009.

66: 259–261.

12 Dreyfus, D. H., Autoimmune disease: a role for new anti-viral therapies?

Autoimmun. Rev. 2011. 11: 88–97.

13 Lanzavecchia, A. and Sallusto, F., Toll-like receptors and innate immu-

nity in B-cell activation and antibody responses. Curr. Opin. Immunol. 2007.

19: 268–274.

14 Browne, E. P., Regulation of B-cell responses by Toll-like receptors.

Immunology 2012. 136: 370–379.

15 Gambuzza, M., Licata, N., Palella, E., Celi, D., Foti Cuzzola, V., Italiano,

D., Marino, S. et al., Targeting Toll-like receptors: emerging therapeutics

for multiple sclerosis management. J. Neuroimmunol. 2011. 239: 1–12.

16 Tomai, M. A., Imbertson, L. M., Stanczak, T. L., Tygrett, L. T. and Wald-

schmidt, T. J., The immune response modifiers imiquimod and R-848 are

potent activators of B lymphocytes. Cell Immunol. 2000. 203: 55–65.

17 Krieg, A. M., Yi, A. K., Matson, S., Waldschmidt, T. J., Bishop, G. A.,

Teasdale, R., Koretzky, G. A. et al., CpG motifs in bacterial DNA trigger

direct B-cell activation. Nature 1995. 374: 546–549.

18 Gonzalez-Navajas, J. M., Lee, J., David, M. and Raz, E., Immunomodula-

tory functions of type I interferons. Nat. Rev. Immunol. 2012. 12: 125–135.

19 Kavanaugh, A., Tomar, R., Reveille, J., Solomon, D. H. and Homburger,

H. A., Guidelines for clinical use of the antinuclear antibody test and

tests for specific autoantibodies to nuclear antigens. American College

of Pathologists. Arch. Pathol. Lab. Med. 2000. 124: 71–81.

20 Remoli, M. E., Gafa, V., Giacomini, E., Severa, M., Lande, R. and Coccia,

E. M., IFN-beta modulates the response to TLR stimulation in human DC:

involvement of IFN regulatory factor-1 (IRF-1) in IL-27 gene expression.

Eur. J. Immunol. 2007. 37: 3499–3508.

21 Zula, J. A., Green, H. C., Ransohoff, R. M., Rudick, R. A., Stark, G. R. and

van Boxel-Dezaire, A. H., The role of cell type-specific responses in IFN-

beta therapy of multiple sclerosis. Proc. Natl. Acad. Sci. USA 2011. 108:

19689–19694.

22 Severa, M., Remoli, M. E., Giacomini, E., Annibali, V., Gafa, V., Lande, R.,

Tomai, M. et al., Sensitization to TLR7 agonist in IFN-beta-preactivated

dendritic cells. J. Immunol. 2007. 178: 6208–6216.

23 Jego, G., Bataille, R. and Pellat-Deceunynck, C., Interleukin-6 is a growth

factor for nonmalignant human plasmablasts. Blood 2001. 97: 1817–1822.

24 Krumbholz, M., Faber, H., Steinmeyer, F., Hoffmann, L. A., Kumpfel, T.,

Pellkofer, H., Derfuss, T. et al., Interferon-beta increases BAFF levels in

multiple sclerosis: implications for B cell autoimmunity. Brain 2008. 131:

1455–1463.

25 Tosato, G., Seamon, K. B., Goldman, N. D., Sehgal, P. B., May, L. T.,

Washington, G. C., Jones, K. D. et al., Monocyte-derived human B-cell

growth factor identified as interferon-beta 2 (BSF-2, IL-6). Science 1988.

239: 502–504.

26 Bauer, J., Ganter, U., Geiger, T., Jacobshagen, U., Hirano, T., Matsuda,

T., Kishimoto, T. et al., Regulation of interleukin-6 expression in cul-

tured human blood monocytes and monocyte-derived macrophages.

Blood 1988. 72: 1134–1140.

27 Tribouley, C., Wallroth, M., Chan, V., Paliard, X., Fang, E., Lamson,

G., Pot, D. et al., Characterization of a new member of the TNF family

expressed on antigen presenting cells. Biol. Chem. 1999. 380: 1443–1447.

28 Jiang, H., Milo, R., Swoveland, P., Johnson, K. P., Panitch, H. and Dhib-

Jalbut, S., Interferon beta-1b reduces interferon gamma-induced antigen-

presenting capacity of human glial and B cells. J. Neuroimmunol. 1995. 61:

17–25.

29 Ramgolam, V. S., Sha, Y., Marcus, K. L., Choudhary, N., Troiani, L.,

Chopra, M. and Markovic-Plese, S., B cells as a therapeutic target for

IFN-beta in relapsing-remitting multiple sclerosis. J. Immunol. 2011. 186:

4518–4526.

30 Ruprecht, C. R. and Lanzavecchia, A., Toll-like receptor stimulation as a

third signal required for activation of human naive B cells. Eur. J. Immunol.

2006. 36: 810–816.

31 Bernasconi, N. L., Traggiai, E. and Lanzavecchia, A., Maintenance of sero-

logical memory by polyclonal activation of human memory B cells. Science

2002. 298: 2199–2202.

32 Tangye, S. G., Avery, D. T., Deenick, E. K. and Hodgkin, P. D., Intrinsic

differences in the proliferation of naive and memory human B cells as

a mechanism for enhanced secondary immune responses. J. Immunol.

2003. 170: 686–694.

33 Green, N. M., Laws, A., Kiefer, K., Busconi, L., Kim, Y. M., Brinkmann, M.

M., Trail, E. H. et al., Murine B cell response to TLR7 ligands depends on

an IFN-beta feedback loop. J. Immunol. 2009. 183: 1569–1576.

34 Bekeredjian-Ding, I. B., Wagner, M., Hornung, V., Giese, T., Schnurr,

M., Endres, S. and Hartmann, G., Plasmacytoid dendritic cells control

TLR7 sensitivity of naive B cells via type I IFN. J. Immunol. 2005. 174:

4043–4050.

35 Douagi, I., Gujer, C., Sundling, C., Adams, W. C., Smed-Sorensen, A.,

Seder, R. A., Karlsson Hedestam, G. B. et al., Human B cell responses to

TLR ligands are differentially modulated by myeloid and plasmacytoid

dendritic cells. J. Immunol. 2009. 182: 1991–2001.

36 Jego, G., Palucka, A. K., Blanck, J. P., Chalouni, C., Pascual, V. and

Banchereau, J., Plasmacytoid dendritic cells induce plasma cell differ-

entiation through type I interferon and interleukin 6. Immunity 2003. 19:

225–234.

37 Hirano, T., Yasukawa, K., Harada, H., Taga, T., Watanabe, Y., Matsuda,

T., Kashiwamura, S. et al., Complementary DNA for a novel human inter-

leukin (BSF-2) that induces B lymphocytes to produce immunoglobulin.

Nature 1986. 324: 73–76.



38 Roldan, E., Garcia-Pardo, A. and Brieva, J. A., VLA-4-fibronectin interac-

tion is required for the terminal differentiation of human bone marrow

cells capable of spontaneous and high rate immunoglobulin secretion.

J. Exp. Med. 1992. 175: 1739–1747.

39 Mackay, F., Silveira, P. A. and Brink, R., B cells and the BAFF/APRIL axis:

fast-forward on autoimmunity and signaling. Curr. Opin. Immunol. 2007.

19: 327–336.

40 Serafini, B., Severa, M., Columba-Cabezas, S., Rosicarelli, B., Veroni, C.,

Chiappetta, G., Magliozzi, R. et al., Epstein-Barr virus latent infection and

BAFF expression in B cells in the multiple sclerosis brain: implications

for viral persistence and intrathecal B-cell activation. J. Neuropathol. Exp.

Neurol. 2010. 69: 677–693.

41 Coccia, E. M., Severa, M., Giacomini, E., Monneron, D., Remoli, M. E.,

Julkunen, I., Cella, M. et al., Viral infection and Toll-like receptor ago-

nists induce a differential expression of type I and lambda interferons

in human plasmacytoid and monocyte-derived dendritic cells. Eur. J.

Immunol. 2004. 34: 796–805.

42 Molnarfi, N., Benkhoucha, M., Bjarnadottir, K., Juillard, C. and Lalive, P.

H., Interferon-beta induces hepatocyte growth factor in monocytes of

multiple sclerosis patients. PLoS One 2013. 7: e49882.

43 Comabella, M., Lunemann, J. D., Rio, J., Sanchez, A., Lopez, C., Julia,

E., Fernandez, M. et al., A type I interferon signature in monocytes is

associated with poor response to interferon-beta in multiple sclerosis.

Brain 2009. 132: 3353–3365.

44 Prinz, M., Schmidt, H., Mildner, A., Knobeloch, K. P., Hanisch, U. K.,

Raasch, J., Merkler, D. et al., Distinct and nonredundant in vivo functions

of IFNAR on myeloid cells limit autoimmunity in the central nervous

system. Immunity 2008. 28: 675–686.

45 Poovassery, J. S. and Bishop, G. A., Type I IFN receptor and the B cell

antigen receptor regulate TLR7 responses via distinct molecular mecha-

nisms. J. Immunol. 2012. 189: 1757–1764.

46 Ciccarelli, O., Bagnato, F., Mainero, C., Salvetti, M., Paolillo, A., Gasperini,

C., Bastianello, S. et al., Antinuclear antibodies and response to IFNbeta-

1a therapy in relapsing-remitting multiple sclerosis. Mult. Scler. 2000. 6:

137–139.

47 Warrington, A. E., Van Keulen, V., Pease, L. R. and Rodriguez, M., Nat-

urally occurring antibodies as therapeutics for neurologic disease: can

human monoclonal IgMs replace the limited resource IVIG? Adv. Exp.

Med. Biol. 2012. 750: 44–55.

48 Buljevac, D., Flach, H. Z., Hop, W. C., Hijdra, D., Laman, J. D., Savelkoul,

H. F., van Der Meche, F. G. et al., Prospective study on the relationship

between infections and multiple sclerosis exacerbations. Brain 2002. 125:

952–960.

49 Correale, J., Fiol, M. and Gilmore, W., The risk of relapses in multiple

sclerosis during systemic infections. Neurology 2006. 67: 652–659.

50 De Keyser, J., Zwanikken, C. and Boon, M., Effects of influenza vaccina-

tion and influenza illness on exacerbations in multiple sclerosis. J. Neurol.

Sci. 1998. 159: 51–53.

51 Merelli, E. and Casoni, F., Prognostic factors in multiple sclerosis: role of

intercurrent infections and vaccinations against influenza and hepatitis

B. Neurol. Sci. 2000. 21: S853–S856.

52 Loebermann, M., Winkelmann, A., Hartung, H. P., Hengel, H., Reisinger,

E. C. and Zettl, U. K., Vaccination against infection in patients with mul-

tiple sclerosis. Nat. Rev. Neurol. 2012. 8: 143–151.

53 Clanchy, F. I. and Sacre, S. M., Modulation of toll-like receptor function

has therapeutic potential in autoimmune disease. Expert. Opin. Biol. Ther.

2010. 10: 1703–1716.

54 Enevold, C., Oturai, A. B., Sorensen, P. S., Ryder, L. P., Koch-Henriksen,

N. and Bendtzen, K., Polymorphisms of innate pattern recognition recep-

tors, response to interferon-beta and development of neutralizing anti-

bodies in multiple sclerosis patients. Mult. Scler. 2010. 16: 942–949.

55 Kawasaki, A., Furukawa, H., Kondo, Y., Ito, S., Hayashi, T., Kusaoi, M.,

Matsumoto, I. et al., TLR7 single-nucleotide polymorphisms in the 3’

untranslated region and intron 2 independently contribute to systemic

lupus erythematosus in Japanese women: a case-control association

study. Arthritis Res. Ther. 2011. 13: R41. doi:10.1186/ar3277.

56 Polman, C. H., Reingold, S. C., Edan, G., Filippi, M., Hartung, H. P.,

Kappos, L., Lublin, F. D. et al., Diagnostic criteria for multiple scle-

rosis: 2005 revisions to the “McDonald Criteria”. Ann. Neurol. 2005. 58:

840–846.

Abbreviation: BAFF: B-cell-activating factor of the TNF family · HD:

healthy donor · RRMS: relapsing remitting MS

Full correspondence: Dr. Eliana Marina Coccia, Department ofInfectious, Parasitic and Immune-mediated Diseases, IstitutoSuperiore di Sanita, Viale Regina Elena 299, 00161 Rome, ItalyFax: +39-06-49903638E-mail: [email protected]

Received: 28/11/2012Revised: 19/3/2013Accepted: 25/4/2013Accepted article online: 2/5/2013


IFN-β therapy modulates B-cell and monocyte crosstalk via TLR7 in multiple sclerosis patients

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Transcript of IFN-β therapy modulates B-cell and monocyte crosstalk via TLR7 in multiple sclerosis patients