Loss of Braking Signals During Inflammation: a Factor Affecting The

ORIGINAL CONTRIBUTION

ONLINE FIRST Loss of Braking Signals During Inflammation A Factor Affecting the Development and Disease Course of Multiple Sclerosis

Francesca Gilli, PhD; Nicole De´sire´e Navone, MSc; Simona Perga, PhD; Fabiana Marnetto, MSc; Marzia Caldano, PharmD; Marco Capobianco, MD; Annalisa Pulizzi, MD; Simona Malucchi, MD; Antonio Bertolotto, MD

Background: In a recent genome-wide transcriptional Main Outcome Measures: Gene expression levels, analysis, we identified a gene signature for multiple scle- relapse rate, and change in Expanded Disability Status rosis (MS), which reverted back to normal during preg- Scale. nancy. Reversion was particularly evident for 7 genes: SOCS2, TNFAIP3, NR4A2, CXCR4, POLR2J, FAM49B, and Results: We found a dysregulated gene pathway STAG3L1, most of which encode negative regulators of (PՅ.006), with a downregulation of genes encoding inflammation. negative regulators. The SOCS2, NR4A2, and TNFAIP3 genes were inversely correlated with both relapse rate Objectives: To corroborate dysregulation of genes, to Յ evaluate the prognostic value of genes, and to study modu- (P .002) and change in Expanded Disability Յ lation of genes during different treatments. Status Scale (P .005). SOCS2 was modulated by both interferon beta and glatiramer acetate, TNFAIP3 was Design: Comparison study. modulated by glatiramer acetate, and NR4A2 was not altered at all. No changes were induced by natali- Setting: Italian referral center for MS. zumab.

Patients: Quantitative polymerase chain reaction mea- Conclusions: We demonstrate that there is a new mo- surements were performed for 274 patients with MS and lecular pathogenic mechanism that underlies the initia- 60 healthy controls. Of the 274 patients with MS, 113 tion and progression of MS. Defects in negative- were treatment-naive patients in the initial stages of their feedback loops of inflammation lead to an overactivation disorder who were followed up in real-world clinical set- of the immune system so as to predispose the brain to tings and categorized on the basis of disease course. The inflammation-sensitive MS. remaining 161 patients with MS received disease- modifying therapies (55 patients were treated with interferon beta, 52 with glatiramer acetate, and 54 with na- Arch Neurol. 2011;68(7):879-888. Published online talizumab) for a mean (SD) of 12 (2) months. March 14, 2011. doi:10.1001/archneurol.2011.32

O INVESTIGATE THE MECHA- were are as follows: suppressor of cyto- nisms behind the regula- kine signaling 2 (SOCS2); tumor necro- tion of inflammation in sis factor, ␣-induced protein 3 (TNFAIP3); Author Affiliations: Clinical multiple sclerosis (MS), we nuclear receptor subfamily 4, group A, Neurobiology Unit, recently studied a physi- member 2 (NR4A2); chemokine (C-X-C Neuroscience Institute Cavalieri ologicalT transitory condition (ie, preg- motif) receptor 4 (CXCR4); the poly- Ottolenghi (Drs Gilli, Navone, nancy) in which the immune system merase (RNA) II (DNA directed) polypep- Perga, Marnetto, Caldano, and strengthens its anti-inflammatory compo- tide J, 13.3kDa (POLR2J); family with se- Bertolotto), Struttura nent1 and the disease frequently goes into quence similarity 49, member B (FAM49B); Complessa Dipartimentale remission.2,3 In particular, we conducted and stromal antigen 3-like 1 (STAG3L1). Ospedaliera: Neurology 2, a genome-wide transcriptional analysis of All genes, with the exception of FAM49B Regional Reference Center for peripheral blood mononuclear cells and STAG3L1, were previously reported Multiple Sclerosis (Drs Gilli, (PBMCs) obtained from women with MS to be associated with autoimmune dis- Navone, Perga, Marnetto, and healthy women before, during, and af- eases and are involved in the regulation Caldano, Capobianco, Pulizzi, 4 5-9 Malucchi, and Bertolotto), ter gestation. We identified 7 genes whose of inflammatory processes. University Hospital San Luigi expression was altered in nonpregnant pa- Even considering that many genes are Gonzaga, Orbassano (Torino), tients, and whose expression reverted to involved in inflammatory responses and Italy. “normal” during gestation. The 7 genes that 3 of the 7 genes do not have a clearly

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©2011 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/28/2021 Table 1. Demographic and Clinical Characteristics of Patients at Baseline and During Therapy

IFN-␤–Treated Patients Treatment-Naive NAT-Treated GA-Treated −Characteristic Patients Patients Patients NAb؉ NAb Sample size, No. 113 54 52 20 35 Sex, No. F8741361124 M261316911 RRMS/CIS 111/2 54/0 52/0 20/0 35/0 Age at start of therapy, y, median (range) 35 (17-65) 34 (18-55) 38 (13-58) 36 (24-59) 38 (24-59) Disease duration at start of therapy, mo, median (range) 17 (2-39) 108 (4-363) 68 (4-359) 27 (1-139) 61 (1-256) EDSS score at start of therapy, median (range) 1.0 (0-2.0) 3.5 (1.5-7.0) 1.5 (0-5.5) 1.5 (0-3.0) 1.5 (0-3.5) RR 2 y before therapy, median (range) 1.1 (0-4) 2.5 (2-6) 1.3 (0-3) 1.2 (0-2) 1.1 (0-2) EDSS score 1 y after therapy, median (range) 3.0 (1.0-7.0) 1.5 (0-5.5) 1.5 (0-3.0) 1.0 (0-3.5) RR 1 y after therapy, median (range) 0.2 (0-2) 0.2 (0-2) 0.2 (0-2) 0.1 (0-1)

Abbreviations: CIS, clinically isolated syndrome; EDSS, Extended Disability Status Scale; GA, glatiramer acetate; IFN-␤, interferon beta; NAb, neutralizing antibodies; NAT, natalizumab; RR, relapse rate; RRMS, relapsing-remitting multiple sclerosis.

defined role in inflammation (POLR2J encodes a sub- METHODS unit of RNA polymerase II, whereas FAM49B and STAG3L1 encode molecules of yet unknown function), PARTICIPANTS it is impressive that the remaining genes encode anti- inflammatory effectors, being indeed downregulated in Our study was approved by the ethical committee of our hos- patients with MS. TNFAIP3 is critical in terminating tu- pital and conducted according to the Declaration of Helsinki. mor necrosis factor–induced NF-␬B responses.10-12 NR4A2 Informed consent was obtained from each individual. A total functions as an inhibitor of inflammatory gene expres- of 274 patients (199 women and 75 men) with definite relapsing- sion,13 SOCS2 inhibits signal transduction induced by cy- remitting MS19 were enrolled. Of these 274 patients, 113 (87 tokines,14 and CXCR4 regulates the trafficking of T- women and 26 men) had never been treated with DMT before regulatory cells.15 Thus, data suggest that, besides the being enrolled, and 161 (112 women and 49 men) had been known molecular mechanisms underlying overactive in- treated with DMT. Of the 161 patients who had had DMT, 55 flammation in MS, there might be an alteration in mecha- were treated with interferon beta (20 patients with Avonex [Bio- gen-Idec, Cambridge, Massachusetts]; 18 patients with Rebif nisms that act to counter-regulate or resolve inflamma- [Merck-Serono, Geneva, Switzerland]; and 17 patients with Be- tory responses. Accordingly, similar mechanisms were taferon [Bayer Schering, Berlin, Germany]), 54 were treated with identified as relevant to the pathology of other neurode- natalizumab (Tysabri; Biogen-Idec, Cambridge, Massachu- generative diseases.16 On the other hand, although in- setts), and 52 were treated with glatiramer acetate (Copaxone; ducers of inflammation may be generated in a disease- sanofi-aventis, Paris, France). Patients were treated for a mean specific manner, there is evidence for a remarkable (SD) duration of 12 (2) months (Table 1). convergence in the mechanisms responsible for the sens- Patients treated with interferon beta were either positive ing and amplification of inflammatory processes that re- (n=20) or negative (n=35) for anti-interferon beta neutraliz- 16 ing antibodies (NAb), whereas all natalizumab-treated pa- sult in the production of neurotoxic mediators. 20,21 In our study, we first aimed at corroborating, in dif- tients were negative for antinatalizumab NAb. Sixty age- matched healthy individuals (39 women and 21 men) were ferent cohorts of subjects, our finding that SOCS2, included as controls. TNFAIP3, NR4A2, CXCR4, POLR2J, FAM49B, and STAG3L1 are dysregulated in patients with MS. To over- CLINICAL FOLLOW-UP AFTER BASELINE come possible epitope-spreading phenomena,17,18 we ana- EVALUATION OF GENE EXPRESSION lyzed an extremely homogeneous cohort of untreated patients with MS in the initial stages of their disorder. Baseline samples were obtained from the treatment-naive co- Furthermore, because our previous gene expression pro- hort during a 4-year period (ie, from October 2004 to Novem- files were analyzed in a female cohort only, we checked ber 2008). After blood samples were obtained, patients were for possible sex-specific differences by comparing gene clinically followed up in our MS center, for at least 2 years and expression profiles in men and women. in real-world clinical settings. For routine clinical monitor- Our second aim was to evaluate the possible prognos- ing, subjects were required to visit the clinic for a baseline evalu- tic value of the 7 genes for the clinical evolution of MS. ation and, every 3 months thereafter, for an evaluation of blood Hence, we analyzed the relationship between gene ex- parameters and to determine whether there were any adverse pression and clinical outcomes, with respect to disabil- effects. Neurologic examinations, with classification using the ity progression and relapse rates (RRs). Finally, we com- Expanded Disability Status Scale (EDSS) and the recording of relapses, were performed every 6 months or in case of need. A pared the specific immunoregulatory gene pathway with relapse was defined as the appearance of a new symptom or a the targeted immunomodulation that appears to be as- worsening of a preexisting symptom, lasting more than 24 hours sociated with some differentially acting disease- and producing a modification in the functional system of the modifying therapies (DMTs), such as interferon beta, gla- EDSS.22 All relapses were treated with high-dose methylpred- tiramer acetate, and natalizumab. nisolone hemisuccinate sodium (15 mg/kg for 10 days).

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73 Patients still 8 Patients discontinued 17 Patients switched 3 Patients switched to 8 Patients switched to 4 Patients converted treated with either treatment because to NAT because of MITO because either AZA or METH to SPMS IFN-β or GA they wish to become aggressive failure of aggressive failure because of adverse pregnant of first-line DMT of first-line DMT events

Group A: Group B: 81 Patients with 20 Patients with nonaggressive aggressive course course of MS of MS

Figure 1. Patient groups with regard to real-world clinical management at baseline and follow-up. AZA indicates azathioprine sodium; DMT, disease-modifying therapy; GA, glatiramer acetate; IFN-␤, interferon beta; METH, methotrexate sodium; MITO, mitoxantrone hydrochloride; MS, multiple sclerosis; and NAT, natalizumab.

After confirmed diagnosis of relapsing-remitting MS, all pa- baseline in the EDSS score per year (⌬EDSS). All statistical analy- tients received first-line DMT (ie, 100 patients received inter- ses were performed with GraphPad PRISM version 4.00 (San Di- feron beta, and 13 patients received glatiramer acetate). At the ego, California). Data were evaluated by means of the Kruskal- end of the follow-up (ie, November 2010), there were 73 pa- Wallis test and the Mann-Whitney U test. Nonlinear regression tients (64.6%) still being treated with either interferon beta or analysis was conducted to determine the relation between mes- glatiramer acetate, whereas 8 patients (7.1%) discontinued treat- senger RNA (mRNA) levels and both RR and ⌬EDSS. All re- ment because they wanted to become pregnant. As a whole, these ported P values were based on 2-tailed statistical tests, with an ␣ 81 patients (71.7%) were considered to have a “nonaggres- level of .05 indicating the threshold of statistical significance. sive” form of the disease. On the contrary, 20 patients (17.7%) were categorized as having first-line DMT that failed completely, because they had experienced at least 2 relapses dur- RESULTS ing the prior year. These patients were switched to second- line treatments, such as natalizumab (n=17) or mitoxantrone UNTREATED PATIENTS WITH MS hydrochloride (n=3), and were considered to have an “aggres- AND HEALTHY CONTROLS sive” form of the disease (Figure 1). First-line DMT had also failed in a second group of 8 patients (7.1%), mostly because of adverse events; these patients Deregulated gene expression was observed in untreated were switched to alternative treatments such as methotrexate patients with MS compared with healthy controls sodium (n=1) or azathioprine sodium (n=7). Also, it was de- (PՅ.007). In particular, a lower mRNA expression of termined during follow-up that 4 patients (3.5%) had devel- CXCR4, SOCS2, TNFAIP3, and NR4A2 was observed in oped secondary progressive MS. These 12 patients were ex- untreated patients with MS (PϽ.001), whereas a higher cluded from the present analysis owing to the possible different mRNA expression of FAM49B, POLR2J, and STAG3L1 was phenotypes and the paucity of subjects in these series. observed in healthy controls (PՅ.007) (Figure 2). To account for potential sex-related differences in gene ex- RNA EXTRACTION AND pression, we also compared mRNA levels in men and MESSENGER RNA QUANTIFICATION women: sex-related differences did not reach statistical significance, considering both untreated patients with MS Whole blood samples, collected into a Tempus vacuette, were (PՆ.08) and healthy controls (PՆ.16). extracted using the ABI Prism 6100 Nucleic Acid PrepStation (Applied Biosystems, Foster City, California), following the manufacturer’s instructions. Total RNA (final concentration, RELATIONSHIP BETWEEN GENE EXPRESSION 5 ng/µL) was reverse-transcribed using random hexamer prim- AND CLINICAL OUTCOME AT FOLLOW-UP ers. Complementary DNA was then used as a template for a blind real-time reverse transcription–polymerase chain reac- First, we confirmed the definition of “aggressive” and “non- Ј tion analysis based on a 5 -nuclease assay. Expression of CXCR4, aggressive” for delineating different clinical courses in pa- TNFAIP3, SOCS2, NR4A2, FAM49B, POLR2J, and STAG3L1 was tients; both the RR and ⌬EDSS were higher in patients who analyzed using Applied Biosystems’ TaqMan gene expression were switched to natalizumab or mitoxantrone therapy, products. Relative expression levels of targets were calculated by the comparative cycle threshold method and by normaliz- compared with patients who were still treated with inter- ing to glyceraldehyde-phosphate-dehydrogenase. feron beta or glatiramer acetate and patients who wanted to become pregnant (PՅ.004) (Table 2). STATISTICAL ANALYSIS To examine whether the dysregulated expression of the genes was associated with a worse clinical course, we Clinical analyses were performed in which we calculated an RR, compared mRNA levels in aggressive and nonaggres- defined as the number of confirmed relapses per patient-year, and sive clinical courses in patients. The comparison showed a rate of disease progression measured by the mean change from that the levels of gene expression for SOCS2 (P=.04),

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©2011 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/28/2021 A B 2.0 0.007 1.8 0.006 1.6 P = .21 1.4 0.005 P = .19 P = .13 1.2 0.004 1.0 0.003 0.8 ‡ † ∗ ‡ ∗ ‡ Relative Expression Relative Expression 0.6 ‡ 0.002 0.4

CXCR4 SOCS2 0.001 0.2 0.0 0.000

C D 3.5 0.10

3.0 0.08 2.5 P = .14 2.0 0.06 † ‡ † ‡ ‡ ‡ ‡ 1.5 0.04 Relative Expression Relative Expression ‡ 1.0 ‡ 0.02 NR4A2

TNFAIP3 0.5

0.0 0.00

E F 0.5 1.4

1.2 ‡ 0.4 † † ‡ † 1.0 ‡ 0.3 = 0.8 = P .23 P .25 P = .26 0.2 0.6 Relative Expression Relative Expression P = .15 0.4 0.1 POLR2J FAM49B 0.2

0.0 0.0

β β

– Patients + Patients Patients b G With GA With NAT NA NAb 1.0 Healthy Controls Treatment-Naive Patients Treated Patients Treated Treated With IFN- Treated With IFN- † 0.8 † † 0.6 †

Relative Expression 0.4 P = .23

0.2 STAG3L1 STAG3L1

0.0

β β

– Patients + Patients Patients With GA With NAT NAb NAb Healthy Controls Treatment-Naive Patients Treated Patients Treated

Treated With IFN- Treated With IFN-

Figure 2. Comparison of mean gene expression levels of (A) CXCR4, (B) SOCS2, (C) TNFAIP3, (D) NR4A2, (E) FAM49B, (F) POLR2J, and (G) STAG3L1 among 60 healthy controls, 113 treatment-naive patients, and 161 patients with multiple sclerosis (MS) who received disease-modifying therapy (55 with interferon beta [IFN-␤], 54 with natalizumab [NAT], and 52 with glatiramer acetate [GA]). Such an analysis disclosed dysregulation for the 7 genes in treatment-naive patients compared with healthy controls (PՅ.007). Treatment with NAT leads to the reversion in expression of 1 of 7 genes (ie, FAM49B ). Treatment with GA therapy leads to the reversion in expression of 5 of 7 genes (ie, TNFAIP3, SOCS2, FAM49B, POLR2J, and STAG3L1). Treatment with IFN-␤ leads to the reversion in expression of 3 of 7 genes (ie, CXCR4, FAM49B, and SOCS2); the latter regulation was observed in patients negative for neutralizing antibodies (NAb−) but not in patients positive for NAb (NAbϩ). *PՅ.05, †PՅ.01, and ‡PՅ.001. P values are calculated for differences between healthy controls and patients with MS (both untreated patients and patients receiving DMT).

NR4A2 (P=.003), and TNFAIP3 (P=.001) were lower in the level of gene expression for POLR2J was higher in pa- patients who had an aggressive course compared with patients who had an aggressive course compared with patient who had a nonaggressive course. On the contrary, tients who had a nonaggressive course (P=.05). On the

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©2011 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/28/2021 Table 2. Demographic and Clinical Characteristics of Patients at Baseline and at the End of Follow-up

Patients Patients Who Wanted Patients Patients Treatment-Naive Treated With to Become Treated With Treated With Patients Who Characteristic Patients GA or IFN-␤ Pregnant NAT or MITO AZA or METH Developed SPMS Sample size, No. 113 73 8 20 8 4 Sex F876181422 M26120662 Treatment at follow-up 18 received GA, 17 received NAT, 7 received AZA, 2 received AZA, 55 received IFN-␤ 3 received MITO 1 received METH 1 received IFN-␤, 1 received MITO Age at sampling, y, median (range) 35 (17-65) 35 (17-65) 30 (26-35) 27 (17-45) 37 (19-50) 42 (35-60) Disease duration at sampling, 17 (2-39) 18 (3-39) 17 (10-25) 10 (2-23) 20 (8-29) 25 (21-30) mo, median (range) EDSS score at sampling, median (range) 1.0 (0-2.0) 1.0 (0-1.5) 0 (0-1.0) 1.0 (1.0-2.0) 1.0 (0-2.0) 1.5 (0-2.0) RR 1 y before sampling, median (range) 1.1 (0-4) 1.0 (0-3) 1.5 (0-3) 2.0 (0-4) 1.0 (0-3) 1.5 (1.0-2.0) ⌬EDSS, median (range) 1.0 (0-4.0) 0 (0-2.0) 2.0 (1.0-4.5)a 1.0 (0-2.5) 2.0 (1.0-4.0) RR, median (range) 0.1 (0-1.3) 0.3 (0-0.6) 1.3 (0.5-6.7)a 0.2 (0-0.6) 0.2 (0-1.3)

Abbreviations: AZA, azathioprine sodium; EDSS, Extended Disability Status Scale; GA, glatiramer acetate; IFN-␤, interferon beta; METH, methotrexate sodium; MITO, mitoxantrone hydrochloride; NAT, Natalizumab; RR, relapse rate; SPMS, secondary progressive multiple sclerosis; and ⌬EDSS, annualized changes in EDSS score. a The ⌬EDSS and RR at follow-up were significantly higher (both PՅ.004) in patients who were switched to NAT and MITO therapy compared with the group of patients still treated with IFN-␤ or GA or patients who wanted to become pregnant.

other hand, the levels of gene expression for FAM49B ap- PATIENTS TREATED proached statistical significance (P=.06), whereas no dif- WITH GLATIRAMER ACETATE ferences in gene expression for CXCR4 and STAG3L1 were detected in the 2 groups of patients (Figure 3). After- In patients treated with glatiramer acetate, the level of ward, we examined the relationship between baseline expression of 5 of 7 genes was shown to be affected by mRNA levels and both RR and ⌬EDSS. Interestingly, the therapy. In particular, TNFAIP3, SOCS2, FAM49B, levels of gene expression for SOCS2, NR4A2, and TNFAIP3 POLR2J, and STAG3L1 reverted their expression to lev- were inversely correlated with both RR and ⌬EDSS. How- els displayed by healthy controls (PՆ.06), whereas glati- ever, variables needed to be log-transformed because the ramer acetate did not affect either NR4A2 or CXCR4 ex- relationship between untransformed variables appeared pression (Figure 2). No differences in gene expression nonlinear. After log transformation of variables, the cor- were observed between men and women. relation coefficient for ⌬EDSS was −0.423 (P=.005) when related to SOCS2 expression, −0.597 (PϽ.001) when re- PATIENTS TREATED WITH NATALIZUMAB lated to NR4A2 expression, and −0.618 (PϽ.001) when related to TNFAIP3 expression (Figure 4). Likewise, cor- In patients treated with natalizumab, 1 of 7 genes (ie, relation coefficients for RRs were −0.392 (P=.002), −0.517 Ͻ Ͻ Ͻ FAM49B) reverted its expression to normal (P .001); (P .001), and −0.586 (P .001), respectively these natalizumab-treated patients maintained the same (Figure 5). levels of CXCR4, POLR2J, NR4A2, STAG3L1, and SOCS2 compared with untreated patients (Figure 2). On the con- PATIENTS TREATED WITH INTERFERON BETA trary, despite the initial downregulation of TNFAIP3, the gene was further downregulated in patients who re- Neutralizing antibodies are capable of abrogating both 20,23,24 25-27 ceived natalizumab therapy (P=.004) compared with un- the biological and clinical action of interferon treated patients (Figure 2C). No significant differences beta. Hence, to study the effect of interferon beta on gene were observed between men and women. expression, we performed analyses on 2 groups of interferon beta–treated patients: NAb-positive and NAb- negative patients. COMMENT Following interferon beta treatment, in NAb- negative patients, 3 of 7 genes (ie, SOCS2, CXCR4, and In a recent genome-wide transcription analysis compar- FAM49B) reverted to expression levels displayed by ing PBMCs from patients with MS and healthy controls healthy controls (PՆ.20). This was not the case for the before, during, and after pregnancy, we have identified remaining genes, which had levels of expression that did 7 genes (ie, CXCR4, SOCS2, TNFAIP3, NR4A2, FAM49B, not change during therapy. By contrast, in NAb- STAG3L1 and POLR2J) whose expression was dysregu- positive patients, expression was comparable to levels de- lated before gestation and reverted by the pregnancy protected in untreated patients with MS (PՅ.04) (Figure 2). cess.4 Of these 7 genes, 3 (ie, POLR2J, FAM49B, and No differences in gene expression were observed be- STAG3L1) do not have a clearly defined role in inflam- tween men and women in both groups. mation, whereas 4 genes (ie, CXCR4, SOCS2, TNFAIP3,

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1.0 0.01 Relative Expression Relative Expression 0.1 0.001 SOCS2 CXCR4

0.01 0.0001

C D 10.0 0.1

1.0 0.01 Relative Expression 0.1 Relative Expression 0.001 NR4A2 TNFAIP3

0.01 0.0001

E F 10.0 10.0

1.0 1.0 Relative Expression 0.1 Relative Expression 0.1 POLR2J FAM49B

0.01 0.01 Aggressive Nonaggressive Controls

G 10.0

Figure 3. Comparison of mean gene expression levels of (A) CXCR4, (B) SOCS2, (C) TNFAIP3, (D) NR4A2, (E) FAM49B, 1.0 (F) POLR2J, and (G) STAG3L1 in healthy controls and in patients with multiple sclerosis who were subdivided into “aggressive” and “nonaggressive” on the basis of their

Relative Expression clinical course after providing a blood sample. Horizontal 0.1 bars indicate median values. STAG3L1

0.01 Aggressive Nonaggressive Controls

and NR4A2) encode negative regulators of inflamma- autoimmunity. Accordingly, in our previous study,4 we tory responses.10-15,28 Previous studies had already shown had observed markedly decreased levels of CXCR4, SOCS2, a consistent association between autoimmune diseases TNFAIP3, and NR4A2 expression in patients with MS, (eg, rheumatoid arthritis, systemic lupus erythemato- which might contribute to the chronic invasive immune sus, and ankylosing spondylitis) and the latter 4 genes.5-9 processes in these patients, resulting from an initial ab- Hence, these genes have been proposed to be critical errant inflammation. The obvious rationale for such an “braking” molecules, acting to control inflammation in observation is that, in patients with MS (and probably

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©2011 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/28/2021 A B 0.010 – 1.0 r = – 0.423 0.009 – 1.5 0.008 – 2.0 0.007 0.006 – 2.5 0.005 – 3.0 SOCS2 0.004 – 3.5 Log Relative Expression 0.003 – 4.0 0.002 SOCS2 0.001 – 4.5 0.000 – 5.0

C D 0.045 0.0 = – 0.040 – 0.5 r 0.597

0.035 – 1.0 – 1.5 0.030

– 2.0 0.025 – 2.5

0.020 NR4A2 – 3.0 Log Relative Expression 0.015 – 3.5 0.010 – 4.0 NR4A2 0.005 – 4.5 0.000 – 5.0

E F 4.0 1.5 r = – 0.618 3.5 1.0

3.0 0.5

2.5 0.0

2.0 – 0.5 TNFAIP3 1.5 – 1.0 Relative Expression Log 1.0 – 1.5

TNFAIP3 0.5 – 2.0

0.0 – 2.5

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 – 1.25 – 1.00 – 0.75 – 0.50 – 0.25 0.00 0.25 0.50 0.75 ΔEDSS Log ΔEDSS

Figure 4. Relationships between annualized changes in Expanded Disability Status Scale score (⌬EDSS) and baseline messenger RNA levels of SOCS2 (A and B), NR4A2 (C and D), and TNFAIP3 (E and F) in 101 patients with multiple sclerosis. B, D, and F, Correlations between ⌬EDSS and gene expression after log transformation of nonnormally distributed variables. PՅ.005 for all correlations.

in patients with autoimmune diseases overall), there is flammation is still highly prevalent29 and the MS im- a dysregulation of molecular mechanisms underlying anti- mune response more homogenous.18 This strict selection inflammatory phenotypes, leading to an overactivation process allowed us to conclude that the altered expres- of the immune system. sion of negative regulators of inflammation is a central As a whole, the results of our present study, analyz- pathogenic mechanism and not only a consequence of ing different sets of treatment-naive patients with MS and the disease progression. On the other hand, this finding healthy controls, confirm this specific gene dysregula- agrees with the finding of a silent MS trait that is asso- tion in MS. Given the immunological variables in MS, it ciated with suppressed expression of critical negative- is significant that the present results stem from the analy- feedback effectors such as nuclear receptors (including sis of an extremely homogeneous cohort of patients in NR4A2),30 which regulate induction and resolution of in- the initial stages of MS. Indeed, all of the patients were flammatory responses, specifically through transrepres- in the initial stages of their disorder, and their levels of sion mechanisms in macrophages and microglia.31 neurological disability were still low. Therefore, pa- Although men and women have genetically identical tients were at a specific stage of the disease at which in- autosomal chromosomes, they could result in sex-

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©2011 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/28/2021 A B 0.010 – 1.0 r = – 0.392 0.009 – 1.5 0.008 – 2.0 0.007 0.006 – 2.5 0.005 – 3.0 SOCS2 0.004 – 3.5 Log Relative Expression 0.003 – 4.0 0.002 SOCS2 0.001 – 4.5 0.000 – 5.0

C D 0.045 – 1.0 r = – 0.517 0.040 – 1.5 0.035 – 2.0 0.030

– 2.5 0.025 – 3.0

0.020 NR4A2 – 3.5 Log Relative Expression 0.015 – 4.0 0.010 NR4A2 0.005 – 4.5

0.000 – 5.0

E F 5.5 1.5 5.0 r = – 0.586 1.0 4.5 4.0 0.5 3.5 0.0 3.0 – 0.5

2.5 TNFAIP3 – 1.0 Relative Expression 2.0 Log 1.5 – 1.5 1.0 TNFAIP3 – 2.0 0.5 0.0 – 2.5

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 – 1.25 – 1.00 – 0.75 – 0.50 – 0.25 0.00 0.25 0.50 0.75 1.00 RR Log RR

Figure 5. Relationships between relapse rate (RR) and baseline messenger RNA levels of SOCS2 (A and B), NR4A2 (C and D), and TNFAIP3 (E and F) in 101 patients with multiple sclerosis. B, D, and F, Correlations between RR and gene expression after log transformation of nonnormally distributed variables. PՅ.002 for all correlations.

specific transcriptional regulation leading to differential files of 113 treatment-naive patients, who, after provid- mRNA products for some genes. Because, in our previ- ing blood samples, were followed up in real-world clini- ous study,4 we analyzed gene expression profiles in women cal settings. In particular, we examined whether there was only, in our present study, we also evaluated possible sex- a relationship between baseline expression and disease related differences, comparing gene expression patterns course, as measured by RR and disease progression. between men and women. Although most of the target Clearly, operational criteria were proposed for differen- genes have been reported to be regulated by estro- tiating clinical courses into aggressive and nonaggres- gens,32,33 we did not find evidence of sex-related differ- sive. Nevertheless, we believe that the group of patients ences in gene expression. defined as having an aggressive disease course is actu- Because detection of differentially regulated genes may ally characterized by a different phenotype of MS. In fact, lead to the identification of new therapeutic targets, we these patients have experienced at least 2 relapses in the next asked whether our gene expression findings could prior 12 months, despite their having received immu- have any clinical relevance for patients with MS. To this nomodulating treatment. This worse disease course is end, we have further analyzed the gene expression pro- likely due to pathogenic mechanisms underlying a par-

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©2011 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/28/2021 ticularly strong inflammation that is not responding to integrin antibody is thought to block leukocyte migra- both interferon beta and glatiramer acetate. Accord- tion across endothelial cells,38 thus changing the ecol- ingly, a recent study34 found an enhanced type I inter- ogy of the cell entry into the brain rather than modulating feron signature in monocytes from interferon beta– their anti-inflammatory actions. Natalizumab-treated pa- treated nonresponsive patients who also had a more tients consistently showed a molecular modulation lim- aggressive disease course. ited to the unknown FAM49B gene. On the contrary, the As a whole, the results of our present study show that already downregulated expression of the TNFAIP3 gene altered gene expression correlates with clinical features. was shown to be further lowered in natalizumab-treated In particular, our findings indicate a correlation be- patients compared with treatment-naive patients. The tween reduced expression of SOCS2, NR4A2, and downregulation of TNFAIP3 may reflect the increase of TNFAIP3 and a worse disease course. Baseline levels were interleukin 2 activation of PBMCs, as observed in pa- indeed lower in patients with a more malignant disease tients during natalizumab therapy.39,40 course than in patients with a more benign disease course, Our findings clearly show that there are partial dif- and also negatively correlated with both RR and ⌬EDSS. ferential overlapping mechanisms of protection be- Given this clinical correlation, it is important to note the tween DMT and pregnancy, because each of the drugs key role played by those genes in critical negative- leads to the reversion in expression of some, but not all, feedback loops of inflammation. SOCS2 is a well- of the pregnancy-related genes. The importance of modu- known negative regulator of cytokines that signal through lating those genes to obtain a clinical improvement is un- the Jak-STAT pathway.14 Likewise, both TNFAIP3 and derlined by our previous observation that women with NR4A2 are part of negative-feedback loops that mediate MS who relapse during pregnancy have a delayed modu- their inhibitory function by downregulating proinflam- lation of gene expression compared with relapse-free matory signaling pathways, including those controlling women. NF-␬B10-12 and IRF3-dependent gene expression.12 NR4A2 In conclusion, our findings suggest that there is a new also functions as an inhibitor of neurotoxic gene expres- molecular pathogenic mechanism that underlies the ini- sion in microglia and astrocytes via a CoREST- tiation and progression of MS. Defects in the negative regu- dependent transrepression pathway.13,16,31 Taken to- lation of inflammatory responses lead to an overactiva- gether, these findings corroborate our initial statement tion of the immune system so as to predispose certain that MS arises from a dysregulation of “braking signals” organ systems to inflammation-sensitive pathologies such in inflammation, rather than merely from an overactive as MS. All this information can be used to design pow- proinflammatory reaction. This observation offers a clear erful new diagnostic tools and can also be used to iden- model by which to consider the clinical data presented tify new therapeutic targets. here: with lower SOCS2, NR4A2, and TNFAIP3 gene expression levels, one can expect worse inflammation and Accepted for Publication: January 4, 2011. thus a worse clinical course. Published Online: March 14, 2011. doi:10.1001 A third aim of our study was to compare gene expres- /archneurol.2011.32 sion with the immunomodulation that appears to be as- Correspondence: Francesca Gilli, PhD, Clinical Neuro- sociated with common DMTs (namely, interferon beta, biology Unit, Neuroscience Institute Cavalieri Otto- glatiramer acetate, and natalizumab). With respect to the lenghi, University Hospital San Luigi Gonzaga, Regione interferon beta–treated patients, we observed a modula- Gonzole 10, I-10043 Orbassano (Torino) Italy (francesca tion of 3 genes (ie, CXCR4, SOCS2, and FAM49B) that [email protected]). reverted to expression levels displayed by healthy con- Author Contributions: Study concept and design: Gilli and trols. Evidence of an actual effect of interferon beta is given Bertolotto. Acquisition of data: Gilli, Perga, Caldano, Ca- by previous studies that have already shown CXCR4 and pobianco, and Malucchi. Analysis and interpretation of data: SOCS2 to be upregulated by interferon alpha.35,36 A fur- Gilli, Navone, Perga, Marnetto, Caldano, Pulizzi, Maluc- ther confirmation is given by the separate analysis of NAb- chi, and Bertolotto. Drafting of the manuscript: Gilli and positive and NAb-negative patients; although a specific Bertolotto. Critical revision of the manuscript for impor- gene modulation was seen in interferon beta–treated, NAb- tant intellectual content: Gilli, Navone, Perga, Marnetto, negative patients, the effect was completely abolished in Caldano, Capobianco, Pulizzi, Malucchi, and Berto- the treated NAb-positive patients, supporting the spe- lotto. Statistical analysis: Gilli and Malucchi. Obtained fund- cific effect of interferon beta on the target genes. ing: Gilli and Bertolotto. Administrative, technical, and ma- Glatiramer acetate is another well-established treat- terial support: Gilli, Navone, Perga, Marnetto, Caldano, ment for relapsing-remitting MS. Herein, glatiramer ac- Capobianco, and Pulizzi. Study supervision: Gilli and Ber- etate was shown to reverse the functional defect of tolotto. TNFAIP3, SOCS2, FAM49B, POLR2J, and STAG3L1.To Financial Disclosure: Drs Gilli, Caldano, Capobianco, our knowledge, there are no published data to date link- Pulizzi, Malucchi, and Bertolotto report that they have ing glatiramer acetate therapy to the regulation of those been reimbursed by Merck-Serono, sanofi-aventis, Bio- genes, and it has been previously reported that glati- gen Dompè, Novartis, and Bayer Schering for attending ramer acetate does not affect CXCR4 expression37 ex- several conferences. Drs Gilli, Marnetto, Caldano, Ca- actly as observed here. pobianco, Pulizzi, Malucchi, and Bertolotto report that Natalizumab controls inflammation in MS through they have received fees for lectures by Merck-Serono, Bio- mechanisms of action that are different from those found gen Dompè, Biogen Idec, Teva, and sanofi-aventis. Dr Ber- in interferon beta and glatiramer acetate. This anti-␣(4)- tolotto reports that he has received funds for research and

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©2011 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/28/2021 for staff members from Merck-Serono, Biogen Dompè, 17. McMahon EJ, Bailey SL, Castenada CV, Waldner H, Miller SD. Epitope spread- Biogen Idec, Bayer Schering, Novartis, and sanofi- ing initiates in the CNS in two mouse models of multiple sclerosis. Nat Med. 2005; 11(3):335-339. aventis. 18. Kidd BA, Ho PP, Sharpe O, et al. Epitope spreading to citrullinated antigens in Funding/Support: This study was supported by a grant mouse models of autoimmune arthritis and demyelination. Arthritis Res Ther. from the Fondazione Italiana Sclerosi Multipla (FISM con- 2008;10(5):R119. tract 2009/R/14). Financial support was also obtained from 19. Polman CH, Reingold SC, Edan G, et al. Diagnostic criteria for multiple sclero- the Fondazione per la Ricerca Biomedica and from the sis: 2005 revisions to the “McDonald Criteria”. Ann Neurol. 2005;58(6):840- 846. San Luigi Gonzaga ONLUS (Organizzazione Non Lucra- 20. Bertolotto A, Malucchi S, Sala A, et al. Differential effects of three interferon be- tiva di Utilità Sociale). tas on neutralising antibodies in patients with multiple sclerosis: a follow up study Role of the Sponsor: Both the sponsors and the phar- in an independent laboratory. J Neurol Neurosurg Psychiatry. 2002;73(2):148- maceutical companies had no role in the design and con- 153. 21. Calabresi PA, Giovannoni G, Confavreux C, et al; AFFIRM and SENTINEL Inves- duct of the study; in the collection, analysis, and inter- tigators. The incidence and significance of anti-natalizumab antibodies: results pretation of the data; or in the preparation, review, or from AFFIRM and SENTINEL. Neurology. 2007;69(14):1391-1403. approval of the manuscript. 22. Schumacker GA, Beebe G, Kibler RF, et al. Problems of experimental trials of Additional Contributions: We thank Rita Guerrieri, RNC, therapy in multiple sclerosis: report by the panel on the evaluation of experi- Marina Panealbo, RNC, Giuliana Savoldi, RNC, and mental trials of therapy in multiple sclerosis. AnnNYAcadSci. 1965;122: 552-568. Angela Zaccaria, RNC, for their nursing assistance dur- 23. Bertolotto A, Gilli F, Sala A, et al. Persistent neutralizing antibodies abolish the ing our study. We also thank Anna Messina, MSEd, and interferon ␤ bioavailability in MS patients. Neurology. 2003;60(4):634-639. Daniele Dell’Anna for their excellent administrative 24. Gilli F, Bertolotto A, Sala A, et al. Neutralizing antibodies against IFN-beta in mul- support. tiple sclerosis: antagonization of IFN-beta mediated suppression of MMPs. Brain. 2004;127(Pt 2):259-268. 25. PRISMS Study Group and the University of British Columbia MS/MRI Analysis REFERENCES Group. PRISMS-4: Long-term efficacy of interferon-beta-1a in relapsing MS. 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