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MULT I P LE SCLEROS I S

Genetic variants associated with autoimmunity driveNFkB signaling and responses to inflammatory stimuliWilliam J. Housley,1 Salvador D. Fernandez,1 Kenneth Vera,1 Sasidhar R. Murikinati,1

Jaime Grutzendler,1 Nicole Cuerdon,2 Laura Glick,2 Phillip L. De Jager,2,3 Mitja Mitrovic,1

Chris Cotsapas,1,3,4 David A. Hafler1,3*

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The transcription factor nuclear factor kB (NFkB) is a central regulator of inflammation, and genome-wide associ-ation studies in subjects with autoimmune disease have identified a number of variants within the NFkB signalingcascade. In addition, causal variant fine-mapping has demonstrated that autoimmune disease susceptibility variantsfor multiple sclerosis (MS) and ulcerative colitis are strongly enriched within binding sites for NFkB. We report thatMS-associated variants proximal to NFkB1 and in an intron of TNFRSF1A (TNFR1) are associated with increased NFkBsignaling after tumor necrosis factor–a (TNFa) stimulation. Both variants result in increased degradation of inhibitorof NFkB a (IkBa), a negative regulator of NFkB, and nuclear translocation of p65 NFkB. The variant proximal toNFkB1 controls signaling responses by altering the expression of NFkB itself, with the GG risk genotype expressing20-fold more p50 NFkB and diminished expression of the negative regulators of the NFkB pathway: TNFa-inducedprotein 3 (TNFAIP3), B cell leukemia 3 (BCL3), and cellular inhibitor of apoptosis 1 (CIAP1). Finally, naïve CD4 T cellsfrom patients with MS express enhanced activation of p65 NFkB. These results demonstrate that genetic variantsassociated with risk of developing MS alter NFkB signaling pathways, resulting in enhanced NFkB activation andgreater responsiveness to inflammatory stimuli. As such, this suggests that rapid genetic screening for variantsassociated with NFkB signaling may identify individuals amenable to NFkB or cytokine blockade.

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INTRODUCTION

Nuclear factor kB (NFkB) was one of the first transcription factorsidentified and is a central regulator of inflammation (1). The canonicalp50/p65 NFkB signaling cascade is critical for activation of immune re-sponses downstream of T and B cell receptors, toll-like receptors, andcytokines, including tumor necrosis factor–a (TNFa) and interleukin-1b (IL-1b). Moreover, alterations in NFkB have been associated with bothautoimmune disease and malignancies (2, 3). Inflammatory autoimmunediseases, which reflect complex interactions between genetic variationand environment, are important systems for genetic investigationof human disease. These diseases share a substantial degree of immu-nopathology, with increased activity of autoreactive CD4+ T cellssecreting inflammatory cytokines and loss of regulatory T cell function(4–7). Multiple sclerosis (MS) is one such autoimmune disease wherethere is chronic inflammation in the central nervous system (CNS), withinfiltration of activated mononuclear cells into the CNS that damagesboth myelin and axons. This complex genetic disease is associated withenvironmental factors that appear to drive a predominantly T cell auto-immune response against CNS antigens (8, 9).

Genome-wide association studies (GWAS) and subsequent targetedgenomic studies have identified 97 variants associated with MS sus-ceptibility (10–12). Although each of these variants contributes only asmall increase in the complex phenotype of disease risk, the biologicalfunction associated with individual allelic variants has been striking(13–17). Many of these variants fall within specific signaling cascades,suggesting that alterations in pathways, rather than individual genes,

1Department of Neurology and Immunobiology, Yale School of Medicine, New Haven,CT 06511, USA. 2Program in Translational NeuroPsychiatric Genomics, Department ofNeurology, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA 02115,USA. 3Broad Institute of Harvard University and Massachusetts Institute of Technol-ogy, Cambridge, MA 02142, USA. 4Department of Genetics, Yale School of Medicine,New Haven, CT 06511, USA.*Corresponding author. E-mail: david.hafler@yale.edu

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may be the key to understanding how individual variants with smallodds ratios result in disease susceptibility (18–20). About 17% (17 of 97)of MS susceptibility variants identified by GWAS fall either within orproximal to NFkB signaling genes, including variants proximal toNFkB1 itself and within TNF receptor 1 (TNFR1) (10, 12, 21).

We recently integrated genetic and epigenetic fine-mapping to iden-tify potentially causal variants in autoimmune disease–associated lociand explore their functions by generating cis-regulatory element mapsfor a spectrum of immune cell types. About 60% of likely causal var-iants map to enhancer-like elements, with preferential enrichment instimulus-dependent CD4+ T cell enhancers. When overlapping causalsingle-nucleotide polymorphisms (SNPs) with 31 transcription factorbinding maps generated by ENCODE, SNPs were strongly enrichedwithin binding sites for immune-related transcription factors, and var-iants associated with different diseases correlate to different combina-tions of transcription factors that control immune cell identity andresponse to stimulation. In patients with MS, SNPs preferentially coin-cide with NFkB-, EBF1 (early B cell factor 1)–, and MEF2A (myocyteenhancer factor 2A)–bound regions (22).

Previous studies have shown that total peripheral blood mono-nuclear cells (PBMCs) from relapsing-remitting MS (RRMS) patients ex-hibit increased levels of active NFkB (23). We similarly observed thatnaïve CD4 T cells from RRMS patients exhibit increased activation ofthe canonical p65 NFkB pathway compared to healthy controls, suggest-ing that this difference is not due to the activation status of the cells. Thus,we hypothesized that the alterations in NFkB signaling seen in patientswith MS are a result of SNPs in the NFkB signaling cascade associatedwith MS susceptibility. Because NFkB signaling is triggered by manyenvironmental stimuli through toll-like receptors, this may represent acritical intersection between genetic and environmental factors result-ing in MS development. However, the impact of autoimmunity-associatedgenetic variants on the biological function of this major transcriptionfactor is unknown.

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It has been shown that mice with a constitutively active form ofp65 NFkB exhibit multiorgan inflammation and die within 3 weeksof birth. However, when these mice with constitutively active p65NFkB were bred back to mice lacking TNFR1, they were protectedfrom inflammation and exhibited only a mild Sjogren’s syndrome–likeocular keratitis, indicating a critical role of the NFkB/TNF pathway inregulating autoimmune disease (24). Variants within or proximal to bothTNFRSF1A (TNFR1) and NFkB1 have been identified by GWAS as beingassociated with risk of developing MS. Therefore, we chose to focus ourstudies on these SNPs to determine whether they alter NFkB responses.

Here, we investigated the effect of MS risk haplotypes on the functionof the NFkB signaling cascade. We demonstrate that both the haplotypewith the variant proximal to NFkB1 (rs228614-G) and that with thevariant within TNFR1 (rs1800693-C) result in enhanced NFkB responsesto TNFa. The rs228614-G risk allele also enhances responses to phorbol12-myristate 13-acetate (PMA), suggesting that this variant stronglycontrols NFkB signaling. Mechanistically, we found that rs228614-Gis associated with a 20-fold increase in p50 NFkB expression and de-creased expression of the negative regulators of NFkB signaling. Thesefindings elucidate a genetic mechanism controlling NFkB responsesthat result in predisposition to developing MS.

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RESULTS

Patients with MS have increased activation of thecanonical NFkB cascadePrevious reports suggest that total PBMCs from patients with MS ex-hibit increased activity of NFkB, consistent with excessive immuneactivation (23, 25). However, it was not possible to determine whetherthis increased NFkB activation is due to up-regulation of NFkB or tothe increased frequency of activated and memory CD4 T cells in pa-tients with MS. To resolve this question, we isolated total PBMCs frompatients with MS and healthy control subjects ex vivo and directlystained for phospho-p65 NFkB. We found that naïve CD4 cells frompatients with MS exhibit significantly higher phospho-p65 NFkB thanthose from age-matched healthy control donors, and this increasedactivation of p65 NFkB was mitigated by treatment (Fig. 1; subjectdemographics are listed in table S1). This increased constitutive ex-pression of phospho-p65 NFkB was repeated in a second cohort ofMS patients and healthy controls (fig. S1). The presence of enhancedactivation of NFkB in naïve CD4 cells demonstrates that this is notdue to an increase in the number of activated or memory cells butis rather due to a hyperactivated state of CD4 cells.

The MS risk variant rs228614 near NFkB1 is associated withincreased NFkB signalingTo determine whether the increased NFkB activation seen in patientswith MS may be due to genetic variation in the NFkB signaling cas-cade associated with disease susceptibility, we next assessed whetherthe MS risk variant proximal to NFkB1 increases NFkB signaling.The SNP rs228614 on chromosome 4 is associated with MS susceptibil-ity (odds ratio of 1.09 per G allele carried; P = 1 × 10−8) and lies near theNFkB1 gene (12). It is part of a haplotype of more than 90 variants intight linkage disequilibrium (LD) that spans the region encoding bothNFkB1 and MANBA (Fig. 2A). Although this strong LD precludesidentifying this specific SNP as the causal variant for disease, we strati-fied donors into those carrying one or two copies of the G risk allele

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(rs228614-G) and correlated NFkB signaling to genotype in totalPBMCs. In addition, we also investigated a second variant, rs7665090,in LD to rs228614 (r2 > 0.8) that was identified by fine-mapping as themost associated variant in the region (10). This allowed us to examinetwo variants within the same region to determine the impact on NFkBsignaling and thus address the difficulty of identifying the causal variantin genetic mapping studies. We used healthy control subjects carryingthe risk or protective variants to avoid the confounding factors of ongoinginflammation and therapeutic intervention seen in patients with MS.

We collected blood samples from healthy donors in our previouslygenotyped biorepository of recallable subjects (Yale PhenogeneticProject) (see demographics in tables S2 and S3). We found that the MSrisk alleles rs228614-G and rs7665090-G are significantly associatedwith an increase in inhibitor of NFkB a (IkBa) degradation afterTNFa stimulation (allelic logistic regression: rs228614, P = 0.00078,and rs7665090, P = 0.01; Fig. 2B). This difference is also significant whencomparing homozygous risk and protective genotypes (Student’s t testbetween GG and AA carriers: rs228614, P = 0.0091; rs7665090, P =0.0089; Fig. 2B). Risk allele homozygotes showed consistently increasedphosphorylation of p65 NFkB (Student’s t test between GG and AAcarriers: rs228614, P = 0.029; rs7665090, P = 0.039; Fig. 2C, represent-ative histogram and gating strategy in fig. S2). A trend of increasedsignaling from both rs228614 and rs7665090 was also observed at 15,30, and 45 min (fig. S3). Thus, the region containing the rs228614 andrs7665090 SNPs modulates TNFa signaling through NFkB.

To determine whether this represents a global impact on NFkB sig-naling or a specific alteration in TNFa signaling, we stimulated total PBMCsfrom subjects with the different genotypes at rs228614 with PMA, anactivator of NFkB independent of TNFa. The risk genotype (GG) resultedin greater degradation of IkBa and phosphorylation of p65 NFkB in naïveCD4 cells after stimulation with PMA, suggesting a modulation of globalNFkB responses rather than to a specific stimulus (Fig. 2D; IkBa, P =0.019 and pNFkB, P = 0.027). To determine whether this region wasalso affecting NFkB signaling inMS patients, we stimulated total PBMCsfrom rs7665090 homozygous risk (GG) and protective (AA) subjectswith TNFa and determined degradation of IkBa and phosphorylation

Fig. 1. Naïve CD4 cells from patients with MS exhibit increased phospho-p65 NFkB. Flow cytometry of PBMCs from age-matched healthy control (HC)

and RRMS patients stained for CD4, CD45RA, CD45RO, and pS529 p65 NFkB.Mean fluorescence intensity (MFI) results of p65 are shown gated on naïveCD4+CD45RA+CD45RO− T cells. Healthy control, n = 34; untreated MS, n = 11;treated MS, n = 25. P value shown for unpaired t test. n.s., not significant.

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of p65 NFkB by flow cytometry. Similar tothe changes in NFkB signaling seen inhealthy controls, the risk variant also re-sulted in increased IkBa degradation (Fig.2E) and phosphorylation of p65 NFkB(Fig. 2F) in naïve CD4 cells in MS pa-tients. We hypothesized that these diver-gent NFkB responses should result indifferential rates of nuclear localizationof p65 NFkB. We tested this by compar-ing healthy donors with AA (protective)and GG (risk) genotype at rs228614. Westimulated CD4 cells with TNFa for 15 or30 min, stained for p65 NFkB and 4′,6-diamidino-2-phenylindole (DAPI), anddetermined nuclear localization of p65.We found that GG carriers demonstrateincreased p65 nuclear localization after30 min, confirming stronger signaling re-sponses (Fig. 3, A and B). Together, thesedata demonstrate that the MS risk haplo-type captured by rs228614 and rs7665090strongly modulates NFkB responses, withthe GG risk genotype resulting in en-hanced NFkB signaling.

rs228614 is associated withchanges in the expression of NFkB1and the negative regulators of theNFkB pathwayWe did not find differences in IkBa andphospho-p65 NFkB expression in restingCD4 cells, suggesting that the effect ofthe rs228614 risk variant is only evidentafter signaling induction (fig. S4). Thissuggests that the underlying causal vari-ant perturbs regulatory elements control-ling stimulus-dependent activation of theNFkB gene, consistent with the observa-tion that most GWAS risk variants altergene regulation rather than structure(26). The haplotype block containingrs228614 spans the coding regions ofNFkB1 and MANBA and the intergenicspace between them, which contains atleast 62 transcription factor binding sites,12 regions of DNA hypersensitivity, 3 in-sulator regions, and multiple putativeenhancer regions active in both T celland myeloid cell lineages (27–33), sug-gestive of important regulatory functions.We therefore looked for changes in ex-pression of p50 NFkB in rs228614 GGand AA genotype carriers by Western blot.

We found that subjects with the GGrisk genotype express p50 NFkB at a 20-foldhigher level than the AA genotype (Fig. 4Aand fig. S5, uncut blot). We also found a

Fig. 2. MS-associated allelic variants proximal to NFkB1 results in increased TNFa and PMAsignaling. (A) Association with MS risk in the region surrounding NFkB. Y axis shows the GWAS –log(P

value) for the allelic test of association as reported in (12). We have highlighted rs228614 andrs7665090, which are the most associated variants in the region. cM, centimorgan. (B) Degradationof IkBa in CD4+CD45RA+CD45RO− T cells after 15 min of TNFa stimulation by flow cytometry in healthycontrols. (C) Phospho-p65 NFkB in CD4+CD45RA+CD45RO− naïve T cells after 15 min of TNFa stimulation byflow cytometry. Results shown normalized to unstimulated T cells. (D) rs228614 CD4+CD45RA+CD45RO−

T cell degradation of IkBa and phosphorylation of p65 NFkB after 15 min of PMA stimulation. (E and F)Degradation of IkBa after 15 min (E) and phosphorylation of p65 NFkB at 15 and 30 min (F) in CD4+

CD45RA+CD45RO− T cells after TNFa stimulation by flow cytometry in MS patients. For (A) to (D):rs228614 GG, n = 4; AG, n = 18; AA, n = 6; rs7665090 AA, n = 8; AG, n = 49; GG, n = 23. For (E) and (F), GG,n = 6; AA, n = 6. P value shown for homozygous unpaired t test.

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commensurate decrease in mRNA levels in total PBMCs of three keynegative regulators of NFkB [TNFa-induced protein 3 (TNFAIP3),B cell leukemia 3 (BCL3), and cellular inhibitor of apoptosis1 (CIAP1)] in the GG homozygotes (Fig. 4B). Together, these resultssuggest that an increase in total p50 NFkB and a loss of negative regula-tion of this pathway are responsible for enhanced signaling. AlthoughIkBa degradation is upstream of NFkB, signaling through NFkB regulatesthe expression of NFkB1 itself, as well as the negative regulators of NFkB.Thus, we demonstrate alterations in the expression of both NFkB1 and thenegative regulators of this pathway, suggesting that the MS risk allelecauses a fundamental shift in the regulation of NFkB signaling.

NFkB responses are stable over time and are not associatedwith age or genderIf the strength of NFkB signaling is primarily mediated by genetic var-iability between individuals and not by changes in environmental or ex-ternal stimuli, wewould expect consistent strength of signal acrossmultipletimepoints.We redrew15 subjects on an average of 6months after the firstblood draw.We found no statistically significant difference between draws(fig. S6). Although there was significant variability between individuals inthe strength ofNFkB signaling, the stability in this variability acrossmulti-ple drawswas consistent with a geneticallymediated threshold for NFkBsignaling.

It has been shown that individuals 65 years or older have an increasein constitutive activation of p65 NFkB (34). The Phenogenetic Proj-

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ect enrolled subjects up to age 56 years at the time of enrollment,and subjects were consented for redraws for up to 4 years. To determinewhether NFkB signaling changes with age, we compared age at blooddraw to NFkB signaling after TNFa stimulation. We found no correla-tion between age and strength of signal by either IkBa degradation orphosphorylation of p65 before the age of 60 years (fig. S7, A and B).This suggests that changes in NFkB signaling associated with aging onlyoccur after 60 years of age. We also found no differences in gender orethnicity in NFkB signaling after TNFa stimulation (fig. S7, C to F).

rs1800693 in TNFR1 is associated with enhanced NFkBresponses to TNFaBecause both NFkB and TNFR1 are important in autoimmune inflam-mation and variants near both NFkB1 (rs266814) and TNFRSF1A(rs1800693) are associated with risk of developing MS (12), we inves-tigated the impact of the TNFRSF1A variant on NFkB signaling. Un-like the NFkB1 locus, where tight LD precludes identifying the likelycausal SNP, rs1800693 alone best explains the association signal inthe TNFRSF1A region and is thus likely causal (10). The variant falls10 base pairs upstream of TNFRSF1A exon 6 in a splice acceptorsite (Fig. 5A). We and others have previously shown that homozy-gous carriers of the rs1800693-C risk allele show loss of exon 6 and apremature stop codon in ~10% of TNFR1 mRNAs (35, 36). Moreover,this variant results in an exaggerated cytokine production responsefrom monocytes after stimulation with TNFa (35), suggesting that this

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transcriptional change results in changesin overall signaling responses. We evalu-ated this hypothesis by stimulatingPBMCs and purified monocytes withTNFa and assessing resultant IkBa deg-radation (see demographics in table S4).We found that carriers of the CC riskgenotype show increased degradationof IkBa after stimulation with TNFa inboth naïve CD4 cells (P = 0.0092; Fig.5B) and monocytes (P = 0.011; Fig. 5C)compared to TT genotype carriers. Unlikethe variant proximal to NFkB1, there wereno differences in signaling after PMAstimulation between the CC and TT gen-otypes (Fig. 5B). These data suggest thatrs1800693 is associated with changes insignaling only when TNFa binds toTNFR1.

TNFa can signal through two recep-tors, TNFR1 and TNFR2. Naïve CD4 cellsprimarily express TNFR1, with TNFR2being up-regulated after activation (37).Our results suggest that altered TNFa-induced signaling is due to rs1800693-Cexerting regulatory effects on TNFR1. Tovalidate this model, we blocked TNFR1and found that TNFa signaling in naïveCD4 cells occurs exclusively throughTNFR1, with no contribution from TNFR2(fig. S8). As such, the changes in signalingobserved in naïve CD4 cells can be at-tributed directly to alterations in TNFR1.

Fig. 3. The rs228614 GG risk genotyperesults in rapid nuclear localization ofp65 NFkB. (A) Representative Imagestreamdata showing nuclear localization of p65

NFkB after 30 min of TNFa stimulation by AmnisImagestreamx in CD4+ T cells. Green, p65 NFkB; pur-ple, DAPI; colocalization, overlap of p65 and DAPI byPearson coefficient. (B) Compilation of rs228614 nu-clear localization of p65 NFkB after TNFa stimulationin CD4+ T cells (GG, n = 5; AA, n = 4). Nuclear local-ization is shown normalized to unstimulated cells.

P value shown for unpaired t test.

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To confirm these changes in NFkB signaling in CD4 cells, we purifiedtotal CD4 cells, stimulated them with TNFa for 15 or 30 min, anddetermined the amount of p65 NFkB nuclear localization. We observedan increase in nuclear localization 15 min after stimulation in car-riers of the CC genotype compared to the TT genotype (Fig. 6, A andB). The CC genotype did not result in changes in the cell surface ex-pression of TNFR1 on naïve CD4 cells, either as a percentage of cellsexpressing TNFR1 or the expression level of TNFR1 on the surface (fig.S9). This suggests that the change in signaling is not due to changes inoverall TNFR1 levels on the cell surface. We and others have shownthat expression of the prematurely terminated transcript missing ex-on 6 caused by the rs1800693-C allele results in altered intracellularaccumulation of TNFR1 in human embryonic kidney (HEK) 293 cellsor HeLa cells (35, 36). We investigated whether this is true in primaryimmune cells directly ex vivo and found that CD14+ monocytes fromCC risk genotype carriers exhibit altered intracellular accumulation ofTNFR1 into punctate structures within the cytoplasm (Fig. 7A, single-stained control, fig. S10). Together, these results demonstrate that thevariant in TNFR1 results in increased signaling to TNFa in both naïveCD4 cells and monocytes and that this altered signaling may be dueto altered localization of TNFR1 within the cell.

rs1800693 in TNFR1 is associated with increased plasmacytokines in healthy controls consistent with levelsseen in MS patientsBecause TNFa signaling drives the expression of inflammatory cyto-kines, we examined whether changes in TNFR1-mediated signalingassociated with the rs1800693-C risk allele result in altered plasma cy-tokines levels. We measured plasma levels for 29 cytokines in healthysubjects and found that CC genotype carriers have increased plasma

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levels of IL-7, IL-8, granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon-inducible pro-tein 10 (IP10), and monocyte chemoattractant protein1 (MCP1) (Fig. 7B and table S5). This is consistentwith our previous findings that IP10 is overexpressedin monocytes after TNFa stimulation in CC genotypecarriers (35). It has been reported that IL-7, GM-CSF,IP10, IL-8, and MCP1 are increased in serum of pa-tients with MS (38–42). In addition, recent twin studieshave shown that serum concentrations of all five ofthese cytokines are highly heritable (43). This demon-strates that the CC homozygous risk genotype altersinflammation levels in healthy control subjects,consistent with that seen in MS.

DISCUSSION

NFkB is a central regulator of inflammation, control-ling the activation, proliferation, and cytokine produc-tion of immune responses. We observed that naïveCD4 T cells from patients with the autoimmune dis-ease MS exhibit increased activation of p65 NFkB,prompting us to investigate the genetic control ofNFkB signaling in the disease. We found that var-iants near genes in the NFkB signaling cascade havelarge effects on NFkB signaling. Specifically, the allelicvariant proximal to NFkB1 controls signaling re-

sponses by altering the expression of NFkB itself, with the GG riskgenotype expressing 20-fold more p50 NFkB. This genotype is asso-ciated with altered NFkB responses to both TNFa and PMA, suggestinga global control of NFkB signaling. Because causal SNPs are stronglyenriched within binding sites and signaling molecules for NFkB, theseresults demonstrate a central role for NFkB in driving the patho-physiology of MS.

The region spanned by the MS risk haplotype near NFkB1 con-tains a large number of gene regulatory elements, as mapped by theENCODE project (27–33). This suggested the hypothesis that the MSrisk allele affects NFkB1 expression and thus alters NFkB signaling. Toinvestigate this potential mechanism, we examined how allelic variationin this region influenced expression of p50 NFkB and found that MSrisk variants are strongly associated with large increases in expression.Consistent with increased NFkB signaling, there was a decrease inexpression of the negative regulators of NFkB, suggesting a global dis-ruption in the NFkB cascade. The central role of NFkB in immuneactivation would suggest that this MS haplotype broadly increases in-flammatory responses, thereby predisposing individuals to autoimmunity.

We recently investigated the overlap of potentially causative SNPswith 31 transcription factor binding maps generated by ENCODE andobserved that they were strongly enriched within binding sites forimmune-related transcription factors (22). Moreover, variants associatedwith different autoimmune diseases correlate with different combina-tions of transcription factors that control immune cell identity andresponse to stimulation. We found that MS SNPs preferentially coin-cide with NFkB-, EBF1-, and MEF2A-bound regions, whereas rheu-matoid arthritis and celiac disease SNPs preferentially coincide withIRF4 (interferon regulatory factor 4) regions (22). Thus, both GWASand epigenetic mapping strongly implicate NFkB as a critical pathway

Fig. 4. rs228614 allelic variant results in increased NFkB1 expression and decreasedexpression of the negative regulators of NFkB. (A) Representative Western blot of p105

and p50 NFkB in total PBMCs and densitometry of total p50 NFkB by Western blot (GG, n = 7;AA, n = 7). (B) mRNA expression by quantitative polymerase chain reaction (qPCR) of BCL3,TNFAIP3, and CIAP1 in total PBMCs (GG, n = 6; AA, n = 7). P value shown for unpaired t test.

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associated with MS. That is, variants associated with MS tend to fall inor near genes involved in NFkB signaling, and secondly, variants asso-ciated with MS frequently fall near known NFkB response elements.We propose a model in which variants associated with NFkB signalingand binding interact to form a cumulative burden on this criticalpathway. The enhanced NFkB activity we observe in naïve CD4 cellssuggests that these cells may have a decreased threshold of activation,thereby making them more susceptible to low levels of stimuli and ul-timately autoimmunity.

We also established that an MS risk variant intronic to TNFRSF1A(TNFR1) is associated with increased NFkB signaling in response toTNFa. We confirmed previous reports that this risk variant results inan altered intracellular accumulation of TNFR1 and extended thesefindings to show that monocytes isolated from peripheral blood showthis altered phenotype (35, 36). Notably, an R92Q mutation in TNFR1is associated with TNFR-associated periodic syndrome (TRAPS), arelapsing-remitting peripheral inflammatory disorder. The R92Qmutation also results in an altered intracellular accumulation of TNFR1and increased responses to inflammatory stimuli (44). This may suggesta common mechanism for altered NFkB-mediated signaling betweenMS and TRAPS.

We have previously reported that CD4 cells from MS patients ex-hibit increased proliferation at low doses of anti-CD3 stimulation com-

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pared to healthy controls (45). These data suggesta decreased threshold for activation of CD4 cells inpatients with MS, consistent with genetic risk var-iants perturbing NFkB pathway genes to create anet increase in signaling through this inflammato-ry cascade.

Treatments that suppress NFkB signaling,such as steroids, are routinely used in MS, al-though their prolonged use is ineffective, perhapsbecause of the many mechanisms associated withchronic steroid usage. Although these studiesmight support the use of NFkB inhibitors inMS, they also suggest that it may be possible todetermine the specific stimuli and downstreamsignaling cascades that may identify novel diseasetreatments. Notably, TNFa blockade by monoclo-nal antibodies or soluble receptors is highly effec-tive in many autoimmune diseases. In contrast,TNFa blockade in patients with MS worsens dis-ease and induces new-onset MS in some patientswith rheumatoid arthritis, Crohn’s disease, andulcerative colitis (46). Moreover, TNFa blockadecan also initiate psoriasis and induce the develop-ment of anti-nuclear or double-stranded DNAantibodies in up to 70% of other autoimmune pa-tients, with some progressing to anti-Rho/La anti-bodies and clinical lupus (46, 47). Blocking TNFain patients with mutations in TNFR1 associatedwith TRAPS also results in increased NFkB signalingand inflammation similar to that seen in MS (48).These data emphasize the clinical importance ofunderstanding the NFkB pathway in MS and otherautoimmune diseases and implicate genetic variantsin the TNF signaling pathway as controlling the al-tered responses to anti-TNFa therapy.

The haplotype block surrounding the intergenic region downstreamof NFkB1 contains more than 90 variants in strong LD, and we wereunable to identify a single most likely causal variant by genetic andepigenetic fine-mapping. In the future, the use of probabilistic iden-tification of causative SNPs in larger cohorts may allow furtherfine-mapping of this region, which may better identify the mostlikely causative SNP (22). In spite of this lack of resolution in thediscovery process, we are able to show that the underlying MS var-iant in this region tagged by the rs228614-G allele is strongly asso-ciated with NFkB responses to TNFa.

We demonstrated that significant interindividual variation exists inNFkB responses, and this variability is partially mediated by geneticvariability in the NFkB signaling cascade. Because many autoimmunediseases and cancers have genetic variants associated with NFkB, it islikely that genetically mediated variability in this pathway is centralto disease risk. In addition, because the variants associated with eachdisorder are different, variation in the NFkB pathway may result inchanges in signaling that are specific to each disease. Finally, theincreased NFkB signaling we observed is consistent with the enhancedactivation of the canonical NFkB pathway observed in naïve CD4 cellsfrom patients with MS. Hyperactivation of NFkB in mice results in rap-id postnatal death from massive multiorgan inflammation not seenin human disease (24, 49). As such, it is likely that dysregulation of

Fig. 5. The TNFR1 variant rs1800693 results in increased TNFa responses. (A) Associationwith MS risk in the region surrounding TNFRSF1A. Y axis shows the GWAS –log(P value) for the

allelic test of association as reported in (10). We have highlighted rs1800693, the most associatedvariant in the region. With further replication data in independent samples, rs228614 meets theGWAS significance threshold of P < 5 × 10−8. (B) Degradation of IkBa after 30 min of TNFa stim-ulation in CD4+CD45RA+CD45RO− T cells (CC, n = 14; TT, n = 20). (C) Degradation of IkBa after30 min of TNFa stimulation in CD14+ monocytes (CC, n = 5; TT, n = 12). P value shown forunpaired t test.

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NFkB represents a necessary factor in the developmentof autoimmunity but that other factors are also re-quired to determine the location and nature of the in-flammatory insult. Current GWAS have identifiedvariants associated with disease susceptibility but donot determine how these variants are affecting diseaseseverity or progression. Given that NFkB signaling iscritical for both inflammation and neuronal degener-ation (50), this pathway may represent a node thatboth predisposes to disease and contributes to diseaseprogression.

In conclusion, naïve CD4 T cells from patientswith MS exhibit increased p65 activation, consistentwith changes in NFkB signaling. We observe equiv-alent changes in healthy controls carrying MS riskvariants, which alter NFkB responses to TNFa, sug-gesting that disease risk is mediated by gene regulatorychanges resulting in altered NFkB signaling. BecauseSNPs causal for MS are enriched in binding sitesand signaling molecules for NFkB, these data dem-onstrate a central role for NFkB in driving the path-ophysiology of MS. Identifying these critical nodesmay suggest novel therapeutics aimed at specifi-cally targeting the underlying causes of diseasewhile leaving systemic immune responses primarilyintact.

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MATERIALS AND METHODS

Study designHealthy subjects between the ages of 18 and 56 yearsat the time of initial assessment were enrolled in theYale Phenogenetic Project and could be recalled up tosix times a year for up to 4 years. Subjects with auto-immune disorders were excluded from the repository.In addition, any subjects exhibiting illness or fever atthe time of draw were excluded. Investigation of theimpact of rs228614 and rs7665090 on phospho-p65NFkB and total IkBa was performed blinded, withthe strength of signal determined before genotypingthe samples. Samples were unblinded, and compari-son of strength of signal to genotype was performedafter unblinding. For blinded studies, power calcula-tions determined that a singleton quantitative trait lo-cus with a variance of 10% and an allele frequency of20% could generate a significance of 0.05 for overallassociation with 50 samples and 0.01 with 90 samples.Because rs228614, rs7665090, and rs1800693 all haveminor allele frequencies of >30%, these studies weresufficiently powered to generate significance. Nuclearlocalization, Western blotting, and qPCR studies wereperformed on recalled samples with specific geno-types. All TNFRSF1A studies were performed onsamples of risk and protective genotypes recalled fromthe Brigham and Women’s Hospital (BWH) Pheno-Genetic Project. All studies were performed to at leastthree biological replicates.

Fig. 6. TNFR1 rs1800693CC risk genotyperesults in increased NFkB nuclear localiza-tion. (A) Representative nuclear localizationimages from TT protective and CC risk geno-

types in CD4+ T cells by Amnis Imagestreamx. Green,p65 NFkB; purple, DAPI; colocalization, overlap ofp65 and DAPI by Pearson coefficient. (B) Compositenuclear localization of p65 NFkB after TNFa stimula-tion in CD4+ T cells (CC, n = 9; TT, n = 9). Nuclear lo-calization is shown normalized to unstimulated cells.

P value shown for unpaired t test.

Fig. 7. The TNFR1 variant rs1800693 results in altered intracellular accumulation ofTNFR1 and plasma cytokines. (A) Confocal microscopy of CD14+ monocytes from the TT

(protective) or CC (risk) variant in the TNFR1 region. Blue, DAPI; green, TNFR1. Two of foursubjects from each genotype are shown. (B) Concentrations of IL-7, IL-8, GM-CSF, MCP1,and IP10 in plasma samples from healthy control subjects with the CC (risk) or TT (protective)genotypes (CC, n = 25; TT, n = 40). P value shown for unpaired t test.

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Blood repositoriesSamples were acquired from the Yale Phenogenetic Project or theBWH PhenoGenetic Project. These samples have been genotypedfor autoimmunity-associated SNPs by Illumina Chip. Blood was drawninto heparin tubes. Samples from Yale were prepared immediately afterblood draw. TNFR1 samples from BWH were shipped overnight withroom temperature gel packs (SAF-T-PAK) and prepared the followingmorning. RRMS from age- and gender-matched blood was drawn atthe Yale MS Clinic and prepared the same as the healthy controlsamples. All samples were compared to samples from the same source(Yale or BHW).

PBMC preparation and cell purificationPBMCs were prepared from whole blood by Ficoll-Hypaque densitygradient centrifugation. For some experiments, total CD4 cells orCD14+ monocytes (containing both CD16+ and CD16−) were purifiedby magnetic negative selection using EasySep magnetic separation kits(Stem Cell Technologies).

Cytokine stimulationTotal PBMCs were rested for 1 hour in RPMI medium without fetalbovine serum (FBS) after isolation. Monocytes were plated overnightin RPMI medium containing 5% FBS, L-glutamine, nonessential aminoacids, sodium pyruvate, and Hepes. Monocytes were washed two timeswith RPMI and rested for 1 hour with RPMI without FBS before stim-ulation. Stimulation was performed with TNFa (50 ng/ml; R&DSystems) or PMA (500 ng/ml; Sigma-Aldrich). Cells were fixed at15, 30, or 45 min with BD fixation buffer (BD Biosciences), washedtwo times with phosphate-buffered saline (PBS), and permeabilizedwith ice-cold BD permeabilization buffer III (BD Biosciences). Cellswere permeabilized overnight at −80 °C. After being washed, the cellswere stained for CD4 phycoerythrin (PE), CD45RA AF700, CD45ROPE-Cy7, phospho-p65 NFkB (pS529) (BD Biosciences), and IkBaA488 (Cell Signaling Technologies). Flow cytometry was performedon a BD LSRII Fortessa (BD Biosciences). Analysis was performedusing FlowJo software (TreeStar).

Nuclear localizationTotal CD4 cells were stimulated with TNFa (50 ng/ml; R&D Systems)for 15 or 30 min. Cells were fixed with 3% formalin for 10 min, washed,and permeabilized with 0.1% Triton X-100 + 2% FBS in PBS (no Ca/Mg).Cells were blocked with Fc Block (eBioscience). p65 NFkB was stainedwith anti–p65 NFkB (Santa Cruz Biotechnology) and fluoresceinisothiocyanate (FITC)–labeled donkey Fab2 anti-rabbit immuno-globulin G (IgG) (Jackson ImmunoResearch). Cells were stained withDAPI (Sigma-Aldrich) nuclear stain for 10 min and washed twice withPBS. Nuclear localization was performed on an Amnis Imagestreamx orAmnis Imagestreamx Mark II at ×40 or ×60 magnification. Nuclearlocalization was determined using Amnis IDEAS software (Amnis)by Pearson coefficient colocalization of DAPI and p65 NFkB.

Western blotTotal PBMCs were lysed with radioimmunoprecipitation assay buffer(Pierce) containing Halt protease inhibitors (Pierce). Total protein wasdetermined by bicinchoninic acid assay (Pierce), and 10 mg of total pro-tein was run per lane. Samples were prepared with loading buffer (LifeTechnologies) and heated for 10 min at 72 °C before running onWesternblot. Samples were run on a 10% bis-tris gel and transferred onto nitro-

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cellulose with the XCELL Western blot transfer apparatus (Life Tech-nologies). Blots were blocked with 1× tris-buffered saline (TBS) and0.1% Tween 20 with 5% (w/v) nonfat dry milk overnight. p105/p50NFkB and actin (Cell Signaling Technology) were stained overnight,washed three times with 1× TBS and 0.1% Tween 20, and labeledwith horseradish peroxidase–linked anti-mouse IgG (Cell SignalingTechnologies). Blots were developed using ECL Prime (GE Healthcare).Densitometry was performed using ImageJ analysis software [NationalInstitutes of Health (NIH)].

Confocal microscopyTwo hundred thousand monocytes were plated overnight in 24-wellplates with poly-L-lysine–coated coverslips. Cells were incubatedovernight, fixed with 3% formalin, and permeabilized with 0.1%Triton X-100 + 2% FBS in PBS (no Ca/Mg). Cells were blocked withFc Block (eBioscience) and a cocktail of 1% FBS, 1% donkey serum,and 1% human serum. TNFR1 was stained with polyclonal TNFR1antibody (R&D Systems) and labeled with FITC-labeled anti-rabbitIgG (Jackson ImmunoResearch). Images were captured on a Leica mi-croscope (Leica Biosystems) and analyzed using ImageJ software (NIH).

LuminexPlasma was prepared by centrifuging total blood at 5000 rpm for 10 minand removing the plasma fraction from the red cell fraction. Plasma wasimmediately frozen at −80 °C until run on a 30-plex Luminex (Millipore).

Quantitative PCRRNAwas extracted fromwhole PBMCs using Qiagen RNeasy PlusMicroKit according to the manufacturer’s instructions. Sample concentrationand purity were determined by spectrophotometer analysis on NanoDrop2000. Complementary DNA was prepared from RNA through reversetranscription–PCR with reverse transcriptase kits from Life Technologies.qPCR was performed using primers for TNFAIP3 (hs00234713_m1),BCL3 (hs00180403_m1), CIAP1 (BIRC2, Hs01112284_m1), and the house-keeping genes HPRT (hs01003267_m1) and b2M (H500984230_m1)with TaqMan Fast Universal PCR Master Mix (No AmpErase UracilN-Glycosylase) (Life Technologies). Samples were run on an ABI Prismquantitative PCR machine (Life Technologies).

StatisticsGenotype-phenotype associations were calculated in two ways: an allelictest as implemented in PLINK (51) and comparing opposite homo-zygote groups by unpaired t test, as implemented in PRISM (GraphPad).In both cases, uncorrected P values were reported.

Study approvalThe study was conducted in compliance with the Declaration ofHelsinki. Before study initiation, approval was obtained from theethics committee of Yale-New Haven Hospital. Informed consent wasreceived from all subjects before inclusion in the study.

SUPPLEMENTARY MATERIALS

www.sciencetranslationalmedicine.org/cgi/content/full/7/291/291ra93/DC1Fig. S1. Confirmation of increased pNFkB in MS patients.Fig. S2. Gating strategy and representative histogram for rs228614 GG (risk) and AA (protec-tive) variants.Fig. S3. rs228614 and rs7665090 proximal to NFkB1 result in increased IkBa degradation afterTNFa treatment.

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Fig. S4. Constitutive total IkBa and phospho-p65 NFkB are similar in rs228614 risk and pro-tective variants.Fig. S5. Full uncut gel for Fig. 4.Fig. S6. TNFa and PMA responses are stable across multiple draws.Fig. S7. Responses to TNFa do not change with age, gender, or ethnicity.Fig. S8. TNFa signals through TNFR1 on naïve CD4 cells.Fig. S9. The rs1800693 CC variant does not change the surface expression of TNFR1.Fig. S10. Representative single-stained control.Table S1. Demographics of healthy subjects and MS samples for Fig. 1 and fig. S1.Table S2. Demographics of rs228614 results from Fig. 2.Table S3. Demographics of rs7665090 from Fig. 2.Table S4. Demographics of rs1800693 from Fig. 5.Table S5. Luminex data from rs1800693 subjects.Table S6. Raw data.

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Acknowledgments: We thank S. Eisenbarth, J. Cruz, and A. Singh for their assistance withsample acquisition. We thank E. Menet and L. Devine for their assistance with cell sortingand Amnis imaging. Funding: This research was supported by grants from the National Insti-tute of Allergy and Infectious Disease (AI045757, AI046130, AI070352, and AI039671), the Na-tional Institute of Neurological Disorders and Stroke (NS24247 and NS067305), the NationalInstitute of General Medical Sciences (GM093080), the National Multiple Sclerosis Society(CA1061-A-18), the Penates Foundation, and the Nancy Taylor Foundation for Chronic Disease.W.J.H. was supported by a training grant from NIH (to D. Schatz, 5T32AI007019-36) and is aPfizer Fellow of the Life Sciences Research Foundation (to W.J.H.). Author contributions: W.J.H.designed, performed, and analyzed experiments and wrote the manuscript. S.D.F., K.V., and S.R.M.performed experiments, and N.C., L.G., and P.L.D. assisted in sample acquisition. M.M. and C.C.analyzed data. J.G. contributed to the confocal microscopy experiments. D.A.H. oversaw theexperiments and wrote the paper. C.C. performed statistical analyses. Competing interests:The authors declare that they have no competing interests.

Submitted 16 February 2015Accepted 13 May 2015Published 10 June 201510.1126/scitranslmed.aaa9223

Citation: W. J. Housley, S. D. Fernandez, K. Vera, S. R. Murikinati, J. Grutzendler, N. Cuerdon,L. Glick, P. L. De Jager, M. Mitrovic, C. Cotsapas, D. A. Hafler, Genetic variants associated withautoimmunity drive NFkB signaling and responses to inflammatory stimuli. Sci. Transl. Med. 7,291ra93 (2015).

ceTranslationalMedicine.org 10 June 2015 Vol 7 Issue 291 291ra93 10

inflammatory stimuliB signaling and responses toκGenetic variants associated with autoimmunity drive NF

Laura Glick, Phillip L. De Jager, Mitja Mitrovic, Chris Cotsapas and David A. HaflerWilliam J. Housley, Salvador D. Fernandez, Kenneth Vera, Sasidhar R. Murikinati, Jaime Grutzendler, Nicole Cuerdon,

DOI: 10.1126/scitranslmed.aaa9223, 291ra93291ra93.7Sci Transl Med

B signaling or inflammatory cytokines.κcontribute to autoimmunity. Patients with these variants may be good candidates for therapies that block either NFactivation threshold of CD4 T cells, making them more responsive to inflammation and thus more likely to

stimulation. These variants, in effect, lower theαB signaling after TNFκvariants that result in increased NF show that at least some multiple sclerosis patients have geneticet al.individuals and not in others. Now, Housley

are supposed to protect. However, it remains unclear why these self-targeted cells become activated in some In patients with autoimmune diseases, such as multiple sclerosis, immune cells attack the tissues that they

Lowering the bar for autoimmunity

ARTICLE TOOLS http://stm.sciencemag.org/content/7/291/291ra93

MATERIALSSUPPLEMENTARY http://stm.sciencemag.org/content/suppl/2015/06/08/7.291.291ra93.DC1

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