Conserved 33-kb haplotype in the MHC class III region regulates chronic arthritis

Anthony C. Y. Yaua, Jonatan Tuncela, Sabrina Haaga, Ulrika Norina, Miranda Houtmanb, Leonid Padyukovb, and Rikard Holmdahla,c,1

aMedical Inflammation Research, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-171 77 Stockholm, Sweden; bRheumatology Unit, Department of Medicine, Karolinska Institutet and Karolinska University Hospital, SE-171 76, Stockholm, Sweden; and cSouthern Medical University, Guangzhou 510515, China

Edited by Philippa Marrack, Howard Hughes Medical Institute, National Jewish Health, Denver, CO, and approved May 16, 2016 (received for review January 12, 2016) Genome-wide association studies have revealed many genetic loci and lymphotoxin-β (LTB) (10). However, so far there is no evi- associated with complex autoimmune diseases. In rheumatoid ar- dence that polymorphism in the MHC-III region really contributes thritis (RA), the MHC HLA-DRB1 is the strongest candidate to the pathogenesis in RA or in its associated animal models. predicting disease development. It has been suggested that other MHC-III is challenging to study because of its high gene density immune-regulating in the MHC contribute to the disease and extensive linkage disequilibrium (LD) (9). Animal models for risk, but this contribution has been difficult to show because of RA are attractive alternatives to human association studies for the strong linkage disequilibrium within the MHC. We isolated gene identifications because the use of these models not only genomic regions in the form of congenic fragments in rats to test overcomes genetic heterogeneity and reduces environmental whether there are additional susceptibility loci in the MHC. By effects but also allows the isolation of specific genetic regions to both congenic mapping in inbred strains and SNP typing in wild study their roles in RA in vivo (14–16). Rat arthritis models such rats, we identified a conserved, 33-kb large haplotype Ltab-Ncr3 in as pristane-induced arthritis (PIA) (17, 18), which meet many of the MHC-III region, which regulates the onset, severity, and chro- the criteria for the diagnosis of RA (19), have been used to nicity of arthritis. The Ltab-Ncr3 haplotype consists of five poly- reveal highly significant linkages to unique chromosomal re- morphic immunoregulatory genes: Lta (lymphotoxin-α), Tnf, Ltb gions, often a much smaller interval with far fewer genes than (lymphotoxin-β), Lst1 (leukocyte-specific transcript 1), and Ncr3 previously postulated for quantitative traits (16, 20, 21). (natural cytotoxicity-triggering 3). Significant correlation We recently established a panel of recombinant strains in the in the expression of the Ltab-Ncr3 genes suggests that interaction MHC region to study the MHC association with T-cell selection of these genes may be important in keeping these genes clustered (22) and showed that RT1-B (the human ortholog is HLA-DQ)in together as a conserved haplotype. We studied the arthritis asso- MHC-II determines the onset and severity of PIA (21). Here, we ciation and the spliceo-transcriptome of four different Ltab-Ncr3 constructed a panel of recombinant strains in MHC-III to study haplotypes and showed that higher Ltb and Ncr3 expression, the association of MHC-III with arthritis on the DA (RT1av1) lower Lst1 expression, and the expression of a shorter splice var- background. By dissecting this gene-dense region, gradually nar- iant of Lst1 correlate with reduced arthritis severity in rats. Inter- rowing down the size of the arthritis-regulating quantitative trait estingly, patients with mild RA also showed higher NCR3 expression (QTL), we identified a conserved 33-kb MHC-III haplo- and lower LST1 expression than patients with severe RA. These data type comprising highly polymorphic, differentially expressed, and demonstrate the importance of a conserved haplotype in the regu- spliced genes that regulates the onset, severity, and chronicity of lation of complex diseases such as arthritis. autoimmune arthritis.

arthritis | major histocompatibility complex | congenic mapping | Significance haplotype | inflammation The role of the MHC region has been a long-standing issue in he MHC, known as the HLA complex in humans, is the most chronic inflammatory diseases, such as , and Tgene-dense and polymorphic region in the it has not been possible to identify the underlying specific poly- with strong associations to many autoimmune diseases (1, 2). morphism. Here, we provide evidence that some of the MHC One such example is rheumatoid arthritis (RA). RA is a chronic association must be explained by how closely linked genes – autoimmune inflammatory joint disorder affecting 0.5 1% of the operate together as haplotype blocks. We identified a conserved general population. The genetic association of RA is mainly with haplotype, Ltab-Ncr3, comprising five genes (lymphotoxin α and MHC polymorphism; non-MHC loci [more than 100 RA-risk β,Tnf, leukocyte-specific transcript 1,andnatural cytotoxicity- loci have been identified in genome-wide association studies triggering receptor 3) within MHC class III, regulating arthritis. We (GWAS)] contribute only to a minor extent (3, 4). found significant coexpression of the Ltab-Ncr3 genes, indicating Recently it has been suggested that six different amino acids in how these genes may work together as a haplotype. Further- HLA-DRB1, HLA-A, HLA-B, and HLA-DPB1 explain most of more, haplotype-specific differences in Ltab-Ncr3 gene expres- the MHC association with RA in seropositive patients (5, 6). In sion and correlate remarkably to susceptibility addition, independent genetic susceptibility for RA has been to arthritis. Our data show that a conserved haplotype within identified in the MHC class III (MHC-III) (7–10). MHC-III is a MHC class III regulates arthritis development. region sandwiched between MHC class I (MHC-I) and MHC class II (MHC-II) in humans. This region consists of many genes Author contributions: A.C.Y.Y., J.T., and R.H. designed research; A.C.Y.Y., J.T., S.H., and U.N. performed research; M.H. and L.P. provided and prepared human samples; A.C.Y.Y. with important immune functions encoding for complement and J.T. analyzed data; and A.C.Y.Y., J.T., S.H., M.H., L.P., and R.H. wrote the paper. , , and heat-shock proteins. Many of these The authors declare no conflict of interest. MHC-III genes have been described as candidate genes in RA, This article is a PNAS Direct Submission. including a gene encoding for TNF (11), which now is a thera- 1To whom correspondence should be addressed. Email: [email protected]. peutic target in RA and other autoimmune diseases (12, 13). This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. Other candidate genes include allograft-inflammatory factor-1 (8) 1073/pnas.1600567113/-/DCSupplemental.

E3716–E3724 | PNAS | Published online June 14, 2016 www.pnas.org/cgi/doi/10.1073/pnas.1600567113 Downloaded by guest on September 29, 2021 Results intervals in this 2.8-Mb region (Fig. 1). We mapped all the MHC PNAS PLUS Identification of a 33-kb Locus in MHC-III That Regulates the Onset congenic and subcongenic strains on the 2004 (Baylor 3.4/rn4) and Severity of Arthritis. We previously identified RT1-B in the assembly which, based on more than 70 widely dispersed SNP MHC-II region as a susceptibility locus for acute PIA and ex- and short tandem repeat (STR) markers as described in the cur- cluded a contribution of the classical MHC-Ia region (21). Al- rent and a previous study (22), offers more accurate annotation though rats of both the MHC congenic strain DA.1HR2, which of the region than the 2012 (RGSC 5.0/rn5) and 2014 (RGSC spans the entire MHC, and the MHC-II congenic strain DA.1HR61 6.0/rn6) assemblies. We assessed acute arthritis in these strains (Fig. 1) were less susceptible to PIA, we observed a small but re- and found that rats of the subcongenic strains DA.1HR7N, producible difference in arthritis protection between these two strains DA.1HR56, DA.1HR56T, DA.1HR56A, and DA.1HR56D de- from day 14 to day 25 (Fig. 2A) (21). This observation suggested veloped significantly milder acute arthritis than DA rats, having a putative second QTL in the nonclassical MHC-Ib and/or the MHC- lower disease score, reduced weight loss, and a delayed disease III region, which was confirmed with the recombinant strain onset, whereas rats of the subcongenic strains DA.1HR4, DA.1HR7, which carries a fragment from DA.1HR2 in the DA.1HR52C, DA.1HR56C, and DA.1HR56E were not pro- MHC-Ib/MHC-III region (Fig. 1 and Table 1). tected (Fig. 1 and Table 1). These results demonstrate the ex- To identify the underlying genetic regulation in DA.1HR7 istence of a 32.7-kb arthritis-regulating QTL in the MHC-III rats, we generated ten subcongenic strains spanning different region (3.655–3.713 Mb) comprising five genes, lymphotoxin-α GENETICS if1 Bat2 Lst1Ncr3 A RT1-CE1 Bat1 Atp6v1g2 Nfkbil1 Abhd16a Ly6g5c Ly6g6f Ng23 G7c G7a Skiv2l Dxo Stk19 C4a C4b Cyp21a1 Tnxa-ps1 Atf6b fkbpl Prrt1 RT1-Db2 RT1-CE10 Ddah2 Clic1 RT1-CE4 RT1-CE5 RT1-CE2 RT1-CE3 Tnf Ltb Lta Bat3 Apom G4 Gpank1 Csnk2b Ly6g5b Ly6g6c Ly6g6d Ly6g6e G6b Ddah2 Msh5 Hspa1b Hspa1a Neu1 Slc44a4 Ehmt2 Zbtb12 C2 Cfb Nelfe Gpsm3 RT1-Ba RT1-Bb RT1-DOb Tap2 Psmb8 Tap1 Psmb9 Agpat1 Rnf5 Ager Pbx2 Notch4 Btnl5 Btnl3a2 Btnl8 Btnl7 Tesb RT1-DMb RT1-DMa RT1-Ha RT1-DOa Rxrb Col11a2 Slc39a7 Hsd17b8 Ring1 RT1-A3 RT1-A2 RT1-A1 Ppt2 Egfl8 Btnl2 RT1-Da RT1-Db1 Brd2 Btnl3 MHC Ib MHC III MHC II MHCIa telomere 3.50 3.75 4.00 4.25 4.50 4.75 5.00 centromere 1.62 5.447 5.976 HR2* 4.315 4.403 4.815 4.834 HR7* HR83 4.047 4.103 4.274 4.301 4.815 4.830 HR7N* HR61* 3.655 3.658 4.583 4.629 HR4 HR62 3.845 3.864 HR52C 3.430 3.452 epytolpah HR56* 3.850 3.863 HR56T* RT1h 3.801 3.806 HR56A*

HR56B 3.711 3.713 HR56D* HR56C 3.635 3.649 HR56E

UR2 3.801 3.806

RT1u RT1u UR2A haplotype

FR2 2.79 4.815 4.834

RT1f RT1f FR9

haplotype 3.736 3.957 FR5

Ltab-Ncr3 RT1-B

Fig. 1. Overview of the genetic mapping of MHC recombinant strains of haplotypes RT1h, RT1u, and RT1f based on the University of California, Santa Cruz (UCSC) Genomic Browser 2004 (Baylor 3.4/rn4) assembly. Genes are depicted at the corresponding position (in megabases) at the top of the figure. The overall organization of class Ia, II, III, and Ib regions is adopted from a previous study (48). Congenic strains are depicted as horizontal bars with horizontal lines indicating intervals of unknown genotype. Congenic fragments with an asterisk exhibit a protective disease phenotype in PIA. The prefixes “HR,”“UR,” and “FR” indicate that the congenic fragments originate from DA.1H (RT1h haplotype), DA.1U (RT1u haplotype), and DA.1F (RT1f haplotype), respectively (Ma- terials and Methods). PIA has been previously reported in DA.1HR2, DA.1HR61, DA.1HR62, DA.1HR83, DA.1FR2, DA.1FR9, and DA.1UR2 rats (21, 22). The MHC arthritis QTLs Ltab-Ncr3 and RT1-B (previously identified) (21), are shaded in brown.

Yau et al. PNAS | Published online June 14, 2016 | E3717 Downloaded by guest on September 29, 2021 Fig. 2. The Ltab-Ncr3 region regulates onset, severity, and chronicity of PIA. Development of PIA in DA.1HR2 (n = 13), DA.1HR61 (n = 12), DA.1HR56D (n = 15), and DA (n = 14) rats with mean arthritis score (A), disease incidence (B), day of disease onset (C), percentage of weight change from day 9 to day 20 after pristane immunization (D), and percentage of weight change from day 79 to day 212 after pristane immunization (E). (F) Comparison of serum AGP levels in DA.1HR56D and DA rats on day 21 after pristane immunization. (G) Mean arthritis score of recipient rats after transfer of pristane-primed T cells from DA.1HR56D (n = 12) and DA (n = 10) donor rats. (H) Mean arthritis score of DA.1HR56 (n = 11) and DA (n = 9) rats after transfer of pristane-primed T cells from DA rats. (G and H) Lymph node cells were harvested from rats 8 d after pristane immunization. Data shown in A, G, and H are mean ± SEM; horizontal lines in C–F represent mean values. (A and B) An asterisk denotes a significant difference between DA.1HR56D and DA; a plus sign denotes a significant difference between DA.1HR61 and DA; a carat denotes a significant difference between DA.1HR2 and DA. (A–H) One symbol indicates P < 0.05; two symbols, P < 0.01; three symbols, P < 0.001 compared with DA, unless otherwise specified. Disease incidence was evaluated by Fisher’s exact test. Other statistics were de- termined with the Mann–Whitney U test.

(Lta), Tnf, Ltb, leukocyte-specific transcript 1 (Lst1), and natural chronic PIA (Fig. 2E). We therefore conclude that DA.1HR56D cytotoxicity-triggering receptor 3 (Ncr3) (hereafter referred to as the and DA.1HR61 regulate the acute and chronic phases of ar- Ltab-Ncr3 region). thritis to different extent. DA.1HR56D rats, which carried the smallest congenic frag- ment harboring the Ltab-Ncr3 region, developed milder acute Ltab-Ncr3 Regulates the Priming of Arthritis. To show whether the PIA than DA rats, with significantly lower disease scores from Ltab-Ncr3 region regulates the priming or the effector phase of day 12 to day 25 after pristane injection (Fig. 2A). In addition, arthritis, we performed reciprocal adoptive transfers of in vivo- DA.1HR56D rats developed arthritis with a lower disease in- primed T cells between the congenic and DA rats (24). When we cidence and delayed disease onset (Fig. 2 B and C). The reduced transferred pristane-primed T cells from congenic and DA donor disease severity in DA.1HR56D rats also was reflected in re- rats to heterozygous recipient rats, we observed that T cells duced weight loss and a lower serum level of the acute-phase originating from congenic rats were less arthritogenic than the T α1-acid glycoprotein (AGP) (Fig. 2 D and F). cells from DA rats, showing that the Ltab-Ncr3 region regulates the priming of arthritis (Fig. 2G). Consistent with the reduced + DA.1HR56D and DA.1HR61 Have Different Effects on Acute and arthritogenicity of the congenic-derived T cells, CD4 Tcells Chronic PIA. To compare the effect sizes of MHC-II and MHC- from congenic rats expressed lower levels of activation markers III on PIA, we assessed PIA in DA.1HR56D, DA.1HR61, and and produced less of the proinflammatory IFN-γ (Fig. DA.1HR2 rats. In the acute phase of PIA (days 10–30) (18), S1), which has been shown to be important in PIA (24). In con- DA.1HR2, DA.1HR61, and DA.1HR56D rats developed milder trast, when T cells were transferred from pristane-primed DA rats PIA than DA rats (Fig. 2 A–D). DA.1HR61 had a stronger pro- to congenic or DA recipients, there was no difference in disease tective effect than DA.1HR56D; this result was not surprising, severity (Fig. 2H), demonstrating that the Ltab-Ncr3 region does considering the association between MHC-II and both experi- not regulate the effector phase of arthritis. mental arthritis and RA patients (21, 23). In the chronic phase of PIA (day >80) (18), both DA.HR2 and Ltab-Ncr3 Is an Evolutionarily Conserved, Disease-Associated DA.1HR56D rats developed significantly milder disease than Haplotype. Studying the genomic sequence of the DA.1HR56D DA rats, whereas DA.1HR61 rats showed no significant disease and DA rats by next-generation sequencing (NGS) showed that protection (Fig. 2A); this result is consistent with our previous the 33-kb Ltab-Ncr3 region is highly polymorphic, with 261 SNPs data (21). Also, DA.1HR2 and DA.1HR56D rats gained signif- (Table S1). When the polymorphisms in the arthritis-protective icantly more weight than both DA.1HR61 and DA rats in DA.1HR56D rats were compared with those in the arthritis-prone

E3718 | www.pnas.org/cgi/doi/10.1073/pnas.1600567113 Yau et al. Downloaded by guest on September 29, 2021 Table 1. Arthritis disease phenotype of MHC-III congenic fragments and DA in the acute phase of PIA PNAS PLUS † ‡ jj †† ‡‡ Strain Origin Haplotype Arthritis score§,¶ Weight change (%)¶,# Day of onset¶, Incidence (%) No.

DA a 30.02 ± 2.053 −9.27 ± 0.86 9.56 ± 0.18 100 18 DA.1HR7* DA.1HR2 h 18.75 ± 4.37** nd 11.50 ± 0.65** 100 4 DA.1HR7N* DA.1HR7 h 18.46 ± 2.86** +0.71 ± 1.38**** 14.25 ± 0.64** 100 13 DA.1HR56* DA.1HR7N h 16.48 ± 2.151*** −4.18 ± 1.25** 12.17 ± 0.43** 96 25 DA.1HR56T* DA.1HR56 h 13.58 ± 4.507** −6.85 ± 2.28** 12.14 ± 0.67 89 9 DA.1HR56A* DA.1HR56T h 17.42 ± 2.896*** −2.17 ± 0.95*** 12.56 ± 0.58** 100 15 DA.1HR56D* DA.1HR56A h 13.10 ± 2.82**** +0.23 ± 1.52**** 12.00 ± 0.53**** 89 18 DA.1HR4 DA.1HR2 h 24.20 ± 4.43 nd 9.40 ± 0.40 100 5 DA.1HR52C DA.1HR7 h 26.88 ± 3.83 nd 10.63 ± 0.80 100 8 DA.1HR56C DA.1HR56A h 25.63 ± 2.51 −12.06 ± 2.13 10.63 ± 0.205 100 19 DA.1HR56E DA.1HR56D h 22.67 ± 4.25 −10.8 ± 1.98 13.75 ± 0.92 100 12 DA.1FR5 DA.1FR2 f 33.62 ± 2.471 nd 9.70 ± 0.62 100 10 DA.1UR2A DA.1UR2 u 22.50 ± 3.74 −9.79 ± 2.64 10.60 ± 0.64 100 10

†Congenic strains with disease protection are indicated with an asterisk. ‡Haplotype names have been abbreviated: h = RT1h, u = RT1u, f = RT1f, a = RT1av1. §Arthritis score indicates the mean disease score at the peak of acute arthritis (approximately day 20). ¶Arthritis score, weight change, and day of onset are shown in mean values ± SEM. **P < 0.05, ***P < 0.01, ****P < 0.001, relative to DA; nd, not determined. Statistics were determined with the Mann–Whitney U test. #Weight change is shown as percentage change on day 20 vs. day 9. jjDay of disease onset is defined as the first day when any clinical signs of arthritis in the rats could be observed. ††Denotes the percentage of animals which develop arthritis for at least two consecutive scoring days. ‡‡ Denotes the number of animals in the group.

DA.1UR2A rats (Table 1 and Table S1), 229 of the 261 SNPs we investigated whether the DA.1HR56D and DA rats responded GENETICS (including five nonsynonymous SNPs in Ltb, Lst1,andNcr3) differently to etanercept treatment. We found that both groups correlated with the arthritis-protective phenotype in DA.1HR56D responded effectively to the etanercept treatment, with a signifi- (Table S1). Using PROVEAN software (25), we examined the cant reduction of arthritis (Fig. 3E). Taken together, these findings potential effect of the five nonsynonymous SNPs in Ltb, Lst1,and suggest that Tnf is not responsible for the disease protection in the Ncr3 on their protein structure. All the nonsynonymous SNPs DA.1HR56D rats. were predicted not to impact the structure and function of the proteins (Table S2). The Ltab-Ncr3 Genes Are Differentially Expressed and Undergo To investigate the degree of conservation in this region, we Alternative Splicing. Variation in is an important typed 13 SNPs in the Ltab-Ncr3 region in nonrelated wild rats mechanism underlying susceptibility to complex diseases (27–30). collected at different locations in three countries. Wild rats are In addition to Tnf, we also studied the gene-expression levels of genetically more diverse than inbred laboratory strains, thus the other four genes in the Ltab-Ncr3 region in the lymph nodes allowing us to assess better the haplotype structure in this region. from naive and immunized rats [on day 5 after pristane injection, Five of the 48 wild rats shared exactly the same allelic variants as because the arthritis protection could be transferred by congenic DA.1HR56D rats; 17 shared exactly the same allelic variants as T cells as early as day 5 (Fig. S1E)]. DA.1UR2A, DA.1FR9, and DA rats; and the remaining 26 wild Gene expression in arthritis-protective DA.1HR56D rats was rats were found to be heterozygous at all these sites (Table 2). compared with the expression levels in DA.1UR2A, DA.1FR9, These findings strongly support the existence of a conserved and DA rats. Both DA.1HR56D and DA.1FR9 rats showed lower haplotype in the Ltab-Ncr3 region. In addition, despite genotyping more than 8,000 congenic rats, no further recombination was detected in the Ltab-Ncr3 region. This result further indicates that Table 2. SNPs in Ltab-Ncr3 haplotype in wild rats the Ltab-Ncr3 region is resistant to recombinations (Fig. S2). Location* SNP type Sequence variant

Excluding Tnf Polymorphism as a Contributor to the Haplotype 3661408 Intronic A C A/C Disease Effect. We considered Tnf as a potential candidate gene 3661469 Intronic G A A/G in the Ltab-Ncr3 haplotype because of its well-recognized role in 3667565 Nonsynonymous A G A/G the pathogenesis and treatment of RA (12). To investigate the 3667652 Nonsynonymous T C C/T potential contribution of Tnf polymorphism, we compared the 3699415 Nonsynonymous T C C/T coding and mRNA expression levels of dif- 3699425 Synonymous C G C/G ferent Ltab-Ncr3 alleles. We found no nonsynonymous SNPs in 3702451 Synonymous G A A/G the Tnf and also found no differential expression of Tnf between 3702600 Nonsynonymous C T C/T strains in the lymph nodes of immunized rats (Fig. S3 A and B). 3706976 Intronic A G A/G Comparing congenic and DA rats, we found that similar levels of 3707078 Nonsynonymous C T C/T TNF were secreted upon LPS stimulation of whole blood har- 3707248 5′ UTR G A A/G vested from naive rats (Fig. 3A) and from rats with active arthritis 3710003 Intergenic T A A/T (Fig. 3B) and also by splenocytes and peritoneal macrophages 3710030 Intergenic AT A/T upon stimulation with LPS, zymosan, polyinosinic-polycytidylic Total number of animals 5 17 26 acid (poly I:C), or concanavalin A (ConA) (Fig. 3 C and D and – Wild rats were collected at various locations in Norway, Sweden, and The Fig. S3 C F). Because anti-TNF therapy is often used for treat- Netherlands. ment of RA and the LTA-TNF region has been associated with *Genomic coordinates are based on the UCSC Genomic Browser 2004 (Baylor the response to treatment with etanercept (a TNF inhibitor) (26), 3.4/rn4) assembly.

Yau et al. PNAS | Published online June 14, 2016 | E3719 Downloaded by guest on September 29, 2021 Fig. 3. Excluding Tnf polymorphism as a contributor to the DA.1HR56D arthritis protection effect. (A) Level of TNF in whole blood from naive DA.1HR56D (n = 8) and DA (n = 9) rats after LPS stimulation for 18 h. (B) Level of TNF in whole blood from PIA (day 16) DA.1HR56T (n = 12) and DA (n = 15) rats after LPS stimulation for 18 h. (C and D) Level of TNF in the supernatant produced by splenocytes (C) and peritoneal macrophages (D) harvested from naive DA.1HR56D (n = 8) and DA (n = 9) rats after stimulation for 24 h with LPS, zymosan, poly I:C, and ConA. (E) Development of PIA in DA.1HR56D (etanercept: n = 9; control: n = 9) and DA (etanercept: n = 10; control: n = 13) rats treated with etanercept or PBS on days 2, 4, and 6 after pristane immunization. All data are rep- resented as mean ± SEM; *P < 0.05 (DA.1HR56D) and ***P < 0.001 (DA) comparing cumulative disease scores of the treatment group vs. the control group. Statistics were determined with the Mann–Whitney U test.

Lta expression (Fig. 4A); however, because DA.1FR9 is not as- higher Ltb and Ncr3 expression and lower Lst1 expression and with sociated with any MHC-III arthritis regulation (21), it is unlikely the expression of a shorter splice variant of Lst1. that the differential Lta expression is associated with the disease One possible advantage of the genes in the Ltab-Ncr3 haplo- protection in DA.1HR56D rats. This conclusion is supported by type clustering together as a conserved haplotype could be en- the finding that Lta was not differentially expressed on day 5 after hanced coordination of gene expression (34, 35). We therefore pristane immunization (Fig. S4A). More compelling was the determined the correlation between the expression of different finding that lymph node cells from DA.1HR56D rats showed genes in the Ltab-Ncr3 haplotype and found a significant positive higher expression of Ltb and Ncr3 and lower expression of Lst1 in correlation in the expression of different Ltab-Ncr3 genes, espe- a strain-specific manner (i.e., relative to DA.1UR2A, DA.1FR9, cially Lta-Lst1 and Lst1-Ncr3 (Spearman’s ρ 0.60 and 0.71, re- and DA rats) in both naive (Fig. 4A) and immunized rats (shown spectively) (Fig. 5). This finding indicates that genes in the region for DA.1HR56D vs. DA rats in Figs. S4 A–D and S5). may directly or indirectly interact with each other in the haplotype. Because MHC-III genes such as LST1 and NCR3 are known to exhibit alternatively spliced isoforms in humans (31, 32), we assessed LTB, LST1, and NCR3 Gene Expression in RA Patients. We studied the the expression of different isoforms in DA.1HR56D, DA.1UR2A, level of expression of LTB, LST1,andNCR3 in whole-blood samples DA.1FR9, and DA rats. We found that different MHC-III congenic from a cohort of 32 RA patients and 92 healthy controls. Overall, the strains expressed different Lst1 and Ncr3 isoforms. Although expression of these genes was increased significantly in blood cells of DA.1UR2A, DA.1FR9, and DA rats expressed the full-length Lst1 RA patients compared with blood cells from healthy controls, in- transcript (NM_022634.2), DA.1HR56D rats expressed only a shorter dicating that these genes could be important for the development of spliced variant (XM_006256080.2) lacking exon 2, which leads to a RA (Fig. 6), as is consistent with previous findings (10, 36). shorter extracellular domain (Fig. 4B). NCR3 is a functional activa- Having shown that the increased expression of Ltb and Ncr3 tion receptor on a subset of rat natural killer (NK) cells (33), and the and reduced expression of Lst1 correlated with reduced arthritis potential importance of NK cells in experimental arthritis is high- severity in PIA, we next investigated whether the expression of lighted by our finding that NK cell depletion reduced the severity of these genes also differed between two extremes, groups of patients arthritis (Fig. S6). We found that although DA.1HR56D rats with relatively mild and severe RA. On the basis of the disease showed the highest expression of Ncr3 full-length transcript activity score (DAS28), we used clinically accepted thresholds to (NM_181822.2) (Fig. 4A), these rats showed the lowest expression of define mild RA (DAS28 ≤ 3.2) and severe RA (DAS28 > 5.1). a Ncr3 splice variant formed through the fusion of exons 3 and 4 The group of patients with mild RA showed lower expression of that results in a longer intracellular domain (XM_006256054.2) LST1 and higher expression of NCR3 than the group of patients (Fig. 4C). Similar splicing patterns of both Lst1 and Ncr3 also were with severe RA (Fig. 6 B and C), similar to our observations in observed in immunized rats (Fig. S4 E–H). both naive and immunized DA.1HR56D rats (Fig. 4A and Fig. S4 Taken together, our results suggest that reduced arthritis severity B–D). However, LTB gene expression did not differ in the groups in DA.1HR56D rats (RT1h Ltab-Ncr3 haplotype) is associated with of patients with mild and severe RA (Fig. 6A).

E3720 | www.pnas.org/cgi/doi/10.1073/pnas.1600567113 Yau et al. Downloaded by guest on September 29, 2021 A PNAS PLUS

B C GENETICS

Fig. 4. Ltab-Ncr3 genes are differentially expressed and undergo alternative splicing. Gene expression in lymph nodes from naive DA.1HR56D (n = 17), DA.1UR2A (n = 8), DA.1FR9 (n = 9), and DA (n = 14) rats. (A) Expression of genes Lta, Ltb, Lst1, and Ncr3.(B, Left) Expression of the Lst1 full-length transcript (NM_022634.2) and Lst1 splice variant without exon 2 (XM_006256080.2). (Right) Gel photo illustrating the expression of the Lst1 full-length transcript and Lst1 splice variant without exon 2. (C) Expression of the Ncr3 full-length transcript with separate exon 3 and 4 (NM_181822.2) and the variant with fused exon 3 and 4 (XM_006256054.2). The reference genes Actb, Arbp, and Hmbs were used for normalization. Data are represented as mean ± SEM; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Statistics were determined with the Mann–Whitney U test.

Discussion only genes/loci that have been positionally mapped with high Although the association of the classical MHC with RA and resolution are Ncf1 (40), Igl (20), and RT1-B (21). Instead of experimental arthritis is well known (5, 21, 37), we show, for the identifying an association between a specific gene/locus and ar- first time to our knowledge, that MHC-III also controls arthritis. In thritis, here we identified the association between a conserved the MHC, we showed that, in addition to the arthritis-regulatory haplotype, Ltab-Ncr3, and arthritis and showed that this haplotype RT1-B locusinMHC-II(21),thereisasecondarthritis-regulatory exists in not only laboratory inbred rat strains but also in the wild QTL in MHC-III that determines the onset, severity, and also the rat population. In fact, comparative analysis of MHC-III gene chronicity of arthritis. We further narrowed down this QTL to sequences between different vertebrates showed that the Ltab- a conserved Ltab-Ncr3 haplotype with five polymorphic, tightly Ncr3 genes have clustered together for millions of years (41). The linked genes. Ltab-Ncr3 genes encode proteins involved in inflammation, and it MHC-III has been a particularly challenging region to study is possible that selection pressure has driven the conservation of for association with complex diseases such as RA (9, 38): The this haplotype structure so that allelic variants of Ltab-Ncr3 genes strong LD in the MHC makes it difficult to distinguish causal of similar functions could operate in cis. This theory is supported variants from linked neutral variants, and therefore studying by our data showing significant correlation in the expression of the human arthritis association within the MHC requires controlling genes in the Ltab-Ncr3 haplotype. For example, we showed sig- for the effects of HLA-DRB1 and other classical MHC alleles nificant correlation in the expression of Lta, Tnf,andLtb in rats. (5). An alternative approach is to study arthritis in animals in a LTA, LTB, and TNF are members of the TNF superfamily im- congenic setting. Because congenic fragments of MHC-III are portant in the organogenesis of lymphoid organs and immune isolated on a fixed genetic background, stratification of the effect responses (42). In addition, we showed a significant correlation in of classical MHC alleles is no longer needed. One possible the expression between Lst1 and Ncr3 in rats, and that correlation reason an association with arthritis was identified in the Ltab- also was found in the blood and synovium of RA patients (36). Ncr3 region in congenic rats but not (in different GWAS) in LST1 and NCR3 genes are located extremely close to each other humans (5, 6) is the difference in the genomic structure of the in both human and rats and are transcribed in the opposite orien- MHC region between humans and rats. In humans, the LTAB- tation. It is possible they share a common regulatory element at the NCR3 region is very close to HLA-B, a classical MHC-I gene 3′ end of their genes controlling their gene expression, which would associated with RA risk (5), and therefore disease association explain the strong correlation in Lst1 and Ncr3 gene expression. from LTAB-NCR3 region is hard to detect because of LD; in rats Furthermore, we showed significant correlation in the expression of the Ltab-Ncr3 region is much further away from classical MHC-I. Lta and Lst1, Lta and Ncr3,andTnf and Lst1, illustrating the Despite the identification of thousands of QTLs controlling complexity of the gene network in the Ltab-Ncr3 haplotype. different phenotypes in both humans and rodents, rather few Haplotype-specific differences in gene expression are, in genes have been identified from these QTLs (39). In the rat, the fact, common across the MHC in humans (43). In the rat, we

Yau et al. PNAS | Published online June 14, 2016 | E3721 Downloaded by guest on September 29, 2021 A BC

D Correlation between Ltab-Ncr3 genes Ncr3 0.8-1.0 Lst1 0.71**** 0.6-0.8 Ltb 0.062 0.19 0.4-0.6 Tnf 0.53** 0.46* 0.25 0.2-0.4 Lta 0.43* 0.28 0.60*** 0.46* 0-0.2

Fig. 5. Ltab-Ncr3 gene expressions are significantly and positively correlated. Pink circles, purple triangles, and filled black circles denote Ltab-Ncr3 gene expression of DA.1UR2A, DA.1FR9, and DA rats, respectively. (A) Correlation analysis of Lta and Lst1 gene expression. (B) Correlation analysis of Tnf and Ltb gene expression. (C) Correlation analysis of Lst1 and Ncr3 gene expression. In A–C each dot represents the fold-change of the corresponding genes of each individual rat (n = 31). Spearman’s correlation coefficient ρ and the P value for each gene pair are indicated in each figure. Reference genes Actb, Arbp,and Hmbs were used as controls for normalization to calculate the fold-change. (D) Summary of correlation showing Spearman’s correlation coefficient ρ between different Ltab-Ncr3 genes. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

showed further that higher Ltb and Ncr3 expression and lower because various factors can complicate comparisons between Lst1 expression are associated with reduced arthritis severity in experimental arthritis models and RA. For instance, subtle ge- the RT1h Ltab-Ncr3 haplotype. In humans, we showed that RA netic differences identified in inbred rodents can be difficult to patients expressed significantly higher levels of LTB, LST1, show in humans because of the genetic heterogeneity and envi- and NCR3 genes than healthy controls. This finding, together ronmental interactions. In addition, different RA treatments with other RA and animal studies (10, 36, 44, 45), indicates could alter the gene-expression profile of the RA patients and that these genes could be important for the development of increase the variation in the groups, thus making the comparison arthritis. One recent study demonstrated the linkage between between rat and human data even more difficult. Future studies the Lymphotoxin/LIGHT axis and IFN responses in RA pa- are needed to demonstrate the roles of these differentially expressed tients and suggested that Lymphotoxin/LIGHT could act as an genes in RA pathogenesis. upstream modulator of IFN-signaling genes in RA (46). Fur- Alternative splicing could be evolutionarily selected to enrich thermore, similar to our observations in DA.1HR56D rats, we transcript diversity in the MHC for a better and more diverse found that the group of patients with mild RA showed lower immune response against pathogens. In humans, specific LST1 expression of LST1 and higher expression of NCR3 than the splice variants are differentially expressed in RA patients and group of patients with severe RA, indicating that LST1 and non-RA controls (36), and here we show that the arthritis-pro- NCR3 could be associated with the severity of arthritis. However, tective congenic rats expressed only the Lst1 splice variant no difference in LTB gene expression, such as we had observed without exon 2 but not the full-length Lst1 transcript with exon 2, in rats, was observed between groups of patients with mild RA which was expressed by other rat strains. In addition, we showed and severe RA. All these data should be taken with caution that Ncr3 transcripts with different lengths of the intracellular

Fig. 6. The level of expression of genes LTB (A), LST1 (B), and NCR3 (C) in patients with mild RA (DAS28 ≤ 3.2, n = 10) and severe RA (DAS28 > 5.1, n = 22) and in healthy controls (n = 92). The reference gene ZNF592 was used for normalization. Data are represented as mean ± SEM; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 for comparison between groups. Statistics were determined with the Mann–Whitney U test.

E3722 | www.pnas.org/cgi/doi/10.1073/pnas.1600567113 Yau et al. Downloaded by guest on September 29, 2021 domain were differentially expressed in the arthritis-protective 1987 criteria for RA (19). Informed consent was obtained from all the partic- PNAS PLUS Ltab-Ncr3 haplotype. In humans, three constitutively expressed ipants. The Stockholm Ethical Review Board approved the study. NCR3 isoforms characterized by intracellular domains of dif- ferent lengths are functionally described as either immunosti- RNA and DNA Extraction. For rat samples, total RNA was extracted using the RNeasy Mini (Qiagen) and was treated with DNase I (Roche). cDNA was mulatory or immunosuppressive (32). Therefore it is possible synthesized using the High Capacity cDNA Reverse Transcription kit (ABI). For that the differential expression of the Ncr3 isoforms in rats might human samples, blood was collected into PAXgene Blood RNA Tubes, and affect the balance of these functions. total RNA was extracted with the PAXgene Blood RNA kit (PreAnalytiX) In conclusion, using a panel of intra-MHC recombinant congenic according to the manufacturer’s protocol. Samples were treated with DNase strains, we identified a 33-kb conserved haplotype consisting of five (PreAnalytiX ) for 20 min at room temperature to avoid contamination with tightly linked immunoregulatory genes in the telomeric end of genomic DNA. RNA was converted into cDNA using the iScript cDNA Syn- MHC-III regulating chronic arthritis. The Ltab-Ncr3 haplotype thesis Kit (Bio-Rad). Rat genomic DNA was isolated by proteinase K digestion showed strong haplotypic-specific differences in both gene expres- (AquaPure Genomic DNA kit; Bio-Rad). sion and alternative splicing, and these differences correlated re- Sequencing. DNA was amplified using specific primers (Eurofins MWG Operon) markably with arthritis susceptibility. Here, we demonstrate that as previously described (22) and was purified using Bio-Gel P-100 poly- congenic mapping in inbred strains and SNP typing in wild rats acrylamide beads. For sequencing of different splice variants, PCR products remain powerful tools in identifying and characterizing conserved were first run on a 1.5% agarose gel at 120 V, and bands of specific size were haplotypes regulating complex diseases such as arthritis. cut out from the gel and purified using the Gel Extraction Kit (Qiagen). Se- quencing was performed with BigDye Terminator 3.1 using 0.4 μM of primer Materials and Methods according to the manufacturer’s instructions (Applied Biosystems). Sequencing Animals. Inbred DA/Ztm rats were obtained from the Zentralinstitut für products were purified by the standard ethanol precipitation method, resus- Versuchstierzucht, and DA/OlaHsd rats were obtained from Harlan Europe. pended in 10 μL Hi-Di formamide (Applied Biosystems), and analyzed on a Rats were maintained by sister–brother mating in a barrier facility at Scheele 3730 DNA analyzer (Applied Biosystems). Genomic DNA for the NGS was Laboratory, Karolinska Institutet, and were specific pathogen-free according obtained from a liver sample harvested from a male DA.1HR2 rat using the to the guidelines of the Federation of European Animal Laboratory Science standard phenol/chloroform extraction method, followed by ethanol pre- Associations. All animals were housed in individually ventilated microisolator cipitation of the DNA. NGS was performed at the Science for Life Laboratory, × cages (Allentown) containing wood shavings (Tapvei) in a climate-controlled Sweden, using Illumina HiSeq. 2500 to generate pair-ended 2 100-bp reads. environment with 14-h /10-h dark cycles and were fed standard rodent See Table S3 for primers for genotyping recombinant strains and wild rats. chow (R70; Lantmännen) with free access to water. GENETICS The generation of the MHC congenic fragments DA.1FR2 (RT1f), DA.1HR2 Quantitative Real-Time PCR. For rat gene expression, quantitative real-time (RT1h), and DA.1UR2 (RT1u) spanning 1.62–5.98 Mb has been described pre- PCR (qRT-PCR) was performed on an ABI 7900 HT system (Applied Biosystems) viously (21, 22, 37). Briefly, congenic strains were originally established on the using SYBR Green (Applied Biosystems) and a two-step PCR protocol (95 °C DA/Ztm background (n >20) and thereafter were backcrossed further (n >5) for 10 min followed by 40 cycles for 95 °C for 10 s and 60 °C for 30 s). Primers were designed using Primer-BLAST (National Center for Biotechnology In- to DA/OlaHsd rats. Congenic strains with MHC haplotype RT1f were derived formation) with sequences retrieved from public databases; only primers from congenic DA.1F (DA.LEW-RT1f) rats (22). Congenic strains with MHC binding at nonpolymorphic locations were used. The expression fold-change haplotype RT1u were derived from DA.1U, generated by introgression of the of each gene was determined by the standard comparative cycle threshold corresponding E3/ZtmRhd fragment on 20. Congenic strains (C ) method after normalization to the geometric mean of the reference with MHC haplotype RT1h were derived from DA.1H rats (established at the T genes Actb (actin, beta), Arbp (attachment region-binding protein), and Hmbs Zentralinstitut für Versuchstierzucht) originating from the KHW strain. Fur- (hydroxymethylbilane synthase). See Table S4 for primers used in qRT-PCR. ther recombinant congenic strains were generated by crossing F1 hybrid rats For human gene expression, qRT-PCR was performed on CFX96 system and were screened for recombination using different microsatellite markers. (Bio-Rad) using TaqMan assays (Applied Biosystems) and a protocol of 50 °C Unless otherwise specified, 8- to 12-wk-old, age- and sex-matched congenic for 2 min, 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C rats and wild-type control rats have been used in all experiments. Wild rat for 1 min. The assays used to study expression of LTB, LST1,andNCR3 were samples were collected in different locations in Norway, Sweden, and the Hs00242739_m1, Hs00705788_s1, and Hs00394809_m1 respectively. The ex- Netherlands. Tail biopsies were stored in ethanol until analysis. pression fold-change of each gene also was determined by the standard

comparative CT method using reference gene ZNF592 (Hs00206029_m1) (47). Disease Induction and Evaluation. PIA was induced by intradermal injection of 100 μL pristane (2,6,10,14-tetramethylpentadecane; Acros Organics) at the T-Cell Transfer. Complete medium was prepared consisting of the following base of the tail in age-matched rats, and arthritis severity was monitored ingredients: DMEM (Gibco) supplemented with 5% (vol/vol) FCS (Gibco), blindly using a macroscopic scoring system (18). One point was given for 50 μM β-mercaptoethanol (Gibco), 10 mM Hepes (Gibco), 10 U/mL penicillin, each inflamed knuckle or toe, and up to five points were given for an af- and 100 μg/mL streptomycin (both from Invitrogen Life Technologies). fected ankle (for a maximum score of 15 per limb and 60 per rat). Rats are Draining lymph node cells from donor rats were harvested and cultured at a described as “protected” if the mean arthritis score is significantly lower 6 cell density of 3 × 10 /mL in complete medium with 3 μg/mL ConA (Sigma- than that of the control. Besides scoring, another arthritis evaluation crite- Aldrich) at 37 °C and 5% CO2. After 65 h, cells were washed, resuspended in rion is the day of disease onset, defined as the first day when any clinical PBS, and injected intravenously into recipient rats. In the pooled cell transfer, signs of arthritis in the rats could be observed. For DA rats, the typical day of lymph node cells from different DA rats were pooled together, and 2 × 107 arthritis onset is day 9 after pristane injection. Weight change, depicted as cells were injected into each recipient rat (DA or congenic). In the individual the percentage change in weight relative to the weight at disease onset, was transfer experiment, cells were not pooled; 3 × 107 cells from each donor rat used as an objective measurement of disease severity. For acute arthritis, the (congenic or DA) were injected into each recipient rat (heterozygous). percentage change in weight between day 20 (the peak of acute PIA) and day 9 was calculated. For chronic arthritis, the percentage change in In Vitro Stimulation Assays and TNF Measurement. Peritoneal macrophages weight between day 212 (the peak of chronic PIA) and day 79 (start of were collected from killed rats by lavage with sterile PBS into the peritoneal cavity chronic PIA) was calculated. The disease marker AGP in serum diluted and were cultured in complete medium (defined above in T-Cell Transfer). Cells 1:20,000 was measured by the AGP ELISA kit (Life Diagnostics). All exper- (3 × 105) were cultured in a 24-well tissue culture plate. After 2 h, super- iments were approved and performed in accordance with the guidelines of natant was removed, leaving adhered macrophages on the culture plate. the Swedish National Board for Laboratory Animals and the European Splenocytes were collected from spleen by mechanical separation through Community Council Directive (86/609/EEC). a mesh followed by erythrocyte lysis. Cells were stimulated for 12 or 24 h with 1–100 ng/mL LPS (Sigma-Aldrich), 100 μg/mL zymosan (Sigma-Aldrich),

Patients and Controls. The RNA-expression experiments used samples from 32 200 μg/mL poly I:C, or 3.0 μg/mL ConA (Sigma-Aldrich) at 37 °C and 5% CO2. RA patients and 92 healthy controls from the Swedish population; controls For whole-blood LPS stimulation, 100 μL heparinized blood in 400 μL com- were selected with consideration of the gender, age, and ethnicity of the plete medium (defined above in T-Cell Transfer) was cultured in a 48-well patient group. RA patients were selected at the Rheumatology Clinic at cell culture plate with 62.5 μg/mL LPS for 18 h. The level of TNF secretion Karolinska University Hospital, and all met American College of Rheumatology in supernatant was determined using a Rat TNF ELISA set (BD OptEIA; BD

Yau et al. PNAS | Published online June 14, 2016 | E3723 Downloaded by guest on September 29, 2021 + Biosciences). Eu3 -conjugated streptavidin (DELFIA; PerkinElmer) was used as each gene pair and its corresponding P value. All other statistical analyses were a secondary reagent, and detection was performed on a Synergy 2 multi- evaluated by Mann–Whitney U test and performed on Prism 6.0. mode plate reader (BioTek). ACKNOWLEDGMENTS. We thank Dr. Tanja Strand for kindly supplying wild TNF Depletion. Etanercept (kindly provided by H. Burkhardt, University rat samples; Prof. Anca Irinel Catrina for help with collection of clinical Hospital Frankfurt and Goethe University, Frankfurt am Main, Germany) samples; Prof. Thomas Hünig for generously supplying anti-rat NKR-P1A/B was administered subcutaneously in 0.2 mL PBS (12.5 mg/mL) on days 2, 4, and (10/78); and Carlos Palestro, Kristina Palestro, Tomasz Klaczkowski, Evelina Wernersson, and Sanna Eklund for excellent animal care. The authors 6 after pristane immunization as previously described (18). Control rats re- would like to acknowledge support from Science for Life Laboratory, the Na- ceived an equal volume of PBS. tional Genomics Infrastructure (NGI), Sweden, the Knut and Alice Wallenberg Foundation, and Uppsala Multidisciplinary Center for Advanced Computa- Bioinformatics. PROVEAN (Protein Variation Effect Analyzer) software was tional Science (UPPMAX) for providing assistance in massively parallel DNA used to predict whether an amino acid substitution has an impact on the sequencing and computational infrastructure. This work was supported by biological function of a protein (25). PROVEAN is available online at provean. grants from the Knut and Alice Wallenberg Foundation, the Swedish Asso- jcvi.org/. ciation against Rheumatism, the Swedish Medical Research Council, and the Swedish Foundation for Strategic Research. The research leading to these results received further funding from the European Community’sSeventh Statistical Analysis. The significance of differences in arthritis disease incidence Framework Program under the Grant Agreements HEALTH-F4-2010-241504 was evaluated by Fisher’s exact test. For correlation of gene expression, in (EURATRANS) and LSHG-Ct-2005-019015 (EURATools), and from the Euro- every dataset we calculated a Spearman correlation coefficient, ρ,for pean Union Innovative Medicine Initiative project BeTheCure.

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