Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2013 Jun 5.
Published in final edited form as: J Immunol. 2011 Feb 14;186(6):3572–3580. doi: 10.4049/jimmunol.1001330

A Polymorphism in the Coding Region of Il12b promotes IL-12p70 and IL-23 Heterodimer Formation

Antonie Zwiers *,†, Ivan J Fuss ‡, Diana Seegers *, Tanja Konijn †, Juan J Garcia-Vallejo †, Janneke N Samson §, Warren Strober ‡, Georg Kraal †, Gerd Bouma *,†
PMCID: PMC3673698  NIHMSID: NIHMS420554  PMID: 21321105

Abstract

Interleukin (IL)-12 and IL-23 are heterodimeric cytokines involved in the induction of Th1 and Th17 immune responses. Previous work indicated that a region on chromosome 11 encoding the IL-12 p40 subunit regulates strain differences in susceptibility to murine trinitrobenzene sulfonic acid (TNBS)-induced colitis. In addition, this region determines strain differences in LPS induced IL-12 responses.

Here we investigated how polymorphisms in the coding region of murine Il12b influence IL-12 and IL-23 heterodimer formation. Transfection studies using constructs containing IL-12p35 linked to either IL-12p40 from the colitis-resistant C57Bl/6 strain or to the polymorphic p40 variant from the colitis-susceptible SJL/J strain demonstrated that SJL/J-derived p40 constructs synthesized significantly more IL-12p70 than constructs harboring the C57Bl/6-p40 variant. This could not be attributed to differences in synthesis rate or secretion implicating a higher affinity of SJL/J derived IL-12p40 for its IL-12p35 subunit. This higher affinity is also associated with increased IL-23 synthesis. In addition, C57Bl/6 mice transgenic for the SJL/J 40 variant synthesized significantly more IL-12p70 and were more prone to develop colonic inflammation than did C57Bl/6 mice transgenic for the C57Bl/6-p40 variant upon LPS challenge. The more efficient binding of the polymorphic Il12b variant to p35 and p19 is most likely due to conformational changes following differential glycosylation as a consequence of the polymorphism.

The high synthesis rate of the mature cytokines resulting from this efficient binding can lead to rapid pro-inflammatory skewing of immune responses and distortion of the homeostatic balance underlying the higher susceptibility for colitis.

Introduction

Crohn’s disease (CD) and ulcerative colitis (UC) are chronic inflammatory disorders of the gastrointestinal tract which together form the two main entities comprising the inflammatory bowel diseases (IBD). There is general consensus that these diseases have their basis in a disturbed mucosal immune response that, in turn, results from the underlying presence of multiple genetic and environmental factors affecting one or both forms of the disease (1-4).

Recent genome wide association studies (GWAS) have established that at least 30 genetic loci are associated with IBD susceptibility (4, 5). These findings confirm the complex genetic basis of IBD already evident from earlier epidemiological, family and twin studies. One of the strongest gene associations observed involves the gene encoding IL-23R, part of the heterodimeric membrane receptor for the proinflammatory cytokine IL-23. The latter drives inflammation via its ability to sustain IL-17 production and is composed of a p40 chain, common to both IL-23 and IL-12 and a p19 chain unique to IL-23 (6). Of particular interest to the present study, the gene encoding the common p40 subunit, IL12B, has also been shown to be genetically linked to IBD susceptibility (7). In addition, a recent pathway analysis of pooled GWAS data not only confirmed these findings but also demonstrated that other genes involved in the IL12/IL23 pathway (containing 19 other genes) are involved in CD susceptibility (8).

The genetic information described above nicely complements previous immunological findings demonstrating that the IL-12/IL-23 signaling pathway has a key role in mediating the inflammation present in CD (9). Indeed, monoclonal antibodies against interleukin-12 may induce clinical responses and remissions in patients with active Crohn’s disease. This treatment is associated with decreases in Th1-mediated inflammatory cytokines at the site of disease. (10). Thus, it is reasonable to postulate that the genetic variation in the genes encoding these cytokines and cytokine receptors acts by causing an exaggeration of the inflammation driven by these factors. However, studies to substantiate this possibility have not yet been reported.

We previously found that differences in susceptibility to experimental trinitrobenzene sulfonic acid (TNBS)-colitis between mouse strains map to at least two regions on the mouse genome, one on chromosome 11 harboring the gene encoding the p40 subunit and another on chromosome 9 (11). In addition, we demonstrated that the colitis-sensitive SJL/J strain exhibits high IL-12p70 responses following intraperitoneal challenge with LPS, whereas colitis-resistant C57Bl/6 mice exhibit low responses and, importantly, these differences are under the control of a gene that is present in the same region of chromosome 11 that was identified as a susceptibility factor for TNBS colitis (11). Taken together, these findings strongly suggested that SJL/J mice have a genetically-determined predisposition to mount high IL-12 responses when challenged with the appropriate bacterial stimulus and that this high IL-12 responsiveness is at the basis of colitis susceptibility. It is unlikely that this had to be attributed to differences in transcription since sequencing of the first kB of the promoter region of Il12b, i.e., the region that harbors key binding sites for transcription factors (12) in both strains of mice failed to reveal distinguishing polymorphisms in any known regulatory region (data not shown).

A possible clue to understanding the differences in responses to LPS between SJL/J and BL/6 mice was that the increased SJL/J IL-12p70 responses were not accompanied by increases in the IL-12p40 response. This suggested that sequence variation in the coding region of Il12b of SJL/J mice leads to more efficient heterodimer formation and thus larger quantities of IL-12p70 because of changed affinities of the p40 chain for the p35 chain. In the present studies we explore this possibility with studies of SJL/J IL-12p40 function under both in vitro and in vivo conditions.

Materials and Methods

Animals

Specific pathogen-free, 5–6-week-old male SJL/J and C57Bl/6 mice were obtained from the National Cancer Institute.

Mice were maintained in the National Institute of Allergy and Infectious Diseases animal holding facilities. Animal use adhered to National Institutes of Health Laboratory Animal Care Guidelines.

DO11.10 mice (OVA-specific TCR transgenic mice) were bred at the Vrije Universiteit University Medical Center. Experiments involving these mice were approved by the Animal Experiments Committee of the Vrije Universiteit University Medical Center.

Reagents

Lipopolysaccharide (LPS) from Salmonella enteritidis was obtained from Sigma (Sigma-Aldrich, St. Louis, MO). Benzyl 2-acetamido-2-deoxy-α-D-galactopyranoside and kifunensine were obtained from Sigma (Sigma-Aldrich, Zwijndrecht, The Netherlands). OVA323–339 peptide was purchased from Sigma (Sigma-Aldrich, Zwijndrecht, The Netherlands). IL-12 p40, IL-12 p70 and IL-23, were determined using enzyme-linked immunosorbent assay kits. The IL-12p40 and IL-12p70 kits were obtained from PharMingen (PharMingen, Alphen a/d Rijn, The Netherlands), the IL-23 kit was purchased from eBioscience (ITK diagnostics, Uithoorn, The Netherlands). IFN-γ, TNF-α, IL-6 and MCP-1 production was measured with the Cytometric Bead Array (CBA). For IFN-γ, the Mouse Th1/Th2 Cytokine Kit was used and for TNF-α, IL-6 and MCP-1 the Mouse Inflammation Kit, both supplied by PharMingen. Restriction enzymes were supplied by New England Biolabs (Westburg, Leiden, The Netherlands).

Induction of TNBS Colitis

For the induction of colitis, mice were first lightly anesthetized with metofane (methoxyflurane; Pitman-Moore, Mundelein, IL). To induce colitis, 3.15 mg TNBS (Sigma Chemical Co., St. Louis, MO) was mixed and dissolved with an equal amount of 100% ethanol. A total volume of 150 μl of the TNBS-ethanol mixture was slowly administered per rectum via a 3.5F catheter equipped with a 1-ml syringe. To ensure distribution of TNBS within the entire colon and cecum, mice were held in a vertical position for 30 seconds after the injection. The mice were killed 4 days after induction of colitis.

IL-12, IL-23 and IFN-γ induction

From each strain, 5 male mice were intraperitoneally injected with a sublethal dose of 300 μg LPS. Six hours after administration, mice were bled and IL-12 p70, IL-12 p40 and IL-23 were measured in the serum. Preparation of spleen cell suspensions for OVA activated IFN-γ induction by IL12p70 was performed as described elsewhere (13). In short, spleens from 8-12 weeks old DO11.10 mice were strained through a 100-μm gauze. Erythrocytes in the splenocyte suspension were lysed by incubation with lysis buffer (150 mM NH4Cl, 1 mM NaHCO3, pH 7.4) for 5 min on ice. Cells were diluted to 5x105 cells/ml and incubated with 100nM OVA323–339 peptide and the appropriate amount of IL-12p70 and/or IL-12p40. After 48 hrs IFN-γ production was measured.

Generation of IL-12p40 polymorphic and non-polymorphic constructs

Full length murine wild type cDNA (GenBank accession number M86671), cloned in a pBluescriptSK+ expression vector (Promega, Madison, WI) was ordered from the ATCC (ATCC number 87595). The polymorphisms at positions 169 and 294 relative to the Methionine start codon generating a Met (C57Bl/6) -> Thr (SJL/J) and a Phe (C57Bl/6) -> Leu (SJL/J), respectively were introduced by using a PCR-based mutagenesis method (QuickChange™ Site-directed Mutagenesis Kit, Stratagene, Cedar creek, TX). Successful mutagenesis was confirmed by sequencing.

Retroviral constructs

The retroviral vector pBMN-I-GFP was purchased from the Nolan Lab (http://www.stanford.edu/group/nolan/). The retroviral constructs, B6n and SJn, were generated by subcloning the murine IL-12p35 gene in the XhoI/NotI site which is located proximal of the Internal Ribosomal Entry Site (IRES) and the C57Bl/6 or SJL/J derived IL 12p40 construct at the NcoI/SalI located distal of the IRES.

Constructs were verified by subsequent sequencing. In one of our clones the first base of the ATG start codon, i.e. the adenosine, and the last nine bases, including the TGA stop codon, of the IL 12p35 gene were deleted. This gave rise to a shift in the reading frame of the IL 12p35 gene, as a consequence of which this clone was only able to produce IL 12p40 and no IL-12p70. This clone was used to make constructs synthesizing only p40 from C57Bl/6 or SJL/J origin, constructs B6p40 and SJp40 respectively.

Transfection, cell culture and inhibition of glycosylation

Retroviral constructs were transfected in the Phoenix packaging cell line obtained form the ATCC (http://www.atcc.org) as previously described (14). 48 – 72 hours after transfection viral supernatants were collected and filtered. Subsequently BW5147 thymoma cells were infected using a centrifugation-facilitated protocol. Cells were grown in RPMI 1640 (HEPES buffered 10mM) containing 10% (v/v) fetal calf serum and 1 % pen/strep. Single cell clones were generated by limiting dilution. Supernatants were collected by centrifugation and positive clones were identified by ELISA.

To block glycosylation, cells were incubated during 4 days in the presence of the N-glycosylation inhibitor kifunensine (Kitasatosporia kifusinense, 2 μg/ml; Calbiochem, USA) or the O-glycosylation inhibitor Benzyl 2-acetamido-2-deoxy-α-D-galactopyranoside (Benzyl- α -GalNAc, 4 mM, Sigma, The Netherlands). Efficiency of inhibition was assessed by flow cytometry using the biotinylated lectins Con A (concanavalin A), PNA (peanut agglutinin) (Vector Laboratories, USA), HPA (Helix pomatia agglutinin) and GNA (Galanthus nivalis agglutinin) (Sigma, The Netherlands). Incubation with kifunensine results in an increase in immature N-glycans, highly reactive with Con A and GNA whereas incubation with Benzyl- α -GalNAc results in an increase in immature O-glycans highly reactive with HPA and PNA.

Generation of IL-12 transgenic mice

An artificial EcoRV restriction site was generated just upstream of the start codon of murine Il12b. At the 3’end a BamHI site was generated surrounding the stop codon. The products were loaded on a 1% agarose gel at 150V for 2 hours, and isolated from the gel with a gel purification kit (Qiagen). The constructs were subsequently cloned into a pBluescript®II KS(+/-) expression vector (Stratagene, Cedar creek, TX) containing a 2.5 kB stretch of the Human Growth Hormone gene (kindly provided by dr. Frank Scheiffele) that was cloned into the BamHI and NotI restriction sites of the vector. Ligation of the vector and insert was performed with a Rapid DNA Ligation Kit (Boehringer Mannheim, Indianapolis, IN). Colonies were tested for the insert with restriction digestion with EcoRV and BamHI and confirmed by sequencing.

A stretch of 1040 basepairs of the p40 promoter (from position -974 relative to the start site of transcription to position + 57; including the first noncoding exon of Il12b (12) was generated by PCR amplification of genomic DNA from both a C57Bl/6 and a SJL/J mouse. Direct sequencing did not reveal genetic variation between the two strains in any regulatory part of the promoter, and therefore, the C57Bl/6 derived construct was used for both transgenic lines. A SalI restriction site was added to the 5’region, whereas an EcoRV site was added tot the 3’end and cloned into the pBluescript vector.

The 4.5 kb transgene constructs were isolated from the pBluescript®II KS(+/-) expression vector after Sal I-Not I double digestion and electroelution, followed by Qiagen column purification (Qiagen Inc., Valencia, CA), and Ethanol precipitation.

C57Bl/6 were purchased from The Jackson Laboratory (Bar Harbor, ME) and bred and maintained under approved protocols at the NIAID Transgenic/Knockout Mouse Facility (TKMF), Frederick, MD. One-cell mouse embryos were flushed from female oviducts by standard methods (15). Male pronuclei were microinjected with the isolated DNA construct, and the embryos were implanted into pseudo-pregnant females.

Transgenic founders were identified by screening tail DNA by PCR amplification, using the following primers: forward 5’-CAC ATC TGC TGC TCC ACA, reverse 5’ - TGA TGA TGT CCC TGA TGA A –3’

Electrophoresis and Western blotting

Non-denaturing SDS-PAGE was performed on 4-12% NuPage Bis-Tris gradient gels (Novex; Invitrogen, Breda, The Netherlands) with MOPS running buffer (Novex; Invitrogen, Breda, The Netherlands) in a X cell II mini gel/blot module (Novex; Invitrogen, Breda, The Netherlands) according to the manufacturers protocol. After electrophoresis the separated bands were transferred onto a 0.45 μm nitrocellulose membrane (Schleicher & Schuell,’s-Hertogenbosch, The Netherlands) in the NuPageTransfer Buffer (Novex; Invitrogen, Breda, The Netherlands) containing 10% methanol per gel. Unoccupied places on the membrane were blocked with 5% skimmed milk in phosphate buffered saline (PBS) containing 0.05% Tween 20 (blocking buffer) for 1 hour at room temperature. Incubation with the primary antibody, rat anti-mouse IL-12 Mab - clone C17-8 - (Endogen/Perbio, Etten-Leur, The Netherlands), was performed over night at room temperature in blocking buffer. After washing with PBS and Tween 20 (0.05%) incubation with peroxidase-labeled goat anti-rat antibody (Brunschwig; Amsterdam, The Netherlands) followed. Antibody binding was detected using a chemiluminescence detection system (ECL; Amersham Biosciences, Roosendaal, The Netherlands) according to the manufacturer’s instructions. Molecular weights were estimated by simultaneous electrophoresis and transfer of fluorescence labeled molecular weight markers (MagicMark Western Standard; Invitrogen, Breda, The Netherlands).

Glycoprotein-lectin Immunosorbent Assay (GLIA)

The glycosyl status of both forms of IL-12p40 was determined with the procedure described by Hampel et al. (16). In short, the capturing antibody from the mouse IL-12p40 kit (PharMingen, Alphen a/d Rijn, The Netherlands) was coupled to polystyrene microtiter ELISA plates. After blocking of unoccupied sites with 1% BSA in Trisbuffer incubation with equimolar amounts of either SJL/J type or C57Bl/6 type p40 followed. The presence of specific glycan moieties was analyzed with a panel of lectins with different specificities (see Table I). The IL-12p40-lectin complex was quantified with ExtraAvidin peroxidase conjugate. Background absorbance was measured by incubating with buffer instead of IL-12p40. Samples were analyzed in pentuplicate.

Table I.

Lectins used for identification of Oligosaccharide content of mutant and WT IL-12p40.

lectin Carbohydrate Binding Specificity
Canavalia ensiformis agglutinin (ConA) TerminalαMan, Manα3[Manα6]Man
Galanthus nivalis (GNA) TerminalαMan
Helix pomatia agglutinin (HPA) αGalNac(terminal)
Maackia amurensis agglutinin (MAA) Neu5Acα(2,3)Gal
Sambucus nigra agglutinin (SNA) Neu5Acα2,6Gal or α2,6GalNAc
Ricinus communis agglutinin (RCA), TerminalβGal
Open in a new tab

(GlcNAc: N-acetylglucosamine; Neu5A: sialic acid; Gal: galactose; GalNac: Nacetylgalactosamine; Man: mannose)

Histologic assessment of Tissues

Colonic and cecal tissue specimens obtained 4 days after induction of colitis were fixed in 10% buffered formalin phosphate (Sigma-Aldrich, St. Louis, MO). The specimens were then embedded in paraffin, cut into sections, and stained with H&E. The degree of inflammation on microscopic cross sections of the colon was graded semiquantitatively from 0 to 4 as described previously (17): 0, no evidence of inflammation; 1, low level of lymphocyte infiltration with infiltration seen in ≤ 10% highpower fields and no structural changes observed; 2, moderate lymphocyte infiltration with infiltration seen in 10%–25% high-power fields, crypt elongation, bowel wall thickening that does not extend beyond the mucosal layer, and no evidence of ulceration; 3, high level of lymphocyte infiltration with infiltration seen in 25%–50% high-power fields, high vascular density, and thickening of the bowel wall that extends beyond the mucosal layer; 4, marked degree of lymphocyte infiltration with infiltration seen in ≥ 50% high-power fields, high vascular density, crypt elongation with distortion, and transmural bowel wall thickening with ulceration.

Statistics

To evaluate whether the medians of the collected data did or did not differ statistically significant from each other the Mann–Whitney U test was used, this was performed using GraphPad Prism version 4.00 for Windows, GraphPad Software, San Diego California USA, (http://www.graphpad.com)

Results

Polymorphisms in the coding region of Il12b promote formation of IL12-p70 heterodimers

As alluded to above, we hypothesized that the increased IL-12 p70 secretion in SJL/J mice is the consequence of more efficient heterodimer formation resulting from genetic polymorphism in Il12b. In agreement with published data, we found that the genomic p40 sequences of SJL/J and C57Bl/6 mice differ at two sites and that these differences gives rise to two amino acid substitutions: Met (C57Bl/6) → Thr (SJL/J) at position 169 and Phe (C57Bl/6) → Leu (SJL/J) at position 294 relative to the methionine startcodon. The Met→ Thr substitution is situated opposite of the plane where p40 and p35 interact (amino acid positions 195-203, 261-268 and 315-322)

Next, to determine if these structural differences in the IL-12p40 from the two strains led to differences in their capacities to form heterodimers we generated bicistronic retroviral vectors harboring the IL12p40 from each strain in conjunction with their common p35 chain and determined the capacity of these vectors to produce IL-12p70. In constructing these vectors we took note of the fact that in IL-12p70 expressing cells the IL-12p40 chain is produced in excess (18); therefore to mimic the physiologic condition as closely as possible we placed the p40 chain in the higher producing cap cistron and the p35 chain in the lower producing IRES cistron. In the studies performed murine BW5147 thymoma cells were transduced with the packaged retroviral constructs and cells containing the constructs were selected by limiting dilution and cultured for 4 days after which the concentrations of secreted IL-12p40 and IL-12p70 were measured and then used to determine the ratio of p70 to free p40 as a measure of the affinity of the p40 chain for the p35 chain. Ratios rather than absolute values of p70 and p40 were used since ratios were independent of inter-assay variation and copy number of the integrated gene. As shown in Figure 1A, the ratio of p70/p40 in clones transfected with SJL/J-derived p40 was consistently higher than in clones transfected with C57Bl/6-derived p40 (p<0.001), regardless of the amount of DNA transfected. This indicates that the SJL/J-derived polymorphic variant of p40 more readily forms p70 heterodimers than does the C57Bl/6-derived p40 variant. The above mentioned measurements could also be explained when a difference exists between the two transfected cell lines in the secretion of either the IL-12p70 or IL-12p40 product. As the constructs used only differ in their IL-12p40 region this difference must in it self be a consequence of the polymorphism. To exclude an effect of differential secretion the IL-12p70 ratio over free p40 was measured both intracellularly as well as extracellularly.

Figure 1.

Figure 1

Open in a new tab

A. IL-12p70 synthesis relative to IL-12p40 synthesis.

IL-12p70 and IL-12p40 concentrations were measured by ELISA in the supernatants of cells transfected with plasmids expressing SJL/J type IL-12p70 (○) or C57Bl/6 type IL-12p70 (●). The results are given as the ratio of IL-12p70 over IL-12p40. Shown are data from individual clones of 1 representative transfection out of 3. From each transfection between 15 to 30 clones of each construct were used per measurement.

B. Intracellular and extracellular IL-12p70/IL-12p40 ratios.

IL-12p70 and p40 concentrations were determined with ELISA in supernatants and cell lysates from transfected cells containing plasmids expressing SJL/J type IL-12p70 and p40 or C57Bl/6 type IL-12p70 and p40. The results are given as the ratio of IL-12p70 over free IL-12p40. Bars represent mean ± SD of one representative experiment out of 2. Each measurement was performed in duplicate. (■ C57Bl/6 type construct, IL-12p40, □ SJL/J type construct).

As is shown in figure 1B, the intracellularly derived ratios showed the same, significantly higher values for the SJL/J type IL-12p40 over the C57Bl/6 construct as was seen earlier with the extracellularly derived data (Extracellular p70 respectively p40 from SJL/J derived constructs: 64159 +/- 12730, 1882 +/- 482 vs. C57Bl/6 derived constructs: 9614 +/- 1910, 630 +/-71 pg/ml. Intracellular p70 respectively p40 from SJL/J derived constructs: 5229 +/- 1506, 291 +/- 2 vs. C57Bl/6 derived constructs: 1108 +/- 243, 125 +/- 9 pg/ml).

This shows that the observed differences cannot be ascribed to differential secretion of either the IL12p40 or the IL-12p70 molecule but are a consequence of preferential heterodimerization with the SJL/J derived type IL-12p40.

Taken together, these in vitro findings provide a molecular basis for our previous in vivo observations demonstrating significantly higher serum IL-p70 responses in SJL/J mice as compared to C57Bl/6 mice upon LPS challenge.

The SJL/J derived IL-12p40 subunit also promotes IL-23 formation

In parallel studies, we investigated whether the polymorphism in the p40 subunit also affects affinity for the p19 subunit and thus the amount of IL-23 produced by SJL/J and C57Bl/6 cells. Mice of both strains were challenged with LPS and serum IL-12p40 and IL-23 levels were measured by ELISA. As can be seen in Figure 2, the SJL/J strain responded with a significantly higher IL-23 serum level as compared with that of the C57Bl/6 strain. In accordance with our previous findings, the IL-12p40 level in the two strains again did not differ (SJL/J 73358 +/- 11560 vs. C57Bl/6 66864 +/- 15455 pg/ml, P=NS). This indicates that the SJL/J- derived IL-12p40 subunit also displays a higher affinity for the p19 subunit. Although it can not be fully excluded that strain specific differences in production of the p19 subunit of IL-23 could be responsible for these results, this is less likely since sequencing of the first kB of the promoter region as well as the coding sequence of the SJL/J IL-23A did not reveal sequence variation as compared to the C57Bl/6 IL-23A (results not shown). In addition, the genetic analysis of both strains (11) was not indicative for linkage with the IL23A subunit on chromosome 10.

Figure 2.

Figure 2

Open in a new tab

Serologic cytokine IL-23 response to administration of LPS in SJL/J and C57Bl/6 mice. Mice were intraperitoneally challenged with a sublethal dose of 300 μg LPS. After six hours mice were bled and IL-23 was measured in the serum by means of ELISA. Bars represent mean ± SD of a representative experiment out of 3, each involving 5 mice per group. (□ SJL/J mice, ■ C57Bl/6 mice)

Biological consequences of the SJL/J polymorphic form of IL-12p40

In addition to an effect on heterodimer formation as shown in the previous experiments, the polymorphic variant might also affect the three-dimensional configuration of the protein and thereby its affinity for the IL-12 receptor. To address this question, we determined the in vitro IFN-γ responses in OVA primed DO11.10 cells, carrying the transgenic TCR for OVA, after culturing them in the presence of IL-12p70 (at 8 ng/ml) derived from the respective constructs. As can be seen from Figure 3, equal amounts of IL-12 p70 from either strain resulted in identical induction of IFN-γ release by DO11.10 splenocytes.

Figure 3.

Figure 3

Open in a new tab

Effect of the polymorphic p40 variants on IFN-γ secretion.

Splenocytes from DO11.10 mice were incubated with OVA peptide in the presence of IL-12p70 and increasing amounts of IL-12p40. After 48 hr IFNγ responses were measured in the supernatants. Bars represent mean ± SEM, each measurement was performed in quadruplicate. (■ = induction by C57Bl/6 type IL-12p70 together with C57Bl/6 derived IL-12p40 amounts as indicated, □ = induction by mutant SJL/J type IL-12p70 together with SJL/J derived IL-12p40 amounts as indicated).

In previous studies it was reported that p40, either as a monomer or as a dimer (p40)2, can act as an inhibitor of IFN-γ synthesis by competing with IL-12p70 for its receptor (19). Thus, in further studies we determined if contructs derived from the two strains expressing different polymorphisms produced p40 with different inhibitory capacities. However, as shown in the Western blot studies depicted in Figure 4, we were limited to the study of p40 monomers since the contructs did not produce dimers. We therefore could only investigate a possible influence of the polymorphisms on the antagonizing effect with the p40 monomer. Adding increasing amounts of either of the two p40 variants resulted in a similar mild, dose-related inhibition of IFN-γ production in the in vitro assay system described above (Figure 3). Thus, the polymorphic variation of p40 has neither an effect on the affinity of p70 for its receptor nor the ability to induce IFN-γ; in addition, free p40 monomers exhibiting different polymorphisms do not have differential effects on inhibition of the IFN-γ response.

Figure 4.

Figure 4

Open in a new tab

Western Blot analysis of the two polymorphic variants of IL-12 p40. Supernatants from transfected cell lines expressing only the SJL/J derived IL-12p40 or the C57Bl/6 derived IL-12p40 were run on 4-12% gradient gels under non – denaturing conditions. After transfer to 0.45 μm nitrocellulose the blotted bands were immunodetected with a specific rat anti-mouse IL-12p40 Mab and subsequently visualized with peroxidase labeled rabbit anti-rat IgG antibodies. On the left hand side a molecular weight standard is displayed

Note: for esthetic reasons individual lanes are shown. The original blot is added as supplementary Figure 1

Polymorphic variants of IL12p40 associated with differential glycosylation

As shown in Figure 4, Western blot analysis revealed that SJL/J-derived p40 has a higher molecular weight as compared to C57Bl/6- derived p40, suggesting that the SJL/J-derived variant is associated with increased glycosylation. This can be either due to enhanced N-glycosylation or, alternatively, it can be the consequence of an additional O-glycosylation event since the Met to Thr change in the SJL/J type of IL-12p40 variant is subjected to O-glycosylation.

To investigate these possibilities we first determined differences in glycosylation of IL12p40 from both strains using a glycoprotein-lectin binding assay. To this end we determined the ability of the two variants to bind to a panel of lectins with different glycoprotein binding specifities (Table I). As shown in Figure 5A, the SJL/J –derived type IL-12p40 showed a significantly higher degree of staining with the SNA lectin than did the C57Bl/6-derived IL-12p40. In contrast the other lectins showed no difference between both types of IL-12p40 (data not shown). Increased binding of the SJL/J-derived variant to SNA is indicative for the presence of more sialic acid units and thus for increased glycosylation as already suggested by the Western blot experiments. Sialic residues can be added to N-linked as well as to O-linked sugars in the Golgi apparatus and therefore this assay did not discriminate between these types of glycosylation.

Figure 5.

Figure 5

Open in a new tab

A. Differences in sialic acid content of both IL-12p40 variants.

Equimolar amounts of either SJL/J type or C57Bl/6 type IL-12p40 were subjected to glycoprotein-lectin immunosorbent assay analysis with a panel of different lectins. Only the results with SNA are shown as the other lectins did not generate a signal above background staining. Results are given as the relative absorbance at 450 nm. Samples were run in pentuplicate, bars represent mean ± SD (□: SJL/J type IL-12p40, ■: C57Bl/6 type IL-12p40).

B. Differences in P70/p40 ratios obtained after incubation with or without glycosylation inhibitors.

IL-12p40 and IL-12p70 ratios were determined in the supernatants of cells transfected with either the SJL/J or C57Bl/6 derived constructs which were cultured in the presence of BGN an inhibitor of O-glycosylation, the solvent for BGN (methanol) or with kifunensine, an inhibitor of N-glycosylation. Bars represent mean ± SD of one representative experiment out of 2, each measurement was performed in duplicate. (■ = ratio’s from C57Bl/6 derived constructs, □ = ratio’s from SJL/J type IL-12p40 derived constructs KIF = kifunensine).

We next investigated the consequences of inhibiting the glycosylation on p40 to p35 binding. Here, we cultured cells transfected with either the SJL/J- or C57Bl/6- derived constructs in the presence of 4 mM Benzyl 2-acetamido-2-deoxy-α-D-galactopyranoside (BGN), an inhibitor of O-glycosylation or with kifunensin, an inhibitor of N-glycosylation, and then, following incubation of the cells for 48 hours (at which time inhibition of glycosylation was evident, see supplemental figure 2), determined the p70/p40 ratios of secreted IL-12. As can be seen from Figure 5B, culturing in the presence of kifunensin resulted in a clear decline in the p70/p40 ratio in IL-12 secreted from cells expressing the SJL/J- derived constructs, albeit not to the level seen with C57Bl/6 constructs. In contrast, culturing in the presence of BGN did result in only slightly decreased ratios, and this decrease could be totally ascribed to the effect of methanol in which BGN, but not kifunensin, is dissolved. This implies that if O-glycosylation is present, it does not participate in the increased heterodimerization seen with SJL/J-derived constructs.

Increased production of IL-12p70 in transgenic C57Bl/6 mice harboring the SJL/J derived IL-12p40 variant

In further studies we determined to what extent the above in vitro studies of cells transduced with retroviruses expressing different p40 variants reflect in vivo expression of IL-12p70 in mice with these variants. We therefore generated transgenic C57Bl/6 mice that carried transgenes expressing either the SJL/J variant or the C57Bl/6 variant of the p40 subunit under the IL-12 p40 promoter. From the several founders initially present for each construct, we have randomly chosen two founders from each transgenic line for our experiments. We challenged these transgenics intraperitoneally with LPS and determined their ability to produce IL-12p70 (as measured in the serum). As shown in Figure 6, mice bearing a transgene expressing the SJL/J variant exhibited a significantly higher level of IL-12p70 synthesis than mice bearing the C57Bl/6 variant, while there is no difference in the IL-12p40 synthesis at the translational level (Figure 6) as well as at the transcriptional level (supplementary Figure 3).

Figure 6.

Figure 6

Open in a new tab

C57Bl/6 transgenic mice harboring the SJL/J variant of the IL-12p40 gene or the C57Bl/6 variant were challenged with a sublethal dose of LPS after which the concentration in serum of IL-12p70 and IL-12p40 was measured by ELISA. Bars represent mean ± SEM. of (□ = IL-12p70 or IL-12p40 synthesized by transgenics carrying the SJL/J type IL-12p40; n= 14. ■ = IL-12p70 or IL-12p40 synthesized by transgenics carrying the C57Bl/6 type IL-12p40; n= 17).

No interlineage differences between founder lines were observed. Because in these mice all parameters at the transcriptional as well as at the translational level are exactly the same, the differences observed are very likely due to enhanced IL-12p70 synthesis resulting from preferential heterodimerization of IL12-p35 with the SJL/J IL12-p40 variant.

Role of the Il12b polymorphism in colitis susceptibility

The above mentioned experiments clearly indicate that the strain differences in IL-12p70 formation can be related to polymorphisms in the coding region of Il12b. This difference in response is likely to be relevant to the difference in TNBS-colitis susceptibility in the two strains since it has been shown that TNBS-colitis is driven by IL-12-mediated responses. In a next series of experiments we sought to further establish this relationship. Hereto, we first determined serum IL-12p70 responses in colitis resistant C57Bl/6 and colitis susceptible SJL/J mice four days after induction of TNBS-colitis. As shown in Figure 7, IL-12 responses were significantly higher in SJL/J mice as compared with C57Bl/6 mice (p<0.05). This higher level can not be accounted for by an initially higher basal level in the SJL/J mice as is clear from the data at day 0, showing no differences present between both strains (Figure 7). Again, similar to the previous LPS challenge studies, the IL-12p40 responses were not different between strains (results not shown).

Figure 7.

Figure 7

Open in a new tab

Serologic cytokine responses to intrarectal administration of TNBS in SJL/J and C57Bl/6 mice.

Mice were instilled with TNBS dissolved in ethanol (day 4) or with ethanol alone (day 0). Four days after the induction of colitis, mice were sacrificed and the serum IL12 responses determined. Bars represent mean ± SD of one representative experiment out of 3, each involving 5 mice per group, (□ SJL/J mice, ■ C57Bl/6 mice).

In a final series of experiments we determined whether C57Bl/6 mice which are normally highly resistant to the induction of TNBS-colitis are rendered susceptible to such induction if they are bearing a transgene expressing the SJL/J- derived p40 variant. To this end, intra-rectal TNBS was administered to transgenic C57BLl/6 mice carrying the SJL/J- derived p40 variant transgene as well as to transgenic C57Bl/6 mice carrying the C57Bl/6-derived variant transgene; in addition, intra-rectal TNBS was administered to control SJL/J mice as well as non-transgenic C57Bl/6 littermates. Three days after the TNBS-challenge mice were sacrificed and evaluated for the presence of colitis. It should be noted upfront that the differences in susceptibility to TNBS colitis are under polygenic control and that the region on chromosome 11 was only one out of several genetic risk loci (11). Thus, in the absence of other genetic risk factors derived from the SJL/J strain, it was to be expected that the transgene would only have limited effect on the trait in the C57Bl/6 strain. As can be seen in Figure 8A, 8 of 10 SJL/J mice developed severe weight loss, whereas in all other groups, weights had normalized at day 3. Remarkably, in the transgenic mice bearing the SJL/J variant, histological signs of mild-moderate inflammation were seen in 8 of 10 mice (Figure 8D), however this did not reach statistical significance. In contrast such abnormalities were seen in only a small proportion of mice bearing the transgene expressing the C57Bl/6-derived p40 variant, while control SJL/J and C57Bl/6 mice were positive and negative respectively for histological signs of inflammation (Figure 8B).

Figure 8.

Figure 8

Open in a new tab

A. Weight loss after induction of colitis by rectal instillation of TNBS at day 0 (mean ± SD relative to day 0 which was set as 100%, each group involved 10 mice). □ = C57Bl/6 mice carrying the SJL/J derived p40 variant, ▲ = C57Bl/6 mice carrying the C57Bl/6 derived p40 variant, ■ = non transgenic littermates ◊ = SJL/J wild type control.

B. Histological scoring of inflammation.

Scoring was done on H&E stained slides. Results are grouped according to their genetic background: SJL/J = nontransgenic SJL/J WT mice, SJL Tg = C57Bl/6 mice carrying the SJL/J derived p40 variant, BL6 Tg = C57Bl/6 mice carrying the C57Bl/6 derived p40 variant and littermates = non transgenic littermates.

Within a bar, each section represents the percentage of mice that either showed; no signs of inflammation (□); mild signs of inflammation ( Inline graphic); moderate signs of inflammation ( Inline graphic); or severe signs of inflammation (■).

C. Histologic analysis of the colons of transgenic, nontransgenic littermates and SJL/J wt mice after TNBS colitis induction. Representative H&E-stained cross sections of colon specimens are shown.

A: represents the normal appearing colon from resistant mice predominantly found in mice carrying the C57Bl/6 derived p40 variant and non transgenic littermates. B and C are examples of the mild (B) to moderate (C) forms of colitis predominantly found in mice carrying the SJL/J derived p40 variant. D shows the severe colitis found in the colitis susceptible SJL/J WT control group.

D. Distribution of histological scoring with the transgenic mouse strains. Scoring was done on H&E stained slides. SJL Tg = C57Bl/6 mice carrying the SJL/J derived p40 variant, BL6 Tg = C57Bl/6 mice carrying the C57Bl/6 derived p40 variant. 0, no evidence of inflammation; 1, mild inflammation; 2 moderate inflammation; 3 severe inflammation. The average score of both groups is indicated with a horizontal line.

Representative histological slides are shown in Figure 8C (a larger magnification is provided in supplementary Figure 4). Thus, transgenic mice carrying the SJL/J derived p40 variant, are more prone to develop an inflammatory reaction in the colonic mucosa upon TNBS challenge, albeit, as predicted, a reaction that was too mild to result in frank clinical signs of colitis.

Discussion

Studies of both Crohn’s disease (CD) and murine models of this disease provide clear evidence that IL-12p40 plays a crucial and non-redundant role in disease pathogenesis. Human mucosal antigen presenting cells from CD patients secrete increased amounts of IL-12p70 (20) and treatment of patients with this disease with anti-IL-12p40 results in marked amelioration of disease. In addition, IL-12p40 is overexpressed by dendritic cells in the two common murine models of Crohn’s disease, TNBS-colitis (21) and cell-transfer colitis (22) and here again, treatment with anti-IL-12p40 is a very effective agent for preventing or treating the inflammation (17). Thus, while some controversy persists as to whether the main down-stream effector cytokine in Crohn’s-like inflammation is IFN-γ or IL-17, the role of IL-12p40 as a component of either IL-12p70 or IL-23, the “master” cytokines supporting the production of these downstream cytokines is unequivocal. More recently an etiological role for the IL-12/IL-23 axis in Crohn’s disease has also been supported by the fact that polymorphisms in both the gene encoding IL-12 p40 (Il12b) and the gene encoding the IL-23 receptor (IL23R) are genetically linked to disease susceptibility; however, the mechanism by which these polymorphisms contribute to disease susceptibility or resistance remains to be determined.

Genetic analysis of experimental colitis has revealed that one of the two loci governing susceptibility to TNBS-colitis in the susceptible SJL/J strain is located on chromosome 11 and harbors the gene for the IL-12p40 subunit of IL-12 (11). Moreover, it was established that this SJL/J locus is associated with higher IL-12 responses than found in C57Bl/6 mice carrying the resistant genotype. Surprisingly, this higher response was not accompanied by a concomitant increase in IL-12p40 synthesis and no differences were observed in the sequence of the regulatory regions of the genes in the two strains. However, the two strains did differ at two sites in the structural part of the gene. Since in previous in vitro mutagenesis experiments it has been shown that amino acid changes in human IL-12 can dramatically influence formation of the mature heterodimer (23) we hypothesized that these structural differences also lead to effects on the formation of the heterodimer and thus contribute to the mechanism of differences in colitis susceptibility of the SJL/J and C57Bl/6 mouse strains.

To test this hypothesis we generated vectors containing both subunits of IL-12 and found that supernatants of cells containing constructs expressing the SJL/J-derived variant of IL-12p40 synthesized significantly greater amounts of IL-12p70 than the constructs expressing the C57Bl/6-derived variant of IL-12p40. These differences are most likely attributable to post-translational events since the transcriptional and translational regime applying to the two constructs are identical. In addition, the differences cannot be ascribed to preferential changes in secretion as suggested by studies showing the mutations in the IL-12p40 chain can influence secretion rates of this protein (24), since we determined that cells expressing both constructs exhibited excellent correlation of intra- and extracellular IL-12p70/p40 ratios. Finally, we also determined that the IL-12p70 expressed by cells bearing both transgenes produced IL-12 that had equivalent capacity to induce synthesis of IFN-γ, indicating that while cells exhibited quantitative differences in IL-12p70 production they did not exhibit qualitative differences in IL-12 function.

In previous studies conducted by Carra et al (25) it was found that interference with N-glycosylation but not O-glycosylation results in alterations in binding of individual chains of heterodimers. Indeed, in studies involving specific inhibition of these forms of glycosylation we confirmed this differential effect with respect to the IL-12p40/IL-12p35 heterodimer formation. However, two caveats to this conclusion should be noted. First, since the Met-Thr variation would predict an extra O-glycosylation site in the SJL/J strain IL-12p40 one cannot completely rule out the presence of an altered O-glycosylation site in this p40 even if this site does not participate in influencing heterodimer formation. Second, we noted that incubation of cells expressing the SJL/J-derived construct with kifunensin, an inhibitor of N-glycosylation, did not completely resolve the increased heterodimer formation seen with this construct. This either implies that the inhibition was not complete or that other mechanisms are at play in SJL/J-type IL-12 p40/p35 (or IL-12p40/p19) heterodimer formation e.g. amino acid-induced alterations in electrostatic interactions between the subunits.

The higher molecular weight of the SJL/J-derived IL-12p40 seen on Western blot may be the consequence of an altered structure in the SJL/J-derived chain which makes the N-glycosylated residues more accessible to secondary glycosylation in the Golgi apparatus. This mechanism applies to the formation of complex-type N-glycans as opposed to the high-mannose type N-glycans since the sugar residues are inaccessible to secondary glycosylation due to protein folding (for a review see (26)). This structural difference is likely to be a consequence of the amino acid differences in the SJL/J strain which are also responsible for the enhanced heterodimer formation involving SJL/J IL-12p40 and p35 (and possibly p19).

Final proof that the increased levels of IL-12p70 synthesis by the SJL/J mice as compared to that of C57Bl/6 mice is due to differences in amino acid composition of the respective IL-12p40 chains came from studies of C57Bl/6 transgenic mice bearing transgenes expressing the p40 chains from the two mouse strains. In particular, the mice bearing the transgene expressing the SJL/J variant of IL-12p40 synthesized significantly more IL-12p70 than did the mice bearing a transgene expressing the C57Bl/6 variant of IL-12p40. Since the two types of transgenic mice were completely similar at the transcriptional and translational level as evidenced for instance by the identical amount of free IL-12p40 synthesized by both transgenic mice after LPS challenge with the only difference being the amino acid composition it follows that this latter is in fact the cause of the differences in the amounts of secreted IL-12p70. It is unlikely that the differences in IL-12 p70 secretion observed in the IL-12 transgenic mice were attributable to different copy number insertion of the transgene, since the amount of p40 secreted and the level of mRNA was similar between the transgenic lines. In addition, we generated transgenic lines from different founders with potential different copy number inserts and the results were similar in these lines (results not shown). Although not directly studied here, a similar mechanism is likely to account for the fact that we observed a higher synthesis rate of IL-23 in LPS-stimulated SJL/J mice as compared to C57Bl/6 mice.

Mucosal homeostasis is a dynamic process that depends on the balance between pro- and anti-inflammatory signals most of which are under the control of genetic factors. Thus, in an individual or strain bearing a genetic trait resulting in altered regulation of the immunologic response, one can expect to see a change in susceptibility to inflammation. In the present study we formally verified this principle by showing that a genetic trait controlled by a locus on chromosome 11 and leading to an increased capacity of IL-12p40 to form heterodimers with IL-12p35 or IL-23p19 to form IL-12 and IL-23 respectively, is in fact associated with a greater susceptibility to induction of TNBS colitis. Thus, in the study relevant to this point we showed that C57Bl/6 mice, normally highly resistant to TNBS-colitis but in this case bearing a transgene expressing SJL/J IL-12p40, developed histological signs of colon inflammation upon administration of TNBS whereas C57Bl/6 mice bearing a transgene expressing the C57Bl/6 IL-12p40 developed little or no colitis.

It should be kept in mind that the C57Bl/6 mice bearing the SJL/J transgene expressing a SJL/J IL-12p40 did not develop a colitis as severe as that observed in the same study of SJL/J mice. It is reasonable to explain this apparent discrepancy by the fact that the genetic susceptibility locus in the transgenic mice was only one of at least two susceptibility loci previously identified as important in the induction of TNBS-colitis. The other identified locus was on chromosome 9 and has subsequently been shown to affect the expression of the gene encoding claudin-18, a protein involved in epithelial barrier integrity (27). Thus, it seems likely that a full degree of susceptibility to TNBS-colitis requires not one, but at least two factors: decreased epithelial barrier function and increased pro-inflammatory effector cytokine synthesis. Ongoing studies with congenic C57Bl/6 Tg mice harboring both the chromosome 9 region derived from the SJL/J strain as well as the IL-12p40 chain will be necessary to provide a definitive verification of this hypothesis.

There has been ongoing interest in the relation between polymorphisms in cytokine genes and the expression of these genes to a large number of immune-mediated diseases. This is seen, for example, in the case of IL-12, where various polymorphisms have been found that correlate with variations in IL-12 levels (28-30) and a substantial number of human diseases have now been genetically linked to the IL-12 gene, including various infectious diseases and autoimmune diseases such as psoriasis (7, 28, 29, 31-33). However most if not all of the studies of polymorphisms relating to cytokine genes relate to polymorphisms in regulatory sequences, i.e., the promoter region or the 3’ UTR region (34). Thus, as far as we know, the present study of IL-12 is the first study to demonstrate that a polymorphism in the coding region of a cytokine gene directly dictates the biological availability of that cytokine and that this, in turn, leads to changes in disease susceptibility. Thus, while earlier more limited studies did not find an association between IBD and IL12B, recent genome-wide association studies have found such an association with human IBD and IL12B (35). Whether or not the mechanism identified in this study underlies this association and thus influences human disease requires further investigation and should be taken into consideration when interpreting findings from GWAS studies.

In conclusion, we have found ample evidence that polymorphisms in the SJL/J-derived IL-12p40 chain lead to an IL-12p40 subunit of IL-12 and IL-23 with a higher affinity for its p35 or p19 subunit counterpart, respectively. This results in an inherently higher synthesis rate of the mature cytokine which contributes to the higher susceptibility for TNBS-colitis found in the SJL/J strain.

Supplementary Material

figures 1-4

Abbreviations used

BGN

Benzyl-2-acetamido-2-deoxy-α-D-galactopyranoside

GWAS

genome wide association studies

IRES

Internal Ribosomal Entry Site

TNBS

trinitrobenzene sulfonic acid

References

  • 1.Bonen DK, Cho JH. The genetics of inflammatory bowel disease. Gastroenterology. 2003;124:521. doi: 10.1053/gast.2003.50045. [DOI] [PubMed] [Google Scholar]
  • 2.Bouma G, Strober W. The immunological and genetic basis of inflammatory bowel disease. Nat Rev Immunol. 2003;3:521. doi: 10.1038/nri1132. [DOI] [PubMed] [Google Scholar]
  • 3.Ekbom A, Montgomery SM. Environmental risk factors (excluding tobacco and microorganisms): critical analysis of old and new hypotheses. Best Pract Res Clin Gastroenterol. 2004;18:497. doi: 10.1016/j.bpg.2003.12.005. [DOI] [PubMed] [Google Scholar]
  • 4.Budarf ML, Labbe C, David G, Rioux JD. GWA studies: rewriting the story of IBD. Trends Genet. 2009;25:137. doi: 10.1016/j.tig.2009.01.001. [DOI] [PubMed] [Google Scholar]
  • 5.Barrett JC, Hansoul S, Nicolae DL, Cho JH, Duerr RH, Rioux JD, Brant SR, Silverberg MS, Taylor KD, Barmada MM, Bitton A, Dassopoulos T, Datta LW, Green T, Griffiths AM, Kistner EO, Murtha MT, Regueiro MD, Rotter JI, Schumm LP, Steinhart AH, Targan SR, Xavier RJ, Libioulle C, Sandor C, Lathrop M, Belaiche J, Dewit O, Gut I, Heath S, Laukens D, Mni M, Rutgeerts P, Van Gossum A, Zelenika D, Franchimont D, Hugot JP, de Vos M, Vermeire S, Louis E, Cardon LR, Anderson CA, Drummond H, Nimmo E, Ahmad T, Prescott NJ, Onnie CM, Fisher SA, Marchini J, Ghori J, Bumpstead S, Gwilliam R, Tremelling M, Deloukas P, Mansfield J, Jewell D, Satsangi J, Mathew CG, Parkes M, Georges M, Daly MJ. Genome-wide association defines more than 30 distinct susceptibility loci for Crohn’s disease. Nat Genet. 2008;40:955. doi: 10.1038/NG.175. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Yen D, Cheung J, Scheerens H, Poulet F, McClanahan T, McKenzie B, Kleinschek MA, Owyang A, Mattson J, Blumenschein W, Murphy E, Sathe M, Cua DJ, Kastelein RA, Rennick D. IL-23 is essential for T cell-mediated colitis and promotes inflammation via IL-17 and IL-6. J Clin Invest. 2006;116:1310. doi: 10.1172/JCI21404. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Wellcome, Trust, Case, Control, and Consortium. Genome-wide association study of 14,000 cases of seven common diseases and 3,000 shared controls. Nature. 2007;447:661. doi: 10.1038/nature05911. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Wang K, Zhang H, Kugathasan S, Annese V, Bradfield JP, Russell RK, Sleiman PM, Imielinski M, Glessner J, Hou C, Wilson DC, Walters T, Kim C, Frackelton EC, Lionetti P, Barabino A, Van Limbergen J, Guthery S, Denson L, Piccoli D, Li M, Dubinsky M, Silverberg M, Griffiths A, Grant SF, Satsangi J, Baldassano R, Hakonarson H. Diverse genome-wide association studies associate the IL12/IL23 pathway with Crohn Disease. Am J Hum Genet. 2009;84:399. doi: 10.1016/j.ajhg.2009.01.026. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Cho JH. The genetics and immunopathogenesis of inflammatory bowel disease. Nat Rev Immunol. 2008;8:458. doi: 10.1038/nri2340. [DOI] [PubMed] [Google Scholar]
  • 10.Mannon PJ, Fuss IJ, Mayer L, Elson CO, Sandborn WJ, Present D, Dolin B, Goodman N, Groden C, Hornung RL, Quezado M, Yang Z, Neurath MF, Salfeld J, Veldman GM, Schwertschlag U, Strober W. Anti-interleukin-12 antibody for active Crohn’s disease. N Engl J Med. 2004;351:2069. doi: 10.1056/NEJMoa033402. [DOI] [PubMed] [Google Scholar]
  • 11.Bouma G, Kaushiva A, Strober W. Experimental murine colitis is regulated by two genetic loci, including one on chromosome 11 that regulates IL-12 responses. Gastroenterology. 2002;123:554. doi: 10.1053/gast.2002.34752. [DOI] [PubMed] [Google Scholar]
  • 12.Murphy TL, Cleveland MG, Kulesza P, Magram J, Murphy KM. Regulation of interleukin 12 p40 expression through an NF-kappa B half-site. Mol Cell Biol. 1995;15:5258. doi: 10.1128/mcb.15.10.5258. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Samsom JN, van Berkel LA, van Helvoort JM, Unger WW, Jansen W, Thepen T, Mebius RE, Verbeek SS, Kraal G. Fc gamma RIIB regulates nasal and oral tolerance: a role for dendritic cells. J Immunol. 2005;174:5279. doi: 10.4049/jimmunol.174.9.5279. [DOI] [PubMed] [Google Scholar]
  • 14.Kinsella TM, Nolan GP. Episomal vectors rapidly and stably produce high-titer recombinant retrovirus. Hum Gene Ther. 1996;7:1405. doi: 10.1089/hum.1996.7.12-1405. [DOI] [PubMed] [Google Scholar]
  • 15.Hogan BL, Blessing M, Winnier GE, Suzuki N, Jones CM. Growth factors in development: the role of TGF-beta related polypeptide signalling molecules in embryogenesis. Dev Suppl. 1994:53. [PubMed] [Google Scholar]
  • 16.Hampel DJ, Kottgen B, Dudenhausen JW, Kottgen E. Fetal fibronectin as a marker for an imminent (preterm) delivery. A new technique using the glycoprotein lectin immunosorbent assay. J Immunol Methods. 1999;224:31. doi: 10.1016/s0022-1759(99)00006-x. [DOI] [PubMed] [Google Scholar]
  • 17.Neurath MF, Fuss I, Kelsall BL, Stuber E, Strober W. Antibodies to interleukin 12 abrogate established experimental colitis in mice. J Exp Med. 1995;182:1281. doi: 10.1084/jem.182.5.1281. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Snijders A, Hilkens CM, van der Pouw Kraan TC, Engel M, Aarden LA, Kapsenberg ML. Regulation of bioactive IL-12 production in lipopolysaccharide-stimulated human monocytes is determined by the expression of the p35 subunit. J Immunol. 1996;156:1207. [PubMed] [Google Scholar]
  • 19.Gillessen S, Carvajal D, Ling P, Podlaski FJ, Stremlo DL, Familletti PC, Gubler U, Presky DH, Stern AS, Gately MK. Mouse interleukin-12 (IL-12) p40 homodimer: a potent IL-12 antagonist. Eur J Immunol. 1995;25:200. doi: 10.1002/eji.1830250133. [DOI] [PubMed] [Google Scholar]
  • 20.Fuss IJ, Becker C, Yang Z, Groden C, Hornung RL, Heller F, Neurath MF, Strober W, Mannon PJ. Both IL-12p70 and IL-23 are synthesized during active Crohn’s disease and are down-regulated by treatment with anti-IL-12 p40 monoclonal antibody. Inflamm Bowel Dis. 2006;12:9. doi: 10.1097/01.mib.0000194183.92671.b6. [DOI] [PubMed] [Google Scholar]
  • 21.Becker C, Dornhoff H, Neufert C, Fantini MC, Wirtz S, Huebner S, Nikolaev A, Lehr HA, Murphy AJ, Valenzuela DM, Yancopoulos GD, Galle PR, Karow M, Neurath MF. Cutting edge: IL-23 cross-regulates IL-12 production in T cell-dependent experimental colitis. J Immunol. 2006;177:2760. doi: 10.4049/jimmunol.177.5.2760. [DOI] [PubMed] [Google Scholar]
  • 22.Powrie F, Leach MW, Mauze S, Caddle LB, Coffman RL. Phenotypically distinct subsets of CD4+ T cells induce or protect from chronic intestinal inflammation in C. B-17 scid mice. Int Immunol. 1993;5:1461. doi: 10.1093/intimm/5.11.1461. [DOI] [PubMed] [Google Scholar]
  • 23.Yoon C, Johnston SC, Tang J, Stahl M, Tobin JF, Somers WS. Charged residues dominate a unique interlocking topography in the heterodimeric cytokine interleukin-12. Embo J. 2000;19:3530. doi: 10.1093/emboj/19.14.3530. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Ha SJ, Chang J, Song MK, Suh YS, Jin HT, Lee CH, Nam GH, Choi G, Choi KY, Lee SH, Kim WB, Sung YC. Engineering N-glycosylation mutations in IL-12 enhances sustained cytotoxic T lymphocyte responses for DNA immunization. Nat Biotechnol. 2002;20:381. doi: 10.1038/nbt0402-381. [DOI] [PubMed] [Google Scholar]
  • 25.Carra G, Gerosa F, Trinchieri G. Biosynthesis and posttranslational regulation of human IL-12. J Immunol. 2000;164:4752. doi: 10.4049/jimmunol.164.9.4752. [DOI] [PubMed] [Google Scholar]
  • 26.Freeze HH. Genetic defects in the human glycome. Nat Rev Genet. 2006;7:537. doi: 10.1038/nrg1894. [DOI] [PubMed] [Google Scholar]
  • 27.Zwiers A, Fuss IJ, Leijen S, Mulder CJ, Kraal G, Bouma G. Increased expression of the tight junction molecule claudin-18 A1 in both experimental colitis and ulcerative colitis. Inflamm Bowel Dis. 2008 doi: 10.1002/ibd.20695. [DOI] [PubMed] [Google Scholar]
  • 28.Morahan G, Huang D, Wu M, Holt BJ, White GP, Kendall GE, Sly PD, Holt PG. Association of IL12B promoter polymorphism with severity of atopic and non-atopic asthma in children. Lancet. 2002;360:455. doi: 10.1016/S0140-6736(02)09676-9. [DOI] [PubMed] [Google Scholar]
  • 29.Morahan G, Huang D, Ymer SI, Cancilla MR, Stephen K, Dabadghao P, Werther G, Tait BD, Harrison LC, Colman PG. Linkage disequilibrium of a type 1 diabetes susceptibility locus with a regulatory IL12B allele. Nat Genet. 2001;27:218. doi: 10.1038/84872. [DOI] [PubMed] [Google Scholar]
  • 30.Zwiers A, Seegers D, Heijmans R, Koch A, Hampe J, Nikolaus S, Pena AS, Schreiber S, Bouma G. Definition of polymorphisms and haplotypes in the interleukin-12B gene: association with IL-12 production but not with Crohn’s disease. Genes Immun. 2004;5:675. doi: 10.1038/sj.gene.6364131. [DOI] [PubMed] [Google Scholar]
  • 31.Altare F, Lammas D, Revy P, Jouanguy E, Doffinger R, Lamhamedi S, Drysdale P, Scheel-Toellner D, Girdlestone J, Darbyshire P, Wadhwa M, Dockrell H, Salmon M, Fischer A, Durandy A, Casanova JL, Kumararatne DS. Inherited interleukin 12 deficiency in a child with bacille Calmette-Guerin and Salmonella enteritidis disseminated infection. J Clin Invest. 1998;102:2035. doi: 10.1172/JCI4950. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Picard C, Fieschi C, Altare F, Al-Jumaah S, Al-Hajjar S, Feinberg J, Dupuis S, Soudais C, Al-Mohsen IZ, Genin E, Lammas D, Kumararatne DS, Leclerc T, Rafii A, Frayha H, Murugasu B, Wah LB, Sinniah R, Loubser M, Okamoto E, Al-Ghonaium A, Tufenkeji H, Abel L, Casanova JL. Inherited interleukin-12 deficiency: IL12B genotype and clinical phenotype of 13 patients from six kindreds. Am J Hum Genet. 2002;70:336. doi: 10.1086/338625. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Randolph AG, Lange C, Silverman EK, Lazarus R, Silverman ES, Raby B, Brown A, Ozonoff A, Richter B, Weiss ST. The IL12B gene is associated with asthma. Am J Hum Genet. 2004;75:709. doi: 10.1086/424886. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Bouma G, Crusius JB, Oudkerk Pool M, Kolkman JJ, von Blomberg BM, Kostense PJ, Giphart MJ, Schreuder GM, Meuwissen SG, Pena AS. Secretion of tumour necrosis factor alpha and lymphotoxin alpha in relation to polymorphisms in the TNF genes and HLA-DR alleles. Relevance for inflammatory bowel disease Scand. J Immunol. 1996;43:456. doi: 10.1046/j.1365-3083.1996.d01-65.x. [DOI] [PubMed] [Google Scholar]
  • 35.Parkes M, Barrett JC, Prescott NJ, Tremelling M, Anderson CA, Fisher SA, Roberts RG, Nimmo ER, Cummings FR, Soars D, Drummond H, Lees CW, Khawaja SA, Bagnall R, Burke DA, Todhunter CE, Ahmad T, Onnie CM, McArdle W, Strachan D, Bethel G, Bryan C, Lewis CM, Deloukas P, Forbes A, Sanderson J, Jewell DP, Satsangi J, Mansfield JC, Cardon L, Mathew CG. Sequence variants in the autophagy gene IRGM and multiple other replicating loci contribute to Crohn’s disease susceptibility. Nat Genet. 2007;39:830. doi: 10.1038/ng2061. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

figures 1-4
NIHMS420554-supplement-figures_1-4.doc (3.6MB, doc)

RESOURCES