Natural genetic variation profoundly regulates gene expression in immune cells and dictates susceptibility to CNS autoimmunity.

Regulation of gene expression in immune cells is known to be under genetic control, and likely contributes to susceptibility to autoimmune diseases such as multiple sclerosis (MS). How this occurs in concert across multiple immune cell types is poorly understood. Using a mouse model that harnesses the genetic diversity of wild-derived mice, more accurately reflecting genetically diverse human populations, we provide an extensive characterization of the genetic regulation of gene expression in five different naive immune cell types relevant to MS. The immune cell transcriptome is shown to be under profound genetic control, exhibiting diverse patterns: global, cell-specific and sex-specific. Bioinformatic analysis of the genetically controlled transcript networks reveals reduced cell type specificity and inflammatory activity in wild-derived PWD/PhJ mice, compared with the conventional laboratory strain C57BL/6J. Additionally, candidate MS-GWAS (genome-wide association study candidate genes for MS susceptibility) genes were significantly enriched among transcripts overrepresented in C57BL/6J cells compared with PWD. These expression level differences correlate with robust differences in susceptibility to experimental autoimmune encephalomyelitis, the principal model of MS, and skewing of the encephalitogenic T-cell responses. Taken together, our results provide functional insights into the genetic regulation of the immune transcriptome, and shed light on how this in turn contributes to susceptibility to autoimmune disease.

Diubiquitin (Ubd) is a susceptibility gene for virus-triggered autoimmune diabetes in rats.

Genetic studies of type 1 diabetes (T1D) have been advanced by comparative analysis of multiple susceptible and resistant rat strains with a permissive class II MHC haplotype, RT1(u). LEW.1WR1 (but not resistant LEW.1W or WF) rats are susceptible to T1D induced by a TLR3 agonist polyinosinic:polycytidylic acid followed by infection with parvovirus. We have mapped genetic loci for virus-induced T1D susceptibility, identifying a major susceptibility locus (Iddm37) near the MHC. The Iddm37 homologs on mouse and human chromosomes are also diabetes linked. We report that a major effect gene within Iddm37 is diubiquitin (Ubd). Gene expression profiling of pancreatic lymph nodes in susceptible and resistant rats during disease induction showed differences in Ubd transcript abundance. The LEW.1WR1 Ubd promoter allele leads to higher inducible levels of UBD than that of LEW.1W or WF. Using zinc-finger nucleases , we deleted a segment of the LEW.1WR1 Ubd gene and eliminated its expression. UBD-deficient rats show substantially reduced diabetes after viral infection. Complementary studies show that there may be another diabetes gene in addition to Ubd in the Iddm37 interval. These data prove that Ubd is a diabetes susceptibility gene, providing insight into the interplay of multiple genes and environmental factors in T1D susceptibility.

Fine-mapping quantitative trait loci affecting murine external ear tissue regeneration in the LG/J by SM/J advanced intercross line.

External ear hole closure in LG/J mice represents a model of regenerative response. It is accompanied by the formation of a blastema-like structure and the re-growth of multiple tissues, including cartilage. The ability to regenerate tissue is heritable. An F34 advanced intercross line of mice (Wustl:LG,SM-G34) was generated to identify genomic loci involved in ear hole closure over a 30-day healing period. We mapped 19 quantitative trait loci (QTL) for ear hole closure. Individual gene effects are relatively small (0.08 mm), and most loci have co-dominant effects with phenotypically intermediate heterozygotes. QTL support regions were limited to a median size of 2 Mb containing a median of 19 genes. Positional candidate genes were evaluated using differential transcript expression between LG/J and SM/J healing tissue, function analysis and bioinformatic analysis of single-nucleotide polymorphisms in and around positional candidate genes of interest. Analysis of the set of 34 positional candidate genes and those displaying expression differences revealed over-representation of genes involved in cell cycle regulation/DNA damage, cell migration and adhesion, developmentally related genes and metabolism. This indicates that the healing phenotype in LG/J mice involves multiple physiological mechanisms.

Temporal induction of immunoregulatory processes coincides with age-dependent resistance to viral-induced type 1 diabetes.

The dilute plasma cytokine milieu associated with type 1 diabetes (T1D), while difficult to measure directly, is sufficient to drive transcription in a bioassay that uses healthy leukocytes as reporters. Previously, we reported disease-associated, partially IL-1 dependent, transcriptional signatures in both T1D patients and the BioBreeding (BB) rat model. Here, we examine temporal signatures in congenic BBDR.lyp/lyp rats that develop spontaneous T1D, and BBDR rats where T1D progresses only after immunological perturbation in young animals. After weaning, the BBDR temporal signature showed early coincident induction of transcription related to innate inflammation as well as IL-10- and TGF-ß-mediated regulation. BBDR plasma cytokine levels mirrored the signatures showing early inflammation, followed by induction of a regulated state that correlated with failure of virus to induce T1D in older rats. In contrast, the BBDR.lyp/lyp temporal signature exhibited asynchronous dynamics, with delayed induction of inflammatory transcription and later, weaker induction of regulatory transcription, consistent with their deficiency in regulatory T cells. Through longitudinal analyses of plasma-induced signatures in BB rats and a human T1D progressor, we have identified changes in immunoregulatory processes that attenuate a preexisting innate inflammatory state in BBDR rats, suggesting a mechanism underlying the decline in T1D susceptibility with age.

Healing quantitative trait loci in a combined cross analysis using related mouse strain crosses.

Inbred mouse strains MRL and LG share the ability to fully heal ear hole punches with the full range of appropriate tissues without scarring. They also share a common ancestry, MRL being formed from a multi-strain cross with two final backcrosses to LG before being inbred by brother-sister mating. Many gene-mapping studies for healing ability have been performed using these two strains, resulting in the location of about 20 quantitative trait loci (QTLs). Here, we combine two of these crosses (N = 638), MRL/lpr × C57BL/6NTac and LG/J × SM/J, in a single combined cross analysis to increase the mapping power, decrease QTL support intervals, separate multiple QTLs and establish allelic states at individual QTL. The combined cross analysis located 11 QTLs, 6 affecting only one cross (5 LG × SM and 1 MRL × B6) and 5 affecting both crosses, approximately the number of common QTLs expected given strain SNP similarity. Amongst the five QTLs mapped in both crosses, three had significantly different genetic effects, additive in one cross and over or underdominant in the other. It is possible that allelic states at these three loci are different in SM and B6 because they lead to differences in dominance interactions with the LG and MRL alleles. QTL support intervals are 40% smaller in the combined cross analysis than in either of the single crosses. Combined cross analysis was successful in enhancing the interpretation of earlier QTL results for these strains.

Multiple linked quantitative trait loci within the Tmevd2/Eae3 interval control the severity of experimental allergic encephalomyelitis in DBA/2J mice.

Theiler's murine encephalomyelitis virus-induced demyelination (TMEVD) and experimental allergic encephalomyelitis (EAE) are the principal animal models of multiple sclerosis (MS). Previously, we provided evidence that Tmevd2 and Eae3 may represent either a shared susceptibility locus or members of a gene complex controlling susceptibility to central nervous system inflammatory demyelinating disease. To explore the genetic relationship between Tmevd2 and Eae3, we generated a D2.C-Tmevd2 interval-specific congenic (ISC) line and three overlapping interval-specific recombinant congenic (ISRC) lines in which the Tmevd2-resistant allele from BALB/cByJ was introgressed onto the TMEVD-susceptible DBA/2J background. These mice, all H2(d), were studied for susceptibility to EAE elicited by immunization with proteolipid protein peptide 180-199. Compared with DBA/2J mice, D2.C-Tmevd2 mice developed a significantly less severe clinical disease course and EAE pathology in the spinal cord, confirming the existence of Eae3 and its linkage to Tmevd2 in this strain combination. Compared with DBA/2J and D2.C-Tmevd2, all three ISRC lines exhibited clinical disease courses of intermediate severity. Neither differences in ex vivo antigen-specific cytokine nor proliferative responses uniquely cosegregated with differences in disease severity. These results indicate that multiple quantitative trait loci (QTLs) within the Tmevd2/Eae3 interval influence EAE severity, one of which includes a homology region for a QTL found in MS by admixture mapping.

Genetic dissection reveals diabetes loci proximal to the gimap5 lymphopenia gene.

Congenic DRF.(f/f) rats are protected from type 1 diabetes (T1D) by 34 Mb of F344 DNA introgressed proximal to the gimap5 lymphopenia gene. To dissect the genetic factor(s) that confer protection from T1D in the DRF.(f/f) rat line, DRF.(f/f) rats were crossed to inbred BBDR or DR.(lyp/lyp) rats to generate congenic sublines that were genotyped and monitored for T1D, and positional candidate genes were sequenced. All (100%) DR.(lyp/lyp) rats developed T1D by 83 days of age. Reduction of the DRF.(f/f) F344 DNA fragment by 26 Mb (42.52-68.51 Mb) retained complete T1D protection. Further dissection revealed that a 2 Mb interval of F344 DNA (67.41-70.17 Mb) (region 1) resulted in 47% protection and significantly delayed onset (P < 0.001 compared with DR.(lyp/lyp)). Retaining <1 Mb of F344 DNA at the distal end (76.49-76.83 Mb) (region 2) resulted in 28% protection and also delayed onset (P < 0.001 compared with DR.(lyp/lyp)). Comparative analysis of diabetes frequency in the DRF.(f/f) congenic sublines further refined the RNO4 region 1 interval to approximately 670 kb and region 2 to the 340 kb proximal to gimap5. All congenic DRF.(f/f) sublines were prone to low-grade pancreatic mononuclear cell infiltration around ducts and vessels, but <20% of islets in nondiabetic rats showed islet infiltration. Coding sequence analysis revealed TCR Vbeta 8E, 12, and 13 as candidate genes in region 1 and znf467 and atp6v0e2 as candidate genes in region 2. Our results show that spontaneous T1D is controlled by at least two genetic loci 7 Mb apart on rat chromosome 4.

Immunogenetics of a thymus antigen in lymphoma-prone and lymphoma-resistant colonies of wild mice.

The Thy-1 (theta) antigen was identified in wild mice (Mus musculus), the frequency of its alleles was determined in two natural populations of wild mice, and the possible T-cell origin of spontaneous lymphomas was investigated in one of these populations. Reaction patterns for Thy-1 antigen with the use of direct cytoxicity and indirect absorption assays were identical in wild mice and inbred strains. Between 15 and 55 percent of viable spleen cells from healthy young or old wild mice were Thy-1 positive. Spleen, but not brain, cells from older wild mice were less strongly positive. Wild mice from the lymphoma-prone population were polymorphic for Thy-1alpha and Thy-1beta alleles, whereas wild mice from the lymphoma-resistant populations were homozygous for the Thy-1beta allele. Contrary to expectation, a higher frequency of the Thy-1beta allele was noted in the mice with lymphomas. Alleles at two other loci on chromosome number 9 (Mod-1alpha and Trfbeta) were fixed in both populations. The absence of detectable Thy-1 antigen on spleen cells (despite its detectability in undiminished titer in brain tissue) in 80 percent of mice with lymphomas, along with the absence of thymus involvement in the lymphomatous proliferations, suggested that these tumors are derived from an expansion of non-thymus-derived cells.

Diabetes-prone and diabetes-resistant BB rats share a common major diabetes susceptibility locus, iddm4: additional evidence for a "universal autoimmunity locus" on rat chromosome 4.

Diabetes-prone (DP) BB rats develop autoimmune type 1 diabetes spontaneously. At least five loci are linked to disease expression: the major histocompatibility complex (iddm2), two susceptibility loci (iddm4, iddm5), and, possibly, a resistance locus (iddm3). Spontaneous disease also requires homozygosity for lyp/iddm1, which causes lymphopenia. It has not been determined whether lyp/iddm1 is required for predisposition to diabetes autoimmunity in addition to being required for its spontaneous expression. We analyzed backcross rats segregating for diabetes but not lymphopenia using Wistar-Furth (WF) and diabetes-resistant (DR) BB animals. The latter are nonlymphopenic (lyp+/+) and develop diabetes only in response to immunological perturbants. Treatment of (DR-BB x WF)F1 x WF animals (all lyp+/+) using a standard induction protocol caused type 1 diabetes in 58% of progeny. Expression of type 1 diabetes was strongly linked to iddm4. The results suggest that lyp/iddm1 does not determine the predisposition to autoimmunity in BB rats and that iddm4 is a major diabetogenic locus in both DP- and DR-BB animals. The iddm4 gene maps to a region containing several major autoimmunity loci, including aia2, aia3, and cia3. We propose that BB rat diabetes requires 1) class II RT1u (iddm2) for susceptibility, 2) additional loci for disease initiation and progression in response to perturbants, and 3) lyp for spontaneous disease.

Non-major histocompatibility complex-linked diabetes susceptibility loci on chromosomes 4 and 13 in a backcross of the DP-BB/Wor rat to the WF rat.

BB rats are used as models of autoimmune human IDDM. Genetic control of IDDM in both species is complex, including both major histocompatibility complex (MHC)-linked and non-MHC-linked genes. DP-BB rats develop IDDM spontaneously. Expression of disease in these animals requires homozygosity at the lyp locus, which causes lymphopenia. All genetic analyses of BB rat diabetes to date have backcrossed to the DP-BB strain or used (DP-BB x non-BB)F2 animals to ensure that a fraction of progeny are homozygous for lyp. Here we report the analysis of a backcross of the DP-BB rat to the histocompatible WF rat. Neither WF nor (WF x DP-BB)F1 animals develop spontaneous IDDM. However, 95% of (WF x DP-BB)F1 rats and a fraction of (WF x DP-BB) x WF backcross animals readily develop IDDM after treatment with polyinosinic:polycytidylic acid and a cytotoxic anti-RT6.1 monoclonal antibody. Using simple sequence length polymorphism analysis, we have mapped loci on chromosomes 4 and 13 that show significant linkage to IDDM expression and insulitis. The susceptibility locus on chromosome 4 is linked to, but not identical to, lyp. We propose a disease model for the BB rat that requires 1) the RT1u MHC haplotype for disease susceptibility, 2) a new locus on chromosome 4 for disease initiation (as measured by insulitis), 3) a new locus on chromosome 13 for disease progression in response to environmental perturbation, and 4) lyp for spontaneous expression of disease.

Chromosomal location of the regulator of mouse alpha-fetoprotein, Afr-1.

Afr-1 is a gene whose product contributes to the adult regulation of mouse alpha-fetoprotein (AFP). In Afr-1b/b homozygotes, the adult serum levels of AFP are 10- to 20-fold higher than in Afr-1a/a or Afr-1a/b mice. The studies reported here were performed to map the Afr-1 gene. Our results show that Afr-1 resides on mouse chromosome 15, approximately 25 cM from Gdc-1. Afr-1 appears to be located in close proximity to the mouse c-myc oncogene. These results are discussed with respect to the susceptibility or resistance of different BALB/c sublines (which are either Afr-1a or Afr-1b, respectively) to pristane-induced plasmacytomas.

Background and gender effects on survival in the TgN(SOD1-G93A)1Gur mouse model of ALS.

Amyotrophic lateral sclerosis (ALS) is a progressive neuromuscular disorder. While most cases of ALS are sporadic, 10-15% are familial, and of these 15-20% possess a mutation in the gene that codes for the enzyme Cu/Zn superoxide dismutase (SOD1). In families of ALS patients with specific SOD1 mutations, affected members demonstrate significant heterogeneity of disease and a large variation in age of onset and severity, suggesting that there are genetic modifiers of disease expression. Transgenic mice expressing mutant forms of SOD1 demonstrate symptoms similar to those seen in patients with ALS. We have observed in our colony of G93A SOD1 transgenic mice a milder phenotype in mice in a C57BL/6J background than the C57BL/6JxSJL/J hybrid background used by Jackson Laboratories to maintain their colony. To investigate the effect of genetic background on phenotype, we have constructed congenic lines on two genetic backgrounds, C57BL/6J (B6) and SJL/J (SJL). We report the influence of background and gender on the survival of these congenic lines compared to the hybrid C57BL/6JxSJL/J background. The mean survival of G93A SOD1 mice in the hybrid B6/SJL background was 130 days, with females surviving significantly longer than males. When compared to the hybrid B6/SJL background, the survival of mice in the SJL background significantly decreased, and the gender difference in survival was maintained. On the other hand, mean survival in the B6 background significantly increased, and in contrast to the B6/SJL and SJL backgrounds, there was no difference in survival between males and females. Transgene copy numbers were verified in all animals to ensure that any phenotypic differences observed were not due to alterations in copy number. This is the first report of a shortened lifespan when the G93A SOD1 transgene is placed on the SJL/J background and an increased survival with the loss of gender influences when the transgene is placed on the C57BL/6J background.

Cloning of myelin basic protein-reactive T cells from the experimental allergic encephalomyelitis-resistant rat strain, LER.

Rats of the LER inbred strain are resistant to the active induction of experimental allergic encephalomyelitis (EAE), although they are susceptible to adoptively transferred EAE when they are injected with encephalitogenic T cells from EAE-susceptible Lewis rats. The mechanism of resistance remains to be elucidated. We report here that myelin basic protein (MBP)-specific T cells can be cloned from LER rats immunized with MBP, that these CD4+ LER T cells can recognize the encephalitogenic peptide (MBP-EP) and will divide vigorously when it is presented to them, and that these T cells bear V beta 8 + TCR chains. Nevertheless, in contrast to Lewis T cells with the same specificity and TCR beta chains, LER T cells from MBP-EP-specific clones cannot induce EAE when adoptively transferred into naive rats of either strain. Thus, LER T cells can assemble and use a TCR with the canonical encephalitogenic V beta 8.2-D beta-J beta region in response to immunization with MBP, yet they continue to display resistance to EAE.

Susceptibility to actively-induced murine experimental allergic encephalomyelitis is not linked to genes of the T cell receptor or CD3 complexes.

The role of the T cell receptor (TCR) in the genetic control of susceptibility to autoimmune demyelinating diseases remains shrouded in controversy. We have used the CXD2 series of recombinant inbred lines (RIL) and a (B10.S/DvTe x SJL/J) x B10.S/DvTe backcross (BC1) population to test for linkage between susceptibility to actively-induced EAE and the different TCR and CD3 loci. The two populations were inoculated for induction of EAE, phenotyped for both clinical and histological parameters of disease, and genotyped using markers flanking the loci of interest in the CXD2 RIL and an SJL/J allele-specific TCR V beta assay in the BC1 mice. Comparisons between the CXD2 strain distribution pattern (SDP) for disease and the SDPs for the chromosomal regions containing the TCR alpha, beta, gamma, delta, and CD3 delta, epsilon, gamma and zeta loci showed no linkage to these loci. Additional tests between EAE susceptibility and several other immunologically important loci for which the SDPs were known also showed no linkage to the minor lymphocyte-stimulating antigen gene Mlsl, Hc, the gene encoding complement component C5, Cd8a, or Cd5. Furthermore, our data from the BC1 mice demonstrate that the Tcrb locus segregates independent of disease and does not modulate disease severity. We conclude that while autoreactive TCRs are undoubtedly necessary for disease pathogenesis, the principle non-MHC-linked loci controlling susceptibility to murine EAE in BALB/c mice are not linked to any of the individual TCR-CD3 complex genes. Similarly, the major disease genes in the SJL/J mouse are not linked to TCR V beta. Our data cannot, however, preclude the possibility that TCR/CD3 alleles are involved in epigenetic phenomena or susceptibility in other mouse strains or animal systems.

Nitric oxide-induced anti-mitogenic effects in high and low responder rat strains.

Nitric oxide (NO) has multiple biologic functions: in the brain it acts as a neuronal messenger; elsewhere, it causes smooth muscle relaxation, inhibition of platelet aggregation, inhibition of leukocyte adhesion, inhibition of tumor growth, and microbiostasis. Our studies show that production of NO is responsible for the unusual unresponsiveness of BN rat spleen cells to mitogens. NG-monomethyl-L-arginine (NGMMA), a potent competitive inhibitor for NO synthase, reverses this defect. Lysed RBC or NGMMA were shown to enhance mitogen-induced spleen cell proliferation only one- to twofold in Lewis rats (that have normal mitogen responsiveness) but act to stimulate BN rat T cells by 10- to 100-fold. NGMMA-enhanced proliferation was significantly diminished by prior depletion of macrophages. Surprisingly, NO did not inhibit IL-2 production in 48-h cultures of BN rat spleen cells, and exogenous IL-2 was ineffective in releasing NO-mediated suppression. These studies indicate that NO produced by macrophages can completely and reversibly inhibit T cell proliferation. The BN rat appears to be unique in its production of very high levels of NO, making it an especially useful animal model for studying the biologic control and functional consequences of NO generation.

Orientation of the loci encoding RT1.B polypeptides in the major histocompatibility complex of the rat.

The major histocompatibility complex of the rat (RT1) contains genes that code for two class II histocompatibility antigens. The r12 rat strain (WRC) was derived from a mating which revealed a genetic recombination that defines the two loci, RT1.B and RT1.D, that code for the class II antigens. The RT1.B and RT1.D antigens of the RT1 complex are protein dimers consisting of an alpha and beta glycoprotein chain and are homologous to I-A and I-E genes, respectively, in the H-2 complex of the mouse. We have performed Southern blot analysis on liver DNA from the r12 and parental strains to examine the precise location of the recombinant event. After digestion with restriction enzymes, the DNA was separated on agarose gels, blotted onto nitrocellulose, and hybridized with mouse H-2 cDNA probes specific for I-A alpha and beta genes. The pattern of restriction fragment polymorphisms demonstrated that the site of recombination is between the RT1.B alpha and the RT1.B beta genes. As a result of these findings, we suggest that the sequence of genes within the RT1 complex consists of RT1.A ... RT1.B beta ... RT1.B alpha ... RT1.D (alpha, beta) ... Other class I genes, possibly corresponding to mouse Qa and Tla-like genes, were also apparent in these experiments.