Elizabeth P. Blankenhorn

The nature and identification of quantitative trait loci: a community’s view

This white paper by eighty members of the Complex Trait Consortium presents a communitys view on the approaches and statistical analyses that are needed for the identification of genetic loci that determine quantitative traits. Quantitative trait loci (QTLs) can be identified in several ways, but is there a definitive test of whether a candidate locus actually corresponds to a specific QTL?

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.

The Protective Effect of 17β-Estradiol on Experimental Autoimmune Encephalomyelitis Is Mediated through Estrogen Receptor-α

Low-dose estrogen (E2) treatment significantly inhibits the clinical signs and histopathological lesions of experimental autoimmune encephalomyelitis (EAE), and is being used in clinical trials to treat multiple sclerosis. To assess the role of intracytoplasmic estrogen receptors in mediating suppression of EAE, we studied mice with disrupted estrogen receptor-α (Esr1) and -β (Esr2) genes. We demonstrate that the protective effect of E2 is abrogated in B6.129-Esr1tm1Unc mice (Esr1−/−) but not in B6.129-Esr2tm1Unc mice (Esr2−/−). The loss of E2-mediated protection from EAE in Esr1−/− mice immunized with the encephalitogenic MOG-35–55 peptide was manifested phenotypically by the development of severe acute clinical signs and histopathological lesions even in the presence of moderately high serum E2 levels. This is in contrast to C57BL/6 wild-type (WT) mice and Esr2−/− mice in which E2 treatment resulted in comparable serum levels and markedly suppressed clinical signs of EAE and abolished inflammatory lesions in the CNS. This pattern showing a lack of E2-dependent inhibition of EAE in Esr1−/− mice was mirrored by an enhanced rather than a reduced secretion of TNF-α, IFN-γ, and interleukin (IL)-6 in MOG-specific splenocytes and a lack of inhibition of message for inflammatory cytokines, chemokines and chemokine receptors in CNS tissue. These results indicate that the immunomodulatory effects of E2 in EAE are dependent on Esr1 and not Esr2 signaling.

Identification of Genetic Loci Controlling the Characteristics and Severity of Brain and Spinal Cord Lesions in Experimental Allergic Encephalomyelitis

Experimental allergic encephalomyelitis (EAE) is the principal genetically determined animal model for multiple sclerosis (MS), the major inflammatory disease of the central nervous system (CNS). Although genetics clearly play a role in susceptibility to MS, attempts to identify the underlying genes have been disappointing. Considerable variation exists between MS patients with regard to the severity of clinical signs, mechanism of demyelination, and location of CNS lesions, confounding the interpretation of genetic data. A mouse-human synteny mapping approach may allow the identification of candidate susceptibility loci for MS based on the location of EAE susceptibility loci. To date, 16 regions of the mouse genome have been identified that control susceptibility or clinical signs of EAE. In this work, we examined the genetic control of histopathological lesions of EAE in an F2 intercross population generated from the EAE susceptible SJL/J and EAE resistant B10.S/DvTe mouse strains. Composite interval mapping was used to identify 10 quantitative trait loci (QTL), including seven newly identified loci controlling the distribution and severity of CNS lesions associated with murine EAE. QTL on chromosome 10 control lesions in the brain, whereas QTL on chromosomes 3, 7, and 12 control lesions in the spinal cord. Furthermore, sexually dimorphic QTL on chromosomes 2, 9, and 11 control CNS lesions in females, whereas QTL on chromosomes 10, 11, 12, 16, and 19 control lesions in males. Our results suggest that the severity and location of CNS lesions in EAE are genetically controlled, and that the genetic component controlling the character and severity of the lesions can be influenced by sex.

Genetic analysis of the influence of pertussis toxin on experimental allergic encephalomyelitis susceptibility: An environmental agent can override genetic checkpoints

Pertussis toxin (PTX) is a potent ancillary adjuvant used to elicit several different autoimmune diseases, including experimental allergic encephalomyelitis (EAE). To delineate the genetics of PTX effect in EAE, we mapped EAE-modifying (eae-m) loci in cohorts of backcross mice immunized with and without PTX. In this study, we analyzed the genetic basis of EAE susceptibility and severity and the intermediate phenotypes of mononuclear cell infiltration, suppuration, and demyelination. In animals immunized with PTX, one major locus, eae9, controls disease susceptibility and severity. Eae9 also regulates the extent of mononuclear cell infiltration of the spinal cord in male mice. Without PTX, five eae-m loci were noted, including three new loci in intervals on chromosomes 8 (eae14), 10 (eae17), and 18 (eae18). Taken together, these results suggest that eae9 controls the effects of PTX in EAE susceptibility, and is capable of overriding the other genetic checkpoints in the pathogenesis of this disease.

Central histamine H3 receptor signaling negatively regulates susceptibility to autoimmune inflammatory disease of the CNS.

Histamine (HA), a biogenic amine with a broad spectrum of activities in both physiological and pathological settings, plays a key regulatory role in experimental allergic encephalomyelitis, the autoimmune model of multiple sclerosis. HA exerts its effect through four G protein-coupled receptors designated HA receptor H1, H2, H3, and H4. We report here that, compared with wild-type animals, mice with a disrupted HA H3 receptor (H3RKO), the expression of which is normally confined to cells of the nervous system, develop more severe disease and neuroinflammation. We show that this effect is associated with dysregulation of blood–brain barrier permeability and increased expression of MIP-2, IP-10, and CXCR3 by peripheral T cells. Our data suggest that pharmacological targeting of the H3R may be useful in preventing the development and formation of new lesions in multiple sclerosis, thereby significantly limiting the progression of the disease.

Histamine receptor H1 is required for TCR-mediated p38 MAPK activation and optimal IFN-γ production in mice

Histamine receptor H1 (H1R) is a susceptibility gene in both experimental autoimmune encephalomyelitis (EAE) and experimental autoimmune orchitis (EAO), 2 classical T cell-mediated models of organ-specific autoimmune disease. Here we showed that expression of H1R in naive CD4+ T cells was required for maximal IFN-gamma production but was dispensable for proliferation. Moreover, H1R signaling at the time of TCR ligation was required for activation of p38 MAPK, a known regulator of IFN-gamma expression. Importantly, selective reexpression of H1R in CD4+ T cells fully complemented both the IFN-gamma production and the EAE susceptibility of H1R-deficient mice. These data suggest that the presence of H1R in CD4+ T cells and its interaction with histamine regulates early TCR signals that lead to Th1 differentiation and autoimmune disease.

Sexually dimorphic genes regulate healing and regeneration in MRL mice.

The MRL mouse has been shown to display unusual healing properties. In particular, when the ear pinna is hole punched, the hole that is made closes completely without scarring, with reformation of hair follicles and sebaceous glands, and regrowth of cartilage. Initial studies using (MRL/lpr × C57BL/6) F2 and backcross mice showed that this phenomenon is genetically determined and that multiple loci contribute to this quantitative trait. In the present study, with twice as many animals, we have confirmed many of the original heal loci and identified new ones. We have also found that this phenotype is sexually dimorphic in that female mice heal more quickly and more completely than male mice. To test the cause of this difference, we castrated both males and females. Castration of males led to better healing, although ovariectomy did not lead to worse healing in female mice. Finally, most heal loci were shown to be responsible for regulating healing primarily in male animals more than in females, or vice versa. Thus, sex plays a highly significant role in the closure of wounded tissue in this mammalian model of healing and regeneration.

Gender, Age, and Season at Immunization Uniquely Influence the Genetic Control of Susceptibility to Histopathological Lesions and Clinical Signs of Experimental Allergic Encephalomyelitis: Implications for the Genetics of Multiple Sclerosis

Multiple sclerosis (MS), the principal inflammatory demyelinating disease of the central nervous system (CNS), is believed to have an immunopathological etiology arising from gene-environment interactions. In this study, we examined the effect of sex, age, and season at immunization on the susceptibility of (B10.S x SJL/J) F(2) intercross mice to experimental allergic encephalomyelitis (EAE), the foremost animal model of MS. Results of logistic regression analyses suggest that female mice were more likely to exhibit CNS lesions than male mice [odds ratio (OR) = 2.28 for brain lesions; OR = 2.37 for spinal cord (SC) lesions]. Although statistically significant associations were seen between brain and SC lesions and age at the time of injection or month of injection when examined separately; these associations disappeared when controlling for sex in multiple logistic regression analyses. These results suggest that the sex of the mouse is more important in influencing the development of brain and SC lesions than was either age or month of immunization. When examining clinical disease as the endpoint, the OR for the age at immunization is 1.04, indicating that the odds of being affected increase by 4% for each increasing week of age. When controlled for age, the OR for injection in the summer months (July through September) is 1.90, suggesting that the odds of being clinically affected are 90% greater for F(2) intercross animals injected in the summercompared to those injected in the winter to spring months (February through May). In contrast to CNS lesions, the age and season at immunization significantly and independently influenced susceptibility to clinical EAE and did so equally in both males and females. Linkage analysis to eae5, the H2-linked locus controlling susceptibility to clinical disease, was performed using 6- to 12- and >12-week-old cohorts as well as summer and winter/spring cohorts of F(2) mice. Significant linkage of clinical EAE to eae5 was observed with the 6- to 12-week-old and summer populations. In contrast, linkage of clinical EAE to eae5 was not detected with the >12-week-old and winter/spring populations. These results indicate that age and seasonal effects are capable of overriding eae5-dependent genetic control of susceptibility to clinical EAE and have significant implications for the genetics of MS.

Attenuation of Th1 Effector Cell Responses and Susceptibility to Experimental Allergic Encephalomyelitis in Histamine H2 Receptor Knockout Mice Is Due to Dysregulation of Cytokine Production by Antigen-Presenting Cells

Histamine, a biogenic amine with both neurotransmitter and vasoactive properties, is well recognized as an immunomodulatory agent in allergic and inflammatory reactions. It also plays a regulatory role in the development of antigen-specific immune responses. CD4+ T-cells from histamine H1 receptor (H1R)-deficient (H1RKO) mice produce significantly less interferon-gamma and more interleukin (IL)-4 in in vitro recall assays compared to wild-type controls. H1RKO mice are also less susceptible to acute early-phase experimental allergic encephalomyelitis indicating that H1R signaling in CD4+ T cells plays a central role in regulating pathogenic T-cell responses. In this study, we show that mice lacking histamine H2 receptor (H2RKO) are similar to H1RKO mice in that they develop encephalitogen-specific T-cell responses as assessed by proliferation and IL-2 production and present with less severe acute early-phase experimental allergic encephalomyelitis. However, unlike T cells from H1RKO mice, which exhibit a strong Th2 bias, T cells from H2RKO mice do not. Rather, they are uniquely characterized by a significant inhibition of Th1 effector cell responses. Given that both histamine and adjuvants such as pertussis toxin modulate antigen-presenting cell (APC) maturation and function, including T-cell-polarizing activity, we analyzed the cytokines/chemokines secreted by APCs from wild-type, H1RKO, and H2RKO mice. Significant differences in cytokine/chemokine production by APCs from unimmunized and immunized mice were delineated. APCs from H2RKO mice produce significantly less IL-12 and IL-6 and markedly greater amounts of MCP-1 compared to wild-type and H1RKO mice. Because MCP-1 is known to inhibit IL-12 production, the failure of H2RKO mice to generate encephalitogenic Th1 effector cell responses is consistent with inhibition of negative regulation of MCP-1 secretion by H2R signaling in APCs.