Neutralizing Antibody Responses to Viral Infections Are Linked to the Non-classical MHC Class II Gene H2-Ob.

Select humans and animals control persistent viral infections via adaptive immune responses that include production of neutralizing antibodies. The precise genetic basis for the control remains enigmatic. Here, we report positional cloning of the gene responsible for production of retrovirus-neutralizing antibodies in mice of the I/LnJ strain. It encodes the beta subunit of the non-classical major histocompatibility complex class II (MHC-II)-like molecule H2-O, a negative regulator of antigen presentation. The recessive and functionally null I/LnJ H2-Ob allele supported the production of virus-neutralizing antibodies independently of the classical MHC haplotype. Subsequent bioinformatics and functional analyses of the human H2-Ob homolog, HLA-DOB, revealed both loss- and gain-of-function alleles, which could affect the ability of their carriers to control infections with human hepatitis B (HBV) and C (HCV) viruses. Thus, understanding of the previously unappreciated role of H2-O (HLA-DO) in immunity to infections may suggest new approaches in achieving neutralizing immunity to viruses.

Innate immune sensing of retroviral infection via Toll-like receptor 7 occurs upon viral entry.

Innate immune sensors are required for induction of pathogen-specific immune responses. Retroviruses are notorious for their ability to evade immune defenses and establish long-term persistence in susceptible hosts. However, some infected animals are able to develop efficient virus-specific immune responses, and thus can be employed for identification of critical innate virus-sensing mechanisms. With mice from two inbred strains that control retroviruses via adaptive immune mechanisms, we found that of all steps in viral replication, the ability to enter the host cell was sufficient to induce antivirus humoral immune responses. Virus sensing occurred in endosomes via a MyD88-Toll-like receptor 7-dependent mechanism and stimulated virus-neutralizing immunity independently of type I interferons. Thus, efficient adaptive immunity to retroviruses is induced in vivo by innate sensing of the early stages of retroviral infection.

Genetic variation in chromosome Y regulates susceptibility to influenza A virus infection.

Males of many species, ranging from humans to insects, are more susceptible than females to parasitic, fungal, bacterial, and viral infections. One mechanism that has been proposed to account for this difference is the immunocompetence handicap model, which posits that the greater infectious disease burden in males is due to testosterone, which drives the development of secondary male sex characteristics at the expense of suppressing immunity. However, emerging data suggest that cell-intrinsic (chromosome X and Y) sex-specific factors also may contribute to the sex differences in infectious disease burden. Using a murine model of influenza A virus (IAV) infection and a panel of chromosome Y (ChrY) consomic strains on the C57BL/6J background, we present data showing that genetic variation in ChrY influences IAV pathogenesis in males. Specific ChrY variants increase susceptibility to IAV in males and augment pathogenic immune responses in the lung, including activation of proinflammatory IL-17-producing γδ T cells, without affecting viral replication. In addition, susceptibility to IAV segregates independent of copy number variation in multicopy ChrY gene families that influence susceptibility to other immunopathological phenotypes, including survival after infection with coxsackievirus B3. These results demonstrate a critical role for genetic variation in ChrY in regulating susceptibility to infectious disease.

Modeling month-season of birth as a risk factor in mouse models of chronic disease: from multiple sclerosis to autoimmune encephalomyelitis.

Month-season of birth (M-SOB) is a risk factor in multiple chronic diseases, including multiple sclerosis (MS), where the lowest and greatest risk of developing MS coincide with the lowest and highest birth rates, respectively. To determine whether M-SOB effects in such chronic diseases as MS can be experimentally modeled, we examined the effect of M-SOB on susceptibility of C57BL/6J mice to experimental autoimmune encephalomyelitis (EAE). As in MS, mice that were born during the M-SOB with the lowest birth rate were less susceptible to EAE than mice born during the M-SOB with the highest birth rate. We also show that the M-SOB effect on EAE susceptibility is associated with differential production of multiple cytokines/chemokines by neuroantigen-specific T cells that are known to play a role in EAE pathogenesis. Taken together, these results support the existence of an M-SOB effect that may reflect seasonally dependent developmental differences in adaptive immune responses to self-antigens independent of external stimuli, including exposure to sunlight and vitamin D. Moreover, our documentation of an M-SOB effect on EAE susceptibility in mice allows for modeling and detailed analysis of mechanisms that underlie the M-SOB effect in not only MS but in numerous other diseases in which M-SOB impacts susceptibility.-Reynolds, J. D., Case, L. K., Krementsov, D. N., Raza, A., Bartiss, R., Teuscher, C. Modeling month-season of birth as a risk factor in mouse models of chronic disease: from multiple sclerosis to autoimmune encephalomyelitis.

The Y chromosome as a regulatory element shaping immune cell transcriptomes and susceptibility to autoimmune disease.

Understanding the DNA elements that constitute and control the regulatory genome is critical for the appropriate therapeutic management of complex diseases. Here, using chromosome Y (ChrY) consomic mouse strains on the C57BL/6J (B6) background, we show that susceptibility to two diverse animal models of autoimmune disease, experimental allergic encephalomyelitis (EAE) and experimental myocarditis, correlates with the natural variation in copy number of Sly and Rbmy multicopy ChrY genes. On the B6 background, ChrY possesses gene regulatory properties that impact genome-wide gene expression in pathogenic CD4(+) T cells. Using a ChrY consomic strain on the SJL background, we discovered a preference for ChrY-mediated gene regulation in macrophages, the immune cell subset underlying the EAE sexual dimorphism in SJL mice, rather than CD4(+) T cells. Importantly, in both genetic backgrounds, an inverse correlation exists between the number of Sly and Rbmy ChrY gene copies and the number of significantly up-regulated genes in immune cells, thereby supporting a link between copy number variation of Sly and Rbmy with the ChrY genetic element exerting regulatory properties. Additionally, we show that ChrY polymorphism can determine the sexual dimorphism in EAE and myocarditis. In humans, an analysis of the CD4(+) T cell transcriptome from male multiple sclerosis patients versus healthy controls provides further evidence for an evolutionarily conserved mechanism of gene regulation by ChrY. Thus, as in Drosophila, these data establish the mammalian ChrY as a member of the regulatory genome due to its ability to epigenetically regulate genome-wide gene expression in immune cells.

Exacerbation of autoimmune neuroinflammation by dietary sodium is genetically controlled and sex specific.

Multiple sclerosis (MS) is a debilitating autoimmune neuroinflammatory disease influenced by genetics and the environment. MS incidence in female subjects has approximately tripled in the last century, suggesting a sex-specific environmental influence. Recent animal and human studies have implicated dietary sodium as a risk factor in MS, whereby high sodium augmented the generation of T helper (Th) 17 cells and exacerbated experimental autoimmune encephalomyelitis (EAE), the principal model of MS. However, whether dietary sodium interacts with sex or genetics remains unknown. Here, we show that high dietary sodium exacerbates EAE in a strain- and sex-specific fashion. In C57BL6/J mice, exposure to a high-salt diet exacerbated disease in both sexes, while in SJL/JCrHsd mice, it did so only in females. In further support of a genetic component, we found that sodium failed to modify EAE course in C57BL6/J mice carrying a 129/Sv-derived interval on chromosome 17. Furthermore, we found that the high-sodium diet did not augment Th17 or Th1 responses, but it did result in increased blood-brain barrier permeability and brain pathology. Our results demonstrate that the effects of dietary sodium on autoimmune neuroinflammation are sex specific, genetically controlled, and CNS mediated.

Histamine H2 receptor signaling × environment interactions determine susceptibility to experimental allergic encephalomyelitis.

Histamine and its receptors are important in both multiple sclerosis and experimental allergic encephalomyelitis (EAE). C57BL/6J (B6) mice deficient for the histamine H2 receptor (H2RKO) are less susceptible to EAE and exhibit blunted Th1 responses. However, whether decreased antigen-specific T-cell effector responses in H2RKO mice were due to a lack of H2R signaling in CD4(+) T cells or antigen-presenting cells has remained unclear. We generated transgenic mice expressing H2R specifically in T cells on the H2RKO background, and, using wild-type B6 and H2RKO mice as controls, induced EAE either in the presence or absence of the ancillary adjuvant pertussis toxin (PTX), which models the effects of infectious inflammatory stimuli on autoimmune disease. We monitored the mice for clinical signs of EAE and neuropathology, as well as effector T-cell responses using flow cytometry. EAE severity and neuropathology in H2RKO mice expressing H2R exclusively in T cells become equal to those in wild-type B6 mice only when PTX is used to elicit disease. EAE complementation was associated with frequencies of CD4(+)IFN-γ(+) and CD4(+)IL-17(+) cells that are equal to or greater than those in wild-type B6, respectively. Thus, the regulation of encephalitogenic T-cell responses and EAE susceptibility by H2R signaling in CD4(+) T cells is dependent on gene × environment interactions.

The role of genetics in estrogen responses: a critical piece of an intricate puzzle.

The estrogens are female sex hormones that are involved in a variety of physiological processes, including reproductive development and function, wound healing, and bone growth. They are mainly known for their roles in reproductive tissues--specifically, 17ß-estradiol (E2), the primary estrogen, which is secreted by the ovaries and induces cellular proliferation and growth of the uterus and mammary glands. In addition to the role of estrogens in promoting tissue growth and development during normal physiological states, they have a well-established role in determining susceptibility to disease, particularly cancer, in reproductive tissues. The responsiveness of various tissues to estrogen is genetically controlled, with marked quantitative variation observed across multiple species, including humans. This variation presents both researchers and clinicians with a veritable physiological puzzle, the pieces of which--many of them unknown--are complex and difficult to fit together. Although genetics is known to play a major role in determining sensitivity to estrogens, there are other factors, including parent of origin and the maternal environment, that are intimately linked to heritable phenotypes but do not represent genotype, per se. The objectives of this review article were to summarize the current knowledge of the role of genotype, and uterine and neonatal environments, in phenotypic variation in the response to estrogens; to discuss recent findings and the potential mechanisms involved; and to highlight exciting research opportunities for the future.

Systemic lack of canonical histamine receptor signaling results in increased resistance to autoimmune encephalomyelitis.

Histamine (HA) is a key regulator of experimental allergic encephalomyelitis (EAE), the autoimmune model of multiple sclerosis. HA exerts its effects through four known G-protein-coupled receptors: H1, H2, H3, and H4 (histamine receptors; H(1-4)R). Using HR-deficient mice, our laboratory has demonstrated that H1R, H2R, H3R, and H4R play important roles in EAE pathogenesis, by regulating encephalitogenic T cell responses, cytokine production by APCs, blood-brain barrier permeability, and T regulatory cell activity, respectively. Histidine decarboxylase-deficient mice (HDCKO), which lack systemic HA, exhibit more severe EAE and increased Th1 effector cytokine production by splenocytes in response to myelin oligodendrocyte gp35-55. In an inverse approach, we tested the effect of depleting systemic canonical HA signaling on susceptibility to EAE by generating mice lacking all four known G-protein-coupled-HRs (H(1-4)RKO mice). In this article, we report that in contrast to HDCKO mice, H(1-4)RKO mice develop less severe EAE compared with wild-type animals. Furthermore, splenocytes from immunized H(1-4)RKO mice, compared with wild-type mice, produce a lower amount of Th1/Th17 effector cytokines. The opposing results seen between HDCKO and H1-4RKO mice suggest that HA may signal independently of H1-4R and support the existence of an alternative HAergic pathway in regulating EAE resistance. Understanding and exploiting this pathway has the potential to lead to new disease-modifying therapies in multiple sclerosis and other autoimmune and allergic diseases.

Genetic control of estrogen-regulated transcriptional and cellular responses in mouse uterus.

The uterotropic response of the uterus to 17ß-estradiol (E2) is genetically controlled, with marked variation observed depending on the mouse strain studied. Previous genetic studies from our laboratory using inbred mice that are high [C57BL/6J (B6)] or low [C3H/HeJ (C3H)] responders to E2 led to the identification of quantitative trait (QT) loci associated with phenotypic variation in uterine growth and leukocyte infiltration. The mechanisms underlying differential responsiveness to E2, and the genes involved, are unknown. Therefore, we used a microarray approach to show association of distinct E2-regulated transcriptional signatures with genetically controlled high and low responses to E2 and their segregation in (C57BL/6J×C3H/HeJ) F1 hybrids. Among the 6664 E2-regulated transcripts, analysis of cellular functions of those that were strain specific indicated C3H-selective enrichment of apoptosis, consistent with a 7-fold increase in the apoptosis indicator CASP3, and a 2.4-fold decrease in the apoptosis inhibitor Naip1 (Birc1a) in C3H vs. B6 following treatment with E2. In addition, several differentially expressed transcripts reside within our previously identified QT loci, including the ERα-tethering factor Runx1, demonstrated to enhance E2-mediated transcript regulation. The level of RUNX1 in uterine epithelial cells was shown to be 3.5-fold greater in B6 compared to C3H. Our novel insights into the mechanisms underlying the genetic control of tissue sensitivity to estrogen have great potential to advance understanding of individualized effects in physiological and disease states.

Combinatorial roles for histamine H1-H2 and H3-H4 receptors in autoimmune inflammatory disease of the central nervous system.

Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system in which histamine (HA) and its receptors have been implicated in disease pathogenesis. HA exerts its effects through four different G protein-coupled receptors designated H(1)-H(4). We previously examined the effects of traditional single HA receptor (HR) knockouts (KOs) in experimental allergic encephalomyelitis (EAE), the autoimmune model of MS. Our results revealed that H(1) R and H(2) R are propathogenic, while H(3) R and H(4) R are antipathogenic. This suggests that combinatorial targeting of HRs may be an effective disease-modifying therapy (DMT) in MS. To test this hypothesis, we generated H(1) H(2) RKO and H(3) H(4) RKO mice and studied them for susceptibility to EAE. Compared with wild-type (WT) mice, H(1) H(2) RKO mice developed a less severe clinical disease course, whereas the disease course of H(3) H(4) RKO mice was more severe. H(1) H(2) RKO mice also developed less neuropathology and disrupted blood brain barrier permeability compared with WT and H(3) H(4) RKO mice. Additionally, splenocytes from immunized H(1) H(2) RKO mice produced less interferon(IFN)-γ and interleukin(IL)-17. These findings support the concept that combined pharmacological targeting of HRs may be an appropriate ancillary DMT in MS and other immunopathologic diseases.

A genetic locus complements resistance to Bordetella pertussis-induced histamine sensitization.

Histamine plays pivotal role in normal physiology and dysregulated production of histamine or signaling through histamine receptors (HRH) can promote pathology. Previously, we showed that Bordetella pertussis or pertussis toxin can induce histamine sensitization in laboratory inbred mice and is genetically controlled by Hrh1/HRH1. HRH1 allotypes differ at three amino acid residues with P263-V313-L331 and L263-M313-S331, imparting sensitization and resistance respectively. Unexpectedly, we found several wild-derived inbred strains that carry the resistant HRH1 allotype (L263-M313-S331) but exhibit histamine sensitization. This suggests the existence of a locus modifying pertussis-dependent histamine sensitization. Congenic mapping identified the location of this modifier locus on mouse chromosome 6 within a functional linkage disequilibrium domain encoding multiple loci controlling sensitization to histamine. We utilized interval-specific single-nucleotide polymorphism (SNP) based association testing across laboratory and wild-derived inbred mouse strains and functional prioritization analyses to identify candidate genes for this modifier locus. Atg7, Plxnd1, Tmcc1, Mkrn2, Il17re, Pparg, Lhfpl4, Vgll4, Rho and Syn2 are candidate genes within this modifier locus, which we named Bphse, enhancer of Bordetella pertussis induced histamine sensitization. Taken together, these results identify, using the evolutionarily significant diversity of wild-derived inbred mice, additional genetic mechanisms controlling histamine sensitization.

Vital role for CD8+ cells in controlling retroviral infections.

Antiviral adaptive immune defenses consist of humoral and cell-mediated responses, which together eliminate extracellular and intracellular virus. As most retrovirus-infected individuals do not raise efficient protective antivirus immune responses, the relative importance of humoral and cell-mediated responses in restraining retroviral infection is not well understood. We utilized retrovirus-resistant I/LnJ mice, which control infection with mouse mammary tumor virus (MMTV) and murine leukemia virus (MuLV) via an adaptive immune mechanism, to assess the contribution of cellular responses and virus-neutralizing antibodies (Abs) to the control of retroviral infection. We found that in retrovirus-infected CD8-deficient I/LnJ mice, viral titers exceed the neutralizing capability of antiviral Abs, resulting in augmented virus spread and disease induction. Thus, even in the presence of robust neutralizing Ab responses, CD8-mediated responses are essential for full protection against retroviral infection.

H(1)R expression by CD11B(+) cells is not required for susceptibility to experimental allergic encephalomyelitis.

The histamine H(1) receptor (Hrh1/H(1)R) was identified as an autoimmune disease gene in experimental allergic encephalomyelitis (EAE), the principal autoimmune model of multiple sclerosis (MS). Previously, we showed that selective re-expression of H(1)R by endothelial cells or T cells in H(1)RKO mice significantly reduced or complemented EAE severity and cytokine responses, respectively. H(1)R regulates innate immune cells, which in turn influences peripheral and central nervous system CD4(+) T cell effector responses. Therefore, we selectively re-expressed H(1)R in CD11b(+) cells of H(1)RKO mice to test the hypothesis that H(1)R signaling in these cells contributes to EAE susceptibility. We demonstrate that transgenic re-expression of H(1)R by H(1)RKO-CD11b(+) cells neither complements EAE susceptibility nor T cell cytokine responses highlighting the cell-specific effects of Hrh1 in the pathogenesis of EAE and MS, and the need for cell-specific targeting in optimizing therapeutic interventions based on such genes.

Histamine H(1) receptor signaling regulates effector T cell responses and susceptibility to coxsackievirus B3-induced myocarditis.

Susceptibility to autoimmune myocarditis has been associated with histamine release by mast cells during the innate immune response to coxsackievirus B3 (CVB3) infection. To investigate the contribution of histamine H(1) receptor (H(1)R) signaling to CVB3-induced myocarditis, we assessed susceptibility to the disease in C57BL/6J (B6) H(1)R(-/-) mice. No difference was observed in mortality between CVB3-infected B6 and H(1)R(-/-) mice. However, analysis of their hearts revealed a significant increase in myocarditis in H(1)R(-/-) mice that is not attributed to increased virus replication. Enhanced myocarditis susceptibility correlated with a significant expansion in pathogenic Th1 and Vγ4(+) γδ T cells in the periphery of these animals. Furthermore, an increase in regulatory T cells was observed, yet these cells were incapable of controlling myocarditis in H(1)R(-/-) mice. These data establish a critical role for histamine and H(1)R signaling in regulating T cell responses and susceptibility to CVB3-induced myocarditis in B6 mice.

Genetics of experimental allergic encephalomyelitis supports the role of T helper cells in multiple sclerosis pathogenesis.

OBJECTIVE: The major histocompatibility complex (MHC) is the primary genetic contributor to multiple sclerosis (MS) and experimental allergic encephalomyelitis (EAE), but multiple additional interacting loci are required for genetic susceptibility. The identity of most of these non-MHC genes is unknown. In this report, we identify genes within evolutionarily conserved genetic pathways leading to MS and EAE. METHODS: To identify non-MHC binary and quantitative trait loci (BTL/QTL) important in the pathogenesis of EAE, we generated phenotype-selected congenic mice using EAE-resistant B10.S and EAE-susceptible SJL mice. We hypothesized that genes linked to EAE BTL/QTL and MS-GWAS can be identified if they belong to common evolutionarily conserved pathways, which can be identified with a bioinformatic approach using Ingenuity software. RESULTS: Many known BTL/QTL were retained and linked to susceptibility during phenotype selection, the most significant being a region on chromosome 17 distal to H2 (Eae5). We show in pathway analysis that T helper (T(H))-cell differentiation genes are critical for both diseases. Bioinformatic analyses predicted that Eae5 is important in CD4 T-effector and/or Foxp3(+) T-regulatory cells (Tregs), and we found that B10.S-Eae5(SJL) congenic mice have significantly greater numbers of lymph node CD4 and Tregs than B10.S mice. INTERPRETATION: These results support the polygenic model of MS/EAE, whereby MHC and multiple minor loci are required for full susceptibility, and confirm a critical genetic dependence on CD4 T(H)-cell differentiation and function in the pathogenesis of both diseases.

The postnatal maternal environment affects autoimmune disease susceptibility in A/J mice.

The postnatal maternal environment is known to increase susceptibility to a number of autoimmune diseases. Here we asked whether the postnatal maternal environment could influence autoimmune disease development to day 3 thymectomy (d3tx)-induced autoimmune ovarian disease (AOD) and experimental allergic encephalomyelitis (EAE) in cross-fostered A/J and B6 mice. A/J pups foster-nursed by B6 mothers exhibit an increase in autoimmune disease development while cross-fostering B6 pups on A/J mothers did not alter their susceptibility. The increase in AOD incidence seen in foster-nursed d3tx A/J mice correlated with a decrease in the total number of CD4(+) T cells in the lymph nodes of these animals. Analysis of the cellular composition in the milk revealed that B6 mice shed significantly more maternally derived lymphocytes into their milk compared to A/J mothers. These data suggest that there are maternally derived postnatal factors that influence the development of autoimmune disease in A/J mice.

Replication of beta- and gammaretroviruses is restricted in I/LnJ mice via the same genetic mechanism.

Mice of the I/LnJ inbred strain are unique in their ability to mount a robust and sustained humoral immune response capable of neutralizing infection with a betaretrovirus, mouse mammary tumor virus (MMTV). Virus-neutralizing antibodies (Abs) coat MMTV virions secreted by infected cells, preventing virus spread and hence the formation of mammary tumors. To investigate whether I/LnJ mice resist infection with other retroviruses besides MMTV, the animals were infected with murine leukemia virus (MuLV), a gammaretrovirus. MuLV-infected I/LnJ mice produced virus-neutralizing Abs that block virus transmission and virally induced disease. Generation of virus-neutralizing Abs required gamma interferon but was independent of interleukin-12. This unique mechanism of retrovirus resistance is governed by a single recessive gene, virus infectivity controller 1 (vic1), mapped to chromosome 17. In addition to controlling the antivirus humoral immune response, vic1 is also required for an antiviral cytotoxic response. Both types of responses were maintained in mice of the susceptible genetic background but congenic for the I/LnJ vic1 locus. Although the vic1-mediated resistance to MuLV resembles the mechanism of retroviral recovery controlled by the resistance to Friend virus 3 (rfv3) gene, the rfv3 gene has been mapped to chromosome 15 and confers resistance to MuLV but not to MMTV. Thus, we have identified a unique virus resistance mechanism that controls immunity against two distinct retroviruses.

Network-Based Functional Prediction Augments Genetic Association To Predict Candidate Genes for Histamine Hypersensitivity in Mice.

Genetic mapping is a primary tool of genetics in model organisms; however, many quantitative trait loci (QTL) contain tens or hundreds of positional candidate genes. Prioritizing these genes for validation is often ad hoc and biased by previous findings. Here we present a technique for prioritizing positional candidates based on computationally inferred gene function. Our method uses machine learning with functional genomic networks, whose links encode functional associations among genes, to identify network-based signatures of functional association to a trait of interest. We demonstrate the method by functionally ranking positional candidates in a large locus on mouse Chr 6 (45.9 Mb to 127.8 Mb) associated with histamine hypersensitivity (Histh). Histh is characterized by systemic vascular leakage and edema in response to histamine challenge, which can lead to multiple organ failure and death. Although Histh risk is strongly influenced by genetics, little is known about its underlying molecular or genetic causes, due to genetic and physiological complexity of the trait. To dissect this complexity, we ranked genes in the Histh locus by predicting functional association with multiple Histh-related processes. We integrated these predictions with new single nucleotide polymorphism (SNP) association data derived from a survey of 23 inbred mouse strains and congenic mapping data. The top-ranked genes included Cxcl12, Ret, Cacna1c, and Cntn3, all of which had strong functional associations and were proximal to SNPs segregating with Histh. These results demonstrate the power of network-based computational methods to nominate highly plausible quantitative trait genes even in challenging cases involving large QTL and extreme trait complexity.