Emma H. Wall

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.

Histamine H4 Receptor Optimizes T Regulatory Cell Frequency and Facilitates Anti-Inflammatory Responses within the Central Nervous System

Histamine is a biogenic amine that mediates multiple physiological processes, including immunomodulatory effects in allergic and inflammatory reactions, and also plays a key regulatory role in experimental allergic encephalomyelitis, the autoimmune model of multiple sclerosis. The pleiotropic effects of histamine are mediated by four G protein-coupled receptors, as follows: Hrh1/H1R, Hrh2/H2R, Hrh3/H3R, and Hrh4/H4R. H4R expression is primarily restricted to hematopoietic cells, and its role in autoimmune inflammatory demyelinating disease of the CNS has not been studied. In this study, we show that, compared with wild-type mice, animals with a disrupted Hrh4 (H4RKO) develop more severe myelin oligodendrocyte glycoprotein (MOG)35\x{2013}55-induced experimental allergic encephalomyelitis. Mechanistically, we also show that H4R plays a role in determining the frequency of T regulatory (TR) cells in secondary lymphoid tissues, and regulates TR cell chemotaxis and suppressor activity. Moreover, the lack of H4R leads to an impairment of an anti-inflammatory response due to fewer TR cells in the CNS during the acute phase of the disease and an increase in the proportion of Th17 cells.

Lactogenic hormones stimulate expression of lipogenic genes but not glucose transporters in bovine mammary gland.

During the onset of lactation, there is a dramatic increase in the expression of glucose transporters (GLUT) and a group of enzymes involved in milk fat synthesis in the bovine mammary gland. The objective of this study was to investigate whether the lactogenic hormones mediate both of these increases. Bovine mammary explants were cultured for 48, 72, or 96 h with the following hormone treatments: no hormone (control), IGF-I, insulin (Ins), Ins + hydrocortisone + ovine prolactin (InsHPrl), or Ins + hydrocortisone + prolactin + 17β-estradiol (InsHPrlE). The relative expression of β-casein, α-lactalbumin, sterol regulatory element binding factor 1 (SREBF1), fatty acid synthase (FASN), acetyl-CoA carboxylase α (ACACA), stearyol-CoA desaturase (SCD), GLUT1, GLUT8, and GLUT12 were measured by real-time PCR. Exposure to the lactogenic hormone combinations InsHPrl and InsHPrlE for 96 h stimulated expression of β-casein and α-lactalbumin mRNA by several hundred-fold and also increased the expression of SREBF1, FASN, ACACA, and SCD genes in mammary explants (P < 0.01). However, those hormone combinations had no effect on GLUT1 or GLUT8 expression and inhibited GLUT12 expression by 50% after 72 h of treatment (P < 0.05). In separate experiments, the expression of GLUTs in the mouse mammary epithelial cell line HC11 or in bovine primary mammary epithelial cells was not increased by lactogenic hormone treatments. Moreover, treatment of dairy cows with bovine prolactin had no effect on GLUT expression in the mammary gland. In conclusion, lactogenic hormones clearly stimulate expression of milk protein and lipogenic genes, but they do not appear to mediate the marked up-regulation of GLUT expression in the mammary gland during the onset of lactation.

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

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.

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

The uterotropic response of the uterus to 17β‐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.—Wall, E. H., Hewitt, S. C., Liu, L., del Rio, R., Case, L. K., Lin, C.‐Y., Korach, K. S., Teuscher, C. Genetic control of estrogen‐regulated transcriptional and cellular responses in mouse uterus. FASEB J. 27, 1874–1886 (2013). www.fasebj.org

The effects of milk removal or four-times-daily milking on mammary expression of genes involved in the insulin-like growth factor-I axis.

Frequent milking of dairy cows during early lactation elicits both an immediate increase in milk yield and a partial carryover effect that persists to the end of lactation. We hypothesized that the immediate response would be associated with a local increase in insulin-like growth factor (IGF)-I signaling and a consequent increase in mammary growth. Four multiparous cows were assigned at parturition to unilateral frequent milking [UFM; milking of the left udder half twice daily (2x; 0230 and 1430 h); milking of the right udder half 4 times daily (4x; 0230, 0530, 1430, and 1730 h)]. Mammary biopsies were obtained from both udder halves at 5 d in milk at 0530 h (immediately after 4x glands were milked). Incorporation of [3H]-thymidine into DNA and mammary cell apoptosis were not affected by UFM. Because biopsies were obtained when udder halves were at different postmilking intervals, our results reflected both the acute, transient mammary response to milking and the sustained mammary response to frequent milking treatment. We further hypothesized that the acute, transient response involves mechanisms distinct from those regulating the sustained response to frequent milking. To test that hypothesis, mammary biopsies were obtained from UFM cows (n=5) at 0500 h, when time postmilking was the same for both udder halves. Mammary cell apoptosis was not affected by UFM. Expression of genes involved in the IGF-I axis was analyzed to identify acute responses associated with milking, per se, versus sustained responses to frequent milking treatment. Removal of milk from 4x glands was associated with an acute increase in expression of IGF binding protein-1, -3, and -4 mRNA in 2x glands, whereas IGF-I expression was increased by frequent milking treatment. These effects, however, were significant only for expression of IGF binding protein-3. Expression of IGF-I receptor did not differ because of milking frequency but was higher in both udder halves immediately postmilking, indicating a systemic effect. We conclude that several genes of the IGF-I axis respond to milking, per se, or frequent milking treatment, via at least 3 distinct patterns. Increased milking frequency does not alter mammary cell proliferation or apoptosis at 5 d in milk; however, it may increase the bioavailability of IGF-I in the mammary gland. Moreover, the increase in local expression of IGF-I in 4x udder halves indicates a role for this gene in the immediate milk yield response to frequent milking during early lactation.

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, 17β‐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.—Wall, E. H., Hewitt, S. C., Case, L. K., Lin, C.‐Y., Korach, K. S., Teuscher, C., The role of genetics in estrogen responses: a critical piece of an intricate puzzle. FASEB J. 28, 5042–5054 (2014). www.fasebj.org

Identification of Genetic Determinants of the Sexual Dimorphism in CNS Autoimmunity

Multiple sclerosis (MS) is a debilitating chronic inflammatory disease of the nervous system that affects approximately 2.3 million individuals worldwide, with higher prevalence in females, and a strong genetic component. While over 200 MS susceptibility loci have been identified in GWAS, the underlying mechanisms whereby they contribute to disease susceptibility remains ill-defined. Forward genetics approaches using conventional laboratory mouse strains are useful in identifying and functionally dissecting genes controlling disease-relevant phenotypes, but are hindered by the limited genetic diversity represented in such strains. To address this, we have combined the powerful chromosome substitution (consomic) strain approach with the genetic diversity of a wild-derived inbred mouse strain. Using experimental allergic encephalomyelitis (EAE), a mouse model of MS, we evaluated genetic control of disease course among a panel of 26 consomic strains of mice inheriting chromosomes from the wild-derived PWD strain on the C57BL/6J background, which models the genetic diversity seen in human populations. Nineteen linkages on 18 chromosomes were found to harbor loci controlling EAE. Of these 19 linkages, six were male-specific, four were female-specific, and nine were non-sex-specific, consistent with a differential genetic control of disease course between males and females. An MS-GWAS candidate-driven bioinformatic analysis using orthologous genes linked to EAE course identified sex-specific and non-sex-specific gene networks underlying disease pathogenesis. An analysis of sex hormone regulation of genes within these networks identified several key molecules, prominently including the MAP kinase family, known hormone-dependent regulators of sex differences in EAE course. Importantly, our results provide the framework by which consomic mouse strains with overall genome-wide genetic diversity, approximating that seen in humans, can be used as a rapid and powerful tool for modeling the genetic architecture of MS. Moreover, our data represent the first step towards mechanistic dissection of genetic control of sexual dimorphism in CNS autoimmunity.

Histamine H3 Receptor Integrates Peripheral Inflammatory Signals in the Neurogenic Control of Immune Responses and Autoimmune Disease Susceptibility

Histamine H3 receptor (Hrh3/H3R) is primarily expressed by neurons in the central nervous system (CNS) where it functions as a presynaptic inhibitory autoreceptor and heteroreceptor. Previously, we identified an H3R-mediated central component in susceptibility to experimental allergic encephalomyelitis (EAE), the principal autoimmune model of multiple sclerosis (MS), related to neurogenic control of blood brain barrier permeability and peripheral T cell effector responses. Furthermore, we identified Hrh3 as a positional candidate for the EAE susceptibility locus Eae8. Here, we characterize Hrh3 polymorphisms between EAE-susceptible and resistant SJL and B10.S mice, respectively, and show that Hrh3 isoform expression in the CNS is differentially regulated by acute peripheral inflammatory stimuli in an allele-specific fashion. Next, we show that Hrh3 is not expressed in any subpopulations of the immune compartment, and that secondary lymphoid tissue is anatomically poised to be regulated by central H3R signaling. Accordingly, using transcriptome analysis, we show that, inflammatory stimuli elicit unique transcriptional profiles in the lymph nodes of H3RKO mice compared to WT mice, which is indicative of negative regulation of peripheral immune responses by central H3R signaling. These results further support a functional link between the neurogenic control of T cell responses and susceptibility to CNS autoimmune disease coincident with acute and/or chronic peripheral inflammation. Pharmacological targeting of H3R may therefore be useful in preventing the development and formation of new lesions in MS, thereby limiting disease progression.

Genetic Control of Ductal Morphology, Estrogen-Induced Ductal Growth, and Gene Expression in Female Mouse Mammary Gland

The uterotropic response of the uterus to 17β-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 (C57BL6/J; B6) or low (C3H/HeJ; C3H) responders to E2 led to the identification of quantitative trait loci (QTL) associated with phenotypic variation in uterine growth and leukocyte infiltration. Like the uterus, phenotypic variation in the responsiveness of the mammary gland to E2 during both normal and pathologic conditions has been reported. In the current experiment, we utilized an E2-specific model of mammary ductal growth combined with a microarray approach to determine the degree to which genotype influences the responsiveness of the mammary gland to E2, including the associated transcriptional programs, in B6 and C3H mice. Our results reveal that E2-induced mammary ductal growth and ductal morphology are genetically controlled. In addition, we observed a paradoxical effect of mammary ductal growth in response to E2 compared with what has been reported for the uterus; B6 is a high responder for the uterus and was a low responder for mammary ductal growth, whereas the reverse was observed for C3H. In contrast, B6 was a high responder for mammary ductal side branching. The B6 phenotype was associated with increased mammary epithelial cell proliferation and apoptosis, and a distinct E2-induced transcriptional program. These findings lay the groundwork for future experiments designed to investigate the genes and mechanisms underlying phenotypic variation in tissue-specific sensitivity to systemic and environmental estrogens during various physiological and disease states.