Eric M. Jacobson

Joint Genetic Susceptibility to Type 1 Diabetes and Autoimmune Thyroiditis: from Epidemiology to Mechanisms

Type 1 diabetes (T1D) and autoimmune thyroid diseases (AITD) frequently occur together within families and in the same individual. The co-occurrence of T1D and AITD in the same patient is one of the variants of the autoimmune polyglandular syndrome type 3 [APS3 variant (APS3v)]. Epidemiological data point to a strong genetic influence on the shared susceptibility to T1D and AITD. Recently, significant progress has been made in our understanding of the genetic association between T1D and AITD. At least three genes have been confirmed as major joint susceptibility genes for T1D and AITD: human leukocyte antigen class II, cytotoxic T-lymphocyte antigen 4 (CTLA-4), and protein tyrosine phosphatase non-receptor type 22. Moreover, the first whole genome linkage study has been recently completed, and additional genes will soon be identified. Not unexpectedly, all the joint genes for T1D and AITD identified so far are involved in immune regulation, specifically in the presentation of antigenic peptides to T cells. One of the lessons learned from the analysis of the joint susceptibility genes for T1D and AITD is that subset analysis is a key to dissecting the etiology of complex diseases. One of the best demonstrations of the power of subset analysis is the CTLA-4 gene in T1D. Although CTLA-4 showed very weak association with T1D, when analyzed in the subset of patients with both T1D and AITD, the genetic effect of CTLA-4 was significantly stronger. Gene-gene and genetic-epigenetic interactions most likely play a role in the shared genetic susceptibility to T1D and AITD. Dissecting these mechanisms will lead to a better understanding of the etiology of T1D and AITD, as well as autoimmunity in general.

Molecular amino acid signatures in the MHC class II peptide-binding pocket predispose to autoimmune thyroiditis in humans and in mice

Hashimotos thyroiditis (HT) is associated with HLA, but the associated allele is still controversial. We hypothesized that specific HLA-DR pocket-sequence variants are associated with HT and that similar variants in the murine I-E locus (homologous to HLA-DR) predispose to experimental autoimmune thyroiditis (EAT), a classical mouse model of HT. Therefore, we sequenced the polymorphic exon 2 of the HLA-DR gene in 94 HT patients and 149 controls. In addition, we sequenced exon 2 of the I-E gene in 22 strains of mice, 12 susceptible to EAT and 10 resistant. Using logistic regression analysis, we identified a pocket amino acid signature, Tyr-26, Tyr-30, Gln-70, Lys-71, strongly associated with HT (P = 6.18 × 10−5, OR = 3.73). Lys-71 showed the strongest association (P = 1.7 × 10−8, OR = 2.98). This association was seen across HLA-DR types. The 5-aa haplotype Tyr-26, Tyr-30, Gln-70, Lys-71, Arg-74 also was associated with HT (P = 3.66 × 10−4). In mice, the I-E pocket amino acids Val-28, Phe-86, and Asn-88 were strongly associated with EAT. Structural modeling studies demonstrated that pocket P4 was critical for the development of HT, and pockets P1 and P4 influenced susceptibility to EAT. Surprisingly, the structures of the HT- and EAT-susceptible pockets were different. We conclude that specific MHC II pocket amino acid signatures determine susceptibility to HT and EAT by causing structural changes in peptide-binding pockets that may influence peptide binding, selectivity, and presentation. Because the HT- and EAT-associated pockets are structurally different, it is likely that distinct antigenic peptides are associated with HT and EAT.

A Pilot Randomized, Placebo Controlled, Double Blind Phase I Trial of the Novel SIRT1 Activator SRT2104 in Elderly Volunteers

Background SRT2104 has been developed as a selective small molecule activator of SIRT1, a NAD+-dependent deacetylase involved in the regulation of energy homeostasis and the modulation of various metabolic pathways, including glucose metabolism, oxidative stress and lipid metabolism. SIRT1 has been suggested as putative therapeutic target in multiple age-related diseases including type 2 diabetes and dyslipidemias. We report the first clinical trial of SRT2104 in elderly volunteers. Methods Oral doses of 0.5 or 2.0 g SRT2104 or matching placebo were administered once daily for 28 days. Pharmacokinetic samples were collected through 24 hours post-dose on days 1 and 28. Multiple pharmacodynamic endpoints were explored with oral glucose tolerance tests (OGTT), serum lipid profiles, magnetic resonance imaging (MRI) for assessment of whole body visceral and subcutaneous fat, maximal aerobic capacity test and muscle 31P magnetic resonance spectroscopy (MRS) for estimation of mitochondrial oxidative capacity. Results SRT2104 was generally safe and well tolerated. Pharmacokinetic exposure increased less than dose-proportionally. Mean Tmax was 2–4 hours with elimination half-life of 15–20 hours. Serum cholesterol, LDL levels and triglycerides decreased with treatment. No significant changes in OGTT responses were observed. 31P MRS showed trends for more rapid calculated adenosine diphosphate (ADP) and phosphocreatine (PCr) recoveries after exercise, consistent with increased mitochondrial oxidative phosphorylation. Conclusions SRT2104 can be safely administered in elderly individuals and has biological effects in humans that are consistent with SIRT1 activation. The results of this study support further development of SRT2104 and may be useful in dose selection for future clinical trials in patients. Trial Registration ClinicalTrials.gov NCT00964340

Novel Variant of Thyroglobulin Promoter Triggers Thyroid Autoimmunity through an Epigenetic Interferon α-modulated Mechanism

Autoimmune thyroid diseases (AITD) arise from complex interactions between genetic, epigenetic, and environmental factors. Whole genome linkage scans and association studies have established thyroglobulin (TG) as a major AITD susceptibility gene. However, the causative TG variants and the pathogenic mechanisms are unknown. Here, we describe a genetic/epigenetic mechanism by which a newly identified TG promoter single-nucleotide polymorphism (SNP) variant predisposes to AITD. Sequencing analyses followed by case control and family-based association studies identified an SNP (−1623A→G) that was associated with AITD in the Caucasian population (p = 0.006). We show that the nucleotide substitution introduced by SNP (−1623A/G) modified a binding site for interferon regulatory factor-1 (IRF-1), a major interferon-induced transcription factor. Using chromatin immunoprecipitation, we demonstrated that IRF-1 binds to the 5′ TG promoter motif, and the transcription factor binding correlates with active chromatin structure and is marked by enrichment of mono-methylated Lys-4 residue of histone H3, a signature of active transcriptional enhancers. Using reporter mutations and siRNA approaches, we demonstrate that the disease-associated allele (G) conferred increased TG promoter activity through IRF-1 binding. Finally, treatment of thyroid cells with interferon α, a known trigger of AITD, increased TG promoter activity only when it interacted with the disease-associated variant through IRF-1 binding. These results reveal a new mechanism of interaction between environmental (IFNα) and genetic (TG) factors to trigger AITD.

‘Linkage Analysis of Thyroid Antibody Production: Evidence for Shared Susceptibility to Clinical Autoimmune Thyroid Disease

CONTEXT Epidemiological data suggest a genetic susceptibility to thyroid antibody (TAb) production. OBJECTIVE The objective of the study was to identify genetic loci that are linked with TAb production. DESIGN The design of the study was a whole genome linkage study in families with clustering of thyroid autoimmunity. SETTINGS The study took place at an academic medical center. PARTICIPANTS Participants included 102 multigenerational families (540 individuals) multiplex for autoimmune thyroid disease (AITD) and TAb production. MAIN OUTCOME MEASURES We computed two-point logarithm of odds (LOD) scores and multipoint heterogeneity LOD scores for 400 microsatellite markers spanning the entire human genome at an average distance of 10 cm (approximately 10 Mb). RESULTS Three loci showed evidence for linkage with TAb production: 1) 2q locus, which gave a maximum multipoint heterogeneity LOD score (HLOD) of 2.8 and contained the CTLA-4 gene, previously reported to be linked and associated with clinical AITD; (2) 6p locus (HLOD 2.5), which was the same AITD-1 locus found to be linked with clinical AITD; and (3) 8q locus (HLOD 2.2), which contained the thyroglobulin gene, also previously reported to be linked and associated with AITD. All loci that were linked to TAb were also linked to AITD, suggesting that TAb and AITD share the same genetic predisposition. CONCLUSIONS We conclude that: 1) some of the genes/loci predisposing to TAb and AITD are shared, whereas distinct genes/loci also exist; (2) the presence of TAb in relatives of AITD patients may be associated with increased risk for the development of clinical AITD; and (3) further studies are needed to determine the predictive value of TAb levels for the development of clinical AITD in relatives of patients with familial AITD.

Unexploited therapies in breast and prostate cancer: blockade of the prolactin receptor.

Breast and prostate cancers are hormone-sensitive malignancies that afflict millions of women and men. Although prolactin (PRL) is known as a survival factor that supports tumor growth and confers chemoresistance in both cancers, its precise role in these tumors has not been studied extensively. Growth hormone and placental lactogen also bind PRL receptor (PRLR) and mimic some of the actions of PRL. Blockade of the PRLR represents a novel treatment for patients with advanced breast or prostate cancer with limited therapeutic options. This review discusses different approaches for generating PRLR antagonists. Emphasis is placed on technological advances which enable high-throughput screening for small molecule inhibitors of PRLR signaling that could serve as oral medications.

Apilimod inhibits the production of IL-12 and IL-23 and reduces dendritic cell infiltration in psoriasis.

Psoriasis is characterized by hyperplasia of the epidermis and infiltration of leukocytes into both the dermis and epidermis. IL-23, a key cytokine that induces TH17 cells, has been found to play a critical role in the pathogenesis of psoriasis. Apilimod is a small-molecule compound that selectively suppresses synthesis of IL-12 and IL-23. An open-label clinical study of oral administration of apilimod was conducted in patients with psoriasis. Substantial improvements in histology and clinical measurements were observed in patients receiving 70mg QD. The expression of IL-23p19 and IL-12/IL-23p40 in skin lesions was significantly reduced in this dose group, with a simultaneous increase in IL-10 observed. A decrease in the levels of TH1 and TH17 cytokines/chemokines in skin lesions followed these p19 and p40 changes. In parallel, a reduction in skin-infiltrating CD11c+ dendritic cells and CD3+ T cells was seen, with a greater decrease in the CD11c+ population. This was accompanied by increases in T and B cells, and decreases in neutrophils and eosinophils in the periphery. This study demonstrates the immunomodulatory activity of apilimod and provides clinical evidence supporting the inhibition of IL-12/IL-23 synthesis for the treatment of TH1- and TH17-mediated inflammatory diseases.

Genetically Driven Target Tissue Overexpression of CD40: A Novel Mechanism in Autoimmune Disease

The CD40 gene, an important immune regulatory gene, is also expressed and functional on nonmyeloid-derived cells, many of which are targets for tissue-specific autoimmune diseases, including β cells in type 1 diabetes, intestinal epithelial cells in Crohn’s disease, and thyroid follicular cells in Graves’ disease (GD). Whether target tissue CD40 expression plays a role in autoimmune disease etiology has yet to be determined. In this study, we show that target tissue overexpression of CD40 plays a key role in the etiology of autoimmunity. Using a murine model of GD, we demonstrated that thyroidal CD40 overexpression augmented the production of thyroid-specific Abs, resulting in more severe experimental autoimmune GD (EAGD), whereas deletion of thyroidal CD40 suppressed disease. Using transcriptome and immune–pathway analyses, we showed that in both EAGD mouse thyroids and human primary thyrocytes, CD40 mediates this effect by activating downstream cytokines and chemokines, most notably IL-6. To translate these findings into therapy, we blocked IL-6 during EAGD induction in the setting of thyroidal CD40 overexpression and showed decreased levels of thyroid stimulating hormone receptor-stimulating Abs and frequency of disease. We conclude that target tissue overexpression of CD40 plays a key role in the etiology of organ-specific autoimmune disease.

Employing a Recombinant HLA-DR3 Expression System to Dissect Major Histocompatibility Complex II-Thyroglobulin Peptide Dynamism A GENETIC, BIOCHEMICAL, AND REVERSE IMMUNOLOGICAL PERSPECTIVE

Previously, we have shown that statistical synergism between amino acid variants in thyroglobulin (Tg) and specific HLA-DR3 pocket sequence signatures conferred a high risk for autoimmune thyroid disease (AITD). Therefore, we hypothesized that this statistical synergism mirrors a biochemical interaction between Tg peptides and HLA-DR3, which is key to the pathoetiology of AITD. To test this hypothesis, we designed a recombinant HLA-DR3 expression system that was used to express HLA-DR molecules harboring either AITD susceptibility or resistance DR pocket sequences. Next, we biochemically generated the potential Tg peptidic repertoire available to HLA-DR3 by separately treating 20 purified human thyroglobulin samples with cathepsins B, D, or L, lysosomal proteases that are involved in antigen processing and thyroid biology. Sequences of the cathepsin-generated peptides were then determined by matrix-assisted laser desorption ionization time-of-flight-mass spectroscopy, and algorithmic means were employed to identify putative AITD-susceptible HLA-DR3 binders. From four predicted peptides, we identified two novel peptides that bound strongly and specifically to both recombinant AITD-susceptible HLA-DR3 protein and HLA-DR3 molecules expressed on stably transfected cells. Intriguingly, the HLA-DR3-binding peptides we identified had a marked preference for the AITD-susceptibility DR signatures and not to those signatures that were AITD-protective. Structural analyses demonstrated the profound influence that the pocket signatures have on the interaction of HLA-DR molecules with Tg peptides. Our study suggests that interactions between Tg and discrete HLA-DR pocket signatures contribute to the initiation of AITD.

Lactogens and estrogens in breast cancer chemoresistance

Tumor resistance to chemotherapy in advanced breast cancer is a major impediment to treatment success. Resistance can be induced by the drugs themselves or result from the action of internal factors. The role of hormones in chemoresistance has received little attention. This article focuses on two classes of hormones: lactogens and estrogens. Lactogens include prolactin, growth hormone and placental lactogen, all of which can activate the prolactin receptor. Estrogens include endogenous steroids and nonsteroidal compounds from the environment termed endocrine disruptors, all of which can activate ‘classical’ estrogen receptors (ERα and ERβ), as well as other types of receptors. Both lactogens and estrogens antagonize cytotoxicity of multiple chemotherapeutic agents through complementary mechanisms. The implications of chemoresistance by these hormones to patients with breast cancer, and the potential benefits of developing combinatorial anti-lactogen/anti-estrogen treatment regimens, are discussed.