Immunogenetics of autoimmune thyroid diseases: A comprehensive review

Both environmental and genetic triggers factor into the etiology of autoimmune thyroid disease (AITD), including Graves' disease (GD) and Hashimoto's thyroiditis (HT). Although the exact pathogenesis and causative interaction between environment and genes are unknown, GD and HT share similar immune-mediated mechanisms of disease. They both are characterized by the production of thyroid autoantibodies and by thyroidal lymphocytic infiltration, despite being clinically distinct entities with thyrotoxicosis in GD and hypothyroidism in HT. Family and population studies confirm the strong genetic influence and inheritability in the development of AITD. AITD susceptibility genes can be categorized as either thyroid specific (Tg, TSHR) or immune-modulating (FOXP3, CD25, CD40, CTLA-4, HLA), with HLA-DR3 carrying the highest risk. Of the AITD susceptibility genes, FOXP3 and CD25 play critical roles in the establishment of peripheral tolerance while CD40, CTLA-4, and the HLA genes are pivotal for T lymphocyte activation and antigen presentation. Polymorphisms in these immune-modulating genes, in particular, significantly contribute to the predisposition for GD, HT and, unsurprisingly, other autoimmune diseases. Emerging evidence suggests that single nucleotide polymorphisms (SNPs) in the immunoregulatory genes may functionally hinder the proper development of central and peripheral tolerance and alter T cell interactions with antigen presenting cells (APCs) in the immunological synapse. Thus, susceptibility genes for AITD contribute directly to the key mechanism underlying the development of organ-specific autoimmunity, namely the breakdown in self-tolerance. Here we review the major immune-modulating genes that are associated with AITD and their potential functional effects on thyroidal immune dysregulation.     

New immunomodulatory role of neuropeptide Y (NPY) in Salmo salar leucocytes

Neuropeptide Y (NPY) plays different roles in mammals such as: regulate food intake, memory retention, cardiovascular functions, and anxiety. It has also been shown in the modulation of chemotaxis, T lymphocyte differentiation, and leukocyte migration. In fish, NPY expression and functions have been studied but its immunomodulatory role remains undescribed. This study confirmed the expression and synthesis of NPY in S. salar under inflammation, and validated a commercial antibody for NPY detection in teleost. Additionally, immunomodulatory effects of NPY were assayed in vitro and in vivo. Phagocytosis and superoxide anion production in leukocytes and SHK cells were induced under stimulation with a synthetic peptide. IL-8 mRNA was selectively and strongly induced in the spleen, head kidney, and isolated cells, after in vivo challenge with NPY. All together suggest that NPY is expressed in immune tissues and modulates the immune response in teleost fish.     

Metabolic responses of clam Ruditapes philippinarum exposed to its pathogen Vibrio tapetis in relation to diet

We investigated the effect of brown ring disease (BRD) development and algal diet on energy reserves and activity of enzymes related to energy metabolism, antioxidant system and immunity in Manila clam, Ruditapes philippinarum. We found that algal diet did not impact the metabolic response of clams exposed to Vibrio tapetis. At two days post-injection (dpi), activities of superoxide dismutase and glutathione peroxidase (GPx) decreased whereas activities of nitric oxide synthase (iNOS) and catalase increased in infected clams, although no clinical signs were visible (BRD−). At 7 dpi, activities of several antioxidant and immune-related enzymes were markedly increased in BRD-likely indicating an efficient reactive oxygen species (ROS) scavenging compared to animals which developed clinical signs of BRD (BRD+). Therefore, resistance to BRD clinical signs appearance was associated with higher detoxification of ROS and enhancement of immune response. This study provides new biochemical indicators of disease resistance and a more comprehensive view of the global antioxidant response of clam to BRD development.     

Central role of interferon-beta in thymic events leading to myasthenia gravis

The thymus plays a primary role in early-onset Myasthenia Gravis (MG) mediated by anti-acetylcholine receptor (AChR) antibodies. As we recently showed an inflammatory and anti-viral signature in MG thymuses, we investigated in detail the contribution of interferon (IFN)-I and IFN-III subtypes in thymic changes associated with MG. We showed that IFN-I and IFN-III subtypes, but especially IFN-β, induced specifically α-AChR expression in thymic epithelial cells (TECs). We also demonstrated that IFN-β increased TEC death and the uptake of TEC proteins by dendritic cells.In parallel, we showed that IFN-β increased the expression of the chemokines CXCL13 and CCL21 by TECs and lymphatic endothelial cells, respectively. These two chemokines are involved in germinal center (GC) development and overexpressed in MG thymus with follicular hyperplasia. We also demonstrated that the B-cell activating factor (BAFF), which favors autoreactive B-cells, was overexpressed by TECs in MG thymus and was also induced by IFN-β in TEC cultures.Some of IFN-β effects were down-regulated when cell cultures were treated with glucocorticoids, a treatment widely used in MG patients that decreases the number of thymic GCs.Similar changes were observed in vivo. The injections of Poly(I:C) to C57BL/6 mice triggered a thymic overexpression of IFN-β and IFN-α2 associated with increased expressions of CXCL13, CCL21, BAFF, and favored the recruitment of B cells. These changes were not observed in the thymus of IFN-I receptor KO mice injected with Poly(I:C), even if IFN-β and IFN-α2 were overexpressed.Altogether, these results demonstrate that IFN-β could play a central role in thymic events leading to MG by triggering the overexpression of α-AChR probably leading to thymic DC autosensitization, the abnormal recruitment of peripheral cells and GC formation.     

Screening for thyroid disease and iodine deficiency

The high global prevalence of iodine deficiency and autoimmune thyroid disorders and the mental and physical consequences of these disorders creates a huge human and economic burden that can be prevented, in large part, by early detection and appropriate preventative or therapeutic measures. The availability of sophisticated, sensitive and accurate laboratory testing procedures provides an efficient and effective platform for the application of screening for these disorders. Measurement of urine iodine concentration (UIC) in school children or pregnant women is the recommended indicator for screening populations for iodine deficiency. The severity of the iodine deficiency is classified according to the UIC. Measurement of serum thyrotropin (TSH) as an indicator for population iodine deficiency is used only in neonates and is supplementary to UIC screening. Other indicators such as goitre rates, thyroid function and serum thyroglobulin levels are useful adjunctive but not frontline process indicators. The human and economic benefits of screening for congenital hypothyroidism by measurement of heel-prick TSH have been well documented and justify its universal application. Using this measurement for monitoring population iodine intake is recommended by the World Health Organization but further validation is required before it can be universally recommended. Subclinical thyroid dysfunction is readily detected by current highly sensitive serum TSH assays and its prevalence appears to increase with age, varies with iodine intake and ethnicity and may occur in up to 20% of older age people. Subclinical hyperthyroidism is the less common disorder and screening cannot be justified because of its low prevalence and minimal or insignificant clinical effects. The argument for screening for subclinical hypothyroidism in middle-aged and older women is stronger but lacks evidence of benefit from randomised controlled trials or cost benefit analyses of therapeutic intervention, so it cannot currently be recommended. The publication of recent Clinical Practice Guidelines for management of thyroid disease in pregnancy from the American Endocrine Society and American Thyroid Association provide persuasive arguments for early detection and treatment of overt and subclinical hypothyroidism to prevent obstetric complications and potential neurocognitive disorders in the offspring. Given the indisputable benefits of therapy, the sooner thyroid dysfunction is detected, before or as early as possible in gestation, the more likely there will be a better outcome. Because of the limitations of targeted case detection in women at risk of subclinical hypothyroidism, there has been a gradual shift in opinion to universal TSH screening of all women as soon as practicable in pregnancy. While a positive association exists between the presence of anti-thyroid antibodies and increased pregnancy loss, universal screening of all pregnant women for underlying autoimmune thyroid disease is difficult to justify until there is evidence of beneficial outcomes from randomised controlled trials. Vigorous and liberal targeted case detection remains the recommended strategy to address this problem.     

Echinacea pupurea extracts promote murine dendritic cell maturation by activation of JNK,p38 MAPK and NF-κB pathways

Dendritic cells (DCs) comprise a system of highly professional antigen presenting cells (APCs) which connect innate and adaptive immunity by undergoing dramatic shift in their maturation state. Phytomedicine Echinacea purpurea extracts (EE) could modulate murine dendritic cell fate and function. However, the underlying mechanism of EE on DCs development and maturation remains limited. In this study, immature DCs were induced phenotypic maturation with up-regulated expression of key accessory molecules and the phagocytic activity was decreased after being treated with EE (400 μg/ml) for 48 h. We found that TLR1/2, JNK, p38-MAPK and NF-κB pathways were activated following EE exposure. Notably, JNK activation was demonstrated to be associated with increased IFN-γ response while p38-MAPK pathway exhibited immuno-regulatory effects via induction of IL-10 and TGF-β1. Furthermore, it was verified that NF-κB signaling was responsible for EE-induced synthesis of IFN-γ, IL-12 and TGF-β1, but not for IL-10 induction. These results indicate that EE have the immunomodulatory potency to promote both phenotypic and functional maturation of BMDCs via modulating the activation of JNK, p38-MAPK and NF-κB pathways. Our findings contributed to the current understanding of the immunoregulatory function of EE and the mechanism of DCs maturation.     

Molecular and functional characterization of pigeon (Columba livia) tumor necrosis factor receptor-associated factor 3

Tumor necrosis factor receptor-associated factor 3 (TRAF3) plays a key antiviral role by promoting type I interferon production. We cloned the pigeon TRAF3 gene (PiTRAF3) according to its predicted mRNA sequence to investigate its function. The 1704-bp full-length open reading frame encodes a 567-amino acid protein. One Ring finger, two TRAF-type Zinc fingers, one Coiled coil, and one MATH domain were inferred. RT-PCR showed that PiTRAF3 was expressed in all tissues, with relatively weak expression in the heart and liver. In HEK293T cells, over-expression of wild-type, △Ring, △Zinc finger, and △Coiled coil PiTRAF3, but not a △MATH form, significantly increased IFN-β promoter activity. Zinc finger and Coiled coil domains were essential for NF-κB activation. In chicken HD11 cells, PiTRAF3 increased IFN-β promoter activity and four domains were all contributing. R848 stimulation of pigeon peripheral blood mononuclear cells and splenocytes significantly increased expression of PiTRAF3 and the inflammatory cytokine genes CCL5, IL-8, and IL-10. These data demonstrate TRAF3's innate immune function and improve understanding of its involvement in poultry antiviral defense.     

Multifunctional effects of honokiol as an anti-inflammatory and anti-cancer drug in human oral squamous cancer cells and xenograft

The aim of this study was to investigate anti-inflammatory and anti-cancer effects of honokiol (HK) in two oral squamous cancer cell carcinoma (OSCC) cell lines, HN22 and HSC4, through the regulation of inducible nitric oxide synthase (iNOS) and endoplasmic reticulum resident protein 44 (ERp44). Griess assay, zymography, and quantitative PCR were performed to study iNOS expression and subsequent nitric oxide (NO) production in OSCC cell lines. Liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based proteomic analysis was used to elucidate the proteins associated with ER stress and cellular cytotoxic response induced by HK. Pull-down assay and molecular modeling were performed to better understand how HK interacts with ERp44. In vitro and in vivo experiments in which ERp44 expression was knocked down were performed to better understand the effects of ERp44 on a cellular level and anti-cancer effects of HK. Expression levels of iNOS and subsequent NO secretion were reduced in OSCC cell lines treated with HK. ERp44 was significantly decreased in OSCC cell lines by HK treatment. HK directly bound to ERp44, and ERp44 knock-down significantly inhibited oral cancer cell proliferation and colony formation. Moreover, HK treatment effectively inhibited tumor growth and ERp44 levels in BALB/c nude mice bearing HN22 cell xenografts. Our findings suggest that HK inhibited inflammation and induced apoptosis by suppressing both iNOS/NO and ERp44 expression in HN22 and HSC4 cells and xenograft tumors, and thus could be a potent anti-inflammatory and anti-cancer drug candidate for human oral cancer treatment.     

The toll-like receptor ligands Hiltonol® (polyICLC) and imiquimod effectively activate antigen-specific immune responses in Tasmanian devils (Sarcophilus harrisii)

Devil facial tumour disease (DFTD) describes two genetically distinct transmissible tumours that pose a significant threat to the survival of the Tasmanian devil. A prophylactic vaccine could protect devils from DFTD transmission. For this vaccine to be effective, potent immune adjuvants will be required. Toll-like receptors (TLRs) promote robust immune responses in human cancer studies and are highly conserved across mammalian species. In this study, we investigated the proficiency of TLR ligands for immune activation in the Tasmanian devil using in vitro mononuclear cell stimulations and in vivo immunisation trials with a model antigen. We identified two such TLR ligands, polyICLC (Hiltonol^®) (TLR3) and imiquimod (TLR7), that in combination induced significant IFNγ production from Tasmanian devil lymphocytes in vitro. Immunisation with these ligands and the model antigen keyhole limpet haemocyanin activated robust antigen-specific primary, secondary and long-term memory IgG responses. Our results support the conserved nature of TLR signaling across mammalian species. PolyICLC and imiquimod will be trialed as immune adjuvants in future DFTD vaccine formulations.     

Myelin repair in vivo is increased by targeting oligodendrocyte precursor cells with nanoparticles encapsulating leukaemia inhibitory factor (LIF)

Multiple sclerosis (MS) is a progressive demyelinating disease of the central nervous system (CNS). Many nerve axons are insulated by a myelin sheath and their demyelination not only prevents saltatory electrical signal conduction along the axons but also removes their metabolic support leading to irreversible neurodegeneration, which currently is untreatable. There is much interest in potential therapeutics that promote remyelination and here we explore use of leukaemia inhibitory factor (LIF), a cytokine known to play a key regulatory role in self-tolerant immunity and recently identified as a pro-myelination factor. In this study, we tested a nanoparticle-based strategy for targeted delivery of LIF to oligodendrocyte precursor cells (OPC) to promote their differentiation into mature oligodendrocytes able to repair myelin. Poly(lactic-co-glycolic acid)-based nanoparticles of ∼120 nm diameter were constructed with LIF as cargo (LIF-NP) with surface antibodies against NG-2 chondroitin sulfate proteoglycan, expressed on OPC. In vitro, NG2-targeted LIF-NP bound to OPCs, activated pSTAT-3 signalling and induced OPC differentiation into mature oligodendrocytes. In vivo, using a model of focal CNS demyelination, we show that NG2-targeted LIF-NP increased myelin repair, both at the level of increased number of myelinated axons, and increased thickness of myelin per axon. Potency was high: a single NP dose delivering picomolar quantities of LIF is sufficient to increase remyelination.Impact statementNanotherapy-based delivery of leukaemia inhibitory factor (LIF) directly to OPCs proved to be highly potent in promoting myelin repair in vivo: this delivery strategy introduces a novel approach to delivering drugs or biologics targeted to myelin repair in diseases such as MS.     

Histamine and mast cell activator compound 48/80 are safe but inefficient systemic adjuvants for gilthead seabream vaccination

Histamine has a key role in the regulation of inflammatory and innate immune responses in vertebrates. Gilthead seabream (Sparus aurata L.), a marine hermaphrodite teleost of great commercial value, was the first fish species shown to possess histamine-containing mast cells (MCs) at mucosal tissues. MCs are highly abundant in the peritoneal exudate of gilthead seabream and compound 48/80 (Co 48/80), often used to promote MC activation and histamine release, is able to promote histamine release from gilthead seabream MCs in vitro and in vivo. The aim of the present study was to analyze the effect of histamine and Co 48/80 on the immune responses of gilthead seabream. For this purpose, histamine and Co 48/80 were intraperitoneally injected alone or combined with 10⁹ heat-killed Vibrio anguillarum cells and their effects on head kidney and peritoneal exudate were analyzed. The results indicated that although histamine and Co 48/80 were both able to alter the percentage of peritoneal exudate and head kidney immune cell types, only Co 48/80 increased reactive oxygen species production by peritoneal leukocytes. In addition, histamine, but not Co 48/80, was able to slightly impair the humoral adaptive immune response, i.e. production of specific IgM to V. anguillarum. Notably, both histamine and Co 48/80 reduced the expression of the gene encoding histamine receptor H2 in peritoneal exudate leukocytes. These results show for the first time in fish that although systemic administration of histamine and Co 48/80 is safe, neither compound can be regarded as an efficient adjuvant for gilthead seabream vaccination.     

Paracrine co-delivery of TGF-β and IL-2 using CD4-targeted nanoparticles for induction and maintenance of regulatory T cells

The cytokine milieu is critical for orchestration of lineage development towards effector T cell (Teff) or regulatory T cell (Treg) subsets implicated in the progression of cancer and autoimmune disease. Importantly, the fitness and survival of the Treg subset is dependent on the cytokines Interleukin-2 (IL-2) and transforming growth factor beta (TGF-β). The production of these cytokines is impaired in autoimmunity increasing the probability of Treg conversion to aggressive effector cells in a proinflammatory microenvironment. Therapy using soluble TGF-β and IL-2 administration is hindered by the cytokines' toxic pleiotropic effects and hence bioavailability to CD4⁺ T cell targets. Thus, there is a clear need for a strategy that rectifies the cytokine milieu in autoimmunity and inflammation leading to enhanced Treg stability, frequency and number. Here we show that inert biodegradable nanoparticles (NP) loaded with TGF-β and IL-2 and targeted to CD4⁺ cells can induce CD4⁺ Tregs in-vitro and expand their number in-vivo. The stability of induced Tregs with cytokine-loaded NP was enhanced leading to retention of their suppressive phenotype even in the presence of proinflammatory cytokines. Our results highlight the importance of a nanocarrier-based approach for stabilizing and expanding Tregs essential for cell-immunotherapy of inflammation and autoimmune disease.     

Roles of bovine viral diarrhea virus envelope glycoproteins in inducing autophagy in MDBK cells

Macroautophagy (autophagy) is an evolutionarily conserved control process that maintains cellular homeostasis in eukaryotic cells. Autophagy principally serves an adaptive role to degrade dysfunctional proteins and to clean damaged organelles in response to pathogenic, viral, or microbial infection, nutrient deprivation and endoplasmic reticulum (ER) stress. In previous study, we showed bovine viral diarrhea virus (BVDV) NADL infection induced autophagy and significantly elevated the expression levels of autophagy-related genes, Beclin1 and ATG14, at 12 h post-infection in MDBK cells. However, the specific mechanisms involved in controlling autophagic activity remain unclear. Here, we investigate the effects of BVDV NADL envelope glycoproteins overexpression on inducing autophagy. The results show that viral envelope glycoproteins E^rns and E2 overexpression mediated by lentivirus increase the formation of autophagosome, the percentage of GFP-LC3 puncta-positive cells and the expression levels of Beclin1 and ATG14. Whereas E1 overexpression doesn't affect autophagic activity. Collectively, these findings suggest that the viral envelope glycoproteins E^rns and E2 are involved in inducing autophagy, and provide a mechanistic insight into the regulation of autophagy in viral infected cells.     

MBD2 regulates TH17 differentiation and experimental autoimmune encephalomyelitis by controlling the homeostasis of T-bet/Hlx axis

Unlike genetic alterations, epigenetic modifications are reversible and amenable to pharmacological interventions, which make them appealing targets for clinical therapy. However, little is known about epigenetic regulation in experimental autoimmune encephalomyelitis (EAE). Here we demonstrated that methyl-CpG-binding domain protein 2 (MBD2), an epigenetic regulator, controls autoimmunity and EAE through T-bet/Hlx. Tbx21 and Hlx underwent a DNA methylation turnover upon polarizations and a unique methylation pattern was essential for T_H17 development. Loss of Mbd2 resulted in a defect for reading the information encoded by this methylation turnover, which disrupted the homeostasis of T-bet/Hlx axis and suppressed T_H17 differentiation. DNA demethylation induced similar effect on helper T cell differentiation. Therefore, Mbd2^−/− mice were completely protected from EAE. Pathogenic splenocytes isolated from wild-type mice challenged with MOG35-55 could adoptively transfer disease to Mbd2^−/− mice. In addition, Mbd2^−/− mice reconstituted with unstimulated wild-type splenocytes developed EAE as wild-type mice did. These data would provide novel insights into epigenetic regulation of EAE.     

Ingestion of oats and barley in patients with celiac disease mobilizes cross-reactive T cells activated by avenin peptides and immuno-dominant hordein peptides

Celiac disease (CD) is a common CD4⁺ T cell mediated enteropathy driven by gluten in wheat, rye, and barley. Whilst clinical feeding studies generally support the safety of oats ingestion in CD, the avenin protein from oats can stimulate intestinal gluten-reactive T cells isolated from some CD patients in vitro. Our objective was to establish whether ingestion of oats or other grains toxic in CD stimulate an avenin-specific T cell response in vivo.We fed participants a meal of oats (100 g/day over 3 days) to measure the in vivo polyclonal avenin-specific T cell responses to peptides contained within comprehensive avenin peptide libraries in 73 HLA-DQ2.5⁺ CD patients. Grain cross-reactivity was investigated using oral challenge with wheat, barley, and rye.Avenin-specific responses were observed in 6/73 HLA-DQ2.5⁺ CD patients (8%), against four closely related peptides. Oral barley challenge efficiently induced cross-reactive avenin/hordein-specific T cells in most CD patients, whereas wheat or rye challenge did not. In vitro, immunogenic avenin peptides were susceptible to digestive endopeptidases and showed weak HLA-DQ2.5 binding stability.Our findings indicate that CD patients possess T cells capable of responding to immuno-dominant hordein epitopes and homologous avenin peptides ex vivo, but the frequency and consistency of these T cells in blood is substantially higher after oral challenge with barley compared to oats. The low rates of T cell activation after a substantial oats challenge (100 g/d) suggests that doses of oats commonly consumed are insufficient to cause clinical relapse, and supports the safety of oats demonstrated in long-term feeding studies.     

Distinct functional responses to stressors of bone marrow derived dendritic cells from diverse inbred chicken lines

Differences in responses of chicken bone marrow derived dendritic cells (BMDC) to in vitro treatment with lipopolysaccharide (LPS), heat, and LPS + heat were identified. The Fayoumi is more disease resistant and heat tolerant than the Leghorn line. Nitric Oxide (NO) production, phagocytic ability, MHC II surface expression and mRNA expression were measured. NO was induced in BMDC from both lines in response to LPS and LPS + heat stimulation; Fayoumi produced more NO with LPS treatment. Fayoumi had higher phagocytic ability and MHC II surface expression. Gene expression for the heat-related genes BAG3, HSP25, HSPA2, and HSPH1 was strongly induced with heat and few differences existed between lines. Expression for the immune-related genes CCL4, CCL5, CD40, GM-CSF, IFN-γ, IL-10, IL-12β, IL-1β, IL-6, IL-8, and iNOS was highly induced in response to LPS and different between lines. This research contributes to the sparse knowledge of genetic differences in chicken BMDC biology and function.