cDNA Immunization of Mice with Human Thyroglobulin Generates Both Humoral and T Cell Responses: A Novel Model of Thyroid Autoimmunity

Thyroglobulin (Tg) represents one of the largest known self-antigens involved in autoimmunity. Numerous studies have implicated it in triggering and perpetuating the autoimmune response in autoimmune thyroid diseases (AITD). Indeed, traditional models of autoimmune thyroid disease, experimental autoimmune thyroiditis (EAT), are generated by immunizing mice with thyroglobulin protein in conjunction with an adjuvant, or by high repeated doses of Tg alone, without adjuvant. These extant models are limited in their experimental flexibility, i.e. the ability to make modifications to the Tg used in immunizations. In this study, we have immunized mice with a plasmid cDNA encoding the full-length human Tg (hTG) protein, in order to generate a model of Hashimoto's thyroiditis which is closer to the human disease and does not require adjuvants to breakdown tolerance. Human thyroglobulin cDNA was injected and subsequently electroporated into skeletal muscle using a square wave generator. Following hTg cDNA immunizations, the mice developed both B and T cell responses to Tg, albeit with no evidence of lymphocytic infiltration of the thyroid. This novel model will afford investigators the means to test various hypotheses which were unavailable with the previous EAT models, specifically the effects of hTg sequence variations on the induction of thyroiditis

Naturally occurring mutations in the thyroglobulin gene

Thyroglobulin (Tg) is a large glycoprotein dimer secreted into the follicular lumen. It serves as the matrix for the synthesis of thyroxine (T 4) and triiodothyronine (T3), and the storage of thyroid hormone and iodide. In response to demand for thyroid hormone secretion, Tg is internalized into the follicular cell and digested in lysosomes. Subsequently, the thyronines T4 (approximately 80%) and T3 (approximately 20%) are released into the blood stream. Biallelic mutations in the Tg gene have been identified in several animal species and human patients presenting with goiter and overt or compensated hypothyroidism. In untreated patients, goiters are often remarkably large and display continuous growth. In most instances, the affected individuals have related parents and are homozygous for inactivating mutations in the Tg gene. More rarely, compound heterozygous mutations lead to a loss of function of both alleles. Molecular analyses indicate that at least some of these alterations result in a secretory defect and an endoplasmic reticulum storage disease (ERSD). This review discusses the nature and consequences of naturally occurring Tg gene mutations in humans and several animal species. Recent recommendations for the nomenclature of mutations have led to different numbering systems, an aspect that is discussed in order to clarify discrepancies between different publications.Fil: Vono-Toniolo, Jussara. Universidade de Sao Paulo; Brasil. Northwestern University; Estados UnidosFil: Rivolta, Carina Marcela. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Ciencias Biológicas. Cátedra de Biología Celular y Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Inmunología, Genética y Metabolismo. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Inmunología, Genética y Metabolismo; ArgentinaFil: Targovnik, Hector Manuel. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Ciencias Biológicas. Cátedra de Biología Celular y Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Inmunología, Genética y Metabolismo. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Inmunología, Genética y Metabolismo; ArgentinaFil: Medeiros-Neto, Geraldo. Universidade de Sao Paulo; BrasilFil: Kopp, Peter. Northwestern University; Estados Unido

T cell stimulation.

<p>Splenocytes isolated from immunized (2–3 months after the final boost) and non-immunized C3H/HeNmice were incubated with increasing concentrations of hTg protein for 72 hrs. Splenocytes from hTg immunized/electroporated mice showed significantly increased proliferation, compared to similarly challenged splenocytes from non-immunized mice. The x axis represents the concentration (µg/ml) of hTg which was used to stimulate the splenocytes. All T-cell proliferation experiments were performed in triplicates. Data, which were initially recorded as cpm, were subsequently expressed relative to splenocytes which did not receive thyroglobulin, are shown as mean + SEM.</p

IgG subtypes or anti-mTg antibodies produced by hTg immunized/electroporated mice.

<p>Three, 8-week old C3H/HeN mice received hTg immunization/electroporation while 4 mice received an empty pcDNA plasmid electroporation (denoted by ‘**’). Anti-mouse Tg antibodies of the IgG2a subclass were the most prevalent isotype, followed by IgG1. There were very low levels of anti-mTg antibodies of the IgG2b class. All ELISA experiments were performed in triplicates. Data are presented as mean + SEM.</p

Effects of different means of cDNA delivery on anti-Tg antibody response.

<p>Shown is an ELISA measuring total IgG (all subclasses), directed against murine thyroglobulin (Tg). The results demonstrate a significant anti-Tg response in mice immunized with hTg cDNA. For this analysis, we compared 2 non-immunized mice, the 2 best responders, out of a group of 6 that were immunized with hTg without electroporation, and 6 hTg cDNA immunized/electroporated mice. Relative to directly immunized mice, the use of an electric current (6 pulses total, 60 V/cm, and 50 ms pulse length, with a 200 ms respite between pulses) caused a significant increase in the level of anti-Tg antibodies (p = 0.037). All readings were performed in triplicate. Data are shown as mean + standard error of the mean (SEM). Animals immunized with empty vector (pCDNA3.1+) exhibited no response (data not shown) to mouse Tg.</p

IgG subtypes or anti-mTg antibodies produced by C3H/HeN mice induced with classical EAT by immunizing with hTg protein and adjuvant.

<p>Three, 8-week old C3H/HeN mice were intravenously immunized with 50 µg of hTg in 100 µl of PBS into the tail vein. 2–3 hours later, 20 µl of LPS was injected into the tail vein. Three immunizations, spaced two weeks apart, were administered. Animals were sacrificed two weeks after the third and final round of immunization. At sacrifice, sera were collected and were analyzed for the presence of anti-mTg and the specific isotype of the antibodies. All ELISA experiments were performed in triplicates. Data are shown as mean + SEM.</p

Th1 nature of the Cytokine Profile.

<p>Splenocytes were isolated from immunized/electroporated (2–3 months after the final boost) and non-immunized mice. 2×10⁵ cells were challenged with 0, 5, or 20 µg/ml of human thyroglobulin. Supernatants were collected and were analyzed for the presence of cytokines. hTg-treated splenocytes from immunized/electroporated animals secreted significantly higher levels of interferon-gamma than controls. Mice immunized with empty vector showed no differences compared to the non-immunized controls (data not shown). All readings were performed in triplicate. Data are shown as mean + SEM.</p

The p.A2215D thyroglobulin gene mutation leads to deficient synthesis and secretion of the mutated protein and congenital hypothyroidism with wide phenotype variation

Context: Thyroglobulin (TG) is a large glycoprotein and functions as a matrix for thyroid hormone synthesis. TG gene mutations give rise to goitrous congenital hypothyroidism (CH) with considerable phenotype variation. Objectives: The aim of the study was to report the genetic screening of 15 patients with CH due to TG gene mutations and to perform functional analysis of the p.A2215D mutation. Design: Clinical evaluation and DNA sequencing of the TG gene were performed in all patients. TG expression was analyzed in the goitrous tissue of one patient. Human cells were transfected with expression vectors containing mutated and wild-type human TG cDNA. Results: All patients had an absent rise of serum TG after stimulation with recombinant human TSH. Sequence analysis revealed three previously described mutations (p.A2215D, p.R277X, and g.IVS30+1G>T), and two novel mutations (p.Q2142X and g.IVS46-1G>A). Two known (g.IVS30+1G/p.A2215D and p.A2215D/p.R277X) and one novel (p.R277X/g.IVS46-1G>A) compound heterozygous constellations were also identified. Functional analysis indicated deficiency in TG synthesis, reduction of TG secretion, and retention of the mutant TG within the cell, leading to an endoplasmic reticulum storage disease, whereas small amounts of mutant TG were still secreted within the cell system. Conclusion: All studied patients were either homozygous or heterozygous for TG gene mutations. Two novel mutations have been detected, and we show that TG mutation p.A2215D promotes the retention of TG within the endoplasmic reticulum and reduces TG synthesis and secretion, causing mild hypothyroidism. In the presence of sufficient iodine supply, some patients with TG mutations are able to compensate the impaired hormonogenesis and generate thyroid hormone.Fil: Pardo, Viviane. Universidade de Sao Paulo; BrasilFil: Vono Toniolo, Jussara. Universidade de Sao Paulo; BrasilFil: Rubio, Ileana G. S.. Universidade de Sao Paulo; BrasilFil: Knobel, Meyer. Universidade de Sao Paulo; BrasilFil: Possato, Roberta F.. Universidade de Sao Paulo; BrasilFil: Targovnik, Hector Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Inmunología, Genética y Metabolismo. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Inmunología, Genética y Metabolismo; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; ArgentinaFil: Kopp, Peter. Northwestern University; Estados UnidosFil: Medeiros Neto, Geraldo. Universidade de Sao Paulo; Brasi