Insights into the pathophysiology of Hashimoto thyroiditis: Assessing thyroid Reserve capacity & the role of the Gut micrObiome in thyroid hormone metaboliSm

Autoimmune hypothyroidism is a T-cell mediated autoimmune disease which is
characterized by chronic lymphocytic infiltration of the thyroid gland leading
to progressive and irreversible destruction of thyrocytes. This disease can
either be clinical evident with an absolute hormone deficiency - Hashimoto*s
thyroiditis (HT) - or subclinical, which is defined as an elevated serum TSH
level in combination with the presence of anti-TPO antibodies and a serum free
thyroxine (FT4) level that is within normal range. Despite sufficient serum
thyroid hormone levels, subclinical hypothyroidism is associated with an
increased risk of hypothyroid symptoms and cardiovascular events, particularly
in subjects with a TSH level > 10 mU/L. In this respect, subclinical
hypothyroidism can be seen as an early stage of HT with still sufficient
thyrocytes left to maintain serum thyroid hormone levels.
Hashimoto thyroiditis is the most common endocrine autoimmune disease of which
the prevalence has increased over the past years, from 0.4% to 2.9% during 10
years in the Netherlands alone. Considering this steep increase, novel
preventative and/or therapeutic opportunities are greatly needed as the current
treatment consists of continuous hormone treatment rather than affecting
disease progression.

Need for a dynamic thyroid function test to test thyroid reserve capacity
To test the effect of such a potential new intervention in pathophysiology of
autoimmune hypothyroidism (i.e. reduction of the ongoing gradual thyroid
failure), a standardized outcome measure which assesses the residual thyroid
capacity is needed. To date, the current modern assays for thyroid hormone
levels (serum TSH, FT4 and FT3) represent a static functional test and thus do
not reflect thyroid reserve. Thyroid response upon administration of
recombinant human TSH (rh-TSH; Thyrogen©) could help to determine residual
thyroid function. Such a dynamic function test has already been used in current
clinical practice in the follow-up of patients with differentiated thyroid
carcinoma. Moreover, recent trials showed that a single dose of 0.9mg Thyrogen©
in healthy individuals without thyroid dysfunction leads to a temporary
significant increase in serum thyroid hormone (TH) levels without severe
adverse events. As far we know, yet no published clinical study exists
validating a rh-TSH driven dynamic stimulation test in autoimmune hypothyroid
patients. Thus, the principal aim of this study is to validate a dynamic
thyroid function test to assess thyroid reserve capacity, by measuring maximal
serum FT4 and FT3 response upon an single intramuscular administration of 0.9mg
rh-TSH (Thyrogen©). Therefore, we will include 10 patients in the early stage
of Hashimoto*s thyroiditis (subclinical autoimmune hypothyroidism) and 10
healthy volunteers as a control. We will perform this test twice to assess the
reproducibility.

Gut microbiota involved in thyroid hormone metabolism
The thyroid gland produces both the prohormone thyroxine (T4) and the
biologically active triidothyronine (T3). When secreted into the circulation
they are either bound to proteins (albumin, thyroid binding globulin or
transthyretin) or unbound (free T4 or free T3). Circulating thyroid hormones
(TH) can be metabolized by a number of different pathways of which deiodination
and conjugation of the phenolic hydroxyl group with either sulfate or
glucuronide are the major pathways.
About 20% of daily T4 production appears in feces, predominantly via biliary
excretion of glucuronide conjugates. Conjugation of iodothyronines enhances the
biliary and/or urinary clearance, however, these pathways are reversible.
Specifically, after the rapid excretion of iodothyronine glucuronides (T4G and
T3G) in the bile, these conjugates can be hydrolyzed by intestinal obligatory
anaerobes by the bacterial enzyme *-glucuronidase. This process promotes the
intestinal reabsorption of free thyroid hormone into the enterohepatic
circulation were they can be reutilized. These findings suggest that
glucuronidated iodothyronines may serve as an intestinal thyroid hormone
reserve, which may prevent fluctuation of serum TH levels. Evidence to support
this metabolic symbiosis between host and gut microbiome is demonstrated in
previous studies, showing that in absence of intestinal bacteria (using fecal
suspensions from germfree rats as well as from orally decontaminated rats) only
very little of the glucuronidated iodothyronines were hydrolyzed, resulting in
lower fecal T3 excretion when compared to fecal samples of conventional raised
rats. However, these mentioned findings are mostly identified using rat models
or in-vitro experiments and warrants to be validated in humans.

More recently, two papers have evaluated the gut microbiome composition in
fecal samples of HT patients. Both studies found an association between
alterations in the gut microbiome and HT disease compared to matched healthy
controls. However, it remains unknown whether dysbiosis in those patients also
leads to an alteration in the abovementioned metabolic pathways of
iodothyronines and subsequent enterohepatic circulation. A better understanding
of the (patho)physiological pathway of the gut microbiome involvement in
thyroid hormone metabolism in autoimmune hypothyroidism is needed in search for
possible effective therapeutic options. Therefore, the second aim of this study
is to evaluate the effect of the gut microbiome on thyroid hormone metabolism
in subclinical autoimmune hypothyroid subjects.
Moreover, using a short course of antibiotics to temporarily deplete the gut
microbiota will help to discern if indeed gut microbiota are involved in the
enterohepatic circulation of thyroid hormones in human (as seen by the
decreased fecal T3 and T4 excretion after the antibiotic course). To do so, we
will apply a methodology already published and validated for other studies,
investigating the role of the microbiome in production of metabolites (e.g.
TMAO, ImP)

In conclusion, an altered intestinal microbiota composition has been implicated
to play an important role in (human) metabolism, as well as in autoimmune
diseases. Furthermore, recent studies have shown that HT patients display
changes in the pro- and anti-inflammatory phenotype of immune circulating cells
as (PBMCs) as well as in thyroid tissue. It has been demonstrated that thyroid
tissue of HT subjects highly expressed CD20+ B-cells, CD4+ and CD8+ T-cells
and, surprisingly, FoxP3+ T-regulatory cells, whereas this was not seen in
thyroid tissue of healthy subjects. Therefore, a parallel objective of this
study is to assess the immunological status (T cells, B cells and cytokines in
peripheral blood) of subclinical autoimmune hypothyroid subjects, using imaging
mass cytometry.

Primary objective: To assess and compare the difference in thyroid gland
secretion capacity by measuring maximal FT4 and FT3 response upon intramuscular
administration of 0.9mg Thyrogen assessed by AUC0-48hours in SCT subjects and
healthy controls.

Secondary objectives:
Secondary objectives will be influence of gut microbiome on thyroid hormone
metabolism and effect of thyroid hormone stimulation on inflammatory status.

Gut microbiome composition:
- To assess and compare changes in the gut microbiome composition between SCT
subjects and healthy controls at baseline.
- In addition, microbiota composition will be determined and compared upon
thyroid hormone stimulation and before and after short term antibiotics
course.

Thyroid hormone metabolism:
- To assess and compare changes of fecal excretion of T4 and T3 in SCT subjects
and healthy controls before and after thyroid hormone stimulation.
- In addition, we will determine changes of fecal excretion of T4 and T3 before
and after short term antibiotics course.

Immunologic parameters:
- To assess and compare changes in immunological status, based on FACS on
peripheral blood mononuclear cells (Th1, Th2, Th17, Treg, B cells), cytokines
and markers of thyroid autoimmunity (anti-TPO antibodies) in SCT subjects as
compared to healthy controls at 0h, 5h and 48h after thyroid stimulation and
before and after a short term antibiotics course.

Intestinal transit time
- Evaluate the differences of intestinal transit time as assessed by radiopaque
makers between SCT subjects and healthy controls.

This is a prospective, single-centre intervention study. The study duration is
1.5 month (start seven days before intervention and then lasts 32 days) during
which the faecal sample collection and the dietary questionnaire can be carried
out by the subjects at home. Subjects are requested to visit the AMC 7 times,
including the screening visit. They will spend a total of 20,5h in the AMC
(table 1 and figure 5).
The study is designed to validate a dynamic thyroid function test (TFT) and
evaluate thyroid hormone metabolism after stimulation of the thyroid gland and
link the expected changes in metabolism to the gut microbiome. Therefore, we
will include 10 subclinical autoimmune hypothyroid subjects and 10 healthy
controls. For the dynamic TFT serum thyroid hormone levels will be drawn at
timed intervals after intramuscular administration of 0.9mg Thyrogen. To test
validity of this dynamic TFT, this same regimen is repeated after two weeks.
In discern if gut microbiota are indeed involved in the enterohepatic
circulation of thyroid hormones, the third dynamic TFT test will be preceded by
a short course of an oral antibiotics cocktail to suppress the gut microbiome.
The seven-day antibiotics regimen will consist of metronidazole (500 mg twice
daily) plus ciprofloxacin (500 mg once daily), plus oral vancomycine (500mg
four times daily). In total a volume of 175 ml blood will be drawn per subject
during the complete study period of 3 months.

3x 0.9mg Thyrogen injection intramuscular.

The proposed administration of 0.9mg Thyrogen has previously been shown to be
safe in a clinical study. The recorded adverse events were related to
hyperfunction of the thyroid gland (tachycardia, increased appetite,
restlessness, headache and nausea) and symptoms related to possible thyroid
growth (visual enlargement of the thyroid gland or tenderness). However, these
symptoms and discomfort were of short duration and self-limiting, appearing
between 4h and 48h after injection. Importantly, the subjects of this study
will be monitored during this timeframe with the following visit 48h after
administration. Furthermore, inclusion criteria were chosen to select only
relatively healthy patients without prior cardiovascular disease to limit any
potential risk.

The placing of the intravenous cannula in our study can be an unpleasant
experience for the subjects and may result in (self-limiting) bruising.

Transit-Pellets (radiopaque markers) are used for determing the internal
transit time. On day seven, an abdominal X-ray is made to count the markers (if
still present in digestive tract). In total 3x 0.7mSv per x-ray amounts to
2.1mSv in a year that equals the yearly average background radiation exposure).

For the antibiotics, the most commonly reported adverse reactions are nausea
and diarrhoea. Furthermore, vomiting, headache, taste disorders, allergic
reactions, mycotic superinfections or rupture of the achilles tendon are
rarely mentioned.