Microbiota e TH

Thyroid
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© Mary Ann Liebert, Inc.
DOI: 10.1089/thy.2017.0395
1
Alterations of the gut microbiota in Hashimoto’s thyroiditis
patients
This paper has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
Fuya Zhao,1,+ Jing Feng,1,+ Jun Li,1,+ Lei Zhao,1 Yang Liu,1 Huinan Chen,1 Ye Jin,1 Biqiang
Zhu,1 and Yunwei Wei 2,*
1
M.D, Department of Oncological and Endoscopic Surgery, The First Affiliated Hospital of
Harbin Medical University, Harbin, Heilongjiang 150001, People’s Republic of China, E-mail:
[email protected], [email protected], [email protected], [email protected],
Downloaded by University of Rochester package NERL from online.liebertpub.com at 01/13/18. For personal use only.
Alterations of the gut microbiota in Hashimoto’s thyroiditis patients (DOI: 10.1089/thy.2017.0395)
[email protected], [email protected], [email protected],

[email protected].
2
Ph.D., Department of Oncological and Endoscopic Surgery, The First Affiliated Hospital of
Harbin Medical University, Harbin, Heilongjiang 150001, People’s Republic of China, E-mail:
[email protected].
*
Correspondence and requests for materials should be addressed to Yunwei Wei.
Thyroid
+
These authors contributed equally to this work.
Running title: Hashimoto’s thyroiditis and gut microbiota dysbiosis
Keywords: Hashimoto’s thyroiditis, Gut microbiota, Dysbiosis, Clinical parameters,

Biomarker
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Abstract
Background: Hashimoto’s thyroiditis (HT) is an organ-specific autoimmune disease in

which both genetic predisposition and environmental factors serve as disease triggers.
Many studies have indicated that alterations in the gut microbiota are important
environmental factors in the development of inflammatory and autoimmune diseases. We
systematically performed a comparative analysis of the gut microbiota in HT patients and
healthy controls.
Methods: We first conducted a cross-sectional study of 28 HT patients and 16 matched

healthy controls. Fecal samples were collected, and microbiota were analyzed using 16S
ribosomal RNA gene sequencing. Second, an independent cohort of 22 HT patients and 11
healthy controls was used to evaluate the diagnostic potential of the selected biomarkers.
Results: Similar levels of bacterial richness and diversity were found in the gut microbiota
of HT patients and healthy controls (p = 0.11). A detailed fecal microbiota Mann-Whitney
Thyroid
U-test (Q value < 0.05) revealed that the abundance levels of Blautia, Roseburia,
Ruminococcus_torques_group, Romboutsia, Dorea, Fusicatenibacter and
Eubacterium_hallii_group genera were increased in HT patients, whereas the abundance
levels of Fecalibacterium, Bacteroides, Prevotella_9 and Lachnoclostridium genera were
decreased. A correlation matrix based on the Spearman correlation distance confirmed
correlations among 7 clinical parameters. Additionally, the LEfSe method showed
significant differences in 27 genera between the two groups that were strongly correlated
with clinical parameters. We used the linear discriminant analysis (LDA) value to select the
first 10 species from the 27 different genera as biomarkers, achieving area under the curve
(AUC) values of 0.91 and 0.88 for exploration and validation data, respectively.
Conclusions: Characterization of the gut microbiota in HT patients confirmed that HT

patients have altered gut microbiota and that gut microbiota are correlated with clinical
parameters, suggesting that microbiome composition data could be used for disease
diagnosis. Further investigation is required to better understand the role of the gut
microbiota in the pathogenesis of HT.
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Introduction
Hashimoto’s thyroiditis (HT), also known as chronic lymphocytic thyroiditis, is a

common organ-specific autoimmune disorder characterized by the production of

autoantibodies against thyroid peroxidase (TPO) and thyroglobulin (TG) and infiltration of
the thyroid gland by inflammatory cells, which are often followed by hypothyroidism due
to the destruction of thyroid follicles and eventual fibrous replacement of parenchymal
tissue (1). The clinical diagnosis of HT depends on both physical (typically ultrasonography)
and biochemical abnormalities and serological assessments of autoantibodies (TPO-Ab or

TG-Ab) (2). Although the exact etiology remains unknown, HT is thought to arise from
interactions between genetic susceptibility factors, epigenetic effects and various
environmental factors (3). Studies of twins have provided epidemiological evidence for a
genetic susceptibility to HT. The concordance rate was 55% for monozygotic twins with HT,
but it was 0% for dizygotic twins with HT (4). However, even in identical twins, the
concordance rate was only approximately 50%, emphasizing that other important factors,
Thyroid
such as the environment, including excessive iodine intake, selenium deficiency,

Helicobacter pylori infection, viral infection (hepatitis C virus, human parvovirus B19 and
enteroviruses), vitamin D deficiency, hygiene, and new treatment modalities and chemical
agents, may have important roles in the pathogenesis of HT. Additional unmodifiable
predisposing factors include stress, climate, age and sex (5-7). The potential role of gut
microbiota as an environmental factor that could alter the health status of a host has
recently drawn considerable attention. Emerging evidence suggests a connection between
the gut microbiota and various autoimmune diseases, including systemic lupus
erythematosus (SLE) (8), type 1 diabetes (T1D) (9), rheumatoid arthritis (RA) (10),
inflammatory bowel disease (IBD) (11), psoriasis (12), multiple sclerosis (MS) (13), celiac
disease (14), Bechet's disease (15), Graves' disease (GD) and Graves' orbitopathy (GO) (16-
18).
However, few studies have addressed the link between the gut microbiota and HT,
and these studies have only provided indirect or weak evidence. First, a previous study
indicated that many interrelationships exist between the thyroid gland and the
gastrointestinal tract. Similarities in embryology, phylogeny, and function persist
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throughout adult life even after fragmentation of the primitive thyroglossal duct (19).
Therefore, we hypothesized that thyroid disorders may be associated with alterations of
the gut microbiota. Second, molecular mimicry mechanisms provide the most reasonable
explanation of the role of the gut microbiota in provoking autoimmune disease, i.e., the
emergence of autoreactive clones of T and B lymphocytes as a result of a cross-immune
response to homologous bacterial or viral antigens (20,21). It was recently found that
components of the cells of Bifidobacterium bifidum 791, Bifidobacterium adolescentis 94
BIM, Bifidobacterium longum B379M and Lactobacillus plantarum B-01 selectively bind
human antibodies (TPO-Ab and TG-Ab) and compete with natural antigens (22). Third,
some authors have proposed that disruption of the mucosal barrier exposes submucosal
immune cells to bacterial and dietary antigens and to self-antigens, leading to unfavorable
immune activation or failure of the tolerance reaction with the consequent development
of autoimmune diseases (23,24). In accordance with this hypothesis, morphological
changes in gut epithelial cells, increased intestinal permeability, and intraepithelial
lymphocyte infiltration have been demonstrated in type I diabetes patients and in animal
Thyroid
models (25,26). Interestingly, similar changes have been detected in patients with HT (27),
suggesting a pathogenic role of a leaky gut barrier in the development of HT. Finally, in
non-obese diabetic mice with thyroiditis, polarization toward a prevalent Th1 or Th17
pathway has been reported (28,29). Activation of Toll-like receptors in the development of
thyroiditis (30) and a protective inhibitory effect of Treg cells (31) have also been reported
in this model. However, whether a similar mechanism (i.e., CD4+ Th pathway imbalances)
initiates autoimmune thyroiditis in humans requires further investigation (32).
Therefore, we hypothesized that HT patients have gut microbial dysbiosis that

influences HT development. To test our hypothesis, we analyzed the gut microbiota
composition in euthyroid HT patients. The results showed that HT patients have a distinct
microbiota community profile from that of healthy controls.
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Materials and Methods
Ethics statement
The protocols used in this study were approved by the Ethics Committee of the First
Affiliated Hospital of Harbin Medical University (China). All subjects were informed of the
nature of the study and were asked to provide written informed consent. The procedures
were conducted in accordance with approved guidelines.
Study cohort and recruitment of subjects

There were two study groups. We first conducted a cross-sectional study (exploration
cohort) of 28 HT patients and 16 matched healthy controls. Second, an independent
cohort (validation cohort) composed of 22 HT patients and 11 matched healthy controls
was used to evaluate the diagnostic potential of selected biomarkers. Seventy-seven
participants were enrolled in our study from November 2016 to April 2017, including 50 HT
patients recruited from the outpatient departments of endocrinology at the First Affiliated
Thyroid
Hospital of Harbin Medical University and 27 healthy controls recruited from the health
screening center. The healthy volunteers were matched with the HT groups in terms of
age, sex, and BMI. All HT patients were diagnosed and underwent disease verification by
an endocrinologist based on their histories, physical examination, ultrasound and
biochemistry (2). The inclusion criteria for HT patients were as follows: aged 18 to 65
years and the presence of euthyroidism (normal FT3, FT4, and TSH plasma levels without
hormonal therapy). The following exclusion criteria were applied to all groups: pregnancy;
lactation; cigarette smoking; alcohol addiction; hypertension; diabetes mellitus; lipid
dysregulation; BMI > 27; recent (< 3 months prior) use of antibiotics, probiotics, prebiotics,
symbiotics, hormonal medication, laxatives, proton pump inhibitors, insulin sensitizers or
Chinese herbal medicine; a known history of disease with an autoimmune component,
such as MS, rheumatoid arthritis, IBS, or IBD; and a history of malignancy or any
gastrointestinal tract surgery (e.g., gastrectomy, bariatric surgery, colectomy, ileectomy,
cholecystectomy or appendectomy).
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Sample collection
All subjects were examined in the morning after an overnight fast (≥ 8 h). Peripheral
blood (6 mL) was collected from all subjects and stored in EDTA tubes at 4°C for routine
blood, thyroid function and thyroid autoantibody examinations. In addition, all subjects
were provided with Commode Specimen Collection Kits for stool collection. Fecal samples
were collected by the patients using disposable sterile forceps in the morning, when the
patients had an empty stomach. Basic information, such as acquisition time and patient
name, was recorded on the sample collection box. If the subjects were unable to provide a
stool sample during their stay at the hospital outpatient clinic, they were instructed to
collect the sample at home in accordance with the above methods and to send it to our
laboratory in an ice pack within two hours. Upon collection, each fecal sample was
immediately divided into aliquots, frozen on dry ice, and stored at −80°C un l DNA
extraction.
Thyroid function and thyroid autoantibodies tests

Thyroid
Serum thyrotropin (TSH), free thyroxine (FT4), and free triiodothyronine (FT3) levels
were measured by chemiluminescent immunoassays (Abbott Diagnostics, Tokyo, Japan).
Reference ranges were defined as follows: TSH 0.35–4.94 µIU/L; FT4 0.70–1.48 ng/dl; and
FT3 1.71–3.71 pg/ml. TgAb and TPOAb were measured using chemiluminescent
immunoassays (Beckman Coulter, Fullerton, CA, USA). Reference ranges were defined as
follows: TPO-Ab 0.00-5.61 IU/mL; TG-Ab 0.00-4.11 IU/mL.
gDNA extraction
DNA extraction was performed within one month after sample collection. Bacterial
DNA was extracted from the fecal samples at Novogene Bioinformatics Technology Co.,
Ltd. using a TIANGEN kit according to the manufacturer’s recommendations. DNA
concentration and purity were monitored on 1% agarose gels. After determining the
concentration, DNA was diluted to 1 ng/μL using sterile water. The extracted DNA was
stored at −20°C.
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Amplicon generation and purification
The bacterial genomic DNA was amplified with primers 341F (CCTAYGGGRBGCASCAG)
and 806R (GGACTACNNGGGTATC- TAAT) specific to the V3-V4 hypervariable regions of the
16S rRNA gene (33). All PCR reactions were carried out with Phusion® High-Fidelity PCR
Master Mix (New England Biolabs). An equal volume of 1X loading buffer (containing SYB
green) was mixed with the PCR products and subjected to electrophoresis on 2% agarose
gels for detection. Samples with a bright, primary band between 400-450 bp were chosen
for further experiments. The products of the same sample were combined and subjected
to electrophoresis. DNA of the correct size was purified using a Gel Extraction Kit (Qiagen,
Germany) and quantified using a Qubit instrument (Life Technologies).
Library preparation and sequencing
Sequencing libraries were generated using a TruSeq® DNA PCR-Free Sample

Preparation Kit (Illumina, USA) following the manufacturer's recommendations, and index
Thyroid
codes were added. Library quality was assessed using a Qubit@ 2.0 Fluorometer (Thermo
Scientific) and an Agilent Bioanalyzer 2100 system. Finally, the library was sequenced on
an IlluminaHiSeq2500 platform, and 250-bp paired-end reads were generated.
Paired-end reads assembly and quality control
Paired-end reads were assigned to samples based on their unique barcode and were
truncated by cleaving the barcode and primer sequence. Paired-end reads were merged
using FLASH (V1.2.7), a rapid and highly accurate analysis tool designed to merge paired-
end reads when at least some of the reads overlap the reads generated from the opposite
end of the same DNA fragment; the splicing sequences were termed raw tags.
Quality filtering of the raw tags was performed under specific filtering conditions to
obtain high-quality clean tags according to the QIIME (V1.7.0) quality control process. The
tags were compared with a reference database (the Gold database) using the UCHIME
algorithm to detect chimaera sequences, and the chimaera sequences were then removed.
The effective tags were finally obtained.
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OTU cluster and species annotation
Sequence analysis was performed using Uparse software (Uparse v7.0.1001).

Sequences with ≥ 97% similarity were assigned to the same OTUs. Representative
sequences for each OTU were screened for further annotation. For each representative
sequence, the SILVA128/16S database was used based on the RDP classifier (Version 2.2)
algorithm to annotate taxonomic information. To study the phylogenetic relationships
between different OTUs and differences in dominant species in different samples (groups),
multiple sequence alignment was conducted using MUSCLE software (Version 3.8.31). OTU
abundance information was normalized using the sequence number corresponding to the
sample with the fewest sequences.
Statistical analyses
The analysis of clinical parameters, F/B ratio and Pearson correlation distance results
was performed using Statistical Package for the Social Sciences (SPSS), version 19.0 (SPSS
Thyroid
Inc., 2010 Chicago, IL, USA). Alpha diversity was applied to analyze the complexity of
species diversity in each sample based on 6 indices, including Observed-species, Chao1,
Shannon, Simpson, ACE, and Good’s coverage. These indices were calculated for our
samples using QIIME (Version 1.7.0) based on the rarefied OTU counts and were displayed
using R software (Version 2.15.3). A beta diversity analysis was used to evaluate
differences in the species complexity between samples, and beta diversity-weighted
UniFrac was calculated using QIIME software (Version 1.7.0) based on the rarefied OTU
counts. A Principal Coordinate Analysis (PCoA) was performed to obtain the principal
coordinates and to visualize complex, multidimensional data. The results of the PCoA were
displayed using the WGCNA package, stats package and ggplot2 package in R software
(Version 2.15.3). Differences between the two groups were tested based on a distance
matrix using the nonparametric multivariate analysis test (ANOSIM, used to examine
whether differences between groups were significantly greater than differences within
groups) included in R’s Vegan package.
The microbiota features differentiating the fecal microbiota were characterized

using the LDA effect size (LEfSe) method for biomarker discovery, which emphasizes both
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statistical significance and biological relevance (34). Based on a normalized relative
abundance matrix, LEfSe uses the Kruskal-Wallis rank-sum test to detect features with
significantly different abundance levels between assigned taxa and performs an LDA to
estimate the effect size of each feature. An alpha significance level of 0.05 and an effect
size threshold of 3 were used for all biomarkers discussed in this study. A differential
abundance analysis was performed using the Wilcoxon rank-sum test at the phylum,
family, and genus levels. Microbiome features of healthy controls were compared to those
of patients with HT using Metastats based on the P value and the false discovery rate (Q
value) for non-normal distributions. Only taxa with average abundance levels >1%, P
values < 0.05, and Q values < 0.05 were considered significant (35). Correlations between
variables were computed using the Spearman rank correlation. Statistical analyses were
performed using the SPSS Data Analysis Program (version 16.0; SPSS Inc., Chicago, IL, USA).
To evaluate the discriminatory ability of the prediction model, operating characteristic
curves (receiving operational curve, ROC) were constructed and the area under curve
(AUC) values were calculated.
Thyroid
Results
Study population
All subjects (n = 77) were of Han nationality and were born in northeastern China. The
fecal samples of 50 HT patients and 27 healthy controls were sequenced and analyzed.
Because of traditional customs and the cold climate, typical meals include foods that are
rich in saturated fat and salt, such as pork fat, blood sausage and mutton. The
demographics and clinical parameters of the subjects are summarized in Table 1.
The gut microbiota of HT patients differs from that of healthy controls
To identify whether HT was associated with a change in microbiota diversity, the fecal
samples of HT patients and healthy controls were subjected to pyrosequencing and
statistical analysis. A summary of the microbiota diversity is shown in Table 2, and the
detailed characteristics of each sample are shown in Table S1. A total of 7,182,787 usable
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sequences were obtained from all samples using the IlluminaHiSeq2500 platform (Illumina,
San Diego, CA, USA). Of these, 3,149,257 high-quality sequences were selected, yielding an
average of 71,574 sequences per sample. A total of 784 operational taxonomic units
(OTUs) were delineated at a 97% similarity level. Good’s coverage for the 2 groups was
greater than 99.5%, indicating a satisfactory sequencing depth of the gut microbiota.
Based on the OTU analysis results, the rank-abundance curves for the bacterial
communities of the HT patients and the healthy controls exhibited similar patterns (Fig.
1A). In the analysis of alpha diversity, the Chao1 index considers only species richness.
However, the Shannon diversity index considers both species richness and evenness. The
Wilcoxon rank-sum test results demonstrated that the gut microbiota richness and
diversity of the HT patients were higher than those of the healthy controls, although the
differences were not significant (P > 0.05). Other estimators of community diversity are
shown in Table 2. Rarefaction curve analysis estimates showed that the gut microbiota in
HT patients exhibited species richness (observed OTU number) similar to that of the
healthy controls (Fig. 1B). There was also a trend toward higher overall diversity (Shannon
Thyroid
index) in HT patients compared to healthy controls (Fig. S1). Additionally, a Venn diagram
showing the overlapping OTU data of the two groups was constructed to better evaluate
their shared richness. This analysis revealed that 660 of the 783 OTUs accounting for the
total richness were common to all samples. Therefore, approximately 81 and 42 of the
OTUs were identified in the samples of HT patients and healthy controls, respectively (Fig.
S2).
A taxon-dependent analysis using the Ribosomal Database Project (RDP) classifier was
conducted to describe the composition of the fecal microbiota in HT patients and healthy
controls. Compared with healthy controls, the proportions of Firmicutes and
Actinobacteria were increased and the proportions of Bacteroidetes and Proteobacteria
were decreased in patients with HT (Fig. 1C). At the family and genus levels, the microbiota
composition of the HT patients was also altered compared with that of the healthy
controls (Fig. 1D and S3). To evaluate the extent of similarity between the microbiota
communities, beta-diversity values were calculated using the weighted UniFrac method,
and a principal coordinate analysis (PCoA) was performed. Despite significant inter-
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individual variation, the gut microbiota of the HT patients and the healthy controls were
clearly separated using PCoA (Fig. 1E). The HT-patient samples also showed greater
heterogeneity, with subsets resembling the healthy-control samples. The analysis of group
similarities (ANOSIM) indicated that the differences between the HT patients and the
healthy controls were significant at the phylum and genus levels, with R values of 0.7154
and 0.6653, respectively (P < 0.001, Fig. 1F and S4). These results demonstrate that the
experimental grouping design was reliable.
The above results indicate that similar levels of bacterial richness and diversity were
found in the gut microbiota of HT patients compared with those of healthy controls,
whereas the overall structures of the gut microbiota of the HT and control groups were
significantly different.
The abundance levels of certain bacteria are associated with HT
To identify the specific bacterial taxa associated with HT, we compared the
Thyroid
compositions of the fecal microbiota of HT patients and healthy controls using the linear
discriminant analysis effect size (LEfSe) method. A cladogram representing the structures
of the fecal microbiota and the predominant bacteria in the healthy controls and HT
patients, and the largest differences in the taxa between the two communities are
compared. In total, the LEfSe analysis revealed 40 discriminative features (linear
discriminant analysis (LDA) > 3, P < 0.05, Fig. 2A) at the phylum (n = 3), family (n = 10), and
genus (n = 27) levels. Members of the Bacteroidetes bacterial taxa were enriched in the
healthy-control samples, whereas members of the Firmicutes and Synergistetes were
enriched in the HT-patient samples. Therefore, these taxa may be used as biomarkers to
discriminate HT patients. Changes in the composition of the gut microbiota in the HT-
patient samples were also explored using the Mann-Whitney U-test (a nonparametric test
for two independent groups) at different taxon levels, confining the analyses to the taxa
with average abundance levels > 1%, P values < 0.05, and false discovery rates (Q values) <
0.05. We identified 18 differentially abundant taxa (Table 3). At the phylum level, the
proportion of Firmicutes was higher in the HT-patient samples than that in the healthy-
control samples, whereas the proportion of Bacteroidetes was lower (Q value < 0.05, Fig.
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2B). At the family level, members of Lachnospiraceae were prevalent in the HT-patient
samples, whereas members of Bacteroidaceae, Prevotellaceae, Streptococcaceae and
Peptostreptococcaceae were enriched in the healthy-control samples (Q value < 0.05, Fig.
2B). At the genus level, 11 genera differed dramatically between the HT-patient samples
and the healthy-control samples. The proportions of the Bacteroides, Fecalibacterium,
Prevotella_9 and Lachnoclostridium genera were decreased, whereas the proportions of
the Blautia, Ruminococcus_torques_group, Roseburia, Fusicatenibacter, Romboutsia,
Dorea and Eubacterium_hallii_group genera were increased in the HT-patient samples (Q

value < 0.05, Fig. 2B).
These data indicate that the HT patients had different abundance levels of certain
bacteria in the gut microbiota than the healthy controls. Although bacterial diversity was
not altered dramatically, the aberrant compositions of the fecal microbiota indicated gut
dysbiosis in the patients with HT. The ratio of Firmicutes/Bacteroidetes (F/B) was
significantly higher in the HT-patient samples (9.534 ± 3.207, P < 0.05; Fig. 2C) than that in
Thyroid
the healthy-control samples (3.175 ± 0.8209). A correlation matrix based on Pearson

correlation distance confirmed correlations between the F/B ratio and age and BMI, but no
significant differences (P > 0.05) were detected in the correlations between the healthy
group or the HT group (Fig. 2D, 2E, 2F, 2G).
Clinical parameters correlated with the gut microbiota
The relationships between the gut microbiota and host clinical parameters were
explored. A correlation matrix based on Spearman correlation distance confirmed
correlations among 7 clinical parameters, and 27 genera showed significant differences
between the two groups according to the LEfSe method (Fig. 2A). The results revealed
significant correlations between different genera (not including Subdoligranulum,
Alloprevotella, and Lachnoclostridium) and HT-related clinical diagnostic parameters,
including TPO-Ab and TG-Ab (P < 0.05). Eighteen genera were enriched in the HT patient
group and were positively correlated with TPO-Ab or TG-Ab, while 6 genera were enriched
in the healthy control group and exhibited opposite correlations (Fig. 3A). Additionally,
Alloprevotella was positively correlated with FT4, while Fusicatenibacter exhibited the
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opposite correlation. Romboutsia was negatively correlated with TSH. However, other
environmental factors were not significantly correlated with the gut microbiota, including
FT3, AGE and BMI (P > 0.05).
Predictive model
The LEfSe analysis results revealed significant differences (LDA > 3, P < 0.05) between
the two groups for 27 genera, and the Spearman correlation test confirmed that 24 genera
were significantly correlated with TPO-Ab or TG-Ab. Therefore, these differences in

microbiota could serve as potential novel biomarkers for non-invasive monitoring and
diagnosis of diseases. The performance of the model was assessed using ROC analysis.
Finally, we selected the first 10 species based on the LDA value from the 27 different
genera as biomarkers, achieving an AUC value of 0.91 (Fig. 3C). These species included
members of Bacteroides (LDA = 4.55), Fecalibacterium (LDA = 4.34), Prevotella_9 (LDA =
4.31), Blautia (LDA = 4.31), Eubacterium_hallii_group (LDA = 3.90),
Ruminococcus_torques_group (LDA = 3.74), Streptococcus (LDA = 3.67), Alloprevotella (LDA
Thyroid
= 3.67), Roseburia (LDA = 3.63) and Fusicatenibacter (LDA = 3.62). Identification of the
most prevalent genera in the earlier abundance analysis (Fig. 2B) indicated the robustness
of both analyses. A heat map was generated based on the abundance levels of the first 10
species from the 27 different genera. Hierarchical clustering (Euclidean distance, complete
linkage) shows that the two groups can be clearly separated (Fig. 3B), and HT-patient
samples generally clustered together (Fig. S5). Subsequently, we evaluated the
discriminatory power of the model using a validation cohort of 22 HT patients and 11
healthy controls and achieved an AUC value of 0.88 (Fig. 3D), confirming that the gut-
microbiota-based classifier can distinguish HT patients from controls.
Discussion
To date, no study has indicated a direct association between the gut microbiota and
HT. To bridge this gap, we used the 16S rRNA sequencing technique to characterize the gut
microbiota and found that HT patients exhibit a distinct gut microbiota composition from
that of healthy controls. A Spearman correlation analysis revealed correlations between
altered gut microbiota and various clinical parameters. Moreover, a prediction model was
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proposed based on the LEfSe results and achieved high AUC values for both the
exploration and the validation cohort. These results demonstrate that gut microbiota
dysbiosis may play a role in the development of HT. Notably, all HT subjects in our study
were euthyroid, whereas some previous studies revealed that changes in thyroid hormone
concentrations in hypothyroidism or hyperthyroidism (16,17) may affect the composition
of the gut microbiota. Regarding hypo- and hyperthyroidism, we were unable to reveal an
intrinsic link between HT and the gut microbiota. Therefore, only euthyroid HT patients
were included in our study. However, in future studies, analyzing the gut microbiota at
various thyroxine concentrations may provide a better understanding of the changes in the
gut microbiota at different stages of the disease.
This cross-sectional study revealed that HT patients have greater gut microbiota
richness and diversity (α-diversity) than healthy controls, although the differences did not
differ significantly. In previous studies, increased bacterial diversity was also observed in
hyperthyroid and hypothyroid patients and may be related to bacterial overgrowth in the
Thyroid
intestinal tract (17,36). The similarity of the gut microbiota between HT patients and
healthy controls was analyzed using cluster analysis (PCoA). The bacterial structures were
clustered into two distinct groups, and the HT group displayed relatively high homology,
indicating common characteristics among the HT patients. In our study, at the phylum
level, Firmicutes were more abundant in the HT patients, whereas Bacteroidetes were less
abundant. These findings are consistent with those of a previous study that reported
significant increases in Firmicutes and decreases in Bacteroidetes species in obese, irritable
bowel syndrome (IBS) and MS patients (13,37,38). To the best of our knowledge, the
Firmicutes/Bacteroidetes ratio is considered representative of health status and may
reflect eubiosis of the gastrointestinal tract. In our study, the F/B ratio was significantly
increased in HT patients compared to healthy controls (Fig. 2C), but this increase showed
no correlation with age or BMI (Fig. 2D, 2E, 2F, 2G). In addition, other studies have
indicated that the intestinal flora of IBS patients has a higher F/B ratio (36, 38). Therefore,
we think that an increased F/B ratio corresponds to HT disease status.
At the genus level, we found that the abundance levels of many genera were
decreased in HT patients according to the LEfSe results (Fig. 2A). Bacteroides,
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Faecalibacterium, Prevotella_9, Alloprevotella, Lachnoclostridium, Phascolarctobacterium,
Parabacteroides and Paraprevotella were decreased in HT patients according to the LEfSe
results. In previous studies, some species with decreased abundance levels were found to
play important roles in maintaining human health. Similarly, other studies demonstrated
that Bacteroides may efficiently ferment fibre into acetates and propionates (39) and that
Phascolarctobacterium can produce short-chain fatty acids, which are intestinal epithelial-
specific nutrients and energy components that protect the intestinal mucosal barrier and
reduce inflammation (40). Fecalibacterium may also produce butyrate, which is essential
for energy metabolism and the normal development of colonic epithelial cells, therefore
exhibiting a protective role (41). Other studies have shown that F. prausnitzii supernatant,
which contains a mixture of secreted products, has an anti-inflammatory effect (42) and
may ameliorate colitis in mice by regulating Th17 cell differentiation and inhibiting the
excretion of relevant inflammatory cytokines (43). A recent study indicated that F.
prausnitzii levels were decreased in IBD patients (44). Prevotella and Oscillibacter are also
known to produce anti-inflammatory metabolites, which subsequently reduce Th17
Thyroid
polarization and promote the differentiation of anti-inflammatory Treg/Tr1 cells in the gut
(45). Moreover, decreased Prevotella levels have been reported in diseases such as MS,
autism and T1D (13,46,47). Alloprevotella are obligate anaerobic, non-motile, gram-
negative bacilli. These strains are weakly to moderately saccharolytic and produce acetic
and succinic acids as the end products of fermentation (48). Parabacteroides may reduce
intestinal inflammation by inducing the anti-inflammatory cytokine IL-10 and suppressing
the secretion of the inflammatory cytokines IL-17, IL-6, and IFN-γ (49). Decreases in
Bifidobacterium were also observed in the HT group, although the difference was not
significant (P = 0.5338). Bifidobacterium may beneficially affect a host by augmenting its
intestinal microbial population, possibly inhibiting pathogens. Decreased levels of
Bifidobacterium may lead to poor immune function (50). Therefore, in HT patients,
decreased levels of these species may lead to intestinal mucosal barrier destruction,
resulting in the translocation of bacteria and their products across the mucosal barrier
and, consequently, the activation of immune responses.
Page 16 of 44
16
According to the LEfSe results, the abundance levels of many genera were increased in
HT patients (Fig. 2A).
Blautia, Dorea, Clostridium_sensu_stricto1, Ruminococcus_torques_group,

Ruminococcus_gauvreauii_group, Haemophilus, Eubacterium_hallii_group,
Eubacterium_ruminantium_group, Roseburia, Lachnospiraceae_UCG_001,
unclassified_f_Lachnospiraceae, Butyricicoccus, Streptococcus, Fusicatenibacter,
Anaerostipes, Romboutsia, Erysipelotrichaceae_UCG_003, Coprococcus_2, and
Subdoligranulum were increased in HT patients. Some species with increased abundance

levels were shown to be related to autoimmune or inflammatory diseases in previous

studies. Two Firmicutes, Blautia and Dorea, were more abundant in HT patients than in
healthy controls; however, these species have been both positively (51) and negatively
(52) linked to inflammatory diseases, and members of the genera Blautia and Dorea
exhibited increased abundance levels in MS and Parkinson’s disease (13,53).
Ruminococcus_torques is a mucin-degrading member of the Clostridium_coccoides_group
Thyroid
of Firmicutes in the human intestinal microbiota (54) and has been associated with Crohn’s
disease (55). The Ruminococcus_gauvreauii_group is a novel, strictly anaerobic,
vancomycin-resistant, gram-positive coccus group, and acetic acid is the sole product of
glucose fermentation by these organisms (56). Coprococcus_catus, Eubacterium_hallii and
Anaerostipes_caccae were shown to use lactate as a substrate to produce propionate and
butyrate (57,58). Fusicatenibacter saccharivorans (FS), a member of Clostridium cluster
XIVa, is present at markedly lower levels in active ulcerative colitis (UC) than in quiescent
UC. Lamina propria mononuclear cells (LPMCs) that were incubated with FS produced
higher amounts of IL-10. FS also induced IL-10 production in human LPMCs isolated from
UC patients. These results suggest that human-derived FS suppresses intestinal
inflammation, probably through IL-10 induction, representing a possible future strategy of
IBD bacteriotherapy to encourage these bacteria to colonize the intestines of IBD patients
(59). Phylogenetically, Subdoligranulum is a member of Clostridium leptum and produces
bacteriocins, which are protein toxins that suppress the growth of similar or closely related
bacterial strains (60). Bacteriocins produced by this genus may play a role in suppressing
Bifidobacteria growth during inulin fermentation.
Page 17 of 44
17
Interestingly, the changes in gut microbial structure are similar between IBS and HT
patients. Several recent comprehensive studies of the microbiota in IBS patients have
reported increases in the relative abundance levels of Firmicutes, mainly Clostridium

cluster XIVa (Blautia, Dorea, Eubacterium_rectale_group and Ruminococcaceae),
Ruminococcus_torques_group, Streptococcus and Lachnospiraceae, and their abundance
levels were positively correlated with bowel symptoms (38,61-63). The question of
whether the increased abundance levels of certain species can initiate HT in humans
requires further investigation.

A heat map was used to visualize the relationships between species and clinical
parameters, and the results show that some gut microbiota were notably correlated with
HT-related clinical parameters (TPO-Ab, TG-Ab, FT4 and TSH), indicating that the gut
microbiota are closely related to HT (Fig. 3A). These findings lay the foundation for
research on the interaction between the gut microbiota and a host relative to the
development of HT. We used LEfSe analysis to select ten species based on their LDA values
Thyroid
for use as biomarkers for predicting disease status. A heat map based on the abundance of
the first 10 genera (biomarkers) from 27 different genera was generated. Hierarchical
clustering shows that the two groups could be clearly separated (Fig. 3B) and that HT-
patient samples generally cluster together (Fig. S5), with an AUC value of 0.91 (Fig. 3C).
Subsequently, we also obtained good results in the validation cohort (Fig. 3D), thus
confirming that the gut microbiota can be used to accurately identify patients. Although
this represents a new method for the early diagnosis of HT, large samples are needed to
verify the accuracy of this method.
A key advantage of our study is that we used the 16S rRNA gene sequencing technique
to characterize the gut microbiota of HT patients. Environmental factors were examined
using Spearman correlation analysis, and a novel prediction model was proposed.
However, this study has several limitations that must be addressed in future studies. First,
our study is a single-center, cross-sectional study involving a small number of samples.
Second, we were unable to observe variations among patients at different disease stages
of HT because all patients in our cohort were euthyroid (most patients in our cohort were
new-onset HT patients). Third, although HT patients were age-, sex- and BMI-matched in
Page 18 of 44
18
the analysis, our results may be influenced by other confounding effects, such as stress
and dietary factors. Fourth, microbiota analyses must include studies of the ‘virome’,
‘fungiome’, etc. These factors warrant examination in future studies. Finally, our study
does not include animal experiments or research regarding the possible mechanism linking
gut microbiota dysbiosis to the development of HT. Therefore, further studies are needed
with larger samples, multicenter designs, animal experiments and the use of
innovative research techniques (metagenomics and metabonomics) to explore the
potential causal mechanisms between the gut microbiota and HT, such as intestinal barrier
dysfunction, microbiota translocation and molecular mimicry, to facilitate future scientific

studies and provide guidance for the diagnosis, treatment, and prevention of HT.
Acknowledgements
This study was conducted at the First Affiliated Hospital of Harbin Medical University.
Thyroid
This study was supported by Heilongjiang Provincial Science and Technology Department
Funds for Distinguished Young Scholar JC201416 and the Translational Research Fund of
Translational Medical Centre between China and Russia CR201514. We thank all the staff
and participants for their important contributions.
Disclosure Statement
The authors declare no competing financial interests.
Corresponding Author
Yunwei Wei, Department of Oncological and Endoscopic Surgery, The First Affiliated
Hospital of Harbin Medical University, No. 23, Youzheng Street, Nangang District, Harbin,
Heilongjiang, 150001, People’s Republic of China.
Page 19 of 44
19
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26
Table 1. Clinical and demographic features of HT patients and healthy controls. M/F,
male/female; TG-Ab, thyroglobulin antibody; TPO-Ab, thyroperoxidase antibody; FT3, free
triiodothyronine; FT4, free thyroxine; TSH, thyroid-stimulating hormone; BMI, body mass
index; P/N, positive/negative ratio.
Exploration cohort Validation cohort

HT group Healthy P HT group Healthy P
Parameters (n=28) group valu (n=22) group valu

(n=16) e (n=11) e
Sex (M/F) 3/25 2/14 1.00 3/19 2/9 1.00
0 0
Age (years, mean±SD) 44.29±12.25 44.63±10.33 0.10 43.73±10.9 0.56
45.82±10.70
0 5 7
BMI (kg/m2, 22.14±2.51 22.50±1.18 0.06 22.25±2.38 22.87±1.01 0.08
mean±SD) 3 7
Thyroid
FT3 (pg/mL, mean±SD) 2.78±0.39 2.78±0.46 0.25 2.81±0.38 2.80±0.40 0.61

6 8
FT4 (ng/mL, mean±SD) 1.06±0.14 1.08±0.15 0.92 1.10±0.11 1.04±0.12 0.47
8 2
TSH (μIU/mL, 2.59±0.97 2.63±0.79 0.52 2.87±0.80 2.54±0.83 0.70
mean±SD) 1 6
TPO-Ab (IU/mL, 460.23±341. 0.81±0.36 0.00 530.69±339.2 0.83±0.32 0.00
mean±SD) 38 1 0 1
TPO-Ab>5.61IU/mL 28/0 0/16 0.00 22/0 0/11 0.00
(P/N) 0 0
TG-Ab (IU/mL, 353.47±345. 1.13±0.97 0.00 416.41±361.1 0.82±0.63 0.00
mean±SD) 29 1 7 1
TG-Ab>4.11IU/mL 27/1 0/16 0.00 20/2 0/11 0.00
(P/N) 0 0
Page 27 of 44
27
Table 2. Comparison of phylotype coverage and diversity estimation of the 16S rRNA
gene libraries at 97% similarity based on the pyrosequencing analysis. 1The operational
taxonomic units (OTUs) were defined at a 97% similarity level. 2The coverage percentage
(Good’s) was calculated using Good’s method, and richness estimators (ACE and Chao1)
and diversity indices (Shannon and Simpson) were calculated using the Mothur program.
Sa No. Richness estimator Diversity index

Good’
Grou m No. of of Shanno
s ACE Chao1 Simpson
p pl Reads OTUs 2
95%CI 95%CI n
1
(%) Mean Mean Mean
es Mean
425.07 426.990
Thyroid
2 20030 99.89 434.9 69 - 8- 0.04874

HT 746 437.04 3.9331
8 36 % 8 464.47 464.340 9
04 6
398.13
400.251
39
Cont 1 11462 99.89 413.0 1- 0.06010
703 - 412.88 3.8135
rol 6 21 % 4 439.351 5
443.90
4
05
Page 28 of 44
28
Table 3. Differential taxa abundances between HT-patient and healthy-control samples
at phylum, family and genus levels.
HT Control Log2 HT Control

Species P value Q value Mean Mean fold preval prevale
change ence nce
Phylum
Firmicutes 1.85E- 16 28
1.38E-05 8.26E-01 6.91E-01 0.26

06
Bacteroidetes 4.70E- 16 28
7.05E-06 9.85E-02 2.27E-01 -1.20
07
Family
Firmicutes; 16 28
Lachnospirac 1.28E-06 5.82E-05 4.90E-01 3.30E-01 0.57
eae
Thyroid
Bacteroidetes 16 28
;
1.67E-07 1.52E-05 6.13E-02 1.33E-01 -1.11
Bacteroidace
ae
Bacteroidetes 16 28
;
2.57E-05 5.85E-04 2.82E-02 7.77E-02 -1.46
Prevotellacea
e
Firmicutes; 16 28
Peptostrepto 2.47E-03 3.54E-02 3.37E-02 1.22E-02 1.47
coccaceae
Firmicutes; 16 28
Streptococca 3.28E-03 4.39E-02 2.60E-02 1.50E-02 0.79
ceae
Genus
Page 29 of 44
29
Firmicutes; 16 28
Faecalibacteri 3.70E-03 2.45E-02 9.87E-02 1.51E-01 -0.61
um
Bacteroidetes 16 28
1.67E-07 3.02E-05 6.13E-02 1.33E-01 -1.11
; Bacteroides
Bacteroidetes 16 28
7.35E-05 2.05E-03 1.83E-02 6.01E-02 -1.72
; Prevotella_9
Firmicutes; 16 27
1.19E-05 5.54E-04 9.77E-02 5.86E-02 0.74
Blautia
Firmicutes; 16 28
1.03E-02 4.25E-02 3.98E-02 3.12E-02 0.35
Roseburia
Firmicutes; 16 28
Lachnoclostri 1.28E-02 4.39E-02 2.41E-02 2.85E-02 -0.24
dium
Firmicutes; 16 28
Thyroid
Ruminococcu
1.72E-03 1.50E-02 3.06E-02 2.00E-02 0.61
s_torque_gro
up
Firmicutes; 16 28
5.62E-03 4.12E-02 2.35E-02 1.46E-02 0.68
Romboutsia
Firmicutes; 16 28
6.05E-03 5.84E-02 2.00E-02 1.38E-02 0.53
Dorea
Firmicutes; 16 28
Fusicatenibac 1.63E-06 1.14E-04 1.86E-02 1.00E-02 0.89
ter
Firmicutes; 16 28
Eubacterium_ 2.16E-07 3.02E-05 2.58E-02 1.10E-02 1.24
hallii_group
Page 30 of 44
30

Thyroid
Figure 1. Gut microbiota of HT patients differs from that of healthy controls. Rank-
abundance curves were used to explain species richness and evenness. The bacterial
communities of the healthy controls and HT patients exhibited similar patterns (A).
Rarefaction curves were used to compare the species richness between HT patients and
healthy controls, and the microbiota richness of the HT patients was higher (B). The
relative abundance of faecal bacterial phyla and genera were clustered into two groups,
and the microbiota compositions significantly differed. In this analysis, only phyla and
genera with relative abundances of greater than 1% were included. All OTUs with lower
abundances were grouped as “other” (C, D). A PCoA plot based on the Bray-Curtis distance
matrix. The first two coordinates are plotted with the percentage of variability explained
indicated on the axis. Each point represents a sample, and the colours represent different
groups (E). ANOSIM analysis indicated that the difference between the HT group and the
healthy control group was significant (R = 0.7154, P < 0.001) at the phylum level (F).
Thyroid
Page 31 of 44
of the 16S sequences. LEfSe identified the taxa with the greatest differences in abundance
Figure 2. Taxonomic cladogram obtained using LEfSe analysis and Mann-Whitney U-tests
31
Page 32 of 44
32
between the HT patients and the healthy controls. At the phylum, family and genus level,
the HT patient-enriched taxa are indicated by a positive LDA score (purple), and healthy
control-enriched taxa are indicated by a negative score (red). Only taxa meeting a
significant LDA threshold value of > 3 are shown (A). Comparisons of the relative
abundance at the bacterial phylum, family and genus levels in HT patients and healthy
controls; ‘*’ denotes adjusted Q value < 0.05; ‘**’ denotes adjusted Q value < 0.01; ‘***’
denotes adjusted Q value < 0.001 (B). The F/B ratio in HT patients and healthy controls (C)
；‘#’ denotes adjusted P value < 0.05; Correlations of F/B ratio and age/BMI in HT patients
and healthy controls (D, E, F, G).

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Page 33 of 44
33

Thyroid
Figure 3. Spearman correlation analysis of environmental factors and the predictive

model based on LEfSe analysis results. The relationships among 7 clinical factors and the
relative abundance of top 27 altered genera (Fig. 2B) in HT patients and healthy controls
were estimated using Spearman correlation analysis. Heatmaps showing correlations
between clinical factors and gut microbiota at genus level. Color intensity represent
Page 34 of 44
34
magnitude of correlation. Red = positive correlations; green = negative correlations. ‘*’
denotes adjusted P < 0.05; ‘**’ denotes adjusted P < 0.01; ‘***’ denotes adjusted P <
0.001. TG-Ab, thyroglobulin antibody; TPO-Ab, thyroperoxidase antibody; FT3, free

triiodothyronine; FT4, free thyroxine; TSH, thyroid-stimulating hormone; BMI, body mass
index (A). Heatmap based on the abundance of the first 10 species from the 27 different
genera. Hierarchical clustering (Euclidean distance, complete linkage) shows that two
groups can be clearly separated. The color of the spot corresponds to the Log10-
transformed relative abundance of each genera (B). ROC analysis was used to assess the
predictive model performance among exploration and validation cohorts (C, D). AUC =
0.91 for the exploration cohort (n = 44, red), and AUC = 0.88 for the validation group (n =
33, purple).
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Page 35 of 44
35
Supplementary Information
NERL from online.liebertpub.com at 01/13/18. For personal use only.
Supplemental Table S1: Comparison of phylotypes coverage and diversity estimation of the 16S rRNA gene libraries for individuals at 97%
eviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
similarity from the pyrosequencing analysis.
Sample/ Good’s
No. of No. of ACE ACE Chao1 Chao1 Simpso
Estimator Coverag ACE Chao1 Shannon
Reads OTUs _lci _hci _lci _hci n
s e
10.1089/thy.2017.0395)
HT_EU1 97,842 311 99.90% 383 355.0 428.2 360 337.3 403.0 3.42301 0.099896
3.86750
HT_EU2 98,179 333 99.91% 384 363.4 419.8 388 361.4 439.7 0.053715
6
4.10158
package
HT_EU3 95,353 370 99.89% 430 407.1 468.4 420 396.9 462.5 0.039622
patients (DOI:
7
of Rochester
4.05072
HT_EU4 94,190 381 99.90% 429 409.7 460.6 425 404.2 465.4 0.039828
thyroiditis
6
by University
Thyroid
4.15118
in Hashimoto’s
HT_EU5 89,151 408 99.86% 492 462.8 538.2 511 467.7 587.3 0.030764
8
Alterations of the gut microbiotaDownloaded
4.08133
HT_EU6 83,114 402 99.87% 480 451.7 523.5 483 447.9 545.1 0.037547
1
Page 36 of 44
36
3.94251
HT_EU7 88,541 353 99.88% 430 401.3 476.9 424 392.2 481.5 0.045089
5
3.76114
HT_EU8 90,234 317 99.92% 353 337.2 380.1 353 334.4 391.5 0.064626
2
4.37991
HT_EU9 84,422 467 99.87% 530 507.3 565.7 533 504.2 582.6 0.024481
8
4.03330
HT_EU10 86,162 381 99.87% 520 482.5 572.4 468 430.8 533.8 0.038869
3
10.1089/thy.2017.0395)
4.27039
HT_EU11 94,836 448 99.88% 506 484.8 540.6 516 485.8 569.3 0.029862
2
3.94956
HT_EU12 92,100 352 99.89% 411 388.2 448.6 408 382.4 456.8 0.035951
package
8
patients (DOI:
3.90594
of Rochester
HT_EU13 94,787 386 99.88% 447 423.7 483.6 463 428.5 527.3 0.056967
7
thyroiditis
by University
3.90524
Thyroid
HT_EU14 96,899 357 99.87% 448 415.5 497.8 449 409.4 518.5 0.037363
2
in Hashimoto’s
3.96678
HT_EU15 86,879 353 99.90% 406 384.7 441.3 421 388.3 483.1 0.053383
3
HT_EU16 90,508 392 99.87% 476 446.4 522.1 466 434.5 520.9 3.80507 0.059524
Page 37 of 44
37
1
4.00846
HT_EU17 91,428 318 99.91% 369 347.7 404.1 377 348.1 434.5 0.034636
2
3.77734
HT_EU18 96,849 357 99.88% 435 406.7 480.1 436 401.4 497.6 0.054436
6
HT_EU19 95,456 305 99.90% 363 340.1 402.2 366 337.1 420.9 3.54838 0.073517
3.58183
HT_EU20 89,060 356 99.88% 435 406.4 481.2 433 399.0 494.0 0.089627
3
10.1089/thy.2017.0395)
3.74679
HT_EU21 91,495 311 99.89% 385 356.5 430.2 386 352.0 449.8 0.045577
2
4.18845
HT_EU22 93,789 406 99.88% 478 451.9 519.7 500 458.3 574.3 0.031997
package
6
patients (DOI:
3.78239
of Rochester
HT_EU23 89,305 357 99.88% 429 401.7 471.6 447 406.4 521.0 0.051588
4
thyroiditis
by University
4.08303
Thyroid
HT_EU24 84,464 367 99.90% 421 399.7 456.5 430 400.3 486.2 0.038079
7
in Hashimoto’s
HT_EU25 88,613 345 99.89% 413 386.9 456.0 418 384.7 480.3 3.775012 0.057087
HT_EU26 91,623 396 99.89% 449 428.4 482.5 447 423.5 490.7 4.080734 0.035619
HT_EU27 89,291 370 99.88% 441 414.2 483.2 457 417.9 526.9 3.859233 0.063601
Page 38 of 44
38
HT_EU28 91,879 364 99.89% 435 408.2 479.6 450 410.6 523.8 4.101167 0.041733
Control 4.06443
88,979 366 99.89% 439 411.0 482.9 435 403.8 491.9 0.0379
_1 4
Control 4.10771
91,807 372 99.87% 460 428.1 510.0 460 421.5 527.6 0.032508
_2 6
Control 4.08555
86,790 435 99.85% 528 496.0 575.9 519 484.3 577.1 0.03846
_3 5
Control 3.81395
95,810 346 99.90% 403 380.9 439.9 403 376.5 454.3 0.049085
10.1089/thy.2017.0395)
_4 6
Control 3.75924
94,742 346 99.89% 411 386.0 450.4 406 378.3 456.6 0.05434
_5 2
package
Control 3.84142
patients (DOI:
98,362 377 99.89% 436 413.8 473.0 429 405.5 472.4 0.064732
_6 4
of Rochester
Control 3.73816
thyroiditis
95,643 329 99.91% 384 361.7 421.1 382 356.6 432.2 0.065354
by University
_7 7
Thyroid
Control 4.04782
in Hashimoto’s
86,385 381 99.91% 424 406.5 454.2 431 406.7 477.9 0.050494
_8 5
Control 3.74690
94,606 342 99.89% 417 388.2 462.6 402 374.8 452.3 0.064761
_9 6
Page 39 of 44
39
Control 3.71901
83,546 336 99.92% 374 357.9 403.4 380 357.6 424.7 0.065124
_10 6
Control 3.94750
91,509 353 99.88% 430 401.1 476.8 449 405.6 527.1 0.039995
_11 1
Control 3.46848
86,023 300 99.92% 347 327.4 381.4 349 324.5 398.5 0.08004
_12 4
Control 2.88666
85,189 290 99.90% 350 325.8 391.3 365 329.4 433.3 0.188134
_13 6
10.1089/thy.2017.0395)
Control 3.86847
97,856 340 99.89% 405 379.8 447.4 411 378.3 472.8 0.052748
_14 8
Control 4.16354
87,843 376 99.89% 432 410.3 467.2 425 402.3 466.4 0.0319
package
_15 2
patients (DOI:
of Rochester
Control 3.75721
84,521 311 99.90% 368 345.2 406.4 360 336.7 404.9 0.046101
_16 7
thyroiditis
by University
Thyroid
in Hashimoto’s
Thyroid
(Shannon index) between HT patients and healthy controls.

HT patients and healthy controls. Rarefaction curves comparing the species diversity
Supplemental Figure S1: The overall species diversity of microbiota was similar between
40
Page 40 of 44
Thyroid
Page 41 of 44
microbiota among the two groups.

Supplemental Figure S2: The Venn diagram illustrates the overlap of OTUs in gut
41
Thyroid
color), Bacteroides (green) and eubacterium_rectale_group (sky blue).

healthy controls. Bar colors depicts average proportion of genus level abundance in all
groups of a particular category. Majority of bacteria belonged to Faecalibacterium (red

Supplemental Figure S3: genus level comparison of fecal microbiota in HT patients and
42
Page 42 of 44
Thyroid
Page 43 of 44
extending to 1.5 inter-quartile range (IQR).

box represent the first, second (median) and third quartiles respectively with the whisk
group and between group difference of gut microbiota. The three horizontal lines of the
Supplemental Figure S4: At genus level, ANOSIM analysis was used to comparison of intra-
43
Page 44 of 44
44
Supplemental Figure S5: Heatmap based on the LDA value selected genera. The legends
below the heatmap represent each participant. The color of the spot corresponds to the
Thyroid
Log10-transformed relative abundance of each genera. The Log10-transformed relative
abundance of each genera is indicated by a gradient of colour from green (low abundance)
to red (high abundance). Complete linkage clustering of the samples was based on the
genus-level composition and abundance of the gut microbiota, and HT-patient samples
generally clustered together (Fig. S5).

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Thyroid

Alterations of the gut microbiota in Hashimoto’s thyroiditis patients (DOI: 10.1089/thy.2017.0395)

[email protected], [email protected], [email protected],

Running title: Hashimoto’s thyroiditis and gut microbiota dysbiosis

Keywords: Hashimoto’s thyroiditis, Gut microbiota, Dysbiosis, Clinical parameters,

Background: Hashimoto’s thyroiditis (HT) is an organ-specific autoimmune disease in

Alterations of the gut microbiota in Hashimoto’s thyroiditis patients (DOI: 10.1089/thy.2017.0395)

Methods: We first conducted a cross-sectional study of 28 HT patients and 16 matched

Conclusions: Characterization of the gut microbiota in HT patients confirmed that HT

Hashimoto’s thyroiditis (HT), also known as chronic lymphocytic thyroiditis, is a

common organ-specific autoimmune disorder characterized by the production of

and biochemical abnormalities and serological assessments of autoantibodies (TPO-Ab or

such as the environment, including excessive iodine intake, selenium deficiency,

Therefore, we hypothesized that HT patients have gut microbial dysbiosis that

Study cohort and recruitment of subjects

Thyroid function and thyroid autoantibodies tests

Library preparation and sequencing

Sequencing libraries were generated using a TruSeq® DNA PCR-Free Sample

Paired-end reads assembly and quality control

Sequence analysis was performed using Uparse software (Uparse v7.0.1001).

The microbiota features differentiating the fecal microbiota were characterized

The gut microbiota of HT patients differs from that of healthy controls

The abundance levels of certain bacteria are associated with HT

Dorea and Eubacterium_hallii_group genera were increased in the HT-patient samples (Q

value < 0.05, Fig. 2B).

the healthy-control samples (3.175 ± 0.8209). A correlation matrix based on Pearson

Clinical parameters correlated with the gut microbiota

FT3, AGE and BMI (P > 0.05).

were significantly correlated with TPO-Ab or TG-Ab. Therefore, these differences in

Blautia, Dorea, Clostridium_sensu_stricto1, Ruminococcus_torques_group,

Subdoligranulum were increased in HT patients. Some species with increased abundance

levels were shown to be related to autoimmune or inflammatory diseases in previous

reported increases in the relative abundance levels of Firmicutes, mainly Clostridium

requires further investigation.

dysfunction, microbiota translocation and molecular mimicry, to facilitate future scientific

The authors declare no competing financial interests.

autoimmune hypothyroidism in Danish twins. J Clin Endocrinol Metab 85:536-539.

5. Morohoshi K, Takahashi Y, Mori K 2011 Viral infection and innate pattern

Developments in our Understanding. Horm Metab Res 47:702-710.

exposure on celiac disease autoimmunity in genetically at-risk infants. PLoS One

complex interplay between intestinal microbiota, gut permeability, and mucosal

disruption by an enteric bacterial pathogen accelerates insulitis in NOD mice.

29. Horie I, Abiru N, Nagayama Y, Kuriya G, Saitoh O, Ichikawa T, Iwakura Y, Eguchi K

and obesity in Kazakh school children. Bmc Microbiology 12:283.

induced obesity in rats. Sci Rep 5:14405.

that in healthy children: a case-control study. BMC Medicine,11,1(2013-02-21) 11:1-

Mrazek J, Kopecny J, Verdu EF, Tlaskalova-Hogenova H 2011 Oral administration of

Kaakkola S, Eerola-Rautio J, Pohja M, Kinnunen E, Murros K, Auvinen P 2015 Gut

glycoproteins. J Clin Invest 75:944-953.

human faecal specimen. Int J Syst Evol Microbiol 58:1393-1397.

samples from patients with irritable bowel syndrome. Gastroenterology 141:1792-

predominant irritable bowel syndrome sufferers. BMC Gastroenterol 9:95.

Exploration cohort Validation cohort

Parameters (n=28) group valu (n=22) group valu

FT3 (pg/mL, mean±SD) 2.78±0.39 2.78±0.46 0.25 2.81±0.38 2.80±0.40 0.61

Alterations of the gut microbiota in Hashimoto’s thyroiditis patients (DOI: 10.1089/thy.2017.0395)

Sa No. Richness estimator Diversity index

2 20030 99.89 434.9 69 - 8- 0.04874

HT Control Log2 HT Control

1.38E-05 8.26E-01 6.91E-01 0.26