Journal of Autoimmunity 137 (2023) 103055

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Journal of Autoimmunity
journal homepage: www.elsevier.com/locate/jautimm

Gut microbial signatures and their functions in Behcet’s uveitis and

Vogt-Koyanagi-Harada disease
Qingfeng Wang a, 1, Shuang Wu b, 1, Xingsheng Ye a, Shiyao Tan a, Fanfan Huang a, Guannan Su a,
Aize Kijlstra c, Peizeng Yang a, *
a
The First Affiliated Hospital of Chongqing Medical University, Chongqing Key Laboratory of Ophthalmology, Chongqing Eye Institute, Chongqing Branch (Municipality
Division) of National Clinical Research Center for Ocular Diseases, Chongqing, People’s Republic of China
b
Guangxi Key Laboratory for Polysaccharide Materials and Modifications, School of Marine Sciences and Biotechnology, Guangxi Minzu University, Nanning, People’s
Republic of China
c
University Eye Clinic Maastricht, Maastricht, the Netherlands

A R T I C L E I N F O A B S T R A C T

Handling Editor: M.E. Gershwin Background: A number of public metagenomic studies reveal an association between the gut microbiome and
various immune-mediated diseases including Behcet’s uveitis (BU) and Vogt-Koyanagi-Harada disease (VKH).
Keywords: Integrated-analysis and subsequent validation of these results could be a potentially powerful way to understand
Gut microbiome the microbial signatures and their functions in these two uveitis entities.
Behcet’s uveitis (BU)
Methods: We integrated the sequencing data of our previous metagenomic studies on two major uveitis entities,
Vogt-Koyanagi-Harada disease (VKH)
BU and VKH as well as four other publicly available immune-mediated diseases datasets, including Ankylosing
Metagenomic sequencing
Immune-mediated disease Spondylitis (AS), Rheumatoid Arthritis (RA), Crohn’s disease (CD) and Ulcerative Colitis (UC). Alpha-diversity
and beta-diversity analysis were used to compare the gut microbiome signatures between both uveitis entities
and other immune-mediated diseases and healthy controls. Amino acid homology between microbial proteins
and a uveitogenic peptide of the interphotoreceptor retinoid-binding protein (IRBP)161-180 was investigated using
a similarity search in the NCBI protein BLAST program (BLASTP). Enzyme-linked Immunosorbent Assay (ELISA)
was performed to evaluate the cross-reactive responses of experimental autoimmune uveitis (EAU)-derived
lymphocytes and BU patients-derived peripheral blood mononuclear cells (PBMCs) against homologous peptides.
The area under the curve (AUC) analysis was used to test the sensitivity and specificity of gut microbial
biomarkers.
Results: Depleted Dorea, Blautia, Coprococcus, Erysipelotrichaceae and Lachnospiraceae as well as enriched
Bilophila and Stenotrophomonas were identified in BU patients. An enriched Alistipes along with a lower level of
Dorea were observed in VKH patients. A peptide antigen (SteTDR) encoded by BU specifically enriched Steno­
trophomonas was identified to share homology with IRBP161-180. In vitro experiments showed that lymphocytes
from EAU or PBMCs from BU patients reacted to this peptide antigen as shown by the production of IFN-γ and IL-
17. Addition of the SteTDR peptide to the classical IRBP immunization protocol exacerbated EAU severity. Gut
microbial marker profiles consisted of 24 species and 32 species respectively differentiated BU and VKH from
each other as well as from the other four immune-mediated diseases and healthy controls. Protein annotation
identified 148 and 119 specific microbial proteins associated with BU and VKH, respectively. For metabolic
function analysis, 108 and 178 metabolic pathways were shown to be associated with BU and VKH, respectively.
Conclusions: Our study revealed specific gut microbial signatures and their potentially functional roles in BU and
VKH pathogenesis that differ significantly from other immune-mediated diseases as well as healthy controls.

Abbreviations: BU, Behcet’s uveitis; VKH, Vogt-Koyanagi-Harada disease; RA, Rheumatoid Arthritis; CD, Crohn’s disease; UC, Ulcerative Colitis; IRBP, inter­
photoreceptor retinoid-binding protein; EAU, experimental autoimmune uveitis.
* Corresponding author. The First Affiliated Hospital of Chongqing Medical University, Youyi Road 1, Chongqing, 400016, People’s Republic of China
E-mail address: peizengycmu@126.com (P. Yang).
1
These authors contributed equally to this work.

https://doi.org/10.1016/j.jaut.2023.103055
Received 27 February 2023; Received in revised form 27 April 2023; Accepted 3 May 2023
Available online 18 May 2023
0896-8411/© 2023 Elsevier Ltd. All rights reserved.
Q. Wang et al.
1. Introduction
Uveitis is an intraocular inflammatory disease which can lead to
serious visual impairment worldwide [1]. Although clinical features
vary significantly among different uveitis entities, several risk factors
such as genetic, environmental, infectious and immunological factors
have been shown to play an important role in their pathogenesis [2].
Recently evidence is emerging concerning the role of the gut micro­
biome in the pathogenesis of uveitis [3].
The gut microbiome plays an important role in modulating host
immune homeostasis [4,5]. Dysbiosis of the gut microbiota has been
found in a series of immune-mediated diseases including Ankylosing
Spondylitis (AS), Rheumatoid Arthritis (RA), Crohn’s disease (CD) and
Ulcerative Colitis (UC) using metagenomic sequence (MGS) [6–8].
Studies dealing with the pathogenesis of these immune-mediated dis­
eases have suggested that the gut microbiome contributes to immune
disorders via production of homologous peptides, effector proteins and
metabolites [9–11]. In our previous metagenomic studies, we also
showed that gut microbiome composition was associated with Behcet’s
uveitis (BU) and Vogt-Koyanagi-Harada disease (VKH), which are
common non-infectious uveitis entities [12,13]. BU is an auto­
inflammatory disease causing multisystemic inflammation in various
organs including the eye (uveitis) and skin (lesions and ulcers) [14].
VKH is different from BU and is commonly considered as an autoim­
mune disease directed against melanocyte associated antigens and
which is characterized by bilateral granulomatous panuveitis with sys­
temic manifestations, such as tinnitus, vertigo and meningism [15].
Several studies identified similarities and differences concerning the
pathogenetic mechanisms between BU, VKH and various
immune-mediated diseases including AS, RA, CD and UC [16–19].
However, few studies have focused on the differences of the gut
microbiome composition between these diseases and this was therefore
the subject of the study presented here. With a number of metagenomic
datasets, publicly available in the NCBI sequence read archive (SRA) and
the platform of MICrobiome HELminth INteractions database
(MICHELINdb) [20], this allowed comparative analyses of the meta­
genomic studies on BU and VKH with other immune-mediated diseases.
In this study, we integrated the sequencing data of five metagenomic
studies and identified the gut microbial signatures in two separate
uveitis entities (BU and VKH). Moreover, we identified gut microbial
markers and the functional consequences for these two uveitis entities
based on their specific microbial signatures.
2. Methods
2.1. Datasets
To conduct the comparative analyses of the metagenomic studies on
BU and VKH with other immune-mediated diseases, we obtained the
public metagenomic sequence data from 695 fecal metagenomic sam­
ples across 5 studies and 3 populations from the NCBI SRA [6–8,12,13]
(Table 1). The datasets included the fecal metagenomic samples of VKH,
BU, CD, UC, RA and AS patients as well as healthy controls. The datasets
of VKH, BU, AS and RA were from four different Chinese metagenomic
studies. CD and UC datasets were from one metagenomic study con­
taining patient samples from the Netherlands and USA. We only selected
the active patients from the datasets of metagenomic studies on VKH and
BU. For healthy controls, the controls from the dataset of CD and UC
were excluded since non-CD and UC patients instead of healthy controls
were included as controls in their study [6]. Furthermore, the healthy
controls samples from the four Chinese studies including BU, VKH, RA
and AS were combined for a larger sample size to compare the gut
microbiome compositions between BU or VKH and healthy controls.
Besides the healthy controls, the non-uveitis patient samples including
CD, UC, RA and AS patients from the datasets mentioned above were
also used to further investigate the gut microbial features of BU or VKH
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Journal of Autoimmunity 137 (2023) 103055
Table 1
The studies included in the intergated-analysis.
Diseases Sample Populations PMID
size
BU 24 China 30077182
VKH 71 China 31928124
CD 86 American and 30531976
Netherland
UC 65 American and 30531976
Netherland
AS 51 China 28750650
RA 95 China 26214836
HC 303 China 26214836, 28750650,
30077182, 31928124
BU, Behcet’s uveitis; VKH, Vogt-Koyanagi-Harada disease; CD, Crohn’s disease,
UC, ulcerative colitis; AS, Ankylosing spondylitis; RA, rheumatoid arthritis, HC,
healthy controls; PMID, PubMed unique identifier.
as compared to other immune-mediated diseases. Although public
metagenomic sequence data from different studies differed in their li­
brary sizes (number of reads), read length, and how they were quality
controlled, the variations caused by these technical factors is relatively
small in contrast to biological variation as described in a previous study
[21], suggesting that these variations will not influence our analysis.
2.2. Taxonomical annotation and profiling
Raw sequence data (BioProject accession number PRJNA356102,
PRJNA431482, PRJNA400072, PRJNA375935, PRJNA356225) were
downloaded from SRA (https://www.ncbi.nlm.nih.gov/Traces/st
udy/?) and then converted to fastq files by using the fastq-dump func­
tion from the NCBI SRA Toolkit (https://github.com/ncbi/sratoolkit).
Human genes, sequences of poor quality (mean quality <20) and short
sequences (reads <80 bp) from raw reads were filtered by using the
program KneadData (0.6.1), which combined Trimmomatic (0.39) and
Bowtie2 (2.3.5) [22,23]. MetaPhlan was used for enhanced meta­
genomic taxonomic profiling. The composition (Bacteria, Archaea, Eu­
karyotes and Viruses) and gene abundance profile of microbial
taxonomic units was profiled using MetaPhlan2 (v2.7.2) on the
post-processed reads [24]. Using MetaPhlan2, the annotation rate at
species and strain level in this study were 92.56% ± 6.84% and 46.27%
± 23.39% in the different groups, respectively.
2.3. Alpha- and beta-diversity
Gut microbiome composition data of each sample from MetaPhlan2
was merged and converted to an spf file with predicted read counts by
the Python-based tool included merge_metaphlan_tables.py and meta­
phlan_to_stamp.pl [24]. Shannon diversity index was calculated based
on genera and species abundance profiles using the R package (https://
github.com/const-ae/ggsignif). To identify genera and species showing
different abundances in uveitis and other immune-mediated diseases as
well as healthy controls, the spf file was analyzed by the Statistical
Analysis of Metagenomic Profiles tool (STAMP). The
Benjamini-Hochberg procedure was used to control the false discovery
rate due to multiple testing. False discovery rate (FDR) correction lower
than 0.1 was identified as significant difference.
To compare the gut microbiome composition between diseases and
healthy controls, operational taxonomic units (OTU) at different levels
were separated from the spf files and used to compare the differences of
beta diversity (distance between samples based on differences in OTUs
present in each sample) among different groups. Principal Component
Analysis (PCA) was generated by using R package (https://github.com/
YongxinLiu/MicrobiomeStatPlot/tree/master/223PCA) to identify
structural differences in the microbial community from different dis­
eases and healthy controls based on beta diversity distances. Con­
strained PCoA analysis (CPCoA) was performed to visualize the
Q. Wang et al.
influence from the microbiome on the variants of different diseases and
healthy controls using the R package (https://github.com/YongxinLiu/
MicrobiomeStatPlot/tree/master/222CPCoA).
2.4. Linear discriminant analysis effect size
Linear discriminant analysis effect size (LEfSe), a method for
biomarker discovery, was used to identify the microbial features of BU
and VKH from other diseases and healthy controls. In this study, LEfSe
was performed by an online Galaxy web application (http://huttenhowe
r.sph.harvard.edu/galaxy), in which the alpha value for Kruskal-Wallis
rank-sum test among classes was 0.5, and the threshold on the loga­
rithmic LDA score for discriminative features was 3.0 [25].
2.5. IRBP161-180 peptide homology
To determine the sequence homology between microbial proteins
and a uveitogenic peptide of the interphotoreceptor retinoid-binding
protein (IRBP)161-180, IRBP161-180-SGIPYIISYLHPGNTILHVD was input
as a query sequence using the BLASTP program. Proteins encoded in
cultured and uncultured bacteria represented in the National Center for
Biotechnology Information (NCBI) microbial protein database were
searched for similarity to IRBP161-180 with BLAST p2.3.1 (https://blast.
ncbi.nlm.nih.gov/Blast.cgi).
2.6. Study participants
Eight active BU patients with uveitis were included in this study for
in vitro experiments. Diagnosis of BU was based on the diagnostic
criteria of the international study group for BU [26]. Active BU was
defined according to the presence of active intraocular inflammation.
The study was approved by the Ethics Committee of Chongqing Medical
University. Signed informed consent was obtained from all participants
at the beginning of the study. All procedures were performed in accor­
dance with the Declaration of Helsinki.
2.7. Experimental autoimmune uveitis (EAU) induction
B10.RIII mice were purchased from Jackson Laboratory (Bar Harbor,
ME, USA) and maintained under specific pathogen free (SPF) conditions.
For cytokine secretion assays, EAU was induced in 6–8 week old B10.RIII
mice (males and females) using 50 μg of IRBP161–180 in complete
Freund’s adjuvant (CFA) (Sigma-Aldrich, St. Louis, MO, USA) supple­
mented with 1.0 mg/ml Mycobacterium tuberculosis strain (MTB) as
previously described [27]. For immunization with a low dose of
IRBP161–180, EAU was induced in B10.RIII mice using 25 μg of
IRBP161–180 in CFA. The animal study was approved by the Ethics
Committee of the First Affiliated Hospital of Chongqing Medical
University.
2.8. Real-time PCR and cytokine secretion assays
Peripheral blood mononuclear cells (PBMCs) were purified from
heparinized blood of BU patients with active intraocular inflammation
by human Ficoll-Hypaque density gradient centrifugation. Lymphocytes
from mouse spleen were filtered with cell strainers and purified by
mouse Ficoll-Hypaque density gradient centrifugation. The cells were
cultured in culture medium consisting of RPMI medium 1640, 100 U/ml
penicillin/streptomycin and 10% fetal bovine serum. Total RNA was
isolated by TRIzol reagent (Invitrogen, Carlsbad, CA, USA) from lym­
phoctyes. The PrimeScript RT kit (Takara Biotechnology, Dalian, China)
was used to reverse the extracted RNA into complementary DNA. The
ABI Prism 7500 system on the SYBR Premix (BIO-RAD, CA, USA) was
used to detect and analyze the expression. The relative expression of
target genes was quantified by using the 2-△△Ct method with β-actin as
the internal reference.
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Journal of Autoimmunity 137 (2023) 103055
For cytokine secretion assays, cells (5 × 105/500 μl) were seeded into
each well of a 48-well plate. The supernatants of PBMCs and lympho­
cytes after stimulation with peptides were collected after 3 days. The
production of IFN-γ and IL-17 in the supernatants was quantified using
Duoset ELISA development kits (R&D Systems, MN, USA) in accordance
with the manufacturer’s instructions. For statistical analysis, concen­
trations below the detection limit were converted to a value of 50% of
the lowest point of the calibration curve [28].
2.9. Development of microbial markers
To establish the microbial markers for BU and VKH, extremely low
abundant taxa (mean relative abundance less than 0.001 in any group)
were removed as described previously [12]. The differentially abundant
species in BU or VKH and other groups (including other five diseases and
healthy controls) by LDA were chosen to build a receiver-operating
characteristic (ROC) curve. Area under the curve (AUC) values were
obtained to assess the disease probability values and an AUC higher than
0.7 was considered as a good microbial marker for the disease in
question.
2.10. Protein annotation and metabolic function analysis
To investigate the functional contributions of gut microbiome
composition to the development of these diseases, the proteins of strains
associated with BU and VKH were downloaded from the NCBI database
(https://www.ncbi.nlm.nih.gov/genome/?term=) and annotated by
aligning gene sequences against the proteins in the KEGG Automatic
Annotation Server (KAAS) [29]. Then, these proteins were mapped in
the functional pathways via KAAS to predict their potential functions.
Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis
based on these proteins were also performed via the KEGG and String
database [30]. The shared and specific proteins or pathways were
analyzed by an online Venn figures drawing tool (http://bioinformatics.
psb.ugent.be/webtools/Venn). For metabolic function analysis, un­
mapped reads were translated against the UniRef90 protein reference
database using DIAMOND [31,32]. The generated gene lists are then
collapsed into protein families and enzyme classes/pathways using
MetCyc databases [33].
Availability of data and materials
The datasets supporting the conclusions of this article are included
within the article and additional files. The raw sequence data (Bio­
Project accession number PRJNA356102, PRJNA431482,
PRJNA400072, PRJNA375935, PRJNA356225) were downloaded from
SRA (https://www.ncbi.nlm.nih.gov/Traces/study/?) as mentioned in
the method section.
3. Results
3.1. The comparison of gut microbiome composition between selected
diseases and healthy controls
Two kinds of platforms (Illumina HiSeq 2000 and 4000) and five
studies on three populations (China, Netherlands and America) were
included for the following analysis. To examine the influence from
different platforms and populations on the microbiome composition, we
first performed PCA of samples from these 5 studies in the context of
platforms and populations. We found that there was no significant
segregation of the microbiome among different platforms or populations
indicating that these factors did not influence the following results and
analysis (Supplementary Figs. 1A and B). In addition, PCA of the gut
microbiome could not segregate BU, VKH from other immune-mediated
diseases or healthy controls at genera level (Supplementary Fig. 2,
Supplementary Fig. 3 and Supplementary Fig. 4).
Q. Wang et al. Journal of Autoimmunity 137 (2023) 103055

CPCoA based on the Bray–Curtis distance is another in depth method
to analyze differences of the gut microbiome composition between dis­
eases. This method showed that CPCoA at genera level and at species
level could explain 8.82% (P = 1.0*10^-4) and 3.85% (P = 1.0*10^-4) of
the total variance among these groups, respectively (Fig. 1A and B),
showing that the difference in gut microbiome composition between
these groups is statistically significant.
3.2. Differences of the gut microbiome composition between uveitis and
other immune-mediated diseases as well as healthy controls
The aforementioned results showed a difference of gut microbiome
composition between the selected diseases and healthy controls. A
further study was performed to investigate the different abundances of
genera and species between these diseases based on the alpha diversity.
The results of the Shannon index revealed differences concerning mi­
crobial diversity. VKH showed a much lower microbial diversity as

Fig. 1. The comparison of the gut microbiome composition between patients with various diseases and healthy controls. (A) CPCoA of the gut microbiome
composition between various diseases and healthy controls as based on the Bray–Curtis distance was performed by ANOSIM. (C and D) Circos plot depicting the
relative abundances of top 10 genera and species in six immune mediated diseases. The outer track represents the relative abundances of genera or species from six
diseases. The inner track with different colors represents different genera, species and diseases.

4
Q. Wang et al. Journal of Autoimmunity 137 (2023) 103055

compared to the other five diseases and healthy controls at genera and
species level (Supplementary Figs. 5A and B). BU also showed a lower
microbial diversity as compared with UC and CD but not with AS, RA
and healthy controls (Supplementary Figs. 5A and B). The genera of
Bacteroides, Prevotella, Faecalibacterium, Alistipes, Eubacterium, Bifi­
dobacterium, Ruminococcus, Blautia, Escherichia and Roseburia were
the top 10 with the most significant differences at genera level among
the six investigated diseases. Prevotella copri, Faecalibacterium praus­
nitzii, Alistipes putredinis, Bacteroides stercoris, Bacteroides plebeius,
Eubacterium rectale, Bacteroides uniformis, Bacteroides coprocola,
Escherichia coli and Bacteroides ulgaus were the top 10 with the most
significant differences at species level between these diseases. A Circos
plot depicts the relative abundance of the top 10 genera and species in
these six diseases (Fig. 1C and D).
To further investigate the differences in microbial taxa between these
diseases and healthy controls, we performed a pairwise comparison
between two uveitis entities, as well as between each disease and
healthy controls. In general, there was no difference in taxa between the
two uveitis entities based on the alpha diversity. An increased abun­
dance of Bilophila, Alistipes and Stenotrophomonas was found in BU as
compared with the other four selected diseases or healthy controls,
whereas a lower abundance of Dorea, Blautia, Coprococcus, Erysipelo­
trichaceae and Lachnospiraceae was observed in BU (Fig. 2A–E). An
enriched Alistipes was also identified in VKH as compared with the other
four diseases (Fig. 2F–J), whereas Dorea was decreased in VKH. At the
species level, Eubacterium hallii, Dorea formicigenerans, Dorea long­
icatena and Ruminococcus obeum were found to be decreased in both
VKH and BU (Supplementary Figs. 6A–J).
3.3. Identification of a gut microbial antigen homologous with IRBP161-
180
Immune responses mediated by the retina specific protein IRBP is
thought to be involved in the pathogenesis of BU [34]. To investigate
whether a gut microbial antigen shares homology with IRBP, we
analyzed the specifically enriched gut microbiome in BU patients by
using the NCBI microbial protein database for amino acid sequences
homologous to the dominant pathogenic epitope of IRBP (residues
161–180). The result showed that the highest scoring sequence align­
ment of peptide (SteTDR9-17, YVQPGNTIL) was found in the
TonB-dependent receptor (TDR) encoded by Stenotrophomonas species
and differed from IRBP169–177 by two conserved amino acids (Fig. 3A).
To investigate whether the Stenotrophomonas encoded SteTDR
could induce Th1-, and Th17-related cytokine production in BU patients,
PBMCs were cultured with different concentrations (10, 50, 150 and
300 μg/ml) of SteTDR, 10 μg/ml IRBP161-180 as a positive control and 10
μg/ml human myelin oligodendrocyte glycoprotein (MOG) 35-55 as a
negative control. We found that SteTDR (300 μg/ml) and IRBP161-180

Fig. 2. Differences of the gut microbiome in uveitis and other immune-mediated diseases as well as healthy controls. (A–E) Genera showing significant differences in
the gut microbiome when comparing BU patients with other immune-mediated diseases and healthy controls. (F–J) Genera showing significant differences in the gut
microbiome when comparing VKH with other immune-mediated diseases and healthy controls. Blue, BU; Red, VKH; Green, Healthy controls; Yellow, AS; Purple, RA;
Black, CD; Brown, UC. Differentially abundant genera were identified by Wilcoxon rank sum test (FDR<0.1, corrected by the Benjamini Hochberg for multi­
ple comparison).

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Q. Wang et al. Journal of Autoimmunity 137 (2023) 103055

Fig. 3. Immune responses to IRBP161-180, MOG35-55 and SteTDR in PBMCs from BU patients and splenic lymphocytes from mice undergoing EAU. (A) Schematic
diagram of the peptide-SteTDR homology with IRBP161-180. (B) Cytokine secretion assay by ELISA, the concentrations of IFN-γ and IL-17 in the supernatants of PBMCs
and lymphocytes cultured with different peptides. n = 8 for BU patients. n = 6 for the EAU model. ***P < 0.001,**P < 0.01, *P < 0.05, ns, not significant. Wilcoxon
matched pairs test and paired samples t-test. (C) Clinical and histological uveitis in 25 μg IRBP161-180 group and two peptides mixture group. ***P < 0.001, **P <
0.01, *P < 0.05, Data was analyzed by the Mann–Whitney U test. n = 6 for each group. (D) Comparison of IFN-γ, IL-17, IL-10, MCP-1, IL-1β and TNF-α mRNA
expression between 25 μg IRBP161-180 group and two peptides mixture group, ***P < 0.001, **P < 0.01, *P < 0.05, Data was analyzed by the Mann–Whitney U test.
n = 6 for each group. (E) IRBP161-180-induced production of IFN-γ and IL-17 at protein levels in the 25 μg IRBP161-180 group and two peptides mixture group. **P <
0.01, *P < 0.05, Data was analyzed by the Mann-Whitney U test. n = 6 for each group.

were able to stimulate PBMCs from BU patients to produce more IFN-γ
and IL-17 as compared to the control MOG35-55 peptide group (Fig. 3B).
B10.RIII mice are highly susceptible to the dominant pathogenic
murine epitope of IRBP (residues 161− 180) [35]. We stimulated lym­
phocytes from B10.RIII mice after EAU induction using SteTDR and
found that this peptide could induce EAU-derived lymphocytes to pro­
duce more IFN-γ and IL-17 as compared to the MOG35-55 group (Fig. 3B),
and there was no sex difference in these responses as shown by sex
stratification analysis (Supplementary Fig. 7). Whether the homologous
SteTDR could also induce EAU, was tested by immunizing B10.RIII mice
with SteTDR combined with CFA. However, SteTDR could not induce
EAU in male or female B10.RIII mice even at a dose of 300 μg per mouse
(data not shown). Based on the results of the immune responses
mentioned above, we next questioned whether SteTDR could exacerbate
EAU in vivo. To test this, we immunized mice with a combination of the
SteTDR peptide with a low dose of IRBP161-180. SteTDR (100 μg) was
combined with 25 μg IRBP161-180 and CFA for EAU induction. The two
peptides mixture induced a more severe clinical and histological uveitis
in B10.RIII mice as compared to the 25ug IRBP161-180 group (Fig. 3C).
We then isolated splenic lymphocytes from these two groups and
showed that the mRNA expression of IFN-γ and IL-17 was increased in
the two peptides mixture group as compared to the 25 μg IRBP161-180
group (Fig. 3D). However, there was no significant difference of IL-10
mRNA expression between these two groups. mRNA expression of the
proinflammatory cytokines MCP-1, IL1-β and TNF-α was also increased
in the two peptides mixture group (Fig. 3D). Moreover, the result of
ELISA measurements showed that IRBP161-180-induced production of
IFN-γ and IL-17 at the protein level was increased in the two peptides
mixture group (Fig. 3E) as compared to the 25 μg IRBP161-180 group.
These results indicate that SteTDR derived from the gut microbiota,
shares homology and a stimulatory function with IRBP161-180 thereby
amplifying the antigenicity of IRBP161-180 and exacerbating EAU.
3.4. Identification of gut microbial markers for BU and VKH
As mentioned above, several genera showed significant differences
concerning their abundances when comparing uveitis with the other
immune mediated diseases. A gut microbial marker was developed to
test whether these genera could differentiate both uveitis entities from
the other diseases or healthy controls. After filtering the low abundant
taxa as described in the methods section, each genera including Bilo­
phila, Alistipes, Dorea, Blautia, Coprococcus and Lachnospiraceae was
selected to develop microbial markers for BU to differentiate this disease
from the other five diseases and healthy controls (Fig. 4A). These results
showed that AUCs of the classifiers of Bilophila, Dorea and Blautia were
higher than 70%, except for VKH (Fig. 4B and Table 2). Each genera
including Alistipes and Dorea was also used to develop microbial
markers for VKH. The results showed that AUC of the classifier of Dorea
were higher than 70% in discriminating VKH from the other four im­
mune mediated diseases. However, the marker could not differentiate
VKH from BU. (Fig. 4C and Table 3). These results show that several gut
microbial markers can be used to differentiate both uveitis entities from
the other four selected diseases and healthy controls, whereas other gut
microbial markers should be investigated to differentiate BU from VKH.

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Q. Wang et al. Journal of Autoimmunity 137 (2023) 103055

Fig. 4. Identification of gut microbial markers for BU and VKH. (A) The relative abundances of Bilophila, Alistipes, Dorea, Blautia, Coprococcus and Lachnospiraceae
when comparing uveitis patients with other immune-mediated diseases and healthy controls. (B) The ROC curve based on the relative abundances of Bilophila,
Alistipes, Dorea, Blautia, Coprococcus and Lachnospiraceae between BU and other groups. (C) The ROC curve based on the relative abundances of Alistipes and Dorea
between VKH and other groups.

Table 2
The AUCs of the classifiers between BU and other diseases as well as healthy controls.
AUC (Confidence Interval)

Classfiers VKH AS RA UC CD HC
g. Bilophila 69.13% (56.91%– 81.99% (71.30%– 77.52% (66.29%– 88.24% (80.36%– 91.38% (84.42%– 74.75% (63.90%–
81.34%) 92.67%) 88.75%) 96.12%) 98.33%) 85.61%)
g. Alistipes 57.35% (43.76%– 78.92% (67.75%– 71.97% (59.54%– 82.05% (72.49%– 83.37% (74.69%– 65.98% (53.67%–
70.94%) 90.09%) 84.40%) 91.61%) 93.04%) 78.29%)
g. Dorea 52.02% (38.96%– 91.50% (85.12%– 60.83% (50.41%– 79.42% (70.19%– 81.01% (73.20%– 75.70% (67.87%–
65.09%) 97.89%) 71.26%) 88.86%) 88.82%) 83.53%)
g. Blautia 57.99% (44.87%– 91.83% (85.77%– 81.49% (72.90%– 95.61% (91.38%– 97.53% (94.57%– 70.37% (74.87%–
71.10%) 97.89%) 90.08%) 99.87%) 100.5%) 84.41%)
g. Coprococcus 54.75% (41.47%– 70.59% (58.92%– 62.43% (51.51%– 68.04% (57.23%- 68.51% (58.91%– 63.22% (53.42%–
68.02%) 82.26%) 73.35%) 78.86) 78.11%) 73.01%)
g. Lachnospiraceae 55.53% (43.11%– 82.68% (72.51%– 69.25% (58.44%– 84.68% 89.92% (83.36%– 67.08% (58.71%
67.94%) 92.85%) 80.07%) (76.36–92.99%) 96.48%) 75.45%)
Gut microbial marker profiles 74.83% (63.23%– 98.37% (96.27%– 88.64% (81.09%– 99.55% (98.59%– 99.03% (97.78%– 82.82% (72.85%–
(24 species) 86.43%) 100.5%) 96.20%) 100.5%) 100.3%) 92.78%)

AUC, area under the curve; BU, Behcet’s uveitis; VKH, Vogt-Koyanagi-Harada disease; CD, Crohn’s disease, UC, ulcerative colitis; AS, Ankylosing spondylitis; RA,
rheumatoid arthritis, HC, healthy controls.

7
Q. Wang et al. Journal of Autoimmunity 137 (2023) 103055

Table 3
The AUCs of the classifiers between VKH and other diseases as well as healthy controls.
AUC (Confidence Interval)

Classfiers BU AS RA UC CD HC
g. Alistipes 57.35% (43.76%– 74.70% (66.22%– 66.21% (57.72%– 77.80% (70.21%– 79.60% (72.77%– 59.51% (51.80%–
70.94%) 83.19%) 74.71%) 85.38%) 86.43%) 67.21%)
g. Dorea 52.02% (38.96%– 88.96% (82.93%– 78.34% (71.17%– 79.37% (70.35%– 81.02% (73.61%– 73.30% (64.34%–
65.09%) 94.99%) 85.52%) 88.20%) 88.43%) 79.26%)
Gut microbial marker profiles 76.42% (65.30%– 93.25% (88.67%– 96.35% (93.95%– 97.16% (93.79%– 98.53% (96.53%– 82.99% (78.29%–
(32 species) 87.53%) 97.82%) 98.74%) 100.5%) 100.5%) 87.69%)

AUC, area under the curve; BU, Behcet’s uveitis; Vogt-Koyanagi-Harada disease; CD, Crohn’s disease, UC, ulcerative colitis; AS, Ankylosing spondylitis; RA, rheu­
matoid arthritis, HC, healthy controls.

3.5. Development of gut microbial marker profiles for BU and VKH
To further develop microbial marker profiles for BU and VKH, we
also identified the gut microbiome features of BU and VKH and
compared data between these two uveitis entities and the combined
other groups including the other immune mediated diseases and healthy
controls. Six species including Alitispes spp and Bilophila spp were
enriched in BU, whereas thirty-three species including Ruminococcus
spp, Bifidobacterium spp and Roseburia spp were depleted, respectively
(Fig. 5A). After filtering the low abundant taxa as described in the
methods section, a gut microbial marker profile consisting of twenty-
four species was used to classify BU from the other five diseases and
healthy controls. The AUCs of classifiers based on the profile were all
higher than 70% indicating that the usefulness of this gut microbial
marker profile in differentiating BU from the other five diseases and
healthy controls (Fig. 5C and Table 2).
For VKH, twenty-two species including Prevotella spp, Alistipes spp
and Paraprevotella spp were enriched as compared to the combined
other groups, whereas twenty three species including Ruminococcus spp
were depleted (Fig. 5B). Thirteen species were removed due to their
extremely low abundance, and thirty-two species were used as a gut
microbial marker profile to differentiate VKH from other diseases and
healthy controls. The AUCs of classifiers based on the profile between
VKH and the other groups were also higher than 70% (Fig. 5D and
Table 3). The result also indicated that the classifier was able to
distinguish VKH from the other diseases (BU, AS, RA, CD and UC) and

Fig. 5. Development of gut microbial marker profiles for BU and VKH. (A) Histogram of LDA scores computed for species differentially abundant between BU and the
combined other groups. (B) Histogram of LDA scores computed for species differentially abundant between VKH and the other groups combined. The LDA scores
(log) > 3 are listed. (C) The ROC curve based on the profile consisting of 24 species in BU and other diseases as well as healthy controls. (D) The ROC curve based on
the profile consisted of 32 species for VKH and other diseases as well as healthy controls.

8
Q. Wang et al. Journal of Autoimmunity 137 (2023) 103055

healthy controls. depleted strains were identified (Supplementary Fig. 8). After mapping
these proteins in the functional pathways, a number of pathways showed
an association with these proteins. The results showed that several
3.6. Functional analysis of the gut microbiome features in BU and VKH
proteins were mapped in a series of immune-related pathways including
patients
Th1 and Th2 cell differentiation, natural killer cell mediated cytotox­
icity, tumor necrosis factor (TNF) signaling pathway and hypoxia
To further investigate the functional roles of the gut microbiome in
inducible factor (HIF) signaling pathway (Supplementary Tables 1 and
the pathogenesis of BU and VKH, we compared the gut microbiome
2). Furthermore, two depleted proteins that mapped in the pathway of
features of BU and VKH with the combined other groups at strain level
melanogenesis were specific for VKH (Supplementary Table 2). One
and then functionally annotated the proteins using KAAS. We found that
enriched protein specific for BU was found to be mapped in the Toll and
five strains were enriched in BU, whereas thirty-three strains were
Imd signaling pathway (Supplementary Table 1).
depleted (Fig. 6A). After functional annotation, 834 and 470 proteins
It is known that the gut microbiome contributes to immunoregula­
were identified from the sequences of strains enriched and depleted in
tory function via the cumulative effects of microbial metabolites [36].
the BU patients, respectively. For VKH, 19 strains were enriched and 22
We further carried out a study to perform metabolic function analysis
strains were depleted (Fig. 6B). In these differentially abundant strains,
based on microbial features of BU and VKH patients. The result showed
790 and 536 proteins were identified from the enriched and depleted
that 108 and 178 metabolic pathways were identified associated with
strains, respectively. From the venn figure, 128 specific proteins for BU-
BU and VKH microbial features, respectively (Supplementary Tables 3
enriched strains, 20 specific proteins for BU-depleted strains, 72 specific
and 4). Among these pathways, there were 80 pathways that were
proteins for VKH-enriched strains and 47 specific proteins for VKH-

Fig. 6. Functional analysis of gut microbiome features in BU and VKH. (A) Histogram of LDA scores computed for strains differentially abundant between BU and the
other groups. The LDA scores (log) > 3 are listed. (B) Histogram of LDA scores computed for strains differentially abundant between VKH and other groups. The LDA
scores (log) > 3 are listed. (C) Diversity patterns of metabolic pathway in samples from different groups. The top of each set of stacked bars indicates the total
stratified abundance of the pathway within a single sample (log-scaled). Species and “unclassified” stratifications are linearly (proportionally) scaled within the total
bar height.

9
Q. Wang et al.
shown to be associated with both uveitis entities (Supplementary
Fig. 11). Furthermore, we found that the gut microbiome of uveitis
patients showed a marked enrichment in the peptidoglycan biosynthesis
I, whereas the mevalonate pathway I was depleted (Supplementary
Tables 3 and 4). We also identified 28 and 98 pathways specific for BU
and VKH microbial features, respectively (Supplementary Fig. 11). Of
the pathways specific for BU, the gut microbiome of BU patients was
enriched in L-methionine biosynthesis III and superpathway of L-threo­
nine biosynthesis (Fig. 6C, Supplementary Fig. 9, Supplementary
Table 3). For VKH, we showed that the gut microbiome of VKH patients
was enriched in L-lysine biosynthesis VI and L-lysine biosynthesis III
(Fig. 6C, Supplementary Fig. 10, Supplementary Table 4).
4. Discussion
This study shows that the gut microbial composition in two selected
uveitis entities is significantly different from a selection of four other
immune-mediated diseases. A peptide antigen (SteTDR) encoded by BU-
enriched Stenotrophomonas was identified as a homologous peptide
with IRBP161-180. The specific pathways based on these microbial dif­
ferences were also identified in BU and VKH via functional annotations.
BU and VKH are the major uveitis entities in China which cause sight-
threatening intraocular inflammation [37,38]. In the present study, we
enlarged the sample size of healthy controls and further emphasized the
aberrant abundances of Bilophila, Stenotrophomonas and Alistipes in
the BU and VKH patients, which are consistent with the results of our
previous studies on BU and VKH [12,13]. More importantly, these
genera could differentiate BU or VKH from other diseases including AS,
RA, UC and CD. Bilophila is known as a sulfate-reducing bacterium
(SRB) which inhibits butyrate β-oxidation whereby degraded butyrate
may cause a Treg cell dysfunction [39–41]. In addition, both Bilophila
and Stenotrophomonas are commonly considered as opportunistic
pathogens which produce lipopolysaccharide (LPS) that can trigger an
innate inflammatory response via TLRs [41]. Thus, these opportunistic
pathogens may contribute to the decrease of Treg cells, and the activa­
tion of innate immune responses in BU pathogenesis. Alistipes is usually
considered as a pro-inflammatory bacterium and positively correlated
with increased levels of immune-related indicators (B cell and CD4+ T
cells) [42], which might partly explain the infiltration of CD4+ T cells
and B cells reported in the different lesions of patients with VKH [43].
However, it should be mentioned that some differences were apparent
due to the fact that in our current study we used a larger group of healthy
controls and that we now used another method of taxonomical anno­
tation [12,13].
The gut microbiome has been shown to be involved in a variety of
inflammatory diseases by molecular mimicry with self-antigens. The
group of Rachel Caspi has identified a number of sequences with partial
homology with IRBP161-180 derived from Turicibacter sp H121, which is
a new species of Turicibacter sp found in R161H mice. None of these
sequences were able to induce T cell proliferation from R161H mice
expressing a TCR specific for peptide IRBP161-180 or induce disease upon
immunization of WT mice [44,45]. Different from these latter studies,
we investigated the microbial peptide homology with IRBP161-180 based
on the results of gut microbiome composition in human samples.
Although PBMCs from BU patients and lymphocytes from EAU mice
responded to the IRBP cross-reactive peptide we identified in the Sten­
otrophomonas species, we could not induce disease upon immunization
of B10.RIII mice. The in vitro response as reflected by the release of
IFN-γ and IL-17 was positive, following stimulation with SteTDR but was
significantly lower than that seen with the self-antigen IRBP161-180. This
may be due to the small differences in the amino acid sequence between
SteTDR and IRBP161–180. While our data could not provide direct evi­
dence that a gut microbiome mimic of a self-antigen can induce auto­
immune uveitis by itself, SteTDR was able to play a synergistic effect
whereby its presence could enhance the antigenicity of IRBP in our EAU
model.
10
Journal of Autoimmunity 137 (2023) 103055
Several studies provided evidence that a gut microbiome-based
metagenomic signature may offer a universal utility as a non-invasive
diagnostic test for a number of diseases including schizophrenia,
cirrhosis and nonalcoholic fatty liver disease [46–48]. Previous studies
focused on the differences of the gut microbiome composition between a
disease and a healthy control group. A number of datasets from meta­
genomic studies are publicly available, and this allowed us to show that
the gut microbial signature of uveitis differs from several other immune
mediated diseases. However, it should be kept in mind that these results
are based on a retrospective integrated-analysis and further prospective
studies are needed to validate these findings.
The functional annotations of proteins in the strains provides evi­
dence for a role of the gut microbiome in the pathogenesis of various
immune-mediated diseases. In the present study, we revealed that the
proteins in the enriched and depleted strains from BU and VKH were
mapped in IL-17 signaling pathway and MAPK signaling pathway, which
have been reported earlier to be involved in the pathogenesis of BU and
VKH [15,49–53]. The HIF signaling pathway, for instance, plays an
important role in triggering both innate and adaptive immune response
[54,55]. This finding combined with our results suggested that the mi­
crobial proteins from BU and VKH patients, mapped in these pathways,
might contribute to the activation of innate and adaptive immune re­
sponses involved in the pathogenesis of both uveitis entities [15,56]. In
addition, we found that melanogenesis was a specific pathway for VKH
after functional annotation of proteins encoded by depleted strains in
VKH. This finding is consistent with the fact that VKH is thought to be
caused by an autoimmune reaction against melanocyte-associated an­
tigens and thus affects organs and tissues expressing these antigens,
which for instance explains the clinical feature of vitiligo in VKH. Thus,
the specific pathways identified for BU or VKH might offer clues that
explain their different clinical manifestations and pathogenesis.
Through metabolic pathway analysis, a number of metabolic path­
ways including peptidoglycan biosynthesis I showed associations with
BU and VKH. Previous studies have reported the associations of these
two uveitis entities with the gene polymorphisms of TLRs, which belong
to the pattern recognition receptors recognizing peptidoglycan [57,58].
These findings provide important indications that infectious agents from
the gut may trigger aberrant immune responses and contribute to the
pathogenesis of these diseases. Furthermore, we observed a marked
enrichment of L-lysine biosynthesis VI and L-lysine biosynthesis III in
VKH as well as a marked enrichment of L-methionine biosynthesis III and
superpathway of L-threonine biosynthesis in BU. These observations are
consistent with the results of previous studies showing that the levels of
these metabolites were increased in the synovial fluid and plasma from
BU and VKH patients, respectively [59,60]. Methionine, especially it’s
derivatives (such as N-formyl-methionine), is a potent neutrophil che­
moattractant that can trigger a variety of neutrophil-mediated responses
leading to tissue damage [61], which are considered to play an impor­
tant role in the pathogenesis of BD. Threonine is a target amino acid of
protein kinase for phosphorylation that is able to activate the MAPK
pathway and subsequently enhances inflammatory responses during
immune-mediated disease [62]. These findings suggest that higher
levels of methionine and threonine might be involved in BD pathogen­
esis by enhancing neutrophil activation and inflammatory responses.
L-lysine is necessary to assist T cells in the specific recognition of anti­
gens [63]. High lysine levels may activate or enhance autoimmune re­
sponses, which may contribute to VKH development. These observations
collectively suggest that the dysbiosis of the gut microbiome in BU and
VKH patients may contribute to the development of these two diseases
via release of their metabolites into the circulation.
The gut microbial signatures and their functions in BU and VKH
patients showed in this study also provided a possibility of targeting the
gut microbiome as a tool to prevent or treat uveitis. For example, we
noticed that Lactobacillus and Bifidobacterium species were depleted in
VKH patients, as compared to the other groups. A previous study showed
that a probiotic formulation consisting of Lactobacillus and
Q. Wang et al.
Bifidobacterium had a potential therapeutic effect on experimental
autoimmune uveitis [64]. On the other hand, targeted antibiotic killing
of pathogenic bacteria may also be envisaged as a means to manage
intraocular inflammation in uveitis patients.
We would like to mention the limitations of our study. The biological
factors, including donor characteristics such as age, lifestyle, diet, sex,
genetic background, and diet, potentially introduce considerable vari­
ation among datasets in microbiome composition. With the future
publication of additional microbiome datasets with detailed metadata of
donors, stratification analysis on a specific factor may potentially
address this issue and help us to understand the effects of these factors
on the contribution of gut microbiome to uveitis pathogenesis.
Furthermore, there may be a correlation between gut microbiome and
disease status. Thus, further longitudinal studies comparing early dis­
ease and more advanced disease patient cohorts are needed to investi­
gate the association between disease development and gut microbiome.
In conclusion, our study revealed a disease-specific gut microbiome
composition in uveitis via an integrating metagenomic sequencing data
analysis. Our results also provide evidence that a uveitis-specific gut
microbiome may contribute to the pathogenesis of disease via a cumu­
lative effect of microbial antigens and their metabolites. The observa­
tions shown in the present study are focused on the microbial signatures
in uveitis but a similar approach may also be used for other immune-
mediated diseases.
Author contributions
Q.W. and P.Y. conceived and directed the study. Q.W., S.W., X.Y., S.
T. and F.H analyzed the data. P.Y., G.S. and A.K. reviewed the data
interpretation and helped revise the final versions of the manuscript. All
authors read and approved the final manuscript.
Funding
This study was supported by the National Natural Science Founda­
tion Key Program (81930023), National Natural Science Foundation Key
Program (82230032), National Natural Science Foundation
(82101106), Natural Science Key Project of Chongqing Science and
Technology Bureau (CSTC2021jscx-gksb-N0010), Foundation of
Chongqing (cstc2021jcyj-bsh0055), Chongqing Outstanding Scientists
Project (2019), Chongqing Chief Medical Scientist Project (2018),
Chongqing Key Laboratory of Ophthalmology (CSTC, 2008CA5003) and
Chongqing Science & Technology Platform and Base Construction Pro­
gram (cstc2014pt-sy10002).
Data sharing statement
Raw sequence data (BioProject accession number PRJNA356102,
PRJNA431482, PRJNA400072, PRJNA375935, PRJNA356225) were
downloaded from SRA (https://www.ncbi.nlm.nih.gov/Traces/st
udy/?). The datasets used and/or analyzed during the current study
are available from the corresponding author on reasonable request.
Declaration of competing interest
The authors declare no competing interests.
Data availability
Data will be made available on request.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.jaut.2023.103055.
11
Journal of Autoimmunity 137 (2023) 103055
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