Altınönder et al. Journal of Neuroinflammation
https://doi.org/10.1186/s12974-024-03095-7(2024) 21:126

Journal of Neuroinflammation

Open Access

RESEARCH

Thymic gene expression analysis reveals
a potential link between HIF-1A and Th17/
Treg imbalance in thymoma associatedmyasthenia gravisİlayda Altınönder1†, Mustafa Kaya1†, Sibel P. Yentür1, Arman Çakar2, Hacer Durmuş2, Gülçin Yegen3, Berker Özkan4,Yeşim Parman2, Amr H. Sawalha5 and Guher Saruhan-Direskeneli1*
Abstract
Myasthenia gravis (MG) is an immune-mediated disease frequently associated with thymic changes. Increased T
helper 17 (Th17) cell activity and dysfunctional regulatory T (Treg) cells have been demonstrated in subgroupsof MG. On the other hand, hypoxia-inducible factor 1 (HIF-1) has been shown to regulate the Th17/Treg balance
by inducing Th17 differentiation while attenuating Treg development. To identify the underlying mechanismsof different thymic pathologies in MG development, we evaluated thymic samples from thymoma-associatedmyasthenia gravis (TAMG), MG with hyperplasia (TFH-MG) and thymoma without MG (TOMA) patients. Differential
gene expression analysis revealed that TAMG and TFH-MG cells are associated with different functional pathways.
A higher RORC/FOXP3 ratio provided evidence for Th17/Treg imbalance in TAMG potentially related to increased
HIF1A. The hypoxic microenvironment in thymoma may be a driver of TAMG by increasing HIF1A. These findings
may lead to new therapeutic approaches targeting HIF1A in the development of TAMG.
Keywords Thymoma, Thymus, Myasthenia gravis, Hypoxia-inducible factor-1†

İlayda Altınönder and Mustafa Kaya have contributed equally.

*Correspondence:
Guher Saruhan-Direskeneli
gsaruhan@istanbul.edu.tr
1
Department of Physiology, Istanbul Medical Faculty, Istanbul University,
Istanbul 34093, Turkey
2
Department of Neurology, Istanbul Medical Faculty, Istanbul University,
Istanbul 34093, Turkey
3
Department of Thoracic Surgery, Istanbul Medical Faculty, Istanbul
University, Istanbul 34093, Turkey
4
Department of Pathology, Istanbul Medical Faculty, Istanbul University,
Istanbul 34093, Turkey
5
Division of Rheumatology, Department of Pediatrics, University ofPittsburgh, Pittsburgh, PA, USA

Introduction
Myasthenia gravis (MG) is an immune-mediated neuromuscular disease frequently associated with thymic
changes and autoantibodies against the acetylcholine
receptor (AChR). While the most common concomitant
thymic pathology with MG is thymic follicular hyperplasia (TFH), a smaller subgroup (10–20%) of MG patients
presents with thymoma, which is clinically indistinguishable from TFH-MG.
Thymoma is a rare epithelial tumor of the thymus.
Thymoma patients frequently co-exhibit autoimmune
disorders, of which thymoma-associated myasthenia
gravis (TAMG) is the most common, occurring in 64%
of all thymoma patients [1]. Thymomas are classified© The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use,sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) andthe source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in thisarticle are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not includedin the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you willneed to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The
Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available
in this article, unless otherwise stated in a credit line to the data.

Altınönder et al. Journal of Neuroinflammation(2024) 21:126

into several subtypes according to the morphology of
the tumor cells and the proportion of associated immature T cells (i.e., WHO type A, AB, B1, B2, B3, and otherrare subtypes) [2]. TAMG is more common with type B
than type A or AB thymomas [2–5] and is almost always
accompanied by anti-AChR antibodies, as also frequently
observed in other forms of MG with TFH.
Tolerance breakdown by intrathymic mechanisms,
including abnormal T cell selection and activation, hasbeen intensively investigated in MG [6, 7]. Defective
tolerogenic features (e.g., the lack of AIRE expression)[5, 8–10] and reduced intratumoral generation of regulatory T (Treg) cells [11, 12] have been considered possible
mechanisms for MG development. The analysis of T cellsubset composition in the blood revealed an increased
proportion of mature CD4 + and CD8 + T cells in TFHMG and TAMG compared to healthy controls [13, 14].
Similar results were also reported in thymic samples,showing that the presence of TFH-MG or TAMG cells
was significantly linked to high levels of mature naive
CD4 + T cells [15]. Furthermore, an export of increased
autoreactive CD4 + T cells, accompanied by reduced
numbers of Treg cells, was demonstrated in TAMG incomparison to TFH-MG and thymoma patients [12]. The
functional and effective balance between Treg cells andeffector T cells is lost in the thymus of anti-AChR + TFHMG patients as well [16]. However, there was no significant difference between these groups in the periphery[11, 17, 18]. Finally, an increase in IL-17-related activity and a decrease in Treg cell development have also
been demonstrated in TAMG or thymoma without MG(TOMA) [19, 20]. All these data suggest possible Tregand IL-17-related imbalance and potential activationof Th17 cells in the thymus of MG patients [21]. The
increase in active IL-17-expressing cells in the MG thymus may be a consequence of the impaired ability of Treg
cells to suppress effector T cells. Therefore, “plastic” Treg
cells in AChR + MG may become Th17-like ex-Treg cells
and contribute to the rise in the IL-17 concentration in
the thymus [22].
Solid tumors commonly have a hypoxic microenvironment because of rapid proliferation. To adapt to low O2levels, cells activate several survival pathways, such as
the hypoxia-inducible factor-1 (HIF-1) signaling pathway. HIF-1, as a transcription factor, activates over 100
downstream genes that regulate cell survival, proliferation, metabolism, and angiogenesis. Its α-subunit(HIF-1α) is hydroxylated by prolyl hydroxylase enzymes(PHDs, also known as EGLNs) and degraded by the vonHippel − Lindau protein (pVHL) complex under normal O2 tension. However, under hypoxic conditions,
PHD3 activity is reduced, leading to HIF-1α stabilization and HIF-1 activation [23]. Importantly, HIF-1 has
been shown to modulate Th17/Treg balance by favoring

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Th17 development while inhibiting Treg differentiation.
HIF-1 transcriptionally activates RORγt, a transcription
factor that plays a central role in Th17 differentiation andinduces IL-17 A by cooperating with RORγt and p300.
Moreover, HIF-1 targets FOXP3, the specific transcription factor of Treg cells, for ubiquitination and proteasomal degradation [24].
We hypothesized that the hypoxic tumor microenvironment of thymoma and the subsequent changes in
HIF-1-related mechanisms could have disease-causing or
disease-modulating effects on the development of TAMG
in thymoma patients. In this study, we assessed gene
expression in thymic tissues from TAMG and TFH-MG
patients, as well as TOMA patients, with two different
approaches. Global expression differences and targeted
expression analysis were performed in thymic tissue
samples to confirm disease-related/promoting changes in
MG. We report that HIF-1 might play an important rolein the development of TAMG but not TFH-MG.

Materials and methods
Patients and controls

In this study, thymic tissues from patients undergoing
thymectomy at the Istanbul Medical Faculty Hospital(Istanbul, Turkey) between 2015 and 2021 were used. The
diagnosis of MG was based on clinical presentation andelectrophysiological examination.
Thymic samples from thymectomized MG patientswith pathologically proven thymoma (TAMG, n = 7,
median age: 33, 16–47 years old, 4 women and 3 men)or follicular hyperplasia (TFH-MG, n = 8, medianage: 24, 23–66 years old, all women) were included in
the sequencing experiments. TFH-MG patients wereyounger than TAMG patients. All patients had AChR
antibodies, and half of the patients had immunosuppressive (IS) treatment before thymectomy in both groups (3of 7 thymoma vs. 4 of 8 hyperplasia patients).
The WHO classification was used for the thymomas[25]. Only one patient with type B1 thymoma and 6
patients with B2 thymoma were included in the thymoma
tissues (Table 1).
In the targeted expression analysis group, 38 thymic
tissue samples from 12 TAMG, 14 TFH-MG and 12
thymoma without MG (TOMA) patients were included(Table 2). All MG patients had AChR autoantibodies andhad not received IS treatment at the time of thymectomy.
Among the patient groups, TFH-MG patients were significantly younger than TAMG and TOMA patients(p < 0.001). The proportion of women was slightly higher
in the TFH-MG subgroup (86 vs. 50 and 67%). Written
informed consent was obtained from all participants.
This study was approved by the Institutional EthicalCommittee of Istanbul Medical Faculty and performed

Altınönder et al. Journal of Neuroinflammation(2024) 21:126

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Table 1 Demographic information of the tissue samples (15) included in the sequencing experiments
DNA (ID)
Age at TX
Tissue samples
10,97032
10,971
23
10,68566
10,86642
10,80728
12,17544
12,18333
10,81427
10,85016
12,07422
12,333
17
10,82722
12,07626
12,28747
10,80940

Sex

Anti-AChR titer (nmol/liter)

ImmunosuppressiveGroup

Pathological classification (WHO)

F
M
M
F
F
F
M
F
F
F
F
F
F
F
F

(+)
12.4
4.2
(+)
130
14.3
18
84.4
19.3
7.1
9.8
11.7
8.8
(+)
12.8

no
no
no
no
yes
yes
yes
no
no
no
no
yes
yes
yes
yes

TAMG
TAMG
TAMG
TAMG
TAMG
TAMG
TAMG
TFH-MG
TFH-MG
TFH-MG
TFH-MG
TFH-MG
TFH-MG
TFH-MG
TFH-MG

B2
B2
B1
B2
B2
B2
B2

TX: thymectomy

in accordance with the latest version of the Helsinki
Declaration.
RNA library preparation and sequencing

RNA sequencing libraries were prepared using an Illumina TruSeq RNA Exome kit (Illumina, Inc., San Diego,
CA, USA) according to the manufacturer`s protocol.
The RNA concentration was measured with a Nanodrop 2000c spectrophotometer (Thermo Scientific Inc.,
Waltham, MA, USA). Integrity was assessed using an
Agilent 2200 Tapestation instrument (Agilent Technologies, Santa Clara, CA, USA), and the percentages offragments larger than 200 nucleotides (DV200) were calculated. RNA samples (20 ∼ 100 ng) were used as input
based on the DV200 value. First strand cDNA syntheses
were performed at 25 °C for 10 min, 42 °C for 15 min and
70 °C for 15 min using random hexamers and ProtoScript
II Reverse Transcriptase (New England BioLabs Inc.). In
second-strand cDNA synthesis, the RNA templates were
removed, and a second replacement strand was generated by incorporating dUTP (in place of dTTP to keep
strand information) to generate ds cDNA. The bluntended cDNA was cleaned from the second strand reaction mix with beads. The 3` ends of the cDNA were thenadenylated, followed by the ligation of indexing adaptors.
PCR (15 cycles of 98 °C for 10 s, 60 °C for 30 s and 72 °C
for 30 s) was used to selectively enrich those DNA fragments that had adapter molecules on both ends and to
amplify the amount of DNA in the library.
The library was qualified using an Agilent 2200 Tapestation instrument and quantified using a QuantiFluor
dsDNA System (Promega). A 4-plex pool of libraries
was made by combining 200 ng of each DNA library.
The pooled DNA libraries were then mixed with captureprobes to target regions of interest. Hybridization was
performed by 18 cycles of 1-minute incubation, startingat 94 °C and then decreasing 2 °C per cycle. Then, streptavidin-coated magnetic beads were used to capture probes
hybridized to the target regions. The enriched libraries
were then eluted from the beads and prepared for a second round of hybridization and capture to ensure highspecificity of the capture regions. The enriched libraries were amplified by a second 10 cycles of PCR amplification (98 °C for 10 s, 60 °C for 30 s and 72 °C for 30 s)
followed by bead clean up. The final libraries were validated using Agilent High Sensitivity D1000 ScreenTapeon an Agilent 2200 TapeStation instrument. The size
distribution of the library ranged from approximately
200 bp–1 kb. Libraries were normalized, pooled and subjected to cluster and pair read sequencing for 150 cycles
on a HiSeqX10 instrument (Illumina, Inc. San Diego, CA,
USA) according to the manufacturer’s instructions.
Data analysis

Data were analyzed with the Illumina BaseSpace app
suite(https://www.illumina.com/products/by-type/
informatics-products/basespace-sequence-hub/apps/
rna-seq-alignment.html). The sequencing reads werealigned to Homo sapiens hg19 using STAR aligner.
Salmon was used for quantification of reference genes
and transcripts. Manta was used to detect gene fusions.
GC and mean coverage information for every target wascomputed using Picard. The gene counts, gene FPKMs,
principal component analysis and differential expression
results were produced by DESeq2.
For gene expression, the data were analyzed using oneway ANOVA, and differentially expressed genes (DEGs)
were defined as transcripts with more than 1.2-fold

Altınönder et al. Journal of Neuroinflammation(2024) 21:126

Table 2 Demographic information of the tissue samples
included in the qPCR experiments
DNA (ID)

10,250
10,254
10,685
10,866
10,971
12,282
12,653
12,676
12,71012,711
10,97012,701
Median age(range)10,237
10,617
10,81010,933
10,94812,098
12,23912,274
12,704
10,953
12,334
12,344
Median age(range)10,814
12,02912,04812,074
12,214
12,25012,251
12,31912,32812,662
12,70912,717
10,962
12,333
Median age(range)Patients

Age ofthymectomy

Sex

TAMG 1
TAMG 2
TAMG 3
TAMG 4
TAMG 5TAMG 6
TAMG 7
TAMG 8TAMG 9TAMG 10TAMG 11
TAMG 12

42
39
66
42
23
43
57
45
62
47
32
45
44 (23–66)

M
M
M
F
M
F
F
F
M
M
F
F

WHO
histologicalthymomaclassification
B2
B2B3
B1
B2
B2
B2
B1
B2
B2B1
B2B3
B2B3
B2B1

TOMA 1TOMA 2TOMA 3
TOMA 4
TOMA 5TOMA 6
TOMA 7
TOMA 8TOMA 9TOMA 10
TOMA 11
TOMA 12

59
53
43
43
77
52
61
46
27
62
63
62
56 (27–77)

F
F
F
F
F
F
M
M
F
M
M
F

AB
AB
AB
B2B3
B1B2
B2
B2B3
B2
AB
B1B2
B2B3
B1B2

TFH 1
TFH 2
TFH 3
TFH 4
TFH 5
TFH 6
TFH 7
TFH 8
TFH 9
TFH 10
TFH 11
TFH 12
TFH 13
TFH 14

27
12
25
22
25
26
31
22
32
37
26
21
42
17
25,5 (12–42)

F
F
F
F
F
F
F
F
F
M
M
F
F
F

change (FC) of expression at a false discovery rate (FDR)
adjusted P value less than 0.05.
Pathway analysis of DEGs was performed for geneenrichment analysis using the Ingenuity Pathway

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Analysis tool (IPA 8.0, Ingenuity Systems, and Redwood
City, CA).
RNA extraction and cDNA synthesis

Thymic tissues were preserved first in RNAlater® andthen stored in liquid nitrogen. RNA was extracted fromwhole thymic tissues following the protocol recommended by the manufacturer (Zymo Research Direct-zol
Miniprep Kit). The RNA pellet was dissolved in waterand stored at – 80 °C until cDNA synthesis.
All RNA samples were quantified using a spectrophotometer (NanoDrop, ND-2000, Thermo Fisher Scientific).
The ratio of absorbance at 260 nm and 280 nm was used
to determine RNA purity. Samples with a ratio ranging
between 1.7 and 2.1 were included in the relative quantitative PCR (qPCR) experiments.
cDNA was synthesized through a reverse transcription reaction according to the manufacturer’s protocol(Thermo Scientific) and stored at − 20 °C until qPCR.
Quantitative PCR (qPCR)

qPCR was performed on a LightCycler 480 (Roche Laboratories) with SYBR green using 30 ng (3 µl) cDNA, 0.5 µlof 10 µM each primer and 6.5 µl Mastermix (SensiFAST™
SYBR® No-ROX Kit, Meridian Bioscience). Relative
expression of selected genes was evaluated by comparing
to 18 S and HPRT as house-keeping genes by real-time
qPCR. The experimental run protocol was as follows:
preincubation at 95 °C for 8 min; amplification for 45
cycles (15 s at 95 °C, then 5 s at 30 °C, final extension for
10 s at 72 °C); and final melting curve analysis. For eachgene, the specificity of the PCR product was assessed byverifying a single peak in melting curve analysis.
The list of the genes and the primers (Sentromer DNA
Technologies) used in the study is shown in Supplementary Table 1.
Statistical analysis

Efficiencies for individual samples in qPCR were calculated using the LinRegPCR program [26]. Relative
gene expression was calculated using E−ΔCT. Differences
between two different groups were compared by unpaired(Mann‒Whitney U test) nonparametric tests. Values ofp < 0.05 were considered significant. The results are presented as the mean ± SEM.

Results
Global sequencing of thymic tissues of TAMG and TFH-MG
patients

Although the clinical picture of AChR + MG can be phenotypically similar in both TAMG and TFH-MG, thymic
pathologies implicate differential effects of the thymus.
To compare the possible effects of pathologies in these
disease subgroups, tissue samples from 7 TAMG andAltınönder et al. Journal of Neuroinflammation(2024) 21:126

Table 3A Pathways deteceted with differentially expressed
genes
Name
Fc Receptor-mediated Phagocytosis in
Macrophages and Monocytes
IL-8 Signaling
Pyridoxal 5’-phosphate Salvage Pathway
Remodeling of Epithelial Adherence Junctions
Macropinocytosis Signaling

p value
6.44E− 04

Overlap
3.2% 3/94

5.54E− 03
6.28E− 03
6.66E− 03
8.25E− 03

1.5% 3/200
3.0% 2/66
2.9% 2/68
2.6% 2/76

8 TFH-MG patients were evaluated in this study. The
TFH-MG group consisted of women, whereas there
was no significant dominance of women in the TAMG
group. TAMG patients were 9 years older than TFH-MG
patients. In total, 43% and 50% of patients were treatedwith IS agents in the TAMG and TFH-MG groups before
thymectomy. As the proportions of patients on treatment
were similar in the thymoma and follicular hyperplasia
groups, the treatment effect was not evaluated separately
in the groups.
RNA sequencing and comparison between expressed
genes revealed 51 DEGs in the whole group. Amongthese genes, the expression of 24 was higher in TAMG,
whereas 27 genes were upregulated in TFH-MG tissue
samples (Supplementary Table 2).
To independently validate gene expression changes inthe thymus, 3 DEGs with FC > 1.8 and detailed/functionalannotation (TF, GATA3, FCLR2 and FLT4) were selected
for further validation by qPCR using RNA samples from
16 MG patients (8 TAMG and 8 TFH-MG). The expression levels of these four mRNAs in TAMG cells were
not significantly different from those in TFH-MG cells,

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among which TF, GATA3, FCLR2 and FLT4 displayed
1.8-, 0.55-, 0.14-, and 1.89-fold differences in expression,respectively. Out of the 4 DEGs evaluated, only FCLR2
was significantly different between cases and controls.
However, the TFH-MG group had higher expression of
FCLR2 than the TAMG group (3.1 vs. 0.5, p = 0.015).
Different pathways are associated with TAMG and TFH-MG
cells

To better understand the role of differentially expressed
genes in underlying mechanisms in TAMG, we performed pathway analyses for upregulated mRNAs with
an absolute fold-change ≥ 2 and identified pathways
known to be important in TAMG [27]. The analysis ofthese 51 genes showed high connectivity between transcripts with activation of immune function, inflammation, and enrichment of canonical pathways, such as
TREM1 and Toll-like receptor signaling (Table 3A).
Gene enrichment analysis of these findings implicated
ESR1 (estrogen receptor alpha)-, NFKB (nuclear factor
kappa B)-, HNF4A (hepatocyte nuclear factor 4 alpha)-,and EGLN3 (PHD3, prolyl hydroxylase enzymes)-related
activities as increased in TAMG patients compared to
TFH-MG patients (Table 3B).
Targeted gene analysis reveals increased HIF1A expressionin TAMG

Increased Th17 and decreased Treg cell activity were
demonstrated in TAMG [19, 20]. HIF-1 transcriptionallyactivates RORγt, favoring Th17 cells while inhibiting Treg
cells by targeting FOXP3 for degradation [24]. PHD3
(EGLN3) is a prolyl hydroxylase enzyme that regulates

Table 3B Some of the differentially expressed genes between TAMG and THP-MG samples and the related pathways derived on these
genes
Genes
TBC1D3D (TBC1 domain family member 3D)
TBC1D3I (TBC1 domain family member 3I)
TBC1D3K (TBC1 domain family member 3 K)
HSD17B3 (hydroxysteroid 17-beta dehydrogenase 3)
LRRK2 (leucine rich repeat kinase 2)
MRNIP (MRN complex interacting protein)
ACCS (1-amino-cyclopropane-1-carboxylate synthase homolog)
FAM153A (family with sequence similarity 153 member A)
OSBPL10 (oxysterol binding protein like 10)
POU6F1 (POU class 6 homeobox 1)
FLT4 (fms related receptor tyrosine kinase 4)
PRKCE (protein kinase C epsilon)
FCRL2 (Fc receptor like 2)
SNORA71D (small nucleolar RNA, H/ACA box 71D)
SNORA8 (small nucleolar RNA, H/ACA box 8)
PPFIBP2 (PPFIA binding protein 2)
PPIP5K1 (diphosphoinositol pentakisphosphate kinase 1)

log2 (Fold
Change)
25.45
25.45
25.45
3.51
1.36
1.75
1.19
2.77
2.27
2.15
1.86
1.43
3.35
5.84
3.65
1.27
1.2

Std. Err.
Log2
2.98
2.98
2.98
0.87
0.36
0.34
0.32
0.68
0.61
0.51
0.38
0.36
0.81
1.51
0.97
0.31
0.3

q Value

Pathways

0.000
0.000
0.000
0.024
0.044
0.000
0.049
0.022
0.049
0.015
0.001
0.029
0.020
0.036
0.046
0.024
0.022

GLN3 (PHD3, prolyl hydroxylase
enzymes)
HNF4A (hepatocyte nuclear factor 4 alpha)

NFKB (nuclear factor kappa B)

ESR1 (estrogen receptor alpha)Altınönder et al. Journal of Neuroinflammation(2024) 21:126

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Fig. 1 Relative expression of HIF1A (TAMG n = 9, TFH-MG n = 13) and related EP300 (TAMG n = 9, TFH-MG n = 14) and EGLN3 (TAMG n = 9, TFH-MG n = 13)
genes in thymic samples of TAMG and TFH-MG patients. *p = 0.049

Fig. 2 Relative expression of selected genes for IL-17-related activity: RORC (TAMG n = 12, TFH-MG n = 13), STAT3 (TAMG n = 13, TFH-MG n = 14) and CCR6(TAMG n = 12, TFH-MG n = 13) expression in thymic samples of patients. *p = 0.039

HIF-1 levels by hydroxylating HIF-1α subunits when
O2 is available [28]. Increases in PDH3 mRNA levels
have been demonstrated in hypoxia [29]. Under hypoxic
conditions, HIF-1α is stabilized and binds to hypoxiaresponsive elements (HREs) in the promoters of its target
genes. HIF-1 requires its coactivator p300 (encoded byEP300) to exert its transcriptional activity [30].
Increased EGLN3 (PHD3) expression in the sequence
analysis of TAMG samples supported the involvement of
the HIF-1 signaling pathway in TAMG. To validate thisfinding, we measured HIF1A, EGLN3 and EP300 expression in thymic tissue samples from TAMG or TFH-MG
patients by using qPCR. HIF1A expression was significantly increased (2.6-fold higher) in TAMG cells compared to that in TFH-MG cells (p = 0.049). Interestingly,
the overexpression of HIF1A correlated with its downstream signaling pathway. The expression levels of EP300
and EGLN3 were both 1.9-fold higher in TAMG cells
than in TFH-MG cells, without reaching significance(Fig. 1).
The Th17 lineage is involved in TAMG

The expression of Th17-related genes was analyzed toinvestigate whether elevated levels of HIF-1 may have

led to increased Th17 involvement in TAMG. RORγt is a
Th17 lineage-specific transcription factor and is encodedby the RORC gene. STAT3 is one of the transcription factors that regulates RORγt and IL17A [31], while CCR6 isa surrogate Th17 marker [32]. RORC expression was significantly increased in TAMG cells compared to that in
TFH-MG cells (1.8-fold, p = 0.039). Moreover, genes performing as drivers of the Th17 response revealed a trend
for an increase in expression levels. STAT3 and CCR6
expression in TAMG cells was 3.2-fold and 2.8-foldhigher than that in TFH-MG cells (Fig. 2).
Treg-related genes are underexpressed in TAMG

Since HIF-1 inhibits Treg differentiation [24], we performed a similar analysis for Treg-related genes. In thethymus, IL-2 is essential for Treg differentiation [33].
The expression of IL2 was significantly decreased in
TAMG cells compared to that in TFH-MG cells (0.16fold, p = 0.047). Similarly, FOXP3 expression in TAMG
revealed a decreasing trend compared to that in TFHMG (0.19-fold) (Fig. 3). The expression levels of Tregrelated molecules, such as CTLA-4 and GITR, weresimilar in both groups (Supplementary Fig. 1).

Altınönder et al. Journal of Neuroinflammation(2024) 21:126

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Fig. 3 Relative expression of selected genes for Treg-related activity: IL2 (TAMG n = 10, TFH-MG n = 13) and FOXP3 (TAMG n = 11, TFH-MG n = 13) in thymic
samples of patients. *p = 0.047

Fig. 4 Relative expression of selected genes for Treg-related activity: IL2(TAMG n = 10, TFH-MG n = 13) and FOXP3 (TAMG n = 11, TFH-MG n = 13) inthymic samples of patients. *p = 0.047

Impairment of the Th17/Treg balance in TAMG

This expression analysis indicated a disturbance of theTh17/Treg balance in the pathogenesis of TAMG. To
support this finding, we evaluated both changes in conjunction by analyzing the ratios of the relative expression of RORC to FOXP3 and HIF1A to FOXP3. The ratiosof both RORC and HIF1A to FOXP3 were significantly
higher in TAMG cells than in TFH-MG cells (p = 0.003
and p < 0.001, Fig. 4).
No difference in expression levels between TAMG and
TOMA

To rule out/dissect thymoma-related changes, we also
compared the gene expression in a thymoma (TOMA)
cohort with that in the TAMG samples. We did notobserve any statistically significant differences in gene
expression between TAMG and TOMA (Supplementary
Fig. 2).

Discussion
Thymoma is a rare epithelial tumor that originates fromthe thymus and is frequently associated with MG. The
role of thymoma in the development of concomitant
MG is not fully understood. As thymic follicular hyperplasia or even thymic age-dependent atrophy can also be
observed alongside MG development, thymus pathologies may not be inducing but only accompanying thedisease. In this study, the thymus was approached as
the decisive organ for disease development, and thymic
pathologies were compared with each other to determine
possible effects on the disease. By RNA-sequencing analysis of thymic samples, several differentially expressed
genes and pathways were detected, which were then partially confirmed by expression analysis. Thymoma-relatedchanges have pointed to HIF-1 upregulation at the transcriptional level, which may be the driver of the previously described IL-17/Treg imbalance in the thymus.
Thymoma tissue has been investigated intensively for
its role in autoimmunization against autoantigens in MG.
Skeletal muscle antigens have been reported as potential autoantibody targets in TAMG [34], and incomplete
autoantigens, proteins cross-reacting with AChR, titin,
and ryanodine receptor epitopes have also been demonstrated in MG [10, 35–37]. A further comprehensive
analysis of thymic epithelial tumors revealed that TAMG
was associated with intratumoral overexpression of genesthat show sequence similarity with major autoimmune
targets as well [38]. Similar to our results, the expression of genes involved in immunity showed no significant
differences between TAMG and TOMA in the TCGA
thymoma study [38]. In a more recent report utilizing
single-cell RNA-seq and immunohistological examination of MG-type thymoma specimens, neuromuscular
expression was shown to be limited in a subpopulationof medullary thymic epithelial cells (mTECs). Therefore,
TAMG develops in an atypical immune microenvironment that leads to B cell maturation and ectopic expression of neuromuscular antigens by epithelial cells [39].

Altınönder et al. Journal of Neuroinflammation(2024) 21:126

Increased expression of neuromuscular antigens could
not be demonstrated in the present study. The relatively
low number of samples and heterogeneity of cellularcomponents in whole thymic tissue may account for the
discordance with the reported findings. Although notsignificant, gender and age differences among the study
groups might have also influenced the different genetic
expression patterns in the present study.
A previous analysis of functional pathways with metabolic and immunological relevance has also shown differences in TAMG compared to TOMA. The comparison of
expression profiles of simple thymoma with TAMG identified candidate genes/pathways [27]. The link between
the nuclear factor-κB/autoimmune regulator pathways
and MG pathogenesis was supported in this study bygene enrichment analysis. Further analysis of thymic cellgroups for their contribution to this pathway is needed.
Potentially relevant functional pathways were also
compared among thymoma histological subtypes (typeAB vs. B2 thymomas) in the TCGA data set, showing thatdifferent molecular pathways are upregulated in different thymoma histotypes [40]. In the present sample set,
the group was relatively homogenous, and differences
between thymic histotypic pathologies could not be
evaluated.
Targeted expression analysis in this study revealed
HIF1A upregulation in TAMG, potentially driving thepreviously described imbalance between Th17 cells andTregs in the thymus. HIF-1 is a heterodimeric transcription factor consisting of a highly regulated oxygen-sensitive HIF-1α subunit (HIF1A) and a constitutively present
β subunit (HIF1B) [41]. The hypoxic microenvironment
and the distorted immune landscape of thymoma may be
responsible for the failure of T cell tolerance and TAMG
initiation. In a recent study, HIF3A was also differentiallyexpressed in thymoma patients with MG [42], pointing to
the relationship between hypoxia and TAMG. Increased
expression of HSP60 demonstrated in thymoma tissues
may also be involved in the response of the tumor tissue
to hypoxic stress [10]. Indeed, HIF1A expression was also
increased in TAMG cells compared to that in TFH-MG
cells in the present study. As reported earlier as a risk
factor [43], TAMG patients were older than TFH-MG
patients in this cohort. In the TFH-MG group, only early
onset MG patients were included, as the older patientsare not be thymectomized. The observed difference may
be due to the age difference between the groups, but thefact that this difference was not observed in the TOMA
group emphasizes the role of the thymus in the development of MG in TAMG.
HIF-1-related mechanisms are regulated by hydroxylation with EGLN2 and EGLN3.
The VHL gene encodes pVHL, which is a part of theE3 ubiquitin ligase complex and controls the cellular

Page 8 of 10

levels of HIF-1α. Intracellular prolyl hydroxylases (PHD2,
PHD3) hydroxylate HIF-1α under normoxic conditions.
The VHL protein complex binds to the hydroxylated HIF
subunits, causing the degradation of HIF-1α. Von Hippel‒Lindau (VHL) disease is a hereditary condition witha germline pathogenic variation of the VHL gene. Lossof function of the VHL complex in VHL disease causes
HIF-1α accumulation and thus pseudohypoxia. Cases
with both VHL disease and MG have been reported
recently [44]. Although these cases might be coincidental, a link between VHL disease and MG with overlapping molecular mechanisms has also been proposed.
Another indirect finding in the same direction has beenreported in TAMG. USP29, a deubiquitylase that stabilizes HIF-1 and posttranscriptionally regulates RORγt,was increased in TAMG patients [45]. Thus far, the associations of HIF-1-related mechanisms with the pathogenesis of TAMG need further clarification.
The present study has certain limitations. First, thenumber of samples included in the sequencing group
was limited, and the proportion of patients receiving
immunosuppressive (IS) treatment was relatively high.
However, as the proportion of patients with treatment
was similar in the TFH-MG and TAMG groups, treatment itself is unlikely to affect the results in the wholegroup. Second, the present findings are at the transcriptional level and need further verification at the protein
level. Thirdly, the age-related changes in the thymic tissue could not be ruled out in the sample groups, as theages of the patients were different in the study goups
directly inherent to the disease subgroups.In conclusion,
we suggest that the hypoxic environment of thymoma
is a potential driver of TAMG but not TFH-MG. HIF1A
upregulation in thymic tissues of TAMG patients correlated with a higher RORC/FOXP3 ratio, indicating its
role in Th17/Treg imbalance. Further studies focusing on
the causality between HIF-1 and TAMG development are
warranted. A better understanding of the link between
hypoxic changes and the thymoma microenvironment
could help in the development of novel treatment strategies for TAMG.
This study was supported by TÜBİTAK (116S317)
and I.U. BAP (36227and36288).
Abbreviations
MG	myasthenia gravis
TOMA	thymoma without myasthenia gravis
TAMG	thymoma-associated myasthenia gravis
TFH	thymic follicular hyperplasia

Supplementary Information

The online version contains supplementary material available at https://doi.
org/10.1186/s12974-024-03095-7.
Supplementary Material 1

Altınönder et al. Journal of Neuroinflammation(2024) 21:126

Page 9 of 10

Author contributions
İ.A., M.K. and S.P.Y. have performed all experiments, analyzed the data andwrote the manuscript. A.Ç., H.D., Y.P. have chosen the patients, planned thestudy. G.Y. has evaluated the pathology and contributed to the analysis.
B.O. has performed the surgery and selection the tissue samples. A.H.S.
has performed the analysis of the sequencing data and evaluation of thedata. G.S.D has planned and followed all the steps of the study. All authors
contributed to the manuscript and reviewed it.

12. Strobel P, Rosenwald A, Beyersdorf N, Kerkau T, Elert O, Murumagi A, Sillanpaa
N, Peterson P, Hummel V, Rieckmann P, et al. Selective loss of regulatory T cells
in thymomas. Ann Neurol. 2004;56:901–4.
13. Hoffacker V, Schultz A, Tiesinga JJ, Gold R, Schalke B, Nix W, Kiefer R, MullerHermelink HK, Marx A. Thymomas alter the T-cell subset composition in the
blood: a potential mechanism for thymoma-associated autoimmune disease.
Blood. 2000;96:3872–9.
14. Buckley C, Douek D, Newsom-Davis J, Vincent A, Willcox N. Mature, long-lived
CD4 + and CD8 + T cells are generated by the thymoma in myasthenia gravis.
Ann Neurol. 2001;50:64–72.
15. Strobel P, Helmreich M, Menioudakis G, Lewin SR, Rudiger T, Bauer A, Hoffacker V, Gold R, Nix W, Schalke B, et al. Paraneoplastic myasthenia gravis correlates with generation of mature naive CD4(+) T cells in thymomas. Blood.
2002;100:159–66.
16. Balandina A, Saoudi A, Dartevelle P, Berrih-Aknin S. Analysis of
CD4 + CD25 + cell population in the thymus from myasthenia gravis patients.
Ann N Y Acad Sci. 2003;998:275–7.
17. Luther C, Poeschel S, Varga M, Melms A, Tolosa E. Decreased frequency of
intrathymic regulatory T cells in patients with myasthenia-associated thymoma. J Neuroimmunol. 2005;164:124–8.
18. Kohler S, Keil TOP, Hoffmann S, Swierzy M, Ismail M, Ruckert JC, AlexanderT, Meisel A. CD4(+) FoxP3(+) T regulatory cell subsets in myasthenia gravis
patients. Clin Immunol. 2017;179:40–6.
19. Chen Y, Zhang XS, Wang YG, Lu C, Li J, Zhang P. Imbalance of Th17 and Tregs
in Thymoma may be a pathological mechanism of myasthenia gravis. Mol
Immunol. 2021;133:67–76.
20. Cebi M, Cakar A, Erdogdu E, Durmus-Tekce H, Yegen G, Ozkan B, Parman Y,
Saruhan-Direskeneli G. Thymoma patients with or without myasthenia gravis
have increased Th17 cells, IL-17 production and ICOS expression. J Neuroimmunol. 2023;381:578129.
21. Gradolatto A, Nazzal D, Truffault F, Bismuth J, Fadel E, Foti M, Berrih-Aknin S.
Both Treg cells and tconv cells are defective in the Myasthenia gravis thymus:
roles of IL-17 and TNF-alpha. J Autoimmun. 2014;52:53–63.
22. Villegas JA, Bayer AC, Ider K, Bismuth J, Truffault F, Roussin R, Santelmo N,
Le Panse R, Berrih-Aknin S, Dragin N. Il-23/Th17 cell pathway: a promisingtarget to alleviate thymic inflammation maintenance in myasthenia gravis. J
Autoimmun. 2019;98:59–73.
23. Masoud GN, Li W. HIF-1alpha pathway: role, regulation and intervention forcancer therapy. Acta Pharm Sin B. 2015;5:378–89.
24. Dang EV, Barbi J, Yang HY, Jinasena D, Yu H, Zheng Y, Bordman Z, Fu J, Kim Y,
Yen HR, et al. Control of T(H)17/T(reg) balance by hypoxia-inducible factor 1.
Cell. 2011;146:772–84.
25. Marx A, Chan JKC, Chalabreysse L, Dacic S, Detterbeck F, French CA, Hornick
JL, Inagaki H, Jain D, Lazar AJ, et al. The 2021 WHO classification of tumors of
the Thymus and Mediastinum: what is New in Thymic Epithelial, Germ Cell,and mesenchymal tumors? J Thorac Oncol. 2022;17:200–13.
26. Ruijter JM, Ramakers C, Hoogaars WM, Karlen Y, Bakker O, van den Hoff MJ,
Moorman AF. Amplification efficiency: linking baseline and bias in the analysis of quantitative PCR data. Nucleic Acids Res. 2009;37:e45.
27. Guo F, Wang CY, Wang S, Zhang J, Yan YJ, Guan ZY, Meng FJ. Alteration ingene expression profile of thymomas with or without myasthenia gravis
linked with the nuclear factor-kappaB/autoimmune regulator pathway tomyasthenia gravis pathogenesis. Thorac Cancer. 2019;10:564–70.
28. Majmundar AJ, Wong WJ, Simon MC. Hypoxia-inducible factors and the
response to hypoxic stress. Mol Cell. 2010;40:294–309.
29. Appelhoff RJ, Tian YM, Raval RR, Turley H, Harris AL, Pugh CW, Ratcliffe PJ,
Gleadle JM. Differential function of the prolyl hydroxylases PHD1, PHD2,
and PHD3 in the regulation of hypoxia-inducible factor. J Biol Chem.
2004;279:38458–65.
30. Lee JW, Bae SH, Jeong JW, Kim SH, Kim KW. Hypoxia-inducible factor(HIF-1)alpha: its protein stability and biological functions. Exp Mol Med.
2004;36:1–12.
31. Capone A, Volpe E. Transcriptional regulators of T Helper 17 cell differentiation in Health and Autoimmune diseases. Front Immunol. 2020;11:348.
32. Annunziato F, Cosmi L, Santarlasci V, Maggi L, Liotta F, Mazzinghi B, Parente E,Filì L, Ferri S, Frosali F, et al. Phenotypic and functional features of human Th17
cells. J Exp Med. 2007;204:1849–61.
33. Chinen T, Kannan AK, Levine AG, Fan X, Klein U, Zheng Y, Gasteiger G, Feng Y,
Fontenot JD, Rudensky AY. An essential role for the IL-2 receptor in T(reg) cellfunction. Nat Immunol. 2016;17:1322–33.
34. Gilhus NE. Myasthenia Gravis. N Engl J Med. 2016;375:2570–81.

Funding
This study was supported by TÜBİTAK (116S317) and I.U. BAP (36227).
Data availability
All datasets can be accessed by request from the authors.

Declarations
Ethical approval
This study was approved by the Institutional Ethical Committee of Istanbul
Medical Faculty and performed in accordance with the latest version of theHelsinki Declaration.
Competing interests
None of the authors has any potential financial conflict of interest related tothis manuscript.
Received: 21 August 2023 / Accepted: 7 April 2024

References
1. Zekeridou A, McKeon A, Lennon VA. Frequency of synaptic autoantibody
accompaniments and neurological manifestations of Thymoma. JAMA
Neurol. 2016;73:853–9.
2. Travis WD, Brambilla E, Burke AP, Marx A, Nicholson AG. Introduction to the2015 World Health Organization Classification of Tumors of the lung, Pleura,
Thymus, and heart. J Thorac Oncol. 2015;10:1240–2.
3. Cufi P, Dragin N, Ruhlmann N, Weiss JM, Fadel E, Serraf A, Berrih-Aknin S, Le
Panse R. Central role of interferon-beta in thymic events leading to myasthenia gravis. J Autoimmun. 2014;52:44–52.
4. Cufi P, Soussan P, Truffault F, Fetouchi R, Robinet M, Fadel E, Berrih-Aknin S, Le
Panse R. Thymoma-associated myasthenia gravis: on the search for a pathogen signature. J Autoimmun. 2014;52:29–35.
5. Strobel P, Murumagi A, Klein R, Luster M, Lahti M, Krohn K, Schalke B, Nix W,
Gold R, Rieckmann P, et al. Deficiency of the autoimmune regulator AIRE
in thymomas is insufficient to elicit autoimmune polyendocrinopathy syndrome type 1 (APS-1). J Pathol. 2007;211:563–71.
6. Marx A, Pfister F, Schalke B, Saruhan-Direskeneli G, Melms A, Strobel P. The
different roles of the thymus in the pathogenesis of the various myasthenia
gravis subtypes. Autoimmun Rev. 2013;12:875–84.
7. Marx A, Porubsky S, Belharazem D, Saruhan-Direskeneli G, Schalke B, Strobel
P, Weis CA. Thymoma related myasthenia gravis in humans and potential
animal models. Exp Neurol. 2015;270:55–65.
8. Scarpino S, Di Napoli A, Stoppacciaro A, Antonelli M, Pilozzi E, Chiarle R,
Palestro G, Marino M, Facciolo F, Rendina EA, et al. Expression of autoimmune
regulator gene (AIRE) and T regulatory cells in human thymomas. Clin Exp
Immunol. 2007;149:504–12.
9. Suzuki E, Kobayashi Y, Yano M, Fujii Y. Infrequent and low AIRE expression inthymoma: difference in AIRE expression among WHO subtypes does notcorrelate with association of MG. Autoimmunity. 2008;41:377–82.
10. Iacomino N, Scandiffio L, Conforti F, Salvi E, Tarasco MC, Bortone F, Marcuzzo
S, Simoncini O, Andreetta F, Pistillo D, et al. Muscle and muscle-like Autoantigen expression in Myasthenia gravis Thymus: possible molecular hint for
autosensitization. Biomedicines. 2023;11:732.
11. Luther C, Adamopoulou E, Stoeckle C, Brucklacher-Waldert V, Rosenkranz
D, Stoltze L, Lauer S, Poeschel S, Melms A, Tolosa E. Prednisolone treatment
induces tolerogenic dendritic cells and a regulatory milieu in myasthenia
gravis patients. J Immunol. 2009;183:841–8.

Altınönder et al. Journal of Neuroinflammation(2024) 21:126

35. Marx A, Wilisch A, Schultz A, Greiner A, Magi B, Pallini V, Schalke B, Toyka K, Nix
W, Kirchner T, Muller-Hermelink HK. Expression of neurofilaments and of atitin epitope in thymic epithelial tumors. Implications for the pathogenesis ofmyasthenia gravis. Am J Pathol. 1996;148:1839–50.
36. Mygland A, Kuwajima G, Mikoshiba K, Tysnes OB, Aarli JA, Gilhus NE. Thymomas express epitopes shared by the ryanodine receptor. J Neuroimmunol.
1995;62:79–83.
37. Schultz A, Hoffacker V, Wilisch A, Nix W, Gold R, Schalke B, Tzartos S, MullerHermelink HK, Marx A. Neurofilament is an autoantigenic determinant inmyasthenia gravis. Ann Neurol. 1999;46:167–75.
38. Radovich M, Pickering CR, Felau I, Ha G, Zhang H, Jo H, Hoadley KA, Anur P,
Zhang J, McLellan M, et al. The Integrated genomic Landscape of Thymic
epithelial tumors. Cancer Cell. 2018;33:244–58. e210.
39. Yasumizu Y, Ohkura N, Murata H, Kinoshita M, Funaki S, Nojima S, Kido K,
Kohara M, Motooka D, Okuzaki D, et al. Myasthenia gravis-specific aberrant
neuromuscular gene expression by medullary thymic epithelial cells inthymoma. Nat Commun. 2022;13:4230.
40. Yamada Y, Weis CA, Thelen J, Sticht C, Schalke B, Strobel P, Marx A. Thymoma
Associated Myasthenia Gravis (TAMG): Differential expression of functional
pathways in relation to MG Status in different Thymoma Histotypes. Front
Immunol. 2020;11:664.
41. Semenza GL. Hypoxia-inducible factor 1 (HIF-1) pathway. Sci STKE 2007,
2007:cm8.

Page 10 of 10

42. Lee MC, Hsiao TH, Chuang HN, Lee LW, Chi PL, Tsai HM, Mao CL, Hsu
CP. Molecular profiling of thymoma with myasthenia gravis: risk factorsof developing myasthenia gravis in thymoma patients. Lung Cancer.
2020;139:157–64.
43. Lefeuvre CMJ, Payet CA, Fayet OM, Maillard S, Truffault F, Bondet V, Duffy D, deMontpreville V, Ghigna MR, Fadel E et al. Risk factors associated with myasthenia gravis in thymoma patients: the potential role of thymic germinal centers.
J Autoimmun 2020, 106.
44. Norata D, Peri M, Giammalva GR, Lupica A, Paolini F, Incorvaia L, Badalamenti G, Gristina V, Galvano A, Russo A, et al. Immunological aspects of Von
Hippel-Lindau Disease: a Focus on Neuro-Oncology and Myasthenia Gravis.
Diagnostics (Basel). 2023;13:144.
45. Xia D, Pan L, Fu X, Meng Y, Li M, Wang W, Wang Z. Deubiquitinase USP29
correlates RORgammat expression and its association with thymoma myasthenia gravis. Am J Transl Res. 2022;14:8632–9.

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