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\begin{document}

\title{Interleukin 17 receptor E identifies heterogeneous T helper 17 cells in
peritoneal fluid of severe endometriosis patients}



\author[1]{Yanping Jiang}%
\author[1]{Lu Wang}%
\author[1]{Yaqin Peng}%
\author[1]{Jian Qin}%
\author[1]{Aili Tan}%
\author[1]{Shujun Wang}%
\affil[1]{Renmin Hospital of Wuhan University}%


\vspace{-1em}



  
  \date{May 29, 2024}


\begingroup
\let\center\flushleft
\let\endcenter\endflushleft
\maketitle
\endgroup





\selectlanguage{english}
\begin{abstract}
Endometriosis is a chronic inflammatory disorder resulting in pelvic
pain and infertility. The role of T helper 17 (Th17) cells in
endometriosis remains elusive. In this study, through detecting CXCR3,
CCR4, CCR10, CCR6, interleukin-17 Receptor E (IL-17RE), and CD27, live
ROR\selectlanguage{greek}γ\selectlanguage{english}t-and-IL-17A-expressing Th17 cells were distinguished and sorted
from peritoneal fluid (PF) of patients with stage III and IV
endometriosis. Furthermore, we found that IL-17RE and CD27 were the
labels of heterogeneous PF Th17 subsets, i.e. IL-17RE-CD27- subset,
IL-17RE+CD27- subset, and IL-17RE+CD27+ subset. The former two subsets
expressed higher IL-17A, GM-CSF, and IL-22 and were more proliferative
than the latter subset. RNA-Seq analysis on IL-17RE+ Th17 subset and
IL-17RE- Th17 subset revealed up-regulation of genes involved in
oxidative phosphorylation and electron transport chain in IL-17RE+ Th17
subset relative to IL-17RE- Th17 subset. Consistently, the IL-17RE+ Th17
subset produced more adenosine triphosphate (ATP) and reactive oxygen
species (ROS) than IL-17RE- Th17 subset. In conclusion, this study
provides a novel method to detect and isolate live PF Th17 cells from
endometriosis patients and unveils the functional and metabolic
heterogeneity of PF Th17 subsets. Therefore, it sheds light on the
elucidation of molecular mechanisms that modulate the function of
pathological Th17 cells in endometriosis.%
\end{abstract}%



\sloppy


\textbf{Interleukin 17 receptor E identifies heterogeneous T helper 17
cells in peritoneal fluid of severe endometriosis patients}

Yanping Jiang\textsuperscript{1}, Lu Wang*\textsuperscript{1} ,Yaqin
Peng\textsuperscript{1}, Jian Qin\textsuperscript{2}, Aili
Tan\textsuperscript{1}, Shujun Wang\textsuperscript{1}

*Co-first authors

1. Department of Gynaecology, Renmin Hospital of Wuhan University,
Wuhan, Hubei 430060

2. Renmin Hospital of Wuhan University, Wuhan, Hubei 430060

\emph{Correspondence to:}Professor Yanping Jiang, Department of
Gynaecology, Renmin Hospital of Wuhan University, 238 Jiefang Road,
Wuhan, Hubei 430060, P.R. China E-mail: jiang\_yanping@whu.edu.cn

\emph{\textbf{Running title:} IL-17RE marks Th17 heterogeneity in
endometriosis}

\textbf{Abstract:} Endometriosis is a chronic inflammatory disorder
resulting in pelvic pain and infertility. The role of T helper 17 (Th17)
cells in endometriosis remains elusive. In this study, through detecting
CXCR3, CCR4, CCR10, CCR6, interleukin-17 Receptor E (IL-17RE), and CD27,
live ROR\selectlanguage{greek}γ\selectlanguage{english}t-and-IL-17A-expressing Th17 cells were distinguished and
sorted from peritoneal fluid (PF) of patients with stage III and IV
endometriosis. Furthermore, we found that IL-17RE and CD27 were the
labels of heterogeneous PF Th17 subsets, i.e. IL-17RE-CD27- subset,
IL-17RE+CD27- subset, and IL-17RE+CD27+ subset. The former two subsets
expressed higher IL-17A, GM-CSF, and IL-22 and were more proliferative
than the latter subset. RNA-Seq analysis on IL-17RE+ Th17 subset and
IL-17RE- Th17 subset revealed up-regulation of genes involved in
oxidative phosphorylation and electron transport chain in IL-17RE+ Th17
subset relative to IL-17RE- Th17 subset. Consistently, the IL-17RE+ Th17
subset produced more adenosine triphosphate (ATP) and reactive oxygen
species (ROS) than IL-17RE- Th17 subset. In conclusion, this study
provides a novel method to detect and isolate live PF Th17 cells from
endometriosis patients and unveils the functional and metabolic
heterogeneity of PF Th17 subsets. Therefore, it sheds light on the
elucidation of molecular mechanisms that modulate the function of
pathological Th17 cells in endometriosis.

\textbf{Keywords:}Endometriosis, T helper 17 (Th17) cells, Interleukin
17~Receptor E, CD27, Oxidative phosphorylation, Electron transport
chain.

\textbf{Introduction}

Featuring the presence of endometrial tissue outside the uterine cavity,
endometriosis is a chronic inflammatory disorder resulting in pelvic
pain and infertility {[}1{]}. Various immune cell types including
neutrophils, macrophages, natural killer (NK) cells, dendritic cells,
and T lymphocytes play significant roles in the initiation and
progression of endometriosis {[}2{]}. Immune cells secret biochemical
factors that facilitate endometriotic cell division, invasion, as well
as angiogenesis {[}3{]}. However, the exact functions and regulatory
mechanisms of these immune cells have not been thoroughly understood.

Previous findings suggest that imbalanced T cell subsets and/or T cell
dysfunction significantly contribute to the elevation of pathological
cytokines and inflammatory reactions at endometriosis tissues. Cytokines
released by peritoneal T cells are predominantly T helper 2 (Th2)
cytokines in endometriosis tissue {[}4{]}. Regulatory T cell deficiency
seems to promote local inflammation, angiogenesis, and facilitate the
attachment and growth of endometrial implants {[}5{]}. Recent studies
have revealed the role of T helper 17 (Th17) cells in endometriosis
pathophysiology. Th17 cells are defined by the secretion of IL-17A, a
pro-inflammatory cytokine crucial for the development of inflammatory
and autoimmune diseases. The proportion of Th17 cells in endometriosis
lesions is higher than that in the normal endometrium {[}6{]}, and their
high proportion in peritoneal fluid (PF) is related to the severity of
endometriosis {[}7{]}. Additionally, the functional features and
underlying regulatory mechanisms of Th17 cells in endometriosis are yet
to be completely understood. It is, therefore, important to isolate live
Th17 cells from blood, lymphoid organs, PF, and endometriosis lesions to
disclose the molecular characteristics critical to their activities.
Unfortunately, previous and current Th17 studies in endometriosis
patients rely on intracellular staining of IL-17A for flow cytometry
analysis, making the isolation of live Th17 cells impractical. A novel
approach to sort live Th17 cells is in urgent demand for endometriosis
research. Several research groups have used an array of chemokine
receptors to dissect T cell subsets including Th17 cells in other
diseases {[}8-11{]}. However, whether these receptors apply to Th17
detection in endometriosis remains untested.

In the present study, we use a set of cell surface markers to detect and
sort live Th17 cells from PF of patients with advanced/severe
endometriosis. We found that CCR4, CCR6, IL-17RE, and CD27 defined live
PF Th17 cells. Moreover, RNA-Seq analysis revealed significant distinct
transcript profiles of PF Th17 subpopulations. Therefore, our data
disclose the heterogeneity of PF Th17 cells in endometriosis.

\textbf{Materials and methods}

\textbf{Patients}

This study was approved by the Research Ethics Committee of Renmin
Hospital of Wuhan University with written informed consent obtained from
all participants. Between April 2018 and November 2020 at the Department
of Gynaecology, Renmin Hospital of Wuhan University, 48 women with stage
III-IV endometriosis according to the revised American~Fertility~Society
score (r-AFS) (aged 28-35, mean age=30.3 years) were recruited for
analysis.

\textbf{Enrichment of mononuclear cells from PF}

Under general anesthesia, PF was collected from the pouch of Douglas at
the start of laparoscopy. Any blood-containing PF was discarded and not
included in the study. PF mononuclear cells were overlaid on the
lymphocyte separation medium (Sigma-Aldrich) and enriched by
density-gradient centrifugation at 400 rcf~for 20 minutes. Cells in the
interface were harvested, washed with PBS, and incubated in 200 \selectlanguage{greek}μ\selectlanguage{english}l of
red blood cell~lysis buffer (ThermoFisher) for 15 minutes at room
temperature to remove any residual red blood cells. The cells were then
washed with PBS and suspended in PBS containing 2 mM~EDTA~and 0.5\%
bovine serum albumin (BSA) for flow cytometry.

\textbf{Flow cytometry}

For Th17 detection and sorting, the following antibodies were used:
APC/Cy7 anti-CD3 (HIT3a), FITC anti-CD4 (A161A1), PE anti-CCR4 (L291H4),
PE/Cy7 anti-CXCR3 (G025H7), PerCP/Cy5.5 anti-CCR6 (29-2L17), APC
anti-CCR10 (6588-5), PE anti-CD14 (63D3), PE anti-CXCR3 (G025H7), PE
anti-CCR10 (6588-5), PE/Cy7 anti-CCR4 (L291H4), APC/Cy7 anti-CD27
(M-T271), Pacific blue anti-IL-17A (BL168), and Pacific blue anti-GM-CSF
(BVD2-21C11) were purchased from BioLegend. APC anti-IL-17RE (FAB8358A)
was purchased from R\&D Systems. To stain cell surface markers, PF
mononuclear cells were incubated with 2 \selectlanguage{greek}µ\selectlanguage{english}g/ml of each antibody for 30
min on ice before analysis and sorting on a BD FACSAria Flow Cytometer
(BD biosciences). To stain intracellular cytokines after surface marker
staining, cells were fixed with 200 \selectlanguage{greek}μ\selectlanguage{english}l of 2\% paraformaldehyde for 20
minutes, followed by permeabilization in 1 ml of 90\% methanol-PBS on
ice for 25 min. Cells were then incubated with 5 \selectlanguage{greek}µ\selectlanguage{english}g/ml of each antibody
for 1 hour at room temperature, respectively. Cells were washed and
suspended in PBS for analysis on an LSRII flow cytometer with a UV laser
(BD biosciences).

\textbf{RNA-Seq}

PF Th17 cell subpopulations were sorted from 5 patients and pooled for
RNA-Seq. Cellular RNA was extracted using the Arcturus~PicoPure
RNA~Isolation Kit~(ThermoFisher). The quality and quantities of RNA
samples were measured with the QIAxcel RNA QC Kit v2.0 (Qiagen)
following the manufacturer's instructions.~One microgram RNA at the
concentration \textgreater{} 200 ng/\selectlanguage{greek}µ\selectlanguage{english}l was subjected to library
construction, sequencing, reads quality check, alignment, clustering,
expression assessment, Gene Ontology (GO) enrichment analysis, and Kyoto
Encyclopedia of Genes and Genomes (KEGG) enrichment analysis by the
Wuhan SeqHealth Co., Ltd. The Collibri\textsuperscript{TM} Stranded RNA
Library Prep Kit for Illumina (ThermoFisher), HiSeq 2000 Sequencing
System (Illumina), TopHat v2.1.1, HTSeq v0.12.4, DEGseq2, and
Hypergeometric test were used for the above procedures, respectively.

\textbf{Validation of DEGs by quantitative polymerase chain reaction
(qPCR)}

cDNA preparation was achieved using the PrimeScript 1st strand cDNA
Synthesis Kit (Takara Bio). qPCR was implemented using the SYBR
Green~Quantitative RT-PCR (Sigma-Aldrich) on an Azure Cielo 3 Real-Time
qPCR System (Azure Biosystems). The PCR cycle condition was 50\selectlanguage{ngerman}°C for 2
minutes, 95°C for 10 minutes, 95°C for 15 seconds and 60°C for 30
seconds in 40 cycles, then 72°C for10 minutes. After normalization to
\selectlanguage{greek}β-\selectlanguage{english}actin mRNA level, the relative transcript amounts were determined by
the 2\selectlanguage{greek}\textsuperscript{-ΔΔCt}\selectlanguage{english} method. The primer sequences are displayed
in \textbf{Supplementary Table 1\&2} .\selectlanguage{english}
\begin{longtable}[]{@{}ll@{}}
\toprule
\begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
\textbf{Table. 1 Twenty Up-regulated Metabolic Genes in
IL-17RE\textsuperscript{+} Th17 Cells}\strut
\end{minipage} & \begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
\textbf{Table. 1 Twenty Up-regulated Metabolic Genes in
IL-17RE\textsuperscript{+} Th17 Cells}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
\textbf{Gene}\strut
\end{minipage} & \begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
\textbf{Protein}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
\emph{NDUFB5}\strut
\end{minipage} & \begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
NADH dehydrogenase~{[}ubiquinone{]}~1 beta subcomplex subunit 5\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
\emph{IDH1}\strut
\end{minipage} & \begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
Isocitrate dehydrogenase {[}NADP+{]} Cytoplasmic\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
\emph{MT-CO1}\strut
\end{minipage} & \begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
Cytochrome c oxidase subunit 1\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
\emph{UQCRC1}\strut
\end{minipage} & \begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
Cytochrome b-c1 complex subunit 1\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
\emph{NDUFS6}\strut
\end{minipage} & \begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
NADH dehydrogenase {[}ubiquinone{]} iron-sulfur protein 6\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.48\columnwidth}\raggedright\strut
\emph{SDHAF4}\strut
\end{minipage} & \begin{minipage}[t]{0.48\columnwidth}\raggedright\strut
\subsubsection*{Succinate dehydrogenase assembly factor
4}

{\label{succinate-dehydrogenase-assembly-factor-4}}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
\emph{ATPAF1}\strut
\end{minipage} & \begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
ATP synthase mitochondrial F1 complex assembly factor 1\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
\emph{CYDB}\strut
\end{minipage} & \begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
Cytochrome bd-I ubiquinol oxidase subunit 2\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
\emph{SDHD}\strut
\end{minipage} & \begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
Succinate dehydrogenase {[}ubiquinone{]} cytochrome b small
subunit\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
\emph{PGK1}\strut
\end{minipage} & \begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
Phosphoglycerate kinase 1\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
\emph{UQCRB}\strut
\end{minipage} & \begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
Cytochrome b-c1 complex subunit 7\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
\emph{NDUFC2}\strut
\end{minipage} & \begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
NADH dehydrogenase {[}ubiquinone{]} 1 subunit C2\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
\emph{NDUFA5}\strut
\end{minipage} & \begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
NADH dehydrogenase {[}ubiquinone{]} 1 alpha subcomplex subunit 5\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
\emph{MT-CO2}\strut
\end{minipage} & \begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
Cytochrome c oxidase subunit 2\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
\emph{NDUFAF6}\strut
\end{minipage} & \begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
NADH dehydrogenase {[}ubiquinone{]} complex I, assembly factor 6\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
\emph{ATPAF1A}\strut
\end{minipage} & \begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
ATP synthase mitochondrial F1 complex assembly factor 1\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
\emph{MT-ND3}\strut
\end{minipage} & \begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
NADH-ubiquinone oxidoreductase chain 3\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
\emph{SDH8}\strut
\end{minipage} & \begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
Succinate dehydrogenase assembly factor 4\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
\emph{COX10}\strut
\end{minipage} & \begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
Protoheme IX farnesyltransferase\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
\emph{NDUFA3}\strut
\end{minipage} & \begin{minipage}[t]{0.47\columnwidth}\raggedright\strut
NADH dehydrogenase {[}ubiquinone{]} 1 alpha subcomplex subunit 3\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}\selectlanguage{english}
\begin{longtable}[]{@{}ll@{}}
\toprule
\textbf{Table. 2 Twenty Down-regulated Metabolic Genes in
IL-17RE\textsuperscript{+} Th17 Cells} & \textbf{Table. 2 Twenty
Down-regulated Metabolic Genes in IL-17RE\textsuperscript{+} Th17
Cells}\tabularnewline
\textbf{Gene} & \textbf{Protein}\tabularnewline
\emph{MT-ND3} & NADH-ubiquinone oxidoreductase chain 3\tabularnewline
\emph{DLAT} & Dihydrolipoyllysine-residue acetyltransferase component of
pyruvate dehydrogenase complex\tabularnewline
\emph{MT-ATP6} & F-ATPase protein 6\tabularnewline
\emph{IDH3B} & Isocitrate dehydrogenase {[}NAD{]} subunit
beta\tabularnewline
\emph{CYP51A1} & Lanosterol 14-alpha demethylase\tabularnewline
\emph{HMGCS1} & Hydroxymethylglutaryl-CoA synthase,
cytoplasmic\tabularnewline
\emph{ACADL} & Long-chain specific acyl-CoA dehydrogenase\tabularnewline
\emph{COQ5} & 2-methoxy-6-polyprenyl-1,4-benzoquinol
methylase\tabularnewline
\emph{GCDH} & Glutaryl-CoA dehydrogenase\tabularnewline
\emph{TPI1} & Triosephosphate isomerase\tabularnewline
\emph{LPCAT4} & Lysophospholipid acyltransferase\tabularnewline
\emph{PPAP2A} & Lipid phosphate phosphohydrolase 1\tabularnewline
\emph{DHCR24} & Delta(24)-sterol reductase\tabularnewline
\emph{HMGCL} & Hydroxymethylglutaryl-CoA lyase\tabularnewline
\emph{ACADM} & Medium-chain specific acyl-CoA
dehydrogenase\tabularnewline
\emph{GLB1L} & Beta-galactosidase-1-like protein\tabularnewline
\emph{ENPP6} & Glycerophosphocholine
cholinephosphodiesterase\tabularnewline
\emph{SGPL1} & Sphingosine-1-phosphate lyase 1\tabularnewline
\emph{GAL3ST1} & Galactosylceramide sulfotransferase\tabularnewline
\emph{BPGM} & Bisphosphoglycerate mutase\tabularnewline
\bottomrule
\end{longtable}

\textbf{Cell cycle analysis}

Sorted Th17 subpopulations were fixed in ice-cold 70\% ethanol for 2.5
hours. Cells were centrifuged for 5 min at 300 rcf at
4\textsuperscript{o}C. Ethanol was removed and cells were rinsed once
with ice-cold Hank's balanced salt solution (HBSS) containing
Mg\textsuperscript{2+} and Ca\textsuperscript{2+}. After that, cells
were suspended in 0.5 ml of ice-cold HBSS containing
Mg\textsuperscript{2+} and Ca\textsuperscript{2+}. The cell suspension
was mixed with an equal volume of HBSS containing 2 mg Hoechst 33342
(Molecular Probes) and 4 mg Pyronin Y (Sigma-Aldrich) and incubated for
20 minutes in the dark. Cells were then loaded on the flow cytometer for
cell cycle analysis.

\textbf{Adenosine triphosphate (ATP) assay}

The ATP amount evaluation was conducted using the Luminescent ATP
Detection Assay Kit (ab113849, Abcam) following the manufacturer's
protocol.

\textbf{2, 7-dichlorodihydrofluorescein diacetate (H2DCFDA) staining}

Reactive oxygen species~(ROS) in Th17 cells were assessed by H2DCFDA
staining. Th17 cells were incubated in pre-warmed PBS containing 10 \selectlanguage{greek}μ\selectlanguage{english}M
H2DCFDA (ThermoFisher) for 15 minutes at 37\selectlanguage{ngerman}°C. After two washes with
PBS, cells were suspended in PBS and the green fluorescence was measured
on the flow cytometer.

\textbf{Statistics}

Each experiment was independently performed 2 or 3 times. The data were
shown as mean ± standard deviation unless specified. The Student's
t-test, non-parametric Mann-Whitney~U test or Kruskal-Wallis~test was
used to analyze the statistical significance. A \emph{p} -value
\textless{} 0.05 was regarded as significant.

\textbf{Results}

\textbf{The phenotype of PF IL-17-producing T cells in endometriosis}

Several cell surface markers have been applied to distinguish T cell
subsets {[}12, 13{]}. To characterize the phenotype of PF Th17 cells, we
first detected the expression of these markers on IL-17-producing T
cells in PF of endometriosis patients. As shown in \textbf{Figure 1A} ,
a substantial PF CD3\textsuperscript{+} IL-17-producing T cell
population was observed. Further detection revealed that these cells
were CD4\textsuperscript{+}CXCR3\textsuperscript{-} \textbf{(Figure 1B)}
. Based on the expression of CCR6 and CCR4, these cells can be divided
into CCR6\textsuperscript{+}CCR4\textsuperscript{+} and
CCR6\textsuperscript{+}CCR4\textsuperscript{-} subpopulations,
respectively \textbf{(Figure 1B \& 1C)} . Based on the expression of
IL-17RE and CD27, these cells can be divided into
IL-17RE\textsuperscript{+}CD27\textsuperscript{-} subset,
IL-17RE\textsuperscript{+}CD27\textsuperscript{+} subset,
IL-17RE\textsuperscript{-}CD27\textsuperscript{+} subset (very few or
almost absent in some samples), and
IL-17RE\textsuperscript{-}CD27\textsuperscript{-} subset \textbf{(Figure
1B \& 1D)} .

Because IL-17-producing T cells might involve both Th17 and Th1/17
cells, we needed to accurately discriminate these two populations to
isolate live Th17 cells for further investigations. To this end, we
analyzed PF CD3\textsuperscript{+}CD4\textsuperscript{+} T
cells\textbf{(Figure 2A)} for the expression of CCR6, CCR4, CXCR3, and
CCR10. As shown in \textbf{Figure 2B} , within PF
CD3\textsuperscript{+}CD4\textsuperscript{+} T cells, four
subpopulations were seen based on the expression of CCR4 and CXCR3:
CCR4\textsuperscript{+}CXCR3\textsuperscript{-} cells,
CCR4\textsuperscript{+}CXCR3\textsuperscript{+} cells,
CCR4\textsuperscript{-}CXCR3\textsuperscript{+} cells, and
CCR4\textsuperscript{-}CXCR3\textsuperscript{-} cells. We then sorted
the four subpopulations and measured the expression of T cell subset
master regulators, i.e. T-bet (Gene name TBX21), GATA3, and ROR\selectlanguage{greek}γ\selectlanguage{english}t. As
shown in \textbf{Figure 2C} , T-bet was highly expressed in
CCR4\textsuperscript{-}CXCR3\textsuperscript{+} cells and GATA3 was
highly expressed in
CCR4\textsuperscript{+}CXCR3\textsuperscript{-}cells, whereas ROR\selectlanguage{greek}γ\selectlanguage{english}t was
also significantly CCR4\textsuperscript{+}CXCR3\textsuperscript{-}
cells. This suggested that
CCR4\textsuperscript{+}CXCR3\textsuperscript{-} cells contained both Th2
and Th17 cells. Interestingly,
CCR4\textsuperscript{-}CXCR3\textsuperscript{+} T cells, which expressed
high T-bet, also expressed moderate levels of ROR\selectlanguage{greek}γ\selectlanguage{english}t, suggesting that
this subpopulation harbored Th1 and Th1/17 cells.

We then further dissected
CCR4\textsuperscript{+}CXCR3\textsuperscript{-} T cells based on the
expression of CCR6 and CCR10. As shown in \textbf{Figure 2D} , four
subpopulations were observed:
CCR6\textsuperscript{+}CCR10\textsuperscript{-} cells,
CCR6\textsuperscript{+}CCR10\textsuperscript{+} cells,
CCR6-CCR10\textsuperscript{+} cells, and
CCR6\textsuperscript{-}CCR10\textsuperscript{-} cells. We then sorted
these cells to check the expression of T-bet, GATA3, and ROR\selectlanguage{greek}γ\selectlanguage{english}t. T-bet
expression was ubiquitously low in all four subpopulations, while GATA3
was highly expressed in the
CCR6\textsuperscript{-}CCR10\textsuperscript{-}
subpopulation\textbf{(Figure 2E)} . ROR\selectlanguage{greek}γ\selectlanguage{english}t was robustly expressed in the
CCR6\textsuperscript{+}CCR10\textsuperscript{-} subpopulation but low in
the other three subpopulations \textbf{(Figure 2E)} . Therefore, Th17
cells were
CCR6\textsuperscript{+}CCR4\textsuperscript{+}CXCR3\textsuperscript{-}CCR10\textsuperscript{-}.

To precisely study live PF Th17 cells, we needed to introduce more
fluorophore-conjugated antibodies to label Th17 cells. However, it is
difficult for conventional flow cytometers without ultraviolet lasers to
recognize more than 6 fluorophores. Therefore, we came up with a new
method to distinguish live Th17 cells. PE-conjugated anti-CD14 antibody,
anti-CXCR3 antibody, and anti-CCR10 antibody were used together to
exclude CD14\textsuperscript{+} macrophages, CXCR3\textsuperscript{+}Th1
cells, CXCR3\textsuperscript{+} Th1/17 cells, and other non-Th2 and
non-Th17 T cells \textbf{(Figure 3A)} . Within the
CD14\textsuperscript{-}CXCR3\textsuperscript{-}CCR10\textsuperscript{-}cells,
CD4\textsuperscript{+} T cells were divided into four subpopulations
according to the expression of CCR6 and CCR4:
CCR6\textsuperscript{+}CCR4\textsuperscript{-} cells,
CCR6\textsuperscript{+}CCR4\textsuperscript{+} cells,
CCR6\textsuperscript{-}CCR4\textsuperscript{+} cells, and
CCR6\textsuperscript{+}CCR4\textsuperscript{+} cells \textbf{(Figure
3A)} . Evaluation of T cell subset master regulators indicated that
ROR\selectlanguage{greek}γ\selectlanguage{english}t was exclusively highly expressed in the
CCR6\textsuperscript{+}CCR4\textsuperscript{+} subpopulation while GATA3
was predominantly expressed in the
CCR6\textsuperscript{-}CCR4\textsuperscript{+}
subpopulation\textbf{(Figure 3B)} . Consistently, analysis of IL-17A
expression confirmed that the
CCR6\textsuperscript{+}CCR4\textsuperscript{+}subpopulation was Th17
cells \textbf{(Figure 3C \& 3D)} . Previous studies have reported the
significance of IL-17RE and CD27 to Th17 activity {[}14, 15{]}.
Therefore, we further dissected the
CCR6\textsuperscript{+}CCR4\textsuperscript{+} subpopulation based on
the expression of IL-17RE and CD27. As shown in \textbf{Figure 3E} , the
CCR6\textsuperscript{+}CCR4\textsuperscript{+} subpopulation was divided
into three subsets: IL-17RE\textsuperscript{+}CD27\textsuperscript{-}
cells, IL-17RE\textsuperscript{+}CD27\textsuperscript{+} cells, and
IL-17RE\textsuperscript{-}CD27\textsuperscript{-} cells. The
IL-17RE\textsuperscript{+}CD27\textsuperscript{-} subset and
IL-17RE\textsuperscript{+}CD27\textsuperscript{+} subset expressed
higher IL-17A than the
IL-17RE\textsuperscript{-}CD27\textsuperscript{-}subset \textbf{(Figure
3F)} . However, their ROR\selectlanguage{greek}γ\selectlanguage{english}t expression was comparable \textbf{(Figure
3G)} . Therefore, IL-17RE and CD27 signify the heterogeneity of PF Th17
cells.

Assessment of the expression of other Th17-related cytokines indicated
that in comparison with the
IL-17RE\textsuperscript{-}CD27\textsuperscript{-} subset, the
IL-17RE\textsuperscript{+}CD27\textsuperscript{-} subset and
IL-17RE\textsuperscript{+}CD27\textsuperscript{+} subset produced more
GM-CSF \textbf{(Figure 4A \& 4B)} as well as IL-22 mRNA \textbf{(Figure
4C)} . Furthermore, evaluation of cell cycle using Hoechst 33342 and
Pyronin Y demonstrated that the
IL-17RE\textsuperscript{+}CD27\textsuperscript{-} subset and
IL-17RE\textsuperscript{+}CD27\textsuperscript{+} subset had more cells
in G1 phase and S-G2/M phase than the
IL-17RE\textsuperscript{-}CD27\textsuperscript{-} subset, suggesting
that the former two subsets were more proliferative than the
latter\textbf{(Figure 4D)} . Therefore, IL-17RE\textsuperscript{+} Th17
cells were more pro-inflammatory than IL-17RE\textsuperscript{-} Th17
cells. CD27 seemed not profoundly influencing Th17 activity because no
significant difference was seen between the
IL-17RE\textsuperscript{+}CD27\textsuperscript{-} subset and
IL-17RE\textsuperscript{+}CD27\textsuperscript{+} subset.

To deeply understand the molecular traits of PF
IL-17RE\textsuperscript{+} Th17 cells and IL-17RE\textsuperscript{-}Th17
cells, we sorted these cells to conduct the RNA-Seq
analysis\textbf{(Figure 5A)} . Compared with IL-17RE\textsuperscript{-}
Th17 cells, a total of 5288 differentially expressed transcripts (DETs)
were identified in IL-17RE\textsuperscript{+} Th17 cells, including 3866
up-regulated DETs and 1422 down-regulated DETs \textbf{(Figure 5B \&
5C)} . The GO biological process (BP) enrichment results showed
significant associations with respiratory electron transport chain,
oxidative phosphorylation, and ATP synthesis coupled transport,
etc.\textbf{(Figure 5D)} . GO cellular component (CC) analysis showed
notable associations with respiratory chain, ribosome, and ribosomal
subunit, etc. \textbf{(Figure 5E)} . GO molecular function (MF) analysis
showed remarkable associations with structural molecular activity,
structural constituent of ribosome, RNA binding, NADH dehydrogenase
(ubiquinone) activity, and other metabolism-related activity
\textbf{(Figure 5F)} . KEGG analysis also enriched DETs in certain KEGG
terms such as ribosome, oxidative phosphorylation, olfactory
transduction, and glycolysis/gluconeogenesis \textbf{(Figure 5G)} .
Hence, the expression of genes for OXPHOS and ETC seemed to be
remarkably increased in IL-17RE\textsuperscript{+} Th17 cells relative
to IL-17RE\textsuperscript{-} Th17 cells.

Analysis of metabolism-related DETs revealed that in comparison to PF
IL-17RE\textsuperscript{-} Th17 cells, PF IL-17RE\textsuperscript{+}Th17
cells up-regulated the expression of genes involved in OXPHOS and ETC,
including \emph{NDUFB5} ,\emph{MT-CO1} ,\emph{UQCRC1} , and\emph{NDUFS6}
, etc. \textbf{(Figure 6A and Table.1)} . The up-regulation of these
genes was validated by qRT-PCR \textbf{(Figure 6B)} . Since OXPHOS and
ETC are crucial for the generation of ATP and ROS, we quantified ATP and
ROS in the two Th17 subsets. As shown in \textbf{Figure 6C} ,
IL-17RE\textsuperscript{+} Th17 cells produced more ATP than
IL-17RE\textsuperscript{-} Th17 cells. H2DCFDA staining suggested more
ROS generation in IL-17RE\textsuperscript{+} Th17 cells relative to
IL-17RE\textsuperscript{-} Th17 cells \textbf{(Figure 6D)} . Therefore,
IL-17RE\textsuperscript{+} Th17 cells and IL-17RE\textsuperscript{-}Th17
cells are metabolically different.

\textbf{Discussion}

In the current study, we developed a method to sort live Th17 cells from
PF of severe endometriosis patients to evaluate the function of
pathological Th17 cells. We are the first to use CXCR3, CCR4, and CCR6
isolate PF Th17 cells from endometriosis patients. In the future, it
would be necessary to detect more surface markers to precisely pinpoint
Th17 cells. Furthermore, detection of the above chemokine receptors
would recognize Th1, Th2, and Th1/Th17 subsets and thus greatly
facilitates research on T cell biology at endometriosis lesions.

Th17 cells are heterogeneous and possess a remarkable degree of
plasticity {[}16{]}. Our study indicates that PF Th17 cells are
heterogeneous according to the expression of CD27 and IL-17RE. CD27 is a
member of TNF receptor family expressed on CD4\textsuperscript{+} T
cells at the priming or effector stage, while its ligand, CD70, is
expressed on activated dendritic cells and lymphocytes {[}17{]}. Recent
research suggests its inhibitory effect on Th17 cells through repressing
transcription of IL-17 and CCR6 {[}15{]}. IL-17RE forms a receptor
complex with IL-17RA to bind to IL-17C to potentiate the Th17 cell
response {[}14{]}. IL-17C is preferentially secreted by epithelial cells
in the lung, skin, and colon {[}18{]}. Interestingly, the Human Protein
Atlas describes high IL-17C expression in the endometrium
(\emph{https://www.proteinatlas.org/ENSG00000124391-IL17C/tissue}),
strongly suggesting the existence of IL-17C in endometriosis lesions.
Our ongoing research is investigating the levels of IL-17C in PF and
endometriosis tissues. Besides, our data indicate that IL-17RE rather
than CD27 marks a more active Th17 subpopulation, because
IL-17RE\textsuperscript{+} Th17 cells expressed more Th17-related
cytokines and were more proliferative than
IL-17RE\textsuperscript{-}Th17 cells. A possible explanation is that
IL-17RE-triggered signaling overcomes CD27-induced signaling.

Through RNA-Seq, we found that most of the top 20 up-regulated metabolic
genes in IL-17RE\textsuperscript{+} Th17 cells encode enzymes involved
in OXPHOS and ETC, while most of the down-regulated metabolic genes were
associated with other metabolic pathways. It has been known that
mitochondrial respiration is enhanced during Th17 differentiation and
OXPHOS is essential for Th17 cell pathogenic signature gene expression
{[}19{]}. Besides, Th17 cells have limited capacity to promote
glycolysis to react to metabolic stresses and they rely on OXPHOS for
the production of energy and cytokines {[}20{]}. Indeed, we observed
higher pro-inflammatory activity associated with higher ATP and ROS in
IL-17RE\textsuperscript{+} Th17 cells. This suggests that OXPHOS and ETC
support the pathogenicity of PF IL-17RE\textsuperscript{+} Th17 cells in
endometriosis. Our data also poses an interesting question: whether the
IL-17C/IL-17RE axis results in the up-regulation of OXPHOS and
ETC-related genes. This could be answered in future studies.

What remains unknown is the origin of the heterogeneous PF Th17 subsets.
Are they a CD4\textsuperscript{+} T cell population at different
maturation statuses? Or do they arise from different
CD4\textsuperscript{+} T cell subsets under distinct microenvironment
factors? Lineage tracing of Th17 cells in animal models and single-cell
transcriptome sequencing might give the answer.

In conclusion, this study provides a novel method to detect and isolate
live PF Th17 cells from endometriosis patients and unveils the
functional and metabolic heterogeneity of PF Th17 subsets. Furthermore,
the data of other differentially expressed genes shed light on the
elucidation of other molecular mechanisms that modulate the function of
pathological Th17 cells in endometriosis.

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\textbf{Figure captions}

\textbf{Figure 1. The phenotype of PF IL-17-producing T cells in
endometriosis. (A)} Representative FLOW CYTOMETRY plots showing
IL-17-producing T cells in endometriosis PF. Single cells were gated
among the whole events, followed by gating lymphocytes based on FSC-A
and SSC-A. CD3\textsuperscript{+}IL-17A\textsuperscript{+} cells were
then observed in lymphocytes. Numbers in the quadrants indicate the
percentages of cell populations. \textbf{(B)} Detection of indicated
surface markers in PF IL-17-producing T cells. Isotype: isotype antibody
control. Ab: specific antibody for corresponding markers.
\textbf{(C)}Frequencies of
CCR6\textsuperscript{+}CCR4\textsuperscript{+} and
CCR6\textsuperscript{+}CCR4\textsuperscript{-} subpopulations in
IL-17-producing T cells. \textbf{(D)} Frequencies of
IL-17RE\textsuperscript{+}CD27\textsuperscript{-},
IL-17RE\textsuperscript{+}CD27\textsuperscript{+},
IL-17RE\textsuperscript{-}CD27\textsuperscript{+}, and
IL-17RE\textsuperscript{-}CD27\textsuperscript{-} subset in
IL-17-producing T cells. N=5 individuals per group.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

\textbf{Figure 2. Identification of live PF Th17 cells according to CCR6
and CCR4. (A)} Gating of CD3\textsuperscript{+}CD4\textsuperscript{+} T
cells in endometriosis PF and normal blood. PF: peritoneal fluid. PB:
peripheral blood. \textbf{(B)} T cell subsets based on the expression of
CCR4 and CXCR3. PB: blood CD3\textsuperscript{+}CD4\textsuperscript{+} T
cells as control. \textbf{(C)} Relative mRNA levels of indicated master
regulators in CCR4-and-CXCR3-defined T cell subsets.
\textbf{(D)}Subpopulations in
CCR4\textsuperscript{+}CXCR3\textsuperscript{-} T cells based on the
expression of CCR6 and CCR10. \textbf{(E)} Relative mRNA levels of
indicated master regulators in the subpopulations described in (D). *:
\emph{p} \textless{}0.05; **:\emph{p} \textless{}0.01; ***: \emph{p}
\textless{}0.001. N=6 individuals per group. Kruskal-Wallis~test.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

\textbf{Figure 3. Identification of live PF Th17 subsets according to
IL-17RE and CD27.} (A) Subsets in CD14-CXCR3-CCR10-CD4+ cells based on
the expression of CCR6 and CCR4. (B) Relative mRNA levels of indicated
master regulators in the subpopulations described in (A). PB: blood
CD3+CD4+ T cells as control. R6: CCR6. R4: CCR4. N=3 per group. (C) Flow
cytometry plots showing IL-17A expression in the subpopulations
described in (A). (D) Frequencies of IL-17A+ cells in the 4
subpopulations. (E) Expression of IL-17RE and CD27 on the surface of the
CCR6+CCR4+ subpopulation. (F) Relative mRNA levels of IL-17A in
IL-17RE+CD27- cells, IL-17RE+CD27+ cells, and IL-17RE-CD27- cells. (G)
Relative mRNA levels of ROR\selectlanguage{greek}γ\selectlanguage{english}t in the same three subsets as (F). ). *:
p\textless{}0.05; **: p\textless{}0.01; ***: p\textless{}0.001. N=5 per
group in (D) to (G). Kruskal-Wallis test.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

\textbf{Figure 4. Functions of PF Th17 subsets. (A \& B)} GM-CSF
expression in IL-17RE\textsuperscript{+}CD27\textsuperscript{-} subset,
IL-17RE\textsuperscript{+}CD27\textsuperscript{+} subset, and
IL-17RE\textsuperscript{-}CD27\textsuperscript{-} subset. Representative
flow cytometry plots are shown in (A), and statistics of the frequencies
of GM-CSF\textsuperscript{+} cells are shown in (B).
\textbf{(C)}Relative mRNA levels of IL22 in the three subsets. N=5 per
group. Kruskal-Wallis~test. \textbf{(D)} Flow cytometry plots showing
cell cycle measured by staining with Hoechst 33342 and Pyronin Y.
*:\emph{p} \textless{}0.05; **: \emph{p} \textless{}0.01.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image5/image5}

\end{center}
\end{figure}

\textbf{Figure 5. RNA-Seq analysis of PF IL-17RE+ Th17 cells and
IL-17RE- Th17 cells.} (A) Gating and sorting of PF CCR6+CCR4+IL-17RE+
Th17 cells and CCR6+CCR4+IL-17RE- Th17 cells. (B) Volcano plot
displaying the DETs. (C) Cluster analysis of DETs. 17RE-: IL-17RE- Th17.
17RE+: IL-17RE+ Th17. (D to F) Enrichment of up-regulated DETs for GO
terms in the category of biological process terms (D), cellular
component terms (E), and molecular function terms (F). (G) Enrichment of
up-regulated DETs for KEGG terms.

\textbf{Figure 6. OXPHOS and ETC in PF IL-17RE\textsuperscript{+} Th17
cells and IL-17RE\textsuperscript{-} Th17 cells. (A)} Heat map of~top 40
metabolism-related DETs (FDR \textless{} 0.05). \textbf{(B)} Validation
of DETs in (A) by qRT-PCR. N=5\textasciitilde{}7 per group. The data are
presented as min to max. \textbf{(C)} ATP levels in
IL-17RE\textsuperscript{+} Th17 cells and IL-17RE\textsuperscript{-}Th17
cells. N=6 per group. \textbf{(D)} ROS levels measured by H2DCFDA
staining. N=5 per group. *: p\textless{}0.05; **: p\textless{}0.01; ***:
p\textless{}0.001. Mann-Whitney~U test.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image6/image6}

\end{center}
\end{figure}

All data included in this study are available upon request by contact
with the corresponding author.

Conflicts of interest

The authors declare no conflict of interest with regard to the
publication of this paper.

There are no funders to report for this submission.

Author contributions:

Yanping Jiang performed experiment and the data analyses, and wrote the
manuscript;

Lu Wang performed the experiment and wrote the manuscript;

Yanping Jiang and Lu Wang contributed equally to this work and should be
considered co-first authors.

Yaqin Peng contributed significantly to analysis and manuscript
preparation;

Jian Qin contributed to the conception of the study

Aili Tan helped perform the analysis with constructive discussions.

Shujun Wang helped perform data analyses

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