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 Figure 1A , a substantial PF
CD3+ IL-17-producing T cell population was observed.
Further detection revealed that these cells were
CD4+CXCR3- (Figure 1B) .
Based on the expression of CCR6 and CCR4, these cells can be divided
into CCR6+CCR4+ and
CCR6+CCR4- subpopulations,
respectively (Figure 1B & 1C) . Based on the expression of
IL-17RE and CD27, these cells can be divided into
IL-17RE+CD27- subset,
IL-17RE+CD27+ subset,
IL-17RE-CD27+ subset (very few or
almost absent in some samples), and
IL-17RE-CD27- subset (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+CD4+ T cells(Figure 2A) for the expression of CCR6, CCR4, CXCR3, and CCR10.
As shown in Figure 2B , within PF
CD3+CD4+ T cells, four
subpopulations were seen based on the expression of CCR4 and CXCR3:
CCR4+CXCR3- cells,
CCR4+CXCR3+ cells,
CCR4-CXCR3+ cells, and
CCR4-CXCR3- 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 RORγt. As
shown in Figure 2C , T-bet was highly expressed in
CCR4-CXCR3+ cells and GATA3 was
highly expressed in CCR4+CXCR3-cells, whereas RORγt was also significantly
CCR4+CXCR3- cells. This suggested
that CCR4+CXCR3- cells contained
both Th2 and Th17 cells. Interestingly,
CCR4-CXCR3+ T cells, which expressed
high T-bet, also expressed moderate levels of RORγt, suggesting that
this subpopulation harbored Th1 and Th1/17 cells.
We then further dissected
CCR4+CXCR3- T cells based on the
expression of CCR6 and CCR10. As shown in Figure 2D , four
subpopulations were observed:
CCR6+CCR10- cells,
CCR6+CCR10+ cells,
CCR6-CCR10+ cells, and
CCR6-CCR10- cells. We then sorted
these cells to check the expression of T-bet, GATA3, and RORγt. T-bet
expression was ubiquitously low in all four subpopulations, while GATA3
was highly expressed in the
CCR6-CCR10- subpopulation(Figure 2E) . RORγt was robustly expressed in the
CCR6+CCR10- subpopulation but low in
the other three subpopulations (Figure 2E) . Therefore, Th17
cells were
CCR6+CCR4+CXCR3-CCR10-.
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+ macrophages, CXCR3+Th1 cells, CXCR3+ Th1/17 cells, and other non-Th2 and
non-Th17 T cells (Figure 3A) . Within the
CD14-CXCR3-CCR10-cells, CD4+ T cells were divided into four
subpopulations according to the expression of CCR6 and CCR4:
CCR6+CCR4- cells,
CCR6+CCR4+ cells,
CCR6-CCR4+ cells, and
CCR6+CCR4+ cells (Figure
3A) . Evaluation of T cell subset master regulators indicated that RORγt
was exclusively highly expressed in
the
CCR6+CCR4+ subpopulation while GATA3
was predominantly expressed in the
CCR6-CCR4+ subpopulation(Figure 3B) . Consistently, analysis of IL-17A expression
confirmed that the CCR6+CCR4+subpopulation was Th17 cells (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+CCR4+ subpopulation based
on the expression of IL-17RE and CD27. As shown in Figure 3E ,
the CCR6+CCR4+ subpopulation was
divided into three subsets:
IL-17RE+CD27- cells,
IL-17RE+CD27+ cells, and
IL-17RE-CD27- cells. The
IL-17RE+CD27- subset and
IL-17RE+CD27+ subset expressed
higher IL-17A than the IL-17RE-CD27-subset (Figure 3F) . However, their RORγt expression was
comparable (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-CD27- subset, the
IL-17RE+CD27- subset and
IL-17RE+CD27+ subset produced more
GM-CSF (Figure 4A & 4B) as well as IL-22 mRNA (Figure
4C) . Furthermore, evaluation of cell cycle using Hoechst 33342 and
Pyronin Y demonstrated that the
IL-17RE+CD27- subset and
IL-17RE+CD27+ subset had more cells
in G1 phase and S-G2/M phase than the
IL-17RE-CD27- subset, suggesting
that the former two subsets were more proliferative than the latter(Figure 4D) . Therefore, IL-17RE+ Th17 cells
were more pro-inflammatory than IL-17RE- Th17 cells.
CD27 seemed not profoundly influencing Th17 activity because no
significant difference was seen between the
IL-17RE+CD27- subset and
IL-17RE+CD27+ subset.
To deeply understand the molecular traits of PF
IL-17RE+ Th17 cells and IL-17RE-Th17 cells, we sorted these cells to conduct the RNA-Seq analysis(Figure 5A) . Compared with IL-17RE- Th17
cells, a total of 5288 differentially expressed transcripts (DETs) were
identified in IL-17RE+ Th17 cells, including 3866
up-regulated DETs and 1422 down-regulated DETs (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.(Figure 5D) . GO cellular component (CC) analysis showed notable
associations with respiratory chain, ribosome, and ribosomal subunit,
etc. (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 (Figure 5F) .
KEGG analysis also enriched DETs in certain KEGG terms such as ribosome,
oxidative phosphorylation, olfactory transduction, and
glycolysis/gluconeogenesis (Figure 5G) . Hence, the expression
of genes for OXPHOS and ETC seemed to be remarkably increased in
IL-17RE+ Th17 cells relative to
IL-17RE- Th17 cells.
Analysis of metabolism-related DETs revealed that in comparison to PF
IL-17RE- Th17 cells, PF IL-17RE+Th17 cells up-regulated the expression of genes involved in OXPHOS and
ETC, including NDUFB5 ,MT-CO1 ,UQCRC1 ,
andNDUFS6 , etc. (Figure
6A and Table.1) . The up-regulation of these genes was validated by
qRT-PCR (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 Figure 6C , IL-17RE+ Th17
cells produced more ATP than IL-17RE- Th17 cells.
H2DCFDA staining suggested more ROS generation in
IL-17RE+ Th17 cells relative to
IL-17RE- Th17 cells (Figure 6D) . Therefore,
IL-17RE+ Th17 cells and IL-17RE-Th17 cells are metabolically different.