Comprehensive analysis utilizing flow cytometry and immunohistochemistry reveals inflammatory changes in local endometrial and systemic dendritic ...
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Human Reproduction, Vol.36, No.2, pp. 415–428, 2021
Advance Access Publication on December 12, 2020 doi:10.1093/humrep/deaa318
ORIGINAL ARTICLE Reproductive biology
Comprehensive analysis utilizing flow
cytometry and immunohistochemistry
reveals inflammatory changes in local
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endometrial and systemic dendritic
cell populations in endometriosis
A.J. Hey-Cunningham1, C. Wong1, J. Hsu2, P.D. Fromm2, G.J. Clark2,
F. Kupresanin2, E.J. Miller1, R. Markham1, and H.M. McGuire3,*
1
The University of Sydney Obstetrics, Gynaecology and Neonatology, Central Clinical School, Faculty of Medicine and Health, The
University of Sydney, Sydney, NSW, Australia 2Dendritic Cell Research Group, ANZAC Research Institute, The University of Sydney,
Sydney, NSW, Australia 3Ramaciotti Facility for Human Systems Biology and Discipline of Pathology, Charles Perkins Centre, The
University of Sydney, Sydney, NSW, Australia
*Correspondence address. Ramaciotti Facility for Human Systems Biology and Discipline of Pathology, Charles Perkins Centre, The
University of Sydney, Sydney, NSW 2006, Australia. E-mail: helen.mcguire@sydney.edu.au
Submitted on March 16, 2020; resubmitted on October 15, 2020; editorial decision on October 22, 2020
STUDY QUESTION: What are the detailed endometrial tissue specific and systemic dendritic cell (DC) subset disturbances in
endometriosis?
SUMMARY ANSWER: This study confirms myeloid DC (mDC) and plasmacytoid DC subsets are readily identified in endometrial tissue
and shows both endometrial and circulating differences in DC populations in women with endometriosis, with disease stage-specific
relationships evident locally in the endometrium.
WHAT IS KNOWN ALREADY: Immune factors in the uterus, the peritoneal environment and systemically are implicated in the
pathogenesis and progression of both endometriosis and infertility. While there is some evidence that endometrial DC populations are
altered in endometriosis, DC subset involvement in both the endometrium and peripheral blood have not been comprehensively
investigated so the functional consequences have been unknown.
STUDY DESIGN, SIZE, DURATION: This prospective cross-sectional cohort study compares circulating and endometrial DC popula-
tions in women of reproductive age with and without endometriosis (n ¼ 55 and 30, respectively), wherein each participant donated
samples at a single time point. Study participants were surveyed for menstrual cycle phase, American Society for Reproductive Medicine
(ASRM) endometriosis disease stage and fertility status (where possible).
PARTICIPANTS/MATERIALS, SETTING, METHODS: Peripheral blood samples were processed into mononuclear cells for analysis
by flow cytometry, and endometrial samples were analysed by immunohistochemistry and dissociated into single-cell suspension for flow
cytometry.
MAIN RESULTS AND THE ROLE OF CHANCE: In the endometrium of women with endometriosis, IRF-8þ cells were increased
during the proliferative phase (P ¼ 0.014), total DC proportions increased in the secretory phase (P ¼ 0.038) and normal menstrual cyclical
fluctuations in CD1cþ and IRF-8þ cells blunted; indicative of a consistently inflammatory tissue environment. The inflammatory changes in
CD141þ and IRF-8þ populations in the endometrium of women with endometriosis were particularly evident in more advanced ASRM
stages of the disease (respective P-values 0.032 and 0.045). There was also evidence of systemic inflammation in women with endometri-
osis, with increased circulating CD141þ mDC proportions (overall P ¼ 0.040, secretory phase P ¼ 0.021).
LARGE SCALE DATA: N/A.
LIMITATIONS, REASONS FOR CAUTION: As is common in this type of study, one of the main limitations was small sample
numbers, particularly during the menstrual phase of the cycle.
C The Author(s) 2020. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved.
V
For permissions, please email: journals.permissions@oup.com
416 Hey-Cunningham et al.
WIDER IMPLICATIONS OF THE FINDINGS: Further phenotyping of local and circulating immune cell subtypes is critical to improving
understanding of endometriosis pathogenesis and immune contributions to infertility associated with the disease.
STUDY FUNDING/COMPETING INTEREST(S): This research was financially supported by a Sydney Medical School and Balnaves
Foundation Kick Start Grant and the Department of Obstetrics, Gynaecology and Neonatology at The University of Sydney. The authors
have no conflicts of interest to declare.
Key words: blood / dendritic cells / endometrium / endometriosis / menstrual cycle / inflammation / flow cytometry /
immunohistochemistry
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..
Introduction ..
..
the tissue, they have specific functions linked to their anatomical site.
Whilst populations of CD1cþ mDCs, CD141þ mDC can be found in
Endometriosis is a chronic, inflammatory gynaecological disease defined ..
.. most non-lymphoid tissue, there are generally few pDC, which can be
by growth of tissue resembling the endometrium outside the uterus .. distinguished definitively as CD304þ by cytometric staining, and collec-
(Ulukus et al., 2006). Endometriosis affects 10–15% of women in their ..
.. tively with cDC1 cells identified by transcription factor IRF-8 expres-
reproductive years and can result in reduced fertility and debilitating .. sion by immunohistochemistry (Collin and Bigley, 2018). pDCs can be
pain (Giudice, 2010). The most widely accepted theory of its patho-
..
.. further subdivided by CD2 to identify a subset demonstrating particu-
genesis is that endometrial tissue shed at menstruation is refluxed .. lar resistance to stress (Bryant et al., 2016). CD1cþ mDCs are usually
through the Fallopian tubes and subsequently implants at ectopic sites,
..
.. the predominant tissue DC subset capable of sensing most challenges.
forming endometriotic lesions (Sampson, 1927). Supporting this the- ..
ory, the eutopic endometrium of women with endometriosis has been
.. On the uptake of apoptotic cells, CD1cþ mDCs become polarized in
.. a tolerogenic state to maintain homeostasis (Hasegawa and
shown to be different to endometrium from women without the dis- ..
.. Matsumoto, 2018). However, with sufficient stimuli, they have excel-
ease (Sharpe-Timms, 2001; Al-Jefout et al., 2009). There is increasing ..
.. lent capacity to prime a naı̈ve CD4þ T cells. An inflammatory environ-
evidence that immune factors in the uterus, the peritoneal environ-
.. ment provides sufficient stimuli to trigger maturation of CD1cþ DC
ment and even systemically contribute to pathogenesis and progres- .. leading to a productive immune response. If these triggers remain, the
sion of endometriosis. The failure of the immune system to clear shed ..
.. inflammation in the tissue environment can become chronic. The other
and refluxed endometrial tissue, and to protect the peritoneum from .. main mDC population, distinguished by expression of CD141, also
its adhesion and invasion, is implicated in the establishment of endo- ..
.. known as thrombomodulin, is very rare but has a notable ability to
metriosis (Berbic and Fraser, 2011). ..
.. cross present antigen to stimulate CD8þ T cells, along with secretion
Dendritic cells (DCs) are ‘professional’ antigen presenting cells
.. of a range of proinflammatory cytokines including IL-12 (Canavan
which capture potentially harmful foreign or displaced molecules and ..
cells (antigens) and present them to lymphocytes to stimulate a tar- .. et al., 2018). Within a lineage negative, human leukocyte antigen
.. (HLA)-DRþ gate there is a clear CD16þ population that does not ex-
geted immune response. DCs are involved in the establishment and ..
maintenance of tolerance (Banchereau and Steinman, 1998). They are .. press CD14 (Fromm et al., 2020). Whether this is a CD16 DC (as we
.. have referred to herein) or a differentiated monocyte, the population’s
exquisitely primed to sense and respond to their environment. In the ..
periphery, DC circulate in an immature form of those found in the tis-
.. existence and presence may reflect inflammatory microenvironments,
.. so has also been investigated in this study.
sues. These immature DC are efficient at antigen capture but in the ..
absence of other maturation signals, they maintain homeostasis (Collin
.. Immune mechanisms and specific immune cell populations, including
.. DC, play crucial roles in the female reproductive tract (Lee et al.,
et al., 2013). Within non-lymphoid tissues migratory DC can be found ..
whose function is to sense and take up foreign antigen before migrat-
.. 2015; Marron et al., 2019). There is immunohistochemical evidence
..
ing through the lymphatics to the lymph nodes (Hasegawa and .. that endometrial DC populations may be altered in women with en-
Matsumoto, 2018).
.. dometriosis. In particular, it has been shown by immunohistochemistry
..
Within the human female reproductive tract, the immune system is .. that endometrial density of immature DC (CD1aþ) is increased, but
.. mature DC (CD83þ) are decreased in women with endometriosis
engaged in the complex challenge of enabling successful reproduction ..
while protecting against infections and disease (Wira et al., 2015). This .. compared to women without the disease (Schulke et al., 2009). In our
.. early studies of circulating DC in women with endometriosis, we used
environment constantly changes as part of the menstrual cycle which ..
results in exposure of tissue DC to cell debris through apoptotic cell .. CD11cþ flow cytometric staining on CD14 cells to define mDCs,
.. and observed an indication of changes in menstrual cyclical patterns of
death. DC present must sense and distinguish these changes from
... mDC populations compared to women without endometriosis
those resulting from infection and disease. To do this, DC express a ..
variety of pattern recognition receptors capable of recognizing molecu- .. (Maridas et al., 2014). Specifically, CD141þ mDCs were found to de-
..
lar patterns expressed by pathogens or danger signals associated with .. crease between secretory and menstrual phases in women with endo-
inflammation or necrosis caused by tissue damage as well as receptors .. metriosis only. Beyond these preliminary studies of DC in women with
..
that recognize homeostatic cell death (Rodriguez-Garcia et al., 2018). .. endometriosis, animal model research has indicated that DC modulate
The main DC subsets in blood include plasmacytoid DC (pDC), .. the formation of endometriotic lesions (Fainaru et al., 2008; Pencovich
..
and myeloid DC (mDC) subpopulations of conventional DC1 (cDC1); .. et al., 2014; Stanic et al., 2014). In the context of endometriosis patho-
CD141þ mDC and conventional DC2 (cDC2); CD1cþ mDCs (Collin
.. genesis, DC populations may contribute to the evasion of immune re-
..
et al., 2013). When DC subsets migrate from the peripheral blood to . sponse by endometrial cells displaced outside the uterine cavity and
Dendritic cell subsets in endometriosis 417
their subsequent development into endometriotic lesions at ectopic
Table I Participant and sample characteristics.
locations (Schulke et al. 2009; Berbic and Fraser, 2011; Pencovich
et al., 2014). Participant characteristic Group
Beyond the implication of DC in endometriosis, DC disturbances in
Endometriosis No endometriosis
the female reproductive tract have been shown in relation to fertility No. (%) No. (%)
problems such as recurrent miscarriage and recurrent implantation fail- ......................................................................................................
ure (Askelund et al., 2004; Tirado-González et al., 2010; Liu et al., Number 55 (100) 30 (100)
2018). This is relevant to endometriosis given that 30–50% of women Menstrual cycle phase
with endometriosis are infertile or sub-fertile, and endometriosis is Menstrual 6 (11) 2 (7)
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present in 20–50% of women with infertility (Strathy et al., 1982; Proliferative 24 (44) 16 (53)
Evans and Decherney, 2017). While it is clear that endometriosis is as- Secretory 25 (46) 12 (40)
sociated with reduced fertility, the exact mechanisms behind this and r-ASRM* endometriosis stage
the nature of any immune involvement are unknown (Giudice et al., Stage I 11 (20) –
2002; Evans and Decherney, 2017). Stage II 12 (22) –
Application of specialist immunology to endometriosis has been lim- Stage III 16 (29) –
ited, and immune mechanisms in endometriosis-associated infertility Stage IV 14 (26) –
remain largely unknown, meaning current understanding of the adap- Unknown 2 (4) –
tive immune response in endometriosis is limited. Peripheral blood None – 30 (100)
allows ready access to a DC population that can be assessed. By un- Fertility status
derstanding the relationship between blood and tissue DC we may be Fertile 10 (18) 5 (16)
able to monitor immune outcomes in a less invasive manner. Infertile 32 (58) 23 (77)
Therefore, the aim of this study was to characterize DC populations Unknown 13 (24) 2 (7)
in the blood and endometrium of women with and without endome- Sample type a
triosis, and to investigate any links to fertility status. Blood (flow cytometry) 27 (49 ) 10 (33)
Endometrium (flow cytometry) 18 (33 5 (17)
Endometrium 44 (80) 29 (97)
(immunohistochemistry)
Materials and methods
*Revised American Society for Reproductive Medicine.
This study was approved by the human research ethics committees of a
Refer to Supplementary Fig. S1 for pairing of samples from study participants and
Sydney Local Health District (Royal Prince Alfred Hospital Zone; Supplementary Tables SI–SIV for details of sample use across the study.
Protocol No X12-0344, HREC/12/RPAH/525 and SSA/13/RPAH/
36) and University of Sydney (Protocol No 2013/112). ..
.. Flow cytometry
..
Participants .. Immediately prior to surgery, blood samples (4–8 ml) were collected
.. from a portion of participants (as per Table I) into Vacutainer Cell
Participants for this cross-sectional study were adult women of repro- ..
.. Preparation Tubes with Sodium Heparin (BD Biosciences, USA) and
ductive age (18–43 years) with laparoscopically confirmed absence or ..
presence of endometriosis, recruited from gynaecological operating
.. peripheral blood mononuclear cells (PBMCs) separated according to
.. manufacturer’s instructions. Briefly, PBMCs were isolated within Cell
theatre lists at Royal Prince Alfred Hospital (total n ¼ 85; median age ..
.. Preparations Tubes using a thixotropic gel and Ficoll-hypaque (BD
¼ 35, interquartile range (IQR) ¼ 8, range ¼ 21–43; participant and .. Biosciences, USA), by centrifuging without brake, washed twice in
sample details provided in Table I). Informed consent was obtained ..
.. phosphate buffered saline, and proceeded to flow cytometry staining
from all participants. A detailed clinical history (including age, menstrual .. without cryopreservation.
cycle history and symptoms, fertility history, pelvic and related pain
..
.. Endometrial curettings (100 mg) were collected into buffer (phos-
symptoms, previous gynaecological diagnoses and surgeries, current .. phate buffered saline with 0.5% w/v Bovine Serum Albumin), promptly
..
medications and family history of gynaecological and autoimmune dis- .. transported to the laboratory and mechanically dissociated with
ease) was recorded at the time of recruitment in a standard data col- .. gentleMACSTM (MiltenyiBiotec, Germany). Samples were then enzy-
..
lection form. Women on any hormonal or immune-suppressive .. matically dissociated using 5% w/v Collagenase IV (Gibco by Life
therapy, with a history of autoimmune disease or recent infection ..
.. Technologies, Australia) and filtered to remove larger tissue fragments
(within 2 weeks) were excluded from the study. Blood and endome- .. and debris. Red blood cells were lysed with Red Cell Lysis solution
trial tissue samples were collected as was practicable; collection of
..
.. (MiltenyiBiotec, Germany).
matched blood and endometrial samples was not always possible; .. Prepared PBMC and endometrial cell suspensions were washed and
..
Supplementary Fig. S1 provides detail on what samples were available .. stained with a live/dead cell discriminator (Live/Dead Fixable Violet
from which participants. Endometrial dating was performed by an ex- .. Stain Kit; Invitrogen, USA). Following further washing, samples were
..
perienced gynaecological histopathologist according to the histological .. stained with a panel of monoclonal antibodies conjugated to fluoro-
appearance of the endometrium (Noyes et al., 1950).
.. chromes (Table II) that included pan leucocyte markers, CD45, lineage418 Hey-Cunningham et al.
Table II Details of fluorescent-conjugated antibodies used for blood and endometrial cell staining for flow cytometry.
Antibody Identifies Fluorochrome (blood Fluorochrome Clone Supplier
staining) (tissue staining)
............................................................................................................................................................................................................................
CD45 All leukocytes – FITC HI30 BD Pharmingen, USA
CD3 T cells (lineage marker) Pacific Blue Pacific Blue HIT3a iCyt, USA
CD14 Monocytes and macro- V450 V450 M5E2 BD Horizon, USA
phages (lineage marker)
CD19 B cells (lineage marker) V450 V450 HlB19 BD Horizon, USA
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CD20 B cells (lineage marker) Pacific Blue Pacific Blue 2h7 iCyt, USA
CD56 NK cells (lineage Pacific Blue Pacific Blue HCD56 iCyt, USA
marker)
HLA-DR MHC-II on APC APCCy7 APCCy7 L243 BD Biosciences, USA
CD11c Myeloid DC PECy7 PECy7 B-ly6 BD Pharmingen, USA
CD1c Myeloid DC 2 FITC eF710 L161 eBioscience, USA
CD141 Myeloid DC 1 PE PE BDCA-3 Miltenyi, Germany
CD16 Novel myeloid DC V500 V500 3G8 BD Horizon, USA
CD304 Plasmacytoid DC APC APC BDCA-4 Miltenyi, Germany
CD2 Novel plasmacytoid DC PECy5 – RPA-2.10 BD Pharmingen, USA
markers (CD3, CD19, CD20, CD14 and CD56) as well as HLA-DR
.. Slides were blocked (Dual Endogenous Enzyme Block and Protein
..
to allow identification of the CD45þ, Lineage-, HLA-DRþ DC popula- .. Block Serum-Free, Dako, Denmark), followed by primary antibody ap-
tions which contribute to less than 1% of the peripheral blood leuco- ... plication. Antibodies used were monoclonal mouse anti-human CD1c
..
cytes. Further subset discrimination into CD304þ pDC (with .. antibody (clone 2F4; Abcam, UK) at 1/100 dilution to identify the
subsequent CD2þ gating in PBMC samples) and CD11cþ mDC was .. mDC subpopulation; and polyclonal rabbit anti-human interferon regu-
..
followed by identification of three mDC subsets, CD16þ (Fromm .. latory factor IRF-8 (Sigma-Aldrich, Sweden) at 1/100 dilution to iden-
et al. 2020), CD1cþ and CD141þ (Fig. 1). This analysis of DC popu-
.. tify pDC along with mDC1 cells (IRF-8 expression is localized to the
..
lations adheres to the current international consensus on DC nomen- .. nuclei of DCs, mononuclear phagocytes and macrophages; Tamura
..
clature (Bryant et al., 2016; Fromm et al., 2016; Villani et al., 2017). Of .. et al., 2000; Aliberti et al., 2003). EnVision FLEXþ Mouse and Rabbit
note, the recent single-cell transcriptomic analysis undertaken by Villani .. linkers (Dako, Denmark) were used to amplify the antibody signals
..
et al. (2017) also characterized a further DC subset as CD11c and .. (for CD1c and IRF-8, respectively). Positive cells were visualized with
negative for another standard pDC identification marker, CD123, so
.. EnVisionþ Dual Link (Dako, Denmark) and Diaminobenzidine
..
for completeness, we have included an equivalent quantification of .. (DABþ) Substrate Chromogen System (Dako, Denmark). The sec-
CD11cCD304 DCs in our flow cytometric analysis. Stained cells
.. tions were then counterstained with Mayer’s Haematoxylin, dehy-
..
were then fixed using Cytofix buffer (BD Biosciences, USA). .. drated and cover-slipped. Staining with mouse IgG1 and rabbit
..
Labelled cells were analysed by flow cytometry using the .. polyclonal isotype controls (for CD1c and IRF-8, respectively) as well
FACSCanto II flow cytometer (BD Biosciences, USA). Single colour .. as diluent only controls (no antibodies) was negative.
..
compensation was performed with CompBeads (BD Bioscience, .. Slide analysis was conducted using Olympus BXS1 microscope
USA). Data were analysed using FlowJo version 10.6.1 software (Tree
.. (Olympus, Japan) coupled with a DP 70 camera (Olympus, Japan). Up
..
Star, USA) to distinguish mDC and pDC subpopulations (Fig. 1). .. to 20 randomly selected fields of view (FOV) filled with intact endo-
.. metrium were captured under 400 magnification depending on total
..
Immunohistochemistry .. tissue area available (median FOV number ¼ 12; range 5–20 FOV).
..
In order to better characterize endometrial DC populations, immuno- .. DCs were identified according to their morphology and staining char-
.. acteristics. Counts were expressed as density per mm2. Counting was
histochemical staining was performed for distinct myeloid and plasma- ..
cytoid cells. Endometrial samples were fixed in neutral buffered
.. validated over all FOV for 10 randomly selected slides by two sepa-
.. rate, blinded investigators with good correlation (Pearson correlation
formalin and embedded in paraffin. Sections were cut at 4 mm, and ..
mounted onto silanized slides (Dako, Denmark). Following deparaffini-
.. coefficient ¼ 0.96, 95% confidence interval ¼ 0.95–0.97, P < 0.001).
..
zation and rehydration, slides were treated with antigen retrieval solu- ..
.. Statistical analyses
tion (High pH Target Retrieval Solution, pH 9 (Dako, Denmark) for ..
CD1c; Low pH Target Retrieval Solution, pH 6.1 (Dako, Denmark) .. Statistical analyses were performed using Statistical Package for the
..
for IRF-8). Autostainer Plus Model S3400 (Dako, Denmark) was used .. Social Sciences (SPSS) Version 24 software (IBM, USA). The distribu-
to perform all immunostaining.
.. tion of variables was determined with a one-Sample Kolmogorov–Dendritic cell subsets in endometriosis 419
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Figure 1. Representative cytometry showing FlowJo gating strategy to quantify dendritic cell (DC) populations. (A) Circulating
and (B) endometrial, briefly all leukocytes were selected based on size and complexity parameters, and CD45þ for endometrial samples; single-cell
events were selected from forward scatter; live, Lin-* HLADRþ DC were selected; CD11cþ myeloid DC, CD304þ plasmacytoid DC and
CD11cCD304 dendritic cell subpopulations identified; CD141þ, CD1cþ and CD16þ subpopulations of CD11cþ myeloid DC identified; and
separation of CD2þ/ subpopulations of CD304þ plasmacytoid DC in blood. *Lin refers to the combined staining of lineage markers CD3, CD19,
CD20, CD14 and CD56.
Smirnov test. Normally distributed data were compared across all .. observed between IRF-8þ cell density and the number of FOV
..
three menstrual phases using the one-way ANOVA, and between two .. counted (rs ¼ 0.28, P ¼ 0.016, n ¼ 73). Therefore, for further analyses,
phases using independent samples t-tests, with summary data reported
.. a median split of the data was performed (into 5–12 FOV counted
..
as means and SDs. Skewed and proportional data were compared .. (52% of samples) and 13þ FOV (48% of samples)) and analyses re-
..
across all three menstrual phases and revised American Society for .. peated within each of these two strata.
Reproductive Medicine (r-ASRM) endometriosis stages using the .. Correlations between DC populations were examined using
..
Kruskal–Wallis test, and between two phases/stages using the non- .. Spearman’s correlation coefficient as data were skewed or propor-
parametric Mann–Whitney U-test, and summary data reported as .. tional. The significance threshold was set as P < 0.01 for these analyses
..
medians and IQRs. If there were too few data points for an IQR then .. to account for multiple comparisons.
this was reported as n/a. Differences were considered to be statisti-
..
..
cally significant at P-values of less than 0.05. ..
Circulating and endometrial total DC proportions of leukocytes
..
.. Results
measured by flow cytometry did not show much variation during the ..
.. For a cohort of women with confirmed diagnosis of endometriosis or
menstrual cycle or between groups. Therefore, analyses of flow cy- ..
tometry data were reported for DC subsets expressed as proportions .. not, flow cytometry was performed on PBMC and/or endometrial tis-
..
of parent DC populations total. .. sue samples when available in order to examine the immune associa-
Endometrial CD1cþ and IRF-8þ cell density data were explored .. tions with endometriosis and fertility (Supplementary Fig. S1). To
..
for any correlation with the number of FOV counted using Spearman’s .. complement this analysis, and to examine in situ cellular relationships,
correlation coefficients (rs) as overall data were skewed in distribution. .. immunohistochemical assessment of paraffin embedded endometrial
..
There was a small but statistically significant positive correlation . tissue was performed. At a cohort level, there were no significant420 Hey-Cunningham et al.
..
differences in age between endometriosis group or fertility status .. proportion of live, single leukocytes measured by flow cytometry
(Supplementary Fig. S2). .. across the participants which had both analyses performed, n ¼ 16
..
Using multiparametric flow cytometry, human DC populations were .. (Fig. 3), showing concordant results for our cross-platform analysis.
identified in the blood and/or endometrium of all participants accord- ... We confirmed this correlation was significant even when limited to as-
ing to the international consensus on DC subsets with overall propor- .. sessment of endometriosis only group, n ¼ 11, or secretory cycle
..
tions/cell densities provided in Table III. Immunohistochemical staining .. phase, n ¼ 9 (data not shown). In terms of relationships between cir-
for IRF-8, the best available formalin fixed tissue staining for identifica-
.. culating and tissue immune effects, we did not observe any statistically
..
tion of pDCs along with mDC1 cells, identified cells characterized by .. significant correlations in DC populations matched across blood and
.. tissue as interrogated by flow cytometry (n ¼ 20 for the paired analy-
oval or round, brown nuclear staining with a size of 3–10 mm ..
..
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(Fig. 2A and B). Whilst IRF-8þ DCs were rare in the endometrium, sis). Equivalent comparison for IRF-8 histology correlation to endome-
.. trial pDC proportion of live, single leukocytes was not possible due to
they were scattered through the endometrial stroma. In some sam- ..
ples, 5–10 IRF-8þ DC were clustered as part of what appeared to be .. small n in 13þ FOV counted stratum (n ¼ 6 for the paired analysis),
.. also with acknowledgement that IRF8 staining identifies both pDCs
small immune cell aggregates. Results reported below are for the 13þ ..
FOV counted stratum for IRF-8þ DC densities. Staining for CD1c to
.. and cDC1 cells (Collin and Bigley, 2018).
..
identify CD1cþ mDCs, showed nucleated cells of 10–20 mm display- .. We confirmed that no significant correlations were observed be-
.. tween age and quantification of DC populations as measured by both
ing DC morphology with visible processes extending from the cell ..
bodies (Fig. 2C and D). Discrete CD1cþ mDC were present through .. flow cytometry and immunohistochemistry in the study cohort. In or-
.. der to assess the effect endometriosis and fertility may have on DC
the endometrial stroma, infiltrating into the basal aspect of some en- ..
dometrial glands (as shown in Fig. 2C), and sometimes located directly .. populations, we first set out to establish how proportions and cell
.. densities of different populations varied during the menstrual cycle and
adjacent to vessel endothelium. ..
Of note, endometrial CD1cþ DC density measured by immunohis-
.. between groups.
..
tochemistry significantly correlated with endometrial CD1cþ mDC ..
.. Dendritic cell populations during the
..
.. menstrual cycle in women with and
Table III Overall proportions and cell densities of studied ..
.. without endometriosis
dendritic cell populations in blood and endometrium. ..
Median and interquartile range (IQR) are provided for all .. Circulating populations
data (proportional measures or skewed in distribution). .. Circulating DC populations did not significantly change throughout the
..
.. menstrual cycle in the study population overall (all data available in
DCa measure Median IQR .. Supplementary Table SI). The study was not powered to examine cir-
...................................................................................................... ..
Blood (n ¼ 37) .. culating DC populations during the normal menstrual cycle as there
.. were no samples from women without endometriosis during the men-
Total lineage- HLADRþ DC 2.00 1.50 ..
(% live, single leukocytes) .. strual phase. In contrast to the overall observation of no significant
.. changes in circulating DC populations during the menstrual cycle, in
pDC (% total DC) 14.70 13.15 ..
CDb2þ pDC (%pDC) 11.90 11.30
.. women with endometriosis alone, lineage-HLADRþ blood DCs nega-
..
mDC (% total DC) 69.76 16.00 .. tive for CD11c and CD304 were significantly decreased as a propor-
.. tion of total DCs between the secretory phase and menstruation
CD16þ mDC (%mDC) 52.67 22.03 ..
CD1cþ mDC (%mDC) 30.01 16.79
.. (Fig. 4A). There was also an apparent trend for the number of blood
.. CD141 mDCs to be lower in menstruation compared to the secre-
CD141þ mDC (%mDC) 1.56 2.00 ..
.. tory phase (Fig. 4B) in women with endometriosis.
CD11c CD304 DC (% total DC) 7.01 6.97 ..
Endometrium (n ¼ 23 for flow cytometry, n ¼ 73 for .. Endometrial populations
immunohistochemistry)
..
.. In the endometrium, utilizing flow cytometry we found the CD1cþ
Total lineage-HLAcDRþ DC 4.40 2.32 .. proportion of mDC was significantly lowered during menstruation
(% live, single, CD45þ leukocytes)
..
.. (Fig. 5A). In addition to the observation of lowest endometrial CD1cþ
pDC (% total DC) 15.19 11.24 .. proportions of mDC populations during menstruation in the study
..
mDC (% total DC) 46.60 18.51 .. population overall; in women with endometriosis alone, endometrial
CD16þ mDC (%mDC) 10.97 8.37 ..
.. CD1cþ proportion of mDC was lowest during menstruation and high-
CD1cþ mDC (%mDC) 28.72 15.45 .. est during the proliferative phase (Fig. 5A).
CD141þ mDC (% mDC) 3.80 5.23
..
.. By histology, the CD1cþ population was also modulated by phase,
CD11c CD304 DC (% total DC) 33.60 31.60 .. however only reaching significance in women without endometriosis.
..
IRF-8þ cell density per mm2 2.93 2.64 .. Endometrial CD1cþ cell density was significantly higher in the secre-
CD1cþ cell density per mm2 6.35 8.86
.. tory compared to proliferative phase (Fig. 5B) in women without en-
..
.. dometriosis. Endometrial IRF-8þ cell density also appeared to change
a
DC ¼ dendritic cell; m, myeloid; p, plasmacytoid. .. during the menstrual cycle in women without endometriosis with spe-
b
CD ¼ cluster of differentiation. ..
c
HLA ¼ human leukocyte antigen. .. cific comparisons between phases demonstrating the major modula-
.. tion was an elevation during menstrual phase (menstrual vsDendritic cell subsets in endometriosis 421
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Figure 2. Representative images of IRF-81 in proliferative phase endometrium. (A) From a woman with endometriosis and (B) with-
out endometriosis. Yellow circles in contain small immune cell aggregates. CD1cþ cells in secretory phase endometrium samples from (C) a woman
with endometriosis and (D) without endometriosis. Yellow box insert shows a zoomed image of a stained dendritic cell (DC) with visible processes
extending from the cell body. Yellow arrow in (C) points to a CD1cþ myeloid DC (mDC) infiltrating into the basal aspect of an endometrial gland.
Positive cells are stained brown with diaminobenzidine þ Chromogen (400 magnification).
proliferative, t ¼ 5.33, df ¼ 7, P ¼ 0.001; secretory vs menstrual,
.. endometriosis. We next sought to further characterize DC popula-
..
t ¼ 4.02, df ¼ 4, P ¼ 0.016; it should be noted that menstrual n ¼ 1; .. tions in women with endometriosis by direct comparison to women
..
and as such has not been indicated on Fig. 5C). In women with endo- .. without endometriosis (all data available in Supplementary Table SII).
metriosis, the endometrial densities of IRF-8þ and CD1cþ cells mea- .. In women with endometriosis, blood CD141þ proportion of mDC
..
sured by immunohistochemistry did not significantly change during the
... was overall significantly higher than in women without endometriosis
menstrual cycle. .. (Fig. 4B). This difference was particularly evident in the secretory phase
.. (Fig. 4B).
..
Dendritic cell populations in women with .. There were no statistically significant differences in circulating DC
..
compared to women without .. populations between the r-ASRM stages of endometriosis (all data
.. available in Supplementary Table SIII).
endometriosis ..
..
Circulating populations .. Endometrial populations
The exploration of differences across menstrual cycle phases revealed .. In women with endometriosis, endometrial IRF-8þ cell density was
..
some significant DC population differences in women with . significantly higher than in women without endometriosis during the422 Hey-Cunningham et al.
..
.. Dendritic cell populations in women with a
..
.. history of infertility compared to fertile
.. women
..
..
.. We queried the data for differences in DC populations between
.. women with and without a history of infertility. No statistically signifi-
..
.. cant differences in DC populations were observed between women
.. with and without a history of infertility (all data available in
..
.. Supplementary Table SIV). The power of these analyses was limited
.. by small numbers of fertile participants.
..
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..
..
..
.. Discussion
..
..
.. This study has characterized DC populations in the blood and endo-
.. metrium of women with and without endometriosis, including the
..
.. most comprehensive range mDC and pDC populations in the endo-
.. metrium to date. For the first time, this study has allowed us to assess
..
.. DC populations not only by endometriosis presence or absence but
.. also by r-ASRM disease stage. We have shown that a range of DC
..
Figure 3. Correlation between endometrial CD1c1 den- .. populations are altered in women with endometriosis, particularly in
dritic cell (DC) density measured by immunohistochemis- .. the endometrium, both compared to women without endometriosis
try (x axis) and endometrial CD1c1 myeloid DC (mDC)
..
.. and between r-ASRM endometriosis stages. These novel findings high-
proportion of live, single leukocytes determined by flow cy- ..
tometry (y axis). n ¼ 16 for study participants from whom endo- .. light the importance of the endometrial immune environment in endo-
.. metriosis. DCs are modified by their tissue environment, meaning that
metrium was available for both immunohistochemical and flow ..
cytometric analyses. .. analysis of endometrial resident DC populations is key to understand-
.. ing endometrial-related conditions such as endometriosis. Our obser-
..
.. vations have important implications for the understanding of the
.. immune aspects of endometriosis, revealing new insights relevant to
..
proliferative phase (Fig. 5C). During the secretory phase, total tis- .. disease pathogenesis and symptom generation. Another novel consid-
sue DC proportion (determined by flow cytometry as CD45þ, lin- .. eration has been the documented annotation of our study cohort for
..
eage markers negative and HLA-DRþ) was statistically significantly .. fertility status, which by and large is not reported. The most striking
higher in the endometrium of women with endometriosis than .. aspect, and unfortunately largely limited our analysis power was the
..
without (Fig. 5D). In secretory phase endometrium, mDC, as .. observation that infertility was indeed widespread, a terrible and well-
gated by CD11cþ and lineage- HLADRþ CD11c CD304 DC ..
.. characterized detriment of endometriosis.
proportions of total DC (gated as CD45þ, lineage markers nega- .. While we examined both circulating and endometrial DC popula-
tive and HLADRþ) also were trending to significance in women
..
.. tions, and for flow cytometry experiments had larger blood than tissue
with compared to without endometriosis ((i) tissue mDC propor- .. sample numbers, the main findings of this research relate to endome-
..
tion total DC lower in endometriosis, Fig. 5E; (ii) tissue CD11c .. trial DC. We have performed the most comprehensive characteriza-
CD304 DC proportion of total DC higher in endometriosis, .. tion of mDC and pDC subpopulations in the endometrium to date
..
Fig. 5F). .. (Rodriguez-Garcia et al., 2017, 2018), including concurrent analysis of
Endometrial DC populations in women with endometriosis showed .. CD1cþ, CD141þ and CD16þ mDC populations for the first time. It
..
some subtle differences between r-ASRM disease stages (all data avail- .. is critical to look at the endometrium in endometriosis due to the
able in Supplementary Table SIII). In particular, endometrial CD141þ .. pathogenesis of the disease, associated endometrial anomalies and
..
proportions of mDC significantly varied between r-ASRM stages .. knowledge that tissue environment modifies DC population phenotype
(Fig. 6A). There was also a trend for endometrial IRF-8þ cell density
.. and function (Rodriguez-Garcia et al., 2017, 2018). In particular, we
..
per mm2 to be different by r-ASRM endometriosis stage (Fig. 6B). .. observed that in the endometrium of women with endometriosis, IRF-
When compared across simplified categories of minimal-mild (Stages
..
I–II) and moderate–severe (Stages III–IV) endometriosis, endometrial
... 8þ DC density was significantly increased during the proliferative
.. phase, and total DC proportion of leukocytes was significantly in-
IRF-8þ cell density was significantly higher in minimal-mild compared .. creased during the secretory phase compared to women without en-
..
to moderate–severe disease (Fig. 6B). These distinct observations .. dometriosis. DC are recruited to inflammatory tissues (Boltjes and
around CD141þ and IRF-8þ identified cells are interesting indeed .. Van Wijk, 2014) and the increase of IRF-8þ DC during the prolifera-
..
given the understanding that IRF-8 captures identification of both .. tive phase and total DC during the secretory phase in women with en-
pDCs and cDC1 cells, as is identified by CD141 expression (Collin .. dometriosis is consistent with this.
..
and Bigley, 2018). While the cross-platform nature of these findings .. We also observed a possible blunting of normal menstrual cyclical
mean we can only infer pDCs may account in part with the relative
.. changes in endometrial DC populations in women with endometriosis.
..
deficiency of IFR-8þ cells in more severe disease. . In the endometrium of women without endometriosis,Dendritic cell subsets in endometriosis 423
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Figure 4. Graphs and representative flow cytometry dot plots showing dendritic cell (DC) populations during the menstrual
cycle with data points colour coded by women with and without endometriosis. (A) Circulating CD11c CD304 DC as a proportion
of total DC and (B) CD141þ myeloidDC (mDC) as a proportion of mDC Bars show median value and error bars show interquartile range.
..
immunohistochemistry showed density of CD1cþ and IRF-8þ popula- .. We therefore were surprised to observe across the r-ASRM stages
tions significantly fluctuated during the menstrual cycle, but these .. of endometriosis (I–IV), CD141þ proportion of mDC showed overall
..
effects were absent in endometriosis. This would be consistent with .. significant differences, such that they were higher in Stages II and III
endometrial microenvironment changes associated with the normal
.. than I and IV. Additionally, IRF-8þ endometrial density was significantly
..
menstrual cycle (Berbic and Fraser, 2013), including cycling of DC .. lower in minimal-mild disease (Stages I–II) compared to moderate-
..
chemo-attractants, extensively reviewed by Tiberio et al (2018). In .. severe disease (Stages III–IV). CD141þ mDCs have an enhanced abil-
contrast, as we have observed, cyclical effects are lost as the milieu .. ity to take up dead and necrotic cells (Collin et al., 2013). It may be
..
becomes consistently inflamed in endometriosis. The uterine environ- .. that in mild-moderate endometriosis, these cells are locally enriched
ment in women with endometriosis is inflammatory, with altered den-
.. as they attempt to clear shed and ectopic endometrial cells. However,
..
sity and function of a range of immune cell populations and altered .. in severe disease, this immune response may become exhausted and
..
expression of substances which influence the immune response .. inflammation chronic (Riccio et al., 2018), reflected in reduced
(Symons et al., 2018; Vallvé-Juanico et al., 2019). .. CD141þ mDC and increased IRF-8þ density in the endometrium.
Within the endometrium of women with endometriosis, there ... While the implications of these immunological observations in rela-
..
was some evidence of DC population differences between r-ASRM .. tion to the endometrial environment and endometriosis disease stage
disease stage classifications. The r-ASRM classification of endometri-
.. classification are somewhat unclear at present, what is apparent is that
..
osis is a surgical staging system based on the extent and location .. sample numbers are not everything in this aspect. We had small num-
..
of the disease (American Society for Reproductive Medicine, .. bers of clinically well-characterized samples in each menstrual cycle
1997). While longstanding and widely in clinical use, there are con- .. phase and endometriosis disease stage, which was sufficient to show
..
troversies over the utility of r-ASRM disease staging, given the lack .. interesting differences by immunological activity and phenotype.
of correlation with symptoms, prognosis, biological or scientific .. Furthermore, we would be fascinated to understand whether profiles
..
parameters (Johnson et al., 2017). . observed could be a reflection of disease development, advancement424 Hey-Cunningham et al.
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Figure 5. Graphs and representative flow cytometry dot plots showing endometrial dendritic cell (DC) subsets colour coded by
women with and without endometriosis, organized by menstrual cycle phase. (A) CD1cþ as a proportion of myeloid dendritic cells
(mDC) measured by flow cytometry, (B) CD1cþ cell density per mm2 and (C) IRF-8þ cell density per mm2 during the menstrual cycle, with n ¼ 1
each for menstrual analysis, (D) total DC (gated as CD45þ, lineage markers negative and HLADRþ) as a proportion of total leukocytes, (E) mDC
(CD11cþ gated within DCs) as a proportion of total DC and (F) Lineage- HLADRþ CD11c CD304 DC as a proportion of total DC. Bars show
median value and error bars show interquartile range.Dendritic cell subsets in endometriosis 425
..
.. enhancing cytotoxic T cell responses through their important cross-
.. presentation role (Canavan et al. 2018). This remarkable finding indi-
..
.. cates that endometriosis may be driving reminiscent inflammatory
... processes. Beyond this, in women with endometriosis, blood CD11c
.. CD304 DC proportions of total DC significantly decreased and
..
.. CD141 proportion of mDC tended to decrease between the secre-
.. tory phase and menstruation. From the current study, it was unclear
..
.. whether these decreases reflect normal biology due to a lack of sam-
.. ples from women without endometriosis during menstruation.
..
..
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Interestingly, a significant decrease in circulating CD141þ % of periph-
.. eral leukocytes between secretory phase and menstruation in women
..
.. with endometriosis has been previously demonstrated, alongside no
.. significant cyclical changes in control samples (Maridas et al., 2014).
..
.. CD141þ DC have been shown to be enriched in inflammatory
.. environments which result in their activation and increased migratory
..
.. capacity. In these environments, they can produce more pro-
.. inflammatory cytokines such as tumour necrosis factor-a and inter-
..
.. feron-c which enhances T-cell activation (Canavan et al., 2018), an
.. intriguing functionality to be explored in future studies.
..
.. There is growing evidence of systemic inflammation in endometri-
..
.. osis, in addition to the local inflammation in the uterus and peritoneal
.. cavity (Monsanto et al., 2016; Nematian et al., 2017). Having identified
..
.. these differences, investigating the activation status of these cells and
.. the inflammatory milieu in women with endometriosis will provide in-
..
.. sight into the contributions DC make to pathogenesis. The inflamma-
.. tory environment in endometriosis is linked to pain sensitization and
..
.. generation of pain symptoms (Howard, 2009). There are now an in-
.. creasing number of immune-based therapies successfully in use for a
..
.. range of other chronic inflammatory conditions, meaning it is of partic-
.. ular relevance to understand inflammation in endometriosis and to
..
.. determine whether this may be a future therapeutic target for the
.. condition.
..
.. Understanding of endometriosis pathogenesis remains elusive; how-
.. ever, immunology is thought to make pivotal local (uterine and perito-
..
.. neal) and systemic contributions to its genesis and generation of
.. common symptoms and morbidities such as pain, infertility and poor
Figure 6. Graphs and representative flow cytometry dot
..
.. pregnancy outcomes (Vallvé-Juanico et al., 2019). Combined with
plots showing endometrial dendritic cell (DC) populations. .. established immune, including DC, roles in the female reproductive
(A) CD141þ as proportions of myeloid DC (mDC) and (B) IRF-8 ..
.. tract in relation to fertility problems such as recurrent miscarriage and
density per mm2 in women with endometriosis by different revised ..
American Society for Reproductive Medicine (r-ASRM) disease .. recurrent implantation failure (Askelund et al., 2004; Tirado-González
stages.
.. et al., 2010; Liu et al., 2018); it is hypothesized that the immune phe-
..
.. notype is disturbed in infertile women with endometriosis. We did not
.. observe any statistically significant differences in DC populations be-
..
.. tween women with and without a history of infertility in this study.
or treatment as reflected in the time the samples were collected. In fu- .. The power of our analyses were limited by small numbers of fertile
..
ture, further investigation and more focused phenotypic analysis of im- .. participants and further research is required to investigate the true
mune cell subtypes in the endometrium, such as with mass cytometry, .. nature of the immune contributions to endometriosis-associated
..
will be essential capture the complexities of the endometrial immune .. infertility.
niche and relationships to reproductive disorders. Longitudinal studies .. As is common in this type of study, one of the main overall limita-
..
will also help unravel phenotype relationships explored. .. tions was small sample numbers, particularly during the menstrual
In addition to the range of tissue DC population changes in endo- .. phase of the cycle. This is the shortest phase of the menstrual cycle
..
metriosis described above, we observed some alterations in circulating .. and often an undesirable time for gynaecological surgery from a patient
DC populations indicative of heightened systemic inflammation. In par- .. perspective, making sample collection particularly challenging. Another
..
ticular, circulating CD141þ mDC proportions, which are usually very .. contributing factor to low endometrial tissue sample numbers for flow
rare, were significantly increased in women with endometriosis com- .. cytometric experiments was the novelty of our approach, performing
..
pared to women without the disease, both overall and during the se- .. the most comprehensive characterization of mDC and pDC subpopu-
cretory phase. CD141þ mDCs are usually linked with viral infection,
.. lations in the endometrium to date. Optimization of our protocol for426 Hey-Cunningham et al.
..
identification of endometrial DC populations included testing of differ- .. thank you to all the gynaecology surgical staff at Royal Prince Alfred
ent approaches for mechanical and enzymatic tissue dissociation, spe- .. Hospital (Sydney) and women who generously gave their time and pro-
..
cific identification of leukocytes, live/dead cell discrimination, DC .. vided biological samples.
staining and fixation. Analysis of rare cells in the endometrium presents ...
a unique set of challenges underpinned by the scientific benefits pro- ..
.. Authors’ roles
vided by a large sample volume versus the clinical and ethical difficulties ..
of obtaining tissue (Rodriguez-Garcia et al., 2018). The optimization .. A.J.H.-C.—substantial contributions to study conception and design,
..
process necessarily used a substantial number of tissue samples which .. analysis and interpretation of data; drafting of the bulk of the article
were not included in the final participant cohort for which the results
.. and revising it critically for important intellectual content; and final ap-
..
.. proval of the version to be published. C.W.—involvement in study
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have been reported here.
Here we have reported the most comprehensive characterization of
.. conception and design and substantial contributions to data acquisition;
..
DC populations in the blood and endometrium of women with and .. contribution to initial draft of materials and methods section; and final
without endometriosis, assessing differences not only by endometriosis
.. approval of the version to be published. J.H.—key role in analysis and
..
presence or absence but also by r-ASRM disease stage and fertility sta- .. interpretation of data; drafting of article sections and critical revisions
.. for important intellectual content; and final approval of the version to
tus for the first time. We have shown evidence of endometrial and ..
systemic inflammation in women with endometriosis. In the endome- .. be published. P.D.F.—involvement in study conception and design and
.. substantial contributions to data acquisition; critical revisions of manu-
trium of women with endometriosis, IRF-8þ cells were increased dur- ..
ing the proliferative phase, total DC proportions increased in the .. script; and final approval of the version to be published. G.J.C.—role in
..
secretory phase and normal menstrual cyclical fluctuations in CD1cþ .. study conception and design; drafting DC biology content for the man-
and IRF-8þ cells blunted; indicative of a consistently inflammatory tis- .. uscript; critical revisions of manuscript; and final approval of the version
..
sue environment. The inflammatory changes in CD141þ and IRF-8þ .. to be published. F.K.—involvement in study conception and design and
populations in the endometrium of women with endometriosis were .. substantial contributions to data acquisition; critical revisions of manu-
..
particularly evident in more advanced ASRM stages of the disease. .. script; and final approval of the version to be published. E.J.M.—role in
These novel observations highlight the importance of the endometrial
.. study conception and design; contributions to sample collection and
..
immune environment in endometriosis. There was also evidence of .. data acquisition; critical revisions of manuscript; and final approval of
systemic inflammation in women with endometriosis, with increased
.. the version to be published. R.M.—role in study conception and de-
..
circulating CD141þ mDC proportions. Further phenotyping of local .. sign; critical revisions of manuscript; and final approval of the version to
.. be published. H.M.M.G.—substantial contributions to analysis and in-
and circulating immune cell subtypes is critical to improving under- ..
standing of endometriosis pathogenesis and immune contributions to .. terpretation of data; role in drafting the article and driver of revising it
.. critically for important intellectual content; and final approval of the
infertility associated with the disease. ..
.. version to be published.
..
..
..
Supplementary data .. Funding
..
Supplementary data are available at Human Reproduction online. .. This research was financially supported by a Sydney Medical School
.. and Balnaves Foundation Kick Start Grant and the Department of
..
.. Obstetrics, Gynaecology and Neonatology at The University of
..
Data availability .. Sydney.
..
The data underlying this article will be shared on reasonable request
..
.. Conflict of interest
to the corresponding author. ..
..
.. The authors have no conflicts of interest to declare.
..
Acknowledgements ..
..
The authors thank Professor Ian S. Fraser (Department of Obstetrics ..
and Gynaecology, The University of New South Wales), Dr Marina
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