Enhanced local and systemic inflammatory cytokine mRNA expression in women with endometriosis evokes compensatory adaptive regulatory mRNA response that mediates immune suppression and impairs cytotoxicity

Endometriosis is a disease characterized by ectopic implantation of endometrium and impaired immune responses. To explore its pathogenic mechanisms, we studied the local and systemic cytokine mRNA profiles and their role in the immunity of patients with endometriosis and healthy controls.


| INTRODUC TI ON
Endometriosis, affecting 10% of women worldwide, is an estrogen-dependent disease-causing chronic abdominal pain and infertility in women of reproductive age. It results from implantation and subsequent growth of endometrial tissue outside the uterine cavity. 1,2 The most widely accepted hypothesis for the development of pelvic endometriosis is ectopic dissemination of endometrial tissue through retrograde menstruation, first proposed in the 1920s. 3,4 However, although approximately 90% of women have evidence of retrograde menstruation, 5 only about 10% develop endometriosis.
Several factors are likely to influence the susceptibility to the disease. Despite long-time research, endometriosis is still an enigma and its cause(s) are so far unknown.
It is suggested that aberrant immunological mechanisms causing a dysfunction of immune cells and mediators are involved in the pathogenesis of endometriosis. The ectopically disseminated endometrium is allowed to escape immune surveillance. It implants, proliferates, and invades the underlying tissue, forming painful endometriotic lesions that grow under the hormonal influence of the menstrual cycle. 2 Endometriosis is considered a benign disease, but has many features in common with tumours such as clonal proliferation, dissemination, and tissue invasion. 6 Research suggests heritability for endometriosis, estimated to be around 50% in twin studies. 7 Cytokines may play a significant role in the current understanding of the pathogenesis of endometriosis by modulating the patients' immune system toward acceptance of ectopic implantation of endometrial tissue thus allowing chronic disease progression. 2,8 Cytokines, secreted by a variety of cells, are small proteins/peptides that are key mediators of intercellular communication. The ability of the immune system to regulate immunity and inflammation, promote or prevent cell growth and movement and exert immune surveillance is associated with different cytokine profiles. These are operating locally and mediate a variety of immune responses, denoted T helper (Th) 1, Th2, inflammatory, and T regulatory response. A cytokine profile dominated by Th1 cytokines such as IFNγ and IL15 promotes a Th1 response, that is, cytotoxicity; a Th 2 cytokine profile dominated by IL 4 promotes a Th2 response, that is, a humoral response, while an inflammatory cytokine profile including IL1β, IL6, IL8, tumour necrosis factor (TNF)α, and LTA/TNFβ promotes inflammation, and a To understand the interplay and contribution of cytokines in the overall immune response in endometriosis, here, using real-time quantitative RT-PCR, we investigated the cytokine profiles locally in the endometriotic and endometrial tissues, and systemically in peripheral blood mononuclear cells (PBMC), and compared them to the cytokine profiles in healthy controls. We studied the simultaneous relative mRNA expression for a broad panel of 11 cytokines, defining the specific immune responses described above. In addition, using immunohistochemistry with monoclonal antibodies, we analyzed the presence of immune cells in endometriotic lesions focusing on T regulatory cells.

| Materials
The

| Isolation of peripheral blood mononuclear cells (PBMC) from patients and healthy donors
PBMC were isolated from endometriosis patients and healthy controls within 24 hours from sample collection using Lymphoprep (Nycomed) gradient centrifugation as previously described. 10,11 The interphase containing lymphocytes and macrophages were collected, washed, counted, and kept frozen at −80°C until use. Representative photomicrographs are shown in Figure 1.

| Total RNA extraction and real-time quantitative reverse transcription-polymerase chain reaction (RT-qPCR)
The gene expression analysis of cytokines was performed by realtime RT-qPCR following the MIQE requirements. 12

| RNA extraction
For RNA isolation, about 30 mg of each tissue sample was minced by lancet into small pieces and further processed according to a standard protocol described below.
Total RNA from tissue biopsies and PBMC was extracted using TRIsol and RNeasy Mini kit, respectively. PBMC samples were immediately disintegrated in 350 µl of lysis buffer at room temperature according to RNeasy Mini manual without DNase treatment.

| Overall quality evaluation of isolated total RNA
RNA yield (on average 245 ng/µl) and purity (on average A 260 /A 280 = 1.6) were assessed in 2 µl of RNA sample by using NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific Inc, USA).

| Reverse transcription
For each tissue/cell sample, 400 ng of total RNA in reaction volume 20 µL was transcribed into cDNA using High-Capacity cDNA Reverse Transcription Kit according to the manufacture manual.
60 µL of sterile Milli-Q water was added to every sample to adjust cDNA concentration equal to 5 ng/µL total RNA.

| Processing of data and statistical analyses
To analyze the data, a comparative or ∆∆Ct method was used with an array of relative quantities (RQ) for each study group. The ratios were calculated by dividing the group average of each study group by a reference group average. A non-parametric bootstrap test was used to evaluate the statistical significance of the ratios compared to the null hypothesis that the ratios were less or equal to one. Pvalues ≤ .05 were considered significant.

| Patients and controls
A brief summary describing the patients and healthy controls is presented in Tables 1 and 2

| CD4 + CD25 ++ T regulatory cells are abundant in endometriotic lesions
To study the presence/absence of T regulatory (Treg) cells in endo-  Table S1 cells of tr1 type that are known to scarcely express FoxP3, or (b) The anti-FoxP3 antibodies did not perform in IHC staining. IHC stainings of CD4 + CD25 ++ T regs in serial sections of 3 representative samples of endometriotic tissue are shown in Figure 1. In Figure 2A and in contrast to the local inflammatory response (Figure 2A), there was an upregulation of TNFα as well.

The local cytokine mRNA response in patients' endometrium and endometriotic lesions suggests an enhanced local inflammation and priming of the adaptive T regulatory response in the endometriotic lesions.
In the next step, after comparing the local and systemic cytokine mRNA profile in endometriosis patients, we specifically analyzed the local cytokine profile in endometriotic lesions compared to patient endometrium ( Figure 3A) and in patient endometrium compared to endometrium from healthy controls ( Figure 3B). As can be seen, the Combining the results in Figure 3A Figure 3A compared to sixfold in Figure 3B), lost/slightly downregulated TGFβ1 mRNA expression and simultaneously 5-20 times upregulated IL-2 mRNA (shown in Figure 3A).

| D ISCUSS I ON
Immune mechanisms are proposed to play a role in the development of endometriosis. In contrast to healthy women, patients suffering from endometriosis appear to have a decreased cell-mediated immunity with suppressed T-and NK-cell cytotoxicity. 15 Thus, several studies support our results of upregulated Treg cytokines but there are also results that show no difference compared to controls. 19,33 The reasons for these discrepancies are not known but might be due to differences in the disease duration and treatment, the experimental set-up, the methods used, and the timing of the sample collection. To try to accurately reflect the natural course of endometriosis, our samples were collected at the time of the disease diagnosis and the vast majority of the patients (all but 6) were not hormonally treated.

CO N FLI C T S O F I NTE R E S T
There are no conflicts of interests.