Dados do Trabalho

Title

Evaluation of interferon beta and inflamossome AIM2 protein expression in deep endometriosis lesion

Objective

Endometriosis is described as an estrogen dependent and progesterone resistant inflammatory disease, categorized into four stages (minimal, mild, moderate and severe). Endometriosis is characterized by the presence of eutopic tissue outside the uterine cavity, in which, accompanied by a failure of the innate and adaptative immune system, acts on the release of pro-inflammatory cytokines, leading to endometrial cells to implant and proliferate in the peritoneal cavity and organs, resulting in endometriosis development and progression. Previous studies have already described an increase in the inflammatory response related to downregulation of some protein markers, such as interferon. Recently, it was found a link between the inflammasome complex gene expression and endometriosis. Therefore, the aim of this experimental project is to evaluate the protein expression of interferon beta type 1 (INF-1) and inflammasome absent in melanoma 2 (AIM2) in endometriotic lesions and compare it with eutopic endometrium and non-endometriosis endometrium. This study was approved by the project management for research – SGPP (3617-18), has a certificate of presentation of ethics assessment (CAE 05776919.3.0000.0071) and grants 2019/24351, São Paulo Research Foundation (FAPESP).

Methods

For the development of this project immunofluorescence reaction was used in samples of endometrial tissues from patients with and without endometriosis and in lesions of deep endometriosis. The patients were divided between endometriosis group (10 eutopic endometrium and 10 lesions) and control group (10 non-endometriosis endometrium). The selection criteria encompassed women between 18 and 50 years old that were submitted to laparoscopy surgery for endometriosis excision or pain evaluation and were excluded patients with autoimmune diseases, infections or neoplastic diseases. For the immunofluorescence reaction primary antibody used were anti-INF-1 1:150 (BS-0787R - Thermofisher, São Paulo, Brazil) 1 mg/ml 100uL, and the anti-AIM2 antibody 1:150 (MA5-38442 - Thermofisher, São Paulo, Brazil) 1mg/ml 100uL. The secondary antibody was 1:400 Alexa Fluor 488 (a11008 – Thermofisher, São Paulo, Brazil), and 1:400 Alexa Fluor 647 (ab150115 –Abcam, São Paulo, Brazil), for nucleus staining DAPI (D1306 – Thermofisher, São Paulo, Brazil) was used. Samples were immersed on ice during the surgery and freeze -80 º Cwith the OCT tissue tek. Slices were cut with cryostat (Leica – RM2125 RTS, Buffalo Grove, IL United States of America) in a thickness of 5um. Immunofluorescesce reaction was analyzed by a Confocal microscopy (Zeiss AIM-SYSTEM 2501000723) and 9 images were taken per slide (Zen Black – Carl Zeiss 2.3 SP1 FP1 65 bit). To evaluate protein concentration, pixel concentration (ratio between INFB/DAPI and AIM2/DAPI) of each antibody was measured using Zen 2.5 blue edition (Carl Zeiss Microscopy GmbH, 2018).

Results

No difference between samples were observed for INF-B and AIM2 among groups (p>0,05).

Conclusion

Although IFNB1 mRNAwas described as elevated in endometriosis tissue and peritoneal fluid, its protein expression seems not to be altered in endometriotic lesions compared to eutopic and non- endometriosis endometrium. It’s important to state that INFB1 has a very short half-life detected inserum, which might be extrapolated to tissue, thus making harder to detect subtle differences between groups that however, would have great biological impact on cells behaver.

Keywords

Infertility; Inflammation; Immunology; Immunofluorescence; Endometriosis.