O-031 Activation of NLRP3 inflammasome enhances neutrophil extracellular traps in women with stage III/IV endometriosis: convergence of multiple signaling portray association between genetics and lesion type
Authors: Chakravarty B , Kalapahar S , Sharma S , Chattopadhyay R , Ghosh S , Chakraborty M , Chakraborty R , Bose U , Chakraborty P 
Published: June 2022

Abstract

Study question
Do NOD-like receptor pyrin-containing 3 (NLRP3) signaling affect neutrophil extracellular traps (NET)-osis in women with stage III/IV endometriosis?

Summary answer
Inflammasome activation whether due to pathogen (disease itself) or damage-associated molecular pattern (through NET) might be one mechanism involved in establishing perturbed endometrium in endometriosis.

What is known already
Endometriosis is characterized by number of processes like vascularization, hypernociception, and fibrosis, cardinal cause being inflammation. Recently, expression of inflammasome components, including NLRP3 and apoptosis signal-regulating kinase has been demonstrated in human endometrium cueing involvement in uterine innate immunity. Moreover, a novel extracellular killing mechanism, NET, is documented to reflect an inflammatory status in deep infiltrating endometriosis. Since, endometriosis demonstrates similarities with chronic inflammatory and autoimmune disorders; we postulated inflammatory responses in endometriosis may become modulated through a feed-forward loop of NET-induced specific cytokine production thus providing insights against potentiality of endometriotic cells to limit progression of the disease.

Study design, size, duration
Twenty-two consented women (24-39 years) with endometriosis (Group A) (Stages III–IV) (based on ASRM-2018 guidelines) and age-matched counterpart/s of male sub-fertility (Group B; control), free of uterine abnormalities (n = 18) were recruited between January to December 2021 from Institute of Reproductive Medicine, Kolkata. Eutopic endometrium, were collected from women undergoing diagnostic laparoscopy (Group A) or by curettage from women undergoing endometrial ablation (Group B). Serum was collected during window-of-implantation (LH + 7) for both the group/s.

Participants/materials, setting, methods
Pro-(IFN-g, TNF-a, IL-6, TGF-b) and anti-inflammatory (IL-10, IL-13, IL-4, IL-5) cytokines was evaluated by western blot. Expression level of inflammasome-related proteins (NLRP3, ASC, CASPASE1, PYCARD, IL-1-b) and mRNA expression was estimated by western-blot and quantitative-real-time PCR (qRT-PCR) respectively from tissue biopsies in Group A and B. SYTOX® green assay by flow-cytometry and neutrophil-elastase activity by immunofluorescence was done to quantify and characterize NET production in blood monocytes. P &lt; 0.05 was considered statistically significant.

Main results and the role of chance
The mean (±SD) age of study population was 31.6±5.2 years. Hyperestrogenic milieu possibly stimulated (p &lt; 0.001) pro-inflammatory molecules (IFN-g, TNF-a, IL-6, TGF-b) in endometriosis as observed by western-blot and qRT-PCR. A significant up-regulation (p &lt; 0.001) was observed in relative mRNA expression of NLRP3 and PYCARD gene in Group A. However, caspase 1 expression documented non-significant variation in biopsies from endometriosis patients. Western blot corroborated the finding/s. The outcome of NLRP3 activation was supported by increased (p &lt; 0.002) mRNA expression of IL-1b. NETs were detected significantly higher (p &lt; 0.01) in 54.54% (12/22) patients in group A compared to control (16.66%; (3/18)). Moreover, quantification of NETs showed a significantly higher amount in endometriosis compared to group B (0.097 vs. 0.02, p &lt; 0.03).
Spermann-rank correlation by SPSS version22 revealed positive correlation between IL-1b with NLRP3 (r = 0.56, p&lt; 0.001), PYCARD (r = 0.11, p&lt; 0.01); and caspase1 (r = 0.16, p&lt; 0.01) and IL-1b (r = 0.47, p&lt; 0.01) with NET-positive cell/s in endometriosis. Moreover, posterior cul-de-sac lesions correlated positively with NET-positive cell/s (r = 0.29; p &lt; 0.01) cueing to possible prognostic marker/s. In summary, a coordinated-fashioned action of components of NLRP3 inflammasome machinery may regulate production of NET/s forming a complex network allowing communication between cell-types in order to maintain viability and development of endometrial lesion/s.

Limitations, reasons for caution
Our findings need to be replicated in larger study cohort/s, especially stratified by severity of endometriosis sub-groups and in women of different ethnicities. Another limitation is the missing information about NETs in normal functioning human endometrium throughout the menstrual cycle.

Wider implications of the findings
The proposed study aims to understand potential role of NLRP3 inflammasome complex as a “double-edged sword” in the development and pathophysiology of endometriosis. Therapeutic alternatives that aim to re-balance the pro- to anti-inflammatory milieu in the endometrium should consider the inflammasome as part of the equation.

Trial registration number
Not applicable