Innate Immunity, MIcrobiota and Inovative Treatments in Endometriosis

Introduction Endometriosis is a chronic inflammatory, polygenic, and multifactorial disease affecting approximately 10% of women of reproductive age, corresponding to over one million women in France. Endometriosis profoundly impairs the health and quality of life of affected individuals and carries a significant socio-economic burden, making it a major public health concern. To date, the pathogenesis and prognostic factors of disease progression remain poorly understood. Despite current treatment options, which are based on hormonal therapy or surgery, resistance and recurrence are frequent, underscoring the urgent need for innovative therapeutic strategies.

The hypothesis of retrograde menstruation of endometrial cells, among other proposed theories, appears insufficient to fully explain the development of the disease. Immunological factors may be implicated. Endometriosis is characterized by the presence of endometriotic tissue outside the uterine cavity - within the peritoneal cavity or at distant sites - forming lesions that, like eutopic endometrium, contain infiltrating immune cells, with varying compositions across menstrual cycle phases. Although few data remain, the literature points to several key mechanisms:

Inflammation and Innate Immunity: Dendritic cells (DC), pivotal in initiating and orchestrating immune responses, appear to be present in different proportions and exhibit altered phenotypes in endometriotic tissue compared to healthy tissue. Macrophages, essential for phagocytosis, tissue repair, and the resolution of inflammation, also show functional and phenotypic modulation. In particular, efferocytosis-their ability to clear apoptotic cells-is impaired, and an imbalance in M1/M2 polarization has been described, potentially facilitating menstrual cell escape. The local microenvironment is characterized by altered cytokine and chemokine profiles, with interleukin-8 (IL-8) potentially playing a key role via its angiogenic and chemoattractant properties. Natural Killer (NK) cells exhibit disrupted expression patterns of activating and inhibitory Killer-cell Immunoglobulin-like Receptors (KIRs), alongside diminished IFN - gamma (InterFeroN) secretion and degranulation capacity.

Microbiota: It is now well established that the immune system maintains a dynamic interaction with the gut microbiota at the mucosal barrier, with reciprocal modulation of local and systemic immunity. Furthermore, endometriosis is a hormone-sensitive pathology, and it is noteworthy that microbiota could increase plasma estrogen levels via β-glucuronidase-expressing bacteria. Other link with the microbiota, in mice, the treatment with antibiotics decrease the pathology. It is now well known that the dysbiosis can lead to systemic inflammation and subsequently contribute to inflammatory diseases development. A limited number of studies suggest a potential role for the intestinal microbiota in the initiation and/or promotion of endometriosis. Patients frequently exhibit gut dysbiosis, marked by reduced microbial diversity and an altered Firmicutes/Bacteroidetes ratio.

Resolution of Inflammation: Endometriosis may be associated with defective resolution of inflammation. Resolutive pharmacology involves the use of pro-resolving factors to exert a therapeutic effect by accelerating or stimulating the resolution of inflammation.

The biotechnology company Med'Inn'Pharma has developed a complex pro-resolving biological drug consisting of a macrophage supernatant that has eliminated apoptotic cells. This drug candidate has been used effectively in various experimental models of unresolved chronic inflammatory diseases, such as rheumatoid arthritis, multiple sclerosis and inflammatory bowel disease. In collagen-induced arthritis (a relevant model for rheumatoid arthritis), this drug candidate induces the reprogramming of macrophages towards a pro-resolving profile. The same applies to dendritic cells, with a significant reduction in the expression of costimulatory molecules.

The interplay between local inflammation, the gut microbiota, and disease progression remains incompletely elucidated. A comprehensive phenotypic and functional characterization of immune cells-particularly innate immune cells (dendritic cells, macrophages, NK cells)-in parallel with microbiome profiling and clinical outcome data, may yield novel insights into disease mechanisms and support the development of pro-resolutive therapeutic strategies.

Study Design This will be a monocentric (at Grenoble University Hospital), open-label, prospective experimental study with a control arm.

The primary objective is to identify immune biomarkers associated with endometriosis. The primary endpoint is the characterization and comparison of innate immune cells (both tissue-resident and circulating) across different biological compartments (blood, peritoneal fluid, biopsies), and between women with and without endometriosis ("endometriosis" vs "control" groups).

For that, we will perform immunophenotyping using flow cytometry on cells from each tissue (blood, endometrium, peritoneum, peritoneal lavage, and, for the endometriosis group, endometriotic lesions). A panel of approximately twenty fluorochrome-conjugated antibodies will be used to characterize the phenotype and activation status of each cell type within each tissue, including dendritic cells, macrophages, natural killer cells, and other innate immune cells. Cellular functionality will also be assessed by analyzing culture supernatants using the Luminex technique.

Secondary objectives include:

1. Identification of immune biomarkers associated with clinical outcomes in endometriosis.
2. Characterization of the immunogenetic KIR/HLA (Human Leukocyte Antigen) system in both study groups.
3. Analysis of stromal cells, apoptosis, macrophage efferocytosis, and their responsiveness to pro-resolutive factors in both groups.
4. Identification of a characteristic bacterial gut microbiota profile at diagnosis in women with endometriosis versus controls, and/or profiles associated with one-year clinical outcomes (favorable vs unfavorable), as well as temporal microbiota trajectories over one year in relation to clinical response.

Secondary endpoints include:

1. Description and comparison of innate immune cell populations (tissue and circulating) based on one-year clinical outcome (favorable vs unfavorable).

Immunogenetic profiling of HLA-A, -B, -C and KIR alleles by Next-Generation Sequencing (NGS), compared between women with and without endometriosis.

Stromal cell analysis:

• Comparison of stromal cell phenotypes, susceptibility to apoptosis, and macrophage efferocytosis capacity (using monocyte-derived macrophages) between cases and controls.
• Assessment of pro-resolutive factors' impact on macrophage efferocytosis.

Gut microbiota profiling:

Comparison of microbial community composition using 16S rDNA sequencing (relative abundances, Shannon and Bray-Curtis indices), and its evolution at 12 months (cases only), in relation to the presence of endometriosis and clinical outcome at one year.

Study Population:

The study will include women undergoing surgery for endometriosis versus control women undergoing benign gynecological surgery with no known history or intraoperative evidence of endometriosis. Patients should be young (18 to 42 years old) to ensure that, unfortunately, they may have endometriosis with predominantly inflammatory rather than fibrotic lesions. Furthermore, patients must not be undergoing immune modulation with antibiotics or immunosuppressive agents for the purposes of the study.

Study Procedures:

Women in the endometriosis group will undergo collection of endometriotic lesions, adjacent tissue, eutopic endometrium, and peritoneal lavage during surgery. Controls will provide biopsies of eutopic endometrium, unaffected peritoneum, and peritoneal lavage. These samples are collected by a gynecologic surgeon specialized in endometriosis.

For both groups, peripheral blood samples will be collected during routine care by the nurse and self stool samples obtained at baseline (Day 0). For the endometriosis group, a second stool sample will be collected at 12 months (M12).

No investigational treatment will be administered. But given the extent of biopsies performed during surgery, French legislation classifies this study as a Class 2 interventional study. The study has been approved by an ethics committee in accordance with French regulatory requirements.

A clinical evaluation will be performed at inclusion for all participants and repeated at one year for the endometriosis group. Participation for control group subjects is limited to the day of surgery, whereas endometriosis group subjects will be followed for 12 months.

The clinical evaluation focus on the medical history, the pain, the treatment. She is realized by surgeon.

All biological samples (blood, biopsies, peritoneal lavage, stool) will be processed and stored (plasma, cells, DNA, tissue) in a dedicated biobank for subsequent analysis. All samples will be collected at CHUGA.

Statistical Analysis

Given the expected sample size, appropriate non-parametric tests will be used. For each group, qualitative variables will be expressed as counts and percentages, and quantitative variables as mean ± standard deviation or median with interquartile range depending on the distribution. Comparisons will be conducted using Friedman, Wilcoxon, Mann-Whitney, or Student's t-tests, depending on data distribution, number of groups, and pairing status. Log-transformation will be applied if normality is not met. Primary comparisons will focus on immune cell parameters across tissues and study groups, including:

• Frequencies of immune cell subsets,
• Activation/maturation status,
• TLR ligand responses,
• KIR/HLA genotype combinations,
• Stromal cell apoptosis,
• Macrophage efferocytosis capacity,
• Gut microbiota composition (α-diversity: Shannon and Simpson indices; β-diversity: Bray-Curtis dissimilarity).

To control for multiple comparisons, Bonferroni correction may be applied. This exploratory study aims to identify candidate biomarkers and immunological signatures associated with endometriosis and disease recurrence, thereby generating hypotheses for future investigations.

Conclusion Endometriosis imposes a substantial burden on affected women and society. While no direct benefit is anticipated for participants, this study aims to advance the understanding of biological-especially immunological-mechanisms underpinning the disease. The results may support the identification of novel therapeutic targets, including agents that promote inflammation resolution. This research will help define an immunological signature of endometriosis, establish links with the gut microbiota, and assess the preclinical efficacy of a pro-resolutive biologic compound as a potential disease-modifying therapy.