Atopobium vaginae And Porphyromonas somerae Induce Proinflammatory Cytokines Expression In Endometrial Cells: A Possible Implication For Endometrial Cancer?

(Figure 2) showed that all the cytokines detected in the culture supernatant of endometrial cells upon co-culture with bacteria were indeed activated at the transcriptional level, as indicated by the upregulation of the correspondent mRNAs compared to control uninfected cells or cells co-cultured with killed bacteria. In particular, IL1α and IL1β RNAs resulted respectively 6.8-fold and 8-fold more expressed in the presence of Atopobium than in controls (endometrial cells uninfected or exposed to killed bacteria) or in Lactobacillus and Porphyromonas co-cultured cells (respectively 1.1 and 1 folds with Lactobacillus, and 1 and 1.3 folds with Porphyromonas); IL8 resulted upregulated only by Lactobacillus (16 folds) and not by Atopobium/Porphyromonas; IL17α was activated only by Atopobium (9.5 folds) and Porphyromonas (8.2 folds); similarly, TNFα was upregulated by Atopobium and Porphyromonas (16.3 and 3 folds, respectively), but not by Lactobacillus (1.1 folds). Interestingly, in addition to the cytokines analyzed by ELISA and confirmed at the transcriptional level, other transcripts resulted significantly altered by co-culture with Atopobium and Porphyromonas, including CCL13 (9 folds with P. somerae only), CCL8 (15 folds with A. vaginae and 18 folds with P. somerae), CXCL2 (9 folds with A. vaginae and 11 folds with P. somerae), IL22 (11 folds with A. vaginae and 16 folds with P. somerae) and IL9 (15 folds with A. vaginae and 20 folds with P. somerae). However, CCL13 was also upregulated by Lactobacillus as well (9 folds), suggesting a nonspecific stimulation associated with the mere presence of bacteria, rather than with a specific bacterial species. CCL13 (also known as Monocyte Chemoattractant Protein 4, MCP4) can be induced by IL1 and TNFα and is indeed involved in many chronic diseases, including allergies. By contrast, the other chemokines that resulted altered by Atopobium or Porphyromonas co-culture have been reported to be associated with cancer progression. In particular, CCL8 has been associated with melanoma progression and dissemination,13 enhances breast cancer cell motility and is upregulated in tumor-associated macrophages (TAM) in breast and endometrial cancer patients, favoring tumor invasion.14 The expression of CXCL2 is increased in ovarian and endometrial carcinomas,15,16 it is induced by TNFα and induces chemoresistance.17 IL22 promotes progression in breast cancer18 and endometrial cell proliferation by stimulating CCL2 and IL8,19 IL9 is upregulated in endometriosis,20 and its expression was reported to be upregulated via IL-17α in endometrial adenocarcinoma cells,10 and to prevent apoptosis, promoting proliferation and metastasis in pancreatic cancer.21 In conclusion, our in vitro observations show for the first time that certain bacteria have the ability to induce expression of proinflammatory cytokines and chemokines by endometrial cells, and suggest that their presence in the uterine environment might be associated with the establishment of conditions promoting inflammation. One limitation of our study is the use of a cell line, rather than endometrial primary cells derived from healthy tissue, and future studies are needed to investigate host-microbiota interactions using primary endometrial cells and different bacterial loads, to better understand the impact of microorganisms on the local endometrial microenvironment and its mechanisms. However, our preliminary observations provide a base for additional knowledge in this field, and strengthen the hypothesis of a significant role of Atopobium and Porphyromonas species in endometrial tumor onset and/or progression, providing also a starting point for future researches focusing on the impact of uterine microbiota on uterine physiology and pathologies, and hopefully potentially useful tools for diagnosis and effective clinical interventions. Disclosure The authors report no conflicts of interest in this work.

1. Baker JM, Chase DM, Herbst-Kralovetz MM. Uterine microbiota: residents, tourists, or invaders? Front Immunol. 2018;9:208. doi:10.3389/fimmu.2018.00208 2. Chen C, Song X, Wei W, et al. The microbiota continuum along the female reproductive tract and its relation to uterine-related diseases. Nat Commun. 2017;8(1):875. doi:10.1038/s41467-017-00901-0 3. Cicinelli E, Matteo M, Tinelli R, et al. Prevalence of chronic endometritis in repeated unexplained implantation failure and the IVF success rate after antibiotic therapy. Hum Reprod. 2015;30(2):323–330. doi:10.1093/humrep/deu292 4. Pelzer ES, Willner D, Buttini M, Huygens F. A role for the endometrial microbiome in dysfunctional menstrual bleeding. Antonie Van Leeuwenhoek. 2018;111(6):933–943. doi:10.1007/s10482-017-0992-6 5. Walther-Antonio MR, Chen J, Multinu F, et al. Potential contribution of the uterine microbiome in the development of endometrial cancer. Genome Med. 2016;8(1):122. doi:10.1186/s13073-016-0368-y 6. Mert I, Walther-Antonio M, Mariani A. Case for a role of the microbiome in gynecologic cancers: clinician’s perspective. J Obstet Gynaecol Res. 2018;44(9):1693–1704. doi:10.1111/jog.13701 7. Wang Y, Wang Y, Duan X, Wang Y, Zhang Z. Interleukin-1 receptor-associated kinase 1 correlates with metastasis and invasion in endometrial carcinoma. J Cell Biochem. 2018;119(3):2545–2555. doi:10.1002/jcb.26416 8. Litmanovich A, Khazim K, Cohen I. The role of interleukin-1 in the pathogenesis of cancer and its potential as a therapeutic target in clinical practice. Oncol Ther. 2018;6(2):109–127. doi:10.1007/s40487-018-0089-z 9. Hirata T, Osuga Y, Hamasaki K, et al. Interleukin (IL)-17A stimulates IL-8 secretion, cyclooxygensase-2 expression, and cell proliferation of endometriotic stromal cells. Endocrinology. 2008;149(3):1260–1267. doi:10.1210/en.2007-0749 10. Ahn SH, Edwards AK, Singh SS, Young SL, Lessey BA, Tayade C. IL-17A contributes to the pathogenesis of endometriosis by triggering proinflammatory cytokines and angiogenic growth factors. J Immunol. 2015;195(6):2591–2600. doi:10.4049/jimmunol.1501138 11. Garcia FU, Chen HL, Yang Y, Pace JL, Hu XL, Hunt JS. Tumor necrosis factor-alpha mRNA and protein in endometrial tumors: analysis by in situ hybridization and immunocytochemistry. Hum Pathol. 1994;25(12):1324–1331. doi:10.1016/0046-8177(94)90093-0 12. Smith HO, Stephens ND, Qualls CR, et al. The clinical significance of inflammatory cytokines in primary cell culture in endometrial carcinoma. Mol Oncol. 2013;7(1):41–54. doi:10.1016/j.molonc.2012.07.002 13. Barbai T, Fejos Z, Puskas LG, Timar J, Raso E. The importance of microenvironment: the role of CCL8 in metastasis formation of melanoma. Oncotarget. 2015;6(30):29111–29128. doi:10.18632/oncotarget.5059 14. Cassetta L, Fragkogianni S, Sims AH, et al. Human tumor-associated macrophage and monocyte transcriptional landscapes reveal cancer-specific reprogramming, biomarkers, and therapeutic targets. Cancer Cell. 2019;35(4):588–602 e510. doi:10.1016/j.ccell.2019.02.009 15. Furuya M, Suyama T, Usui H, et al. Up-regulation of CXC chemokines and their receptors: implications for proinflammatory microenvironments of ovarian carcinomas and endometriosis. Hum Pathol. 2007;38(11):1676–1687. doi:10.1016/j.humpath.2007.03.023 16. Kavandi L, Collier MA, Nguyen H, Syed V. Progesterone and calcitriol attenuate inflammatory cytokines CXCL1 and CXCL2 in ovarian and endometrial cancer cells. J Cell Biochem. 2012;113(10):3143–3152. doi:10.1002/jcb.24191 17. Palacios-Arreola MI, Nava-Castro KE, Castro JI, Garcia-Zepeda E, Carrero JC, Morales-Montor J. The role of chemokines in breast cancer pathology and its possible use as therapeutic targets. J Immunol Res. 2014;2014:849720. doi:10.1155/2014/394127 18. Rui J, Chunming Z, Binbin G, Na S, Shengxi W, Wei S. IL-22 promotes the progression of breast cancer through regulating HOXB-AS5. Oncotarget. 2017;8(61):103601–103612. doi:10.18632/oncotarget.22063 19. Guo Y, Chen Y, Liu LB, et al. IL-22 in the endometriotic milieu promotes the proliferation of endometrial stromal cells via stimulating the secretion of CCL2 and IL-8. Int J Clin Exp Pathol. 2013;6(10):2011–2020. 20. Jorgensen H, Hill AS, Beste MT, et al. Peritoneal fluid cytokines related to endometriosis in patients evaluated for infertility. Fertil Steril. 2017;107(5):1191–1199 e1192. doi:10.1016/j.fertnstert.2017.03.013 21. Hu B, Qiu-Lan H, Lei RE, Shi C, Jiang HX, Qin SY. Interleukin-9 promotes pancreatic cancer cells proliferation and migration via the miR-200a/beta-catenin axis. Biomed Res Int. 2017;2017:2831056. doi:10.1155/2017/2831056 © 2019 The Author(s). This work is published and licensed by Dove Medical Press Limited. The full terms of this license are available at https://www.dovepress.com/terms and incorporate the Creative Commons Attribution - Non Commercial (unported, 3.0) License. By accessing the work you hereby accept the Terms. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. For permission for commercial use of this work, please see paragraphs 4.2 and 5 of our Terms.