BACKGROUND: Human papillomavirus (HPV) infection is the central factor for cervical cancer, whereas epithelial immune mechanisms contribute to the progression of HPV infection and its associated lesions. The authors evaluated the expression of indoleamine 2,3-dioxygenase (IDO) and tryptophan 2,3-dioxygenase (TDO) in cervicovaginal samples from women with normal cervical epithelium or with different degrees of squamous intraepithelial lesions (SILs) and cervical cancer. METHODS: IDO expression was analyzed by immunocytochemistry in liquid-based cytology samples from 165 women, of whom 42 had cervical changes subclassified as low-grade SIL (n = 6), high-grade SIL (n = 30), or squamous cell carcinoma (SCC) (n = 6), and 123 had negative Papanicolaou smears. IDO and TDO expression also were analyzed by immunohistochemistry, and HPV and other genital pathogens were evaluated by polymerase chain reaction analysis. RESULTS: Low IDO expression was observed in normal cervical epithelium irrespective of HPV status. Increased numbers of IDO-positive squamous cells and IDO-positive leukocytes were observed in women with SIL or SCC. TDO expression was detected in leukocytes infiltrating the stroma around intraepithelial or invasive cervical lesions. Higher IDO levels were detected in organotypic epithelial cultures established from keratinocytes transduced with the HPV16 E6/E7 oncoproteins. CONCLUSIONS: The upregulation of IDO expression in leukocytes and squamous cells in HPVassociated SIL and SCC suggests that immunosuppressive mechanisms involving tryptophan metabolism may have a role in cervical carcinogenesis. Although previous studies have suggested the role of IDO in HPV pathogenesis, this is the first evidence of TDO involvement in the process. Furthermore, the current data emphasize the role of leukocytes, especially neutrophil-like cells, as an IDO source.We gratefully acknowledge the collaboration of Luiz Carlos Zeferino, Júlio Cesar Teixeira, and Luiz Bahamondes for their assistance to the women included in this study and Maria Cecília Costa and Fabrícia Gimenez for their assistance with polymerase chain reaction analyses.Additional supporting information may be found in the online version of this article.

Introduction: Bacterial vaginosis, a modification of the vaginal microbiota, is positively associated with the acquisition of certain genital infections, including cervical human papillomavirus (HPV) infection. In turn, HPV infection is the chief etiologic agent in the development of cervical cancer. Although HPV infection is widely prevalent, only few infected women will develop cervical cancer, suggesting that environmental or host factors contribute to malignancy. In this study, we are interested in identifying if the enzyme indoleamine-2,3-dioxygenase (IDO) has a role in the mechanism by which BV contributes to HPV infection and cervical lesion. IDO catalyzes the oxidation of the essential amino acid tryptophan (Trp) to kynurenine. IDO expression is induced by IFN-gamma and has been linked to T-cell immunosuppression and immune tolerance. IDO also plays an important role in host–pathogen interactions during viral and bacterial infections and on immune-escape of cancer cells. Methods: This cross-sectional study included 296 women attending the Women's Hospital - CAISM/UNICAMP. The case group was composed of women who had a confirmed histologic diagnosis of cervical intraepithelial neoplasia (CIN) grade 1, 2, or 3 or squamous cell carcinoma (SCC). The control group was composed of women who presented at least two previous normal Pap smears. The levels of IDO expression were analyzed by immunocytochemistry in liquid-based cytology samples from 196 women. Score 1 and 2 (0 to 50% of DAB stained squamous cells) were considered as mild expression and Score 3 and 4 (51 to 100% of DAB stained squamous cells) were considered as strong expression. Immunocytochemical analysis considered both atypical and typical squamous in the counting. The Gram-stained slides were analyzed by Nugent criteria. We used the Linear Array Genotyping HPV kit for detection of 37 types of low- and high-risk HPVs. Multiplex PCR was performed for the diagnosis of Chlamydia trachomatis (Ct), Neisseria gonorrhoeae (Ng), and Trichomonas vaginalis. Study variables were evaluated by odds ratio (OR) with a 95% confidence interval (CI) and Qui-square test and significance level was defined at 5%. Results: BV was positively associated with genital infection by HPV (p = 0.02), Trichomonas vaginalis (p=0.001) and endocervicitis by Ct and Ng (p= 0.000003). We found a higher frequency of strong expression of IDO (74%, 31/42) in women with BV when compared with women without BV (44%, 60/137 and p= 0.0007). Although the expression of IDO was slightly greater for HPV infection associated with BV (62%, 15/24), and women with CIN1 and CIN3 had a slightly higher frequency of IDO expression, these associations were not statistically significant. No correlation was found for IDO and CIN2, CIN3, or SCC. Conclusion: Here we show that IDO is upregulated in BV but is not associated with the severity of cervical lesion. We also found that BV is positively associated with genital infection by HPV. We suggest that upregulation of IDO in BV could be one of the cofactors that lead to an increased risk of HPV coinfection, whose importance is attenuated during cervical lesion progression. Ongoing studies are focused on the analysis of IDO expression, specifically in the atypical cells of cervical lesions. Citation Format: Paloma Almeida Venancio, Silvya Stuchi Maria-Engler, Marcia Edilaine Lopes Consolaro, Luisa Lina Villa, Sophie Françoise Derchain, Ana Campa, Michelle Garcia Discacciati. Upregulation of IDO in bacterial vaginosis: Role in HPV coinfection [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr A46.

À Deus por ser minha força, me guardar e por tornar essa conquista possível. Aos meus pais, não tenho palavras para agradecer tudo que sempre fizeram por mim. Vocês sempre estão ao meu lado, me encorajando e acreditando em mim. Agradeço pelas marmitinhas e por me levarem no ponto de manhã rs. Ao meu irmão Pierre, por ser tão carinhoso. Amo vocês! Ao meu futuro marido Eric, obrigada amor por estar comigo e sonhar comigo. Essa conquista é nossa, você me motivou todos os dias à ir em frente sem medo e mostrou que tudo seria capaz se acreditasse. Você é meu alicerce, amo você! Agradeço aos meus tios, Priscila, Valmir e Luciano pelo incentivo e carinho. E aos meus primos Blenner, Enzo e Guilherme. Vocês são muito especiais para mim. À minha amiga Larissa, por ter me incentivado à entrar no mestrado e por todo carinho e cuidado que tem comigo. À minha orientadora Ana Campa, agradeço pelos ensinamentos e pela dedicação. Foi muito bom esses anos juntas, sou muito grata pela confiança, incentivo e por ter acreditado em mim. Você é uma pessoa maravilhosa. À minha coorientadora Michelle, agradeço muito pelo carinho e paciência que me ensinou, e pelos puxões de orelha, sem você esse trabalho não seria possível. Obrigada por tudo, principalmente pela confiança que teve em mim para prosseguir com seu trabalho. Às minhas amigas Cláudia e Babi, pela nossa amizade e por toda força. À minha amiga Aline, pelas boas risadas no telefone até altas horas e por sempre me lembrar que ter fé é importante. À Andressa, uma grande amiga que ganhei durante essa trajetória, obrigada pelas noites em claro juntas estudando, pela paciência e por tudo que me ensinou. Agradeço demais por me ajudar com a imagem da dissertação. Às minhas cunhadas, sogra e a Soraia pelo incetivo e apoio nas horas difíceis. Às minhas companheiras de Lab: Branqs, Maysa, Mar e Carmem. Obrigada por tudo meninas, sinto um carinho especial por cada uma, agradeço por tudo que me ensinaram, também pelas risadas, cafezinhos e gordices. Agradeço pela paciência e até pelos puxões de orelha que fizeram toda diferença e me ajudaram a crescer mais como pessoa. Desejo muito sucesso a todas vocês! À Sika, mais que uma técnica, uma amiga e mãe, obrigada pelas conversas e por toda ajuda. E a Fabi, por ser sempre tão legal comigo. À Profa. Silvya Stuchi, muito obrigada por ter me acolhido tão bem no seu laboratório e colaborado muito com o desenvolvimento deste trabalho. Aos colegas do Lab de citopatologia: Érika, Débora, Dani, Vanessa e Luís. E agradeço também a técnica Silvia, por toda paciência e ensinamentos. Aos professores membros da minha banca de qualificação: Profa. Márcia Consolaro, Prof. Enrique Boccardo e Profa. Sandra Farsky. Agradeço ainda aos colegas de departamento e funcionários. Ao apoio financeiro do CNPq, CAPES e FAPESP.