Table_1_Protozoan-Viral-Bacterial Co-Infections Alter Galectin Levels and Associated Immunity Mediators in the Female Genital Tract.xls

Co-infections with sexually transmittable pathogens are common and more likely in women with disturbed vaginal bacteriome. Among those pathogens, the protozoan parasite Trichomonas vaginalis (TV) is most common after accounting for the highly persistent DNA viruses human papillomavirus (HPV) and genital herpes. The parasitic infection often concurs with the dysbiotic syndrome diagnosed as bacterial vaginosis (BV) and both are associated with risks of superimposed viral infections. Yet, the mechanisms of microbial synergisms in evading host immunity remain elusive. We present clinical and experimental evidence for a new role of galectins, glycan-sensing family of proteins, in mixed infections. We assessed participants of the HIV Epidemiology Research Study (HERS) at each of their incident TV visits (223 case visits) matched to controls who remained TV-negative throughout the study. Matching criteria included age, race, BV (by Nugent score), HIV status, hysterectomy, and contraceptive use. Non-matched variables included BV status at 6 months before the matched visit, and variables examined at baseline, within 6 months of and/or at the matched visit e.g. HSV-2, HPV, and relevant laboratory and socio-demographic parameters. Conditional logistic regression models using generalized estimating equations calculated odds ratios (OR) for incident TV occurrence with each log10 unit higher cervicovaginal concentration of galectins and cytokines. Incident TV was associated with higher levels of galectin-1, galectin-9, IL-1β and chemokines (ORs 1.53 to 2.91, p <0.001). Galectin-9, IL-1β and chemokines were up and galectin-3 down in TV cases with BV or intermediate Nugent versus normal Nugent scores (p <0.001). Galectin-9, IL-1β and chemokines were up in TV-HIV and down in TV-HPV co-infections. In-vitro, TV synergized with its endosymbiont Trichomonasvirus (TVV) and BV bacteria to upregulate galectin-1, galectin-9, and inflammatory cytokines. The BV-bacterium Prevotella bivia alone and together with TV downregulated galectin-3 and synergistically upregulated galectin-1, galectin-9 and IL-1β, mirroring the clinical findings of mixed TV–BV infections. P. bivia also downregulated TVV+TV-induced anti-viral response e.g. IP-10 and RANTES, providing a mechanism for conducing viral persistence in TV-BV co-infections. Collectively, the experimental and clinical data suggest that galectin-mediated immunity may be dysregulated and exploited by viral–protozoan–bacterial synergisms exacerbating inflammatory complications from dysbiosis and sexually transmitted infections.

性传播病原体合并感染较为常见，且在阴道菌群失调的女性中发生率更高。在这类病原体中，原生动物寄生虫阴道毛滴虫（Trichomonas vaginalis, TV）是仅次于高持续性DNA病毒人乳头瘤病毒（human papillomavirus, HPV）和生殖器疱疹病毒的最常见病原体。该寄生虫感染常与被诊断为细菌性阴道病（bacterial vaginosis, BV）的菌群失调综合征合并发生，且二者均与继发性病毒感染的风险升高相关。然而，微生物协同作用逃避宿主免疫的具体机制仍不明晰。

我们提供了临床与实验证据，证明半乳凝素（galectins）——一类糖基感知蛋白家族——在混合感染中发挥全新作用。我们对HIV流行病学研究（HIV Epidemiology Research Study, HERS）的参与者进行了评估，针对每一次首次确诊TV感染的就诊记录（共223例病例就诊），匹配了研究全程TV检测均为阴性的对照个体。匹配标准包括年龄、种族、细菌性阴道病状态（通过Nugent评分判定）、HIV感染状态、子宫切除史及避孕方式使用情况。未纳入匹配的变量包括匹配就诊前6个月的细菌性阴道病状态，以及基线、匹配就诊前后6个月内或匹配就诊当时检测的各类变量，例如HSV-2、HPV及相关实验室指标与社会人口学参数。

我们采用广义估计方程构建条件logistic回归模型，计算了宫颈阴道分泌物中半乳凝素与细胞因子每升高1个log10单位时，首次发生TV感染的比值比（odds ratios, OR）。研究发现，首次确诊TV感染与半乳凝素-1、半乳凝素-9、IL-1β及趋化因子水平升高显著相关（比值比范围1.53~2.91，p<0.001）。与Nugent评分正常的TV感染者相比，合并细菌性阴道病或Nugent评分处于中间区间的TV感染者体内半乳凝素-9、IL-1β及趋化因子表达上调，而半乳凝素-3表达下调（p<0.001）。在TV-HIV合并感染个体中，半乳凝素-9、IL-1β及趋化因子表达上调，而在TV-HPV合并感染个体中则表达下调。

体外实验中，阴道毛滴虫与其内共生体毛滴虫病毒（Trichomonasvirus, TVV）及细菌性阴道病相关细菌协同作用，上调半乳凝素-1、半乳凝素-9及炎症细胞因子的表达。单独的细菌性阴道病相关细菌栖组织普雷沃菌（Prevotella bivia），以及其与阴道毛滴虫联合作用时，均可下调半乳凝素-3的表达，并协同上调半乳凝素-1、半乳凝素-9及IL-1β的表达，这与TV-BV混合感染的临床观测结果一致。栖组织普雷沃菌还可下调TVV+TV诱导的抗病毒免疫应答（如IP-10与RANTES），为TV-BV合并感染中病毒持续存在提供了潜在机制。

综上，实验与临床数据表明，半乳凝素介导的免疫功能可能出现失调，并被病毒-原生动物-细菌协同作用所利用，进而加重由菌群失调与性传播感染引发的炎症并发症。