Study On Vaginal Pathogens And TLR2/4/MyD88 And Releted Cytokines In Pathogenesis Of Aerobic Vaginitis

Aerobic vaginitis(AV) is a common vaginitis proposed recently, but neither its pathogens nor pathogenesis is clear. Our previous study showed that Lactobacillus decreased and Gram positive/negative aerobes increased, with significantly higher vaginal IL-1β/6/8 in AV patients. However,some questions are still to be answered, including which kind of Lactobacillus decreased and which kind of aerobes increased. And pathogenesis about cytokines production and relationship between cytokines and AV are unknown. In addition, studies on infection have shown that toll-like receptor(TLR) 2 and TLR4 play a key role in identifying peptidoglycan of Gram positive bacteria and lipopolysaccharide of Gram negative bacteria, respectively, activating TLR2/4 signaling pathway and leading to inflammation, in which Myeloid differentiation factor 88(My D88) is a key intracellular adaptor. What’s more, TLR2/4 are expressed in vaginal epithelia, and aerobes increase with proinflammatory cytokines in AV. All these facts indicate that pathogens could increase the expression of inflammatory cytokines by activating the TLR2/4 signaling pathway to cause AV. It should be aware that high throughput sequencing stands out in analyzing vaginal flora, and liquid chips method is superior to enzyme linked immunosorbent assay in evaluating diverse cytokines. Therefore, in the current study, the pathogens of AV, TLR2/4/My D88 and related cytokines will be discussed in three sections.Section One Analysis of vaginal flora in AV patients compared with healthy womenObjective Investigate on vaginal flora in healthy women, and analyze vaginal microbiota changes in AV patients by high throughput sequencing, so as to explore the aerobes increasing and certain Lactobacillus decreasing related to AV.Methods 53 AV outpatients were recruited at Tianjin Medical University General Hospital between December 2014 and December 2015; 53 healthy women was enrolled as controls at Health examination center of the hospital in the corresponding period. The vaginal rinsing samples were used for investigating on the differences in vaginal flora through sequencing of 16 S r RNA gene hypervariable V4 region amplified PCR products.Results(1) Effective tags of group AV and controls were 3,195,798 and 3,264,786, clustering with 2459 Operational Taxonomic Units(OTU). Average numbers of OTUs in group AV were higher than controls(P<0.001), finding as many as 25, 51, 110, 224, 483 and 719 in taxonomy of kingdom, phylum, class, order, family, genus and species. In addition, 626 and 516 species were found in AV group and controls, respectively.(2) Microbial abundance and diversity differed significantly between group AV and controls(R=0.4571; A=0.1737; P=0.001). Firmicutes(97.90%) dominated in controls, which decreased dramatically in group AV(58.19%, P<0.001). Instead, Actinobacteria, Bacteroidetes, Proteobacteria and Fusobacteria increased in group AV(P<0.001).(3) 18 species of Lactobacillus were found in controls. L.crispatus and L.iners were main flora in controls(94.55%), which followed by 13 species with abundance more than 0.1%, including G.vaginalis(1.01%), L.delbrueckii(0.99%), L.gasseri(0.62%), S.anginosus(0.38%), A.urinae(0.28%), S.agalactiae(0.23%), P.intermedia(0.22%), A.vaginae(0.16%), Sneathia.sp(0.16%), L.vaginalis(0.14%), S.alactolyticus(0.13%).(4) The same 18 species of Lactobacillus were found in AV group, but L.crispatus decreased notably(10.54%, P<0.001), and there was no distinction in L.iners between the two groups(P=0.191). Furthermore, several aerobes and facultative anaerobes rised, such as S.anginosus, S.agalactiae, A.urinae, S. alactolyticus, K.pneumoniae, S.mitis, E.faecalis, Gemella.sp, S.epidermidis, G.vaginalis and so on(P<0.05), as well as some obligate anaerobes including A.vaginae, P.intermedia, A.prevotii, etc.(P<0.05).(5)Mycoplasm existed more in AV patients(χ2 8.116, P=0.017), but its abundance was less than 0.1%. Carrier rate of Mycoplasma genitalium, Mycoplasma hominis and Ureaplasma urealyticum in AV group and controls were 32.08%(17/53) vs. 3.77%(2/53), 52.83%(28/53) vs. 52.83%(28/53), and 84.91%(45/53) vs. 62.26%(33/53), respectively.Conclusions(1) Distribution of vaginal flora in healthy women tended to be centered, mainly dominated by L.crispatus and L.iners. Besides, other bacteria in normal flora also including S.anginosus, S.agalactiae, A.urinae, S. alactolyticus, K.pneumoniae, S.mitis, E.faecalis, Gemella.sp, S.epidermidis, G.vaginalis and so on.(2) Relative abundances of L.crispatus in AV patients decreased significantly. There are distinguished individual differences and the diversity in vaginal flora of AV,which implied that pathogens in AV might be not a specific bacteria, but a rising group of aerobes and facultative anaerobes, such as S.anginosus, S.agalactiae, A.urinae, S. alactolyticus, K.pneumoniae, S.mitis, E.faecalis, Gemella.sp, S.epidermidis, G.vaginalis and so on.Section Two Expression and significance of TLR2/4/My D88 in the pathogenesis of AVObjective The aim of this study is to assess TLR2/4/My D88 m RNA in the innate immune system TLR2/4 signaling pathway.Methods The vaginal epithelia from the mid-vagina were collected from participants including 29 AV patients and 30 healthy women from August 2015 to January 2016. The difference of TLR2, TLR4 and My D88 m RNA levels between AV patients and healthy women were measured. A correlation of m RNA levels between My D88 and TLR2/4 were also analyzed. In addition, m RNA levels of TLR2, TLR4 and My D88 among mild AV, moderate AV and severe AV were assessed. The role of TLR2/4 signaling pathway and inflammatory mediators release of vaginal mucosa played in AV was evaluated.Results m RNA levels of TLR2, TLR4 and My D88 in epithelia of AV patients were higher than those in controls(P<0.001). In addition, My D88 m RNA levels were positively correlated to TLR2/4 m RNA levels(P<0.05). m RNA levels of TLR2, TLR4 and My D88 in severe AV were higher than mild AV(P<0.05); m RNA levels of My D88 in severe AV were higher than moderate AV(P<0.05).Conclusions Aerobes may activate TLR2/4 signaling pathway to cause AV and involve with severity.Section Three Analysis of cytokine concentrations in vagina of AV patientsObjective To investigate the concentration of cytokines in vagina of AV patients, analyze the correlation between the expression of TLR2/4/My D88 and the production of cytokines, and explore its role in the development of AV.Methods The vaginal rinsing samples were collected from participants including 19 AV patients and 20 healthy women from September 2015 to December 2015.The supernatants were measured for IL-1β, IL-2, IL-5, IL-6, IL-8, IL-12p70, IL-17,IFN-γ,TNF-α(proinflammatory cytokines) and IL-4, IL-10, IL-13(anti-inflammatory cytokines) by liquid chips, to identify cytokines increasing significantly in vaginal microbiota. Additionally, cytokines were compared among groups among mild AV, moderate AV and severe AV, to analyze the correlation between severity in AV and proinflammatory cytokines. What’s more, the relations between the m RNA level of TLR2, TLR4 and My D88 and cytokines were also assessed.Results Concentration in vagina of IL-1β, IL-6, IL-8, IL-12p70, IL-17, TNF-α increased significantly in group AV(P<0.05), among which IL-1β, IL-6, IL-8, IL-17, TNF-α levels were higher in severe AV than mild AV(P<0.05). The concentrations of IL-1β, IL-6, IL-12p70 and IL-17 were positively related to the levels of TLR2, TLR4 and My D88 m RNA(P<0.05).Conclusions Disorders of immunity in vaginal microenvironment occurred in AV, which was characterized by higher proinflammatory cytokines including IL-1β, IL-6, IL-8, IL-12p70, IL-17 and TNF-α. It seemed that proinflammatory cytokines increased highly in severe AV, which might be involved with individual variations in severity. TLR2/4-My D88 signaling pathway and related proinflammatory cytokines may play a role in AV.