Lactobacillus rhamnosus GG attenuates the pathology of Chlamydial muridarium in the upper genital tract in mice

Abstract

Objectives: Chlamydia trachomatis is a pathogen which can cause hydrosalpinx and tubal fibrosis when infecting the urogenital tract. However, the mechanism is still not clear. There is growing evidence that the gut microbiota is associated with the pathogenesis of both intestinal and extra-intestinal disorders, such as cardiovascular disease, hepatocirrhosis, allergy, respiratory tract infection, polycystic ovary syndrome, endometriosis, and bacterial vaginitis. Lactobacillus rhamnosus GG (LGG) is one of the most extensively studied and widely used probiotic bacteria, the benefits of LGG including the treatment in gastrointestinal disorders and immunomodulation are well demonstrated, and it can also alleviate hypersensitivity reaction and diarrhoea, inhibit a variety of respiratory and urogenital diseases. Chlamydia muridarium (Cm) infection is a good model for the study on human Chlamydia pathogenicity in genitourinary tract. The mice infected with Cm were used as animal models to preliminarily explore the mechanism for the effect of LGG on upper reproductive tract infection in the mice, and to provide experimental basis for the pathogenesis of Chlamydia trachomatis genitourinary tract infection and the new idea for the treatment of Chlamydia trachomatis infection.

Methods: Five to six weeks-old C57BL/6J mice were divided into 2 groups: An experimental group and a control group. The experimental group were administrated with 5×10⁸ colony forming units (CFU) LGG for 19 consecutive days, while the control group were feed PBS. The mice in the 2 group were subcutaneously injected with 2.5 mg progesterone on Day 9 and infected with 1×10⁵ inclusion body forming unit of Cm via the vaginal tract on Day 14. Vaginal and rectal swabs were taken every 7 days to infect HeLa cells for 24 hours, then the indirect immunofluorescence assay was used and the number of inclusion bodies of Chlamydia were calculated. Mice were euthanized on Day 14 and Day 63 after Cm inoculation, the vaginal tracts were dissected, and the tissue homogenates were prepared to culture the pathogens for 24 hours. The Cm bearing capacity in the bilateral uterine horn, tubal ovary, and cervical vaginal tissues in the 2 groups were calculated. The spleen cells were harvested to assay the intracellular IFN-γ, IL-5, and IL-17 by flow cytometry. On Day 63 after the Chlamydia infection, the pathology injury in the bilateral uterine horn and oviduct was observed, and the pathological sections and HE staining in the various part of genital tract were performed. The inflammatory cell infiltration and lumen dilatation was assessed. The specific IgM and IgG in sera were detected by indirect ELISA on Day 14 and 63 after infection.

Results: There was no effect of LGG on the clearing of Cm from the urogenital tract, the Chlamydia ascending to fallopian tube or the uterine horn, and the organism dissemination and colonization to the gastrointestinal tract (all P>0.05). On Day 14 after Cm infection via the vagina, the IL-17 expression level in the experimental group was significant decreased than that in the control group (t=2.486, P<0.05), but there was no significant difference between the 2 groups in the CD4⁺ T rate in spleen and IgM and IgG levels in serum after Cm intravaginal infection (all P>0.05). On Day 63 after Cm infection, there was no difference in the severity of inflammation in the uterine horns and fallopian tubes between the 2 groups (P>0.05), but the dilation of the fallopian tubes and hydrosalpinx was attenuated in the experimental group compared with the control group (P<0.05).

Conclusions: Oral administration of LGG has no effect on inhibiting Cm ascending to upper genital tract and preventing the dissemination and colonization of Cm to the gastrointestinal tract, which also cannot affect the secretion of specific IgM and IgG in sera. Oral administration of LGG can suppress the production of IL-17 in the spleen cells and attenuate hydrosalpinx development when following Cm intravaginal infection in mice.

Keywords: Chlamydial muridarium; Lactobacillus rhamnosus GG; hydrosalpinx; interleukin-17.

图1

Cm 感染后小鼠下生殖道衣原体载量 Figure 1 Loading capacity of Chlamydia after Cm infection in lower genital tract in mice

图2

Cm 感染后第14天小鼠生殖道衣原体载量 Figure 2 Loading capacity in genital tract on the 14th day after Cm infection in mice C+V: Vagina and cervix; L-UH: Left uterine horn; R-UH: Right uterine horn; L-OV: Left oviduct; R-OV: Right oviduct.

图3

Cm 感染后小鼠肠道的衣原体载量 Figure 3 Loading capacity of Chlamydia after Cm infection in gastrointestinal tract in mice

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口服LGG对 Cm 感染小鼠生殖道病理损伤的影响 Figure 4 Effect of oral administration of LGG on genital tract pathology in mice after intravaginal Cm infection A: Gross pathology of genital tract. The white arrow indicates the oviducts with hydrosalpinges. B: Hydrosalpinx score. *P<0.05 vs the control group.

图5

口服LGG对 Cm 感染后第14天小鼠脾脏T淋巴分泌细胞因子的调节 Figure 5 Regulation of oral LGG to cytokines secretion in mouse spleen T lymphocytes after intravaginal Cm infection A: Intracellular IFN-γ in CD4⁺ T cells; B: Intracellular IFN-γ in CD8⁺ T cells; C: Intracellular IL-5 in CD4⁺ T cells; D: Intracellular IL-17 in CD4⁺ T cells. *P<0.05 vs the control group.

图6

口服LGG对 Cm 感染小鼠特异性IgM和IgG表达水平的影响 Figure 6 Effect of oral LGG on specific IgG and IgM expression in sera after intravaginal Cm infection