Lactobacillus gasseri LGV03 isolated from the cervico-vagina of HPV-cleared women modulates epithelial innate immune responses and suppresses the growth of HPV-positive human cervical cancer cells

Abstract

Persistent human papillomavirus (HPV) infections is necessary for the development of cervical cancers. An increasing number of retrospective studies have found the depletion of Lactobacillus microbiota in the cervico-vagina facilitate HPV infection and might be involved in viral persistence and cancer development. However, there have been no reports confirming the immunomodulatory effects of Lactobacillus microbiota isolated from cervico-vaginal samples of HPV clearance in women. Using cervico-vaginal samples from HPV persistent infection and clearance in women, this study investigated the local immune properties in cervical mucosa. As expected, type I interferons, such as IFN-α and IFN-β, and TLR3 globally downregulated in HPV+ persistence group. Luminex cytokine/chemokine panel analysis revealed that L. jannaschii LJV03, L. vaginalis LVV03, L. reuteri LRV03, and L. gasseri LGV03 isolated from cervicovaginal samples of HPV clearance in women altered the host's epithelial immune response, particularly L. gasseri LGV03. Furthermore, L. gasseri LGV03 enhanced the poly (I:C)-induced production of IFN by modulating the IRF3 pathway and attenuating poly (I:C)-induced production of proinflammatory mediators by regulating the NF-κB pathway in Ect1/E6E7 cells, indicating that L. gasseri LGV03 keeps the innate system alert to potential pathogens and reduces the inflammatory effects during persistent pathogen infection. L. gasseri LGV03 also markedly inhibited the proliferation of Ect1/E6E7 cells in a zebrafish xenograft model, which may be attributed to an increased immune response mediated by L. gasseri LGV03.

Keywords: Antiviral immune response; Common cervicovaginal microbiota; HPV clearance; IRF3 and NF-κB signaling; Lactobacillus gasseri LGV03.

Graphical abstract

Fig. 1

TLR and cytokine expression levels in cervical cells of HPV+ clearance and HPV+ persistence patient. Relative expression of target genes was normalized to GAPDH (2^−ΔCt). Box plots represent 10–90 percentile; Mann-Whitney U test; *p < 0.05; **p < 0.01.

Fig. 2

Effect of L. jensenii LJV03, L. vaginalis LVV03, L. reuteri LRV03, and L. gasseri LGV03 on viability and host epithelial immune response of Ect1/E6E7 and CaSki cells. (A) Effects of L. jensenii LJV03, L. vaginalis LVV03, L. reuteri LRV03, and L. gasseri LGV03 on Ect1/E6E7 and CaSki cells proliferation. (B) Immune cytokines/chemokines released from Ect1/E6E7 and CaSki cells after exposure to L. jensenii, L. vaginalis, L. reuteri, and L. gasseri (1 × 10⁷ CFU/mL) for 48 h were measured by Luminex. Heat map depicts fold change by color, p-value by asterisks, and concentration (pg/mL) of each analyte by the number value present in each corresponding box. one-way analysis of variance (ANOVA); *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. ND: Non-Detectable.

Fig. 3

Characterization and whole genome sequencing of L. gasseri LGV03. (A) L. gasseri LGV03 colonies on MRS agar cultured anaerobically after 48 h at 37 °C. (B) L. gasseri LGV03 colonies after culturing for 48 h on Columbia blood agar. 1 (negative control): Listeria innocua (CICC 10,417); 2 (positive control): Staphylococcus aureus (CICC 10,473); 3: L. gasseri LGV03. (C) Gram staining properties of L. gasseri LGV03. (D) Scanning electron microscope observation of L. gasseri LGV03. (E) The graphical circular map of the L. gasseri LGV03 genome. (F) COG function classification of L. gasseri LGV03. (G) Scanning electron microscope analysis of Ect1/E6E7 cells where the L. gasseri LGV03 adheres to the surface monolayer of cells. Red arrow indicates attachment of L. gasseri LGV03 to Ect1/E6E7 cells.

Fig. 4

Antiviral activities of L. gasseri LGV03 in human ectocervical Ect1/E6E7 cells. After Ect1/E6E7 cells were treated with L. gasseri LGV03 and Poly (I:C), the expression of IFN-α, IFN-βIL-6, MCP-1, IL-8, and IL-1β were analyzed by RT-PCR. Cells treated with either Poly (I:C) or medium alone were used as positive and negative controls, respectively. The positive control was used for comparison of L. gasseri LGV03 treated groups. The mean differences among the different superscript letters (a, b, C) were significant at 0.05 level. The results are expressed as mean ± SE and represent data from three independent experiments (n = 3 in each experiment).

Fig. 5

Effect of L. gasseri LGV03 on NF-κB and IRF3 pathways in human ectocervical Ect1/E6E7 cells induced by poly (I:C) treatment. Ect1/E6E7 cells were pre-stimulated with L. gasseri LGV03 for 48 h, and then stimulated with Poly (I:C) for 12 h.

Fig. 6

In vivo immunomodulatory effect of L. gasseri LGV03 in zebrafish. (A) DiI-labeled Ect1/E6E7 cells (EC, red) were implanted into the yolk sac of zebrafish embryos, and different concentrations of L. gasseri LGV03 (1 × 10⁶ CFU/mL, 1 × 10⁷ CFU/mL) were added. Fluorescence intensity (B) and fluorescent area (C) of xenografted Ect1/E6E7 cells were analyzed quantitatively. Fluorescent microscopy images of Ect1/E6E7 cells in zebrafish xenografts at 0 h and 72 h. The results are presented as the mean ± SE (n = 5). one-way analysis of variance (ANOVA); **p < 0.01, ***p < 0.001, ****p < 0.0001 compared to control.

Fig. 7

Effect of L. gasseri LGV03 on the ROS and NO production in the zebrafish larvae. The levels of ROS (A) and NO (B) generation were measured through image analysis and fluorescence microscopy. Experiments were performed in triplicates (n = 15 per treatment), and the data are expressed as mean ± SE. Different superscript letters (a, b and ab) indicate significant differences (p <0.05) among the groups.

Fig. 8

Proposed mechanism involved in the antiviral activity of L. gasseri LGV03 in vitro. The possible immunomodulatory activity of L. gasseri LGV03 in Ect1/E6E7 cells after stimulation with poly (I:C). Arrows indicate up and down-regulation of cytokines/chemokines, and anti-viral factors. (+): upregulation, (-): down-regulation.