A unique insight into the MiRNA profile during genital chlamydial infection

Abstract

Background: Genital C. trachomatis infection may cause pelvic inflammatory disease (PID) that can lead to tubal factor infertility (TFI). Understanding the pathogenesis of chlamydial complications including the pathophysiological processes within the female host genital tract is important in preventing adverse pathology. MicroRNAs regulate several pathophysiological processes of infectious and non-infectious etiologies. In this study, we tested the hypothesis that the miRNA profile of single and repeat genital chlamydial infections will be different and that these differences will be time dependent. Thus, we analyzed and compared differentially expressed mice genital tract miRNAs after single and repeat chlamydia infections using a C. muridarum mouse model. Mice were sacrificed and their genital tract tissues were collected at 1, 2, 4, and 8 weeks after a single and repeat chlamydia infections. Histopathology, and miRNA sequencing were performed.

Results: Histopathology presentation showed that the oviduct and uterus of reinfected mice were more inflamed, distended and dilated compared to mice infected once. The miRNAs expression profile was different in the reproductive tissues after a reinfection, with a greater number of miRNAs expressed after reinfection. Also, the number of miRNAs expressed each week after chlamydia infection and reinfection varied, with weeks eight and one having the highest number of differentially expressed miRNAs for chlamydia infection and reinfection respectively. Ten miRNAs; mmu-miR-378b, mmu-miR-204-5p, mmu-miR-151-5p, mmu-miR-142-3p, mmu-miR-128-3p, mmu-miR-335-3p, mmu-miR-195a-3p, mmu-miR-142-5p, mmu-miR-106a-5p and mmu-miR-92a-3p were common in both primary chlamydia infection and reinfection. Pathway analysis showed that, amongst other functions, the differentially regulated miRNAs control pathways involved in cellular and tissue development, disease conditions and toxicity.

Conclusions: This study provides insights into the changes in miRNA expression over time after chlamydia infection and reinfection, as well as the pathways they regulate to determine pathological outcomes. The miRNAs networks generated in our study shows that there are differences in the focus molecules involved in significant biological functions in chlamydia infection and reinfection, implying that chlamydial pathogenesis occurs differently for each type of infection and that this could be important when determining treatments regime and disease outcome. The study underscores the crucial role of host factors in chlamydia pathogenesis.

Keywords: Chlamydia infection; Chlamydial pathogenesis; Differential expression; TFI; miRNAs.

Fig. 1

Histopathological assessment of genital tract of mice infected once with Chlamydia. a. Representative image of the endometrium from an uninfected mouse. A single layer of columnar epithelia (arrow), with basally located nuclei lining the endometrium. The underlying lamina propria has scattered spindled cells, some capillaries, and occasional leukocytes. Hematoxylin and eosin (HE) stain. Scale bar = 20 μm. b and c. Representative image of uterine endometrium with increased presence of eosinophils after the first and second weeks of infection. The lamina propria is mildly hypercellular, with loosely arranged spindled stromal cells and increased numbers of eosinophils around an endometrial gland (Gl). HE stain. Scale bar 20 μm. d-f. Representative images of inflammed uterine endometrium with necrosis after 4 weeks of infection. d. The uterine lumen (asterisks) is filled with necrotic cellular debris. The mucosa has widespread epithelial apoptotic necrosis (arrowheads). The lamina propria is moderately hypercellular, with increased numbers of neutrophils, eosinophils, and few lymphocytes (arrows). HE stain. Scale bar 20 μm

Fig. 2

Histopathological assessment of genital tract of mice infected twice with Chlamydia. a. Representative image of the endometrium from an uninfected mouse. A single layer of columnar epithelia (arrow), with basally located nuclei lining the endometrium. The underlying lamina propria has scattered spindled cells, some capillaries, and occasional leukocytes in a pale basophilic myxomatous matrix. (Endometrial gland = Gl). HE stain. Scale bar 50 μm. b. Representative image of uterine endometrium with periglandular fibrosis after 1 week of reinfection. Increased numbers of neutrophils (asterisk) and spindled cells (fibroblasts, arrow) are arranged around endometrial glands (Gl). HE stain. Scale bar 50 μm. c. Representative image of uterine endometrium with increased lymphocyte presence after 2 weeks of reinfection. Large numbers of lymphocytes (arrows) obscure and expand the tissues around an endometrial gland (Gl). HE stain. Scale bar 50 μm. d and e. Representative image of uterine endometrium with increased lymphocyte presence after 4 weeks of reinfection with lymphocytic endometritis. Large numbers of lymphocytes, with fewer plasma cells (long arrows) and occasional neutrophils (short arrow) obscure and expand the tissues around an endometrial gland (Gl). Arrowheads point to foci of glandular apoptotic necrosis. Capillaries = Ca. HE stain. Scale bars 50 and 20 μm, respectively. f. Representative image of uterine endometrium with increased lymphocyte presence and lymphoid follicle formation (arrows) after 8 weeks of reinfection. An expansive lymphoid follicle obscures and expands the tissues of the deep stratum spongiosum and adjacent myometrium. HE stain. Scale bar 50 μm

Fig. 3

Further Histopathological assessment of genital tract of mice infected twice with Chlamydia. a. Representative, longitudinal image of an uninfected mouse uterus (U), oviduct (Ovi), and ovary (Ova). A single layer of columnar epithelia (arrow), with basally located nuclei lining the endometrium (arrow). HE stain. Scale bar 500 μm. b and c. Representative, sub-gross (b) and close up (c) images of a mouse uterus with marked cystic endometrial hyperplasia (arrows) 8 weeks after reinfection. Branched, papillary projections (arrowheads) of endometrium extend into the cystically dilated uterine lumen (asterisk). HE stain. Scale bars are 15 mm and 200 μm, respectively

Fig. 4

Heat map and Cluster analysis showing the numbers of differentially expressed miRNAs after chlamydia infection and reinfection. a. Heat map showed that there is a difference in miRNA expression profile 1 week after chlamydia infection and reinfection. R script was used to create the heatmap for each set of sample comparison. b. Heat map showed that there is a difference in miRNA expression profile 2 weeks after chlamydia infection and reinfection. R script was used to create the heatmap for each set of sample comparison. c. Heat map showed that there is a difference in miRNA expression profile 4 weeks after chlamydia infection and reinfection. R script was used to create the heatmap for each set of sample comparison. d. Heat map showed that there is a difference in miRNA expression profile 8 weeks after chlamydia infection and reinfection. R script was used to create the heatmap for each set of sample comparison. e. Venn diagram of differentially expressed miRNAs after chlamydia infection. Figure compares the differentially expressed microRNAs 1, 2, 4 and 8 weeks after infection. The numbers in the Venn diagram represents the number of distinct and common microRNAs in the different weeks of infection. There were no common microRNAs expressed in all weeks of infection. p-values here were adjusted by false discovery rate. f. Venn diagram of differentially expressed miRNAs after chlamydia reinfection. Figure compares the differentially expressed microRNAs 1, 2, 4 and 8 weeks after infection. The numbers in the Venn diagram represents the number of distinct and common microRNAs in the different weeks of infection. There were no common microRNAs expressed in all weeks of infection. p-values here were adjusted by false discovery rate. g. Venn Diagram comparing miRNAs expressed during chlamydia infection and reinfection. Figure compares all the differentially expressed microRNAs in chlamydia infection and reinfection. The numbers in the Venn diagram represents the number of distinct and common microRNAs. There were 10 common microRNAs expressed in both chlamydia infection and reinfection. p-values here were adjusted by false discovery rate

Fig. 5

Pathways predicted to be regulated by miRNAs differentially expressed in mice after chlamydia infection and reinfection. a. Pathways predicted to regulate physiological system development after chlamydia infection and reinfection. This analysis was determined using Ingenuity pathway analysis software (IPA). miRNAs used in the analysis were from the list derived after FDR correction. b. Pathways predicted to regulate diseases and disorders after chlamydia infection and reinfection. This analysis was determined using Ingenuity pathway analysis software (IPA). miRNAs used in the analysis were from the list derived after FDR correction. c. Pathways predicted to regulate molecular and cellular functions after chlamydia infection and reinfection. This analysis was determined using Ingenuity pathway analysis (IPA). miRNAs used in the analysis were from the list derived after FDR correction