A unique insight into the MiRNA profile during genital chlamydial infection.

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.

The immunoregulatory role of alpha enolase in dendritic cell function during Chlamydia infection.

BACKGROUND: We have previously reported that interleukin-10 (IL-10) deficient dendritic cells (DCs) are potent antigen presenting cells that induced elevated protective immunity against Chlamydia. To further investigate the molecular and biochemical mechanism underlying the superior immunostimulatory property of IL-10 deficient DCs we performed proteomic analysis on protein profiles from Chlamydia-pulsed wild-type (WT) and IL-10-/- DCs to identify differentially expressed proteins with immunomodulatory properties. RESULTS: The results showed that alpha enolase (ENO1), a metabolic enzyme involved in the last step of glycolysis was significantly upregulated in Chlamydia-pulsed IL-10-/- DCs compared to WT DCs. We further studied the immunoregulatory role of ENO1 in DC function by generating ENO1 knockdown DCs, using lentiviral siRNA technology. We analyzed the effect of the ENO1 knockdown on DC functions after pulsing with Chlamydia. Pyruvate assay, transmission electron microscopy, flow cytometry, confocal microscopy, cytokine, T-cell activation and adoptive transfer assays were also used to study DC function. The results showed that ENO1 knockdown DCs had impaired maturation and activation, with significant decrease in intracellular pyruvate concentration as compared with the Chlamydia-pulsed WT DCs. Adoptive transfer of Chlamydia-pulsed ENO1 knockdown DCs were poorly immunogenic in vitro and in vivo, especially the ability to induce protective immunity against genital chlamydia infection. The marked remodeling of the mitochondrial morphology of Chlamydia-pulsed ENO1 knockdown DCs compared to the Chlamydia-pulsed WT DCs was associated with the dysregulation of translocase of the outer membrane (TOM) 20 and adenine nucleotide translocator (ANT) 1/2/3/4 that regulate mitochondrial permeability. The results suggest that an enhanced glycolysis is required for efficient antigen processing and presentation by DCs to induce a robust immune response. CONCLUSIONS: The upregulation of ENO1 contributes to the superior immunostimulatory function of IL-10 deficient DCs. Our studies indicated that ENO1 deficiency causes the reduced production of pyruvate, which then contributes to a dysfunction in mitochondrial homeostasis that may affect DC survival, maturation and antigen presenting properties. Modulation of ENO1 thus provides a potentially effective strategy to boost DC function and promote immunity against infectious and non-infectious diseases.

Interleukin-10 modulates antigen presentation by dendritic cells through regulation of NLRP3 inflammasome assembly during Chlamydia infection.

Interleukin-10 (IL-10) has been implicated in susceptibility to genital chlamydial infection and the development of tubal pathologies. IL-10 limitation also resulted in the rapid elicitation of immune responses against Chlamydia, and decreased levels of IL-10 correlated with protective anti-Chlamydia immunity. To investigate the molecular basis for these effects, we compared the reproductive pathologies and fertility rates in Chlamydia-infected wild-type (WT) and IL-10-knockout (IL-10(-/-)) mice; we also analyzed the expression of the Toll-like receptor (TLR)/interleukin-1 receptor (IL-1R) superfamily, IL-1ß production, NLRP3 inflammasome assembly and activation, and the immunostimulatory capacity and apoptotic predilection of Chlamydia-exposed dendritic cells (DCs) from WT and IL-10(-/-) mice. Our results revealed that, in addition to the rapid clearance of infection, genitally infected IL-10(-/-) mice were protected from tubal pathologies and infertility, whereas WT (IL-10(+/+)) mice were not. Chlamydia-pulsed IL-10(-/-) DCs expressed larger numbers of TLR4/IL-1R molecules and had enhanced IL-1ß production. In addition, NLRP3 inflammasome assembly was suppressed in IL-10(-/-) DCs through the inhibition of the P2X purinoceptor 7 (P2X7) receptor (P2X7R), an ATP-gated ion channel, and a decrease in intracellular Ca(2+) levels, which inhibited DC apoptosis. Thus, the potent immunostimulatory capacity of IL-10-deficient DCs is due, at least in part, to the suppression of the intracellular inflammasome assembly, which prevents DC apoptosis, allowing efficient antigen presentation. The results indicate that IL-10 deficiency enables efficient antigen presentation by DCs for rapid and enhanced immune activation against Chlamydia, which results in rapid microbial clearance, which prevents tubal pathologies during infection. Our finding has important implications for the induction of protective immunity against Chlamydia and other infectious and noninfectious diseases by vaccines.

The emerging role of ASC in dendritic cell metabolism during Chlamydia infection.

Chlamydia trachomatis is a bacterial agent that causes sexually transmitted infections worldwide. The regulatory functions of dendritic cells (DCs) play a major role in protective immunity against Chlamydia infections. Here, we investigated the role of ASC in DCs metabolism and the regulation of DCs activation and function during Chlamydia infection. Following Chlamydia stimulation, maturation and antigen presenting functions were impaired in ASC-/- DCs compared to wild type (WT) DCs, in addition, ASC deficiency induced a tolerant phenotype in Chlamydia stimulated DCs. Using real-time extracellular flux analysis, we showed that activation in Chlamydia stimulated WT DCs is associated with a metabolic change in which mitochondrial oxidative phosphorylation (OXPHOS) is inhibited and the cells become committed to utilizing glucose through aerobic glycolysis for differentiation and antigen presenting functions. However, in ASC-/- DCs Chlamydia-induced metabolic change was prevented and there was a significant effect on mitochondrial morphology. The mitochondria of Chlamydia stimulated ASC-/- DCs had disrupted cristae compared to the normal narrow pleomorphic cristae found in stimulated WT DCs. In conclusion, our results suggest that Chlamydia-mediated activation of DCs is associated with a metabolic transition in which OXPHOS is inhibited, thereby dedicating the DCs to aerobic glycolysis, while ASC deficiency disrupts DCs function by inhibiting the reprogramming of DCs metabolism within the mitochondria, from glycolysis to electron transport chain.