Identification, characterisation and expression analysis of natural killer receptor genes in Chlamydia pecorum infected koalas (Phascolarctos cinereus)

BACKGROUND: Koalas (Phascolarctos cinereus), an iconic Australian marsupial, are being heavily impacted by the spread of Chlamydia pecorum, an obligate intracellular bacterial pathogen. Koalas vary in their response to this pathogen, with some showing no symptoms, while others suffer severe symptoms leading to infertility, blindness or death. Little is known about the pathology of this disease and the immune response against it in this host. Studies have demonstrated that natural killer (NK) cells, key components of the innate immune system, are involved in the immune response to chlamydial infections in humans. These cells can directly lyse cells infected by intracellular pathogens and their ability to recognise these infected cells is mediated through NK receptors on their surface. These are encoded in two regions of the genome, the leukocyte receptor complex (LRC) and the natural killer complex (NKC). These two families evolve rapidly and different repertoires of genes, which have evolved by gene duplication, are seen in different species. METHODS: In this study we aimed to characterise genes belonging to the NK receptor clusters in the koala by searching available koala transcriptomes using a combination of search methods. We developed a qPCR assay to quantify relative expression of four genes, two encoded within the NK receptor cluster (CLEC1B, CLEC4E) and two known to play a role in NK response to Chalmydia in humans (NCR3, PRF1). RESULTS: We found that the NK receptor repertoire of the koala closely resembles that of the Tasmanian devil, with minimal genes in the NKC, but with lineage specific expansions in the LRC. Additional genes important for NK cell activity, NCR3 and PRF1, were also identified and characterised. In a preliminary study to investigate whether these genes are involved in the koala immune response to infection by its chlamydial pathogen, C. pecorum, we investigated the expression of four genes in koalas with active chlamydia infection, those with past infection and those without infection using qPCR. This analysis revealed that one of these four, CLEC4E, may be upregulated in response to chlamydia infection. CONCLUSION: We have characterised genes of the NKC and LRC in koalas and have discovered evidence that one of these genes may be upregulated in koalas with chlamydia, suggesting that these receptors may play a role in the immune response of koalas to chlamydia infection

Chemokine response induced by Chlamydia trachomatis in prostate derived CD45+ and CD45- cells.

The role of innate cells and their receptors within the male genital tract remains poorly understood. Much less is known about the relative contribution of different genital tract cells such as epithelial/stromal cells and resident leucocytes. In this study, we examined innate immune responses to Chlamydia trachomatis by prostate epithelial/stromal cells and prostate resident leucocytes. Murine prostate primary cultures were performed and leucocyte and epithelial/stromal cells were sorted based on surface protein expression of CD45 by magnetism-activated cell sorting or fluorescence-activated cell sorting. Prostate derived CD45- and CD45+ cells were infected with C. trachomatis and chemokine secretion assayed by ELISA. Similar experiments were performed using prostate CD45+ and CD45- cells from myeloid differentiation factor 88 (Myd88(-/-)) mice or toll-like receptor (Tlr2(-/-)) and Tlr4(mutant) double-deficient mice. Moreover, a TLR-signalling pathway array was used to screen changes in different genes involved in TLR-signalling pathways by real-time PCR. Prostate derived CD45- and CD45+ cells responded to chlamydial infection with the production of different chemokines. Both populations expressed genes involved in TLR signalling and required to respond to pathogen-associated molecular patterns and to C. trachomatis infection. Both populations required the adaptor molecule MYD88 to elicit chemokine response against C. trachomatis. TLR2-TLR4 was essential for chemokine production by CD45+ prostate derived cells, but in their absence, CD45- cells still produced significant levels of chemokines. We demonstrate that C. trachomatis is differentially recognised by prostate derived CD45+ and CD45- cells and suggest that diverse strategies are taking place in the local microenvironment of the host in response to the infection

Innate immunity in the male genital tract: Chlamydia trachomatis induces keratinocyte-derived chemokine production in prostate, seminal vesicle and epididymis/vas deferens primary cultures.

Chlamydia trachomatis is an intracellular pathogen that infects mucosal epithelial cells, causing persistent infections. Although chronic inflammation is a hallmark of chlamydial disease, the proinflammatory mechanisms involved are poorly understood. Little is known about how innate immunity in the male genital tract (MGT) responds to C. trachomatis. Toll-like receptors (TLRs) are a family of receptors of the innate immunity that recognize different pathogen-associated molecular patterns (PAMPs) present in bacteria, viruses, yeasts and parasites. The study of TLR expression in the MGT has been poorly investigated. The aim of this work was to investigate the keratinocyte-derived chemokine (KC) response of MGT primary cultures from C57BL/6 mice to C. trachomatis and different PAMPs. KC production by prostate, seminal vesicle and epididymis/vas deferens cell cultures was determined by ELISA in culture supernatants. TLR2, 3, 4 and 9 agonists induced the production of KC by all MGT primary cultures assayed. In addition, we analysed the host response against C. trachomatis and Chlamydia muridarum. Chlamydial LPS (cLPS) as well as C. trachomatis and C. muridarum infection induced KC secretion by all MGT cell cultures analysed. Differences in KC levels were observed between cultures, suggesting specific sensitivity against pathogens among MGT tissues. Chemokine secretion was observed after stimulation of seminal vesicle cells with TLR agonists, cLPS and C. trachomatis. To our knowledge, this is the first report showing KC production by seminal vesicle cells after stimulation with TLR ligands, C. trachomatis or C. muridarum antigens. These results indicate that different receptors of the innate immunity are present in the MGT. Understanding specific immune responses, both innate and adaptive, against chlamydial infections, mounted in each tissue of the MGT, will be crucial to design new therapeutic approaches where innate and/or adaptive immunity would be targeted

Chlamydia trachomatis neither exerts deleterious effects on spermatozoa nor impairs male fertility

ABSTARCT: Chlamydia trachomatis is the most prevalent sexually transmitted bacterial infection. However, whether Chlamydia trachomatis has a negative impact on sperm quality and male fertility is still controversial. Herein, we report the effects on sperm quality of the in vitro exposure of spermatozoa to Chlamydia trachomatis, and also the effects of male genital infection on male fertility using an animal model. Human and mouse sperm were obtained from healthy donors and cauda epididimys from C57BL/6 mice, respectively. Highly motile human or mouse spermatozoa were in vitro exposed to C. trachomatis (serovar E or LGV) or C. muridarum, respectively. Then, sperm quality parameters were analyzed. Moreover, male fertility of Chlamydia muridarum infected male C57BL/6 mice was assessed. Human or murine sperm in vitro exposed to increasing bacterial concentrations or soluble factors from C. trachomatis or C. muridarum, respectively, did not show differences in sperm motility and viability, apoptosis, mitochondrial membrane potential, DNA fragmentation, ROS production and lipid peroxidation levels, when compared with control sperm (p > 0.05). Moreover, no differences in fertility parameters (potency, fecundity, fertility index, pre- and post-implantation loss) were observed between control and infected males. In conclusion, our results indicate that Chlamydia spp. neither directly exerts deleterious effects on spermatozoa nor impairs male fertility