A novel murine model mimicking male genital Neisseria species infection using Neisseria musculi†.

With ~78 million cases yearly, the sexually transmitted bacterium Neisseria gonorrhoeae is an urgent threat to global public health due to continued emergence of antimicrobial resistance. In the male reproductive tract, untreated infections may cause permanent damage, poor sperm quality, and subsequently subfertility. Currently, few animal models exist for N. gonorrhoeae infection, which has strict human tropism, and available models have limited translatability to human disease. The absence of appropriate models inhibits the development of vital new diagnostics and treatments. However, the discovery of Neisseria musculi, a mouse oral cavity bacterium, offers much promise. This bacterium has already been used to develop an oral Neisseria infection model, but the feasibility of establishing urogenital gonococcal models is unexplored. We inoculated mice via the intrapenile route with N. musculi. We assessed bacterial burden throughout the male reproductive tract, the systemic and tissue-specific immune response 2-weeks postinfection, and the effect of infection on sperm health. Neisseria musculi was found in penis (2/5) and vas deferens (3/5) tissues. Infection altered immune cell counts: CD19+ (spleen, lymph node, penis), F4/80+ (spleen, lymph node, epididymus), and Gr1+ (penis) compared with noninfected mice. This culminated in sperm from infected mice having poor viability, motility, and morphology. We hypothesize that in the absence of testis infection, infection and inflammation in other reproductive is sufficient to damage sperm quality. Many results herein are consistent with outcomes of gonorrhoea infection, indicating the potential of this model as a tool for enhancing the understanding of Neisseria infections of the human male reproductive tract.

Viral-vectored boosting of OmcB or CPAF-specific T cell responses fail to enhance protection from Chlamydia muridarum in infection immune mice and elicits a non-protective CD8-dominant response in naïve mice.

A vaccine is needed to combat the Chlamydia epidemic. Replication-deficient viral vectors are safe and induce antigen-specific T cell memory. We tested the ability of intramuscular immunization with modified vaccinia virus Ankara (MVA) or Chimpanzee Adenovirus (ChAd) expressing chlamydial outer membrane protein, OmcB, or the secreted protein, CPAF, to enhance T cell immunity and protection in mice previously infected with plasmid-deficient Chlamydia muridarum CM972, and to elicit protection in naïve mice. MVA.OmcB or MVA.CPAF increased antigen-specific T cells in CM972-immune mice ∼150 and 50-fold respectively but failed to improve bacterial clearance. ChAd.OmcB/MVA.OmcB prime-boost immunization of naïve mice elicited a CD8-dominant T cell response that failed to protect. ChAd.CPAF/ChAd.CPAF prime-boost also induced a CD8-dominant response with a marginal reduction in burden. Challenge of ChAd.CPAF-immunized mice genetically deficient in CD4 or CD8 T cells showed that protection was entirely CD4-dependent. CD4-deficient mice had prolonged infection, while CD8-deficient mice had higher frequencies of CPAF-specific CD4 T cells, earlier clearance, and reduced burden compared to wild-type controls. These data reinforce the essential nature of the CD4 T cell response in protection from chlamydial genital infection in mice and the need for vaccine platforms that drive CD4-dominant responses.

Stability and antigenicity of Chlamydia muridarum major outer membrane protein antigen at body temperature.

Chlamydia is an obligate intracellular bacterial pathogen responsible for disease and infertility across multiple species. Currently vaccines are being studied to help reduce the prevalence of this disease. The main advantage of protein subunit vaccines is their high degree of safety although this is traded off with the requirement for multiple booster doses to achieve complete protection. Although in certain populations the booster dose can be difficult and costly to administer, development of delayed vaccine delivery techniques, such as a vaccine capsule, could be the solution to this problem. One of the main drawbacks in this technology is that the antigen must remain stable at body temperature (37 °C) until release is achieved. Here we elucidate the stability of a recombinant chlamydial major outer membrane protein (MOMP) antigen and assess its antigenic and immunogenic properties after subjecting the antigen to 37 °C for four to six weeks. Through in vitro and in vivo assessment we found that the aged chlamydial MOMP was able to produce equivalent humoral and cell-mediated immune responses when compared with the unaged vaccine. It was also found that vaccines formulated with the aged antigen conferred equivalent protection against a live infection challenge as the unaged antigen. Thus ageing chlamydial MOMP antigens at 37 °C for four to six weeks did not cause any significant structural or antigenic/immunogenic degradation and recombinant C. muridarum MOMP is suitable for use in a delayed vaccine delivery system.

Haematopoietic innate interleukin 17A production drives immunopathology in female mouse genital Chlamydia muridarum infection.

Chlamydia trachomatis infection is the leading cause of bacterial urogenital infection and has been demonstrated to drive inflammation and scarring of the reproductive tract. Recent studies have identified key triggers of proinflammatory adaptive immune responses driven by innate leukocytes and epithelia driving immunopathology. Utilizing chimeric mouse models, we investigated the definitive source and role of IL17 and IL17 signalling receptors during early Chlamydia muridarum infection of the female urogenital tract. Bone marrow transplants from wild-type (WT) and IL17A-/- mice to recipients demonstrated equivocal infection kinetics in the reproductive tract, but interestingly, adoptive transfer of IL17A-/- immune cells to WT recipients resulted in no infertility, suggesting a haematopoietic (as opposed to tissue) source of IL17 driving immunopathology. To further delineate the role of IL17 in immunopathology, we infected WT and IL17 receptor A (IL17RA)-/- female mice and observed a significant reduction in immunopathology in IL17RA-/- mice. WT bone marrow transplants to IL17RA-/- recipient mice prevented hydrosalpinx, suggesting signalling through IL17RA drives immunopathology. Furthermore, early chemical inhibition of IL17 signalling significantly reduced hydrosalpinx, suggesting IL17 acts as an innate driver of disease. Early during the infection, IL17 was produced by γδ T cells in the cervico-vagina, but more importantly, by neutrophils at the site of infertility in the oviducts. Taken together, these data suggest innate production of IL17 by haematopoietic leukocytes drives immunopathology in the epithelia during early C. muridarum infection of the female reproductive tract.

IgG exacerbates genital chlamydial pathology in females by enhancing pathogenic CD8+ T cell responses.

Chlamydia trachomatis infections are an important sexually transmitted infection that can lead to inflammation, scarring and hydrosalpinx/infertility. However, infections are commonly clinically asymptomatic and do not receive treatment. The underlying cause of asymptomatic immunopathology remains unknown. Here, we demonstrate that IgG produced during male infection enhanced the incidence of immunopathology and infertility in females. Human endocervical cells expressing the neonatal Fc Receptor (FcRn) increased translocation of human IgG-opsonized C. trachomatis. Using total IgG purified from infected male mice, we opsonized C. muridarum and then infected female mice, mimicking sexual transmission. Following infection, IgG-opsonized Chlamydia was found to transcytose the epithelial barrier in the uterus, where it was phagocytosed by antigen-presenting cells (APCs) and trafficked to the draining lymph nodes. APCs then expanded both CD4+ and CD8+ T cell populations and caused significantly more infertility in female mice infected with non-opsonized Chlamydia. Enhanced phagocytosis of IgG-opsonized Chlamydia significantly increased pro-inflammatory signalling and T cell proliferation. As IgG is transcytosed by FcRn, we utilized FcRn-/- mice and observed that shedding kinetics of Chlamydia were only affected in FcRn-/- mice infected with IgG-opsonized Chlamydia. Depletion of CD8+ T cells in FcRn-/- mice lead to a significant reduction in the incidence of infertility. Taken together, these data demonstrate that IgG seroconversion during male infection can amplify female immunopathology, dependent on FcRn transcytosis, APC differentiation and enhanced CD8 T cell responses.

Pathogenic NKT cells attenuate urogenital chlamydial clearance and enhance infertility.

Urogenital chlamydial infections continue to increase with over 127 million people affected annually, causing significant economic and public health pressures. While the role of traditional MHCI and II peptide presentation is well defined in chlamydial infections, the role of lipid antigens in immunity remains unclear. Natural killer (NK) T cells are important effector cells that recognize and respond to lipid antigens during infections. Chlamydial infection of antigen-presenting cells facilitates presentation of lipid on the MHCI-like protein, CD1d, which stimulates NKT cells to respond. During urogenital chlamydial infection, wild-type (WT) female mice had significantly greater chlamydial burden than CD1d-/- (NKT-deficient) mice, and had significantly greater incidence and severity of immunopathology in both primary and secondary infections. WT mice had similar vaginal lymphocytic infiltrate, but 59% more oviduct occlusion compared to CD1d-/- mice. Transcriptional array analysis of oviducts day 6 post-infection revealed WT mice had elevated levels of Ifnγ (6-fold), Tnfα (38-fold), Il6 (2.5-fold), Il1ß (3-fold) and Il17a (6-fold) mRNA compared to CD1d-/- mice. In infected females, oviduct tissues had an elevated infiltration of CD4+ -invariant NKT (iNKT) cells, however, iNKT-deficient Jα18-/- mice had no significant differences in hydrosalpinx severity or incidence compared to WT controls. Lipid mass spectrometry of surface-cleaved CD1d in infected macrophages revealed an enhancement of presented lipids and cellular sequestration of sphingomyelin. Taken together, these data suggest an immunopathogenic role for non-invariant NKT cells in urogenital chlamydial infections, facilitated by lipid presentation via CD1d via infected antigen-presenting cells.

Prophylactic and therapeutic vaccination protects sperm health from Chlamydia muridarum-induced abnormalities.

Chlamydia is the most common bacterial sexually transmitted infection worldwide and it is widely acknowledged that controlling the rampant community transmission of this infection requires vaccine development. In this study, for the first time, we elucidate the long-term response to male mouse chlamydial vaccination with chlamydial major outer membrane protein (MOMP) and ISCOMATRIX (IMX) both prophylactically and in a novel therapeutic setting. Vaccination significantly reduced and, in some cases, cleared chlamydial burden from the prostates, epididymides, and testes, which correlates with high IgG and IgA tires in tissues and serum. Important markers of sperm health and fertility were protected including sperm motility and proteins associated with fertility in men. Within splenocytes, expression of IFNγ, TNFα, IL17, IL13, IL10, and TGFß were changed by both infection and vaccination within CD4 and CD8 T cells and regulatory T cells. Within the testicular tissue, phenotypic and concentration changes were observed in macrophages and T cells (resident and transitory). This revealed some pathogenic phenotypes associated with infection and critically that vaccination allows maintenance of testicular homeostasis, likely by preventing significant influx of CD4 T cells and promoting IL10 production. Finally, we demonstrated the testes contained immature (B220+) B cells and mature (CD138+) Chlamydia-specific plasma cells. Thus, through vaccination, we can maintain the healthy function of the testes, which is vital to protection of male fertility.

Primary oral vaccination followed by a vaginal pull protects mice against genital HSV-2 infection.

PROBLEM: HSV-2 infected more than 491 million people aged 15-49 world-wide in 2016. The morbidity associated with recurrent infections and the increased risk of HIV infection make this a major health problem. To date there is no effective vaccine. Because HSV-2 ascends to the dorsal route ganglion within 12-18 h of infection, an effective vaccine will need to elicit a strong local resident CD8+ T cell response to prevent the infection from becoming life-long. METHOD OF STUDY: Using a mouse model we investigated the potential of oral immunization with a novel lipid adjuvant (LiporaleTM ) followed by local vaginal application of an inflammatory agents to protect against primary HSV-2 infections. RESULTS: Oral vaccination of mice with live-attenuated HSV-2 in Liporale followed by vaginal application of DNFB or CXCL9/10 led to recruitment of tissue-resident CD8+ memory cells into the genital epithelia. This prime and pull vaccination strategy provided complete protection against wild-type HSV-2 challenge and prevented viral dissemination to the spinal cords. CONCLUSIONS: Activation of mucosal immunity by oral immunization, combined with induction of transient local genital inflammation can recruit long-lived tissue resident CD8+ T cells into the genital epithelium, providing significant protection against primary HSV-2 infection.

Controlled release vaccine implants for delivery of booster immunisations.

Most current animal vaccine regimes involve a primary vaccination followed sometime later by a booster vaccination. This presents challenges when vaccinating difficult to access animals such as livestock. Mustering livestock to deliver a vaccine boost is costly and stressful for animals. Thus, we have produced a platform system that can be administered at the same time as the priming immunisation and delivers payload after an appropriate delay time to boost the immune response, without need for further handling of animals. A 30 × 2 mm osmotically triggered polymer implant device with burst-release characteristics delivered the booster dose of a tetanus vaccine. Blood samples were collected from an experimental group that received the priming vaccine and implant on day 0 and control group that received the initial vaccine (tetanus toxoid) and then a bolus dose 28 days later via subcutaneous injection. The two groups showed identical weight gain curves. T cell proliferation following in vitro stimulation with antigen was identical between the two groups at all time points. However, serum IgG antibody responses to the tetanus toxoid antigen were significantly higher in the control group at weeks 8 and 12. The implant capsules stayed at the site of implantation and at week 12 there was evidence of tissue integration. No local reactions at the implant site were observed, other than mild thickening of the skin in half of the experimental group animals and no other adverse health events were recorded in either group.

Chlamydia pneumoniae can infect the central nervous system via the olfactory and trigeminal nerves and contributes to Alzheimer's disease risk.

Chlamydia pneumoniae is a respiratory tract pathogen but can also infect the central nervous system (CNS). Recently, the link between C. pneumoniae CNS infection and late-onset dementia has become increasingly evident. In mice, CNS infection has been shown to occur weeks to months after intranasal inoculation. By isolating live C. pneumoniae from tissues and using immunohistochemistry, we show that C. pneumoniae can infect the olfactory and trigeminal nerves, olfactory bulb and brain within 72 h in mice. C. pneumoniae infection also resulted in dysregulation of key pathways involved in Alzheimer's disease pathogenesis at 7 and 28 days after inoculation. Interestingly, amyloid beta accumulations were also detected adjacent to the C. pneumoniae inclusions in the olfactory system. Furthermore, injury to the nasal epithelium resulted in increased peripheral nerve and olfactory bulb infection, but did not alter general CNS infection. In vitro, C. pneumoniae was able to infect peripheral nerve and CNS glia. In summary, the nerves extending between the nasal cavity and the brain constitute invasion paths by which C. pneumoniae can rapidly invade the CNS likely by surviving in glia and leading to Aß deposition.

DNA damage contributes to transcriptional and immunological dysregulation of testicular cells during Chlamydia infection.

Chlamydia is the most commonly reported sexually transmitted bacterial infection, with 127 million notifications worldwide each year. Both males and females are susceptible to the pathological impacts on fertility that Chlamydia infections can induce. However, male chlamydial infections, particularly within the upper reproductive tract, including the testis, are not well characterized. In this study, using mouse testicular cell lines, we examined the impact of infection on testicular cell lineage transcriptomes and potential mechanisms for this impact. The somatic cell lineages exhibited significantly fragmented genomes during infection. Likely resulting from this, each of the Leydig, Sertoli and germ cell lineages experienced extensive transcriptional dysregulation, leading to significant changes in cellular biological pathways, including interferon and germ-Sertoli cell signalling. The cell lineages, as well as isolated spermatozoa from infected mice, also contained globally hypomethylated DNA. Cumulatively, the DNA damage and epigenetic-mediated transcriptional dysregulation observed within testicular cells during chlamydial infection could result in the production of spermatozoa with abnormal epigenomes, resulting in previously observed subfertility in infected animals and congenital defects in their offspring.

Chronic testicular Chlamydia muridarum infection impairs mouse fertility and offspring development†.

With approximately 131 million new genital tract infections occurring each year, Chlamydia is the most common sexually transmitted bacterial pathogen worldwide. Male and female infections occur at similar rates and both cause serious pathological sequelae. Despite this, the impact of chlamydial infection on male fertility has long been debated, and the effects of paternal chlamydial infection on offspring development are unknown. Using a male mouse chronic infection model, we show that chlamydial infection persists in the testes, adversely affecting the testicular environment. Infection increased leukocyte infiltration, disrupted the blood:testis barrier and reduced spermiogenic cell numbers and seminiferous tubule volume. Sperm from infected mice had decreased motility, increased abnormal morphology, decreased zona-binding capacity, and increased DNA damage. Serum anti-sperm antibodies were also increased. When both acutely and chronically infected male mice were bred with healthy female mice, 16.7% of pups displayed developmental abnormalities. Female offspring of chronically infected sires had smaller reproductive tracts than offspring of noninfected sires. The male pups of infected sires displayed delayed testicular development, with abnormalities in sperm vitality, motility, and sperm-oocyte binding evident at sexual maturity. These data suggest that chronic testicular Chlamydia infection can contribute to male infertility, which may have an intergenerational impact on sperm quality.

Chlamydia muridarum Can Invade the Central Nervous System via the Olfactory and Trigeminal Nerves and Infect Peripheral Nerve Glial Cells.

Chlamydia pneumoniae can infect the brain and has been linked to late-onset dementia. Chlamydia muridarum, which infects mice, is often used to model human chlamydial infections. While it has been suggested to be also important for modelling brain infection, nervous system infection by C. muridarum has not been reported in the literature. C. pneumoniae has been shown to infect the olfactory bulb in mice after intranasal inoculation, and has therefore been suggested to invade the brain via the olfactory nerve; however, nerve infection has not been shown to date. Another path by which certain bacteria can reach the brain is via the trigeminal nerve, but it remains unknown whether Chlamydia species can infect this nerve. Other bacteria that can invade the brain via the olfactory and/or trigeminal nerve can do so rapidly, however, whether Chlamydia spp. can reach the brain earlier than one-week post inoculation remains unknown. In the current study, we showed that C. muridarum can within 48 h invade the brain via the olfactory nerve, in addition to infecting the trigeminal nerve. We also cultured the glial cells of the olfactory and trigeminal nerves and showed that C. muridarum readily infected the cells, constituting a possible cellular mechanism explaining how the bacteria can invade the nerves without being eliminated by glial immune functions. Further, we demonstrated that olfactory and trigeminal glia differed in their responses to C. muridarum, with olfactory glia showing less infection and stronger immune response than trigeminal glia.

Group B Streptococcus serotypes Ia and V induce differential vaginal immune responses that may contribute to long term colonization of the female reproductive tract.

PROBLEM: Group B Streptococcus (GBS) is a common colonizer of the female genital tract at the time of pregnancy and has been associated with severe neonatal infections. Despite trials for GBS vaccines already being underway, the factors influencing vaginal GBS colonization and clearance are currently poorly understood. METHOD OF STUDY: Within this study, we investigated the host immune responses to GBS infections in mice that affect GBS vaginal colonization and clearance. Cervicovaginal swabs were used to measure vaginal GBS persistence, and vaginal cytokine responses were measured using the BioPlex® system. Lymphocytes isolated from spleens were stimulated with UV-killed GBS to examine systemic cellular responses. Additional in vitro cellular experiments using human vaginal epithelial cells were also performed, examining the effect pregnancy level hormones had on GBS adhesion, invasion, and cytokine responses. RESULTS: We observed significant differences in the ability of GBS serotype V infections to persist, compared with GBS serotype Ia vaginal infections. Vaginal cytokine response examination identified temporal changes in cytokine production (IL10, IFNγ, IL6, IL1ß, and TNFα) in relation to GBS serotype and clearance or colonization. Lymphocyte proliferation assays also revealed robust cellular immune responses to GBS vaginal infections irrespective of clearance or colonization. In vitro human cellular analyses also identified that vaginal epithelial cell line cytokine production was suppressed in the presence of hormones despite no alteration in adhesion/invasion. CONCLUSION: Here, we establish previously unknown, serotype specific, temporal immune responses which may be associated with vaginal GBS colonization or clearance in the female genital tract.

Detection of chlamydia infection within human testicular biopsies.

STUDY QUESTION: Can Chlamydia be found in the testes of infertile men? SUMMARY ANSWER: Chlamydia can be found in 16.7% of fresh testicular biopsies and 45.3% of fixed testicular biopsies taken from a selection of infertile men. WHAT IS KNOWN ALREADY: Male chlamydial infection has been understudied despite male and female infections occurring at similar rates. This is particularly true of asymptomatic infections, which occur in 50% of cases. Chlamydial infection has also been associated with increased sperm DNA damage and reduced male fertility. STUDY DESIGN, SIZE, DURATION: We collected diagnostic (fixed, n = 100) and therapeutic (fresh, n = 18) human testicular biopsies during sperm recovery procedures from moderately to severely infertile men in a cross-sectional approach to sampling. PARTICIPANTS/MATERIALS, SETTING, METHODS: The diagnostic and therapeutic biopsies were tested for Chlamydia-specific DNA and protein, using real-time PCR and immunohistochemical approaches, respectively. Serum samples matched to the fresh biopsies were also assayed for the presence of Chlamydia-specific antibodies using immunoblotting techniques. MAIN RESULTS AND THE ROLE OF CHANCE: Chlamydial major outer membrane protein was detected in fixed biopsies at a rate of 45.3%. This was confirmed by detection of chlamydial DNA and TC0500 protein (replication marker). C. trachomatis DNA was detected in fresh biopsies at a rate of 16.7%, and the sera from each of these three positive patients contained C. trachomatis-specific antibodies. Overall, C. trachomatis-specific antibodies were detected in 72.2% of the serum samples from the patients providing fresh biopsies, although none of the patients were symptomatic nor had they reported a previous sexually transmitted infection diagnosis including Chlamydia. LIMITATIONS, REASONS FOR CAUTION: No reproductively healthy male testicular biopsies were tested for the presence of Chlamydia DNA or proteins or Chlamydia-specific antibodies due to the unavailability of these samples. WIDER IMPLICATIONS FOR THE FINDINGS: Application of Chlamydia-specific PCR and immunohistochemistry in this human male infertility context of testicular biopsies reveals evidence of a high prevalence of previously unrecognised infection, which may potentially have a pathogenic role in spermatogenic failure. STUDY FUNDING/COMPETING INTEREST(S): Funding for this project was provided by the Australian NHMRC under project grant number APP1062198. We also acknowledge assistance from the Monash IVF Group and Queensland Fertility Group in the collection of fresh biopsies, and the Monash Health and co-author McLachlan (declared equity interest) in retrieval and sectioning of fixed biopsies. E.M. declares an equity interest in the study due to financing of fixed biopsy sectioning. All other authors declare no conflicts of interest. TRIAL REGISTRATION NUMBER: N/A.

Hematogenous dissemination of Chlamydia muridarum from the urethra in macrophages causes testicular infection and sperm DNA damage†.

The incidence of Chlamydia infection, in both females and males, is increasing worldwide. Male infections have been associated clinically with urethritis, epididymitis, and orchitis, believed to be caused by ascending infection, although the impact of infection on male fertility remains controversial. Using a mouse model of male chlamydial infection, we show that all the major testicular cell populations, germ cells, Sertoli cells, Leydig cells, and testicular macrophages can be productively infected. Furthermore, sperm isolated from vas deferens of infected mice also had increased levels of DNA damage as early as 4 weeks post-infection. Bilateral vasectomy, prior to infection, did not affect the chlamydial load recovered from testes at 2, 4, and 8 weeks post-infection, and Chlamydia-infected macrophages were detectable in blood and the testes as soon as 3 days post-infection. Partial depletion of macrophages with clodronate liposomes significantly reduced the testicular chlamydial burden, consistent with a hematogenous route of infection, with Chlamydia transported to the testes in infected macrophages. These data suggest that macrophages serve as Trojan horses, transporting Chlamydia from the penile urethra to the testes within 3 days of infection, bypassing the entire male reproductive tract. In the testes, infected macrophages likely transfer infection to Leydig, Sertoli, and germ cells, causing sperm DNA damage and impaired spermatogenesis.