Anti-chlamydial effects of azelastine hydrochloride and the impact of the histamine H1 receptor on chlamydial development.

Introduction. Azelastine hydrochloride, a second-generation histamine H1 receptor (H1R) antagonist, exhibits anti-chlamydial effects against Chlamydia trachomatis (CT) in HeLa cells (genital infection model).Hypothesis/Gap Statement. Non-antibiotic pharmaceutical interactions with CT are an understudied field and the anti-chlamydial effects of azelastine are a potential interaction requiring further elucidation.Aim. To explore the underlying anti-chlamydial mechanisms of azelastine.Methodology. We assessed the specificity of azelastine for the chlamydial species and host cell type, the timing of azelastine application and whether the anti-chlamydial effects could be reproduced with different H1R-modulating compounds.Results. We observed similar anti-chlamydial azelastine effects for Chlamydia muridarum as well as for an ocular CT strain in human conjunctival epithelial cells (ocular infection model). Pre-incubating host cells with azelastine before infection mildly reduced chlamydial inclusion numbers and infectivity. Incubation of cells with azelastine initiated concomitantly with the chlamydial infection, or initiated several hours post-infection, reduced inclusion size, number and infectivity, and altered chlamydial morphology. These effects were strongest when azelastine was added shortly after or with the infection. Azelastine effects were not alleviated by increased concentrations of culture medium nutrients. Additionally, we did not observe anti-chlamydial effects when incubating cultures either with a different H1R antagonist or agonist, indicating that azelastine effects are probably H1R-independent.Conclusion. Accordingly, we conclude that azelastine anti-chlamydial effects are not restricted to a specific chlamydial species, strain or culture model, and are probably not mediated by H1R antagonism. Thus, it appears likely that off-target mechanisms of azelastine may explain our observations.

Neisseria gonorrhoeae Coinfection during Chlamydia muridarum Genital Latency Does Not Modulate Murine Vaginal Bacterial Shedding.

Chlamydia trachomatis and Neisseria gonorrhoeae are the most frequently reported agents of bacterial sexually transmitted disease worldwide. Nonetheless, C. trachomatis/N. gonorrhoeae coinfection remains understudied. C. trachomatis/N. gonorrhoeae coinfections are more common than expected by chance, suggesting C. trachomatis/N. gonorrhoeae interaction, and N. gonorrhoeae infection may reactivate genital chlamydial shedding in women with latent (quiescent) chlamydial infection. We hypothesized that N. gonorrhoeae would reactivate latent genital Chlamydia muridarum infection in mice. Two groups of C. muridarum-infected mice were allowed to transition into genital latency. One group was then vaginally inoculated with N. gonorrhoeae; a third group received N. gonorrhoeae alone. C. muridarum and N. gonorrhoeae vaginal shedding was measured over time in the coinfected and singly infected groups. Viable C. muridarum was absent from vaginal swabs but detected in rectal swabs, confirming C. muridarum genital latency and consistent with the intestinal tract as a C. muridarum reservoir. C. muridarum inclusions were observed in large intestinal, but not genital, tissues during latency. Oviduct dilation was associated with C. muridarum infection, as expected. Contradicting our hypothesis, N. gonorrhoeae coinfection did not reactivate latent C. muridarum vaginal shedding. In addition, latent C. muridarum infection did not modulate recovery of vaginal viable N. gonorrhoeae. Evidence for N. gonorrhoeae-dependent increased C. muridarum infectivity has thus not been demonstrated in murine coinfection, and the ability of C. muridarum coinfection to potentiate N. gonorrhoeae infectivity may depend on actively replicating vaginal C. muridarum. The proportion of mice with increased vaginal neutrophils (PMNs) was higher in N. gonorrhoeae-infected than in C. muridarum-infected mice, as expected, while that of C. muridarum/N. gonorrhoeae-coinfected mice was intermediate to the singly infected groups, suggesting latent C. muridarum murine infection may limit PMN response to subsequent N. gonorrhoeae infection. IMPORTANCE Our work builds upon the limited understanding of C. muridarum/N. gonorrhoeae coinfection. Previously, N. gonorrhoeae infection of mice with acute (actively replicating) vaginal C. muridarum infection was shown to increase recovery of viable vaginal N. gonorrhoeae and vaginal PMNs, with no effect on C. muridarum vaginal shedding (R. A. Vonck et al., Infect Immun 79:1566-1577, 2011). It has also been shown that chlamydial infection of human and murine PMNs prevents normal PMN responses, including the response to N. gonorrhoeae (K. Rajeeve et al., Nat Microbiol 3:824-835, 2018). Our findings show no effect of latent genital C. muridarum infection on the recovery of viable N. gonorrhoeae, in contrast to the previously reported effect of acute C. muridarum infection, and suggesting that acute versus latent C. muridarum infection may have distinct effects on PMN function in mice. Together, these studies to date provide evidence that Chlamydia/N. gonorrhoeae synergistic interactions may depend on the presence of replicating Chlamydia in the genital tract, while chlamydial effects on vaginal PMNs may extend beyond acute infection.

Refinement of water-filtered infrared A (wIRA) irradiations of in vitro acute and persistent chlamydial infections.

Water-filtered infrared A (wIRA) alone or in combination with visible light (VIS) exerts anti-chlamydial effects in vitro and in vivo in acute infection models. However, it has remained unclear whether reduced irradiation duration and irradiance would still maintain anti-chlamydial efficacy. Furthermore, efficacy of this non-chemical treatment option against persistent (chronic) chlamydial infections has not been investigated to date. To address this knowledge gap, we evaluated 1) irradiation durations of 5, 15 or 30 min in genital and ocular Chlamydia trachomatis acute infection models, 2) irradiances of 100, 150 or 200 mW/cm2 in the acute genital infection model and 3) anti-chlamydial activity of wIRA and VIS against C. trachomatis serovar B and E with amoxicillin (AMX)- or interferon γ (IFN-γ)-induced persistence. Reduction of irradiation duration reduced anti-chlamydial efficacy. Irradiances of 150 to 200 mW/cm2, but not 100 mW/cm2, induced anti-chlamydial effects. For persistent infections, wIRA and VIS irradiation showed robust anti-chlamydial activity independent of the infection status (persistent or recovering), persistence inducer (AMX or IFN-γ) or chlamydial strain (serovar B or E). This study clarifies the requirement of 30 min irradiation duration and 150 mW/cm2 irradiance to induce significant anti-chlamydial effects in vitro, supports the use of irradiation in the wIRA and VIS spectrum as a promising non-chemical treatment for chlamydial infections and provides important information for follow-up in vivo studies. Notably, wIRA and VIS exert anti-chlamydial effects on persistent chlamydiae which are known to be refractory to antibiotic treatment.

Neisseria gonorrhoeae Limits Chlamydia trachomatis Inclusion Development and Infectivity in a Novel In Vitro Co-Infection Model.

Chlamydia trachomatis (Ct) and Neisseria gonorrhoeae (Ng) are the most common bacterial sexually transmitted infections (STIs) worldwide. The primary site of infection for both bacteria is the epithelium of the endocervix in women and the urethra in men; both can also infect the rectum, pharynx and conjunctiva. Ct/Ng co-infections are more common than expected by chance, suggesting Ct/Ng interactions increase susceptibility and/or transmissibility. To date, studies have largely focused on each pathogen individually and models exploring co-infection are limited. We aimed to determine if Ng co-infection influences chlamydial infection and development and we hypothesized that Ng-infected cells are more susceptible to chlamydial infection than uninfected cells. To address this hypothesis, we established an in vitro model of Ct/Ng co-infection in cultured human cervical epithelial cells. Our data show that Ng co-infection elicits an anti-chlamydial effect by reducing chlamydial infection, inclusion size, and subsequent infectivity. Notably, the anti-chlamydial effect is dependent on Ng viability but not extracellular nutrient depletion or pH modulation. Though this finding is not consistent with our hypothesis, it provides evidence that interaction of these bacteria in vitro influences chlamydial infection and development. This Ct/Ng co-infection model, established in an epithelial cell line, will facilitate further exploration into the pathogenic interplay between Ct and Ng.

Maraviroc, celastrol and azelastine alter Chlamydia trachomatis development in HeLa cells.

Introduction . Chlamydia trachomatis (Ct) is an obligate intracellular bacterium, causing a range of diseases in humans. Interactions between chlamydiae and antibiotics have been extensively studied in the past.Hypothesis/Gap statement: Chlamydial interactions with non-antibiotic drugs have received less attention and warrant further investigations. We hypothesized that selected cytokine inhibitors would alter Ct growth characteristics in HeLa cells.Aim. To investigate potential interactions between selected cytokine inhibitors and Ct development in vitro.Methodology. The CCR5 receptor antagonist maraviroc (Mara; clinically used as HIV treatment), the triterpenoid celastrol (Cel; used in traditional Chinese medicine) and the histamine H1 receptor antagonist azelastine (Az; clinically used to treat allergic rhinitis and conjunctivitis) were used in a genital in vitro model of Ct serovar E infecting human adenocarcinoma cells (HeLa).Results. Initial analyses revealed no cytotoxicity of Mara up to 20 µM, Cel up to 1 µM and Az up to 20 µM. Mara exposure (1, 5, 10 and 20 µM) elicited a reduction of chlamydial inclusion numbers, while 10 µM reduced chlamydial infectivity. Cel 1 µM, as well as 10 and 20 µM Az, reduced chlamydial inclusion size, number and infectivity. Morphological immunofluorescence and ultrastructural analysis indicated that exposure to 20 µM Az disrupted chlamydial inclusion structure. Immunofluorescence evaluation of Cel-incubated inclusions showed reduced inclusion sizes whilst Mara incubation had no effect on inclusion morphology. Recovery assays demonstrated incomplete recovery of chlamydial infectivity and formation of structures resembling typical chlamydial inclusions upon Az removal.Conclusion. These observations indicate that distinct mechanisms might be involved in potential interactions of the drugs evaluated herein and highlight the need for continued investigation of the interaction of commonly used drugs with Chlamydia and its host.

Chlamydia suis is associated with intestinal NF-κB activation in experimentally infected gnotobiotic piglets.

Chlamydia suis intestinal infection of single-animal experimental groups of gnotobiotic newborn piglets was previously reported to cause severe, temporary small intestinal epithelium damage. We investigated archived intestinal samples for pro-inflammatory nuclear factor kappa B (NF-κB) activation, Interleukin (IL)-6 and IL-8 production and immune cell influx. Samples were collected 2, 4 and 7 days post-inoculation with C. suis strain S45/6 or mock inoculum (control). Increased nuclear localization of epithelial NF-κB, representative of activation, in the jejunum and ileum of C. suis-infected animals, compared to uninfected controls, began by 2 days post-infection (dpi) and persisted through 7 dpi. Infected animals showed increased production of IL-8, peaking at 2 dpi, compared to controls. Infection-mediated CD45-positive immune cell influx into the jejunal lamina propria peaked at 7 dpi, when epithelial damage was largely resolved. Activation of NF-κB appears to be a key early event in the innate response of the unprimed porcine immune system challenged with C. suis. This results in an acute phase, coinciding with the most severe clinical symptoms, diarrhea and weight loss. Immune cells recruited shortly after infection remain present in the lamina propria during the recovery phase, which is characterized by reduced chlamydial shedding and restored intestinal epithelium integrity.

Water Filtered Infrared A and Visible Light (wIRA/VIS) Irradiation Reduces Chlamydia trachomatis Infectivity Independent of Targeted Cytokine Inhibition.

Chlamydia trachomatis is the major cause of infectious blindness and represents the most common bacterial sexually transmitted infection worldwide. Considering the potential side effects of antibiotic therapy and increasing threat of antibiotic resistance, alternative therapeutic strategies are needed. Previous studies showed that water filtered infrared A alone (wIRA) or in combination with visible light (wIRA/VIS) reduced C. trachomatis infectivity. Furthermore, wIRA/VIS irradiation led to secretion of pro-inflammatory cytokines similar to that observed upon C. trachomatis infection. We confirmed the results of previous studies, namely that cytokine secretion (IL-6, IL-8, and RANTES/CCL5) upon wIRA/VIS treatment, and the subsequent reduction of chlamydial infectivity, are independent of the addition of cycloheximide, a host protein synthesis inhibitor. Reproducible cytokine release upon irradiation indicated that cytokines might be involved in the anti-chlamydial mechanism of wIRA/VIS. This hypothesis was tested by inhibiting IL-6, IL-8, and RANTES secretion in C. trachomatis or mock-infected cells by gene silencing or pharmaceutical inhibition. Celastrol, a substance derived from Trypterygium wilfordii, used in traditional Chinese medicine and known for anti-cancer and anti-inflammatory effects, was used for IL-6 and IL-8 inhibition, while Maraviroc, a competitive CCR5 antagonist and anti-HIV drug, served as a RANTES/CCL5 inhibitor. HeLa cell cytotoxicity and impact on chlamydial morphology, size and inclusion number was evaluated upon increasing inhibitor concentration, and concentrations of 0.1 and 1 µM Celastrol and 10 and 20 µM Maraviroc were subsequently selected for irradiation experiments. Celastrol at any concentration reduced chlamydial infectivity, an effect only observed for 20 µM Maraviroc. Triple dose irradiation (24, 36, 40 hpi) significantly reduced chlamydial infectivity regardless of IL-6, IL-8, or RANTES/CCL5 gene silencing, Celastrol or Maraviroc treatment. Neither gene silencing nor pharmaceutical cytokine inhibition provoked the chlamydial stress response. The anti-chlamydial effect of wIRA/VIS is independent of cytokine inhibition under all conditions evaluated. Thus, factors other than host cell cytokines must be involved in the working mechanism of wIRA/VIS. This study gives a first insight into the working mechanism of wIRA/VIS in relation to an integral component of the host immune system and supports the potential of wIRA/VIS as a promising new tool for treatment in trachoma.

Novel Chlamydia species isolated from snakes are temperature-sensitive and exhibit decreased susceptibility to azithromycin.

Chlamydia species have recently been recognized as emerging pathogens in snakes. However, isolation of novel snake chlamydiae is critical and their growth characteristics are largely unknown. In this study, two novel chlamydial species are described: Chlamydia serpentis and Chlamydia poikilothermis, isolated after attempts on 23 cloacal and choanal swabs from 18 PCR-positive captive snakes originating from different Swiss snake collections. Isolation success, growth curve and infectivity rates over a 48-hour time period were dependent on temperature (37 °C for C. serpentis, 28 °C for C. poikilothermis). C. serpentis and C. poikilothermis were sensitive to tetracycline and moxifloxacin during evaluation by in vitro antibiotic susceptibility assay but intermediate to resistant (2-4 µg/ml) to azithromycin. Whole genome sequencing of the isolates provided proof of the novel species status, and gives insights into the evolution of these branches of genus Chlamydia.

Water-filtered infrared A reduces chlamydial infectivity in vitro without causing ex vivo eye damage in pig and mouse models.

Repeated ocular infections with Chlamydia trachomatis trigger the development of trachoma, the most common cause of infectious blindness worldwide. Water-filtered infrared A (wIRA) has shown positive effects on cultured cells and human skin. Our aim was to evaluate the potential of wIRA as a possible non-chemical treatment for trachoma patients. We both modeled ocular chlamydial infections using C. trachomatis B to infect human conjunctival epithelial cells (HCjE) and studied the effects of wIRA on non-infected ocular structures with two ex vivo eye models. We focused on the temperature development during wIRA irradiation in cell culture and perfused pig eyes to exclude potentially harmful side effects. Furthermore, cell viability of HCjE and cytotoxicity in mouse retina explants was analyzed. We demonstrated a significant wIRA-dependent reduction of chlamydial infectivity in HCjE cells. Moreover, we observed that wIRA treatment of HCjE prior to infection was sufficient to inhibit chlamydial infectivity and that visible light enhances the effect of wIRA. Irradiation did not reduce cell viability and there was no indication of retinal damage post treatment. Additionally, temperatures during wIRA exposure did not markedly exceed physiological eye temperatures, suggesting that hyperthermia-related lesions are unlikely. For clinical applications, further exploration of wIRA as a non-chemical treatment device in an experimental animal model is essential.

18S Ribosomal RNA Evaluation as Preanalytical Quality Control for Animal DNA.

The 18S ribosomal RNA (rRNA) gene is present in all eukaryotic cells. In this study, we evaluated the use of this gene to verify the presence of PCR-amplifiable host (animal) DNA as an indicator of sufficient sample quality for quantitative real-time PCR (qPCR) analysis. We compared (i) samples from various animal species, tissues, and sample types, including swabs; (ii) multiple DNA extraction methods; and (iii) both fresh and formalin-fixed paraffin-embedded (FFPE) samples. Results showed that 18S ribosomal RNA gene amplification was possible from all tissue samples evaluated, including avian, reptile, and FFPE samples and most swab samples. A single swine rectal swab, which showed sufficient DNA quantity and the demonstrated lack of PCR inhibitors, nonetheless was negative by 18S qPCR. Such a sample specifically illustrates the improvement of determination of sample integrity afforded by inclusion of 18S rRNA gene qPCR analysis in addition to spectrophotometric analysis and the use of internal controls for PCR inhibition. Other possible applications for the described 18S rRNA qPCR are preselection of optimal tissue specimens for studies or preliminary screening of archived samples prior to acceptance for biobanking projects.

Penicillin G-Induced Chlamydial Stress Response in a Porcine Strain of Chlamydia pecorum.

Chlamydia pecorum causes asymptomatic infection and pathology in ruminants, pigs, and koalas. We characterized the antichlamydial effect of the beta lactam penicillin G on Chlamydia pecorum strain 1710S (porcine abortion isolate). Penicillin-exposed and mock-exposed infected host cells showed equivalent inclusions numbers. Penicillin-exposed inclusions contained aberrant bacterial forms and exhibited reduced infectivity, while mock-exposed inclusions contained normal bacterial forms and exhibited robust infectivity. Infectious bacteria production increased upon discontinuation of penicillin exposure, compared to continued exposure. Chlamydia-induced cell death occurred in mock-exposed controls; cell survival was improved in penicillin-exposed infected groups. Similar results were obtained both in the presence and in the absence of the eukaryotic protein translation inhibitor cycloheximide and at different times of initiation of penicillin exposure. These data demonstrate that penicillin G induces the chlamydial stress response (persistence) and is not bactericidal, for this chlamydial species/strain in vitro, regardless of host cell de novo protein synthesis.

Damage/Danger Associated Molecular Patterns (DAMPs) Modulate Chlamydia pecorum and C. trachomatis Serovar E Inclusion Development In Vitro.

Persistence, more recently termed the chlamydial stress response, is a viable but non-infectious state constituting a divergence from the characteristic chlamydial biphasic developmental cycle. Damage/danger associated molecular patterns (DAMPs) are normal intracellular components or metabolites that, when released from cells, signal cellular damage/lysis. Purine metabolite DAMPs, including extracellular ATP and adenosine, inhibit chlamydial development in a species-specific manner. Viral co-infection has been shown to reversibly abrogate Chlamydia inclusion development, suggesting persistence/chlamydial stress. Because viral infection can cause host cell DAMP release, we hypothesized DAMPs may influence chlamydial development. Therefore, we examined the effect of extracellular ATP, adenosine, and cyclic AMP exposure, at 0 and 14 hours post infection, on C. pecorum and C. trachomatis serovar E development. In the absence of de novo host protein synthesis, exposure to DAMPs immediately post or at 14 hours post infection reduced inclusion size; however, the effect was less robust upon 14 hours post infection exposure. Additionally, upon exposure to DAMPs immediately post infection, bacteria per inclusion and subsequent infectivity were reduced in both Chlamydia species. These effects were reversible, and C. pecorum exhibited more pronounced recovery from DAMP exposure. Aberrant bodies, typical in virus-induced chlamydial persistence, were absent upon DAMP exposure. In the presence of de novo host protein synthesis, exposure to DAMPs immediately post infection reduced inclusion size, but only variably modulated chlamydial infectivity. Because chlamydial infection and other infections may increase local DAMP concentrations, DAMPs may influence Chlamydia infection in vivo, particularly in the context of poly-microbial infections.

Encephalitozoon intestinalis infection increases host cell mutation frequency.

BACKGROUND: Microsporidia are obligate intracellular opportunistic fungi that cause significant pathology in immunocompromised hosts. However, 11 percent of immunocompetent individuals in the general population are microsporidia-seropositive, indicating that severe immune suppression may not be a prerequisite for infection. Encephalitozoon intestinalis is transmitted in contaminated water and initially infects gastro-intestinal enterocytes, leading to diarrheal disease. This organism can also disseminate to many other organs. A recent report suggests that microsporidia can establish persistent infections, which anti-fungal treatment does not eradicate. Like other intracellular pathogens, microsporidia infection stresses the host cell and infected individuals have elevated hydrogen peroxide and free radical levels. FINDINGS: As oxidative stress can lead to DNA damage, we hypothesized that E. intestinalis-infection would increase host cell nuclear mutation rate. Embryo fibroblasts from Big BlueTM transgenic mice were E. intestinalis-infected and host nuclear mutation frequency was determined by selection of temperature-sensitive c-II gene mutant λ phage. The host mutation frequency in E. intestinalis-infected cultures was 2.5-fold higher than that observed in either mock-infected cells or cells infected with UV-inactivated E. intestinalis spores. CONCLUSIONS: These data provide the first evidence that microsporidia infection can directly increase host cellular mutation frequency. Additionally, some event in the microsporidia developmental cycle between host cell attachment and parasitophorous vacuole formation is required for the observed effect. As there is considerable evidence linking infection with other intracellular pathogens and cancer, future studies to dissect the mechanism by which E. intestinalis infection increases host mutation frequency are warranted.