Characterization of pathogenic CD8 + T cells in Chlamydia - infected OT1 mice

Chlamydia trachomatis is a leading infectious cause of infertility in women due to its induction of lasting pathology such as hydrosalpinx. Chlamydia muridarum induces mouse hydrosalpinx because C. muridarum can both invade tubal epithelia directly (as a 1 st hit) and induce lymphocytes to promote hydrosalpinx indirectly (as a 2 nd hit). In the current study, a critical role of CD8 + T cells in chlamydial induction of hydrosalpinx was validated in both wild type C57BL/6J and OT1 transgenic mice. OT1 mice failed to develop hydrosalpinx partially due to the failure of their lymphocytes to recognize chlamydial antigens. CD8 + T cells from naïve C57BL/6J rescued the recipient OT1 mice to develop hydrosalpinx when naïve CD8 + T cells were transferred at the time of infection with Chlamydia . However, when the transfer was delayed for 2 weeks or longer after the chlamydial infection, naïve CD8 + T cells no longer promoted hydrosalpinx. Nevertheless, Chlamydia -immunized CD8 + T cells still promoted significant hydrosalpinx in the recipient OT1 mice even when the transfer was delayed for 3 weeks. Thus, CD8 + T cells must be primed within 2 weeks after chlamydial infection to be pathogenic but once primed, they can promote hydrosalpinx for >3 weeks. However, Chlamydia -primed CD4 + T cells failed to promote chlamydial induction of pathology in OT1 mice. This study has optimized an OT1 mouse-based model for revealing the pathogenic mechanisms of Chlamydia -specific CD8 + T cells.

A comprehensive approach for microbiota and health monitoring in mouse colonies using metagenomic shotgun sequencing

Background Health surveillance of murine colonies employed for scientific purposes aim at detecting unwanted infection that can affect the well-being of animals and personnel, and potentially undermine scientific results. In this study, we investigated the use of a next-generation sequencing (NGS) metagenomic approach for monitoring the microbiota composition and uncovering the possible presence of pathogens in mice housed in specific pathogen-free (SPF) or conventional (non-SPF) facilities. Results Analysis of metagenomic NGS assay through public and free algorithms and databases allowed to precisely assess the composition of mouse gut microbiome and quantify the contribution of the different microorganisms at the species level. Sequence analysis allowed the uncovering of pathogens or the presence of imbalances in the microbiota composition. In several cases, fecal pellets taken from conventional facilities were found to carry gene sequences from bacterial pathogens (Helicobacter hepaticus, Helicobacter typhlonius, Chlamydia muridarum, Streptococcus pyogenes, Rodentibacter pneumotropicus, Citrobacter rodentium, Staphylococcus aureus), intestinal protozoa (Entamoeba muris, Tritrichomonas muris, Spironucleus muris) nematoda (Aspiculuris tetraptera, Syphacia obvelata), eukaryotic parasites (Myocoptes musculinus) and RNA virus (Norwalk virus). Thus, the use of NGS metagenomics can reduce the number of tests required for the detection of pathogens and avoid the use of sentinel mice. Conclusions In summary, in comparison with standard approaches, which require multiple types of test, NGS assay can detect bacteria, fungi, DNA and RNA viruses, and eukaryotic parasites from fecal pellets in a single test. Considering the need to protect animal well-being and to improve the success and reproducibility of preclinical studies, this work provides the proof-of-concept that the use of NGS metagenomics for health monitoring of laboratory mice is a feasible and dependable approach, that is able to broaden the current concept of health monitoring of laboratory mice from “pathogen surveillance” to a more inclusive “microbiota surveillance”.

Induction of Protection in Mice against a Chlamydia muridarum Respiratory Challenge by a Vaccine Formulated with the Major Outer Membrane Protein in Nanolipoprotein Particles

Chlamydia trachomatis is a sexually transmitted bacterium that infects over 130 million individuals worldwide annually. To implement a vaccine, we developed a cell-free co-translational system to express the Chlamydia muridarum major outer membrane protein (MOMP). This approach uses a nanolipoprotein particles (tNLP) made from ApoA1 protein, amphiphilic telodendrimer and lipids that self-assemble to form 10–25 nm discs. These tNLP provide a protein-encapsulated lipid support to solubilize and fold membrane proteins. The cell-free system co-translated MOMP and ApoA1 in the presence of telodendrimer mixed with lipids. The MOMP-tNLP complex was amenable to CpG and FSL-1 adjuvant addition. To investigate the ability of MOMP-tNLP+CpG+FSL-1 to induce protection against an intranasal (i.n.) C. muridarum challenge, female mice were vaccinated intramuscularly (i.m.) or i.n. and i.m. simultaneously 4 weeks apart. Following vaccination with MOMP-tNLP+CpG+FSL-1, mice mounted significant humoral and cell-mediated immune responses. Following the i.n. challenge, mice vaccinated with MOMP-tNLP+CpG+FSL-1 i.n. + i.m. group were protected as determined by the percentage change in body weight and by the number of C. muridarum inclusion forming units (IFU) recovered from the lungs. To our knowledge, this is the first time a MOMP-based vaccine formulated in tNLP has been shown to protect against C. muridarum.

Reduced uterine tissue damage during Chlamydia muridarum infection in TREM-1,3 deficient mice

Genital infections with Chlamydia trachomatis can lead to uterine and oviduct tissue damage in the female reproductive tract. Neutrophils are strongly associated with tissue damage during chlamydial infection, while an adaptive CD4 T cell response is necessary to combat infection. Activation of triggering receptor expressed on myeloid cells-1 (TREM-1) on neutrophils has previously been shown to induce and/or enhance degranulation synergistically with TLR-signaling. Additionally, TREM-1 can promote neutrophil transepithelial migration. In this study, we sought to determine the contribution of TREM-1,3 in immunopathology in the female mouse genital tract during Chlamydia muridarum infection. Relative to control mice, trem1,3 -/- mice had no difference in chlamydial burden or duration of lower genital tract infection. We also observed a similar incidence of oviduct hydrosalpinx 45 days post-infection in trem1,3 -/- compared to WT mice. However, compared to WT, trem1,3 -/- mice developed significantly fewer uterine horn hydrometra. Early in infection, trem1,3 -/- mice displayed a notable decrease in the number of uterine glands containing polymorphonuclear cells and uterine horn lumens had fewer neutrophils, with increased G-CSF. Trem1,3 -/- mice also had reduced erosion of the luminal epithelium. These data indicate TREM-1,3 contributes to transepithelial neutrophil migration in the uterus and uterine glands, promoting the development of uterine hydrometra in infected mice.

Encapsulation of Recombinant MOMP in Extended-Releasing PLGA 85:15 Nanoparticles Confer Protective Immunity Against a Chlamydia muridarum Genital Challenge and Re-Challenge

Recently we reported the immune-potentiating capacity of a Chlamydia nanovaccine (PLGA-rMOMP) comprising rMOMP (recombinant major outer membrane protein) encapsulated in extended-releasing PLGA [poly (D, L-lactide-co-glycolide) (85:15)] nanoparticles. Here we hypothesized that PLGA-rMOMP would bolster immune-effector mechanisms to confer protective efficacy in mice against a Chlamydia muridarum genital challenge and re-challenge. Female BALB/c mice received three immunizations, either subcutaneously (SC) or intranasally (IN), before receiving an intravaginal challenge with C. muridarum on day 49 and a re-challenge on day 170. Both the SC and IN immunization routes protected mice against genital challenge with enhanced protection after a re-challenge, especially in the SC mice. The nanovaccine induced robust antigen-specific Th1 (IFN-γ, IL-2) and IL-17 cytokines plus CD4+ proliferating T-cells and memory (CD44high CD62Lhigh) and effector (CD44high CD62Llow) phenotypes in immunized mice. Parallel induction of antigen-specific systemic and mucosal Th1 (IgG2a, IgG2b), Th2 (IgG1), and IgA antibodies were also noted. Importantly, immunized mice produced highly functional Th1 avidity and serum antibodies that neutralized C. muridarum infectivity of McCoy fibroblasts in-vitro that correlated with their respective protection levels. The SC, rather than the IN immunization route, triggered higher cellular and humoral immune effectors that improved mice protection against genital C. muridarum. We report for the first time that the extended-releasing PLGA 85:15 encapsulated rMOMP nanovaccine confers protective immunity in mice against genital Chlamydia and advances the potential towards acquiring a nano-based Chlamydia vaccine.

Modulation of Immune Response to Chlamydia muridarum by Host miR-135a

Previously, our laboratory established the role of small, noncoding RNA species, i.e., microRNA (miRNA) including miR-135a in anti-chlamydial immunity in infected hosts. We report here chlamydial infection results in decreased miR-135a expression in mouse genital tissue and a fibroblast cell line. Several chemokine and chemokine receptor genes (including CXCL10, CCR5) associated with chlamydial pathogenesis were identified in silico to contain putative miR-135a binding sequence(s) in the 3’ untranslated region. The role of miR-135a in the host immune response was investigated using exogenous miR-135a mimic to restore the immune phenotype associated with decreased miR-135a following Chlamydia muridarum (Cm) infection. We observed miR-135a regulation of Cm-primed bone marrow derived dendritic cells (BMDC) via activation of Cm-immune CD4+ T cells for clonal expansion and CCR5 expression. Using a transwell cell migration assay, we explore the role of miR-135a in regulation of genital tract CXCL10 expression and recruitment of CXCR3+ CD4+ T cells via the CXCL10/CXCR3 axis. Collectively, data reported here support miR-135a affecting multiple cellular processes in response to chlamydial infection.

Active hexose correlated compound restores the gene expression and protein secretion of protective cytokines of immune cells in a murine stress model during Chlamydia muridarum genital infection

Chlamydia trachomatis genital infection is the most common bacterial sexually transmitted disease worldwide. Previously we reported that cold-induced stress results in immune suppression of mice that subsequently leads to increased intensity of Chlamydia muridarum genital infection. Furthermore, we demonstrated that stressed mice orally fed with active hexose correlated compound (AHCC) have reduced shedding of C. muridarum from the genital tract. However, the mechanism of AHCC on reducing the organ load and changes in the immune response in the stress model is not well known. This study evaluated infection and changes in immunological parameters of stressed AHCC-fed mice with or without C. muridarum genital infection. We hypothesized that AHCC feeding to stressed mice restores the protective immune function and reduces susceptibility to C. muridarum genital infection. Results show that oral feeding of stressed mice with AHCC resulted in decreased shedding of C. muridarum from the genital tract, reduced production of plasma catecholamines, increased expression of T-bet and reduced GATA-3 in CD4+ T cells, increased production of IL-12 and IFN-γ, and reduced production of IL-4 in CD4+ T cells, and enhanced expression of surface markers and co-stimulatory molecules of CD4+ T cells, bone marrow-derived dendritic cells (BMDCs), and natural killer cells. Co-culturing of mature BMDCs with splenic CD4+ T cells led to the increased and decreased production of T-helper 1 and T-helper2 cytokines, respectively. Overall, our results show that AHCC fosters the restoration of Th1 cytokine production while reducing Th2 cytokine production, which would promote C. muridarum clearance in the murine stress model.

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.

Chlamydia muridarum Alleviates Colitis via the IL-22/Occludin Signal Pathway

Ulcerative colitis (UC) is the most common inflammatory bowel disease, and its incidence has increased in recent years. Recent clinical and experimental data indicate that gut microbiota plays a pivotal role in the pathogenesis of UC. Chlamydia establishes a stable and persistent colonization in the gastrointestinal tract without apparent pathogenicity to gastrointestinal or extragastrointestinal tissues. However, the detailed effects of Chlamydia on the gastrointestinal tissue remain unknown. The primary aim of this study is to investigate the effects of Chlamydia muridarum (C. muridarum) on development of colitis induced by dextran sodium sulfate (DSS) and the underlying molecular mechanism. The results suggested that C. muridarum significantly improved colitis symptoms—including weight loss, disease activity index, colon length, and histopathological changes in the colon caused by DSS—and alleviated the reduced expression of interleukin-22 and occludin in the colonic tissue due to DSS administration. Furthermore, the absence of IL-22 completely prevented C. muridarum from alleviating colitis and significantly decreased the levels of occludin, an important downstream effector protein of IL-22. These findings suggest that C. muridarum ameliorates ulcerative colitis induced by DSS via the IL-22/occludin signal pathway.