Respiratory infections are one of the most common causes of illness and morbidity in neonates worldwide. In the acute phase infections are known to cause wide-spread peripheral inflammation. However, the inflammatory consequences to the critical neural control centres for respiration have not been explored. Utilising a well characterised model of neonatal respiratory infection, we investigated acute responses within the medulla oblongata which contains key respiratory regions. Neonatal mice were intranasally inoculated within 24 h of birth, with either Chlamydia muridarum or sham-infected, and tissue collected on postnatal day 15, the peak of peripheral inflammation. A key finding of this study is that, while the periphery appeared to show no sex-specific effects of a neonatal respiratory infection, sex had a significant impact on the inflammatory response of the medulla oblongata. There was a distinct sex-specific response in the medulla coincident with peak of peripheral inflammation, with females demonstrating an upregulation of anti-inflammatory cytokines and males showing very few changes. Microglia also demonstrated sex-specificity with the morphology of females and males differing based upon the nuclei. Astrocytes showed limited changes during the acute response to neonatal infection. These data highlight the strong sex-specific impact of a respiratory infection can have on the medulla in the acute inflammatory phase.

To begin to optimize the immunization routes for our reported PLGA-rMOMP nanovaccine [PLGA-encapsulated Chlamydia muridarum (Cm) recombinant major outer membrane protein (rMOMP)], we compared two prime-boost immunization strategies [subcutaneous (SC) and intramuscular (IM-p) prime routes followed by two SC-boosts)] to evaluate the nanovaccine-induced protective efficacy and immunogenicity in female BALB/c mice. Our results showed that mice immunized via the SC and IM-p routes were protected against a Cm genital challenge by a reduction in bacterial burden and with fewer bacteria in the SC mice. Protection of mice correlated with rMOMP-specific Th1 (IL-2 and IFN-γ) and not Th2 (IL-4, IL-9, and IL-13) cytokines, and CD4+ memory (CD44highCD62Lhigh) T-cells, especially in the SC mice. We also observed higher levels of IL-1α, IL-6, IL-17, CCL-2, and G-CSF in SC-immunized mice. Notably, an increase of cytokines/chemokines was seen after the challenge in the SC, IM-p, and control mice (rMOMP and PBS), suggesting a Cm stimulation. In parallel, rMOMP-specific Th1 (IgG2a and IgG2b) and Th2 (IgG1) serum, mucosal, serum avidity, and neutralizing antibodies were more elevated in SC than in IM-p mice. Overall, the homologous SC prime-boost immunization of mice induced enhanced cellular and antibody responses with better protection against a genital challenge compared to the heterologous IM-p.

We have demonstrated previously that TNF-α-producing CD8+ T cells mediate chlamydial pathogenesis, likely in an antigen (Ag)-specific fashion. Here we hypothesize that inhibition of Ag-specific CD8+ T cell response after immunization and/or challenge would correlate with protection against oviduct pathology induced by a protective vaccine regimen. Intranasal (i.n.) live chlamydial elementary body (EB), intramuscular (i.m.) live EB, or i.n. irrelevant antigen, bovine serum albumin (BSA), immunized animals induced near-total protection, 50% protection, or no protection, respectively against oviduct pathology following i.vag. C. muridarum challenge. In these models, we evaluated Ag-specific CD8+ T cell cytokine response at various time-periods after immunization or challenge. The results show protective efficacy of vaccine regimens correlated with reduction of Ag-specific CD8+ T cell TNF-α responses following i.vag. chlamydial challenge, not after immunization. Depletion of CD4+ T cells abrogated, whereas adoptive transfer of Ag-specific CD4+ T cells induced the significant reduction of Ag-specific CD8+ T cell TNF-α response after chlamydial challenge. In conclusion, protective anti-chlamydial vaccine regimens induce Ag-specific CD4+ T cell response that mediate early inhibition of pathogenic CD8+ T cell response following challenge and may serve as a predictive biomarker of protection against Chlamydia -induced chronic pathologies.

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.

Chlamydia muridarum (Cm), an intracellular bacterium of historical importance, was recently rediscovered as moderately prevalent in research mouse colonies. Cm was first reported as a causative agent of severe pneumonia in mice about 80 y ago, and while it has been used experimentally to model Chlamydia trachomatis infection of humans, there have been no further reports of clinical disease associated with natural infection. We observed clinical disease and pathology in 2 genetically engi- neered mouse (GEM) strains, Il12rb2 KO and STAT1 KO, with impaired interferon-γ signaling and Th1 CD4+ T cell responses in a colony of various GEM strains known to be colonized with and shedding Cm. Clinical signs included poor condition, hunched posture, and poor fecundity. Histopathology revealed disseminated Cm with lesions in pulmonary, gastrointestinal, and urogenital tissues. The presence of Cm was confirmed using both immunohistochemistry for Cm major outer membrane protein-1 antigen and in situ hybridization using a target probe directed against select regions of Cm strain Nigg. Cm was also found in association with a urothelial papilloma in one mouse. These cases provide additional support for excluding Cm from research mouse colonies.

Chlamydiae are obligate intracellular bacterial pathogens that may cause genital pathology via induction of destructive host immune responses. Human-adapted Chlamydia trachomatis causes inflammatory disease in human hosts but is easily cleared in mice, and mouse-adapted Chlamydia muridarum establishes a productive and pathogenic infection in murine hosts. While numerous anti-chlamydial host resistance factors have been discovered in mice and humans alike, little is known about host factors promoting host fitness independent of host resistance. Here, we show that interferon-inducible immunity-related GTPase M (Irgm) proteins function as such host factors ameliorating infection-associated sequalae in the murine female genital tract, thus characterizing Irgm proteins as mediators of disease tolerance. Specifically, we demonstrate that mice deficient for all three murine Irgm paralogs (pan-Irgm-/-) are defective for cell-autonomous immunity to C. trachomatis, which correlates with an early and transient increase in bacterial burden and sustained hyperinflammation in vivo. In contrast, upon infection of pan-Irgm-/- mice with C. muridarum, bacterial burden is unaffected, yet genital inflammation and scarring pathology are nonetheless increased, demonstrating that Irgm proteins can promote host fitness without altering bacterial burden. Additionally, pan-Irgm-/- mice display increased granulomatous inflammation in genital Chlamydia infection, implicating Irgm proteins in the regulation of granuloma formation and maintenance. These findings demonstrate that Irgm proteins regulate pathogenic immune responses to Chlamydia infection in vivo, establishing an effective infection model to examine the immunoregulatory functions and mechanisms of Irgm proteins.
OBJECTIVE: In response to genital Chlamydia infection, the immune system mounts a proinflammatory response to resist the pathogen, yet inflammation must be tightly controlled to avoid collateral damage and scarring to host genital tissue. Variation in the human IRGM gene is associated with susceptibility to autoinflammatory diseases but its role in ameliorating inflammatory diseases caused by infections is poorly defined. Here, we use mice deficient for all three murine Irgm paralogs to demonstrate that Irgm proteins not only provide host resistance to Chlamydia infections but also limit associated inflammation in the female genital tract. In particular, we find that murine Irgm expression prevents granulomatous inflammation, which parallels inflammatory diseases associated with variants in human IRGM. Our findings therefore establish genital Chlamydia infection as a useful model to study the roles for Irgm proteins in both promoting protective immunity and limiting pathogenic inflammation.

The protein basic helix-loop-helix family member e40 (BHLHE40) is a transcription factor recently emerged as a key regulator of host immunity to infections, autoimmune diseases and cancer. In this study, we investigated the role of Bhlhe40 in protective T cell responses to the intracellular bacterium Chlamydia in the female reproductive tract (FRT). Mice deficient in Bhlhe40 exhibited severe defects in their ability to control Chlamydia muridarum shedding from the FRT. The heightened bacterial burdens in Bhlhe40-/- mice correlated with a marked increase in IL-10-producing T regulatory type 1 (Tr1) cells and decreased polyfunctional CD4 T cells co-producing IFN-γ, IL-17A and GM-CSF. Genetic ablation of IL-10 or functional blockade of IL-10R increased CD4 T cell polyfunctionality and partially rescued the defects in bacterial control in Bhlhe40-/- mice. Using single-cell RNA sequencing coupled with TCR profiling, we detected a significant enrichment of stem-like T cell signatures in Bhlhe40-deficient CD4 T cells, whereas WT CD4 T cells were further down on the differentiation trajectory with distinct effector functions beyond IFN-γ production by Th1 cells. Altogether, we identified Bhlhe40 as a key molecular driver of CD4 T cell differentiation and polyfunctional responses in the FRT against Chlamydia.

Chlamydia vaccine approaches aspire to induce Th1 cells for optimal protection, despite the fact that there is no direct evidence demonstrating Th1-mediated Chlamydia clearance from the female reproductive tract (FRT). We recently reported that T-bet-deficient mice can resolve primary Chlamydia infection normally, undermining the potentially protective role of Th1 cells in Chlamydia immunity. Here, we show that T-bet-deficient mice develop robust Th17 responses and that mice deficient in Th17 cells exhibit delayed bacterial clearance, demonstrating that Chlamydia-specific Th17 cells represent an underappreciated protective population. Additionally, Th2-deficient mice competently clear cervicovaginal infection. Furthermore, we show that sensing of IFN-γ by non-hematopoietic cells is essential for Chlamydia immunity, yet bacterial clearance in the FRT does not require IFN-γ secretion by CD4 T cells. Despite the fact that Th1 cells are not necessary for Chlamydia clearance, protective immunity to Chlamydia is still dependent on MHC class-II-restricted CD4 T cells and IL-12p40. Together, these data point to IL-12p40-dependent CD4 effector maturation as essential for Chlamydia immunity, and Th17 cells to a lesser extent, yet neither Th1 nor Th2 cell development is critical. Future Chlamydia vaccination efforts will be more effective if they focus on induction of this protective CD4 T cell population.

The Pgp3 subunit vaccine elicits immune protection against Chlamydia trachomatis infection, but additional adjuvants are still required to enhance its immunoprotective efficacy. Flagellin can selectively stimulate immunity and act as an adjuvant. In this research, the FliC-Pgp3 recombinant was successfully expressed and purified. Tri-immunization with the FliC-Pgp3 vaccine in Balb/C mice induced rapid and persistent germinal center B-cell response and Tfh differentiation, promoting a significantly higher IgG antibody titer compared to the Pgp3 group. FliC-Pgp3 immunization primarily induced Th1-type cellular immunity, leading to higher levels of IFN-γ, TNF-α, and IL-2 secreted by CD4+ T cells than in Pgp3-vaccinated mice. Chlamydia muridarum challenge results showed that FliC-Pgp3-vaccinated mice exhibited more rapid clearance of Chlamydia muridarum colonization in the lower genital tract, ensuring a lower hydrosalpinx rate and cumulative score. Histological analysis showed reduced dilation and inflammatory infiltration in the oviduct and uterine horn of FliC-Pgp3-vaccinated mice compared to the PBS and Pgp3 control. Importantly, tri-immunization with FliC-Pgp3 effectively activated CD4+ T cells and dendritic cells, as confirmed by the adoptive transfer, resulting in better immune protection in recipient mice. In summary, the novel FliC-Pgp3 chimeric is hoped to be a novel vaccine with improved immunoprotection against Chlamydia muridarum.

Following an oral inoculation, Chlamydia muridarum descends to the mouse large intestine for long-lasting colonization. However, a mutant C. muridarum that lacks the plasmid-encoded protein pGP3 due to an engineered premature stop codon (designated as CMpGP3S) failed to do so even following an intrajejunal inoculation. This was because a CD4+ T cell-dependent immunity prevented the spread of CMpGP3S from the small intestine to the large intestine. In the current study, we found that mice deficient in IL-22 (IL-22-/-) allowed CMpGP3S to spread from the small intestine to the large intestine on day 3 after intrajejunal inoculation, indicating a critical role of IL-22 in regulating the chlamydial spread. The responsible IL-22 is produced by CD4+ T cells since IL-22-/- mice were rescued to block the CMpGP3S spread by donor CD4+ T cells from C57BL/6J mice. Consistently, CD4+ T cells lacking IL-22 failed to block the spread of CMpGP3S in Rag2-/- mice, while IL-22-competent CD4+ T cells did block. Furthermore, mice deficient in cathelicidin-related antimicrobial peptide (CRAMP) permitted the CMpGP3S spread, but donor CD4+ T cells from CRAMP-/- mice were still sufficient for preventing the CMpGP3S spread in Rag2-/- mice, indicating a critical role of CRAMP in regulating chlamydial spreading, and the responsible CRAMP is not produced by CD4+ T cells. Thus, the IL-22-producing CD4+ T cell-dependent regulation of chlamydial spreading correlated with CRAMP produced by non-CD4+ T cells. These findings provide a platform for further characterizing the subset(s) of CD4+ T cells responsible for regulating bacterial spreading in the intestine.