Regulation of chlamydial spreading from the small intestine to the large intestine by IL-22-producing CD4+ T cells.

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.

Expression activation of over 70% of Chlamydia trachomatis genes during the first hour of infection.

The obligate intracellular bacterium Chlamydia has a unique developmental cycle that alternates between two contrasting cell types. With a hardy envelope and highly condensed genome, the small elementary body (EB) maintains limited metabolic activities yet survives in extracellular environments and is infectious. After entering host cells, EBs differentiate into larger and proliferating reticulate bodies (RBs). Progeny EBs are derived from RBs in late developmental stages and eventually exit host cells. How expression of the chlamydial genome consisting of nearly 1,000 genes governs the chlamydial developmental cycle is unclear. A previous microarray study identified only 29 Chlamydia trachomatis immediate early genes, defined as genes with increased expression during the first hour postinoculation in cultured cells. In this study, we performed more sensitive RNA sequencing (RNA-Seq) analysis for C. trachomatis cultures with high multiplicities of infection. Remarkably, we observed well over 700 C. trachomatis genes that underwent 2- to 900-fold activation within 1 hour postinoculation. Quantitative reverse transcription real-time PCR analysis was further used to validate the activated expression of a large subset of the genes identified by RNA-Seq. Importantly, our results demonstrate that the immediate early transcriptome is over 20 times more extensive than previously realized. Gene ontology analysis indicates that the activated expression spans all functional categories. We conclude that over 70% of C. trachomatis genes are activated in EBs almost immediately upon entry into host cells, thus implicating their importance in initiating rapid differentiation into RBs and establishing an intracellular niche conducive with chlamydial development and growth.

Analyzing RNA-Seq data from Chlamydia with super broad transcriptomic activation: challenges, solutions, and implications for other systems.

BACKGROUND: RNA sequencing (RNA-Seq) offers profound insights into the complex transcriptomes of diverse biological systems. However, standard differential expression analysis pipelines based on DESeq2 and edgeR encounter challenges when applied to the immediate early transcriptomes of Chlamydia spp., obligate intracellular bacteria. These challenges arise from their reliance on assumptions that do not hold in scenarios characterized by extensive transcriptomic activation and limited repression. RESULTS: Standard analyses using unique chlamydial RNA-Seq reads alone identify nearly 300 upregulated and about 300 downregulated genes, significantly deviating from actual RNA-Seq read trends. By incorporating both chlamydial and host reads or adjusting for total sequencing depth, the revised normalization methods each detected over 700 upregulated genes and 30 or fewer downregulated genes, closely aligned with observed RNA-Seq data. Further validation through qRT-PCR analysis confirmed the effectiveness of these adjusted approaches in capturing the true extent of transcriptomic activation during the immediate early phase of chlamydial infection. CONCLUSIONS: This study highlights the limitations of standard RNA-Seq analysis tools in scenarios with extensive transcriptomic activation, such as in Chlamydia spp. during early infection. Our revised normalization methods, incorporating host reads or total sequencing depth, provide a more accurate representation of gene expression dynamics. These approaches may inform similar adjustments in other systems with unbalanced gene expression dynamics, enhancing the accuracy of transcriptomic analysis.

Preclinical screen for protection efficacy of chlamydial antigens that are immunogenic in humans.

To search for subunit vaccine candidates, immunogenic chlamydial antigens identified in humans were evaluated for protection against both infection and pathology in a mouse genital tract infection model under three different immunization regimens. The intramuscular immunization regimen was first used to evaluate 106 chlamydial antigens, which revealed that two antigens significantly reduced while 11 increased genital chlamydial burden. The two infection-reducing antigens failed to prevent pathology and 23 additional antigens even exacerbated pathology. Thus, intranasal mucosal immunization was tested next since intranasal inoculation with live Chlamydia muridarum prevented both genital infection and pathology. Two of the 29 chlamydial antigens evaluated were found to prevent genital infection but not pathology and three exacerbate pathology. To further improve protection efficacy, a combinational regimen (intranasal priming + intramuscular boosting + a third intraperitoneal/subcutaneous boost) was tested. This regimen identified four infection-reducing antigens, but only one of them prevented pathology. Unfortunately, this protective antigen was not advanced further due to its amino acid sequence homology with several human molecules. Two pathology-exacerbating antigens were also found. Nevertheless, intranasal mucosal priming with viable C. muridarum in control groups consistently prevented both genital infection and pathology regardless of the subsequent boosters. Thus, screening 140 different chlamydial antigens with 21 repeated multiple times in 17 experiments failed to identify a subunit vaccine candidate but demonstrated the superiority of viable chlamydial organisms in inducing immunity against both genital infection and pathology, laying the foundation for developing a live-attenuated Chlamydia vaccine.

IL-23 receptor signaling licenses group 3-like innate lymphoid cells to restrict a live-attenuated oral Chlamydia vaccine in the gut.

An IFNγ-susceptible mutant of Chlamydia muridarum is attenuated in pathogenicity in the genital tract and was recently licensed as an intracellular Oral vaccine vector or intrOv. Oral delivery of intrOv induces transmucosal protection in the genital tract, but intrOv itself is cleared from the gut (without shedding any infectious particles externally) by IFNγ from group 3-like innate lymphoid cells (ILC3s). We further characterized the intrOv interactions with ILC3s in the current study, since the interactions may impact both the safety and efficacy of intrOv as an oral Chlamydia vaccine. Intracolonic inoculation with intrOv induced IFNγ that in return inhibited intrOv. The intrOv-IFNγ interactions were dependent on RORγt, a signature transcriptional factor of ILC3s. Consistently, the transfer of oral intrOv-induced ILC3s from RORγt-GFP reporter mice to IFNγ-deficient mice rescued the inhibition of intrOv. Thus, IFNγ produced by intrOv-induced ILC3s is likely responsible for inhibiting intrOv, which is further supported by the observation that oral intrOv did induce significant levels of IFNγ-producing LC3s (IFNγ+ILC3s). Interestingly, IL-23 receptor knockout (IL-23R-/-) mice no longer inhibited intrOv, which was accompanied by reduced colonic IFNγ. Transfer of oral intrOv-induced ILC3s rescued the IL-23R-/- mice to inhibit intrOv, validating the dependence of ILC3s on IL-23R signaling for inhibiting intrOv. Clearly, intrOv induces intestinal IFNγ+ILC3s for its own inhibition in the gut, which is facilitated by IL-23R signaling. These findings have provided a mechanism for ensuring the safety of intrOv as an oral Chlamydia vaccine and a platform for investigating how oral intrOv induces transmucosal protection in the genital tract.

Type-I Interferon Signaling Protects against Chlamydia trachomatis Infection in the Female Lower Genital Tract.

We have previously shown that Chlamydia trachomatis is significantly inhibited during the early stage of infection in the female mouse lower genital tract and the anti-C. trachomatis innate immunity is compromised in the absence of cGAS-STING signaling. Since type-I interferon is a major downstream response of the cGAS-STING signaling, we evaluated the effect of type-I interferon signaling on C. trachomatis infection in the female genital tract in the current study. The infectious yields of chlamydial organisms recovered from vaginal swabs along the infection course were carefully compared between mice with or without deficiency in type-I interferon receptor (IFNαR1) following intravaginal inoculation with 3 different doses of C. trachomatis. It was found that IFNαR1-deficient mice significantly increased the yields of live chlamydial organisms on days 3 and 5, providing the 1st experimental evidence for a protective role of type-I interferon signaling in preventing C. trachomatis infection in mouse female genital tract. Further comparison of live C. trachomatis recovered from different genital tract tissues between wild type and IFNαR1-deficient mice revealed that the type-I interferon-dependent anti-C. trachomatis immunity was restricted to mouse lower genital tract. This conclusion was validated when C. trachomatis was inoculated transcervically. Thus, we have demonstrated an essential role of type-I interferon signaling in innate immunity against C. trachomatis infection in the mouse lower genital tract, providing a platform for further revealing the molecular and cellular basis of type-I interferon-mediated immunity against sexually transmitted infection with C. trachomatis.

Induction of Transmucosal Protection by Oral Vaccination with an Attenuated Chlamydia.

Chlamydia muridarum has been used to study chlamydial pathogenesis because it induces mice to develop hydrosalpinx, a pathology observed in C. trachomatis-infected women. We identified a C. muridarum mutant that is no longer able to induce hydrosalpinx. In the current study, we evaluated the mutant as an attenuated vaccine. Following an intravaginal immunization with the mutant, mice were protected from hydrosalpinx induced by wild-type C. muridarum. However, the mutant itself productively colonized the mouse genital tract and produced infectious organisms in vaginal swabs. Nevertheless, the mutant failed to produce infectious shedding in the rectal swabs following an oral inoculation. Importantly, mice orally inoculated with the mutant mounted transmucosal immunity against challenge infection of wild-type C. muridarum in the genital tract. The protection was detected as early as day 3 following the genital challenge infection and the orally immunized mice were protected from any significant pathology in the upper genital tract. However, the same orally immunized mice failed to prevent the colonization of wild-type C. muridarum in the gastrointestinal tract. The transmucosal immunity induced by the oral mutant was further validated in the airway. The orally vaccinated mice were protected from both lung infection and systemic toxicity caused by intranasally inoculated wild-type C. muridarum although the same mice still permitted the gastrointestinal colonization by the wild-type C. muridarum. These observations suggest that the mutant C. muridarum may be developed into an intracellular oral vaccine vector (or IntrOv) for selectively inducing transmucosal immunity in extra-gut tissues.

Chlamydia Deficient in Plasmid-Encoded Glycoprotein 3 (pGP3) as an Attenuated Live Oral Vaccine.

Despite the extensive efforts, there is still a lack of a licensed vaccine against Chlamydia trachomatis in humans. The mouse genital tract infection with Chlamydia muridarum has been used to both investigate chlamydial pathogenic mechanisms and evaluate vaccine candidates due to the C. muridarum's ability to induce mouse hydrosalpinx. C. muridarum mutants lacking the entire plasmid or deficient in only the plasmid-encoded pGP3 are highly attenuated in inducing hydrosalpinx. We now report that intravaginal immunization with these mutants as live attenuated vaccines protected mice from hydrosalpinx induced by wild type C. muridarum. However, these mutants still productively infected the mouse genital tract. Further, the mutant-infected mice were only partially protected against the subsequent infection with wild type C. muridarum. Thus, these mutants as vaccines are neither safe nor effective when they are delivered via the genital tract. Interestingly, these mutants were highly deficient in colonizing the gastrointestinal tract. Particularly, the pGP3-deficient mutant failed to shed live organisms from mice following an oral inoculation, suggesting that the pGP3-deficient mutant may be developed into a safe oral vaccine. Indeed, oral inoculation with the pGP3-deficient mutant induced robust transmucosal immunity against both the infection and pathogenicity of wild type C. muridarum in the genital tract. Thus, we have demonstrated that the plasmid-encoded virulence factor pGP3 may be targeted for developing an attenuated live oral vaccine.

Interleukin-27 (IL-27) Promotes Chlamydial Infection in the Female Genital Tract.

Intravaginal infection of mice with Chlamydia muridarum has been used for investigating the mechanisms of Chlamydia trachomatis-induced pathogenicity and immune responses. In the current study, the mouse model was used to evaluate the impact of interleukin-27 (IL-27) and its receptor signaling on the susceptibility of the female genital tract to chlamydial infection. Mice deficient in IL-27 developed significantly shortened courses of chlamydial infection in the female genital tract. The titers of live Chlamydia recovered from the genital tract of IL-27-deficient mice declined significantly by day 7 following intravaginal inoculation. These observations suggest that IL-27 may promote chlamydial infection in the female mouse genital tract. This conclusion was validated using IL-27 receptor (R)-deficient mice. Further, the reduction in chlamydial burden corelated with the increase in gamma interferon (IFN-γ) and IL-17 in the genital tract tissues of the IL-27R-deificent mice. However, depletion of IFN-γ but not IL-17 from the IL-27R-deificent mice significantly increased the chlamydial burden, indicating that IL-27 may mainly suppress IFN-γ-mediated immunity for promoting chlamydial infection. Finally, knockout of IL-27R from T cells alone was sufficient for significantly shortening the infectious shedding courses of Chlamydia in the mouse genital tract. The above-described results have demonstrated that Chlamydia can activate IL-27R signaling in Th1-like cells for promoting its infection in the female genital tract, suggesting that attenuating IL-27 signaling in T cells may be used for enhancing genital tract immunity against chlamydial infection.

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 first hit) and induce lymphocytes to promote hydrosalpinx indirectly (as a second 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 mice 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 naive C57BL/6J mice rescued the ability of recipient OT1 mice to develop hydrosalpinx when naive 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 Chlamydia infection, naive CD8+ T cells no longer promoted hydrosalpinx. Nevertheless, CD8+ T cells from mice immunized against Chlamydia 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 Chlamydia 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 optimized an OT1 mouse-based model for revealing the pathogenic mechanisms of Chlamydia-specific CD8+ T cells.