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.

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.

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.

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.

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.

Induction of transmucosal protection by oral vaccination with an attenuated Chlamydia.

Chlamydia muridarum has been used to study chlamydial pathogenesis since 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 challenge infection and the 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 intr acellular o ral v accine vector (or IntrOv) for selectively inducing transmucosal immunity in extra-gut tissues.

Regulation of chlamydial colonization by IFNγ delivered via distinct cells.

The mouse-adapted pathogen Chlamydia muridarum (CM) induces pathology in the mouse genital tract but fails to do so in the gastrointestinal tract. CM is cleared from both the genital tract and small intestine by IFNγ delivered by antigen-specific CD4+ T cells but persists for a long period in the large intestine. The long-lasting colonization of CM in the large intestine is regulated by IFNγ delivered by group 3 innate lymphoid cells (ILC3s). Interestingly, the ILC3-delivered IFNγ can inhibit the human pathogen Chlamydia trachomatis (CT) in the mouse endometrium. Thus, IFNγ produced/delivered by different cells may selectively restrict chlamydial colonization in different tissues. Revealing the underlying mechanisms of chlamydial interactions with IFNγ produced by different cells may yield new insights into both chlamydial pathogenicity and mucosal immunity.

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.

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.

Evidence for cGAS-STING Signaling in the Female Genital Tract Resistance to Chlamydia trachomatis Infection.

Sexually transmitted Chlamydia trachomatis can ascend to the upper genital tract due to its resistance to innate immunity in the lower genital tract. C. trachomatis can activate the cGAS-STING signaling pathway in cultured cells via either cGAS or STING. This study was designed to evaluate the role of the cGAS-STING pathway in innate immunity against C. trachomatis in the mouse genital tract. Following intravaginal inoculation, C. trachomatis significantly declined by day 5 following a peak infection on day 3, while the mouse-adapted Chlamydia muridarum continued to rise for >1 week, indicating that C. trachomatis is susceptible to the innate immunity in the female mouse genital tract. This conclusion was supported by the observation of a similar shedding course in mice deficient in adaptive immunity. Thus, C. trachomatis can be used to evaluate innate immunity in the female genital tract. It was found that mice deficient in either cGAS or STING significantly increased the yields of live C. trachomatis bacteria on day 5, indicating an essential role of the cGAS-STING signaling pathway in innate immunity of the mouse genital tract. Comparison of live C. trachomatis bacteria recovered from different genital tissues revealed that the cGAS-STING-dependent immunity against C. trachomatis was restricted to the mouse lower genital tract regardless of whether C. trachomatis was inoculated intravaginally or transcervically. Thus, we have demonstrated an essential role of the cGAS-STING signaling pathway in innate immunity against chlamydial infection, laying a foundation for further illuminating the mechanisms of the innate immunity in the female lower genital tract.

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.

The role of an enzymatically inactive CPAF mutant vaccination in Chlamydia muridarum genital tract infection.

Chlamydia trachomatis urogenital tract infection causes pelvic inflammatory disease and infertility, increases the risk of co-infection with HPV and HIV. Chlamydial vaccination is considered the most promising approach to prevent and control its infection. Among various chlamydial vaccine candidates, chlamydial protease-like activity factor (CPAF) have been reported to provide robust protective immunity against genital chlamydial infection in mice with reduced vaginal shedding and oviduct pathology. However, CPAF is a serine protease which has enzymatical activity to degrade a large number of substrates. In order to increase the safety of CPAF vaccine, in this study, we used a mutant CPAF that is deficient in enzymatical activity to determine whether proteolytic activity of CPAF affect its vaccine efficacy. The wild type or mutant CPAF immunization causes a significant lower chlamydial shedding from the vaginal and resolve the infection as early as day 20, compared to day 28 in adjuvant control mice. More important, reduced upper reproductive tract pathology were also observed in these two groups. The mutant or wild type CPAF immunization induced not only robust splenic IFN-γ and serum IgG2a but also sIgA secretion in the vaginal fluids. Furthermore, neutralization of chlamydia with immune sera did not provide protection against oviduct pathology. However, adoptive transfer of CD4+ splenocytes isolated from the mutant or wild type CPAF immunized mice resulted in a significant and comparable reduced oviduct pathology. Our results indicate mutant CPAF vaccination is as same efficacy as wild type, and the protection relies on CD4+ T cells, which will further promote the development of CPAF as clinical chlamydial vaccine.

Gastrointestinal Chlamydia-Induced CD8+ T Cells Promote Chlamydial Pathogenicity in the Female Upper Genital Tract.

Chlamydia is known to both ascend to the upper genital tract and spread to the gastrointestinal tract following intravaginal inoculation. Gastrointestinal Chlamydia was recently reported to promote chlamydial pathogenicity in the genital tract since mice intravaginally inoculated with an attenuated Chlamydia strain, which alone failed to develop pathology in the genital tract, were restored to develop hydrosalpinx by intragastric coinoculation with wild-type Chlamydia. Gastrointestinal Chlamydia promoted hydrosalpinx via an indirect mechanism since Chlamydia in the gut did not directly spread to the genital tract lumen. In the current study, we further investigated the role of CD8+ T cells in the promotion of hydrosalpinx by gastrointestinal Chlamydia. First, we confirmed that intragastric coinoculation with wild-type Chlamydia promoted hydrosalpinx in mice that were inoculated with an attenuated Chlamydia strain in the genital tract 1 week earlier. Second, the promotion of hydrosalpinx by intragastrically coinoculated Chlamydia was blocked by depleting CD8+ T cells. Third, adoptive transfer of gastrointestinal Chlamydia-induced CD8+ T cells was sufficient for promoting hydrosalpinx in mice that were intravaginally inoculated with an attenuated Chlamydia strain. These observations have demonstrated that CD8+ T cells induced by gastrointestinal Chlamydia are both necessary and sufficient for promoting hydrosalpinx in the genital tract. The study has laid a foundation for further revealing the mechanisms by which Chlamydia-induced T lymphocyte responses (as a 2nd hit) promote hydrosalpinx in mice with genital Chlamydia-triggered tubal injury (as a 1st hit), a continuing effort in testing the two-hit hypothesis as a chlamydial pathogenic mechanism.

Chlamydia Spreads to the Large Intestine Lumen via Multiple Pathways.

Chlamydia in the genital tract is known to spread via the blood circulation system to the large intestine lumen to achieve long-lasting colonization. However, the precise pathways by which genital Chlamydia accesses the large intestine lumen remain unclear. The spleen was recently reported to be critical for chlamydial spreading. In the current study, it was found that following intravaginal inoculation with Chlamydia, mice with and without splenectomy both yielded infectious Chlamydia on rectal swabs, indicating that the spleen is not essential for genital Chlamydia to spread to the gastrointestinal tract. This conclusion was validated by the observation that intravenously inoculated Chlamydia was also detected on the rectal swabs of mice regardless of splenectomy. Careful comparison of the tissue distribution of live chlamydial organisms following intravenous inoculation revealed redundant pathways by which Chlamydia can reach the large intestine lumen. The intravenously inoculated Chlamydia was predominantly recruited to the spleen within 12 h and then detected in the stomach lumen by 24 h, in the intestinal lumen by 48 h, and on rectal swabs by 72 h. These observations suggest a potential spleen-to-stomach pathway for hematogenous Chlamydia to reach the large intestine lumen. This conclusion was supported by the observation made in mice under coprophagy-free condition. However, in the absence of spleen, hematogenous Chlamydia was predominantly recruited to the liver and then simultaneously detected in the intestinal tissue and lumen, suggesting a potential liver-to-intestine pathway for Chlamydia to reach the large intestine lumen. Thus, genital/hematogenous Chlamydia may reach the large intestine lumen via multiple redundant pathways.

Chlamydia overcomes multiple gastrointestinal barriers to achieve long-lasting colonization.

Chlamydia trachomatis (CT) is frequently detected in the human gastrointestinal (GI) tract despite its leading role in sexually transmitted bacterial infections in the genital tract. Chlamydia muridarum (CM), a model pathogen for investigating CT pathogenesis in the genital tract, can also colonize the mouse GI tract for long periods. Genital-tract mutants of CM no longer colonize the GI tract. The mutants lacking plasmid functions are more defective in colonizing the upper GI tract while certain chromosomal gene-deficient mutants are more defective in the lower GI tract, suggesting that Chlamydia may use the plasmid for promoting its spread to the large intestine while using the chromosome-encoded factors for maintaining its colonization in the large intestine. The plasmid-encoded Pgp3 is critical for Chlamydia to resist the acid barrier in the stomach and to overcome a CD4+ T cell barrier in the small intestine. On reaching the large intestine, Pgp3 is no longer required. Instead, the chromosome-encoded open reading frames TC0237/TC0668 become essential for Chlamydia to evade the group 3-like innate lymphoid cell-secreted interferon (IFN)γ in the large intestine. These findings are important for exploring the medical significance of chlamydial colonization in the gut and for understanding the mechanisms of chlamydial pathogenicity in the genital tract.

Regulation of Chlamydia spreading from the small intestine to the large intestine via an immunological barrier.

The obligate intracellular bacterium Chlamydia is a genital tract pathogen that can also colonize the gastrointestinal tract for long periods. The long-lasting colonization is dependent on chlamydial spreading from the small intestine to the large intestine. We previously reported that a mutant Chlamydia was able to activate an intestinal barrier for blocking its own spreading to the large intestine. In the current study, we used the mutant Chlamydia colonization model to confirm the intestinal barrier function and further to determine the immunological basis of the barrier with gene-deficient mice. Recombination activating gene 1-/- mice failed to block the mutant Chlamydia spreading, while mice deficient in toll-like receptors, myeloid differentiation primary response 88 or stimulator of interferon genes still blocked the spreading, suggesting that the intestinal barrier function is dependent on lymphocytes that express antigen receptors. Mice deficient in CD4, but not CD8 nor µ chain failed to prevent the chlamydial spreading, indicating a protective role of CD4+ cells in the intestinal barrier. Consistently, adoptive transfer of CD4+ T cells reconstituted the intestinal barrier in CD4-/- mice. More importantly, CD4+ but not CD8+ T cells nor B cells restored the intestinal barrier function in recombination activating gene 1-/- mice. Thus, CD4+ T cells are necessary and sufficient for maintaining the intestinal barrier function, indicating that the spread of an intracellular bacterium from the small intestine to the large intestine is regulated by an immunological barrier. This study has also laid a foundation for further illuminating the mechanisms by which a CD4+ T cell-dependent intestinal barrier regulates bacterial spreading in the gut.

Effectiveness of Platelet-Rich Plasma in the Prevention of Chlamydia-Induced Hydrosalpinx in a Murine Model.

Chlamydia trachomatis (C. trachomatis) is a major pathogen implicated in the formation of hydrosalpinx in the female reproductive tract. In mice, a related strain of Chlamydia, Chlamydia trachomatis (C. trachomatis) can induce almost 100% bilateral hydrosalpinx. This model was used as a hydrosalpinx induction model to test whether oviduct delivery of platelet-rich plasma (PRP) can attenuate chlamydia induction of hydrosalpinx in a mouse model. Mice were infected intravaginally with Chlamydia muridarum organisms, and 21 days after the infection, PRP was instilled into the lumen of one oviduct, and a sham instillation with phosphate buffer solution was performed on the contralateral oviduct. Mice were then sacrificed at designated time points after infection for oviduct pathologic evaluation including incidence, severity, and histopathologic grade of chronic inflammation. Oviduct instillation of PRP was associated with a 36% reduction in the incidence of hydrosalpinx and a 33% reduction in severity compared with sham. The median grade of chronic inflammation on histopathology was significantly lower with PRP instillation compared with sham and control. No differences were observed in vaginal or rectal shedding of C. muridarum between the test group and the control group. In short, the results suggest that oviduct instillation of PRP can significantly reduce the incidence and severity of C. muridarum-induced hydrosalpinx without affecting chlamydial infection courses in CBA/J mice.

Adoptive Transfer of Group 3-Like Innate Lymphoid Cells Restores Mouse Colon Resistance to Colonization of a Gamma Interferon-Susceptible Chlamydia muridarum Mutant.

The obligate intracellular bacterium Chlamydia muridarum can colonize the mouse colon for a long period, but a gamma interferon (IFN-γ)-susceptible mutant clone fails to do so. Nevertheless, the mutant's colonization is rescued in mice deficient in interleukin-7 receptor (IL-7R) (lacking both lymphocytes and innate lymphoid cells [ILCs]) or IFN-γ but not in mice lacking recombination-activated gene 1 (Rag1-/- mice) (lacking adaptive immunity lymphocytes), indicating a critical role of ILC-derived IFN-γ in regulating chlamydial colonization. In the current study, we have used an adoptive transfer approach for further characterizing the responsible ILCs. First, intestinal ILCs isolated from Rag1-/- mice were able to rescue IL-7R-deficient mice to restrict the colonization of the IFN-γ-susceptible Chlamydia muridarum mutant. Second, the responsible ILCs were localized to the intestinal lamina propria since ILCs from the lamina propria but not the intraepithelial compartment conferred the restriction. Third, lamina propria ILCs enriched for RORγt expression but not those negative for RORγt rescued the IL-7R-deficient mice to restrict mutant colonization, indicating a critical role of group 3-like ILCs (ILC3s) since RORγt is a signature transcriptional factor of ILC3s. Fourth, a portion of the ILC3s expressed IFN-γ, thus defined as ex-ILC3s, and the transfer of the ex-ILC3s conferred colon resistance to mutant Chlamydia muridarum colonization in IFN-γ-deficient mice. Finally, genetically labeled RORγt-positive (RORγt+) ILCs were able to inhibit mutant colonization. Thus, we have demonstrated that ILC3s are sufficient for regulating chlamydial colonization, laying a foundation for further revealing the mechanisms by which an obligate intracellular bacterium activates colonic ILC3s.