Regulation of chlamydial colonization by IFNγ delivered via distinct cells

Abstract

The mouse-adapted pathogen Chlamydia muridarum induces pathology in the mouse genital tract but fails to do so in the gastrointestinal tract. C. muridarum 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 C. muridarum in the large intestine is regulated by IFNγ delivered by group 3 innate lymphoid cells (ILC3s). Interestingly, the ILC3s-delivered IFNγ can inhibit the human pathogen C. trachomatis 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.

1. Chlamydia is a professional colonizer of mucosal tissues

1a. Chlamydial infection of mucosal tissues in the eye and urogenital tract

The medical significance of investigating Chlamydia trachomatis (CT) lies in CT’s ability to infect both human ocular and genital mucosal tissues. Ocular infection with CT causes trachoma that may lead to preventable blindness [1] while the infection of urogenital tract with CT is a leading cause of sexually transmitted bacterial infections [2]. While the health care burden caused by trachoma has been greatly reduced in most parts of the world [3], the rates of CT-caused sexually transmitted infection continue to climb (https://www.cdc.gov/std/statistics/2020/default.htm).

While the precise mechanisms of CT pathogenesis remain unclear, the mouse-adapted C. muridarum (CM) has been used for investigating CT pathogenesis in the genital tract because intravaginal inoculation with CM can induce pathology in the upper genital tract similar to those observed in CT-infected women [4, 5]. Studies based on the mouse genital tract infection with CM model have revealed pathogenic roles of numerous chlamydial factors with some encoded by chlamydial plasmid [6] while others in the chlamydial chromosome [7–9]. For example, a CM clone with loss-of-function mutations in chromosome-encoded hypothetical open reading frames TC0237/TC0668, which is designated as G28.51.1 or IntrOV (intracellular oral delivery vehicle), is both attenuated in pathogenicity in the mouse genital tract and defective in colonizing the gastrointestinal (GI) tract [8, 10–13].

1b. Chlamydia can infect other mucosal tissues and undergo systemic spreading

Besides the ocular and urogenital mucosal tissues, CT also infects the mucosal tissues in the airway and GI tract. For example, neonatal pneumonia caused by CT via vertical transmission from the mother’s birth canal has been reported [14] and CT-associated pneumonia is frequently diagnosed in children [15, 16]. More importantly, CT is routinely detected in the human GI tract [17, 18] although it remains unknown whether CT in the GI tract is detrimental or beneficial to human health. What is known is that no significant gut pathologies have been associated with any CT strains that are non-lymphogranuloma venereum (LGV) [19]. Nevertheless, CT in the GI tract has been proposed to serve as a reservoir to seed repeated infections in the genital tract [20]. However, there is no experimental evidence supporting this hypothesis, although it is conceivable that GI CT may be passed onto the genital tract via human behavior. In mice, GI CM fails to spread to the genital tract despite its long-lasting colonization in the GI tract [21]. On the contrary, mouse genital CM can spread to the GI tract [22] via systemic pathways [23–25]. It is worth pointing out that the transient systemic dissemination followed by inoculation with CM at any mucosal site described by Perry et al [26] is distinct from the selective spreading of CM to the lumen of the GI tract [22, 25]. Upon mucosal inoculation with CM, CM enters the blood circulation system and disseminates systemically. Live CM organisms can be recovered from tissue homogenates of many different organs for about 2 weeks. However, when swabs are taken from different mucosal lumens for monitoring chlamydial burden, no live organisms can be detected in the swabs from mucosal tissues other than the initial inoculation site and the GI tract. Thus, although hematogenous chlamydial organisms are able to reach all mucosal tissues, they may fail to cross the mucosal barriers to cause lumenal infection. Two weeks later, the systemic infection is cleared. However, live CM continues to be detected in the lumen of the inoculation site and the GI tract, suggesting that the hematogenous CM is capable of selectively entering the gut lumen during the bacteremia phase [23]. The questions on how and where the hematogenous CM crosses the gut mucosal barrier to reach the lumen remain to be answered. It is possible that hematogenous CM may first exit the blood circulation system to reach the gut lamina propria and then cause basolateral infection of enterocytes. After intracellular replication, the progeny infectious CM elementary bodies may be released into the epic side of the gut epithelial cells. This basolateral infection and epic release model may explain how hematogenous CM uses the spleen-stomach route to reach the lumen of the GI tract [25]. Alternatively, hematogenous CM may also use a liver-bile duct-intestine pathway to reach the gut lumen. Although the liver-bile duct-intestine pathway is obvious, it is still unclear why hematogenous CM selectively crosses the gastric mucosal epithelial layer to reach the lumen of stomach but not the other mucosal organs. Even though hematogenous CM can enter the lumen of GI tract via two routes, the spreading is considered highly selective and probably tightly regulated. This is because examples of hematogenous pathogens entering the gut lumen are rare. For example, hematogenous hookworm is known to exit into the airway lumen first and then access to the lumen of the GI tract via coughing and swallowing (https://www.cdc.gov/parasites/hookworm/biology.html). Regardless how the hematogenous CM spreading into the lumen of GI tract is regulated, the spreading has at least two obvious consequences: First, once CM reaches the lumen of the GI tract, it can colonize the colon for long periods [21–23], which may serve as a reservoir for seeding repeated infections elsewhere possibly via host behavior-dependent pathways. Second, after spreading from the genital lumen to the lumen of GI tract, CM may induce profibrotic immune responses that may exacerbate the pathogenicity of CM in the genital tract [27–29]. The profibrotic response is likely mediated via CM antigen-specific CD8+ T cells [28, 30]. It is worth pointing out that despite its long-lasting colonization in the GI tract, CM does not cause any significant pathology to the GI tract [21]. Surprisingly, when a naïve mouse is orally inoculated with CM first, the GI CM induces transmucosal protection against subsequent chlamydial infection in both the genital tract [31] and the airway [32]. Thus, depending on the order of mucosal tissue exposure to Chlamydia, the GI Chlamydia may either promote or prevent chlamydial pathogenicity in the genital tract. It is worth investigating whether exposure of CT in the GI tract of neonates during birth delivery can also function as oral vaccination, resulting in protective immunity against subsequent CT infection in extra-gut mucosal tissues in their later lives. This hypothesis is consistent with the fact that there is no reported association of neonatal GI pathology with CT despite the emergence of CT-associated ocular and airway pathology in neonates.

1c. Chlamydia has developed distinct adaptation/fitness levels with different mucosal tissues

Chlamydial infection of mucosal tissue in the airway often causes acute pneumonia, which is cleared without significant long-term sequelae. This appears to be true with both CT infection in humans and CM in mice. Although CT has been reported to cause pneumonia in neonates [14] and children [16], there is no significant association of CT with chronic pathology in the airway. Similarly, when a sufficient dose of CM is intranasally inoculated into mice, acute pneumonia is induced. However, mice can completely recover in 2 to 3 weeks. Upon intranasal inoculation, the lung burden of live CM peaks in a week followed by complete clearance of live CM by 2 to 3 weeks. However, intranasal inoculation with a high dose of CM is lethal and mice often die within 2 weeks, indicating limited adaptation or fitness between Chlamydia and the airway.

Chlamydial colonization in the genital mucosal tissues of women and female mice is eventually cleared but may still induce inflammation that may lead to long-lasting pathology. Most women suffering from CT-associated tubal infertility are no longer positive for CT nucleic acids in their vaginal swabs although their immune responses to CT remain detectable [33]. Similarly, mice that are induced to develop hydrosalpinx by genital inoculation with CM no longer harbor live CM in the genital tract tissues by the time when hydrosalpinx is examined [4]. Live CM is often cleared from the genital tract in 4 to 5 weeks (comparing to the clearance of live CM from the airway within 2 to 3 weeks). The gradual clearance of chlamydial infection and the tissue pathology left behind suggest that these organisms have partially adapted to this tissue environment. Thus, Chlamydia may have achieved partial fitness with genital mucosal tissues.

On the contrary, CM colonization in the mouse GI tract is long-lasting but nonpathogenic [21, 31]. Although it is still unknown whether CT colonizes human GI tract for a long period, there is no reported association of significant GI tract pathology with non-LGV serovars of CT [18]. Consistently, although CT has been reported to cause both neonatal conjunctivitis and pneumonia, there is no report on neonatal GI tract diseases caused by the vertically transmitted CT although the chance of the birth canal CT to enter the GI tract of a neonate would be similar to that of the CT exposure to the neonatal ocular and airway mucosal tissues. The nonpathogenic colonization in the GI tract suggests that Chlamydia has fully adapted to and achieved complete fitness with the GI tract mucosal tissues.

A series of recent studies have provided new insights into the chlamydial interactions with mucosal tissues in the GI tract [34]. Following an oral inoculation, CM is cleared from the stomach within a week [35] and from the small intestine within a month [36] but persists in the large intestine for a much longer period [21]. The long-lasting colonization is mainly restricted to the large intestine. The above tissue distribution pattern of Chlamydia in the GI tract is shared by many gut microbiota species, which has led to the hypothesis that Chlamydia may be a commensal species in the GI tract. Thus, the CM-mouse gut interaction model may be used to address the long-standing question on how microbiota species maintain their long-lasting colonization in host tissue without causing significant pathology. When mutant CM clones and gene knockout mice are used to identify chlamydial and host factors involved in chlamydial interactions with different regions of the GI tract, it is revealed that chlamydial plasmid genes are required for chlamydial survival in the stomach and small intestine [35, 37–40] while some chlamydial chromosomal genes required for chlamydial colonization in the large intestine [10, 12]. Both innate [11, 13] and adaptive [36] immune responses may regulate CM colonization in the GI tract.

1d. Chlamydia may have acquired the capability of invading mucosal tissues during its adaptation to the GI tract.

It is believed that the strategies developed by Chlamydia to colonize the GI tract may make it a successful sexually transmitted agent in the genital tract. Although in humans the relationship between the GI CT and genital CT remains unknown, in mice, the oral-fecal route is more efficient than genital mucosal contact in transmitting CM, allowing CM to adapt to the GI tract more efficiently than the genital tract. Consistently, CM virulence factors identified so far are more important for CM to colonize the GI tract than the genital tract, supporting the idea that the virulence factors of CM are initially selected for adaption to GI mucosal tissues. The urinary pathogenic E. coli may have acquired its skills to persist in the urinary tract from its interactions with the GI tract [41], which serves as another example of the general concept that the GI tract-adapted microbes are enabled to infect extra-gut mucosal tissues.

Although Chlamydia may have well adapted to the GI tract mucosal tissues, it is still recognized as a stress by extra-gut mucosal tissues. This is probably due to the unique environmental cues confronted by different mucosal tissues, which drives different mucosal tissues to develop different mechanisms. Thus, while the gut-adapted Chlamydia can infect extra-gut mucosal tissues, it does not share the same level of fitness as it does with the GI tract. The environmental cues received by the airway may be dramatically different from those confronted by the GI tract. As a result, CT and CM can cause acute pneumonia in humans and mice respectively, resulting in rapid clearance of Chlamydia by the hosts that survive the infection. In contrast, the environmental cues received by the urogenital and GI mucosal tissues are likely similar (but not identical). The GI tract-adapted Chlamydia may find partial fitness in the genital tract. As a result, CT and CM can persist longer in the genital mucosal tissues of humans and mice respectively although chronic pathology is left behind. Uncovering the underlying mechanisms involved in chlamydial interactions with different mucosal tissues, particularly the GI tissues versus the genital tissues, may provide novel insights into both chlamydial pathogenicity and mucosal immunity.

2. The role of IFNγ in host defense against chlamydial infection

IFNγ is a powerful and multi-functional cytokine that participates in multiple biological processes [42], including host defense against microbial infections [43]. The role of IFNγ in host defense against chlamydial infection can be directly demonstrated in cultured cells [44]. Cells treated with IFNγ prior to or during chlamydial infection significantly restrict chlamydial replication. The cell culture finding has been reproduced in mice. Neutralization of endogenous IFNγ increased mouse susceptibility to intravenous CT infection while the supplementation of exogenous IFNγ decreased mouse susceptibility [44–46]. The anti-chlamydial activity of IFNγ has been reproducibly demonstrated in a more medically relevant intravaginal infection with CM model [47]. Although wild type mice can clear CM infection from the genital tract within ~1 month following an intravaginal inoculation [4], mice deficient in either IFNγ or IFNγ receptor are no longer able to control CM infection in the genital tract. Interestingly, in a mouse small intestine colonization model, CM is also cleared from the small intestine within 1 month following an oral inoculation [21] but IFN-γ deficiency significantly prolongs the chlamydial persistence in the small intestine [36]. The role of IFNγ in controlling chlamydial infection in the airway or other mucosal tissues has also been demonstrated in mice [48, 49]. There is a strong correlation of IFNγ with the control of CT infection in women [50].

Extensive efforts have been made to reveal the mechanisms by which IFNγ inhibits Chlamydia [51–54]. IFNγ receptor-triggered signaling can lead to both cell-intrinsic or autonomous responses such as phago-lysosomal fusion and autophagy, or cell-extrinsic responses that are dependent on the production of new proteins like cytokines, chemokines, and antimicrobial peptides. Both mechanisms are important for IFNγ to control chlamydial infection. IFNγ-enhanced small GTPase activation [55, 56], IFNγ-induced modification of chlamydial inclusion membrane [57] and IFNγ-driven autophagy [53] responses have been shown to reduce chlamydial infectivity. Further, IFNγ-induced expression of indoleamine-2,3-dioxygenase (IDO) may be inhibitory to chlamydial growth by starving for tryptophan [58] while IFNγ-enhanced expression of chemokines and antimicrobial peptides have been shown to restrict chlamydial infection [59]. A recent study has shown that antibody-dependent immunity against genital CM is also facilitated by IFNγ [54] since IFNγ is necessary for activation of the effector cell population that functions in antibody-mediated immunity against CM in mice. However, more studies are required to reveal the precise mechanism.

3. Chlamydial evasion of IFNγ immunity

Since IFNγ-mediated immunity is effective in clearing chlamydial infection, Chlamydia has in turn evolved strategies to evade the same immunity in order to survive in the infected host cells. CM has been shown to evade growth restriction imposed by IFNγ-induced irgb10 [60] while CT is able to resist inclusion ubiquitination induced by IFNγ in human epithelial cells [61]. However, it is not clear what mechanisms enable Chlamydia to evade IFNγ-mediated immunity. As the genetic manipulation of Chlamydia has become easier [62, 63], chlamydial mutagenesis libraries have been used for identifying chlamydial virulence factors [64]. For example, the Nelson group has recently screened a CM temperature sensitive mutagenesis library for susceptibility to IFNγ treatment, which has identified 31 IFNγ-sensitive mutants. One of the mutants contains a missense mutation in a putative chlamydial inclusion membrane protein, TC0574 [65]. Surprisingly, this mutant still maintains resistance to IFNγ-induced cell autonomous mechanisms but is no longer able to prevent apoptosis of IFNγ-treated host cells, which Chlamydia is known to prevent [66]. Thus, it is likely that TC0574 may promote chlamydial resistance to IFNγ treatment by inhibiting host cell apoptosis. Further characterization of the remaining temperature sensitive mutants that are IFNγ-susceptible should reveal novel chlamydial strategies for evading IFNγ immunity.

Chen et al has reported another approach for identifying chlamydial genes required for chlamydial resistance to immunity in vivo [7]. This approach is based on repeated in vitro passaging of CM in HeLa cell cultures, which allows CM organisms to accumulate mutations in genes that are nonessential for chlamydial replication in HeLa cells. Since these in vitro non-essential genes may be required for chlamydial resistance to mucosal immunity in mice, the corresponding CM mutants may no longer be able to colonize a given mucosal tissue in mice. Whole genome sequencing of the mutants has identified several prominent mutations including those in hypothetical genes tc0237 and tc0668. When a clone that carries loss of function mutations in both tc0237 and tc0668 genes (the clone was designated as G28.51.1 or IntroV) was compared with its isogenic control clones in mice, it was found that G28.51.1 was not only highly attenuated in pathogenicity in the upper genital tract [8] but also deficient in colonizing the GI tract [10]. Nevertheless, G28.51.1 still maintained significant colonization in the genital tract. This led to the hypothesis that CM colonization in the GI tract may contribute to chlamydial pathogenicity in the genital tract, laying the foundation for the so called two-hit model [29]. The deficiency of G28.51.1 in colonizing the GI tract was further mapped to the large intestine. Since G28.51.1 was rescued to colonize the large intestine of mice deficient in IFNγ [11, 12], this clone has been designated as an IFNγ-susceptible mutant. Both CT0237 and TC0668 have been hypothesized to promote chlamydial survival in the large intestine by helping CM evade IFNγ immunity.

IFNγ-susceptible mutants are useful tools for both identifying chlamydial factors responsible for evading IFNγ immunity and characterizing the IFNγ immunity, including revealing the ontology of the cells responsible for delivering IFNγ to Chlamydia-infected cells in different host mucosal tissues.

4. IFNγ required for inhibiting chlamydial infection at different mucosal tissue sites is delivered by different cells.

While IFNγ plays a large part in anti-chlamydial immunity, IFNγ is produced by multiple cell types. It will be interesting to determine the cell types required for most efficiently delivering IFNγ to combat chlamydial infections in different mucosal tissues. During innate immunity NK cells, groups 1 and 3 innate lymphoid cells (ILC1s & ILC3s), NK T cells and gamma/delta T cells are the major sources of IFNγ. During adaptive immunity the major IFNγ-producing cells are CD4+ T cells, although other lymphocytes such as CD8+ T cells and B cells may also produce IFNγ. It is conceivable that IFNγ produced by innate immunity cells plays a dominant role during the early phase of infection while IFNγ produced by adaptive immunity cells plays a dominant role during the late phase of the infection. However, it is not clear whether different IFNγ-producing cells are preferred for delivering IFNγ to Chlamydia-infected cells in different mucosal tissues.

During CM infection in the female mouse genital tract, the shedding of live CM continues to rise and peaks on days 7–14 after infection and the entire infection course lasts about one month. This indicates that CM can successfully evade the innate immunity regardless of whether IFNγ is produced by innate cells. However, the final clearance of CM infection from the genital tract is dependent on IFNγ since mice deficient in IFNγ but not any other cytokines tested failed to clear the infection. The responsible IFNγ appears to be produced by CD4+ T cells since mice deficient in either MHC class II or CD4+ T cells (but not MHC class I, CD8+ T cells or B cells) largely reproduced the phenotype of mice deficient in IFNγ [67, 68]. By using mice deficient in IFNγ from CD4+ T cells only, a recent study has shown that CD4+ T cells incapable of producing IFNγ can still offer significant protection against CM infection in the female genital tract [69], suggesting that besides IFNγ, CD4+ T cells may produce other effector molecules for inhibiting CM in the female genital tract. This conclusion is consistent with an early observation that genital immunity to CM is dependent CD4+ T cells through both IFNγ-dependent and -independent pathways [70]. However, a recent study has revealed that during primary infection with CM, the IFNγ-producing CD4+ T cells or Th1 cells are dispensable for the clearance of CM from female mouse reproductive tract. This is because mice lacking either T-bet-expressing CD4+ T cells or IFNγ are still able to significantly reduce CM burden [71]. This observation may suggest that CM can evade IFNγ delivered by CD4+ T cells during primary infection. Nevertheless, more studies are required for identifying the IFNγ-independent mechanisms responsible for clearing CM primary infection.

In the GI tract CM can only colonize the stomach for ~1 week [35] and the small intestine for ~1 month [36]. Only CM that reaches the large intestine can persist for months [21]. The clearance of CM from the stomach is dependent on gastric acid but not IFNγ or any other known immune mechanisms. However, the clearance of CM from the small intestine requires IFNγ secreted by CD4+ T cells since mice deficient in α/β T cells or CD4⁺ T cells, but not CD8⁺ T cells, can significantly prolong CM infection in the small intestine [36]. Consistently, IFN-γ-producing CD4⁺ but not CD8⁺ T cells from immunized donor mice are sufficient for eliminating CM from the mouse small intestine. Thus, IFN-γ-producing CD4⁺ T cells are both necessary and sufficient for clearing CM from the small intestine.

The next question is why the IFNγ-producing CD4⁺ T cells that are capable of clearing CM infection from the small intestine within 30 days of the infection, fail to terminate chlamydial infection in the large intestine in the same mice. Is it possible that the IFNγ-producing CD4⁺ T cells may not be able to deliver IFNγ to CM-infected cells in the large intestine? This hypothesis is consistent with the knowledge that the large intestine maintains high levels of regulatory T cells [72]. Using the CM-large intestine interaction model, recent studies revealed CM interactions with IFNγ-producing ILC3s (see next section for detail).

The role of IFNγ in preventing systemic dissemination of CM has long been recognized. To identify the cellular origin of IFNγ, Poston et al [73] used a spread-deficient mutant (CM001) in combination with knockout mice. It was found that the responsible IFNγ was not produced by T cells but required B cell cooperation. However, the precise cellular source remains unknown. A recent study suggests that helper ILC1s and killer ILC1 NK cells may be required for producing the IFNγ for limiting the systemic spreading of CM [69]. In the mouse airway infected with CM, IFNγ produced by both CD4+ T cells and NK cells are necessary for protection [48, 74]. The role of IFNγ produced by NK cells has also been demonstrated in the mouse genital tract [59].

5. ILC3s provide a unique source of IFNγ for interacting with Chlamydia-infected mucosal cells in both the large intestine and endometrium

5a. IFNγ⁺ILC3s are the main source of IFNγ responsible for interacting with CM in the large intestine

ILCs are recognized as critical components of mucosal immunity [75]. ILC3s, expressing RORγt and IL-17/22 as their signature transcriptional factor and cytokines, respectively, can respond to diverse signals from microbes, food, and host signals via different receptors. A unique feature of ILC3s is its flexibility in differentiating into IFNγ-producing cells, to become IFNγ⁺ILC3s or ex-ILC3s [76]. Different microbes may induce IFNγ⁺ILC3s via distinct mechanisms [77].

Using the IFNγ-susceptible mutant clone G28.51.1, it was determined that ILC3s were the major cellular source of IFNγ in the GI tract [34]. Following intracolon inoculation into Rag1 knockout mice, G28.51.1 is still prevented from colonization, suggesting that the IFNγ responsible for inhibiting G28.51.1 is likely produced by innate immune cells. Indeed, depletion of IFNγ from Rag1 knockout mice or mice deficient in both Rag2 (hence lacking conventional lymphocytes) and IL-2Rcγ (IL-2 receptor common γ chain, hence lacking all lymphoid cells including ILCs) fully restored colonization of G28.51.1 [11]. Thus, ILCs are the cellular source of IFNγ responsible for inhibiting G28.51.1. Since mice deficient in RORγt, a signature transcriptional factor of ILC3s, fully restored colonization of G28.51.1 [11, 12], it was concluded that the IFNγ responsible for inhibiting G28.51.1 was most likely produced by ILC3s. Consistently, adoptive transfer of intestinal lamina propria ILCs enriched for RORγt expression from donor mice restored the recipient mice deficient in IL-7R or IFNγ to inhibit G28.51.1 [13]. When genetically labeled RORγt+ILC3s were used as donor cells, the colonization of G28 was completely prevented in the large intestine of the recipient mice deficient in IFNγ. Thus, IFNγ-producing ILC3s are both necessary and sufficient for interacting with Chlamydia in the mouse large intestine.

5b. IFNγ⁺ILC3s are necessary for inhibiting CT in the mouse endometrium.

Sexually transmitted CT ascends to infect endometrium, leading to pathology in the upper genital tract. IFNγ+ILC3s have been recently shown to increase endometrial resistance against CT infection [78]. When CT was inoculated into mouse endometrium via a transcervical inoculation, live CT recovered from vaginal swabs or endometrial tissues peaked on day 3 and then declined in mice with or without deficiency in adaptive immunity. Interestingly, removal of IL-2 receptor common gamma chain (IL-2Rγc) from adaptive immunity-deficient mice significantly compromised the endometrial resistance to CT infection, indicating a critical role of innate lymphoid cells (ILCs) in controlling CT infection. Furthermore, mice deficient in RORγt or T-bet became more susceptible to endometrial infection with CT, suggesting that ILC3s are involved in the endometrial innate immunity against CT. Thus, IFNγ+ILC3s likely mediate the endometrial immunity against CT infection. This conclusion is supported by the observation that depletion of NK1.1+ cells from adaptive immunity-deficient mice both significantly reduced IFNγ and increased CT burden in the endometrial tissue.

While the above experiments have demonstrated a functional requirement for IFNγ+ILC3s to control CT infection in the endometrium, it remains unknown whether adoptive transfer of IFNγ+ILC3s can increase endometrial resistance to CT infection. This experiment may be technically challenging since the number of IFNγ+ILC3s is extremely low in the female genital tract, with ~500 cells in total [79]. Alternatively, it will be interesting to test whether transfer of intestinal IFNγ+ILC3s can enhance endometrial resistance to CT.

6. Concluding remarks

IFNγ has been investigated extensively for its anti-chlamydial activity. Chlamydia is known to infect multiple mucosal tissues and IFNγ is produced by multiple types of cells. In the current manuscript, we have attempted to address whether different cell types are required for delivering IFNγ to Chlamydia-infected cells in different mucosal tissues. IFNγ+ILC3s play a critical role in regulating CM colonization in the large intestine and inhibiting CT infection in the endometrium while CD4+ Th1-produced IFNγ may be responsible for eventually clearing CM infection in the small intestine or the female genital tract. IFNγ produced by circulating cells such as CD4+ T cells, NK cells and NKT cells is essential for preventing chlamydial systemic spreading. The IFNγ-producing cells may render IFNγ with the ability to selectively target chlamydial infection in different tissues. Thus, IFNγ-producing cells may be induced/enhanced therapeutically for attenuating chlamydial pathogenicity. It is worth pointing out that the mouse model-based findings described above may not necessarily be applicable to humans. Nevertheless, the mechanisms learnt from mouse studies may provide information for guiding the design of clinical studies. It will be worth investigating whether induction of IFNγ+ILC3s can enhance endometrial resistance to and/or improve vaccine efficacy against sexually transmitted CT infection in women [80].

Outstanding questions.

1. What is the relationship between the varied chlamydial fitness levels in different mucosal tissues and the chlamydial evasion of IFNγ delivered by different cell types? Does evasion of both IFNγ+ILC3s and antigen-specific IFNγ+T cells indicate complete fitness?

2. Why is the inhibition of the IFNγ-susceptible CM mutant in the large intestine dependent on IFNγ produced by ILC3s but not ILC1s, NKs or T cells? The regulatory T cells (Tregs) may selectively suppress the function of T cells. How do the Tregs differentiate ILC1s from ILC3s? Can ILC3s access to Chlamydia-infected cells more efficiently or produce co-factors that synergize with IFNγ for more efficient inhibition of Chlamydia?

3. IFNγ+ILC3s are effective against CT in mouse endometrium. What is the role of IFNγ+ILC3s in regulating CT infection in other parts of the female mouse genital tract? Can IFNγ+ILC3s also inhibit CT infection in the genital tract of women?

4. Besides IFNγ, the role of other effectors in clearing chlamydial infection in the female genital tract has recently been recognized. How to separate the overlapping effector mechanisms delivered by different lymphoid cells? Mice with cell type-specific knockout or overexpression of a target molecule are useful tools.

Highlights.

1. Chlamydia invades multiple mucosal tissues of humans and animals. IFNγ is the most potent cytokine effector that inhibits chlamydial infection in various mucosal tissues, which has driven Chlamydia to evolve genes for evading IFNγ-mediated immunity. It is hypothesized that IFNγ required for inhibiting chlamydial infection in different mucosal tissues is delivered by different types of cells.

2. Chlamydia displays varied adaptation/fitness levels with different mucosal tissues, with limited fitness in the airway, partial fitness in the genital tract, and complete fitness in the gut. Within the mouse gastrointestinal (GI) tract, the complete fitness of Chlamydia muridarum (CM) is restricted to the large intestine since CM is eventually cleared from the small intestine as it is from the genital tract while maintaining long-lasting colonization in the large intestine.

3. The clearance of CM from the small intestine and genital tract depends on IFNγ produced by antigen-specific CD4+ T cells. However, the same IFNγ-producing CD4+ T cells fail to clear CM from the large intestine.

4. Instead, CM colonization in the large intestine is regulated by IFNγ produced by ILC3s (or IFNγ+ILC3s) since IFNγ+ILC3s are both necessary and sufficient for inhibiting the colonization of an IFNγ-susceptible CM mutant.

5. IFNγ+ILC3s may provide a unique source of IFNγ for accessing the chlamydia-infected cells. Consistently, IFNγ+ILC3s are required for inhibiting C. trachomatis (CT) infection in the mouse endometrium.