Immune Response to Chlamydia

Abstract

Following the chlamydial exposure, a series of events occur in the host belonging to the innate and adaptive immune systems. The first line of defense against chlamydial infections is mucosal secretions contain various antimicrobial peptides. The complement system that can be part of defense is triggered by elementary bodies of Chlamydiae. Chlamydiae that escape from the complement system infect the epithelial cells. Chlamydiae are protected from phagolysosome fusion by generating inclusion formation. However, they are recognized by pattern recognition receptors (PRR), mainly Toll-like receptor 2. Chlamydia-PRR interaction can be resulted by cytokine/chemokine secretion. The first innate immune cells that reach the infection site are natural killer (NK) cells and neutrophils. The most important contribution of NK cells to this pathogen is the production of high levels of IFNγ. Neutrophils are effective in reducing the load of Chlamydia and shortening the duration of infection. The relationship of neutrophils with pathology is also discussed. Recognition of MHC class II-restricted Chlamydia peptides presented by dendritic cells via CD4 T cells initiates an adaptive immune response. IFNγ-mediated Th1 immune response is essential for Chlamydia clearance. CD8 T cells, which are fewer in numbers, have been suggested that they are the main cause of infection-related immunopathology. B cells and antibodies were found to be particularly effective in preventing reinfection.

1. Introduction

Chlamydia genera belong to the Chlamydiae phylum, Chlamydia class, Chlamydiales order, and Chlamydiaceae family according to phylogenetic classification. Chlamydiae are Gram-negative non-motile obligate intracellular bacteria with biphasic developmental cycle. They appear in two different forms called the elementary body (EB) and the reticulate body (RB). EB in the extracellular environment infects susceptible cells. After cell invasion, the disulfide bonds of EB are reduced and the bacteria transform into the RB form. This intracellular form, which is responsible for the persistence of the disease, is metabolically active, replicative, and non-infectious [1, 2, 3, 4]. Many species of chlamydia occur as disease agents in humans and animals. The two main species responsible for disease in humans are Chlamydia trachomatis and Chlamydia pneumoniae. In addition to these two species, which are strictly human pathogens, Chlamydia psittaci, whose natural host are birds, also causes infection as a result of transmission to humans [5, 6]. C. pneumoniae is primarily associated with bronchitis and pneumonia. Acute infections are usually asymptomatic or mildly clinical. Reinfection is common. In recent years, there has been evidence that C. pneumoniae causes some inflammatory diseases. Chronic bronchitis, asthma, atherosclerotic cardiovascular diseases, and cerebrovascular diseases are among the diseases associated with chronic C. pneumoniae infection [7]. C. trachomatis is grouped into various serovars according to its outer membrane genotype. Serovars A, B, Ba, and C mainly infect the conjunctival epithelium, causing trachoma defects. Recurrent infections can result in corneal damage and blindness [8]. Serovars D, Da, E, F, G, Ga, H, I, Ia, J, and K are causative agents of urogenital chlamydial infection [9]. It causes proctitis and urethritis in men. In women, it causes urethritis, cervicitis, endometritis, and salpingitis. Chronic pelvic pain, ectopic pregnancy, and infertility are common complications, accounting for 20–50% of non-gonococcal urethritis cases [10, 11]. L1, L2, L2a, and L3 serovars spread to the inguinal lymph nodes and cause lymphogranuloma venereum (LGV) [12]. Although sensitive tests such as PCR can be used for the diagnosis of chlamydial infections, their asymptomatic nature delays their diagnosis and increases their spread in the community. Sequelae increase in the long term as a result of not applying timely and appropriate antibiotherapy [13]. Chlamydial infections continue to be an interesting research topic due to their impact on public health. The necessity to understand all aspects of chlamydial infections necessitates the examination of host responses. Various stages of the innate and adaptive immune response have been studied in numerous studies, both in humans and in animals. In this topic, immunological events that occur in the host after chlamydial transmission are summarized based on the literature.

2. Innate immune response

2.1 Physical barriers

Mucosal barriers are physical barriers that form the first line of defense against Chlamydial invasion. They are formed by epithelial cells and the substances they secrete. Mucosal secretions contain various antimicrobial peptides [14].

2.2 Complement system

The complement system, which forms the humoral arm of native immunity, is activated by being stimulated by the EBs. While opsonins such as C3b, which are formed as a result of complement activation, contribute to the removal of EBs, it has been shown that C3a has an immune modulatory effect [15].

Chlamydia that escapes the effect of complement infects epithelial cells. Thanks to the inclusion, they escape phagolysosome fusion, but they cannot avoid being recognized by pattern recognition receptors (PRRs) [16]. The PRRs are an important part of the innate immune response against chlamydia. PRRs are proteins that recognize conserved motifs associated with pathogens called pathogen-associated molecular patterns (PAMPs). More than 20 types of PRRs have been identified in humans, found in epithelial cells as well as innate and adaptive immune cells. PRRs can be located in the cytoplasm or surface of cells [17]. Chlamydial PAMPs are recognized by both intracellular and extracellular PRRs since chlamydia is found in the host cell in the form of reticulate body (RB) and released out of the cell in the form of EB. After PRR activation, soluble antimicrobials, chemokines, and proinflammatory cytokines are secreted from the related cells. The prominent PRRs in chlamydial infections are TLRs, especially Toll-like receptors 2 (TLR2) [18], nucleotide-binding oligomerization domain-like receptors or NOD-like receptors (NLRs) [19], stimulator of interferon genes (STING) [20] and CD14 [21].

TLRs recognize chlamydial components such as lipopolysaccharide (LPS), lipoprotein, Heat Shock Proteins (HSP) [22, 23]. TLR2 is located around inclusion during chlamydial infection. Intracellular signal transmission occurs after recognition of its ligand (LPS, HSP60). In a study demonstrating the role of TLR2 and its adapter myeloid differentiation primary response protein 88 (MYD88), LPS isolated from C. trachomatis has been known to activate nuclear factor-kB (NF-kB) via TLR2 [24]. Studies in human embryonic kidney 293 (HEK293) cells revealed that TLR2 and its adapter protein MyD88 are required for interleukin-8 (IL-8) production [25]. Darville et al. showed that macrophages from TLR2 knockout mice secrete significantly less IL-6 and TNF-α in response to infection than those from wild-type mice [26].

Where TLR2 is most common in the female genital tract in the uterine tubes and inside the cervix. TLR4 is frequently encountered in the uterine tubes and endometrium [27]. In the study by Bulut et al., it was shown that TLR4-mediated recognition of chlamydial LPS and chlamydial HSP60 during C. pneumoniae infection is associated with dendritic cell (DC) maturation and cytokine release [23]. STING, a cytosolic PRR, is activated by recognizing dsDNA, cyclic di-AMP, or di-GMP. STING activation leads to the production of type-I interferon (IFN) [9]. It has been shown that cyclic di-AMP produced by C. trachomatis activates STING and causes IFN-β production in infected cells [28]. However, the roles of type-I IFNs in chlamydial infections are not yet clearly understood. In Chlamydia muridarum genital infection of genetically deficient mice with type-I IFN receptors, chlamydial shedding and duration of infection were found to be reduced, and there was less chronic oviduct pathology [29]. Type-I IFNs have also been associated with PID and infertility in human studies [30]. These findings suggest that type-I IFNs have a negative effect on chlamydial infection.

CD14, a PRR found in monocytes and macrophages, acts as a receptor for bacterial LPS [21] and mediates the secretion of proinflammatory cytokine during infection with C. trachomatis.

NOD-like receptors interact with the LPS and PGN of intracellular bacteria [19]. In a study using HEK293 cells, it was shown that dead C. pneumoniae was unable to activate the NOD1 or NOD2 PRRs, indicating that live bacteria are necessary for their stimulation [31].

2.3 Innate immune cells

2.3.1 Neutrophils

Neutrophils are the first immune cells to reach the site of infection [32]. Although they cannot clear the infection on their own, it is predicted that they have effects that reduce the burden of Chlamydia and limit the spread in the initial period of the infection [33]. Studies reporting that neutrophils inactivate C. trachomatis in vitro support this view [34]. Some studies do not confirm this assumption. In one study, C. muridarum load in the genital tract of neutrophil-depleted mice was 10 times higher than in wild-type mice, while C. trachomatis was shown to be eliminated in the same time period in both groups [33]. It has been found to increase chlamydial replication through MYD88-dependent signaling [35].

The relationship of neutrophils with pathology has also been the subject of research. In animal model studies, neutrophils are found to be associated with the development of tissue damage as well as contributing to the development of adaptive immune response [36, 37].

Neutrophils are very short-lived cells compared to other immune cells. They survive for about 5 hours before spontaneous apoptosis. How Chlamydia can persist in such a short-lived cell has been the subject of research. Although uninfected granulocytes become apoptotic within 10 hours, survival of infected granulocytes for up to 90 hours has revealed that Chlamydia can delay neutrophil apoptosis [38]. In a study in which primary human neutrophils were infected with C. pneumoniae in vitro, it was reported that the infection activates the ERK1/2 and PI3K/Akt survival signaling pathway, delaying neutrophil apoptosis, and thus prolonging their survival [39]. It has also been shown that granulocyte-macrophage colony-stimulating factor, which supports neutrophil activation and survival, is secreted from epithelial cells infected with C. trachomatis [40].

It is thought that the prolongation of neutrophil lifespan may have a negative effect on the outcome of chlamydial infection due to the cytokines they secrete causing tissue damage [41]. In a study performed with human fallopian tube tissue culture, the addition of an IL-1 receptor antagonist prevented tissue damage due to C. trachomatis. This study provides evidence that IL-1, a cytokine released mainly by neutrophils and monocytes, causes tissue damage in the genital tract [42].

One of the many mechanisms that Chlamydia spp. uses to break innate immune responses and ensure their persistence is that it causes neutrophil dysfunction. The chlamydial protease-like activity factor (CPAF) affects defense mechanisms such as oxidative burst and formation of extracellular traps by targeting the neutrophil surface receptor formyl peptide receptor 2 [43]. As a result, it is suggested that neutrophils with prolonged lifespans but weakened functions contribute to the pathogenesis of chronic chlamydial infections.

2.3.2 Natural killer cells

Natural killer (NK) cells are a group of innate cells involved in the response against cancer, viral infections, and intracellular bacteria [44]. Their role during chlamydial infection has been studied in various studies [32]. In mice inoculated intravaginally with C. muridarum, Tseng and Rank determined that NK cells reached the site of infection within 12–24 hours after inoculation [32, 45].

NK cells produce high levels of interferon-γ (IFN-γ). Hook and colleagues showed that interleukin-18 released from human epithelial and IL-12 produced by dendritic cells after being stimulated by C. trachomatis cell (DC) stimulate IFN-γ production in NK cells in vitro [46]. IFN-γ is important for the inhibition of Chlamydia growth, as well as one of the main cytokines important for the induction of a Th1 immune response. Animal studies in which NK cells have been experimentally destroyed highlight the importance of these cells. In the study by Tseng and Rank, antibody responses were investigated after intravaginal C. muridarum inoculation in mice and wild-type mice treated with anti-NK-cell antibody. In the humoral response to Chlamydia in NK cells depleted mice, Th2-associated antibody IgG1 was found to be significantly higher, while Th1-associated IgG2a antibodies were dominant in mice that did not receive anti-NK-cell antibody treatment. In conclusion, the absence of NK cells was associated with decreased TH1 response and exacerbation of the course of infection [32, 45]. In another study, it was shown in vitro that IL-12 secretions were decreased and their CD4 T cell-stimulating capacity decreased in DC obtained after intranasal inoculation of C. muridarum in mice with NK cells depleted. In addition, DC cells transferred from NK cell-depleted mice to naive mice failed to induce Th1-mediated immune response against intranasal C. muridarum infection [47]. These findings indicate that IFN-γ secreted by the NK cell in the early stages of infection shifts the immune response toward Th1 instead of Th2.

2.3.3 Macrophages

Studies show that macrophages migrate to sites of chlamydial infection [48]. They are attracted to the infection site by chemokines and cytokines secreted from infected epithelial cells [49, 50]. They recognize chlamydial PAMPs through the PRRs they carry, primarily TLR and NOD-like receptors. Chlamydia enters macrophages via phagocytosis or receptor-mediated endocytosis [51, 52] and proinflammatory cytokines are secreted [22, 53]. Degradation of ingested bacteria with lysosomes ensures the elimination of bacteria. M66 Host cell autophagy, a process by which cells degrade cytoplasmic proteins and organelles, also makes bacteria the target of lysosomes. There are studies showing that autophagy is important for the clearance of C. trachomatis [54, 55]. Far fewer forms of chlamydial RB have been detected in macrophages compared to epithelial cells. Although chlamydia infects macrophages, it does not create a niche for intracellular replication. The reasons for this may be the failure of C. trachomatis to inhibit phagosome-lysosome fusion and autophagy [54, 56]. Furthermore, autophagy indirectly enhances cell-mediated and humoral responses against Chlamydia by increasing antigen presentation to T cells as supports [57, 58]. It is also important to note that IFN-γ both enhances autophagy and causes upregulation of MHC class II molecules [59].

2.3.4 Mast cells and eosinophils

After infection of mast cells with Chlamydia, cytokines such as TNF-α and IL-4 are released. As a result of these cytokines opening tight junctions, infiltration of the airways with immune cells occurs. This situation has a negative effect on the spread of Chlamydia [60, 61].

Eosinophils secrete IL-4 in the upper genital tract during genital C. trachomatis infection. It has been reported that this cytokine indirectly promotes the proliferation of endometrial stromal cells. In addition, it is thought that IL-4 may regulate the development of Th2 immune response after C. trachomatis infection [62].

2.4 Cytokines of the innate immune response

In various animal and human studies, it has been shown that proinflammatory cytokines such as TNF-α IL-8, IL-1, and GM-CSF are associated with the development of tissue damage during the innate immune response to C. trachomatis infection [27, 29].

2.5 Dendritic cell

The DCs, which are professional antigen presenting cells (APC), have been shown to activate both CD4 and CD8 T cells through MHC class I/II presentation in Chlamydial infections [63, 64]. In a murine model, DCs appear to harbor infectious C. muridarum but can still present antigen to T cells. TLR2, STING, and NLRs in DC lead to the production of proinflammatory cytokines such as IL-6, TNF-a, CCR7, CXCL10, IL-1a, and IL-12 after uptake of Chlamydia. These cytokines ensure DC maturation and optimal antigen presentation [65]. Cytokines produced by DC and process of processing and presenting antigens to T cells determine the Th1/Th2 balance of the adaptive response during chlamydial infection. Upon preferential antigen uptake, preferential production of IL-12 from DC occurs. IL-12 activates naive CD4 T lymphocytes and enables them to differentiate toward the Th1 subgroup [66]. In a study, when DCs stimulated with recombinant chlamydial proteins were adoptively transferred to mice, the predominantly produced antibody became Th2-associated antibody IgG1 [65]. In the study by He et al., Th1 cells were highly activated when Th2-associated cytokine IL-10 knockout DC was stimulated and adoptively transferred [67]. In another related study, Lu and Zhong incubated bone marrow-derived DCs with heat-killed C. trachomatis and showed that a Th1 response developed after nasal infection of mice with live C. trachomatis [68]. DCs provide a link between innate and adaptive immunity in the control of chlamydial infection. Chlamydiae limit MHC class I/II expression in antigen presenting cells to cope with the immune response at this stage [69]. Chlamydial protease-activating factor (CPAF) released into the cytogel by C. trachomatis has been shown to inhibit MHC molecules by degrading the MHC class I transcription factor RFX-5 and the MHC class II transcription factor USF-1 [70, 71].

3. Adaptive immune response

3.1 T cell

Research by Rank et al. in athymic mice demonstrated the importance of T lymphocytes for chlamydial immunity. In this study, after inoculation of C. muridarum intravaginally in mice, chronic infection occurred in athymic mice, while wild-type controls were able to eliminate the infection within 20 days [32].

T cells cannot recognize pathogen antigens without MHC molecules. MHC II molecules are only found on professional antigen presenting cells, including DC, macrophage, B cell, while MHC I molecules are expressed on the surface of all nucleated cells. CD4 T cells recognize antigens presented in MHC class II and CD8 T cells are activated by MHC class I antigen complexes [72]. In fact, both T cell subsets have been shown to recognize C. trachomatis antigens such as outer membrane protein 2 (Omp2), polymorphic outer membrane protein D (POMP-D), MOMP, heat shock protein 60 (HSP60), chlamydial protease-activating factor (CPAF), PmpG, PmpF, and RpIF [58].

4. CD4 t cell

CD4 T cells recognize extracellular antigens from proteins endocytosed by APCs and degraded by endosome proteases. During chlamydial infection, APCs such as DCs and macrophages acquire exogenous chlamydial antigens by phagocytizing EBs in the extracellular space or by capturing infected cells harboring RBs. After phagocytosis, APC cleaves chlamydial components and the peptide-MHC II complex is assembled. This complex is then transferred to the cell surface, where it is recognized by the TCR in CD4 T cells [72].

T cells are detected at the site of infection in mice and humans. The recruitment of CD4 T cells to the infection site occurs by the release of various chemokines as well as the regulation of some surface and adhesion molecules [49, 73, 74, 75]. Post-infection APCs also migrate to regions of CD4 T cells. Here, clonal expansion of CD4 T cells recognizing chlamydial antigens is achieved (S. G. [48]).

CD4 T cells play a critical role during chlamydial infection. Evidence from murine non-MHC II models has demonstrated the importance of CD4 T cells in clearing the disease (R. P. [76]). Gondek et al., in their study of murine upper genital C. trachomatis infection model studies, suggested that CD4 T cells are necessary and sufficient for clearance of Chlamydia and protection against reinfection [77].

When the cellular immune response against C. pneumoniae was examined, proliferation and activation of both CD4 and CD8 T cells were detected during primary infection. However, only the activation of CD4 T cells was detected in the later stage of the infection [78].

CD4 T cells differentiate into subtypes as a result of upregulation of transcription factors that increase the production of specific cytokines after antigen recognition [79]. For example, Th1 cells, which are characterized by the production of large amounts of proinflammatory cytokines, especially IFN-γ, are particularly important for clearance of viral infections and intracellular bacteria [80]. In the context of infection by intracellular bacteria such as Chlamydia, the predominant T cell subset expected to be present is Th1 cells. As stated earlier in the relevant section of this article, Th1 subtype differentiation in CD4+ cells occurs following the production of IFN-γ and IL-12 by innate immune cells early during infection [47, 81].

4.1 T-helper1 responses

Evidence from mouse models indicates that the Th1 subtype is of particular importance in Chlamydia clearance [48]. Observation of increased susceptibility to chlamydial infection in the absence of IL-12 [82, 83] or IFN-γ receptor [84] emphasizes that IFN-γ-producing CD4 T cells are protective against Chlamydia. However, some evidence suggests that a polyfunctional response involving IFN-γ as well as TNF-α can increase immunity [85].

Th1 cells not only activate phagocytic macrophages, but also direct humoral immunity. At the end of the process in which B cells are activated, Th1-related antibodies such as IgG2a and IgG3 are secreted by plasma cells [25, 86, 87]. In addition, the cytotoxic effect of CD4 T cells has also been demonstrated [84].

Th1 responses against C. pneumonia predominate, especially during reinfection. Even in mice genetically predisposed to Th2 responsiveness during primary infection, Th1 responses were elicited during reinfection and increased IFN-γ production [88].

4.2 Other T-helper responses

Although the predominant CD4 cells are Th1 in chlamydial infection, other T-helper types such as Th2, Th17, Th22, and Th9 have also been detected. However, the role they play during chlamydial infection cannot be definitively determined [80]. For example, the production of IgG1 antibodies is induced by Th2 cells. However, there is evidence that the Th2 response is not protective and even associated with pathology. The Th2 response during human ocular infection has been associated with disease progression and pathology [89]. Transfer of chlamydia-specific Th2 clones failed to protect mice from genital infection [90]. Another T-helper (Th17) is thought to contribute to the formation of Th1 immunity, but has been associated with both protection and pathogenesis in the mouse model [91, 92].

4.3 Memory CD4 T cells

Memory CD4 T cells are traditionally grouped into two groups: central memory (Tcm) and effector memory (Tem), while CD4 Tcm cells are primarily found in the circulation and lymphatic tissues; peripheral, non-lymphoid tissues host CD4 Tem cells [93]. Therefore, CD4 Tem cells are thought to play a dominant role in clearing genital chlamydial infections. Recently, it has been discovered that a third subset of memory T cells is important in tissue-specific immune responses. Unlike TEM, which recirculates into the lymphatics and blood after pathogen clearance, these cells that remain in non-lymphoid peripheral tissue after pathogen clearance are called tissue resident memory T cells (Trms). Even in the absence of persistent antigen, Trms persist in peripheral tissues for a long time [93]. These cells are found in epithelial tissues in areas that interface with the environment, such as the gut, lungs, skin, reproductive system [94]. They act as the first line of defense when re-exposure to pathogens. They can respond to pathogenic attack faster than other subsets of memory T cells that need tissue traffic.

During secondary C. trachomatis infection in mice, memory T cells coming from the circulation to the upper genital tract mucosa together with the CD4 Trm cells present in the tissues provide optimum clearance. Circulating memory T cells contribute to the clearance of secondary infection. However, it has been shown that they cannot clear secondary C. trachomatis infection alone without Trm cells [95]. Studies have shown that memory T cells proliferate more rapidly in response to antigen during secondary infection [8, 96].

5. CD8 T cells

CD8 T cells are associated with MHC I. MHC I is expressed in all nucleated cells. Cytosolic proteins, which may originate from intracellular pathogens, are degraded by the proteasome. The degradation product peptides are loaded into the binding groove of MHCI at the end of the process involving TAP and a chaperone protein, tapasin. The MHC I-peptide complex is then exported to the surface of the cell [72]. TCRs on CD8 T cells recognize the endogenous antigen presented on MHC I. The results of this recognition are the expression of various effector cytokines, including IFN-γ, and the release of cytotoxic granzyme and perforin molecules that can lead to target cell death [97]. Because they can kill infected cells, CD8 T cells are thought to play an important role in the immune response to intracellular pathogens.

The role of CD8 T cells in chlamydial infections is controversial. While a broader CD8 T cell response was expected against Chlamydia, an intracellular pathogen, it was determined that the CD8 T cell response against C. muridarum in the genital tract was much lower than CD4 cells in mouse experiments. Despite their small number, CD8 T cells are known to migrate to the site of infection, and both human and mouse CD8 T cells have been shown to destroy Chlamydia-infected cells [98]. It has been shown that CD8 T cells recognizing trachomatis proteins class I accessible protein-1 (Cap1) and cysteine-rich protein A (CrpA) can kill target cells in an antigen-dependent manner [99, 100]. However, there is evidence to suggest that CD8 T cells are not essential for clearance of Chlamydia. It has been observed in past studies that CD8−/− and perforin-deficient mice clear the infection at the same rate as wild-type mice [82, 84]. Murthy and colleagues have also shown in a more recent study that CD8 T knockout mice exhibited similar clearances of C. muridarum as wild-type mice following vaginal exposure. In the same study, less hydrosalpinx formation in CD8 T knockout mice is remarkable in terms of the relationship between pathogenesis and CD8 T [101]. It has been suggested that they are primarily responsible for the immunopathology associated with chlamydial Infection [102]. The association of CD8 T cells with pathogenesis has also been reported in a macaque model [103]. Although CD8 T cells are not critical for C. trachomatis elimination and may even cause chlamydial sequelae, antigen-specific CD8 T cell clones can localize to the genital tract and contribute to clearance of infection through IFN-γ production [98].

During C. pneumoniae infection, it is observed that the infection progresses rapidly in the absence of CD8 T cells. Unlike C. trachomatis infection, CD8 T cells have been suggested to play a very important role in protection against C. pneumonia [104].

5.1 Memory CD8 T cells

The memory T cell population formation process of CD8 T cells during C. trachomatis infection differs from the responses detected against acute infection agents. Some expansion of CD8 T cells is observed during primary infection in mouse models. However, the fact that C. trachomatis CrpA antigen-specific CD8 T cells does not proliferate in the expected number and rate during secondary infection with C. trachomatis indicates insufficient formation of memory CD8 T cells [105]. While the elimination of the infected cell will deprive the organism of its intracellular niche, with the deterioration of the adaptive immune response, both the infections cannot be cleared and permanent immunity is not formed.

Differential programming of memory CD8 T cells when stimulated by agents such as C. trachomatis that cause persistent infection is attributed to the environment at the onset of infection. Namely, for the activation of Chlamydia-specific naïve T cell clones, both T cell receptors must recognize Chlamydia-derived peptides presented by dendritic cells, and co-stimulatory molecules on the dendritic cell must interact with those on the T cell. Interactions of some of these co-stimulatory molecules cause upregulation of the T cell response, while others cause downregulation of the T cell response [81]. One of the inhibitory interactions is the binding of programmed death ligand 1 (PD-L1) on the dendritic cell with PD-1 on the T cell [106]. A study of murine infection with C. trachomatis found PD-L1 upregulation in the uterus and PD-L1 upregulated in in vitro infected cells. As a result, CD8 T cell expansion is impaired and the development of CD8 memory responses is inhibited. This upregulation leads CrpA-specific CD8 T cells to the Tcm phenotype, which is found in secondary lymphoid organs and lymphatic vessels but has limited effect in peripheral tissues, instead of the Tem phenotype, which contributes to clearance of pathogens in peripheral tissues. When antibodies that block the interaction of PD-1 with PD-L1 were used during primary infection, or when knockout animals were used in both molecules, there was a marked increase in the number of T cells responding to secondary C. trachomatis infection, with more IFN-γ producing CD8 T cells. The memory CD8 T cell population shifted toward the Tem phenotype, resulting in faster clearance of infection [105].

On the other hand, there are studies suggesting that C. muridarum CD8 T cell response contributes to the pathology [101, 107, 108]. For this reason, it has been suggested that PD-L1-mediated inhibition may be a mechanism that prevents cell-mediated uterine pathology by CD8 T cells [101]. In the study by Peng et al., immuno-inhibitory molecules TIM3 and PD-L1 were blocked in C. muridarum-infected mice. As a result, it was observed that uterus and oviduct pathology increased [109]. This finding reveals that immunoinhibitory molecules regulate inflammation by preventing T cell activation and cytokine production.

5.2 Interferon-gamma

IFN-γ, which is released from both innate cells such as macrophages and NK cells and CD4 and CD8 T cells in response to chlamydial infection, is a critical cytokine for inhibiting chlamydial growth [104]. Gamma interferon is responsible for the upregulation of some interferon-induced genes that may help control intracellular bacterial replication in infected epithelial cells [110]. As a result, some protective mechanisms emerge in infected cells. Iron metabolism, a critical mineral for Chlamydia, is blocked [111, 112]. Expression of the tryptophan-decyclizing enzyme indoleamine-2,3-dioxygenase (IDO) is induced, which breaks down tryptophan necessary for the survival of most Chlamydia species. Also, IFN-γ enhances the phagocytic abilities of macrophages and also ingestion and destruction of C. trachomatis [71, 113].

The effect of IFN-γ on tryptophan metabolism was reviewed by Vasilevsky et al. during C. trachomatis infection in humans and the IFN-γ signaling cascade leads to upregulation of the IDO enzyme in genital tract epithelial cells. The enzyme catalyzes the breakdown of tryptophan to N-formylkynurenine and kynurenine, thereby disrupting intracellular tryptophan stores [58]. C. trachomatis deprived of this essential amino acid has been shown to die due to tryptophan starvation. There are also chlamydia species that have adapted to tryptophan starvation by transforming into the non-replicating persistent form. After IFN-γ removal and subsequent tryptophan production, these persistent forms rapidly become replicative [76, 114, 115]. Since some genital tract strains also express tryptophan synthase, they can overcome tryptophan depletion by producing their tryptophan using exogenous indole [116]. In addition, kynurenine, a by-product of tryptophan catabolism, inhibits host CD4 T cells, thus reducing IFN-γ production and ultimately limiting its overall production. It has been reported that it can lead to the re-activation of C. trachomatis [110].

6. B cells and antibodies

B cells support the immune response in a variety of ways. Effector mechanisms such as antibody-mediated neutralization and opsonization [117], antibody-dependent cellular cytotoxicity (ADCC) [118], induction of phagocytosis, and antigen presentation to CD4 T cells by binding of antigen-antibody complexes to Fc receptors in APC [102] have been identified.

The role of B cells in the immune response against Chlamydia has been the subject of many studies. It is known that many C. trachomatis proteins, including the major outer membrane protein, induce the formation of specific antibodies [119]. It has also been shown in vitro that anti-chlamydial antibodies are neutralizing [71, 117]. However, there is evidence to suggest that B cells play an important role in the secondary memory response rather than the primary infection. It was determined that primary genital infection with C. muridarum in mice lacking B cells did not show a different course than in wild-type mice [120], while mice with B cell deficit were more susceptible to reinfection [121]. Mice that cleared primary genital tract infection were found to be resistant to reinfection even after experimental depletion of CD4 and CD8 T cells. It was observed that B cell-deficient mice were unable to resolve the secondary infection after CD4 T cell depletion [122]. It has been reported that passive immune serum transfer to naïve mice does not provide protection, but CD4 T cells prepared from antigen-experienced mice and immune serum together provide optimum protection [123]. The protective effects of the antibodies are likely due to their ability to activate Th1 cells and enhance cellular immune responses [124]. The detection of high antibody titers associated with infertility rather than infection control in epidemiological studies indicates that the humoral response may also have negative effects [125]. However, data on pathogenic antibodies are limited. One of the antibodies discussed in relation to its contribution to pathology is anti-HSP antibodies. HSPs are a group of chaperones, proteins that ensure the correct folding of intracellular proteins: They are found in both eukaryotic and prokaryotic organisms. Its levels increase when cells are exposed to temperature rises, oxidative stress, and inflammation. HSPs, mainly HSP60, are produced by C. trachomatis during infection. HSP60 has high immunogenicity. It is quite similar to human HSP60. Therefore, it is suggested that it may trigger an autoimmune response that leads to pathology. High antibody titers against HSP60 were found to be associated with pelvic inflammatory disease, ectopic pregnancy, and trachoma scar [126].

The effects elicited by the response to HSP60 during C. trachomatis infection are thought to be similar in C. pneumonia infection. It has been suggested that HSP60 released from infected epithelium and macrophages during recurrent and persistent infection with C. pneumonia produces immunopathological results [127].

Antibodies against C. pneumonia are also used in seroepidemiological studies investigating the relationship between C. pneumoniae infection and inflammatory diseases. It has been suggested that IgA levels are a better marker than IgG, especially in terms of detecting chronic infection [128].