Tissue macrophages (Mφ) generally engulf bacteria early in the infection when they are outside the host cells. It is known that C. trachomatis entry into the monocyte macrophages is less efficient when compared to epithelial cells. Moreover, ROS-mediated microbicidal activity is unable to kill the engulfed bacteria, and only IFN-γ stimulated, highly activated phagocytes with sufficient effector molecules can effectively kill bacteria (17, 60) ( Figure 1 ). Experiments by Chen, et al. in 2017 showed that both LPS/ATP and murine chlamydial infection of mouse bone marrow-derived macrophages (BMDM) could lead to caspase-1 cleavage and IL-1β released. However, unlike LPS/ATP, the inflammatory molecular switch RIP3 was not involved in the activation of NLRP3 inflammatory vesicles in murine Chlamydia-stimulated BMDM, suggesting that chlamydial infection leads to caspase-1 cleavage and IL-1β release by a different mechanism than LPS/ATP (61). Two years later, further experiments showed that lipopolysaccharide (LPS) of C. trachomatis in BMDM, unlike other Gram-negative bacteria, blocked downstream signaling of TRIF and MyD88 classical inflammatory pathways and failed to activate non-classical inflammatory pathways mediated through caspase-11 to evade innate immunity. This study explored the mechanism in more depth: trachoma LPS, while effectively binding CD14, cannot effectively induce TLR4/MD-2 complex dimerization or endocytosis, implying that the classical pathway is blocked at its source. It is speculated that this phenomenon may be related to the fact that lipid A of chlamydial LPS is penta-acylated, rather than hexa-acylated (25).

Macrophages are divided into two phenotypes, M1 and M2: IFN-γ/LPS-induced M1 and selectively activated, IL-4-induced M2. M1 has a strong capacity to engulf and kill bacteria that triggers an inflammatory response by releasing chemokines and pro-inflammatory cytokines. And M2 cells inhibit inflammatory responses or participate in tissue repair and fibrosis. Buchacher T et al. showed that positive Chlamydia pneumoniae (Cpn) lipopolysaccharide staining and quantification of 16 S rDNA were significantly higher in M2 than in M1. A large number of intact perinuclear inclusions were found in M2-type macrophages, whereas rupture of inclusion bodies occurred in M1-type macrophages (62). The Chlamydia muridarum and murine bone-marrow derived macrophages showed that although M1 could mediate IFN-γ to control infection, it could not eliminate intracellular chlamydia (63). This suggests that M2-type macrophages provide a better environment for Cpn to survive. It has been shown that Nutlin-3 inhibits Chlamydia abortus growth and affects TNF-α secretion in M1 in a dose-dependent manner (64). In addition to improving the survival environment within macrophages, another important escape mechanism is the regulation of apoptosis. Utilizing CRISPR/Cas9 technology, Amy T.Y. Yeung and his colleagues hypothesized that deletion of the IL-10/IL-10R signaling pathway may resist macrophage apoptosis and thus sustain chlamydial growth (65). In addition, Chlamydia is expelled from the host cell by extrusion release, forming an inclusion-like structure called extrusions. Extrusions retard macrophage killing and eventually release infectious EB from macrophages, which facilitates the spread of EB to more distant sites. Extrusions are also able to survive longer in the extracellular environment than free C. trachomatis EB (66). This chlamydia-specific cell exit is likely a self-interested ability shaped by thousands of years of host-pathogen interactions. In summary, chlamydial survival in macrophages is suitable but limited. The restricted growth pattern is often associated with lysosomal trafficking, perforin-2 release, and nutrient starvation. Further work is needed to investigate the mechanisms of chlamydia-macrophage interactions, as well as to confirm them in vivo. This may, in some ways, increase the clearance susceptibility of the macrophage and decrease its ability to assist in chlamydial transmission.

Neutrophil (NE) is the most abundant innate immune cells and the first leukocytes recruited to infected tissues. Neutrophils kill microorganisms primarily by phagocytosis and activation of the NADPH oxidase machinery (67). When neutrophils encounter pathogens, they activate their bactericidal reservoir to produce reactive oxygen species (ROS) and neutrophil extracellular traps (NETs). NETs are DNA structures decorated with cytoplasm, granules and nucleoproteins, and they can capture microorganisms including bacteria, viruses and 50 species of fungi. A variety of microorganisms are known to activate neutrophils and induce the formation of NETs. However, in order to evade or survive in neutrophils, some pathogens (68), such as C. trachomatis, have evolved mechanisms to degrade extracellular chromatin traps by secreting effector nucleases and proteases. It has been shown that chlamydial protease-like activating factor (CPAF) directly affects PMN survival and that formyl peptide receptor 2 (FPR2) is a key target of CPAF. In addition, C. trachomatis is able to reverse the short-lived neutrophils and delay apoptosis by activating ERK1/2 and PI3K/Akt survival signaling pathways (69); a key protein in human intracellular defense against Chlamydia is the tryptophan-degrading enzyme indoleamine 2,3-dioxygenase (IDO), but its activation does not inhibit chlamydial survival in neutrophils (70).

In pelvic inflammatory disease caused by C. trachomatis, interleukin1α(IL-1α) and type I interferon released upon the death of infected inflammatory cells favored the pathogen over the host. Persistent infection and chronic antigenic stimulation impair the ability of T cells to produce IFN-γ, thereby attenuating the protective response of Th1 and Th2 (4). In addition, Chlamydia-infected neutrophils exhibit high levels of extracellular ATP, which can act as a damage-associated molecular pattern (DAMP) to activate submucosal macrophage NLRP3 inflammatory vesicles, thereby driving damaging immunopathology (71). To some extent, these immune evasion mechanisms suggest that C. trachomatis infection can be overcome by the use of CPAF inhibitors, vaccines with Th1-inducing adjuvants providing antigens, etc.

Classical dendritic cell (cDC) is the bridge between innate and adaptive immunity, and it is currently recognized as the most powerful specialized antigen-presenting cell in the body. From immature DC (iDC) to mature DC, it becomes progressively less capable of antigen uptake and processing (phagocytosis) and progressively more capable of antigen presentation. DCs are the sole initiators of initial T-cell activation (72). C. trachomatis, as an exogenous antigen, is internalized and processed by the APC, then processed by the proteasome in the cytoplasm, and finally activates CD4⁺T lymphocytes mainly through MHC-II molecules on the surface of DC. DC cells with the efficient presentation of cross-antigens activate CD8⁺T lymphocytes via surface-expressed MHC class I molecules in response to innate antigens in cells already infected with C. trachomatis. Moreover, CD8α⁺ dendritic cells were found to present antigen to CD8⁺T cells and induce Th1 production in mice at a better level than CD8α^- using the adoptive transfer method (73). It has been shown that C. trachomatis hijacks the DC endocytic recycling system by recruiting Rab proteins associated with the recycling pathway around the inclusions, which leads to adverse changes in MHC-I intracellular transport (54).

As previously described, Chlamydia exits infected host cells via extrusion formation, which confers an infectious advantage to Chlamydia and promotes its survival within macrophages (74). It was found that the extrusion formation also prolonged bacterial survival within dendritic cells and altered the initial innate immune signaling of dendritic cells. Protective immunity against Chlamydia is primarily driven by IFN-γ producing T cells. Unlike macrophages, phagocytosis of extrudates leads to rapid apoptosis of DCs through a caspase 3/7-dependent mechanism, whereas exposure to free Chlamydia does not undergo apoptosis, directly preventing the initiation of adaptive immune responses (75). In 2017, Khamia Ryans et al. demonstrated that alpha enolase (ENO1) deficiency affects DC survival, maturation, and antigen presentation characteristics in vivo and in vitro experiments. Thus, modulation of ENO1 facilitates the enhancement of DC function, which could be used as an immunotherapeutic strategy to generate long-term immunity against Chlamydia-induced tubal lesions (76). In fact, using a combination of affinity chromatography and tandem mass spectrometry, it has previously been shown that DCs transferred with the chlamydial antigenic peptide Ags: PmpG1_25-500, RplF, or PmpE/F-2_25-575 overtly induced significant protective immunity against pulmonary and genital tract infections. Among them, PmpG1_25-500 is the most immunoprotective and could be a candidate for T cell protein-based subunit vaccines (77).

In addition to neutrophils, granulocytes include eosinophils, basophils and mast cells ( Figure 2 ), which are rare in the study of C. trachomatis now. Eosinophils have unique chemokine receptors on their surface, and Vicetti Miguel et al. revealed in mouse experiments that IL-4-producing eosinophils promoted endometrial stromal cell (ESC) proliferation during primary Chlamydia infection, thereby repairing endometrial tissue induced by genital pathogens (78). In addition, a recent pathological study on endometriosis and endometritis revealed a significant increase in not only eosinophils but also basophils (79). And statistically, histological analysis of sexually transmitted infections caused by C. trachomatis in gay men presenting with proctitis revealed a scarcity of eosinophils (80). Mast cells are less frequent in studies of C. trachomatis infections and more frequent in allergic diseases. In a recent study of Chlamydia pneumoniae (C. pneumoniae), Norika Chiba et al. constructed mast cell-deficient mice (Wsh) and then found that the lungs of Wsh mice were more efficient at clearing Chlamydia pathology than those of WT mice. This suggests that the presence of mast cells exacerbates the inflammatory response and increases mortality, because it promotes the infiltration of immune cells into the air and provides a more favorable environment for C. pneumoniae to multiply (81).

The predominant innate lymphoid-like cells are natural killer cells (NKs), which do not have specific antigen recognition receptors like TCR and BCR or pattern recognition receptors like Mφ and DC. In the case of infection, killer activated receptors (KAR) and killer inhibitory receptors (KIR) on the surface of NK cells bind to target cell surface ligands (MHC-I), resulting in the deletion or downregulation of MHC-I molecules and abnormal expression or up-regulation of non-MHC-I molecules. At this point, the inhibitory signal is absent and the activating signal predominates, thereby initiating killing (82). MHC-I is down-regulated, and MICA (MHC class I-related protein A) is up-regulated on cells infected by C. trachomatis, which can be recognized by NK cells. Moreover, specific combinations of NKG2D (NK cell-activated receptor) and MICA alleles may promote escape of C. trachomatis from NK cell-mediated immune responses more effectively than others in different individuals, which may be a factor contributing to individual prognostic differences in female genital tract infections (83).

In addition to NK, other innate lymphoid cells that do not express specific antigen receptors and whose activation does not depend on recognition of antibodies are named ILCs, which are classified as ILC-1, ILC-2, and ILC-3. ILCs have no direct killing capacity, and their response depends on the activation of cytokines released by other cells, as they have only cytokine receptors. Activated ILCs also release corresponding cytokines to amplify the intensity of the previous immune response to perform an immunological function (84). Both ILC-1 and ILC-3 can secrete IFN-γ. IFN-γ produced by a variety of cells is known to be effective in clearing different sites or species of Chlamydia. IFN-γ+ILC-3 plays an important role in regulating colonization of the colon by Chlamydia muridarum and inhibiting endometrial chlamydial infection. IFN-γ produced by circulating cells such as NK cells and NKT cells prevents the spread of Chlamydia (85). Hong Xu and colleagues demonstrated that mouse ILCs significantly promote endometrial innate immunity in adaptive immunodeficient mice through interferon-dependent mechanisms, and that the role of ILC-3 is more important (86) in mice with C. trachomatis inoculation.

In addition to the frequently studied models of reproductive tract infection, gastrointestinal C. trachomatis is currently considered a seed bank for recurrent reproductive tract infections. Koprivsek et al. suggested in a murine model of Chlamydia muridarum-induced gastrointestinal infection that Chlamydia could evade IFN-γ from ILC-3 but not NK and maintain its long-term colonization in the colon (87). Furthermore, IFN-γ induced downregulation of c-Myc, a key regulator of host cell metabolism, in a STAT1-dependent manner, suggesting that c-Myc expression rescued the survival of C. trachomatis. IFN-γ caused the persistence of epithelial Chlamydia through infiltrative secretion by T cells and NK cells, as found in a pathological study of trachoma scarring (88, 89). Concerning therapy, it has been documented that NK cells are potential therapeutic targets in inflammatory bowel disease (IBD), atherosclerosis (AS), pulmonary arterial hypertension (PAH), and other inflammatory diseases (31). “NK therapies” are known to be widely used in cancer treatment. A nanoparticles (NP) therapy against the C. trachomatis antigen: Nanoparticles composed of poly (D,L-lactide-co-glycolide) (PLGA) have attracted much attention because of their biodegradability, biocompatibility and good colloidal stability. Co-delivery of C. trachomatis MOMP and immunostimulant (IS) with PLGA particles can prevent systemic adverse effects of immune boosters and activate dendritic cells and natural killer cells, thus enhancing the therapeutic effect of antigen-loaded PLGA particles (90).

Both Natural killer T cells (NKT cells) and γδ T cells belong to the atypical T cell family, and antigen recognition by these atypical T cells is not restricted by MHC class I and class II molecules (91). NKT cells are lymphocytes that express both the NK cell surface marker CD56 (NK1.1 in mice) and the T cell surface marker TCRαβ-CD3 complex. NKT cells are usually divided into type I and type II. Type I NKT cells are called semi-invariant NKT cells (iNKT) and recognize glycolipid and lipid antigens presented to them by the CD1d molecule, which respond rapidly to danger signals and pro-inflammatory cytokines (92). However, C. trachomatis infection can downregulate the CD1d molecule in human penile urothelial cells, which is associated with the CPAF protein. Activated type I NKT can promote the maturation and differentiation of DCs into CD8+/CD103+ DCs, which can further activate T cell differentiation into functional Th1 or Tc1-like peptide antigen-specific T cells. Activated antigen-specific CD4⁺Th1 and CD8⁺Tc1 can suppress bacterial infection (93, 94). However, CD1d-restricted NKT cells can regulate the immune response to chlamydial infection and cause immunopathological damage. Recent studies have shown that wild-type (WT) female mice have a significantly higher chlamydial burden than CD1d-/-(NKT-deficient) mice, suggesting that NKT cells delay chlamydial clearance and exacerbate immunopathology such as tubal effusion and obstruction. In contrast, there is no significant difference in the severity or incidence of tubal effusion in Jα18-/-(iNKT-deficient) mice compared with WT controls. Thus, non-invariant NKT cells have an immunopathogenic role in urogenital tract chlamydial infection (95). This partly explains a seemingly contradictory earlier study that NK T cells triggered pathological Th2 responses during chlamydial infection (96). It has also been shown that the activation of NKT triggers a pathology also associated with disruption of the CXCL13-CXCR5 axis. In this process, activated NKT cells increase chronic inflammation in the upper genital tract of mice by secreting cytokines or chemokines to recruit neutrophils and dendritic cells (97). These results suggest that NK T cells show protective Th1 immunity and pathological Th2 immunity in their role against Chlamydia.

γδT cells are known to bridge the gap between innate and adaptive immunity. γδT cells represent a small proportion of the population and are mainly distributed in mucosal tissues such as the peritoneal cavity, intestine and lung. They are the first to be recruited for mucosal infections. Experiments have shown that IL-17A plays a protective role against Chlamydia pulmonary infection, and it is produced rapidly but transiently by γδT cells in the early stages and mainly by Th17 in the later stages. Although the depletion of γδT cells led to a decrease in CD80 expression and an increase in IL-10 production by DCs, it had little effect on IL-12 production. There was no effect on type 1 T-cell responses after γδT cells depletion. In contrast, the decrease in IL-1α was more pronounced, suggesting that γδT cells could play a supportive but non-essential role in host defense against Chlamydia pulmonary infection (98, 99).

B cells are divided into B-1 cells, which mediate the innate immune response, and B-2 cells, which mediate the adaptive immune response. Depending on the presence or absence of surface CD5 molecules, B-1 cells can be further subdivided into B-1a (CD5+) and B-1b (CD5-). B-1 is mainly found in the peritoneal cavity, pleural cavity and lamina propria of the intestine. Most of the B cells are restricted to B-1 cells, which can be activated by TI-Ag (bacterial LPS, etc.) and produce antibodies with low specificity and do not produce memory cells. Mouse B-1 cells are thought to be the primary producers of natural antibodies to IgM (100, 101), which are independent of foreign antigen stimulation and mediate mucosal immunity below the mucosal lamina propria. Furthermore, in response to antigenic stimulation, it is estimated that 50% of serum IgA and IgG3 are also derived from B-1a cells (101). Lack of BCR diversity on the surface of B-1 cells and reconstitution of IgM-BCR complexes may explain the antigen-specific responses of self-reactive B-1 cells in response to infection by various pathogens, including bacteria, viruses, fungi and parasites (102). At the onset of infection, B-1a cells also spontaneously secrete IL-10 stimulated by lipopolysaccharide (LPS), GM-CSF and IL-3 (103). These natural antibodies and cytokines can protect the host from infection or reduce bacterial burden. In addition, B-1a cells are efficient antigen-presenting cells that provide effective signals to T cells through the co-stimulatory molecule CD80/CD86, which is constitutively expressed on B-1a cells (104). B-1 cells play an important role in assisting M1-type macrophages in the killing of Encephalitozoon cuniculi and in reducing its immune escape mechanism (105). In conclusion, the study of trends in B-1 cells and inflammation may lead to a paradigm shift toward sustainable treatment of various inflammatory diseases (101). C. trachomatis infection is an inflammatory disease and it is known that Chlamydia can colonize the gastrointestinal tract for long periods of time (106). Although B-1 cells have been little studied in C. trachomatis, this may, provide new ideas for C. trachomatis research.