Insights into innate immune cell evasion by Chlamydia trachomatis.

C. trachomatis , a specific intracellular pathogen, is closely associated with human epidemic diseases. According to the World Health Organization, in 2020, an estimated 129 million people would be infected with C. trachomatis ( 1 ). There are 19 serovars of C. trachomatis . In addition to trachoma, inclusion conjunctivitis, and infantile pneumonia(serovars A to C), it also causes more serious genitourinary tract infections(serovars D to K) and lymphogranuloma venereum(serovars L). The former is highly curable and can be treated with large-scale azithromycin ( 2 ), while the latter is insidious and chronic both in men and women ( 3 ). Although sensitive to antibiotics, their therapeutic benefit is limited mainly because of silent (asymptomatic) and recurrent infections attributable to immune evasion and the development of partial immunity from these infections. In the long term, these recurrent infections are thought to be contributing to the development of pelvic inflammatory disease ( 4 ), tubal infertility ( 5 ), cervical cancer ( 6 ), adverse pregnancy, miscarriage ( 7 ) in women, urethritis, epididymitis, orchitis, prostatitis, proctitis ( 8 ) or reactive arthritis ( 9 ) in men. These outcomes are thought to be culminating either alone or as a complex combination of Chlamydial pathogenesis, deficient or inferior immunological memory- immune escape and immunopathology. Chlamydia lives in membrane-coated vacuoles, called inclusion, which protect it from the humoral immune response. It has been found that Chlamydia obtains nutrients (amino acids, lipids ( 10 ), iron ( 11 )) mainly from the host cell, and acquires ATP ( 12 ) entirely from the host. Chlamydia takes self-serving measures to deal with nutritional crisis, one of which is to limit the detection of innate immunity ( 13 ). NF-κB plays an important role in the inflammatory response. Surprisingly, no obvious signs of NF-κB activation were detected in Chlamydia -infected cells ( 14 ). C. trachomatis blocks NF-κB signaling through effector ChlaDUB1 reversal of IκB-α ubiquitination ( 15 ) and CPAF-mediated p65/RelA degradation ( 16 ). However, the in vivo role of CPAF in NF-κB signaling has not been proven. Inflammatory damage caused by chlamydial infection is largely due to both innate and adaptive immunity. Chlamydial infection stimulates host cells to produce interleukins, interferons, and tumor necrosis factor, which play a dual role in infection ( 17 , 18 ). Consequent to infection of the upper genital tract, infiltration of neutrophils and monocytes that are responsible for potentially deleterious inflammation along with the bystander T cells both with the potential to cause immunopathology ( 19 ). Highly potent antibodies may also cause corresponding immunopathology through mechanisms such as activation of complement ( 20 ), ADCC (antibody-dependent cellular cytotoxicity) ( 21 ), and the emergence of a type IV hypersensitivity reaction ( 22 ). In addition, immune escape of Chlamydia is also an important reason for chronic recurrent infections, which will be described primarily in this review. Recent studies indicate that C. trachomatis uses effector molecules like GarD that helps it to escape immune surveillance via avoiding ubiquitination and proteosomal degradation ( 23 , 24 ). Recent findings indicate that Chlamydial lipopolysaccharide (LPS) ( 25 ) and lipooligosaccharide (LOS) ( 26 ) do not readily trigger innate inflammatory pathways, thus avoiding early innate recognition and promoting its survival and multiplication. In summary, the key to maintaining the intracellular survival and persistence of C. trachomatis is to escape from the host’s innate immune cells. In recent years, it has been found that properly functioning immune cells have the potential to treat disease, and immune responses are often associated with the course of disease. When the body is in the midst of a persistent infection or the immune cells are not functioning properly, the ability of the immune system to clear the infection is declining. Then a critical point is reached where the immune cells are unable to clear the infected cells, and the disease occurs. The development of a novel vaccine against C. trachomatis infection benefits greatly from research on cancer immunotherapy ( 27 ) and experimental vaccinations against intracellular diseases. In fact, after H Su et al. immunized mice intravenously with bone marrow-derived dendritic cells stimulated in vitro by dead chlamydia , DC was able to efficiently perform its functions of bacterial phagocytosis and antigen presentation. The results showed that this method of immunization produced protective immunity against Chlamydia infection in the female genital tract comparable to that following in vivo infection ( 28 ). Karunakaran et al. have employed DCs pulsed with chlamydial immunopeptides in immunizations by adoptive transfer. Immunized mice developed Th1 protective immunity and partially resisted chlamydial lung and genital infections ( 29 ). However, there is a long way to go in the development of immune cell-targeted biologics in chlamydial immunity and immunopathology for prevention or treatment ( 30 , 31 ).

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.

Currently, the interaction between various innate immune cells and C. trachomatis is still a challenging research topic, and both compete and promote each other in the course of a long-term battle ( Figure 3 ). The complex set of mechanisms involved in the killing of innate immune cells against Chlamydia infection and Chlamydia immune escape from host cells can be broadly classified as follows: C. trachomatis evades the pursuit of innate immune cells. Pro-inflammatory cytokines secreted by C. trachomatis -infected cervical epithelial cells attract innate immune cells to the site of infection. Chlamydia protease-like activating factor (CPAF), which targets the cleaved NE surface receptor formyl peptide receptor 2 (FPR2), blocks the formation of neutrophil extracellular traps (NETs) and inhibits downstream reactive oxygen species (ROS) production, which paralyzes murine polymorphonuclear neutrophil (PMNs) activation. Chlamydia -infected NE produces elevated levels of extracellular ATP, adenosine triphosphate (ATP) that binds to P2X purinocreceptor 7 (P2X7R) and activates the NLRP3 inflammasome, thereby contributing to macrophage-associated immunopathology. Chlamydia is released from epithelial cells by extrusion and then forms extrusions that are taken up by Mφ. Interferon-induced GTPases are known to promote inclusions ubiquitination, leading to premature inclusion lysis. Bacterial lipid antigens are presented to iNKT cells via CD1d molecules on the surface of Mφ and DC. Activated NKT and NK promote DC maturation through the release of IFN-γ and positive feedback from cell-to-cell interactions. Rab proteins involved in the DC endocytic cycle are recruited around the inclusions and impede MHC-I intracellular trafficking. Notably, MICA upregulation occurs in parallel with MHC class I downregulation, affecting the sensitivity of C. trachomatis -infected cells to NK cell activity. Firstly, resisting the phagocytic bactericidal effect of the phagocyte. C. trachomatis resists phagocytes through secreting specific effector proteins that evade the capture of NETs and prevent the activation of Mφ. In addition, the ability of nascent inclusions to evade fusion with phagocytic lysosomes is also related to these effector proteins, for example, IncE disrupts retromer and lysosome function by binding SNXs 5 and 6 (sorting nexin). However, the mechanism is not clear ( 38 ). Secondly, blocking the activation of the lymphocytes. The persistent presence of C. trachomatis in innate immune cells may due to the pressure exerted by T lymphocyte-mediated immunity, which is the primary defense mechanism of the host against C. trachomatis infection. This prompts C. trachomatis to interfere with host antigen presentation by downregulating MHC molecules on the surface of target cells, including downregulation of MHC class I molecules that impede CD8 + CTL activation and downregulation of IFN-γ-induced MHC class II molecules that impede CD4 + T lymphocyte activation. Thirdly, reproducing in immune cells (anti-apoptosis). In vitro experiments have shown that the surface structures of EB, like LPS and certain proteins, promote endocytosis of Chlamydia by susceptible cells ( 25 ). For example, the pore protein OmpA of the C. trachomatis outer membrane and the plasmid-encoded Pgp3 respectively inhibit apoptosis by targeting the pro-apoptotic proteins Bax and Bak ( 107 ), or block the activation of the apoptotic signaling pathway ( 108 ), which facilitates the pathogen to use the host cells for nutrition to multiply in and survival. In addition, C. trachomatis converts from RBs to AB (aberrant body) by changing the expression of HSP60, outer membrane proteins and LPS when it enters a crypt-infected state under the influence of external stresses (antibiotic use, iron deficiency or co-infection). This process is convenient for C. trachomatis to escape the anti-infective immune response of the host. ABs can be converted back to RBs in an active state, then RBs transformed into infectious EBs and released from the target cells when the external pressure is reduced or removed. This release mechanism is associated with the CPAF protein ( 32 , 109 ). Fourth, inducing the immune cells to apoptosis or directly killing immune cells. Host exposure to Chlamydia infection is known to exhibit high levels of metabolism, including sugar metabolism, nucleotide metabolism, etc. , which is attributed to the reproduction-dependent nature of the bacteria. Moreover, Chlamydia infection causes excessive production of reactive oxygen species (ROS), causing oxidative DNA damage, resulting in single-strand breaks and even double-strand breaks, which can severely damage host cells. For example, Chlamydia not only causes macrophage foam ( 110 ), but also stimulates macrophages to produce TNF-α and induce apoptosis in neighboring T cells ( 111 , 112 ). Hydrogen sulfide (H2S)-mammalian endogenous signaling gas transmitter is reported to exert protective effects on various innate immune cells against damage from ROS, immune or inflammatory hyperactivation, and also to control differentiation, maturation or polarization of immune cells (e.g. M2 polarization of macrophages) ( 113 ). Nowadays, there are more studies on the interaction between C. trachomatis and classical innate immune cells, such as Mφ, NE and DC, but few studies on ILCs and ILLs. And the detailed aspects of how C. trachomatis evades innate immune cell pursuit need to be explored in depth by further techniques. Cellular immunotherapies have been reported to be emerging in the field of cancer ( 114 ), but this has rarely been studied in pathogenic infections. Therefore, an in-depth understanding of the interaction between Chlamydia and innate immune cells will provide further therapeutic interventions to combat this intractable epidemic.

Unlike other bacteria, C. trachomatis has a unique biphasic developmental cycle. In the initial step, non-replicating elementary bodies (EBs) bind to the host acetyl heparan sulfate proteoglycan (HSPG) and primarily the receptor tyrosine kinase (RTK), injecting various effector proteins called C. trachomatis secretory proteins (CtSPs) into the cytoplasm via the type III secretion system (T3SS) or other secretory mechanisms ( 32 , 33 ). After entry, they differentiate into non-infectious replicating reticulate bodies (RBs) within parietal vesicles called inclusions, and RB-secreted inclusions membrane proteins (Incs) are incorporated into the membranes of the compartment ( 34 ). The interaction of C. trachomatis proteins with host proteins involves altering vesicular transport in extracellular vesicles, regulating cell survival pathways, and suppressing the innate host immune response. The Chlamydial effector proteins interfere with the host’s innate immune response, such as INCs, TepP ( 35 ), CPAF, Pgp3 ( 36 ), Pgp4 ( 37 ) and 60 other proteins ( 38 ), which promote intracellular survival of Chlamydia and limit the host response to infection. RBs continue to differentiate in inclusions and, at later stages, asynchronously undergo secondary differentiation to create new EBs. Studies have shown that C. trachomatis divides by a polarized budding mechanism, rather than binary fission ( 39 ). In the final process, intracellular EBs are release by two pathways described so far: lysis cell destruction or exit by extrusion formation. The particular form of cyclic propagation from EB to RB to EB occurs repeatedly in neighboring cells of the host, which takes approximately 36-48h to complete a developmental cycle. The first line of defense in the human immune system is the skin and mucous membranes, and the second line is the phagocytes and bactericidal substances in the body fluids, which together constitute innate immunity. C. trachomatis genital serovars have a tropism for columnar epithelial cells located on mucosal surfaces ( 40 , 41 ). On the one hand, host epithelial cells recognize the invasion of C. trachomatis antigens by surface receptors, endosomal receptors, and innate immune factors. These antigens are first blocked by the mucosal barrier and neutralized by mucosal antibodies. Microbiota namely lactobacillus along with lactoferrin confer an important mucosal defense in the cervicovaginal region ( 42 ). Upon breach of this barrier, the infected cells release cytokines and chemokines ( 43 ) that serve to recruit cells like neutrophils and monocytes as well as others that serve to curtail the infection and limit its spread ( 44 ). Macrophages engulf the bacteria and produce pro-inflammatory factors ( 45 ); IFN-γ secreted by NK cells not only kills infected host cells but also induces an immune response to Th1 ( 46 , 47 ); When infection leads to the development of antigen-specific immunity, CD4 + T cells along with B cells produce immunity where chlamydia -specific Th1 CD4 + T cells and antibodies are considered protective ( 48 , 49 ), whereas CD8 + T cell response is considered non-essential or even pathogenic ( 50 , 51 ). These cells interact and collaborate to clear C. trachomatis . On the other hand, this infection stimulates the establishment of immunogenicity is necessary to generate a good protective immune response. Chlamydia has evolved to evade immunity as well as actively subvert it by inhibiting the cytokines and chemotactic proteins produced by epithelial cells ( 52 , 53 ). It interferes with the antigen-presenting function of antigen-presenting cells (APCs) (downregulation of MHC class I and II molecules) ( 54 , 55 ), regulating specific cytokines with multiple effects (IL-18, IFN-γ, TNF-α), and anti-apoptosis (increased cell survival signaling and CPAF release) ( 56 – 58 ). In addition, recent studies have shown that intracellular RBs can enter uninfected neighboring cells via tunneling nanotubes (TNT) ( 59 ). This allows Chlamydia to remain unexposed to body fluids, thus evading to some extent the pursuit of various cytokines in body fluids.

XW: Writing – review & editing, Writing – original draft. HW: Writing – review & editing. CF: Writing – review & editing. ZL: Writing – review & editing.