**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].
