Sexually transmitted
Chlamydia trachomatis infections continue to be a major public health concern due to high infection rates and long-term reproductive morbidities [
1,
2]. While antibiotic treatment is generally efficient in clearing the infection [
3], treatment failure and reoccurring infections are still common [
4]. Protective immunity against
C. trachomatis reinfections has been associated with interferon gamma (IFN-γ) production in several human studies [
5‐
7]. Various in vitro studies with
C. trachomatis-infected cell lines have also shown the important role of IFN-γ in eliminating infection [
8,
9], due to the induction of the enzyme indoleamine 2,3-dioxygenase 1 (IDO1) [
10,
11]. IDO1 is responsible for the catabolism of tryptophan to kynurenine [
12]. Depriving this essential amino acid from the tryptophan auxotroph
C. trachomatis have an inhibitory effect on infection in vitro. While high IFN-γ levels were shown to eradicate the infection, lower levels can drive urogenital
C. trachomatis to enter their persistence form, characterized by in vitro aberrant, non-infectious bodies [
13].
C. trachomatis aberrant bodies were also identified in infected women, and were associated with low IFN-γ levels in the genital tract [
14]. Furthermore, low vaginal tryptophan levels were shown to be associated with spontaneous resolution of
C. trachomatis infections in women [
15]. Although chlamydial death due to tryptophan depletion via IFN-γ-induced IDO1 axis, has been well characterized in vitro, relatively few studies have measured IDO1 expression levels and its enzymatic activity in the actual infection site [
16]. In a recent study, IFN-γ, tryptophan and kynurenine levels were measured in vaginal secretions of women who were infected with
C. trachomatis (single or repeated infection) versus uninfected women [
17]. It was reported that higher kynurenine to tryptophan ratios (kyn/trp) were associated with current, single or repeated
C. trachomatis infections. Specifically, it was found that women with repeated
C. trachomatis infection had significantly higher kynurenine levels in their vaginal secretions. High kyn/trp ratios however, did not correlate with the low IFN-γ levels measured from vaginal secretions of the same women. IDO1-IFN-γ axis, which is a well-known important antimicrobial mechanism [
18,
19], is also responsible for downregulation of the pro-inflammatory response in the host [
24‐
30]. The depletion of tryptophan, which is necessary for the survival and proliferation of T cells, causes their arrest in the G1 phase of the cell cycle [
25]. This is partly due to the induction of stress response triggered by GCN2, a stress kinase that is activated by the elevation in uncharged tRNAs [
24]. Tryptophan depletion can also inhibit T cell proliferation through inactivation of the mTOR pathway [
23]. Another mechanism is through tryptophan catabolites, such as 3-hydroxyanthranilic and quinolinic acids that can induce T-cell apoptosis [
22,
27]. Other kynurenine derivatives and kynurenine itself can induce the differentiation of naïve T cells to regulatory T cells (Tregs) [
21], through the activation of the AhR [
20]. IDO1 is inducible in different immune cells as well as mucosal epithelial cells, and is regulated by a complex immune signal cascade [
19]. Aside from IFN-γ stimulus, there are other cytokines which induce IDO1 expression, such as by IFN-α, IFN-β, TGF-β, TNF-α and IL-1β [
28‐
30]. In addition, lipopolysaccharide (LPS), extracted from cell membranes of gram-negative bacteria, was shown to induce IDO1 expression in IFN-γ-KO mice [
31]. The LPS-stimulated IDO1-induction was shown to be largely dependent on TNF-α. Furthermore, IDO1 can act as a signal transducer that contributes to long-term tolerogenic phenotype of plasmacytoid dendritic cells (pDCs) in response to the immune-regulatory cytokine transforming growth factor β (TGF-β) [
29]. TGF-β-conditioned pDCs induced forkhead box P3 (FoxP3), which is an essential transcription factor for the immuno-suppressive function of Tregs [
32], whereas IFN-γ conditioning induced T cell apoptosis and suppressed proliferation. These data have led us to hypothesize that low IFN-γ levels in repeatedly infected women, that had high kynurenine levels [
17], may be due to downregulation of the cytokine by the immune-regulatory properties of IDO1, as a result of recurrent chlamydial infections.
C. trachomatis was shown to have several immune evasion mechanisms that allow it to establish infection, which can often persist for long periods of time without any symptoms [
33]. These mechanisms include reduced inflammatory responses elicited by the chlamydial LPS, reduced adaptive immune response and enhanced survival inside the host cell (reviewed in [
34]). Here, we have focused on a selection of immune genes, IDO1, IFN-γ, TGF-β1 and FoxP3, to examine the regulatory immune response during
C. trachomatis single versus repeated infection and post antibiotic treatment, in a cohort of a previously published study [
17]. In addition, the expression levels of these genes were measured in vitro using a
C. trachomatis infected endometrium cell line (ECC1) co-cultured with PBMCs, with or without azithromycin treatment. We found that in women with repeated infections and in women who have cleared their infection after antibiotic treatment, immune-regulatory markers, IDO1, TGF-β1 and FoxP3 were significantly upregulated in comparison to
C. trachomatis negative women and in those with single current infection. We also show that IDO1 induction was not accompanied by elevated IFN-γ levels. Although IFN-γ-induced IDO1 activity and tryptophan depletion was previously proposed to be an anti-chlamydial mechanism, our data suggest that repeated
C. trachomatis infections, as well as antibiotic treatment, can induce IDO1 in an IFN-γ independent manner, that contribute to downregulation of the immune response. These mechanisms may assist the pathogen to avoid immunity and establish an infection.