Background
Chlamydia trachomatis causes the most frequent sexually transmitted bacterial infections [
1‐
3], which, if untreated, can lead to complications characterized with tubal inflammatory damages, including pelvic inflammatory diseases, ectopic pregnancy and infertility [
1,
4,
5]. Although both intracellular replication of
C. trachomatis organisms and host responses to
C. trachomatis antigens may significantly contribute to inflammatory pathologies [
6‐
9], the precise pathogenic mechanisms of
C. trachomatis-induced diseases remain unknown. In addition, there is still no licensed
C. trachomatis vaccine [
10] despite the urgent need and extensive efforts in searching for such a vaccine.
Previous immunological studies, mainly based on a
C. muridarum intravaginal infection mouse model, have revealed that a Th1-dominant cell-mediated immunity is required for protection against Chlamydia urogenital tract infection [
10‐
12]. It is also hypothesized that excessive and/or prolonged cellular (particularly CD + 8 T cell) responses may contribute to tubal pathologies during chlamydial infection [
13,
14]. However, how these protective and pathogenic cellular responses are regulated remains unknown. Both Tim-3 (T cell immunoglobulin and mucin domain 3) and PD-1 (Programmed death one) are negative regulators of T cell responses [
15,
16]. We evaluated the role of these two negative regulatory signaling pathways in chlamydial urogenital infection in the current study.
Tim-3-mediated signal inhibits both CD4+ Th1 and CD8+ T cell responses, which may prevent unintended tissue inflammation [
17]. However, inappropriate activation of Tim-3 signals may lead to premature T cell exhaustion, thus, permitting persistent or chronic infection [
18‐
21]. Tim-3 has emerged as a promising therapeutic target to correct abnormal immune function in several autoimmune and chronic inflammatory conditions [
22]. PD-1 is an inducible molecule on activated T and B lymphocytes and plays a critical role in controlling lymphocyte activation and maintaining peripheral tolerance [
19,
23]. PD-L1, the primary regulatory counter-receptor for PD-1 in the peripheral tissues is broadly inducible in various tissues and cell types [
23‐
26]. The interaction between PD-1 and PD-L1 plays a critical role in determining the fate of T-cell activation and tolerance during T-cell priming [
23]. Like Tim-3, inappropriate activation of PD-1 signaling can lead to immune suppression and persistent/chronic infection [
27]. For example, PD-1-PD-L1 pathway has been shown to impair Th1 immune response in the late stage of infection with
Mycobacterium bovis bacillus Calmette-Guérin, thereby facilitating the bacterial persistence in the host [
28]. Decrease in the exhaustion markers PD-1 and TIM-3 in T cells correlates with reduction of
Mycobacterium tuberculosis load in the lungs [
29]. Thus, blocking PD-1 signaling pathway may prevent persistent infection.
However, Targeting the PD-1-PD-L1 pathway alone does not always result in complete restoration of T cell function [
30]. Double blocking with neutralization antibodies against both Tim-3 and PD-L1 has been shown to restore T cell function in both solid tumor-bearing mice [
31] and mice chronically infected with viruses [
32], leading to controlling tumor growth and viral infection respectively. Thus, in the current study, we used a combined blocking approach to assess the effect of Tim-3 and PD-L1 signaling pathways on Chlamydia infection in a
C. muridarum intravaginal infection mouse model. We found that the
C. muridarum organism shedding time course after an intravaginal infection was not altered despite the double blocking. However, the tubal pathology following the
C. muridarum infection was more severe in mice treated with neutralization antibodies targeting both Tim-3 and PD-L1. These observations suggest that Tim-3 and PD-L1 signaling may play an important role in reducing pathologies in the upper genital tract after chlamydial infection.
Materials and methods
Mouse infection, antibody treatment and titration of live organism shedding
C. muridarum Nigg strain (also called MoPn) organisms used in the current study were grown in HeLa cells (ATCC, Manassas, VA 20108), purified and titrated as described previously [
9]. Female Balb/c mice were purchased at the age of 6 to 8 weeks old from Charles River Laboratories, Inc. (Wilmington, MA). Each mouse was inoculated intravaginally with 2 × 10
4 IFUs of live
C. muridarum organisms as described previously [
9]. After infection, the mice were treated with either neutralization antibodies or isotype control IgG for 12 days as following: For the anti-Tim-3 + PD-L1 group, each mouse was injected intraperitoneally (i.p.) with 100 μg of anti-Tim-3 (clone# 8B.2 C12; Rat IgG1, cat#16-5871, eBioscience, San Diego, CA) on days 0 (the same day of infection), 2 and 4, 200 μg of anti-PD-L1 (clone 10 F.9 G2, Rat IgG2b, cat#124309, Biolegend, San Diego, CA) on days 0, 3, 6, 9 and 12. For the control group, each mouse was similarly injected with the equivalent amounts of isotype-matched rat IgGs following the same injection schedule. After the intravaginal infection, vaginal swabs were taken once every 7 days until two consecutive negative detection results were obtained from the same mouse. The chlamydial organisms released from swabs were inoculated onto HeLa cell monolayers in duplicates as described previously [
8]. An immunofluorescence assay was used to quantitate live organisms (expressed as inclusion forming units or IFUs) from each swab as described previously [
9]. The number of IFUs/swab was converted into log
10 and the log
10 IFUs were used to calculate means and standard deviation for each group at each time point.
Evaluating mouse genital tract tissue pathologies and histological scoring
Mice were sacrificed 60 days after infection and the mouse urogenital tract tissues were isolated. Before the tissues were removed from mice, an
in situ gross examination was performed for evidence of hydrosalpinx formation and any other abnormalities. The excised tissues were processed for making serial sections. Efforts were made to include cervix, both uterine horns and oviducts as well as lumenal structures of each tissue in each section. The sections were stained with hematoxylin and eosin (H&E) and assessed by a board certified pathologist (I-T.Y) blinded to mouse treatment for severity of inflammation and pathologies based on the modified schemes established previously [
8]. The uterine horns and oviducts were scored separately. Scoring for dilatation of uterine horn or fallopian tube: 0, no significant dilatation; 1, mild dilatation of a single cross section; 2, one to three dilated cross sections; 3, more than three dilated cross sections; and 4, confluent pronounced dilation. Scoring for inflammatory cell infiltrates (at the chronic stage of infection, the infiltrates mainly contain mononuclear cells): 0, no significant infiltration; 1, infiltration at a single focus; 2, infiltration at two to four foci; 3, infiltration at more than four foci; and 4, confluent infiltration. Scores assigned to individual mice were calculated into means ± standard errors for each group of animals.
Immunofluorescence assay
For titrating IFUs from mouse swab samples, HeLa cells grown on glass coverslips in 24 well plates were inoculated with serially diluted swab samples and processed for immunofluorescence assay 24 h after infection. The cell samples were labeled with Hoechst (blue, Sigma) for visualizing DNA and a rabbit anti-chlamydial chaperon cofactor antibody (unpublished data) plus a goat anti-rabbit IgG conjugated with Cy2 (green; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) for visualizing chlamydial inclusions. The immuno-labeled cell samples were quantitated and used for image analysis and acquisition as described previously [
33,
34]. For titrating anti-
C. muridarum antibodies in mouse serum samples, the
C. muridarum-infected HeLa cells were processed and added with serially diluted mouse serum samples. After the primary antibody binding, a goat anti-mouse IgG conjugated with Cy3 (red; Jackson ImmunoResearch Laboratories) was used to visualize the mouse antibody binding to
C. muridarum organisms. The antibody titer was defined as the highest dilution that still showed positive fluorescence labeling and expressed as log10 dilutions for statistic analyses.
Enzyme-Linked Immunosorbent Assay (ELISA)
Cytokines from mouse vaginal swabs and mouse splenocyte culture supernatants (after in vitro restimulation) were measured using Bio-plex Pro™ mouse cytokine standard Group I 23-plex (cat# 171I5001, BIO-RAD, Hercules, CA) and Group II 9-plex (cat# 171I6001) kits by following the manufacturer's instruction. Since all 32 cytokine beads are uniquely labeled, they were mixed and used in the same assays. For swab samples, after the C. muridarum organism titration, each of the left over swab samples was diluted 1:2 in PBS containing 10% fetal bovine serum. 50 ul of the diluted sample was added to each reaction well containing the 32 cytokine beads (each type of beads is covalently coated with a capture antibody specifically recognizing one cytokine). To prepare the splenocyte samples, splenocytes were harvested from mice 60 days after C. muridarum infection and stimulated in vitro with UV-inactivated C. muridarum organisms (elementary bodies or EBs) or medium alone for 3 days. The culture supernatants were diluted 1:2 and 50 ul were used to react with the 32 cytokine beads. The bead capture antibody-bound cytokines were detected with biotinylated detection antibodies and phycoerythrin fluorescence-conjugated Avidin. The fluorescence-associated with each cytokine was quantitated using a Bio-Plex Luminex system (BIO-RAD). The concentrations of each cytokine were determined based on the standards measured in the same plate and sample dilution factors using Bio-Plex Manager software (BIO-RAD) and expressed as pg/ml. The 32 cytokines analyzed were: IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12 (p40), IL-12 (p70), IL-13, IL-17, Eotaxin, G-CSF, CM-CSF, IFN-γ, KC, MCP-1, MIP-1α, MIP-1β, RANTES, and TNFα, IL-15, IL-18, Basic FGF, LIF, M-CSF, MIG, MIP-2, PDGF-BB and VEGF.
Statistical analysis
ANOVA Test (
http://www.physics.csbsju.edu/stats/anova.html) was performed to analyze the IFU, cytokine & antibody titration data from multiple groups and a two-tailed Student
t test (Microsoft Excel) to compare two given groups. For qualitative data analyses, the Chi-square or Fisher's Exact tests were used.
Discussion
Using a combined blocking of Tim-3 and PD-L1, we assessed the role of Tim-3 and PD-L1 signaling pathways in chlamydial infection. Despite the double blockade, the time course of live organism shedding from the lower genital tract after
C. muridarum infection was not significantly altered. Since the double blocking with neutralization antibodies targeting both Tim-3 and PD-L1 has been shown to effectively suppress both Tim-3 and PD-L1 signaling pathways [
31,
32], the above result suggests that Tim-3- and PD-1-meditaed negative regulation signaling pathways might not significantly suppress host immunity against chlamydial infection in lower genital tract. This conclusion is consistent with the observations that treatment with anti-Tim3 and PD-L1 neutralizing antibodies did not alter either the lower genital tract local cytokine production or the systemic humoral or cellular responses to
C. muridarum infection. The fact that the antibody treatment did not affect the balance of Th1 versus Th2 responses may explain the failure of the antibody blocking to impact the lower genital tract infection time course. However, the treatment of mice with anti-Tim-3 and anti-PD-1 neutralization antibodies did significantly enhance pathologies in the upper genital tract, demonstrating that the antibody treatment was effective in blocking of Tim-3 and PD-L1 signaling pathways. More importantly, this observation suggests that Tim-3- and PD-L1-mediated negative regulation signaling pathways may play significant roles in reducing pathogenic responses induced by chlamydial infection in the upper genital tract. Apparently, this negative regulation didn't make the lower genital tract more susceptible to chlamydial infection since removal of the negative regulation did not reduce live chlamydial organism shedding.
Given the multifaceted roles of Tim-3 and PD-L1 signaling pathways, it is unexpected that double blocking of these two pathways did not alter
C. muridarum infection time course in the lower genital tract. The combined blocking of Tim-3 and PD-L1 has been shown to fully restore the functionality of exhausted T cells expressing both Tim-3 and PD-1 in tumor-bearing animals and chronically infected hosts with viruses [
31,
32]. Under these chronic pathogenic conditions, the negative regulatory roles of Tim-3 and PD-1 may be heightened or exacerbated. The impact of inhibition of the exacerbated negative regulation can be more sensitively detected immunologically and clinically. However, intravaginal infection with
C. muridarum is an acute infection. The role of Tim-3 and PD-1 in acute infection is not as well understood as in chronic infection. A recent study has shown that Th1 immune responses at the lower genital tract are regulated by IL-10-producing dendritic cells [
35], which may be independent of Tim-3- or PD-1-mediated mechanisms. In addition, it has been demonstrated that interaction of Tim-3 on T cells with its ligand Galectin9 (Gal9) on macrophages may promote clearance of intracellular
Mycobacterium tuberculosis [
36]. Tim3-Gal9 interaction leads to macrophage activation and stimulates bactericidal activity by inducing caspase-1-dependent IL-1β secretion [
36]. Thus, Tim-3 may also play a positive role in controlling chlamydial infection. Blocking Tim-3 signaling with neutralizing antibodies remove both the negative and positive roles of Tim-3 in chlamydial infection, resulting in null net effect on the live organism shedding time course. However, since the double antibody blocking treatment affected neither the lower genital tract local cytokine nor the systemic adaptive immune responses to
C. muridarum infection, it is more likely that the negative regulation mediated by Tim-3 and PD-L1 did not significantly inhibit host immunity during the acute phase of
C. muridarum in the lower tract.
The double antibody treatment significantly enhanced tubal inflammatory pathology, which is consistent with the overall concept that the negative regulation mediated by Tim-3 and PD-L1 plays an important role in minimizing inflammatory damages caused by excessive and prolonged immune responses [
26,
37]. What is the cellular basis of the tubal pathology-exacerbating responses negatively regulated by Tim-3 and PD-L1? Although Tim-3 and PD-1 can negatively regulate both CD4+ Th1 and CD8+ T cells, under various chronic infection or tumor-bearing conditions, the frequency of antigen-specific CD8+ T cells positive for both TIM-3 and PD-1 is the highest [
16,
19,
21]. Double blocking with neutralization antibodies to Tim-3 and PD-1 could fully restore the functionality of the exhausted CD8+ T cells. Thus, it is assumed that the enhanced anti-tumor or anti-viral effects restored by the anti-Tim-3 and PD-1 antibodies were due to the removal of negative regulation from CD8+ T cells. Although the cellular basis of inflammatory responses involved in tubal pathology after chlamydial infection is still not clear, some previous studies have correlated CD8+ responses with pathogenic responses in the upper genital tract [
13]. Indeed, mice deficient in MHC class I or CD8 displayed normal infection time courses in the lower genital tract [
12,
38] but developed less severe pathology in the upper genital tract. TNFα produced by CD8+ T cells may contribute to the tubal pathologies [
13]. It is possible that Tim-3 and PD-L1 may mainly target antigen-specific CD8+ T cells during chlamydial infection as observed during mycobacterial infection [
28].
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
BP & CL carried out most experiments. LT measured cytokines & IY evaluated pathology. ZH, YW & GZ conceived of the study, and participated in its design and drafted the manuscript. All authors read and approved the final manuscript.