Chlamydia trachomatis is the leading infectious cause of blindness worldwide. Persistent infection or recurrent infection with C. trachomatis, can stimulate a fibrotic immune response that leads to deposition of scar tissue on the upper tarsal conjunctiva and the development of a dry eye syndrome. The progressive development of scar tissue can lead to entropion and trachomatous trichiasis (TT), which if left untreated may result in damage to the cornea causing opacity, low vision, and blindness (Mabey et al., 2003). There are an estimated eight million individuals currently suffering from TT with about 1.3 million irreversibly blind as a result (Mariotti et al., 2009).

Chlamydia trachomatis is also the most common bacterial sexually transmitted infection. It is frequently asymptomatic, especially in women and can lead to sub-fertility, chronic pelvic inflammatory disease, ectopic pregnancy, and sterility (WHO, 2007). Although both ocular and genital chlamydial infections are treatable with antibiotics, persistent, and recurring infection is common. A licensed vaccine against C. trachomatis for use in the human population is not yet available despite continued efforts since the 1960s. Recent developments (reviewed by Rockey et al., 2009) have yielded encouraging results in some animal models; however, the development of an effective vaccine requires further understanding of the basic features of the human immune response to C. trachomatis infection. Indeed work in mice has suggested that chlamydial vaccine induced immunity without pathology can be achieved and this is distinct from immunity induced by infection that results in pathology (Igietseme et al., 2009). However in naturally exposed populations only a minority develops the pathological sequelae of infection, suggesting that the immunological features of naturally immune individuals may be equally distinct.

In animal models of infection it is well established that CD4+ T helper type 1 (Th1) cells are essential for the clearance of primary chlamydial infection (Igietseme et al., 2009). In particular, IFNγ plays an important role in the clearance of chlamydial infection. In mice and in humans its production is associated with protection from re-infection (Wang et al., 1999; Cohen et al., 2005). However, uncontrolled inflammatory responses, in part driven by IFNγ production, result in pathology. Regulatory T cells (Tregs), induction of regulatory cytokines such as IL-10 or the activation of type 2 (Th2) responses counteract the inflammatory environment and contribute to restoring homeostasis (Yang et al., 1999; Wang et al., 2005). Conversely unchecked type 2 responses are frequently associated with chronic inflammation and infection (Wynn, 2004) and hampering Th2 responses have been implicated in chlamydial infections and induced disease (Holland et al., 1993, 1996; Gondek et al., 2009). In addition some immune mediated fibrotic diseases, such as schistosomula induced hepatic fibrosis, are also dominated by Th2-cytokine responses (Chiaramonte et al., 2003). The pathogenic changes observed at the site of infection in chlamydial genital and ocular disease are also due to development of fibrotic tissue yet convincing evidence of a significant role for polar CD4+ Th2 responses or alternative macrophage activation are lacking (Holland et al., 2010; Natividad et al., 2010). Many groups have now demonstrated the important role of immunoregulatory subsets of CD4+ T cells in preventing immune damage during viral, bacterial, and parasitic infections, and specifically in persistent infections at mucosal surfaces (Maloy and Powrie, 2001; Belkaid and Tarbell, 2009) but these have not been extensively studied in human chlamydial disease.

One subset of CD4+ T cells that has key functions in immune homeostasis are Tregs (CD4+CD25+FOXP3+). The regulatory function of these natural and adaptive Tregs may be mediated by production of TGF-β and IL-10 or by cognate interaction (Hori et al., 2003; Fontenot et al., 2005). Antigen-specific IL-10 secreting type-1 T regulatory cells (T_R1) which do not express FOXP3 have also been described in vivo following chronic antigenic stimulation (Vieira et al., 2004), and develop in parallel with Th1 cells in several chronic infectious diseases (McGuirk et al., 2002). Murine studies have established that FOXP3 + regulatory cells can secrete both IFNγ and IL-10 (Stock et al., 2004) and in humans regulatory function can also be exerted via perforin dependent lysis (Grossman et al., 2004). Thus, although most work has focused on cells with a classic Treg phenotype (CD4+CD25+FOXP3+), cells from CD4+CD25−, CD8+, and NKT subsets can all exert regulatory function (Jiang and Chess, 2004). There is also evidence that CD4+ Th1 cells can exhibit self-regulatory properties and cytokine profiles, obviating the need to explain disease in terms of a separate regulatory class of T cells (Anderson et al., 2007; Jankovic et al., 2007).

We have previously demonstrated a relative Chlamydia-specific hypo-responsiveness associated with chronic ocular infection (Holland et al., 1993; Bailey et al., 1995), and described imbalances in antigen-specific secretion of both IL-10 and IFNγ in cultured peripheral blood mononuclear cell (PBMC) from scarred subjects (Holland et al., 1996). We have also found that IFNγ, IL-10, and perforin expression in the conjunctiva is associated with the clinical signs of active trachoma (Burton et al., 2004); that susceptibility to scarring sequelae is affected by haplotypic variation at the IL-10 locus (Natividad et al., 2005, 2008), that contributes to variation in IL-10 expression; and that the expression of FOXP3 is up-regulated in individuals who have recently resolved ocular C. trachomatis infection (Faal et al., 2006).

In animal models of viral ocular infection, several groups have identified an important role for Tregs in modulating the severity of disease (Suvas et al., 2004; Nesburn et al., 2007). Aside from FOXP3, other potential makers of regulatory T cells have been identified in the search to phenotypically define Tregs. Most commonly the relative expression of the IL-7 receptor (CD127) is used as a fourth surface marker (Finney et al., 2010), the low expression of which is regarded as further evidence of Tregs. Two other common markers with demonstrated independent down regulatory properties are Cytotoxic T Lymphocyte Antigen 4 (CTLA4) and Glucocorticoid-induced tumor necrosis factor-receptor related protein (GITR) both of which have been linked to the suppressive mechanism of Tregs (Zheng et al., 2004; Nocentini and Riccardi, 2005). Moreover, in rabbits with ocular HSV infection, cells in the conjunctiva, which expressed high levels of FOXP3, GITR, and CTLA4, were effectively able to suppress the effector function of HSV-specific CD4+ and CD8+ T cells (Nesburn et al., 2007).

In this study we compared the in vitro immune responses of individuals with TT and controls with normal eyes from trachoma endemic communities, since we expected that immune responses from these individuals would be the most polar. We investigated the immune responsiveness of several chlamydial antigens that had not previously been measured in individuals from trachoma endemic communities. Cytokine levels in supernatants from proliferation assays were quantified for IL-5, IL-10, IL-12p70, IL-13, IFNγ, and TNFα. Immune responses were also assessed using flow cytometry, where cells were stained for IFNγ and IL-10, as well as Treg cell markers FOXP3, CTLA4, and GITR. To investigate which cells were the major sources of IFNγ, we further stained for markers that included CD8 and CD3−CD56+ cells in PBMC isolated from young and adult individuals from the same trachoma endemic environment.

Both TT cases and controls had lymphoproliferative responses that were above background when stimulated with EBs, Enolase, PmpD, PmpG, and Omp2 antigens. Responses to some of these antigens have been investigated as putative vaccine candidates (Coler et al., 2009; Goodall et al., 2001a,b) and studied in human infection. More recently, a number of chlamydial antigens such as LcrE (CPn0324/CT089) and chlamydial protease-like activating factor (CPAF/CT858) have been proposed as potential vaccine candidates and their responsiveness has been tested in animals and humans (Olsen et al., 2006, 2007, 2010; Thorpe et al., 2007; Barker et al., 2008; Follmann et al., 2008). Although most of the TT study participants were able to elicit proliferative responses to the chlamydial antigens, the degree of proliferation was not significantly different between individuals with severe scarring disease and controls without disease. As a general trend responses to each antigen were higher in cases with TT, in contrast to our previous findings (Holland et al., 1993, 1996). However, in previous studies higher proportions of individuals with trachomatous scarring were still infected with C. trachomatis and were at earlier stages of disease, where C. trachomatis driven fibrotic responses could still be progressing. We also previously found that serum antibody responses in those with trachomatous scarring were higher compared to controls whereas in this cohort C. trachomatis serovar A antibody responses were equal between the groups. This suggested that both groups of individuals had experienced similar levels of exposure to ocular infection and that there had been little recent infection. This changing environment could explain why we did not detect any significant differences compared with our own previous findings (Harding-Esch et al., 2009b).

Although MOMP constitutes up to 60% of an EB it nevertheless contains a significant proportion of the total antigenic repertoire (Wang et al., 2010). It is the most abundant surface exposed protein of EBs, is highly conserved across C. trachomatis serovars containing a large number of T cell specific epitopes (Kim and DeMars, 2001; Nunes et al., 2010) and anti-MOMP antibodies have been shown to neutralize C. trachomatis in vitro (Cotter et al., 1995). Because of these properties it has been widely studied as a putative vaccine candidate (Zhang et al., 1997). More recently native MOMP (nMOMP) has been used to immunize monkeys and successfully induced high titers of neutralizing antibodies that reduced the infectious ocular burden within the first 2 weeks of infection. Despite the promising results nMOMP immunization had no lasting effects on the course of clinical disease or burden and duration of infection (Kari et al., 2009). In our experiments MOMP induced only weak responses in the majority of individuals. Antibodies specific to PmpD have also been shown to neutralize C. trachomatis in vitro regardless of serotype (Crane et al., 2006), and Omp2 has been shown to be recognized by Chlamydia-specific human CD8+ T cells (Gervassi et al., 2004). PmpG which is part of the outer membrane complex and may be involved in the invasion of the host cell by C. trachomatis was also tested. Each of these antigens induced responses but these were not significantly different between cases and controls. Thus far, with the exception of MOMP and hsp60, no other antigens have been evaluated by measuring cell mediated immune responses in individuals with trachoma. Skwor et al. (2010) have tested human tear fluid for antibody reactivity to a number of antigens (cHSP60, CPAF, and CT795) and found that IgG responses to CPAF were associated with TT cases and responses to all three antigens were elevated during inflammation, but it remains unclear how this might relate to cellular responses and requires further investigation.

Interleukin-10 and IFNγ have been extensively studied in mice and humans with respect to chlamydial infection and IL-10 has been found to be associated with the persistence of infection, while IFNγ has been linked to its resolution (reviewed in Yang, 2003). One study using IFNγ knockout mice infected with C. muridarum found that production of IL-5 was elevated and correlated with more severe disease (Wang et al., 1999). Another study found that when murine dendritic cells were infected with C. trachomatis, the subsequent production of IL-12 and TNFα correlated with containment of the bacteria (Knight et al., 1995). Furthermore, studies have shown that mice infected with C. muridarum that received CD11c+ CD8α+ cells had increased amounts of proinflammatory cytokines such as IFNγ, TNFα, and IL-12 and decreased production of the Th2 cytokines IL-4, IL-5, and IL-13, which correlated with protection against infection. Our results show that in peripheral blood of individuals exposed to C. trachomatis, most chlamydial antigens tested were able to produce above background levels of IL-10, IL-12, IFNγ, and TNFα cytokines after 6 days of culture. Only PmpD and Enolase were able to stimulate IL-5 and IL-13 cytokine responses respectively. However, there were no clear indications that any particular antigen or cytokine profile was significantly different between cases and controls. This suggests that a larger population study is required with follow-up and repeat testing, or that the methods employed in vitro are not sufficiently sensitive to detect differences in these particular cytokines.

Whilst the roles of IL-10 and IFNγ have been intensively studied in chlamydial disease, there has been little investigation of regulatory T cells in mediating the response to C. trachomatis. Our results show that Tregs (CD4+CD25+FOXP3+) and the co-stimulatory surface markers with inhibitory or regulatory functions (CTLA4 and GITR) are up-regulated in response to stimulation with chlamydial EBs. We also found higher levels of CD4+CD25+FOXP3+ and CD4+GITR+ T cells in TT cases compared to controls (although these differences were not statistically significant). Other authors have suggested that the CD4+CD25+FOXP3− can also represent an effector population (Burl et al., 2010); whilst these were increased over co-stimulation alone by stimulation with EB, the levels were not significantly different between TT cases and unaffected controls. Interestingly, we found that IFNγ produced in response to C. trachomatis EB was not exclusive to CD4+ lymphocytes, and the in vitro nature of the stimulation with non-viable EBs would not efficiently favor stimulation of CD8+ T cells. Given that <50% of the total IFNγ production appeared to be from CD4+ T cells, we investigated using flow cytometry which cells were responsible for the early IFNγ production. We found that even with a restricted sample size and therefore statistical power, CD3−CD56+ NK cells were a major source of very early IFNγ production (within the first 18 h of in vitro restimulation). Since the magnitude of this response increased with age and the frequency and duration of trachoma decrease with age (Bailey et al., 1999; Faal et al., 2006), it is possible that these cells play a significant role in ocular disease and infection. Such responses are not unique and are in line with those observed for responses following vaccination or as a result of protozoan or viral infection (Horowitz and Riley, 2010; Horowitz et al., 2010a,b; McCall et al., 2010). Interestingly, in subjects with active trachoma and infection, host gene transcriptional profiles in the conjunctiva suggest a previously unappreciated role for NK cells (Natividad et al., 2010). These initial in vitro experiments suggest that NK or NK-like cells are rapidly induced by EB and supports the suggestion that lymphocytes other than CD4+ T cells contribute a significant proportion of IFNγ in the periphery. EB stimulation also leads to the early activation of other non-CD4+ T cells along with IL-10 production. We did not investigate the phenotypes of the cells that are activated or produce IL-10 and this warrants further more detailed investigation. Some in vitro studies have suggested that stimulated T cells rapidly acquire CD56 when strongly stimulated through the CD3/TCR complex with additional IL-2 (Kelly-Rogers et al., 2006) and that the phenotypic profile of NK cells is heterogeneous. Since we stained for both surface and internalized CD3 to identify the classical NK cell population (CD3−CD56+; Meier et al., 2005; Titanji et al., 2008; Li et al., 2009; Linn et al., 2009) we exclude this possibility as an in vitro artifact and suggest that the interaction with antigen-experienced T cells should be investigated further in response to chlamydial infection.

The onset of TT usually occurs well into adulthood and previous studies have revealed that C. trachomatis is rarely detected in the conjunctiva at that point (Burton et al., 2007). Intriguingly, TT cases continue to progress toward more severe trichiasis and eventually blindness despite their peripheral immune response being comparable to that of individuals with equal exposure to infection but without the clinical signs or sequelae of trachoma. This would suggest that adaptive T cell memory responses in the periphery do not reflect clinical disease status and that other factors such as innate inflammatory responses involving polymorphs (such as neutrophils) and host epithelial cells, continue to drive fibrosis or recurrent trichiasis. This is supported by several previous studies linking conjunctival gene expression and trichiasis (Burton et al., 2010, 2011; Holland et al., 2010). Other factors such as the compromised ocular surface and secondary bacterial infection also contribute to continued inflammation and disease. Thorough sampling of the immune response at the site of infection and a more in-depth examination of the interaction of adaptive T cell responses and NK cell responses may help further probe the mechanisms of immunopathogenesisof trachoma.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflictof interest.