Figures
Abstract
Background
Chlamydia trachomatis is responsible for trachoma, the primary cause of preventable blindness worldwide. Plans to eradicate trachoma using the World Health Organization's SAFE program (Surgery, Antibiotics, Facial Cleanliness and Environment Improvement) have resulted in recurrence of infection and disease following cessation of treatment in many endemic countries, suggesting the need for a vaccine to control infection and trachomatous disease. Vaccine development requires, in part, knowledge of the mucosal host immune responses in both healthy and trachomatous conjuctivae—an area of research that remains insufficiently studied.
Methodology/Principal Findings
We characterized 25 secreted cytokines and chemokines from the conjunctival mucosa of individuals residing in a trachoma endemic region of Nepal using Luminex X100 multiplexing technology. Immunomodulating effects of concurrent C. trachomatis infection were also examined. We found that proinflammatory cytokines IL-1β (r = 0.259, P = 0.001) and TNFα (r = 0.168, P<0.05) were significantly associated with trachomatous disease and concurrent C. trachomatis infection compared with age and sex matched controls from the same region who did not have trachoma. In support of these findings, anti-inflammatory cytokine IL-1 receptor antagonist (IL-1Ra) was negatively associated with chronic scarring trachoma (r = −0.249, P = 0.001). Additional cytokines (Th1, IL-12p40 [r = −0.212, P<0.01], and Th2, IL-4 and IL-13 [r = −0.165 and −0.189, respectively, P<0.05 for both]) were negatively associated with chronic scarring trachoma, suggesting a protective role. Conversely, a pathogenic role for the Th3/Tr1 cytokine IL-10 (r = 0.180, P<0.05) was evident with increased levels for all trachoma grades. New risk factors for chronic scarring trachoma included IL-6 and IL-15 (r = 0.259 and 0.292, respectively, P<0.005 for both) with increased levels for concurrent C. trachomatis infections (r = 0.206, P<0.05, and r = 0.304, P<0.005, respectively). Chemokine protein levels for CCL11 (Eotaxin), CXCL8 (IL-8), CXCL9 (MIG), and CCL2 (MCP-1) were elevated in chronic scarring trachoma compared with age and sex matched controls (P<0.05, for all).
Conclusions/Significance
Our quantitative detection of previously uncharacterized and partially characterized cytokines, a soluble cytokine receptor, and chemokines for each trachoma grade and associations with C. trachomatis infections provide, to date, the most comprehensive immunologic evaluation of trachoma. These findings highlight novel pathologic and protective factors involved in trachomatous disease, which will aid in designing immunomodulating therapeutics and a vaccine.
Author Summary
Trachoma, a disease of antiquity dating back to the 16th century B.C.E., predominates among developing countries, where it remains the primary cause of preventable blindness worldwide. In trachoma, recurrent Chlamydia trachomatis bacterial infections during childhood are thought to result in inflammation and subsequent conjunctival scarring that can progress to trichiasis (TT; chronic trachoma; inversion of ≥1 eyelash that touches the globe of the eye). The trachomatous follicular grade (TF; active disease) is a self-limiting disease, suggesting the coexistence of protective inflammatory proteins. The trachomatous inflammatory grade (TI; active disease) is more likely to progress to trachomatous scarring (TS; chronic trachoma). To date, there are only a handful of studies that have examined the immune response in trachoma, and these were primarily based on gene expression. Characterizing quantified conjunctival mucosal immune differences for secreted proteins among individuals with no, active, and chronic trachoma may identify protein biomarkers associated with protection versus disease, which would greatly aid our understanding of the immunopathogenesis of trachoma. In this study, we characterized 25 cytokine and chemokine proteins for all trachoma grades. We identified eight cytokines and chemokines as risk factors for chronic trachoma and four as protective. Together, these findings further characterize the immunopathologic responses involved during trachoma, which will likely aid in the design of a vaccine and immunomodulating therapeutics for trachoma.
Citation: Skwor TA, Atik B, Kandel RP, Adhikari HK, Sharma B, Dean D (2008) Role of Secreted Conjunctival Mucosal Cytokine and Chemokine Proteins in Different Stages of Trachomatous Disease. PLoS Negl Trop Dis 2(7): e264. https://doi.org/10.1371/journal.pntd.0000264
Editor: Albert I. Ko, Weill Medical College of Cornell University, United States of America
Received: December 18, 2007; Accepted: June 17, 2008; Published: July 16, 2008
Copyright: © 2008 Skwor et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This research was supported by Public Health Service grants R01 EY/AI12219 (to DD) and R01 AI039499 (to DD), both from the National Institutes of Health. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Trachoma, the leading global cause of preventable blindness, has plagued populations for thousands of years [1]. There are over 360 million trachoma cases of whom ∼6 million are blind [2],[3]. Limited access to clean water and inadequate sanitary conditions provides ideal conditions for the persistence of this endemic disease caused by the obligate intracellular bacterium Chlamydia trachomatis. Additionally, C. trachomatis is the leading bacterial cause of sexually transmitted diseases (STD) throughout the world [4], causing chronic conditions such as arthritis [5], infertility and ectopic pregnancy [6]. The economics and health burden from trachoma alone claims billions of dollars in productivity loss in already poverty stricken countries [7]. Recognizing this, the World Health Organization (WHO) proposed the SAFE program (Surgery, Antibiotics, Facial cleanliness and Environmental improvement) in 2001 with the goal of eradicating blinding trachoma by the year 2020.
In trachoma endemic villages, disease progression is associated with repeated C. trachomatis infections of the conjunctivae, which can eventually lead to scarring and blindness from trachomatous trichiasis (TT; ≥1 in-turned eyelash) [1]. Recently, we and others have demonstrated that the S and A components of the SAFE program results in recurrent C. trachomatis infections and disease in endemic countries 6–24 months following cessation of treatment [8],[9],[10],[11],[12],[13]. Additionally, the difficulty in implementing and sustaining the F and E programs in resource poor countries suggests that a vaccine is the most effective method of control.
Designing efficacious vaccine candidates requires, in part, characterization of host inflammatory risk factors versus protective immunological components that are associated with the different grades of trachoma. These include follicular trachomatous inflammation (TF); intense trachomatous inflammation (TI); trachomatous conjunctival scarring (TS), trachomatous trichiasis (TT) and corneal opacity (CO) [14]. A limited number of animal models and human studies have begun to elucidate the host immune response in trachoma. The use of Giemsa stains and immunohistochemistry has characterized active trachoma (TF, TI or both) in young children as an epithelial and stromal infiltrate containing neutrophils [15], macrophages, dendritic cells, and B and T lymphocytes [16]. Physiologic characterization has been obtained from non-human primate models where trachoma-induced immunopathology consisted of conjunctival follicles containing B cells coexisting with a rich T lymphocyte population in the perifollicular area [17],[18]. These T lymphocytes were associated with macrophages, and epithelial and stromal cells. The quantity and size of trachoma-related follicles have been shown to directly correlate with the frequency of repeated C. trachomatis infections in the non-human primate model [17].
Characterization of intense inflammation during early stages of trachomatous disease exhibits elevated transcriptional levels of the proinflammatory cytokines TNFα and IL-1β, and amplified expression in the presence of C. trachomatis infection in human populations [19],[20],[21]. These studies also linked elevated Th1 cytokines IL-12 and IFNγ mRNA levels in conjunctival samples with TI cases [19],[20],[21],[22] and diminished levels in the presence of scarring [19]. No mRNA expression was detected for the Th2 cytokines IL-4 and IL-5 for any trachoma grade [19],[21]. However, elevated IL-10 mRNA levels were apparent in TF and TI cases who were also infected with C. trachomatis [21],[22]. Together these data suggest Th1 polarization as protective and IL-10 as a risk factor for exacerbating disease progression.
As trachomatous scarring develops, there is a diminished lymphocytic proliferative response to C. trachomatis elementary bodies (EBs) in both cynologus monkeys [23] and humans [24],[25]. Significantly elevated levels of T cytotoxic/suppressor cells compared to helper T lymphocytes have been shown in monkeys with chronic ocular infections [17],[23],[26]. Similarly, this immunosuppressive polarized environment was mirrored in infected TS cases where conjunctival IFNγ, TNFα, and IL-12 mRNA expression levels were decreased compared to levels among TF/TI cases, although IL-1β and TGFβ1 levels were consistently elevated for all grades of trachoma [19]. However, these studies have not examined the post-transcriptional and –translational regulation of these cytokines and their respective protein levels, making it difficult to assess their relevance to trachoma [27],[28],[29],[30]. To date, only one study has evaluated protein levels in which TS cases were shown to have significantly elevated TNFα levels compared with controls [31].
While the above data have started to elucidate our understanding of the immune responses in trachoma, the lack of cytokine protein data prevent us from determining the immunological modulators associated with successive disease severity and identifying protective versus pathologic risk factors. Furthermore, resolving infection and disease relies on localizing and attracting effective leukocyte populations to the infected site. Chemokines are a group of small molecular weight proteins (8–12 kDa) responsible for such a task. However, there have been no studies of chemokines and their association with trachoma. To begin to address these deficiencies, we quantitatively analyzed 25 secreted conjunctival mucosal cytokine and chemokine proteins in a trachoma endemic Nepali population. We detected previously uncharacterized and partially characterized cytokines, a soluble cytokine receptor, and chemokines for each grade of trachoma, and the effect of C. trachomatis infections on their production. Our data provide the most comprehensive immunological evaluation associated with trachoma to date.
Methods
Study population, trachoma grading, and ethics
This cross-sectional study included 208 individuals residing in a trachoma endemic region of Southwestern Nepal that were age and sex matched post hoc using a stratified sampling method. Samples from mutually exclusive trachoma grades (TF/TI, TS, TT, and Normal) were placed into separate strata by age (5 year spread) and sex. Samples were randomly selected (using a table of random numbers) from each age and gender stratum for each trachoma grade and matched with randomly selected samples from each age and gender stratum for normals. Verbal informed consent was obtained from all study subjects following the institutional review board approval by Children's Hospital and Research Center at Oakland, CA, and the Nepal Netra Jhoti Shang. Verbal consent was documented on an information sheet by the team member who consented each individual who agreed to be a study subject. Grading of the upper tarsus of each study subject was performed according to a modified grading scale previously described by WHO [14]. Briefly, the grades were: no evidence of trachoma characterized by ≤4 follicles on the lower 2/3 of the upper tarsal conjunctiva (Normal denoted as T0), follicular and/or intense trachomatous inflammation (TF/TI), trachomatous conjunctival scarring (TS), trachomatous trichiasis (TT) and TT with inflammation (TT/TI). An agreement between two of three independent readers (authors DD and RK, and Dr. Tracey Hessel) on final grading was made for each patient.
Sample collection
Conjunctival mucosal secretions were obtained by applying a sterile Weck-cel sponge (Medtronic Inc., Minneapolis, MN) to the inner canthus of each eye and allowing the swab to reach saturation. Samples were placed in a sterile eppendorf tube. A Dacron swab (Remel Inc., Lenexa, KS) was used to sample the upper tarsal and lower conjunctivae of each eye and was placed in M4-RT media (Micro Test Inc., Lilburn, GA) immediately following collection. All swabs were kept on ice for no longer than eight hours until transfer to liquid nitrogen tanks and then to Children's Hospital Oakland Research Institute, Oakland, CA and stored at −80°C until analyzed.
Detection of C. trachomatis in conjunctival samples
DNA was isolated from a M4-RT media containing the conjunctival swabs as previously described [32] following manufacturer's instructions. Samples were defined as positive at an OD450 nm>0.8 and negative at an OD450 nm<0.2 [32]. Equivocal samples were defined as those that fell within an OD450 nm of 0.2 to 0.8, and were further evaluated using an in-house validation PCR test to assess the presence or absence of chlamydiae as we have described previously [10]. Briefly, DNA from equivocal and negative and positive control samples were amplified using primers flanking the ompA gene. The presence of a 1200 bp band corresponding to the positive C. trachomatis DNA control, while the negative control was negative, defined an equivocal sample as positive for C. trachomatis.
Detection of cytokine and chemokine protein levels in conjunctival mucosal samples
Mucosal sponges were thawed on ice, and 80 µl of resuspension fluid [50 mM Tris, 0.15 M NaCl, 10 mM CaCl2, serine and cysteine protease inhibitors (Protease Inhibitor Cocktail tablets, Roche Diagnostics, Mannheim, Germany) at pH 7.5] was applied to each sponge. Fluid was extracted, and insoluble protein separated via centrifugation at 10,000×g for 10 min at 4°C.
Samples were applied to a Human Cytokine/Chemokine 25-plex [IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8 (CXCL8), IL-10, IL-12p40, IL-13, IL-15, IL-17, IFNα, IFNγ, TNFα, GM-CSF, CCL2 (MCP-1), CCL3 (MIP-1α), CCL4 (MIP-1β), CCL11 (Eotaxin), CXCL10 (IP-10), CXCL9 (MIG), CCL5 (RANTES), IL-1Ra, and IL-2R] 96 well plate assay (Biosource International, Inc., Camarillo, CA) following manufacturer's instructions. Briefly, 25-Plex beads were vortexed and sonicated to disperse aggregates, and washed using a vacuum manifold not exceeding 5 psi. Subsequently, 25-Plex beads were incubated with 50 µl of sample or standards for 2 h on an orbital shaker at 500 rpm. Wells were aspirated and washed as described above through the vacuum manifold. Biotinylated detector antibodies were added and incubated on the orbital shaker for 1 h with subsequent washes. After the addition of Streptavidin-RPE, the plates were analyzed using a Luminex X100 instrument (Luminex Technologies, Inc) to determine the quantities of each protein. Calibration was performed before each run, and all 25 cytokines/chemokines were gated (9050–12050) to eliminate bead aggregates and debris associated with the samples. Luminex analysis was set to 50 µl/sample and 80 events/bead. Standard curves were assessed from duplicates consisting of all 25 cytokines/chemokines using a five parameter logistic modeling system. Contiguous dilutions (8 three-fold followed by 3 two-fold dilutions) were applied to the standards, and resuspension fluid was used to determine background. Sensitivity levels were determined for each cytokine/chemokine as the lowest dilution, two standard deviations above background. Sensitivity levels were as follows: TNFα (0.2 pg/ml), IL-1β (1.9 pg/ml), IL-1Ra (14–31560 pg/ml), IL-6 (2.6 pg/ml), IL-4 (1.0 pg/ml), IL-5 (0.8 pg/ml), IL-13 (2.3 pg/ml), IL-12p40 (2.2 pg/ml), IFNγ (0.9 pg/ml), IL-10 (0.3 pg/ml), IL-2 (0.9 pg/ml), IL-2R (4.5 pg/ml), IL-15 (2.1 pg/ml), IFNα (4.2 pg/ml), IL-7 (16.2 pg/ml), IL-17 (5.6 pg/ml), Eotaxin (0.3 pg/ml), GM-CSF (2.7 pg/ml), IL-8 (2.7 pg/ml), MCP-1 (12.0 pg/ml), MIG (9.9 pg/ml), IP-10 (4.0 pg/ml), MIP-1α (4.3 pg/ml), MIP-1β (5.5 pg/ml) and RANTES (8.9 pg/ml).
Statistical analysis
We used a stratified sampling method to divide the population into mutually exclusive groups based on disease grade (TF/TI vs T0, TS vs T0, etc) as described above. Cytokine and chemokine data, comparing particular disease grades versus age and sex matched controls, were assessed for normality by Shapiro-Wilk W test to determine the appropriate statistical tests. Two-sample Wilcoxon rank-sum (Mann-Whitney) test was performed when p values were <0.05 from the Shapiro-Wilk test. Student t test with equal variance was used when p values were >0.05 (Figures 1–4). Significance for the association between cytokine/chemokine production and different trachoma grades was determined using multiple logistic regression and adjusting for age and C. trachomatis infection for the TF/TI cases. Statistical analysis of immuno-modulating effects by C. trachomatis for trachoma cases or controls was determined using frequency of cytokine or chemokine detection as the dependent variable adjusting for age. We used Spearman's rank test to quantify the association between individual cytokines/chemokines and different trachoma grades and infection. All statistics were performed using Stata 9.0 software (Stata Corp, College Station, TX).
Concentrations of conjunctival mucosal cytokines and chemokines among individuals residing in a trachoma endemic region of Nepal were analyzed using multiplexing Luminex technology (see methods). The data are grouped by three categories: (A) proinflammatory cytokines, (B) Th1/Th2/Th3 cytokines and (C) chemokines, representing individual conjunctival mucosal secretory concentrations from TF/TI cases (n = 28) and age and sex matched controls (T0, n = 28). Lines designate the geometric means of each group. Statistical differences for cytokine and chemokine concentrations between cases with TF/TI and controls with T0 were denoted by * P<0.05 or ** P<0.01, Wilcoxon rank-sum test.
Cytokine and chemokine concentrations within conjunctival mucosal secretions from Nepali patients exhibiting TS were compared to age and sex matched controls. The data are grouped into significant difference within four categories: (A) proinflammatory cytokines, (B) Th1/Th2/Th3 cytokines, (C) IL-2 cytokine family, and (D) chemokines, representing individual conjunctival mucosal secretory concentrations from TS patients (n = 18) and age and sex matched controls (T0, n = 18). Lines designate geometric means of each group. Statistical differences for cytokine and chemokine concentrations between TS and T0 was denoted * P<0.05, ** P<0.01 and *** P<0.001, Wilcoxon rank-sum test or Student t test when Shapiro-Wilk W test ≥0.05.
Cytokine and chemokine concentrations within conjunctival mucosal secretions from Nepali patients exhibiting TT were compared to age and sex matched controls. The data are grouped into significant difference within three categories: (A) proinflammatory cytokines, (B) IL-2 cytokine family and (C) chemokines, representing individual conjunctival mucosal secretory concentrations from TT patients (n = 20) and age and sex matched controls (T0, n = 20). Lines designate geometric means of each group. Statistical differences between TT and TO was denoted * P<0.05 or ** P<0.01, Wilcoxon rank-sum test.
Cytokine and chemokine concentrations within conjunctival mucosal secretions from Nepali patients exhibiting TT/TI were compared to age and sex matched controls. The data are grouped into four categories with significant differences: (A) proinflammatory cytokines, (B) Th1/Th2/Th3, (C) IL-2 cytokine family, and (D) chemokines, representing individual conjunctival mucosal secretory concentrations from TT/TI patients (n = 38) and age and sex matched controls (T0, n = 38). Lines designate geometric means of each group. Statistical differences between TT/TI and T0 was denoted * P<0.05, ** P<0.01 and *** P<0.001, Wilcoxon rank-sum test.
Results
Clinical and microbiologic characteristics of the study population
Our Nepali study population comprised individuals with TF/TI, TS, TT, and TT/TI who were age and sex matched with identical numbers of individuals without disease (T0) from the same community (Table 1). We assessed the prevalence of C. trachomatis infection with different grades of trachoma. Table 1 shows that there was an inverse association between C. trachomatis infection and age [P = 0.003; OR = 0.97 (0.95–0.99)] while infection was directly associated with TF/TI cases [(P = 0.017; OR = 4.4 (1.30–14.92)]. The presence of chronic trachoma defined as any grade demonstrating scarring (TS, TT and TT/TI) was associated with age [P<0.001; OR = 1.20 (1.12–1.30)].
Pro-inflammatory mediators in conjunctival mucosal secretions are associated with trachomatous inflammation
We found significantly elevated protein concentrations of the pro-inflammatory cytokine TNFα for TF/TI cases compared to age and sex matched controls (T0) (Figure 1A, P = 0.003) but not for the pro-inflammatory cytokine IL-1β. For the pleiotropic cytokine IL-6, which has been linked to acute and chronic inflammation [33], we found a greater percentage of TF/TI cases (50%) that produced IL-6 compared with controls (25%) (Table S1).
A previous study suggested a protective effect with elevated Th1, IL-12 and IFNγ, mRNA expression for cases with TF/TI, but not for those with scarring [19]. At the protein level, we did not find the same association for TF/TI cases. For Th2 cytokines, we detected IL-4 and IL13 protein levels in over 60% of TF/TI cases compared to controls. However, minimal differences in protein concentrations were observed between the two groups (data not shown). The anti-inflammatory and Th2 cytokine IL-10, which is also referred to as a Th3/T regulatory cytokine 1 (Tr1), was significantly associated with TF/TI cases (Figure 1B, P = 0.005) where the mean represented a three-fold higher concentration compared with controls.
We found no significant elevation in any of the IL-2 family of cytokines and receptors (IL-2, IL-7, IL-15, and IL-2R) for the TF/TI cases (Table S1 and data not shown).
We found minimal differences in conjunctival mucosal chemokine protein levels among TF/TI cases except for the CXC family where significantly elevated levels of IL-8 and MCP-1 were found among TF/TI cases compared to controls (Figure 1C; P<0.05 and P<0.005, respectively). These chemokines are primarily associated with attracting and activating neutrophils, monocytes and T lymphocytes [34], which have all been linked to the immunopathology associated with TF/TI [15],[16].
Elevated pro-inflammatory cytokine and chemokine proteins in conjunctival mucosal secretions predominate in trachomatous scarring cases
Cases with scarring had significantly elevated levels of the proinflammatory cytokines TNFα and IL-1β compared to controls (Figure 2A, P = 0.001 and P<0.05, respectively). Levels of IL-6 were barely detected among these cases. However, there was an overall greater frequency of detectable IL-6 protein levels among TS cases (Table S1, 17% TS vs 0% T0), which was similar to the TF/TI cases mentioned above.
The Th2 cytokine IL-13 was decreased in TS cases compared with controls (Table S1, P<0.005), while IL-12p40 was elevated (Figure 2B, P<0.05), suggesting a slight polarization towards the Th1 phenotype. Elevation in the Th3/Tr1 cytokine IL-10 protein levels for TS cases, although not quite significant (Figure 2B, P = 0.051), suggested immuno-regulatory patterns coexisting with Th1. A distinguishing finding for TS cases was the significant protein elevation of an IL-2 family cytokine, IL-15 (Figure 2C, P<0.05). This cytokine is produced by a plethora of cell types, including epithelial, monocytic and dendritic cells, and is involved in the activation of effector memory T lymphocytes [35].
Significantly elevated levels of Th1 CC chemokines, MIP-1α and MIP-1β (Figure 2D, P<0.01 and P<0.001 respectively), and Th2 chemokines, MCP-1 and Eotaxin (Figure 2D, P<0.01) were found for the TS cases compared with controls.
Elevated IL-6 protein levels in conjunctival mucosal secretions predominate among trachomatous trichiasis cases
Conjunctival mucosal secretions demonstrated minimal differences at the protein level among proinflammatory and Th1, Th2 and Th3 cytokines for TT cases (data not shown). However, IL-6 protein levels were significantly elevated among cases with TT compared to controls (Figure 3A, P<0.01) as in the other trachoma grades. For the IL-2 family of cytokines, minimal differences were observed except for significantly elevated concentrations of IL-15 (Figure 3B, P<0.05), which further suggests that this cytokine is a risk factor for chronic trachoma.
For chemokines, TT cases had increased MIG protein levels compared with controls (Figure 3C, P = 0.05), which were not observed in other trachoma grades.
Low levels of conjunctival mucosal IL-12p40 and IL-1Ra distinguish trachomatous trichiasis with inflammation cases from controls
To our knowledge, the inflammatory response in TT/TI cases has been characterized only once for a limited number of cytokine mRNA expression levels [19]. In our study, the most novel finding was the significant reduction in IL-1Ra protein for cases with TT/TI (Figure 4A, P<0.001) compared to controls, further supporting a chronic inflammatory environment for this grade. This is the first time an IL-1β antagonist has been characterized in trachomatous disease. In agreement with an inflammatory environment, significantly higher protein levels of IL-1β were also evident in TT/TI cases compared to controls (Figure 4A, P<0.05).
For Th1, Th2 and Th3 cytokines, TT/TI cases had significantly reduced protein levels for the Th1 cytokine IL-12p40 (Figure 4B, P<0.001) and for the Th2 cytokine IL-4 (Figure 4B, P<0.05). There was a trend for elevated protein levels for the Th3/Tr1 cytokine IL-10, similar to what was observed in most grades of trachoma (Table S1 and data not shown). Among the IL-2 family, the TT/TI cases had elevated levels of IL-2 and IL-15 compared to controls (Figure 4C, P<0.05).
In agreement with the decreased Th1 response, CC chemokines MIP-1α and MIP-1β were significantly lower for TT/TI cases compared to controls (Table S1, P<0.05 for both). However, CXC chemokines, IL-8 and MCP-1, remain elevated (Figure 4D, P<0.05).
Elevated Th1 and Th2 cytokines and IL-1Ra levels in conjunctival mucosal secretions distinguish trachomatous inflammation from scarring grades of trachoma
Figure 5A shows that TF/TI cases had a higher frequency of TNFα production compared to all other grades, although the difference was only significant between TF/TI and TT/TI (P<0.05), likely due to small patient numbers for TS and TT. A reverse trend was apparent for IL-1β, which was supported by the significant reduction in its soluble antagonist receptor (IL-1Ra) concentrations in the TT/TI cases compared with TF/TI and TS cases (data not shown; P<0.05 and P<0.005 respectively).
(A) The TF/TI cases had significantly elevated TNFα mucosal protein production compared to the TT/TI cases (P<0.05) and higher IL-6 mucosal production than TS (P<0.05). (B) TF/TI cases had significantly elevated production of IL-4 compared to TT/TI cases (P<0.01). Further, IL-4 production was detected more frequently for TS cases compared to TT (P<0.01) and TT/TI cases (P<0.01). Diminished IL-13 production was evident for TT/TI cases compared to TF/TI cases (P = 0.005). TF/TI cases had a significantly higher production of IFNγ compared to TT/TI cases (P<0.05). Ratios were determined by dividing cases with detectable levels of cytokines and chemokines by the total number of cases in each group. Statistics were based on multiple logistic regression adjusting for C. trachomatis infection, age and sex.
Cytokines involved in Th1 and Th2 phenotypes showed a consistent reduction for all grades (TF/TI to TT/TI) (Figure 5B) with TF/TI and TS cases demonstrating more frequent IL-4 production over TT/TI and TT cases (Figure 5B, P<0.01, respectively). This correlated with decreased Th2, IL-13, and Th1, IFNγ and IL-12p40, cytokines for TT/TI cases (Figure 5B; P<0.05; IL-12p40: OR = 0.80 (0.66–0.97), P<0.05 TT/TI versus TS). Among the IL-2 family of cytokines and receptor, decreased IL-2R levels were evident in TT/TI cases compared with TF/TI cases, although these differences were not significant (P = 0.082, data not shown). However, these data correlated with elevated IL-2 production in TT/TI compared to TF/TI cases (Table S1), further supporting IL-2 as a risk factor for the development of TT/TI.
A significant reduction in IP-10 levels was found for TT/TI cases compared with TF/TI cases (OR = 0.9980 (0.9961–0.9998), P<0.05). Though not significant, a reverse trend was found for MCP-1 levels.
Elevated protein levels of chemokines, pro-inflammatory and Th3/Tr1 cytokines and lower levels of Th1 and Th2 cytokines in conjunctival mucosal secretions characterized chronic trachoma compared with controls
The effects of chronic trachoma (TS, TT and TT/TI) on inflammatory components were elucidated by grouping these grades and comparing them against their age and sex matched controls. Inflammatory patterns for cases with chronic trachoma compared with controls are shown in Table 2 and Table S2; significantly elevated TNFα, IL-1β and IL-6 concentrations and diminished concentrations of the anti-inflammatory cytokine IL-1Ra were found in chronic trachoma cases compared with controls.
In characterizing host Th1, Th2 and Th3 cytokine levels, IL-4, IL-13 and IL-12p40 were negatively associated with chronic trachoma cases compared with controls (Table 2). However, elevated IL-10 levels were associated with chronic cases (Table 2), further supporting an association with the Th3/Tr1 phenotype and the role of IL-10 as a risk factor for chronic trachoma. Members of the IL-2 cytokine family, IL-2 and IL-15, were also significantly associated with chronic trachoma cases compared with controls (Table 2).
Among the chemokine family, chronic trachoma was significantly associated with elevated levels of Eotaxin, IL-8, MCP-1 and MIG compared with controls (Table 2).
Influence of C. trachomatis infections on cytokine, cytokine receptor and chemokine protein levels in conjunctival mucosal secretions for all grades of trachoma
Table 3 shows the frequency of cytokine, chemokine and cytokine receptor protein concentrations comparing C. trachomatis infected and uninfected patients for trachoma cases (all grades) and for controls. TNFα and IL-6 were significantly associated with C. trachomatis infections for chronic trachoma cases (Table 3 and S3) with similar patterns for IL-1β, though not significant (Table S3 and S4). This phenotype was not evident among controls (Table 3).
Minimal differences were observed for Th1 and Th2 cytokines in the presence or absence of C. trachomatis infections (Table S3 and S4). However, elevated IL-10 levels persisted in the presence of C. trachomatis infections regardless of trachoma grade (Table 3). Additionally, C. trachomatis infections appeared to effect the IL-2 family of cytokines with a significant association of elevated IL-2 concentrations with controls (Table 3); elevated IL-15 was associated with trachoma (Table 3 and Table S3).
In the chemokine family, C. trachomatis infection was associated with increased levels of Th1-associated chemokines MIP-1β and RANTES (Table 3). Independent of trachoma grade, MIG was significantly associated with infection (OR = 2.61 (1.25 to 5.45), P = 0.011).
Discussion
While there is some important research on cytokine mRNA expression in association with trachoma, our fundamental knowledge of the host immune response has been limited by a lack of quantitative protein data, especially for those cytokines and chemokines that are post-transcriptionally and –translationally modified, in association with each grade of trachoma. The goal of this study was to validate previous gene expression findings and to characterize novel inflammatory cytokine and chemokine protein concentrations in mucosal conjunctival secretions and how each may influence inflammation for the different grades of trachomatous disease. Additionally, we further characterized immuno-regulatory effects induced by concurrent C. trachomatis infection.
Our Nepali study population showed a significant inverse association of C. trachomatis infection with age and a direct association with TF/TI cases agreeing with our previous findings and those of others [19],[22],[32].
Proinflammatory cytokines have previously been associated with acute and chronic trachoma [36],[37]. We and others [19],[20] found elevated TNFα levels among TI and chronic trachoma cases and also higher levels when both C. trachomatis infection and trachoma were present. Additionally, in our study, elevated IL-1β levels were significantly associated with chronic trachoma cases but not with infection. Previous studies have demonstrated elevated IL-1β mRNA levels during both disease and infection [19],[20]. These differences may be due to post-translational modifications of IL-1β, thus supporting both findings but suggesting the need for quantitative protein studies for confirmation. The previous associations of these cytokines with the induction of scarring-associated proteins, matrix metalloproteins (MMPs) and collagen [19],[20],[21], suggest plausible mechanisms for the development and progression of chronic trachoma. This chronic inflammatory phenotype is further supported in our study by the significantly decreased levels of the anti-inflammatory cytokine IL-1Ra. Recently, Hvid et al. characterized the role of IL-1Ra and IL-1α in a human fallopian tube organ culture model [38]. C. trachomatis infection resulted in destruction of ciliated and secretory cells within the tubes. However, when co-cultured with IL-1Ra, tissue destruction was minimal while IL-1α exacerbated disease pathology. In trachoma, the prolonged production of TNFα and IL-β together with a reduction in IL-1Ra inhibitory pathways may promote the development of scarring and progression to TT.
The pleiotropic cytokine IL-6 has been associated with chronic trachoma [33]. One previous study evaluated IL-6 gene expression in trachoma but the findings were inconclusive due to detectable mRNA levels in only two patients [19]. We found elevated IL-6 production for all grades of trachoma. Additionally, we demonstrated for the first time a significant association of C. trachomatis infection with elevated IL-6 protein levels in both inflammatory and chronic trachoma. Our findings are supported by a previous study demonstrating elevated IL-6 production in the fallopian tubes of macaques after repeated infection with C. trachomatis [39]. Murine studies, however, have provided conflicting data. One study showed an increased bacterial burden and mortality associated with pulmonary infection with the mouse pneumonitis strain (MoPn; now referred to as Chlamydia muridarum) in IL-6 -/- KO mice [40]. In the mouse genital tract model, however, there was an absence of any pathological or bacterial complications when IL-6 -/- KO mice were infected with C. muridarum [41]. Darville et al., found that TLR2 -/- KO mice exhibited decreased levels of IL-6 and oviduct pathology during C. muridarum -genital tract infection [42], further supporting IL-6 as a risk factor for chronic trachoma.
Considering the obligate intracellular nature of C. trachomatis and previous studies, host cell-mediated immunity (Th1) appears to be critical in eliciting protection against Chlamydia-associated diseases. Transcriptional studies have associated elevated Th1 cytokines, IL-12 and IFNγ, mRNA expression with C. trachomatis infection for cases with TF/TI [19],[21] but decreased levels with scarring [19]. Wang et al. demonstrated elevated endocervical IL-12 protein concentrations in adolescents prior to resolution of their C. trachomatis infection [43], further supporting a role for IL-12 in host defense against infection. In our study, we found minimal differences in IL-12p40 concentrations for TF/TI compared with controls but significantly decreased mucosal levels associated with scarring. Additionally, TT/TI cases had lower levels for both Th1 cytokines, IFNγ and IL-12p40, compared to TF/TI. Together, these data suggest that Th1 cytokines are protective immunologic factors against disease progression. While Th2 cytokines are known to be associated with humoral mediated immunity, previous studies have been inconclusive due to undetectable transcriptional levels of these cytokines [19],[21]. In our study, the Th2 cytokines, IL-4 and IL-13, displayed similar patterns to the Th1 cytokines, suggesting that both Th1 and Th2 cytokines may be protective factors against chronic sequelae.
The negative associations of Th1 and Th2 cytokines with chronic trachoma suggest a potential role for Th3/Tr1. The previous categorization of IL-10 as an anti-inflammatory Th2 cytokine has recently been extended given the documented association with T-regulatory (Tregs) cells [44]. Kinjyo et al. characterized IL-10 as a Th3/Tr1 cytokine, showing the over production of IL-10 and TGFβ in the absence of a Th2 polarizing gene, SOC3, the suppressor of cytokine signaling [45]. Recently, Faal et al. found elevated levels of a T cell regulatory gene, forkhead box 3 (FOXP3), during active trachoma (TF/TI) [22]. This was associated with elevated IL-10 and indoleamine-2, 3-dioxygenase (IDO) mRNA expression levels. IDO levels have been associated with immune tolerance and regulatory pathways [46], which have lead to outgrowth of secondary pneumococcal infections [47]. Additionally, Mark et al. demonstrated diminished FOXP3 and IL-10 levels with early clearing of C. trachomatis infection in a murine model [48]. In our study, IL-10 was overproduced during all grades of trachoma and with C. trachomatis infection. These findings are supported by previous IL-10 studies where elevated mRNA expression was similarly associated with trachoma and infection [20],[21],[22]. In the genital tract, elevated levels of IL-10 have been found in infertile women with documented C. trachomatis infections [49] and in macaques that were repeatedly infected with C. trachomatis [39]. These collective data support the association of IL-10 with a Th3/Tr1 phenotype and suggest that IL-10 may be a major risk factor for chronic trachoma associated with C. trachomatis infection. In contrast, Hvid et al. demonstrated reduced ex vivo fallopian tube pathology with C. trachomatis infection in the presence of excess IL-10, suggesting a protective effect [38] at least during the early stages of infection in the female genital tract. Further investigations are needed to clearly define the role of IL-10 for each trachoma grade.
The involvement of T lymphocytes in the perifollicular area of conjunctival follicles suggests an active involvement of the IL-2 family of cytokines, IL-2, IL-2R, and IL-15, in trachoma. Our IL-2 findings showed an association with chronic trachoma and also agree with a previous study that found an association of IL-2 mRNA expression, although at low levels, with TI and C. trachomatis infection [19]. In this study, we demonstrated higher concentrations of IL-15 in cases with scarring and in cases with concurrent C. trachomatis infection. IL-15 shares the IL-2Rβ and IL-2Rγ receptors with IL-2, and, therefore, has overlapping bioactivity, which includes the stimulation of proliferation of activated T cells and natural killer (NK) cells [50],[51]. Similar trends in elevated IL-15 protein have been found in the lymph nodes of chronically infected HIV patients [52]. In a murine model, IL-15 KO mice have been shown to have significantly diminished acute and chronic colitis compared to wild-type mice [53], further suggesting that IL-15 production is required to sustain chronic infections. Previous studies have also shown elevated IL-15 mRNA expression in patients with C. trachomatis-induced arthritis compared to healthy controls [54]. Immunopathology studies in cynologus monkeys have demonstrated elevated ratios of CD8+ to CD4+ T cell populations in C. trachomatis infected naïve compared to orally immunized monkeys [55], suggesting that an over abundance of CD8+ T cells are a risk factor for disease. Recently, IL-15 was linked to extended CD8+ memory T lymphocyte survival rates in mice [56]. IL-15 has also been associated with Treg proliferation [57]. These data support IL-2 and IL-15 as risk factors for chronic trachoma, especially with concurrent C. trachomatis infection, which may be associated with abundant CD8+ T lymphocyte populations.
Cellular infiltration resulting in typical trachomatous follicles suggests a major role for chemokines in the progression of disease. However, to our knowledge, there has been no characterization of these proteins in trachoma. Most chlamydial studies that have evaluated chemokines focused on murine and human genital tract infections. In our study, the significant elevation of CXC chemokine IL-8 protein levels for all trachoma grades agrees with previous findings where an abundance of neutrophil populations were present in conjunctival swabs from individuals with trachoma [15]. A chlamydicidal role for neutrophils within the conjunctiva has been demonstrated in one in vitro study by Yong et al. [58]. However, further studies are needed to characterize these findings in vivo. Our findings for IL-8 are also supported by a murine study where the murine form of IL-8, MIP-2, had prolonged production along with neutrophil infiltration and pathology in the genital tract of BALB/c and C3H/HeN mice but not in the more resistant C57BL/6 mouse strain [59]. We found pronounced protein levels for MCP-1 for all grades of trachoma, but lower MIP-1α production for TT/TI cases compared to controls. In addition, there was a trend for elevated MCP-1 in TT/TI compared to TS and TF/TI cases, further associating MCP-1 with chronic grades of disease. These findings are supported by a murine study that found elevated MIP-1α and decreased MCP-1 levels among C57BL/6 mice that had a shorter course of infection [60], suggesting that these patterns of chemokine production are protective. Another murine study exhibited elevated levels of Th1 associated chemokines, RANTES, IP-10 and MIG, with chronic, upper genital tract infections compared to lower genital tract infections for C. muridarum [61]. Our study also demonstrated elevated MIG production among chronic trachoma cases with elevated RANTES levels among those who were also infected with C. trachomatis.
In conclusion, we characterized the secreted cytokines, chemokines, and cytokine receptor associated with immunopathology for each grade of trachoma and determined the immunomodulating effects of concurrent C. trachomatis infection. Our studies are in agreement with others who demonstrated Th1 cytokines as protective and Th3/Tr1 cytokine IL-10 as a possible risk factor for chronic trachoma. Additionally, we reconfirmed previous gene expression studies linking elevated proinflammatory cytokines IL-1β and TNFα with C. trachomatis infections, and IL-1β as a strong risk factor for chronic trachoma. Our findings expanded on the IL-1β linkage by demonstrating for the first time, to our knowledge, inverse levels of its antagonist, IL-1Ra, in association with TT, suggesting that IL-Ra is a protective factor against chronic sequelae. We also identified two new risk factors, IL-6 and IL-15, which were associated with chronic trachoma with significantly elevated levels evident with concurrent C. trachomatis infection. Currently, we are elucidating signal transduction pathways affiliated with these cytokines and chemokines during C. trachomatis infections and their possible inter- and intra-cellular roles during disease. In addition, future vaccine design will likely need to take into consideration the immune responses we have characterized and to ensure a vaccine has the desired protective outcome.
Supporting Information
Table S1.
Frequency of cytokine and chemokine conjunctival mucosal production for different grades of trachoma.
https://doi.org/10.1371/journal.pntd.0000264.s001
(0.10 MB DOC)
Table S2.
Correlation of cytokine and chemokine conjunctival mucosal protein production with chronic scarring trachoma.
https://doi.org/10.1371/journal.pntd.0000264.s002
(0.03 MB DOC)
Table S3.
Association of C. trachomatis infection with cytokine and chemokine conjunctival mucosal protein levels for normal, active and chronic trachoma grades.
https://doi.org/10.1371/journal.pntd.0000264.s003
(0.08 MB DOC)
Table S4.
Correlation of cytokine and chemokine conjunctival mucosal protein production with C. trachomatis infections for cases with all grades of trachomatous disease and for controls.
https://doi.org/10.1371/journal.pntd.0000264.s004
(0.05 MB DOC)
Acknowledgments
We would like to express our sincerest gratitude to the people of Kapilvastu District, Lumbini Zone, Nepal, who made this research possible. Additionally, we are grateful to Dr. Alexander Lucas for his suggestions on data presentation.
Author Contributions
Conceived and designed the experiments: TAS DD. Performed the experiments: TAS DD. Analyzed the data: TAS BA DD. Wrote the paper: TAS DD. Clinical grading: RPK HKA BS DD.
References
- 1.
Dean D (1997) Chlamydial Infections. In: Conner D, editor. Pathology of Infectious Diseases: Appleton and Lange. pp. 473–507.
- 2. Dawson CR, Schachter J (1985) Strategies for treatment and control of blinding trachoma: cost-effectiveness of topical or systemic antibiotics. Rev Infect Dis 7: 768–773.
- 3. Thylefors B, Negrel AD, Pararajasegaram R, Dadzie KY (1995) Global data on blindness. Bull World Health Organ 73: 115–121.
- 4. Gerbase AC, Rowley JT, Mertens TE (1998) Global epidemiology of sexually transmitted diseases. Lancet 351 Suppl 3: 2–4.
- 5. Schumacher HR Jr, Magge S, Cherian PV, Sleckman J, Rothfuss S, et al. (1988) Light and electron microscopic studies on the synovial membrane in Reiter's syndrome. Immunocytochemical identification of chlamydial antigen in patients with early disease. Arthritis Rheum 31: 937–946.
- 6. Neuer A, Lam KN, Tiller FW, Kiesel L, Witkin SS (1997) Humoral immune response to membrane components of Chlamydia trachomatis and expression of human 60 kDa heat shock protein in follicular fluid of in-vitro fertilization patients. Hum Reprod 12: 925–929.
- 7. Frick KD, Basilion EV, Hanson CL, Colchero MA (2003) Estimating the burden and economic impact of trachomatous visual loss. Ophthalmic Epidemiol 10: 121–132.
- 8. Melese M, Chidambaram JD, Alemayehu W, Lee DC, Yi EH, et al. (2004) Feasibility of eliminating ocular Chlamydia trachomatis with repeat mass antibiotic treatments. JAMA 292: 721–725.
- 9. Atik B, Thanh TT, Luong VQ, Lagree S, Dean D (2006) Impact of annual targeted treatment on infectious trachoma and susceptibility to reinfection. Jama 296: 1488–1497.
- 10. Zhang H, Kandel RP, Sharma B, Dean D (2004) Risk factors for recurrence of postoperative trichiasis: implications for trachoma blindness prevention. Arch Ophthalmol 122: 511–516.
- 11. West ES, Mkocha H, Munoz B, Mabey D, Foster A, et al. (2005) Risk factors for postsurgical trichiasis recurrence in a trachoma-endemic area. Invest Ophthalmol Vis Sci 46: 447–453.
- 12. West SK, Munoz B, Mkocha H, Holland MJ, Aguirre A, et al. (2005) Infection with Chlamydia trachomatis after mass treatment of a trachoma hyperendemic community in Tanzania: a longitudinal study. Lancet 366: 1296–1300.
- 13. Broman AT, Shum K, Munoz B, Duncan DD, West SK (2006) Spatial clustering of ocular chlamydial infection over time following treatment, among households in a village in Tanzania. Invest Ophthalmol Vis Sci 47: 99–104.
- 14. Thylefors B, Dawson CR, Jones BR, West SK, Taylor HR (1987) A simple system for the assessment of trachoma and its complications. Bull World Health Organ 65: 477–483.
- 15. Yoneda C, Dawson CR, Daghfous T, Hoshiwara I, Jones P, et al. (1975) Cytology as a guide to the presence of chlamydial inclusions in Giemsa-stained conjunctival smears in severe endemic trachoma. Br J Ophthalmol 59: 116–124.
- 16. el-Asrar AM, Van den Oord JJ, Geboes K, Missotten L, Emarah MH, et al. (1989) Immunopathology of trachomatous conjunctivitis. Br J Ophthalmol 73: 276–282.
- 17. Whittum-Hudson JA, Taylor HR, Farazdaghi M, Prendergast RA (1986) Immunohistochemical study of the local inflammatory response to chlamydial ocular infection. Invest Ophthalmol Vis Sci 27: 64–69.
- 18. Patton DL, Taylor HR (1986) The histopathology of experimental trachoma: ultrastructural changes in the conjunctival epithelium. J Infect Dis 153: 870–878.
- 19. Bobo L, Novak N, Mkocha H, Vitale S, West S, et al. (1996) Evidence for a predominant proinflammatory conjunctival cytokine response in individuals with trachoma. Infect Immun 64: 3273–3279.
- 20. Faal N, Bailey RL, Sarr I, Joof H, Mabey DC, et al. (2005) Temporal cytokine gene expression patterns in subjects with trachoma identify distinct conjunctival responses associated with infection. Clin Exp Immunol 142: 347–353.
- 21. Burton MJ, Bailey RL, Jeffries D, Mabey DC, Holland MJ (2004) Cytokine and fibrogenic gene expression in the conjunctivas of subjects from a Gambian community where trachoma is endemic. Infect Immun 72: 7352–7356.
- 22. Faal N, Bailey RL, Jeffries D, Joof H, Sarr I, et al. (2006) Conjunctival FOXP3 expression in trachoma: do regulatory T cells have a role in human ocular Chlamydia trachomatis infection? PLoS Med 3: e266.
- 23. Young E, Taylor HR (1984) Immune mechanisms in chlamydial eye infection: cellular immune responses in chronic and acute disease. J Infect Dis 150: 745–751.
- 24. Bailey RL, Holland MJ, Whittle HC, Mabey DC (1995) Subjects recovering from human ocular chlamydial infection have enhanced lymphoproliferative responses to chlamydial antigens compared with those of persistently diseased controls. Infect Immun 63: 389–392.
- 25. Holland MJ, Bailey RL, Hayes LJ, Whittle HC, Mabey DC (1993) Conjunctival scarring in trachoma is associated with depressed cell-mediated immune responses to chlamydial antigens. J Infect Dis 168: 1528–1531.
- 26. Young E, Taylor HR (1986) Immune mechanisms in chlamydial eye infection. Development of T suppressor cells. Invest Ophthalmol Vis Sci 27: 615–619.
- 27. Fernandez MC, Walters J, Marucha P (1996) Transcriptional and post-transcriptional regulation of GM-CSF-induced IL-1 beta gene expression in PMN. J Leukoc Biol 59: 598–603.
- 28. Turner M, Chantry D, Buchan G, Barrett K, Feldmann M (1989) Regulation of expression of human IL-1 alpha and IL-1 beta genes. J Immunol 143: 3556–3561.
- 29. Fan J, Heller NM, Gorospe M, Atasoy U, Stellato C (2005) The role of post-transcriptional regulation in chemokine gene expression in inflammation and allergy. Eur Respir J 26: 933–947.
- 30. Bamford RN, Battiata AP, Waldmann TA (1996) IL-15: the role of translational regulation in their expression. J Leukoc Biol 59: 476–480.
- 31. Conway DJ, Holland MJ, Bailey RL, Campbell AE, Mahdi OS, et al. (1997) Scarring trachoma is associated with polymorphism in the tumor necrosis factor alpha (TNF-alpha) gene promoter and with elevated TNF-alpha levels in tear fluid. Infect Immun 65: 1003–1006.
- 32. Hessel T, Dhital SP, Plank R, Dean D (2001) Immune response to chlamydial 60-kilodalton heat shock protein in tears from Nepali trachoma patients. Infect Immun 69: 4996–5000.
- 33. Gabay C (2006) Interleukin-6 and chronic inflammation. Arthritis Res Ther 8 Suppl 2: S3.
- 34. Luster AD (1998) Chemokines–chemotactic cytokines that mediate inflammation. N Engl J Med 338: 436–445.
- 35. Budagian V, Bulanova E, Paus R, Bulfone-Paus S (2006) IL-15/IL-15 receptor biology: a guided tour through an expanding universe. Cytokine Growth Factor Rev 17: 259–280.
- 36. Popa C, Netea MG, van Riel PL, van der Meer JW, Stalenhoef AF (2007) The role of TNF-alpha in chronic inflammatory conditions, intermediary metabolism, and cardiovascular risk. J Lipid Res 48: 751–762.
- 37. Dinarello CA, Wolff SM (1993) The role of interleukin-1 in disease. N Engl J Med 328: 106–113.
- 38. Hvid M, Baczynska A, Deleuran B, Fedder J, Knudsen HJ, et al. (2007) Interleukin-1 is the initiator of fallopian tube destruction during Chlamydia trachomatis infection. Cell Microbiol.
- 39. Van Voorhis WC, Barrett LK, Sweeney YT, Kuo CC, Patton DL (1997) Repeated Chlamydia trachomatis infection of Macaca nemestrina fallopian tubes produces a Th1-like cytokine response associated with fibrosis and scarring. Infect Immun 65: 2175–2182.
- 40. Williams DM, Grubbs BG, Darville T, Kelly K, Rank RG (1998) A role for interleukin-6 in host defense against murine Chlamydia trachomatis infection. Infect Immun 66: 4564–4567.
- 41. Perry LL, Feilzer K, Caldwell HD (1998) Neither interleukin-6 nor inducible nitric oxide synthase is required for clearance of Chlamydia trachomatis from the murine genital tract epithelium. Infect Immun 66: 1265–1269.
- 42. Darville T, O'Neill JM, Andrews CW Jr, Nagarajan UM, Stahl L, et al. (2003) Toll-like receptor-2, but not Toll-like receptor-4, is essential for development of oviduct pathology in chlamydial genital tract infection. J Immunol 171: 6187–6197.
- 43. Wang C, Tang J, Crowley-Nowick PA, Wilson CM, Kaslow RA, et al. (2005) Interleukin (IL)-2 and IL-12 responses to Chlamydia trachomatis infection in adolescents. Clin Exp Immunol 142: 548–554.
- 44. Seo N, Hayakawa S, Takigawa M, Tokura Y (2001) Interleukin-10 expressed at early tumour sites induces subsequent generation of CD4(+) T-regulatory cells and systemic collapse of antitumour immunity. Immunology 103: 449–457.
- 45. Kinjyo I, Inoue H, Hamano S, Fukuyama S, Yoshimura T, et al. (2006) Loss of SOCS3 in T helper cells resulted in reduced immune responses and hyperproduction of interleukin 10 and transforming growth factor-beta 1. J Exp Med 203: 1021–1031.
- 46. Munn DH, Mellor AL (2007) Indoleamine 2,3-dioxygenase and tumor-induced tolerance. J Clin Invest 117: 1147–1154.
- 47. van der Sluijs KF, Nijhuis M, Levels JH, Florquin S, Mellor AL, et al. (2006) Influenza-induced expression of indoleamine 2,3-dioxygenase enhances interleukin-10 production and bacterial outgrowth during secondary pneumococcal pneumonia. J Infect Dis 193: 214–222.
- 48. Marks E, Verolin M, Stensson A, Lycke N (2007) Differential CD28 and Inducible Costimulatory Molecule Signaling Requirements for Protective CD4+ T-Cell-Mediated Immunity against Genital Tract Chlamydia trachomatis Infection. Infect Immun 75: 4638–4647.
- 49. Reddy BS, Rastogi S, Das B, Salhan S, Verma S, et al. (2004) Cytokine expression pattern in the genital tract of Chlamydia trachomatis positive infertile women - implication for T-cell responses. Clin Exp Immunol 137: 552–558.
- 50. Kennedy MK, Glaccum M, Brown SN, Butz EA, Viney JL, et al. (2000) Reversible defects in natural killer and memory CD8 T cell lineages in interleukin 15-deficient mice. J Exp Med 191: 771–780.
- 51. Picker LJ, Reed-Inderbitzin EF, Hagen SI, Edgar JB, Hansen SG, et al. (2006) IL-15 induces CD4 effector memory T cell production and tissue emigration in nonhuman primates. J Clin Invest 116: 1514–1524.
- 52. Biancotto A, Grivel JC, Iglehart SJ, Vanpouille C, Lisco A, et al. (2007) Abnormal activation and cytokine spectra in lymph nodes of people chronically infected with HIV-1. Blood 109: 4272–4279.
- 53. Yoshihara K, Yajima T, Kubo C, Yoshikai Y (2006) Role of interleukin 15 in colitis induced by dextran sulphate sodium in mice. Gut 55: 334–341.
- 54. Gerard HC, Wang Z, Whittum-Hudson JA, El-Gabalawy H, Goldbach-Mansky R, et al. (2002) Cytokine and chemokine mRNA produced in synovial tissue chronically infected with Chlamydia trachomatis and C. pneumoniae. J Rheumatol 29: 1827–1835.
- 55. Whittum-Hudson JA, Prendergast RA, Taylor HR (1986) Changes in conjunctival lymphocyte populations induced by oral immunization with Chlamydia trachomatis. Curr Eye Res 5: 973–979.
- 56. Sato N, Patel HJ, Waldmann TA, Tagaya Y (2007) The IL-15/IL-15Ralpha on cell surfaces enables sustained IL-15 activity and contributes to the long survival of CD8 memory T cells. Proc Natl Acad Sci U S A 104: 588–593.
- 57. Clark RA, Kupper TS (2007) IL-15 and dermal fibroblasts induce proliferation of natural regulatory T cells isolated from human skin. Blood 109: 194–202.
- 58. Yong EC, Klebanoff SJ, Kuo CC (1982) Toxic effect of human polymorphonuclear leukocytes on Chlamydia trachomatis. Infect Immun 37: 422–426.
- 59. Darville T, Andrews CW Jr, Sikes JD, Fraley PL, Rank RG (2001) Early local cytokine profiles in strains of mice with different outcomes from chlamydial genital tract infection. Infect Immun 69: 3556–3561.
- 60. Darville T, Andrews CW Jr, Sikes JD, Fraley PL, Braswell L, et al. (2001) Mouse strain-dependent chemokine regulation of the genital tract T helper cell type 1 immune response. Infect Immun 69: 7419–7424.
- 61. Maxion HK, Kelly KA (2002) Chemokine expression patterns differ within anatomically distinct regions of the genital tract during Chlamydia trachomatis infection. Infect Immun 70: 1538–1546.