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Cell Biology International (2011) 35, 793–798 (Printed in Great Britain)
Ferritin heavy chain-mediated iron homoeostasis regulates expression of IL-10 in Chlamydia trachomatis-infected HeLa cells
Harsh Vardhan, Rishein Gupta, Rajneesh Jha, Apurb Rashmi Bhengraj and Aruna Mittal1
Institute of Pathology, Indian Council of Medical Research ICMR, Safdarjung Hospital Campus, Post Box No. 4909, New Delhi110029, India


Chlamydia trachomatis is the leading cause of sexually transmitted infection worldwide, in which disease outcome is determined by the balance between pro- and anti-inflammatory host immune responses. Iron plays important roles in regulation and enhancement of various pro- and anti-inflammatory cytokines. Earlier studies have established essentiality of iron in C. trachomatis infection; however, there is lack of study wherein modulatory effect of iron regulated protein [FHC (ferritin heavy chain)] in regulation of anti-inflammatory cytokine IL (interleukin)-10 has been investigated. In this study, immunoblotting results showed the up-regulation of FHC in C. trachomatis-infected HeLa cells in comparison with mock (in vitro control). Further secretory IL-10 level was significantly increased (P<0.001) or decreased (P<0.001) in response to iron supplementation [FAC (ferric ammonium citrate)] and depletion [DFO (deferoxamine)], respectively. However, in C. trachomatis-infected HeLa cells, levels of IL-10 remain higher, irrespective of availability of iron in comparison with their respective control. These results showed that secretion of IL-10 and expressions of FHC have concordance. Further, to understand interdependence of IL-10 and iron homoeostasis (regulation), the levels of IL-10 were compared with iron-responsive GFP (green fluorescent protein) expression in HeLa-229 cells. The mean fluorescent intensities of GFP were in accordance with levels of IL-10 in C. trachomatis-infected cells. These results showed the association of secreted IL-10, FHC and iron homoeostasis in C. trachomatis-infected HeLa-229 cells. This study provides insight into host–Chlamydia interaction at the crossroad of iron metabolism and immune responses and may help in realizing the potential of iron homoeostasis modulators in treatment of chronic chlamydial infection.


Key words: Chlamydia trachomatis, deferoxamine, ferritin, IL-10, iron response element, transferrin receptor

Abbreviations: DFO, deferoxamine, EB, elementary body, EMEM, Earl's modified Eagle's medium, FAC, ferric ammonium citrate, FCS, fetal calf serum, FHC, ferritin heavy chain, GFP, green fluorescent protein, HBSS, Hanks balanced salt solution, hpi, hours postinfection, IL, interleukin, IRE, iron-responsive element, IRPs, iron regulatory proteins, MFI, mean fluorescence intensity, moi, multiplicity of infection, TfR, transferrin receptor

1To whom correspondence should be addressed (email amittal_cp@rediffmail.com).


1. Introduction

Chlamydia trachomatis is an obligate intracellular bacterium that causes a variety of human and animal diseases, affecting principally mucosal epithelial surfaces in the eye and genital tract. Chlamydial disease manifestation is more of host immune reactions rather than Chlamydia itself. These reactions result in chronic inflammation with dense lymphocytic and plasma cell infiltration, occasional granuloma formation and fibrotic scarring of the mucosa, ultimately leading to blindness and fallopian tubal obstruction (Schachter, 1978; Ward, 1995).

It has long been accepted that iron is the key determinant of infection state of Chlamydia and is centrally placed at the crossroad of immune responses and infections (Fresno et al., 1997; Dill and Raulston, 2007). Cellular iron availability alters the proliferation and activation of immune cells and is able to modulate immune effector pathways and cytokine activities (Bahia-Oliveira et al., 2009; Dagvadorj et al., 2009; Vardhan et al., 2009b). Moreover, iron is directly involved in cytotoxic immune defense mechanisms, where iron is needed to catalyse the formation of the hydroxyl radical (OH) via Fenton reaction (Gray et al., 2002). Therefore, cells and pathogens regulate iron homoeostasis by controlling transport, export and storage of iron. TfR (transferrin receptor) and ferritin are iron transporter and iron-storage protein, respectively, that plays a key role in cellular iron homoeostasis and are regulated by availability of iron (Rouault, 2002).

Besides iron, cytokines are also involved in transcriptional and/or posttranscriptional regulation of TfR and FHC (ferritin heavy chain) synthesis (Harrison and Arosio, 1996). Moreover, FHC is potent immunosuppressive protein, which induces IL (interleukin)-10 secretion (Gray et al., 2002). Several studies using various pathogenic infection models have shown that IL-10 plays an important role in balancing the protective and pathological immune responses during intracellular infection (Gazzinelli et al., 1996; Linke et al., 1996; Hunter et al., 1997). The C. trachomatis-associated immunopathological changes are the product of pro- or anti-inflammatory responses. C. trachomatis infection induces production of many pro- and anti-inflammatory cytokines, in which IL-10 is the key determinant of disease pathology (Yang et al., 1998). Earlier reports suggest that IL-10 plays a negative regulatory role in the immune responses, which inhibits host clearance of chlamydial infection (Yang et al., 1996).

Although many studies have shown induction of anti-inflammatory cytokines in chlamydial infections, the interaction of IL-10 with intracellular redox regulator iron and its homoeostasis is still in infancy and need to be studied. Elucidation of association between iron homoeostasis and temporal expression of IL-10 shall give a clue to find effective strategy to control C. trachomatis infection.

2. Materials and methods

Unless otherwise stated, all the reagents were purchased from Sigma–Aldrich and antibodies from Santa Cruz Biotechnology. Plasticwares and glasswares for tissue culture were obtained from Greiner.

2.1. Culture of C. trachomatis

Laboratory reference of C. trachomatis serovar D (D/UW-3/Cx) was propagated in HeLa-229 cells as described previously (Schachter and Wyrick, 1994). The EBs (elementary bodies) were purified and stored at 80°C in sucrose–phosphate–glutamate medium (pH 7.0). The inoculums were confirmed to be free from contamination with Mycoplasma spp. using specific kits (Mycoplasma detection kit, Takara).

2.2. Cytokine assays using ELISA

Levels of secreted IL-10 in culture supernatants were assayed, using ELISA kit (Pierce Biotechnology Inc, Rockford, USA) having a minimum detection limit of 2 pg/ml. All the experiments were performed in triplicate following the manufacturer's instructions, and experiments were conducted three times to confirm reproducibility.

2.3. IRE-GFP (iron-responsive element–green fluorescent protein) construct and its transfection in HeLa-229 cells

IRE-containing plasmid was a kind gift from Dr Michael Kiebler. The construct was a modified pd2EGFP-N1 vector containing ferritin IRE, in place of indigenous CMV (cytomegalovirus) promoter, ahead of NLS (nuclear localization sequence)–GFP cassette. In abundance of iron, cytoplasmic IRPs (iron regulatory proteins) lose their binding activity to IRE, thus no translation blockage occurs, and expression of GFP is registered as green fluorescence in FL-1 detectors (Macchi et al., 2003).

HeLa-229 cells were grown in six-well tissue culture plates (5×105 cells/well) in EMEM (Earl's modified Eagle's medium) containing 10% FCS (fetal calf serum) for 24 h until subconfluence was reached. Subconfluent monolayer was washed twice with HBSS (Hanks balanced salt solution) and transfected with 1 μg construct using 3 μl HD Fugene (Roche Healthcare) transfection reagents as per manufacturer's instructions followed by addition of maintenance media (EMEM+10% FCS). Subsequently transfected cells were placed in humidified incubator at 37°C in 5% CO2 environment for 24 h prior to infection with C. trachomatis. Transfection of cells was confirmed by fluorescence microscope equipped with 488 nm excitation and 525 nm emission filters.

2.4. Flow cytometry of transfected (IRE–GFP) HeLa-229 cells

Transfected cells were washed with HBSS and infected with chlamydial EBs at moi (multiplicity of infection) of 2. For homogenous infection, tissue culture plates were placed on a shaker for 2 h at 35°C after addition of serum-free media containing EBs. Media containing unbound EBs were aspirated and supplemented with complete EMEM containing 10% FCS. Infected HeLa-229 cells were incubated at 37°C with 5% CO2 in a humid environment. Thereafter, at 18 hpi (hours postinfection) media were aspirated and replaced with fresh media containing 50 μM DFO (deferoxamine) and 1 mM FAC (ferric ammonium citrate) in respective wells with their controls. After 6 h of addition of DFO and FAC, cells were detached and acquired using flow cytometer (BD FACS Caliber) in FL-1 channel. For negating autofluorescence, the same pool of untransfected cells was used, and the appropriate setting was used for further acquisition and analysis. Flow histogram was analysed for mean fluorescent intensity using FCS V3 express (DeNovo Inc). Data represented was originated from three independent experiments, and each experiment was performed in triplicate.

2.5. Immunoblotting of proteins

C. trachomatis-infected HeLa-229 cells were washed with PBS and subsequently treated with protein lysis buffer (0.5% Nonidet P-40, 150 mM NaCl, 0.1% SDS, 50 mM NaF, 1 mM Na3VO4 and 1 mM phenyl methyl sulfonyl fluoride) containing the complete protease-inhibitor cocktail (Roche Diagnostics). Protein concentrations were determined by the Bradford protein assay (BioRad Laboratories) using BSA as standard. Extracted proteins (40 μg) were electrophoresed on SDS/polyacrylamide gels and transferred to PVDF membranes (BioRad Laboratories); the membranes were then reversibly stained with Ponceau S to confirm complete transfer. Membranes were blocked with 5% non-fat dry milk in PBS–Tween 20 and incubated with rabbit anti-IgGs against TfR-1 (1:1000) and FHC (1:1000); they were further incubated with the goat anti-rabbit IgG conjugated with horseradish peroxidase (1:3000). Subsequently, they were developed using DAB (di-aminobenzamide) as the detection agent and scanned using Image master II scanner (GE Healthcare). Scanned images were quantified by Image J software (NIH) and represented as fold changes considering mock as 1. Reproducibility of immunoblot was confirmed in three biological replicates, and the mean was represented in bar diagram.

2.6. Statistical analysis

Statistical analysis was carried out with the GraphPad Prism software (version 5.0). Differences were tested for statistical significance by one-way ANOVA (analysis of variance), and P<0.05 was considered as significant.

3. Results

3.1. Up-regulation of FHC in C. trachomatis-infected HeLa cells

FHC is an acute phase anti-inflammatory protein, which gets up-regulated in response to infection with pathogenic bacteria. Therefore, determination of FHC expression in C. trachomatis-infected HeLa cells would help to understand its role in immunomodulation. Immunoblotting was performed, and results showed significant (P<0.001) higher expression of FHC in C. trachomatis-infected cells in comparison with mock-infected cells (Figure 1). C. trachomatis- and mock-infected HeLa cells were treated with FAC (1 mM) to determine effect of iron supplementation on FHC expression. Results showed significant (P<0.001) up-regulation of FHC in mock; however, in C. trachomatis-infected cells, changes were less significant (P<0.01) in comparison with mock and C. trachomatis control, respectively. Further, to assess the effect of iron deprivation, intracellular iron chelator DFO (50 μM) was added to mock- and C. trachomatis-infected cells. Significant (P<0.005) decrease in FHC expression was observed in mock-infected cells in comparison with untreated mock; however, C. trachomatis-infected cells showed non-significant decline in comparison with untreated C. trachomatis-infected cells (Figure 1). These results inferred that expression of FHC in C. trachomatis-infected cells was less dependent on availability of iron; rather, it was responsive to C. trachomatis.

3.2. Level of secreted IL-10 in supernatants of C. trachomatis-infected cells

To understand immunomodulatory effect of FHC, secretory level of anti-inflammatory IL-10 was assessed in C. trachomatis and mock-infected cells in the presence and absence of iron. C. trachomatis-infected HeLa cells secreted significantly (P<0.001) a higher level of IL-10 in comparison with untreated mock-infected HeLa cells (Figure 2). On addition of iron supplement FAC, a significant increase in IL-10 levels were observed in both mock (P<0.001)- and C. trachomatis (P<0.01)-infected cells when compared with untreated mock and C. trachomatis-infected cells, respectively. Moreover, changes were more significant in mock (P<0.001) than C. trachomatis (P<0.01)-infected cells. When mock-infected cells were treated with iron chelator DFO, a significant decrease in IL-10 level was observed in comparison with untreated mock. In contrast, DFO-treated C. trachomatis-infected cells showed a lower degree (P<0.01) of decrease in IL-10 level in comparison with C. trachomatis control (Figure 2). Thus, these results showed association of IL-10 secretions with iron supplementation and depletion in mock-infected HeLa cells. However, C. trachomatis-infected HeLa cells did not show the same pattern for secreted IL-10, in contrast with the mock-infected cells. These results are in accordance with the expression of FHC as observed in immunoblotting. Moreover, it is important to investigate the effect of C. trachomatis on regulatory mechanism, which controls expression of FHC with temporal secretion of IL-10 to find the relation between cause and effect.

3.3. Concerted IL-10 secretion and IRE–IRP-regulated GFP expression

The level of IL-10 in mock- and C. trachomatis-infected cells was compared with MFI (mean fluorescence intensity) of GFP as observed in flourocytometry (BD FACaliber). The level of IL-10 was increased on addition of iron supplement FAC and decreased on addition of iron chelator DFO in mock-infected cells (Figure 3). Similarly, in mock-infected cell, iron-regulated GFP expression (MFI) was increased (P<0.005) in the case of iron supplementation with FAC, and decrease was evident (P<0.005) in the case of iron chelation with DFO. However, in C. trachomatis-infected cells, there was significant (P<0.0005) increase in the level of IL-10 observed in comparison with mock-infected HeLa cells, which was concomitant with apparent increase in MFI of GFP (Figure 3). In C. trachomatis-infected cells, increase in IL-10 was observed irrespective of iron supplementation with FAC and iron depletion with DFO in comparison with C. trachomatis in vitro control. When these results were compared with GFP expression (MFI) of C. trachomatis-infected cells, a similar trend of consistently elevated MFI was observed, irrespective of treatment with FAC and DFO (Figure 3). These results further showed the similar trend in expression pattern of IL-10, FHC and GFP, thereby suggesting the association in C. trachomatis infected cells.

4. Discussion

Chlamydiae have shown the ability to modulate host pathways in favour of its own survival. In this study, we have shown that Chlamydia-induced increase in IL-10 secretion is associated with FHC expression in HeLa cells. Iron plays a vital role in key processes of host–pathogen interaction and innate immune system. Free iron ion catalyses many reactions, which generate free radicals of oxygen that is toxic to pathogen as well as for the host if not controlled. The balance of inflammatory reaction is becoming a key determinant in disease pathology. Iron metabolism and immune responses are closely interconnected. This is due to divergent regulatory effects of the metal on immune cell proliferation and on the effectiveness of cellular immune effector pathways (Tilg et al., 2002; Ludwiczek et al., 2003; Weiss, 2005).

In this study, we did comparative analysis of temporal levels of IL-10-, FHC- and IRE-regulated GFP expression as MFI. The analysis showed the direct association of temporal level of IL-10, FHC expression and expression of GFP (MFI) as FL-1 shift was seen in the flow histogram. In the presence of FAC and DFO, mock-infected cells showed significant increase and decrease in the IL-10 level and FHC expression. Similar increased and decreased expressions of GFP (MFI) were observed in flow cytometric analysis. In contrary, C. trachomatis-infected HeLa-229 cells showed non-responsiveness to iron supplementation (FAC) and depletion (DFO) and appeared with consistently increased levels of IL-10, FHC and MFI in comparison with mock control. Further, these results have inferred that levels of IL-10 are independent on the availability (supplementation and depletion) of iron; moreover, it specifically depends on IRE-regulated FHC expression in C. trachomatis-infected HeLa-229 cells. Hence, these results showed the association of temporal levels of IL-10, FHC and iron homeostasis in C. trachomatis-infected cells. In our earlier study, we have shown that the attenuated regulatory control of iron homoeostasis is responsible for higher expression of FHC in C. trachomatis-infected HeLa-229 cells (Vardhan et al., 2009a). Further, these results indicate that higher expression of FHC could be an acute response of C. trachomatis, mediated through suppression of immune responses by IL-10 to complete their developmental cycle. In our study, significantly higher levels of IL-10 were detected by ELISA in Chlamydia-infected cells compared with control cells. In earlier studies, IL-10 has been shown to be related to the suppression of immune reactions (Bahia-Oliveira et al., 2009; Dagvadorj et al., 2009). The function of IL-10 production induced by pathogen exposure may have an opponent effect on host–pathogen interaction. On one hand, IL-10 may lead to dysfunctional immune protection, thus providing an opportunity for immune evasion by the microbes (McGuirk and Mills, 2002; Ocana-Morgner et al., 2003; Jeong et al., 2009). On the other hand, IL-10 production may be beneficial to the host, given the fact that IL-10 is essential in regulating immune responses and thus preventing an excessive inflammatory response that may be detrimental (Akbari et al., 2001; Higgins et al., 2003). Certain microbial products can induce IL-10-producing cells that show that an immunoregulatory function has been reported in several studies (Pulendran et al., 2001; Romagnoli et al., 2004; Han et al., 2006; Srivastava et al., 2008). IL-10 effectively suppresses production of proinflammatory cytokines, generation of a reactive oxygen intermediate and expression of surface MHC class II and co-stimulatory molecules such as B7 (Shibata et al., 1998). The unique immunostimulatory function of IL-10 is broadly dependent on the microenvironment and cytokine milieu (Shibata et al., 1998; Herrero et al., 2003).

Further, the association of cytokine and iron homoeostasis is a widely reported and reviewed phenomenon (Luft, 2009). Alternatively, it has been extensively reported that cytokines influence the posttranscriptional control of iron homoeostasis by modulating the binding affinity of IRP-1 and IRP-2 to specific RNA stem loop structures, termed IREs (Pantopoulos and Hentze, 1995; Hentze et al., 2004; Kell, 2009). This study suggests the interdependence of IL-10 levels and iron homoeostasis in C. trachomatis-infected HeLa-229 cells, which is linked through FHC. This data further showed the ability of Chlamydia to modulate interwoven host regulatory pathways for its existence. Thus, this study provides a stepping stone to understand possible interdependence of cytokine and iron homoeostasis in C. trachomatis infection in vitro. Extrapolation of these finding may help to develop effective treatment strategy against C. trachomatis-infected cases pertaining to complex disease outcome.

Author contribution

Harsh Vardhan, Rishien Gupta and Aruna Mittal were involved in the conception, design of experiments, analysis of data and interpretation of results and drafting of the manuscript. Harsh Vardhan performed the major experiments in flow cytometry, ELISA and transfection. Rajneesh Jha helped in the flow cytometry and ELISA and Apurb Rashmi Bhengraj helped in the transfection and cell culture experiments.

Acknowledgments

Authors are thankful to Dr Michael Keibler for the desired construct.

Funding

The authors wish to thank the Indian Council of Medical Research (ICMR, India) for financial assistance to Harsh Vardhan and Rajneesh Jha in the form of fellowship and to the University Grant commission (UGC) India for providing fellowship to Rishien Gupta and Apurb Rashmi Bhengraj. This study was funded by the intramural project grant by the Indian Council of Medical Research.

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Received 29 June 2010/13 February 2011; accepted 17 March 2011

Published as Cell Biology International Immediate Publication 17 March 2011, doi:10.1042/CBI20100463


© The Author(s) Journal compilation © 2011 Portland Press Limited


ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
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