Ferritin heavy chain-mediated iron homoeostasis regulates expression of IL-10 in Chlamydia trachomatis-infected HeLa cells.

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.

Higher expression of ferritin protects Chlamydia trachomatis infected HeLa 229 cells from reactive oxygen species mediated cell death.

Apoptosis plays an important role in modulating the pathogenesis of a variety of infectious diseases. Chlamydial infection protects cells against different forms of apoptosis: extrinsic, intrinsic, and granzyme B mediated. Redox reactions are central to the life and death decision of cells and pathogens and an intimate relationship exists between oxidative stress and iron metabolism. The link between redox status and ferritin was largely unexplored in chlamydia-infected cells. In the present study, we showed that Chlamydia trachomatis (CT) infection induced FHC protein in HeLa cells. FHC induction by CT-infected cells stably expressing FHC blunted ROS production compared with mock infected cells, and the infected cells were relatively resistant to apoptosis induced by H2O2. We also demonstrated that endogenous FHC overexpression correlates well with the stabilization of the mitochondrial membrane potential in CT-infected cells. Increased expression of FHC is independent of iron supplementation (FAC) and depletion (DFO) in CT-infected cells. These data suggest that FHC up-regulation is an acute response of HeLa cells against CT infection and that FHC exerts anti-apoptotic activity against oxidative stress.

Decreased susceptibility to azithromycin and doxycycline in clinical isolates of Chlamydia trachomatis obtained from recurrently infected female patients in India.

BACKGROUND: Recurrent genital Chlamydia trachomatis infection often results in serious sequelae and has a major impact on reproductive health. MATERIALS AND METHODS: Recurrent infections were determined in symptomatic female patients. In vitro susceptibility assay was performed for azithromycin and doxycycline using the cell culture technique against 21 clinical isolates obtained from C. trachomatis-positive patients including those who were recurrently infected. RESULTS: Thirteen isolates (61.9%) were found to be susceptible to azithromycin and doxycycline with minimum inhibitory concentration (MIC) values ≤0.125 and ≤0.25 µg/ml, respectively. Eight isolates (38%) were found to be less susceptible to the drugs. Two of them had MICs of 8 µg/ml for both the drugs and could not be completely eradicated as observed by minimum bactericidal concentration assay. CONCLUSIONS: Decreased antibiotic susceptibility to the current first-line drugs (azithromycin and doxycycline) for chlamydial infection treatment was observed in isolates obtained from recurrently infected patients.

Persistently elevated level of IL-8 in Chlamydia trachomatis infected HeLa 229 cells is dependent on intracellular available iron.

Chlamydia trachomatis is a leading cause of sexually transmitted infection worldwide and responsible for myriad of immunopathological changes associated with reproductive health. Delayed secretion of proinflammatory chemokine interleukin (IL)-8 is a hallmark of chlamydial infection and is dependent on chlamydial growth. We examined the effect of iron chelators on IL-8 production in HeLa 229 (cervix epitheloid cell, CCL2) cells infected with C. trachomatis. IL-8 production was induced by Iron chelator DFO and Mimosine, however, synergy with chlamydial infection was obtained with DFO only. Temporal expression of proinflammatory secreted cytokines IL-1beta, TNF-alpha, and IL-8 did not show synchrony in Chlamydia trachomatis infected cells. Secretion of IL-8 from Hela cells infected with C. trachomatis was not dependent on IL-1 beta and TNF- alpha induction. These results indicate towards involvement of iron in chlamydia induced IL-8 production.

Serovar-specific immune responses to peptides of variable regions of Chlamydia trachomatis major outer membrane protein in serovar D-infected women.

The role of major outer membrane protein (MOMP) variable regions in the interaction of chlamydiae and host cells has been evaluated and their role in neutralization of antibodies has been clearly demonstrated. There are also studies that delineate the contribution of these regions to the cell-mediated immune response of the host and suggest that serovar E elicits serovar-specific immune responses in infected humans. However, further studies with other serovars are required to confirm these findings and to elucidate the role and importance of serovar-specific responses of variable regions of MOMP in other serovars. We, therefore, performed a detailed analysis of the humoral and cellular immune responses against the serovar D-specific variable segments (VS) of MOMP in women infected with Chlamydia trachomatis. We found that VS4 elicits significantly higher responses (both humoral and cellular) than other VS peptides (VS1, VS2 and VS3). VS4 elicited significantly higher (P < 0.0001) proliferative responses, interferon-gamma levels (P < 0.0001) as well as higher prevalence (P < 0.0001) of IgG antibodies against VS4 in serovar D-infected patients as compared to patients infected with other serovars, suggesting its role in serovar-specific immune responses.

Differing effects of azithromycin and doxycycline on cytokines in cells from Chlamydia trachomatis-infected women.

Chlamydial infection of the lower genital tract usually spreads to the upper genital tract and is then responsible for more serious consequences, such as infertility, ectopic pregnancy, pelvic pain, and pelvic inflammatory disease. Genital infection with Chlamydia trachomatis and the resulting cytokine response largely determines the outcome of infection and disease. To date, studies showing comparative effects of azithromycin and doxycycline treatment for C. trachomatis infection in women with reproductive sequelae like infertility and their effect on immune molecules like cytokines are lacking. Hence, our objective was to study the effect of azithromycin and doxycycline in vitro on cytokines in cells from C. trachomatis-positive fertile and infertile women as well as their efficacy in C. trachomatis infection. Fertile and infertile women with primary and recurrent C. trachomatis infection attending the gynecology outpatient department of Safdarjung Hospital, New Delhi, India, were enrolled. Enzyme-linked immunosorbent assay and real-time reverse transcription-polymerase chain reaction was performed for evaluating cytokines in cells stimulated with chlamydial elementary bodies (EBs) in the presence and absence of antibiotics (azithromycin and doxycycline). C. trachomatis-infected women were also followed up to assess the efficacy of azithromycin and doxycycline. We observed inhibition of cytokines (interleukin [IL]-1beta (ß), IL-6, IL-8, IL-10, and tumor necrosis factor-alpha) in the presence of azithromycin in EB-stimulated cells from both fertile and infertile women with primary and recurrent C. trachomatis infection. However, in presence of doxycycline, inhibition of cytokines (IL-1ß and IL-6) was only observed in stimulated cells from fertile women with primary C. trachomatis infection. The clinical efficacy of azithromycin was also better than doxycycline in recurrent C. trachomatis infection in women with complications such as infertility. Overall, this study suggests that azithromycin treatment with broader immunomodulatory effects may be preferable to doxycycline for the treatment of recurrent C. trachomatis infection associated with infertility.

Chlamydia pneumoniae heat shock protein 60 is associated with apoptotic signaling pathway in human atheromatous plaques of coronary artery disease patients.

BACKGROUND: Chlamydia pneumoniae heat shock protein (HSP) 60 is known to contribute to the activation of inflammation. In addition, there are contradictory reports on C. pneumoniae and their role in activation of pathways (apoptotic/antiapoptotic/necrosis) in coronary artery disease (CAD). Hence, more studies are required to know the actual role of C. pneumoniae in activation of apoptotic/antiapoptotic/necrosis pathways. METHODS AND RESULTS: In this study, two sets of patient groups (cHSP60 positive and cHSP60 negative) were included and gene expression was studied by cDNA micro array and real time polymerase chain reaction arrays. Expression of Caspase-3, 8, 9, c-FLIP, PPAR-γ, PGC-1α, and Gsk-3b were also evaluated at protein level by immunoblotting. In cHSP60 positive CAD patients significantly higher (p<0.001) mRNA expression was found for CCL4, CXCL4, CXCL9, IL-8, CD40LG, CD8, TGFß1, TGFß2, APOE, EGR1, CTGF, APOB, LDLR, LPA, and LPL, whereas significantly lower (p<0.001) mRNA expression was detected for CD4, IL1F10, IFNA2, and IL-10 as compared to cHSP60 negative CAD patients. Additionally, at protein level expression of Caspase-3 (p=0.027), 8 (p=0.028), and 9 (p=0.037) were higher and c-FLIP (p=0.028) and PPAR-γ (p=0.95) expression were comparable in cHSP60 positive CAD patients compared to cHSP60 negative CAD patients. CONCLUSION: Genes/proteins of pre-apoptotic caspase dependent/independent pathways, chemokines, and inflammatory cytokines receptors were significantly up-regulated in human atheromatous plaques of cHSP60 positive CAD patients suggesting an association of cHSP60 with CAD.

Azithromycin treatment modulates the extracellular signal-regulated kinase mediated pathway and inhibits inflammatory cytokines and chemokines in epithelial cells from infertile women with recurrent Chlamydia trachomatis infection.

Epidemiological and animal model studies suggest that sequelae of genital Chlamydia trachomatis infection are more often associated with second or subsequent infections than with initial infection. Further, in order to establish an acute or long-term persistent infection, C. trachomatis develops several strategies to circumvent host immune responses. Hence, resolution of the C. trachomatis infection may require modulation of host factors especially during persistent or chronic infection. Moreover, azithromycin treatment has been reported to possess anti-inflammatory properties but its mechanism of action is still not elucidated. Therefore, in order to better understand the effect of azithromycin in chronic conditions, our aim was to study changes in expression of key genes associated with inflammatory cytokines and receptors, mitogen-activated protein kinase (MAPK) signaling pathway, and apoptosis pathway before and after therapy with azithromycin in infertile women with recurrent C. trachomatis infection. Real-time polymerase chain reaction was performed to study inflammatory cytokines and receptors, MAPK signaling pathway, and apoptosis pathway before and after therapy with azithromycin in infertile women with recurrent C. trachomatis infection. Further, effect of azithromycin on activation of extracellular signal-regulated kinase was studied in epithelial cells by western blotting. Chemokine (C-C motif) ligand 2 (CCL2), CCL5, chemokine (C-X-C motif) ligand 1 (CXCL1), CXCL5, CXCL9, interleukin-1B (IL-1B), IL-8, baculoviral IAP repeat-containing 3 (BIRC3), myeloid cell leukemia sequence 1 (MCL1), and MAPK1 were downregualted after azithromycin treatment. In addition, phosphorylation of extracellular signal-regulated kinase was inhibited after azithromycin treatment in epithelial cells obtained from women with recurrent infection. Hence, our data suggest that azithromycin with its properties apart from antibacterial activity may contribute to its therapeutic potential in treatment of chronic recurrent infection in infertile women.