Apurb Rashmi Bhengraj

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.

PROTECTIVE OR PATHOGENIC IMMUNE RESPONSE TO GENITAL CHLAMYDIAL INFECTION IN WOMEN-A POSSIBLE ROLE OF CYTOKINE SECRETION PROFILE OF CERVICAL MUCOSAL CELLS

Little is known about genital mucosal immune response to chlamydial infection in women with or without sequelae (Chlamydia positive women with or without fertility disorders as infertility and multiple spontaneous abortions). Cervical lymphocytes were stimulated with chlamydial EBs and cytokine secretion was determined by ELISA, RT-PCR and ELISPOT assays. Stimulated cervical cells from women with fertility disorders (FD) secrete significantly (P<0.05) higher levels of IL-1beta, IL-6, IL-8 and IL-10 and cells from fertile women secrete significantly higher levels of IL-12 and IFN-gamma compared to other groups. RT-PCR analysis showed similar results for IFN-gamma and IL-12. For IL-10 and IL-4, mRNA expression levels were significantly higher (P<0.05) in cells obtained from women with FD compared to other groups. Results for ELISPOT assay were similar as those of RT-PCR. The results suggest that cytokine secretion profile of cervical cells may decide whether infection does not hamper fertility or will develop fertility disorder.

Expression of TLR 2, TLR 4 and iNOS in cervical monocytes of Chlamydia trachomatis-infected women and their role in host immune response.

Citation Agrawal T, Bhengraj AR, Vats V, Salhan S, Mittal A. Expression of Toll‐like receptors (TLR) 2, TLR 4 and inducible nitric oxide synthase (iNOS) in cervical monocytes of Chlamydia trachomatis‐infected women and their role in host immune response. Am J Reprod Immunol 2011; 66: 534–543

Potential of a novel polyherbal formulation BASANT for prevention of Chlamydia trachomatis infection

The effect of a novel polyherbal formulation BASANT on Chlamydia trachomatis was studied. In vitro sensitivity testing was done by direct exposure of C. trachomatis (pre-infection incubation with BASANT) and exposure of C. trachomatis within HeLa 229 cells (post-infection incubation with BASANT). Pre-infection incubation of standard serovar D/UW-3/Cx with BASANT showed complete inhibition after 60, 30 and 15 min of incubation at concentrations of 12, 30 and 60 microg/mL, respectively. In the post-infection incubation, 8-10 microg/mL of BASANT showed complete inhibition of standard serovar D/UW-3/Cx as well as of five clinical isolates of C. trachomatis after 48 h of incubation. BASANT also inhibited a clinical isolate obtained from a doxycycline treatment failure patient at a concentration of 30 microg/mL. Both assays with standard and clinical isolates showed that BASANT has antimicrobial activity against C. trachomatis, suggesting the potential clinical utility of BASANT for the prevention of C. trachomatis infection by the sexual route.

Chlamydia trachomatis Alters Iron-Regulatory Protein-1 Binding Capacity and Modulates Cellular Iron Homeostasis in HeLa-229 Cells

Chlamydia trachomatis (CT) is the leading cause of diseases related to reproductive health and iron plays important role in chlamydial pathogenesis. Iron homeostasis in chlamydia-infected cells is not clear thus far. This study shows that expression of the transferrin receptor (TfR) is downregulated, whereas expression of the ferritin heavy chain is upregulated in CT-infected HeLa-229 cells. Expression of iron-regulatory protein (IRP)-1 predominates over IRP-2 in infected cells. In infected cells, attenuated binding activity of IRP-iron responsive elements (IREs) is observed using the electrophoretic mobility-shift assay. These results suggest that iron homeostasis is modulated in CT-infected HeLa cells at the interface of acquisition and commensal use of iron.

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.

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 (β), 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-1β 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.

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.

Chlamydia trachomatis: TLR4-mediated recognition by human dendritic cells is impaired following oestradiol treatment.

Abstract Genital Chlamydia trachomatis infection creates a substantial reproductive health burden in women. The high incidence of asymptomatic infection often precludes timely antibiotic therapy to control the sequelae of infection, and therefore a vaccine is required. Dendritic cells (DC) are now being used as an adjuvant for vaccine development; however, the fate of C. trachomatis in human DC and differential regulation of cytokine secretion remains unclear. Hence, an in vitro study was performed using C. trachomatis (serovar D) elementary body (EB)-pulsed, monocyte-derived DCs co-cultured with autologous CD4+ T cells. Secreted cytokines were measured to assess the protective/pathogenic immune response. The effect of P-oestradiol in the modulation of DC function and on Tolllike receptor (TLR) gene expression was also studied. Elementary body-pulsed DCs showed induction of protective Th1 immune response with upregulation of TLR4 expression, secretion of interleukin (IL)-6, IL-12 and interferon (LFN)-γ, together with upregulation of major histocompatibility complex (MHC) class II, CD83 and CD86. When co-cultured with autologous CD4+ T cells, DCs presented chlamydial antigens efficiently, as shown by proliferation of T cells and secretion of IL-2 and IFNγ, which provide a protective immune response. However; pretreatment of cells with oestradiol significantly reduced TLR4 expression and upregulated IL-10 secretion, modulating the Th1 immune response to a Th2-type response, which may lead to pathogenesis.

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.