Localization of TLR2 and MyD88 to Chlamydia trachomatis Inclusions
2005; Elsevier BV; Volume: 281; Issue: 3 Linguagem: Inglês
10.1074/jbc.m510182200
ISSN1083-351X
AutoresCatherine M. O’Connell, И. А. Ионова, Alison J. Quayle, Alberto Visintin, Robin R. Ingalls,
Tópico(s)Urinary Tract Infections Management
ResumoChlamydia trachomatis is an obligate intracellular Gram-negative pathogen and the etiologic agent of significant ocular and genital tract diseases. Chlamydiae primarily infect epithelial cells, and the inflammatory response of these cells to the infection directs both the innate and adaptive immune response. This study focused on determining the cellular immune receptors involved in the early events following infection with the L2 serovar of C. trachomatis.We found that dominant negative MyD88 inhibited interleukin-8 (IL-8) secretion during a productive infection with chlamydia. Furthermore, expression of Toll-like receptor (TLR)-2 was required for IL-8 secretion from infected cells, whereas the effect of TLR4/MD-2 expression was minimal. Cell activation was dependent on infection with live, replicating bacteria, because infection with UV-irradiated bacteria and treatment of infected cells with chloramphenicol, but not ampicillin, abrogated the induction of IL-8 secretion. Finally, we show that both TLR2 and MyD88 co-localize with the intracellular chlamydial inclusion, suggesting that TLR2 is actively engaged in signaling from this intracellular location. These data support the role of TLR2 in the host response to infection with C. trachomatis. Our data further demonstrate that TLR2 and the adaptor MyD88 are specifically recruited to the bacterial or inclusion membrane during a productive infection with chlamydia and provide the first evidence that intracellular TLR2 is responsible for signal transduction during infection with an intracellular bacterium. Chlamydia trachomatis is an obligate intracellular Gram-negative pathogen and the etiologic agent of significant ocular and genital tract diseases. Chlamydiae primarily infect epithelial cells, and the inflammatory response of these cells to the infection directs both the innate and adaptive immune response. This study focused on determining the cellular immune receptors involved in the early events following infection with the L2 serovar of C. trachomatis.We found that dominant negative MyD88 inhibited interleukin-8 (IL-8) secretion during a productive infection with chlamydia. Furthermore, expression of Toll-like receptor (TLR)-2 was required for IL-8 secretion from infected cells, whereas the effect of TLR4/MD-2 expression was minimal. Cell activation was dependent on infection with live, replicating bacteria, because infection with UV-irradiated bacteria and treatment of infected cells with chloramphenicol, but not ampicillin, abrogated the induction of IL-8 secretion. Finally, we show that both TLR2 and MyD88 co-localize with the intracellular chlamydial inclusion, suggesting that TLR2 is actively engaged in signaling from this intracellular location. These data support the role of TLR2 in the host response to infection with C. trachomatis. Our data further demonstrate that TLR2 and the adaptor MyD88 are specifically recruited to the bacterial or inclusion membrane during a productive infection with chlamydia and provide the first evidence that intracellular TLR2 is responsible for signal transduction during infection with an intracellular bacterium. Chlamydia trachomatis is an obligate intracellular bacterial pathogen associated with a variety of human diseases. The species of C. trachomatis can be divided into biovars, such as the trachoma biovar and the lymphogranuloma venereum biovar; these are further divided into serovars. In the developing world, serovars A-C are associated with trachoma, one of the oldest diseases caused by C. trachomatis infection, and the leading cause of preventable blindness in the world (reviewed in Ref. 1Munoz B. West S. Epidemiol. Rev. 1997; 19: 205-217Crossref PubMed Scopus (54) Google Scholar). Serovars D-K are also of the trachoma biovar and are all associated with genital tract disease as well as conjunctivitis and infant pneumonia. The World Health Organization reported 89 million new cases of genital Chlamydia infections in 1995, demonstrating the extent of this infection worldwide and hinting at the economic burden it has on healthcare (2Beagley K.W. Timms P. J. Reprod. Immunol. 2000; 48: 47-68Crossref PubMed Scopus (74) Google Scholar). In 2003, 877,478 cases of infection with C. trachomatis were reported to the CDC from 50 states and the District of Columbia (3Centers for Disease Control and Prevention Sexually Transmitted Disease Surveillance 2003 Supplement, Chlamydia Prevalence Monitoring Project. U.S. Department of Health and Human Services, Centers for Disease Control and Prevention, Atlanta, GA2004: 4Google Scholar), making it the most common bacterial sexually transmitted infection. The lymphogranuloma venereum (LGV) 2The abbreviations used are: LGVlymphogranuloma venereumEBelementary bodyRBreticulate bodym.o.i.multiplicity of infectionCFPcyan fluorescent proteinYFPyellow fluorescent proteinGFPgreen fluorescent proteinEGFPenhanced GFPTLRToll-like receptorLPSlipopolysaccharideIL-1βinterleukin-1βShEChuman papillomavirus 16/E6E7 immortalized ectocervical epithelial cell lineHEKhuman embryonic kidney cellsFBSfetal bovine serumELISAenzyme-linked immunosorbent assayPBSphosphate-buffered saline. 2The abbreviations used are: LGVlymphogranuloma venereumEBelementary bodyRBreticulate bodym.o.i.multiplicity of infectionCFPcyan fluorescent proteinYFPyellow fluorescent proteinGFPgreen fluorescent proteinEGFPenhanced GFPTLRToll-like receptorLPSlipopolysaccharideIL-1βinterleukin-1βShEChuman papillomavirus 16/E6E7 immortalized ectocervical epithelial cell lineHEKhuman embryonic kidney cellsFBSfetal bovine serumELISAenzyme-linked immunosorbent assayPBSphosphate-buffered saline. serovars L1, L2, and L3 are also sexually transmitted but are associated with a more invasive disease known as lymphogranuloma venereum (4Schachter J. Holmes K.K. Sparling P.F. Mård P.A. Lemon S.M. Stamm W.E. Piot P. Wasserheit J.N. Sexually Transmitted Diseases. McGraw-Hill, New York1999: 391-405Google Scholar). Although LGV is uncommon in the developed world, there have been recent outbreaks of LGV proctitis in the Netherlands and Western Europe (5Gotz H.M. Ossewaarde J.M. Nieuwenhuis R.F. van der Meijden W.I. Dees J. Thio B. de Zwart O. van de Laar M.J. Ned. Tijdschr. Geneeskd. 2004; 148: 441-442PubMed Google Scholar, 6Nieuwenhuis R.F. Ossewaarde J.M. Gotz H.M. Dees J. Thio H.B. Thomeer M.G. den Hollander J.C. Neumann M.H. van der Meijden W.I. Clin. Infect. Dis. 2004; 39: 996-1003Crossref PubMed Scopus (260) Google Scholar). lymphogranuloma venereum elementary body reticulate body multiplicity of infection cyan fluorescent protein yellow fluorescent protein green fluorescent protein enhanced GFP Toll-like receptor lipopolysaccharide interleukin-1β human papillomavirus 16/E6E7 immortalized ectocervical epithelial cell line human embryonic kidney cells fetal bovine serum enzyme-linked immunosorbent assay phosphate-buffered saline. lymphogranuloma venereum elementary body reticulate body multiplicity of infection cyan fluorescent protein yellow fluorescent protein green fluorescent protein enhanced GFP Toll-like receptor lipopolysaccharide interleukin-1β human papillomavirus 16/E6E7 immortalized ectocervical epithelial cell line human embryonic kidney cells fetal bovine serum enzyme-linked immunosorbent assay phosphate-buffered saline. The unique developmental cycle of the chlamydiae sets them apart from other bacterial species. C. trachomatis primarily infects columnar epithelial cells, undergoing a complex intracellular lifecycle that is required for its survival. The lifecycle begins with the attachment of the infectious elementary body (EB) to the host cell, followed by cell entry. Once it has entered the cell, the surrounding plasma membrane becomes the inclusion membrane. The EB subsequently converts to the replicative reticulate body (RB) and begins to reproduce by binary fission. The chlamydial inclusion, which neither fuses with lysosomes nor acidifies, then traffics to the Golgi region where it can intercept sphingolipid-containing vesicles (reviewed in Ref. 7Hackstadt T. Fischer E.R. Scidmore M.A. Rockey D.D. Heinzen R.A. Trends Microbiol. 1997; 5: 288-293Abstract Full Text PDF PubMed Scopus (155) Google Scholar). The RBs grow and divide within this unique cellular inclusion. At some point late in the infectious process, RBs asynchronously begin to differentiate back to EBs. 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To further examine the role of specific TLRs in the host response to a productive chlamydia infection, we used an immortalized human cervical epithelial cell line, as well as transfected HEK293 cells to determine which members of the Toll-like receptor (TLR) family might be involved in the early events following attachment and invasion of chlamydia. We found that expression of MyD88 and TLR2 was required for cellular activation following infection with C. trachomatis, as measured by the secretion of the chemokine IL-8. In contrast, we found expression of TLR4/MD-2 had only a minor effect on IL-8 production by HEK293 cells infected with C. trachomatis. Cell activation required a productive chlamydia infection, and the use of UV-irradiated bacteria or treatment of infected cells with chloramphenicol following infection abrogated the response. In contrast, treatment with ampicillin, which effectively halts the differentiation of the organism, did not inhibit cytokine production, suggesting that bacterial replication and differentiation to at least the reticulate body phase was also required for cell activation. Finally, we found that TLR2 was tightly associated with the bacteria during the intracellular phase and that MyD88 was recruited along with TLR2 to the inclusion membrane. These data suggest that intracellular TLR2 is responsible for the initiation of signal transduction events during infection with C. trachomatis. Reagents—Phosphate-buffered saline and Trypsin-Versene Mixture (trypsin-EDTA) were obtained from BioWhittaker (Walkersville, MD), and fetal calf serum was obtained from HyClone (Logan, UT). Recombinant human IL-1β and tumor necrosis factor-α were purchased from R&D Systems (Minneapolis, MN). Synthetic lipopeptide Pam3-Cys-Lip, which contains a tripalmitoyl-S-glyceryl cysteine at the amino terminus, was based on the amino-terminal sequence of the Neisseria gonorrhoeae F62 H.8/Lip protein (sequence CGGEKAAEAPAAEAS) (47Fisette P.L. Ram S. Andersen J.M. Guo W. Ingalls R.R. J. Biol. Chem. 2003; 278: 46252-46260Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar) was purchased from EMC Microcollections (Tuebingen, Germany). LPS purified from Escherichia coli K235 was purchased from List Biologicals (Campbell, CA). Contaminating endotoxin-associated proteins were removed by phenol re-extraction as described previously (48Manthey C.L. Vogel S.N. J. Endotoxin. Res. 1994; 1: 84Crossref Scopus (96) Google Scholar, 49Hirschfeld M. Ma Y. Weis J.H. Vogel S.N. Weis J.J. J. Immunol. 2000; 165: 618-622Crossref PubMed Scopus (957) Google Scholar). Ampicillin and chloramphenicol were purchased from Sigma-Aldrich. Cell Culture—A human papillomavirus 16/E6E7 immortalized ectocervical epithelial cell line (ShEC) was engineered from human ectocervix collected as discarded surgical material at University Hospital, New Orleans, LA, with approval from the Institutional Review Board. The specimen was from a 35-year-old woman who had undergone hysterectomy for metrorrhagia. Ectocervix was dissected out from an area distant from the transformation zone, and the ectocervical margins were confirmed on frozen sections. Small pieces of tissue were placed epithelial side down in 6-well plates and cultured in keratinocyte serum-free medium (Invitrogen) supplemented with bovine pituitary extract and epidermal growth factor (as recommended and supplied by the manufacturer), and CaCl2 to a final concentration of 0.4 mm. Out-growth of epithelial cells occurred within 10 days, and cells were then passaged twice before transduction with a retroviral vector (LXSN-16E6E7) packaged by the amphotropic fibroblast line PA17 (50Fichorova R.N. Anderson D.J. Biol. Reprod. 1999; 60: 508-514Crossref PubMed Scopus (169) Google Scholar). Cells with integrated vector were selected by resistance to the neomycin analogue G418 and were passaged more than 20 times prior to experiments. The expression of the cytokeratin CK13 and lack of expression of secretory component confirmed the ectocervical derivation of the line. Cells were maintained in keratinocyte serum-free medium as described above with the addition of 20 μg of gentamicin per milliliter (Invitrogen). Human embryonic kidney (HEK) 293 cell lines stably expressing fluorescent TLR chimeric proteins were a gift from Dr. Douglas Golenbock and have been previously described (51Latz E. Visintin A. Lien E. Fitzgerald K.A. Monks B.G. Kurt-Jones E.A. Golenbock D.T. Espevik T. J. Biol. Chem. 2002; 277: 47834-47843Abstract Full Text Full Text PDF PubMed Scopus (363) Google Scholar). The following cell lines were used in these studies: HEK293 expressing cyan fluorescent protein (CFP)- or yellow fluorescent protein (YFP)-tagged TLR2; HEK293 cells expressing YFP-tagged TLR4 and MD-2; and HEK293 cells expressing the empty vector pcDNA3. HEK293 cells expressing YFP-tagged TLR2 or YFP-tagged TLR4, with CFP-tagged MyD88, were engineered by transiently transfecting HEK/TLR2YFP or HEK/TLR4YFP/MD2 with the plasmid pcDNA3 containing CFP-tagged MyD88 (gift of Drs. Douglas Golenbock and Terje Espevik) using Gene Juice (Novagen, San Diego, CA) according to the manufacturer's instructions. Cells were used for infection experiments on day 2 post-transfection. All HEK293 cell lines were cultured in Dulbecco's modified Eagle's medium (BioWhittaker) supplemented with 10% FBS (HyClone) and 20 μg of gentamicin per milliliter. Retroviral Transduction—ShEC cells stably expressing green fluorescent protein (GFP)-tagged MyD88 dominant-negative construct or GFP alone were engineered by retroviral transduction. The cDNA corresponding to amino acids 152-296 of human MyD88 (a kind gift of Dr. Marta Muzio (52Muzio M. Ni J. Feng P. Dixit V.M. Science. 1997; 278: 1612-1615Crossref PubMed Scopus (967) Google Scholar)) was PCR-amplified and cloned in the XhoI (5′)-BamHI (3′) sites of pEGFP-N1 (BD Biosciences-Clontech). The resulting cDNA encodes for a dominant negative version of MyD88 consisting of its TIR (Toll Interleukin 1 Resistance) domain, fused in-frame with the enhanced green fluorescent protein (ΔMyD88EGFP). The retroviral vector rΔMyD88EGFP was generated by subcloning the ΔMyD88EGFP cassette into the XhoI-NotI sites of the retroviral vector CLRCX- (53Wang R.F. Wang X. Johnston S.L. Zeng G. Robbins P.F. Rosenberg S.A. Cancer Res. 1998; 58: 3519-3525PubMed Google Scholar). Infectious retroviral particles were produced by transiently co-transfecting the packaging cell line 293gag/pol (∼1 × 106 cells) with the retroviral plasmid (3 μg) and a plasmid encoding the vesicular stomatitis virus glycoprotein envelope protein (0.5 μg). The supernatant (3 ml) was then collected, filtered through a 0.22-mm nitro-cellulose filter, and applied overnight to the target cells (5 × 105) in an equal amount of fresh medium containing 20 μg of Polybrene per milliliter (Sigma). Cells were allowed to recover for 2 days and re-infected. An identical backbone retrovirus encoding for the EGFP alone was used to construct a control cell line. Following transduction, cells were expanded and subjected to two rounds of positive selection using a MoFlo™ Ultra-High Speed Cell Sorter (DakoCytomation, Ft. Collins, CO) in enrichment mode to select for cells with high levels of GFP fluorescence. Bacterial Culture—C. trachomatis L2/434/Bu was used for these studies (54Schachter J. Smith D.E. Dawson C.R. Anderson W.R. Deller Jr., J.J. Hoke A.W. Smartt W.H. Meyer K.F. J. Infect. Dis. 1969; 120: 372-375Crossref PubMed Scopus (30) Google Scholar, 55Schachter J. Meyer K.F. J. Bacteriol. 1969; 99: 636-638Crossref PubMed Google Scholar). Bacteria were routinely cultured in L929 or McCoy cells. Cells were infected at an approximate multiplicity of infection (m.o.i.) of 0.5-1 before being centrifuged for 1 h at 37 °C. The cell culture medium was then removed and replaced with Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated FBS, 20 μg of gentamicin per milliliter, and 0.1 μg of cycloheximide per milliliter. Infected cells were harvested into sucrose-phosphate-glutamate buffer at 40 h post infection, sonicated, and stored at -80 °C until further use. Bacteria were subsequently titrated by the plaque assay (56Banks J. Eddie B. Schachter J. Meyer K.F. Infect. Immun. 1970; 1: 259-262Crossref PubMed Google Scholar, 57Oaks E.V. Wingfield M.E. Formal S.B. Infect. Immun. 1985; 48: 124-129Crossref PubMed Google Scholar) using the L2 mouse fibroblast cell line. Chlamydia stocks tested negative for mycoplasma by PCR (58Ossewaarde J.M. de Vries A. Bestebroer T. Angulo A.F. Appl. Environ. Microbiol. 1996; 62: 328-331Crossref PubMed Google Scholar). To prepare UV-inactivated bacteria, chlamydial suspensions were placed under a 30-watt UV lamp at 30 cm for 30 min. Inactivation was confirmed by the failure to develop inclusions following infection of HeLa cells. Infection Studies—All antibiotics were removed from cell culture medium 3 days prior to infection with chlamydia. For stimulation assays, cells were plated in 24-well tissue culture dishes at a density of 105 cells per well. Infection with C. trachomatis L2 was carried out by overlaying cells with an m.o.i. of 1-5. Other soluble stimuli were added as noted. Cells were incubated for 18-40 h at 37 °C/5% CO2. Supernatant was harvested in 1.5-ml microcentrifuge tubes, spun for 5 min at 4 °C to pellet any cellular debris, transferred to clean tubes, and frozen at -80 °C. Culture supernatants were assayed for IL-8 using a DuoSet ELISA kit from R&D Systems, or IL-1β using an Eli-pair ELISA kit from Cell Sciences (Norwood, MA). Optical density was measured using a Bio-Kinetics microplate reader (Bio-Tek Instruments, Winooski, VT). All data points were assayed in triplicate and reported as the mean ± S.D. Confocal Microscopy Studies—HEK293 cells were plated at a density of 7.5 × 105 cells per dish on glass bottom 35-mm tissue culture dishes (MatTek Corp., Ashland, MA) coated with 300 μg/ml Type 1 Rat Tail Collagen (BD Biosciences) 1 day prior to infection. For all confocal studies, infections were carried out by overlaying cells with an m.o.i. of 1-5. For antibody staining, cells were fixed and permeabilized with ice-cold methanol for 15 min and blocked for 5 min with PBS containing 5% FBS. Chlamydiae were stained using monoclonal antibody against chlamydia LPS (gift of Dr. You-Xun Zhang) followed by Alexa-647-conjugated goat anti-mouse IgG (Molecular Probes, Eugene, OR). Cells were incubated for 20 min with each antibody, with three washes of PBS/5% FBS in between. Antibodies were used at a concentration of 10 μg/ml. Following the final antibody stain, cells were washed three times with PBS/5% FBS and mounted to a coverslip using SlowFade Light (Molecular Probes). Dishes were viewed under fluorescent confocal microscopy using a Zeiss LSM 510 Axiovert confocal laser scanning microscope equipped with an argon and helium laser, using a 63× objective. Dual color images were consecutively scanned using one active laser per scan. Epithelial Cells Secrete IL-8 in an MyD88-dependent Manner in Response to Productive
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