SIGIRR Attenuates Toll-Like Receptor 4,5,9-mediated Immune Responses In Human Airway Epithelial Cells

Background and ObjectiveToll-like receptors (TLRs) exist on human airway epithelial cells (HAEC) and contribute to the initiation of innate immunity during respiratory infection with pathogenic microorganisms. Single immunoglobulin interleukin-1 receptor-related protein (SIGIRR) is a member of the Toll-interleukin-1 receptors (TIRs) that can negatively modulate immune response. We carried out studies to characterize SIGIRR modulation of TLR-mediated immune response in HAEC and define its mechanisms of action.Methods1. Cell cultureThe human lung mucoepidermoid carcinoma cell line (NCI-H292) was cultured in RPMI 1640 supplemented with 10% fetal bovine serum in the presence of penicillin (100U/ml) /streptomycin (100μg/ml) at 37℃in a humidified chamber with 5% CO2. For serum deprivation, confluent cells were washed twice with phosphate-buffered saline (PBS) and re-cultured in RPMI 1640 with 0.2% fetal bovine serum. The recombinant stable cell lines were cultured with 400μg/ml G418 Sulfate in the medium.2. Plasmid construction and transfectionThe SIGIRR gene was cloned into the pEGFP-Nl expression plasmid placing the SIGIRR in-frame with the N-terminus of the EGFP protein, with a six-amino acid linker of sequence LGCRRW. SIGIRR DNA fragments were generated by PCR from the plasmid pReceiver-Lv19, using the following pair of primers:5'-TAG CTA GAA TTC TGA TGC CAG GTG TCT GTG AT-3' (forward) and 5'-AGT CAG GAT CCC GCA TAT CAT CC T TGG ACA CC-3'(reverse). The PCR product was digested with EcoR I/BamH I restriction enzymes and inserted into pEGFP-N1 vector to produce the recombinant plasmid SIGIRR-EGFP. Cells were transfected with SIGIRR-EGFP or the empty vector control pEGFP-N1 by using Lipofectamine 2000TM transfection reagent according to the manufacturer's instructions, and the stably transfected cell lines were selected with 800μg/ml G418.3. RNA extraction and real-time quantitative RT-PCRTotal RNA was isolated with TRIZOL reagent according to the manufacturer's protocol. The expression of TLR4,5,9 and SIGIRR mRNA in H292 cells were assessed by real-time quantitative RT-PCR as described, including both a negative control and a housekeeper gene control. Oligonucleotide primers were from TaKaRa Bio Inc. SIGIRR mRNA was compared in two stable overexpressed H292 cell lines by using a double standard curves relative quantitation and the results were normalized against Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an endogenous control. Real-time reverse transcription was performed on Rotor-Gene 3000. The PCR parameters were set as 95℃for 2 min, followed by 40 cycles of 95℃for 5 s and 60℃for 20 s. All reactions were performed in triplicate, and reports were generated by Rotor-Gene Real-Time Analysis Software 6.0.4. ImmunocytochemistryTransfected or untransfected H292 cell monolayers were processed for analysis by confocal microscopy. At 48 hrs post-transfection, cell monolayers were fixed with 4% paraformaldehyde in PBS, incubated with 5μg/ml DAPI, and mounted on slides. Stably transfected cells were fixed with 4% paraformaldehyde for 20 min, and permeabilized with 0.1% Triton X-100 for 30 min. Cells were incubated with primary antibody (1:250) overnight at 4℃, rinsed in PBS, then incubated with secondary antibody conjugated to Rhodamine (TRITC) at 1:1,000 dilution for 90 min at 37℃. After several rinses, cells were counterstained with DAPI for 2 min and mounted on glass slides. Samples were observed using a confocal Leica TCS SP5.5. Inflammatory cytokines assaysH292 cells stably transfected with SIGIRR-EGFP or pEGFP-N1 and untransfected control cells were plated at 1×106 cells/well in 12-well flat bottom tissue culture plates. When growth reached 70-80% confluence, the cells were cultured for a further 24 hrs in serum deprivation conditions, as described above. Finally, the cells were treated for 6 hrs with different stimuli at various concentrations, including Flagellin (1,0.2, and 0.05μg/ml), LPS (1, 0.2, and 0.05μg/ml) and CpG DNA 2006 (1,0.2, and 0.05μM). Subsequently, concentrations of IL-6 and TNF-a in the culture supernatants were measured by enzyme-linked immunosorbent assay (ELISA). At least three sets of replicates were·examined for each experimental group.6. Western-blot analysisH292 cells untransfected and transfected stably with the indicated plasmids were treated with different stimuli including LPS (1ug/ml), Flagellin (0.1μg/ml) and CpG DNA 2006 (0.5μM) for 24hrs. At 0,1,6, or 24 hrs post-stimulation, the expression of TLR4,5,9 and SIGIRR were detected. Briefly, cells were harvested, washed in cold PBS buffer, pelleted, and lysed in ice-cold lysis buffer (30mM Tris-HCl, pH7.5,150mM NaCl,1% Nonidet P-40 and 1mM PMSF) for 30 min. Cell debris was pelleted by centrifugation for 10 min at 13,000×g. Supernatants were separated on 10% SDS-PAGE, transferred to supported nitrocellulose membrane, and blocked in a 5% solution of nonfat dry milk prepared in 1×PBS and 0.05% Tween 20. Blots were incubated with primary antibody diluted in PBS overnight at 4℃, washed three times for 10 min each with PBS, detected with horseradish peroxidase-conjugated secondary antibody diluted 1:5,000 in PBS+5% nonfat milk, and developed using the enhanced chemiluminescence method following the manufacturer's protocol.7. Co-immunoprecipitationsThe transfected and untransfected H292 cells were stimulated for 6hrs with LPS (1μg/ml), or Flagellin (0.1μg/ml), or CpG DNA 2006 (0.5μM), followed by lysis in Triton containing lysis buffer as described. Cell extracts were incubated with 1μg SIGIRR antibody or normal rabbit serum (negative control) overnight at 4℃with 20μl protein A-Sepharose beads. After incubation, the beads were washed four times with lysis buffer, separated by SDS-PAGE, transferred to Immobilon-P membranes, and analyzed by immunoblotting.8. Statistical analysisAll results are presented as means±SD; Student t tests were used to compare 2 groups, or ANOVA was used with the Tukey's multiple comparison tests for multiple groups. Values of P<0.05 were regarded as statistically significant. Statistical analyses were performed using the Statistical Package for the Social Sciences (SPSS) statistical software for Windows, version 11.0. Results1. Localization of SIGIRR in HAEC.Cellular localization of the fusion protein SIGIRR-EGFP was analyzed in H292 cells by confocal assay. The fluorescence signal of the EGFP-fused SIGIRR protein was observed at 48 hrs post-transfection. SIGIRR-EGFP fluorescent signals superimposed at the plasma and nuclear membrane. In contrast, the EGFP fluorescence in cells transfected with empty vector pEGFP-N1 diffusely localized throughout both cytoplasmic and nuclear compartments but not at the plasma membrane. As a control, the wild type H292 (wtH292) cells did not emit any detectable fluorescence.The cellular localization of SIGIRR was further analyzed in transfected H292 cells by immunostaining assay with anti-SIGIRR antibody as the primary Ab. Positive staining signals appeared to only correspond with outer membranes of H292 cells and SIGIRR-EGFP fusion protein located mainly on the plasma membranes of SIGIRR overexpressed H292 cells. SIGIRR labeled with EGFP or TRITC was apparently colocalized. Positive staining signals were not found in negative control without the addition of primary antibody. These results indicated that SIGIRR was expressed on the plasma membrane of human airway epithelial cell line, H292 cells.2. SIGIRR suppressed proinflammatory cytokines production in response to LPS, Flagellin and CpG DNA 2006 in HAEC.To determine whether overexpression of SIGIRR affected TLR4,5,9-mediated innate immune response, we selected the stably transfected cell line RH292-2 for further assay. RH292-2 overexpressed the target gene SIGIRR substantially more than other stably transfected and control cells as analyzed by real-time RT-PCR. Subsequently, levels of proinflammatory cytokines IL-6 and TNF-αwere assayed by ELISA from culture supernatants of the RH292-2 cells that were treated with the indicated concentrations of LPS, Flagellin, or CpG DNA 2006 for 6hrs. WtH292 cells, and H292 cells transfected with empty vector and stimulated with the same factors were taken as controls. Results showed that the protein expression of IL-6 and TNF-αin RH292-2 cells decreased significantly as compared to the controls, and no significant difference was found between either control, the untransfected H292 cells and the H292 cells transfected with empty vector. Results indicated that SIGIRR was a common suppressor in TLR4,5,9-mediated signaling pathways by negatively regulating IL-6 and TNF-α. inflammatory gene expression.3. SIGIRR did not modulate TLR4,5,9 expression in HAEC.Since overexpression of SIGIRR can attenuate TLR4,5,9-mediated proinflammatory signals, we investigated whether TLR4,5,9 expressions were also down-regulated in SIGIRR overexpressed HAEC. The mRNA and protein levels of TLR4,5,9 did not exhibit significant differences between SIGIRR-overexpressed H292 cells and control cells. Furthermore, the protein levels of TLR4,5,9 in different cells treated by LPS (1ug/ml), Flagellin (0.1μg/ml) or CpG DNA 2006 (0.5μM) for 24hrs were measured, respectively. The results showed that the protein levels of TLR4,5,9 decreased obviously in response to stimulation with cognate ligands for 1hr, but the protein levels increased gradually in a time-dependent manner up to 24hrs, indicating overexpression of SIGIRR did not affect the TLR4,5,9 expression in HAEC. Moreover, the protein expression pattern of SIGIRR in each experimental group of cells was similar to TLR4,5,9 following ligand stimulation.4. SIGIRR interacted with MyD88 following TLR4,5,9 activation by their cognate ligands in HAEC.As MyD88 is believed to be the key adaptor protein of most TLR-mediated signaling pathways, we next examined whether MyD88 was involved in the SIGIRR impaired TLR4,5, 9-mediated inflammation signal. Cell extracts from the wtH292 and H292 transfectants stimulated with LPS (1μg/ml), Flagellin (0.1μg/ml) and CpG DNA 2006 (0.5μM) for 1 hr were co-immunoprecipitated with anti-SIGIRR antibody, followed by Western-blot analysis with antibody against MyD88 or SIGIRR.Minimal MyD88 interacted with SIGIRR in wtH292 cells in the absence of stimulation. However, obvious amounts of MyD88 complexed with SIGIRR could be detected upon TLR ligand stimulation. Moreover, MyD88 combined with SIGIRR in RH292 cells was much more than that detected in wtH292 or EH292. Taken together, the results demonstrated that overexpression of SIGIRR attenuated the TLR4,5, 9-mediated inflammation signals through competing for the association of MyD88 with TLRs after ligand stimulations in HAEC.ConclusionIn summary, the results obtained in this study revealed that overexpression of SIGIRR in HAEC stimulated with either LPS, Flagellin or CpG DNA resulted in attenuated production of the inflammatory mediators IL-6 and TNF-α. This attenuation was not the result of lower TLR4,5 nor 9 expression, but rather a sequestration of MyD88 to SIGIRR.