Interaction Between Candida Albicans And Human Oral Epithelial Cell In Vitro

PURPOSEOropharyngeal candidiases are the most common forms of mucosal fungal infections and are primarily caused by Candida albicans, a dimorphic fungal. Clinical and experimental observations suggest that local immunity is important in host defense against candidiasis. Accordingly, cytokines and chemokines are present at the oral mucosa during C. albicans infections. Since mucosal epithelial cells produce a variety of cytokines and chemokines in response to microorganisms and since C. albicans is closely associated with mucosal epithelial cells as a commensal, we sought to identify cytokines produced by oral epithelial cell lines in response to C. albicans.Host defense mechanisms against mucosal candidiasis are not well understood. Although cell -mediated immunity (CMI) is considered important for protection against mucosal and/or systemic candidiasis, the role of systemic CMI against oral candidiasis has been challenged to various degrees. Thus, investigations have shifted to the role of local oral CMI. Based on a questionable role for CMI against Candida at the oral mucosa, recent attention has focused on innate immunity. Epithelial cells have recently been recognized as playing active roles in mucosal immune responses, including antigen presentation antimicrobial activity and cytokine production in response to microorganisms .METHODS1. Candida albicans cultrure. C. albicans ATCC 90028 was grown on Sab-ouraud - dextrose agar at 37 . The blastoconidia were collected, washed twicewith sterile PBS, and suspended in PBS at a concentration of 10~6 CFU/ml for use.2. Oral epithelial cells culture. KB cells were maintained in 90% Eagle minimal essential medium with nonessential amino acids and Earle's balanced salt solution (Sigma) supplemented with 10% FBS and 1% penicillin -streptomycin. Both cell lines were maintained at 37 C and 5% CO2 and were passaged every 3 to 4 days.3. Co - culture of Candida albicans with oral epithelial cells, epithelial ceDs were co - cultured with Candida at different ratio of in separate wells for 0, 24, 48, 72, and 96 h . Control included epithelial cells cultured alone in separate well. At each time point, the epithelial cell - Candida co - culture and the control cultures were aspirated from a new set of individual wells and centrifuged for 5 min at 800 x g. Thereafter, the supernatants were collected and stored at-70 C until assayed. Epithelial cells were also evaluated for viability at each time point and observed to be similar to those in the pre - culture conditions.4. Cytokine and chemokine analysis of co -culture supernatants. Supernatants were assayed for proinflammatory (IL - 8 TNF - α, MCP - 1, and ICAM- 1) cytokines by enzyme - linked immunosorbent assay ( ELISA). Standard curves were generated by using the respective recombinant human cytokines as previously described.5. Microscopic examination of Candida albicans and adhesion assay. Observe shapes and structures of cultured KB cells and C. albicans with phase contrast microscope, scan electron microscope ( SEM) and transmission electron microscope ( TEM). The adhesion assay was set up in 12 well polystyrene plates. After co - cultured 8,12,24,48h, the media were discarded and the cells were washed by PBS twice. The proportion of epithelial cell - associated C. albicans was determined at each time point under scan electron microscope (SEM).6. Flow cytometric analysis for cell cycle. KB cells cultured with C. albicans were digested by 0.25% tripsin after that PI was added. Then suspension was subjected to FACS Flow cytometry for detecting the DNA content. The experiment was repeated three times.7. Detection of IL - 8 , MCP -1 ,TNF - a and ICAM - 1 mRNA expression in KB by reverse transcriptase - polymerase chain reaction (RT - PCR). KB cells were exposed to C. albicans for 24h. Subsequently, total RNA was isolated from cells using TRLzol reagent according to the manufactures' instrumentioons . The PCR conditions were: initial denaturation 3 min, 94℃, denaturation 30 sec,94℃,annealing 1 min,55℃,extension lmin,72℃,for 35 cycles; followed by a 5 min elongation step at 72℃。 Primer sequences were : IL - 8: forward 5' - GTG CAG AGG GTT GTG GAG - 3', reverse 5' - GTG AGG TAA GAT GGT GGC T-3';TNF -α:forward 5'-TCT CGA ACC CCG AGT GAC AA -3', reverse 5'-TAT CTC TCA GCT CCA CAC CA -3';ICAM - 1 forward 5'- AGG TGT GAT ATC CGG TAG A - 3', reverse 5' - CCT TCT AAG TGG TTG GAA CA -3';MCP - 1 :forward 5'- CTG AGC CTG GAC AAA GAC AC -3', reverse 5'-GGG TGC CAT AGA TAA ACT G - 3';PCR products were eletrophoresed on 2% agarose gel and visualized by ethidium bromide staining.RESULTS1. Observation of KB cells and C. albicans form under phase contrast microscope. After co - culturing for 48h, C. albicans formed ture hyphae and adhere to the KB cell. KB cell became round and the contacting disappeared, but co -cultured with C. albicans the killed , KB cell not changed.2. Observation of KB cells and C. albicans form under electron microscope. After co - culturing forl2, 24,48h C. albicans formed to hyphae and adhere to the KB cell. Some C. albicans penetrate the KB cell membrane and migrate into the intercells. KB cell became round and the connecting disappeared, washed the discrete C. albicans with PBS for three times, the adhesion between C. albicans and Kb cells were increased when time passed under the scan electron microscope but co - cultured with C. albicans the killed , KB cell not changed.3. Effects of C. albicans on the expression of IL -8, TNF - α, ICAM -1 and MCP -1 in cultured KB cells. C. albicans induced KB cells to express IL-8, TNF -α, ICAM - 1 and MCP - 1 at gene level, only found IL - 8 and...