The Effect Of Supernatant Derived From Trichophyton Rubrum Grown In The Nail Medium On The Innate Immune Response Of HaCaT

[Background]Trichophyton rubrum are superficial fungi characteristically confined to the dead keratinized tissues. These observations suggest that keratinocyte might play a crucial role in the natural defense against T. rubrum and only through a cell-contact-free manner in most cases. Thus the soluble fungal substances might play an even more important role in the immunomodulation than the insoluble compounds located in the fungal cell was suggested. Analysis has indicated that the culture supernatant of T. rubrum can effectively stimulate immune response of keratinocytes. However, all the culture mediums used in these studies are complete medium that containing simple and absorbable nutrients, in which dermatophyte can directly utilize all they need without the adherence and invasion of the host keratin. The immunogenicity and pathogenicity of dermatophyte living in such a cultivation are distinct from the in vivo infection, might not be able to reflect the immune response of keratinocyte to the free pathogen-associated molecular patterns released by the T. rubrum during in vivo infection. In this study, the medium containing keratin as a sole carbon and nitrogen source were chose to cultivate T. rubrum in vitro, in order to mimic the actual condition that T. rubrum infect the host. In nail medium, T. rubrum must adhere and invade keratin to live. By this way, we investigate the immune response of keratinocyte to the free pathogen-associated molecular patterns released by T. rubrum growth in keratin culture medium.[Object]To analyze the effect of T. rubrum culture supernatant derived from T. rubrum grown in the medium on the immune response of keratinocyte, and compare the difference between two strains.[Methods]1. Two strains of T. rubrum (T1a, TXHB) were used in this study. T. rubrum strain T1a was obtained from China Medical Microbiological Culture Collection Center; TXHB was isolated from a tinea corporis patient. The mineral-salt liquid-culture medium contains nails as the only carbon and nitrogen source making the nail powder suspension medium. After the vibrant cultivation, the supernatant of the T. rubrum was obtain by ultrafiltration and sterile filtration.2. The free β-glucan concentration in supernatant of T1a and TXHB were determined using the Fungal glucan G-test reagent kit.3. Protein concentrations of the supernatant were determined by BCA protein quantitative method, then select a suitable dosage of T. rubrum culture supernatant to stimulate HaCaT by the morphological method with different concentrations of T. rubrum culture supernatant.4. The expressions of host defense genes:pattern recognition receptors (Dectin-1, TLR2, TLR4, DC-SIGN), the intracellular signaling molecules CARD9, the antimicrobial protein of healthy human skin RNase 7 and the neutrophils chemokine IL-8 were assessed by quantitative PCR (qPCR) after the HaCaT was stimulated with the culture supernatants from two strains of T. rubrum.[Results]1. The T. rubrum strains Tla and TXHB released 87.530±37.581 pg/ml and 15.747±6.453 pg/ml of β-glucan respectively to the media.2. At the final concentrations of T. rubrum proteins in medium concentrations were higher than 40μg/ml, Tla supernatant significantly increase the number of cells detachment from the culture flask or cells with atypical polynuclear, which implies the cytotoxic effects began. Then morphological examination of HaCaT exposed to supernatant of T. rubrum with the concentration of 20 μg/ml within 48 hours revealed that cell viability was not likely affected, thus the HaCaT was stimulated by T. rubrum culture supernatant in which the final concentration of T. rubrum proteins was 20 μg/ml.3. The mRNA expressions of TLR2, TLR4 and CARD9 were significantly up-regulated within 6 h application of both supernatants in HaCaT cells (p<0.05). HaCaT cells were more responsive to Tla than TXHB. The expression of CARD9 by Tla were upregulated at 6 h and 24 h, longer than 6 h by TXHB (p<0.05). The mRNA expression of DC-SIGN was slightly up-regulated after application of Tla supernatant for 3 and 6 h (p<0.05), the increases were 2-4 times higher than in the control groups, while the slightly increase happened at 24 h with TXHB supernatant (p<0.05). The mRNA expression of Dectin-1 was slightly up-regulated after application of Tla supernatant for 6 h (p<0.05), and then downregulated after 48 h (p<0.05), while no significant change was seen with TXHB supernatant. IL-8 was minimally upregulated applied with Tla supernatant for 6 h, the increase was 2 times higher than in the control group (p<0.05), and was moderatly declined at 48 h with TXHB supernatant (p<0.05). Moderately decrease was detected at 24 h and 48 h for RNase 7 at mRNA level respectively in response to Tla except TXHB, decreased approximately to half of the control group (p<0.05)[Conclusions]1. The culture supernatant of T. rubrum could active the innate immune response of keratinocytes directly, but the responses were mild and short-lasting which may account for the chronic and recurrent nature of T. rubrum infection.2. The culture supernatant of T. rubrum contain (3-glucan, the most common kind of PAMPs, but the amounts in both supernatants were too scarce to induce and maintain the immune response of keratinocyte.3. Different strains of T. rubrum could exist intraspecific diversity in the immunomodulation of keratinocyte.