Abstract
Purpose :
Thymic stromal lymphopoietin (TSLP) is a cytokine that plays diverse roles in the regulation of immune response. TSLP can be released by human epithelial cells in response to microbes, trauma, or inflammation. Innate immunity plays vital important role in the early stage of corneal anti-fungal immunity. It consists of pattern recognition receptors (PRRs), inflammatory factors, antimicrobial peptides, NK cells, macrophage and many other immune cell. This study aims to figure out the mechanism of how TSLP regulates the corneal innate immunity against Aspergillus fumigatus infection.
Methods :
For the in vitro study, human corneal epithelium cells (HCECs) were cultured and pre-treated with small interfering RNA (siRNA) for 48h prior to incubated with A. fumigatus hyphae suspension for 12h. The supernate and cells were collected to determine the protein levels of TSLP, IL-6, IL-8, hBD2, CAMP by ELISA. For the in vivo study, C57/B6 mice pre-treated with siRNA and recombined mouse TSLP protein, then were scratched and incubated with A. fumigatus hyphae suspension on the right corneas. At different time posted to infection, TSLP, TLR2, TLR4 were determined by Western blot and immunofluorescent staining. Corneal infection evaluation determined by clinical score. IL-6, IL-8, hBD2, CAMP were determined by qPCR and ELISA.
Results :
In vitro, TSLP secretion gradually increased after 1hpi, 3hpi, 6hpi and reach the peak at 12hpi. In vivo, corneal infection were observed at 12hpi, 1dpi, 3dpi, 5dpi and 10dpi. TSLP were secreted to the epithelial layer by MCECs, reach the peak at 1dpi. The expressions of TLR2/TLR4 were increased. Clinical score indicated the most severe infection symptom was at 3dpi. When pre-treated with rTSLP by subconjunctival injection 4h prior to incubation, clinical score showed rise tendency, TLR2/TLR4 were highly expressed. Pre-treated HCECs and C57/B6 with TSLP siRNA resulted in degradation of clinical score, MPO and TLR2/TLR4 expression. HCECs secreted less in IL-6, IL-8 but more in hBD2 and CAMP.
Conclusions :
The current findings demonstrate that TSLP participates in corneal anti-fungal innate immunity by regulating the expression of TLR2/TLR4, aggravated the infection as pro-inflammatory cytokine. SiRNA of TSLP blocked the neutrophil infiltration, increased the antimicrobial peptide expression and enhanced the innate immune defense.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.