Nucleotide oligomerization domain 2 contributes to the innate immune response in THCE cells stimulated by Aspergillus fumigatus conidia

Abstract

Aim: To investigate the expression of nucleotide oligomerization domain 2 (NOD2) in the immortalized human corneal epithelial cell line (THCE), and its role in the innate immune response triggered by inactive Aspergillus fumigatus (Af) conidia.

Methods: The normal THCE cells were investigated as controls. After incubation with inactive Af conidia for 0.5, 2, 4, 6, and 8 hours, THCE cells were harvested, mRNA expression of NOD2 and receptor interacting protein 2 (RIP2) was detected by RT-PCR. Intracellular proteins including NOD2, NF-κB and proinflammatory cytokines such as TNF-α, IL-8, IL-6 in the cell supernatant were analyzed by ELISA.

Results: Our data indicate that NOD2 expressed in the normal THCE cells. After triggered by the inactive Af conidia, the expression of NOD2, RIP2 mRNA and the secretion of NOD2, NF-κB, TNF-α, IL-8, IL-6 both increased in a time-depended manner, and reached the peak point at 4, 6, 6, 4, 6, 6, 4 hours, respectively. And after pretreated with NOD2 neutralizing antibody, the expression of RIP2, NF-κB, TNF-α, IL-8 both decreased dramatically at the peak point, while the secretion of IL-6 changed little.

Conclusion: The results of this study suggest that NOD2 exists and expresses in the THCE cells, and contributes to the innate immune responses triggered by inactive Af conidia by induction of proinflammatory cytokines such as TNF-α and IL-8 through the NF-κB pathway.

Keywords: Aspergillus fumigatus; corneal epithelial cell; innate immune response; nucleotide oligomerization domain 2.

Figure 1. Microscopic of Af conidia and THCE cells

A: Smear of purified Af conidia observed under light microscope (40×); B and C: THCE cells observed under inverted microscope (100×), at 6 hours and 48 hours respectively.

Figure 2. Af conidia induced expressions of NOD2, RIP2, NF-κB, TNF-α, IL-8 and IL-6 in THCE cells

A and B: The synthesis of NOD2 and RIP2 mRNA in THCE cells stimulated by inactive Af conidia, measured by relative intensity; C, D and E: The concentration of NOD2, NF-κB, TNF-α, IL-8 and IL-6 in THCE cells stimulated by inactive Af conidia, measured by ELISA at 0.5, 2, 4, 6, 8 hours. Values represent means±SD. ^aP<0.05, ^bP<0.01 vs control, n=6.

Figure 3. Decreased expressions of RIP2, NF-κB, TNF-α and IL-8 in stimulated THCE cells preincubated with NOD2 neutralizing antibody

A and B: The synthesis of RIP2 mRNA in stimulated THCE cells preincubated with NOD2 neutralizing antibody decreased significantly than in stimulated THCE cells without NOD2 antibody, measured using relative intensity; C to E: The concentration of NF-κB, TNF-α and IL-8 decreased in blocked THCE cells, measured by ELISA; F: The concentration of IL-6 changed little in blocked THCE cells. Values represent means±SD. ^aP<0.05, ^bP<0.01 vs unblocking, n=6.