Abstract
Purpose :
The tear film of patients with dry eye disease associated with Sjogren’s syndrome has high levels of TNFα and IFNγ. In non-ocular mucosal epithelial cells, these cytokines have been shown to upregulate complement proteins C3, C4, and C factor B (CfB). The purpose of this proof-of-concept study was to assess whether immortalized human conjunctival epithelial (HCjE) cells upregulate C3, C4, and CfB gene expression in response to TNFα and IFNγ.
Methods :
HCjE cells were differentiated in DMEM/F12 + 10 ng/ml EGF + 10% heat-inactivated FBS for 48 hours prior to stimulating with TNFα or IFNγ (0 to 10,000 pg/ml). After three hours, total RNA was extracted using the RNeasy Mini Kit (Qiagen), and cDNA was generated with the Maxima First Strand cDNA Synthesis Kit (ThermoFisher). PCR amplification was performed with Absolute qPCR Master Mix (ThermoFisher) with FAM-labeled primers specific for C3, C4, and CfB, each in duplex with VIC-labeled primers for GAPDH. Amplified gene products were normalized to GAPDH and internal plate controls prior to calibrating to vehicle-treated control samples. All experiments consisted of two experimental replicates and two technical replicates.
Results :
mRNA transcripts for C3, C4, and CfB were readily detectable from HCjE extracts with expression values corresponding to high (mean CT = 18.93 ± 1.15), low (mean CT = 29.92 ± 0.78), and medium (mean CT = 23.84 ± 1.43), respectively. Following TNFα stimulation, gene expression for C3 (2.25 fold change, p = 0.03), C4 (1.72 fold change, p < 0.01), and CfB (2.61 fold change, p = 0.03) revealed a dose-dependent upregulation. IFNγ invoked no response in C3 or CfB gene expression but stimulated a dose-dependent increase in that for C4 (2.51 fold change, p < 0.001).
Conclusions :
We have previously reported that HCjE cells are independently capable of producing a fully functional complement system. This report adds a critically important relationship that the conjunctival complement system is dynamic and adversely influenced by local inflammation. In the presence of TNFα and IFNγ, the complement system is primed such that C3, C4, and CfB expression is upregulated. This priming can theoretically lead to a more robust inflammatory response in the presence of a complement activator, such as immune complexes associated with Sjogren’s syndrome. Future work is aimed to validate these findings in human subjects.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.