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Li-Fong Seet, Li Zhen Toh, Stephanie Chu, Tina Wong; Upregulation of RelB in the Post-Operative Conjunctiva and its Role in Regulating Inflammation. Invest. Ophthalmol. Vis. Sci. 2018;59(9):121.
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© ARVO (1962-2015); The Authors (2016-present)
The nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) transcription factors are prototypical regulators of proinflammatory gene expression. This study aims to determine their involvement in inflammation during conjunctival wound healing.
Experimental surgery was performed as described for the mouse model of glaucoma filtration surgery (GFS). Blebs and contralateral, unoperated conjunctival tissues were harvested on days 2, 7, 14 and 21 post-surgery to assess the expression of NF-κB members. Mouse conjunctival fibroblasts were treated with tumor necrosis factor-alpha (TNF-A) to determine its effect on NF-κB expression. Fibroblasts were transfected with small interfering RNA (siRNA) targeted at Relb to downregulate RelB expression. Scrambled siRNA was used as control. mRNA and protein expression were determined by real-time PCR and immunoblotting respectively. Cytokine production by cells transfected with siRNA or co-treated with TNF-A was determined using the MCYTOMAG-70K multiplex assay. 3 independent experiments were performed for all in vitro studies. Statistical analyses were performed using one-way ANOVA with post-hoc Bonferroni adjustments.
Compared to Nfkb1, Nfkb2, Rela and cRel, Relb mRNA was the most upregulated in day 2, 7, and 14 blebs (p<0.05, n=5). However, RelB protein was upregulated in day 2 (1.79-fold, p=0.0027) but not day 7 tissues (n=3). In cells, TNF-A induced significantly higher RelB expression at both mRNA (mean 3.59-fold, p<0.05) and protein (mean 3.69-fold, p<0.05) levels compared to other NF-κB members. si-RelB transfection downregulated fibroblast RelB mRNA and protein by mean 2.38- and 2.46-folds respectively. si-RelB also suppressed TNF-A induction of RelB mRNA and protein by mean 2.91- and 1.52-folds respectively. RelB silencing in fibroblasts resulted in increased CCL2 (2.28-fold), and VEGF (1.84-fold) but reduced CXCL1 (0.46-fold). In the presence of TNF-A, si-Relb further increased CCL2 and VEGF, as well as other cytokines including M-CSF, GM-CSF, leukemia inhibitor factor (LIF) and IL-15 by mean 1.4 to 1.6-fold increases over TNF-A-induced levels.
The selectively high upregulation of RelB in the day 2 operated conjunctiva indicates a predominant role for this NF-κB member in the inflammatory response. Our data also suggest that elevated conjunctival RelB is likely to be involved in mitigating inflammation in GFS.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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