Abstract Rationale Th2-driven airway inflammation in allergic asthma depends on sustained expression of the master transcription factor GATA3 and its downstream cytokines. The RNA-binding protein HuR (ELAVL1), a ubiquitously expressed post-transcriptional regulator, binds AU-rich elements within target mRNAs to enhance their stability and translation. HuR is markedly upregulated in activated CD4⁺ T cells and contributes to chronic allergic inflammation by stabilizing Th2-related transcripts. We hypothesized that pharmacologic HuR inhibition with the small-molecule inhibitor KH-3 would suppress GATA3 expression, dampen Th2 cytokine networks, attenuate airway inflammation, and improve lung function in vivo, with translational confirmation in human lung CD4⁺ T cells. Methods C57BL/6 mice were sensitized and challenged with house-dust-mite (HDM) extract and treated intraperitoneally with KH-3 (100 mg/kg) or vehicle during the challenge phase. Airway resistance was assessed using the Buxco system. BALF, lung tissue, and spleen samples were analyzed by qPCR, ELISA, histopathology, and flow cytometry for inflammatory cells, cytokines, and transcription factors. Gata3 mRNA stability was evaluated by actinomycin D treatment of purified murine lung CD4⁺ T cells to determine KH-3 effects on transcript decay. For translational validation, CD4⁺ T cells were isolated from digested control human lungs using magnetic bead column separation, pretreated with KH-3 or inactive analog KH-3B (5 µM), then activated with anti-CD3/CD28 before mRNA stability assays. Results KH-3 treatment significantly reduced airway inflammation and improved airway resistance compared with HDM controls. BALF analysis showed marked decreases in total inflammatory cells and Th2 cytokines (IL-4, IL-5, IL-13). Lung homogenates revealed reduced GATA3 expression and Th2 cytokine transcripts, whereas TBX21 and RORC were unaffected, confirming Th2 specificity. In vivo actinomycin D experiments demonstrated accelerated decay of Gata3 and Th2 cytokine mRNAs in KH-3-treated mice, validating HuR-dependent mRNA stabilization. Histologic examination showed decreased peribronchial inflammation in KH-3-treated mice. In our GATA3 knock-in mice lacking the HuR-binding site, splenic CD4⁺ T cells exhibited reduced GATA3 and Th2 cytokine mRNA and protein levels, reinforcing this mechanism. In human lung CD4⁺ T cells (n = 4 donors), KH-3 pretreatment decreased GATA3 mRNA stability and reduced IL-4 and IL-13 production without altering TBX21 and RORC expression. Conclusions HuR stabilizes GATA3 and Th2 cytokine transcripts to sustain allergic inflammation and airway hyperresponsiveness. Pharmacologic HuR inhibition by KH-3 reduces Th2 cytokine production, inflammation, and airway resistance in vivo and suppresses GATA3-dependent signaling ex vivo in human lung CD4⁺ T cells, establishing HuR as a promising therapeutic target in allergic asthma. This abstract is funded by: NIH
Atasoy et al. (Fri,) studied this question.