Abstract
Interleukin-3 (IL-3) receptor α (IL-3Rα) is the α subunit of the ligand-specific IL-3R and initiates intracellular signaling in response to IL-3. IL-3 amplifies proinflammatory signaling and cytokine storm in murine sepsis models. Here we found that RNFT2 (RING finger transmembrane-domain containing protein 2, also TMEM118), a previously uncharacterized RING finger ubiquitin E3 ligase, negatively regulated IL-3-dependent cellular responses through IL-3Rα ubiquitination and degradation in the proteasome. In vitro, IL-3 stimulation promoted IL-3Rα proteasomal degradation dependent on RNFT2, and we identified IL-3Rα lysine 357 as a ubiquitin acceptor site. We determined that LPS priming reduces RNFT2 abundance, extends IL-3Rα half-life, and sensitizes cells to the effects of IL-3, acting synergistically to increase proinflammatory signaling. In vivo, IL-3 synergized with LPS to exacerbate lung inflammation in LPS and Pseudomonas aeruginosa-challenged mice; conversely, IL-3 neutralization reduced LPS-induced lung injury. Further, RNFT2 overexpression reduced lung inflammation and injury, whereas Rnft2 knockdown exacerbated inflammatory responses in LPS-induced murine lung injury. Last, we examined RNFT2 and IL-3Rα in human lung explants from patients with cystic fibrosis and also showed that IL-3 is elevated in mechanically ventilated critically ill humans at risk for acute respiratory distress syndrome. These results identify RNFT2 as a negative regulator of IL-3Rα and show a potential role for the RNFT2/IL-3Rα/IL-3 axis in regulating innate immune responses in the lung.
Highlights
Interleukin-3 (IL-3), a cytokine that regulates hematopoiesis [1], amplifies cytokine storm in murine sepsis [2], and in septic humans elevated circulating IL-3 is associated with mortality [2]
In humans we show that IL-3 was elevated in subjects at risk for acute respiratory distress syndrome (ARDS) and that IL-3Rα and RING finger transmembrane-domain containing protein 2 (RNFT2) abundance are inversely correlated in lung homogenates from lung explants in patients with cystic fibrosis
MLE cells were treated with CHX, and IL-3Rα half-life was observed to be approximately 6 hours; we found CHX-induced IL-3Rα degradation was prevented by the proteasome inhibitor MG132 but not by the lysosomal inhibitor leupeptin (Figure 1A)
Summary
Interleukin-3 (IL-3), a cytokine that regulates hematopoiesis [1], amplifies cytokine storm in murine sepsis [2], and in septic humans elevated circulating IL-3 is associated with mortality [2]. We show a previously uncharacterized mechanism regulating IL-3Rα abundance through the ubiquitin/proteasome system (UPS), directed by the E3 ligase RING finger transmembrane-domain containing protein 2 (RNFT2). IL-3–initiated intracellular signaling is dependent on receptor subunits IL-3Rα and IL-3Rβ [1, 6]. The β subunit IL-3Rβ, known as the β-common receptor (βc), is a shared receptor subunit that detects several colony-stimulating factor cytokines, including IL-3, GM-CSF, and IL-5 [1, 3, 6]. Depending on the stimulus (IL-3, GM-CSF, or IL-5) and the cell type, several intracellular signaling pathways can be activated.
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