IL-3 Maintains Activation of the p90S6K/RPS6 Pathway and Increases Translation in Human Eosinophils

Abstract

IL-5 is a major therapeutic target to reduce eosinophilia. However, all of the eosinophil-activating cytokines IL-5, IL-3, and GM-CSF are typically present in atopic diseases including allergic asthma. Due to the functional redundancy of these 3 cytokines on eosinophils and the loss of IL-5 receptor on airway eosinophils, it is important to take IL-3 and GM-CSF into account to efficiently reduce tissue eosinophil functions. Moreover, these 3 cytokines signal through a common β-chain receptor, and yet differentially affect protein production in eosinophils. Notably, the increased ability of IL-3 to induce production of proteins such as semaphorin-7A without affecting mRNA level suggests a unique influence by IL-3 on translation. The purpose of this study is to identify the mechanisms by which IL-3 distinctively affects eosinophil function compared to IL-5 and GM-CSF, with a focus on protein translation. Peripheral blood eosinophils were used to study intracellular signaling and protein translation in cells activated with IL-3, GM-CSF or IL-5. We establish that, unlike GM-CSF or IL-5, IL-3 triggers prolonged signaling through activation of ribosomal protein (RP) S6 and the upstream kinase, p90S6K. Blockade of p90S6K activation inhibited phosphorylation of RPS6 and IL-3-enhanced semaphorin-7A translation. Furthermore, in an allergen-challenged environment, in vivo phosphorylation of RPS6 and p90S6K was enhanced in human airway compared to circulating eosinophils. Our findings provide new insights into the mechanisms underlying differential activation of eosinophils by IL-3, GM-CSF, and IL-5. These observations place IL-3 and its downstream intracellular signals as novel targets that should be considered to modulate eosinophil functions.

Proteomic and Phosphoproteomic Changes Induced by Prolonged Activation of Human Eosinophils with IL-3

“…DAVID cluster analysis indicates that the hyper-phosphorylated proteins are mostly implicated in intracellular signaling, cytoskeleton organization, RNA binding, regulation of protein translation, and intracellular movement (Table 4 and Supplemental Tables 5, 6, 7 and 8). The present phospho-proteome confirms our previous published study, in which we demonstrated continuous phosphorylation of RPS6KA1 (RSK1), RPS6, and EIF4B by a 14 h activation of EOS with IL-3 (7) . Additionally, we previously found that IL-3-prolonged RPS6KA1 phosphorylation was critical for SEMA7A and FCGR2B translation and protein accumulation (7, 8) , and as described above, SEMA7A and FCGR2B were both highly up-regulated by IL-3.…”

“…IL-3-enhanced IL3RA is in agreement with the previous finding showing that the surface receptor for IL-3 is enhanced on EOS by a prolonged (~20 h) activation with the ß-chain cytokines (IL-5, GM-CSF, or IL-3) while the amount of surface receptors for IL-5 and GM-CSF are decreased or unchanged, respectively, in the same conditions (22, 23) . The enhanced amount of both IL3RA and CSF2RB explains the unique ability for IL-3 compared to IL-5 and GM-CSF to sustain intracellular signaling in EOS (7, 24) .…”

GM-CSF and IL-3 Modulate Human Monocyte TNF-α Production and Renewal in In Vitro Models of Trained Immunity

“…To understand the regulation of TNF-α production in primed monocytes, we compared intracellular TNF-α protein levels, TNF mRNA, and primary transcript levels upon LPS stimulation for 1 and 4 h. Although in several experimental models priming of monocyte and macrophages results in chromatin remodeling (20, 22–24, 26–30), our data support a posttranscriptional regulation of TNF-α production in primed monocytes. Posttranscriptional mechanisms, including modulation of mRNA stability and translation (33–35), are increasingly recognized as critical regulators of gene and protein expression also in immune cells (25, 34, 36, 37, 54, 55). Our results demonstrate that inhibition of SIRT2 during priming and of p38 or ERK1/2 (the latter only for GM-CSF primed cells) during LPS stimulation reduces intracellular TNF-α protein levels without significantly modulating TNF RNA levels.…”

IL‐3 up‐regulates and activates human eosinophil CD32 and αMβ2 integrin causing degranulation

“…In addition, IL-3 is elevated in serum in poorly controlled asthmatic patients, and airway IL-3-positive cells are increased with asthma severity [25, 26]. Furthermore, segmental allergen challenge in mild asthma leads to elevated levels of IL-3 in the BAL fluid [20, 27], and while airway eosinophils display reduced surface α chain for the IL-5 receptor, they show increased IL-3 receptor compared to blood eosinophils [12, 28]. …”