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. 2020 Feb 13;5(3):e133652.
doi: 10.1172/jci.insight.133652.

The RNFT2/IL-3Rα axis regulates IL-3 signaling and innate immunity

Yao Tong  1   2 , Travis B Lear  1   3   4 , John Evankovich  1   4 , Yanwen Chen  4   5 , James D Londino  1   6 , Michael M Myerburg  1 , Yingze Zhang  1 , Iulia D Popescu  1 , John F McDyer  1 , Bryan J McVerry  1   3 , Karina C Lockwood  4 , Michael J Jurczak  7 , Yuan Liu  1   4   8 , Bill B Chen  1   4   9
Affiliations

The RNFT2/IL-3Rα axis regulates IL-3 signaling and innate immunity

Yao Tong et al. JCI Insight. .

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.

Keywords: Inflammation; Innate immunity; Pulmonology; Ubiquitin-proteosome system.

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Conflict of interest statement

Conflict of interest: The authors have declared that no conflict of interest exists.

Figures

Figure 1
Figure 1. IL-3Rα is degraded in the proteasome in response to IL-3.
(A) Immunoblot analysis of IL-3Rα protein abundance in MLE cells treated with cycloheximide (CHX) with or without MG-132 or leupeptin in a time-dependent manner. Data and mean ± SEM of 3 independent experiments. (B) Immunoblotting of IL-3Rα-V5 HIS pull-down (HIS PD) from MLE cells after coexpression of IL-3Rα-V5 and HA-ubiquitin and treatment with MG132 or leupeptin. (C) Immunoblot analysis of MLE cells transfected with WT or lysine mutant (K357R) IL-3Rα and treated with CHX. Data and mean ± SEM of 3 independent experiments. (D) Immunoblotting of IL-3Rα protein abundance from MLE cells treated with recombinant IL-3 protein (rIL-3) in a dose- or time-dependent manner. (E) Immunoblot analysis of IL-3Rα protein amount from MLE cells treated with rIL-3 with or without MG-132 or leupeptin in a time-dependent manner. Data and mean ± SEM of 3 independent experiments. (F) Immunoblot analysis of IL-3Rα-V5 protein amount from MLE transfected with WT or K357R mutant IL-3Rα and treated with rIL-3 treatment. Data and mean ± SEM of 3 independent experiments. (G) Immunoblot analysis of MLE cells transfected with IL-3Rα or empty plasmid and challenged with or without rIL-3. Data and mean ± SEM of 3 independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001 by F test (A, C, E, F) or by 1-way ANOVA with Tukey’s post hoc test (G).
Figure 2
Figure 2. RING finger E3 ligase RNFT2 regulates IL-3Rα stability and IL-3 signaling.
(A) Immunoblot analysis of IL-3Rα from MLE cells transfected with LacZ or 1 of the other 10 randomly selected RING finger E3 ligases. IL-3Rα/actin densitometry normalized to LacZ treatment is shown below IL-3Rα bands. (B) Immunoblotting of IL-3Rα from MLE cells transfected with a dose course of RNFT2. Data and mean ± SEM of 3 independent experiments. (C) Immunoblot analysis of MLE cells following expression of RNFT2-HA and immunoprecipitation (IP) of RNFT2-HA. (D) Immunoblotting of in vitro ubiquitination assay involving the full complement of ubiquitination machinery (E1, E2, ubiquitin, ATP, Mg2+) and RNFT2 protein. (E) Immunoblot analysis of in vivo ubiquitination assay in MLE cells expressing IL-3Rα-V5-HIS with HA-ubiquitin and RNFT2 before MG132 treatment and HIS tag pull-down of IL-3Rα-V5-HIS. (F) Immunoblotting of in vitro protein binding assay between V5-tagged ubiquitin E2 enzymes (Ub E2) and immunoprecipitated RNFT2 protein (IP RNFT2). TnT, in vitro transcription and translation. (G) Immunoblot analysis of MLE cells expressing RNFT2-HIS-V5 before HIS pull-down of RNFT2. (H) Immunoblot analysis of MLE cells cotransfected with WT or K357R IL-3Rα lysine mutants with or without RNFT2. Data and mean ± SEM of 3 independent experiments. *P < 0.05, ***P < 0.001, and ****P < 0.0001 by 1-way ANOVA with Dunnett’s post hoc test (B) or by 1-way ANOVA with Tukey’s post hoc test (H).
Figure 3
Figure 3. RNFT2 regulates IL-3Rα protein levels and IL-3Rα–dependent inflammatory signaling.
(A) Immunoblotting of MLE cells transfected with empty or RNFT2 plasmid before IL-3 treatment. Data and mean ± SEM of 3 independent experiments. (B) Immunoblot analysis of MLE cells transfected with control siRNA or Rnft2 siRNA before rIL-3 treatment. Data and mean ± SEM of 3 independent experiments. (C) Immunoblotting of MLE cells transfected with empty or RNFT2 plasmid and challenged with or without rIL-3. Data and mean ± SEM of 3 independent experiments. (D) Immunoblot analysis of MLE cells transfected with Rnft2 siRNA or control siRNA and then challenged with or without rIL-3. Data and mean ± SEM of 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by F test (A and B) or by 1-way ANOVA with Tukey’s post hoc test (C and D).
Figure 4
Figure 4. IL-3 augments proinflammatory cellular responses to LPS through IL-3Rα and RNFT2.
(A–C) Immunoblot analysis of IL-3Rα (A), RNFT2 (B), and IL-3Rβ (C) protein abundances in MLE cells treated with LPS at the indicated times. Blots are representative of 3 independent experiments. Quantified data are mean ± SEM from all experiments (n = 3). (D) Reverse transcription-quantitative PCR (RT-qPCR) analysis of IL-3Rα mRNA expression in MLE cells treated with LPS at the indicated times. Quantified data are mean ± SEM from all experiments (n = 3). (E) Immunoblot of IL-3Rα in MLE cells treated with CHX and control or LPS, as indicated. Blots are representative of 3 independent experiments. Quantified data are mean ± SEM from all experiments (n = 3). (F and G) ELISA analysis of IL-6 and CXCL1 in supernatants from MLE cells transfected with IL-3Rα or empty plasmid and challenged with LPS and rIL-3, as indicated. Data are mean ± SEM from all experiments. (H) ELISA analysis of CXCL1 in MLE cells transfected with RNFT2 or empty plasmid before treatment with LPS with or without rIL-3. Data and mean ± SEM of 3 independent experiments. (I) ELISA analysis of CXCL1 in MLE cells transfected with control siRNA or Rnft2 siRNA before treatment with LPS with or without rIL-3. Data and mean ± SEM of 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by F test (E), by 1-way ANOVA with Dunnett’s post hoc test (A–D), or by 1-way ANOVA with Tukey’s post hoc test (F–I).
Figure 5
Figure 5. The RNFT2/IL-3Rα axis regulates lung innate immunity and inflammation in vivo.
(A and B) Immunoblot and densitometry analysis of RNFT2, IL-3Rα, and IL-3Rβ from mice intratracheally treated with control or LPS, as indicated. Data and mean ± SEM of 4 mice per group. (C) Cell concentration, (D) total protein analysis, and (E–G) ELISA analysis of (E) IL-6, (F) TNF-α, and (G) IL-1β from BALF of mice intratracheally treated with Lenti-empty or Lenti-RNFT2 and then treated with LPS and PBS or rIL-3, as indicated. Data and mean ± SEM pooled of 3 mice per group are from 2 independent experiments. (H) Cell concentration, (I) total protein analysis, and (J) ELISA analysis of IL-6 from BALF of mice intratracheally treated with Lenti-control shRNA or Lenti-RNFT2 shRNA and then treated with LPS and PBS or rIL-3, as indicated. Data and mean ± SEM pooled of 3 mice per group are from 2 independent experiments. (K and L) Histological analysis of lung samples from mice treated as indicated. Images are representative of all independent experiments. Scale bar: 100 μm. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by Student’s t test (B) or by 1-way ANOVA with Tukey’s post hoc test (C–J).
Figure 6
Figure 6. The IL-3Rα/RNFT2 signaling axis is relevant in human lung disease.
(A) “Densitometric analysis of RNFT2 and IL3Rα immunoblotting from Human CF subjects lung explants with Pseudomonas colonization (n = 12). (B) ELISA analysis of plasma IL-3 in mechanically ventilated patients without lung injury (n = 4) or at risk for ARDS (ARFA; n = 16). Subject classification was blinded to the operator. Bars and error bars represent median and IQR, respectively. Data were analyzed by Mann-Whitney U test. *P < 0.05. (C and D) Linear regression of plasma IL-3 versus plasma TNF-α (C) and IL-1β (D) in subjects ARFA (n = 16); ***P < 0.001; F-test of Linear Regression, Pearson’s R2 goodness of fit. (E and F) Linear regression of RNFT2 gene expression normalized to control patients versus plasma TNF-α (E) and IL-1β (F) in subjects ARFA (n = 15).
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