c-CBL/LCK/c-JUN/ETS1/CD28 axis restrains childhood asthma by suppressing Th2 differentiation

Abstract

Background: Asthma is a common immune disease with high morbidity in children. Type 2 inflammation is the center of asthma development, and mainly mediated by a subset of CD4 + T cells, T helper 2 (Th2) cells. Excess Th2 differentiation was generally associated with asthmatic attack. Casitas B-lineage lymphoma (c-CBL) was reported to involved in T cell development and databank showed its decreased expression in CD4 + T cells from peripheral blood of asthmatic children. This study aims to investigate the role of c-CBL in childhood asthma and Th2 differentiation, and explore the underlying mechanism.

Methods: We collected peripheral blood samples from clinical childhood asthma cases and healthy controls, and determined c-CBL expression in CD4 + T cells. Asthma was induced in neonatal mice by ovalbumin (OVA) intraperitoneal injection and aerosol inhalation, and c-CBL expression in CD4 + T cells from peripheral blood and spleen was measured. Gain-of-function experiments was performed to confirm the effects of c-CBL on Th2 differentiation in vitro. Finally, c-CBL was delivered into asthmatic mice via lentivirus infection to verify its effects on experimental asthma.

Results: c-CBL was lowly expressed in CD4 + T cells from asthmatic children than those of healthy controls. Similarly, it was downregulated in CD4 + T cells from peripheral blood and spleen of asthma mice. Overexpression of c-CBL restrained lung pathological injury and type 2 inflammation in experimental asthmatic mice. Gain-of-function experiments demonstrated that c-CBL inhibited Th2 differentiation of CD4 + T cells from healthy children, and mediated the ubiquitination of lymphocyte cell-specific protein-tyrosine kinase (LCK). LCK acted as a kinase to phosphorylate and activate c-JUN, which was predicted to bind promoter sequence of CD28 by bioinformatic analysis. Dual-luciferase reporter assay verified that c-JUN and ETS1 synergically enhanced transcription of CD28, and this transcription activation was aggravated by LCK overexpression.

Conclusion: c-CBL alleviated asthma and suppressed Th2 differentiation by facilitating LCK ubiquitination, interrupting c-JUN activation and CD28 expression in vivo and in vitro. c-CBL/LCK/c-JUN/ETS1/CD28 axis was partially involved in childhood asthma, and may provide novel insights for clinical treatment for asthma.

Keywords: Asthma; Inflammation; LCK; Th2 cells; c-CBL.

Fig. 1

c-CBL was decreased in CD4 + T cells from asthmatic children and neonatal mice. (A) Process of CD4 + T cell isolation from childhood asthma cases and healthy children. (B and C) The expression of c-CBL in CD4 + T cells from blood of asthmatic children and healthy control was determined by western blot and real-time PCR. (D) The percentage of Th2 cells in CD4 + T cells. (E) The correlation analysis between CD4 + T cell percentage and c-CBL expression in CD4 + T cells from childhood asthma cases and healthy controls. (F). The asthma induction and treatment in neonatal mice. G. HE staining of lung tissue from asthmatic and control mice. (H and I) The mRNA level of c-CBL in CD4 + T cells from peripheral blood or spleen of control and asthma mice. (OVA, ovalbumin; AW, airway; BV, blood vessel; the scale bar represented as 100 μm; **p < 0.01, ***p < 0.001)

Fig. 2

c-CBL facilitated Th2 differentiation of CD4 + T cells. (A) The CD4 + T cells were induced to Th2 differentiation, and the percentage of Th2 cells was determined by flow cytometry. (B and C) The expression of c-CBL in CD4 + T cells with or without Th2 induction was assessed by real-time PCR and western blot. (D) c-CBL was overexpressed in CD4 + T cells by lentivirus infection, and the efficiency was confirmed. (E and F) The Th2 cell percentage in CD4 + T cells with Th2 differentiation induction or/and c-CBL overexpression was evaluated by flow cytometry. (G and H) The contents of Th2 markers IL-5 and IL-13 in supernatant were detected by ELISA. (I). The expression of Th2 driving molecule GATA3. (*p < 0.05, **p < 0.01, ***p < 0.001)

Fig. 3

c-CBL bound to LCK and mediated its ubiquitination and degradation. (A) The binding between c-CBL and LCK was confirmed by co-IP in 293T cells. (B) The interaction between c-CBL and LCK was measured in CD4 + T cells with or without Th2 differentiation induction. (C) The LCK expression in Th2 cells after c-CBL overexpression. (D) The ubiquitination of LCK was assessed in Th2 cells after c-CBL overexpression. (E) The protein level of LCK in c-CBL-overexpressed Th2 cells with CHX treatment for different times. (CHX, cyclohexane; **p < 0.01, ***p < 0.001)

Fig. 4

LCK phosphorylated c-JUN and enhanced its transcription activation on CD28. (A) The expression of LCK in CD4 + T cells with or without Th2 differentiation induction. (B) The expression and phosphorylation (Ser73) of c-JUN in CD4 + T cells with or without Th2 differentiation. (C) The binding of LCK and c-JUN in CD4 + T cells. (D) The expression efficiency of LCK-overexpressed lentivirus in Th2 cells. (E) The expression and phosphorylation (Ser73) of c-JUN after LCK overexpression. (F and G) The nuclear level of c-JUN in Th2 cells after LCK overexpression. (H). The expression of CD28 in Th2 cells after LCK overexpression. (I) The potential binding sites of ETS1 and c-JUN to CD28 promoter sequence. (J) The conservative binding sequence of transcription factors ETS1 and c-JUN. (K) Dual-luciferase reporter assay was used to assess the binding of ETS1 and c-JUN to potential sites of CD28 promoter sequence. (L) Dual-luciferase reporter assay with or without LCK overexpression. (**p < 0.01, ***p < 0.001)

Fig. 5

The effect of c-CBL on Th2 differentiation was abolished by LCK overexpression. (A and B) The Th2 cell percentage after overexpression of c-CBL or/and LCK was determined by flow cytometry. (C and D) The content of Th2 markers IL-5 and IL-13 in supernatant was measured by ELISA. (E) The mRNA level of CD28 after overexpression of c-CBL or/and LCK. (*p < 0.05, **p < 0.01, ***p < 0.001)

Fig. 6

c-CBL alleviated experimental asthma in neonatal mice. The overexpression of c-CBL via lentivirus intraperitoneal injection in experimental childhood asthma mice. (A-C) The expression of c-CBL and LCK in CD4 + T cells from peripheral blood of mice. (D-F) The expression of c-CBL and LCK in CD4 + T cells from spleen of mice. (G) HE, PAS and Masson staining were performed to detect the pathological change, glycogen and collagen in lung tissues. (OVA, ovalbumin; AW, airway; BV, blood vessel; HE, hematoxylin-eosin; PAS, Periodic acid-schiff; the scale bar represented as 100 μm; *p < 0.05, **p < 0.01, ***p < 0.001)

Fig. 7

c-CBL suppressed Th2 cell activity in asthmatic mice. (A-E) The numbers of inflammatory cells, neutrophils, eosinophils, macrophages and lymphocytes in BALF were counted by Giemsa staining. (F and G) The OVA-specific IgE levels in BALF and peripheral blood from asthmatic mice with or without c-CBL overexpression were assessed by ELISA. (H-J) The contents of Th2 markers IL-4, IL-5 and IL-13 in BALF. (K and L) The Th2% in lung tissue. (OVA, ovalbumin; BALF, bronchoalveolar lavage fluid; *p < 0.05, **p < 0.01, ***p < 0.001)

Fig. 8

The mechanism of c-CBL/LCK/c-JUN/ETS1/CD28 regulation on childhood asthma. c-CBL catalyzed the ubiquitination of degradation of LCK; LCK mediated the phosphorylation of c-JUN; phosphorylated c-JUN and ETS1 synergistically promoted the transcription of CD28; CD28 homodimers facilitated the Th2 differentiation and asthma development