Allergy Asthma Immunol Res.  2018 Sep;10(5):543-554. 10.4168/aair.2018.10.5.543.

Bcl11b Regulates IL-17 Through the TGF-β/Smad Pathway in HDM-Induced Asthma

• Chen S
• Han Y
• Chen H
• Wu J
• Zhang M

• KMID: 2418051
• DOI: http://doi.org/10.4168/aair.2018.10.5.543

Abstract

PURPOSE
T helper (Th) 17 cells play a critical role in the development of asthma, but the underlying mechanism of how interleukin (IL)-17 is regulated in allergic airway inflammation is poorly understood. In this study, we investigated the impact of Bcl11b on Th17 response in asthma.
METHODS
Blood samples from patients with mild asthma (MA) and severe asthma (SA) were collected. Expression of Bcl11b, IL-4, IL-5, IL-13, IL-17A and transforming growth factor (TGF)-β1 were determined in CD4+ T cells and plasma by polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA). Relative mRNA and protein levels of Bcl11b, IL-17A and genes involved in the TGF/Smad signaling pathway were examined by PCR, ELISA and western blot analysis in house dust mite (HDM)-challenged mice. Ectopic expression of Bcl11b in HDM-stimulated primary mouse splenocytes was achieved by nucleofection of Bcl11b expression plasmid.
RESULTS
We found significantly decreased Bcl11b but increased IL-17A and TGF-β1 expression in patients with asthma and a strongly negative correlation between Bcl11b and these 2 cytokines in SA patients. Similar expression patterns of Bcl11b, IL-17A and TGF-β1 were also found in mice with HDM-induced allergic airway inflammation. We demonstrated further that Smad2/3 phosphorylation was increased in HDM-challenged mice and that ectopic expression of Bcl11b in HDM-stimulated primary mouse splenocytes reduced Smad2 phosphorylation and IL-17 expression.
CONCLUSIONS
Our findings demonstrate a potential effect of Bc111b in controlling IL-17-mediated inflammation in asthma and suggest that Bc111b may be a useful therapeutic target for asthma.

Keyword

Asthma; Bcl11b; IL-17A

MeSH Terms

Animals
Asthma*
Blotting, Western
Cytokines
Ectopic Gene Expression
Enzyme-Linked Immunosorbent Assay
Humans
Inflammation
Interleukin-13
Interleukin-17*
Interleukin-4
Interleukin-5
Interleukins
Mice
Phosphorylation
Plasma
Plasmids
Polymerase Chain Reaction
Pyroglyphidae
RNA, Messenger
T-Lymphocytes
Transforming Growth Factors
Cytokines
Interleukin-13
Interleukin-17
Interleukin-4
Interleukin-5
Interleukins
RNA, Messenger
Transforming Growth Factors

Figure

• 

  Fig. 1 Levels of Th2 and Th17 cytokines in HDM-induced mouse model of asthma. (A) The design of the HDM-induced mouse model of asthma. (B) The numbers of lymphocytes, eosinophils, neutrophils and macrophages in the BALF were evaluated 48 hours after the last HDM challenge. (C) The ARH to Mch were measured 24 hours after the last HDM challenge. (D) The levels of HDM-specific IgE and IgG1 in BALF were measured by ELISA. (E) IL-4, IL-5, and IL-13 levels in BALF were quantified by ELISA. (F) IL-17A and TGF-β1 levels in BALF were measured by ELISA. The results are representative of 3 independent experiments (n = 3–5). Data represent mean ± standard error of the mean. Th, T helper; HDM, house dust mite; BALF, bronchoalveolar lavage fluid; AHR, airway hyperreactivity; Mch, methacholine; Ig, immunoglobulin; ELISA, enzyme-linked immunosorbent assay; IL, interleukin; ns, not significant. *P < 0.05, †P < 0.01, ‡P < 0.001.

• 

  Fig. 2 Expression levels of Bcl11b, Th2 and Th17 cytokines from patients with asthma. (A) IL-4, IL-5, and IL-13 levels in the plasma of patients with MA (n = 25), SA (n = 15) and NC (n = 15) were quantified by ELISA. (B) IL-17A and TGF-β1 levels in the plasma of MA, SA and NC were measured by ELISA. (C) The mRNA copies of Bcl11b in purified CD4+ T cells were normalized by 105 β2M using TaqMan based real-time PCR. (D) The relative mRNA expression of IL-17A and TGF-β1 in purified CD4+ T were normalized by the 2−ΔΔCt method using SYBR GREEN based real-time PCR. (E) The correlation between Bcl11b copies and relative IL-17A mRNA levels from patients with SA. (F) The correlation between Bcl11b copies and relative TGF-β1 mRNA levels from patients with SA. Data represent mean ± standard error of the mean. Th, T helper; IL, interleukin; MA, mild asthma; SA, severe asthma; NC, normal controls; ELISA, enzyme-linked immunosorbent assay; TGD, transforming growth factor; PCR, polymerase chain reaction; ns, not significant. *P < 0.05, †P < 0.01, ‡P < 0.001.

• 

  Fig. 3 Expression of Bcl11b, IL-17A and the TGF-β/Smad signaling pathway in HDM-treated mice. (A) The relative mRNA expression of Bcl11b, IL-17A and TGF-β1 in CD4+ T cells were normalized by 2−ΔΔCt method using SYBR GREEN based real-time PCR. (B) The protein expression of Bcl11b, Smad2, pSmad2, Smad3, pSmad3, and Smad4 in CD4+ T were determined by western Blot. (C) Quantification of western blotting results. Band intensities were measured using Image J. The results are representative of 3 independent experiments (n = 3–5). Data represent mean ± standard error of the mean. IL, interleukin; TGF, transforming growth factor; HDM, house dust mite; PCR, polymerase chain reaction; ns, not significant. *P < 0.05, †P < 0.01, ‡P < 0.001.

• 

  Fig. 4 Effect of Bcl11b on TGF-β1/Smad signaling and IL-17 expression in vitro. (A) The relative mRNA expression of Bcl11b in HDM-cultured splenocytes from naïve BALB/c mice were normalized by 2−ΔΔCt method using SYBR GREEN based real-time PCR (pI2: pIRES2-EGFP vetor control; pBcl11b: pIRES2-EGFP-Bcl11b plasmid; TGF-β1: TGF-β1 stimulation; pBcl11b + TGF-β1: pBcl11b + TGF-β1 treatment). (B) The relative mRNA expression of IL-17A and TGF-β1 in cultured lymphocytes were normalized by 2−ΔΔCt method using SYBR GREEN based real-time PCR. (C) The relative mRNA expression of Smad2 and Smad3 in cultured cells were normalized by 2−ΔΔCt method using SYBR GREEN based real-time PCR. (D) IL-17A levels in the supernatant were measured by ELISA. (E) The protein expression of Bcl11b, Smad2, pSmad2, Smad3, and pSmad3 in cultured cells were determined by western Blot. (F) Quantification of western Blot results. Band intensities were measured using Image J. The results are representative of the 3 independent experiments (n = 3–5). Data represent mean ± standard error of the mean. TGF, transforming growth factor; HDM, house dust mite; PCR, polymerase chain reaction; ELISA, enzyme-linked immunosorbent assay; ns, not significant. *P < 0.05, †P < 0.01, ‡P < 0.001.

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