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. 2018 May 25;13(5):e0197491.
doi: 10.1371/journal.pone.0197491. eCollection 2018.

Regulator of calcineurin 1 differentially regulates TLR-dependent MyD88 and TRIF signaling pathways

Zheng Pang  1 , Robert D Junkins  2 , Renee Raudonis  2 , Adam J MacNeil  2 , Craig McCormick  2   3 , Zhenyu Cheng  1   2 , Tong-Jun Lin  1   2   3   4
Affiliations

Regulator of calcineurin 1 differentially regulates TLR-dependent MyD88 and TRIF signaling pathways

Zheng Pang et al. PLoS One. .

Abstract

Toll-like receptors (TLRs) recognize the conserved molecular patterns in microorganisms and trigger myeloid differentiation primary response 88 (MyD88) and/or TIR-domain-containing adapter-inducing interferon-β (TRIF) pathways that are critical for host defense against microbial infection. However, the molecular mechanisms that govern TLR signaling remain incompletely understood. Regulator of calcineurin-1 (RCAN1), a small evolutionarily conserved protein that inhibits calcineurin phosphatase activity, suppresses inflammation during Pseudomonas aeruginosa infection. Here, we define the roles for RCAN1 in P. aeruginosa lipopolysaccharide (LPS)-activated TLR4 signaling. We compared the effects of P. aeruginosa LPS challenge on bone marrow-derived macrophages from both wild-type and RCAN1-deficient mice and found that RCAN1 deficiency increased the MyD88-NF-κB-mediated cytokine production (IL-6, TNF and MIP-2), whereas TRIF-interferon-stimulated response elements (ISRE)-mediated cytokine production (IFNβ, RANTES and IP-10) was suppressed. RCAN1 deficiency caused increased IκBα phosphorylation and NF-κB activity in the MyD88-dependent pathway, but impaired ISRE activation and reduced IRF7 expression in the TRIF-dependent pathway. Complementary studies of a mouse model of P. aeruginosa LPS-induced acute pneumonia confirmed that RCAN1-deficient mice displayed greatly enhanced NF-κB activity and MyD88-NF-κB-mediated cytokine production, which correlated with enhanced pulmonary infiltration of neutrophils. By contrast, RCAN1 deficiency had little effect on the TRIF pathway in vivo. These findings demonstrate a novel regulatory role of RCAN1 in TLR signaling, which differentially regulates MyD88 and TRIF pathways.

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Conflict of interest statement

The authors have no competing interests related to this manuscript.

Figures

Fig 1
Fig 1. RCAN1 deficiency upregulates MyD88-NF-κB-mediated cytokine and chemokine production but downregulates TRIF-IRF-ISRE-mediated cytokine and chemokine production in BMMs during P. aeruginosa LPS stimulation.
Wild-type (+/+) and RCAN1-deficient (-/-) BMMs were stimulated with 200 ng/ml P. aeruginosa LPS for 3 h, 6 h, 12 h, 24 h or left untreated (NT). Cell supernatants were collected for the determination of IL-6 (A), TNF (B), MIP2 (C), IFNβ (D), RANTES (E) and IP-10 (F) secretion by ELISA (n = 3 ± SEM, *p<0.05, **p<0.01, ***p<0.001 ****p<0.0001).
Fig 2
Fig 2. RCAN1 deficiency elevates IκBα phosphorylation in vitro following P. aeruginosa LPS challenge.
Wild-type (+/+) and RCAN1-deficient (-/-) BMMs were stimulated with 200 ng/ml P. aeruginosa LPS for 3 h, 6 h, 12 h and 24 h or left untreated (NT). Cell lysates were subjected to Western blot analysis for phospho- and total IKKα, IKKβ and IκBα, as well as actin as a loading control. Blots are representative of three independent experiments (A). Densitometry analysis of phosphorylated IKKα (B), IKKβ (C) and IκBα (D) was normalized to their total protein respectively (n = 3 ± SEM, *p<0.05, ***p<0.001).
Fig 3
Fig 3. RCAN1 deficiency enhances NF-κB activity in vitro following P. aeruginosa LPS challenge of mouse BMMs.
Wild-type (+/+) and RCAN1-deficient (-/-) BMMs were treated with 200 ng/ml P. aeruginosa LPS for 2 h, 4 h, 6 h, 8 h or left untreated (NT). Nuclear proteins were extracted and subjected to EMSA by incubation with 32P-labeled NF-κB DNA probe (A). Data are representative of three individual experiments. Scan densitometry was performed for analysis of NF-κB activity (B), and data are expressed as fold change (n = 3 ± SEM, *p<0.05).
Fig 4
Fig 4. RCAN1-deficient BMMs display diminished IRF7 mRNA and protein expression.
Wild type (+/+) and RCAN1-deficient (-/-) BMMs were treated with 200 ng/ml P. aeruginosa LPS for various time points or left untreated (NT). The total RNA isolated from these cells was reverse transcribed to cDNA and subjected to real-time quantitative PCR for IRF3 (A), and IRF7 (B) gene expression. The gene expression was normalized to housekeeping control gene HPRT. Cell lysates were immunoblotted to measure IRF3, IRF7 and actin protein levels. Immunoblots are representative of three independent experiments (C). Densitometry analysis of IRF3 and IRF7 levels was normalized to actin (D, E), and data are presented as fold change (n = 3 ± SEM, **p<0.01, ****p<0.0001).
Fig 5
Fig 5. RCAN1 deficiency impairs TRIF-IRF-ISRE activity in vitro during P. aeruginosa LPS stimulation.
Wild-type (+/+) and RCAN1-deficient (-/-) BMMs were treated with 200 ng/ml P. aeruginosa LPS for 2 h, 4 h, 6 h, 8 h or left untreated (NT). Nuclear proteins were extracted and subjected to EMSA by incubation with 32P-labeled ISRE DNA probe (A). Data are representative of three individual experiments. Nuclear extracts from wild-type (+/+) BMMs treated with 200 ng/ml P. aeruginosa LPS for 2 h were incubated with or without specific antibodies to IRF3 and IRF7 for 1 h or 50 X unlabeled ISRE probe for 30 min at room temperature before EMSA experiment using the 32P-labeled ISRE probe (B). Data are representative of three individual experiments. Scan densitometry was performed for analysis of ISRE activity (C, D), and data are expressed as fold change. Cell nuclear extracts from NT and LPS 2 h stimulated wild-type and RCAN1-deficient BMMs were subjected to transcription factor ELISA for determining IRF7 activity (E). (n = 3 ± SEM *p<0.05, **p<0.01).
Fig 6
Fig 6. RCAN1-deficient mice display enhanced MyD88 pathway-mediated proinflammatory cytokine and chemokine production in response to P. aeruginosa LPS stimulation in vivo.
Wild-type (+/+) and RCAN1-deficient (-/-) mice were treated intranasally with 1 μg P. aeruginosa LPS per gram of body weight, or an equivalent volume of saline as a control (NT) for 4 h or 24 h. After 4 h or 24 h, lung tissues and BALF were collected for determination of IL-6 (A, B) and TNF (C, D), MIP2 (E, F) production by ELISA. (n = 9 ± SEM, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).
Fig 7
Fig 7. RCAN1 deficiency has minor effects on TRIF-IRF-ISRE-regulated cytokine and chemokine production following P. aeruginosa LPS stimulation in lung.
Wild-type (+/+) and RCAN1-deficient (-/-) mice were administered intranasally with 1 μg P. aeruginosa LPS per gram of body weight, or an equivalent volume of saline as a control (NT) for 4 h or 24 h. After 4 h or 24 h, lung tissues and BALF were collected for the determination of IFNβ (A, B), RANTES (C, D) and IP-10 (E, F) production by ELISA (n = 9 ± SEM, *p<0.05).
Fig 8
Fig 8. RCAN1-deficicent mice show increased activity of transcription factor NF-κB in vivo following P. aeruginosa LPS challenge.
Wild-type (+/+) and RCAN1-deficient (-/-) mice were challenged intranasally with 1 μg P. aeruginosa LPS per gram of body weight, or an equivalent volume of saline as a control (NT) for 4 h or 24 h. Nuclear proteins were extracted from lung tissues and subjected to EMSA by incubation with 32P-labeled NF-κB DNA probe (A). Data are representative of six individual experiments. Scan densitometry was performed for analysis of NF-κB activation (B), and data are expressed as fold change versus wild-type untreated lungs (n = 6 ± SEM, ****p<0.0001).
Fig 9
Fig 9. RCAN1 deficiency does not significantly affect TRIF-IRF-ISRE activity in lung following P. aeruginosa LPS challenge.
Wild-type (+/+) and RCAN1-deficient (-/-) mice were challenged intranasally with 1 μg P. aeruginosa LPS per gram of body weight, or an equivalent volume of saline as a control (NT) for 4 h or 24 h. Nuclear proteins were extracted from lung tissues and subjected to EMSA by incubation with 32P-labeled ISRE DNA probe (A). Data are representative of six individual experiments. Nuclear proteins from the lungs of wild-type (+/+) mice treated with P. aeruginosa LPS for 4 h were incubated with or without specific antibodies to IRF3 and IRF7 for 1 h or 50 X unlabeled ISRE probe for 30 min at room temperature before EMSA experiment using the 32P-labeled ISRE probe (B). Data are representative of five individual experiments. Scan densitometry was performed for analysis of ISRE activity (C, D), and data are expressed as fold change. n = 6 ± SEM (C). n = 5 ± SEM *p<0.05, **p<0.01, ***p<0.001 (D).
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This work was supported by funding from Natural Sciences and Engineering Research Council of Canada and Canadian Institutes of Health Research.

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