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IL-21-mediated non-canonical pathway for IL-1β production in conventional dendritic cells.

Wan CK, Li P, Spolski R, Oh J, Andraski AB, Du N, Yu ZX, Dillon CP, Green DR, Leonard WJ - Nat Commun (2015)

Bottom Line: IL-21 does not induce Il1b expression in CD4(+) T cells, with differential histone marks present in these cells versus cDCs.IL-21-induced IL-1β processing in cDCs does not require caspase-1 or caspase-8 but depends on IL-21-mediated death and activation of serine protease(s).These results demonstrate lineage-restricted IL-21-induced IL-1β via a non-canonical pathway and provide evidence for its importance in vivo.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Immunology and the Immunology Center, National Heart, Lung and Blood Institute, National Institutes of Health, 10 Center Drive, Building 10, Room 7B05, Bethesda, Maryland 20892-1674 USA.

ABSTRACT
The canonical pathway for IL-1β production requires TLR-mediated NF-κB-dependent Il1b gene induction, followed by caspase-containing inflammasome-mediated processing of pro-IL-1β. Here we show that IL-21 unexpectedly induces IL-1β production in conventional dendritic cells (cDCs) via a STAT3-dependent but NF-κB-independent pathway. IL-21 does not induce Il1b expression in CD4(+) T cells, with differential histone marks present in these cells versus cDCs. IL-21-induced IL-1β processing in cDCs does not require caspase-1 or caspase-8 but depends on IL-21-mediated death and activation of serine protease(s). Moreover, STAT3-dependent IL-1β expression in cDCs at least partially explains the IL-21-mediated pathologic response occurring during infection with pneumonia virus of mice. These results demonstrate lineage-restricted IL-21-induced IL-1β via a non-canonical pathway and provide evidence for its importance in vivo.

No MeSH data available.


Related in: MedlinePlus

IL-21 induces Il1b mRNA expression via STAT3.(a) cDCs were rested 1 h, treated with 100 ng ml−1 IL-21 at the indicated time points, and Il1b mRNA assessed relative to Rpl7 by reverse transcription–PCR (RT–PCR). Shown is one of two similar experiments. (b) Il21r+/+ and Il21r−/− cDCs were treated with IL-21 for 5 h and Il1b mRNA assessed. (c) cDCs were rested 1 h, treated with 100 ng ml−1 IL-21 or LPS at the indicated time points and Il1b mRNA assessed relative to Rpl7 by RT–PCR. Shown are the combined results of two independent experiments. (d) cDCs were incubated with 10 μM MLN120B for 1 h, treated with IL-21 or LPS for 4 h and Il1b mRNA assessed. (e) cDCs were rested 1 h, stimulated with 100 ng ml−1 IL-21 or LPS for 30 min, and the expression of phosphorylated and total IκBα was determined. Shown is one of two similar experiments. (f,g) cDCs from Stat3+/+ and Stat3−/− mice were treated with IL-21 (f) or LPS (g) as in b, and Il1b mRNA assessed. In g, NS, P=0.3. Data in b and d–g are representative of three experiments; error bars are technical duplicates of the representative experiment; shown is expression relative to Rpl7 assessed by RT–PCR. (h) Human peripheral blood DCs were rested 16 h and stimulated with IL-21 for 15 min. pSTAT3 in CD1c+ cells was measured by flow cytometry. Shown are data representative of five samples in three experiments. (i) Human CD1c+ cDCs were rested 16 h, stimulated with IL-21 for 4 h, and IL1B mRNA relative to ACTB was assessed by RT–PCR. Data are from three experiments (six total samples); error bars are means±s.e.m. Statistical analysis was performed by Student's t-test.
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f1: IL-21 induces Il1b mRNA expression via STAT3.(a) cDCs were rested 1 h, treated with 100 ng ml−1 IL-21 at the indicated time points, and Il1b mRNA assessed relative to Rpl7 by reverse transcription–PCR (RT–PCR). Shown is one of two similar experiments. (b) Il21r+/+ and Il21r−/− cDCs were treated with IL-21 for 5 h and Il1b mRNA assessed. (c) cDCs were rested 1 h, treated with 100 ng ml−1 IL-21 or LPS at the indicated time points and Il1b mRNA assessed relative to Rpl7 by RT–PCR. Shown are the combined results of two independent experiments. (d) cDCs were incubated with 10 μM MLN120B for 1 h, treated with IL-21 or LPS for 4 h and Il1b mRNA assessed. (e) cDCs were rested 1 h, stimulated with 100 ng ml−1 IL-21 or LPS for 30 min, and the expression of phosphorylated and total IκBα was determined. Shown is one of two similar experiments. (f,g) cDCs from Stat3+/+ and Stat3−/− mice were treated with IL-21 (f) or LPS (g) as in b, and Il1b mRNA assessed. In g, NS, P=0.3. Data in b and d–g are representative of three experiments; error bars are technical duplicates of the representative experiment; shown is expression relative to Rpl7 assessed by RT–PCR. (h) Human peripheral blood DCs were rested 16 h and stimulated with IL-21 for 15 min. pSTAT3 in CD1c+ cells was measured by flow cytometry. Shown are data representative of five samples in three experiments. (i) Human CD1c+ cDCs were rested 16 h, stimulated with IL-21 for 4 h, and IL1B mRNA relative to ACTB was assessed by RT–PCR. Data are from three experiments (six total samples); error bars are means±s.e.m. Statistical analysis was performed by Student's t-test.

Mentions: We previously performed microarray analysis to identify genes that are regulated by IL-21 in cDCs16. Surprisingly, although IL-21 inhibits the LPS-induced IL-1β expression in BMDCs15, we observed a significant induction of Il1b mRNA by IL-21 in cDCs. Transcription of Il1b mRNA is induced in response to IL-1 itself or microbial products acting via toll-like receptors2, but Il1b induction by IL-21 was not anticipated. Therefore, we validated this result by reverse transcription–PCR and confirmed that IL-21 indeed rapidly (within 30 min) induced Il1b mRNA (Fig. 1a) and that this induction did not occur in Il21r−/− cells (Fig. 1b), excluding the possibility that it resulted from contaminating endotoxin. We compared the effect of IL-21 with the classical Il1b-inducing ligand LPS and found that IL-21 more robustly induced Il1b mRNA expression in cDCs than did LPS (Fig. 1c). Because NF-κB is known to be critical for the induction of Il1b mRNA expression by LPS8, we treated cDCs with an IκB kinase β inhibitor, MLN120B17 and this strongly inhibited LPS-induced, but not IL-21-induced, Il1b mRNA expression (Fig. 1d), indicating that the effect of IL-21 was independent of NF-κB. Correspondingly, LPS treatment of cDCs induced phosphorylation of IκBα, which promotes its ubiquitination and degradation, and allows nuclear translocation of NF-κB, whereas IL-21 had no effect (Fig. 1e and Supplementary Fig. 1). Because IL-21 can activate STAT3 in cDCs16, we next crossed Stat3-floxed mice to CD11c-Cre transgenic mice to delete Stat3 in cDCs (herein denoted as Stat3−/− mice), and this markedly decreased IL-21-induced (Fig. 1f) but not LPS-induced (Fig. 1g) Il1b mRNA expression. Importantly, when we tested human cDCs isolated from peripheral blood, IL-21 also induced tyrosine phosphorylation of STAT3 (Fig. 1h) and augmented IL1B mRNA expression (Fig. 1i) in human cDCs. Thus, IL-21 uses a distinctive, STAT3-dependent pathway for the induction of Il1b mRNA expression in cDCs.


IL-21-mediated non-canonical pathway for IL-1β production in conventional dendritic cells.

Wan CK, Li P, Spolski R, Oh J, Andraski AB, Du N, Yu ZX, Dillon CP, Green DR, Leonard WJ - Nat Commun (2015)

IL-21 induces Il1b mRNA expression via STAT3.(a) cDCs were rested 1 h, treated with 100 ng ml−1 IL-21 at the indicated time points, and Il1b mRNA assessed relative to Rpl7 by reverse transcription–PCR (RT–PCR). Shown is one of two similar experiments. (b) Il21r+/+ and Il21r−/− cDCs were treated with IL-21 for 5 h and Il1b mRNA assessed. (c) cDCs were rested 1 h, treated with 100 ng ml−1 IL-21 or LPS at the indicated time points and Il1b mRNA assessed relative to Rpl7 by RT–PCR. Shown are the combined results of two independent experiments. (d) cDCs were incubated with 10 μM MLN120B for 1 h, treated with IL-21 or LPS for 4 h and Il1b mRNA assessed. (e) cDCs were rested 1 h, stimulated with 100 ng ml−1 IL-21 or LPS for 30 min, and the expression of phosphorylated and total IκBα was determined. Shown is one of two similar experiments. (f,g) cDCs from Stat3+/+ and Stat3−/− mice were treated with IL-21 (f) or LPS (g) as in b, and Il1b mRNA assessed. In g, NS, P=0.3. Data in b and d–g are representative of three experiments; error bars are technical duplicates of the representative experiment; shown is expression relative to Rpl7 assessed by RT–PCR. (h) Human peripheral blood DCs were rested 16 h and stimulated with IL-21 for 15 min. pSTAT3 in CD1c+ cells was measured by flow cytometry. Shown are data representative of five samples in three experiments. (i) Human CD1c+ cDCs were rested 16 h, stimulated with IL-21 for 4 h, and IL1B mRNA relative to ACTB was assessed by RT–PCR. Data are from three experiments (six total samples); error bars are means±s.e.m. Statistical analysis was performed by Student's t-test.
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f1: IL-21 induces Il1b mRNA expression via STAT3.(a) cDCs were rested 1 h, treated with 100 ng ml−1 IL-21 at the indicated time points, and Il1b mRNA assessed relative to Rpl7 by reverse transcription–PCR (RT–PCR). Shown is one of two similar experiments. (b) Il21r+/+ and Il21r−/− cDCs were treated with IL-21 for 5 h and Il1b mRNA assessed. (c) cDCs were rested 1 h, treated with 100 ng ml−1 IL-21 or LPS at the indicated time points and Il1b mRNA assessed relative to Rpl7 by RT–PCR. Shown are the combined results of two independent experiments. (d) cDCs were incubated with 10 μM MLN120B for 1 h, treated with IL-21 or LPS for 4 h and Il1b mRNA assessed. (e) cDCs were rested 1 h, stimulated with 100 ng ml−1 IL-21 or LPS for 30 min, and the expression of phosphorylated and total IκBα was determined. Shown is one of two similar experiments. (f,g) cDCs from Stat3+/+ and Stat3−/− mice were treated with IL-21 (f) or LPS (g) as in b, and Il1b mRNA assessed. In g, NS, P=0.3. Data in b and d–g are representative of three experiments; error bars are technical duplicates of the representative experiment; shown is expression relative to Rpl7 assessed by RT–PCR. (h) Human peripheral blood DCs were rested 16 h and stimulated with IL-21 for 15 min. pSTAT3 in CD1c+ cells was measured by flow cytometry. Shown are data representative of five samples in three experiments. (i) Human CD1c+ cDCs were rested 16 h, stimulated with IL-21 for 4 h, and IL1B mRNA relative to ACTB was assessed by RT–PCR. Data are from three experiments (six total samples); error bars are means±s.e.m. Statistical analysis was performed by Student's t-test.
Mentions: We previously performed microarray analysis to identify genes that are regulated by IL-21 in cDCs16. Surprisingly, although IL-21 inhibits the LPS-induced IL-1β expression in BMDCs15, we observed a significant induction of Il1b mRNA by IL-21 in cDCs. Transcription of Il1b mRNA is induced in response to IL-1 itself or microbial products acting via toll-like receptors2, but Il1b induction by IL-21 was not anticipated. Therefore, we validated this result by reverse transcription–PCR and confirmed that IL-21 indeed rapidly (within 30 min) induced Il1b mRNA (Fig. 1a) and that this induction did not occur in Il21r−/− cells (Fig. 1b), excluding the possibility that it resulted from contaminating endotoxin. We compared the effect of IL-21 with the classical Il1b-inducing ligand LPS and found that IL-21 more robustly induced Il1b mRNA expression in cDCs than did LPS (Fig. 1c). Because NF-κB is known to be critical for the induction of Il1b mRNA expression by LPS8, we treated cDCs with an IκB kinase β inhibitor, MLN120B17 and this strongly inhibited LPS-induced, but not IL-21-induced, Il1b mRNA expression (Fig. 1d), indicating that the effect of IL-21 was independent of NF-κB. Correspondingly, LPS treatment of cDCs induced phosphorylation of IκBα, which promotes its ubiquitination and degradation, and allows nuclear translocation of NF-κB, whereas IL-21 had no effect (Fig. 1e and Supplementary Fig. 1). Because IL-21 can activate STAT3 in cDCs16, we next crossed Stat3-floxed mice to CD11c-Cre transgenic mice to delete Stat3 in cDCs (herein denoted as Stat3−/− mice), and this markedly decreased IL-21-induced (Fig. 1f) but not LPS-induced (Fig. 1g) Il1b mRNA expression. Importantly, when we tested human cDCs isolated from peripheral blood, IL-21 also induced tyrosine phosphorylation of STAT3 (Fig. 1h) and augmented IL1B mRNA expression (Fig. 1i) in human cDCs. Thus, IL-21 uses a distinctive, STAT3-dependent pathway for the induction of Il1b mRNA expression in cDCs.

Bottom Line: IL-21 does not induce Il1b expression in CD4(+) T cells, with differential histone marks present in these cells versus cDCs.IL-21-induced IL-1β processing in cDCs does not require caspase-1 or caspase-8 but depends on IL-21-mediated death and activation of serine protease(s).These results demonstrate lineage-restricted IL-21-induced IL-1β via a non-canonical pathway and provide evidence for its importance in vivo.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Immunology and the Immunology Center, National Heart, Lung and Blood Institute, National Institutes of Health, 10 Center Drive, Building 10, Room 7B05, Bethesda, Maryland 20892-1674 USA.

ABSTRACT
The canonical pathway for IL-1β production requires TLR-mediated NF-κB-dependent Il1b gene induction, followed by caspase-containing inflammasome-mediated processing of pro-IL-1β. Here we show that IL-21 unexpectedly induces IL-1β production in conventional dendritic cells (cDCs) via a STAT3-dependent but NF-κB-independent pathway. IL-21 does not induce Il1b expression in CD4(+) T cells, with differential histone marks present in these cells versus cDCs. IL-21-induced IL-1β processing in cDCs does not require caspase-1 or caspase-8 but depends on IL-21-mediated death and activation of serine protease(s). Moreover, STAT3-dependent IL-1β expression in cDCs at least partially explains the IL-21-mediated pathologic response occurring during infection with pneumonia virus of mice. These results demonstrate lineage-restricted IL-21-induced IL-1β via a non-canonical pathway and provide evidence for its importance in vivo.

No MeSH data available.


Related in: MedlinePlus