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An IL-9 fate reporter demonstrates the induction of an innate IL-9 response in lung inflammation.

Wilhelm C, Hirota K, Stieglitz B, Van Snick J, Tolaini M, Lahl K, Sparwasser T, Helmby H, Stockinger B - Nat. Immunol. (2011)

Bottom Line: We found that during papain-induced lung inflammation, IL-9 production was largely restricted to innate lymphoid cells (ILCs).IL-9 production by ILCs depended on IL-2 from adaptive immune cells and was rapidly lost in favor of other cytokines, such as IL-13 and IL-5.Blockade of IL-9 production via neutralizing antibodies resulted in much lower expression of IL-13 and IL-5, which suggested that ILCs provide the missing link between the well-established functions of IL-9 in the regulation of type 2 helper T cell cytokines and responses.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Immunology, Medical Research Council National Institute for Medical Research, Mill Hill, UK.

ABSTRACT
Interleukin 9 (IL-9) is a cytokine linked to lung inflammation, but its cellular origin and function remain unclear. Here we describe a reporter mouse strain designed to map the fate of cells that have activated IL-9. We found that during papain-induced lung inflammation, IL-9 production was largely restricted to innate lymphoid cells (ILCs). IL-9 production by ILCs depended on IL-2 from adaptive immune cells and was rapidly lost in favor of other cytokines, such as IL-13 and IL-5. Blockade of IL-9 production via neutralizing antibodies resulted in much lower expression of IL-13 and IL-5, which suggested that ILCs provide the missing link between the well-established functions of IL-9 in the regulation of type 2 helper T cell cytokines and responses.

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IL-9 in ILC is induced by IL-2 but not IL-25, IL-33 or TSLPa) IL-9 cytokine concentration in the supernatant of MACS sorted ILC (black bars) and cells expressing lineage markers (Lin+, white bars) isolated from the lungs of papain challenged IL9CreR26ReYFP mice and stimulated in vitro overnight with P+I, IL-2, IL-25, IL-33 or TSLP. b) Concentration of IL-9 in the supernatant of sorted CD4+ cells (grey bars), CD25− ILC (white bars) CD25+ ILC black bars) and c) sorted eYFP+ ILC cultured overnight as in a. d) Quantitative PCR analysis of the expression of transcripts for IL-4 (Il4), IL-5 Iil5), IL-6 (Il6), IL-9 (Il9) and IL-13 (Il13) in sorted CD4+, CD25− ILC, CD25+ ILC, eYFP+ ILC. mRNA expression was normalized to the expression of Hprt1 (encoding hypoxanthine guanine phosphoribosyl transferase) and ispresented relative to CD4+ expression levels. Data represents at least three (a,b,c) or two (d) independent experiments (mean ±SEM in a,b.c). P+I: stimulated with PdBU and ionomycin; ctrl: un-stimulated.
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Figure 3: IL-9 in ILC is induced by IL-2 but not IL-25, IL-33 or TSLPa) IL-9 cytokine concentration in the supernatant of MACS sorted ILC (black bars) and cells expressing lineage markers (Lin+, white bars) isolated from the lungs of papain challenged IL9CreR26ReYFP mice and stimulated in vitro overnight with P+I, IL-2, IL-25, IL-33 or TSLP. b) Concentration of IL-9 in the supernatant of sorted CD4+ cells (grey bars), CD25− ILC (white bars) CD25+ ILC black bars) and c) sorted eYFP+ ILC cultured overnight as in a. d) Quantitative PCR analysis of the expression of transcripts for IL-4 (Il4), IL-5 Iil5), IL-6 (Il6), IL-9 (Il9) and IL-13 (Il13) in sorted CD4+, CD25− ILC, CD25+ ILC, eYFP+ ILC. mRNA expression was normalized to the expression of Hprt1 (encoding hypoxanthine guanine phosphoribosyl transferase) and ispresented relative to CD4+ expression levels. Data represents at least three (a,b,c) or two (d) independent experiments (mean ±SEM in a,b.c). P+I: stimulated with PdBU and ionomycin; ctrl: un-stimulated.

Mentions: IL-25, IL-33 or thymic stromal lymphopoietin (TSLP) or the combination of these cytokines did not induce substantial IL-9 production from ILC isolated from papain challenged mice (Supplementary Fig. 5c). However, culture of ILC in the presence of IL-2 resulted in IL-9 protein levels similar to those seen after stimulation with PdBU and ionomycin (Fig. 3a). CD25, the high affinity chain of the IL-2 receptor, is expressed on a proportion of eYFP+ ILC and ILC responded to IL-2 stimulation with IL-9 production. To test if CD25 expression can be used to identify ILC poised for expression of IL-9 in wild-type B6 mice, we sorted CD25+ ILC and CD25− ILC and tested their IL-2-induced IL-9 protein production in comparison to that of total CD4+ T cells from papain-challenged mice (Fig.3b). IL-2-mediated IL-9 production was mainly found in CD25+ ILC, suggesting an important role of IL-2 signalling for the production of IL-9 from ILC (Fig. 3b). No IL-9 production was detectable from CD4+ T cells. IL-9 expression in eYFP+ ILC isolated from IL9CreR26ReYFP reporter mice was likewise dependent on IL-2 (Fig. 3c). Analysis of cytokine mRNA expression comparing lung-infiltrating unstimulated total CD4+ T cells (set as reference standard) with CD25+ or CD25− ILC from wild-type mice or eYFP+ ILC showed substantially increased expression of TH2-type cytokines, such as IL-5, IL-6 and IL-13, in CD25+ ILC and especially in eYFP+ ILC from the IL-9 reporter mice (Fig. 3d), emphasising their activated state. Expression of IL-9 was increased in CD25+ ILC, in comparison to total CD4+ T cells or CD25− ILC. IL-9 mRNA was even higher in eYFP+ ILC from reporter mice, suggesting the eYFP specificity for reporting IL-9 expression. These data indicate the dependency of IL-9 production on the availability of IL-2.


An IL-9 fate reporter demonstrates the induction of an innate IL-9 response in lung inflammation.

Wilhelm C, Hirota K, Stieglitz B, Van Snick J, Tolaini M, Lahl K, Sparwasser T, Helmby H, Stockinger B - Nat. Immunol. (2011)

IL-9 in ILC is induced by IL-2 but not IL-25, IL-33 or TSLPa) IL-9 cytokine concentration in the supernatant of MACS sorted ILC (black bars) and cells expressing lineage markers (Lin+, white bars) isolated from the lungs of papain challenged IL9CreR26ReYFP mice and stimulated in vitro overnight with P+I, IL-2, IL-25, IL-33 or TSLP. b) Concentration of IL-9 in the supernatant of sorted CD4+ cells (grey bars), CD25− ILC (white bars) CD25+ ILC black bars) and c) sorted eYFP+ ILC cultured overnight as in a. d) Quantitative PCR analysis of the expression of transcripts for IL-4 (Il4), IL-5 Iil5), IL-6 (Il6), IL-9 (Il9) and IL-13 (Il13) in sorted CD4+, CD25− ILC, CD25+ ILC, eYFP+ ILC. mRNA expression was normalized to the expression of Hprt1 (encoding hypoxanthine guanine phosphoribosyl transferase) and ispresented relative to CD4+ expression levels. Data represents at least three (a,b,c) or two (d) independent experiments (mean ±SEM in a,b.c). P+I: stimulated with PdBU and ionomycin; ctrl: un-stimulated.
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Related In: Results  -  Collection

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Figure 3: IL-9 in ILC is induced by IL-2 but not IL-25, IL-33 or TSLPa) IL-9 cytokine concentration in the supernatant of MACS sorted ILC (black bars) and cells expressing lineage markers (Lin+, white bars) isolated from the lungs of papain challenged IL9CreR26ReYFP mice and stimulated in vitro overnight with P+I, IL-2, IL-25, IL-33 or TSLP. b) Concentration of IL-9 in the supernatant of sorted CD4+ cells (grey bars), CD25− ILC (white bars) CD25+ ILC black bars) and c) sorted eYFP+ ILC cultured overnight as in a. d) Quantitative PCR analysis of the expression of transcripts for IL-4 (Il4), IL-5 Iil5), IL-6 (Il6), IL-9 (Il9) and IL-13 (Il13) in sorted CD4+, CD25− ILC, CD25+ ILC, eYFP+ ILC. mRNA expression was normalized to the expression of Hprt1 (encoding hypoxanthine guanine phosphoribosyl transferase) and ispresented relative to CD4+ expression levels. Data represents at least three (a,b,c) or two (d) independent experiments (mean ±SEM in a,b.c). P+I: stimulated with PdBU and ionomycin; ctrl: un-stimulated.
Mentions: IL-25, IL-33 or thymic stromal lymphopoietin (TSLP) or the combination of these cytokines did not induce substantial IL-9 production from ILC isolated from papain challenged mice (Supplementary Fig. 5c). However, culture of ILC in the presence of IL-2 resulted in IL-9 protein levels similar to those seen after stimulation with PdBU and ionomycin (Fig. 3a). CD25, the high affinity chain of the IL-2 receptor, is expressed on a proportion of eYFP+ ILC and ILC responded to IL-2 stimulation with IL-9 production. To test if CD25 expression can be used to identify ILC poised for expression of IL-9 in wild-type B6 mice, we sorted CD25+ ILC and CD25− ILC and tested their IL-2-induced IL-9 protein production in comparison to that of total CD4+ T cells from papain-challenged mice (Fig.3b). IL-2-mediated IL-9 production was mainly found in CD25+ ILC, suggesting an important role of IL-2 signalling for the production of IL-9 from ILC (Fig. 3b). No IL-9 production was detectable from CD4+ T cells. IL-9 expression in eYFP+ ILC isolated from IL9CreR26ReYFP reporter mice was likewise dependent on IL-2 (Fig. 3c). Analysis of cytokine mRNA expression comparing lung-infiltrating unstimulated total CD4+ T cells (set as reference standard) with CD25+ or CD25− ILC from wild-type mice or eYFP+ ILC showed substantially increased expression of TH2-type cytokines, such as IL-5, IL-6 and IL-13, in CD25+ ILC and especially in eYFP+ ILC from the IL-9 reporter mice (Fig. 3d), emphasising their activated state. Expression of IL-9 was increased in CD25+ ILC, in comparison to total CD4+ T cells or CD25− ILC. IL-9 mRNA was even higher in eYFP+ ILC from reporter mice, suggesting the eYFP specificity for reporting IL-9 expression. These data indicate the dependency of IL-9 production on the availability of IL-2.

Bottom Line: We found that during papain-induced lung inflammation, IL-9 production was largely restricted to innate lymphoid cells (ILCs).IL-9 production by ILCs depended on IL-2 from adaptive immune cells and was rapidly lost in favor of other cytokines, such as IL-13 and IL-5.Blockade of IL-9 production via neutralizing antibodies resulted in much lower expression of IL-13 and IL-5, which suggested that ILCs provide the missing link between the well-established functions of IL-9 in the regulation of type 2 helper T cell cytokines and responses.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Immunology, Medical Research Council National Institute for Medical Research, Mill Hill, UK.

ABSTRACT
Interleukin 9 (IL-9) is a cytokine linked to lung inflammation, but its cellular origin and function remain unclear. Here we describe a reporter mouse strain designed to map the fate of cells that have activated IL-9. We found that during papain-induced lung inflammation, IL-9 production was largely restricted to innate lymphoid cells (ILCs). IL-9 production by ILCs depended on IL-2 from adaptive immune cells and was rapidly lost in favor of other cytokines, such as IL-13 and IL-5. Blockade of IL-9 production via neutralizing antibodies resulted in much lower expression of IL-13 and IL-5, which suggested that ILCs provide the missing link between the well-established functions of IL-9 in the regulation of type 2 helper T cell cytokines and responses.

Show MeSH
Related in: MedlinePlus