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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 promotes cytokine expression from ILCa) Flow cytometry of lung cells isolated from papain challenged mice, treated with isotype control or neutralising IL-9-specific antibodies 30 min before the last papain re-challenge, stained for lineage markers and Thy1.2 (upper panel) and gated on ILC and stained for Sca-1 and CD25 (lower panel). b) Absolute number of ILC in the lung and e) absolute number of Sca-1+ CD25+ ILC in the lung of papain challenged mice 3 days after re-challenge and antibody treatment **p=0.001. d) Quantitative PCR analysis of the expression of transcripts for IL-9R in sorted naïve T cells and B and CD25+ ILC isolated from the lungs of papain challenged mice. mRNA expression is presented relative to the expression of Hprt1. f) Cytokine concentration in the supernatant of sorted CD25+ ILC stimulated with or without IL-9 in vitro overnight *p=0.04, **p=0.009 g) Cytokine concentration in the lung homogenate and BALF of papain challenged wt mice treated with IL-9-specific or isotype control antibodies 3 days after the last papain re-challenge**p=0.002, ***p=0.0002. Numbers in gates indicate percent cells in each. (Treatment regimes are shown in Supplementary Fig.9). .Data represents at least two (e) or three independent experiments with four mice (b,c,d,g) in each experimental group or three groups of pooled cells from two mice (f) (mean ±SEM in c,d,f,g).
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Figure 7: IL-9 promotes cytokine expression from ILCa) Flow cytometry of lung cells isolated from papain challenged mice, treated with isotype control or neutralising IL-9-specific antibodies 30 min before the last papain re-challenge, stained for lineage markers and Thy1.2 (upper panel) and gated on ILC and stained for Sca-1 and CD25 (lower panel). b) Absolute number of ILC in the lung and e) absolute number of Sca-1+ CD25+ ILC in the lung of papain challenged mice 3 days after re-challenge and antibody treatment **p=0.001. d) Quantitative PCR analysis of the expression of transcripts for IL-9R in sorted naïve T cells and B and CD25+ ILC isolated from the lungs of papain challenged mice. mRNA expression is presented relative to the expression of Hprt1. f) Cytokine concentration in the supernatant of sorted CD25+ ILC stimulated with or without IL-9 in vitro overnight *p=0.04, **p=0.009 g) Cytokine concentration in the lung homogenate and BALF of papain challenged wt mice treated with IL-9-specific or isotype control antibodies 3 days after the last papain re-challenge**p=0.002, ***p=0.0002. Numbers in gates indicate percent cells in each. (Treatment regimes are shown in Supplementary Fig.9). .Data represents at least two (e) or three independent experiments with four mice (b,c,d,g) in each experimental group or three groups of pooled cells from two mice (f) (mean ±SEM in c,d,f,g).

Mentions: To investigate the potential physiological consequences of IL-9 expression during papain-induced airway inflammation, we treated wild-type mice with neutralising IL-9-specific antibodies 30 minutes before the last papain challenge (Fig. 7a). The number and composition of cells infiltrating the lungs and airways of papain-challenged mice remained unchanged upon IL-9 neutralisation at this stage (data not shown) and the total number of ILC did not change significantly (Fig. 7b,c). However, we observed a drastic phenotypic change in the ILC population three days after papain challenge and IL-9 neutralisation. While ILC from papain-challenged mice treated with isotype control antibodies displayed high levels of CD25 and Sca-1, ILC from IL-9 neutralised mice showed a substantial reduction in CD25+ Sca-1+ double positive cells (Fig. 7b,d). The IL-9 receptor (IL-9R) was reported to be expressed on Ih2 cells29. To address if ILC recovered from the lung of papain-challenged mice expressed the IL-9R, we sorted CD25+ ILC and tested IL-9R expression compared to B cells, known to express the IL-9R, and naïve T cells that do not express the receptor. CD25+ ILC showed IL-9R expression similar to B cells (Fig. 7e), implying that IL-9 could directly affect ILC. To investigate if exposure to IL-9 is able to change the cytokine profile of ILC, we cultured sorted CD25+ ILC in the presence or absence of IL-9. Addition of IL-9 markedly increased the ILC production of IL-5, IL-6 and IL-13 (Fig. 7f), suggesting that IL-9 could provide an additional activation signal important for cytokine expression. To address if IL-9 has a similar impact on IL-5 and IL-13 expression in vivo, we analysed the lung homogenate and broncheo-alveolar lavage fluid (BALF) of papain-challenged mice treated with neutralising IL-9-specific or isotype control antibodies three days after challenge and IL-9 neutralisation. In IL-9-neutralized mice, IL-5 and IL-13 expression was reduced to less than half of the levels recovered from isotype control treated mice, indicating that IL-9 indeed has an additive effect on IL-5 and IL-13 expression in vivo (Fig. 7g). Thus, it appears that IL-9 has a feedback effect on ILC, resulting in increased production of IL-5 and IL-13.


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 promotes cytokine expression from ILCa) Flow cytometry of lung cells isolated from papain challenged mice, treated with isotype control or neutralising IL-9-specific antibodies 30 min before the last papain re-challenge, stained for lineage markers and Thy1.2 (upper panel) and gated on ILC and stained for Sca-1 and CD25 (lower panel). b) Absolute number of ILC in the lung and e) absolute number of Sca-1+ CD25+ ILC in the lung of papain challenged mice 3 days after re-challenge and antibody treatment **p=0.001. d) Quantitative PCR analysis of the expression of transcripts for IL-9R in sorted naïve T cells and B and CD25+ ILC isolated from the lungs of papain challenged mice. mRNA expression is presented relative to the expression of Hprt1. f) Cytokine concentration in the supernatant of sorted CD25+ ILC stimulated with or without IL-9 in vitro overnight *p=0.04, **p=0.009 g) Cytokine concentration in the lung homogenate and BALF of papain challenged wt mice treated with IL-9-specific or isotype control antibodies 3 days after the last papain re-challenge**p=0.002, ***p=0.0002. Numbers in gates indicate percent cells in each. (Treatment regimes are shown in Supplementary Fig.9). .Data represents at least two (e) or three independent experiments with four mice (b,c,d,g) in each experimental group or three groups of pooled cells from two mice (f) (mean ±SEM in c,d,f,g).
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Figure 7: IL-9 promotes cytokine expression from ILCa) Flow cytometry of lung cells isolated from papain challenged mice, treated with isotype control or neutralising IL-9-specific antibodies 30 min before the last papain re-challenge, stained for lineage markers and Thy1.2 (upper panel) and gated on ILC and stained for Sca-1 and CD25 (lower panel). b) Absolute number of ILC in the lung and e) absolute number of Sca-1+ CD25+ ILC in the lung of papain challenged mice 3 days after re-challenge and antibody treatment **p=0.001. d) Quantitative PCR analysis of the expression of transcripts for IL-9R in sorted naïve T cells and B and CD25+ ILC isolated from the lungs of papain challenged mice. mRNA expression is presented relative to the expression of Hprt1. f) Cytokine concentration in the supernatant of sorted CD25+ ILC stimulated with or without IL-9 in vitro overnight *p=0.04, **p=0.009 g) Cytokine concentration in the lung homogenate and BALF of papain challenged wt mice treated with IL-9-specific or isotype control antibodies 3 days after the last papain re-challenge**p=0.002, ***p=0.0002. Numbers in gates indicate percent cells in each. (Treatment regimes are shown in Supplementary Fig.9). .Data represents at least two (e) or three independent experiments with four mice (b,c,d,g) in each experimental group or three groups of pooled cells from two mice (f) (mean ±SEM in c,d,f,g).
Mentions: To investigate the potential physiological consequences of IL-9 expression during papain-induced airway inflammation, we treated wild-type mice with neutralising IL-9-specific antibodies 30 minutes before the last papain challenge (Fig. 7a). The number and composition of cells infiltrating the lungs and airways of papain-challenged mice remained unchanged upon IL-9 neutralisation at this stage (data not shown) and the total number of ILC did not change significantly (Fig. 7b,c). However, we observed a drastic phenotypic change in the ILC population three days after papain challenge and IL-9 neutralisation. While ILC from papain-challenged mice treated with isotype control antibodies displayed high levels of CD25 and Sca-1, ILC from IL-9 neutralised mice showed a substantial reduction in CD25+ Sca-1+ double positive cells (Fig. 7b,d). The IL-9 receptor (IL-9R) was reported to be expressed on Ih2 cells29. To address if ILC recovered from the lung of papain-challenged mice expressed the IL-9R, we sorted CD25+ ILC and tested IL-9R expression compared to B cells, known to express the IL-9R, and naïve T cells that do not express the receptor. CD25+ ILC showed IL-9R expression similar to B cells (Fig. 7e), implying that IL-9 could directly affect ILC. To investigate if exposure to IL-9 is able to change the cytokine profile of ILC, we cultured sorted CD25+ ILC in the presence or absence of IL-9. Addition of IL-9 markedly increased the ILC production of IL-5, IL-6 and IL-13 (Fig. 7f), suggesting that IL-9 could provide an additional activation signal important for cytokine expression. To address if IL-9 has a similar impact on IL-5 and IL-13 expression in vivo, we analysed the lung homogenate and broncheo-alveolar lavage fluid (BALF) of papain-challenged mice treated with neutralising IL-9-specific or isotype control antibodies three days after challenge and IL-9 neutralisation. In IL-9-neutralized mice, IL-5 and IL-13 expression was reduced to less than half of the levels recovered from isotype control treated mice, indicating that IL-9 indeed has an additive effect on IL-5 and IL-13 expression in vivo (Fig. 7g). Thus, it appears that IL-9 has a feedback effect on ILC, resulting in increased production of IL-5 and IL-13.

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