|An IL-9 fate reporter demonstrates the induction of an innate IL-9 response in lung inflammation.|
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|PMID: 21983833 Owner: NLM Status: MEDLINE|
|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.|
|Christoph Wilhelm; Keiji Hirota; Benjamin Stieglitz; Jacques Van Snick; Mauro Tolaini; Katharina Lahl; Tim Sparwasser; Helena Helmby; Brigitta Stockinger|
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|Type: Journal Article; Research Support, Non-U.S. Gov't Date: 2011-10-09|
|Title: Nature immunology Volume: 12 ISSN: 1529-2916 ISO Abbreviation: Nat. Immunol. Publication Date: 2011 Nov|
|Created Date: 2011-10-20 Completed Date: 2011-12-14 Revised Date: 2013-06-27|
Medline Journal Info:
|Nlm Unique ID: 100941354 Medline TA: Nat Immunol Country: United States|
|Languages: eng Pagination: 1071-7 Citation Subset: IM|
|Division of Molecular Immunology, Medical Research Council National Institute for Medical Research, Mill Hill, UK.|
|APA/MLA Format Download EndNote Download BibTex|
Antibodies, Blocking / administration & dosage
Cytokines / immunology, metabolism*
Genes, Reporter / genetics
Interleukin-9 / genetics, immunology, metabolism*
Lymphocyte Activation / drug effects
Lymphocytes / immunology, metabolism*, pathology
Mice, Inbred C57BL
Papain / administration & dosage
Pneumonia / chemically induced, immunology*
Th2 Cells / drug effects, immunology*
|MC_U117512792//Medical Research Council; U.1175.02.003.00003(60535)//Medical Research Council; U117512792//Medical Research Council|
|0/Antibodies, Blocking; 0/Cytokines; 0/Interleukin-9; EC 220.127.116.11/Papain|
Journal ID (nlm-journal-id): 100941354
Journal ID (pubmed-jr-id): 21750
Journal ID (nlm-ta): Nat Immunol
nihms-submitted publication date: Day: 7 Month: 9 Year: 2011
Electronic publication date: Day: 9 Month: 10 Year: 2011
pmc-release publication date: Day: 9 Month: 4 Year: 2012
Volume: 12 Issue: 11
First Page: 1071 Last Page: 1077
PubMed Id: 21983833
|Interleukin 9 fate reporter reveals induction of innate IL-9 response in lung inflammation|
|Jacques Van Snick§|
*Division of Molecular Immunology, MRC National Institute for Medical Research, Mill Hill, UK
$Division of Molecular Structure, MRC National Institute for Medical Research, Mill Hill, UK
§Ludwig Institute for Cancer Research, Brussels, Belgium
#London School of Hygiene and Tropical Medicine Keppel Street, London, UK
&Twincore, Institut für Infektionsimmunologie, Hannover, Germany
|@present address: Laboratory of Immunology and Vascular Biology, Department of Pathology, Stanford University School of Medicine, Stanford, California, USA
Interleukin 9 (IL-9) is a pleiotropic cytokine that is expressed at elevated levels in the lungs of asthmatic patients1,2. Blocking antibodies are currently in clinical trials as potential therapies for atopic disease3. Similar results have been obtained in mouse models, where specific over-expression of IL-9 in lungs results in the induction of an asthma-like phenotype6-8 and blockage of IL-9 signalling reduces airway inflammation4,5. An important function attributed to IL-9 in lung physiology is the induction of mucus production, goblet cell hyperplasia and other features of airway remodelling9,10, functions that were also attributed to IL-1311 as well as IL-5 via the regulation of eosinophils12. IL-9 is also involved in protective immunity to helminth infections, indicated by the enhanced kinetics of worm expulsion seen in IL-9 transgenic mice13,14 and the susceptibility to helminth infection upon IL-9 depletion15.
The cellular source of IL-9 in the context of airway inflammation has been mainly attributed to T cells16-18. Activated CD4+ T cells from the T helper cell 2 subset (TH2) were believed to comprise the majority of IL-9 producing cells. However, substantial IL-9 production is induced in CD4+ T cells differentiating in vitro in the presence of IL-4 and TGF-β, but not in the context of IL-4 alone19. Thus, IL-9 is not a TH2 cytokine. In addition to T cells, eosinophils and mast cells also produce IL-920-22.
Novel cellular sources for the secretion of TH2-type cytokines have been recently discovered. These cell types show striking similarities to lymphoid tissue inducer cells (LTi cells), do not express known lineage markers, are responsive to both IL-25 and IL-33 and play a protective role during helminth infections23-29. Such lineage negative (Lin−) cells display some LTi-like properties, such as IL-7 receptor expression, but lack CD4 and Rorγt expression and have a different cytokine expression profile. Therefore, they were either termed natural helper cells (NHCs)28, nuocytes27, innate helper type 2 (Ih2) cells29 or multipotent progenitors (MPPs)26. Nuocytes and MPPs reside in mesenteric lymph nodes and spleen, while NHCs were found in the fat associated lymphoid tissue and Ih2 cells are dispersed throughout the body, with the highest numbers recovered from the liver. This subsets of newly identified Lin− cells, or innate lymphoid cells type 2 (ILC2s)30, are characterised by the secretion of high amounts of the TH2 cytokines IL-5, IL-6 and IL-13 after induction with IL-25 or IL-33, which is strongly indicative of a potential involvement in airway inflammation.
Here we show the induction of IL-9 producing ILC identified by an IL-9 specific reporter in a model of papain-induced airway inflammation. ILC were the major source of IL-9 and IL-9 production was transient and dependent on IL-2 from adaptive immune cells. While IL-9 expression waned quickly, ILC continued to produce IL-13 and IL-5. IL-9 was found to facilitate IL-5 and IL-13 production from ILC, while neutralisation of IL-9 reduced the levels of IL-5 and IL-13 after papain challenge. Our findings indicate a previously unrecognized mechanism for the induction of IL-9 from ILC and a potential involvement of IL-9 in allergic lung diseases via the promotion of IL-5 and IL-13 production in ILC.
Despite the demonstration that a subset of in vitro generated CD4+ T cells can secrete IL-9, the cell types producing this cytokine in vivo, under physiological conditions remain unidentified. This is partially due to the technical difficulties associated with ex vivo intracellular staining for IL-9. We generated an IL-9 fate reporter BAC transgenic mice that expresses the Cre recombinase under the control of the endogenous IL-9 locus (IL9Cre mice) (Supplementary Fig. 1). To visualize the Cre activity we bred the IL9Cre mice with reporter mice expressing enhanced yellow fluorescent protein (eYFP) under the control of the endogenous Rosa 26 promoter (termed R26ReYFP). In IL9CreR26ReYFP mice the fluorescent reporter permanently labels cells that had expressed the IL-9 gene, irrespective of the current production status for this cytokine. Analysis of tissues from non-immune IL9CreR26ReYFP reporter mice did not reveal any eYFP+ cells in the hematopoietic compartment of peripheral lymph nodes, mesenteric lymph nodes, spleen, lung, liver, skin and lamina propria of small and large intestine (Supplementary Fig. 2).
In vitro stimulation of FACS purified naïve CD4+ T cells with TGFβ and IL-4 generated a population of ‘TH9’ cells that were detectable by intracellular staining for IL-9 as well as eYFP expression (Supplementary Fig. 3a). In line with recently published reports31, IL-9 expression in T cell cultures was transient. No eYFP induction occured under TH1, TH2 or iTreg conditions, albeit a proportion of eYFP producing cells was induced under TH17 conditions (Supplementary Fig. 3b). However, only about 10% of the cells detected by intracellular cytokine staining had turned on the eYFP gene, suggesting incomplete reporting of IL-9 expressing cells. Our data show that induction of Cre transcripts from the BAC construct was considerably lower than the induction of endogenous IL-9 transcripts. This may add to the observed under-reporting of the BAC transgene in this reporter line (Supplementary Fig.3c). Despite this limitation, IL9CreR26ReYFP mice comprise a useful tool for the detection of IL-9 production in vivo.
Because IL-9 expression has been linked to lung inflammation, we studied the IL-9 expression pattern in IL9CreR26ReYFP mice in a setting of airway inflammation. Papain, a protease with adjuvant activity that leads to asthma-like responses32 induces a strong TH2 phenotype upon subcutaneous injection33 and has been used to induce experimental airway inflammation34,35. Intranasal challenge of IL9CreR26ReYFP reporter mice (Fig. 1a) with papain induces a robust TH2-type lung inflammation as monitored by cell influx in the lungs (Fig. 1b) and cytokine production in the airways (Fig.1c). Notably, papain induced airway inflammation resulted in IL-9 expression in the lung (Fig. 1c) and analysis of reporter mice revealed that most, if not all, eYFP expression was found in non-T cells (Fig. 1d). Further phenotyping of these eYFP+ cells showed that they were not related to any defined haematopoietic lineage (Fig. 1e). No eYFP+ Lin− cells were recovered from the lungs of mice challenged with PBS as control (Fig. 1f).
To address the phenotypic resemblance of the eYFP+ cells that accumulated in papain-induced inflammation with the recently identified natural helper cells, Ih2 cells, nuocytes or MPPs cells26-29, we further characterized their surface expression profile. Although eYFP+ Lin− cells did not exactly resemble any of the reported cell types, they shared the expression of Thy1.2 and CD45 and the majority also showed expression of the IL-33 receptor (IL-33R), while expression of Sca-1, major histocompatibility complex (MHC) class II and CD25 were more heterogeneously distributed (Fig. 1g). We did not detect substantial IL-7R or c-Kit expression in eYFP+ cells directly after papain challenge (data not shown). Hereafter these cells will be referred to as innate lymphoid cells (ILC) 36. Thus, our data identified ILC as the major source of IL-9 in this model of lung inflammation.
We next examined the cytokine expression profile from eYFP+ ILC by intracellular cytokine staining 24 h after the last papain challenge. Following a short 2.5h pulse with PdBU and ionomycin, all eYFP+ ILC expressed IL-13 and the majority expressed IL-5, but only a minority were producing IL-4 (Fig. 2a). However, no IL-9 expression could be recalled in these cells by intracellular cytokine staining (Fig. 2a). IL-9 expression in CD4+ T cells in vitro suggested short-lived IL-9 production. To investigate whether IL-9 expression from ILC is similarly curtailed in papain-mediated airway inflammation, we performed a kinetic analysis of cytokine secretion after papain re-challenge in wild-type C57B6 mice. The percentage of IL-9+ ILC recovered from the lungs of wild-type mice after an acute papain challenge changed rapidly over time, with peak expression observed 6 to 12 h after challenge, and a rapid decline by 24 h (Fig. 2b,c). Analysis of IL-9 protein expression at 6, 12 or 24 h after papain challenge supported the assumption of transient IL-9 expression, with peak protein expression levels observed after 12 h (Fig. 2d). ILC isolated from reporter mice showed eYFP expression co-localized with intracellular IL-9, if analysed 12 h after papain re-challenge (Supplementary Fig. 4). Thus, ILC induced in papain lung inflammation rapidly lose IL-9 protein expression in favour of IL-5 and IL-13 expression.
To test the capacity of ILC to up-regulate IL-9 expression upon stimulation with Toll-like receptor (TLR) ligands, we cultured MACS purified ILC cells (defined by the depletion of cells expressing the lineage markers TCRβ, TCRγδ, CD11c, CD11b, Gr-1, CD19, DX5, NK1.1 and Ter119) and Lin+ cells isolated from papain challenged wild-type mice (Supplementary Fig. 5a) in the presence of PdBU and iononomycin or TLR ligands. None of the tested TLR-ligands was able to induce IL-9 production in ILC or in Lin+ cells, whereas overnight stimulation with PdBU and ionomycin (rather than a 2.5 h stimulation) induced IL-9 protein expression in ILC (Supplementary Fig. 5b). This suggested that IL-9 can be induced or re-induced in ILC upon extended stimulation or in response to factors expressed by other cell types upon PdBU and ionomycin stimulation.
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.
Although neither IL-25 nor IL-33 were able to induce IL-9 production from ILC, previous analysis did not test the capacity of these cytokines to induce recruitment of ILC and the potential for subsequent IL-9 induction following exposure to IL-2. To test whether IL-25 and IL-33-induced ILC were able to produce IL-9 upon IL-2 stimulation we intranasally treated wild-type mice with IL-25 or IL-33. Intranasal challenge with IL-33 was much more potent than IL-25 treatment in inducing the accumulation of ILC in the lung (Fig. 4a, b). Although IL-25 challenge resulted in the induction or recruitment of ILC producing IL-13 (Fig 4a and c), this was again higher after IL-33 application. However, while no IL-9-positive cells were detected by intracellular cytokine staining, IL-33 induced high IL-9 production from ILC upon IL-2 stimulation (Fig. 4d). Additionally, IL-33-primed ILC were also more potent producers of IL-5 and IL-13 than IL-25-primed ILC (Fig. 4d). IL-33 was also more potent to induce eYFP+ ILC upon intranasal administration (Fig. 4e) and such ILC expressed IL-13 and displayed similar surface markers as the eYFP+ ILC generated during papain-induced lung inflammation (Supplementary Fig. 6). In order to compare IL-33 and papain-induced ILC with other ILC2 cell types, we investigated the induction of eYFP expression in nuocytes in the mesenteric lymph nodes after intraperitoneal injection of IL-25 and IL-33, NHCs in the FALT and Ih2 cells in the liver. IL-33, but not IL-25 administration induced a small percentage of eYFP+ nuocytes, while no substantial NHCs or Ih2 cells were detected (Supplementary Fig. 7). Thus, the induction of ILC competent to produce IL-9 is mainly driven by IL-33.
ILC are present in Rag1−/− mice, which lack T and B cells, but not in mice lacking the common gamma chain (IL2rg−/− mice)27-29. To investigate whether the population of IL-9-expressing cells is independent of RAG expression but dependent on signalling via the common gamma chain we analysed IL-9 expression in the lungs of papain-challenged wild-type, Rag1−/− and Rag2−/−IL2rg−/− mice. IL-9 production was reduced by 10-fold in the lungs of Rag1−/− mice as compared to wild-type mice and was absent in Rag2−/−IL2rg−/− mice (Fig. 5a). IL-5 and IL-13 protein expression was also reduced in Rag1−/− mice, in agreement with previous reports that IL-13-expressing cells in the mesenteric lymph nodes were not maintained during helminth infection in Rag1−/− mice27. However, isolation of ILC from the lungs of papain-challenged wild-type, Rag1−/− and Rag2−/−IL2rg−/− mice, followed by overnight stimulation with IL-2, resulted in similar IL-9 protein expression from ILC of wild-type and Rag1−/−(Fig. 5b). In contrast ILC isolated from Rag2−/−IL2rg−/− mice failed to produce IL-9 upon IL-2 stimulation (Fig. 5b). These results suggest that IL-9 production in ILCs upon papain challenge in vivo is dependent on the presence of adaptive immune cells, although the intrinsic capacity of ILCs to produce IL-9 is still intact in Rag1−/− mice.
To address if IL-2 is important for IL-9 expression upon papain challenge in vivo we treated wild-type mice with neutralising IL-2-specific antibodies 30 min before the last papain challenge (Fig. 5c). IL-2 neutralization resulted in significant reduction of IL-9 expression in papain-treated mice, but not in isotype treated control mice (Fig 5d). Other TH2 cytokines like IL-4, IL-5, IL-6 and IL-13 were not affected by IL-2 neutralisation, at least at the early timepoint of 18 h.
To further investigate if reduced IL-9 protein expression in Rag1−/− mice is caused by a lack of IL-2, papain-challenged Rag1−/− mice were treated intranasally with IL-2 or PBS 3 h after the last papain re-challenge (Fig. 5e). IL-2, but not PBS treatment, restored IL-9 expression in Rag1−/− mice to wild-type levels (Fig. 5f). Intranasal application of IL-2 alone, without papain challenge, did not result in IL-9 production (Fig 5f). Total ILC numbers in Rag1−/− mice were only slightly, but not significantly reduced in comparison to wild-type mice and IL-2 application did not result in elevated numbers of ILC (Fig. 5g). These results suggest that IL-9 protein production by ILC is critically dependent on the provision of IL-2 by adaptive immune cells.
To investigate whether ILC are the critical producers of IL-9 during papain-induced lung inflammation we performed intracellular cytokine staining for IL-9 and IL-13 in ILC and CD4+ T cells. The majority of intracellular IL-9 was expressed in ILC, although we detected a minor contribution from CD4+ T cells (Fig. 6a, b). While similar numbers of ILC and CD4+ T cells are recovered from the lungs of papain-challenged mice (Fig. 6c), IL-9 and IL-13 expression stems almost exclusively from ILC (Fig. 6d).
To further explore the relative contribution of ILC or CD4+ T cells to IL-9 production in papain-challenged mice, we adoptively transferred either ILC or CD4+ T cells wild-type sorted from the lung homogenate of papain-challenged intra-tracheally into Rag2−/−IL2rg−/− hosts. In this setting, only transferred cells are able to respond to IL-2 and produce IL-9. Transfer was followed by papain and IL-2 challenge. Whereas very little IL-9, IL-5 and IL-13 were detectable after CD4+ T cell transfer, ILC transfer resulted in substantially elevated levels of IL-9, IL-5 and IL-13 (Fig. 6e). In contrast, sensitisation and challenge of IL9CreR26ReYFP mice with ovalbumin, a model in which T cells were reported to be the major producers of IL-916-18, resulted in eYFP expression largely restricted to CD4+ T cells rather than ILC (Supplementary Fig.8). Thus, ILC and not CD4+ T cells are the major producers of IL-9, 5 and 13 in this particular model.
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.
Several studies have implicated IL-9 in lung inflammation, although the cell type responsible was not identified due to technical difficulties related to intracellular staining for IL-9. In order to address the cellular source of IL-9 production in vivo, we generated an IL-9 fate reporter strain. Initial in vitro tests on T cell differentiation established faithful reporter induction under ‘TH9’ conditions.
Even under TH9 conditions only about 10% of the IL-9 producers were marked by eYFP, suggesting incomplete reporter activity. There are several potential reasons for this limitation. Firstly, it is possible that in vitro conditions for TH9 induction are not efficient, similar to what was seen in the IL-17 Cre fate reporter37 that effectively induces eYPF in TH17 cells generated in vivo, but only marks about 30% of in vitro generated TH17 cells. The second possibility is that incomplete reporting could be due to the BAC transgenic construct itself. The IRES-Cherry sequence contained in the construct was disabled due to a premature translational start site in the IRES sequence possibly diminishing translation from the ATG in the cherry gene. It is possible that the presence of a dysfunctional DNA sequence in the construct interferes with reporter induction. Indeed comparative analysis of transcripts for Cre vs endogenous IL-9 transcripts showed substantially lower Cre induction, pointing to transcriptional dysregulaton of the BAC transgene. However, we did not observe aberrant eYFP expression in a wide range of cell types, so despite the possibility that not all IL-9 producers were labelled by eYFP, we nevertheless could identify a proportion of such cells using this reporter.
Four types of ILC displaying similar surface markers and high amounts of the TH2 cytokines IL-5 and IL-1 have recently been described26-29. The eYFP+ ILC identified in our reporter system shared Thy1.2 surface marker expression with nuocytes, NHCs, MPPs and Ih2 cells and IL-33R and Sca-1 with nuocytes, NHCs and MPPs. Like nuocytes they expressed MHC class II and shared CD25 expression with NHCs. The majority of eYFP+ ILC produced IL-5 and IL-13 but not IL-4 upon restimulation. Although eYFP+ ILC displayed alterations in surface marker expression compared with previously identified ILC types, we cannot exclude that the cells we identified are identical or similar to NHCs, MPPs or nuocytes. We rather propose that changes in surface marker expression reflect activation status and probably tissue distribution.
In contrast to the reported induction of the ILC cytokines IL-5, IL-6 and IL-13 by IL-25 or IL-33, IL-9 was not directly induced by the latter two cytokines, but instead depended on IL-2. However, IL-33- but not IL-25- priming either recruited or induced the ILC population for IL-2 triggered IL-9 production. It was previously reported that IL-9 induction in human T cells is dependent on IL-238,39; however, we did not find similar effects in sorted mouse T cells from papain-challenged mice. Because in most studies T cell purity was assessed by CD2 staining and IL-9 was measured by ELISA, the possibility of an ILC contamination was not excluded.
While ILC are present in papain-treated Rag1−/− mice, IL-9 expression was considerably reduced compared to papain-treated wild-type animals, but could be restored by IL-2 application. This suggests that cells absent in Rag1−/− mice, such as T cells or NKT cells, could be the most important source of IL-2 to support IL-9 production by ILC.
IL-9 expression influenced the expression of other ILC cytokines, since ILC, like lh2 cells, express high levels of IL-9R transcripts29 and neutralisation of IL-9 in the context of papain challenge led to dramatic surface phenotype changes in ILC with reduction of both CD25 and Sca-1. ILC displaying CD25 showed higher expression levels of IL-5, IL-6 and IL-13 than CD25− ILC, suggesting that CD25 expression, like in T cells, could reflect activation. Sca-1 expression is upregulated upon inflammation on hematopoietic stem cells40 and increased Sca-1 expression on ILC could likewise indicate inflammatory activation. Neutralisation of IL-9 during papain-induced lung inflammation resulted in reduced levels of IL-13 and IL-5 when analysed 3 days after challenge. Since maintenance of IL-5 and IL-13 are linked to IL-9 and IL-9 induction requires IL-2, lack of IL-2 signaling might also affect the amounts of IL-5 and IL-13. Although blockade of IL-2 resulted in reduction of IL-9 early after papain challenge (18h), there was no effect on IL-5 and IL-13 at this time point. IL-5 and IL-13 expression might be affected at later timepoints. Our data might explain the observation that although nuocytes are initially induced and accumulate in the mesenteric lymph nodes of N. brasiliensis infected Rag1−/− mice, their numbers are not maintained in the absence of T cells27. In this system nuocytes were defined by acute reporting of IL-13 expression. We find reduced IL-13 expression, but not reduced numbers of ILC in the lungs of papain-challenged Rag1−/− mice. In contrast to an acute cytokine reporter, the advantage of the fate reporter is to allow detection of cells even after loss of cytokine expression. Thus, IL-2 mediated IL-9 expression might be the missing link for sustained IL-13 expression in nuocytes and ILC in general. Culture of NHC with IL-2 alone or in combination with IL-33 promotes IL-13 expression28, further strengthening IL-2 as the most likely connection to the adaptive immune system.
Spontaneous IL-9 induced airway inflammation is observed in IL-9 transgenic mice6-8, but is ablated if IL-9 transgenic mice are backcrossed onto an IL-13 deficient background6, indicating that IL-9 acts via expression of IL-13. Also IL-13 producing ILC have been described to cause lung pathology during influenza infection; however, the involvement of IL-9 was not investigated in this study41.
IL-9 effects on intestinal mucosa were reported to be IL-13 dependent42. Furthermore blocking IL-5 in IL-9 transgenic mice reduced eosinophil recruitment8, suggesting that IL-5 production in this spontaneous airway inflammation is also linked with IL-9. In agreement with these observations our data show increased IL-13 and IL-5 production in ILC upon IL-9 stimulation, suggesting that IL-9 mediated airway inflammation may be the result of enhanced IL-13 and IL-5 expression from ILC. It appears that IL-9 production has to be curtailed and tightly regulated to prevent lung pathology. Thus, understanding the mechanism of transient IL-9 regulation might prove essential to recognize the pathological function of IL-9 during airway inflammation. Taken together, our data suggest plasticity and a highly dynamic cytokine network in ILC.
We would like to thank Drs M. Wilson and A. Potocnik for advice and discussion and the Division of Biological Services for breeding and maintenance of our mouse strains. This work was funded by the Medical Research Council UK Grant Ref.No. U117512792. T.S. and K.L. are funded by the SFB 587. C.W. designed and carried out the experiments and wrote the manuscript, K.H. gave advice and contributed to experiments, B.S.purified neutralizing antibodies, K.L. and T.S. provided help and advice for the BAC construct, J.V.S. provided reagents, M.T. did the BAC injections, H. H. provided advice and B.S. designed experiments and wrote the manuscript.
For visualization of Cre-mediated recombination, IL9Cre mice were backcrossed to C57Bl/6.Rosa26eYFP mice generating IL9CreR26ReYFP mice. The reporter mice used in this study had been backcrossed to C57Bl/6 (B6) mice for at least six generations. C57Bl/6, Rag1-deficient mice (Rag1−/−) and Rag2-IL2rg-deficient mice (Rag2−/−IL2rg−/−) were bred in our animal facility and all mice were kept under specified pathogen free conditions. All animal experiments were done according to institutional guidelines (NIMR Ethical Review panel) and Home Office regulations.
C57Bl/6, Rag1−/−, Rag2−/−IL2rg−/− or IL9Cre were anaesthetized with isoflurane and exposed intranasally to 25 μg papain (Calbiochem) in 50 μL PBS on day 0, 3 and 6. Seven days later mice were re-challenged intranasally with 25 μg papain in PBS. In some experiments papain challenged mice were treated i.v. with 75 μg neutralising IL-2-specific antibodies (JES6-5H4) 30 minutes before papain re-challenge. Control animals received IgG isotype control antibodies. 18-24 h after the last challenge bronchoalveolar lavage was performed by flushing out the lung with 0.5 ml PBS via a tracheal cannula. Lung homogenate was obtained by meshing diced lung fragments through a 40μm cell strainer and the cell free supernatant was stored at −20°C for cytokine analysis. Isolated cells were further purified using a 37.5% percoll gradient. Cells were stained for surface antigens or stimulated with PdBU/ionomycin for 2.5 h if intracellular cytokine staining was performed.
4 h after the last papain or PBS challenge Rag1−/− mice were intranasally treated with 500 ng recombinant mouse IL-2 (R&D systems) in 35μl PBS. Control animals received PBS only.
Wt B6 mice were intranasally challenged with 500ng recombinant mouse IL-25 (eBioscience) or recombinant mouse IL-33 (Biolegend) on day 0, 1 and 3. Control animals received PBS only. Mice were euthanized on day 4 and lung cells isolated for analysis.
Papain challenged mice were injected i.v. with 25 μg of neutralising IL-9-specifc antibody (MM9C1) 30 minutes before the last papain re-challenge. IgG isotype control antibodies were delivered to control animals. Three days after treatment with IL-9-specific antibodies, mice were culled, lung cells isolated and cell free lung homogenate analysed for cytokine expression.
Lung cells isolated from papain challenged mice were purified by negative sorting on an AutoMACS Pro (Miltenyi) with a combination of monoclonal antibodies against CD4, CD8α CD11b, CD11c, CD49b, TCR-β, TCR-γδ, CD19, Ter119, Nk1.1 and Gr-1 coupled to APC followed by incubation with APC-specific magnetic microbeads (Miltenyi). MACS purified ILC or Lin+ were cultured in 96-well plates at the adjusted density of 5×103 cells in 30 μl culture medium overnight in the presence of PdBU and ionomycin (both at 500 ng/ml). In some experiments cells were cultured in the presence of LPS (100 ng/ml), CpG (10 μg/ml), PolyI:C (2 μg/ml), Pam3CSK4 (10 μg/ml) or R848 (10 μg/ml). Cells were also stimulated with IL-2 (40 ng/ml), IL-25 (50 ng/ml), IL-33 (50 ng/ml) or TSLP (50 ng/ml). Cytokine production in the cell free supernatant was measured by using a bead based cytokine detection assay (FlowCytomix (Bender Medsystems) or CBA (BD Biosciences) for IL-9).
CD4+ cells were sorted by flow cytometry from total lung cells isolated from papain challenged mice based on the expression of the CD4 surface marker. CD25+/CD25− ILC were sorted on the absence of CD4, CD8α, CD11b, CD11c, DX5, CD49b, TCR-β, TCR-γδ, CD19, Ter119, NK1.1 and Gr-1 but expression of Thy1.2 and absence or presence of CD25 respectively. EYFP+ ILC were sorted from total lungs of papain challenged IL9CreR26ReYFP mice on the expression of Thy1.2 and eYFP but absence of CD4. CD4+, CD25+/CD25− ILC and eYFP+ ILC were cultured in the presence of IL-2, IL-25, IL-33 and TSLP overnight at the adjusted density of 5×103 in 30μl culture volume.
ILC and CD4+ T cells were sorted from lungs of papain challenged mice. 1×105 ILC or CD4+ T cells, respectively were intratracheally transferred to Rag2−/−IL2rg−/− mice. 30 min later, mice were intranasally challenged with 25 μg of papain followed by 500 ng recombinant mouse IL-2 2-3 hours post papain challenge. 15 h after papain challenge mice were sacrificed and lung cells stained for surface antigens and stimulated with PdBU/ionomycin for 2.5 h for intracellular cytokine staining. Lung homogenate was analysed for cytokine production.
CD4+, CD25+/CD25− ILC and eYFP+ ILC were sorted by flow cytometry and RNA was extracted using Trizol and reverse transcribed with Omniscript (Qiagen) according to the manufacturer’s protocol. The cDNA served as template for the amplification of genes of interest and the housekeeping gene (Hprt1) by real-time PCR, using TaqMan Gene Expression Assays (Applied Biosystems, Foster City, CA), universal PCR Master Mix (Applied Biosystems, Warrington, UK) and the ABI-PRISM 7900 Sequence detection system (Applied Biosystems, Foster City, CA). Target gene expression was calculated using the comparative method for relative quantification upon normalisation to Hprt gene expression.
A two-tailed Student’s t-test was used for all statistical analysis. * p≤ 0.05, **p ≤ 0.01, *** p≤0.001.
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