Bars show the mean??SEM (n?=?3). migration involved the activation of ERK1/2, p38, Akt, JNK and NF-B, while CXCL16-induced ILC2 migration involved the activation of ERK1/2, p38 and Akt. These data support the hypothesis that epithelium-derived IL-25 and IL-33 induce lung accumulation of ILC2s, while IL-33 exerts a direct chemotactic effect in this process. Although ILC2s express the chemokine receptors CXCR6 and CCR9, only CXCL16, the ligand of CXCR6, exhibits a direct chemoattractant effect. Key Daunorubicin words: ILC2s, Asthma, Accumulation, IL-33, IL-25, Chemokine Subject terms: Chemokines, Daunorubicin Innate lymphoid cells, Interleukins Introduction Currently, three groups (1-3) of innate lymphoid cells (ILCs) have been delineated based on their transcription factor requirements, cytokine production profiles and roles in immunity.1C3 Among these subsets of ILCs, some have the potential to play a prominent role in parasitic immunity and allergy.1C3 In particular, ILC2s express the transcription factor GATA-3 and produce copious amounts of the type 2 cytokines IL-5, IL-9 and IL-13 as well IL-6, IL-10, GM-CSF and small quantities of IL-4,4,5 which likely participate in mucosal defense against parasites but also potentially contribute to airway allergic inflammation. ILC2s develop from common lymphoid progenitors (CLPs) and lack classic hematopoietic lineage markers and are thus defined as lineage-negative. These cells express Thy-1 (CD90), T-cell co-stimulator (ICOS), Sca-1, IL-7R, CD25, CD117, IL-25 receptor (IL17BR), and IL-33 receptor (T1/ST2), leading to the frequent use of these markers to identify and isolate ILC2s.4C6 Since the discovery of ILCs, their presence has been reported in the murine and human respiratory tracts.7C9 In particular, ILC2s have been reported to accumulate in the lung tissue and airway mucosa and contribute to type 2 inflammatory responses in mice following Rabbit polyclonal to ZNF268 infection with influenza virus and challenge with multiple allergens, including imaging at the 30-h time point. *p?<?0.05 (n?=?3 for each group) Effects of IL-33 or CXCL16 on the activation of the ERK, p38, JNK, Akt, and NF-B pathways in ILC2s IL-33 binds to ST2 and initiates the recruitment of Mal or MyD88 signaling adaptor molecules, leading to the activation of downstream pathways (including NF-B and MAPKs).23,24 In murine peritoneal macrophages, human aortic smooth muscle cells and human umbilical vein endothelial cells, CXCL16 activated ERK1/2, p38, and PI3K/Akt, respectively.21,25,26 To determine whether IL-33 or CXCL16 can activate these signaling pathways in ILC2s, we treated ILC2s with IL-33 (60?ng/ml) or CXCL16 (10?ng/ml) (the optimal concentrations Daunorubicin of IL-33 and CXCL16 in vitro in the transwell assay) for 10?min. Flow cytometric analysis revealed that IL-33 markedly increased the phosphorylation of ERK1/2, p38, Akt, and NF-B and, to a lesser extent, that of JNK. CXCL16 induced marked phosphorylation of ERK1/2 and p38 and, to a lesser extent, that of Akt (Fig.?6a). Furthermore, we observed that the specific inhibitors U0126-EtOH, SB203580, SP600125, LY294002, and BAY 11-7082 markedly reduced the phosphorylation of their specific targets in the ILC2s (Fig.?6a). Open in a separate window Fig. 6 a Flow cytometric analysis showing IL-33-induced Daunorubicin and CXCL16-induced phosphorylation of Erk1/2, p38, JNK, Akt, and NF-B. Red lines: IL-33 or CXCL16 stimulation; black lines: PBS stimulation; blue lines: IL-33 or CXCL16 stimulation in the presence of inhibitors of Erk1/2 (U0126-EtOH), p38 (SB203580), JNK (SP600125), Akt (LY294002), and NF-B (BAY 11-7082). b Inhibitors of Erk1/2 (U0126-EtOH), p38 (SB203580), JNK (SP600125), Akt (LY294002), and NF-B (BAY 11-7082) significantly blocked the migration of ILC2s in response to IL-33 and CXCL16 alone. *p?<?0.05 compared with positive control To further analyze the relevance of these signaling pathways for ILC2 migration, migration assays were performed with IL-33 or CXCL16 in the presence of various signaling pathway inhibitors. As shown in Fig.?6b, IL-33-induced ILC2 migration was significantly blocked by U0126-EtOH, SB203580, SP600125, LY294002, and BAY 11-7082, suggesting the involvement of both the MAPK and NF-B pathways in ILC2 chemotaxis toward IL-33 in vitro. CXCL16-induced ILC2 migration was significantly blocked by U0126-EtOH, SB203580 and LY294002, suggesting the involvement of ERK, p38 and Akt signaling in CXCL16-induced ILC2 migration. Pulmonary ILC2s in CXCL16-challenged and CCL25-challenged mice Having shown that IL-33 and CXCL16, but not IL-25 or CCL25, applied to the airway mucosa can attract ILC2s from the peripheral circulation to the lung parenchyma, we next investigated whether these mediators applied locally to the airway mucosa were able to influence the infiltration of ILC2s into the airway mucosa and lumen in vivo..