4C)

4C). This phenomenon was associated with elevated CX3CL1 levels in the airways of IP?/? mice and treatment with a neutralizing antibody to CX3CL1 reduced IFN- production by the lung NK cells. Remarkably, IP?/? mice were less responsive to HDM challenge than WT counterparts since intranasal instillation of RO3280 the allergen induced markedly reduced levels of airway eosinophils, CD4+ lymphocyte infiltration and mucus production, as well as depressed levels of CCL2 chemokine and Th2 cytokines. NK cells were responsible for such attenuated responses since depletion of NK1.1+ cells in IP?/? mice restored both the HDM-induced lung inflammation and ILC2 numbers, while transfer of CD3?NK1.1+ NK cells into the airways of WT hosts suppressed the inflammatory response. Collectively, these data demonstrate a hitherto unknown role for PGI2 in regulating the number and properties of NK cells resident in lung tissue and reveal a role for NK cells in limiting lung tissue ILC2s and preventing allergic inflammatory responses to inhaled HDM allergen. effects of NK cells on allergic lung inflammation, NK1.1+ cells were depleted using anti-NK1.1 antibody. The anti-NK1.1 MoAb (PK136 obtained from the American Type Culture Collection (ATCC), Manassas, VA) was purified by protein-A affinity chromatography (28). Briefly, C57BL/6 WT and IP?/? mice were injected i.p with 250 g anti-NK1.1 antibody or control IgG Bglap (mouse IgG2a) 24h prior to the start of HDM challenge and then twice weekly for a period of 2 weeks (on days -1, 3, 6, 10 and 13). Mice were challenged with PBS (control) or HDM allergen on days 0, 7 and 14 with the allergic inflammatory response characterized on day 16. In certain experiments, na?ve IP?/? or WT mice were similarly treated with the anti-NK1.1 or control mAb, twice weekly over a period of 2 weeks. After treatment the number of NK cells remaining in lungs and spleen was determined by enumerating CD3?NK1.1+NKp46+ cells. Purification of splenic NK cells and transfer to the airways NK cells were purified by magnetic cell sorting (MagCellect, R&D Systems) of spleen cells prepared from WT or IP?/? mice. Sorted cells were selected on the basis of being CD3?NK1.1+ and purity was checked by measuring the proportion of CD3?NK1.1+ cells (87C92% over three experiments). Purified splenic NK cells (5105) were suspended in RPMI (lacking FBS) and instilled directly into the airways of WT mice by the oropharyngeal route in a RO3280 30l volume 24h after the start of HDM allergen sensitization period as detailed above. Purified NK cells were pretreated with 10ng/ml of IL-2 prior to transfer into hosts to maintain cell viability and function. Sham control mice received an equivalent suspension media alone by the oropharyngeal route. Mice were subsequently challenged with 50g of HDM RO3280 allergen on days 7 and 14 and the airway inflammatory characterized on day 16. Examination of the eosinophilic infiltration into the airways was determined by cell differential counts and measuring cell-associated EPO activity. Flow Cytometry Cells (LMC, BALF or splenic cells) were FcR blocked using 2.4G2 antibody (ATCC) and stained with combinations of the following mouse conjugated mAb (all purchased from BioLegend): allophycocyanin (APC) or FITC anti-CD3, APC/Cy7 anti-CD4, PE anti-CD8a, APC-Cy7 anti-CD19, APC or PE anti-CD49b DX5 (pan-NK cells) or Ly49a, FITC or APC anti-NK1.1 (PK136), PE or APC anti-CD335 (NKp46), PE or APC CD27, PE anti-CD94 (NKG2), FITC or PE anti-NKG2D (CD314), APC or PE anti-CD11c, PE or APC/Cy7 anti-I-A/I-E, APC/Cy7 anti-Ly6G, APC or APC/Cy7 anti-Ly6C, APC/Cy7 anti-Ly-6G/Ly6C (Gr1), PE, FITC or Brilliant Violet 421 anti-CD11b, APC or PE anti-F4/80. In addition, PE anti-Siglec-F (BD Biosciences, to stain RO3280 eosinophils), Alexa Fluor? 647 anti-Dectin-2 (AbD Serotec), and PE or APC anti-CX3CR1 (R&D Systems) mAb were used. For intracellular staining, FITC anti-granzyme B, APC anti-granzyme A, and APC anti-IFN-, (all antibodies from BioLegend) were utilized and cells stained intracellularly as previously (29). Flow cytometric acquisition was performed on a FACSAria II (BD Biosciences, San Jose, CA) by 4-color analysis using FACSDiVa software and FlowJo, and a minimum of 50,000 live, single-cell events collected. Lung ILC2s were lineage negative (Lin?) cells that stained with Thy1.2/CD90.2 (AlexaFluor 405), CD45 (CLA) (APC/Cy7), and IL-33R/ST2 (APC) mAb (all from.