Lipoxin A4 regulates natural killer cell and type 2 innate lymphoid cell activation in asthma

Abstract

Asthma is a prevalent disease of chronic inflammation in which endogenous counterregulatory signaling pathways are dysregulated. Recent evidence suggests that innate lymphoid cells (ILCs), including natural killer (NK) cells and type 2 ILCs (ILC2s), can participate in the regulation of allergic airway responses, in particular airway mucosal inflammation. We have identified both NK cells and ILC2s in human lung and peripheral blood in healthy and asthmatic subjects. NK cells were highly activated in severe asthma, were linked to eosinophilia, and interacted with autologous eosinophils to promote their apoptosis. ILC2s generated antigen-independent interleukin-13 (IL-13) in response to the mast cell product prostaglandin D2 alone and in a synergistic manner with the airway epithelial cytokines IL-25 and IL-33. Both NK cells and ILC2s expressed the pro-resolving ALX/FPR2 receptors. Lipoxin A4, a natural pro-resolving ligand for ALX/FPR2 receptors, significantly increased NK cell-mediated eosinophil apoptosis and decreased IL-13 release by ILC2s. Together, these findings indicate that ILCs are targets for lipoxin A4 to decrease airway inflammation and mediate the catabasis of eosinophilic inflammation. Because lipoxin A4 generation is decreased in severe asthma, these findings also implicate unrestrained ILC activation in asthma pathobiology.

Fig. 1. NK cells are activated in asthma

Peripheral venous blood was obtained from healthy subjects and individuals with mild and severe asthma. (A) NK cells were identified as a NKp46⁺CD3⁻ cell population in a lymphocyte morphology gate based on SSC and FSC characteristics (violet box, left panel) and NK cells as a percentage of total lymphocytes were enumerated (right panel), bar indicates mean value. (B) NK cells were further phenotyped by flow cytometry analysis of CD56 expression for CD56^bright and CD56^dim subsets (violet boxes, left panel) in samples from healthy and asthmatic subjects (right panel). (C) Paired samples of BALF and blood were obtained on a subset of severe asthmatics. Representative FACS plots show an enriched NKp46^bright cell population in BALF compared to PBMCs (left panel). BALFs were also enriched for CD56^bright subset of NK cells (n=5) (right panel). (D) Representative FACS histograms showing CD69 expression in peripheral blood NK cells (left panel) that was upregulated in severe asthma (middle panel), in particular in the CD56^dim subset (right panel). (E) Representative FACS histograms showing NKG2D expression in peripheral NK cells (left panel) that was upregulated in severe asthma (right panel). Values are expressed as mean ± s.e.m.; *, P < 0.05, compared with healthy subjects (one-way ANOVA); §, P < 0.0001, compared with PBMCs (one-way ANOVA); **, P < 0.05, compared with healthy and mild asthmatic subjects (one-way ANOVA).

Fig. 2. NK cells interact with eosinophils

(A–C) The percentage of peripheral blood eosinophils in severe asthma was correlated to (A) the percentage of peripheral blood NK cells; Spearman r=0.86, P < 0.05. (B) NK cell CD69 expression; Pearson r=0.50, P < 0.05, and (C) NK cell NKG2D expression, Spearman r=0.59, P < 0.01. (D–F) Incubation (4h, 37°C) of autologous peripheral blood granulocytes with NK cells (1:5 ratio) triggered increased granulocyte apoptosis compared to granulocytes incubated alone by (D) morphological appearance on cytospin (arrows, apoptotic granulocytes) and (E,F) by FACS criteria (Annexin V⁺7-AAD⁻) for (E) eosinophils (Eos) and (F) neutrophils (PMN) in samples of peripheral blood from healthy subjects and individuals with mild or severe asthma. Results are expressed as mean ± SEM.; n=5 individual healthy donors, n=5 mild asthmatic donors, n=5 severe asthmatic donors; *, P < 0.05, compared with granulocytes alone and **, P < 0.05 compared with healthy donors (Student’s t-test).

Fig. 3. NK cells express pro-resolving receptors and respond to LXA₄

(A) Flow cytometry histograms for anti-ALX (FPRL1) Ab (white) and isotype control (gray) for peripheral blood NK cells (left panel) and MFI for ALX expression on peripheral NK cells in healthy subjects and mild and severe asthmatic subjects (right panel). (B) Flow cytometry histograms for anti-CMKLR1 Ab (white) and isotype control (gray) for peripheral blood NK cells (left panel) and MFI for CMKLR1 expression on peripheral NK cells in healthy subjects and mild and severe asthmatic subjects (right panel). *, P < 0.05, compared with healthy and mild asthmatic subjects (one-way ANOVA). (C, D) Autologous granulocytes and NK cells from healthy subjects were selectively exposed to lipoxin A₄ (LX, 100 nM, 15 min, 37°C) in the absence or presence of the ALX/FPR2 antagonist (WRW4, 230nM) prior to co-incubation and the percent change in apoptosis was determined for (C) eosinophils (Eos) and (D) neutrophils (PMN) (see Methods). Results are expressed as mean ± SEM; n≥4; **, P < 0.05, compared with control (one-way ANOVA); §, P < 0.05, compared with NK(LX).

Fig. 4. ILC2 are present in peripheral blood of healthy and asthmatic subjects and express pro-resolving receptors

(A–D) Flow cytometry gating strategy for the identification of helper ILC and the ILC2 subset. PBMCs were depleted of most T and B lymphocytes and monocytes, and a lymphoid morphology cell population was selected based on their forward and side scatter characteristics. (A) Flow cytometry analysis showing the presence of a Lin⁻ cell population (violet box, CD3⁻TCRαβ⁻TCRγδ⁻CD19⁻CD14⁻CD16⁻CD34⁻CD123⁻CD11c⁻) different from NK cells (Lin⁻CD56⁺), left panel; identification of a Lin⁻CD127⁺CD117^+/− population, right panel. (B) Lin⁻CD127⁺CD117^+/− expressed NK cell receptors, including CD161, left panel; NKG2D, right panel, and (C) NKp46. (D) ILC2 were identified as a subpopulation of Lin⁻CD127⁺ cells that also expressed CRTH2. (E) The number of Lin⁻CD127⁺CRTH2⁺ ILC2 cells was determined in healthy and mild and severe asthmatic subjects. (F) ILC2 also expressed the pro-resolving receptors ALX (left panel) and CMKLR1 (right panel) on their surface (see Methods).

Fig. 5. IL-13 production by peripheral blood ILC2 is enhanced by PGD₂and inhibited by lipoxin A₄

IL-13 was quantitated by ELISA in the cell-free supernatants from incubations with (A) Lin⁻CD127⁺ cells, CD56^bright NK cells and CD56^dim NK cells that were sorted by flow cytometry and cultured for 24h alone (Control) or with PMA (50 ng/ml) and A23187 (500 ng/ml). Results are expressed per 1,000 cells. (B) In separate incubations, IL-13 release was determined for Lin⁻CD127⁺ cells that were incubated (15 min, 37°C) with lipoxin A₄ (100 nM) or vehicle (0.1% ethanol) prior to (24h, 37°C) media alone, IL-2 (2 ng/ml) (IL-2), IL-2 (2 ng/ml), IL-25 (50 ng/ml) and IL-33 (50 ng/ml) (IL-2/IL-25/IL-33), IL-2 (2 ng/ml) and PGD₂ (100 nM) (IL2/PGD₂), or IL-2 (2 ng/ml), IL-25 (50 ng/ml), IL-33 (50 ng/ml) and PGD₂ (100 nM) (IL-2/IL-25/IL-33/PGD₂). (C) To assess PGD₂ receptor signaling, ILCs were incubated with IL-2/IL-25/IL-33/PGD₂ in the presence of a DP1 receptor antagonist (BWA868C, aDP1) or DP2 receptor antagonist (BAYu3405, aDP2). Some cells were exposed to IL-2/IL-25/IL-33 in conjunction with a selective DP1 receptor agonist (BW245C, DP1) or DP2 receptor agonist (15(R)-methyl-PGD₂, DP2) without PGD₂. (D) To determine the role of ALX/FPR2 signaling, cells were exposed (15 min, 37°C) to the receptor antagonist WRW4 (aALX/FPR2) prior to LXA₄ and IL-2/IL-25/IL-33/PGD_2. Results are expressed as the percent increase from control (mean ± s.e.m.); n≥3 individual healthy donors; *, P < 0.01, compared with control; **, P < 0.05, compared with IL-2; §, P < 0.05, compared with IL-2/IL-25/IL-33, §§, P < 0.05, compared with IL-2/IL-25/IL-33/PGD₂; ***, P < 0.05, compared with vehicle.

Fig. 6. Innate lymphoid cells are present in human lung

Immune cells were assessed in the distal lung using immunofluorescent staining with antibodies for cKit, CD161, and perforin to identify cells with the staining characteristics of ILC (cKit⁺, CD161⁺) and NK cells (cKit⁻, CD161⁺, perforin⁺). Cell nuclei were stained with DAPI (blue). Panels on the left show cells that stain positively for ckit (green, Panel A) and CD161 (red, Panel B). When images A and B are merged, cells stain for ckit and CD161 (yellow, Panel C) or ckit alone (green, Panel C). Light microscopy images taken with 20X objective, and 100X objective for inset. Panels on the right show cells that that stain positively for ckit (green, Panel D) or Perforin (white, Panel D). Panel E depicts cells that are positive for Perforin (white), CD161 (red), or both Perforin and CD161 (white and red). When images D and E are merged, cells are present in Panel F that stains for Perforin and CD161 (white and red), Perforin alone (white), ckit alone (green), or ckit and CD161 (yellow). Confocal images taken with 60X objective. Inset images provide higher power views of the cells, and correspond numerically with the lower power images. Br, bronchioles