Cysteinyl leukotriene receptor 2 drives lung immunopathology through a platelet and high mobility box 1-dependent mechanism

Abstract

Cysteinyl leukotrienes (cysLTs) facilitate eosinophilic mucosal type 2 immunopathology, especially in aspirin-exacerbated respiratory disease (AERD), by incompletely understood mechanisms. We now demonstrate that platelets, activated through the type 2 cysLT receptor (CysLT₂R), cause IL-33-dependent immunopathology through a rapidly inducible mechanism requiring the actions of high mobility box 1 (HMGB1) and the receptor for advanced glycation end products (RAGE). Leukotriene C₄ (LTC₄) induces surface HMGB1 expression by mouse platelets in a CysLT₂R-dependent manner. Blockade of RAGE and neutralization of HMGB1 prevent LTC₄-induced platelet activation. Challenges of AERD-like Ptges^-/- mice with inhaled lysine aspirin (Lys-ASA) elicit LTC₄ synthesis and cause rapid intrapulmonary recruitment of platelets with adherent granulocytes, along with platelet- and CysLT₂R-mediated increases in lung IL-33, IL-5, IL-13, and bronchoalveolar lavage fluid HMGB1. The intrapulmonary administration of exogenous LTC₄ mimics these effects. Platelet depletion, HMGB1 neutralization, and pharmacologic blockade of RAGE eliminate all manifestations of Lys-ASA challenges, including increase in IL-33, mast cell activation, and changes in airway resistance. Thus, CysLT₂R signaling on platelets prominently utilizes RAGE/HMGB1 as a link to downstream type 2 respiratory immunopathology and IL-33-dependent mast cell activation typical of AERD. Antagonists of HMGB1 or RAGE may be useful to treat AERD and other disorders associated with type 2 immunopathology.

Figure 1.. Induction of HMGB1 surface expression by platelets in response to exogenous and endogenous LTC₄.

Platelet rich plasma (PRP) was obtained from the indicated mouse strains and stimulated with various agonists. The CD41+ platelet gate was analyzed by flow cytometry with Abs specific for HMGB1 (left panels) or CD62P (right panels) along with corresponding isotype controls. A. Time dependent inductions of surface HMGB1 and CD62P by LTC₄, LTD₄, and LTE₄ at the indicated concentrations. B. Comparison of LTC₄ versus thrombin stimulation for surface expressions of HMGB1 and CD62P. C. Effect of Cysltr2 deletion on LTC₄-induced HMGB1 and CD62P expression by platelets. D. Induction of HMGB1 and CD62P expression by platelet-derived LTC₄ converted from exogenous LTA₄. Results in A-D are mean ± SEM from three independent experiments that included a minimum of 3 mice per group.

Figure 2.. Effects of RAGE or TLR4 blockade on LTC₄-induced platelet activation.

PRP was obtained from the indicated mouse strains and stimulated with LTC₄ at the indicated concentrations or with thrombin (0.5 U/ml). Some samples were treated with the selective RAGE antagonist FPS-ZM1, and others with the TLR4 antagonist LPS-RS at the indicated doses prior to stimulation. A. Effects of RAGE blockade on LTC₄induced surface expression of HMGB1 (left) and CD62P (right) by CD41+ platelets stimulated for 30 min. B. Effects of RAGE and TLR4 blockade on expression of HMGB1 (left) and CD62P (right) by platelets in PRP stimulated for 30 min by LTC₄ or thrombin. C. Concentrations of CXCL7 (left) and TXB₂ (right) detected by ELISA in the supernatants from PRP stimulated as in B. D. Effects of RAGE and TLR4 antagonists on surface expression of HMGB1 (left) and CD62P (right) by platelets in PRP stimulated by LTA₄ (to elicit conversion by platelet LTC₄S) or LTC₄ at the indicated doses. Results in A-D are mean ± SEM from three independent experiments that included a minimum of 3 mice per group.

Figure 3.. Effects of RAGE blockade, HMGB1 neutralization, and CysLT₂R antagonism on physiologic response of AERD-like Ptges−/− mice to Lys-ASA inhalation challenge.

Df-primed Ptges−/− mice were treated with the indicated Abs, antagonists, or corresponding isotype and vehicle controls. Twenty-four hours later, mice were anaesthetized, sedated, mechanically ventilated and challenged by aerosoled Lys-ASA or PBS control. A. Maximum change in R_L for the indicate groups of mice monitored for 45 min after Lys-ASA challenge. B. Levels of HMGB1 collected in BAL fluids from the same mice as in A. C. Levels of cysLTs in the BAL fluids. D. Levels of CXCL7 in BAL fluids. E. Levels of MMCP-1, F. histamine and G. PGD₂ in the BAL fluids from the same mice as in A-D. Results are mean ± SEM from two independent experiments using a total of 10 mice in each group.

Figure 4.. Recruited platelets account for the Lys-ASA-induced increment in HMGB1.

Lungs were collected from PBS- or Lys-ASA-challenged Ptges^−/− mice treated with a platelet-depleting anti-CD41 Ab or isotype control. A. Lung sections were stained with anti-CD41 mAb or an isotype control. Infiltrating platelets are identified by the red staining. B. Western blotting of lung lysates from Ptges^−/− mice of the indicated treatment groups for CD61 as a surrogate marker for recuited platelets. A blot from a representative individual experiment (top) and quantitative densitometry from two separate experiments (bottom) are shown. C. BAL fluid levels of HMGB1 from mice challenged with Lys-ASA or PBS after treatment with anti-CD41 or isotype. Results in A are from single mice representative of at least 5/group in three different experiments. Results in C are mean ± SEM from at least 10 mice/group from two separate experiments.

Figure 5.. Rapid platelet-dependent increase in lung IL-33 induced by Lys-ASA challenge.

Df-primed Ptges−/− mice were treated with the indicated Abs, antagonists, or corresponding isotype and vehicle controls. A. Levels of IL-33 (left), IL-5 (middle) and IL-13 (right) proteins detected in homogenates of lungs from the indicated groups of Ptges^−/− mice with and without platelet depletion collected 45 min after PBS- or Lys-ASA challenge. B. Effects of treatment with the indicated Abs and antagonists on Lys-ASAinduced increases in IL-33, IL-5, and IL-13 detected in lysates of lungs from Ptges^−/− mice. C. Western blotting for IL-33 protein in supernatants and pellets of washed platelets from naïve WT mice subjected to the indicated stimuli for 1 h. Results in A and B are mean ± SEM from at least 10 mice/group from two separate experiments. Results in C are representative of three different experiments. Results from experiments using Ptges^−/− platelets yielded similar results.

Figure 6.. Exogenously administered LTC₄ induces a rapid platelet-dependent increase in lung IL-33 and type 2 cytokine production.

OVA-sensitized WT mice received three aerosol challenges with 0.1% OVA. Some mice received intranasal administration of LTC₄ (2.2 nmol) preceding each OVA challenge by 12 h. The indicated mice were then treated with a platelet-depleting anti-CD41 Ab or isotype one day before receiving either an additional intranasal dose of LTC₄ or PBS control 1 h before euthanasia. A. BAL fluid total cells (left) and eosinophils (right). B. BAL fluid levels of HMGB1 (top row), CXCL7 and TXB₂ (middle row) from the same mice as in A. C. Levels of IL-33 protein detected in homogenates from the lungs of mice in the indicated groups. D. Levels of IL-5 and IL-13 from the same homogenates as in C. E. Numbers of ILC2s detected in single cell suspensions from the same mice. F. Western blots of whole lung lysates showing changes in CD61 as a surrogate for platelet recruitment. Results in A-E are from 10 mice per group from 2 independent experiments. Results in F are from a representative blot (left), with quantitative densitometry (right) from 5 mice/group.

Figure 7.

Schematic showing putative initiating (CysLT₂R-dependent) and downstream (CysLT₁R and CysLT₃R) dependent effects of cysLTs in driving the mechanism responsible for reactions to aspirin. Platelet CysLT2R is essential to initiate HMGB1/RAGE signaling that permits platelet activation and conjugation to granulocytes. Platelets adherent to granulocytes and activated via CysLT₂R/HMGB1/RAGE provide a pool of IL-33 that drives MC activation and a secondary surge of cysLTs to act at cognate receptors, and also activates ILC2s to promote additional type 2 immunopathology.