Regulation of neutrophil secretion by the lysosomal amino acid transporter, cystinosin

Abstract

Neutrophil exocytosis is an important immune response, but secretion must be tightly regulated because exacerbated release of the neutrophil toxic mediators is injurious to the host and mediates inflammation. Here we show that neutrophil exocytosis of azurophilic granules is regulated by the cystine lysosomal transporter cystinosin. Cystinosin localized at azurophilic granules and lysosomes in neutrophils. Isolated bone‐marrow‐derived Ctns−/− neutrophils showed exacerbated azurophilic granule secretion under unstimulated conditions, suggesting dysregulation of a cell‐intrinsic mechanism. The defect was specific for azurophilic granules because secretion of gelatinase granules was not affected. Using phalloidin staining, we show that Ctns−/− neutrophils have decreased F‐actin, suggesting disruption of the azurophilic granule‐specific actin barrier. In addition, phalloidin‐based staining showed increased filopodial protrusion formation in Ctns−/− neutrophils, thus affecting cell morphology. Induction of either actin polymerization or depolymerization by jasplakinolide (JK) or cytochalasin, respectively, followed by stimulation with the bacteria‐derived peptide, fMLF, showed that secretion was still significantly increased in Ctns−/− neutrophils, suggesting mechanistic defects beyond impaired actin remodeling. Using Total Internal Reflection Fluorescence Microscope (TIRFM) and Super‐Resolution Radial Fluctuations (SRRF), we show increased numbers of docked granules at the exocytic actin zone in Ctns−/− neutrophils. Immunofluorescence analysis showed increased colocalization of the small GTPase Rab27a with its effector JFC1 in Ctns−/− neutrophils, suggesting that Rab27a drives the increased exocytosis observed in Ctns−/− neutrophils. This was confirmed by treatment with the Rab27a‐JFC1 binding‐specific inhibitor Nexinhib20 (NEI20), which decreased granule docking and impaired secretion of the azurophilic granule marker myeloperoxidase (MPO) in unstimulated and stimulated Ctns−/− neutrophils. Combined treatment with NEI20 and JK further decreased MPO secretion in Ctns−/− neutrophils suggesting the uncoupling of Rab27a activation and actin depolymerization in Ctns−/− neutrophils. This was confirmed by the observation that the number of docking granules decreases in response to the combined treatment. Blocking ERM (Ezrin/Radixin/Moesin) also decreased the number of docked azurophilic granules at the exocytic active zone and impaired MPO secretion in Ctns−/− neutrophils, suggesting the participation of ERM on azurophilic granule exocytosis. In vivo, we found elevated levels of azurophilic granule cargo in plasma from cystinosin‐deficient mice, and Ctns−/− mice showed increased neutrophilic tissue infiltration, denoting systemic activation. Our data suggest that cystinosin is a negative regulator of exocytosis and that neutrophil‐mediated inflammation may play a significant role in disease progression in cystinosis.Support or Funding InformationCRFNIH