Differential dysregulation of granule subsets in WASH-deficient neutrophil leukocytes resulting in inflammation

Dysregulated secretion in neutrophil leukocytes associates with human inflammatory disease. The exocytosis response to triggering stimuli is sequential; gelatinase granules modulate the initiation of the innate immune response, followed by the release of pro-inflammatory azurophilic granules, requiring stronger stimulation. Exocytosis requires actin depolymerization which is actively counteracted under non-stimulatory conditions. Here we show that the actin nucleator, WASH, is necessary to maintain azurophilic granules in their refractory state by granule actin entrapment and interference with the Rab27a-JFC1 exocytic machinery. On the contrary, gelatinase granules of WASH-deficient neutrophil leukocytes are characterized by decreased Rac1, shortened granule-associated actin comets and impaired exocytosis. Rac1 activation restores exocytosis of these granules. In vivo, WASH deficiency induces exacerbated azurophilic granule exocytosis, inflammation, and decreased survival. WASH deficiency thus differentially impacts neutrophil granule subtypes, impairing exocytosis of granules that mediate the initiation of the neutrophil innate response while exacerbating pro-inflammatory granule secretion.

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Data analysis
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Life sciences study design
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Sample size
Sample size was determined by power analysis. For in vivo studies, by power analyses, we predicted that 15 mice/group will allow for an incidence decrease of 50% in the protected groups to be statistically significant, where the α and desired statistical power levels are 0.05 and 0.88, respectively. Based on our previous studies of neutrophil function using similar mouse models we determined the sample size for colocalization, secretion and ROS assays. For example, as described in references: The atypical small GTPase GEM/Kir is a negative regulator of the NADPH oxidase and NETs production through macroautophagy. Johnson  Data exclusions All data were included. For statistical outliers we used Grubb's test (alpha=0.05). If present, outliers were included and indicated in the figures. None of the animals were excluded from the analysis.

Replication
Experiments were performed utilizing distinct samples (3 or more independent mice) and experiments were repeated accordingly. For experiments other than those involving mice, i.e. secretion studies using human neutrophils, TR-FRET studies and pull-down assays, all attempts were successful. Reproducibility was determined using appropriate statistical methods which are described in detail for each of these methods, in the manuscript. A p value <0.05 was considered statistically significant.
Randomization Most experiments in this manuscript compare cells from wild type and KO mice. All samples were processed and analyzed simultaneously. All sample are split between vehicle or drug/stimuli groups. For in vivo studies , animals in each litter are randomly assigned to the drug or placebo group. For secretion analysis using human neutrophils, the sample was split in two and neutrophils from the same donor were either treated with experimental or control conditions/stimuli, so covarieates are not relevant in these experiments. For other studies not involving mice, for example TR-FRET assays and pull-down assays, the same cell line was split and treated either under control or experimental conditions.

Blinding
For hematological, cytokine and some MPO ELISA studies, the investigators were blinded to group allocation. Because Wash-cKo mice are neutropenic and this is very evident during cell isolation, experiments comparing WT and Wash-cKO isolated neutrophils were not blinded to group allocation. For other experiments, including pull-down assays and TR-FRET the experimentalist was not blinded to group allocation, however, the experiments were repeated independently by different investigators with the same outcome.

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Laboratory animals
The Washc1flox/flox /Vav-cre+ mouse conditional knockout model, lacking WASH expression in the haemopoietic linage Washc1haemo, hereon Wash-cKO) was generated as previously described58. Washfl/fl/Mrp8-Cre+ mice (hereon Wash-deltaPMN) were generated by crossing the Washfl/fl mice with the Mrp8-Cre+ (Tg-S100A8-cre,ires-EGFP)1Ilw mice which expresses Cre recombinase under the promoter of the neutrophil specific cargo S100A8 (Mrp8), and directs bicistronic Cre and EGFP protein expression to CD11b+, Ly6G+, granulocytes (The Jackson Laboratory). The characterization of this model was performed by genotyping using Transnetyx technology for the detection of both the the presence of the deleted allele and the Cre allele, PCR for confirmation, flow cytometry and immunoblotting analysis of WASH expression in Washfl/fl/Mrp8-Cre+ mice (Supplemental Fig. 14ac). Experimental males and females, 6 to 10-week old mice and sex and age-matched control mice were used in this study.

Wild animals
No wild animals were used in the study.
Field-collected samples No field collected samples were used in the study.

Ethics oversight
All animal studies were performed in compliance with the Department of Health and Human Services Guide for the Care and Use of Laboratory Animals. All studies were conducted according to National Institutes of Health and institutional guidelines and with approval of the animal review boards at The Scripps Research Institute.
Note that full information on the approval of the study protocol must also be provided in the manuscript.

Human research participants
Policy information about studies involving human research participants

Population characteristics
The experiments interrogate mechanisms using human neutrophils isolated from peripheral blood. This was the only involvement of human subjects in the proposed research. Donors for phlebotomy were selected by the staff of the Normal Blood Donor Services (NBDS) at The Scripps Research Institute. Representative racial and gender composition was drawn from the normal donor pool of the NBDS. The Principal Investigator has no contact with the blood donor and receives number-coded vials. Samples from adult volunteers (18 to 55-year old) of both sexes and any race were requested and used for these studies. The NBDS blood pool is derived from the employees of The Scripps Research Institute and includes the following individuals: 70% female, 30% male, 8% Hispanic, 5% Asians, 3% Afro-American, 0% Native Americans. This project seeks to answer basic cellular and molecular questions concerning neutrophil exocytosis. Therefore the issue of women, children and minority subjects is not germane to this basic research project. Consent was sought by NBDS staff or (for pheresis) staff at the San Diego Blood Bank.

Recruitment
Recruitment was performed by by the staff of the Normal Blood Donor Services (NBDS) at The Scripps Research Institute. Representative racial and gender composition was drawn from the normal donor pool of the NBDS. The Principal Investigator has no contact with the blood donor and receives number-coded vials. Volunteers of both sexes and any race were requested for these studies.
The Normal Blood Donor Services welcomes all healthy individuals in the community who are readily available during blood drawing hours of 6:00am -11:00am. Individuals volunteers either find the on-line call by themselves or respond to notices and fliers which are regularly sent out to recruit donors from the community. The recruitment of donors is not limited to Scripps Research employees, but to others in the community. All eligible donors are initially screened for infectious diseases prior to being enrolled in the program, and on a yearly basis after the initial screen to remain active in the program. The pool of donors is comprehensive and therefore there are no biases identified in this pool.
The Annual Donor Screen consists of the following:

Ethics oversight
Human neutrophils were isolated from normal donor's blood by Ficoll density centrifugation as previously described (Ref. 59). All procedures regarding human subjects have been reviewed and approved by the Human Subjects Committee at The Scripps Research Institute and were conducted in accordance with the requirements set forth by the mentioned Human Subjects Committee and in accordance to NIH guidelines. Informed consent was provided by the donors.
Note that full information on the approval of the study protocol must also be provided in the manuscript.

Flow Cytometry
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