University of Birmingham Consequences of cathepsin C inactivation for membrane exposure of proteinase 3, the target antigen in autoimmune vasculitis

Membrane-bound proteinase 3 (PR3) is the main target antigen of anti-neutrophil cytoplasmic autoantibodies (ANCA) in granulomatosis with polyangiitis (GPA), a systemic small-vessel vasculitis. Binding of ANCA to PR3 triggers neutrophil activation with the secretion of enzymatically active PR3 and related neutrophil serine proteases, thereby contributing to vascular damage. PR3 and related proteases are activated from proforms by the lysosomal cysteine protease cathepsin C (CatC) during neutrophil maturation. We hypothesized that pharmacological inhibition of CatC provides an effective measure to reduce PR3 and has therefore implications as a novel therapeutic approach in GPA. We first studied PR3 in neutrophils from 21 patients with Papillon-Lefèvre syndrome (PLS), a genetic form of CatC deficiency. PLS neutrophil lysates showed a largely reduced, but still detectable (0.5-4%) PR3 activity when compared to healthy control cells. Despite extremely low levels of cellular PR3, the amount of constitutive PR3 expressed on the surface of quiescent neutrophils, and the typical bimodal membrane distribution pattern, was similar to what was observed in healthy neutrophils. However, following cell activation, there was no significant increase in the total amount of PR3 on PLS neutrophils, whereas the total amount of PR3 on healthy neutrophils was significantly increased. We then explored the effect of pharmacological CatC inhibition on PR3 expression in normal neutrophils using a potent cell permeable CatC inhibitor and a CD34 hematopoietic stem cell model. Human CD34 hematopoietic stem cells were treated with the inhibitor during neutrophil differentiation over 10 days. We observed strong reductions in PR3, cellular PR3 protein and proteolytic PR3 activity whereas neutrophil differentiation was not compromised. Granulomatosis with polyangiitis (GPA) is a systemic small-vessel vasculitis most commonly affecting the upper and lower respiratory tract and kidneys (1,2). The main target autoantigen in GPA is the neutrophil serine protease (NSP) proteinase 3 (PR3) (EC 3.4.21.76) (3,4). GPA patients develop antineutrophil cytoplasmic autoantibodies to PR3 (PR3-ANCA) that bind to membrane-bound PR3 (PR3) on the neutrophil surface (5,6). The membrane expression of PR3 is mediated by a hydrophobic patch at the protease surface, which is not conserved in other related NSPs, such as human neutrophil elastase (HNE), cathepsin G (CG) and neutrophil serine protease 4 NSP-4 (7-9). Binding of PR3-ANCA to PR3 on cytokine-primed neutrophils induces cell activation resulting in neutrophil extracellular traps (NETs) production, and in granule protein and superoxide release (10,11). Secreted active proteases, including PR3 and related NSPs, exert proteolytic activity on endothelial cells thereby contributing to vascular necrosis (5,12). NETs are known to be directly implicated in ANCA induction as well as endothelial damage (13). There is no treatment for GPA that is based on disease-specific mechanisms and the current protocols involve combined administration of steroids with either cyclophosphamide or rituximab (14,15). These standard treatments are associated with toxicity highlighting the need to develop novel, more specific therapeutic strategies (16). Cathepsin C (CatC) (EC 3.4.14.1), also known as dipeptidyl peptidase I, is a lysosomal amino peptidase belonging to the papain superfamily of cysteine peptidases (17). CatC catalyzes the cleavage of two residues from the N-termini of peptides and proteins. CatC, which is ubiquitously expressed in mammals is considered to be a major intracellular processing enzyme. High concentrations of CatC are detected in immune defense cells including neutrophils, mast cells, lymphocytes and macrophages. CatC is the physiological activator of several immune cell-associated serine proteases such as NSPs (18,19). NSPs are synthetized as inactive zymogens containing a di-propeptide in the myeloblast/promyelocyte stage in the bone marrow (8,20). The proforms mature in this very early developmental stage, induced by CatC, through the cleavage of the Nterminal di-propeptide. The cleavage of the dipropeptide by CatC results in a re-orientation

Nephrology and Intensive Care Medicine, Charité-Universitätsmedizin, Berlin, Germany Keywords: proteinase 3, cathepsin C, neutrophil, granulomatosis with polyangiitis, Papillon-Lefèvre syndrome ABSTRACT Membrane-bound proteinase 3 (PR3 m ) is the main target antigen of anti-neutrophil cytoplasmic autoantibodies (ANCA) in granulomatosis with polyangiitis (GPA), a systemic small-vessel vasculitis. Binding of ANCA to PR3 m triggers neutrophil activation with the secretion of enzymatically active PR3 and related neutrophil serine proteases, thereby contributing to vascular damage. PR3 and related proteases are activated from proforms by the lysosomal cysteine protease cathepsin C (CatC) during neutrophil maturation. We hypothesized that pharmacological inhibition of CatC provides an effective measure to reduce PR3 m and has therefore implications as a novel therapeutic approach in GPA. We first studied PR3 in neutrophils from 21 patients with Papillon-Lefèvre syndrome (PLS), a genetic form of CatC deficiency. PLS neutrophil lysates showed a largely reduced, but still detectable (0.5-4%) PR3 activity when compared to healthy control cells. Despite extremely low levels of cellular PR3, the amount of constitutive PR3 m expressed on the surface of quiescent neutrophils, and the typical bimodal membrane distribution pattern, was similar to what was observed in healthy neutrophils. However, following cell activation, there was no significant increase in the total amount of PR3 m on PLS neutrophils, whereas the total amount of PR3 m on healthy neutrophils was significantly increased. We then explored the effect of pharmacological CatC inhibition on PR3 expression in normal neutrophils using a potent cell permeable CatC inhibitor and a CD34 + hematopoietic stem cell model. Human CD34 + hematopoietic stem cells were treated with the inhibitor during neutrophil differentiation over 10 days. We observed strong reductions in PR3 m , cellular PR3 protein and proteolytic PR3 activity whereas neutrophil differentiation was not compromised.
Granulomatosis with polyangiitis (GPA) is a systemic small-vessel vasculitis most commonly affecting the upper and lower respiratory tract and kidneys (1,2). The main target autoantigen in GPA is the neutrophil serine protease (NSP) proteinase 3 (PR3) (EC 3.4.21.76) (3,4). GPA patients develop antineutrophil cytoplasmic autoantibodies to PR3 (PR3-ANCA) that bind to membrane-bound PR3 (PR3 m ) on the neutrophil surface (5,6). The membrane expression of PR3 is mediated by a hydrophobic patch at the protease surface, which is not conserved in other related NSPs, such as human neutrophil elastase (HNE), cathepsin G (CG) and neutrophil serine protease 4 NSP-4 (7)(8)(9). Binding of PR3-ANCA to PR3 m on cytokine-primed neutrophils induces cell activation resulting in neutrophil extracellular traps (NETs) production, and in granule protein and superoxide release (10,11). Secreted active proteases, including PR3 and related NSPs, exert proteolytic activity on endothelial cells thereby contributing to vascular necrosis (5,12). NETs are known to be directly implicated in ANCA induction as well as endothelial damage (13). There is no treatment for GPA that is based on disease-specific mechanisms and the current protocols involve combined administration of steroids with either cyclophosphamide or rituximab (14,15). These standard treatments are associated with toxicity highlighting the need to develop novel, more specific therapeutic strategies (16).
Cathepsin C (CatC) (EC 3.4.14.1), also known as dipeptidyl peptidase I, is a lysosomal amino peptidase belonging to the papain superfamily of cysteine peptidases (17). CatC catalyzes the cleavage of two residues from the N-termini of peptides and proteins. CatC, which is ubiquitously expressed in mammals is considered to be a major intracellular processing enzyme. High concentrations of CatC are detected in immune defense cells including neutrophils, mast cells, lymphocytes and macrophages. CatC is the physiological activator of several immune cell-associated serine proteases such as NSPs (18,19). NSPs are synthetized as inactive zymogens containing a di-propeptide in the myeloblast/promyelocyte stage in the bone marrow (8,20). The proforms mature in this very early developmental stage, induced by CatC, through the cleavage of the Nterminal di-propeptide. The cleavage of the dipropeptide by CatC results in a re-orientation and remodeling of three surface loops within the activation domain of the protein and renders the S1 pocket of the active-site accessible to substrates (21). After processing, the active proteases are stored in cytoplasmic granules.
CatC is synthetized as a 60-kDa single chain pro-form containing an exclusion domain, a propeptide, a heavy chain and a light chain (22). Pro-CatC which is a dimer, can be efficiently activated by proteolysis with CatL and S in vitro (22). The initial cleavages liberate the propeptide from the catalytic region. Subsequently, a further cleavage occurs between the heavy chain and the light chain which form a papain-like structure (22,23). Xray images of mature CatC structures revealed that the exclusion domain, the heavy chain and the light chain are held together by non-covalent interactions (17). Mature CatC is a tetramer formed by four identical monomers with their active site clefts fully solvent exposed. The presence of the exclusion domain blocks the active site beyond the S2 pocket and it is responsible for the diaminopeptidase activity of CatC (17,24).
Loss of function mutations in the CatC gene (CTSC) results in Papillon-Lefèvre syndrome (PLS) (OMIM: 245000) (25,26), a rare autosomal recessive disease affecting 1 to 4 persons per million (27,28). PLS involves an aggressive pre-pubertal periodontitis, leading to complete tooth loss in adolescence and palmoplantar keratoderma. More than 75 mutations have been identified in PLS, with missense and nonsense mutations being the most frequent, but small deletions, insertions and splice site mutations have also been reported (29). The presumptive diagnosis of PLS can be made by clinical signs and symptoms, but confirmation requires CTSC sequencing. Analysis of urinary CatC in suspected patients can be also used as an early, simple and easy diagnostic test (30). Roberts et al. (31) demonstrated a variety of neutrophil defects in PLS patients, arising downstream of the failure to activate NSPs by CatC. These functional defects included failure to produce NETs, reduced chemotaxis and exaggerated cytokine and reactive oxygen species release. Pham et al. (19) also studied neutrophils from PLS patients and observed that the loss of CatC activity was associated with strong reduction in the proteolytic activity of NSPs. In addition, only very low protein amounts of PR3 and related NSPs were detected in PLS neutrophils (32)(33)(34). Thus, it is conceivable that mimicking the genetic situation in PLS neutrophils by pharmacological CatC inhibition in bone marrow precursor cells would provide an attractive therapeutic strategy in GPA to eliminate major PR3-related disease mechanisms, including the PR3-ANCA autoantigen itself. However, the effect of CatC inactivation on PR3 that is presented on the neutrophil surface where it becomes accessible to anti-PR3 antibodies is not known.
In this work, we investigated the consequences of CatC inactivation on membrane-expression of PR3. First, we quantified the residual proteolytic activity of CatC and PR3 in white blood cell (WBC) lysates or isolated neutrophils from PLS patients. Second, we studied the membraneexpression of PR3 on PLS neutrophils. Finally, we used a potent synthetic cell permeable nitrile inhibitor to evaluate the effect of pharmacological CatC inhibition on membrane-PR3 expression in normal neutrophils generated from human CD34 + progenitor cells.

CatC in blood cells from PLS patients
Blood samples were collected from 13 PLS patients from European, Asian and African countries. PLS diagnosis was firmly established by genetic testing. These patients carried either premature stop codon, missense, nonsense or frameshift mutations in their CTSC (Table I). Blood from 8 additional patients with clinically suspected PLS was obtained. These patients showed typical symptoms of early-onset periodontitis and hyperkeratosis (Fig. 1A). WBC lysates from these patients and from healthy controls differed by their protein profile as observed by SDS-PAGE (Fig. 1B). CatC activity was assayed in peripheral WBC lysates or in purified neutrophils using a CatC-selective FRET substrate in the presence or absence of the selective nitrile CatC inhibitor (L)-Thi-(L)-Phe-CN. Strong CatC activity was observed in control neutrophils and was completely abrogated by the specific CatC inhibitor. In contrast, we did not detect any CatC activity in samples, from genetically and clinically diagnosed PLS patients ( Table I and Fig. 1C), nor any CatC protein in cell lysates from PLS patients using a specific anti-CatC Ab (Fig.  1D). CatC activity and the CatC antigen were also absent in the urine of all PLS patients, unlike healthy controls (Fig. 1C, D). Thus, the CatC deficiency of all 7 patients was confirmed as described in (30) and the mutations were identified by CTSC gene sequencing for some of these patients (supp. Fig. 2, Table I). Once CatC deficiency was clearly established, we studied the fate of PR3 in samples from PLS patients.

Proteolytically active PR3 in blood cells from PLS patients
WB analysis of white blood cell or neutrophil lysates from PLS patients showed that low amounts of the PR3 antigen were still present in all PLS samples ( Fig. 2A and  supp.Fig. 4A). We checked that residual PR3 was enzymatically active by incubating PLS samples with purified exogenous alpha-1proteinase inhibitor (1PI) (35) and observing the appearance of an additional ~75 kDa band corresponding to the irreversible complex between active PR3 and its inhibitor (Fig. 2B). In contrast to mature PR3, inactive pro-PR3 did not form any irreversible complex with 1PI (Fig. 3). We confirmed the presence of proteolytically active PR3 in permeabilized PLS neutrophils using an activity-based probe (Bt-PEG66-PYDA(O-C6H4-4-Cl)2) selective for PR3 and a fluorescent streptavidin derivative to reveal the formation of irreversible complexes (Fig. 2C). We also measured PR3 activity in supernatants of PLS cells activated with the A23187 calcium ionophore using ABZ-VAD(nor)VADYQ-EDDnp as a substrate (Fig.  2D). PR3 activity in PLS cell supernatants was about 1/20 that in controls cells and was almost totally abrogated by the PR3-specific inhibitor Bt-PYDA P (O-C6H4-4-Cl)2. We found that 10 to 20 times more cell lysate proteins from PLS patients (2.5 to 5 µg/well) as compared to healthy controls (0.25 to 0.5 µg/well), were required to achieve similar PR3 activity values. Enzymatic activities in PLS cells and healthy control cells, measured on ABZ-VAD(nor)VADYQ-EDDnp substrate, were almost completely inhibited by the PR3 inhibitor ( Fig. 4A, B, C). From these results, we estimated that blood cells of PLS patients contained from 0.5 to 4% of the PR3 activity in healthy controls cells ( Fig. 4D and Table I).
Marginal, but still detectable activity of CG was also observed in PLS samples using the appropriate selective FRET substrate (data not show). Next, we checked whether PR3 was present at the cell surface of PLS neutrophils.

Membrane surface expression of PR3 on PLS neutrophils
Since the membrane expression of PR3 depends on the activation status of neutrophils, we analyzed both quiescent neutrophils from two local patients (PLS 13 and PLS 14) 30 min after blood collection and neutrophils from foreign blood samples collected 24-72 h before the analysis and thus inevitably activated during shipping. As a control for the latter conditions, cells from either the parents or healthy individuals of the patient's country-of-origin were collected at the same time. The flow cytometry analysis of PR3 m using anti-PR3 mAbs CLB12.8 showed that constitutive PR3 m was present in significant amounts on quiescent PLS neutrophils and showed the typical bimodal pattern with low (PR3 m(low) ) and high (PR3 m(high) ) subsets. After cell activation with a calcium ionophore (A23187), we observed a single homogeneous PR3 population but no significant increase of the total amount of PR3 m at the surface of PLS neutrophils whereas the bimodal pattern was conserved on control cells and the total amount of PR3 increased significantly (Fig. 5). We also found a single PR3 m -presenting neutrophil population in PLS cells that were spontaneously and inevitably activated during transit (experiments with cells from 9 different PLS patients, Fig. 6 and supp. Fig. 4). The PR3 mean fluorescence intensity of these PLS neutrophils was 26 ± 8 % of the intensity found in controls. Thus, the absence of CatC in PLS patients affected constitutive PR3 expression at the surface of quiescent neutrophils only marginally but largely reduced the PR3 antigen exposure on the neutrophil surface of activated cells. Next, we assessed whether an early treatment of neutrophil precursor cells by a CatC inhibitor, would diminish PR3 m expression, cellular PR3 amount and proteolytic activity.

Membrane surface expression of PR3 on neutrophils generated from human CD34 + progenitor cells in the presence of a CatC inhibitor
We differentiated human CD34 + HSC isolated from umbilical cord blood into neutrophils in the presence or absence of a potent cell permeable cyclopropyl nitrile CatC inhibitor (IcatC) (32). Expression of the neutrophil surface markers CD16, CD66b, and CD11b was assessed by flow cytometry during the 10 day differentiation period and confirmed neutrophil differentiation (Fig. 7A). At day 10, a typical bimodal PR3 m expression pattern was observed by flow cytometry with a PR3 mpositive neutrophil subset of approximately 30-40%. Importantly, CatC inhibition with the pharmacological compound IcatC did not affect neutrophil differentiation, but eliminated the bimodal PR3 m expression pattern leaving only marginal PR3 m amounts on the cell surface (Fig. 7A). The mean fluorescence intensity values for PR3 m were reduced to 17±5 % by IcatC at day 10 (p<0.01). The analysis by immunoblotting of cell lysates of differentiated neutrophils, showed that the amount of PR3 protein was strongly reduced after treatment with the CatC inhibitor (Fig. 7B) and that residual PR3 was enzymatically inactive (Fig.  7C). Thus, inhibition of CatC in progenitor cells reduces both cellular and membrane PR3. This PR3 reduction was even stronger than that observed in neutrophils from PLS patients with genetic CatC deficiency.

Discussion
Neutrophils are key actors in the pathogenesis of ANCA-associated vasculitis (6). Neutrophil activation results in the production of NETs bearing PR3 (11). NETsassociated PR3 is presented to dendritic cells triggering the production of PR3-ANCA by Bcells (6). In addition, PR3 is the only NSP that is presented constitutively on the surface of circulating blood neutrophils and remains partly bound to the neutrophil surface following cell activation. These findings contribute to the important role of PR3 as the main target antigen in GPA and related vasculitis. Interaction of circulating PR3-ANCA with PR3 m initiates the activation of circulating neutrophils and thus triggers necrotizing inflammation (6). Because neutrophils from PLS patients that lack CatC activity do not produce NETs (31,34) and contain only marginal levels of NSPs (19,33), it is reasonable to assume that blocking NETosis and/or eliminating PR3 by interfering with CatC activity pharmacologically would ultimately reduce vascular inflammation. Thus, in vivo inhibition of CatC by a synthetic cell permeable inhibitor that mimicks the conditions observed in PLS patients, could have therapeutic potential by reducing PR3-ANCA production, neutrophil activation, endothelial cell necrosis and inflammation. We have compared here the effects of genetic and pharmacological CatC inactivation on the fate of soluble PR3 and PR3 m .
Because CatC is pathologically inactivated by gene mutations in PLS patients, we first used WBC lysates from 21 PLS, 17 of these patients with established missense, frameshift or nonsense mutations, to investigate the fate of cellular and membrane PR3. As expected, we observed neither CatC activity nor immunoreactive CatC protein in PLS cell lysates irrespective of the underlying CatC mutations. It may be surprising that mutated CatC with a missense mutation was neither detected in cell lysates nor in the urine of PLS patients (30). However, our observation corroborates a previous report from the literature (34) and gives support to the conclusion that missense mutations in the CTSC gene abrogates the constitutive secretion of CatC and triggers its degradation in intra-or extra cellular compartments.
The loss of CatC activity in PLS patients was associated with a severe reduction in the activity and the amounts of NSPs (19,(32)(33)(34). We also found that PR3 activity in PLS WBC lysates was strongly lowered to 1-4% of that in control lysates, but was still detectable. We employed several approaches to ensure that the measured proteolytic activity was indeed due to PR3. We used a highly sensitive and specific PR3 substrate, a specific PR3 inhibitor that totally abrogated the enzymatic activity, and finally, a selective PR3 activity-based probe to detect active PR3 in PLS neutrophils. The results clearly confirmed the presence of residual active PR3 in PLS neutrophils, suggesting that CatC is the main, but not the sole protease involved in the activation of pro-NSPs. This observation is consistent with data from Roberts et al. who demonstrated the presence of some LL37 in stimulated neutrophil supernatants from PLS patients, LL37 being a product of PR3 cleavage of human cathelicidin 18 (31).
We then investigated whether or not the unprocessed pro-PR3 was still present in PLS cell lysates. For this purpose, we exploited the property of 1PI to form irreversible complexes with proteolytically active PR3 but not with its proform. Following incubation of PLS cell lysates with 1PI, almost all immunoreactive PR3 formed irreversible complexes with the inhibitor suggesting that only residual mature PR3 was present in cell lysates. Having ensured that the absence of the pro-PR3 was not due to the cell lysis procedure, we conclude that most of pro-PR3 was degraded very early in PLS neutrophil precursors. Our data are compatible with the notion that a small amount of the pro-PR3 can be early processed into an active protease by one or several aminopeptidase(s) other than CatC. These enzymes remain to be identified.
Human PR3 is expressed constitutively in a bimodal manner with two populations of neutrophils presenting either high (PR3 m(high) ) or low (PR3 m(low) ) amounts of the protease on their surface (36,37). The level of PR3 m on resting neutrophils and the percentage of PR3 m expressing neutrophils is stable over time for a given individual (5,37). In spite of the low PR3 level in PLS cell lysates, we observed that PR3 m was present at the surface of resting PLS neutrophils showing a typical bimodal distribution similar to control neutrophils. This observation suggests that the expression of constitutive PR3 m on resting neutrophils is independent of intracellular PR3 levels and remains stable even when CatC is inactive. Activating cells from PLS patients with the calcium ionophore A23187 resulted in a PR3 increase on the neutrophil surface but this increase was significantly smaller than that observed in control cells. Thus, the genetic inactivation of CatC results in a dramatic decrease of PR3 within intracellular granules but does not interfere with the constitutive expression of proteolytically inactive PR3 (38,39) at the surface of quiescent PLS neutrophils. This data suggests a different intracellular storage site and a different intracellular pathway for constitutive and induced PR3 m . Unexpectedly, and in contrast to control cells, no bimodal PR3 m expression pattern was observed in activated PLS cells. This observation was made in both spontaneously activated cells during shipping and with a pharmacological compound. We have no obvious explanation for this finding at the moment.
We showed previously that a two-step amplification/differentiation protocol of human CD34 + hematopoietic stem cells obtained from umbilical cord blood results in differentiated neutrophils (40). We used this model system to investigate the production and the fate of PR3 in the presence of a CatC nitrile inhibitor, IcatC (23). A subset of PR3 m -positive cells was detectable by flow cytometry at day10 whereas a second cell subset remained negative, consistent with a bimodal expression typically seen with blood neutrophils. Differentiation of CD34 + HSC into neutrophils in the presence of the CatC inhibitor IcatC did not alter the expression of the neutrophil surface markers CD16b, CD66b and CD11b but resulted in strong reduction of intracellular and membrane PR3. Pharmacological CatC inhibition eliminated PR3 from normal neutrophils more effectively than mutated CatC in PLS neutrophils. It is conceivable that additional aminopeptidases exist in blood neutrophils that were absent in CD34 + HCS-derived neutrophils or that the potent IcatC inhibitor inhibited CatC together with additional proteases involved in the activation of the pro-PR3. Our current observations in PLS cells and our recent work reporting the complete disappearance of HNE in bone marrow cells from healthy donors pulse-chased in presence of IcatC supports the latter hypothesis (32).
To conclude, we showed here that CatC is the major but not the unique pro-PR3 processing protease in neutrophils since low amounts of proteolytically active PR3 are still present in neutrophils of CatC deficient individuals. Treating CD34 + hematopoietic stem cells with the CatC inhibitor ICatC resulted in an almost total absence of intracellular PR3 and PR3 m in stem cellsderived neutrophils. The elimination of the PR3-ANCA target antigen supports the notion that pharmacological CatC inhibition provides an alternative therapeutic strategy for reducing neutrophil-mediated vascular inflammation in auto-immune vasculitis. We previously showed that a prolonged IcatC administration in the macaque resulted in an almost complete elimination of PR3 and NE (32). Unlike humans however, macaques do not display constitutive PR3 at the surface of their circulating neutrophils and therefore cannot be used as a relevant model of GPA. Only clinical studies in GPA patients will answer the question whether or not a CatC inhibitor may function as a PR3-ANCA antigen suppressor.

Experimental Procedures
Blood collection-Blood samples were collected from 21 PLS patients from European countries (Germany, the UK, Italy, France, Hungary), from Asian countries (India, Saudi Arabia) and from Egypt. The 13 healthy volunteers were from France, India, Italy, Saudi Arabia and Egypt. 2-15 mL peripheral blood samples from healthy control donors and patients with PLS were collected into EDTA K2 preservative tubes by peripheral venipuncture. Samples were taken giving informed consent. Red blood cells lysis took place with 0.1 mM EDTA, 10 mM KHCO3, 150 mM NH4Cl and white cells pelleted with centrifugation for 5 min at 400 x g.

Differentiation of CD34 + hematopoietic stem cells from umbilical cord blood into neutrophils-Umbilical cord blood samples were taken giving informed consent.
Mononuclear cells were obtained from anticoagulated cord blood by centrifugation over a LSM1077 (PAA, Pasching, Austria) gradient at 800 x g for 20 min. Cells were washed and stained using the CD34 + progenitor isolation kit (Miltenyi, Bergisch-Gladbach, Germany) and sorted according to the manufacturer's instructions. CD34 + cells were cultivated in stem span serum free medium (Cell Systems, St. Katharinen, Germany) supplemented with Penicillin/Streptomycin, 100 ng/mL SCF, 20 ng/ml TPO and 50 ng/mL FLT3-L (Peprotech, London, UK) for expansion. Neutrophil differentiation was performed in RPMI with 10% FCS, 10 ng/mL G-CSF (Peprotech), and either DMSO control or 1 µM IcatC. Medium was changed every other day. We PR3phenotyped the neonatal neutrophils obtained from the freshly harvested umbilical cords by flow cytometry prior to the CD34 + HSC isolation. We selected only cord blood where the neonatal neutrophils showed a clear bimodal membrane PR3 pattern.
Flow cytometry-WBC from PLS patients or healthy controls were resuspended in PBS and a blocking step was performed with 5% bovine serum albumin (BSA), 2.5 mM EDTA in PBS for 15 min at 4°C. Or WBC were fixed with 2% paraformaldehyde and permeabilized with 0,5% Triton-X 100 in PBS and non-specific binding sites were blocked with 5% BSA. Flow cytometry analyses were performed using a MACSQuant analyzer (Miltenyi Biotec, Bergisch-Gladbach, Germany) and VenturiOne software (Applied Cytometry, Sheffield, United Kingdom). These analyses were performed using the following Abs: (Santa Cruz Biotechnology, Heidelberg, Germany) or the secondary antibody FITCconjugated IgG1 Fab2 (DAK-GQ1, 5µg/mL) (Dako, Hamburg, Germany). Dead cells were stained with Viobility 405/520 Fixable Dye (1:200) (Miltenyi Biotec, Bergisch-Gladbach, Germany). The gating strategies used are described in supp. Fig. 1. The compensation was performed using VenturiOne software.

Genetic analysis-
Extraction of genomic DNA (salting out procedure): Peripheral blood samples were obtained from the patients and both parents (if available) after informed consent had been given according to NRC guidelines. Genomic DNAs were prepared as previously described by (44) with some additional modifications described by (45).
PCR amplification of CTSC gene exons: For analysis of CTSC mutations, eight different specific amplifications using CTSC gene specific primers carried out on the genomic DNA according to Toomes et al., (26), except for the newly developed primer pairs for exons 1 and the 5´ half of exon 7.
The amplification conditions were as follows: 2 min at 95°C for one cycle, followed by 35 cycles of 30 s at 94°C, 30 s at the annealing temperature of the primers (53°C for exons 7a and 7b, 54.5°C for exon2, 55.2°C for exons 1 and 6, 56.6°C for exon 3, 57.2°C for exon 4 and 58°C for exon 5), and 1 minute at 72°C in a thermal cycler (Agilent Technologies SureCycler 8800) (46). Five microliters aliquots of the PCR products were analyzed by 2% agarose gel electrophoresis.
Mutation analysis: PCR products were purified using the QIA Quick PCR Purification kit (Qiagene) followed by bidirectional sequenced using the ABI Prism Big Dye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems) and the sequencing reaction products were separated on an ABI Prism 310 Genetic Analyzer (Applied Biosystems). Alignment of sequenced results used NCBI genomic sequence NG_008365.1 and reference cDNA sequence NM_000348.3 for result interpretation.
Acknowledgments: This work was supported by the "Ministère de l'Enseignement Supérieur et de la Recherche", the "Région Centre-Val de Loire" (Project BPCO-Lyse). This project has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement No 668036 (RELENT). Responsibility for the information and views set out in this study lies entirely with the authors. BK acknowledges the "Alexandre von Humboldt Foundation" for a short term institutional research training grant (2016, Comprehensive Pneumology Center, Munich). The authors thank Lise Vanderlynden (INSERM U-1100) for technical assistance.

Figure 5.
Membrane expression of PR3 on quiescent and chemically activated PLS neutrophils. WBC from PLS siblings (local patients P13 and P14) and their mother were activated using calcium ionophore (A23187) 30 min after blood collection. Both viable quiescent PLS (red dotted line) and control (blue dotted line) cells showed expression of PR3 on their surface. After chemical activation (continuous lines), membrane PR3 was largely increased on control cells while PR3 on PLS neutrophils was almost the same as on quiescent cells. We used Viobility 405/452 Fixable Dye to discriminate between live and apoptotic/dead cells. Flow cytometry revealed 81±5% viable neutrophils in all samples. No statistically significant difference between quiescent and activated neutrophils was observed (t test). P: PLS patient. Numbers indicate mean fluorescent intensity values.  Effect of pharmacological CatC inhibition on PR3 expression and proteolytic activity in neutrophil-differentiated CD34 + HSC. CD34 + HSC were differentiated over 10 days into neutrophils in the presence of DMSO buffer control (Bu, blue) or 1 µM of the CatC inhibitor (IcatC, red). (A) Flow cytometry indicates that differentiating cells acquired the typical neutrophil surface markers CD16, CD66b, and CD11b, together with a bimodal membrane PR3 phenotype. Color lines represent staining with the specific antibodies and dotted lines represent the corresponding isotype control. A representative of five independent differentiation experiments is shown. (B) PR3 protein was assessed in cell lysates (7.0 µg/lane) by immunoblotting at the indicated time points using a specific anti-PR3 antibody. PR3 protein was strongly induced during CD34 + HSC differentiation and this effect was significantly reduced with IcatC. A representative western blot and the densitometric analysis from five Supp. Figure 1. Gating strategies. WBC were isolated from EDTA-whole blood by red blood cells lysis. T cells and dead cells were excluded using anti-CD3 Ab and Viobility 405/520 Fixable Dye. The remaining cells were analyzed for the expression of CD15, a neutrophil marker. The percentages of cells in each of the specified gates are indicated.
Supp. Figure 4. Membrane expression of PR3 on PLS neutrophils activated during transportation. (A) PR3 expression on spontaneously activated neutrophils was investigated by flow cytometry. Controls were randomly selected in the local environment of the patient. (B) Mean fluorescence intensity values for PR3 m on PLS and healthy neutrophils. Bar indicates mean ± SEM value of each condition and asterisks the p-value of t test (p< 0.001,***). C: control (n=3), P: PLS patient (n=7).