A rare CTSC mutation in Papillon-Lefèvre Syndrome results in abolished serine protease activity and reduced NET formation but otherwise normal neutrophil function.

Papillon-Lefèvre Syndrome (PLS) is an autosomal recessive monogenic disease caused by loss-of-function mutations in the CTSC gene, thus preventing the synthesis of the protease Cathepsin C (CTSC) in a proteolytically active form. CTSC is responsible for the activation of the pro-forms of the neutrophil serine proteases (NSPs; Elastase, Proteinase 3 and Cathepsin G), suggesting its involvement in a variety of neutrophil functions. In PLS neutrophils, the lack of CTSC protease activity leads to inactivity of the NSPs. Clinically, PLS is characterized by an early, typically pre-pubertal, onset of severe periodontal pathology and palmoplantar hyperkeratosis. However, PLS is not considered an immune deficiency as patients do not typically suffer from recurrent and severe (bacterial and fungal) infections. In this study we investigated an unusual CTSC mutation in two siblings with PLS, a 503A>G substitution in exon 4 of the CTSC gene, expected to result in an amino acid replacement from tyrosine to cysteine at position 168 of the CTSC protein. Both patients bearing this mutation presented with pronounced periodontal pathology. The characteristics and functions of neutrophils from patients homozygous for the 503A>G CTSC mutation were compared to another previously described PLS mutation (755A>T), and a small cohort of healthy volunteers. Neutrophil lysates from patients with the 503A>G substitution lacked CTSC protein and did not display any CTSC or NSP activity, yet neutrophil counts, morphology, priming, chemotaxis, radical production, and regulation of apoptosis were without any overt signs of alteration. However, NET formation upon PMA-stimulation was found to be severely depressed, but not abolished, in PLS neutrophils.

Introduction

Neutrophil granulocytes are phagocytic white blood cells that are critical components of the inflammatory process and fight against invading pathogens. They are loaded with a wide variety of antimicrobial compounds and proteolytic enzymes that contribute to microbial killing but also risk damaging endogenous tissues if not properly regulated [1]. Among these enzymes, the neutrophil serine proteases (NSPs) are three structurally related enzymes, Human Neutrophil Elastase (HNE), Proteinase 3 (PR3), and Cathepsin G (CTSG) [2]. In the human population, a number of hereditary symptoms exist that affect neutrophil function and most of these are more or less severe immune deficiencies characterized by recurrent and severe bacterial and fungal infections. In addition, some immune deficiencies are characterized by an inability to regulate inflammation. For instance, patients with chronic granulomatous disease (CGD) are not only plagued by infections but also by a variety of, often aseptic, inflammatory disorders [3, 4]. This indicates that neutrophils are not only critical microbial killers but that they are also important regulators of inflammatory processes.

One hereditary neutrophil defect is the rare Papillon-Lefèvre Syndrome (PLS) with an estimated prevalence of one to four persons per million in the general population [5]. PLS is caused by loss-of-function mutations in the CTSC gene resulting in the lack of enzymatic activity of the exo-cysteine protease Cathepsin C (CTSC) [6]. This protease is critical for the activation of the proforms of NSPs in neutrophils, thus PLS neutrophils are deficient in NSP activity [7]. The NSPs are all formed as enzymatically inactive zymogens; after proteolytic removal of two N-terminal amino acids by CTSC they are stored as active granule enzymes during the promyelocyte stage [2, 8]. The activity of NSPs has been thought to be vital for the ability of neutrophils to perform microbial killing and to degrade microbes. PLS neutrophils have been shown to kill bacteria in vitro in a similar capacity to neutrophils from healthy controls [9]. Additionally, PLS neutrophils have been found to have less directional chemotactic accuracy to fMLF as well as reduced capacity to produce neutrophil extracellular traps (NETs) upon PMA stimulation [10, 11]. Despite NSP activity being widely held as important enzymes for the killing and degradation of phagocytosed microbes, individuals with PLS are typically not plagued by recurring opportunistic infections. The major clinical finding in PLS is a remarkably aggressive form of periodontitis with manifestation already in the primary dentition, which may lead to complete edentulousness in young adults [12]. Periodontitis is not a typical infectious disease, but rather a chronic inflammatory condition of the tooth-supporting tissues. The condition is triggered by oral bacteria residing in the gingival pockets and it is characterized by progressive destruction of tooth supporting structures [13, 14].

Multiple different mutations in the CTSC gene may result in PLS and it is unclear whether different mutations result in different levels of disease severity or altered neutrophil function. Previous reports on PLS neutrophils have demonstrated normal morphology and differential counts of circulating white blood cells [9], reduced production of NETs triggered with phorbol myristate acetate (PMA) [9,10], defective chemotaxis and increased PMA induced reactive oxygen species (ROS) production [10]. Residual CTSC and NSP activity has also been reported for certain patients with PLS [15, 16].

In this report, we describe an unusual 503A>G mutation in CTSC in two siblings with PLS (family A) and characterize several basic neutrophil functions with special emphasis on CTSC and NSP activity. These findings were compared with data from two other patients with PLS (siblings of family B) with a previously described CTSC mutation (755A>T), as well as from a small cohort of healthy individuals. We found that the 503A>G CTSC mutation resulted in abolished CTSC and NSP activity, quite similar to the 755A>T mutation. The basic neutrophil functions investigated were largely found to be within the range displayed by healthy controls, apart from PMA-triggered NET formation that was potently reduced and delayed, but not completely absent, in PLS neutrophils.

Patients and methods

Clinical description

Four patients, treated at the Specialist Clinic of Periodontics, Region Västra Götaland, Gothenburg, Sweden, from two families (A and B) with PLS were recruited for this study.

The two siblings from family A were at the time of the most recent sampling 32 and 28 years old. They had experienced periodontitis since early childhood with a rapid loss of tooth supporting structures and teeth. Both siblings became edentulous in their teens and the older sibling was restored with implants. All implants were then affected by peri-implantitis, and at the time of sampling >50% of the bone support was lost, and the implants in the upper jaw were scheduled for removal and have since been removed. The younger sibling remained edentulous. Another two siblings in family A were clinically healthy. The siblings were of Iraqi descent with parents who were closely related, consanguine.

Family B was of ethnic Somali descent, but the children were born in Sweden. No known consanguinity was reported between the parents. The patients of family B were 11 and 9 years old at the time of sampling and had been in specialist care for periodontal pathology since the age of two. Both had lost all their deciduous teeth and exhibited severe bone loss at the recently erupted permanent teeth.

In addition to periodontal pathology, all patients displayed the PLS-typical palmoplantar localized hyperkeratosis. Besides these classical symptoms, all four were systemically healthy, except that one patient in family B had experienced an episode of a purulent skin cyst, which had to be surgically removed.

Blood samples

The study was approved by the Regional Ethical Review Board in Gothenburg, Sweden (Dnr: 118–16, 544–17). Peripheral blood was sampled through venipuncture and collected in sample tubes with anti-coagulant (Heparin, or EDTA for genetic analysis). Samples from patients as well as healthy controls without periodontitis were collected after written informed consent was given by the individuals or their legal guardians. For the healthy controls, twelve volunteers aged between 25 and 55 years old were recruited among personnel and students at the at the Institute of Odontology, Sahlgrenska Academy at University of Gothenburg. All attended regular dental checkups, and none reported periodontitis.

Genetic sequencing and analysis

For family A, the DNA was extracted from whole blood samples collected in EDTA tubes with the help of a GenElute Blood Genomic DNA kit (# NA2010-1KT, Sigma-Aldrich, St Louis, US-MO). Whole genome sequencing was performed at the Core Facilities, Sahlgrenska Academy, University of Gothenburg, Sweden. Afterwards, the data was processed with the help of the Bioinformatics Core Facility, Sahlgrenska Academy, University of Gothenburg, Sweden. First the quality of the data was assessed with FastQC (version 0.11.2) and then Samtools (version 1.3.1) was used to sort, index and assess the mapping statistics. Paired end reads were aligned to the human reference genome hg19 (GRCh37, RefSeq assembly accession GCF_000001405.13) using a Burrows-Wheeler Aligner (BWA mem version, BWA 0.7.13) [17]. Duplicates were removed with Picard (version 2.2.4). With the help of the Genome Analysis ToolKit (GATK, version 3.1–1) realignment and variant calling was performed [18]. With the Haplotype Caller tool, the variant calling was performed according to GATK best practice, applying the following quality filters for SNPs: QD < 2.0, MQ < 40.0, FS > 60.0, ReadPosRankSum < -8.0, MQRankSum < -12.5, and for indels: QD < 2.0, FS > 200.0, ReadPosRankSum < -20.0. With the known gene database the variants were annotated by using the ANNOVAR tool [19]. Additionally, ANNOVAR was used to annotate exonic variants with functional predictions with the help of the following tools: whole-exome SIFT [20], PolyPhen2 HDIV [21], PolyPhen2 HVAR [21], LRT [22], MutationTaster [23], MutationAssessor [24], FATHMM [25], MetaSVM [26], MetaLR [26], VEST [27], CADD [28], GERP++ [29], ClinVar [30], PhyloP [31, 32] and SiPhy scores [33] from dbNSFP (version 2.6) [34].

For family B, blood samples collected in EDTA tubes were whole exome sequenced and analyzed (as part of a clinical investigation) at the Department of Clinical Genetics at Sahlgrenska Hospital, Gothenburg, Sweden.

Neutrophil isolation

Neutrophils were isolated from peripheral blood samples of patients and healthy controls based on the isolation method described by Bøyum et al. [35]. In short, with the help of dextran, the erythrocytes were precipitated and a Ficoll-Paque gradient was used to separate and then remove the peripheral blood mononuclear cells (PBMCs) from the granulocytes. Remaining erythrocytes in the granulocyte fraction were lysed with distilled water. After washing with Krebs Ringer phosphate buffer (KRG) the samples were diluted in KRG with Calcium (1 mM) and kept at 4°C for further use.

Neutrophil lysates

Neutrophils isolated from peripheral blood samples were diluted in KRG with Calcium (1 mM) to 5x106 cells/ml, pelleted and lysed in 0.1% Triton X-100. Vortexing and centrifugation at 15871 rcf for 25 min at 4°C was used to mechanically lyse the cells. Afterwards, the lysates were diluted in KRG with Calcium (1 mM) and the cellular debris was removed by centrifugation (short mode, max. 21,130 rcf, 30 s, 4°C). The resulting samples, all at a concentration of 2.5x106 cell equivalents/ml, were stored at -80°C.

Neutrophil cytospins

The isolated neutrophil samples were diluted to a concentration of 1x106 cells/ml and then fixed on a microscope slide through cyto-centrifugation at 130 rcf for 5 min (THARMACspin CS1, Waldsolms, Germany). After drying, the microslides were stained with Giemsa and May-Grünwald staining (both from Sigma-Aldrich) and micrographs were taken at 100x magnification with a microscope (Objective: Olympus UPlan FL U 100x/1.30 Oil Ph3 ∞/0.17/FN26.5, Microscope: BX41, LRI Olympus, Camera: Olympus DP71, Tokyo, Japan).

Neutrophil activation assay and flow cytometry

Whole blood samples (540 μl) were supplemented with TNFα (#T6674, Sigma-Aldrich) to a final concentration of 10 ng/ml or left unstimulated. After incubation at 37°C for 20 min, the samples were transferred into FACS Lysing solution (diluted 1:10 in dH2O, #349202, BD Biosciences, Franklin Lakes, US-NJ), vortexed and placed on ice for 15 min before washing with PBS (277 rcf, 10 min, 4°C). After aspiration of the supernatant, the cells were resuspended in FACS Lysing solution (1:10, BD Biosciences) and the incubation and washing steps were repeated as previously. The cell pellets were resuspended in PBS and antibodies (all from BD Biosciences) were added in accordance with the dilutions recommended by the manufacturer; 1:20 for CD35 PE (#559872) and CD11b APC (#333143), and 1:40 for CD62L PE (#341012). One control per donor sample was left unlabeled. The samples were incubated in the dark at 4°C for 30 min and washed one more time in PBS before measurement in a flow cytometer (Accuri C6, BD Biosciences). The results were analyzed with the help of FlowJo 10 (BD Biosciences).

Protease activity

Protease activity was assessed in a plate reader (BMG Labtech, Ortenberg, Germany) with fluorogenic substrates; H-GR-AMC hydrochloride salt (# I-1215.0050, Bachem, Bubendorf, Switzerland) for CTSC activity, Abz-APEEIMRRQ-EDDnp (# SNE-3230-v, Peptides International, Louisville, US-KY) for HNE activity, Abz-VADXRDR-EDDnp (# SNP-3232-v, Peptides International) for PR3 activity, and Abz-EPFWEDQ-EDDnp (# SFR-3231-v, Peptides International) for CTSG activity. The NSP substrates are currently available from vivitide (www.vivitide.com).

The samples were diluted in KRG with Calcium (1 mM), added to black 96 well culture plates (Thermo Scientific, Waltham, US-MA) and incubated at 37°C for 15 min. Afterwards, the pre-incubated samples were mixed 1:1 with the respective substrate-buffer mix to their final substrate-specific concentration as shown in Table 1. Cell equivalent normalization was used to achieve comparability between the lysates. The fluorescence intensity (FI) of each sample was measured in triplicate every 3 minutes for at least 30 minutes under regular shaking of the plate and at the fluorophore-specific wavelengths according to Hamon et al. and Attucci et al. [36, 37] (Table 1). The design of the protease activity system was based on a system developed by Korkmaz et al. [38]. Briefly, the substrates were provided in abundance to avoid saturation of the fluorescence increase and allow for a longer linear increase in FI. Linear regression was performed on the change of FI over time for the linear segment of the kinetic curve (0–12 min). The calculated slope of the linear regression was then normalized according to the cell equivalents used, which in turn can be related to the amount of protease activity in the sample, allowing for comparison of the protease activity in samples from different individuals.

Table 1

Parameters for protease activity assay.

Protease	Substrate concentration (μM)	Buffer	Lysate dilution	Measurement (nm)a
		Material Concentration
Cathepsin C (CTSC)	1500.0	Na Acetate    50 mM	1:50	Exc.: 355±15
				Em.: 460±20
		NaCl       30 mM
		EDTA      10 mM
		DTT        2 mM
Neutrophil Elastase (HNE)	5.0	HEPES      50 mM	1:20	Exc.: 320±15
		NaCl      750 mM		Em.: 420±20
		Igepal CA-630 0.05 vol%
		pH         7.4
Proteinase 3 (PR3)	5.0	HEPES      50 mM	1:300	Exc.: 320±15
		NaCl      750 mM		Em.: 420±20
		Igepal CA-630 0.05 vol%
		pH          7.4
Cathepsin G (CTSG)	20.0	HEPES      50 mM	1:10	Exc.: 320±15
		NaCl     100 mM		Em.: 420±20
		Igepal CA-630 0.01 vol%
		pH        7.4

aFilter settings for excitation (Exc.) and emission (Em.) wavelength for the fluorescence measurement of the substrates. Peak wavelength±range.

Immunoblotting for CTSC

The presence of CTSC was tested through immunoblotting of samples prepared from isolated neutrophils. The neutrophils (50 μl, 5x106 cells/ml) were mixed with dH2O, LDS Sample Buffer (Invitrogen, Carlsbad, US-CA) and Reducing Agent (Invitrogen). Triton X-100 was added to a final concentration of 0.5% and AEBSF (Thermo Scientific) to a final concentration of 1 mM. The samples were vortexed for 30 s, boiled at 90°C for 5 min and stored at -80°C. Recombinant human CTSC (rhDPPI; UNIZYME Laboratories A/S, Horsholm, Denmark) was mixed with dH2O, LDS Sample Buffer as a control and boiled together with the frozen samples at 90°C for 5 min. Samples, normalized on basis of cell number (each lane was loaded with 31,250 cell equivalents and the CTSC control with 1.0 ng/lane), were then separated on a Bolt bis-tris plus 4–12% gel (Invitrogen) in a mini gel tank electrophoresis bath (Invitrogen) filled with MES SDS running buffer (Invitrogen) with a PowerEase300W power source (Invitrogen) and blotted on a transfer membrane (Invitrogen) in an iBlot2 dry blotting system (Life Technologies, Waltham, US-MA). The membrane was blocked with 5% (w/v) BSA in 0.05% TWEEN in PBS and subsequently incubated with the mouse monoclonal Anti-cathepsin C Antibody (D-6) (1:250; sc-74590, Santa Cruz Biotechnology, Dallas, US-TX) and the secondary Polyclonal Rabbit Anti-Mouse Ig conjugated to HRP (1:500; P 0260, Dako Denmark A/S, Glostrup, Denmark). After labelling with the antibodies, the membranes were developed for 3 min with a WB enhanced chemiluminescence (ECL) kit (Thermo Scientific) and scanned in a Gel scanner (ChemiDoc XRS, Bio-Rad Laboratories, Hercules, US-CA).

NADPH-oxidase derived reactive oxygen species production

The production of extracellular and intracellular reactive oxygen species (ROS) was measured in a chemiluminescence (CL)-based system [39] as CL activity in a ClarioStar plate reader (BMG Labtech) for family A or Biolumat LB 9505 (Berthold Co., Wildbad, Germany) for family B. The assay was performed in a white 96-well microtiter plate (volume 200 μl) or polypropylene tubes, respectively. For extracellular ROS measurements, neutrophils (100 μl, 5x106 cells/ml) were mixed with Isoluminol (5.64 μM, Sigma-Aldrich) and horseradish peroxidase (HRP, 0.2 U/ml, Roche Diagnostics, Basel, Switzerland). For intracellular measurements, neutrophils (100 μl, 5x106 cells/ml) were mixed with Luminol (5.64 μM, Sigma-Aldrich), superoxide dismutase (SOD, f0.5 U/ml, Worthington Biochemical Corp., Lakewood, US-NJ) and Catalase (20 U/ml, Worthington). After mixing, the samples were pre-incubated for 5 min at 37°C. A background base line was then established before adding PMA (50 nM, Sigma-Aldrich) to the wells. The ROS production was measured for a total of 20 min or until the bulk of the reaction had concluded.

PMA-induced NETosis

The NET formation was visualized according to a protocol by Björnsdóttir et al. [40]. In short, neutrophils (2.5x105 cells/coverslip) were allowed to settle on polylysine coated coverslips (Knittel Glass, Braunschweig, Germany) and incubated in cell culture medium supplemented with PMA (50 nM, Sigma-Aldrich) for 3 h at 37°C and 5% CO2. Control cells were incubated without PMA. The cells were fixed with 4% PFA at room temperature for 30 min and subsequently washed with PBS. The slides were blocked in PBS supplemented with 10% Donkey Serum (Abcam, Cambridge, UK) and 2% bovine serum albumin (BSA, Sigma-Aldrich) and incubated with rabbit-anti-human MPO (1:2000 in blocking buffer, # A0398, Agilent, Santa Clara, US-CA) for 30 min at room temperature [11]. After washing, the samples were incubated with donkey-anti-rabbit AF647 (1:500 in blocking buffer, # A-31573, Molecular Probes, Waltham, US-MA) for 30 min at room temperature and washed a final time. The coverslips were mounted on slides with mounting medium containing DAPI (# P36935, Invitrogen) and imaged in a microscope (Objective: Olympus PlanApo 40x/0,95 ∞/0,11–0,23, Microscope: LRI Olympus BX41, Camera: Olympus DP71).

To quantify NETosis, neutrophils were suspended in phenol red-free RPMI culture medium (Gibco, Waltham, US-MA) supplemented with Sytox Green DNA stain (1.25 μM, Molecular Probes). The mixture was placed in black 96 well culture plates (Thermo Scientific) at a concentration of 5x104 cells/well. PMA (Sigma-Aldrich) was added to some wells at a concentration of 50 nM. The plates were incubated for 4 h at 37°C and 5% CO2. The fluorescence (excitation 485 nm, emission 535 nm) was measured at indicated time-points in a ClarioStar plate reader (BMG Labtech) [41].

Neutrophil apoptosis

The regulation of apoptosis was investigated as described by Christenson et al. [42]. In short, the neutrophils were suspended in RPMI cell culture medium (Gibco) supplemented with 10% Fetal Bovine Serum (GE Life Sciences, Chicago, US-IL) and 1% penicillin-streptomycin (PEST, Life Technologies) at a concentration of 5x106 cells/ml. The cell suspension was split into different tubes and supplemented with either purified anti-human CD95 (Fas) antibody (10 μg/ml, # 305704, BioLegend, San Diego, US-CA), lipopolysaccharides (LPS) from E. coli (100 ng/ml, # L2880, Sigma-Aldrich) or left unstimulated. The neutrophils were incubated at 37°C, 5% CO2 for 20 h.

After incubation, samples were centrifuged at 277 rcf for 10 min and the pellet washed with 2 ml Annexin V-Buffer (1 mM HEPES, 14 mM NaCl, 250 μM CaCl2) and centrifuged again. The supernatant was replaced with a cell staining solution of 105 μl Annexin V-Buffer, 1.5 μl Annexin-V-Fluos (Roche Diagnostics) and 5 μl 7-Aminoactinomycin D (7-AAD, BD Biosciences). The mix was incubated at room temperature in the dark for 10 min before cells were immediately analyzed in a flow cytometer (Accuri C6, BD Biosciences). Neutrophils that stained positive for Annexin-V but negative for 7-AAD were classified as apoptotic and the percentage of apoptotic cells in the sample was calculated with the number of total cell events.

Chemotaxis

Isolated neutrophils were resuspended in KRG with Calcium (1 mM) and 0.3% BSA to a final concentration of 2x106 cells/ml. 30 μl of the cell suspension (6x104 cells) were loaded on top of a 3-μm polycarbonate chemotaxis membrane with a hydrophobic mask (ChemoTX Disposable Chemotaxis System, Neuro Probe Inc., Gaithersburg, US-MD). The neutrophils were allowed to migrate through the membrane towards buffer, KRG with Calcium (1 mM) and 0.3% BSA, and buffer supplemented with 10 nM fMLF into the provided clear 96 well plate inside an incubator at 37°C, 5% CO2 for 90 min. After lysing the cells with 2% cetyltrimethylammonium in PBS with 2% BSA, the myeloperoxidase activity was measured as absorbance at 450 nm with the help of a peroxidase reagent (OPD, Sigma-Aldrich) in phosphate citrate buffer (0.05 M, pH 5) in a plate reader (CLARIOstar, BMG Labtech). All samples were run as triplicates and normalized to control wells with the maximum number of cells (6x104 cells).

Statistical analysis

Statistical analysis of the data was performed with Prism 8.0 (GraphPad Software, San Diego, US-CA). Values of p<0.05 were regarded as statistically significant. The specific statistical tests used are stated were applicable.

Results

Genetic and initial findings

A genetic analysis was performed, and the patients of family A (PLS 1 and 2) were found homozygous for the gene variant GCF_000001405.13(CTSC):g.88042469T>C, c.503A>G, p.(Tyr168Cys) (submitted to the European Genome-phenome Archive and stored with the EGA ID: EGAS00001005040). This is a variation (rs750898600 in the ensemble database) at position 88042469 (according to the reference genome hg19) leading to a 503A>G substitution in exon 4 of the CTSC gene, which is expected to result in an amino acid replacement from tyrosine to cysteine at position 168 of the CTSC protein. This substitution was predicted to be deleterious for protein function by the Polyphen2 HDIV, Polyphen2 HVAR, LRT, MutationTaster and FATHMM method, among others. This mutation is rare with only sporadic heterozygote individuals reported, giving rise to different allele frequencies in different databases; gnomAD: C = 0.000032/1 [43], TOPMed: C = 0.000016/2 [44], and no reports in ALFA [45]. We only found this genetic variant entered in genetic databases for heterozygous carriers, and to the best of our knowledge, data of only one patient with PLS homozygous for 503A>G has been previously published by Sørensen et al. [9] without submission to genetic databases. It was not possible for us to genotype unaffected relatives in family A.

PLS 3 and 4 of family B were whole exome analyzed for diagnostic purposes and were both homozygous for the variant NM_001814.5:c.755A>T, p.(Gln252Leu), resulting in a 755A>T transversion in exon 5 of the CTSC gene (rs104894207 in the ensembl database). This mutation is identical to that of patients of a family described in both Toomes et al. and Hewitt et al. [7, 46] (Table 2) and it is predicted to result in an amino acid change from glutamine to leucine in position 252 of the CTSC protein. In addition, the mother of PLS 3 and 4 participated in the genetic analysis and was heterozygous for the same variant.

Table 2

Overview of the PLS patients.

Patients		Dental status/diagnosis	Mutation
			HGVSa	SNPb
Family A	PLS 1	Edentulous	GCF_000001405.13(CTSC):	rs750898600
	PLS 2	Peri-implantitis	g.88042469T>C, c.503A>G, p.(Tyr168Cys)
Family B	PLS 3	Periodontitis	NM_001814.5(CTSC):	rs104894207
	PLS 4		c.755A>T, p.(Gln252Leu)

aNomenclature of the Human Genome Variation Society (HGVS);

bSingle nucleotide polymorphism (SNP).

Peripheral blood samples from all patients displayed apparently normal leukocyte counts with normal levels of circulating neutrophils that presented with normal size, morphology and granularity as based on microscopy and flow cytometry (Fig 1A and 1B).

Fig 1

Cytospins of isolated neutrophils and flow cytometry scatter plots of leukocytes samples.

(A) Representative micrographs of isolated neutrophils from PLS 2 and a healthy control. Neutrophils were fixed on glass coverslips with a cytocentrifuge and stained with Giemsa and May-Grünwald staining. Magnification, 100x. Scale bars, 10 μm. (B) Whole blood samples were treated with FACS lysing solution and measured in a flow cytometer. Presented here through forward- and side-scatter for size and granularity, respectively. The gates highlight the granulocyte population in the samples.

Cathepsin C; activity and presence

Neutrophils isolated from peripheral blood samples of the patients in family A were used to prepare lysates in order to assess enzymatic activity of CTSC. We included neutrophil lysates from family B (n = 2) as well as those from a small cohort of healthy control donors (n = 12) with no history of periodontal problems.

A fluorescence-based activity assay was used to measure the activity of CTSC where an enzyme-specific substrate (H-GR-AMC) was exposed to neutrophil lysates; the proteolytic activity of CTSC cleaves this substrate to generate free AMC, the fluorescence of which was measured over time (Fig 2A). Neutrophil lysates from family A were completely devoid of CTSC activity (Fig 2B), indicating that the 503A>G mutation is a complete loss-of-function mutation. Similarly, CTSC activity was completely absent in neutrophil lysates from family B. Among healthy donors, a pronounced interindividual variation in CTSC activity was found even when all lysates were assayed side-by-side on a single plate (Fig 2B).

Fig 2

Fluorescence-based protease activity assay for CTSC activity of neutrophil lysates.

(A) A representative result of CTSC activity assay with lysates from neutrophil samples from a healthy control, PLS 1, and a lysate-free buffer control. Symbols are means ± standard deviations (of three technical repeats) and lines represent the result of the linear regression. (B) Protease activities in neutrophils of patients with PLS (family A, n = 2 and family B, n = 2) and healthy controls (n = 12) resulting from the linear regression of the fluorescence measurement on the linear section of the kinetic graph (0–12 min). Each symbol represents mean per individual (of three technical repeats) and the horizontal line represents the mean of all samples from the indicated groups.

Having showed the absence of CTSC activity, we wanted to investigate whether CTSC protein could be found in the neutrophils of family A. Immunoblotting for CTSC demonstrated that neutrophils from both patients of family A lacked CTSC, whereas control samples displayed clear bands at the expected molecular weight, around 23kD (Fig 3).

Fig 3

Immunoblot of CTSC on neutrophil lysates of family A and two healthy controls.

Neutrophil lysates (31,250 cell equivalents/sample) from patients of family A and two healthy controls are shown. Recombinant CTSC (1.0 ng/lane) is shown as a positive control. The blot is one out of? similar blots.

Activities of downstream serine proteases

We next quantified the enzymatic activity of the NSPs downstream of CTSC using the following substrates: Abz-APEEIMRRQ-EDDnp for HNE, Abz-VADXRDR-EDDnp for PR3, and Abz-EPFWEDQ-EDDnp for CTSG. Like our results for CTSC activity (Fig 2B), levels of neutrophil NSP activity were highly variable within our cohort of healthy donors (Fig 4). Cell lysates from all patients with PLS lacked NSP activity and there were no apparent differences between samples from family A and B (Fig 4).

Fig 4

Fluorescence-based protease activity assay for NSP activity of neutrophil lysates.

(A, C, E) Representative results of HNE, PR3 and CTSG activity assays, respectively, with neutrophils lysates (in triplicate) of one healthy control, PLS 1, and lysate-free buffer control. Symbols are means ± standard deviation and lines represent the linear regressions of the fluorescence measurement on the linear section of the kinetic graphs (0–12 min). (B, D, F) Protease activities of neutrophils from patients with PLS (family A, n = 2 and B, n = 2) and healthy controls (n = 12) resulting from the linear regression of the fluorescence measurement (0–12 min). Each symbol represents mean per individual (of three technical repeats) and the horizontal line represents the mean of all samples from the indicated groups.

Functional assessment of PLS neutrophils

Having confirmed that PLS neutrophils with the 503A>G CTSC mutation indeed lacked NSP activity, we next wanted to explore whether this would entail any alterations in basic neutrophil functions. Therefore, neutrophils of patients from family A were tested in a variety of functional in vitro assays. The neutrophils were primed with the help of TNFα and responded normally to stimulation by translocating complement receptors from the granule membranes to the cell surface and shedding surface L-selectin (Fig 5A). The PLS neutrophils from family A responded normally with regards to in vitro chemotaxis towards fMLF (Fig 5B). Neutrophils from PLS 2 displayed more pronounced chemotaxis towards fMLF than PLS 1, but both responses were clearly within the range displayed by cells from healthy blood donors (Fig 5B).

Fig 5

Activation of neutrophils in whole blood samples, chemotaxis of neutrophils and apoptosis regulation of neutrophils.

(A) Histograms of flow cytometry analysis showing the ability of the neutrophils to regulate the surface marker expression during activation. Results shown are from the patients of family A (PLS 1 & 2) and one representative healthy control. Neutrophils were left unstimulated or activated with TNFα (10 ng/ml) for 20 min at 37°C and labelled for extracellular markers CD62L, CD11b and CD35. (B) Bar diagram summarizing the migration for family A and healthy controls (n = 7). Isolated neutrophils were allowed to migrate through a polycarbonate chemotaxis membrane with a pore size of 3 μm towards buffer or fMLF (10 nM) for 90 min at 37°C, 5% CO2. The migrated cells were lysed, and the number of cells calculated by measuring the total amount of MPO-activity per well relative to a well corresponding to the total amount of cells. (C) Diagram showing the percentage of apoptotic cells after incubation with indicated stimulus for patients of family A (experiment for PLS 2 performed twice on different occasions), family B, and healthy controls (n = 10). Isolated neutrophils were placed in cell-culture buffer substituted with anti-apoptotic LPS, pro-apoptotic CD95 or left unstimulated for 20 h of incubation at 37°C, 5% CO2 before measurement by flow cytometry. Cells that stained positive for Annexin-V but negative for 7-AAD were classified as apoptotic.

The ability of the PLS neutrophils to regulate cell death was tested with the help of anti-apoptotic (LPS) or pro-apoptotic (anti-CD95) factors [42, 47]. Neutrophils from all four patients and ten healthy controls were incubated and analyzed on several occasions. In the case of PLS 2, we had the opportunity to perform the experiment on two separate occasions. From this limited data we conclude that the PLS neutrophils respond normally with decreased apoptosis in response to LPS and increased apoptosis when stimulated with anti-CD95 (Fig 5C). Only the cells from PLS 2 seem to display a lower level of spontaneous apoptosis, below the range observed in the 12 healthy controls, and these cells also did not respond to the same extent to anti-apoptotic LPS as did neutrophils form other individuals (Fig 5C). Importantly, the suppressed spontaneous neutrophil apoptosis displayed by PLS 2 was not observed for any other patient with PLS of neither family A nor B.

PLS neutrophils were able to mount a seemingly normal respiratory burst upon stimulation with PMA; both when measuring extracellular release of ROS and when measuring ROS produced intracellularly. Based on our earlier findings [48], we know that the ROS produced by neutrophils varies greatly in the general population; with this in mind we conclude that the radical responses of the PLS neutrophils fall within the range of the biological variation (Fig 6).

Fig 6

Extra- and intracellular ROS responses upon PMA stimulation.

(A, B) Isolated neutrophils of patients from family A and B as well as one healthy control were suspended in buffer substituted with Isoluminol and HRP and stimulated with PMA. The extracellular ROS production was recorded as chemiluminescence (CL) emitted by the system. (C, D) Isolated neutrophils of patients from family A and B as well as one healthy control were suspended in buffer substituted with luminol and SOD+catalase and stimulated with PMA. The intracellular ROS production was recorded as CL emitted by the system.

Finally, we investigated in vitro NETs release in response to PMA stimulation, a functional response reported to be altered in PLS neutrophils as it is dependent on HNE activity [11]. Control neutrophils typically form NETs from 2 hours after stimulation, but up until 3 hours of incubation with PMA only very few PLS neutrophils from family A had produced NETs as visualized microscopically (Fig 7A). The Sytox green assay (Fig 7B) confirmed this observation, showing that PMA-triggered NET formation was virtually absent <3 h. This is in line with previous reports on defect PMA-triggered NET formation by PLS neutrophils [9, 10]. However, upon prolonged incubation, PMA stimulated PLS neutrophils from PLS 1 and 2 in fact released significantly (p = 0.044, Paired Student’s t-test, PLS unstimulated vs. PLS+PMA, >3h, n = 2) more NETs than unstimulated PLS cells (Fig 7B). The experiment was performed on two separate occasions, for two different healthy controls, once for PLS 1 and twice for PLS 2. These data show that even though PMA triggered NETs release is severely hampered, the process is not completely absent in PLS neutrophils.

Fig 7

NETosis of neutrophils upon PMA stimulation.

(A) Micrographs showing neutrophils on coverslips after 3 h of incubation. Isolated neutrophils were allowed to settle on coverslips and incubated with a cell culture buffer with or without PMA (50 nM) for 3 h at 37°C, 5% CO2. After fixation, the cells were stained with the DNA-dye DAPI (blue) and anti-MPO stain (green) to highlight the formation of NET-structures. Magnification, 40x. Scale bars, 50 μm. Representative images are shown for PLS 2 and one healthy control with or without PMA stimulation. (B) Graphs showing the amount of extracellular DNA during incubation of neutrophils with PMA. Representative results for PLS neutrophils of family A (PLS 2) and one healthy control. Isolated neutrophils were suspended in cell culture medium with the DNA-dye Sytox Green (1.25 μM), with or without PMA (50 nM). The cells were cultured for 4 h at 37°C, 5% CO2 and fluorescence intensity from extracellular DNA was recorded regularly. Means ± standard deviations of triplicates are shown.

Discussion

In this study we characterized a rare CTSC mutation in two siblings with PLS from different aspects, e.g., protease activity, neutrophil structure and function. With the help of whole genome sequencing on the patients of family A we identified a missense mutation in the CTSC gene, more specifically a 503A>G transversion in exon 4 predicted to cause an amino acid substitution at position 168 from tyrosine to cysteine. In databases linking mutations to human disease, this mutation was not previously reported. We also did not find any reported homozygous individuals in genome databases such as gnomAD. Beyond the siblings described here, we are only aware of one patient with PLS homozygous for this exact mutation [9]. Besides a control cohort of healthy volunteers, we had the opportunity to compare the patients of family A to patients of another family also suffering from PLS with a 755A>T mutation in the CTSC gene, a mutation earlier described in Toomes et al. and Hewitt et al. [7, 46]. Over 50 different types of mutations of the CTSC gene have been classified as PLS [10], identified as, e.g., missense, nonsense or acceptor splice site mutations [7]. These mutations have been reported to either lead to absence or inactivation of the CTSC protease [10], ultimately leading to activity levels unable to maintain maturation of the NSPs. It is generally accepted that PLS is not an immune deficiency in the classical sense as an increased susceptibility to opportunistic infections is not widely observed [49, 50]. This was also the case in our patient group, except for one patient in family B that had one reported episode of a purulent skin cyst.

As expected in patients with PLS, neither CTSC activity nor downstream NSP activity was detected in neutrophil lysates and there were no differences between cells from family A and family B. Furthermore, no CTSC protein was found in neutrophil lysates from family A. This indicates that the 503A>G mutation indeed is a complete loss-of-function mutation that does not result in a stable, functional protein. Neutrophils with a 503A>G mutation, identical to that found in family A, have been studied previously [9] and found to lack the CTSC and the NSPs. This fits well with the complete absence of CTSC protein and NSP activity reported here. When quantifying protease activities of the healthy volunteers in our control cohort, a wide interindividual variation was observed for CTSC as well as for NSP activity. Whether this substantial variation in, e.g., PR3 activity among healthy control neutrophils (Fig 4C and 4D) is the result of differences in cellular PR3 amounts, different PR3 variants, or levels/activity of endogenous PR3 inhibitors we cannot say, but it is intriguing that a set number of neutrophils from healthy blood donors can display such wide variation in terms of protease activity. Regardless of this wide variation among healthy controls, it was clear that PLS samples stood out and lacked measurable protease activities altogether. In fact, PLS lysates were most often indistinguishable from cell-free buffer controls and sometimes displayed readings even lower than buffer controls.

Like earlier described PLS mutations, the neutrophils from family A were normal in abundance and appearance. One recent study that investigated neutrophil function in patients with PLS found that patient cells displayed reduced velocity and chemotactic accuracy towards fMLF, as compared to control neutrophils [10]. In another paper the same authors found that neutrophils from chronic non-PLS periodontitis also present with lower speed, velocity and directional accuracy towards fMLF as compared to healthy controls [51], which makes it hard to determine if it is the lack of NSP activity or the periodontal disease activity that affect the directional migration of PLS neutrophils. In our hands, the net-migration of PLS neutrophils was well within the range displayed by healthy control neutrophils. Protease activities as well as the magnitude of functional responses of primary neutrophils of healthy donors showed substantial interindividual variation. This remarkable variation has been previously described, e.g., regarding the magnitude of ROS production [48]. That points to the importance of including a substantial cohort of healthy controls when testing whether patient cells function abnormally and is especially important when studying rare diseases where the size of the patient groups is naturally limited. PMA-triggered ROS production of PLS neutrophils have been previously reported to be increased as compared to control neutrophils; a conclusion based on data from four patients and three healthy controls [10]. We did not observe any clear-cut differences regarding ROS production and when comparing our patient data with a larger set of controls [48] it was clear that all four PLS samples were well within the normal range of responses. Based on our functional assays, we observed no clear-cut differences that were common for all patients with PLS, or for both patients of one family. Thus, these data indicate that lack of CTSC and NSP activity does not alter neutrophil priming, chemotaxis towards fMLF, regulation of apoptosis or PMA-induced ROS production. Among these assays, only the spontaneous neutrophil apoptosis displayed by PLS 2 was outside the range of healthy controls. This feature was however not shared by any other patient with PLS and is thus likely not the direct result of abolished protease activity. It is possible that the prolonged and severe peri-implantitis experienced by PLS 2 is reflected as systemically suppressed spontaneous neutrophil apoptosis as is the case during, e.g., sepsis and autoinflammation [47, 52]. In previous work, it has been speculated that an overall heightened anti-apoptotic milieu already leads to a prolonged life span in neutrophils, thus anti-apoptotic stimulants like LPS cannot reduce the occurrence of spontaneous apoptosis further [47, 53]. This would fit well with the weak response to LPS displayed by neutrophils from PLS 2.

One neutrophil function described by many to be dependent on HNE activity, and thus defective in PLS neutrophils, is NET formation in response to PMA stimulation [10, 11]. In line with this, we observed a potently delayed as well as reduced NET formation by neutrophils from all patients with PLS. Our previous work showed that PLS neutrophils (from PLS 2) are perfectly able to form NETs when stimulated with the membrane-disturbing protein PSMα2 [40] and it is thus clear that NETs may be induced in both HNE-dependent and independent ways. Thus, it is more correct to say that PLS neutrophils are deficient in terms of PMA-triggered NET formation, as opposed to being deficient for NET formation in general. The importance of NETs, both ROS or HNE dependent, for human health is not clear, but it is interesting to note that though both CGD and PLS are deficient for PMA-induced in vitro NETs, only CGD is characterized by frequent infections. This observation could be taken in support of the view that the susceptibility to infections in CGD has little to do with the inability of CGD neutrophils to form NETs in response to PMA. It is also unlikely that a dysfunctional NETs response in PLS is linked to the periodontal pathology of these patients, since CGD is not typically characterized by periodontal inflammation. Furthermore, PMA-triggered NET formation is also dependent on intracellular ROS production and MPO activity [54, 55] and since these processes were not dysfunctional in PLS neutrophils, the lack of HNE activity is the likely explanation to the reduced levels of PMA-induced NETs formed by PLS neutrophils. Importantly, even though PLS neutrophils displayed reduced NET formation in response to PMA, significant NET formation was in fact detected upon longer incubation times (>3 h) in comparison to PMA-free controls. Thus, HNE activity is a strong influence on the production of PMA-triggered NETs, but it is not indispensable. However, we cannot completely rule out that residual HNE activity in the PLS neutrophils, below the detection limit of our assay, can explain the fact that PLS neutrophils form some NETs after being stimulated with PMA.

In conclusion, in this study we demonstrate that the 503A>G CTSC mutation identified in family A compares to previously described PLS mutations in terms of aggressive periodontal pathology and characteristics of neutrophil functions. The changes in neutrophil function caused by CTSC mutations seem limited to the absence of protease activity and the diminished formation of NETs upon PMA stimulation.

(PDF)

Click here for additional data file.

4 Aug 2021

PONE-D-21-22412

A rare CTSC mutation in Papillon-Lefèvre Syndrome results in abolished serine protease activity and reduced NET formation but otherwise normal neutrophil function

PLOS ONE

Dear Dr. Sanchez Klose,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Specific revisions must address issues raised by reviewers, especially:

- CTSC protein levels in neutrophils by western blot or flow cytometry including potential differences between families A and B.

- changes in figures (dot plots instead of box/whiskers) and in manuscript text as suggested by reviewers

-better delineate the effect of new mutation on NET by comparing NET release induced by other physiological agonists and in comparison with other patients with other PLS mutations.

Please submit your revised manuscript by Sep 18 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Charaf Benarafa, D.V.M., Ph.D.

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

1. When submitting your revision, we need you to address these additional requirements.

Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. We note that the grant information you provided in the ‘Funding Information’ and ‘Financial Disclosure’ sections do not match.

When you resubmit, please ensure that you provide the correct grant numbers for the awards you received for your study in the ‘Funding Information’ section.

3. We note that you have stated that you will provide repository information for your data at acceptance. Should your manuscript be accepted for publication, we will hold it until you provide the relevant accession numbers or DOIs necessary to access your data. If you wish to make changes to your Data Availability statement, please describe these changes in your cover letter and we will update your Data Availability statement to reflect the information you provide.

4. Please note that in order to use the direct billing option the corresponding author must be affiliated with the chosen institute. Please either amend your manuscript to change the affiliation or corresponding author, or email us at plosone@plos.org with a request to remove this option.

5. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

Reviewer #2: N/A

Reviewer #3: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: In this manuscript the authors examined two siblings with PLS due to a rare mutation in CTSC, 503A>G and compared them to another family of PLS previously described. Although this mutation is unusual, it has been previously described (ref 9). Also, one of the siblings, PLS2’s ability to form NET was previously reported (ref 41). Otherwise, the authors suggested that the neutrophil morphology, priming, chemotaxis, ROS production, and regulation of apoptosis were unchanged but NET formation upon PMA stimulation was severely depressed. These phenotypes were previously reported in PLS patients. PLS2, however, displayed more pronounced chemotaxis towards fMLF than PLS1 and lower level of spontaneous apoptosis. Given that most neutrophil phenotypes are similar to previously described PLS mutations, it would have been interesting to understand why PLS2 behaves differently. Otherwise, there is no or minimal gain in understanding of PLS offered by this manuscript.

Other points:

- Why not present NSP activities for all four PLS patients individually instead of box and whisker plots for each family?

- Also, why use box and whisker plots for figure 3 but not for figure 2?

- In figure 4, why was comparison to PLS3 and 4 not presented for all experiments?

- In figure 6, why was NET formation shown only for PLS2? Do PLS1, 3, and 4 neutrophils have the same delayed response to PMA as PLS2? What about NET formation to PSMa2?

Reviewer #2: Klose and coworkers described and analyzed four PLS patients from two families (A,B) with two different homozygous mutations in the cathepsin C gene. The mutation found in family A is an extremely rare allele deposited in some databases, and only one individual with homozygosity for the same Y168C residue substitution has been discovered in a previous paper. The second variation, Q252L, was reported to occur in one affected member of another family 20 years ago, but two other missense changes were also found in the CTSC gene of this individual. Hence the important work of Klose et al. underscores the clinical significance of these homozygous changes in the CTSC sequence and presents a variety of functional and biochemical data for neutrophils derived from new patients.

To improve the manuscript, the following points should be addressed.

Line 21: replace “indicated to be involved” by “suggesting its involvement in a …”

The statement refers to CTSC and its impact on neutrophil functions. The three NSPs may be present in active form at low levels in some neutrophil populations and could be converted by other enzymes during maturation or after release.

Line 22: delete “however” and “consequentially”

Line 44: Homozygosity of CTSC mutations is frequently observed in consanguineous families and in small ethnic populations with a high degree of inbreeding. Other genetic variations may modify and contribute to the clinical manifestations and the functional impairments of neutrophils. To explain all manifestations as the result of a monogenic disease is an oversimplification.

Line 59: insert “to” killing and to degrade

Line 63: It is inappropriate to equal cathepsin G (Ctsg) and elastase (Ela2) deficient mice with PLS patients. Double deficient mice do not acquire infections spontaneously. The differences observed between wild type and double deficient mice are only seen under extreme experimental conditions titrating these mice with increasing doses of bacteria until they die. The authors should compare PLS patients with Ctsc-deficient (Ctsc-null) mice which are healthier than affected humans and do not suffer from periodontitis. Azurocidin is completely absent in all rodents by contrast to humans and may play a human-specific function which missing in PLS. The state of the research should be reported correctly.

Line 448: Ela2-deficient or CTSG-deficient mice are not more susceptible to inflammations and infections than wild type mice under normal housing conditions. Differences are only seen under extreme experimental challenges. This interpretation of animal models gives a wrong impression to your readers, I am afraid.

Line 506: please modify this sentence to make its content clear “how important NET formation induced by persistent high ROS levels and executed by HNE is in vivo

Line 514: the authors are obviously very familiar with the JCI paper of Sorensen OE et al. from 2014, in which eosinophil peroxidase was found to be strongly reduced in PLS neutrophils (supplemental table). Have they indeed convinced themselves independently that peroxidase activities and peroxidase levels were unaltered in the neutrophils of the patients they have studies?

Reviewer #3: In their manuscript, Sanchez Klose and collegues study a CTSC mutation that is associated with Papillon-Lefèvre Syndrome. The reported a new mutation in CTSC and characterise it’s impact on neutrophil functions. The manuscript is interesting and well-``written, and I believe interesting for Plos One readership. I have a few comments that I hope will be helpful to the authors.

Major points:

It is unclear what are the consequence of the mutation. Is this mutation affecting the stability/half-life or expression of CTSC? A simple Western Blot probing for CTSC would be very appropriate here to confirm that the protein expression is not affected by the mutation.

Figure 4C): It seems that CD95 is not increasing much apoptosis (over basal level of death in untreated cells). Would an earlier time point be more appropriate to measure cell death?

Minor points:

Line 152: What is the calcium concentration?

Figure 6A: A quantification of the microscopy slides would be relevant here. Would it be possible to report the proportion of cells generating NETs upon PMA treatment?

It would be intereting to discuss more the effect of Y168C on the protease activity. Why is it affecting the activity? Could you suggest an effect based on CTSC structure?

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

28 Oct 2021

Reviewer #1:

In this manuscript the authors examined two siblings with PLS due to a rare mutation in CTSC, 503A>G and compared them to another family of PLS previously described. Although this mutation is unusual, it has been previously described (ref 9). Also, one of the siblings, PLS2’s ability to form NET was previously reported (ref 41). Otherwise, the authors suggested that the neutrophil morphology, priming, chemotaxis, ROS production, and regulation of apoptosis were unchanged but NET formation upon PMA stimulation was severely depressed. These phenotypes were previously reported in PLS patients. PLS2, however, displayed more pronounced chemotaxis towards fMLF than PLS1 and lower level of spontaneous apoptosis. Given that most neutrophil phenotypes are similar to previously described PLS mutations, it would have been interesting to understand why PLS2 behaves differently. Otherwise, there is no or minimal gain in understanding of PLS offered by this manuscript.

Other points:

- Why not present NSP activities for all four PLS patients individually instead of box and whisker plots for each family?

Response: We have changed the graphs to individual scatter dot plots.

- Also, why use box and whisker plots for figure 3 but not for figure 2?

Response: We have changed the plots.

- In figure 4, why was comparison to PLS3 and 4 not presented for all experiments?

Response: The differences in presented data comes from limited availability of patients for sample donation. Also, the limited amounts of blood/PMNs retrieved restricts the experiments that can be performed on one given occasion. Furthermore, patients of family B are children, thus the sample volumes and patient access have been limited.

- In figure 6, why was NET formation shown only for PLS2? Do PLS1, 3, and 4 neutrophils have the same delayed response to PMA as PLS2? What about NET formation to PSMa2?

Response: Thank you for that feedback, we would have liked to include more experiments as well, but the aforementioned limitations also apply here. On top of that we show only PLS 2 as the result was representative of family A. We now clarified that in the text and made it clear that also neutrophils from PLS 1 demonstrate the delayed response. Regarding NET formation through PSMa2, we have previously shown that neutrophils from PLS2 indeed form NETS in response to PSMa2, to the same extent as do neutrophils form healthy controls. In the text we refer to this work (Björnsdottir H et al., Front. Immunol., 8, 257 (2017)).

Reviewer #2:

Klose and coworkers described and analyzed four PLS patients from two families (A,B) with two different homozygous mutations in the cathepsin C gene. The mutation found in family A is an extremely rare allele deposited in some databases, and only one individual with homozygosity for the same Y168C residue substitution has been discovered in a previous paper. The second variation, Q252L, was reported to occur in one affected member of another family 20 years ago, but two other missense changes were also found in the CTSC gene of this individual. Hence the important work of Klose et al. underscores the clinical significance of these homozygous changes in the CTSC sequence and presents a variety of functional and biochemical data for neutrophils derived from new patients.

To improve the manuscript, the following points should be addressed.

Line 21: replace “indicated to be involved” by “suggesting its involvement in a …”

The statement refers to CTSC and its impact on neutrophil functions. The three NSPs may be present in active form at low levels in some neutrophil populations and could be converted by other enzymes during maturation or after release.

Response: We have changed the sentence accordingly.

Line 22: delete “however” and “consequentially”

Response: This has been amended.

Line 44: Homozygosity of CTSC mutations is frequently observed in consanguineous families and in small ethnic populations with a high degree of inbreeding. Other genetic variations may modify and contribute to the clinical manifestations and the functional impairments of neutrophils. To explain all manifestations as the result of a monogenic disease is an oversimplification.

Response: We strongly agree with the reviewer that patients with PLS could be a very heterogeneous group with respect to different hereditary predispositions and clinical symptoms. Therefore, it is added value that we often compare 2 distinct PLS families. We have clarified the language throughout the manuscript to make that clearer and to not overinterpret the direct influence of CTSC mutations on especially the clinical manifestations.

Line 59: insert “to” killing and to degrade

Response: Thank you, the change was made.

Line 63: It is inappropriate to equal cathepsin G (Ctsg) and elastase (Ela2) deficient mice with PLS patients. Double deficient mice do not acquire infections spontaneously. The differences observed between wild type and double deficient mice are only seen under extreme experimental conditions titrating these mice with increasing doses of bacteria until they die. The authors should compare PLS patients with Ctsc-deficient (Ctsc-null) mice which are healthier than affected humans and do not suffer from periodontitis. Azurocidin is completely absent in all rodents by contrast to humans and may play a human-specific function which missing in PLS. The state of the research should be reported correctly.

Response: We agree that the mouse models are not reflective of the specific phenotype present in patients with PLS, and the insights from animal experiments are not easy to translate to a human setting. Therefore, we decided to remove all mentioning of murine models from our manuscript.

Line 448: Ela2-deficient or CTSG-deficient mice are not more susceptible to inflammations and infections than wild type mice under normal housing conditions. Differences are only seen under extreme experimental challenges. This interpretation of animal models gives a wrong impression to your readers, I am afraid.

Response: We agree and we have therefore removed all mentioning of murine models from the revised manuscript.

Line 506: please modify this sentence to make its content clear “how important NET formation induced by persistent high ROS levels and executed by HNE is in vivo

Response: The paragraph about NET formation has been changed for clarity.

Line 514: the authors are obviously very familiar with the JCI paper of Sorensen OE et al. from 2014, in which eosinophil peroxidase was found to be strongly reduced in PLS neutrophils (supplemental table). Have they indeed convinced themselves independently that peroxidase activities and peroxidase levels were unaltered in the neutrophils of the patients they have studies?

Response: Yes, we have in fact read that particular paper a number of times, but this intriguing detail had escaped our notice and we did not perform any direct experiments on peroxidase (EPO or MPO) activities and levels. Since the ROS method employed for determining intracellular ROS production is completely dependent on MPO activity (Björnsdottir H et al., Free Radical Biology and Medicine, 89 (2015)), the fact that PLS neutrophils are within the normal range of responses, indicate that at least MPO activity of the cells are normal. As we argue in the manuscript, a much bigger patient and control group would be necessary to make clear assessments on subtle differences of ROS production and peroxidase activities.

Reviewer #3:

In their manuscript, Sanchez Klose and collegues study a CTSC mutation that is associated with Papillon-Lefèvre Syndrome. The reported a new mutation in CTSC and characterise it’s impact on neutrophil functions. The manuscript is interesting and well-``written, and I believe interesting for Plos One readership. I have a few comments that I hope will be helpful to the authors.

Major points:

It is unclear what are the consequence of the mutation. Is this mutation affecting the stability/half-life or expression of CTSC? A simple Western Blot probing for CTSC would be very appropriate here to confirm that the protein expression is not affected by the mutation.

Response: We agree with the reviewer that it would be very interesting to figure out the concrete effect of the mutation. For that reason, we have performed the WB as suggested, and the data clearly demonstrate that protein expression is in fact severely affected by the mutation and both patients of family A lack CTSC protein. We have now added one representative blot to the manuscript (new figure 3) and discuss these data in the result as well as discussion section of the revised manuscript.

The figure shows a western blot of recombinant human CTSC, neutrophil lysates of patients with PLS (PLS 1 and 2) as well as two healthy controls. The band for CTSC is expected around 23 kDa.

Figure 4C): It seems that CD95 is not increasing much apoptosis (over basal level of death in untreated cells). Would an earlier time point be more appropriate to measure cell death?

Response: A valid point. Shorter incubations would have been better to see CD95-induced acceleration of apoptosis, but 20 h is typically the time point where roughly half of the incubated neutrophils (without any additions) are apoptotic which enable us to detect both decrease (+LPS) and increase (+CD95) in the same experimental setup.

Minor points:

Line 152: What is the calcium concentration?

Response: The calcium concentration (1mM) has been added.

Figure 6A: A quantification of the microscopy slides would be relevant here. Would it be possible to report the proportion of cells generating NETs upon PMA treatment?

Response: For quantification, we decided to rely on the fluorescent DNA probe Sytox-green to measure the amount of NETs released over time by measuring the amount of extracellular DNA in the samples with the help of a plate reader. The microscopy slides were used to illustrate the appearance of NETs as well as the presence of MPO. By this we get both qualitative (microscopy) and quantitative (Sytox) data for NET formation. Such combination of two methods is commonly employed also by others.

It would be intereting to discuss more the effect of Y168C on the protease activity. Why is it affecting the activity? Could you suggest an effect based on CTSC structure?

Response: An interesting point. We have tried to find structural information related to this position (168), but even though CTSC has been crystallized and structurally defined (see, e.g., Uniprot, #P53634), the amino acids around 168 are located in between two beta strands and appear to not be structurally defined. However, as based on our immunoblotting for CTSC it seems that the (mutant) protein is not expressed in neutrophils from patients with PLS (see response above and new fig 3). We can at present not state the reason why the mutated protein is absent, but this this finding of course explains why no CTSC activity was measured from these cells.

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

9 Dec 2021

A rare CTSC mutation in Papillon-Lefèvre Syndrome results in abolished serine protease activity and reduced NET formation but otherwise normal neutrophil function

PONE-D-21-22412R1

Dear Dr. Sanchez Klose,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Charaf Benarafa, D.V.M., Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: All comments have been addressed

Reviewer #3: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: Yes

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: N/A

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #2: Yes

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #2: Yes

Reviewer #3: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: (No Response)

Reviewer #3: My comments have been addressed to a reasonable extend. I have no other concerns regarding this manuscript.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #2: No

Reviewer #3: No

13 Dec 2021

PONE-D-21-22412R1

A rare CTSC mutation in Papillon-Lefèvre Syndrome results in abolished serine protease activity and reduced NET formation but otherwise normal neutrophil function

Dear Dr. Sanchez Klose:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Prof. Dr. Charaf Benarafa

Academic Editor

PLOS ONE