Reduced ELANE and SLPI expression compromises dental pulp cell activity

Abstract Background Patients with ELANE variants and severe congenital neutropenia (SCN) commonly develop oral complications. Whether they are caused only by low neutrophil count or the combination of neutropenia and aberrant dental cells is unknown. Methods Genetic variant was identified with exome sequencing. Dental pulp cells isolated from the SCN patient with an ELANE mutation were investigated for gene expression, enzyme activity, proliferation, colony formation, wound healing, apoptosis, ROS, attachment, spreading and response to lipopolysaccharide. Results ELANE cells had diminished expression of ELANE and SLPI and reduced neutrophil elastase activity. Moreover, ELANE cells exhibited impaired proliferation, colony forming, migration, attachment and spreading; and significantly increased ROS formation and apoptosis, corresponding with increased Cyclin D1 and MMP2 levels. The intrinsic levels of TGF‐β1 and TNF‐α were significantly increased; however, IL‐6, IL‐8 and NF‐kB1 were significantly decreased in ELANE cells compared with those in controls. After exposure to lipopolysaccharide, ELANE cells grew larger, progressed to more advanced cell spreading stages and showed significantly increased SLPI, TNF‐α and NF‐kB1 and tremendously increased IL‐6 and IL‐8 expression, compared with controls. Conclusion This study, for the first time, suggests that in addition to neutropenia, the aberrant levels and functions of ELANE, SLPI and their downstream molecules in pulp cells play an important role in oral complications in SCN patients. In addition, pulp cells with diminished neutrophil elastase and SLPI are highly responsive to inflammation.


| INTRODUC TI ON
Severe congenital neutropenia (SCN) is a heterogenous group of haematological disorders characterized by impaired promyelocytic proliferation and maturation with an absolute neutrophil count <500 cells/µL. Patients affected with SCN are prone to recurrent, often life-threatening, infections that usually occur in the mucous membranes, skin and oral cavity. 1 Neutrophils are the mainstay innate immune cells that are responsible for ensuring a healthy periodontal tissue. 2 Common oral manifestations of SCN are gingivitis, periodontitis, ulcers and early tooth loss. 3 More than 24 genes are associated with SCN; however, variants in ELANE account for >50% of SCN cases. 4 ELANE encodes neutrophil elastase (NE), which exhibits antimicrobial effects. During infection, neutrophils release NE that cleaves the extracellular matrix protein elastin and modulates the expression of several cytokines, chemokines and growth factors. 5 ELANE variants can cause NE misfolding, mistrafficking, and mislocalization, stress response pathway induction and promyelocyte apoptosis. 6 Excess NE activity results in tissue damage.
A secretory leucocyte protease inhibitor (SLPI), a natural NE inhibitor, acts to counterbalance NE in a dose-dependent manner. 7 SLPI has multifunctional properties including antiprotease, antibacterial, antiviral and anti-inflammatory functions. 8,9 SLPI is also involved in wound healing and cancer metastasis. 10 Currently, the expression and pathogenic role of SLPI and ELANE in dental cells remain elusive.
The diseased cell phenotype is physiologically and molecularly different from that of healthy cells. They are good models to investigate molecular pathomechanisms and explore new and better biomarkers for early disease detection, including when signs and symptoms are barely discernable. We speculated that an imbalance in NE and SLPI might be involved in orodental inflammation and believed that the dental cells obtained from a patient with SCN and ELANE variant could be used to prove our hypothesis. Here, we identified that the diminished ELANE and SLPI expression, as well as NE activity regulate the behaviour, survival and inflammatory responses of dental pulp cells. After exposure to liposaccharide, the patient's pulp cells exhibited significantly higher inflammatory cytokine expression. Our findings suggest that ELANE and SLPI are important regulators in the inflammatory response of dental pulp cells.

| Subject
This study was approved by the Institutional Review Board, Faculty of Medicine, Chulalongkorn University (IRB 316/63) and in accordance with the 1964 Helsinki Declaration and its later amendments.
Written informed consent was obtained from the patient or a legal guardian. Genotype and dental phenotypes analyses were performed as previously described. [11][12][13]

| Mutation analysis
Genomic DNA was extracted from peripheral blood leucocytes of the patients using Puregene Blood Kit (Qiagen) and sent for exome sequencing using lllumina Hiseq 2000 Sequencer (Macrogen). The sequences were aligned to the University of California Santa Cruz (UCSC) hg19 using Burrows-Wheeler Aligner (http://bio-bwa.sourc eforge.net/). Downstream process was performed by SAMtools (samtools.sourceforge.net/) and annotated against dbSNP and 1000 Genomes. The variants were filtered using the following criteria: (a) The identified variant was verified by PCR-Sanger sequencing.

| Characterization of a patient's teeth
Clinical and radiographic examination were performed. The patient's deciduous upper central incisor was extracted due to tooth mobility and scanned using specimen micro-computed tomography and compared with three incisors from age-matched healthy individuals. After the pulp tissues were explanted, the tooth samples were scanned with Specimen Micro-CT35 (SCANCO Medical). Ten spots in the enamel and ten spots in the dentin areas were selected to quantify their mineral densities.

| Cellular and molecular characteristics of ELANE dental pulp cells
Dental pulp cells were isolated from the patient's deciduous upper central incisors (ELANE cells). They were investigated in comparison with three lines of dental pulp cells from the deciduous incisors of age-matched healthy individuals. Briefly, the teeth were rinsed with PBS. The pulp tissues were removed and cut into 1 × 1 mm pieces and placed in a 35-mm culture dish (Corning). The explants were maintained in growth medium composed of Dulbecco's Modified Eagle Medium (DMEM) containing 10% foetal bovine serum (Gibco), 1% L-glutamine (Gibco) and 1% penicillin and streptomycin (Gibco), cells play an important role in oral complications in SCN patients. In addition, pulp cells with diminished neutrophil elastase and SLPI are highly responsive to inflammation. incubated in a humidified environment at 37℃ and 5% CO 2 . Cells from passages 4-6 were used in the experiments. All experiments were performed at least three times.

| Mesenchymal markers
The isolated cells were characterized using flow cytometry. The and CD73 (Cat no. 21270733, ImmunoTools) was investigated.

| Immunofluorescence microscopy
Cells were cultured on a Chamber Slide TM at density 5 × 10 3 cells/well, with/without 0.1 µg/mL lipopolysaccharide (LPS) for 24 h. The cells were washed and fixed in 4% ice-cold paraformaldehyde at room temperature for 10 min, then rinsed three times for 5 min in PBST consisting of 10 mM phosphate-buffered saline (PBS) and 0.1% tritonX100  anti-goat or goat anti-mouse HRP-conjugated secondary antibodies were incubated (R&D systems; 1:2500) for 2 h at room temperature. The membranes were treated with SuperSignal TM West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific) and analysed by the Amersham TM Imager 680 (GE Healthcare). The intensity of the bands was quantified using ImageJ software.

| Neutrophil elastase (NE) activity
NE activity was measured using a fluorometric neutrophil elastase activity assay kit (Cat# ab204730, Abcam) following modified manufacturer's instructions. A single-cell suspension of ELANE cells and controls were seeded with/without 0.1 µg/mL of LPS in 96-well plates using non-phenol red DMEM with 10% FBS at a density of 2 × 10 4 cells/well and cultured at 37℃ in 5% CO 2 for 24 h. After 24 h, neutrophil elastase substrate was added into each well. The activity was measured using fluorescent microplate readers at Ex/ Em = 380/500 nm in a kinetic mode, every 2 min at 37℃ for 10-20 min. Cells with no substrate added were used as a background control. The amount of NE was calculated: ΔRFU 380/500nm = (RFU 2 -RFU BG ) -(RFU 1 -RFU BG ). RFU 1 , the sample reading at time T 1 ; RFU 2 , the sample reading at time T 2 ; RFU BG , the background control sample.

| Real-time polymerase chain reaction (Realtime PCR)
Total cellular RNA was isolated by RiboEx total RNA isolation solution (GeneAll). The concentration of the isolated RNA was measured using Thermo Scientific Nanodrop one (Thermo Fisher scientific).
iScript Reverse Transcription (Bio-rad) was used for converting RNA into cDNA. Real-time PCR was performed with SYBR green detection system (FastStart Essential DNA Green Master, Roche Diagnostic) and MiniOpticon real-time PCR system (Bio-Rad, Hercules). Primer sequences are shown in Table S1.

| Colony-forming assay
Single-cell suspensions (passages 4-6) were seeded in six-well plates at a density of 300 cells per well and cultured in DMEM with 10% FBS at 37℃ in 5% CO 2 . After 14 days, the cells were fixed in 100% methanol for 10 min and stained with 0.1% crystal violet. Colonies containing more than 50 cells were counted.

| Wound healing assay
Cells were cultured in 12-well plates at a density of 1 × 10 4 cells per well in triplicate until a confluent monolayer was formed. A 200-µL pipette tip was used to make a scratch approximately 100 µm wide.
The cells were then washed with phosphate-buffered saline (PBS) and cultured in DMEM medium with 1% FBS at 37℃ for 48 h. Microphotographs were taken at 0, 24 and 48 h after scratching using a Zeiss microscope equipped with a digital camera. The scratchwound closure percentage was determined using ImageJ software.
The wound healing rate (%) = ((the area of primary wounds-the area of healing)/the area of primary wounds)) x 100.

| Cell apoptosis
Cell apoptotic rate was detected using a FITC Annexin V apoptosis detection kit with propidium iodide (PI) (Cat#640914, BioLegend) following the manufacturer's instructions. Briefly, confluent monolayer cells were harvested and washed with PBS. Then, the cells were resuspended in 100 µL binding buffer containing 5 µL Annexin V-FITC and 10 µL PI, incubated for another 15 min at room temperature in the dark, and 300 µL binding buffer was added. Flow cytometric analysis was performed using FACSCalibur flow cytometer (Becton Dickinson).
The Mann-Whitney U test was chosen because it is distribution-free and proper for experiments with small sample sizes.

| Clinical investigations
The patient was a 7-year-old girl. She was diagnosed with severe congenital neutropenia (SCN) at 10 months old due to persistent neutropenia and decreased myeloid series with arrested myelocyte maturation in the bone marrow. The patient's medical condition and genotype were previously reported. 13 Briefly, the patient had  Figure 1D). The deciduous upper left central incisor was extracted due to mobility at 6 years of age ( Figure 1E). Microscopic images demonstrated hypomineralized spots on the enamel ( Figure 1F). Micro-CT revealed significantly reduced mineral density in the patient's enamel compared with that of controls ( Figure 1G).

| Severely downregulated ELANE and SLPI expression, and NE activity
ELANE cells and controls expressed mesenchymal stem cell markers comprising CD45, CD73, CD90 and CD105, but not CD45, indicating that these cells were mesenchymal cells ( Figure S1). We investigated whether ELANE mutation affected ELANE expression in the dental pulp cells of the SCN patient and observed that ELANE cells had a significantly diminished ELANE expression compared to controls (Figure 2A). Correspondingly, the fluorometric neutrophil elastase activity assay indicated that ELANE cells had significantly decreased NE activity compared with controls ( Figure 2B). Western blots showed that the NE protein level was significantly reduced in ELANE cells compared with that in controls ( Figure 2E, F, Figure S3, Table S2), although the immunofluorescence showed no significant difference in NE expression between ELANE cells and controls ( Figure 2D). Furthermore, ELANE cells exhibited significantly downregulated mRNA and protein expression of SLPI, the natural inhibitor of neutrophil elastase (Figure 2C, E, G, Figure S3, Table S2).

| Impaired proliferation, colony formation and migration
SLPI is involved in multiple aspects of cell behaviours including prolif-  Figure 2H).
Accordingly, the mRNA expression of Cyclin D1, a gene involved in cell proliferation, was significantly reduced in ELANE cells compared with controls ( Figure 2I). In addition, the colony-forming unit (CFU) assay illustrated that the ELANE cell CFUs were significantly fewer compared with controls ( Figure 2J, K). We next evaluated cell mi-  Figure 2N).

| Induced apoptosis and reactive oxygen species (ROS) formation
We speculated that the attenuated proliferation, colony forming and migration in ELANE cells was associated with increased apoptosis and ROS formation. We observed that ELANE cells had a higher number of apoptotic (annexin V-positive) and a higher number of dead cells (annexin/propidium iodide-positive) than controls ( Figure 3A). ELANE cells exhibited significantly higher percentages of cells in early, late and total apoptosis compared with controls ( Figure 3B). ROS formation was also significantly increased in ELANE cells compared with controls ( Figure 3C and Figure S2).

| Reduced cell attachment and spreading
Cell attachment and spreading are important for cell communication in tissue development and maintenance. Altered cell attachment can lead to tissue destruction as reported in many diseases and cancers. 15 Thus, we analysed whether the reduced ELANE cell proliferation was associated with delayed cell attachment and spreading. In vitro cell attachment and spreading were evaluated using scanning electron microscopy. At 30 min, ELANE cells were round with filopodia and/or lamellopodia that were shorter than those of controls. At  Figure 3D and Figure S5).
Next, we compared the percentage of ELANE and control cells in each cell attachment stage and observed that the majority of ELANE cells at 2 h were at stage 1, while the majority of controls were at stage 3 ( Figure 3E). At 6 h, most ELANE cells were at stage 2, while most controls had entered stage 3 and 4 ( Figure 3F).

| Upregulated inflammatory responses to LPS exposure
To   Figure 4D, E). The SLPI levels in treated ELANE cells and controls were significantly upregulated compared with their corresponding untreated cells; however, the level in treated ELANE cells was significantly lower than those in treated controls ( Figure 4F).
Regarding inflammatory gene marker expression, the LPS-treated ELANE and control cells exhibited significantly higher TNFα expression compared with untreated cells ( Figure 4G). The NF-kB1 levels after LPS treatment were significantly increased in all cells except one control (Figure4H). Conversely, LPS-treated ELANE cells exhibited lower TGF-ß1 expression (0.46-fold change), while controls had a higher expression than untreated cells (1.67-fold change) ( Figure 4I and

| DISCUSS ION
Neutrophils are the major immune cells that play a critical role in the acute inflammatory response and host defences against bacterial infections. The consequence of neutropenia is increased infection susceptibility. Phenotypically, the prime features associated with SCN are dental and periodontal inflammation. Other orodental findings are recurrent dental infection, mouth ulcers, hypomineralized enamel and dental caries. Genotypically, the patient was identified with a de novo heterozygous 12-bp inframe insertion in the ELANE gene. This mutation is located in the peptidase S1 domain.
Our genotype-phenotype correlation found that the SCN patients who harboured ELANE variants had more severe periodontal diseases compared with the cyclic neutropenia patients with ELANE variants or the SCN patients with variants in other genes, such as HAX1. 16 Correspondingly, our SCN patient had early-onset and rapidly progressive periodontitis resulting in severe tooth mobility and premature tooth loss.  also inhibits the TNFα-induced caspase-3 activation associated with apoptosis in monoblast cell lines and peripheral blood monocytes. 9 These findings indicate that the reduced SLPI expression in ELANE cells could lead to apoptosis through upregulated TGF-β1 and TNFα expression together with increased ROS production.
NF-κB serves as a central inflammatory mediator, and its dysregulation results in various inflammatory diseases. SLPI modulates inflammatory cytokines, such as TNFα, TGF-β1, NF-kB1, IL-6 and IL-8, which are involved in NF-κB and TGFβ signalling. 34 In the NF-κB pathway, SLPI has an anti-inflammatory effect by downregulating TNFα expression, 35  activity. 36 Because NF-κB activation is crucial in many cellular processes, tightly regulated NF-κB signalling is required to fine-tune the inflammatory responses. Counteracting an NF-κB response is essential to prevent persistent NF-κB activation, which may lead to chronic inflammation or tumour development. This is modulated by a number of negative regulators of NF-κB, such as IKK1 and A20 (also known as TNFAIP3), A20-binding inhibitor of NF-κB, TNF receptorassociated factor 1, cylindromatosis and microRNAs, 37 some of which under the control of NF-κB and thus act in a negative feedback loop. In addition, there is cross-talk between NF-κB signalling and the TGFβ pathway that involve SMAD7 and TAK1. 38 NF-κB has a negative feedback to suppress TGFβ signalling by activating inhibitory SMAD7. 39 Our study shows that ELANE cells have a significant Understanding cellular physiology is vital for studying human diseases, leading to a better understanding of pathophysiology and pathogenesis from cellular and molecular perspectives. Correlating cellular level changes to the phenotypic level elucidates disease pathomechanisms and leads to new approaches for early disease detection and treatment.
To conclude, the dental pulp cells with diminished ELANE and SLPI expression demonstrate reduced proliferation, migration, attachment, spreading, colony formation and wound healing, while having significantly elevated ROS, apoptosis and inflammatory responses. In addition to neutropenia that leads to oral infection, we show that severely downregulated neutrophil activity and ELANE and SLPI expression in dental cells could be involved in orodental infections. This study demonstrates that ELANE and SLPI play roles in proliferation, survival and inflammation in dental pulp cells. Kevin A. Tompkins for language revision of the manuscript.