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. Author manuscript; available in PMC: 2026 Jan 1.
Published in final edited form as: J Allergy Clin Immunol. 2024 Sep 17;155(1):188–198. doi: 10.1016/j.jaci.2024.09.001

NETs activate Notch- γ secretase signaling in hidradenitis suppurativa

Christopher B Oliveira 1, Jorge Romo-Tena 1, Eduardo Patino-Martinez 1, Alexandra Woo 1, Angel S Byrd 2,3, Dongwon Kim 3, Ginette A Okoye 2,3, Mariana J Kaplan 1, Carmelo Carmona-Rivera 1,*
PMCID: PMC11700766  NIHMSID: NIHMS2023470  PMID: 39265876

Abstract

Background:

Hidradenitis suppurativa (HS) is an inflammatory chronic skin disorder of unknown etiology characterized by inflamed abscess-like nodules and boils resulting in sinus tract formation, tissue scarring and massive infiltration of neutrophils. Multiple lines of evidence have highlighted the potential association between alterations in the Notch pathway and HS pathogenesis, but the mechanisms remain incompletely characterized.

Objective:

Herein, we aim to elucidate the role of neutrophil extracellular traps in Notch-γ-secretase, signaling.

Methods:

Twenty-six HS lesional tissues, primary HS macrophages and skin fibroblasts were interrogated by quantitative PCR, western blot, and Elisa analyses. γ-Secretase, and TACE activities were measured in HS skin lesions, macrophages and skin fibroblasts. Immunofluorescence and RNAscope analyzes were performed in HS and control skin.

Results:

A prominent presence of Notch ligands, DLL4 and JAG2 were detected at the protein and mRNA levels in HS skin lesion when compared to control. Levels of DLL4, JAG1, cit-H3-DNA and γ-secretase activity correlated with HS disease severity. Additionally, significantly elevated levels of Notch ligands and γ-secretase activity were found in dissected sinus tracts when compared to the rest of HS tissue. Immunofluorescence microscopy in HS skin lesions showed activation of Notch 1 signaling in macrophages and skin fibroblasts. Neutrophil extracellular traps (NETs) purified from HS patients displayed elevated levels of DLL4. HS-NETs activated the Notch pathway in macrophages and dermal fibroblasts isolated from HS patients. HS skin fibroblasts displayed elevated levels of CD90 and DPP4 in association with increased migratory capacity and Notch activation. Inhibition of Notch decreased migratory capacity and pro-fibrotic markers in HS fibroblasts.

Conclusion:

These data support a pathogenic connection between NETs, Notch- γ-secretase activation and the release of pro-fibrotic molecules that promote dysregulation of macrophages and skin fibroblasts in HS. Unveiling the relevance of these molecular events not only expands our understanding of HS but also opens new venues for the development of targeted therapies to address the fibrotic complications of advanced stages of HS.

Keywords: NETs, γ-secretase, Notch, fibroblast, hidradenitis suppurativa

Capsule Summary:

This study links elevated notch ligands and gamma secretase activity in HS lesions and sinus tracts to a NET-induced notch pathway activation in macrophages and fibroblasts, highlighting implications for fibrotic complications and putative therapeutic strategies.

Graphical Abstract

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Introduction

Hidradenitis suppurativa (HS) is an inflammatory skin condition characterized by recurrent painful nodules, boil-like lesions located in intertriginous areas such as the axillae and groin, which ultimately results in the formation of sinus tracts as the disease progresses. The condition often leads to the development of persistent, recurrent lesions and can significantly impact the quality of life of those affected. HS is estimated to affect 0.05– 4% of the population, afflicting skin of colored women in a 3:1 ratio(1, 2).

Unlike sporadic cases, reported cases of familial HS suggest a role for specific genetic mutations or other genetic factors in the development of this disease(3). Among the genetic factors implicated in familial HS, mutations in the γ-secretase complex have emerged as key players in this condition (4, 5). The γ-secretase complex, responsible for the proteolytic cleavage of various substrates including Notch receptors, has been linked to aberrant signaling pathways that contribute to HS pathogenesis (6, 7). The Notch signaling pathway is highly conserved evolutionarily and initiates through the binding of a Notch receptor with its ligands (Delta or Jagged) (8). This is followed by a series of cleavages triggered initially by ADAM-family metalloproteases (ADAM10 or TACE), then by γ-secretase, allowing the release of the Notch intracellular domain (NICD), which translocate to the nucleus and regulates gene expression (8–10). Dysregulated Notch signaling has been associated with aberrant immune responses and inflammation, but it remains elusive if this pathway is operational in HS pathogenesis.

In HS, activated neutrophils are highly abundant in the lesions and are prone to form neutrophil extracellular traps (NETs)(11). NETs are considered web-like structures of DNA, proteolytic and antimicrobial enzymes and anti-microbial proteins typically contained inside the neutrophil’s granules. In HS, aberrant NET formation and degradation have been observed and it has been hypothesized that this phenomenon contributes to the perpetuation of inflammation and dysregulated adaptive immune responses(12, 13). Whether NETs and their cargo can activate Notch- γ-secretase signaling and contribute to HS pathology has not been previously studied.

In the current study, we identified that NETs activate Notch- γ-secretase signaling in macrophages and skin fibroblasts. We also found that this activation was accompanied by increased migratory capacity of skin fibroblasts and their stimulation to release pro-fibrotic molecules. Furthermore, γ-secretase activity and Notch ligands were elevated in skin biopsies of HS in association with disease activity, indicating a putative pathogenic inflammatory pathway initiated by neutrophils in this disease.

Materials and Methods

Patient specimens

Healthy controls were recruited by advertisement. Sera and skin tissue were collected under the NA_00013177 and NA_00031269 studies approved by the Johns Hopkins (JH) University Institutional Review Board (IRB) as well as IRB-19-MED-50 approved by the Howard University Office of Regulatory Research Compliance. All individuals gave written informed consent. Normal (non-HS) and lesional HS skin were obtained from surgical resections and from JH’s tissue bank obtained from patients undergoing surgical removal of HS skin. Visual assessment of the patient at the time of surgery as well as clinical and histopathological diagnosis were verified. Samples were stored at −80 °C.

Antibodies

Rabbit monoclonal Dpp4/cd26 (D6D8k) (cat no. 67138, cell signaling), cleaved NOTCH-1clone D3B8 (Val1744, cat no. 4147, cell signaling), rabbit polyclonal DLL4 (PA5-97664, Thermo Fisher), anti-vimentin clone VI-10 (ab20346, abcam), anti-CD90/thy1 clone EPR3133 (ab133350, abcam), anti-CD68 (ab201340), JAG2 polyclonal antibody (cat. No. PA5-47188, Invitrogen, 1:1000), HES-1 monoclonal antibody (cat. No. sc-166410 (clone E-5), Santa cruz biotechnology, 1:1000), anti- presenilin 1 (cat no. PA5-119872, Invitrogen), anti APH1A (cat no. 11643, Proteintech) and anti-Nicastrin (D38F9, Cell Signaling) were used for Western blot or immunofluorescence assays. γ-secretase inhibitor, DAPT was purchased from Tocris (cat no. 2634).

NET isolation

NETs were isolated as previously described(14). Briefly, HS neutrophils were resuspended in RPMI without phenol red and plated in 24-well tissue culture plates. Cells were incubated for 3 hours at 37°C. Supernatants were removed and NETs were digested with micrococcal nuclease (10 U/ml; Thermo Fisher Scientific) resuspended in RPMI for 15 min at 37°C. Supernatants were collected and centrifuged at 5000 rpm for 5 min at 4°C. NET supernatants were transferred to a fresh tube and stored at −80°C until used.

Detection of NETs, Notch, macrophages and fibroblasts in skin

Frozen sections (15 um) were permeabilized with 0.2% Triton X for 10 min. Then sections were blocked with 10% bovine serum albumin (BSA) for 1 hour at room temperature. Where indicated, anti-CD68 (1:200, Abcam), anti-vimentin (1:200, Abcam), anti- citrullinated histone H4 (citrulline 3) (1:1,000, EMD Millipore) and anti-active Notch-1(1:200, Abcam) antibodies diluted in 5% BSA were added to sections and placed at 4C overnight. Slides were washed three time with PBS. Sections were incubated with Alexa fluor 555- anti rabbit or 488 donkey anti-mouse IgG (Invitrogen) secondary antibody (1:400) for 1 hour at room temperature. Nuclei were counterstained with (1:1,000) Hoechst for 10 min at room temperature. After 5 washed with PBS, tissue samples were sealed with a coverslip containing ProLong Gold solution (Thermo Fisher Scientific). Images were acquired on a Zeiss LSM780 confocal laser-scanner.

NET remnants detection

citH3/DNA complexes were quantifie4d by ELISA as previously described(11). Briefly, 10 ug of healthy control or HS total skin lysate were loaded in a 96-well plate pre-coated with anti-cit-H3 antibody (ab5103). After overnight incubation, the plate was incubated with anti-dsDNA Ab (Millipore) for 1 hour at RT, then washed 4 times with PBS-T and incubated with anti-mouse HRP-conjugated antibody for 1 h at RT. The absorbance was measured at 450 nm, and values were calculated as optical density.

RNAscope

Frozen slides were fixed in 4% PFA for 1h at room temperature. After three washes with PBS, tissues were dehydrated with increasing concentrations of ethanol (50, 70, 100%) at room temperature for 5 min each. Tissues were permeabilized using pepsin in 0.1 HCL for 30 min at 37C. After two washes with PBS, tissues were hybridized using THY1-C4 probe diluted in TSA buffer for 2 hours at 40C. After two washes with SSC, probes were amplified according to manufacturer’s recommendations (Advanced Cell Diagnostics). Then amplified probe was labeled for chromogenic detection. Nuclei were counterstained using DAPI (1:1000) for 10 min at room temperature. Samples were sealed using Prolong Gold mounting solution (Thermo Fisher Scientific). Images were acquired on a Zeiss LSM780 confocal laser-scanner.

Isolation of protein from tissue

Protein from tissues or cells were obtained as previously described(11). Briefly, pulverized tissues were resuspended in RIPA buffer supplemented with a cocktail of protease inhibitors (Roche). After an hour of incubation at 4°C, samples were centrifuged for 10 min at 14,000 rpm, and supernatant was transferred to a fresh Eppendorf tube. Proteins were quantified using BCA kit (ThermoFisher) according to the manufacturer’s instructions.

qPCR analysis

RNA was isolated from tissue section and cells as previously describes(11). Briefly, pulverized tissue or cell pellets were resuspended TRI Reagent (Sigma-Aldrich). RNA was isolated using the Direct-zol RNA MiniPrep Kit (Zymo Research) according to the manufacturer’s instructions. Total RNA (300–500 ng) was reverse- transcribed using iScript RT single-strand complementary DNA (cDNA) (Bio-Rad). qPCR was performed using the TaqMan Gene Expression Master Mix (Thermo Fisher Scientific), human GAPDH primers (Hs99999905_m1), and sequence-specific primers for JAG1(Hs01070032_m1), JAG 2 (Hs01000104_g1), DLL4 (Hs01117332_g1), COL1A1 (Hs00164004_m1), ACTA2 (Hs00426835_g1), THY1 (Hs00174816_m1) and FN1 (Hs01549976_m1), PSEN1 (Hs00997789_m1), APH1A (Hs01046142_g1), NCSTN (Hs00299716_m1), PSENEN (Hs00708570_s1). Fold difference was calculated using the DCt equation.

Western blot

Isolated proteins from tissue, macrophages and fibroblasts were quantified using BCA kit (ThermoFisher) according to the manufacturer’s instructions. Equal amounts of total protein were resolved in a 4 to 12% gradient bis-tris gel (Invitrogen), transferred onto a nitrocellulose membrane, and blocked with 10% BSA for 30 min at room temperature. After overnight incubation with primary antibodies, membranes were washed three times with PBS-Tween (PBS-T) and incubated with secondary antibody coupled to IRDye 680 or 800CW. Membranes were developed using a Li-COR Odyssey Clx scanner.

γ-secretase and TACE activity assay

γ-secretase activity were measured incubating samples with γ-secretase substrate (Calbiochem,cat no. 565764) and TACE activity as measured using the SensoLyte 520 TACE (alpha secretase) activity assay kit fluorometric (cat no AS-72085, AnaSpec) as per the manufacturer’s protocol. Briefly, cells were grown in 96-well black plates coated with NETs. After 24–48h, cells were lysed with RIPA buffer and fluorescently labeled substrates for γ-secretase and TACE were added to each well. Plates were incubated at 37°C for 2–4h. For tissue, 5 ug of total protein were used. Fluorescence was measured using a Biotek Synergy H1 plate reader. Activity was expressed as relative fluorescence units.

Generation of M1 macrophages

Human CD14+ monocytes from control and HS patients were isolated from PBMCs using positive selection with MACs columns (Miltenyi). Purified cells were resuspended in RPMI containing 10% BSA and incubated with 50ng/mL (Peprotech) of GM-CSF for 5 days. 96-well plates were coated with NETs overnight. Polarized macrophages were then transferred to the 96-well plate and incubated for 24–48h hours. Supernatants were collected and Elisa against IL23 was performed. Cells were lysed and analyzed by western blot and for enzyme activity.

Fibroblast isolation

Primary HS skin fibroblasts were obtained from axillae skin tissue. After washing the tissue with PBS, skin was cut in small pieces and plate in a 6-well plate with DMEM supplemented with 15% FBS and Pen strep ate 37C. After 5–8 days, fibroblasts began to grow out of the tissue. Tissue was removed, and fibroblasts were allowed to proliferate.

Migratory/Invasiveness assay

Skin fibroblasts were seeded on 12-well cell culture plates (100,000 cells/well) and allowed to create a monolayer. A wound was generated with a micropipette tips. Cells were washed with PBS and incubated in media at37°C overnight. Pictures were taken before and after wound generation using the ECHO Lite brightfield microscope. Images were analyzed using ImageJ.

Statistical analysis

Data were analyzed using GraphPad Prism Version 8.1.1 (La Jolla, CA). For samples with non-Gaussian distribution, Mann-Whitney U test was used. One-way analysis of variance (ANOVA) Kruskal-Wallis test (Dunn’s multiple comparison test) was used to compare parameters among groups. Pearson correlation was used for all non-categorical statistics. All analyses were considered statistically significant at p < 0.05.

Results

Notch ligands and γ-secretase activity are increased in HS skin.

Accumulating evidence has highlighted the potential association between alterations in Notch signaling and inflammatory responses in HS; therefore, we investigated whether Notch signaling pathway was dysregulated in this disease. Gene expression analysis showed a significant upregulation of the Notch ligands, DLL4 and JAG1, in HS patients skin lesion when compared to control samples (Figure 1A, B). Moreover, upregulation of DLL4 (r=0.4677, p=0.0339) and JAG1 (r= 0.5054, p=0.0229) were associated with disease severity, as assessed by Hurley Staging. Globally, no significant difference was detected for the Notch ligand JAG2 gene expression (Figure 1 C), although was found to be significantly increased in samples from Stage III HS (Figure 1C). Western blot analysis of HS skin demonstrates elevated levels of JAG2 and DLL4 proteins in chronic stages of the disease, when compared to control skin samples (Figure 1D). Since these ligands activate Notch signaling, we measured the activity of α-secretase (TACE) and γ-secretase in HS skin lesion homogenates and found them to be significantly elevated when compared to control skin (Figure 1 E, F), especially in more chronic stages of the disease (Stage II and III). Further, activity of γ-secretase correlated with disease activity (r=0.3933, p=0.0286; Figure 1E). These results suggest that the Notch pathway is activated in HS skin and correlates with disease activity and progression. To further investigate the role of Notch signaling in HS pathogenesis and whether its dysregulation was associated with specific features of HS, we dissected sinus tract-rich regions (Tract), associated with significant scarring, and compared them to HS samples with none or minimal sinus tract development (No Tract) from the same patients. Quantitative PCR analysis showed that the expression of JAG1, JAG2 and DLL4 were significantly upregulated in HS tissue with high sinus tract burden when compared to tissue with no tracts (Figure 1G–I). The activity of γ-secretase and TACE were also significantly increased in tissue with high sinus tract burden when compared to HS tissue with no tracts (Figure 1J, K), suggesting that Notch may be involved in sinus tract formation. Taken together, these results suggest that Notch signaling is activated in HS.

Figure 1. Notch ligands and signaling are increased in HS tissue and sinus tracts.

Figure 1.

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RNA was isolated from homogenized HS lesional tissue and control tissue. qPCR analysis of HS lesions (n=16) and control skin (n=7) for (A) DLL4, (B) JAG1 and (C) JAG2. Results are the mean +/− SEM, Mann-Whitney U test analysis was used. Gene expression found in HS tissue was stratified by Hurley stage (Stage I: n=4; Stage II: n=6; Stage III: n=6). Results are shown as mean +/− SEM, Kruskal-Wallis analysis was used; *p<0.05 (D) Detection of DLL4, JAG2, activated Notch1 and GAPDH proteins in lysates from control skin (n=2) and HS lesions Stage I (n=4), Stage II (n=3) and Stage III (n= 4) by immunoblot (IB). (E) γ-secretase and (F) TACE activities were measured in lysates from HS skin lesion (n=26) and control skin (n=4). Mann-Whitney U test analysis was used. Measurements were stratified by Hurley stage (Stage I: n=5; Stage II: n=11; Stage III: n=10). Results are the mean +/− SEM, Kruskal-Walli’s analysis was used; *p<0.05. HS skin tissues rich in sinus tracts were dissected and RNA isolated. qPCR analysis of HS lesion with (n=8) or without sinus tract (n= 4–6) was performed to detect (G) JAG1, (H) JAG2 and (I) DLL4. (J) γ-secretase and (K)TACE activities were measured in HS tissue with (n=5–8) or without (n=5–6) sinus tracts. Results are the mean +/− SEM. Mann-Whitney U test analysis was used, *p<0.05, **p<0.01.

NET remnants correlate with γ-secretase activity.

Loss-of-function mutation in subunits of γ-secretase (PSEN1, APH1A, NCSTN, PSENEN) has been reported in familial HS(15). While most of the HS samples tested here displayed elevated γ-secretase activity, we found that other samples had low γ-secretase activity (Figure 2A). Therefore, we hypothesized that those with low γ-secretase activity would harbor alterations in γ-secretase subunits. To test this hypothesis, we performed PCR in cDNA generated from HS tissues with high or low γ-secretase activity. We found that the expression of γ-secretase subunit PSEN1 and APH1A were absent or reduced in skin samples from individuals with low γ-secretase activity (Figure 2B), while those with high γ-secretase activity displayed normal levels of these subunits. Western blot analysis corroborated the absence or reduced levels of subunits, presenilin 1 and/or APH1A (Figure 2C). Of note, the levels of NET remnants in tissue were elevated in HS samples with high γ-secretase activity, while no significant differences were seen in those with low γ-secretase activity when compared to control samples (Figure 2D). Moreover, levels of NET remnants significantly correlated with γ-secretase and TACE activities (Figure 2 E, F), suggesting that NETs may be able to activate γ-secretase in HS patients with intact γ-secretase complex.

Figure 2. NET remnants correlate with γ-secretase activity in HS.

Figure 2.

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(A) γ-secretase activities measured in lysates from HS patients were divided into low (n=4) or high (n=10) compared to control. Mann-Whitney U test analysis was used. Polymerase chain reaction (PCR) was performed in cDNA generated from HS and control tissues with high or low γ-secretase activity. (B) Gel displays the presence of bands corresponding to gene products of γ-secretase complex (PSEN1, APH1A, NCTN, PSENEN). GAPDH was used as loading control. (C) Western blot analysis of HS and control tissue extracts against presenilin, APH1A and Nicastrin. (D) Levels of citrullinated histone H3 (cit-H3)- DNA in control (n=4), HS with low γ-secretase activity (n=4), and HS with high γ-secretase activity (n=10). Results are the mean +/− SEM, Kruskal-Wallis analysis was performed *p<0.05. Correlation of NET remnants (citH3-DNA) and (E) γ-secretase or (F) TACE activities measured in lysate from HS skin lesion.

NETs activate Notch-1 and γ-secretase in macrophages.

Given that Notch modulates macrophage polarization and activation, we tested whether Notch signaling was activated in macrophages in HS lesions. Confocal analysis demonstrated that CD68+ macrophages co-localized with activated Notch-1 in HS skin lesions from Stage I, II and III, when compared to control skin tissues (Figure 3A). Peripheral macrophages from HS displayed elevated levels of HES-1, a molecule typically activated during Notch signaling, when compared to control macrophages (Figure 3B). As shown in Fig 3A, most of the co-localization of Notch-1 with macrophages was in areas of highly dense extracellular DNA resembling NETs. Indeed, immunofluorescence analysis showed that CD68+ macrophages coexist with NETs (citH4) in HS skin tissue (Figure 3C). Moreover, Western blot analysis of spontaneously generated NETs from HS patients displayed elevated levels of DLL4, a natural ligand of Notch, when compared to PMA-generated NETs from healthy volunteers (Figure 3D). Thus, we hypothesized that NETs containing Notch-ligand may activate Notch- γ-secretase signaling in macrophages. To test this hypothesis, control and HS macrophages were incubated with spontaneously generated NETs from HS neutrophils. Analysis showed that TACE and γ-secretase activities were significantly increased in control and HS monocyte-derived macrophages after incubation with NETs (Figure 3E, F). This was accompanied by increased detection of activated form of Notch-1 and HES-1 by Western blot analysis (Figure 3G). Of note, NETs induced the release of IL-23 by control macrophages, a cytokine implicated in HS pathogenesis (16, 17) (Figure 3H). Taken together, these results indicate that NETs contain Notch ligands such as DLL4 and therefore can trigger activation of Notch-1 signaling in macrophages in HS skin lesions.

Figure 3. NET-associated DLL4 activates Notch-1 in macrophages.

Figure 3.

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(A)Representative confocal images of activated Notch-1 (red) and macrophages (CD68, green) in control skin and HS skin lesions from Stages I-III. Original magnification 20x. Images are representative of 2 samples per group. (B) Detection of HES-1 in control and HS macrophages by Western blot. GAPDH was used as a loading control. (C) Representative confocal images of NETs (citH4, red) and macrophages (CD68, green) in control skin and HS skin lesion from Stages I-III. Hoechst was used to counterstain nuclei in blue. Original magnification 20x. Images are representative of two samples per group. (D) Western blot analysis of spontaneously generated NETs from HS neutrophils and PMA-generated NETs from controls (Ctrl) against DLL4 and myeloperoxidase (MPO). Lower panel: Densitometry analysis levels of DLL4 and MPO in NETs from controls (n=6) and HS (n=2). (E) TACE activity was measured in control macrophages treated with NETs isolated from HS neutrophils. Results are expressed as relative fluorescence units (RFU) and are the mean +/− SEM. Mann-Whitney U test analysis was used, **p<0.01. (F) γ-secretase activity measured in control and HS macrophages (HS-1 and HS-2) in the absence or presence of HS-NETs for 24h. Results are the mean +/− SEM. Mann-Whitney U test analysis was used, *p<0.05. (G) Western blot analysis of Control and HS macrophages in the absence or presence of HS-NETs for 24h. Tubulin was used as a loading control. (H) ELISA quantification of IL-23 in supernatants from control macrophages treated or untreated with HS NETs for 48h. Results are the mean +/− SEM. Mann-Whitney U test analysis was used, *p<0.05.

Notch-1 is activated in HS skin fibroblasts.

To further confirm activation of Notch signaling in HS, we quantified the expression of other molecules regulated by this pathway. Notch signaling modulates THY-1/CD90 expression in fibroblasts (18).Quantitative PCR analysis showed a significant upregulation of THY-1 in HS skin lesions when compared to control (Figure 4A). RNAscope analysis performed in HS and control skin corroborated the presence and distribution of THY-1(Figure 4B). THY-1 expression was mainly localized in the dermis of HS Stage I-III skin lesions when compared to control skin (Figure 4B). Furthermore, Western blot analysis demonstrated significant expression of CD90 in HS skin lesions when compared to controls (Figure 4C). Confocal analysis demonstrated co-localization of activated Notch-1 in skin fibroblasts (Figure 4D). These data suggested that Notch is activated in HS skin fibroblasts and may regulate CD90 expression. Western blot analysis of HS skin fibroblasts showed that Notch-1 is activated when compared to control skin fibroblasts (Figure 4E). CD90 was also elevated in 2/3 of sample tested, suggesting a role for Notch-1 signaling in the upregulation of CD90. We also detected elevated levels of DPP4, a marker of a subset of skin fibroblasts, in HS skin fibroblasts when compared to control. Wound-healing studies showed that HS fibroblasts display an enhanced migratory capacity when compared to control skin fibroblasts (Figure 4F). HS fibroblasts displayed upregulation of profibrotic genes such as COL1A1 and FN1, while no difference was not observed for ACTA2 (Figure 4G). To demonstrate the involvement of γ-secretase in some of these features, HS fibroblasts were treated with a γ-secretase inhibitor, DAPT. Western blot analysis demonstrated that the levels of CD90 and DPP4 were decreased in HS skin fibroblasts after treatment with γ-secretase inhibitor (Figure 4H). The reduction in CD90 and DPP4 was accompanied with a significant decrease in their invasiveness/migratory capacity after inhibition of γ-secretase (Figure 4I). Together, these data suggest that Notch-1- γ-secretase signaling is activated in HS fibroblasts and modulates their migratory capabilities.

Figure 4. Notch-1 is activated in HS skin fibroblasts.

Figure 4.

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(A) qPCR analysis of THY-1 in control (Control, n= 6) and HS (n= 26) tissue. Results are the mean +/− SEM. Mann-Whitney U test analysis was used, **p<0.01. (B) Representative confocal images of the detection of THY-1 mRNA (red) in control skin and HS skin lesion from Stage I-III using RNAscope. Nuclei are in blue. Original magnification 20x. Images are representative of 2 HS patients per group. (C) Detection of CD90 and GAPDH in control skin (n=2) and HS skin lesions Stage I (n=3), Stage II (n=3) and Stage III (n= 4) by immunoblot (IB). Right panel: Densitometry analysis levels of CD90 and GAPDH in control and HS tissue. Results are the mean +/− SEM. Mann-Whitney U test analysis was used, *p<0.05. (D) Representative confocal images of activated Notch-1 (red) and fibroblasts (vimentin, green) in control skin and HS skin lesions from Stage I-III. Original magnification 40x. Images are representative of 2 samples per group. (E) Western blot analysis of lysates from control and HS fibroblasts. GAPDH was used as loading control. (F) Representative images of 2 independent experiments of the migration assay of control and HS skin fibroblasts over a 24h period. Right panel Quantification of the wound area using ImageJ. Kruskal-Wallis analysis was used. (G) qPCR analysis for the expression of COL1A1, ACTA2 and FN1 in control and HS fibroblasts. (H) Western blot analysis for the expression of CD90 and DPP4 protein s in HS fibroblast after treatment with γ-secretase inhibitor (DAPT). Tubulin was used as loading control. (I) Representative images of three independent experiments of the migration assay of HS fibroblast treated with DAPT. Original magnification is 10x. Right panel Quantification of wound area using ImageJ. Kruskal-Wallis analysis was used. **p<0.01.

NETs activate γ-secretase in HS fibroblasts and promote an aggressive fibroblast phenotype.

We then investigated whether NETs were implicated in Notch- γ-secretase activation in skin fibroblasts. Primary fibroblasts isolated from control and HS skin were incubated with spontaneously generated HS NETs. TACE and γ-secretase activities were significantly increased in control and HS fibroblasts after incubation with HS NETs (Figure 5 A, B), suggesting that these structures activate Notch- γ-secretase pathway in dermal fibroblasts. This finding was accompanied by upregulation of CD90 and DPP4 in control skin fibroblasts incubated with NETs (Figure 5C). ELISA analysis showed that supernatants of control skin fibroblasts incubated with HS NETs contain significantly more collagen and fibronectin, when compared to untreated skin fibroblasts (Figure 5D, E). We then tested the invasiveness/migratory capacity of control fibroblasts incubated with HS NETs for 4 days. An enhanced migratory capacity was displayed by control fibroblasts incubated with HS NETs when compared to controls without NETs (Figure 5F). These observations suggest that NETs modulate migratory capacity of skin fibroblasts in HS.

Figure 5. NETs activate Notch-1 in dermal fibroblasts and promote a pro-fibrotic phenotype.

Figure 5.

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(A) TACE activity was measured in control fibroblasts treated with HS NETs. Results are expressed as relative fluorescence units (RFU) and are the mean +/− SEM. Mann-Whitney U test analysis was used (B) γ-secretase activity was measured in control and HS skin fibroblasts in the absence or presence of HS-NETs at 24h of co-culture. Results are the mean +/− SEM. Mann-Whitney U test analysis was used, *p<0.05, *** p< 0.001. (C) Western blot analysis of control dermal fibroblasts treated with HS-NETs for 48h. Tubulin was used a loading control. (D-E) Elisa analyzes for collagen (COL1A1) and Fibronectin (FN1) in supernatants of control dermal fibroblasts treated with HS-NETs in the presence or absence of γ-secretase inhibitor, DAPT. Results are the mean +/− SEM of four independent experiments. Kruskal-Wallis analysis was used. **p<0.01. (F) Representative images of 24 migration assay of control fibroblasts treated with HS-NETs. Results are representative of two independent experiments. Original magnification of the images is 10x Right panel: Quantification of wound area using ImageJ. Kruskal-Wallis analysis was used. *p<0.05, **p<0.01.

Discussion

Accumulating evidence suggest that NETs are implicated in the pathogenesis of HS(11–13). Here, we evaluated the potential crosstalk between NETs, γ-secretase activation and the ensuing functional consequences of Notch pathway activation in primary macrophages and skin fibroblasts. We found that NET-mediated Notch signaling activation in macrophages stimulates the release of IL23, a cytokine involved in the inflammatory responses and chronic inflammatory disorders(16). In the context of HS, elevated levels of IL23 may contribute to the sustained skin inflammation (17, 19).

Gamma secretase’s role in proteolytic processing, particularly of Notch receptors, has implications for cell fate determination and differentiation, a process known to be dysregulated in HS(15, 20–22). The paradigm of Notch in HS pathogenesis has been associated with inhibition of γ-secretase activity by genetic mutations and environmental factors such as obesity and smoking(15, 23, 24). However, other studies have reported increased inflammation associated with increased Notch activity (25–27). The heightened expression of γ-secretase and Notch ligands in the lesional HS skin suggests a potential involvement of Notch signaling in the perpetuation of the inflammatory and fibrotic responses characterizing this disease(28). The discovery that NETs can activate γ-secretase in primary macrophages and skin fibroblasts sheds additional light on new regulatory mechanism in HS potentially leading to skin inflammation and fibrosis in this disease. The ability of NETs to induce γ-secretase activity implies a direct interaction or modulation of the local microenvironment that requires further experimental exploration into the intricate molecular pathways linking neutrophil activation and γ-secretase regulation in this disease. Moreover, the observation that HS patients with impaired γ-secretase displayed low levels of NET complexes, while those without apparent γ-secretase impairment had significantly increased NETs in skin lesions, suggests the possibility of two different subsets within HS patients: those with impaired γ-secretase due to genetic mutations, and those with increased γ-secretase activity and enhanced NET formation. This subgrouping of HS needs to be validated in larger cohorts and warrants future investigations.

Skin fibroblasts are essential component of the skin’s structural framework and play a crucial role in wound healing and tissue repair(29, 30). The heightened fibroblast migratory capacity induced by NETs can lead to abnormal tissue remodeling, perpetuating the cycle of inflammation and tissue damage characteristic of HS(31). The observed increase in migratory capacity in primary skin fibroblast suggest a potential role of γ-secretase in cell motility, which may contribute to the dissemination of inflammatory signals within the tissue microenvironment. Furthermore, the release of profibrotic molecules such as collagen and fibronectin imply a link between NETs and tissue remodeling and fibrosis in HS (32, 33).

The induction of profibrotic molecules by NETs through γ-secretase activation has significant implications for the tissue remodeling observed in HS. Fibrosis, characterized by excessive deposition of collagen and other matrix proteins, alters the normal architecture of the skin. This fibrotic remodeling not only contributes to the persistence of inflammation but also leads to the formation of sinus tracts and scarring, hallmarks of advanced stages of HS(32). Understanding the mechanisms linking NETs, γ-secretase and fibrosis in HS provides potential targets for therapeutic interventions. Strategies aimed at modulating NET formation could be explored to disrupt the vicious cycle of inflammation and fibrosis in HS lesions.

Our study was performed with samples mostly from African American subjects, an understudied population. It is important that future studies include other ethnic groups that are affected by HS, to determine whether the effect seen in this study can be recapitulated (34–38). Our study has several limitations, including a small sample size for each Hurley stage and the absence of genotyping for HS patients with low γ-secretase activity, which hinders our ability to establish definitive genetic associations. Additionally, we did not examine mutations in intronic or promoter regions of γ-secretase subunits, which could be significant. We also did not analyze lesional samples from patients with known γ-secretase gene mutations, precluding any firm conclusions about the correlation between γ-secretase activity and NETs. Although we observed that patients with low γ-secretase activity have reduced mRNA and protein expression, our findings challenge the notion that γ-secretase is inactive in HS. This suggests that NETs might activate γ-secretase and influence immune and structural cell functions. Further research is necessary to investigate the role of NETs in HS and other skin fibrotic conditions.

In summary, this study establishes a connection between NETs, elevated γ-secretase activity, activation of Notch1 signaling in macrophages and skin fibroblasts and the subsequent release of pro-fibrotic molecules. These finding deepen our understanding of HS pathogenesis and open putative, new avenues for therapeutic interventions aimed at disrupting the inflammatory and fibrotic cascades observed in this debilitating skin disorder. Further research into the specific molecular mechanisms and clinical translation of these findings hopefully holds promise for advancing the management of HS.

Key messages:

  • NETs activate Notch- γ secretase signaling.

  • HS patients with impaired γ secretase activity display low levels of NETs.

  • HS patients with increased γ secretase activity display elevated levels of NETs.

  • NETs activate skin fibroblasts and promote the release of pro-fibrotic proteins.

Acknowledgments and Funding:

This study was supported by the Intramural Research Program, NIAMS/NIH, ZIA AR041199 (MJK), the Danby Hidradenitis Suppurativa Foundation Grant (CC-R) and the Skin of Color Society CDA (CC-R).

Abbreviation used:

NETs

neutrophil extracellular traps

HS

Hidradenitis suppurativa

TACE

Tumor necrosis factor-alpha converting enzyme

DLL4

delta-like ligand 4

JAG

Jagged

cit-H3

citrullinated histone H3

DDP4

Dipeptidyl peptidase 4

Footnotes

Conflict of Interest: ASB consultant for Senté, Inc.and Sonoma Biotherapeutics. GAO serves on the advisory boards for Pfizer, Unilever, Janssen, UCB, Novartis, Sanofi-Genzyme, Lilly, AbbVie, Vaseline Healing Program, Dermatology Foundation, and the HS Foundation. All other authors declare no conflict of interest.

Ethics statement: The study was approved by the Johns Hopkins (JH) University Institutional Review Board (IRB) and the Howard University Office of Regulatory Research Compliance. All participants signed a written informed consent according to the Declaration of Helsinki.

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Data availability:

Data generated during this study is available upon request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

Data generated during this study is available upon request.

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