Detection of native, activated Notch receptors in normal human apocrine‐bearing skin and in hidradenitis suppurativa

Notch signalling has generated considerable interest as a pathogenetic factor and a drug target in a range of human diseases. The gamma‐secretase complex is crucial in the activation of Notch receptors by cleaving the intracellular domain allowing nuclear translocation. In recent years several mutations in gamma‐secretase components have been discovered in patients with familial hidradenitis suppurativa (HS). This has led to hypotheses that impaired Notch signalling could be an important driver for HS in general, not only in the monogenic variants. However, no study has examined in situ Notch activation per se in HS, and some reports with conflicting results have instead been based on expression of Notch receptors or indirect measures of Notch target gene expression. In this study we established immunostaining protocols to identify native, activated Notch receptors in human skin tissue. The ability to detect changes in Notch activation was confirmed with an ex vivo skin organ model in which signal was reduced or obliterated in tissue exposed to a gamma‐secretase inhibitor. Using these methods on skin biopsies from healthy volunteers and a general HS cohort we demonstrated for the first time the distribution of active Notch signalling in human apocrine‐bearing skin. Quantification of activated NOTCH1 & NOTCH2 revealed similar levels in non‐lesional and peri‐lesional HS to that of healthy controls, thus ruling out a general defect in Notch activation in HS patients. We did find a variable but significant reduction of activated Notch in epidermis of lesional HS with a distribution that appeared related to the extent of surrounding tissue inflammation.

[7] The Notch signalling pathway in vertebrates consists of five ligands (JAG1, JAG2, DLL1, DLL3 and DLL4) that bind four Notch receptor paralogues: NOTCH1, NOTCH2, NOTCH3 and NOTCH4. 8series of proteolytic events occurs when a receptor is activated, with the final event being cleavage of the intracellular domain (NICD) by γ-secretase. 9The activated NICD translocate to the nucleus and interacts with the transcriptional regulator CBF1/Su(H)/ Lag-1 to directly modulate the expression of target genes such as the hairy and enhancer of split (HES) gene and hairy and enhancer of split with YRPW motif (HEY) family members. 10The activation of Notch signalling processed by γ-secretase is known as canonical Notch activation. 5γ-secretase is a multiprotein complex consisting of four different protein subunits, presenilin (PSEN1/2), presenilin enhancer-2, (PSENEN), nicastrin (NCST) and anterior pharynx defective 1 (APH-1). 11It functions as a protease cleaving transmembrane proteins, and there are several known substrates. 12Notch receptors are likely key substrates, and gamma-secretase inhibitors are useful to assess canonical Notch signalling. 1 In the skin, Notch signalling participates in epidermal homeostasis, balancing the growth arrest and differentiation of basal progenitors 13,14 and inducing terminal differentiation and cornification of the keratinocytes. 15In the hair follicle, it regulates the fate of stem cells. 16e role of cutaneous Notch signalling in inflammation is complex and context-dependent.Simultaneous lack of epidermal Notch1 and 2 signalling results in an atopic dermatitis-like inflammation in mouse models. 17On the other hand, epidermal Notch1 signalling appears to be an important stress/injury signal driving the recruitment of innate lymphoid cells. 18Moreover, we have previously shown that Notch1 signalling facilitates pro-inflammatory endothelial cell activation in a model of murine skin inflammation. 19dradenitis suppurativa is a chronic inflammatory disorder of the skin.It is characterized by recurrent painful nodules and abscesses in apocrine-bearing areas, and in later stages anatomically changing lesions like fistulas, sinus tracts and scarring. 20Symptoms can be debilitating with a higher impact on life quality 21 and a higher comorbidity burden compared to more common dermatoses like psoriasis and eczema. 22The name implies that the disease originates in the sweat gland, but we now know that the initiating event is occlusion of the follicular infundibulum. 23Whether this initial occlusion is due to a primary keratinocyte defect 24 or occurs secondary to an immune dysfunction is not known. 25Nevertheless, inflammatory signalling is important in disease perpetuation, and this is reflected by the approval of both TNF-and IL-17-inhibitors for treatment of moderate to severe HS. 26,27 First-line treatment often involves the use of topical and systemic antibiotics, and their efficacy is likely attributed to both their antimicrobial and anti-inflammatory properties. 28For chronic disease a combination of medical and surgical treatment is often indicated.Genetic factors influence disease risk, and a twin study reported a narrow sense heritability of 77%. 29In 2010, mutations in genes coding for members of the gamma-secretase enzyme were found in six different Chinese Han families affected by the disease. 30merous subsequent studies have reported new mutations, the majority located within the NCSTN subunit. 31The establishment of mutations of gamma-secretase components in familial HS led to the hypothesis that the disease was a result of defective Notch signalling. 32This viewpoint found support in murine studies, with formation of epidermal cysts in γ-secretase-deficient mice, and a similar phenotype with deficiency in both NOTCH1 and 2. 33 Also, NCSTN knock-out led to hyperkeratinisation and inflammation with increased expression of the cytokines IL17A, TNFα and IL36A, 34 all known to be important in HS.[37] So far, no study has directly assessed active Notch signalling in HS in vivo.There are some contradicting reports using immunohistochemistry and qPCR showing either reduced 38,39 or increased 40 expression of NCSTN and Notch receptors (NOTCH1-3).However, the expression of Notch receptors does not necessarily reflect their activation status.Others have looked at Notch target genes, 41 which can reflect receptor activation, yet they can also be regulated through Notch-independent pathways. 42,43 more directly assess Notch activation in normal human apocrine-bearing skin and HS skin, we report results using commercially available antibodies to identify native, activated Notch receptors in formalin-fixed, paraffin-embedded tissues.The ability of the antibodies to detect changes in Notch activation was established in ex vivo cultured human skin explants exposed to γ-secretase inhibitors.

| Patient samples
Patient samples were obtained from a biobank at the Department of Rheumatology, Dermatology and Infectious Diseases at Oslo University Hospital.As healthy controls, we included seven biopsies of normal axillary skin from individuals without known inflammatory skin disorders.For HS we included 20 patients with biopsies from lesional, peri-lesional (2 cm from active lesion) and non-lesional skin (>10 cm away from active lesion). 44Patient characteristics are shown in Table 1.Informed written consent was obtained upon inclusion.The biobank and the study were approved by the Regional Committee for Medical Research Ethics, South East Norway (REK2016/119 & REK 2019/320).

| Skin organ culture
Skin explants were harvested from abdominal plastic surgery resections (n = 3) after informed written consent, in accordance with the Declaration of Helsinki Principles and protocols approved by the Regional Committee for Medical Research Ethics, South East (5 mM DMSO, Sigma #D4540-500 mL) and incubated at 37°C in 5% CO 2 atmosphere overnight(18-20 h).All samples were formalinfixed and paraffin-embedded.
Ventana Discovery protocols are available upon request.All reagents for automatic staining were purchased from Ventana Medical Systems.Slides were washed in warm, soapy water and counterstained with haematoxylin before mounting.
All antibodies for immunostainings and working concentrations are specified in Table 2. Concentration-and isotype-matched irrelevant antibodies were used to control for non-specific signal.

| Statistical evaluation of stainings
Tissue sections were examined with a Nikon Eclipse E400 brightfield microscope and scanned using Pannoramic Midi slide scanner (3DHISTECH).Automatic counting of cells with positive nuclear signal were generated with QuPath software 45 for NICD1 and HES1.
For NOTCH2, the variable signal intensity made automatic counting difficult, and we instead counted cells with Notch positive nuclei manually.Nuclei were considered positive when a uniform brown nuclear signal was observed that exceeded that of the surrounding cytoplasm.Statistical analyses were performed with GraphPad Prism (version 9.3.1).The data distribution was visualized through scatter plots, and group comparisons were conducted using nonparametric tests, with Kruskal-Wallis test followed by a Dunn's post hoc analysis.can be visualized by antibodies to cleavage-site specific epitopes 46 that produce a nuclear signal and directly reflect receptor activation (Figure 1A,C).An alternative strategy to indirectly detect na-

body (clone D3B8) recognizes NOTCH1 only when cleaved at
7][48] The specificity of this antibody has already been demonstrated in Western blot by its ability to recognize the transfected intracellular domain of NOTCH1, but not NOTCH2, 3, or 4, in lysates of 293T cells. 47Moreover, its specificity in immunohistochemistry has previously been validated in human tumour xenografts with activating Notch1 mutations, where the signal was strongly reduced when xenograft-bearing mice were given the gamma-secretase inhibitor DBZ (dibenzazepine). 46In line with previous observations, 46 we observed an epidermal nuclear NICD1 signal in normal human formalin-fixed skin (Figure 1E).Using this antibody, we assessed the (H) epidermal signalling. 49In control samples (Figure 1G) we observed a clear NICD1 signal that appeared to increase somewhat compared to the signal in skin formalin-fixed immediately after sampling (Figure 1E).By contrast, this signal almost completely disappeared in the DAPT-treated samples (Figure 1I), in line with successful inhibition of NOTCH1 cleavage.As the antibody also cross-reacts with mouse NICD1 we further confirmed antibody specificity in mice exposed to DBZ (10 μmol/kg, ip), a GSI more suited for in vivo administration, observing an obliteration of signal in spleens harvested 6 h after DBZ-treatment (Figure S1A,B).
These observations confirm antibody specificity and demonstrate that endogenous activation of NOTCH1 by gamma-secretase-mediated Val1744-cleavage is inhibited in our model system at levels sufficiently pronounced to be detected by immunostaining.
On these grounds, we concluded that our model is suitable to test the ability of less-well characterized antibodies to detect changes in Notch receptor activation status.

| NOTCH2
Despite testing a range of commercial antibodies designed to specifically target NICD2, we identified none that revealed a nuclear signal that was reduced in tissues exposed to DAPT.We therefore turned to antibodies targeting the C-terminus of NOTCH2, following the strategy depicted in Figure 1B,D.This approach identified a rabbit monoclonal antibody (clone D76A6, Cell Signalling Technologies) that produced the expected mixed nuclear/membranous signal pattern.This antibody was previously used to detect NOTCH2 in mouse tumours in the presence or absence of DBZ. 50In a similar manner, we observed that DAPT-treatment of human skin explants resulted in pronounced reduction in the number of cells with a nuclear signal, and corresponding increase in membrane signal, confirming inhibition of constitutive NOTCH2 activation (Figure 1F,H,J).[53][54] In agreement with previously reported results, 54 the D76A6 clone also produced a strong nuclear signal in the marginal zone of murine spleen, consistent with NOTCH2 activation in B-cells S1C,E).This nuclear signal was clearly reduced in response to DBZ-treatment, and instead we observed a membranous signal pattern (Figure S1D).The specificity of the staining was further confirmed by obliteration of signal in mouse spleens (Figure S1F) harvested 24 h after intraperitoneal injection of blocking antibodies that specifically prevents the activation of NOTCH2. 55

| NOTCH3 and 4
No or few commercially available antibodies target human NICD3 or NICD4, and those available failed to work in our immunohistochemistry protocols.We therefore tested a selection of antibodies targeting the C-terminus of NOTCH3 and 4. Here, we identified a mouse monoclonal antibody against NOTCH3 (clone 487CT6.9.2, available from LifeSpan Biosciences and others) and a mouse monoclonal antibody against NOTCH4 (L5C5, available from Cell Signalling Technologies) with signal in agreement with the literature, exhibiting strong signal for NOTCH3 in vascular smooth muscle cells 56,57 (Figure S2C,D) and NOTCH4 in vascular endothelial cells 58,59 (Figure S4L).In human epidermis, the signal for NOTCH3 by 487CT6.9.2 (Figure S4A-F) and NOTCH4 by L5C5 (Figure S4G-I) was predominantly cytoplasmic/membranous, with little or no nuclear signal.The lack of nuclear signal in the epidermis made it difficult to evaluate the effect of gamma-secretase inhibition (Figure S3D-I), and we were therefore unable to conclusively validate the ability of these antibodies to accurately detect activated NOTCH3 and NOTCH4.Thus, we could not conclude whether these antibodies were suitable to detect changes in epidermal gammasecretase activity.

| HES1
We also stained for HES1, a Notch target gene, finding only partial reduction in HES1 after overnight inhibition in DAPT-treated samples (Figure S3A-C,J).This observation underpins the importance of cautious use of HES1 as a direct marker for Notch activation.

| Constitutive Notch activation in normal apocrine-bearing skin
No previous reports exist of constitutive Notch activation in normal apocrine-bearing human skin.To this end we collected samples of axillary skin of healthy controls (n = 7) and used our validated approaches for active Notch-detection as well as HES1.
In the epidermal compartment, including interfollicular epidermis and hair follicles, we observed keratinocytes positive for NICD1 predominately located in the lower part of stratum spinosum (Figure 2A).NOTCH2 appeared to be expressed throughout the epidermis, but a positive nuclear signal was mainly observed in the suprabasal cells, including the stratum granulosum (Figure 2F).

The combined expression of activated NOTCH1 and NOTCH2
(Figure 2A,F) corresponded to the widespread HES1-expression in the suprabasal epidermis (Figure 2K).Staining for NOTCH3 and NOTCH4 both produced a cytoplasmatic/membranous pattern with sparse nuclear signal mainly confined to the suprabasal layers of epidermal structures (Figure S4A,G).
In sebaceous glands we observed only a few scattered NICD1positive basal cells, while no differentiated sebocytes displayed active NOTCH1 signalling (Figure 2C).On the other hand, NOTCH2 was strongly expressed throughout the gland, but nuclear signal was only seen in a proportion of sebocytes, with no signal in undifferentiated basal cells (Figure 2H).Staining for NOTCH3 gave a cytoplasmatic/ membranous pattern with no certain nuclear signal (Figure S4C), while NOTCH4 showed a strong nuclear signal throughout the sebaceous gland (Figure S4I).HES1 was strongly positive in all sebocytes (Figure 2M).
In sweat glands there appeared to be specific differences between the wide-lumen simple columnar epithelium of the apocrine glands and the narrow-lumen stratified epithelium of glandular ducts.
Although all structures were strongly HES1-positive (Figure 2N), Finally, the Notch signal in the dermal vessels corresponded to the known Notch distribution of the vasculature, with positive, although weak, NICD1 in endothelial cells (Figure 2E) and NOTCH2 expression in the pericytes (Figure 2J).In summary, we show distinct patterns of Notch activation in various structures of normal apocrine-bearing human skin.

| Notch activation in HS
Next, we wanted to examine the extent of Notch activation in biopsies from HS patients.To this end we obtained matched nonlesional, peri-lesional and lesional biopsies of HS-patients (N = 20) for analyses.All patients had moderate to severe HS, and the majority did not have a known family history (Table 1).If reduced Notch activation is an early pathogenic event in HS, then one would expect either reduced baseline signal (non-lesional skin), or a reduced signal in early/future HS-lesions (peri-lesional skin).In our cohort, the distribution of NICD1, nuclear NOTCH2 and HES1 in non-lesional and peri-lesional biopsies appeared to be very similar to that of healthy controls (Figure 3).Due to a scarcity of adnexal structures for quantitative assessment we chose to quantify only the epidermal signal, and found no significant changes between the groups (Figure 3D,H,L).Although we were unable to quantify Notch activity in the adnexal structures, qualitative examination of hair follicles, sebaceous glands, sweat glands as well as blood vessels in samples where these were detected did not reveal any clear differences in the distribution between healthy controls and HS patients Lesional HS skin is marked by severe anatomic changes including epidermal hyperplasia, massive inflammatory infiltrates and dermal tunnels, making comparisons with non-inflamed skin difficult, espe-

| DISCUSS ION
Notch signalling has generated considerable interest as a pathogenic factor and an attractive drug target in a wide range of human diseases. 60e discovery of loss-of-function mutations in gamma-secretase in patients with familial hidradenitis suppurativa in 2010 30 led to speculations that reduced activation of Notch could be a unifying factor in the pathophysiology of this debilitating disease. 7However, so far, no stud- Next, we assessed the signal distribution in pan-Notch-inhibited human tissues, which also allowed an evaluation of the ability to detect changes in Notch activity.Indeed, our immunohistochemistry approach detected a strong reduction of nuclear NOTCH1 and NOTCH2-related signal in ex vivo DAPT-treated human skin.
Unfortunately, we were unable to confirm a reduction in nuclear signal for NOTCH3 and NOTCH4 ex vivo after DAPT treatment, despite showing signal distributions in human skin that is biologically plausible.There could be a number of reasons for this, but the main explanation is likely the low level of nuclear positive cells in normal epidermis, which hampered the ability to detect changes after DAPT treatment.
We next employed the validated methods for Notch activation on human skin samples.Reports of Notch receptor localisation in normal human skin is sparse, 6,15,61 and none have systematically assessed Notch activation.We know that different anatomical areas of the skin encompass not only structural differences but also show variability in the immunological milieu. 62,63This underpins the importance of using relevant control tissue for the disease under investigation.As hidradenitis suppurativa favours apocrine-bearing areas, we obtained axillary skin biopsies from healthy controls, and used these to map the distribution of Notch activation in steady state.
We found that Notch activation was predominantly seen in suprabasal layers in epidermis and hair follicles.NOTCH2 displayed a higher degree of nuclear signal in the more distal layers compared to the more proximal signal of activated NOTCH1 (NICD1).e-g i-k d h The distribution of NOTCH2 is markedly different from the basal expression reported in the most cited study on Notch receptor distribution in human skin. 15Our well validated findings are however in line with in vitro studies showing that NOTCH1 is a regulator of proliferation, while NOTCH2 is more important in terminal differentiation of human keratinocyte. 64These spatial differences in NOTCH1 and NOTCH2 activation also extended to adnexal structures where NOTCH1 activation was confined to the basal cells of sebaceous glands and the double lined epithelium of the sweat gland ducts, whereas active NOTCH2 was seen in a subset of differentiated sebocytes and the luminal cells of the sweat gland ducts.
Interestingly, NOTCH4 was the only Notch receptor showing a nuclear distribution in the secretory part of apocrine gland and this corresponded to a strong HES1-signal in the same cells.The role of Notch signalling in the homeostasis and function of sweat glands is not known, and our findings, together with recent reports showing that NOTCH4 polymorphisms increases the susceptibility to common skin diseases, warrants future functional studies on cutaneous NOTCH4. 65,66antification of Notch activity in skin biopsies from patients with hidradenitis suppurativa revealed no evidence that deficient Notch signalling is an early pathogenic event, as judged by detection of activated NOTCH1, NOTCH2 as well as the target gene HES1.
If a general reduction in cutaneous Notch signalling was a central and general feature of the disease, we would expect to either find an altered baseline signal (non-lesional) or a reduced signal in early/ future HS skin (peri-lesional).Peri-lesional skin is assumed to represent the early inflammatory changes seen in the HS. 67,68Although we observed no apparent alteration in Notch activity, we cannot rule out that biologically relevant changes may exist below the threshold of our methods.Moreover, as this study does not address familial HS, it does not rule out reduced Notch signalling in patients with confirmed gamma-secretase mutations, and future studies should employ our methodology to investigate Notch activation in cohorts of mutation positive patients.
Our findings align with several reports that question whether reduced Notch signalling serve as a unifying mechanism in HS. 41 Mechanistic in vitro experiments on gamma-secretase mutations have not been able to demonstrate a defect in enzyme activity, thus questioning whether haploinsufficiency is sufficient to affect Notch signalling. 69Even if an enzymatic defect was present, gamma-secretase has more than 90 other known substrate that could also mediate biological effects. 70Supporting this, three of the identified sequence variants in 2017 did not impact the canonical Notch pathway 71 and an in silico analysis found several other GS substrates to be altered in HS. 72 Although we did not show altered Notch activation in non-lesional or peri-lesional skin, in lesional epidermis there was a significant decrease in Notch activation.The reduction in active NOTCH1 and NOTCH2 and the target gene HES1 appeared to be mainly confined to areas overlying severe dermal inflammatory infiltrates, while areas with less inflammation showed similar Notch distribution to that of non-lesional skin.We thus believe that the reduced Notch activation most likely is secondary to the inflammation rather than a primary event.However, whether a reduction in Notch signalling can have a role in regulating skin inflammation in HS should be addressed experimentally in future studies.Our findings of reduced HES1 in lesional HS skin contrast that of a recent report examining publicly available transcriptome data that found no change in the expression of HES1 or the Notch receptors. 41This illustrates the difference of whole tissue transcriptome analysis to that of spatially resolved protein detection.Others have observed an increased level of NOTCH1, NOTCH2 and NOTCH3 receptor on both mRNA and protein level in both peri-lesional and lesional HS skin. 40An altered level of Notch receptor expression in diseased tissue is an interesting finding which warrant further investigations, but it does not necessarily reflect Notch activation.The regulation of the Notch pathway is complex and not fully understood. 2,60In situ measurement of Notch signalling has been an issue of concern for decades.Detection of NICD1 using immunohistochemistry has been successfully employed in the past, and is believed to reflect the ongoing signal. 73,74e limitation of this study is mainly related to the inherent limitations of the method of immunohistochemistry.Although we have performed a thorough validation, there are several technical factors that can influence such immunostainings.Currently, there are no other methods that allow for spatially resolved detection of Notchactivity in situ in human tissue, and transgenic methods available for murine studies are not compatible with human studies. 73It is important to appreciate that the signal in the immunohistochemistry staining has an unknown threshold for detection.In other words, while a cell without a nuclear signal can be assumed to have a lower level of signalling than a cell with a positive nuclear signal, the method cannot be used to state the absence of receptor activation or as a measurement of the absolute amount of activated receptor present in the cell.
Beyond human skin and HS, Notch signalling has generated considerable interest as a drug target in a wide range of diseases.
However, global Notch targeting by gamma-secretase inhibitors is limited by severe gastrointestinal toxicity. 75By contrast, selective inhibition of individual ligands and receptors appears to promise therapeutic efficacy in the absence of side effects. 76To assess the translational relevance of preclinical studies, it therefore becomes highly interesting to assess the cell type-specific distribution of human Notch ligands and receptors in health and disease.By testing and validating a panel of primary antibodies, we here provide a tool for scientists to detect native, activated Notch receptors in a range of human tissues.
In conclusion, this study describes methods to detect native, activated Notch receptors in human skin, and for the first time define the distribution of active Notch signalling in apocrine-bearing skin.
We find no evidence that deficient Notch signalling is an early disease marker of hidradenitis suppurativa.

1 |TA B L E 2
Figures were produced in Adobe Illustrator CS6 and AdobeInDesign C26.
tive receptor signalling is to use antibodies targeting the C-terminal domains of Notch receptors.Detection of such epitopes can be expected to produce a mixed nuclear/membranous signal, where the nuclear signal is presumed to represent the cleaved, activated fraction of receptors and the membranous signal to represent the uncleaved, membrane-bound fraction of receptors (Figure 1B,D).
extent of Notch inhibition in our ex vivo human skin model in which biopsies were cultured overnight in growth medium containing either the gamma-secretase inhibitor DAPT or DMSO as vehicle.The ex vivo model is in our experience suitable for assessing changes in F I G U R E 1 Strategies to detect activated Notch receptors by immunostaining.Notch receptor activation includes proteolytic cleavage and nuclear translocation of the intracellular domain of the receptor (A,B).Primary antibodies used in this manuscript (indicated in red) were specific to either the cleavage-site of the intracellular receptor domain (A) or epitopes in the C-terminal domain (B).Detection of the activated receptor by the methods shown in panel A and B can therefore be assumed to produce a nuclear (C) or a mixed nuclear/membranous signal (D), respectively (signal indicated in red).Micrographs (E-J) demonstrate immunostaining with anti-NICD1 (E, G, I), a cleavage-site specific antibody, and anti-NOTCH2 (F, H, J), a primary antibody specific for the C-terminal domain.The antibodies ability to detect Notch activation were evaluated by ex vivo cultured human skin explants exposed to the γ-secretase inhibitor DAPT.Micrographs shows human skin biopsies directly formalin fixed immediately after harvesting (E, F), explants cultured in DMSO control medium (G, H) and explants exposed to DAPT (I, J).Scalebar = 20 μm.Quantitation of NICD1 (K) and nuclear Notch2 (L) signal of directly formalin fixed (dFF), DMSO control and DAPT treated explants was performed.Individual datapoint represent positive nuclei in the entire interfollicular epidermis (positive cells/mm 2 ).(*p = <0.05).
the simple columnar epithelium displayed neither active NOTCH1 (Figure 2D, lower panel), nor active NOTCH2 (Figure 2I, lower panel) signalling.Interestingly, the only Notch receptor exhibiting a nuclear signal in the apocrine gland was NOTCH4 (Figure S4K), possibly explaining the expression of HES1 in the same cells (Figure 2N, lower panel).In the double-layered glandular ducts, luminal cells displayed nuclear NOTCH2 signal (Figure 2I, upper panel), whereas NICD1 was found in a subset of basal cells (Figure 2D, upper panel).
as quantification is based on areas of epidermal tissue.When assessing NICD1, NOTCH2 and HES1 we did however observe a pattern where areas of severe dermal inflammatory infiltrates corresponded to less Notch activation in the overlying epidermis, as illustrated in Figure 4.When comparing overall NICD1 and HES1 epidermal signal, there was a significant reduction in lesional biopsies compared to both healthy controls, non-lesional and peri-lesional HS skin (Figure 4B,C).
have assessed Notch activation per se in HS tissue, and conclusions have instead been based on observations of general Notch receptor expression or indirect measures of Notch target genes.In this study, we established methods to detect native activated Notch receptors beyond that of NICD1 through immunohistochemical staining of human skin biopsies.We substantiated the specificity F I G U R E 3 Detection of activated Notch in non-lesional and peri-lesional HS skin compared to healthy control skin.Representative images of epidermis stained for NICD1 (A, B, C), NOTCH2 (E, F, G) and HES1 (I, J, K) in healthy control skin (A, E, I), non-lesional skin (B, F, J) and peri-lesional skin (C, G, K).Scalebar 20 μm.Quantitation of nuclear signal (positive cells/mm 2 ) are shown for NICD1 (D), NOTCH2 (H) and HES1 (L).Data are presented as dot plots with individual datapoints, means (horizontal lines) and standard deviation (error bars).C = healthy control skin.NL, non-lesional skin.PL, peri-lesional skin.HS, Hidradenitis suppurativa.ns, non-significant.by a number of criteria: first, we ensured that the immunohistochemical signal indicating receptor activation corresponded well to what can be expected from the literature, using CD20-positive follicular B cells, alpha smooth muscle actin-positive perivascular cells, and vascular endothelial cells as positive controls for NOTCH2, NOTCH3 and NOTCH4-activation, respectively.

F I G U R E 4
Detection of activated Notch in lesional HS skin.Panel (A) shows an entire tissue slide from lesional HS with an area of high (D) and low (H) degree of dermal inflammation.Panel (D-K) correspond to magnified view of boxed area of epidermis in panel A. illustrating reduced nuclear Notch signal in epidermis overlying dermis with high (E-G) degree of inflammation compared to low (I-K) degree of inflammation.Immunostaining was performed for NICD1 (E, I), NOTCH2 (F, J) and HES1 (G, K).Scalebar 100 μm.Panel (B) and (C) shows quantification of epidermal nuclear signal (positive cells/mm 2 ) of NICD1 (B) and HES1 (C) in healthy controls, non-lesional, peri-lesional and lesional tissue.Data in panel (B) and (C) are represented as scatter plot with individual datapoints, including means (horizontal line) and standard deviations (error bar).*p = <0.05,**p = <0.01,***p = <0.001.C, healthy control skin.NL, non-lesional skin.PL, peri-lesional skin.HS, Hidradenitis suppurativa.
AUTH O R CO NTR I B UTI O N SOlav Sundnes, Guttorm Haraldsen, Johanna Hol Fosse and Anastasia Renzi designed the study.Olaf Harald Antonsen andS U PP O RTI N G I N FO R M ATI O NAdditional supporting information can be found online in the Supporting Information section at the end of this article.

Figure S1 .
Figure S1.Detection of NICD1 and NOTCH2 in mouse tissues.Murine spleens were immunostained for NICD1 (A, B) or NOTCH2 (C-F).Micrographs (A, B) show a positive nuclear signal for NICD1 in spleens of control animals (A) that was obliterated in biopsies harvested 6 h after DBZ treatment (B).Micrographs (C, D) show NOTCH2 signal in spleens of mice injected with vehicle (C) and mice injected with DBZ (D), showing a clear reduction in nuclear signal in DBZ-treated mice that instead feature a membranous signal pattern (D).Micrographs (E-F) show NOTCH2 signal in spleens of mice treated with isotype (E) or NOTCH2 blocking (F) antibodies, demonstrating obliteration of nuclear NOTCH2 signal upon specific NOTCH2 inhibition.The boxed area in the panels corresponds with the magnified view of the selected area.DBZ, dibenzasepine.

Figure S2 .
Figure S2.NOTCH2 and NOTCH3 in human tonsil.Human tonsils were stained for NOTCH2 and the B-cell marker CD20 and examined for expression of nuclear NOTCH2 (A) and the co-expression of nuclear NOTCH2 (purple) and CD20 (turquoise) (B).Micrographs of immunostained human tonsil show the distribution of NOTCH3 (C) and the co-expression of NOTCH3 and α-SMA (NOTCH3-purple, α-sma-yellow) (D).Strong NOTCH3 expression was observed in α-SMA-positive perivascular cells.The boxed area in the panels corresponds with the magnified view of the selected area.

Figure S5 .
Figure S5.Detection of activated Notch in hair follicle and sebaceous glands of non-lesional and peri-lesional HS skin