Lesional Inflammatory Profile in Hidradenitis Suppurativa Is Not Solely Driven by IL-1

Abstract

Immune dysregulation is strongly implicated in the pathogenesis of hidradenitis suppurativa (HS) (Vossen et al., 2018Vossen A.R.J.V. van der Zee H.H. Prens E.P. Hidradenitis suppurativa: a systematic review integrating inflammatory pathways into a cohesive pathogenic model.Front Immunol. 2018; 9: 2965Crossref PubMed Scopus (82) Google Scholar). This is demonstrated by the presence of inflammation in both lesional and perilesional HS skin (Kelly et al., 2015Kelly G. Hughes R. McGarry T. van den Born M. Adamzik K. Fitzgerald R. et al.Dysregulated cytokine expression in lesional and nonlesional skin in hidradenitis suppurativa.Br J Dermatol. 2015; 173: 1431-1439Crossref PubMed Scopus (151) Google Scholar, Van der Zee et al., 2012Van der Zee H.H. De Ruiter L. Boer J. Van Den Broecke D.G. Den Hollander J.C. Laman J.D. et al.Alterations in leucocyte subsets and histomorphology in normal-appearing perilesional skin and early and chronic hidradenitis suppurativa lesions.Br J Dermatol. 2012; 166: 98-106Crossref PubMed Scopus (98) Google Scholar, Witte-Händel et al., 2019Witte-Händel E. Wolk K. Tsaousi A. Irmer M.L. Mößner R. Shomroni O. et al.The IL-1 pathway is hyperactive in hidradenitis suppurativa and contributes to skin infiltration and destruction.J Invest Dermatol. 2019; 139: 1294-1305Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). However, our current understanding of HS pathophysiology, with follicular occlusion and rupture as the primary events, is under scrutiny. A complex immune-driven pathogenesis, initiated by an initial aberrant immune response to a possibly dysbiotic cutaneous microbiome, is suspected, with follicular occlusion occurring as a secondary phenomenon (Frew, 2019Frew J.W. Commentary: hidradenitis suppurativa: A systematic review integrating inflammatory pathways into a cohesive pathogenic model.Front Immunol. 2019; 10: 302Crossref PubMed Scopus (9) Google Scholar). It is presumed that commensal bacterial products like lipopolysaccharide, peptidoglycan, and bacterial DNA and RNA, as well as complex keratin filaments released after follicular rupture, promote the simultaneous activation of multiple auto-inflammatory pathways such as membrane and cytosolic innate receptors, complement, and the NLRP inflammasome. The production of IL-1 is suspected to drive this inflammation by the induction of various chemokines and cytokines such as TNF, IL-6, IL-8, and IL-23, resulting in the recruitment of T helper type (Th) 1, Th17, and other inflammatory cells into the lesional HS infiltrate (Contassot et al., 2012Contassot E. Beer H.D. French L.E. Interleukin-1, inflammasomes, autoinflammation and the skin.Swiss Med Wkly. 2012; 142: w13590PubMed Google Scholar, Vossen et al., 2018Vossen A.R.J.V. van der Zee H.H. Prens E.P. Hidradenitis suppurativa: a systematic review integrating inflammatory pathways into a cohesive pathogenic model.Front Immunol. 2018; 9: 2965Crossref PubMed Scopus (82) Google Scholar, Witte-Händel et al., 2019Witte-Händel E. Wolk K. Tsaousi A. Irmer M.L. Mößner R. Shomroni O. et al.The IL-1 pathway is hyperactive in hidradenitis suppurativa and contributes to skin infiltration and destruction.J Invest Dermatol. 2019; 139: 1294-1305Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). The objective of this study was to investigate the role of IL-1 in HS. Using an ex vivo skin model, we aimed to mimic the inflammatory cytokine profile of lesional HS skin by stimulation of perilesional HS skin with IL-1α and IL-1β (Vossen et al., 2019Vossen A. Ardon C.B. van der Zee H.H. Lubberts E. Prens E.P. The anti-inflammatory potency of biologics targeting TNF-α, interleukin (IL)-17A, IL-12/23 and CD 20 in hidradenitis suppurativa: an ex vivo study.Br J Dermatol. 2019; 181: 312-323Crossref Scopus (24) Google Scholar). Skin punch biopsies, HS lesional (HSL; actively inflamed, nonfluctuating, indurated, erythemous nodules) and normal-appearing HS perilesional (HSP; 2 cm away from an active lesion, void of any HS-related lesions/activity), were obtained from 10 patients with HS (Hurley stage 1, n = 2; Hurley stage 2, n = 6; and Hurley stage 3, n = 3), and healthy control biopsies (NN) (n = 5) were obtained from submammary skin from breast reduction surgery (n = 3) or from peri- or infra-umbilical skin from abdominoplasty discard (n = 2). For HS samples, according to the opt-out principle used in the Erasmus University Medical Center, no informed consent is required for the use of surgical discard tissue for research purposes. Control skin samples were obtained from healthy individuals in the Saint Franciscus Hospital in Rotterdam, the Netherlands. All healthy volunteers provided written informed consent. Ethical review board approval was given by the IRB Erasmus University Medical Center Rotterdam (NL45264.078.13) HSL and HSP 3-mm biopsies were cultured for 24 hours in a transwell insert using complete Iscove’s Modified Dulbecco’s Medium culture medium (Supplementary Materials and Methods). HSP and NN samples were also stimulated with human recombinant IL-1α 10 ng/ml (200-LA, R&D Systems, Minneapolis, MN) and human recombinant IL-1β 10 ng/ml (201-LB, R&D Systems), separately. After culture, pro-inflammatory cytokine protein levels in the culture media and mRNA expression in the skin biopsies (HSP and NN samples only) were subsequently analyzed using the Luminex multiplex assay (LXSAHM-16, R&D Systems) and real-time qPCR, respectively (Supplementary Materials and Methods). The lower limit of quantification was imputed when protein concentrations (pg/ml) in the supernatant were below the detection limit. For the primary objective, the relative protein and mRNA expression levels for HSP skin stimulated with IL-1α and IL-1β relative to unstimulated HSP skin (only culture medium) were calculated and expressed as fold change. The Wilcoxon signed rank test for paired nonparametric comparisons was used to compare stimulated conditions to culture medium that was set to 1.00. Results obtained from HSL and HSP skin were compared with those of five NN. Additionally, protein concentrations in the culture media of HSL skin and HSP skin were separately compared with NN skin using the nonparametric Mann-Whitney U test to increase dimensionality of the data. Statistical analyses were conducted using SPSS Statistics 24.0 (IBM Corporation, Armonk, NY). A two-sided P-value < 0.05 was considered significant. Overall, 62.5% (10 of 16) of the inflammatory proteins were significantly elevated in HSP skin compared with NN skin (Supplementary Table S1). After stimulation with IL-1α or IL-1β, 40.0% (6 of 15) and 78.6% (11 of 14), respectively, of the inflammatory proteins were significantly elevated in NN skin compared with 7.1% (1 of 14) and 38.5% (5 of 13) in HSP skin (Table 1, Supplementary Table S2). This is illustrated by the combined effect on TNF-α, IFN-γ, IL-17A, and IL-12/23p40 protein levels and IL-6, IL-23p19, and HBD-2 mRNA expression levels in stimulated HSP and NN skin (Figure 1, Supplementary Table S3). Overall, IL-1β induced a stronger response than IL-1α in both NN and HSP skin. Altogether, cytokine levels in HSP skin were substantially elevated compared with NN skin that further induction by IL-1 in HSP skin was negligible.Table 1Cytokine Release in the Supernatant (Fold Change) by NN and HSP Skin after Stimulation with IL-1α and IL-1β for 24 HoursNumberProteinNN Skin + IL-1α (n = 5)P-valueNN Skin + IL-1β (n = 5)P-valueHSP Skin + IL-1α (n = 10)P-valueHSP Skin + IL-1β (n = 10)P-valueFold ChangeMedian (IQR)Fold ChangeMedian (IQR)Fold ChangeMedian (IQR)Fold ChangeMedian (IQR)1CCL-208.4 (5.8–9.2)<0.0514.4 (7.1–51.2)<0.050.9 (0.7–1.9)0.7991.2 (0.7–2.4)0.2852IL-1β9.6 (5.3–11.0)<0.05NC–1.2 (0.8–2.0)0.241NC–3TNF-α11.3 (5.2–14.6)<0.0521.1 (9.7–24.3)<0.051.1 (1.0–1.6)0.2031.1 (0.7–1.8)0.3744IL-17E7.9 (4.5–11.2)<0.0521.0 (9.6–24.2)<0.051.2 (0.9–2.2)0.2142.6 (2.0–4.3)<0.015IL-211.5 (1.1–1.7)0.0682.1 (1.8–2.5)<0.051.1 (1.0–1.2)0.1101.3 (1.1–1.4)0.0596IL-271.0 (1.0–1.0)1.00011.3 (7.9–14.6)<0.051.2 (0.7–6.3)0.2602.9 (1.7–9.3)<0.057IL-63.3 (3.0–7.7)<0.053.3 (2.1–7.7)<0.05NA–NA–8IL-1021.6 (11.5–37.2)<0.0527.8 (14.1–35.7)<0.051.2 (0.8–1.9)0.1691.1 (0.6–1.6)0.2859IL-17A1.5 (1.5–1.5)0.0743.8 (3.4–4.3)<0.050.7 (0.5–1.2)0.3330.8 (0.5–0.9)0.38610IL-331.2 (0.7–1.3)0.8931.1 (0.9–2.0)0.2251.3 (0.9–1.9)0.0741.2 (1.1–1.8)0.05911IFN-γ1.0 (1.0–1.0)1.0004.7 (3.8–4.7)<0.051.0 (0.7–1.0)0.9172.5 (1.4–4.5)<0.0512IL-51.0 (1.0–1.0)0.3172.7 (2.7–2.7)<0.051.5 (1.0–2.2)<0.052.1 (1.7–2.6)<0.0513IL-12/23p401.4 (1.3–1.6)0.0802.6 (2.5–3.0)<0.051.1 (1.0–1.2)0.2852.0 (1.6–2.2)<0.0114IL-221.1 (1.0–1.3)0.3451.3 (1.1–1.5)0.0800.9 (0.6–1.0)0.0661.0 (0.6–1.0)0.31415IL-311.0 (1.0–1.0)1.0001.0 (1.0–157.9)0.1571.0 (1.0–2.6)0.14157.4 (1.0–157.9)<0.0516IL-12p70ND–ND–ND–ND–All cytokines1.5 (1.0–5.8)<0.0013.5 (2.0–12.2)<0.0011.1 (0.8–1.6)0.0021.5 (1.0–2.6)<0.001Abbreviations: HSL, hidradenitis suppurativa lesional; HSP, hidradenitis suppurativa perilesional; IQR, interquartile range; NA, not analyzed; NC, not calculated; ND, not detected; NN, healthy control.IL-1α: 10 ng/ml diluted in culture media. IL-1β: 10 ng/ml diluted in culture media. HSP: normal-appearing skin 1–2 cm away from an active HS lesion. Luminex assay (LXSAHM-16, R&D Systems, Minneapolis, MN). NA samples: IL-6 was out of range above the calibration line in most samples in the culture media condition. Open table in a new tab Abbreviations: HSL, hidradenitis suppurativa lesional; HSP, hidradenitis suppurativa perilesional; IQR, interquartile range; NA, not analyzed; NC, not calculated; ND, not detected; NN, healthy control. IL-1α: 10 ng/ml diluted in culture media. IL-1β: 10 ng/ml diluted in culture media. HSP: normal-appearing skin 1–2 cm away from an active HS lesion. Luminex assay (LXSAHM-16, R&D Systems, Minneapolis, MN). NA samples: IL-6 was out of range above the calibration line in most samples in the culture media condition. In this study, we aimed to mimic the lesional HS cytokine protein and gene expression profiles in HSP skin and NN skin by stimulation with IL-1α and IL-1β. As expected, we found that cytokines in NN skin could be proficiently induced. However, background cytokine levels in HSP skin were substantially higher than NN skin to such an extent that there was generally no additional induction by IL-1. The upregulation of pro-inflammatory cytokines in HSP skin may be the result of a plateau after an in vivo stimulation phase. However, the cytokines that were elevated in HSP skin showed even higher levels in HSL skin, probably because of synergism with other cytokines in vivo. This trend was seen for most of the known HS-associated cytokines such as TNF-α, IL-1β, and CCL-20, which is in accordance with current literature (Kelly et al., 2015Kelly G. Hughes R. McGarry T. van den Born M. Adamzik K. Fitzgerald R. et al.Dysregulated cytokine expression in lesional and nonlesional skin in hidradenitis suppurativa.Br J Dermatol. 2015; 173: 1431-1439Crossref PubMed Scopus (151) Google Scholar, Van der Zee et al., 2012Van der Zee H.H. De Ruiter L. Boer J. Van Den Broecke D.G. Den Hollander J.C. Laman J.D. et al.Alterations in leucocyte subsets and histomorphology in normal-appearing perilesional skin and early and chronic hidradenitis suppurativa lesions.Br J Dermatol. 2012; 166: 98-106Crossref PubMed Scopus (98) Google Scholar, Witte-Händel et al., 2019Witte-Händel E. Wolk K. Tsaousi A. Irmer M.L. Mößner R. Shomroni O. et al.The IL-1 pathway is hyperactive in hidradenitis suppurativa and contributes to skin infiltration and destruction.J Invest Dermatol. 2019; 139: 1294-1305Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). Moreover, the trend of higher levels in HSL than in HSP skin was also seen for IL-33, a cytokine that has not previously been described in HS. IL-33 is a member of the IL-1 superfamily and functions as an alarmin for tissue necrosis and stimulates mast cells, dendritic cells, Th1 cells, neutrophils, and macrophages to produce a broad range of cytokines (Cayrol and Girard, 2018Cayrol C. Girard J.P. Interleukin-33 (IL-33): a nuclear cytokine from the IL-1 family.Immunol Rev. 2018; 281: 154-168Crossref PubMed Scopus (364) Google Scholar, Vossen et al., 2018Vossen A.R.J.V. van der Zee H.H. Prens E.P. Hidradenitis suppurativa: a systematic review integrating inflammatory pathways into a cohesive pathogenic model.Front Immunol. 2018; 9: 2965Crossref PubMed Scopus (82) Google Scholar). In addition, IL-5 levels were found to be significantly elevated in HSL skin compared with NN skin. IL-5, not previously demonstrated to be elevated in HS lesions, is not only a growth factor for eosinophils but also for B-cells (Takatsu et al., 2009Takatsu K. Kouro T. Nagai Y. Interleukin 5 in the link between the innate and acquired immune response.Adv Immunol. 2009; 101: 191-236Crossref PubMed Scopus (86) Google Scholar). The latter are known to be massively present in HSL skin (Van der Zee et al., 2012Van der Zee H.H. De Ruiter L. Boer J. Van Den Broecke D.G. Den Hollander J.C. Laman J.D. et al.Alterations in leucocyte subsets and histomorphology in normal-appearing perilesional skin and early and chronic hidradenitis suppurativa lesions.Br J Dermatol. 2012; 166: 98-106Crossref PubMed Scopus (98) Google Scholar). Moreover, eosinophils are also commonly present in HSL skin and may be partly responsible for the frequently reported symptom of itch (Vossen et al., 2017Vossen A.R.J.V. Schoenmakers A. van Straalen K.R. Prens E.P. van der Zee H.H. Assessing pruritus in hidradenitis suppurativa: a cross-sectional study.Am J Clin Dermatol. 2017; 18: 687-695Crossref PubMed Scopus (35) Google Scholar). The major strength of our study is the use of a standardized ex vivo transwell culture system. However, possible limitations encompass the relatively small sample size, the lack of combined stimulation with IL-1α + IL-β, inclusion of other important cytokines like TNF-α and IL-17, and the lack of the use of distant uninvolved HS skin. Another limitation could be that the use of skin from abdominoplasty surgery as NN may have underestimated the levels of Th17/Th1 cytokine expression in the NN skin biopsies, as this region has a lower density of apocrine glands compared with the apocrine-rich skin of the axillae or groin (Jenei et al., 2019Jenei A. Dajnoki Z. Medgyesi B. Gáspár K. Béke G. Á Kinyó et al.Apocrine gland-rich skin has a non-inflammatory IL-17-related immune milieu, that turns to inflammatory IL-17-mediated disease in hidradenitis suppurativa.J Invest Dermatol. 2019; 139: 964-968Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). As a result, the fold change upon stimulation could have been overestimated in the NN skin biopsies. Future experiments using multiple additive or synergistic cytokine cocktails will shed light on the apparent plateau of cytokine stimulation in HSP skin. In conclusion, this study reveals the auto-inflammatory nature of HS, characterized by the spontaneous increased production in perilesional skin of a broad range of inflammatory cytokines. No data sets were generated during this study. Allard R. J. V. Vossen: http://orcid.org/0000-0002-1448-5972 Kelsey R. van Straalen: http://orcid.org/0000-0003-3305-3814 Edwin F. Florencia: http://orcid.org/0000-0001-9005-4464 Errol P. Prens: http://orcid.org/0000-0002-8158-660X The authors state no conflict of interest. Prior presentation: 6 February 2019, 9th European Hidradenitis Suppurativa Foundation conference. Ethical review board approval: IRB Erasmus University Medical Center Rotterdam (NL45264.078.13). Conceptualization: EP; Data Curation: EF; Formal Analysis: AV, KvS; Methodology: EF, EP; Investigation: EF; Visualization; KvS; Writing - Original Draft Preparation: AV, KvS; Writing - Review and Editing: AV, EP, KvS. The 3-mm biopsies were immediately cultured in a transwell system (Netwell; Costar, Cambridge, MA). In brief, biopsies were placed in punched-out holes in the transwell membrane of a 12-well plate with the epidermis exposed to the air and the dermis immersed in 1 ml Iscove’s Modified Dulbecco’s Medium (Gibco, Paisley, United Kingdom) containing 0.5% human AB serum, penicillin (100 U/ml), and streptomycin (100 U/ml) with or without IL-1α (10 ng/ml) and IL-1β (10 ng/ml). Skin biopsies were incubated for 24 hours at 37 °C in an atmosphere of 5% CO2 and 98% humidity. After harvesting, the biopsies were placed in 250 μl lysis buffer containing 1% β-Mercaptoethanol. Both the supernatants and the biopsies were transferred to a polypropylene tube and stored at −80 °C until further analysis. The following 16 inflammation-related cytokines were simultaneously measured in the culture media using a customized bead-based Multi-Analyte Profiling assay (Luminex, R&D Systems, Minneapolis, MN): CCL-20, TNF-α, IFN-γ, IL-1ß, IL-5, IL-6, IL-10, IL-12/23p40, IL-17A, IL-17E, IL-21, IL-22, IL-23p19, IL-27, IL-31, and IL-33. Assays were used according to the manufacturers’ protocol. A dilution factor of 2 was used for all assays. Total mRNA was extracted using the GenElute Mammalian Total RNA Miniprep Kit (Sigma-Aldrich, St. Louis, MO). RNA was treated with 0.1 U/μl DNAse (Invitrogen, Carlsbad, CA) and cDNA was subsequently synthesized using 1 μg total RNA template, with SuperScript II reverse transcriptase, random hexamer primers (Invitrogen) and oligo(dT)15 (Promega, Madison, WI). Primers and probes were designed and based on the Universal Probe library (Roche Applied Science, Indianapolis, IN). Real-time qPCR was performed for four genes using the ViiA7 sequence-detection system (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA), IL-1β, IL-6, IL-23p19, and HBD-2, with ABL1 as reference housekeeping gene. Gene expression analysis was performed with QuantStudio Real-Time quantitative PCR Software version 1.3 (Applied Biosystems). Primer efficiency and calibration curves were determined for each primerset in 10-fold dilution steps in duplicate and issued based on predetermined standard values.Supplementary Table S1Inflammatory Cytokine Levels in the Supernatant after Culturing of NN, HSP, and HSL Skin for 24 HoursNumberProteinLLOQNN Skin (n = 5)HSP Skin (n = 10)HSL Skin (n = 10)P-valueP-valuepg/mlMedian (IQR), pg/mlMedian (IQR), pg/mlMedian (IQR), pg/mlHSP vs NNHSL vs NN1CCL-205.88.3 (5.8–10.8)683 (303–1,376)3,538 (2,627–4,242)<0.001<0.0012IL-1ß0.90.9 (0.9–2.2)166 (44–399)604 (432–942)<0.001<0.0013TNF-α0.60.6 (0.6–1.7)39.2 (19.6–91.4)101.5 (47.4–184.4)<0.001<0.0017IL-17E15.815.8 (15.8–15.8)229 (71–332)383 (332–421)<0.01<0.0018IL-2110.414.1 (14.1–19.8)32.2 (22.6–37.0)35.1 (27.9–39.4)0.01<0.00111IL-2710.1ND62.4 (10.1–147.9)198.8 (156.4–241.0)0.075<0.0014IL-64.57,345 (3,142–8,065)24,301 (12,098–24,301)1n = 6 out of range above the calibration line.24,301 (24,301–24,301)2n = 9 out of range above the calibration line.0.0010.0015IL-102.12.1 (2.1–2.1)441 (180–1,108)452 (282–784)0.0010.0016IL-17A3.856.1 (56.1–56.1)1,712 (557–6,214)1,693 (544–3,755)<0.010.0019IL-3316.025.0 (18.0–26.0)46.9 (33.2–61.0)92.1 (62.6–112.4)<0.050.00112IFN-γ7.3ND14.3 (7.3–34.1)37.2 (34.1–49.4)0.129<0.0113IL-52.53.3 (2.5–5.5)2.5 (2.5–2.5)6.8 (6.8–7.9)0.129<0.0110IL-12/23p4037.71,192 (1,116–1,305)1,931 (1,566–2,431)1,993 (1,795–2,457)<0.05<0.0514IL-2216.827.9 (24.8–38.3)39.0 (27.9–126.3)36.8 (28.3–67.3)0.1650.25415IL-310.2ND0.2 (0.2–0.2)4.9 (0.2–9.7)0.5940.12916IL-12p7015.3NDNDND1.0001.000Abbreviations: HSL, hidradenitis suppurativa lesional; HSP, hidradenitis suppurativa perilesional; IQR, interquartile range; LLOQ, lowest limit of quantification; ND, not detected; NN, healthy control.HSP skin: normal-appearing skin ± 2 cm away from an active HS lesion. Luminex assay (LXSAHM-16, R&D Systems, Minneapolis, MN).1 n = 6 out of range above the calibration line.2 n = 9 out of range above the calibration line. Open table in a new tab Supplementary Table S2Inflammatory Cytokine Levels in the Supernatant after Stimulation with IL-1α and IL-1β for 24 hoursProteinNN Skin + IL-1α (n = 5)NN Skin + IL-1β (n = 5)HSP Skin + IL-1α (n = 10)HSP Skin + IL-1β (n = 10)Median (IQR), pg/mlMedian (IQR), pg/mlMedian (IQR), pg/mlMedian (IQR), pg/ml1CCL-2099.31 (69.95–144.94)101.68 (83.47–324.87)620.14 (266.90–1776.97)793.85 (308.15–2063.95)2IL-1β11.72 (9.42–32.50)NC357.53 (93.90–554.66)NC3TNF-α8.91 (8.91–8.91)15.76 (14.80–16.73)52.88 (20.80–99.68)48.45 (18.87–88.11)4IL-17E177.21 (124.62–177.21)382.76 (331.89–433.37)280.72 (190.21–331.89)583.86 (408.01–633.77)5IL-2121.65 (21.65–23.55)29.29 (25.46–40.89)33.14 (29.29–37.01)37.98 (31.69–43.81)6IL-2710.11 (10.11–10.11)113.88 (79.68–147.92)96.78 (53.83–173.37)215.67 (147.92–283.23)7IL-624301.08 (24301.08 –24301.08)24301.08 (24301.08–24301.08)24301.08 (24301.08–24301.08)NA8IL-1068.74 (45.42–78.18)74.93 (58.30–81.85)622.68 (203.27–917.70)501.87 (230.19–645.59)9IL-17A82.53 (82.53–240.82)240.82 (214.46–372.51)1186.93 (280.31–3401.68)0.1436.49 (359.33–4121.42)10IL-3322.45 (16.96–33.21)30.10 (27.02–32.18)53.41 (43.21–80.93)57.19 (34.78–80.08)11IFN-γ7.33 (7.33–7.33)34.07 (27.74–34.07)17.87 (9.12–32.49)55.36 (37.15–81.53)12IL-52.51 (2.51–2.51)6.78 (6.78–6.78)5.54 (5.54–5.54)8.47 (6.78–9.78)13IL-12/23p401749.29 (1565.83–1930.83)3024.03 (2972.18–3299.13)2074.80 (1611.88–2505.48)3901.36 (3152.86–4307.60)14IL-2232.42 (27.89–36.85)39.76 (36.85–41.20)36.06 (24.81–70.36)48.20 (28.98–74.51)15IL-310.16 (0.16–0.16)0.16 (0.16–25.27)0.16 (0.16–13.33)21.50 (2.54–41.13)16IL-12p70NDNDNDNDAbbreviations: HSL, hidradenitis suppurativa lesional; HSP, hidradenitis suppurativa perilesional; IQR, interquartile range; NA, not analyzed; NC, not calculated; ND, not detected; NN, healthy control.IL-1α: 10 ng/ml diluted in culture media. IL-1β: 10 ng/ml diluted in culture media. HSP: normal-appearing skin 1–2 cm away from an active HS lesion. Luminex assay (LXSAHM-16, R&D Systems, Minneapolis, MN). NA: IL-6 was out of range above the calibration line in most samples in the culture media condition. NC: IL-1ß was added to the culture media. Open table in a new tab Supplementary Table S3mRNA Expression (Fold Change) in NN and HSP Skin after Stimulation with IL-1α and IL-1ß for 24 hoursNumberGeneNN Skin + IL-1α (n = 5)P-valueNN Skin + IL-1ß (n = 5)P-valueHSP Skin + IL-1α (n = 10)P-valueHSP Skin + IL-1ß (n = 10)P-valueFold Change, Median (IQR)Fold Change, Median (IQR)Fold Change, Median (IQR)Fold Change, Median (IQR)1IL-1ß12.7 (12.3–17.0)<0.055.8 (5.2–11.0)<0.051.9 (0.9–2.6)<0.052.1 (1.4–2.5)<0.052IL-62.4 (1.3–2.7)0.1382.1 (2.0–2.3)0.0800.7 (0.5–1.3)0.6460.5 (0.4–2.2)0.9593IL-23p191n = 4 for condition NN skin + IL-1α and NN skin + IL-1ß because of lack of cDNA for RT qPCR analysis.6.0 (4.6–11.5)0.0686.0 (5.1–8.4)0.0682.6 (1.9–6.2)<0.012.5 (1.8–3.9)<0.014HBD-22n = 4 for condition NN skin + IL-1α because of lack of cDNA for RT qPCR analysis.11.2 (7.2–26.2)0.06816.6 (16.5–24.0)<0.051.8 (1.5–3.3)<0.051.9 (1.7–2.2)<0.01All genes6.0 (2.7–12.7)<0.0016.0 (2.3–16.5)0.0011.8 (0.9–3.0)<0.0011.8 (0.9–2.7)<0.001Abbreviations: HSL, hidradenitis suppurativa lesional; HSP, hidradenitis suppurativa perilesional; IQR, interquartile range; NN, healthy control.IL-1α: 10 ng/ml diluted in culture media. IL-1ß: 10 ng/ml diluted in culture media. HSP: normal-appearing skin ± 2 cm away from an active HS lesion.1 n = 4 for condition NN skin + IL-1α and NN skin + IL-1ß because of lack of cDNA for RT qPCR analysis.2 n = 4 for condition NN skin + IL-1α because of lack of cDNA for RT qPCR analysis. Open table in a new tab Abbreviations: HSL, hidradenitis suppurativa lesional; HSP, hidradenitis suppurativa perilesional; IQR, interquartile range; LLOQ, lowest limit of quantification; ND, not detected; NN, healthy control. HSP skin: normal-appearing skin ± 2 cm away from an active HS lesion. Luminex assay (LXSAHM-16, R&D Systems, Minneapolis, MN). Abbreviations: HSL, hidradenitis suppurativa lesional; HSP, hidradenitis suppurativa perilesional; IQR, interquartile range; NA, not analyzed; NC, not calculated; ND, not detected; NN, healthy control. IL-1α: 10 ng/ml diluted in culture media. IL-1β: 10 ng/ml diluted in culture media. HSP: normal-appearing skin 1–2 cm away from an active HS lesion. Luminex assay (LXSAHM-16, R&D Systems, Minneapolis, MN). NA: IL-6 was out of range above the calibration line in most samples in the culture media condition. NC: IL-1ß was added to the culture media. Abbreviations: HSL, hidradenitis suppurativa lesional; HSP, hidradenitis suppurativa perilesional; IQR, interquartile range; NN, healthy control. IL-1α: 10 ng/ml diluted in culture media. IL-1ß: 10 ng/ml diluted in culture media. HSP: normal-appearing skin ± 2 cm away from an active HS lesion. Ex Vivo Models and Interpretation of Mechanistic Studies in Hidradenitis SuppurativaJournal of Investigative DermatologyVol. 140Issue 7PreviewCurrent ex vivo and animal models of hidradenitis suppurativa (HS) display issues with fidelity and validity to human disease. Vossen et al.’s Transwell culture system holds potential to reliably assess mechanistic pathways in HS. Consideration of the sites of control tissue and comparison of the Transwell model against skin explant models would increase the validity of this method of inquiry. Full-Text PDF Open Archive