Our retrospective study was conducted on archived cutaneous biopsies of patients with SS who were diagnosed by dermatologists at Maharaj Nakorn Chiang Mai Hospital and Srinagarind Hospital over an eight-year period (2012–2020). The diagnosis of SS was reevaluated and confirmed via histological evaluation. Clinical history and relevant data including sex, age, and underlying conditions associated with SS were collected. According to the clinical setting, SS is categorized as classical (idiopathic), malignancy-associated, drug-induced, and AOID-associated. Cases of AOID were diagnosed when a patient met all of the following criteria: (i) they were an adult (over 18 years of age) who presented with disseminated opportunistic infections that were supposed to be from defects in cell-mediated immunity; (ii) the exclusion of other immunocompromised statuses — including human immunodeficiency virus, malignancy, or as a result of receiving immunosuppressive drugs — was confirmed; and (iii) there was a demonstration of the antibodies to IFN-γ using dot enzyme-linked immunosorbent assay (ELISA) (Maharaj Nakorn Chiang Mai Hospital) (12) or via inhibitory ELISA (Srinagarind Hospital) (13). The control group included normal skin tissue samples of amputated specimens from the accidents or individuals who underwent excisions of benign skin tumors presenting with neither clinical nor pathological findings of neutrophilic dermatoses.

Formalin-fixed paraffin-embedded (FFPE) tissues were sectioned at a thickness of 5 µm and stained with hematoxylin and eosin (H&E) using standard histological laboratory methods. For IHC staining and analysis, FFPE blocks were cut into 5 μm sections and heated at 60°C for 1 h in a dry oven to soften the paraffin. Briefly, the sections were deparaffinized with xylene and rehydrated using graded ethanol in water. Antigen retrieval was performed by CC1 (prediluted, pH 8.0) antigen retrieval solution (Ventana) performed on the Benchmark ULTRA automated slide Stainer for 32 minutes at 37°C. The sections were incubated with primary antibodies, at manufacturer’s recommended dilution for 32 min at 37°C. IHC staining was performed using a Ventana BenchMark ULTRA autostainer using a standard protocol. The following primary antibodies were used: mouse monoclonal anti-human IL-1β [2H12] (sc130323; Santa Cruz Biotechnology, Inc., USA, 1:50 dilution), rabbit polyclonal anti-human IL-6 (ab6672; Abcam, Cambridge, MA, USA, 1:100 dilution), rabbit polyclonal anti-human IL-17A (ab79056; Abcam, 1:100 dilution), rabbit monoclonal anti-human IFN-γ (ab218426; Abcam, 1:50 dilution), and mouse monoclonal anti-human TNF-α [Clone 28401] (mab610, R&D Systems, Minneapolis, MN, USA, 1:100 dilution). The Ultraview Universal DAB IHC detection kit was used for the visualization reaction (12 min), followed by counterstaining with hematoxylin and blue reagent. The slides were gently washed, dehydrated in graded ethanol and xylene, and mounted on coverslips onto microscope slides using a mounting medium. As positive controls for antibodies, human lymph node, lung, and kidney tissues were used to establish staining for IL-1β, IL-6 and IFN-γ, respectively, while human tonsil tissue was used for IL-17 and TNF-α staining.

Digital images of the IHC-stained slides were acquired using an Aperio Scanscope CS2 whole-slide scanner (Leica Biosystems, Nussloch, Germany) interfaced with Aperio ImageScope version 12 software (Leica Biosystems, Wetzlar, Germany). Regarding the histopathological pattern of SS as a dense and diffuse dermal neutrophilic infiltrate, the corresponding dermal region was outlined using the pen tool in Aperio ImageScope software. The optimized positive pixel count algorithm version 9 embedded in the Aperio ImageScope software was used to quantify positive IHC staining as described elsewhere (14). In brief, the algorithm classifying each pixel into negative (n; blue), weak-positive (wp; yellow), medium-positive (p; orange), and strong-positive (sp; red) bins, by a threshold of intensity values: In (total intensity of negative) = (220,255); Iwp (intensity threshold of weak-positive pixels) = (175,220); Ip (intensity threshold of medium-positive pixels) = (100,175); Isp (intensity threshold of strong-positive pixels) = (0,100), respectively. These values have an inverse association with the stain darkness, where a high intensity represents unstained pixels, while low intensity represents strong-positive pixels (15). Furthermore, the positivity percentage is given by the total number of positive pixels divided by the number of total pixels (negative and positive) in the analyzed area and multiplied by 100 (16). Quantitative image analysis was also performed using the sum of intensity values for all negative, weak-, medium-, and strong-positive pixels (In+Iwp+Ip+Isp) divided by the number of total pixels (Ntotal), presenting as an intensity score. Two independent blinded observers (P.C. and T.D.) evaluated serial sections. All measurements were repeated at least three times, and the mean values were determined.

SPSS version 23.0 (IBM Corp. 2015. Armonk, NY, U.S.A.) and GraphPad Prism (version 8.0; GraphPad Software, San Diego, CA, U.S.A.) were used to analyze the results. Categorical data were analyzed using the chi-square test and are presented as n (%). Data were analyzed via a one-way analysis of variance (ANOVA) and Student’s t test, as appropriate, and were displayed as mean ± standard deviation (SD). To account for multiple comparisons, the Tukey test was used (17). Values of p < 0.05 were considered statistically significant.

A total of 94 biopsy specimens from 94 patients were included in this study. Of these, 42 had AOID-associated SS (23 men and 19 women; age range 28-76), and 52 had SS without underlying AOID (20 men and 32 women; age range 26-83) ( Table 1 ). The median age of SS onset was 54.6 ± 8.8 and 56.0 ± 12.3 years among AOID and non-AOID, respectively. Most cases (43/52, 82.7%) of SS without AOID were classical or idiopathic, 13.5% (7/52) were associated with an underlying malignancy, and only two (3.8%) had drug-induced SS. Unsurprisingly, we noted that AOID-associated SS had significantly higher number of cases with a history of NTM infection when compared to non-AOID (92.9% vs 17.3%, p < 0.001).

All tissue samples from patients with SS — with or without AOID — and controls were examined. Representative images of H&E staining are shown in Figure 1 (top panels). Marked papillary dermal edema and dense inflammatory cell infiltration, consisting mainly of neutrophils, were evident in SS with and without AOID. Next, we determined the expression of various inflammatory cytokines in the biopsy samples using IHC. Positive IHC staining and the corresponding annotated whole-slide images for IFN-γ (middle panels, Figure 1 ) and IL-17 (bottom panels, Figure 1 ) were observed in diffuse and strong patterns in the dermis of both AOID- and non-AOID-associated SS. Image analysis of IHC staining using the percentage positivity score revealed significantly increased IFN-γ and IL-17 expressions in both AOID-associated SS (p < 0.001 and p < 0.001, respectively) and non-AOID-associated SS (p < 0.001 and p < 0.001, respectively) compared to those in the control group ( Table 2 ; Figure 2A ). Similarly, the staining intensity score confirmed significantly increased IFN-γ and IL-17 expressions in both AOID-associated SS (p < 0.001 and p < 0.001, respectively) and non-AOID-associated SS (p < 0.001 and p < 0.001, respectively) when compared to those in the controls. The results of quantitative image analysis, as well as the representative images of IHC staining for other cytokines, are presented in Table 2 ; Supplementary Figure S1 .

Given that opportunistic infections, and particularly NTM, could be a potential cause of AOID-associated SS by triggering inflammatory conditions (8), we investigated the effects of NTM infection on the expression of various cytokines using IHC analysis. A significant increase in IHC positivity in both IFN-γ and IL-17 in AOID-associated SS with NTM (p = 0.003 and p < 0.001, respectively) and without NTM (p < 0.001 and p < 0.001, respectively) was observed compared to the control group ( Table 3 ; Figure 2B ). Similarly, the staining intensity score significantly increased IFN-γ and IL-17 expression in both SS with NTM (p < 0.001 and p < 0.001, respectively) and without NTM (p < 0.001 and p < 0.001, respectively) compared to that of the controls. However, no pronounced differences were observed between the NTM-infected and non-NTM-infected groups for any of the cytokines.

A subtype analysis based on the etiology of SS was also performed to investigate differential cytokine expression through quantitative analysis. In both classic- and malignant-subtypes of non-AOID-associated SS, both IFN-γ (%positivity: p = 0.001 and p < 0.001, respectively; intensity score: p < 0.001 and p < 0.001, respectively) and IL-17 (%positivity: p < 0.001 and p = 0.006, respectively; intensity score: p = 0.002 and p = 0.031, respectively) were significantly differentially expressed compared with those in controls ( Table 4 ; Figure 2C ). There was a marked increase of IFN-γ in the drug-subtype of non-AOID-associated SS (%positivity: p = 0.023).