Interleukin‐36 cytokines are overexpressed in the skin and sera of patients with bullous pemphigoid

Bullous pemphigoid (BP) is an autoimmune bullous disease, characterized by autoantibodies targeting BP180 and BP230. The role of interleukin (IL)‐36, a potent chemoattractant for granulocytes, in BP remains elusive.The expression of IL‐36 cytokines (IL‐36α, β, γ) and their antagonists (IL‐36Ra and IL‐38) was analysed in the skin and serum samples of patients with BP (n = 31), psoriasis (n = 10) and healthy controls (HC) (n = 14) by quantitative polymerase chain reaction and enzyme linked immunosorbent assay, respectively. Skin and serum levels of all cytokines were correlated with the Bullous Pemphigoid Disease Area Index (BPDAI) score and with the serum concentration of pathogenic antibodies.IL‐36α, IL‐36β, IL‐36γ and IL‐36Ra were significantly (p < 0.05) overexpressed in BP skin compared to HC, without remarkable differences relative to psoriasis skin. The expression of IL‐38 was significantly (p < 0.05) higher in BP compared to psoriasis skin.IL‐36α and γ, but not β, serum concentrations were significantly (p < 0.05) higher in BP compared to HC. IL‐36γ was significantly (p < 0.05) more expressed in the serum of psoriasis patients than BP. The serum concentration of IL‐36Ra and IL‐38 were similar between BP and HC, while IL‐38 serum levels were significantly (p < 0.05) higher in BP compared to psoriasis patients. Serum IL‐36α correlated significantly with BPDAI (r = 0.5 p = 0.001).IL‐36 agonists are increased in BP patients, both locally and systemically. Serum IL‐36α might represent a potential biomarker for BP. An inefficient balance between IL‐36 agonists and antagonists is likely to occur during BP inflammation.


| Patients and controls
The study was conducted in accordance with the Declaration of Helsinki and received approval by local Institutional Review Boards.
Patients with a diagnosis of BP (n = 34) were recruited retrospectively in two tertiary referral centres in Italy (Florence and Rome). Diagnosis of BP was made according to current guidelines. 17 Demographic data, clinical activity calculated with Bullous Pemphigoid Disease Area Index (BPDAI) and immunoserological parameters of the patients are summarized in Table 1. BP was defined as mild with a BPDAI ≤19 and moderate to severe with a BPDAI ≥20. 18 Leftover diagnostic materials including lesional skin biopsies  Table S1.

| Evaluation of IL-36 mRNA expression in skin biopsies
Total RNA from skin biopsies was isolated by using a RNeasy Lipid

| Statistical analysis
Comparisons between three groups were performed using the Kruskal-Wallis test followed by Dunn's multiple comparisons test.
Comparisons between two groups were made using the Mann- and IL-36 γ were higher in BP patients with moderate to severe disease than those with mild disease, although the differences were not significant ( Figure S1).
With regard to the cytokines with antagonistic activity, our results showed a strong upregulation of IL36Ra in the skin lesions of BP patients compared to HC (p = 0.0005), which was similarly observed in the skin lesions of Pso patients (p = 0.004). In BP skin we found an increase, yet not significant, of IL-38, compared to HC skin (p = 0.28). Intriguingly, the mRNA level of this antiinflammatory cytokine was significantly higher in BP compared to Pso skin (p < 0.0001) ( Figure 1D,E). Skin expression of IL-38 was reduced in BP patients with moderate to severe disease than those with mild disease, although the results were not significant ( Figure S1).

| Expression of IL-36 molecules in the sera of patients with BP
In the serum, BP patients showed a significantly higher concentra- No other significant correlations emerged between IL-36 molecules and either BPDAI or the titre of circulating antibodies ( Figure 2F). neutrophil-derived proteases, such as cathepsin G, elastase and proteinase-3. These proteases are present in neutrophil extracellular traps (NETs). 29 NETs are detectable in the blister fluid of BP lesions as well as in the patients' sera. 30 Collectively, these findings suggest that IL-36 agonists can be potentially activated during the inflammatory response of BP.

| CON CLUS I ON S AND PER S PEC TIVE S
In this study, we observed that patients with BPDAI≥20 showed Remarkably, IL-38 was found to be overexpressed both in the skin

S U PP O RTI N G I N FO R M ATI O N
Additional supporting information can be found online in the Supporting Information section at the end of this article.