This was a retrospective cohort study conducted from April 2020 to December 2021. BP patient data were collected from the Eighth Affiliated Hospital of Sun Yat-sen University, Peking Union Medical College Hospital, and the University of Hong Kong Shenzhen Hospital. All BP patients fulfilled the recognized diagnostic criteria (20) based on clinical presentation, histopathological findings, and direct or indirect immunofluorescence test results. Moderate-to-severe BP was defined as an affected body surface area (BSA) > 10% or a skin erosion/blister total score on the BPDAI > 15, a urticaria/erythema score on the BPDAI > 20 (21). According to the medical record, patients receiving dupilumab with or without systemic corticosteroid were included in the dupilumab group, while patients receiving systemic corticosteroid only were included in the conventional group. Patients without regular follow-up for 24 weeks were excluded. Medical records and photographs were reviewed. We retrieved demographic characteristics, any underlying diseases, the lesional features, histological and immunological findings, laboratory data, doppler vascular ultrasonography, bone densitometry, the drugs used, the responses at weeks 4 and 24, and AEs during a 24-week regimen. Initially missing data were obtained by calling patients or their guardians. This study was approved by the Ethics Committee Institutional Board of our local hospital. Written informed consent was obtained from all participants.

Six plasma samples from three BP patients in the dupilumab group were collected before and after dupilumab injections. Patient 1 received both a dupilumab injection and a maintenance dose of corticosteroids after BP recurrence. Patients 2 and 3 were treated with dupilumab alone after primary BP attacks (details were summarized in supplemental patient information). Differentially expressed proteins (DEPs) were analyzed. Tandem mass tagging (TMT) coupled with liquid chromatography and tandem mass spectrometry (LC–MS/MS) were used to explore the proteomes. Supplementary Methods 1 provides detailed information on plasma collection and purification, exosome depletion, peptide labeling, LC–MS/MS and bioinformatics analyses, and database searching.

Serum BP180 IgG antibody, TARC, ICOSL and S100A12 levels in 40 plasma samples were quantitated by ELISAs in 20 BP patients (10 in the dupilumab group and 10in the conventional group) at baseline and at week 4 aftertreatment. All ELISAs followed the manufacturers’ instructions [BP180 (MBL, Nagoya, Japan), TARC (R&D Systems, Minneapolis, MN, USA), ICOSL (Cusabio Biotech Co., Wuhan, China), and S100A12 (Cusabio Biotech Co.)].

A Student’s t-test, Chi-Squared T test, Mann-Whitney U test, and Wilcoxon test, were performed as appropriate using SPSS ver. 26.0 software (IBM Corp., Armonk, NY, USA). A p-value < 0.05 was considered statistically significant.

Twenty patients with moderate-to-severe BP who received dupilumab alone or combined with conventional drugs and 20 age-, sex-, and disease severity-matched BP patients who received corticosteroid were recruited. All patients had been prescribed a background topical corticosteroid more than 1 week with poor clinical improvement. Among 40 patients, 22patients are newly diagnosed BP patients, and 18patients are recurrent ones. In conventional group, 10 of 20 patients received prior treatment with systemic corticosteroids before this recurrence, while in dupilumab group, 9 of 20 patients were treated with systemic corticosteroids or IVIG before. Notably, ten patients received dupilumab alone in the dupilumab group. Six patients in dupilumab group receiving systemic corticosteroid got poor outcomes after 1 week treatment and then received injections of dupilumab. The initial dupilumab dose was 600 mg followed by 300 mg weekly or every 1–4 weeks on 4 to 10 occasions, and the initial dosage of corticosteroid varied from 0.05mg/Kg to 2mg/Kg. By contrast, the initial dosage of corticosteroid in conventional group was varied from 0.2mg/Kg to 1mg/Kg prednisone depending on the disease severity and then was decreased at 2.5-10mg every 1-8 weeks in the follow-up. The baseline data are listed in Table 1 . The details of dupilumab or prednisolone treatment protocol were listed in supplemental patient information.

Of the 20 patients in the dupilumab group, twelve attained a CR with six on dupilumab alone. Eight patients achieved a PR with four on dupilumab alone ( Figure 1A ). In the conventional group, ten patients attained a CR and ten attained a PR ( Supplemental Figure 1 ). The therapeutic efficacy did not differ between the two groups (p =0.751) ( Figure 1B ). Notably, dupilumab alone efficiently treated both initial episode and recurrences of pemphigoid. In the 20 patients on dupilumab, a CR was attained by 16/20, a PR by 4/20 at weeks 12 and a CR was attained by 17/20, a PR by 3/20 at 24 respectively. While in the conventional group, a CR was attained by 15/20 patients, a PR by 5/20 at week 12and a CR by 20/20 at week 24.

The cumulative corticosteroid dose decreased in the dupilumab group by week 4 when a CR or PR was attained (dupilumab group222.75 ± 454.48mg; conventional group 859.63 ± 379.32mg, p<0.01) ( Figure 1C ), and by week 24 (dupilumab group 835.25 ± 1373.31mg; conventional group 3189.33 ± 999.69mg, p<0.01). It is important to highlight that 12 of 20 in dupilumab group received dupilumab without corticosteroid. Further details are provided in supplemental patient information. The AE rate in the conventional group (26 AEs in fourteen patients) was significantly higher than in the dupilumab group (6 AEs in five patients) during the 24 weeks of treatment ( Figure 1D ). What’s more, AEs in the conventional group were more severe than that in the dupilumab group. According to CTCAE criteria, among 26AEs in the conventional group, twelve AEs were classified as grade 1, eleven were grade 2, and three were grade 3, while in the dupilumab group, two AEs were classified as grade 1, and four were grade 2. AEs were summarized in supplemental patient information.

The percentage of eosinophils (EOS%) data at baseline and week4 were collected from 10 individuals in the dupilumab group and 10 in the conventional group whose serum anti-BP180 IgG antibody were detected. The EOS% decreased as BP became controlled after 4 weeks in both groups. In dupilumab group, the EOS% were15.25 ± 15.41% and 0.59 ± 0.95% before and after treatment, p = 0.013. In conventional group, the EOS% were 10.24 ± 11.50% and 0.28 ± 0.37% before and after treatment, respectively, p = 0.021. There EOS% level at baseline (p=0.430) and the reduction from baseline at week 4 (p=0.452) were similar between the groups. However, eight patients (five in the conventional group and three in the dupilumab group) evidenced normal eosinophil counts at baseline. The anti-BP180 antibody level decreased when a CR was attained at 4 weeks of treatment with conventional agents (before 102.70 ± 34.37 U/mL and after 48.60 ± 35.74U/mL, p <0.001) but not in the dupilumab group (before 119.73 ± 48.65 U/mL and after 116.66 ± 50.56 U/mL, p = 0.66) ( Figure 2A ). There was no statistically significant difference of anti-BP180 antibody level in the baseline between the groups (p=0.378). In the conventional group, the reduction from baseline of anti-BP180 antibody level was greater than in the dupilumab group at week 4 (p<0.001). Thus, dupilumab and corticosteroids have similarly effect in decreasing the EOS%.

Proteome profiling of six samples from 3 BP patients in dupilumab group (three samples before and three after treatment) revealed 879 unique proteins. Compared to the baseline, we detected 26 down-regulated and 4 up-regulated proteins at week 4 (Supplementary identified DEPs information). The roles played by several significantly down-regulated proteins, including ICOSL (before 109.65 and after 90.08, p = 0.025), CR2 (before 110.38 and after 89.67, p = 0.03), PRG2 (before 118.70 and after 81.40, p = 0.013), and S100A12 (before 110.31 and after 90.08, p = 0.01) are well understood ( Figure 3D ).

Gene ontology (Go) analysis was performed to comprehensively evaluate the plasma DEPs after dupilumab therapy. We explored molecular functions, the biological processes involved, and cellular components. Molecular function analysis revealed that some DEPs were associated with heterodimerization (H4C1, H2BC1, H3-3A, and H2AC20) or homodimerization (JUN, CR2, ADIPOQ, and TKT), calcium ion binding (H1-4, DSG1, TKT, and S100A12), and nucleosomal DNA binding (H3-3A and H1-4). Biological process analysis revealed that other DEPs were involved in nucleosome assembly (H2BC21, H1-4, and H3-3A), the antimicrobial humoral immune response (REG1A, GAPDH, S100A12, and H2BC21), and antibacterial defense (LYZ, S100A12, CHGA, PRG2, and GHV1-69D). Cellular component analysis revealed that some DEPs were enriched in extracellular exosomes (HABC21, PRG2, KPRP, REG1A, H3-3Q, H2AC20, OMD, TKT, UBA52, KRT78, JUP, GAPDH, LYZ, PYGB, FGB, CR2, ICOSL, TALDO1, GGH, PTPRJ, and ALDOB) which engage in intracellular molecule transportation (22), extracellular spaces (FGB, CR2, S100A12, POSTN, GGH, ADIPOQ, CHGA, H2BC21, REG1A, OMD, UBA52, KRT78, and LYZ), extracellular regions (S100A12, GGH, ADIPOQ, CHGA, H1-4, H3-3A, OMD, JUN, LYZ, PYGB, and FGB), the collagen-containing extracellular matrix (PRG2, OMD, FGB, POSTN, and ADIPOQ), and the lumen of a specific type of granule (JUN, LYZ, and GGH) ( Figure 3B ). Protein-protein interaction (PPI) analysis of all DEPs, performed with the aid of STRING, revealed that the top six categories were the extracellular exosome, nucleosome, extracellular space, killing of non-self-cells, nucleosome assembly, and defense against fungi ( Figure 3A ).

Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis disclosed that some DEPs affected neutrophil extracellular trap (NET) formation (H4C1, H2BC21, H3-3A, H2AC20, and FGB), the complement and coagulation cascades (CR2 and FGB), pentose phosphate metabolism (TKT, TALDO1, and ALDOB), and antifolate resistance (GGH) ( Figure 3C ). NET formation and the complement and coagulation cascades may play important roles in BP pathogenesis by promoting antibody production and blister formation (4, 23).

Compared to the pretreatment levels, the TARC levels decreased after treatment in both groups (dupilumab group: before 1,520.92 ± 1,477.20 pg/mL and after 386.86 ± 612.89 pg/mL, p = 0.016; conventional group: before 1,003.58 ± 953.50 pg/mL and after 190.22 ± 127.3 pg/mL, p = 0.017). The TARC levels at baseline and the reductions after treatment were similar in both groups (p = 0.056) ( Figure 2B ). Compared to before treatment, the ICOSL level decreased in both the conventional and dupilumab groups (dupilumab group: before 20.97 ± 24.55 ng/mL and after 2.35 ± 4.67 ng/mL, p = 0.034; conventional group: before 9.95 ± 15.78 ng/mL and after 0.544 ± 0.51 ng/mL, p = 0.085). The ICOSL levels at baseline and the reductions from baseline were similar in both groups (p = 0.316) ( Figure 2C ). The S100A12 levels before and after treatment did not differ between the two groups (dupilumab group: before 370.32 ± 321.04 ng/mL and after 385.49 ± 252.28 ng/mL, p = 0.835; conventional group: before 307.91 ± 225.15 ng/mL and after 505.83 ± 326.75 ng/mL, p = 0.073). The baseline S100A12 levels did not differ between the two groups (p = 0.147) ( Figure 2D ).