Analysis of IL-10 and IL-35 in DPP-4 inhibitor-related bullous pemphigoid

doi:10.21203/rs.3.rs-1634381/v1

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Analysis of IL-10 and IL-35 in DPP-4 inhibitor-related bullous pemphigoid

https://doi.org/10.21203/rs.3.rs-1634381/v1

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The association between immunoregulatory cytokines, such as IL-10 or IL-35, and DPP-4 inhibitor-related bullous pemphigoid (BP) has not been evaluated. Sera were collected from 39 Japanese patients with BP (24 males and 15 females; 77.0 ± 11.1 years old) including 6 patients with non-DPP-4 inhibitor-related BP before treatment in our hospital. Ten healthy Japanese individuals (4 males and 6 females; 42.2 ± 9.37 years old) were enrolled as healthy controls. No significant difference was observed in serum IL-10 levels (BP patients: 7.63 ± 5.03 pg/ml; healthy individuals: 6.88 ± 0.52 pg/ml; DPP-4 inhibitor-related BP: 6.77 ± 0.24 pg/ml; non-DPP-4 inhibitor-related BP: 6.84 ± 0.20 pg/ml; BP vs healthy: P = 0.368; DPP-4 inhibitor-related BP vs non-DPP-4 inhibitor-related BP: P = 0.553), nor in serum IL-35 levels (BP patients: 2.62 ± 0.20 pg/ml; healthy individuals: 2.60 ± 0.17 pg/ml; DPP-4 inhibitor-related BP: 2.63 ± 0.17 pg/ml; non-DPP-4 inhibitor-related BP: 2.63 ± 0.21 pg/ml; BP vs healthy: P = 0.727; DPP-4 inhibitor-related BP vs non-DPP-4 inhibitor-related BP: P = 0.949). Bullous Pemphigoid Disease Area Index (BPDAI) before treatment was not related with serum IL-10 levels (r = 0.159; Fig. 1A), nor with serum IL-35 levels (r = 0.227; Fig. 1B). The number of serum eosinophils was significantly higher in patients with non-DPP-4 inhibitor-related BP (911.3 ± 948.8) than in patients with DPP-4 inhibitor-related BP (476.1 ± 234.0; P = 0.038). DPP-4 is also known as a CD26 molecule expressed on the surface of T lymphocytes. The mean rate of infiltrating CD26⁺ cells was significantly increased in 6 patients with DPP-4 inhibitor-related BP (32.9 ± 7.1) than in 6 patients with non-DPP-4 inhibitor-related BP (15.7 ± 4.4; P = 0.002; Fig. 2). It was reported that the co-engagement of CD3 and CD26 induces the preferential production of IL-10 from human CD4 + T cells, which might reflect the clinical characteristics of faint inflammatory bulla in DPP-4 inhibitor-related BP.

CD26

DPP-4

IL-10

IL-35

bullous pemphigoid

Bullous Pemphigoid (BP) is characterized histologically by subepidermal blister formation induced by the dermal-epidermal junction (DEJ) component. Interleukin (IL)-10 and IL-35 are thought to play an important role in immunoregulatory and autoimmune disease processes [1–3]. Although IL-35 may act as an efficient therapeutic cytokine for various autoimmune diseases [4], the association has not been investigated between IL-35 and BP. Recently, the association has been well discussed between dipeptidyl peptidase-4 (DPP-4) inhibitor and BP [5]. Although DPP-4 is known as a CD26 molecule expressed on the surface of T lymphocytes and other cell types [6], the association has not been evaluated between immunoregulatory cytokines, such as IL-10 or IL-35. This study conducted a comprehensive evaluation of serum IL-10 and IL-35 levels in patients with BP including DPP-4 inhibitor-related BP. This study also evaluated the association between CD26⁺ cells in the dermis around bulla and DPP-4 inhibitor-related BP using immunohistochemical staining.

Sera were collected from 39 Japanese patients with BP (24 males and 15 females; 77.0 ± 11.1 years old) including 6 patients with non-DPP-4 inhibitor-related BP before treatment in our hospital. Ten healthy Japanese individuals (4 males and 6 females; 42.2 ± 9.37 years old) were enrolled as healthy controls. BP was diagnosed by clinical and histopathological features and detection of anti-DEJ antibodies using immunofluorescence staining and/or anti-BP180NC16a antibody. The disease severity of BP was evaluated using the Bullous Pemphigoid Disease Area Index (BPDAI) defined by the International Pemphigoid Committee using skin score [7]. Serum anti-BP180NC16a antibodies were measured by enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s protocol (MBL, Nagoya, Japan).

Serum IL-10 and IL-35 levels were measured by ELISA according to the manufacturer’s protocol [IL-10 (Bender MedSystems, Vienna, Austria) and IL-35 (Cloud-Clone, Wuhan, China)]. The number of CD26⁺ cells in the dermis around bulla in 6 patients with DPP-4 inhibitor-related BP and 6 patients with non-DPP-4 inhibitor-related BP was counted on sections immunohistochemically stained with monoclonal anti-CD26 antibody (ab215711; Abcam plc, Cambridge, UK) in three random grids per section at ⋅200 magnification. The mean frequency of infiltrating CD26⁺ cells (CD26⁺ mononuclear cells/all mononuclear cells) was decided based on these three values.

Statistical analysis was performed using Student’s t-test to compare patients with BP to healthy controls. P values < 0.05 were considered statistically significant. The Pearson product-moment correlation coefficient was used to examine the relationship between two continuous variables. Data were shown as the mean ± standard deviation. The experimental protocol was established according to the Declaration of Helsinki and approved by the Ethics Committee of Shiga University of Medical Science (reference no. R2017-227).

Serum IL-10 and IL-35 levels were measured in 39 patients with BP and 10 healthy controls. No significant difference in serum IL-10 levels in patients with BP (7.63 ± 5.03 pg/ml) compared to healthy individuals (6.88 ± 0.52 pg/ml; P = 0.368) was observed. Serum IL-35 levels in patients with BP (2.62 ± 0.20 pg/ml) also showed no significant difference compared to healthy controls (2.60 ± 0.17 pg/ml; P = 0.727).

We further assessed whether the severity of BP was correlated with serum IL-10 or IL-35 levels. The use of BPDAI to assess the disease severity of BP is widely accepted. No significant relationship was observed between serum IL-10 levels and BPDAI before treatment (r = 0.159; Fig. 1A). Although serum IL-35 levels decreased in BP patients, no significant relationship was also detected between serum IL-35 levels and BPDAI before treatment (r = 0.227; Fig. 1B).

We compared the clinical features, such as anti-BP180NC16a antibody titer, the number of serum eosinophils, and BPDAI, between 6 patients with DPP-4 inhibitor-related BP and 33 patients with non-DPP-4 inhibitor-related BP (Table 1). Regarding the types of the DPP-4 inhibitor, four were taking sitagliptin and two were taking vidagliptin among 6 patients with DPP-4 inhibitor-related BP. Although anti-BP180NC16a antibody titers were lower in patients with DPP-4 inhibitor-related BP, no significant difference was observed. The number of serum eosinophils was significantly higher in patients with non-DPP-4 inhibitor-related BP (911.3 ± 948.8) than in patients with DPP-4 inhibitor-related BP (476.1 ± 234.0; P = 0.038).

Table 1

Clinical and laboratory differences between DPP-4 inhibitor- and non-DPP-4 inhibitor-related BP
	DPP-4 inhibitor-related BP (n = 6)	Non-DPP-4 inhibitor-related BP (n = 33)	P-value
Anti-BP180NC16a antibody titer (U/mL)	67.31 ± 37.40	76.18 ± 54.59	0.706
No. eosinophils (/mm³)	476.1 ± 234.0	911.3 ± 948.8	0.038
BPDAI (erosions/blisters)	14.50 ± 10.98	16.66 ± 13.71	0.717
BPDAI (urticaria/erythema/other)	15.00 ± 9.75	17.54 ± 10.52	0.585
BPDAI (mucosa)	0	0.63 ± 2.24	0.113
BP: Bullous Pemphigoid; BPDAI: Bullous Pemphigoid Disease Area Index

Serum IL-10 and IL-35 levels were compared with regard to DPP-4. Although serum IL-10 levels were lower in patients with DPP-4 inhibitor-related BP (6.77 ± 0.24 pg/ml) than in patients with non-DPP-4 inhibitor-related BP (6.84 ± 0.20 pg/ml; P = 0.553), no significant difference was observed. Serum IL-35 levels in patients with DPP-4 inhibitor-related BP (2.63 ± 0.17 pg/ml) also showed no significant difference compared to patients with non-DPP-4 inhibitor-related BP (2.63 ± 0.21 pg/ml; P = 0.949).

To investigate the association between DPP-4 inhibitor and BP pathologically, we counted the number of CD26⁺ cells in the dermis around bulla on sections immunohistochemically. The mean rate of infiltrating CD26⁺ cells was significantly increased in 6 patients with DPP-4 inhibitor-related BP (32.9 ± 7.1) than in 6 patients with non-DPP-4 inhibitor-related BP (15.7 ± 4.4; P = 0.002; Fig. 2).

The production of pathogenic autoantibodies is key to the development of autoimmune bullous disease. Many immunological steps, including impaired immunotolerance, are needed for autoantibody production. Immunotolerance to self-antigens is essential to protect the host against chronic inflammatory diseases and tissue damage. We previously reported that B10 cells were associated with long-term remission after intravenous immunoglobulin treatment for pemphigus [8]. We also reported that peripheral blood levels of B10 cells in patients with pemphigus but not patients with BP decreased compared to healthy individuals [9]. Because serum IL-10 levels were not evaluated in these two studies, this study measured serum IL-10 levels. As for serum IL-10 levels in patients with BP, no significant increase was observed in a recent study [10], but there was a reported increase [11]. In this study, we found no significant difference in serum IL-10 levels in patients with BP compared to healthy individuals. Moreover, Fig. 1A shows no significant relationship between serum IL-10 levels and BPDAI. These observations indicated that serum IL-10 levels are not usually increased in patients with BP and corresponded to a previous observation that B10 cells in peripheral blood are decreased in pemphigus but not in BP [9].

IL-35 is another immunoregulatory cytokine. Although serum IL-35 levels have been evaluated in various autoimmune diseases, such as rheumatoid arthritis and systemic sclerosis [4], it has not been investigated in BP. No significant difference in serum IL-35 levels was observed between patients with BP and healthy individuals. Figure 1B shows no significant relationship between serum IL-35 levels and BPDAI. Although further investigations are needed regarding IL-35, this immunoregulatory cytokine may not be a candidate of a therapeutic target for BP, unlike other autoimmune diseases.

Because the role of CD26 in DPP-4 inhibitor-related BP has not been evaluated, this study evaluated infiltrating CD26⁺ cells on bullous lesions. Figure 2 showed that the mean frequency of infiltrating CD26⁺ cells on bullous lesions significantly increased in DPP-4 inhibitor-related BP than non-DPP-4 inhibitor-related BP. It is speculated that this increase in CD26⁺ cells might be associated with the clinical characteristic of a non-inflammatory phenotype with less erythema in DPP-4 inhibitor-related BP than that seen in patients with non-DPP-4 inhibitor-related BP [12], because the co-engagement of CD3 and CD26 induces the preferential production of IL-10 in human CD4⁺ T cells [6].

Financial support: None

Conflict of interest: None

Acknowledgments: The patients in this manuscript have given written informed consent to publication of their case details. The authors would like to thank Ms. Yuko Tsukamoto for her help in immunohistochemical analysis.

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No competing interests reported.

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Analysis of IL-10 and IL-35 in DPP-4 inhibitor-related bullous pemphigoid

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