Blister fluid as a source of steroid resistance biomarkers in patients with life-threatening bullous dermatoses: An observational cohort controlled study

Background. Bullous dermatoses are severe life-threatening antibody-induced organ-specific multifactorial diseases resulting from a genetic predisposition. Systemic glucocorticosteroids are the first-line therapy for many bullous dermatoses. However, there is a small though significant proportion of patients who are resistant to basic therapy with systemic glucocorticosteroids. Although studies of the steroid resistance have been performed at the level of receptors and genes in the serum of patients, no studies of this mechanism have been carried out at the level of blister fluid locally in the area of blister formation.

Objectives. To estimate cytokine (interleukin 10, 15, 4, tumor necrosis factor alpha), chemokine (eosinophil chemotactic protein, interleukin 8) profiles and granulysin levels in patients with bullous dermatoses in serum and blister fluid, as well as their possible relationship with steroid resistance.

Methods. The observational cohort controlled study included 67 patients with bullous dermatoses hospitalized between January 2020 and December 2023 in the V.A. Rakhmanov Clinic of Skin and Venereal Diseases of the University Clinical Hospital No. 2 of the I.M. Sechenov First Moscow State Medical University (Sechenov University) and the Department of Allergology and Immunology of the Municipal Clinical Hospital No. 24 of the Moscow City Health Department. The patients were classified into three groups by diagnosis. The first group included patients with pemphigus vulgaris (n = 43), the second was patients with bullous pemphigoid (n = 11), and the third consisted of patients with Stevens — Johnson syndrome and toxic epidermal necrolysis (n = 13). The control group consisted of 43 healthy donors, whose biological material was obtained from the Blood Center of the I.M. Sechenov First Moscow State Medical University (Sechenov University). After a 3-week course of systemic glucocorticosteroids, the groups of patients with bullous dermatoses were stratified into steroid-resistant and steroid-sensitive subgroups, respectively: with pemphigus vulgaris 18/25 patients; with bullous pemphigoid 3/8; with Stevens — Johnson syndrome and toxic epidermal necrolysis 1/12. Due to the small number of patients, the data of steroid-resistant and steroid-sensitive patients with Stevens — Johnson syndrome and toxic epidermal necrolysis were not statistically analyzed. The main indicators of the study comprised interleukins 10, 15, 4, tumor necrosis factor alpha, eosinophil chemotactic protein, interleukin 8, and granulysin levels in serum and blister fluid of patients depending on the presence or absence of steroid resistance. Statistical processing of data was performed using Statistica 10.01 software package (StatSoft, USA), Microsoft Excel 2010 (Microsoft, USA) and IBM SPSS 24.0 software (IBM, USA). Differences were considered statistically significant at p < 0.05.

Results. The concentrations of tumor necrosis factor-α, interleukin 10, granulysin, as well as chemokines — interleukin 8 and eosinophilic protein — were statistically significantly higher in serum in patients with pemphigus vulgaris, Lever’s bullous pemphigoid, and Stevens — Johnson syndrome and toxic epidermal necrolysis compared to the control group (p < 0.001). Significantly lower levels of tumor necrosis factor-α, particularly in blister fluid as opposed to serum, correlated with steroid resistance in patients with pemphigus vulgaris (p < 0.05). Reduced levels of the key neutrophil chemoattractant may indicate impaired recruitment of immune cells to the lesion, thus being a potential biomarker of the refractory course of the disease. Meanwhile, steroid-resistant patients with bullous pemphigoid had statistically significantly high levels of interleukins 4, 8, 15, and granulysin in their blister fluid compared to steroid-sensitive patients (p < 0.05). Due to this, differences in inflammatory marker profiles associated with insensitivity of patients to hormone therapy are emphasized. Importantly, most patients with bullous pemphigoid responded adequately to therapy (8/11) with systemic glucocorticosteroids compared to the pemphigus vulgaris group. In patients with Stevens — Johnson syndrome and toxic epidermal necrolysis, the level of granulysin (median 12,105 ng/mL) in blister fluid was statistically significantly higher compared with pemphigus vulgaris (median 10,842 ng/mL; p = 0.024) and bullous pemphigoid (median 10,335 ng/mL; p = 0.048). The findings suggest that analysis of this biomarker in blister fluid can be applied to the diagnosis as well as differential diagnosis of bullous dermatoses.

Conclusion. This study identified potential predictors of response to therapy with systemic glucocorticosteroids in the blister fluid of patients with pemphigus vulgaris and bullous pemphigoid. In the long-term, the analysis of blister fluid can be used complementary to the histologic method as an express diagnostic test, differential diagnosis of severe bullous dermatoses, real-time monitoring of response to therapy, and prognosis of the severity of the course of these diseases. Additionally, a timely administration of adjuvant therapy to patients’ refractory to systemic steroids would assist in minimizing the risk of complications and side effects.

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