Bullous pemphigoid (BP) is the most common autoimmune subepidermal blistering disease.1 BP affects mostly elderly patients and is associated with significant rates of morbidity and mortality.2 In the past two decades a trend of growing incidence between 1.9 and 4.3-fold has been reported in data from different European countries.3,4 The increased incidence of BP overtime has been associated with an ageing population, with the increased prevalence of neurologic diseases and with the increasing use of drugs associated with BP development, in particular dipeptidyl peptidase-IV (DPP-4) inhibitors.5

Several observations support a pathogenic role of eosinophils in BP. Dermal inflammatory infiltrate rich in eosinophils is a prominent histological feature and peripheral blood eosinophilia has been documented in 50–60% of BP patients.6,7 Moreover, peripheral blood and cutaneous eosinophil levels have been correlated with disease severity.7,8 Eosinophils seem to be key effector cells in BP initiation and progression. In fact, eosinophil skin infiltration is an early event and precedes blister formation.9 Eosinophils degranulation and release of extracellular eosinophil traps and granule proteins promote lesion formation.10,11

Although increasing evidence of contribution of eosinophils for BP pathogenesis accumulate, the influence of circulating and dermal infiltrate eosinophil levels on the phenotype of BP remains poorly understood. The main objective of this study was to evaluate the association between peripheral blood and cutaneous eosinophil levels and the clinical and immunological characteristics of the disease.

A retrospective cohort study was conducted on all patients diagnosed with BP between January 1st, 2011 and December 31st, 2020, at a tertiary referral centre. The study was approved by the Local Ethics Committee.

The diagnosis of BP was established according to the following criteria: (1) suggestive clinical and histopathological picture; (2) linear deposits of IgG and/or C3 along the dermal–epidermal junction by direct immunofluorescence (DIF) microscopy of a perilesional skin biopsy; and (3) the presence of circulating IgG autoantibodies against BP180 and/or BP230, as identified by BP180 NC16A/BP230 enzyme-linked immunosorbent assay (ELISA).

We excluded all patients with an alternative cause for peripheral blood and tissue eosinophilia, including atopic dermatitis, asthma, allergic rhinitis, parasitic infection and lymphoma. All patients treated with drugs that affect eosinophil counts in the moment of BP diagnosis, including systemic steroids and immunosuppressants, were also excluded.

Peripheral blood eosinophil counts prior to the administration of any treatment were reviewed. Peripheral blood eosinophil counts were classified as normal if below 500×106/L, high if between 500 and 1500×106/L and very high if above 1500×106/L. The levels of peripheral blood anti-BP180 NC16A and anti-BP230 IgG autoantibodies were measured by ELISA (Euroimmun, Lűbeck, Germany). Seropositivity was determined based on the cut-off values proposed by the manufacturer (i.e., 20U/ml).

Haematoxylin and eosin (H&E)-stained lesional skin specimens were reviewed in terms of eosinophil levels by three Dermatopathologists (MAG, PEL, LSA). In each sample, representative hot spots with the highest density of eosinophils were identified in the dermal inflammatory infiltrate. In each hop spot the number of eosinophils was counted using a single×400 high power field (HPF) objective (one large magnification area hot spot).

Data was analysed using IBM SPSS Statistics® (Statistical Package for the Social Sciences, version 24, SPSS Inc., Chicago, IL, USA). Categorical variables were presented as frequencies and percentages and were compared with use of Chi-square test. Data found to be non-normally distributed were analysed using the Mann–Whitney U test for independent subgroups and the Wilcoxon test for dependent subgroups. Continuous variables were presented as means and standard deviations. These variables were compared with the use of Student's t-test. All reported P values are two-tailed, with a P value of 0.05 indicating statistical significance.

The current study revealed that peripheral blood and cutaneous eosinophil levels are associated with BP severity and with treatment outcomes. Peripheral blood eosinophilia was associated with extensive disease and with hand and feet involvement. Moreover, it predicted a higher risk of requiring inpatient care and relapsing. Peripheral blood and tissue eosinophil levels were associated with high titles of BP180 NC16A IgG antibodies. Eosinophil-poor inflammatory infiltrates predicted an increased prevalence of mucosal involvement.

Previous studies had provided inconclusive conclusions on the influence of peripheral blood eosinophil levels on clinical and immunological features of BP. Peripheral blood eosinophil levels were recently shown to be positively correlated with peripheral blood levels of both IgG and IgE anti-BP180 and with disease severity.7,12 Kridin reported that patients with BP and eosinophilia had a higher rate of palmoplantar involvement and extensive disease. In contrast, patients with normal eosinophil counts tended to had atypical clinical features including higher mucosal and head/neck involvement.7

Although an inflammatory infiltrate rich in eosinophil is a well-known feature of BP, the relation between tissue eosinophil levels and the presentation of BP is yet to be established. Izumi et al. showed that patients who presented with an inflammatory phenotype with erythema and urticarial plaques had an increased seropositivity of BP180 NC16A IgG and a higher number of infiltrating eosinophils in lesional skin sections.13 Moreover, tissue eosinophil levels have been recently correlated with disease severity.8

The current study supports the association of peripheral blood eosinophilia and high dermal eosinophil counts with BP severity. Likewise, we found an association between disseminated forms of disease and high titles of BP180 NC16A IgG, which are well-known to parallel BP activity. In contrast, we found a significant association between mucosal involvement and lower dermal eosinophil levels in concordance with a recent study reporting a higher prevalence of mucosal involvement in patients with a cell-poor inflammatory infiltrate.14

We observed that patients with high peripheral blood eosinophil levels have an increased risk of requiring inpatient care and relapsing after successful disease control. In contrast, dermal infiltrate eosinophil levels were not associated with treatment outcomes. Therefore, high peripheral blood eosinophil levels may signify a more recalcitrant clinical presentation necessitating more aggressive management. Notably, almost half of the patients who experienced a relapse had high or very high peripheral blood eosinophil levels at the time of relapse. Moreover, we found a strong and significant correlation between peripheral blood eosinophil counts at baseline and at relapse. Our present results suggest that peripheral blood eosinophil levels may be a biomarker of BP relapse. Most cases of clinical relapse of BP occur in the first six months of follow-up. Therefore, monitoring peripheral blood eosinophil counts in this period may allow early detection of relapse, particularly in patients with high baseline peripheral blood eosinophil counts.

Our study disclosed a significant association between the comorbidities of BP patients and both peripheral blood and dermal infiltrate eosinophil levels. Similar to previous studies, diabetic patients treated with DPP-4 inhibitors had lower circulating and dermal infiltrate eosinophil counts.15–17 In contrast, we found that BP patients with accompanying neurologic disease likely present high circulating eosinophil levels and an eosinophil-rich inflammatory infiltrate. While the association of neurologic diseases in BP with high levels of peripheral blood eosinophils had been previously reported, its association with high dermal eosinophil levels was only identified in a recently published study.8,18

A pivotal contribution of eosinophils in BP pathogenesis has long been suggested as increasing evidence of its biological actions accumulate. Increased expression of eotaxin-1 and Interleukin (IL) -5, two key mediators in the recruitment and activation of eosinophils, has been demonstrated in peripheral blood and blister fluid of BP patients.19,20 Following activation, in the presence of BP autoantibodies, eosinophils directly contribute to BP blister formation. Eosinophils degranulate and release granular proteins, such as eosinophil cationic protein, eosinophil extracellular traps and matrix metalloproteinase that directly contribute to BP antigens degradation and dermal–epidermal junction cleavage.10,11

Eosinophils may be a target for new therapeutics in BP.21 In fact, the increasing number of cases of successful treatment of BP with omalizumab supports this strategy.22,23 Other potential emerging drugs for BP treatment include bertilimumab, a fully human monoclonal antibody targeting eotaxin-1, and anti-IL5 monoclonal antibodies, such as mepolizumab and benzalizumab.15,24,25 Determination of peripheral blood and dermal eosinophil levels may have a role in patient selection for these treatments. Although clearly promising, further studies are necessary to evaluate the efficacy and safety of eosinophil-directed therapies in the management of BP.

The main limitation of the current study is its retrospective design. Disease severity evaluation was limited by the lack of data on standardised BPADI index for each patient. Besides, eosinophil tissue levels were evaluated on a single lesional punch biopsy. However, biopsies from all patients were obtained from the most inflammatory skin lesion and count was performed by three Dermatopathologists. Selection of more severe and recalcitrant cases may have arisen as the study was carried out in a tertiary referral centre setting. Nevertheless, our study sheds light on a topic that has not been fully investigated so far. Moreover, it provides novel insight regarding the relation between BP clinical and immunological characteristics and peripheral blood and dermal infiltrate eosinophil levels.

In conclusion, circulating and cutaneous eosinophils levels may be useful biomarkers for disease activity and treatment outcomes in BP. Monitoring peripheral blood eosinophil counts may allow early detection of relapse. In addition, inhibition of eosinophil activity may be a strategy for new therapeutics in BP.

The authors declare that they have no conflict of interest.