Neutrophil-to-lymphocyte ratio and platelet-to-lymphocyte ratio are positively correlated with disease activity of bullous pemphigoid

Bullous pemphigoid (BP) is a complex inflammatory process with elevated levels of autoantibodies, eosinophils, neutrophils, and various cytokines. Hematological inflammatory biomarkers can reflect inflammatory state in various diseases. Up to now, the correlations of hematological inflammatory biomarkers and disease activity of BP remain unknown. The purpose of this study was to clarify the associations between hematological inflammatory biomarkers and disease activity of BP. The levels of neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR), platelet-to-neutrophil ratio (PNR) and mean platelet volume (MPV) of 36 untreated BP patients and 45 age and gender matched healthy controls were detected by routine blood tests. The correlations between hematological inflammatory markers and clinical characteristics of BP were statistically analyzed. The Bullous Pemphigoid Disease Area Index (BPDAI) was used to measure disease activity of BP. The mean levels of NLR, PLR, PNR and MPV in 36 untreated BP patients were 3.9, 157.9, 45.7 and 9.4 fl, respectively. Increased NLR (p < 0.001), PLR (p < 0.01), and MPV (p < 0.001) but decreased PNR (p < 0.001) were observed in BP patients when compared with healthy controls. In BP patients, the levels of NLR were positively correlated to BPDAI Erosion/Blister Scores (p < 0.01); and the levels of NLR and PLR were both positively correlated to BPDAI without Damage Score (both p < 0.05) and BPDAI Total Score (both p < 0.05). No correlation was found in other statistical analyses between hematological inflammatory markers and clinical characteristics in BP patients involved in the present study. Therefore, NLR and PLR are positively correlated with disease activity of BP.

BP is a complex inflammatory process accompanied by elevated levels of autoantibodies, eosinophils, neutrophils, and various cytokines [1,2,[17][18][19].Eosinophils and neutrophils are also implicated in the pathogenesis of BP.Evidence indicated that eosinophils and neutrophils can induce subepidermal split formation at the basement membrane zone (BMZ) in BP [18,[20][21][22][23][24], and are associated with BP disease activity [14,19,25].Therefore, assessing the inflammatory status of BP patients might provide important implications for the treatment and prognosis.
Hematological inflammatory biomarkers (neutrophilto-lymphocyte ratio, NLR; platelet-to-lymphocyte ratio, PLR; platelet-to-neutrophil ratio, PNR; mean platelet volume, MPV) can reflect inflammatory state and have prognostic value in various diseases, including systemic lupus erythematosus, rheumatoid arthritis, fibromyalgia syndrome, ankylosing spondylitis, cutaneous polyarteritis nodosa, Behçet's disease, IgA vasculitis, lichen planus and psoriasis [26][27][28][29][30][31][32][33][34][35].In 2016, Qin et al. [36] proved that the levels of NLR and PLR can reflect inflammatory state and are positively related to the disease activities of systemic lupus erythematosus in 154 untreated patients.In 2021, Lyakhovitsky et al. [26] indicated that the disease severities of 56 pemphigus vulgaris patients were related positively to the levels of NLR and PLR, but negatively to the levels of PNR and MPV.Up to now, the correlations of hematological inflammatory biomarkers with disease activity of BP remain unknown.
In the present study, we detected the levels of NLR, PLR, PNR and MPV in 36 untreated BP patients and 45 healthy controls, evaluated disease activities, and conducted correlation analyses, in order to clarify the associations between hematological inflammatory biomarkers and disease activity of BP.

Patients and controls
Diagnostic criteria of BP: (1) intensely pruritic blisters of the skin, (2) positive linear IgG and/or C3c staining along the BMZ by direct immunofluorescence (DIF), (3) positive IgG and/or C3c staining on the epidermal side of 1 M-NaClsplit normal human skin by indirect immunofluorescence (ssIIF), (4) positive IgG enzyme-linked immunosorbent assay (ELISA) of BP180 NC16A.BP was diagnosed if the first criterion and at least two of the last three criteria were met.Exclusion criteria included pregnancy and hematologic diseases because of their possible impact on hematological parameters.
Thirty-six untreated BP patients diagnosed between August 2020 and April 2022 in the Hospital for Skin Diseases, Chinese Academy of Medical Sciences were included.Untreated whole blood and blister fluid samples of these BP patients were obtained.
In 36 patients with BP, the Bullous Pemphigoid Disease Area Index (BPDAI) was determined at the time when the serum and blister fluid samples were obtained.The BPDAI was determined by a panel of experts on BP and is composed of a score for the degree of erosions/blisters (range 0-120 points), urticaria/erythema (range 0-120 points), damage (range 0-12 points), mucosal erosions/blisters (range 0-120 points), and pruritus (range 0-30 points), with higher scores indicating greater disease activity [37].BPDAI total score (range 0-372 points) is the sum of four components including the BPDAI erosion/blister, BPDAI urticaria/erythema, BPDAI damage, BPDAI mucosal erosion/blister.Age and gender matched 45 healthy individuals without any disease were included in this study and whole blood samples were obtained.In addition, control blister fluid samples (33 cases) were collected from 11 patients with contact dermatitis, 11 patients with papular urticaria, 9 eczema patients and 2 scald patients (Table S1).
This study was performed in adherence with the Declaration of Helsinki guidelines and was approved by the Ethics Committee of Institute of Dermatology and Hospital for Skin Diseases, Chinese Academy of Medical Sciences and Peking Union Medical College (2017-KY-022).Written informed consents were obtained from all involved patients and controls.

ssIIF
The skin tissues taken from normal individuals were washed with aseptic saline solution, and then placed into 1 mol/L NaCl (20-30 °C) for about 24 h, with slight vibration, until the surface of the skin wrinkled, which indicated that the epidermal and dermal layers had been separated.This saltsplit skin was next embedded with ornithine carbamyl transferase and cut into frozen sections with 6 μm thick.Serum samples of BP patients were diluted at 1:10 with PBS, and incubated with salt-split skin sections at 37 °C for 30 min.After thoroughly washing with PBS (10 mM, pH 7.3), the sections were incubated at 37 °C for 30 min with FITC-conjugated sheep anti-human IgG antibody (GTX26866, Gene-Tex, San Antonio, USA) or FITC-conjugated rabbit antihuman C3c antibody (GTX73472, GeneTex, San Antonio, USA) both at a 1:300 dilution with PBS.The fluorescence intensity was assessed by at least two trained dermatologists.

ELISA for detection of IgG anti-BP180 NC16A autoantibody in serum and blister fluid samples
IgG antibodies against BP180 NC16A was measured by MESACUP BP180 TEST (7695CN, MBL, Nagoya, Japan) according to the manufacturer' instruction.The serum 1 3 samples were diluted to 1:101 and the proper dilution of blister fluid samples were 1:20.The cut-off value for IgG anti-BP180 NC16A antibodies in serum samples was 9 U/mL according to the instruction of the manufacturer.The cut-off values of IgG ELISA for blister fluid samples were set on the mean + 3 standard deviation (SD) of blister fluids of control group (OD 450, 0.126).

Routine blood test
The routine blood test of BP patients and healthy controls was measured using the coulter cellular analysis system (UniCel DxH 600, Beckman Coulter, California, USA).Whole blood counts for eosinophil, neutrophil, lymphocyte, platelet, NLR, PLR, PNR and MPV were used for the analyses in the present study.

Statistical analysis
Quantitative variables were expressed as mean ± SD.SPSS 16.0 software (IBM, Chicago, USA) was used for statistical analyses.The statistical differences were evaluated by Independent Samples T test, Pearson Chi-Square, Spearman's rank correlation or Pearson's correlation.P < 0.05 was considered as statistically significant.

Clinical, pathological and immunological characteristics of BP patients and healthy controls
Clinical, pathological and immunological features of untreated BP patients and healthy controls were shown in Table 1.The 36 BP patients included 24 males (66.7%) and 12 females (33.3%) with an average age of 74.6 years.There  1).
For immunofluorescence detection, the positive rates of DIF for linear IgG and/or C3c deposition to BMZ and ssIIF for IgG and/or C3c reactivity with epidermal side were 82.4% (14 of 17 cases) and 100% (36 of 36 cases), respectively (Table 1).
The BPDAI was used to measure disease activities of the 36 BP patients.The mean of BPDAI Total Score of all patients was 51.8 (Table 1).

NLR, PLR, and MPV are increased but PNR is decreased in BP patients when compared with those in healthy controls
Hematological characteristics of 36 untreated BP patients and 45 healthy individuals were presented in Table 2.All the 36 patients had available routine blood test results, and the means of eosinophil counts, neutrophil counts, lymphocyte counts and platelet counts were 1.3 × 10 9 /L, 5.5 × 10 9 /L, 1.7 × 10 9 /L and 222.2 × 10 9 /L, respectively.Compared with healthy controls, the eosinophil counts (p < 0.001) and neutrophil counts (p < 0.001) were increased, and the lymphocyte counts (p < 0.05) were decreased in BP patients.BP patients showed abnormally higher of eosinophils (50.0%, 18 of 36 cases) and neutrophils (30.6%, 11 of 36 cases), and abnormally lower of lymphocytes (19.4%, 7 of 36 cases).The means of NLR, PLR, PNR and MPV in BP patients were 3.9, 157.9, 45.7 and 9.4 fl, respectively.NLR (p < 0.001), PLR (p < 0.01), and MPV (p < 0.001) were increased but PNR (p < 0.001) was decreased in BP patients compared with those in healthy controls.

Correlation analyses
Correlations between hematological inflammatory markers and the clinical characteristics of 36 untreated BP patients were summarized in Table 3.The NLR levels were positively correlated to BPDAI Erosion/Blister Score (p < 0.01) (Table 3).The levels of NLR and PLR were both positively correlated to BPDAI without Damage Score (both p < 0.05) and BPDAI Total Score (both p < 0.05) (Table 3).However, no correlation was found in other statistical analyses of hematological inflammatory markers with the clinical characteristics of untreated BP patients (Table 3).
The extent of elevated peripheral blood eosinophils was positively correlated to BPDAI without Damage Score (p < 0.05) and BPDAI Total Score (p < 0.05), but not to the extent of elevated peripheral blood neutrophils, BPDAI Erosion/Blister Score, BPDAI Urticaria/Erythema Score, BPDAI Damage Score, BPDAI Mucosal Score and BPDAI-Pruritus Score (Table 4).
The levels of IgG anti-BP180 NC16A autoantibody in untreated serum and blister fluid samples were both positively associated with BPDAI Erosion/Blister Score  6, 7).In addition, a strong positive relationship of IgG anti-BP180 NC16A autoantibody levels was found between untreated serum and blister fluid samples (p < 0.001) (Table 7).The extent of elevated peripheral blood eosinophils was positively associated with blister fluid IgG anti-BP180 NC16A autoantibody (p < 0.001) (Table 7), and tended to be positively associated with serum IgG anti-BP180 NC16A autoantibody (p = 0.057) (Table 6).

Limitations
Our study involved a relatively small number of BP patients.Therefore, it is necessary to further validate the potential conclusions in larger cohorts.

Conclusion
In summary, NLR and PLR are positively correlated with disease activity of bullous pemphigoid, indicating that monitoring the levels of NLR and PLR might benefit for evaluation of therapy effect and prognosis of BP patients.

Table 1
Clinical, pathological and immunological characteristics of BP patients and healthy controls BP bullous pemphigoid, M male, F female, BMZ basement membrane zone, DIF direct immunofluorescence, IIF indirect immunofluorescence using normal human skin, ssIIF IIF using 1M-NaCl-split normal human skin, OD optical density, c/o cut-off value, BPDAI Bullous Pemphigoid was no significant difference in age and sex between the BP patients and healthy controls.Histopathologically, 21 of 36 BP patients had pathological reports available, which revealed subepidermal blister formation (81.0%, 17 of 21 cases) and dermal infiltrations of eosinophils (81.0%, 17 of 21 cases) (Table

Table 2
Hematological characteristics of BP patients and healthy controls BP bullous pemphigoid, NLR neutrophil-to-lymphocyte ratio, PLR platelet-to-lymphocyte ratio, PNR platelet-to-neutrophil ratio, MPV mean platelet volume

Table 3
Correlations between hematological inflammatory markers and the clinical characteristics of untreated BP patients p < 0.001, p < 0.01), BPDAI without Damage Score (both p < 0.001) and BPDAI Total Score (both p < 0.001), but not with the extent of elevated peripheral blood neutrophils, BPDAI Urticaria/Erythema Score, BPDAI Damage Score, BPDAI Mucosal Score and BPDAI-Pruritus Score (Tables (