Immunohistochemical Analysis of Differences of Toll-Like Receptor 2, Mast Cells, and Neurofilaments between Granulomatous Rosacea and Non-Granulomatous Rosacea.

BACKGROUND: Granulomatous rosacea is a distinct variant of rosacea because of its unique histopatholiogic findings. However, the pathogenesis of granulomatous rosacea has not yet been clearly demonstrated. AIMS AND
OBJECTIVES: The aim of this study was to investigate the expression of toll-like receptor 2, mast cells, and neurofilaments in the granulomatous rosacea compared with the non-granulomatous rosacea.
MATERIALS AND METHODS: Biopsy specimens were obtained from 12 patients with erythematotelangiectatic rosacea, 11 patients with granulomatous rosacea, and 11 control patients. Biopsy tissue blocks were subjected to immunohistochemical staining using antibodies against toll-like receptor 2, mast cells, and neurofilaments.
RESULTS: In granulomatous rosacea, the expression of mast cells increased significantly, compared to the erythematotelangiectatic rosacea and the control group (P-value = 0.001 and 0.013, respectively). Additionally, the expression of toll-like receptor 2 in the granulomatous rosacea group was higher than that in the control group (P-value = 0.04).
CONCLUSION: The results of this study suggest that the increased expression of mast cells may be a sign of chronic, later stage of granulomatous rosacea compared to the erythematotelangiectatic rosacea. The increased expression of toll-like receptor 2 suggests that cathelicidin-induced neuroimmune pathogenesis also contributes to the pathophysiology of granulomatous rosacea. Copyright:

Introduction

Granulomatous rosacea is thought to be clinically distinct from other types of rosacea in that it does not often exhibit facial erythema, is not confined to facial convexities, and often presents with lesions around the eye and with an asymmetrical distribution.[1] In this study, we investigated the difference in the expression rate of toll-like receptor 2 (TLR2), neuromediators, and mast cells in the erythematotelangiectatic rosacea (ETR), granulomatous rosacea (GR), and normal skin, and examined their role in the etiology of each subtype of rosacea.

Subjects and Methods

Patients who visited the Institute from January 2007 to December 2015 were enrolled. 12 patients with ETR, 12 patients with GR, and 11 control patients (control group) were included [Table 1]. The control group was selected among patients who underwent dermatologic surgery of face in consideration of age and sex between GR and ETR patients. There was no significant difference in general characteristics including age and sex between the selected control group and the GR and ETR groups. For the control group, tissue was obtained from the remnant tissue during the surgery under protocols approved by the University Medical Center and in accordance with the principles expressed in the Declaration of Helsinki. This study was approved by the Institutional Review Board, and the requirement for informed consent was waived. (IRB no, 2018-03-027-002). Rosacea was diagnosed according to the clinical features and histologic findings. Clinical and epidemiological information was evaluated by electronic medical records (EMR). In this study, the authors investigated the diagnosis, classification, and severity criteria according to those described by the National Rosacea Society and classified only one subtype as the most prominent when the criteria for two or more subtypes were satisfied in one patient.[2]

Table 1

General characteristics of patients with GR, ETR, and control patients

Groups	Patient number	Mean duration (months)	Sex ratio (F/M)	Age±standard deviation
GR	11	9.2	11/0	44.9±5.3
ETR	12	8.2	11/1	49.0±10.7
Control	11		11/0	43.2±21.5
Total	34		33/1	45.8±13.9

GR: Granulomatous rosacea, ETR: Erythematelagiectatic rosacea

Skin biopsy

The histological examination was performed with a 3-mm punch. For the ETR type, the area with the most prominent capillary dilatation was selected as the biopsy site. For the GR subtype, the most indurated area was selected as the biopsy site. The punch biopsy tissue blocks were fixed in formalin and then dehydrated before being embedded in paraffin. The blocks were then sliced into 3-μm sections, mounted on glass slides, and stored at −80° until use.

Antibodies and immunohistochemical staining

The following antibodies (Ab) were used for immunohistochemistry: anti-human TLR2 blocking monoclonal antibody, clone TL2.1 (Novus Biologicals, Littleton, CO, USA), anti-human neurofilmanet (NF) proteins L/H (2F11) blocking monoclonal antibody, sc-52350 (Novus Biologicals, Littleton, CO, USA), and anti-human mast cell (MC) tryptase (G3) blocking monoclonal antibody, sc-33676 (Santa Cruz Biotechnology, Texas, USA).

Prior to staining, the tissue blocks were deparaffinized in xylene, rehydrated using decreasing concentrations of ethanol, and incubated with a target retrieval solution for 20 min in a microwave oven to facilitate the binding of Abs. To increase the membrane permeability, the sections were treated with 1% Triton-X-100, followed by hydrogen peroxidase, to reduce the endogenous peroxidase activity. The tissue sections were then treated with the monoclonal murine antibodies against MCs (diluted 1:20) for 2 h. The sections were then incubated with horseradish peroxidase (HRP)-labeled goat anti-mouse polymer for 30 min, and subsequently with 3,3′-diaminobenzidine (DAB) substrate-chromogen for 10 min for Ab detection. The sections were then dehydrated with increasing concentrations of ethanol, washed with xylene, and thereafter mounted and cover slipped.

Immunohistochemical staining analysis

The immunohistochemical stained slides were photographed at a magnification of 200 × under an optical microscope equipped with a digital camera (ToupTek Photonics, Zhejiang, PR China). One dermatologist randomly selected tissue slides from the epidermis toward the dermis and recorded five image files for each slide. The positively stained sections were quantitatively analyzed using the Image J software (Image Processing and Analysis in Java, National Institutes of Health, Bethesda, Md., USA).

Statistical analysis

Statistical analysis was performed using SPSS version 24.0 for Windows (Statistical package for the social sciences, SPSS, Chicago, USA). For immunohistochemistry analysis of each groups, the number of pixels per image was compared amongst the groups using non-parametric Mann-Whitney test. The statistical significance was considered significant when P value ≤0.05.

Results

All the 11 GR-type patients were female, and their age ranged from 38 to 56 years (mean, 44.9 years). In case of the ETR type, of the 12 patients, 11 were female and 1 was male; their age ranged from 28 to 63 years, with a mean age of 49.0 years. All the control subjects were female; their age ranged from 13 and 76 years, with the mean age being 43.2 years. The mean duration of disease was 9.2 months for the GR type and 8.2 months for the ETR type, with P > 0.05 [Table 1].

Table 2 summarizes the median value and interquartile range of the quantitative number of pixels analyzed for immunohistochemical staining for TLR2, MCs, and NFs for each group. The mean value for TLR2 was 57.1 ± 116.7 in the GR group, 85.7 ± 76.3 in the ETR group, and 7.1 ± 30.1 in the control group. The mean value for MCs was 107.8 ± 59.4 in the GR group, 61.8 ± 55.6 in the ETR group, and 55.1 ± 16.9 in the control group. The mean value for NFs was 5.0 ± 1.2 in the GR group, 6.5 ± 2.1 in the ETR group, and 4.0 ± 1.4 in the control group.

Table 2

Quantitative analysis of immunohistochemical staining

	GRa	ETR	Control
TLR2	69.00±103.00	81.00±101.25	7.50±44.00
Difference between groups (P)
GR and ETR	0.786
GR and Control	0.005
ETR and Control	0.002
MC	125.00±98.00	60.50±70.00	54.00±18.00
Difference between groups (P)
GR and ETR	0.013
GR and Control	0.001
ETR and Control	0.525
NF	5.0±1.00	6.5±3.75	4.0±1.00
Difference between groups (P)
GR and ETR		0.151
GR and Control		0.898
ETR and Control		0.151

(Number of pixels per image median value±interquartile range). GR: Granulomatous rosacea, ETR: Erythematelagiectatic rosacea, TLR2: Toll-like receptor 2, MC: Mast cell trypatase, NF: Neurofilament

Expression of tryptase in the GR group was significantly higher than that in the ETR and control groups (P-value = 0.001 and 0.013, respectively). In addition, the expression of TLR2 in the GR group was significantly higher than that in the control group (P-value = 0.04), but there was no significant difference in expression of TLR2 between GR and ETR groups (P-value = 0.519). There was no significant difference between the NF expressions in all the groups. (GR group vs control group: P value = 0.231, GR group vs ETR group: P value = 0.324).

The expression of TLR2 in the ETR group was significantly higher than that of the control group (P-value = 0.04), but the expression of tryptase and NFs in the ETR group showed no difference compared to the control group (P-value = 0.217 and 0.254, respectively).

The localization of MCs, NFs, and TLR2 in control, ETR-, and GR-type facial skin by immunohistochemical staining is shown in Figure 1.

Figure 1

Localization of MCs, NFs, and TLR2 in control, ETR-, GR-type facial skin as shown by immunohistochemistry. Arrows indicate positive cells in each immunohistochemistry staining. ETR: Erythematelagiectatic rosacea. GR: Granulomatous rosacea. MC: Mast cell trypatase. NF: Neurofilament. TLR2: Toll-like receptor 2

Discussion

Rosacea is a common chronic skin disease that mainly affects the central part of the face (cheeks, chin, nose, and forehead). Recurrent episodes of facial redness or permanent facial erythema are characteristic of rosacea.[2] It is later accompanied by the presence of papules, pustules, telangiectasias, and fibrosis, and sometimes, edema. GR is classified as a distinct disease variant of rosacea due to its unique histopathologic findings. Histologic findings are nonspecific in other subtypes, and skin biopsy is not essential for diagnosis. Characteristic finding of granulomatous rosacea is large granulomas of the superficial and mid dermis, which can include a large, central void space surrounded by layers of neutrophils and peripheral histiocytes mixed with lymphocytes.[3] Continuous sections often show debris from Demodex mites or eosinophils at the center of histiocyte collection sites.

The pathogenesis of granuloma formation in granulomatous rosacea has not yet been clearly demonstrated. Jang et al.[4] compared the expression of matrix metalloproteinases (MMPs) in granulomatous and non-granulomatous rosacea and showed significantly increased MMP-9 expression and a minor increase in MMP-2 expression in GR. This data suggests that MMPs induced by ultraviolet right radiation (UVR), particularly MMP-9, play a role in the development of granuloma by promoting tissue remodeling and enhancing inflammatory cell recruitment into the granuloma.

According to our results, the expression of tryptase was higher in the GR-type and ETR-type skin samples than in the normal skin samples, and the GR group showed a higher expression of tryptase than the ETR group. MCs are functionally closely related to both blood vessels and nerves; together, the blood vessels and nerves form a so-called microvascular unit.[5] MCs are key effector cells well known in neurogenic inflammation, immune defense, and fibrosis.[6] A recent study has discussed the important role of MCs in development of rosacea to a chronic stage.[7] Another recent study found that communication between MCs and fibroblasts is associated with skin fibrosis.[8] Chronic inflammation, which is characterized by Th1 cells, macrophages and MCs, results from sustained innate immune and neurogenic stimulation. This chronic inflammation results in the induction of profibrotic growth factors, probably via MCs, and consequently, the activation of myofibroblasts, rearrangement of the extracellular matrix, and finally, fibrosis. Our results clearly demonstrate a significant increase in MC density in GR, in comparison with ETR. These results suggest that GR occurs at a later stage than ETR and may occur when neurogenic inflammation aggravates. However, in our study, the mean duration of GR (9.2 months) showed no statistically significant difference than that of ETR (8.2 months). Further studies showing the correlation between neurogenic inflammation aggravation and GR are needed to confirm this suggestion.

One mechanism by which the skin recognizes the danger is via innate immune pattern recognition receptors, such as TLRs, which recognizes the specific microbial or host impairment products and play the most prominent role in causing inflammation. UVR increases the amount of reactive oxygen species in the skin. Signaling via TLR2 is possible and can spread the kallikrein 5(KLK 5)-cathlicidin inflammatory cascade present in the patient's skin.[9] Our results also showed the increase of TLR2 expression in the skin of patients with granulomatous rosacea, in comparison with healthy skin. The elevation of TLR2 expression in rosacea is well known, but there is no evidence to date that it also increases in GR.[9] Propionibacterium acnes, the most common facial skin symbiotic microorganism, can produce KLK5 and cathelicidin (LL37) in a TLR2-dependent manner. Demodex folliculorum can contribute to the pathogenesis of rosacea by releasing chitin, which can activate TLR2. High prevalence of these mites can increase protease activity. Thus, increased TLR2 expression may be responsible for abnormal expression of KLK5 and cathelicidin, both of which are important in rosacea. In this study, an increase in TLR2 expression was observed in GR, confirming the involvement of the KLK5-cathelicidin cascade in the pathogenesis of GR.

The pathophysiology of granulomatous rosacea is still poorly understood. The results of this study suggest that the increased expression of tryptase may be a sign of chronic, later stage of granulomatous rosacea, compared to the other subtypes. Furthermore, the increased expression of TLR2 suggests that cathelicidin-induced neuroimmune pathogenesis also contributes to the pathophysiology of GR. This study has a few limitations: the number of cases per group, "n", is limited, and a large-scale immunohistochemistry study is needed to confirm the results of this paper regarding the pathophysiology of GR. Also, immunohistochemistry method is hard to evaluate quantitative results. Further studies using quantitative real-time RT-PCR (qRT-PCR) are needed to verify the level of RNA and its respective proteins for more accurate evaluation of the results.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.