Immunohistochemical Expression of Toll-Like Receptor 3 in Verruca Vulgaris

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1 Med. J. Cairo Univ., Vol. 81, No. 1, December: , Immunohistochemical Expression of Toll-Like Receptor 3 in Verruca Vulgaris AHMED A. SALEH, M.D.; FATMA M. EL-ESAWY, M.D. and TAGHREED A. ABDEL AZIZ, M.D.* The Departments of Dermatology & Andrology and Pathology*, Faculty of Medicine, Benha University Abstract Background: Toll-like receptors are expressed on both immune and nonimmune cells and respond to specific components of microbial pathogens, their signaling results in the activation of nuclear factor B (NF-B), which triggers the production of a variety of antimicrobial and proinflammatory cytokines and chemokines. Objective: The purpose of this study was to evaluate the role of Toll-like receptors 3 (TLR 3) in the pathogenesis of verruca vulgaris (VV) by its immnohistochemical expression in the skin lesions of VV patients. Patients and Methods: This study was carried out on 20 patients with verruca vulgaris and 20 apparently healthy volunteers served as controls. All patients and controls were presenting at Benha Dermatology Department's Outpatients Clinic. A written consent was taken from each patient and control before participation in the study. This study was carried out during the period between March 2012 to February A 4mm punch skin biopsies were taken from wart in patient group and from normal skin in control group for histopathological staining and immnohistochemical staining of TLR 3. Results: There was a highly significant increase (p<0.001) in the TLR3 expression, density and staining intensity in all cases as compared with the normal controls. Conclusion: Epidermal TLR3 may have an important role in cutaneous innate immune responses to cutaneous human papillomavirus infections. So, Toll-like receptor agonists represent a promising approach for the treatment of verruca vulgaris. Key Words: Verruca vulgaris Papillomavirus Toll-like receptors 3 Immunohistochemical staining. Introduction PAPILLOMA viruses are a group of small nonenveloped DNA tumor viruses whose infection usually causes benign epithelial lesions (warts) but sometimes also cause malignancies (cervical cancer and anal cancer) [1]. Correspondence to: Dr. Fatma Mohamed El-Esawy fatmaelesawy99@yahoo.com Human papilloma viruses' lesions arise from the proliferation of infected basal cells. When the basal keratinocytes of the host are exposed to the virus through a disruption of the epithelial barrier, for example after minor skin abrasions or during sexual intercourse, infection occurs [2]. Keratinocytes are one of the first cutaneous cell types to encounter various microbes including the papilloma and molluscum viruses. Keratinocyte TLRs are believed to play an important role in pathogen recognition and host innate immune responses in the skin. Toll-like receptor 3, 7, and 9 were shown to recognize different kinds of viruses [3,4]. Tolllike receptors (TLRs) are a family of pattern recognition receptors that are activated by specific components of microbes and certain host molecules [5]. TLRs constitute the first line of defense against many pathogens and play a crucial role in the function of the innate immune system [6]. TLRs have been shown to play an important role in initiation and modulation of adaptive immune responses (via effects on dendritic cells), T helper subset differentiation, and immune tolerance, so they serve as a link between innate and acquired immune responses [7,8]. Toll-like receptor 3 was highly expressed in dengue viral infection [9], Tolllike receptor 3 recognize viral double-stranded RNA [10] and host messenger RNA (mrna) as endogenous ligand [11]. Following activation of TLR by ligands of microbial origin, several reactions are possible. Immune cells can produce signaling factors called cytokines which trigger inflammation. In the case of a bacterial factor, the pathogen might be phagocytosed and digested, and its antigens presented to CD4 + T cells. In the case of a viral factor, the infected cell may shut off its protein synthesis and may undergo programmed cell death (apoptosis). Immune cells that have detected a virus may also release anti-viral factors such as interferon [12]. 923

2 924 Immunohistochemical Expression of Toll-Like Receptor 3 Patients and Methods This case-control study was carried out on 20 patients with verruca vulgaris who had not received treatment for wart one year before the study. They were diagnosed clinically and histopathologically. Twenty age and sex matched apparently healthy volunteers served as controls. All patients and controls were presenting at Benha Dermatology Department's out Patients Clinic, during the period between October 2012 and February 2013, a written consent was taken from each patient and control before participation in the study which was approved by Ethics Committee Human Research of Benha University. The exclusion criteria were: History of keloid or hypertrophic scar, patients under anticoagulant treatment, patients with allergy to lidocaine, or patients with wart only on the joint. Skin biopsy: Four mm punch skin biopsies were taken from wart in patient group and from normal skin in control group, were fixed in 10% neutral buffered formalin, routinely were processed and paraffin embedded. Sections 5µm thickness from paraffin blocks were mounted on plain slides and routinely were stained with hematoxylin and eosin (H&E) and were examined by light microscopy to confirm the clinical diagnosis. Also, sections 4µm thickness from both patients and controls were mounted on positive charged slides for immunohistochemical staining for TLR3. Immunohistochemical staining: Paraffinembedded tissue sections, 3-4 micron thick were mounted on positively-charged slides and heated at 60 C for 30 minutes then deparaffinized and rehydrated through a series of xylene and alcohol before staining. After antigen retrieval with microwave treatment in 1 0mM citrate buffer (Neo-Markers, Cat. # AP-9003), ph 6.0, endogenous peroxidase was blocked with 3% hydrogen peroxide for 15 minutes. Sections were washed 3 times with cold 0.01 M phosphate buffered saline (PBS) after blocking with 10% normal rabbit serum, slides were incubated for 1 hour with tol-3 antibody (Dako Cytomation Norden A/S, Glostrup, Denmark; dilution 1:50), prepared DAB-substrate-chromogenic solution was applied and incubated for minutes until color intensity has been reached. Lastly, sections were counterstained with Mayer's hematoxylin. Positive controls for tol-3 staining were used consisting of normal skin samples previously shown to stain with this antibody. Evaluation of immunohistochemical staining results was done according to Ku et al., [13]. Sections were screened on low power, detection of TLR 3 expression by intensity of staining in the epidermis was graded as follows: 0 for negative, + for weakly positive, ++ for moderately positive, and +++ for strongly positive. In dermis, the staining was graded according to the presence of stained blood vessels and stained cells as follows: 1=staining and 0=negative staining. Statistical analysis: The results were analyzed using SPSS (version 16) statistical package for Microsoft windows. The person correlation coefficient was used for statistical analysis. p-value<0.05 was considered statistically significant. Results This study comprised 20 patients with verruca vulgaris, 8 females and 12 males. Their age ranged from 13 to 45 years with a mean of ±8.49 years. The duration of the disease ranged from 3 to 96 months with a mean of 26.4 ±23.38 months. Two patients had single lesion 10%, 18 patients had multiple lesions 90%. The number of patients who had previous treatments with electrocautery 10 %, chemical cautery 10%, and who didn't have any previous treatments was 80 %. Twenty healthy volunteers served as control, 7 females and 13 males, their ages ranged from 16 to 42 years (Mean 26.2± 10.22). Results of immunohistochemical staining of TLR3: There was a highly significant increase in the TLR3 expression in all cases as compared with the normal controls (p<0.001). Expression of TLR3 was negative in normal human skin specimens (Fig. 1). TLR 3 was diffusely expressed throughout the horny and granular layer of epidermis in verruca vulgaris skin specimens. The expression staining intensity was moderate in two skin specimens and strong in the remaining specimens in patient group (Table 1) and (Figs. 2-7). Table (1): Comparison between TLR 3 expression in the lesional skin of patients and control. TLR 3 Expression Case Control No. % No. % Negative Corrected χ 2 test p value Moderately HS positive Strongly positive Total (p-value<0.001).

3 Ahmed A. Saleh, et al. 925 Immunohistochemical staining of TLR 3 using Avidin-Biotin complex. Fig. (1): A case of normal skin showing negative cytoplasmic expression of TLR 3 antibody in epidermal layers and dermis (strept-avidin-biotin Fig. (2): A case of verruca vulgaris immunostained by anti TLR 3 showing strong expression (brown staining) in all layers of the epidermis (strept-avidin-biotin Fig. (3): A case of verruca vulgaris immunostained by anti TLR 3 showing mild expression (brown staining) in the epidermis and dermis (strept-avidin-biotin method) (IHx200). Fig. (4): A case of verruca vulgaris immunostained by anti TLR 3 showing strong and diffuse expression (brown staining) in the epidermis and dermis (strept-avidin- Biotin) x 200. Fig. (5): A case of verruca vulgaris immunostained by anti TLR 3 showing strong and diffuse expression in the epidermis with prominent hyperkeratosis (streptavidin-biotin) x 200. Fig. (6): A case of verruca vulgaris immunostained by anti TLR 3 showing moderate expression (brown staining) in the basal layer of epidermis (strept-avidin-biotin

4 926 Immunohistochemical Expression of Toll-Like Receptor 3 cutaneous HPV infection. This is in agreement with Ku et al., [13], who evaluated the expression of various TLRs including TLR 3 in skin biopsies of patients with warts using PCR and immunostaining. The PCR and the immunostaining results showed markedly unregulated TLR 3 and 9 mrna in warts and molluscum, indicating the importance of TLR 3 in cutaneous innate immune response to commonly encountered cutaneous viral infections. Fig. (7): A case of verruca vulgaris immunostained by anti TLR 3 showing strong expression (brown staining) in the basal layer of epidermis (strept-avidin-biotin Discussion Warts are caused by double stranded human papilloma virus. Cutaneous viral infections have been proposed to induce localized inflammatory and antiviral responses that are believed to result in the resolution of viral skin lesions [14]. In order to counteract the innate host immune system, viruses should evolve mechanisms that block recognition and signaling through pattern recognition receptors, such as TLRs [15]. TLRs constitute the first line of defense against many pathogens and play a crucial role in the function of the innate immune system [6]. In the present study, TLR3 expression was negative in all control specimens. This is in agreement with Baker et al., [16], who reported that the expression of TLR3 and 4 was not detected in normal human skin. In contrast, Lebre et al., [17] demonstrated that normal keratinocytes constitutively express TLR 2,3, and 4 mrna, but not TLR 7. These different results might be due to the difference in the biopsy site and skin specimens, or to the staining methods. All the patients showed statistically significant expression of TLR 3 when compared with controls. The intensity of expression was moderate in two wart specimens (10%) and strong in the 18 wart specimens (90%). Localization of TLR 3 by immune-histopathological examination revealed that it is more highly expressed throughout the horny, granular layer and basal layer of epidermis more than the dermal cells in the warts specimens. This strong epidermal expression of TLR 3 and the pure epidermal location of warts may explain that TLR 3 may have a role in recognition and response to The rationale for targeting TLRs as a means of treating viral infections is based on the presence of naturally occurring agonist molecules within invading viruses [18]. Poly-ICLC (Hiltonol) is a polyinosinic-polycytidylic acid stabilized with poly-l-lysine and carboxymethylcellulose. It is a very stable double-stranded RNA and potent TLR 3 agonist with a strong IFN-inducing ability [19]. In this study statistical relationships between TLR 3 versus age and sex of patients, disease duration and number of warts were evaluated and were all found to be insignificant. Conclusion: Over expression of TLR 3 in wart skin implicate the role of TLR 3 in recognition and response to cutaneous HPV infection. Furthermore, pharmacological modulation of TLR 3 signaling or function of its downstream chemokines and/or cytokines can alter TLR 3 agonist-induced innate immune response and enhance its antiviral effects. References 1- ZHENG Z.M. and BAKER C.C.: Papillomavirus genome structure, expression, and post-transcriptional regulation. Front Biosci., 1, 11: , SANCLEMENTE G. and GILL D.K.: Human papillomavirus molecular biology and pathogenesis. JEDAV, 16, 3: , LORE K., BETTS M.R., BENCHLEY J.M., et al.: Tolllike receptor ligands modulate dendritic cells to augment cytomegalovirus- and HIV-1-specific T cell responses. J. Immunol., 171: , RUDD B.D., BURSTEIN E., DUCKETT C.S., et al.: Differential role for TLR 3 in respiratory syncytial virusinduced chemokine expression. J. Virol., 79: , ISHII K.J. and AKIRA S.: Innate immune recognition of, and regulation by, DNA. Trends Immunol., 27: , DABBAGH K. and LEWIS D.B.: Toll-like receptor and Th1/Th2 responses. Curr. Opin. Infect. Dis., 16: , AKIRA S., TAKEDA K. and KAISHO T.: Toll-like receptors: Critical proteins linking innate and acquired immunity. Nat. Immunol., 2: , 2001.

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