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Langerhans cells (CD1a and CD207), dermal dendrocytes (FXIIIa) and plasmacytoid dendritic cells (CD123) in skin lesions of leprosy patients
Microbial Pathogenesis 91 (2016) 18e25
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Langerhans cells (CD1a and CD207), dermal dendrocytes (FXIIIa) and plasmacytoid dendritic cells (CD123) in skin lesions of leprosy patients ~o a, Luciana Mota Silva a, Kelly Emi Hirai a, Tinara Leila de Sousa Aara Jorge Rodrigues de Sousa b, Juarez de Souza a, Leonidas Braga Dias Jr. a, Francisca Regina Oliveira Carneiro b, Hellen Thais Fuzii b, ~ es Quaresma a, b, * Juarez Antonio Simo a b
Centro de Ciencias Biologicas e da Saude, Universidade do Estado do Para, Belem, Para, Brazil Nucleo de Medicina Tropical, Universidade Federal do Para, Belem, Para, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history: Received 4 October 2015 Received in revised form 9 November 2015 Accepted 10 November 2015 Available online 27 November 2015
The clinical course of infection with Mycobacterium leprae varies widely and depends on the pattern of the host immune response. Dendritic cells play an important role in the activation of the innate and adaptive immune system and seem to be essential for the development of the disease. To analyze the presence of epidermal dendritic cells (CD1a and CD207), plasmacytoid dendritic cells (CD123) and dermal dendrocytes (factor XIIIa) in lesion fragments of leprosy patients, skin samples from 30 patients were studied. These samples were submitted to immunohistochemistry against CD1a, CD207, FXIIIa, and CD123. The results showed a larger number of Langerhans cells, detected with the CD1a or CD207 marker, dermal dendrocytes and plasmacytoid dendritic cells in patients with the tuberculoid form. A positive correlation was observed between the Langerhans cell markers CD1a and CD207 in both the tuberculoid and lepromatous forms, and between Langerhans cells and dermal dendrocytes in samples with the tuberculoid form. The present results indicate the existence of a larger number of dendritic cells in patients at the resistant pole of the disease (tuberculoid) and suggest that the different dendritic cells studied play a role, favoring an efficient immune response against infection with M. leprae. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Dendritic cells Immunology Immunopathology Mycobacterium leprae
1. Introduction Leprosy is a chronic disease caused by the obligate intracellular bacillus Mycobacterium leprae, which mainly affects the skin and peripheral nerves. The diagnosis and treatment of leprosy are well established in the literature. However, a late diagnosis and treatment can have severe consequences for patients and their contacts due to the great disabling potential of the disease [1e5]. M. leprae is able to infect a large number of individuals; however, few infected individuals become sick because of the low pathogenicity of the microorganism, a property that is not only due to the intrinsic characteristics of the bacillus, but depends primarily on its relationship with the host and the degree of endemicity of the environment. Active infection with M. leprae shows a broad
* Corresponding author. Nucleo de Medicina, Tropical/UFPA, Av. Generalissimo Deodoro 92, Umarizal, Belem, Para, Brazil. E-mail address: [email protected] (J.A.S. Quaresma). http://dx.doi.org/10.1016/j.micpath.2015.11.013 0882-4010/© 2015 Elsevier Ltd. All rights reserved.
clinical spectrum, ranging from paucibacillary disease characterized by the presence of few bacilli to multibacillary disease characterized by a high bacillary load in the lesions [6,7]. The clinical manifestations of leprosy are highly variable and are determined by the host's immune response against the bacillus [8e10]. Depending on the cytokines secreted during infection, T lymphocytes induce the development of milder disease or even cure [11] through a cell-mediated (Th1 type) response, or a response that is not as effective against the bacillus, called humoral or Th2 response [12,13]. The Th1 subpopulation produces IL-2 and IFN-g, cytokines that are responsible for the maintenance of the cell-mediated immune response. IL-2 activates CD4þ lymphocyte receptors and induces natural killer cells to produce IFN-g which, in turn, acts on macrophages to stimulate phagocytosis [8]. The Th2 subpopulation produces immunosuppressive cytokines such as IL-4 and IL-10, which suppress macrophage activity and stimulates B lymphocytes to differentiate into plasma cells which produce immunoglobulins [8].
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Dendritic cells play an important role in the activation of the innate and adaptive immune system and seem to be essential for the development of leprosy, since these cells are specialized in the processing and presentation of antigens, a function that can influence the outcome of infection [14]. Langerhans cells in the steady-state are immature, highly endocytic and form a dense network in the epidermis where they constantly screen the environment for invading antigens. These cells are ideally positioned to detect any pathogen that breaks the skin barrier [15e17]. Langerhans cells express the CD1a molecule, which presents lipid antigens to T cells, and CD207 (langerin), a Ctype lectin that induces the formation of Birbeck granules, a specific endosomal structure of Langerhans cells [18,19]. Dermal dendrocytes are found in the normal dermis where they predominate in the papillary dermis and in the perivascular adventitial layer. However, an increase in these cells is observed in inflammatory and neoplastic diseases. Dermal dendrocytes serve as a tissue reserve of factor XIIIa (FXIIIa) which is important for tissue healing [20], and act together with mast cells in processes of extracellular matrix remodeling [21]. Rarely observed in normal skin, plasmacytoid dendritic cells (pDCs) have a morphology that resembles plasma cells. These cells are abundantly found in lymphoid organs and in blood, and are rapidly recruited to the target site of bacterial or viral infection or inflammation [22e24]. pDCs express the CD123 molecule, the alpha chain of the IL-3 receptor. This marker is expressed at high levels on the surface of these cells since they require IL-3 for their differentiation [25]. pDCs express high levels of TLR7 and TLR9 which are expressed in the endoplasmic reticulum and endosomal membranes. They detect intracellular microbial nucleic acid [22e25]. So far few studies have investigated the concomitant expression of all these markers in skin lesions of patients with leprosy. The objective of the present study was to analyze by immunohistochemistry the presence of epidermal dendritic cells (CD1a and CD207), pDCs (CD123) and dermal dendrocytes (FXIIIa) in skin lesion fragments of leprosy patients, and to associate the presence of these cells with the polar forms of the disease.
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2.3. Quantitative and statistical analysis The presence of a brown deposit in the cytoplasm and around the nucleus of the cell was defined as a positive immunohistochemical reaction. Positive cells were counted under a Zeiss Axioplan microscope (model 456006) equipped with a 40 objective. The arithmetic mean was obtained by blind counting stained cells, by two observers, in five fields comprising an area of 0.0625 mm2, which were randomly chosen in the epidermis and dermis. Fields located in the epidermis were for the Langerhans cell markers (CD1a and CD207), and fields located in the inflammatory infiltrate for the FXIIIa and CD123 markers. The data were stored in Microsoft Excel 2007® spreadsheets for subsequent statistical analysis with the GraphPad Prism 5.0® program. The results were analyzed using the nonparametric ManneWhitney test and Spearman's linear correlation test. The level of significance was set at 95%. 2.4. Ethical aspects The study was approved by the Research Ethics Committee of the Center of Tropical Medicine, Federal University of Para (Permit No. 212.964). 3. Results 3.1. Langerhans cells
2. Material and methods
The use of the langerin (CD207) and CD1a markers revealed the presence of Langerhans cells in the epidermis. These cells had an irregular appearance, and long and thin cytoplasmic prolongations (Fig. 1A and B). Quantitative analysis of CD1a immunostaining showed a significantly (p ¼ 0.0209) larger median number in patients with tuberculoid leprosy (0.6094 ± 0.416 cells/field) compared to those with the lepromatous form (0.2750 ± 0.4164 cells/field) (Fig. 4A). The median number of CD207-positive cells was 1.569 ± 1.055 cells/ field in patients with tuberculoid leprosy and 0.7643 ± 0.6138 cells/ field in lepromatous patients. This difference was statistically significant (p ¼ 0.0373) (Fig. 4C).
2.1. Sample
3.2. Dermal dendrocytes
Thirty paraffin blocks of skin fragments from untreated patients with a diagnosis of leprosy, 16 with the tuberculoid form and 14 with the lepromatous form, collected at partner institutions of the Laboratory of Immunopathology, Center of Tropical Medicine, Federal University of Para, were used. The diagnosis was made by dermatoneurological and histopathological analysis and identification of bacilli in the samples according to the classification of Ridley-Jopling [26].
In skin fragments of leprosy patients, FXIIIa was detected in the dermis, with the observation of a large number of positive cells in the papillary dermis, at the dermoepidermal junction, and around blood vessels and granulomas (Fig. 2). The cytoplasm of these cells had clearly visible dendritic prolongations, which were elongated, spindle-shaped or oval. Quantitative analysis of FXIIIa-positive dermal dendrocytes showed a median number of 9.05 ± 6.646 cells/field in patients with the tuberculoid form and of 5.4 ± 4.704 cells/field in patients with lepromatous leprosy. Despite the larger number of FXIIIapositive dendrocytes in patients with the tuberculoid form, no significant difference was observed between the different poles of the disease (p ¼ 0.1189) (Fig. 4B).
2.2. Immunohistochemistry The samples were obtained from lesion biopsies fixed in 10% formalin, dehydrated, and embedded in paraffin. The blocks were cut into 5-mm thick histological sections with a microtome and stained with hematoxylin-eosin and Fite. Next, new 5-mm histological sections were obtained and mounted on silanized slides. The sections were submitted to immunohistochemistry by the streptavidin-biotin peroxidase method according to the protocol of Quaresma et al. [27]. The following primary monoclonal antibodies were used: anti-CD1a (Abcam ab708, dilution 1:50), anti-CD207 (Abcam ab49730, dilution 1:180), anti-FXIIIa (Abcam ab1834, dilution 1:180), and anti-CD123 (Abcam ab56411, dilution 1:160).
3.3. Plasmacytoid dendritic cells Plasmacytoid dendritic cells were found in the inflammatory infiltrate of lesions and close to blood vessels. The cells were of medium size and had a round or oval and slightly eccentric nucleus (Fig. 3). In the polar forms of leprosy, the median number of pDCs was significantly higher (p ¼ 0.0481) in tuberculoid patients
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Fig. 1. Immunohistochemistry for Langerhans cells in skin lesions of leprosy patients. A: CD1a in tuberculoid form; B: CD1a in lepromatous form; C: CD207 in tuberculoid form and D: CD207 in lepromatous form (400).
Fig. 2. Immunohistochemistry for dermal dendrocyte Factor XIIIa positive in skin lesions of leprosy patients. A: DDFXIIIa in tuberculoid form; B: DDFXIIIa in lepromatous form; C: DDFXIIIa around the granuloma in a sample of patients with tuberculoid form of leprosy (400).
(2.675 ± 1.657 cells/field) compared to those with the lepromatous form (1.471 ± 1.778 cells/field) (Fig. 4D).
3.4. Correlation between dendritic cells Nonparametric analysis revealed a significant strong positive correlation between CD1a and CD207 in lesions of tuberculoid patients (r ¼ 0.7548, p ¼ 0.0007) (Fig. 5A).
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Fig. 3. Immunohistochemistry for plasmacytoid dendritic cells (CD123) in skin lesions of leprosy patients (arrows). A and B: plasmacytoid dendritic cells in tuberculoid form; C and D: plasmacytoid dendritic cells in lepromatous form (400).
Fig. 4. Quantitative analysis (mean and standard deviation) of immunostaining for CD1a, CD207, FXIII and CD123 molecule in patients with polar forms of leprosy (CD1a TT ¼ 0.6094 ± 0.416 cells/field, LL ¼ 0.2750 ± 0.4164 cells/field, p ¼ 0.0209/CD207 TT ¼ 1.569 ± 1.055 cells/field, LL ¼ 0.7643 ± 0.6138 cells/field, p ¼ 0.0373/FXIIIa TT ¼ 9.05 ± 6.646 cells/field, LL ¼ 5.4 ± 4.704 cells/field, p ¼ 0.1189/CD123 TT ¼ 2.675 ± 1.657 cells/field, LL ¼ 1.471 ± 1.778 cells/field, p ¼ 0.0481) (ManneWhitney).
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Fig. 5. Spearman correlation between markers for dendritic cells in tuberculoid form of leprosy. A: Correlation between CD207 and CD1a (r ¼ 0.7548, p ¼ 0.0007); B: Correlation between and FXIIIa CD1a (r ¼ 0.5919, p ¼ 0.0157); C: Correlation between CD123 and CD1a (r ¼ 0.2761, p ¼ 0.3006); D: Correlation between and FXIIIa CD207 (r ¼ 0.4576, p ¼ 0.0747); E: Correlation between CD207 and CD123 (r ¼ 0.01035, P ¼ 0.9697); F: Correlation between CD123 and FXIIIa (r ¼ 0.1477, p ¼ 0.5851).
A significant moderate positive correlation was observed between CD1a and FXIIIa in patients with the tuberculoid form (r ¼ 0.5919, p ¼ 0.0157) (Fig. 5B). There was a weak positive correlation between CD1a and CD123 (r ¼ 0.2761, p ¼ 0.3006) (Fig. 5C) and a moderate positive correlation between CD207 and FXIIIa (r ¼ 0.4576, p ¼ 0.0747) (Fig. 5D) in patients with tuberculoid leprosy, but these correlations were not statistically significant. In tuberculoid patients, the correlation between CD207 and CD123 (r ¼ 0.01035, p ¼ 0.9697) (Fig. 5E) and between FXIIIa and CD123 (r ¼ 0.1477, p ¼ 0.5851) (Fig. 5F) was weak and negative and was not statistically significant. In the lepromatous form, a significant moderate correlation was observed between CD1a and CD207 (r ¼ 0.5585, p ¼ 0.0379) (Fig. 6A). The correlation between CD1a and FXIIIa, CD1a and CD123, CD207 and FXIIIa, and FXIIIa and CD123 was weak and nonsignificant in patients with lepromatous leprosy [r ¼ 0.2869, p ¼ 0.3199 (Fig. 6B); r ¼ 0.08677, p ¼ 0.768, (Fig. 6C); r ¼ 0.2541, p ¼ 0.3806 (Fig. 6D); r ¼ 0.36, p ¼ 0.2061, (Fig. 6F), respectively].
A weak negative and nonsignificant correlation was observed between CD207 and CD123 in patients with the lepromatous form (r ¼ 0.04967, p ¼ 0.8661) (Fig. 6E). 4. Discussion The results of this study showed a significant difference in CD1a and langerin immunostaining between the different poles of leprosy. The number of CD1a stained cells in the epidermis was higher in patients with the tuberculoid form (0.6094 cells/field) compared to lepromatous patients (0.2750 cells/field). Immunostaining for langerin (CD207) was significantly higher (p ¼ 0.0373) in patients with tuberculoid leprosy (1.569 cells/field) compared to patients at the lepromatous pole (0.7643 cells/field). Similar results have been reported by Quaresma et al. [28] who investigated the role of skin dendritic cells in the pathogenesis of leprosy. Langerhans cells were detected throughout the epidermis, with these cells showing variable morphology and staining intensity. A larger number of cells were observed in the tuberculoid form, suggesting that the activation of the immune response in
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Fig. 6. Spearman correlation between markers for dendritic cells in lepromatous form of leprosy. A: Correlation between CD207 and CD1a (r ¼ 0.5585, p ¼ 0.0379); B: Correlation between and FXIIIa CD1a (r ¼ 0.2869, p ¼ 0.3199); C: Correlation between CD1a and CD123 (R ¼ 0.08677, p ¼ 0.768); D: Correlation between and FXIIIa CD207 (r ¼ 0.2541, p ¼ 0.3806); E: Correlation between CD207 and CD123 (r ¼ 0.04967, P ¼ 0.8661); F: Correlation between and FXIIIa CD123 (r ¼ 0.36, p ¼ 0.2061).
leprosy depends on the initial interaction between T cells and antigen-presenting cells. Sieling et al. [29] observed high expression of CD1a by dendritic cells in samples obtained from patients with the tuberculoid form, in contrast to skin lesions of patients with the lepromatous form in which this protein was rarely detected. The present results agree with the study of Miranda et al. [30] who found increased accumulation of Langerhans cells after mycobacterial stimulation. A significant number of these cells were observed in patients with a reversal reaction and erythema nodosum leprosum, similar to the findings seen in tuberculoid lesions. In contrast, the number of Langerhans cells was considerably lower in patients with lepromatous leprosy compared to the other groups. Hunger et al. [31], evaluating the efficiency of Langerhans cells in presenting nonpeptide antigens to T cells through CD1a and langerin, observed the expression of CD1a and CD207 in skin lesions of patients with leprosy. Both markers were coexpressed on cells with dendritic morphology in the epidermis, indicating that langerin and CD1a are present on epidermal Langerhans cells in leprosy lesions and may therefore mediate antigen presentation to
T cells. Dermal dendritic cells langerinþ were also observed in our sample. Although dermal dendritic cells langerinþ are phenotypically very similar to Langerhans cells in the maturation levels and expression of costimulatory molecules, they can be distinguished from Langerhans cells using the CD103 marker (integrin chain aIEL), CD11b (integrin alpha M) and Ep-CAM adhesion molecule (GP40) [31]. Quantitative analysis of FXIIIa-positive dermal dendrocytes in patients with the polar forms of leprosy showed a median number of 9.05 cells/field in patients with the tuberculoid form, whereas the median number was 5.4 cells/field in patients with lepromatous leprosy. No significant difference was observed between the groups studied (p ¼ 0.1189), but the number of FXIIIa-positive dermal dendrocytes was higher in patients with the tuberculoid form compared to those at the lepromatous pole. Similar results have been reported by Quaresma et al. [28]. These authors evaluated the presence of FXIIIa-positive dermal dendrocytes in patients with the polar forms of leprosy and detected these cells, which had a spindle-shaped morphology, mainly along the superficial dermis. There was a clear predominance of dermal dendrocytes in the
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tuberculoid pole of the disease, suggesting that these cells play a determinant role in the clinical course of the disease. The median number of pDCs was significantly higher (p ¼ 0.0481) in leprosy lesions of tuberculoid patients (2.675 cells/ field) compared to those with the lepromatous form (1.471 cells/ field). The presence of CD123-positive pDCs in leprosy was described for the first time by Massone et al. [32] who investigated the distribution of regulatory T cells and pDCs in this disease. Expression of CD123, the marker used for the identification of pDCs, was not detected in any of the biopsies studied, except for two cases of patients with erythema nodosum leprosum in which focal positivity was observed. The authors suggested that pDCs are not involved in the immune response against M. leprae. In another study, Massone et al. [33] immunophenotyped the skin lymphocytic infiltrate of patients co-infected with M. leprae and human immunodeficiency virus (HIV). As in the previous study, CD123positive cells were not detected in the skin samples analyzed. The authors thus extended the results of the previous study, showing that pDCs are not involved in the immunopathogenesis of leprosy, irrespective of the. 4.1. Serological status of the patient The results of the studies of Massone et al. [32,33] not agree with the present findings since CD123-positive cells were detected in both clinical forms of leprosy. The number of these cells was higher in the tuberculoid form, suggesting the participation of pDCs in the cell-mediated immune response against infection with M. leprae. In the present study, nonparametric analysis revealed a significant positive correlation between CD1a and CD207 in tuberculoid (r ¼ 0.7548, p ¼ 0.0007) and lepromatous lesions (r ¼ 0.5585, p ¼ 0.0379) of leprosy patients. This result corroborates the cellular specificity and efficiency of the two antibodies. Similar results have been reported by Seguier et al. [34] who quantified and compared the presence of CD1a- and CD207positive Langerhans cells in human gingival tissue. The authors also observed a correlation between these two markers, with 92% of CD207-positive Langerhans cells coexpressing CD1a, while 82% of CD1a-positive cells coexpressed langerin. The results showed heterogeneity in the phenotype of gingival Langerhans cells and the authors concluded that langerin seems to be the most specific marker for the investigation of these cells. In a study analyzing Langerhans cells and microvessel density in oral squamous cell carcinoma, Santos et al. [35] also found a positive correlation between the two markers. When analyzed separately in the samples studied, the anti-CD1a and anti-CD207 antibodies showed similar efficiency in the identification of Langerhans cells, with the observation of high expression of the two markers in the tuberculoid form. However, comparison of the expression of these markers revealed higher expression of CD207, a finding suggesting phenotypic diversity of these cells. Therefore, when a specific cell type is analyzed, the combination of the two monoclonal antibodies with different binding specificities seems to increase the reliability of the results. Analysis of the CD1a and FXIIIa markers for the identification of Langerhans cells and dermal dendrocytes showed a significant moderate positive correlation (r ¼ 0.5919, p ¼ 0.0157) in patients with the tuberculoid form. The finding of a larger number of these cells in patients with the tuberculoid form may be related to an attempt of the organism to contain M. leprae infection since, in addition to presenting antigens, dendritic cells secrete cytokines such as IL-12 and IL-23 which induce the activation of T cells. The latter, in turn, release proinflammatory cytokines of the Th1 and Th17 types [36e38], promoting a resistance profile that is likely to
have greater control of disease dissemination. The present results showing a larger number of dendritic cells in patients at the resistance pole of the disease indicate an important role of the different dendritic cells studied, favoring an efficient immune response against infection with M. leprae. Disclosure The authors declare no conflict of interest. Acknowledgments This project has been funded in whole or in part with funds from the Conselho Nacional de Pesquisa e CNPq e Brazil, grants 402738/ 2005-5 and 401223/2005-1. References [1] L.R. Freitas, E.C. Duarte, L.P. Garcia, Leprosy in Brazil and its association with characteristics of municipalities: ecological study, 2009e2011, Trop. Med. Int. Health 19 (2014) 1216e1225. [2] W.C. Smith, A. Aerts, Role of contact tracing and prevention strategies in the interruption of leprosy transmission, Lepr. Rev. 85 (2014) 2e17. [3] M.L. Penna, M.A. Grossi, G.O. Penna, Country profile: leprosy in Brazil, Lepr. Rev. 84 (2013) 308e315. [4] S.L. Walker, D.N.J. Lockwood, The clinical and immunological features of leprosy, Br. Med. Bull. 78 (2006) 103e121. [5] N.T. Foss, A.C.F. Motta, Leprosy, a neglected disease that causes a wide variety of clinical conditions in tropical countries, Mem. Inst. Oswaldo Cruz 107 (2012) 28e33. [6] I.M.B. Goulart, G.O. Penna, G. Cunha, Imunopatologia da Hanseníase: a complexidade dos mecanismos da resposta imune do hospedeiro ao Mycobacterium leprae, Rev. Soc. Bras. Med. Trop. 35 (2002) 365e375. [7] K. Suzuki, T. Akama, A. Kawashima, A. Yoshirara, R.R. Yotsu, N. Ishii, Current status of leprosy: epidemiology, basic science and clinical perspectives, J. Dermatol. 39 (2012) 121e129. lez, J.C. Salas-Alanis, J. Ocampo-Candiani, Leprosy. [8] K. Eichelmann, G.E. Gonza an update: definition, pathogenesis, classification, diagnosis, and treatment, Acta Dermosifiliogr. 104 (2013) 554e563. [9] N.T. Foss, A.C. Motta, Leprosy, a neglected disease that causes a wide variety of clinical conditions in tropical countries, Mem. Inst. Oswaldo Cruz 107 (2012) 28e33. [10] L.C. Rodrigues, D.N.J. Lockwood, Leprosy now: epidemiology, progress, challenges, and research gaps, Lancet Infect. Dis. 11 (2011) 464e470. [11] D.J. Cher, T.R. Mosmann, Two types of murine helper T cell clone. II. Delayedtype hypersensitivity is mediated by Th1 clones, J. Immunol. 138 (1987) 3688e3694. [12] P.A. Sieling, R.L. Modlin, Cytokine patterns at the site of mycobacterial infection, Immunobiology 191 (1994) 378e387. [13] M.O. Moraes, C.C. Cardoso, P.R. Vanderborght, A.G. Pacheco, Genetics of host response in leprosy, Lepr. Rev. 77 (2006) 189e202. [14] B.Z. Igyarto, D.H. Kaplan, Antigen presentation by Langerhans cells, Curr. Opin. Immunol. 25 (2013) 115e119. [15] J. Banchereau, F. Briere, C. Caux, J. Davoust, S. Lebecque, K. Palucka, Immunobiology of dendritic cells, Ann. Rev. Immunol. 18 (2000) 767e811. [16] N. Romani, B.E. Clausen, P. Stoitzner, Langerhans cells and more: langerinexpressing dendritic cell subsets in the skin, Immunol. Rev. 34 (2010) 120e141. [17] B.Z. Igyarto, D.H. Kaplan, The evolving function of Langerhans cells in adaptive skin immunity, Immunol. Cell Biol. 88 (2010) 361e365. [18] K. Yoshida, A. Kubo, H. Fujita, M. Yokouchi, K. Ishii, H. Kawasaki, T. Nomura, H. Shimizu, K. Kouyama, T. Ebihara, K. Nagao, M. Amagai, Distinct behavior of human Langerhans cells and inflammatory dendritic epidermal cells at tight junctions in patients with atopic dermatitis, J. Allergy Clin. Immunol. 134 (2014) 856e864. [19] N. Romani, P.M. Brunner, G. Stingl, Changing views of the role of Langerhans cells, J. Investig. Dermatol. 132 (2012) 872e881. [20] E. Hoyo, J. Kanitakis, D. Schmitt, The dermal dendrocyte, Pathol. Biol. 41 (1993) 613e618. [21] H. Sueki, D. Whitaker, M. Buchsbaum, G.F. Murphy, Novel interactions between dermal dendrocytes and mast cells in human skin. Implications for hemostasis and matrix repair, Lab. Investig. 69 (1993) 160e172. [22] M. Cella, D. Jarrossay, F. Facchetti, O. Alebardi, H. Nakajima, A. Lanzavecchia, M. Colona, Plasmacytoid monocytes migrate to inflamed lymph nodes and produce large amounts of type I interferon, Nat. Med. 5 (1999) 919e923. [23] M. Collin, N. Mcgovern, M. Haniffa, Human dendritic cell subsets, Immunology 140 (2013) 22e30. [24] M. Merad, P. Sathe, J. Helft, J. Miller, A. Mortha, The dendritic cell lineage: ontogeny and function of dendritic cells and their subsets in the steady state
K.E. Hirai et al. / Microbial Pathogenesis 91 (2016) 18e25 and the inflamed setting, Annu. Rev. Immunol. 31 (2013) 563e604. [25] J.M. McNiff, D.H. Kaplan, Plasmacytoid dendritic cells are present in cutaneous dermatomyositis lesions in a pattern distinct from lupus erythematosus, J. Cutan. Pathol. 35 (2008) 452e456. [26] D.S. Ridley, M.J. Jopling, Classification of leprosy according to immunity. A five group system, Int. J. Lepr. Other Mycobact. Dis. 34 (1996) 255e273. [27] J.A.S. Quaresma, V.L.R. Barros, C. Pagliari, E.R. Fernandes, F. Guedes, C.F. Takakura, H.F. Andrade Jr., P.F.C. Vasconcelos, M.I.S. Duarte, Revisiting the liver in human yellow fever: virus-induced apoptosis in hepatocytes associated with TGF-b, TNF-a and NK cells activity, Virology 345 (2006) 22e30. [28] J.A.S. Quaresma, M.F.A. Oliveira, A.C.R. Guimar~ aes, E.B. Brito, C. Pagliari, A.C. de Brito, M.B. Xavier, M.I.S. Duarte, CD1a and FXIIIa immunohistochemistry in leprosy: a possible role of dendritic cells in the pathogenesis of Mycobacterium leprae infection, Am. J. Dermatophatol. 31 (2009) 527e531. [29] P.A. Sieling, D. Jullien, M. Dahlem, T.F. Tedder, T.H. Rea, R.L. Modlin, S.A. Porcelli, CD1 expression by dendritic cells in human leprosy lesions: correlation with effective host immunity, J. Immunol. 162 (1999) 1851e1858. , H. Ferreira, [30] A. Miranda, T.P. Amadeu, G. Schueler, F.B. Alvarenga, N. Duppre J.A. Nery, E.N. Sarno, Increased Langerhans cell accumulation after mycobacterial stimuli, Histopathology 51 (2007) 649e656. [31] R.E. Hunger, P.A. Sieling, M.T. Ochoa, M. Sugaya, A.E. Burdick, T.H. Rea, P.J. Brennan, J.T. Belisle, A. Blauvelt, S.A. Porcelli, R.L. Modlin, Langerhans cells utilize CD1a and langerin to efficiently present nonpeptide antigens to T cells, J. Clin. Investig. 113 (2004) 701e708. [32] C. Massone, E. Nunzi, R. Ribeiro-Rodrigues, S. Talhari, C. Talhari, A.P. Schettini,
[33]
[34]
[35]
[36]
[37]
[38]
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J.N. Parente, A.M. Brunasso, M. Puntoni, A. Clapasson, S. Noto, L. Cerroni, T regulatory cells and plasmocytoid dentritic cells in hansen disease: a new insight into pathogenesis? Am. J. Dermatopathol. 32 (2010) 251e256. C. Massone, C. Talhari, S. Talhari, A.M. Brunasso, T.M. Campbell, P. Curcic, L. Cerroni, R. Ribeiro-Rodrigues, Immunophenotype of skin lymphocytic infiltrate in patients co-infected with Mycobacterium leprae and human immunodeficiency virus: a scenario dependent on CD8þ and/or CD20þ cells, Br. J. Dermatol. 165 (2011) 321e328. S. Seguier, A. Bodineau, G. Godeau, B. Pellat, N. Brousse, Langerinþ versus CD1aþ Langerhans cells in human gingival tissue: a comparative and quantitative immunohistochemical study, Arch. Oral Biol. 48 (2003) 255e262. L.C.S. Santos, I.L.O. Nascimento, J.N. Santos, Immunohistochemistry assessment of Langerhans cells and microvascular density in oral squamous cell carcinomas, Full Dent. Sci. 5 (2013) 103e109. F.A. Verreck, T. Boer, D.M. Langenberg, M.A. Hoeve, M. Kramer, E. Vaisberg, R. Kastelein, A. Kolk, A. Waal-Malefyt, T.H. Ottenhoff, Human IL-23-producing type 1 macrophages promote but IL-10 producing type 2 macrophages subvert immunity to (myco)bacteria, Proc. Natl. Acad. Sci. 101 (2004) 4560e4565. S.R. Krutzik, B. Tan, H. Li, M.T. Ochoa, P.T. Liu, S.E. Sharfstein, T.G. Graeber, P.A. Sieling, Y.J. Liu, T.H. Rea, B.R. Bloom, R.L. Modlin, TLR activation triggers the rapid differentiation of monocytes into macrophages and dendritic cells, Nat. Med. 11 (2005) 653e660. F.O. Nestle, P. Meglio, J.Z. Qin, B.J. Nickoloff, Skin immune sentinels in health and disease, Nat. Rev. Immunol. 9 (2009) 679e691.
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Microbial Pathogenesis journal homepage: www.elsevier.com/locate/micpath
Langerhans cells (CD1a and CD207), dermal dendrocytes (FXIIIa) and plasmacytoid dendritic cells (CD123) in skin lesions of leprosy patients ~o a, Luciana Mota Silva a, Kelly Emi Hirai a, Tinara Leila de Sousa Aara Jorge Rodrigues de Sousa b, Juarez de Souza a, Leonidas Braga Dias Jr. a, Francisca Regina Oliveira Carneiro b, Hellen Thais Fuzii b, ~ es Quaresma a, b, * Juarez Antonio Simo a b
Centro de Ciencias Biologicas e da Saude, Universidade do Estado do Para, Belem, Para, Brazil Nucleo de Medicina Tropical, Universidade Federal do Para, Belem, Para, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history: Received 4 October 2015 Received in revised form 9 November 2015 Accepted 10 November 2015 Available online 27 November 2015
The clinical course of infection with Mycobacterium leprae varies widely and depends on the pattern of the host immune response. Dendritic cells play an important role in the activation of the innate and adaptive immune system and seem to be essential for the development of the disease. To analyze the presence of epidermal dendritic cells (CD1a and CD207), plasmacytoid dendritic cells (CD123) and dermal dendrocytes (factor XIIIa) in lesion fragments of leprosy patients, skin samples from 30 patients were studied. These samples were submitted to immunohistochemistry against CD1a, CD207, FXIIIa, and CD123. The results showed a larger number of Langerhans cells, detected with the CD1a or CD207 marker, dermal dendrocytes and plasmacytoid dendritic cells in patients with the tuberculoid form. A positive correlation was observed between the Langerhans cell markers CD1a and CD207 in both the tuberculoid and lepromatous forms, and between Langerhans cells and dermal dendrocytes in samples with the tuberculoid form. The present results indicate the existence of a larger number of dendritic cells in patients at the resistant pole of the disease (tuberculoid) and suggest that the different dendritic cells studied play a role, favoring an efficient immune response against infection with M. leprae. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Dendritic cells Immunology Immunopathology Mycobacterium leprae
1. Introduction Leprosy is a chronic disease caused by the obligate intracellular bacillus Mycobacterium leprae, which mainly affects the skin and peripheral nerves. The diagnosis and treatment of leprosy are well established in the literature. However, a late diagnosis and treatment can have severe consequences for patients and their contacts due to the great disabling potential of the disease [1e5]. M. leprae is able to infect a large number of individuals; however, few infected individuals become sick because of the low pathogenicity of the microorganism, a property that is not only due to the intrinsic characteristics of the bacillus, but depends primarily on its relationship with the host and the degree of endemicity of the environment. Active infection with M. leprae shows a broad
* Corresponding author. Nucleo de Medicina, Tropical/UFPA, Av. Generalissimo Deodoro 92, Umarizal, Belem, Para, Brazil. E-mail address: [email protected] (J.A.S. Quaresma). http://dx.doi.org/10.1016/j.micpath.2015.11.013 0882-4010/© 2015 Elsevier Ltd. All rights reserved.
clinical spectrum, ranging from paucibacillary disease characterized by the presence of few bacilli to multibacillary disease characterized by a high bacillary load in the lesions [6,7]. The clinical manifestations of leprosy are highly variable and are determined by the host's immune response against the bacillus [8e10]. Depending on the cytokines secreted during infection, T lymphocytes induce the development of milder disease or even cure [11] through a cell-mediated (Th1 type) response, or a response that is not as effective against the bacillus, called humoral or Th2 response [12,13]. The Th1 subpopulation produces IL-2 and IFN-g, cytokines that are responsible for the maintenance of the cell-mediated immune response. IL-2 activates CD4þ lymphocyte receptors and induces natural killer cells to produce IFN-g which, in turn, acts on macrophages to stimulate phagocytosis [8]. The Th2 subpopulation produces immunosuppressive cytokines such as IL-4 and IL-10, which suppress macrophage activity and stimulates B lymphocytes to differentiate into plasma cells which produce immunoglobulins [8].
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Dendritic cells play an important role in the activation of the innate and adaptive immune system and seem to be essential for the development of leprosy, since these cells are specialized in the processing and presentation of antigens, a function that can influence the outcome of infection [14]. Langerhans cells in the steady-state are immature, highly endocytic and form a dense network in the epidermis where they constantly screen the environment for invading antigens. These cells are ideally positioned to detect any pathogen that breaks the skin barrier [15e17]. Langerhans cells express the CD1a molecule, which presents lipid antigens to T cells, and CD207 (langerin), a Ctype lectin that induces the formation of Birbeck granules, a specific endosomal structure of Langerhans cells [18,19]. Dermal dendrocytes are found in the normal dermis where they predominate in the papillary dermis and in the perivascular adventitial layer. However, an increase in these cells is observed in inflammatory and neoplastic diseases. Dermal dendrocytes serve as a tissue reserve of factor XIIIa (FXIIIa) which is important for tissue healing [20], and act together with mast cells in processes of extracellular matrix remodeling [21]. Rarely observed in normal skin, plasmacytoid dendritic cells (pDCs) have a morphology that resembles plasma cells. These cells are abundantly found in lymphoid organs and in blood, and are rapidly recruited to the target site of bacterial or viral infection or inflammation [22e24]. pDCs express the CD123 molecule, the alpha chain of the IL-3 receptor. This marker is expressed at high levels on the surface of these cells since they require IL-3 for their differentiation [25]. pDCs express high levels of TLR7 and TLR9 which are expressed in the endoplasmic reticulum and endosomal membranes. They detect intracellular microbial nucleic acid [22e25]. So far few studies have investigated the concomitant expression of all these markers in skin lesions of patients with leprosy. The objective of the present study was to analyze by immunohistochemistry the presence of epidermal dendritic cells (CD1a and CD207), pDCs (CD123) and dermal dendrocytes (FXIIIa) in skin lesion fragments of leprosy patients, and to associate the presence of these cells with the polar forms of the disease.
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2.3. Quantitative and statistical analysis The presence of a brown deposit in the cytoplasm and around the nucleus of the cell was defined as a positive immunohistochemical reaction. Positive cells were counted under a Zeiss Axioplan microscope (model 456006) equipped with a 40 objective. The arithmetic mean was obtained by blind counting stained cells, by two observers, in five fields comprising an area of 0.0625 mm2, which were randomly chosen in the epidermis and dermis. Fields located in the epidermis were for the Langerhans cell markers (CD1a and CD207), and fields located in the inflammatory infiltrate for the FXIIIa and CD123 markers. The data were stored in Microsoft Excel 2007® spreadsheets for subsequent statistical analysis with the GraphPad Prism 5.0® program. The results were analyzed using the nonparametric ManneWhitney test and Spearman's linear correlation test. The level of significance was set at 95%. 2.4. Ethical aspects The study was approved by the Research Ethics Committee of the Center of Tropical Medicine, Federal University of Para (Permit No. 212.964). 3. Results 3.1. Langerhans cells
2. Material and methods
The use of the langerin (CD207) and CD1a markers revealed the presence of Langerhans cells in the epidermis. These cells had an irregular appearance, and long and thin cytoplasmic prolongations (Fig. 1A and B). Quantitative analysis of CD1a immunostaining showed a significantly (p ¼ 0.0209) larger median number in patients with tuberculoid leprosy (0.6094 ± 0.416 cells/field) compared to those with the lepromatous form (0.2750 ± 0.4164 cells/field) (Fig. 4A). The median number of CD207-positive cells was 1.569 ± 1.055 cells/ field in patients with tuberculoid leprosy and 0.7643 ± 0.6138 cells/ field in lepromatous patients. This difference was statistically significant (p ¼ 0.0373) (Fig. 4C).
2.1. Sample
3.2. Dermal dendrocytes
Thirty paraffin blocks of skin fragments from untreated patients with a diagnosis of leprosy, 16 with the tuberculoid form and 14 with the lepromatous form, collected at partner institutions of the Laboratory of Immunopathology, Center of Tropical Medicine, Federal University of Para, were used. The diagnosis was made by dermatoneurological and histopathological analysis and identification of bacilli in the samples according to the classification of Ridley-Jopling [26].
In skin fragments of leprosy patients, FXIIIa was detected in the dermis, with the observation of a large number of positive cells in the papillary dermis, at the dermoepidermal junction, and around blood vessels and granulomas (Fig. 2). The cytoplasm of these cells had clearly visible dendritic prolongations, which were elongated, spindle-shaped or oval. Quantitative analysis of FXIIIa-positive dermal dendrocytes showed a median number of 9.05 ± 6.646 cells/field in patients with the tuberculoid form and of 5.4 ± 4.704 cells/field in patients with lepromatous leprosy. Despite the larger number of FXIIIapositive dendrocytes in patients with the tuberculoid form, no significant difference was observed between the different poles of the disease (p ¼ 0.1189) (Fig. 4B).
2.2. Immunohistochemistry The samples were obtained from lesion biopsies fixed in 10% formalin, dehydrated, and embedded in paraffin. The blocks were cut into 5-mm thick histological sections with a microtome and stained with hematoxylin-eosin and Fite. Next, new 5-mm histological sections were obtained and mounted on silanized slides. The sections were submitted to immunohistochemistry by the streptavidin-biotin peroxidase method according to the protocol of Quaresma et al. [27]. The following primary monoclonal antibodies were used: anti-CD1a (Abcam ab708, dilution 1:50), anti-CD207 (Abcam ab49730, dilution 1:180), anti-FXIIIa (Abcam ab1834, dilution 1:180), and anti-CD123 (Abcam ab56411, dilution 1:160).
3.3. Plasmacytoid dendritic cells Plasmacytoid dendritic cells were found in the inflammatory infiltrate of lesions and close to blood vessels. The cells were of medium size and had a round or oval and slightly eccentric nucleus (Fig. 3). In the polar forms of leprosy, the median number of pDCs was significantly higher (p ¼ 0.0481) in tuberculoid patients
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Fig. 1. Immunohistochemistry for Langerhans cells in skin lesions of leprosy patients. A: CD1a in tuberculoid form; B: CD1a in lepromatous form; C: CD207 in tuberculoid form and D: CD207 in lepromatous form (400).
Fig. 2. Immunohistochemistry for dermal dendrocyte Factor XIIIa positive in skin lesions of leprosy patients. A: DDFXIIIa in tuberculoid form; B: DDFXIIIa in lepromatous form; C: DDFXIIIa around the granuloma in a sample of patients with tuberculoid form of leprosy (400).
(2.675 ± 1.657 cells/field) compared to those with the lepromatous form (1.471 ± 1.778 cells/field) (Fig. 4D).
3.4. Correlation between dendritic cells Nonparametric analysis revealed a significant strong positive correlation between CD1a and CD207 in lesions of tuberculoid patients (r ¼ 0.7548, p ¼ 0.0007) (Fig. 5A).
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Fig. 3. Immunohistochemistry for plasmacytoid dendritic cells (CD123) in skin lesions of leprosy patients (arrows). A and B: plasmacytoid dendritic cells in tuberculoid form; C and D: plasmacytoid dendritic cells in lepromatous form (400).
Fig. 4. Quantitative analysis (mean and standard deviation) of immunostaining for CD1a, CD207, FXIII and CD123 molecule in patients with polar forms of leprosy (CD1a TT ¼ 0.6094 ± 0.416 cells/field, LL ¼ 0.2750 ± 0.4164 cells/field, p ¼ 0.0209/CD207 TT ¼ 1.569 ± 1.055 cells/field, LL ¼ 0.7643 ± 0.6138 cells/field, p ¼ 0.0373/FXIIIa TT ¼ 9.05 ± 6.646 cells/field, LL ¼ 5.4 ± 4.704 cells/field, p ¼ 0.1189/CD123 TT ¼ 2.675 ± 1.657 cells/field, LL ¼ 1.471 ± 1.778 cells/field, p ¼ 0.0481) (ManneWhitney).
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Fig. 5. Spearman correlation between markers for dendritic cells in tuberculoid form of leprosy. A: Correlation between CD207 and CD1a (r ¼ 0.7548, p ¼ 0.0007); B: Correlation between and FXIIIa CD1a (r ¼ 0.5919, p ¼ 0.0157); C: Correlation between CD123 and CD1a (r ¼ 0.2761, p ¼ 0.3006); D: Correlation between and FXIIIa CD207 (r ¼ 0.4576, p ¼ 0.0747); E: Correlation between CD207 and CD123 (r ¼ 0.01035, P ¼ 0.9697); F: Correlation between CD123 and FXIIIa (r ¼ 0.1477, p ¼ 0.5851).
A significant moderate positive correlation was observed between CD1a and FXIIIa in patients with the tuberculoid form (r ¼ 0.5919, p ¼ 0.0157) (Fig. 5B). There was a weak positive correlation between CD1a and CD123 (r ¼ 0.2761, p ¼ 0.3006) (Fig. 5C) and a moderate positive correlation between CD207 and FXIIIa (r ¼ 0.4576, p ¼ 0.0747) (Fig. 5D) in patients with tuberculoid leprosy, but these correlations were not statistically significant. In tuberculoid patients, the correlation between CD207 and CD123 (r ¼ 0.01035, p ¼ 0.9697) (Fig. 5E) and between FXIIIa and CD123 (r ¼ 0.1477, p ¼ 0.5851) (Fig. 5F) was weak and negative and was not statistically significant. In the lepromatous form, a significant moderate correlation was observed between CD1a and CD207 (r ¼ 0.5585, p ¼ 0.0379) (Fig. 6A). The correlation between CD1a and FXIIIa, CD1a and CD123, CD207 and FXIIIa, and FXIIIa and CD123 was weak and nonsignificant in patients with lepromatous leprosy [r ¼ 0.2869, p ¼ 0.3199 (Fig. 6B); r ¼ 0.08677, p ¼ 0.768, (Fig. 6C); r ¼ 0.2541, p ¼ 0.3806 (Fig. 6D); r ¼ 0.36, p ¼ 0.2061, (Fig. 6F), respectively].
A weak negative and nonsignificant correlation was observed between CD207 and CD123 in patients with the lepromatous form (r ¼ 0.04967, p ¼ 0.8661) (Fig. 6E). 4. Discussion The results of this study showed a significant difference in CD1a and langerin immunostaining between the different poles of leprosy. The number of CD1a stained cells in the epidermis was higher in patients with the tuberculoid form (0.6094 cells/field) compared to lepromatous patients (0.2750 cells/field). Immunostaining for langerin (CD207) was significantly higher (p ¼ 0.0373) in patients with tuberculoid leprosy (1.569 cells/field) compared to patients at the lepromatous pole (0.7643 cells/field). Similar results have been reported by Quaresma et al. [28] who investigated the role of skin dendritic cells in the pathogenesis of leprosy. Langerhans cells were detected throughout the epidermis, with these cells showing variable morphology and staining intensity. A larger number of cells were observed in the tuberculoid form, suggesting that the activation of the immune response in
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Fig. 6. Spearman correlation between markers for dendritic cells in lepromatous form of leprosy. A: Correlation between CD207 and CD1a (r ¼ 0.5585, p ¼ 0.0379); B: Correlation between and FXIIIa CD1a (r ¼ 0.2869, p ¼ 0.3199); C: Correlation between CD1a and CD123 (R ¼ 0.08677, p ¼ 0.768); D: Correlation between and FXIIIa CD207 (r ¼ 0.2541, p ¼ 0.3806); E: Correlation between CD207 and CD123 (r ¼ 0.04967, P ¼ 0.8661); F: Correlation between and FXIIIa CD123 (r ¼ 0.36, p ¼ 0.2061).
leprosy depends on the initial interaction between T cells and antigen-presenting cells. Sieling et al. [29] observed high expression of CD1a by dendritic cells in samples obtained from patients with the tuberculoid form, in contrast to skin lesions of patients with the lepromatous form in which this protein was rarely detected. The present results agree with the study of Miranda et al. [30] who found increased accumulation of Langerhans cells after mycobacterial stimulation. A significant number of these cells were observed in patients with a reversal reaction and erythema nodosum leprosum, similar to the findings seen in tuberculoid lesions. In contrast, the number of Langerhans cells was considerably lower in patients with lepromatous leprosy compared to the other groups. Hunger et al. [31], evaluating the efficiency of Langerhans cells in presenting nonpeptide antigens to T cells through CD1a and langerin, observed the expression of CD1a and CD207 in skin lesions of patients with leprosy. Both markers were coexpressed on cells with dendritic morphology in the epidermis, indicating that langerin and CD1a are present on epidermal Langerhans cells in leprosy lesions and may therefore mediate antigen presentation to
T cells. Dermal dendritic cells langerinþ were also observed in our sample. Although dermal dendritic cells langerinþ are phenotypically very similar to Langerhans cells in the maturation levels and expression of costimulatory molecules, they can be distinguished from Langerhans cells using the CD103 marker (integrin chain aIEL), CD11b (integrin alpha M) and Ep-CAM adhesion molecule (GP40) [31]. Quantitative analysis of FXIIIa-positive dermal dendrocytes in patients with the polar forms of leprosy showed a median number of 9.05 cells/field in patients with the tuberculoid form, whereas the median number was 5.4 cells/field in patients with lepromatous leprosy. No significant difference was observed between the groups studied (p ¼ 0.1189), but the number of FXIIIa-positive dermal dendrocytes was higher in patients with the tuberculoid form compared to those at the lepromatous pole. Similar results have been reported by Quaresma et al. [28]. These authors evaluated the presence of FXIIIa-positive dermal dendrocytes in patients with the polar forms of leprosy and detected these cells, which had a spindle-shaped morphology, mainly along the superficial dermis. There was a clear predominance of dermal dendrocytes in the
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tuberculoid pole of the disease, suggesting that these cells play a determinant role in the clinical course of the disease. The median number of pDCs was significantly higher (p ¼ 0.0481) in leprosy lesions of tuberculoid patients (2.675 cells/ field) compared to those with the lepromatous form (1.471 cells/ field). The presence of CD123-positive pDCs in leprosy was described for the first time by Massone et al. [32] who investigated the distribution of regulatory T cells and pDCs in this disease. Expression of CD123, the marker used for the identification of pDCs, was not detected in any of the biopsies studied, except for two cases of patients with erythema nodosum leprosum in which focal positivity was observed. The authors suggested that pDCs are not involved in the immune response against M. leprae. In another study, Massone et al. [33] immunophenotyped the skin lymphocytic infiltrate of patients co-infected with M. leprae and human immunodeficiency virus (HIV). As in the previous study, CD123positive cells were not detected in the skin samples analyzed. The authors thus extended the results of the previous study, showing that pDCs are not involved in the immunopathogenesis of leprosy, irrespective of the. 4.1. Serological status of the patient The results of the studies of Massone et al. [32,33] not agree with the present findings since CD123-positive cells were detected in both clinical forms of leprosy. The number of these cells was higher in the tuberculoid form, suggesting the participation of pDCs in the cell-mediated immune response against infection with M. leprae. In the present study, nonparametric analysis revealed a significant positive correlation between CD1a and CD207 in tuberculoid (r ¼ 0.7548, p ¼ 0.0007) and lepromatous lesions (r ¼ 0.5585, p ¼ 0.0379) of leprosy patients. This result corroborates the cellular specificity and efficiency of the two antibodies. Similar results have been reported by Seguier et al. [34] who quantified and compared the presence of CD1a- and CD207positive Langerhans cells in human gingival tissue. The authors also observed a correlation between these two markers, with 92% of CD207-positive Langerhans cells coexpressing CD1a, while 82% of CD1a-positive cells coexpressed langerin. The results showed heterogeneity in the phenotype of gingival Langerhans cells and the authors concluded that langerin seems to be the most specific marker for the investigation of these cells. In a study analyzing Langerhans cells and microvessel density in oral squamous cell carcinoma, Santos et al. [35] also found a positive correlation between the two markers. When analyzed separately in the samples studied, the anti-CD1a and anti-CD207 antibodies showed similar efficiency in the identification of Langerhans cells, with the observation of high expression of the two markers in the tuberculoid form. However, comparison of the expression of these markers revealed higher expression of CD207, a finding suggesting phenotypic diversity of these cells. Therefore, when a specific cell type is analyzed, the combination of the two monoclonal antibodies with different binding specificities seems to increase the reliability of the results. Analysis of the CD1a and FXIIIa markers for the identification of Langerhans cells and dermal dendrocytes showed a significant moderate positive correlation (r ¼ 0.5919, p ¼ 0.0157) in patients with the tuberculoid form. The finding of a larger number of these cells in patients with the tuberculoid form may be related to an attempt of the organism to contain M. leprae infection since, in addition to presenting antigens, dendritic cells secrete cytokines such as IL-12 and IL-23 which induce the activation of T cells. The latter, in turn, release proinflammatory cytokines of the Th1 and Th17 types [36e38], promoting a resistance profile that is likely to
have greater control of disease dissemination. The present results showing a larger number of dendritic cells in patients at the resistance pole of the disease indicate an important role of the different dendritic cells studied, favoring an efficient immune response against infection with M. leprae. Disclosure The authors declare no conflict of interest. Acknowledgments This project has been funded in whole or in part with funds from the Conselho Nacional de Pesquisa e CNPq e Brazil, grants 402738/ 2005-5 and 401223/2005-1. References [1] L.R. Freitas, E.C. Duarte, L.P. Garcia, Leprosy in Brazil and its association with characteristics of municipalities: ecological study, 2009e2011, Trop. Med. Int. Health 19 (2014) 1216e1225. [2] W.C. Smith, A. Aerts, Role of contact tracing and prevention strategies in the interruption of leprosy transmission, Lepr. Rev. 85 (2014) 2e17. [3] M.L. Penna, M.A. Grossi, G.O. Penna, Country profile: leprosy in Brazil, Lepr. Rev. 84 (2013) 308e315. [4] S.L. Walker, D.N.J. Lockwood, The clinical and immunological features of leprosy, Br. Med. Bull. 78 (2006) 103e121. [5] N.T. Foss, A.C.F. Motta, Leprosy, a neglected disease that causes a wide variety of clinical conditions in tropical countries, Mem. Inst. Oswaldo Cruz 107 (2012) 28e33. [6] I.M.B. Goulart, G.O. Penna, G. Cunha, Imunopatologia da Hanseníase: a complexidade dos mecanismos da resposta imune do hospedeiro ao Mycobacterium leprae, Rev. Soc. Bras. Med. Trop. 35 (2002) 365e375. [7] K. Suzuki, T. Akama, A. Kawashima, A. Yoshirara, R.R. Yotsu, N. Ishii, Current status of leprosy: epidemiology, basic science and clinical perspectives, J. Dermatol. 39 (2012) 121e129. lez, J.C. Salas-Alanis, J. Ocampo-Candiani, Leprosy. [8] K. Eichelmann, G.E. Gonza an update: definition, pathogenesis, classification, diagnosis, and treatment, Acta Dermosifiliogr. 104 (2013) 554e563. [9] N.T. Foss, A.C. Motta, Leprosy, a neglected disease that causes a wide variety of clinical conditions in tropical countries, Mem. Inst. Oswaldo Cruz 107 (2012) 28e33. [10] L.C. Rodrigues, D.N.J. Lockwood, Leprosy now: epidemiology, progress, challenges, and research gaps, Lancet Infect. Dis. 11 (2011) 464e470. [11] D.J. Cher, T.R. Mosmann, Two types of murine helper T cell clone. II. Delayedtype hypersensitivity is mediated by Th1 clones, J. Immunol. 138 (1987) 3688e3694. [12] P.A. Sieling, R.L. Modlin, Cytokine patterns at the site of mycobacterial infection, Immunobiology 191 (1994) 378e387. [13] M.O. Moraes, C.C. Cardoso, P.R. Vanderborght, A.G. Pacheco, Genetics of host response in leprosy, Lepr. Rev. 77 (2006) 189e202. [14] B.Z. Igyarto, D.H. Kaplan, Antigen presentation by Langerhans cells, Curr. Opin. Immunol. 25 (2013) 115e119. [15] J. Banchereau, F. Briere, C. Caux, J. Davoust, S. Lebecque, K. Palucka, Immunobiology of dendritic cells, Ann. Rev. Immunol. 18 (2000) 767e811. [16] N. Romani, B.E. Clausen, P. Stoitzner, Langerhans cells and more: langerinexpressing dendritic cell subsets in the skin, Immunol. Rev. 34 (2010) 120e141. [17] B.Z. Igyarto, D.H. Kaplan, The evolving function of Langerhans cells in adaptive skin immunity, Immunol. Cell Biol. 88 (2010) 361e365. [18] K. Yoshida, A. Kubo, H. Fujita, M. Yokouchi, K. Ishii, H. Kawasaki, T. Nomura, H. Shimizu, K. Kouyama, T. Ebihara, K. Nagao, M. Amagai, Distinct behavior of human Langerhans cells and inflammatory dendritic epidermal cells at tight junctions in patients with atopic dermatitis, J. Allergy Clin. Immunol. 134 (2014) 856e864. [19] N. Romani, P.M. Brunner, G. Stingl, Changing views of the role of Langerhans cells, J. Investig. Dermatol. 132 (2012) 872e881. [20] E. Hoyo, J. Kanitakis, D. Schmitt, The dermal dendrocyte, Pathol. Biol. 41 (1993) 613e618. [21] H. Sueki, D. Whitaker, M. Buchsbaum, G.F. Murphy, Novel interactions between dermal dendrocytes and mast cells in human skin. Implications for hemostasis and matrix repair, Lab. Investig. 69 (1993) 160e172. [22] M. Cella, D. Jarrossay, F. Facchetti, O. Alebardi, H. Nakajima, A. Lanzavecchia, M. Colona, Plasmacytoid monocytes migrate to inflamed lymph nodes and produce large amounts of type I interferon, Nat. Med. 5 (1999) 919e923. [23] M. Collin, N. Mcgovern, M. Haniffa, Human dendritic cell subsets, Immunology 140 (2013) 22e30. [24] M. Merad, P. Sathe, J. Helft, J. Miller, A. Mortha, The dendritic cell lineage: ontogeny and function of dendritic cells and their subsets in the steady state
K.E. Hirai et al. / Microbial Pathogenesis 91 (2016) 18e25 and the inflamed setting, Annu. Rev. Immunol. 31 (2013) 563e604. [25] J.M. McNiff, D.H. Kaplan, Plasmacytoid dendritic cells are present in cutaneous dermatomyositis lesions in a pattern distinct from lupus erythematosus, J. Cutan. Pathol. 35 (2008) 452e456. [26] D.S. Ridley, M.J. Jopling, Classification of leprosy according to immunity. A five group system, Int. J. Lepr. Other Mycobact. Dis. 34 (1996) 255e273. [27] J.A.S. Quaresma, V.L.R. Barros, C. Pagliari, E.R. Fernandes, F. Guedes, C.F. Takakura, H.F. Andrade Jr., P.F.C. Vasconcelos, M.I.S. Duarte, Revisiting the liver in human yellow fever: virus-induced apoptosis in hepatocytes associated with TGF-b, TNF-a and NK cells activity, Virology 345 (2006) 22e30. [28] J.A.S. Quaresma, M.F.A. Oliveira, A.C.R. Guimar~ aes, E.B. Brito, C. Pagliari, A.C. de Brito, M.B. Xavier, M.I.S. Duarte, CD1a and FXIIIa immunohistochemistry in leprosy: a possible role of dendritic cells in the pathogenesis of Mycobacterium leprae infection, Am. J. Dermatophatol. 31 (2009) 527e531. [29] P.A. Sieling, D. Jullien, M. Dahlem, T.F. Tedder, T.H. Rea, R.L. Modlin, S.A. Porcelli, CD1 expression by dendritic cells in human leprosy lesions: correlation with effective host immunity, J. Immunol. 162 (1999) 1851e1858. , H. Ferreira, [30] A. Miranda, T.P. Amadeu, G. Schueler, F.B. Alvarenga, N. Duppre J.A. Nery, E.N. Sarno, Increased Langerhans cell accumulation after mycobacterial stimuli, Histopathology 51 (2007) 649e656. [31] R.E. Hunger, P.A. Sieling, M.T. Ochoa, M. Sugaya, A.E. Burdick, T.H. Rea, P.J. Brennan, J.T. Belisle, A. Blauvelt, S.A. Porcelli, R.L. Modlin, Langerhans cells utilize CD1a and langerin to efficiently present nonpeptide antigens to T cells, J. Clin. Investig. 113 (2004) 701e708. [32] C. Massone, E. Nunzi, R. Ribeiro-Rodrigues, S. Talhari, C. Talhari, A.P. Schettini,
[33]
[34]
[35]
[36]
[37]
[38]
25
J.N. Parente, A.M. Brunasso, M. Puntoni, A. Clapasson, S. Noto, L. Cerroni, T regulatory cells and plasmocytoid dentritic cells in hansen disease: a new insight into pathogenesis? Am. J. Dermatopathol. 32 (2010) 251e256. C. Massone, C. Talhari, S. Talhari, A.M. Brunasso, T.M. Campbell, P. Curcic, L. Cerroni, R. Ribeiro-Rodrigues, Immunophenotype of skin lymphocytic infiltrate in patients co-infected with Mycobacterium leprae and human immunodeficiency virus: a scenario dependent on CD8þ and/or CD20þ cells, Br. J. Dermatol. 165 (2011) 321e328. S. Seguier, A. Bodineau, G. Godeau, B. Pellat, N. Brousse, Langerinþ versus CD1aþ Langerhans cells in human gingival tissue: a comparative and quantitative immunohistochemical study, Arch. Oral Biol. 48 (2003) 255e262. L.C.S. Santos, I.L.O. Nascimento, J.N. Santos, Immunohistochemistry assessment of Langerhans cells and microvascular density in oral squamous cell carcinomas, Full Dent. Sci. 5 (2013) 103e109. F.A. Verreck, T. Boer, D.M. Langenberg, M.A. Hoeve, M. Kramer, E. Vaisberg, R. Kastelein, A. Kolk, A. Waal-Malefyt, T.H. Ottenhoff, Human IL-23-producing type 1 macrophages promote but IL-10 producing type 2 macrophages subvert immunity to (myco)bacteria, Proc. Natl. Acad. Sci. 101 (2004) 4560e4565. S.R. Krutzik, B. Tan, H. Li, M.T. Ochoa, P.T. Liu, S.E. Sharfstein, T.G. Graeber, P.A. Sieling, Y.J. Liu, T.H. Rea, B.R. Bloom, R.L. Modlin, TLR activation triggers the rapid differentiation of monocytes into macrophages and dendritic cells, Nat. Med. 11 (2005) 653e660. F.O. Nestle, P. Meglio, J.Z. Qin, B.J. Nickoloff, Skin immune sentinels in health and disease, Nat. Rev. Immunol. 9 (2009) 679e691.