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Apoptotic activity and Treg cells in tissue lesions of patients with leprosy
Microbial Pathogenesis 76 (2014) 84e88
Contents lists available at ScienceDirect
Microbial Pathogenesis journal homepage: www.elsevier.com/locate/micpath
Apoptotic activity and Treg cells in tissue lesions of patients with leprosy ~ es Quaresma a, b, *, Paulo Cardoso Esteves b, Juarez Antonio Simo ~o b, Jorge Rodrigues de Sousa a, Denise da Silva Pinto a, Tinara Leila de Sousa Aara a Hellen Thais Fuzii a b
Nucleo de Medicina Tropical, Universidade Federal do Para, Belem-Pa, Brazil Centro de Ci^ encias Biologicas e da Saude, Universidade do Estado do Para, Belem-Pa, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history: Received 21 April 2014 Received in revised form 21 June 2014 Accepted 21 July 2014 Available online 12 August 2014
In order to understand the apoptotic response and the participation of Treg cells in the spectral clinical evolution of leprosy, this study evaluated the immunohistochemical expression of caspase-3 and FoxP3 in skin lesions of leprosy patients with the polar forms of the disease. Forty-nine patients with a confirmed diagnosis of the disease were selected, including 27 with the TT form and 22 with the LL form. Quantitative analysis of caspase-3 immunostaining showed a higher expression of this mediator in the LL form (3.409 ± 0.6517 cells/mm2; p ¼ 0.0001). Immunostaining for the transcription factor FoxP3 was higher in the LL form (3.891 ± 0.9294 cells/mm2; p ¼ 0.0001). A moderate correlation between the two markers was observed in the TT form (r ¼ 0.5214; p ¼ 0.005). It can be concluded that Treg cells and apoptosis play an effective role for the host defense response, inducing mechanisms involved in the activation of cascades that interfere with the control of the immune response and cell homeostasis. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Mycobacterium leprae Apoptosis FoxP3 Caspase 3
1. Introduction Leprosy is a chronic infectious-contagious disease caused by the Gram-negative bacillus Mycobacterium leprae, which exhibits a specific tropism for peripheral nerves and skin. The disease is extremely complex, a fact raising questions about the mechanisms involved in the spectral clinical evolution of the disease [1,2]. Leprosy is characterized by a broad spectrum of clinical forms that are associated with alterations in the immune system of the host. According to histopathological criteria, studies suggest that the forms can vary within the evolutionary spectrum of the disease. Leprosy can be divided into five clinical forms according to the classification of Ridley and Jopling: tuberculoid (TT), dimorphictuberculoid (DT), dimorphicedimorphic (DD), dimorphic lepromatous (DL), and lepromatous (LL) leprosy [3e5]. The tuberculoid form of the disease is characterized by an intense immune response mediated by Th1 inflammatory cytokines, which are important for the control of bacterial proliferation.
* Corresponding author. Nucleo de Medicina Tropical-UFPA, Av. Generalissimo Deodoro 92, Umarizal, Belem-Pa 66055-240, Brazil. E-mail addresses: [email protected], [email protected] (J.A.S. Quaresma). http://dx.doi.org/10.1016/j.micpath.2014.07.005 0882-4010/© 2014 Elsevier Ltd. All rights reserved.
In the lepromatous form, the immune response is mediated by Th2 cytokines which suppress cell-mediated immunity [6,7]. Since leprosy is a spectral disease, the host activates a series of responses against the bacillus during progression of the illness [8]. Among the mechanisms involved in the control of the immune response, increasing evidence suggests that programmed cell death (apoptosis) plays an important role in some infectious diseases by regulating the immune response and by directly affecting bacterial proliferation [8,9]. Within this context, several studies have demonstrated the development of the apoptotic process from the participation of certain mediators which interfere with the initiation of the cascade of events that contribute to cell growth and death [10,11]. In a study involving dose-dependent monocyte cultures, expression of TNF-a and BAX from spontaneous cell death was higher in the lepromatous form of the disease [12]. Another study evaluating the phenomenon of apoptosis based on macrophage activity in the polar forms of the disease demonstrated that an increase in the production of enzymatic content by macrophages which degrades the bacterial wall induced an increase of caspase-3 in the tuberculoid form of the disease [13]. In another study investigating the anti-apoptotic effect of M. leprae in Schwann cells, the mycobacterium was found to induce the production of certain
J.A.S. Quaresma et al. / Microbial Pathogenesis 76 (2014) 84e88
growth factors that interfere with the reprogramming process and with the gene expression of immunoregulatory factors such as NFkb, favoring maintenance of the bacillus and persistence of infection [14,15]. Regarding the participation of Treg cells, studies have shown that these cells can induce the immunosuppressive activity of effector cells against intracellular pathogens [16]. In diseases such as leishmaniasis, quantitative analysis of the expression of the transcription factor that regulates Treg cell differentiation (FoxP3) in clinical forms of the disease revealed a higher expression of this marker in acute cutaneous leishmaniasis when compared to chronic cutaneous leishmaniasis [17]. In this respect, Treg cells were found to suppress the response mediated by IFN-g by the production of IL-10 during the course of the disease. In leprosy, analysis of the polar forms of the disease showed an increase in the number of circulating Treg cells in the inflammatory infiltrate of patients with the lepromatous form [18]. In another study evaluating the participation of these cells, it was demonstrated that the suppressive response triggered the release of cytokines that are important for the regulation of the inflammatory response during the tissue repair process. Expression of IL-10 and TGF-b was found to be higher in the lepromatous form of the disease. Studies indicate that the association between Treg cells and apoptosis is important for the induction of mechanisms that control the physiopathological response in different diseases, since both factors interfere with the control of the immune response and of mechanisms that regulate cell differentiation and cell homeostasis [20,21]. In leprosy, few studies have correlated the regulatory factor of Treg cells with mechanisms that induce the activation of apoptosis during progression of the disease. Since this analysis is important for the understanding of the immune response in the presence of activation of mediators that interfere with the survival of the bacillus, the objective of the present study was to investigate the immunohistochemical expression of caspase-3 and FoxP3 in skin lesions of patients with the polar forms of leprosy.
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caspase 3 (ABCAN) and anti-FoxP3 (ABCAN), diluted in 1% bovine serum albumin in PBS. After incubation with the primary antibody, the slides were kept in a dark chamber for 14 h at 4 C. After this period, the sections were immersed in PBS and incubated with the biotinylated secondary antibody (LSAB þ System HRP, DakoCytomation) for 30 min at 37 C in an oven. After fixation, the sections were again immersed in PBS and incubated with the streptavidin-peroxidase complex (LSAB þ System HRP, DakoCytomation) for 30 min at 37 C. The reaction was developed with a chromogen solution consisting of 0.03% diaminobenzidine and 3% hydrogen peroxide. Finally, the sections were stained with Harris hematoxylin for 3 min, hydrated in ethanol, cleared in xylene, and mounted in Entelan. 2.3. Quantitative analysis of immunostained cells For quantitative analysis, the intensity of immunostaining in the tissue was evaluated. Dermal cells exhibiting unequivocal brown staining on a green background characteristic of hematoxylin staining were defined as positive. Immunostained cells were counted using a 10 10 grid (comprising a total area of 0.0625 mm2 of the slide). Five fields were selected randomly in each area of the lesion [17e19]. 2.4. Statistical analysis The results were entered into spreadsheets of the Excel 2007 program. The GraphPad Prism 5.0 program was used for statistical analysis. For univariate analysis, frequencies and measures of central tendency and dispersion were calculated. The association between the cytokines studied and the clinical forms of the disease was evaluated by the ManneWhitney test and Pearson's correlation test. A level of significance of 5% (p 0.05) was adopted for all tests. 2.5. Ethical aspects
2. Materials and methods 2.1. Characterization of the sample Forty-nine patients seen at the Dermatology Outpatient Clinic of the Nucleus of Tropical Medicine, Federal University of Par a (Núcleo e UFPA) and at de Medicina Tropical, Universidade Federal do Para the Marco Health Center School of the State University of Para (Centro de Saúde da Escola do Marco, Universidade do Estado do e UEPA) were diagnosed and classified according to the Para criteria proposed by Ridley and Jopling. Twenty-seven of the patients had tuberculoid leprosy (TT) and 22 had lepromatous leprosy (LL). Paraffin-embedded biopsies were cut into histological sections with a microtome and stained with hematoxylin-eosin for histopathological analysis or submitted to immunohistochemistry. 2.2. Immunohistochemistry The streptavidin-biotin-peroxidase method was used for tissue immunostaining [17]. First, the histological sections were deparaffinized in xylene and dehydrated in ethanol. Endogenous peroxidase activity was blocked by incubating the sections with 3% hydrogen peroxide for 45 min in a dark chamber. Antigen retrieval was performed using 10 Target Retrieval Solution (Dako, code S1699). The slides were incubated in a steamer (Steam Cuisine 700 Hi Speed, T-FAL) for 20 min at 90 C. Next, the slides were immersed in a solution containing 10% skim milk for 30 min to inhibit nonspecific protein binding. The histological sections were then incubated the following primary monoclonal antibodies: anti-
The project was approved by the Ethics and Research boards of the Tropical Medicine Unit of Federal do Para University. 3. Results A significant difference in caspase-3 immunostaining was observed between the polar forms of leprosy. The mean number of immunostained cells was 1.289 ± 0.3523 cells/mm2 in the tuberculoid form and 3.409 ± 0.6517 cells/mm2 in the lepromatous form (p ¼ 0.0001) (Fig. 1A). With respect to the FoxP3 transcription factor of Treg cells, a significant difference in immunostaining was observed between the groups studied. The mean number of immunostained cells was 2.022 ± 0.5416 cells/mm2 in the tuberculoid form and 3.891 ± 0.9294 cells/mm2 in the lepromatous form (p ¼ 0.0001) (Fig. 1B). Pearson's correlation coefficient revealed a moderate positive correlation between the two markers in the tuberculoid form (r ¼ 0.5214; p ¼ 0.005) (Fig. 2A). In the lepromatous form, a weak positive, but nonsignificant, correlation was observed between caspase-3 and FoxP3 (r ¼ 0.1307; p ¼ 0.5622) (Fig. 2B). 4. Discussion In the present study, analysis of caspase-3 immunostaining in the polar forms of leprosy revealed a quantitative increase of the enzyme in the lepromatous form. This finding can be explained by observations showing the participation of mediators that interfere with the development of the apoptotic cascade during the
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Fig. 1. Quantitative analysis of caspase-3 (A) and FoxP3 (B) immunoexpression in the polar forms of leprosy. Observe a predominance of Treg cells and the phenomenon of apoptosis in the lepromatous form of the disease.
suppressive response to the bacillus. In a study investigating the expression of Fas ligand (FasL), it was observed that in patients with multibacillary leprosy the expression of this protein by macrophages induced lymphocyte apoptosis during the cell-mediated response triggered by the bacillus [22,23]. Studies evaluating the role of proteins that are part of the extrinsic apoptotic pathway and that exert an apoptotic effect, such as Bcl-2 associated with Bax, showed that the expression of these proteins by the monocytic cell line was more intense in lesions of patients with the lepromatous form of the disease [24]. In another study evaluating the rate of apoptosis in skin lesions of leprosy patients based on the cytokine TGF-b, it was observed that in the lepromatous form the cytokine acts synergistically with caspase-3 in the regulation of the apoptotic response. During the physiopathological response, the cytokine would increase the activation of kinases involved in the cleavage of caspase-3 and, consequently, in the expression of signals inducing the activation of death domains during the chronic and infectious processes of the disease [25] (see Fig. 3). In agreement with other studies evaluating the role of apoptosis in leprosy by determining the expression of Toll-like receptors in Schwann cells, it was observed that mycobacterial components in the membrane of the bacillus are able to trigger cellular apoptosis in tissue lesions of the lepromatous form by inducing the release of Fas and effector caspases [11,26]. A study investigating the role of IFN-g and iNOS associated with the expression of caspases-1, -3
and -8 showed that, in the development of the mechanisms resulting from the cell-mediated immune response, both IFN-g and iNOS stimulated the activation of caspase-3 in the lepromatous form of the disease [27]. With respect to the role of Treg cells, the present results showed a quantitative increase in the transcription factor FoxP3 in the lepromatous form of leprosy. This finding can be explained by the immune response profile triggered by these cells during suppressive evolution in infectious manifestations of the disease. Studies evaluating mechanisms that regulate the participation of immune response inducers demonstrated that Treg cells stimulate the production of surface markers which influence the regulation of immunosuppressive activity in inflammatory processes. Within this context, it was demonstrated that CD8 þ Treg cells characterized by expressing CD69, CD25, CTLA4 and Foxp3 can induce T cells to produce cytokines such as IL-4, IL-5, IL-13 and TGF-b which influence the development of the adaptive response in the disease, suppressing the Th1 response. In granulomatous lesions found in lymph nodes infected with the mycobacterium, the presence of cells expressing CD8 þ LAG-3 þ CCL4 has been shown to suppress the immune response, creating a cellular environment that favors the maintenance and persistence of infection [28,29]. In a study investigating the role of CD4 þ CD25 þ FoxP3 þ cells, these cells reduced the production of mediators such as TNF-a and IFN-g, which stimulate the release of reactive oxygen and nitrogen
Fig. 2. Linear correlation between the immunoexpression of caspase-3 and FoxP3 in the polar forms of leprosy. There is a tendency of positive correlation between Treg cells and the phenomenon of apoptosis.
J.A.S. Quaresma et al. / Microbial Pathogenesis 76 (2014) 84e88
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Fig. 3. Immunohistochemistry for caspase 3 in tuberculoid (A) and lepromatous (B) forms of leprosy. Immunohistochemistry for regulatory T cells in tuberculoid (C) and lepromatous (D) forms of leprosy. The pattern of positivity is characterized by deposition of brown pigment in the tissue. Magnification 200. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
species in the inflammatory infiltrate of patients with the lepromatous form of the disease. Another study evaluating the role of costimulatory molecules showed that the bacillus reduced the expression of CD28 on the surface of monocytes and lymphocytes in the lepromatous form of the disease. In addition, the mycobacterium interfered with the expression of CD26 and CD86, favoring persistence of the bacillus during the course of the disease [29,30]. Regarding the correlation between apoptotic activity and Treg cells, a moderate synergistic effect between caspase-3 and FoxP3 was observed in the tuberculoid form of the disease. To our knowledge, there are no studies investigating the association between these markers in leprosy. Literature data indicate that Treg cells induce mechanisms that regulate the production of mediators participating in the apoptotic cascade. A study investigating the role of these markers in the different clinical forms of American tegumentary leishmaniasis has shown a positive correlation between caspase-3 and FoxP3 in cutaneous diffuse leishmaniasis (LCD) and cutaneous localized leishmaniasis (LCL) [31]. In that study, a positive association was also observed between caspase-3 and FasL in LCD and LCL. That study lends support to the present findings since leishmaniasis shows a similar clinical aspect during the course of the disease. Therefore, the present results suggest that the combined participation of Treg cells and apoptosis indicates the possibility that these two phenomena interfere with the evolution of the disease, since it creates an immunosuppressive environment against the bacillus, permitting progression to the lepromatous form of the disease. However, further studies are needed to complement the present results. References [1] Santos DS, Duppre NC, Sales AM, Nery JA, Sarno EN, Hacker MA. Kinship and leprosy in the contacts of leprosy patients: cohort at the Souza Araújo outpatient clinic, Rio de Janeiro, RJ, 1987e2010. J Trop Med 2013;2013: 596316. [2] WHO (World Health Organization) Global leprosy situation. Weekly epidemiological record, vol. 86; 2001. p. 389e400. [3] Ishii N. Recent advances in the treatment of leprosy. Dermatol Online J Davis 2003;9:1e18.
[4] Ridley DS, Jopling WH. Classification of leprosy according to immunity: a fivegroup system. Int J Lepr Other Mycobact Dis 1966;34:255e73. [5] Mendonca VA, Costa RD, Melo GEBA, Antunes CM, Teixeira LT. Imunologia da hanseníase. Bras Dermatol 2008;83:343e50. [6] Teles RM, Graeber TG, Krutzik SR, Montoya D, Schenk M, Lee DJ, et al. Type I interferon suppresses type II interferon-triggered human anti-mycobacterial responses. Science 2013;6126:1448e53. [7] Naves Mde M, Ribeiro FA, Patrocinio LG, Patrocinio JA, Fleury RN, Goulart IM. Patients. Lepr Rev 2013;84:85e91. [8] Alcais A, Mira M, Casanova JL, Schurr E, Abel L. Genetic dissection of immunity in leprosy. Curr Opin Immunol 2005;17:44e8. [9] Cardoso CC, Pereira AC, de Sales Marques C, Moraes MO. Leprosy susceptibility: genetic variations regulate innate and adaptive immunity, and disease outcome. Future Microbiol 2011;6:533e49. [10] Fukutomi Y, Maeda Y, Makino M. Apoptosis-inducing activity of clofazimine in macrophages. Antimicrob Agents Chemother 2011;55:4000e5. [11] Mattos KA, Oliveira VG, D'Avila H, Rodrigues LS, Pinheiro RO, Sarno EN, et al. TLR6-driven lipid droplets in Mycobacterium leprae-infected Schwann cells: immunoinflammatory platforms associated with bacterial persistence. J Immunol 2011;187:2548e58. [12] Hernandez MO, Neves I, Sales JS, Carvalho DS, Sarno EN, Sampaio EP. Induction of apoptosis in monocytes by Mycobacterium leprae in vitro: a possible role for tumor necrosis factor-alpha. Immunology 2003;109:156e64. [13] Quaresma JAS, Lima LWO, Fuzii HT, Libonati RMF, Pagliari C, Duarte MIS. Immunohistochemical evaluation of macrophage activity and its relationship with apoptotic cell death in the polar forms of leprosy. Microb Pathog 2010;49:135e40. [14] Burstein E, Duckett CS. Dying for NF-kB? Control of cell death by transcriptional regulation of the apoptotic machinery. Curr Opin Cell Biol 2003;15: 732e7. [15] Pereira RM, Calegari-Silva TC, Hernandez MO, Saliba AM, Redner P, Pessolani MC, et al. Mycobacterium leprae induces NF-kappaB-dependent transcription repression in human Schwann cells. Biochem Biophys Res Commun 2005;335:20e6. [16] Shevach EM. Mechanisms of foxp3þ T regulatory cell-mediated suppression. Immunity 2009;30:636e45. [17] Bourreau E, Ronet C, Darcissac E, Lise MC, Sainte Marie D, Clity E, et al. Intralesional regulatory T-cell suppressive function during human acute and chronic cutaneous leishmaniasis due to Leishmania guyanensis. Infect Immun 2009;77:1465e74. [18] Massone C, Nunzi E, Ribeiro-Rodrigues R, Talhari C, Talhari S, Schettini AP, et al. Regulatory cells and plasmocytoid dentritic cells in hansen disease: a new insight into pathogenesis? Am J Dermatopathol 2010;32:251e6. [19] Palermo ML, Pagliari C, Trindade MA, Yamashitafuji TM, Duarte AJ, Cacere CR, et al. Increased expression of regulatory T cells and downregulatory molecules in lepromatous leprosy. Am J Trop Med Hyg 2012;86:878e83. [20] Singh A, Mohan A, Dey AB, Mitra DK. Inhibiting the programmed death 1 pathway rescues Mycobacterium tuberculosis-specific interferon g-producing
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J.A.S. Quaresma et al. / Microbial Pathogenesis 76 (2014) 84e88 T cells from apoptosis in patients with pulmonary tuberculosis. J Infect Dis 2013;208:603e15. Singh A, Dey AB, Sharma PK, Mohan A, Mitra DK. Foxp3þ regulatory T cells among tuberculosis patients: impact on prognosis and restoration of antigen specific IFN-g producing T cells. PLoS ONE 2012;9:44728. Hsu SM, Raine L, Fanger H. Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures. J Histochem Cytochem 1981;29: 577e80. Mustafa T, Bjune TG, Jonsson R, Pando RH, Nilsen R. Increased expression of fas ligand in human tuberculosis and leprosy lesions: a potential novel mechanism of immune evasion in mycobacterial infection. Scand J Immunol 2001;54:630e9. Brito de Souza VN, Nogueira ME, Belone AF, Soares CT. Analysis of apoptosis and Bcl-2 expression in polar forms of leprosy. FEMS Immunol Med Microbiol 2010;60:270e4. Quaresma JAS, Almeida FA, Souza ATL, Miranda ASLP, Nunes MIM, Fuzii HT, et al. Transforming growth factor b and apoptosis in leprosy skin lesions: possible relationship with the control of the tissue immune response in the Mycobacterium leprae infection. Microbes Infect 2012;14: 696e701.
[26] Oliveira RB, Ochoa MT, Sieling PA, Rea TH, Rambukkana A, Sarno EN, et al. Expression of toll-like receptor 2 on human Schwann cells: a mechanism of nerve damage in leprosy. Infect Immun 2003;71:1427e33. [27] Choi YJ, Kim MH, Choi HY, Myung KB. Apoptosis and the expression of caspase-1, 3, 8, IFN-gamma, and iNOS mRNA in leprosy lesions. Korean Lepr Bull 2003;36:3e22. [28] Simone AJ, Krista EVM, Savage NDL, Tjitske B, Frederic T, Annemieke VDW, et al. Identification of a human CD8þ regulatory T cell subset that mediates suppression through the chemokine CC chemokine ligand 4. Proc Natl Acad Sci U S A 2007;104:8029e34. [29] Suzuki M, Jagger AL, Konya C, Shimojima Y, Pryshchep S, Goronzy JJ. Weyand CMCD8þCD45RAþCCR7þFOXP3þ T cells with immunosuppressive properties: a novel subset of inducible human regulatory T cells. J Immunol 2012;189:2118e30. [30] Palermo ML, Trindade MAB, Duarte AJS, Cacere CR, Benard G. Differential expression of the costimulatory molecules CD86, CD28, CD152 and PD-1 correlates with the host-parasite outcome in leprosy. Mem Inst Oswaldo Cruz 2012;107:167e73. ~es AV, Jesus AACM, Bocca AL, Muniz JMI, Ribeiro SRN. [31] Carneiro FP, Magalha Foxp3 expression in lesions of the different clinical forms of American tegumentary leishmaniasis. Parasite Immunol 2009;31:646e51.
Contents lists available at ScienceDirect
Microbial Pathogenesis journal homepage: www.elsevier.com/locate/micpath
Apoptotic activity and Treg cells in tissue lesions of patients with leprosy ~ es Quaresma a, b, *, Paulo Cardoso Esteves b, Juarez Antonio Simo ~o b, Jorge Rodrigues de Sousa a, Denise da Silva Pinto a, Tinara Leila de Sousa Aara a Hellen Thais Fuzii a b
Nucleo de Medicina Tropical, Universidade Federal do Para, Belem-Pa, Brazil Centro de Ci^ encias Biologicas e da Saude, Universidade do Estado do Para, Belem-Pa, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history: Received 21 April 2014 Received in revised form 21 June 2014 Accepted 21 July 2014 Available online 12 August 2014
In order to understand the apoptotic response and the participation of Treg cells in the spectral clinical evolution of leprosy, this study evaluated the immunohistochemical expression of caspase-3 and FoxP3 in skin lesions of leprosy patients with the polar forms of the disease. Forty-nine patients with a confirmed diagnosis of the disease were selected, including 27 with the TT form and 22 with the LL form. Quantitative analysis of caspase-3 immunostaining showed a higher expression of this mediator in the LL form (3.409 ± 0.6517 cells/mm2; p ¼ 0.0001). Immunostaining for the transcription factor FoxP3 was higher in the LL form (3.891 ± 0.9294 cells/mm2; p ¼ 0.0001). A moderate correlation between the two markers was observed in the TT form (r ¼ 0.5214; p ¼ 0.005). It can be concluded that Treg cells and apoptosis play an effective role for the host defense response, inducing mechanisms involved in the activation of cascades that interfere with the control of the immune response and cell homeostasis. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Mycobacterium leprae Apoptosis FoxP3 Caspase 3
1. Introduction Leprosy is a chronic infectious-contagious disease caused by the Gram-negative bacillus Mycobacterium leprae, which exhibits a specific tropism for peripheral nerves and skin. The disease is extremely complex, a fact raising questions about the mechanisms involved in the spectral clinical evolution of the disease [1,2]. Leprosy is characterized by a broad spectrum of clinical forms that are associated with alterations in the immune system of the host. According to histopathological criteria, studies suggest that the forms can vary within the evolutionary spectrum of the disease. Leprosy can be divided into five clinical forms according to the classification of Ridley and Jopling: tuberculoid (TT), dimorphictuberculoid (DT), dimorphicedimorphic (DD), dimorphic lepromatous (DL), and lepromatous (LL) leprosy [3e5]. The tuberculoid form of the disease is characterized by an intense immune response mediated by Th1 inflammatory cytokines, which are important for the control of bacterial proliferation.
* Corresponding author. Nucleo de Medicina Tropical-UFPA, Av. Generalissimo Deodoro 92, Umarizal, Belem-Pa 66055-240, Brazil. E-mail addresses: [email protected], [email protected] (J.A.S. Quaresma). http://dx.doi.org/10.1016/j.micpath.2014.07.005 0882-4010/© 2014 Elsevier Ltd. All rights reserved.
In the lepromatous form, the immune response is mediated by Th2 cytokines which suppress cell-mediated immunity [6,7]. Since leprosy is a spectral disease, the host activates a series of responses against the bacillus during progression of the illness [8]. Among the mechanisms involved in the control of the immune response, increasing evidence suggests that programmed cell death (apoptosis) plays an important role in some infectious diseases by regulating the immune response and by directly affecting bacterial proliferation [8,9]. Within this context, several studies have demonstrated the development of the apoptotic process from the participation of certain mediators which interfere with the initiation of the cascade of events that contribute to cell growth and death [10,11]. In a study involving dose-dependent monocyte cultures, expression of TNF-a and BAX from spontaneous cell death was higher in the lepromatous form of the disease [12]. Another study evaluating the phenomenon of apoptosis based on macrophage activity in the polar forms of the disease demonstrated that an increase in the production of enzymatic content by macrophages which degrades the bacterial wall induced an increase of caspase-3 in the tuberculoid form of the disease [13]. In another study investigating the anti-apoptotic effect of M. leprae in Schwann cells, the mycobacterium was found to induce the production of certain
J.A.S. Quaresma et al. / Microbial Pathogenesis 76 (2014) 84e88
growth factors that interfere with the reprogramming process and with the gene expression of immunoregulatory factors such as NFkb, favoring maintenance of the bacillus and persistence of infection [14,15]. Regarding the participation of Treg cells, studies have shown that these cells can induce the immunosuppressive activity of effector cells against intracellular pathogens [16]. In diseases such as leishmaniasis, quantitative analysis of the expression of the transcription factor that regulates Treg cell differentiation (FoxP3) in clinical forms of the disease revealed a higher expression of this marker in acute cutaneous leishmaniasis when compared to chronic cutaneous leishmaniasis [17]. In this respect, Treg cells were found to suppress the response mediated by IFN-g by the production of IL-10 during the course of the disease. In leprosy, analysis of the polar forms of the disease showed an increase in the number of circulating Treg cells in the inflammatory infiltrate of patients with the lepromatous form [18]. In another study evaluating the participation of these cells, it was demonstrated that the suppressive response triggered the release of cytokines that are important for the regulation of the inflammatory response during the tissue repair process. Expression of IL-10 and TGF-b was found to be higher in the lepromatous form of the disease. Studies indicate that the association between Treg cells and apoptosis is important for the induction of mechanisms that control the physiopathological response in different diseases, since both factors interfere with the control of the immune response and of mechanisms that regulate cell differentiation and cell homeostasis [20,21]. In leprosy, few studies have correlated the regulatory factor of Treg cells with mechanisms that induce the activation of apoptosis during progression of the disease. Since this analysis is important for the understanding of the immune response in the presence of activation of mediators that interfere with the survival of the bacillus, the objective of the present study was to investigate the immunohistochemical expression of caspase-3 and FoxP3 in skin lesions of patients with the polar forms of leprosy.
85
caspase 3 (ABCAN) and anti-FoxP3 (ABCAN), diluted in 1% bovine serum albumin in PBS. After incubation with the primary antibody, the slides were kept in a dark chamber for 14 h at 4 C. After this period, the sections were immersed in PBS and incubated with the biotinylated secondary antibody (LSAB þ System HRP, DakoCytomation) for 30 min at 37 C in an oven. After fixation, the sections were again immersed in PBS and incubated with the streptavidin-peroxidase complex (LSAB þ System HRP, DakoCytomation) for 30 min at 37 C. The reaction was developed with a chromogen solution consisting of 0.03% diaminobenzidine and 3% hydrogen peroxide. Finally, the sections were stained with Harris hematoxylin for 3 min, hydrated in ethanol, cleared in xylene, and mounted in Entelan. 2.3. Quantitative analysis of immunostained cells For quantitative analysis, the intensity of immunostaining in the tissue was evaluated. Dermal cells exhibiting unequivocal brown staining on a green background characteristic of hematoxylin staining were defined as positive. Immunostained cells were counted using a 10 10 grid (comprising a total area of 0.0625 mm2 of the slide). Five fields were selected randomly in each area of the lesion [17e19]. 2.4. Statistical analysis The results were entered into spreadsheets of the Excel 2007 program. The GraphPad Prism 5.0 program was used for statistical analysis. For univariate analysis, frequencies and measures of central tendency and dispersion were calculated. The association between the cytokines studied and the clinical forms of the disease was evaluated by the ManneWhitney test and Pearson's correlation test. A level of significance of 5% (p 0.05) was adopted for all tests. 2.5. Ethical aspects
2. Materials and methods 2.1. Characterization of the sample Forty-nine patients seen at the Dermatology Outpatient Clinic of the Nucleus of Tropical Medicine, Federal University of Par a (Núcleo e UFPA) and at de Medicina Tropical, Universidade Federal do Para the Marco Health Center School of the State University of Para (Centro de Saúde da Escola do Marco, Universidade do Estado do e UEPA) were diagnosed and classified according to the Para criteria proposed by Ridley and Jopling. Twenty-seven of the patients had tuberculoid leprosy (TT) and 22 had lepromatous leprosy (LL). Paraffin-embedded biopsies were cut into histological sections with a microtome and stained with hematoxylin-eosin for histopathological analysis or submitted to immunohistochemistry. 2.2. Immunohistochemistry The streptavidin-biotin-peroxidase method was used for tissue immunostaining [17]. First, the histological sections were deparaffinized in xylene and dehydrated in ethanol. Endogenous peroxidase activity was blocked by incubating the sections with 3% hydrogen peroxide for 45 min in a dark chamber. Antigen retrieval was performed using 10 Target Retrieval Solution (Dako, code S1699). The slides were incubated in a steamer (Steam Cuisine 700 Hi Speed, T-FAL) for 20 min at 90 C. Next, the slides were immersed in a solution containing 10% skim milk for 30 min to inhibit nonspecific protein binding. The histological sections were then incubated the following primary monoclonal antibodies: anti-
The project was approved by the Ethics and Research boards of the Tropical Medicine Unit of Federal do Para University. 3. Results A significant difference in caspase-3 immunostaining was observed between the polar forms of leprosy. The mean number of immunostained cells was 1.289 ± 0.3523 cells/mm2 in the tuberculoid form and 3.409 ± 0.6517 cells/mm2 in the lepromatous form (p ¼ 0.0001) (Fig. 1A). With respect to the FoxP3 transcription factor of Treg cells, a significant difference in immunostaining was observed between the groups studied. The mean number of immunostained cells was 2.022 ± 0.5416 cells/mm2 in the tuberculoid form and 3.891 ± 0.9294 cells/mm2 in the lepromatous form (p ¼ 0.0001) (Fig. 1B). Pearson's correlation coefficient revealed a moderate positive correlation between the two markers in the tuberculoid form (r ¼ 0.5214; p ¼ 0.005) (Fig. 2A). In the lepromatous form, a weak positive, but nonsignificant, correlation was observed between caspase-3 and FoxP3 (r ¼ 0.1307; p ¼ 0.5622) (Fig. 2B). 4. Discussion In the present study, analysis of caspase-3 immunostaining in the polar forms of leprosy revealed a quantitative increase of the enzyme in the lepromatous form. This finding can be explained by observations showing the participation of mediators that interfere with the development of the apoptotic cascade during the
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Fig. 1. Quantitative analysis of caspase-3 (A) and FoxP3 (B) immunoexpression in the polar forms of leprosy. Observe a predominance of Treg cells and the phenomenon of apoptosis in the lepromatous form of the disease.
suppressive response to the bacillus. In a study investigating the expression of Fas ligand (FasL), it was observed that in patients with multibacillary leprosy the expression of this protein by macrophages induced lymphocyte apoptosis during the cell-mediated response triggered by the bacillus [22,23]. Studies evaluating the role of proteins that are part of the extrinsic apoptotic pathway and that exert an apoptotic effect, such as Bcl-2 associated with Bax, showed that the expression of these proteins by the monocytic cell line was more intense in lesions of patients with the lepromatous form of the disease [24]. In another study evaluating the rate of apoptosis in skin lesions of leprosy patients based on the cytokine TGF-b, it was observed that in the lepromatous form the cytokine acts synergistically with caspase-3 in the regulation of the apoptotic response. During the physiopathological response, the cytokine would increase the activation of kinases involved in the cleavage of caspase-3 and, consequently, in the expression of signals inducing the activation of death domains during the chronic and infectious processes of the disease [25] (see Fig. 3). In agreement with other studies evaluating the role of apoptosis in leprosy by determining the expression of Toll-like receptors in Schwann cells, it was observed that mycobacterial components in the membrane of the bacillus are able to trigger cellular apoptosis in tissue lesions of the lepromatous form by inducing the release of Fas and effector caspases [11,26]. A study investigating the role of IFN-g and iNOS associated with the expression of caspases-1, -3
and -8 showed that, in the development of the mechanisms resulting from the cell-mediated immune response, both IFN-g and iNOS stimulated the activation of caspase-3 in the lepromatous form of the disease [27]. With respect to the role of Treg cells, the present results showed a quantitative increase in the transcription factor FoxP3 in the lepromatous form of leprosy. This finding can be explained by the immune response profile triggered by these cells during suppressive evolution in infectious manifestations of the disease. Studies evaluating mechanisms that regulate the participation of immune response inducers demonstrated that Treg cells stimulate the production of surface markers which influence the regulation of immunosuppressive activity in inflammatory processes. Within this context, it was demonstrated that CD8 þ Treg cells characterized by expressing CD69, CD25, CTLA4 and Foxp3 can induce T cells to produce cytokines such as IL-4, IL-5, IL-13 and TGF-b which influence the development of the adaptive response in the disease, suppressing the Th1 response. In granulomatous lesions found in lymph nodes infected with the mycobacterium, the presence of cells expressing CD8 þ LAG-3 þ CCL4 has been shown to suppress the immune response, creating a cellular environment that favors the maintenance and persistence of infection [28,29]. In a study investigating the role of CD4 þ CD25 þ FoxP3 þ cells, these cells reduced the production of mediators such as TNF-a and IFN-g, which stimulate the release of reactive oxygen and nitrogen
Fig. 2. Linear correlation between the immunoexpression of caspase-3 and FoxP3 in the polar forms of leprosy. There is a tendency of positive correlation between Treg cells and the phenomenon of apoptosis.
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Fig. 3. Immunohistochemistry for caspase 3 in tuberculoid (A) and lepromatous (B) forms of leprosy. Immunohistochemistry for regulatory T cells in tuberculoid (C) and lepromatous (D) forms of leprosy. The pattern of positivity is characterized by deposition of brown pigment in the tissue. Magnification 200. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
species in the inflammatory infiltrate of patients with the lepromatous form of the disease. Another study evaluating the role of costimulatory molecules showed that the bacillus reduced the expression of CD28 on the surface of monocytes and lymphocytes in the lepromatous form of the disease. In addition, the mycobacterium interfered with the expression of CD26 and CD86, favoring persistence of the bacillus during the course of the disease [29,30]. Regarding the correlation between apoptotic activity and Treg cells, a moderate synergistic effect between caspase-3 and FoxP3 was observed in the tuberculoid form of the disease. To our knowledge, there are no studies investigating the association between these markers in leprosy. Literature data indicate that Treg cells induce mechanisms that regulate the production of mediators participating in the apoptotic cascade. A study investigating the role of these markers in the different clinical forms of American tegumentary leishmaniasis has shown a positive correlation between caspase-3 and FoxP3 in cutaneous diffuse leishmaniasis (LCD) and cutaneous localized leishmaniasis (LCL) [31]. In that study, a positive association was also observed between caspase-3 and FasL in LCD and LCL. That study lends support to the present findings since leishmaniasis shows a similar clinical aspect during the course of the disease. Therefore, the present results suggest that the combined participation of Treg cells and apoptosis indicates the possibility that these two phenomena interfere with the evolution of the disease, since it creates an immunosuppressive environment against the bacillus, permitting progression to the lepromatous form of the disease. However, further studies are needed to complement the present results. References [1] Santos DS, Duppre NC, Sales AM, Nery JA, Sarno EN, Hacker MA. Kinship and leprosy in the contacts of leprosy patients: cohort at the Souza Araújo outpatient clinic, Rio de Janeiro, RJ, 1987e2010. J Trop Med 2013;2013: 596316. [2] WHO (World Health Organization) Global leprosy situation. Weekly epidemiological record, vol. 86; 2001. p. 389e400. [3] Ishii N. Recent advances in the treatment of leprosy. Dermatol Online J Davis 2003;9:1e18.
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