In situ expression of M2 macrophage subpopulation in leprosy skin lesions

Acta Tropica 157 (2016) 108–114

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In situ expression of M2 macrophage subpopulation in leprosy skin lesions Jorge Rodrigues de Sousa a , Raphael Primo Martins de Sousa a , Tinara Leila de Souza Aarão a,b , Leonidas Braga Dias Jr. b , Francisca Regina Oliveira Carneiro a , Hellen Thais Fuzii b , Juarez Antonio Simões Quaresma a,b,∗ a b

Tropical Medicine Center, Federal University of Para, Belem 66055240, Para, Brazil Center of Biomedical and Health Sciences, State University of Para, Belem 66055190, Para, Brazil

a r t i c l e

i n f o

Article history: Received 19 November 2015 Received in revised form 12 December 2015 Accepted 7 January 2016 Available online 28 January 2016 Keywords: Macrophage M2 macrophage Immunopathology Clinical aspect

a b s t r a c t The clinical manifestations of the leprosy depend on host immune response and the macrophages are the primary cells involved in this process. M1 and M2 cells exhibited distinct morphology, distinct surface marker profiles, as well as different cytokine and chemokine secretion. Macrophages express receptors such as CD163, CD68, CD206, and costimulatory molecules such as CD80 and CD86, and cytokines that trigger a suppressive or inflammatory response. Thirty-three untreated patients were selected, 17 patients had the tuberculoid leprosy (TT) and 16 had the lepromatous leprosy (LL). We performed immunohistochemistry to detect IL-13, IL-10, TGF-␤, FGF-␤, CD163, CD68, arginase 1. M2 macrophages showed significant differences between the groups studied with increase in the expression of costimulatory molecules (CD68 and CD163), arginase 1 and cytokines (IL-10, IL-13, TGF-␤ and FGF-b) in the LL form. Response of M2 macrophages emerge as an alternative for a better understanding of the innate immunity in the polar forms of leprosy, highlighting the role of cytokines, arginase 1 and costimulatory molecules in the repair and suppressive responses in the lepromatous form of the disease. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Leprosy is a chronic infectious disease caused by Mycobacterium leprae, an acid-alcohol-fast intracellular obligate bacillus, which shows a unique tropism for macrophages, dendritic cells and Schwann cells and causes tissue damage and demyelination in peripheral nerves (Gonc¸alves, 2013; Wheat et al., 2014). Leprosy is considered a granulomatous disease with a broad clinical spectrum (Adams and Krahenbuhl, 1996), and represents a serious public health problem in periphery and developing countries (Gonc¸alves, 2013; Lastória and Abreu, 2014). Clinically, leprosy exhibits different histopathological alterations. In the tuberculoid (TT) form, the cell-mediated response is characterized by the presence of an inflammatory infiltrate accompanied by granuloma formation and giant cells. In the lepromatous (LL) form, the response elicited by the bacillus ranges from the

∗ Corresponding author at: Tropical Medicine Center/UFPA, Av. Generalíssimo Deodoro 92, Umarizal, Belem, Pa 66055190, Brazil. E-mail address: [email protected] (J.A.S. Quaresma). http://dx.doi.org/10.1016/j.actatropica.2016.01.008 0001-706X/© 2016 Elsevier B.V. All rights reserved.

formation of vacuolated cells to diffuse lymphocytic infiltration (Massone et al., 2010; Talhari et al., 2015; Ridley and Jopling, 1966). Regarding the immune response, leprosy causes intense instability of the host defense system that comprises the spectra of the disease. In the TT form, the individual develops a cellular response mediated by Th1 lymphocytes, with the production of cytokines that trigger an inflammatory response. In the LL form, the response is mediated by Th2 lymphocytes that stimulate a suppressive response. In the transient forms, the cell-mediated response oscillates between the dimorphous, tuberculoid dimorphous, and lepromatous forms (Aarão et al., 2014; Fulco et al., 2014; Ridley and Jopling, 1966). Studies have shown that, according to the evolution of socalled spectral diseases, certain cell groups undergo changes in their response pattern depending on their correlation with the resistant and susceptible forms. Within this context, macrophages are part of a group of cells that, when undergoing phenotypic modification, express receptors such as CD163, CD68, CD206, costimulatory molecules such as CD80, CD86, and cytokines that trigger a suppressive or inflammatory response (Ka et al., 2014; Mills, 2012; Ouedraogo et al., 2012). In studies investigating the

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response of macrophages of the classical pathway, known as M1 macrophages, to intracellular pathogens, the cells were found to express higher levels of chemokines of the CXCL and CCR family such as CXCL10, CXCL11 and CCR7, cytokines that stimulate cell-mediated responses such as TNF-␣, IFN-␥, IL-6 and IL-12, and enzymes such as iNOS, which are important for the production of reactive oxygen and nitrogen species and for pathogen destruction (Huang et al., 2015; Mantovani et al., 2004; Montoya et al., 2009; Murray et al., 2014). In leprosy, a study investigating the alternative response mediated by M2 macrophages demonstrated higher expression of CD163, CD209, HLA-DR, IDO and IL-10 in the lepromatous form (Bobosha et al., 2014; Moura et al., 2012). In the tissue response involving the presence of M2 macrophages, there is evidence that the cells stimulate the production of enzymes such as arginases, which induce the production of growth factors such as TGF-␤ and FGF-b. These factors are important for the mechanisms that lead to apoptosis and healing through the regulation of extracellular matrix by fibroblasts, proliferation of endothelial cells and induction of angiogenesis in tissue lesions (Fulco et al., 2014; Barbay et al., 2015; Dzik, 2014; Montoya et al., 2009; Wijnands et al., 2015). To understand the participation of growth factors and cytokines together with enzymes and costimulatory molecules can bring new approaches to the response of macrophages and their behavior in the spectrum of the disease, the present study evaluated the response of M2 macrophages in skin lesions of the LL and TT forms of leprosy.

Table 1 Quantitative analysis of CD163, CD68, arginase 1, IL-10, IL-13, TGF-␤, and FGF-␤ in TT and LL forms.

2. Materials and methods

The results were stored in electronic spreadsheets of the Excel 2007 program. Statistical analysis was performed using the GraphPad Prism 5.0 program. For univariate analysis, frequencies and measures of central tendency and dispersion were obtained. The hypotheses were tested using the Mann–Whitney test and Spearman’s correlation test. A level of significance of 5% (p < 0.05) was adopted for all tests.

2.1. Characterization of the sample Thirty-three untreated patients with a confirmed diagnosis of leprosy according to the classification of Ridley and Jopling (1966) were selected. Seventeen patients had the TT form and 16 had the LL form. For histopathological analysis, 5-␮m histological sections were cut from paraffin-embedded tissue biopsies. The sections were stained with hematoxylin-eosin and submitted to immunohistochemistry.

Markers

TT Leprosy, Mean ± SD

CD68 CD163 Arginase 1 IL-10 IL-13 TGF-␤ FGF b

25.18 15.82 11.88 10.00 7.88 8.94 9.41

± ± ± ± ± ± ±

6.21 4.06 4.15 2.42 3.21 3.13 2.85

LL Leprosy, Mean ± SD 62.81 2185 22.98 21.36 19.81 23.84 25.13

± ± ± ± ± ± ±

8.13 4.76 4.25 3.10 3.97 4.18 3.34

p* <0.0001*** 0.001** <0.0001*** <0.0001*** <0.0001*** <0.0001*** <0.0001***

LL, lepromatous form; TT, tuberculoid form; SD, standard deviation. * Mann–Whitney test (p < 0.05). ** p < 0.05. *** p < 0.0001.

diaminobenzidine in 3% hydrogen peroxide as chromogen solution. The sections were couterstained with Harris hematoxylin for 1 min, dehydrated in an increasing alcohol series, and cleared in xylene. 2.3. Quantitative analysis and photodocumentation Cellular immunostaining with the antibodies was quantified by randomly selecting five fields under a Zeiss Axio Imager Z1 microscope at 400× magnification using a 0.0625 mm2 grid with 10 × 10 subdivisions. 2.4. Statistical analysis

2.5. Ethical aspects The study was approved by the Ethics Committee of the Center of Tropical Medicine (number 212.969).

2.2. Immunohistochemistry

3. Results

Since some studies (Fulco et al., 2014; Barbay et al., 2015) have shown that M2 macrophages expressing high levels of arginase 1 and CD163, and are associated with increased expression of IL-10, TGF-␤ and FGF-b, the biotin-streptavidin peroxidase method was used for immunohistochemistry employing the following primary antibodies: anti-IL-13 (Abcam, ab9576), antiIL-10 (Abcam, ab18499), anti-TGF-␤ (Abcam, ab66043), anti-FGF-b (Abcam, ab16828), anti-CD163 (Novocastra, NCL-L-CD163), antiCD68[Kp1] (Biocare Medical, CM033C), and anti-arginase 1 (Sigma, HPA 003595). First, the tissue samples were deparaffinized in xylene and hydrated in a decreasing alcohol series. Endogenous peroxidase was blocked by incubating the sections in 3% hydrogen peroxide for 45 min. Antigen retrieval was performed in citrate buffer, pH 6.0, for 20 min at 90 ◦ C. Nonspecific proteins were blocked by incubating the sections in 10% skim milk for 30 min. The histological sections were then incubated with the primary antibodies diluted in 1% bovine serum albumin for 14 h. After this period, the slides were washed in 1X PBS and incubated with the biotinylated secondary antibody (LSAB, DakoCytomation) in an oven for 30 min at 37 ◦ C. The slides were washed again in 1X PBS and incubated with streptavidin-peroxidase (LSAB, DakoCytomation) for 30 min at 37 ◦ C. The reactions were developed using 0.03%

Clinical manifestations showed symptoms of impaired skin and nerves. The lepromatous forms presented numerous, symmetric, poorly delimited macules, papules, nodules with diffuse infiltration with many bacilli. Tuberculoid leprosy showed few lesions, localized with asymmetric distribution, well-defined with sharp borders of infiltrated plaques, often hypopigmented with no detected bacilli. The pattern of immunohistochemical staining showed marked macrophages in the cytoplasm to CD68, CD163 and arginase 1. The pattern of cytokines showed positive areas in cellular and extracellular area (Figs. 1 and 2). Quantitative analysis of immunostaining for M2 macrophages showed significant differences between the groups studied. An increase in the expression of cytokines (IL-10, IL-13, TGF-␤ and FGF-b), receptor molecules (CD68 and CD163) and arginase 1 was observed in the lepromatous form of the disease (Table 1). Analysis of linear correlations in the lesions of LL patients showed a strong positive correlation between CD163 × IL-10 (r = 0.7847, p = 0.0003) (Fig. 3A), a moderate correlation between arginase 1 × CD163 (r = 0.6018, p = 0.0136) (Fig. 3B) and arginase 1 × IL-13 (r = 0.5721, p = 0.0206) (Fig. 3C), a strong correlation between arginase 1 × TGF-␤ (r = 0.7458, p = 0.0009) (Fig. 3D),

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Fig. 1. Immunohistochemistry for CD68 (A and B), CD163 (C and D), and arginase 1 (E and F) in lesions of patients with the TT and LL forms of leprosy, respectively. Note the staining pattern present in granulomas characterized by a brown deposit. (400×).

and a moderate correlation between IL-13 × TGF-␤ (r = 0.5292, p = 0.0361) (Fig. 3E) and IL-10 × TGF-␤ (r = 0.6308, p = 0.0088) (Fig. 3F). 4. Discussion Leprosy is a spectral disease in which the bacillus provokes changes in the tissue environment and modifies the course of the immune response over the spectrum of the disease (Cardoso et al., 2011; Fulco et al., 2007). Macrophages play a key role in the response to M. leprae, undergoing differentiation and polarization into the classical pathway consisting of M1 macrophages

and their inflammatory mediators, and into M2 macrophages and their subtypes that exhibit tissue repair properties, producing antiinflammatory cytokines (Sica and Mantovani, 2012; Wang et al., 2014). In the present study, an increase was observed in the expression of CD163 and CD68 in lesions of lepromatous patients, especially in the inflammatory infiltrate consisting of foamy macrophages with vacuoles contained large numbers of bacilli. Within this context, the expression of CD163 confirms the presence of M2 macrophages in the susceptible form of the disease, while several studies reported that CD163 is one of the main surface markers used to identify sub-

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Fig. 2. Immunohistochemistry for IL-10 (A and B), IL-13 (C and D), TGF-␤ (E and F), and FGF-b (G and H) in lesions of patients with the TT and LL forms of leprosy, respectively. Positive staining is observed in the granulomatous infiltrate located in the dermis. (200×).

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Fig. 3. Linear correlation between CD163, arginase 1 and cytokines in the LL form.

populations of macrophages belonging to the alternative pathway (Buechler et al., 2013; Carli et al., 2015). CD163 is a 130 kDa membrane glycoprotein that consists of a single transmembrane segment and a short cytoplasmic tail composed of 49 amino acid residues. The molecule is a member of the scavenger receptor family, which recognizes hemoglobin–haptoglobin complexes (Etzerodt and Moestrup, 2013; Thomsen et al., 2013). In leprosy, one of the theories suggests that expression of the receptor by macrophages is a key point for the recognition and entry of M. leprae into the cell, but the mechanisms underlying this process need to be elucidated (Fabriek et al., 2009; Moura et al., 2012). Another interesting aspect of the participation of CD163 in the lepromatous form of the disease is the demonstration that increased expression of this receptor is positively correlated with the expression of IDO and CD209, as well as with mechanisms that trigger an anti-inflammatory response and with an increase of iron

stores in phagosomal vacuoles, which contribute to mycobacterial viability and survival (Alcaraz et al., 2003; Moura et al., 2012). The present study also showed a quantitative increase in arginase 1 in the lepromatous form of the disease. Within this context, the increase in this enzyme may play a crucial role in the response of M2 macrophages since arginase 1 is one of the main enzymes participating in the mechanisms involved in the regeneration and repair of tissue damage (Caldwell et al., 2010; Wynn and Barron, 2010). Studies have shown that arginase 1 competes directly with iNOS for l-arginine, which serves as a substrate for the production of polyamines and proline. The latter is an essential amino acid involved in the synthesis of collagen and in the production of growth factors (Das et al., 2010; Hesse et al., 2001). With respect to the increase in IL-10 in the susceptible form of the disease, the response of the cytokine denotes the path that the protein follows in the anti-inflammatory pathway. IL-10 is the main protein that inhibits the activation of inflammatory macrophages,

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the expression of MHCII, and the production of important cytokines that trigger a microbicidal response, including TNF-␣, IFN-␥, and IL-12 (Richardson et al., 2015; Sieling et al., 1993). Another possible response of IL-10 in the alternative pathway may be associated with the induction of angiogenesis because of the response of M2a macrophages (Jetten et al., 2014; Mosser and Edwards, 2008). The increase in the expression of IL-13 during the immunopathological course of the disease might be associated with the regulation of arginase 1 and the differentiation, phenotypic modification and activation of M2 macrophages (McWhorter et al., 2013; Lawrence and Natoli, 2011). Other studies have emphasized that the response of this cytokine may be related to the formation of immune complexes and the development of a Th22 lymphocyte response (Silveira et al., 2015). Regarding the response of TGF-␤ and FGF-␤, the fact that may explain the increase in these markers involves the response of growth factors in tissue lesions. In this respect, both factors may regulate the participation of M2a, M2b and M2c macrophages and different cellular functions that interfere with the processes of healing, migration, cell division, proliferation, differentiation, and induction of angiogenesis (Bakhshayesh et al., 2012; Jetten et al., 2014; Martinez et al., 2008; Presta et al., 2009; Silveira et al., 2015). A recent study suggested FGF-␤ to serve as an immunomodulator in the response against intracellular pathogens, improving phagocytosis, cytokine production and the metabolism of reactive oxygen and nitrogen species in infected macrophages (Wang et al., 2015). Correlation analysis was performed to better understand the effect of CD163 and arginase 1 in conjunction with cytokines and growth factors. Our results showed a positive correlation between CD163 and IL-10, demonstrating that the latter directly influences the expression of the receptor in the susceptible form of the disease. Within this context, studies have shown that IL-10 is able to regulate the gene expression of CD163, indicating that this cytokine contributes to maintain elevated levels of CD163 in M2 macrophages in the lepromatous form (Buechler et al., 2000; Moura et al., 2012). The synergistic effect observed between arginase 1 and CD163 and between arginase 1 and TGF-␤ in lesions of LL patients suggests that the association between the receptor, enzyme and growth factor influences the activity of M2 macrophages, interfering with the suppressive response and cell proliferation (Barros et al., 2013; Gordon, 2003; Vilas-Boas et al., 2010). The association observed between TGF-␤ and IL-13 and between arginase 1 and IL-13 supports the fact that the synergistic action of cytokines can interfere with the tissue repair response. In this respect, studies have shown that the combined action of IL-13, TGF␤ and arginase 1 stimulates the production of factors that influence the remodeling, production of collagenases and differentiation of fibroblasts in tissue lesions (Gordon, 2003; Pearce and MacDonald, 2002; Wynn and Barron, 2010). The moderate correlation found between IL-10 and TGF-␤ in the LL form shows relationship with increased of suppressive immune response for negative regulation of inflammatory mediators such as iNOS, TNF-␣ and IFN-␥ by macrophages (Attia et al., 2014; Venturini et al., 2011). This study concludes that M2 macrophages emerge as an alternative for a better understanding of the innate immune response in the polar forms of leprosy.

Conflict of interest The authors report no conflict of interest.

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Acknowledgment We thank the National Council for Scientific and Technological Development-Brazil for financial support to the project, grant number 481020/2012-8.

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