Manuscrito em preparação

Contribution of metalloproteinase 1 and 3 in remodeling tissue of lesions in cutaneous leishmaniasis patients

Plos One (manuscrito em preparação, vide Normas de Publicação no ANEXO I e a carta ao Editor, no ANEXO II).

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Contribution of metalloproteinase 1 and 3 in remodeling tissue of lesions in cutaneous leishmaniasis patients

Camilla S Paixão1, Rúbia S Costa1, Tais Campos1, Andréa S Magalhães1, Juliana Almeida1, Paulo R L Machado1,2, Edgar M. Carvalho1,2,3, Lucas P Carvalho1,2,3 and Sara Passos1,2

1

Serviço de Imunologia, Complexo Hospitalar Professor Edgard Santos, Universidade Federal da Bahia.

2

Programa de Pós-Graduação em Ciências da Saúde, Universidade Federal da Bahia.

3

Instituto Gonçalo Moniz, Fundação Oswaldo Cruz (FIOCRUZ), Salvador, Bahia.

Corresponding Author: Sara Passos. Rua João das Botas, S/N 5andar. Canela. Salvador- Bahia – Brazil. CEP 40.110-160. saratpassos@hotmail.com

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ABSTRACT.

Cutaneous leishmaniasis (CL) due to infection by Leishmania braziliensis is characterized by high levels of TNF, IFN and cell infiltrate composed by macrophages, monocytes, dendritic cells, CD4+T and CD8+T cells. In addition to these cells, the metalloproteinases, with its ability to degrade extracellular matrix components, can participate in the formation of the lesion. The activity of MMPs is extremely regulated, and its action is controlled by the inhibitor TIMP. To evaluate the production of MMP- 1 and MMP-3 and the effects of TNF, IL-6, IL-1β, IFN on the production of them in patients with CL. Peripheral blood mononuclear cells were obtained from healthy subjects and patients with CL to determine the frequency of CD4+, CD8+ cells and subsets of monocytes and the expression of MMPs and CD147 inducer. Cell culture was performed in the presence or absence of SLA, anti-IFN-γ, anti-TNF, anti-IL-6 and anti- IL-1β and, levels of MMP-1 and MMP-3 were determined by ELISA. Production of MMP-3 was higher in cultures stimulated with SLA from CL patients compared to healthy subjects, although no difference was observed in the production of MMP-1 between these groups. PBMC cultured from patients with CL in the presence of anti- IFN- produces more MMP-1 compared to healthy individuals and, an increase of MMP-1 and TIMP-1 in biopsies from patients with CL. After neutralization of TNF, IL- 6 and IL-1β the level of MMP-1 was increased in patients with CL, however no increase of MMP-3 was observed when we neutralize those cytokines. Meanwhile, CD147 is more expressed in CD8+T cells from CL patients compared to healthy subjects. These observations suggest that these MMPs can participate in the process of tissue remodeling in lesions from patients with L. braziliensis infection.

INTRODUCTION.

Leishmaniasis is an infectious disease caused by protozoa of the genus Leishmania, transmitted to humans by the bite of sandflies infected with the parasite (DESJEUX, 1996) and characterized by ulcerative lesions of the skin and mucous membranes. The lesions in patients with cutaneous leishmaniasis (CL) are characterized by the presence of inflammatory infiltrate composed by T and B cells, macrophages, Langerhans cells and plasmocytes (MARETTI-MIRA, et al., 2010). In those lesions high production of TNF and IFN are found and these cytokines can contribute to the destruction of Leishmania sp and due to exacerbated the response inducing tissue damage and

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participating in the pathogenesis of the disease (GOMES-SILVA 2007, BACELLAR, 2002-2002 CROSS, RIBEIRO DE JESUS 1998). TNF is a cytokine known for mediating the pathology of LC using mechanisms such as expression of matrix metalloproteinases (MMPs) and increase in citotoxicity. The MMPs are enzymes known to their ability to degrade extracellular matrix components (MEC), participating in both physiological processes, such as morphogenesis, development and repair of tissues, as in pathological processes, such as inflammation (Nagase H 1991, Baugh JA 2001, Gupta S 2002, Lehmann W 2005). The activity of MMPs is extremely regulated, because an imbalance between the production of metalloproteinase and its inhibitors TIMPs can lead to excessive degradation of matrix with subsequent tissue destruction (MURPHY, 2008), being the dissemination or persistence of pathogens arising from the breakdown of tissue barriers and the creation of places bad accessed by host immune cells.

The mechanisms that lead to production of MMPs and its contribution to the development of tissue lesions in cutaneous leishmaniasis are not yet well understood. The identification of the mechanisms of action of MMPs in the development of the lesion in CL is very important for the understanding of the pathogenesis of the disease (CAMPOS et al., 2014).) and the development of new therapeutic approaches. As the MMPs participate in both physiological and pathological processes, is very important identify the MMPs producing cells in infection by L. braziliensis, as well as the effect of pro-inflammatory cytokines in expression of MMP-1 and MMP-3 in cutaneous leishmaniasis patients.

MATERIAL and METHODS.

Patients. Participants of the present study includes 27 cutaneous leishmaniasis (CL) patients from the L. braziliensis transmission area of Corte de Pedra, Bahia state, Brazil, and 09 healthy subjects (HS) living in areas not exposure to Leishmania. Diagnosis for CL was performed by a positive parasite culture or PCR as previously described (Machado PR, 2010).

Soluble Leishmania Antigen (SLA) preparation. SLA was prepared with an isolate of L.

braziliensis as previously described (Reed, SG 1987). Briefly, promastigotes

ressuspended in lysis solution (Tris, HCL, EDTA and leupeptin) were immersed in liquid nitrogen, and thawed at 37uC. After freezer-thaw procedure, they were sonicated

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and the disrupted parasites were centrifuged at 14,000G. The supernatant was filtered and assayed for protein concentration. SLA was used at a concentration of (5 mg/ml). Cultures of peripheral blood mononuclear cells and biopsies. Peripheral blood mononuclear cells (PBMC) were isolated from heparinized venous blood by Ficoll- Hypaque (GE Healthcare Biosciences AB, Sweden) gradient centrifugation. After washing three times in 0.9% NaCl, PBMC was adjusted to 3x106 cells/ml in 1 ml of RPMI-1640 (Gibco Laboratories, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco Laboratories, South America Invitrogen), 10 IU/ml penicillin and 100 mg/ml streptomycin. Cells were placed on 24 wells plates and incubated for 72 hours in the presence or absence of SLA (5 mg/ml) or anti-TNF antibody (5 ug/ml) or anti-IFN antibody (10 ug/ml) or anti-IL-6 antibody (10 ug/ml) or anti-IL-1β antibody (10 ug/ml) (R&D systems, Minneapolis, MN), as indicated in figures. Biopsies from L. braziliensis patients and HS were cultured in complete RPMI media without stimuli. Tissue from CL patients and HS were cultured in RPMI for 12 hours. Supernatants from PBMC and biopsies were collected and stored at -70C. The levels of MMP-1, MMP-3 and TIMP-1 (R&D Systems, Minneapolis, MN) were measured by ELISA (R&D Systems) according to the manufactures instructions. The results are expressed in pg/ml.

Statistical analysis. Mann-Whitney was used to compare HS and CL groups; Kruskal- Wallis test (nonparametric test) was used to compare the CL and HS groups; Wilcoxon matched pair test (paired and nonparametric t test) was used to analyze PBMC cultures in different conditions within the same group of individuals. Comparisons were considered statistically significant when P< 0.05. All p values represented are two- sided.

Ethical considerations. This work was approved by the Ethics and Research Committee from Federal University of Bahia. All subjects provided witted informed consent; in case of illiterate subjects, a thumb print plus signature of an independent witness were used.

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RESULTS.

MMP-1 and MMP-3 are produced by PBMC and biopsy cells from CL patients and healthy subjects. MMPs are enzymes that degrade extracellular matrix and in high levels MMPs may cause tissue damage (Elkington PT 2005). Exaggerated inflammatory responses lead to tissue damage and ulcer development in CL (Ribeiro-de-Jesus A 1998). MMP-1 and MMP-3 levels were determined in supernatants of peripheral blood mononuclear cells of healthy subjects and patients with CL (Figure 1A and B) and biopsy from the same groups (Figure 1C, D and E). Soluble Antigen of Leishmania (SLA) was added to cultures of PBMC and observe that the production of MMP-3 was greater in cultures stimulated with SLA in CL patients compared to healthy subjects (HS), however there was no statistical difference in the production of MMP-1 between CL and HS. When we compare the production of metalloproteinasis in the presence or absence of SLA, we observed a significant increase in the stimulated cultures with CL patients (Figure A and B). Evaluating the production of MMP-1, MMP-3 and TIMP-1 on the biopsies from CL patients and HS we found a significance of MMP-1 and TIMP- 1 producing in CL patients compared with HS (Figure 1C and E).

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Figure 1. Production of MMP-1, MMP-3 and TIMP-1 in PBMC and biopsy from cutaneous leishmaniasis patients and healthy subjects. Levels of MMP-1 (A) and MMP- 3 (B), were determined in culture of PBMC with media or Soluble Leishmania Antigen (SLA) (5ug/ml) for 72 hours in patients with CL (n = 19) and healthy subjects (n = 09). The levels of MMP-1(C), MMP-3 (D) and TIMP-1 (E) were determined by ELISA in culture of biopsy from patients with CL (N = 11) and healthy subjects biopsy (N = 4). The results were presented in pg/mL. Mann-Whitney U test *P < 0.05 **P < 0.01.

Relevant contribution of TNF and IFN- in the production of MMP-1 and MMP-3 in healthy subjects and patients with CL. CL patients presents high levels of TNF and IFN- during infection (Ribeiro-de-Jesus A 1998). The production of MMP-1 and MMP-3 were determined by ELISA in the presence or absence of SLA and SLA adding anti-TNF (Figure 2A) and anti-IFN-γ (Figure 2B). We observed that production of MMP-1 was greater in cultures stimulated with SLA with anti-IFN-γ in CL patients compared to healthy subjects. However there was no statistical difference in the production of these cytokines MMP-3 when neutralized. When we compare the production of MMP-1 and MMP-3 in patients with LC under different conditions of

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stimuli, noted increased levels of MMP-1 in culture stimulated with SLA and SLA with anti-TNF and SLA with anti-IFN-γ.

Figure 2. Contribution of TNF and IFN-γ in the production of MMP-1 and MMP-3 in patients with cutaneous leishmaniasis. PBMC from patients with CL (n = 11) and HS (n = 05) cultured with media or stimulated with SLA (Soluble Leishmania Antigen) (5ug/ml) with anti-TNF (10ug/mL) and anti-IFN-(10ug/mL)for 72 hours and MMPs present in the supernatant of cultures were measured by ELISA. The results were presented in pg/ml. Statistical comparisons were performed using the Kruskal-Wallis test and post-test Dunns and Willcoxon test *P<0.05, ** P<0.01, ***P<0.001.

Role of IL-1β and IL-6 in the production of MMP-1 and MMP-3 in patients with cutaneous leishmaniasis. Cytokines such as TNF, TGF-β (MARRETI-MIRA, 2010; COSTA, 2008) and IL-1 (COSTA et al, 2008) are known to stimulate the expression and activation of MMPs, while IL-10 expression and activation decreases, IL-1 β (PRATO et al., 2008; NEE et al., 2004) and IFN-γ (MARETTI-MIRA, 2010) have varying effects. In a study with DNA Microarray in biopsy of patients with CL was demonstrated high expression of MMP-1, MMP-3, MMP-9 and MMP-12 highly expressed in patients with CL (CAMPOS et al., 2014). MMP-1 and MMP-3 levels were determined in peripheral blood mononuclear cells of healthy subjects and patients with

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CL by ELISA after 72 hours of culture in the presence or absence of SLA, SLA with anti-IL-1β (Figure 3A) and SLA with anti-IL-6 (Figure 3B). When we compare the production of MMP-1 in patients with CL under different conditions of stimuli we saw increased levels of MMP-1 in culture stimulated with SLA and SLA with IL-β 1 and SLA with anti-IL-6. However there was no statistical difference in the production of these cytokines MMP-3 when neutralized.

Figure 3. Production of MMP-1 and MMP-3 in patients with CL after blocking IL-1β and IL-6. PBMC from patients with CL (N = 11) and HS (N = 05) were cultured in media or stimulated with Soluble Leishmania Antigen (SLA) (5ug/ml) with anti-IL-β 1(10ug/mL) or anti-IL-6(10ug/mL) for 72 hours and MMPs present in the supernatant were measured by ELISA. The results were presented in pg/ml. Statistical comparisons were performed using the Kruskal-Wallis test and post-test Dunns and Willcoxon test *P<0.05 **P< 0.01, ***P<0.001.

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CD147 is expressed mainly by lymphocytes during CL infection. It was shown that monocytes subsets and lymphocytes are important in the immune response of cutaneous leishmaniasis patients (Coffman et al. 1985 and Passos S et al., 2014). CD147 is a molecule present on the cell surface of leukocytes and epithelial cells and has the role to induce production of metalloproteinases. Following the strategic gate for monocytes based on size and granularity of the cells (figure 4A) and the CD14 and CD16 markers for different subsets of monocytes (Figure 4B), the evaluation showed by flow citometry that CD147 expression is higher in HS compared with CL patients (Figure 4C). For instance, the same evaluation was performed for lymphocytes by flow citometry using a strategic gate (Figure 4D) and stained for CD4+ / CD8+T (Figure 4E) the evaluation showed higher expression of CD147 by CD8+T cells by CL patients (Figure 4F).

Figure 4. Expression of CD147 in monocytes and lymphocytes from CL patients and HS by flow cytometry. Ex-vivo analysis of CD147 expression in subpopulations of monocytes (A, B and C) and lymphocytes (D, E and F) from patients with CL and HS. (A) representative plot from a CL patient identifying the subpopulations of monocytes as classic (CD14+ CD16+), intermediate (CD14+ CD16+) and non-classical (CD14+

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CD16++). (B) frequency of subpopulations of monocytes in healthy subjects (HS) (N = 6) and patients with cutaneous leishmaniasis (CL) (N = 11). (C) frequency of CD147 in sub-populations of monocytes from CL patients and HS. (D) Strategic gate for lymphocytes and stain for CD4+ and CD8+ specific cells. (E) Expression of CD4+ and CD8+T cells from CL patients (N=11) and HS (N=06). (F) Expression of CD147 in lymphocytes from CL patients and HS lymphocytes. Non-parametric tests Kruskal- Waills and Dunns post test was used to compare three continuous variables and Mann- Whitney test was used to analyze the statistical differences between groups * P<0.05, ** P<0.01, *** P<0.001.

Levels of MMP-1 or MMP-3 presents a negative correlation to the size of lesion from CL patients in PBMC or biopsy of CL patients. Its already known that MMPs can be involved with tissue remodeling (…). Due to the high levels of MMP-1 and MMP-3 found in the peripheral blood from CL patients, we evaluate if there is a correlation between those metalloproteinases with size of the lesion from CL patients. We observed a negative correlation between MMP1 and MMP3 and the size of the lesion (r) (Figure 5A and B) and for the biopsies correlation between MMP1 and MMP3 and the size of the lesion (r) (Figure 5C and D).

CD8 12.7% CD4

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Figure 5. Correlation between the lesion size from PBMC and biopsy from patients with CL and the levels of MMP-1 and MMP-3. The PBMC data were obtained from 27 patients with cutaneous leishmaniasis (A and B) and 10 biopsies from CL patients (C and D). The levels of MMP-1 and 3 were expressed in pg/ml. The data were analyzed by Spearman correlation. Statistical significance with P< 0.05 and (r =-0.1667, r =- 0.0606, r=0.1459 and 0.0975), respectively.

DISCUSSION.

The CL caused by L. braziliensis is characterized by an inflammatory response with participation of neutrophils, mononuclear cells and high levels of IFN-γ and TNF. These cytokines contribute to the destruction of Leishmania, but induce tissue damage, participating in the development of ulcers observed in LC (Gomes-Silva 2007, Bacellar, 2002-2002, Cross of Jesus 1998). Some researchers have studied the participation of cytokines and chemokines that contribute to inflammatory response in patients with cutaneous leishmaniasis as CXCL-9, CXCL-10 and CCL-2 in the recruitment of

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leukocytes to inflammatory (Conrad, Strauss-Ayali et al., 2007; George, Zhang et al., 2011; Willenborg et al., 2012; Brito et al., 2014; Guimarães et al., 2015). While macrophages, monocytes and epithelial cells, cells are also involved in the immune response in leishmaniasis and express metalloproteinases, enzymes with properties related to degradation and remodeling of the extracellular matrix. In this study we investigated the expression of metalloproteinases MMP-1, MMP-3 and its inhibitor TIMP-1 in the peripheral blood and the injury of patients with CL, and, for the first time, we suggest how these metalloproteinases can be involved on the re-epithelization of the ulcer from cutaneous leishmaniasis patients infected by L. braziliensis. MMP-1 and MMP-3 have been documented by perform functions of tissue degradation and cell migration, as well as participate in the immune response by clivage cytokines and chemokines (Li et al., 2000; Vanhoutte et al., 2006; Kupai et al., 2010; Polyakova et al., 2010). Several studies have suggested that these metalloproteinases are involved in initial state of the tumor progression in human and murine model, although participate in inflammatory processes, they are also related to the repair of ulceration of intestinal mucosa as observed in infection caused by E. histolytica (Coussens et al., 1996; Sternlicht et al., 2000; Thibeauxet al., 2014). The MMPs are able to degrade extracellular matrix constituents, being secreted in response to external stimuli and inflammatory cytokines, mainly by macrophages and neutrophils (Brenner et al., 1989). In vitro, the migration of T cells and dendritic cells depends on specific stimuli is coordinated by metalloproteinase 9 (Xia et al., 1996). In this study, we found high production of MMP-1 and MMP-3 in PBMC of patients with CL. The expression of MMP-1 is directly related by different genetic polymorphisms. In genomic study with biopsies of patients with CL with infection by L. braziliensis was identified to 36 times higher expression of the gene for MMP-1 when compared with healthy subjects (Arakaki et al., 2009; Nancy et al., 2015). Although its role may lead to degradation of collagen (Coussens et al., 1996; Sternlicht et al., 2000; Xin, Wang et al., 2014; Almeida et al., 2016) so, an increased production of MMP-1 and MMP-3 in peripheral blood monocyte biopsies and MMP-1 and TIMP-1 is indicative for a tissue remodeling activity. This can be justified because the catalytic activity of collagenase-1 depends on the presence of MMP-1 for degradation of the injured tissue and the migration of keratinocytes over the dermal matrix for tissue reconstruction (Santoro; Galdino, 2005; Rohani & Parks, 2015). The production of MMP-3 in turn, explains one of their main actions that is releasing cell surface molecules that activate the colagenases, particularly

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MMP-1, to develop tissue repair and formation of angiogenesis (Sternlichtet al., 2000). These metalloproteinases interact with extracellular matrix proteins and, together, control the mobilization of keratinocytes to the site of injury.

REFERENCES.

DESJEUX, PHILIPPE. Leishmaniasis: public health aspects and control.Clinics in dermatology, v. 14, n. 5, p. 417-423, 1996.

MARETTI‐MIRA, A. C. et al. Therapeutic failure in American cutaneous leishmaniasis is associated with gelatinase activity and cytokine expression.Clinical & Experimental Immunology, v. 163, n. 2, p. 207-214, 2011.

GOMES‐SILVA, A. et al. Can interferon‐γ and interleukin‐10 balance be associated with severity of human Leishmania (Viannia) braziliensis infection?. Clinical & Experimental Immunology, v. 149, n. 3, p. 440-444, 2007.

BACELLAR, OLÍVIA et al. Up-regulation of Th1-type responses in mucosal leishmaniasis patients. Infection and immunity, v. 70, n. 12, p. 6734-6740, 2002. MURPHY, GILLIAN; NAGASE, HIDEAKI. Progress in matrix metalloproteinase research. Molecular aspects of medicine, v. 29, n. 5, p. 290-308, 2008.

CAMPOS, TAÍS M. et al. Matrix Metalloproteinase 9 Production by Monocytes is Enhanced by TNF and Participates in the Pathology of Human Cutaneous Leishmaniasis. 2014.

MACHADO, PAULO R. et al. Miltefosine in the treatment of cutaneous leishmaniasis caused by Leishmania braziliensis in Brazil: a randomized and controlled trial. PLoS Negl Trop Dis, v. 4, n. 12, p. e912, 2010.

Elkington PT, O'Kane CM, Friedland JS. The paradox of matrix metalloproteinases in infectious disease. Clin Exp Immunol. Oct;142(1):12-20, 2005.

48

RIBEIRO-DE-JESUS, A. et al. Cytokine profile and pathology in human leishmaniasis. Brazilian Journal of Medical and Biological Research, v. 31, n. 1, p. 143-148, 1998.

COSTA, JULIANA DIAS et al. In vitro evidence for metallopeptidase participation in hepatocyte damage induced by Leishmania chagasi-infected macrophages. Acta tropica, v. 106, n. 3, p. 175-183, 2008.

PRATO, MAURO et al. Phagocytosis of haemozoin (malarial pigment) enhances metalloproteinase-9 activity in human adherent monocytes: role of IL-1beta and 15- HETE. Malar J, v. 7, n. 1, p. 157, 2008.

NEE, LARINE et al. TNF-alpha and IL-1beta-mediated regulation of MMP-9 and TIMP-1 in human glomerular mesangial cells. Nephron Experimental nephrology, v. 107, n. 2, p. e73-e86, 2007.

VISSE, R. e H. Nagase. Matrix metalloproteinases and tissue inhibitors of metalloproteinases: structure, function, and biochemistry. Circ Res, v.92, n.8, May 2, p.827-39. 2003.

YE, SHU et al. Progression of coronary atherosclerosis is associated with a common genetic variant of the human stromelysin-1 promoter which results in reduced gene expression. Journal of Biological Chemistry, v. 271, n. 22, p. 13055-13060, 1996. YU, X. F., Han, Z. C. (2006). Matrix metalloproteinases in bone marrow: roles of gelatinases in physiological hematopoiesis and hematopoietic malignancies. Histology and Histopathology. 21: 519-531.

ZIEGLER-HEITBROCK, L., P. Ancuta, et al. Nomenclature of monocytes and dendritic cells in blood. Blood, v.116, n.16, Oct 21, p.e74-80. 2010.

KASINRERK, W.; Tokrasinwit, N.; Phunpae, P. CD147 monoclonal antibodies induce homotypic cell aggregation of monocytic cell line U937 via LFA-1/ICAM-1 pathway. IMMUNOLOGY-OXFORD-, v. 96, p. 184-192, 1999.

NOVAIS, Fernanda O. et al. Genomic profiling of human Leishmaniabraziliensis