Expression of annexin A 1 in Leishmania-infected skin and its correlation with histopathological features

Introduction: The aim of this study was quantify annexin A1 expression in macrophages and cluster of differentiation 4 (CD4)+ and cluster of differentiation 8 (CD8)+ T cells from the skin of patients with cutaneous leishmaniasis (n=55) and correlate with histopathological aspects. Methods: Infecting species were identifi ed by polymerase chain reaction-restriction fragment length polymorphism, and expression of annexin A1 was analyzed by immunofl uorescence. Results: All patients (n = 55) were infected with Leishmania braziliensis. Annexin A1 was expressed more abundantly in CD163+ macrophages in infected skin (p < 0.0001) than in uninfected skin. In addition, macrophages in necrotic exudative reaction lesions expressed annexin A1 at higher levels than those observed in granulomatous (p < 0.01) and cellular lesions p < 0.05). This difference might be due to the need to clear both parasites and necrotic tissue from necrotic lesions. CD4+ cells in cellular lesions expressed annexin A1 more abundantly than did those in necrotic (p < 0.05) and granulomatous lesions (p < 0.01). Expression in CD8+ T cells followed the same trend. These differences might be due to the pervasiveness of lymphohistiocytic and plasmacytic infi ltrate in cellular lesions. Conclusions: Annexin A1 is differentially expressed in CD163+ macrophages and T cells depending on the histopathological features of Leishmania-infected skin, which might affect cell activation.

American cutaneous leishmaniasis is a non-contagious infection of the skin and mucosa by parasitic protozoans of the genus Leishmania (1) (2) (3) .It is transmitted through female Phlebotomus and Lutzomyia sand flies (4) .The infection is diagnosed based on a compendium of clinical, epidemiological, and laboratory characteristics (2) .
The host immunological response determines to a signifi cant extent whether the infection persists or is cleared has high relevance for determining cure or persistence (5) (6) .In early infection, no macroscopic pathological changes occur in the epidermis (7) .Following initial acute infl ammation, the infection progresses to a silent phase lasting a few weeks to months, during which the parasite proliferates without clinical manifestation.The silent phase ends with extensive infl ammation and lesion formation at the infection site (8) .Lesion healing and parasite clearance correlate with a preponderance of chronic rather than acute infl ammatory cells in the infected tissue (9) .However, while a T cell-mediated response is essential to clear parasites in most cases (10) , the dogma Th1 = good/Th2 = bad is somewhat inadequate (11) .For instance, hyperinfl ammatory collateral damage from T helper type 1 (Th1) response has been reported, along with a variable T helper type 2 (Th2) response dependent on cytokine release and other T cells (12) (13) .Moreover, Cardoso et al. (14) demonstrated that cluster of differentiation 8 (CD8) + T cells in patients with subclinical Leishmania braziliensis infection secrete interferon gamma (IFN-γ) to activate macrophages and facilitate parasite clearance.Notably, Pereira-Carvalho et al. (15) showed that T cells maintain activation levels at approximately 2 years after the end of therapy, and lymphocytes from well-healed lesions recognize leishmanial stimuli and proliferate upon exposure.Taken together, the data indicate that the immune response against Leishmania is very complex, and it is essential to understand better the processes at the infection site and the molecules present at infl ammation site.Annexin A1 (ANXA1), also known as lipocortin-1, is a 37-kDa calcium-and phospholipid-binding protein involved in several biological processes, including suppression of infl ammation (16) (17) (18) .Indeed, ANXA1 modulates leukocyte extravasation to the site of infl ammation and regulates cytokine release in acute, chronic, or systemic infl ammation (19) (20) .

METHODS
Its role in adaptive immunity is poorly understood; however, it has been shown to inhibit proliferation and differentiation of T cells by modulating T cell receptor signaling (18) .To help defi ne the role of ANXA1 in adaptive immunity, we analyzed its expression in CD163 + macrophages, CD4 + , and CD8 + T cells from skin biopsies of patients with cutaneous leishmaniasis, and investigated its correlation with histopathological features.This study is important for shows the relevance of ANXA1 dynamics in the immune system regulation during cutaneous leishmaniasis.

Patients
This cross-sectional study was performed in 55 patients with symptomatic cutaneous leishmaniasis who were treated at Julio Müller University Hospital [Hospital Universitário Julio Müller (HUJM)], Cuiabá, State of Mato Grosso, Brazil, between February 2012 and November 2013.Patients were of both sexes, aged 18-80 years.Patients who presented other infectious or immunosuppressive diseases, as well as those who were already being treated for leishmaniasis were excluded.Twenty patients undergoing surgery for colorectal cancer were used as control, and skin biopsies were collected at the incision site.
The Ambulatory of American Tegumentary Leishmaniasis/ HUJM is used in the State of Mato Grosso to diagnose and treat leishmaniasis.To confi rm infection, patients received clinical examination, as well as histopathological and parasitological tests suggestive of Leishmania infection, including blades from aspirate, shave, and smear of injured tissue, as well as cultures from aspirate and biopsy of the lesion.Following diagnosis, infecting species were identifi ed by polymerase chain reactionrestriction fragment length polymorphism (PCR-RFLP).

Histopathology
Patient received local anesthesia and lesions were previously disinfected.Tissue samples were obtained by biopsy with a 4-mm punch.Tissues were then immersed in 10% formol, dehydrated through a gradient of crescent alcohol, clarifi ed in xylene, and embedded in paraffi n.Samples were sectioned at 3μm by using a HYRAX M60 microtome (Carl Zeiss, Germany), deparaffi nized in xylene, hydrated in decreasing concentrations of alcohol, and stained with hematoxylin-eosin.Lesions were then scored by pathologists who were blinded to the study according to the criteria defi ned by Magalhães (21) for cellular exudative reaction (CER), granulomatous exudative reaction (GER), necrotic exudative reaction (NER), granulomatous-necrotic exudative reaction (GNER), and tuberculoid exudative reaction (TER).

DNA extraction from skin biopsies
Deoxyribonucleic acid (DNA) was extracted using Wizard TM Genomic DNA Purifi cation kit (Promega, WI, USA) from skin biopsies frozen and stored at -80°C.DNA was quantifi ed using NanoDrop®.

Statistical analyses
We compared ANXA1 expression using one-way analysis of variance (ANOVA) and Bonferroni's post-hoc test in GraphPad Prism v. 5.01 for Windows (La Jolla, CA, USA); Values were considered signifi cant and displayed as symbols in the fi gures as: one symbol, p value below 0.05; two symbols, p value below 0.01; three symbols, p value below 0.001.

Ethical considerations
Participation was voluntary, and patients signed informed consent forms of their own cognizance.This study was approved by the Research Ethical Committee of Julio Müller University Hospital (625-CEP-HUJM/2009).
ANXA1 was also mainly expressed in the cytoplasm of CD8 + and CD4 + T cells (Figures 4A-F).Expression in CD8 + cells was higher in cellular lesions (121.3 ± 9.0U) than in granulomatous (76.0 ± 11.4AU, p < 0.05) and necrotic lesions (77.0 ± 10.4AU, p < 0.05).Similarly, expression in CD4 + cells was higher in cellular (123.9 ± 11.6AU) than in granulomatous (87.7 ± 5.2AU, p < 0.05) and necrotic lesions (53.7 ± 15.7AU, p < 0.01) (Figure 4G and Figure H).As in CD163 + macrophages, expression in CD8 + and CD4 + T cells did not correlate with age of the lesion, with r 2 0.0267 and 0.00488, respectively (Figure 4I  and Figure J).Accordingly, expression did not correlate with age of specifi c lesions, with r 2 0.0274, 0.0213, and 0.1011 for CD8 + T cells in granulomatous, cellular, and, necrotic lesions, respectively, and 0.04981, 0.0024, and 0.0859 for CD4 + T cells.In this study, the patients were infected with L. braziliensis alone; notably, patients examined by Carvalho et al (4) in HUJM/ UFMT, Cuiabá, Brazil were also infected by L. braziliensis alone, suggesting that this species is the most prevalent in different areas in Brazil.
ANXA1 has pleiotropic and pluripotent effects in several biological processes, including infl ammation and tumorigenesis (19) (20) (23) .However, the role of ANXA1 in adaptive immunity is poorly defi ned.Published data indicate that ANXA1 expression is lower in lymphocytes than in neutrophils and monocytes (24) (25) , and imply that ANXA1 modulates T cell-mediated immune response (26) by activating specifi c signaling pathways and suppressing transcription factors (26) (27) .
The host immune elicited by Leishmania has been widely studied, and was shown to be extremely complex and variable (11) (28) .However, the role of ANXA1 in this response is unknown.Our data demonstrate that skin macrophages from patients with cutaneous leishmaniasis express ANXA1 more abundantly than do macrophages from uninfected skin, major histocompatibility complex those observed in other leukocytes at steady state and during acute (29) or chronic infl ammation (30) .Abundant expression at infection sites also suggests that the protein may be involved in phagocytosis of parasites.Indeed, the literature shows that ANXA1 regulates phagocytosis, macrophage differentiation, and activation (20) (31) (32) (33) by inducing the expression of CD40, CD54, CD80, CD84, major histocompatibility complex class II (MHCII), and CCR7 (33) .Finally, Collins et al (34) detected ANXA1 in vacuoles containing L. mexicana, implying that the protein might facilitate vesicle fusion with endosomes.
The literature also suggests that ANXA1 in phagocytes facilitates clearance of apoptotic cells (32) .Indeed, macrophages in ANXA1-defi cient mice have reduced capacity to clear apoptotic bodies (31) .Notably, Tzelepis et al (35) reported that these mice are highly susceptible to Mycobacterium tuberculosis, and that ANXA1 expression is signifi cantly downregulated in infected dendritic cells, suggesting that suppression of ANXA1 is a critical mechanism for immune evasion by Mycobacterium tuberculosis.In our patients, ANXA1 expression is more abundantly expressed in necrotic lesions than in cellular or granulomatous lesions, implying that expression in necrotic lesions is stimulated by the need to clear both parasites and necrotic tissue.Indeed, several studies have demonstrated that ANXA1 expression can be precisely calibrated depending on the stimulus, of which glucocorticoids (36) and tumor necrosis factor alpha (TNF-α) (18) are the most extensively characterized.
We found ANXA1 to be expressed in CD4 + and CD8 + T cells as well, suggesting that the protein is upregulated during the immunological response to Leishmania.T cells in cellular lesions express ANXA1 robustly, presumably due to widespread infl ammation, as indicated by accumulation of lymphohistiocytic and plasmacytic infi ltrate, edema, and absence of granuloma.ANXA1 has been described in the literature as a key regulator of T cell activation and migration to infl ammatory sites (29) (30) (33) , and of signaling pathways (p38, ERK MAPK, Akt, and NF-κB) that control production of cytokines such as TNF-α, INF-γ, IL-2, and IL-17 (37) (38) .ANXA1 also regulates the differentiation and proliferation of lymphocytes (18) .For instance, ANXA1 was recently demonstrated to regulate the differentiation of Th0 cells into Th1 (33) , although Th2 cells predominate in ANXA1-defi cient animals (37) .Finally, ANXA1 expression did not correlate with the age of the lesion, suggesting that expression might be regulated by pro-infl ammatory mediators at infl amed sites, as has been reported (18) (36) .
Our study evaluates the differential ANXA1 expression in different histophatological lesions of patients with cutaneous leishmaniasis (CL).The results were very positive; however, all patients were infected with Leishmania brasiliensis.It is possible that other parasite species shows a different pattern of expression.
In summary, our data show for the fi rst time that ANXA1 is differentially expressed in macrophages and T cells in lesions due to leishmaniasis, and expression is dependent on the histopathological characteristics of the lesion.We anticipate that future studies will further clarify the regulatory mechanism of cell action of ANXA1 during Leishmania infection.