Generation of Multinucleated Giant Cells due to Leishmania (V.) braziliensis Infection

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INTRODUCTION
A Leishmaniasis is a disease caused by the parasite Leishmania and is transmitted by a dipteran of the genus Lutzomyia or Phlebotomus.Leishmaniasis is a growing public health concern and is considered endemic in 88 countries.Leishmaniasis is one of the most neglected tropical diseases whose etiological agent is intramacrophage protozoa, which maintains its life cycle through transmission between the sandfly and a mammalian host.2][3][4] There are three varieties of leishmaniasis: cutaneous leishmaniasis, mucosal leishmaniasis, and visceral leishmaniasis.According to previous research, these varieties are associated with pathological clinical manifestations. 1,2ishmania (V.) braziliensis produces the mucocutaneous leishmaniasis known as espundia, which represents a dreaded complication that causes disfigurement in the patient and is typical for the most part in South America.[3][4] The complexity of leishmaniasis is due to the union of the countless feasible combinations of different syndromes of the disease, species and geographical areas where the infection is obtained, consequently, each combination varies according to its clinical manifestation, ease of diagnosis, natural history and response to treatment. 5 leishmaniasis, human is the accidental host and canids and rodents are natural hosts, being this disease endemic to areas of the tropics, subtropics and southern Europe, in environments ranging from tropical forests in the Americas to deserts in Asia.western, and from rural to peri-urban areas. 5,6e of the species of particular concern is Leishmania (V.) braziliensis.There is evidence that Leishmania (V.) braziliensis can lead to mucosal leishmaniasis in 5% of patients, affecting the upper respiratory tract and destruction of adjacent tissues. 5,6These studies suggest that patients are at high risk of coinfections and may suffer further health complications.Multinucleated giant cells were first described by Langhans. 7The formation of multinucleated cells in healthy people can be found in bone tissue. 8owever, multinucleated giant cells (MGCs) in nonskeletal tissues are the result of chronic inflammation due to the presence of foreign material that is poorly digestible or persistent pathogens that are not eliminated for various reasons.These MGCs have a role in innate immunity that includes extracellular matrix remodeling associated with granuloma formation; another function of them is to participate in the elimination of cellular debris in apoptosis during certain infections. 9,10[13][14][15][16] Macrophages play several important roles in host defense as they have the ability to ingest and destroy microorganisms, remove dead tissue and initiate the process of tissue repair, and produce cytokines that induce and regulate inflammation.These cells that are part of innate immunity and that are the link with acquired immunity present recognition receptors including those of the toll type (TLR, tolllike receptor) and those of the NOD type (NLR, ABSTRACT Background: Multinucleated giant cells were first described by Langhans(MGCs), these MGCs have a role in innate immunity that includes extracellular matrix remodeling associated with granuloma formation; another function of them is to participate in the removal of cellular debris in apoptosis during certain infections.Objective: Generate multinucleated giant cells due to Leishmania (V.) braziliensis infection.Methods: For infection macrophage/parasite ratio of 1 to 10 using the RAW 264.7-line (5 x10 5 /mL) and Leishmania (V.) braziliensis metacyclic promastigotes (50 x10 5 /mL) was a strain maintained in culture Schneider liquid medium, supplemented with 20% fetal bovine serum and 10 000 U/10 mg/mL penicillin streptomycin at pH 7 and 25 °C in the laboratory.Incubation was continued for 4 days, and microscopic observation at 1000X was performed at 24 hours and 96 hours, respectively.Results: In our study an MGC conversion was observed at 96 hours (50% ± 28.2).Conclusions: In vitro MGC model could be used to study the physiopathology of MGC generation by Leishmania (V.) braziliensis infection.Key words: Leishmania (V.) braziliensis, Multinucleated Giant Cells, Infection, Immunity, Macrophage.
NOD-like receptor) are a large family of innate receptors that detect DAMP and PAMP in the cytosol of cells and initiate signals that promote inflammation.[16] Macrophages have the ability to activate by two different pathways, one is the classical activation pathway and the other is the alternative activation pathway, this allows them to serve different functions.[16] M1 macrophages engulf and kill microorganisms, kill tumor cells, and present antigens to T cells to trigger a specific adaptive immune response.In addition, they secrete anti-tumor mediators such as IL-1β, IL-6, IL-12, IL-18, IL-23, tumor necrosis factor alpha (TNF-α) and express high levels of the major complex of type I and II histocompatibility.[16] The cell line RAW264.7 cells, are originally derived from tumors induced with Abelson leukemia virus.In most tumors, macrophages are considered to be of the M2 type, due in part to the absence of M1 signals, providing a microenvironment that favors tumor growth.In addition, M2 TAMs (Tumor Associated Macrophages) secrete many cytokines, chemokines, proteases and growth factors that promote tumor angiogenesis, thus favoring tumor spread and metastasis, thanks to nutrients and oxygen supplied to the tumor by the new blood vessels.These M2 macrophages are the perfect aid in this process, especially the wound healing subset, as they are primed to remodel tissue and produce Vascular Endothelial Growth Factor (VEGF). 16idence suggest that MGCs have been observed in several bacterial infections.[23] Morphologically multinucleated giant cells are generally classified into Langhans giant cells and foreign body giant cells (FBGCs).Langhans giant cells are characterized by having a relatively small number of nuclei, less than 20, which are arranged in a circular peripheral fashion within the giant cell.FBGCs usually present a large number of nuclei, greater than 20 and irregularly located throughout the giant cell. 9,13nghans giant cells are regularly seen in granulomas with epithelioidlike macrophages and are associated with indigestible particles of organisms surrounded by a necklace of mononuclear leukocytes that are essentially lymphocytes.FBGCs are observed at the tissue-material interface of medical devices implanted in soft and hard tissues. 17ditionally, FBGCs have been implicated in biodegradation events of polymeric medical devices.FBGC and macrophages shape the foreign body reaction at the tissue-device interface and are surface area dependent.Tissues that are used as vascular grafts show high densities of FBGC, whereas flat surfaces such as those found in breast implants exhibit only a layer of one or two macrophage cells and FBGC at the tissue-material interface. 9,13ere are apparent differences in the mechanisms of Leishmania clearance elaborated by human macrophages, such is the case that reactive nitrogen species play a minor role in the control of leishmaniasis, studies have indicated that infection with amastigotes results in limited production. of superoxide, one possible explanation for why superoxide might play a limited role in the control of leishmaniasis, is that infection with the amastigotes form of the parasite results in limited superoxide production. 23e aim of this study was to determine the formation of multinucleated giant cells due to in vitro infection by Leishmania (V.) braziliensis.

Growth calibration curve of mouse macrophage commercial cell line RAW264.7
The optimal day of cell growth was determined on a logarithmic scale related to its optimal metabolic state, performing a daily count in a Neubauer chamber using the formula: Cells/mL =[ (# cells counted) (Dilution factor) (1/Volume factor)]x 1000 .The reading was carried out until the next day, when the growth peak was reached on the cellular logarithmic scale.

Growth calibration curve of Leishmania (V.) braziliensis
The optimal day of parasite growth was determined on a logarithmic scale related to its optimal metabolic state, performing a daily count in a Neubauer chamber using the formula: Parasites/mL = [ (# parasites counted) (Dilution factor) (1/Volume factor)]x 1000.The reading was carried out until the next day, when the growth peak was reached on the parasite logarithmic scale.

Cell infection
For infection 24-well sterile polystyrene plates were used and each well contained a macrophage/parasite ratio of 1 to 10 using the RAW 264.7-line (5 x10 5 /mL) and Leishmania (V.) braziliensis metacyclic promastigotes (50 x10 5 /mL) was a strain maintained in culture Schneider liquid medium, supplemented with 20% fetal bovine serum and 10 000 U/10 mg/mL penicillin streptomycin at pH 7 and 25 °C in the laboratory.Incubation was continued for 4 days, and microscopic observation at 1000X was performed at 24 hours and 96 hours, respectively, using slides with cavities which were fixed at room temperature in a laminar flow cabinet for 5 minutes and, then, stained with Giemsa stain.The reading at 24 hours and 96 hours was performed in triplicate and 100 fields were observed for each reading.

Statistical analysis
From the triplicates, the percentages of infected cells at 24 and 96 hours were calculated.Similarly, MGCs percentages were calculated at the evaluated hours with standard deviation.

RESULTS
Results are shown in table 1 and figures 1 to 3. where bone marrow-derived macrophages (BMDM) infected with L. donovani were co-cultured with erythrocytes, this led to the cells having a higher rate of hemophagocytosis compared to uninfected bone marrow-derived macrophages (BMDM).Furthermore, BMDMs infected with L. donovani and stimulated with GM-CSF or IFN-γ showed higher rates of hemophagocytosis, 24,25 inferring that this mechanism is manipulated by the parasite as a survival mechanism within the MGCs.In our study it was observed that 24 hours after infection there were no MGCs with an average infection rate of 25%±3 (Figure 3), and, in the case of observation at 96 hours, an infection rate of 83%±14.7 was determined with 50%± 28.2 of MGCs (Figure 3), in optimal growth scales of the macrophage and the parasite on the fourth and fifth day respectively.(Figures 1 and 2) One study found that at 72 hours infection of macrophages by donovani y L. major generated MGCs of MGCs 39.2% ± 4.2% and 36.2% ± 6.9% respectively. 25However, this study found that infection was 83%±14.7 and MGCs generated by infection of L. (V.) braziliensis at 96 hours was 50%±28.2.This result differ from a previous study were the percentage in our study was higher. 25 vivo, the presence of MGCs infected by Leishmania may serve as an indicator of relapse and hemophagocytosis processes, as previously observed in studies. 10,24This finding is consistent with a previous study where 71.43% of patients with mucosal leishmaniasis exhibited MGCs with granuloma formation and chronic inflammatory reactions. 26[29] In one study they found that BMDMs treated with cytokines including GM-CSF, IL-4 and IFNγ promoted MGC formation in Leishmania donovani infection whereas BMDMs not treated with cytokines or only stimulated either by IL-4 or by INFγ did not show apparent MGC formation.In addition, cytokines including GM-CSF, IL-4, and IFNγ are inducers of multinucleation and the formation of phenotypically distinct MGCs.These results coincide with those of Fais et al. and it would also be the case of our research that after 96 hours the presence of MGC was observed, this could be due to stimulation with cytokines including GM-CSF, IL-4 and IFNγ that promoted the formation of MGC. 25,29 mentioned in the literature, the formation of granulomas with MGCs has been described in animals such as horses. 30This suggest that the generation of MGCs is a reaction to limit the dissemination of etiological agents in mammals.However, recent reports showed that MGCs are implicated in pathological processes such as sarcoidosis and rheumatoid arthritis. 31,32ior studies have shown that MGCs were found in cases of visceral and cutaneous leishmaniasis 33,34 as well as In Vivo infections in murine models such as the BALB/c. 35,36In this study, the formation of MGCs was induced In Vitro by L. (V.) braziliensis.
In an investigation, it was reported that histological studies of the spleens of mice infected with promastigotes of L. donovani showed hemophagocytosis in macrophages with high infection with amastigotes.In addition to being heavily infected, the multinucleated giant cell (MGC) phenotype was prominent in those hemophagocytes.Multinucleated macrophages represented 15.0 ± 6.2% of the total splenic macrophages, 35 in our experimental work they were carried out    The table illustrates that the percentages of infection increased as time elapsed.In the case of the generation of MGCs, they were only generated at 96 hours with a value of 50% ± 28.2.
in vitro with the cells of the commercial mouse macrophage cell line RAW264.7,originally derived from induced tumors.with Abelson's leukemia virus, ATCC (ATCC TIB-71), a percentage of 50% ± 28.2 of MGC generated by Leishmania (V.) braziliensis was obtained.
The multinuclear phenotype was highest in hemophagocytes where 60.4 ± 5.8% of mouse splenic hemophagocytes were multinucleated. 35ome multinucleated macrophages were also found in both liver and bone marrow tissues of mice, the proportion in those tissues being lower than that in the spleen, 35 this would be because the spleen is a larger lymphoid organ to remove.particulate matter in the blood and concentrates hardening and microorganisms.
In addition, the spleen presents the white pulp is the main lymphatic tissue of the spleen.It is the accumulation of lymphocytes around an arterial vessel.This aggregation of lymphocytes constitutes the lymphatic tissue known as the periarterial lymphatic sheath and is the first to react if microorganisms reach the spleen through the bloodstream.The red pulp is made up of splenic venous sinuses and cords (of Billroth), linings of splenic macrophages around the sinuses.The central artery continues from the white pulp and enters the red pulp in the form of capillaries. 37In visceral leishmaniasis, the spleen is infected by Leishmania, therefore it is possible to expect that MGC can also be found in this organ in In Vivo infections, so it is necessary to determine the percentage and pathophysiology of this type of cells.
A possible explanation for this might be that the formation of MGCs is due to the interaction and recognition between macrophages and cellular structures.It has been suggested that MGCs did not form when latex microspheres were administered to macrophages, indicating that phagocytic capacity and MGC formation are linked to distinct pathways. 25

CONCLUSION
In our study an MGC conversion was observed at 96 hours (50% ± 28.2) which would be a reaction due to cytokine overstimulation with IFNˠ trying to control parasitaemia and an overactivation of macrophages.In addition, in vitro MGC model could be used to study the physiopathology of MGC generation by Leishmania (V.) braziliensis infection.
Rojas-Jaimes J, et al.Generation of Multinucleated Giant Cells due to Leishmania (V.) braziliensis InfectionDISCUSSIONA previous study has shown how RAW 264.7 MGCs infected with Leishmania donovani perform hemophagocytosis of red blood cells and stimulate Leishmania survival through the glycoprotein SIRPα (a molecule involved in "don't eat me" cell recognition), in which a decrease of SIRPα expression was found in infected cells performing hemophagocytosis.These results coincide with those of Hong et al.

Figure 1 :
Figure 1: Growth per day of Leishmania (V.) braziliensis *The optimal growth of metacyclic promastigotes for the infection test was given in logarithmic phase x 10 5 /mL on the third day.

Figure 2 :
Figure 2: Growth per day of mouse macrophage commercial cell line RAW264.7 cells *The optimal growth of mouse macrophage commercial cell line RAW264.7 cells for the infection test was given in logarithmic phase x 10 5 /mL on the second day.