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Efficacy of Bothrops Jararaca Venom on the Changes in Immune Functions and TH1/TH2 Cytokine Balance in Murine Macrophages

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Efficacy of Bothrops Jararaca Venom on the Changes in Immune Functions and TH1/TH2 Cytokine Balance in Murine Macrophages Efficacy of Bothrops jararaca venom on the changes in immune functions and TH1/TH2 cytokine balance in murine macrophages Authors: Abstract Background: Hemorrhage, intravascular coagulation and Nuñez Valderrama RP.1 cardiovascular shock are effects commonly observed in Arteaga Figueroa L.1 victims bitten by Bothrops snakes. The severity of the envenomation is dependent on the immunological 2 Zucatelli Mendonça R. condition of the victim. This study was designed to Petricevich VL.1* evaluate the effects of Botrhops jararaca venom on macrophage functions and TH1 / TH2 cytokine balance. 1 Facultad de Medicina, Universidad Methods: Various amounts of B. jararaca venom were used, and the activation of macrophages was determined Autónoma del Estado de Morelos by cytotoxicity assays, hydrogen peroxide production, (UAEM), Laboratorio de Inflamación cell expansion, and phagocytosis. The release of y Toxicología, Calle Iztaccihuatl cytokines presents in macrophages treated with B. jararaca venom were measured by ELISA. Esquina Leñeros, Colonia Volcanes, Results: The results showed that the Bothrops jararaca 62350 Cuernavaca, MOR, México. venom altered the function of peritoneal macrophage cultures, increasing cytotoxicity and production of H O . 2 Instituto Butantan São Paulo – 2 2 The venom was able to inhibit cell expansion and Brasil phagocytosis. The effects of the venom on immunological mediator production in peritoneal 1* Correspondence author: macrophage cultures resulted in an increase in TNF-α, IL-1β and IL-6 production at 24 hours of treatment. The Vera L. Petricevich; E-mail: maximum production of NO and IFN-γ was observed at [email protected] 24–48 and 48-72 hours, respectively. The elevated production of IL-4 and -10 was observed from 24 up to 120 hours after venom exposition. Keywords: venom, Conclusion: The combined data suggest that Bothrops immunomodulator, activation, jararaca venom has an immunomodulatory effect for the first 24 hours; of venom treatment was observed a mediators balance between TH1 and TH2 cytokines. After this time period with a TH2-dominant response in the TH1/Th2 cytokines. Medical Research Archives, Vol. 4, Issue 7, November 2016 Efficacy of Bothrops jararaca venom on the changes in immune functions and TH1/TH2 cytokine balance in murine macrophages 1. Background conditions including severe poisoning The envenomation caused by a snake (14,15). bite from Bothrops jararaca is a common As part of the inflammatory process, medical problem in South America (1). The tissue damage after a snake bite occurs venom of these snakes has been described when the substances released in the affected to have proteolytic action, with coagulant, area activate macrophages, which begin to cytotoxic and rhabdomyolytic effects (2). devour damaged tissues (16-18). The more Recent studies have shown that Bothrops intense the inflammatory reaction is, the jararaca venom (VBj) is a complex mixture greater the tissue injury, because the first of compounds that induce various line of immune defense is macrophages, biological activities and has been which get activated by the inflammatory previously noted to be involved in process. After an injury or infection disordered homeostasis of the inflammatory macrophages can produce many factors that response associated with envenomation influence their own physiology (19,20). (3-10). Immune system activation during They exhibit different phenotypes, which envenomation attempts to maintain alter their cell morphology, antigen homeostasis of the victim’s body; and expression and their various surface septic shock can develop as a result of proteins that trigger responses to excessive immune responses, with the environmental signals (21,22). There are release of associated immune mediators two general phenotypic categories into (11). Various studies have shown that which macrophages are classified: classical Bothrops jararaca venom is able to induce activation (M1) and alternative activation an increase in leukotriene B4, thromboxane (M2) (17,23). A2, interleukin-6 (IL-6) and tumor necrosis M1 macrophages can also be factor (TNF-α) and in various adhesion generated transiently, in response to stimuli molecules (12,13). Another mediator by TH1 cytokines, such as interferon- observed after envenomation is nitric oxide, gamma (IFN-γ), TNF-α and IL-6, as well as which has been associated with a number of Copyright 2016 KEI Journals. All Rights Reserved Page │2 Medical Research Archives, Vol. 4, Issue 7, November 2016 Efficacy of Bothrops jararaca venom on the changes in immune functions and TH1/TH2 cytokine balance in murine macrophages by microbial triggers. Macrophages M1 are associated with processes of resolution or essential components in the host defense chronic inflammation (17). response, and their activation must be Macrophages play key roles in the tightly controlled because excessive immune system by acting as antigen- cytokines and other mediators can lead to presenting cells, producing cytokines and damage to the host tissues (24). In contrast, also having a very important role as M2 macrophages are involved in parasite phagocytic cells, with a high capacity that containment and immunoregulatory can be activated by soluble mediators or functions by reducing secretion of other substances as part of the host response proinflammatory cytokines (24). These to pathogens. The phagocytic process cells are activated by IL-4, IL-13, involves the participation of various cellular glucocorticoids, immune complexes, and components and, is critical for nonspecific IL-10 – all factors representative of the and specific host immune responses (26). TH2 type immune response. Early Different factors are able to stimulate the production of IL-4 leads resident phagocytic response, and the response can macrophages to enter the M2 program, and be induced to varying levels (27). Stimuli of is associated stimulation of characteristic phagocytosis include interleukins IL-1 and arginase activity (25). Another IL-6, while decreases in phagocytic characteristic observed in M2 macrophages function are often due to disorders of is the secretion of high amounts of IL-10 peroxidation (28,29). Three important and low levels of IL-12; they also have cellular factors/processes are critical to efficient phagocytic activity and high phagocytosis: a) cytoskeletal elements, expression of scavenging molecules. which mediate ingestion, b) maturation of Disease states are often associated with the vacuole and c) the inflammatory dynamic changes in the activation of response (30). The activation of appropriate macrophages, with M1 cells being involved effectors of the immune response determine in the initiation and maintenance of the differentiation of precursor cells into inflammation, and M2 cells being TH1, TH2, TH17, T-regulatory T cells Copyright 2016 KEI Journals. All Rights Reserved Page │3 Medical Research Archives, Vol. 4, Issue 7, November 2016 Efficacy of Bothrops jararaca venom on the changes in immune functions and TH1/TH2 cytokine balance in murine macrophages (TH0/Treg), TH9, and follicular TH cells, Nacional de Salud Publica (Cuernavaca, and granulocyte/macrophage colony México). The animal studies were evaluated stimulating factor (GM-CSF) (31). The and approved by the Committee for Basic objective of the present study was to Research and the Clinical School of evaluate the efficacy of Bothrops jararaca Medicine, with registration number venom on peritoneal macrophage 41MM2013-2. The animals were sustained activation. and used under strict ethical conditions according to international 2. METHODS recommendations. 2.1 Chemicals, reagents and 2.4 Peritoneal cells: The peritoneal buffers: RPMI-1640 media, Actinomycin cells from groups of females BALB/c mice D, orthophenyldiamine (OPD), fetal calf were obtained by the method previously serum (FCS), and recombinant TNF were described by Cohn & Benson, 1965 (32). purchased from Sigma (St. Louis, MO, Briefly, the peritoneal cavity of each animal USA). Cytokines (recombinant, antibodies was washed with RPMI-1640 medium and for capture and detection) were purchased the cell suspension obtained was from BD Biosciences Pharmingen (USA). centrifuged at 400 rpm for 5 min. The cell pellet recovered was adjusted to a 2.2 Venom: Lyophilized venom concentration of 1 X 106 cells/mL in RPMI- from Bothrops jararaca (VBj) was obtained 1640 with 10% FCS and was then from Dr. R. Zucatelli Mendonça (Instituto distributed onto microplates. After 2 hours Butantann – SP – Brasil), and stored at -20 of incubation at 37 °C and 5% CO the °C. The lyophilized venom used in different 2 culture media was removed and cells were assays always was reconstituted in RPMI- exposed to different amounts of VBj and 1640 media. cultivated under the same conditions for the 2.3 Animals: Females BALB/c indicated experimental times. mice (6 – 8 weeks old, weighing 15 to 20 g) were purchased from Bioterio – Instituto Copyright 2016 KEI Journals. All Rights Reserved Page │4 Medical Research Archives, Vol. 4, Issue 7, November 2016 Efficacy of Bothrops jararaca venom on the changes in immune functions and TH1/TH2 cytokine balance in murine macrophages 2.5 Activation assays at 37 °C with 5% CO2 and then 10 µL of 2.5.1 Cytotoxic assay: 1M NaOH were added to each well and the Macrophages cultures were treated with optical density was measured with a 620 different amounts of VBj, and incubated for nm filter using a microplate reader. The various times periods. The media was then absorbance
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