Reactive Nitrogen Intermediates Production by Macrophages of Mycobacterium bovis- Infected Goats and Supplemented with Dyhidroxyvitamin D3 in vivo

Problem statement: Tuberculosis (TB) remains one of the world’s major health problems. To evaluate in vivo indices of cellular sensitization, antigen-induced Reactive Nitrogen Intermediates (RNI) responses by blood mononuclear cells from Mycobacterium bovis BCG-infected goats supplemented with 1, 25 dyhidroxyvitamin D3 [1, 25-(OH)2D3]. Approach: An experimental, longitudinal and comparative study was planned. Twelve animals of goat cattle 20-to 24-month-old sannen selected themselves. Five samplings were made, previous to the inoculation (zero d), 3, 7, 14 and 21 d after applying the treatments. The mononuclear cells by the Ficoll-hypaque method were obtained. The RNI, nitrites and nitrates (NO2 − and NO3 ) were quantified by Enzyme-Linked Immunosorbent Assay (ELISA). Results: The treatment with the 1, 25(OH)2D3 stimulated the NO3 synthesis indicating, that by itself it is a good modulador of the micobacterial replication and in the treatment with M. bovis-BCG vaccine increased as a result to the treatment with 1,25(OH)2D3. The exhibition to M. bovis-BCG vaccine with the treatment with 1, 25(OH)2D3 was able to increase answer NO3 − in exposed animals. Conclusion: The 1, 25(OH)2D3 stimulated in vivo the production of RNI in goats exposed to M. bovis BCG vaccine.


INTRODUCTION
Tuberculosis (TB) remains one of the world's major health problems and the leading cause of death from a single infectious agent called Mycobacterium tuberculosis, the primary causative agent [1] . TB is a zoonotic disease that often becomes chronic with high rate of recurrence. The World Health Organization has declared TB a global emergency, the first disease so designated M. tuberculosis infects a third of the worldwide population to the year, in spite of the efforts of public health to control their dissemination [2,3] , the majority of such infections remain clinically latent [4] , the pathogeneicity include its ability to resist the harsh environment of the host macrophage and to persist within immunocompetent hosts [5] . The entry and survival of Mycobacterium in macrophages are central to the pathogenesis of TB. In addition, the ability of the macrophage to resist the growth of the microorganisms is dependent on the activation state of the macrophage. One essential component of tubercular host defense includes Nitric Oxide (NO). Waters, Palmer et al. [12] determined that there are two essential components of tubercular host defense include NO and Tumor Necrosis Factor alpha (TNF-α). Stimulation of inducible NO Synthase (iNOS) in macrophages and subsequent generation of RNI are potent mechanisms for mycobacterial killing [6,7] . Interestingly, it has been associated with risk of dissemination and mortality the absence of iNOS [8] . Furthermore, it has been used, NO inhaled like treatment for TB [9] . Therefore, iNOS synthesis is essential for maintaining stationary-level infection [10] , has been investigated iNOS presence in biopsies exhibiting dermal nerves from patients with untreated leprosy [11] . NO is readily produced by Mycobacterium-induced Peripheral Blood Mononuclear Cells (PBMC) from M. bovis-infected cattle. NO responses play an important role in organism and host defense. NO metabolism of the host is markedly altered in all infections. In recent reports, NO concentrations were found to be increased in culture supernatants and this is gaining wide acceptance for use in TB diagnosis [12] . Evaluation of NO response may prove useful for diagnosis of bovine TB, nitrite (NO 2 − ) is the stable oxidation product of NO and the amount of NO 2 − within culture supernatants is indicative of the amount of NO produced by cells in culture [12] . The 1, 25-(OH) 2 D3 is a powerful stimulus for the production of NO by macrophages, which are indicative of the defense of the host against the TB [13] . Has been considered that the active principle of vitamin D, the 1, 25 Dihydroxyvitamin D3 [1,25-(OH) 2 D3], it has turned to be a powerful regulator of the immune response.
Additionally, it has been observed that 1,25-(OH) 2 D3 suppresses to the growth of Mycobacterium in the macrophages through a dependent mechanism of NO [14] . An important point is that 25-OH-D3 is turned to 1,25-(OH) 2 D3 within the macrophage and the proportion of conversion increases within the alveolar macrophages and in followed human monocites of the stimulation with IFN-γ [15,16] , it is probable that the 1,25-(OH) 2 D3 triggers a dependent mechanism of NO that increases the destruction of Mycobacterium [17,18] . The M. bovis Bacillus Calmette-Guerin (BCG) vaccine, an attenuated strain of M. bovis, was developed for control of human TB more than 70 years ago and is still the only TB vaccine available. The protection against TB requires the induction of Th1 immune response, but studies with new-born animal have shown that they preferentially develop Th2-Type responses following immunization and are deficient in Th1 responses [19] . As a result, the bacterial transcription changes of the Th1mediated immune response are likely induced, directly or indirectly, by NO generated by infected macrophages [20] . BCG vaccination at birth induced a high level of immunity and that the sensitization of very young animals to antigens of environmental mycobacteria by 6 weeks of age did not affect the effectiveness of BCG [19] . Characteristic changes in RNI metabolism, are an integral part of the cellular immune assays. Weatherby et al. [21] , have confirmed that macrophages are among the first cells in innate resistance to intracellular microbial pathogens, they have been determining that the cytokines gamma-Interferon (IFN-γ), Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) and TNF-α activate macrophages to resist the growth of intracellular pathogens during the improvement in the production of antimicrobials molecules, including the RNI and the Reactive Oxigen Intermediates (ROI) [8,21] , proinflammatory cytokines such as interleukin (IL-12) play critical roles in the induction of host resistance to Mycobacterium [22] . In addition, the activated macrophages can turn the L-arginina to NO 2 − in the presence of the iNOS enzyme, with the development of a cytotoxic activity against tumor cells [23] and in front of bacterial infections [24] . The RNI are protective agents in infectious processes as VIH, Helicobacter pilori, M. tuberculosis, malaria and infections of respiratory tract and urinary, also have been observed in a great diversity of autoimmune diseases and chronic inflammatory diseases [25] . Additionally, as an end product in the reaction of iNOS has been determined the presence of nitrotyrosine in lung and tissues several, finding very similar levels [26] . Nevertheless, the targets of RNI in Mycobacterium are unknown [27] . Different studies have raised that the exhibition to the M. bovis BCG vaccine alters the specific immune response, it causing an increase of the immune response mediated by Th1 cells and an stimulation of the immune response Th2. In addition, Flynn et al. [28] demonstrated that Mycobacterium reactivation occurs if the production of Reactive Nitrogen Intermediates (RNI) is inhibited in a murine model of latency [28] . However, little is known about the role of these intermediates during latent infections. Different studies have been raised that the exhibition to the M. bovis BCG vaccine alters the specific immune response, it causing an increase of the immune response mediated by Th1 cells and a stimulation of the immune response Th2. In addition, Flynn et al. [28] demonstrated that Mycobacterium reactivation occurs if the production of Reactive Nitrogen Intermediates (RNI) is inhibited in a murine model of latency [28] . However, little is known about the role of these intermediates during latent infections. It has been observed which in infections of M. bovis the 1, 25-(OH) 2 D3 accelerates the specific production of and RNI [29] . But, it is necessary to consider the development of new investigations to clarify these discoveries. The aim of the present study was to quantify RNI production by macrophages from M. bovis-BCG infected goats supplemented with 1, 25-(OH) 2 D3.

Animals and immunizations:
Twelve goats were 20-to 24-month-old sannen. They were maintained with a diet supplemented with grain; they were supplied with water ad libitum.  [31] . The macrophages (5×10 5 cellsmL −1 ) were dispensed to sixwell plates, 2 mL −1 well. The cells were allowed to adhere for 48 h, was removed the supernatant from adherent cells and were resuspended in cool PBS by scraping, the viability of the cells was assessed and the cell suspension was obtained in a microcentrifuge tube (Eppendorf Axygen®), centrifuged (5 min, 1,500 rpm) to separate the cells from the PBS. The cells were suspended in an adequate cryoprotection it was constituted by 90% of fetal bovine serum and 10% of Di-Methyl-Sulfoxide (DMS) and later were stored at-70°C for the subsequent analyses. Sterilize DMSO was by filtration through a 0.2 µm cellulose membrane and was stored in small quantities (2 mL).

Measurement of Reactive Nitrogen Intermediates (RNI):
The RNI concentrations were obtained prior and after vaccination and challenge. RNI were measured spectrophotometrically as the stable metabolite NO 2 − according to the Griess method (1% sulfanilamide and 0.1% naphthylethylenediamine dihydrochloride in 2% phosphoric acid) [32] . NO [32] . Nitrite concentrations were calculated directly from the nitrite standard curve. To determine nitrate concentration, OD nitrite was subtracted from OD nitrate before comparison with the nitrate standard curve. Medium alone was used to calculate the assay background level and this was subtracted from all data [14] . In order to determine whether there was any association between nitrites or nitrates responses and protection against experimental infection with M. bovis, these RNI were measured at regular intervals during the vaccine trial.

Statistical method:
A design at random with repeat measures in the periods was used completely [33] . The procedure that it was used was PROC MIXED (SAS, 1999). The used statistical model was the following one: Concentrations of nitrite were determined by comparison with a standard of sodium nitrite. All samples were assayed in duplicate (version 8.0; SAS Institute Inc., Cary, North, Carolina).

RESULTS
Differences were observed in the nitrites and nitrates responses ( Fig. 1 and 2).

DISCUSSION
Vitamin D is known for its beneficial effects in diseases with strong Th1 responses, perhaps by altering Th1/Th2 balance in vivo [34] . The monocites [35] and the dendritic cells [36] express the receivers constituently of the 1,25(OH) 2 D3. However, has been observed that 1,25(OH) 2 D3 treatment inhibited chemokine-induced migration of T cells [37] . In this study, it was observed that NO 3 − concentration was significantly higher in animals with M. bovis-BCG plus 1,25(OH) 2 D3 compared with NO 2 − concentration. In addition, an increase in the NO 3 − production was observed with the challenge to the alone 1,25(OH) 2 D3. Indicating with that it is a good regulate of the micobacterial replication. In addition to this asseveration, has been determined that the active metabolite of the vitamin D is the 1,25(OH) 2 D3, that is a powerful regulator of the immune response [38] . Interestingly, in a previous study was determined that it is possible that RNI and ROI can inhibit the bactericidal replication (bacteriostatic) but not eradicate the bacterium (bactericidal) [39] . This agrees with studies [40] that demonstrated that the reactivation with M. tuberculosis happens if the RNI production is inhibited. The NO is a found unstable molecule in the biological systems and quickly is turned NO 2 − and NO 3 − . However, the amount of NO 2 − within culture supernatants is indicative of the amount of NO produced by cells in culture [12] .  [41] are generated. Has been considered that while most virulent strains of Mycobacterium are more susceptible to RNI [32] . The NO in the macrophages acts like a powerful microbicidal to destroy to the ingested microorganisms. We obtained a constant elevation of the concentration of NO 3 − to the course of the period of the study arriving at the 21 day to a concentration of 80.94 µ M −l . There are evidences of endogenous NO 3 − production, especially in answer to inflammatory stimuli where the NO 3 − production in vitro could be induced by the macrophages in the presence of lipopolysaccharide [42] . Therefore, it is deduced that the production of RNI in macrophages is essential for the defense of the host, mainly to exert bactericidal actions.
In addition, it was demonstrated that the stimulation of iNOS in macrophages and the subsequent generation of RNI are powerful mechanisms to kill the Mycobacterium [42] . Was reported that the first time that Mycobacterium induces mRNA for iNOS, iNOS protein, NO and peroxinitrite in human monocyte/macrophage cultures [31] . Moreover, Waters et al. [12] , have investigated 49 cattle with no history of TB and infected with M. bovis, they reported that NO concentration was found to be significantly higher in control group than those of healthy animals and they concluded that NO concentrations were altered probably by the effects of some immunocytokines as host defense elements of organism during infection. However, in culture supernatants exists IFN-γ that increased macrophage NO 2 − production. In addition, IFN-γ was found to be the critical mediator of NO production [43] . Protective immunity against intracellular bacteria such as TB species depends on the interplay between various T-cell subsets and cytokines. Cellmediated immunity to TB is likely to include the production of cytokines that activate macrophages and lymphocytes for production of anti-tuberculosis activities. In this study we determined that 1,25(OH) 2 D3 therapy during acute TB had beneficial effects both clinically and in modulating the systemic humoral and cellular immune response for increased host defense. The present study is the first to assess the effectiveness of BCG administered to goats to induce immune responses and protection against challenge with virulent M. bovis.

CONCLUSION
In conclusion, the 1,25(OH) 2 D3 stimulates in vivo the production of RNI in goats exposed to M. bovis BCG vaccine. Clearly, the high RNI levels observed were associated with the increased of Th1-mediated immune response and can help to study of many antitubercular treatments. RNI diagnosis can provide new clues about the different clinical outcomes after Mycobacterium infection. Further studies are necessary to determine the importance of RNI in cases with tuberculosis.