Nitric Oxide Not Apoptosis Mediates Differential Killing of Mycobacterium bovis in Bovine Macrophages

To identify the resistance phenotype against Mycobacterium bovis in cattle, we used a bactericidal assay that has been considered a marker of this trait. Three of 24 cows (12.5%) were phenotyped as resistant and 21 as susceptible. Resistance of bovine macrophages (MΦ) to BCG challenge was evaluated for its association with SLC11A1 GT microsatellite polymorphisms within 3′UTR region. Twenty-three cows (95.8%) had a GT13 genotype, reported as resistant, consequently the SLC11A1polymorphism was not in agreement with our bactericidal assay results. MΦ of cows with resistant or susceptible phenotype were challenged in vitro with virulent M. bovis field strain or BCG, and nitric oxide production, bacterial killing and apoptosis induction were measured in resting and LPS-primed states. M. bovis field strain induced more apoptosis than BCG, although the difference was not significant. Resistant MΦ controlled better the replication of M. bovis (P<0.01), produced more nitric oxide (P<0.05) and were slightly more prone to undergo apoptosis than susceptible cells. LPS pretreatment of MΦ enhanced all the functional parameters analyzed. Inhibition of nitric oxide production with n G-monomethyl-L-arginine monoacetate enhanced replication of M. bovis but did not modify apoptosis rates in both resistant and susceptible MΦ. We conclude that nitric oxide production not apoptosis is a major determinant of macrophage resistance to M. bovis infection in cattle and that the influence of SLC11A1 gene 3′UTR polymorphism is not associated with this event.


Introduction
Bovine tuberculosis is a disease caused by Mycobacterium bovis that was recognized more than 100 years ago and still affects the global cattle industry [1]. Control of bovine tuberculosis is actually driven by the ''test and slaughter'' strategy, whereby bovine PPD reactor cattle are identified as infected and slaughtered [2]. In addition to the threat for public health, bovine tuberculosis has a significant impact on agriculture causing worldwide annual losses of $3 billion due to depopulation of herds, herd trade restrictions and reduced agricultural productivity, particularly in the developing world and in some developed countries where a residual persistence of infection that cannot be eliminated because of the presence of wildlife reservoirs [3][4][5].
M. bovis is an intracellular pathogen that resides mainly in MW. Both innate and acquired mechanisms of immunity play important roles in the control of infection. T cell-macrophage interactions have great relevance in the development of protective immunity in bovine tuberculosis and in the generation of immune response parameters utilized in diagnostic tests, such as the specific release and measurement of IFN-c that has the property to activate MW to be more bactericidal [6]. The bactericidal efficacy of MW is considered the basis of innate immunity to tuberculosis [7,8] and a phenotypic marker of cattle resistance to M. bovis infection [9].
It is well known that pathogenic mycobacteria induce MW to undergo cell death which is modulated by host resistance to mycobacterial infection [10,11]. Monocytes/MW from mice resistant to Mycobacterium tuberculosis are more prone to undergo apoptosis after infection than those from susceptible mice that largely die by necrosis [12,13]. Likewise mouse models, widespread apoptosis of blood monocytes induced by mycobacteria is observed in resistant PPD-positive healthy subjects, as opposed to patients with active TB in which cells are induced to necrosis [14,15]. Macrophage apoptosis is a host strategy against mycobacteria by depriving the intracellular niches required by bacteria, by limiting bacterial replication and by promoting T cell crosspriming by antigen-presenting cells [16][17][18]. For instance, apoptosis of monocytes infected with M. bovis BCG and Mycobacterium avium-Mycobacterium intracellulare exposed to ATP + and H 2 O 2 respectively resulted in loss in viability of intracellular bacilli [19,20]. Apoptosis is modulated by pathogenic mycobacterial strains and is a correlate of virulence to avoid innate host defense. Virulent strains of M. tuberculosis induce more necrosis and less apoptosis than the attenuated/avirulent ones [21][22][23]. Virulent field strains of M. bovis induce bovine MW to die by apoptosis in a time-dependent manner by a mechanism independent of caspases that is influenced by bacterial load and macrophage soluble factors [24,25]. TNFa release of bovine activated MW by M. bovis alone or in combination with LPS or IFN-c is strongly suggested to play a role in apoptosis induction [6,26,27]. Nitric oxide (NO), produced in bovine MW [6,28], plays a major role in the killing of intracellular pathogens by MW, and it has also been involved in the regulation of cell death [27,29]. Previous results from our group have suggested that MW with a resistant phenotype, produce more NO and are more prone to undergo apoptosis after M. bovis infection than cells with a susceptible phenotype, and that such functional differences are abrogated by the IL-4 effect [30]. In addition, bovine macrophage resistance to intracellular pathogens was initially linked to a polymorphism within the 39untranslated region of SLC11A1 (Nramp1) gene [31], but eventually was refuted to be associated to M. bovis or Brucella abortus resistance in cattle with different genetic backgrounds and naturally challenged under field conditions [32,33].
Altogether, these observations have facilitated us to explore the implication of nitric oxide and apoptosis in the performance of bovine MW on resistant (R) and susceptible (S) phenotype. In this study, we demonstrated that R MW produced more nitric oxide and were slightly more prone to undergo apoptosis than S cells. The blockade of nitric oxide production enhanced the replication of M. bovis in both R an S cells but had no effect on apoptosis induction. We conclude that nitric oxide, not apoptosis, is a major determinant of macrophage resistance to M. bovis infection in cattle and that the SLC11A1 gene 39UTR polymorphism is not associated in this event.

Animals
Twenty-four Holstein-Friesian females 2 to 7 years old were used in this study as blood cell donors for phenotyping as resistant or susceptible to in vitro M. bovis bacille Calmette-Guerin (BCG) infection under macrophage bactericidal assay. Blood samples were collected from the jugular vein of each cow under aseptic conditions using approved safe manual restraint to preserve the animal welfare. All cattle were housed under uniform conditions and nutritional regimens at the Center for Practical Teaching and   Research in Animal Production and Health (FMVZ-UNAM, CEPIPSA, Topilejo, Tlalpan, Mexico City.). The cattle were randomly selected from a tuberculosis-free dairy herd (not infected, exposed or vaccinated; status officially declared since 1998) and all animals were negative for the single intradermal comparative cervical tuberculin (SICCT) test for M. bovis and M. avium antigens.

Bacteria
We used the attenuated BCG Danish strain and the virulent M. bovis 9926 and 129QP field strains isolated from tuberculous lesions of PPD positive cattle. Bacteria were grown at 37uC under shaking conditions in Middlebrook 7H9 broth with 0.05% Tween 80 and supplemented with 10% of OADC enrichment (Becton Dickinson, Cockeysville MD) to mid-log phase to standardize the growth-replication state of cells. BCG strain reached the mid-log phase six days earlier (8th day) than field strains (14th day). Bacteria were suspended in CRPMI, passed twice through a 27gauge needle and stored at 280uC in 1 mL aliquots. Inoculums were titrated by plating CFU serial dilutions on Middlebrook 7H11 medium (Difco Laboratories, Detroit MI) plus 10% of OADC.

Bovine Monocyte-derived MW
For monocyte isolation, 150 ml of whole blood was collected in acid citrate dextrose (ACD) buffer. MW were obtained from peripheral blood monocytes by the method of Campbell et al., with slight modifications [34]. Blood mixed with ACD was centrifuged at 11006g for 20 min at room temperature (RT). Buffy coats were suspended with an equal volume of 13 mM sodium citrate in PBS pH 7.4, and carefully layered onto 0.5 volumes of Percoll suspension (Pharmacia, Uppsala, Sweden) at specific density of 1.077 adjusted with 130 mM trisodium citric acid, 5% BSA and PBS. Layers were centrifuged at 10006g for 30 min at RT. Leukocytes were recovered and washed three times with 5% autologous plasma in PBS citrate for 10 min at 3506g and resuspended at 5610 6 cells/ml in CRPMI (RPMI 1640 plus 2 mM L-glutamine, 0.1 mM non-essential aminoacids, 1 mM sodium piruvate and 20 mM sodium bicarbonate) (Gibco BRL, Life Technologies, Grand Island, NY) containing 4% autologous serum, and 5 ml were placed into 50 ml Teflon flasks (Nalgene, Nalge Nunc, Naperville IL) and cultured for 4 h at 37uC and 5% CO 2 . Non-adherent cells were removed by three washes with prewarmed PBS, and adhered monocytes were cultured at the above conditions in CRPMI with 12.5% autologous serum for 12 days. MW were harvested by repeated gently pipetting after chilling flasks on ice for 45 min and suspended at densities indicated on each experiment. Macrophage priming was achieved by culturing cells with 100 ng/mL of Lipopolysaccharide (LPS) from E. coli

Macrophage Infection and Macrophage Bactericidal Assay
MW were infected with BCG or M. bovis virulent strains for 4 h at a multiplicity of infection (MOI) of 10 mycobacteria (CFU) per macrophage and centrifuged at 2006g for 10 min. Macrophage bactericidal assay was performed with 1610 4 MW per well in Nunc MiniTrays (Nalge Nunc International, Rochester, NY) [9]. After 4 h, non-ingested and non cell-attached bacteria were removed by four washes with RPMI. Initial count of mycobacterial uptake was quantified by plating serial dilutions of cell suspension after lysis with 0.5% Tween 20. Intramacrophage  mycobacterial growth was assessed at 24 h postinfection. The number of ingested bacteria was calculated by dividing total CFU by total MW. Mycobacterial intracellular survival was represented by mycobacterial growth index and was calculated by dividing the number of CFU at end of assay (24 h) by the number of CFU at start of assay (0 h) expressed as percentage. A value of 65% or less of bacterial survival for M. bovis BCG has been correlated with actual numbers of cattle designated as resistant (R), while higher values correlate with designation of susceptible (S) and therefore is considered a phenotypic marker of the resistant trait [9]. MW from the three resistant and three susceptible cattle were used for further functional comparative analysis.

Genotyping of SLC11A1(Nramp1) 39UTR Polymorphism
Blood samples collected into tubes containing EDTA were used for genomic DNA extraction and genotyping. Genotyping of all 24 cows was performed by Single Stranded Conformation Analysis (SSCA). One hundred ng of genomic DNA, was amplified using primers specific for the 39 untranslated region of the bovine SLC11A1 (Nramp1) gene (nucleotide positions 1814-1989). The forward primer (59-AAGGCAGCAAGACAGACAGG-39) and reverse primer (59-ATGGAACTCACGTTGGCTG-39) were used as previously described [33]. PCR was performed as previously described [33]. The PCR products were denatured at 94uC for 5 min in formamide solution (95% formamide, 10 mM EDTA pH 8.0, 0.5% Xilene cyanol, 0.5% bromofenol blue) (Sigma Chemical CO, St Luis,MO), run in a 6% acrylamide gel at 55 V for 150 min, and silver stained (Silver Stain Plus kit, cat. 161-0449, BioRad, Hercules, CA) on the gel. DNA samples of genotypically resistant and susceptible cattle to standardized virulent Brucella abortus in vivo challenge were used as controls for the assay [35]. DNAs of two susceptible and two resistant cows were subcloned and sequenced.

Measurement of Nitric Oxide and Apoptosis
Nitrite (NO 2 ), as an indicator of NO production, was determined in 100 ml cell culture supernatants of 5610 4 MW with 100 ml of Griess reagent (Sigma Chemical Company, St. Louis, MO). NO 2 content was calculated from a NaNO 2 standard curve.

Statistical Analyses
The association between phenotype and allele polymorphism of the 39UTR SLC11A1 (Nramp1) gene was verified by Fisher's exact test. Differences among susceptible and resistant phenotypes and between M. bovis strains were tested for statistical significance by one-way ANOVA. All tests were performed using GraphPad Prism version 5.00 for Windows (GraphPad Software, San Diego California USA, www.graphpad.com). A p value equal or less than 0.05 was considered to be statistically significant.

Herd Screening for Resistant Cattle through Macrophage Bactericidal Response
According to Qureshi T, et al. a cut-off point of 65% of BCG survival was established to designate an animal as resistant (R) or susceptible (S). In our study, MW of 3/24 cattle (12.5%) restricted BCG intracellular replication on levels equal or lesser than 65% thus they were classified as R, otherwise cattle were classified as S (21/24; 87.5%). Virulent M. bovis grew to a larger extent in all bovine MW but R MW were more efficient in controlling intracellular growth than S cells (P,0.05) (Figure 1). These differences were not related to phagocytosis since all MW ingested a similar number of mycobacterial UFC regardless of their phenotype (data not shown).

SLC11A1 (Nramp1) 39Untranslated Region Polymorphisms and their Association with Resistance to M. bovis in Cattle
Resistance of bovine MW to BCG challenge was evaluated for the association with SLC11A1 GT microsatellite polymorphisms within 39UTR region. SSCA results showed only one susceptible genotype among all 24 cows analyzed, corresponding to the heterozygous GT 13 /GT 14 microsatellite repeats in 39untranslated region confirmed by DNA sequencing (Figure 2). Given the proportion of susceptible (21/24) and resistant (3/24) phenotypes into the herd and the very small frequency of susceptible genotype (1/24) among the same sample population, it was obvious that there is no association between the SLC11A1-resistant allele and the SLC11A1-susceptible allelle with the resistant or susceptible phenotypes, respectively (Table 1).

Nitric Oxide Production by M. bovis-infected R and S Bovine MW
Infection of bovine MW with BCG or virulent M. bovis revealed a functional difference in controlling growth of both strains among cattle. Levels of NO released in response to M. bovis were significantly increased compared to levels released by uninfected cells (,1 mM), but were much higher in R (,45 mM) MW than in S cells (,18 mM) (p,0.05). LPS pretreatment, used as an unbiased stimulus of iNOS, induced large NO release (,40 mM) in R and S MW without revealing any differences between them, however LPS pretreatment followed by M. bovis infection revealed a higher NO production by the R (,60 mM) MW compared to S cells (,49 mM) without significant differences (p.0.05). Neutralizing NO production with MMLA in M. bovis and/or LPS treatments confirmed that NO production was dependent and specific for the stimulus (Figure 3).

Nitric Oxide and Macrophage Resistance to M. bovis in Cattle
NO production by R and S bovine MW was M. bovis and/or LPS dependent, but the differences were significant only in response to M. bovis. NO amount was related to the macrophage microbicidal phenotype. We sought to determine if M. bovis replication was in a direct relationship with NO production by MW. Enhancing NO production with LPS stimulus (as shown in figure 3) significantly decreased M. bovis replication in MW (Fig. 4), with higher reduction in R MW (3 fold reduction, from 95% to 29% bacterial survival) than in S cells (2 fold reduction: from 145% to 72% bacterial survival) (p,0.05). Blocking NO by the addition of MMLA in M. bovis infected MW substantially reduced their inherent ability to restrict M. bovis replication (Fig. 4), resulting in a ,2 fold increase in bacterial survival in R MW (from 95% to 177%; p,0.05). However, R MW pre-stimulated with LPS and NO blocked with MMLA had a reduced increase in M. bovis replication (,1.4 fold, from 95% to 138%; p.0.05) while resting or LPS-stimulated S MW had only 1.29 and 1.13 fold increases respectively (from 145% to 187% and 164% bacterial survival; p.0.05) (Fig. 4).

Macrophage Apoptosis and M. bovis Resistance
Many more cells with chromatin condensation were observed in M. bovis-infected MW than uninfected cells, an indication of apoptosis induced by M. bovis that was confirmed by specific labeling of DNA fragmentation (nick ends) by TUNEL ( Figure 5). Given that we observed differences in MW controlling M. bovis replication, we sought to determine any correlation of apoptosis with mycobacterial control by comparing proportions of R and S M. bovis-infected MW with apoptosis. Numbers of MW with    Figure 6).

Nitric Oxide Effect in Apoptosis Induction of M. bovisinfected Bovine MW
Induction of apoptosis in M. bovis-infected bovine MW was associated with the release of high levels of nitrite in the culture medium at 24 h post-infection. Given that bovine MW differentially controlled M. bovis replication in a NO dependent fashion and that the proportion of macrophage undergoing apoptosis agreed with NO levels, we sought to determine the association of NO with apoptosis induction of M. bovis-infected MW. Blocking NO by the inclusion of MMLA did not modify the response of R or S MW to die upon M. bovis infection (Fig. 7).

Discussion
Infection with M. bovis is a major health problem causing zoonoses and economical losses in cattle industry in many countries [2,36]. M. bovis infects and replicates within the hostile intracellular environment of MW as a mechanism of persistence by avoiding the immune system, or as a mechanism of dissemination to other cells and tissues by inducing macrophage to die by necrosis [37], therefore the response elicited by MW after the encounter of M. bovis is crucial in determining the outcome with infection whereby they are potentially involved in innate resistance of cattle to tuberculosis.
Limited information is available on the inherent ability of MW to control M. bovis replication through bactericidal products (NO) and other mechanisms implicated in resistance to infection, and relatively little is known about the differences of these factors between MW having a differential control over M. bovis. We used the innate macrophage ability to restrict BCG replication as a correlate with natural resistance to intracellular pathogens measured by a microbicidal assay [9] in order to select resistant and susceptible cattle as cell donors for the experiments. Using this assay in a sample of a dairy herd (n = 24 cows), we identified 3/24 (12.5%) with a phenotype of resistance. The proportion of cattle detected as resistant in our experimental in vitro conditions using microbicidal assay, agree with the frequency of natural disease resistant cattle detected at slaughter, supporting the usefulness of the assay [33]. In that study, cattle were tuberculin-skin test negative animals that lived in herds with naturally-occurring bovine tuberculosis which were constantly exposed to M. bovis but no tuberculosis-like lesions were observed at necropsy and the M. bovis-culture was negative [38].
A microsatellite (GT)13 repeat at the 39UTR of bovine SLC11A1 (Nramp1) gene was initially related with resistance of cattle to M. bovis and other intracellular pathogens [35,39]. By further investigation of the M. bovis bactericide response in MW, we were able to identify improved phenotyping of previously selected R and S cattle. SSCA indicated that homozygous alleles with microsatellite lengths of 13 GT dinucleotide repeats (resistant genotype) were found in all three cows resistant to in vitro challenge with BCG matching perfectly, while heterozygous alleles with microsatellite lengths of 13/14 GT dinucleotide repeats (susceptible genotype) were found in only one of the 21 cattle susceptible to in vitro challenge by M. bovis BCG, while the other 20 susceptible cattle had a resistant genotype, therefore we were unable to assign the correct phenotype to the genotype in S cattle under our conditions. One possible reason for this is that we selected a phenotype based on the performance of the microbicidal assay not the genotype of our dairy herd. Previous standardized in vivo wild type B. abortus challenge studies in unvaccinated, unexposed 180 day pregnant first calf heifers revealed that microsatellite lengths of  13 GT dinucleotide repeats were found in cattle resistant to challenge by B. abortus, while microsatellite lengths of 14, 15, and 16 GT dinucleotide repeats were found in cattle susceptible to B. abortus challenge. However a minority cattle with # 13 GT were susceptible, and a majority $ 14 GT cattle were resistant, thus the association of natural resistance was not perfect for the GT polymorphisms of SLC11A1 in these studies [35]. The same conclusion was drawn in other studies on polymorphisms within this region of the gene that from cattle of different genetic backgrounds were not associated with disease resistance to B. abortus or M. bovis in cattle under field conditions [32,33,40]. The explanation maybe the discrepancies of standardized (a single specific CFU dose) vs. natural field (discontinuous dose and multiple challenges) challenge differences in assigning phenotypes vs. genotypes, rather than failing of the gene in supporting resistance. Analysis of immune correlates related to natural resistance to Brucella revealed a differential response in macrophage activation between resistant and susceptible cattle, along with the associated segregation of specific alleles of bovine SLC11A1 as observed in R cattle in the present study.
The frequency of natural resistance to brucellosis was shown to be 18% in cross-bred cattle [41]. Although we did not screen a large number of cattle, our results identified 12.5% (3/24) of cattle with the resistant phenotype suggesting that the percentage of natural disease resistance against Mycobacterium bovis in cattle is not high as well, thus limiting the possibility of identifying R animals. In this study, we included the three R individuals available and matched with an equal size sample of S animals for functional comparisons on the basis that they all were of the same breed, similar age and belonged to the same herd with the same housing conditions and that no substantial differences were detected. In addition, three independent replicas were performed for each experiment, each one including three internal repetitions to reduce the impact of individual and experimental variations. The mechanisms involved in differential control of M. bovis between R and S MW were investigated by comparing responses under these circumstances. We primarily identified the role of NO in the M. bovis killing response of resistant MW. It was previously shown that NO generated by M. bovis infection in bovine MW do not reach levels that affect mycobacterial replication [6,42]. Previous works also indicated the requirement of priming with an individual or combinatory stimulus of IFN-c and LPS to block the replication of mycobacteria, but the effect apparently is not determined by the release of NO, as neutralizing NO, via the inclusion of the inhibitor MMLA, had no impact on the intramacrophage replication of mycobacteria [6,43]. We observed that NO release was substantially induced in LPS-primed MW alone or after M bovis infection with significantly higher levels in resistant compared to susceptible MW. This suggested that NO generation is a key process in anti-mycobacterial activity in resistant MW. Indeed priming with LPS enhanced macrophage resistance to M. bovis infection which was abrogated by blocking NO synthesis with MMLA incorporation, and rendered resistant cells as permissive as untreated control cells and also transformed susceptible cells to an even more permissive state, suggesting that additional differential factors are involved in mycobacterial replication blocking than only NO. In addition, LPS had no effect on bacterial uptake in cells. These findings are partially in disagreement with data generated in previous reports about the role of NO in blocking M. bovis replication [6,43]. The situation is more complex in functional studies of cells involving intrinsic phenotypic differences. Although our data suggest that phenotypic resistance against M. bovis is strongly associated to NO release, as a mechanism of generation of maximal bactericidal activity, other possible soluble factors as TNF-a or other mechanisms including iron deprivation [20,44,45] may be involved, however their role in bacterial killing require further evaluation in the bovine model.
In our system, MW with enhanced mycobactericidal activity also had enhanced chromatin condensation, whereas those that allowed M. bovis replication had fewer apoptotic counts. This suggested a role for macrophage apoptosis in macrophage resistance to M. bovis and a link with NO release. Pathways of apoptosis induction in MW via the generation of NO have been described [26]. The exact bactericidal mechanisms activated for apoptosis in the infected macrophage remain undefined, but may be related to calcium and ATP dependent effects [17,20].
Previous works have suggested that induction of bovine macrophage apoptosis is closely linked to the emergence of macrophage resistance to M. bovis replication, which is dependent on TNF-a release [6,30]. Virulent M. bovis has been shown to induce release of higher levels of pro-inflammatory mediators by bovine MW, compared to levels released upon BCG infection. Among these mediators are TNF-a, IL-12 and NO, whose release is significantly enhanced by IFN-c prior to infection and by bovine Natural Killer cells direct contact [6,43]. We previously reported that bovine macrophage apoptosis is induced by live M. bovis and by its cellular components [24,25].
It has been shown that pathogenic mycobacteria induce less apoptosis in monocytes/MW than avirulent mycobacteria, as a mechanism of evasion of host immune response. Virulent M. tuberculosis induces less apoptosis and more necrosis of human monocyte/MW than attenuated M. tuberculosis H7Ra or BCG [10,11,23]. Rojas et al. (1997) showed that avirulent M. tuberculosis H37Ra induced lesser macrophage death and NO production than virulent M. tuberculosis H37Rv in mouse MW [12]. These data provide more evidence that macrophage apoptosis is a mechanism of innate resistance to mycobacterial infection. It has been recently found that after apoptosis of M. tuberculosis-infected MW, the apoptotic cell debris harboring the bacterium are rapidly taken up by uninfected MW and delivered to the lysosomal compartment and M. tuberculosis is killed, indicating that apoptosis itself is not intrinsically bactericidal but facilitates presentation of bacteria sequestered within an apoptotic body to be presented to other MW, thus indirectly functioning as a microbicidal mechanism [46].
The role of macrophage apoptosis in immune resistance against mycobacteria has been studied in great detail in mouse models. Genetically resistant mouse MW responses to a standardized challenge with intracellular pathogens (B10R, Nramp +/+ , sst1 R ), exhibit apoptosis after M. tuberculosis infection at superior levels than susceptible MW (B10S, Nramp 2/2 , sst1 S ) and correlate with generation of NO and TNF-a and demonstrate better restriction of M. tuberculosis [12,13,47]. In addition, people who are infected with M. tuberculosis but do not develop active disease exhibit higher apoptosis than necrosis of monocytes/MW induced by M. tuberculosis or its components compared to people who progress to a clinical outcome and experience more necrosis than apoptosis [14,15].
In our natural field model, for all conditions studied resistant MW had higher apoptosis values than susceptible ones, however only a few were significantly superior as were in controlling intracellular growth of M. bovis. Although our data suggested a relationship between NO release and apoptosis induction, blocking NO by inclusion of MMLA did not abrogate apoptosis induction by M. bovis neither in resistant nor in susceptible MW. Even though macrophage apoptosis was induced at a different level in susceptible and resistant MW, the role in macrophage resistance to M. bovis was inconclusive and requires further definition. In conclusion, our results strongly suggest a key role for NO in bovine macrophage resistance to M. bovis.