José A. Gutiérrez-Pabello

Upregulation of Thymosin β-10 by Mycobacterium bovis Infection of Bovine Macrophages Is Associated with Apoptosis

ABSTRACT Bovine macrophages underwent apoptosis as a result of infection with a Mycobacterium bovis field strain. Macrophages infected with a multiplicity of infection (MOI) of 25:1 developed chromatin condensation and DNA fragmentation at 4 h and 8 h, respectively, whereas changes in chromatin condensation induced by MOIs of 10:1 and 1:1 required more time and had a reduced number of apoptotic cells. Not only infected macrophages underwent apoptosis, but also uninfected bystander macrophages became apoptotic. Increased differential expression of thymosin β-10 was identified in M. bovis-infected bovine macrophages by differential display reverse transcriptase PCR. Phagocytosis of latex beads had no effect on the expression of thymosin β-10, whereas bacterial suspensions upregulated thymosin β-10 expression, suggesting that M. bovis or mycobacterial products are essential in the process. Heat-inactivated M. bovis induced a slight increase in thymosin β-10 mRNA, whereas live virulent and attenuated M. bovis organisms increased the gene expression almost twofold. A mouse macrophage cell line (RAW 264.7) overexpressing the bovine thymosin β-10 transgene had spontaneous apoptosis at a higher rate (66.5%) than parental cells (4.7%) or RAW cells harboring the empty vector (22.8%). The apoptotic rates of the overexpressing cells were significantly higher when compared with both the empty vector transfected (P < 0.01) and parental cells (P < 0.001). Our evidence suggests that upregulation of thymosin β-10 in M. bovis-infected macrophages is linked with increased cell death due to apoptosis.

Apoptosis-Inducing Factor Participation in Bovine Macrophage Mycobacterium bovis-Induced Caspase-Independent Cell Death

ABSTRACT Mycobacterium tuberculosis complex species survive and replicate in phagosomes of the host cell. Cell death (CD) has been highlighted as one of the probable outcomes in this host-pathogen interaction. Previously, our group demonstrated macrophage apoptosis as a consequence of Mycobacterium bovis infection. In this study, we aimed to identify the contribution of apoptotic effector elements in M. bovis-induced CD. Bovine macrophages were either infected with M. bovis (multiplicity of infection, 10:1) or treated with an M. bovis cell extract (CFE). Structural changes compatible with CD were evaluated. Chromatin condensation was increased three times by the CFE. On the other hand, a terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assay demonstrated that levels of DNA fragmentation induced by M. bovis and CFE were 53.7% ± 24% and 38.9% ± 14%, respectively, whereas control cells had a basal proportion of 8.9% ± 4.1%. Rates of DNA fragmentation were unaffected by the presence of the pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp (z-VAD). Cells treated with 100 μg of CFE for 12 h had a fivefold decrease in the level of mitochondrial outer membrane permeabilization compared to that of untreated cells. Neither M. bovis infection nor CFE treatment induced activation of caspase 3, 8, or 9. Translocation of apoptosis-inducing factor (AIF) to the nucleus was identified in 32% ± 3.5% and 26.3% ± 4.9% of M. bovis-infected and CFE-treated cells, respectively. Incubation of macrophages with z-VAD prior to infection did not alter the percentage of cells showing AIF translocation. Our data suggest that M. bovis-induced CD in bovine macrophages is caspase independent with AIF participation.

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.

Salmonella Serotype Typhimurium Infection of Bovine Peyer’s Patches Down-Regulates Plasma Membrane Calcium-Transporting ATPase Expression

To identify host genes differentially expressed during Salmonella enterica serotype Typhimurium infection, an RNA differential display was made with total RNA extracted from ileal loops that were infected with Salmonella Typhimurium 2.5 h after infection. Down-regulated cDNA was identified in bovine Peyers patches after infection that was highly homologous to a human plasma membrane calcium-transporting ATPase (PMCA). Differential expression of PMCA, evaluated by Northern analysis, was found to have more than a 4.6-fold decrease in expression of mRNA (size, approximately 5.1 kb). PMCA mRNA was detected by in situ hybridization exclusively within epithelial cells in the Peyers patches. cDNA (4.4 kb) was amplified by rapid amplification of cDNA ends, cloned, and sequenced and showed a high homology to hPMCA. Bovine PMCA is down-regulated in epithelial cells of Peyers patches after infection with Salmonella Typhimurium and, subsequently, may influence cellular calcium levels that contribute to the inflammatory processes in the pathogenesis of diarrhea.

Divergent macrophage responses to Mycobacterium bovis among naturally exposed uninfected and infected cattle

Mycobacterium bovis, the causative agent of bovine tuberculosis (TB), is a successful pathogen that remains an important global threat to livestock. Cattle naturally exposed to M. bovis normally become reactive to the M. bovis‐purified protein derivative (tuberculin) skin test; however, some individuals remain negative, suggesting that they may be resistant to infection. To better understand host innate resistance to infection, 26 cattle from herds with a long history of high TB prevalence were included in this study. We investigated the bactericidal activity, the production of reactive oxygen and nitrogen species and the TB‐related gene expression profile after in vitro M. bovis challenge of monocyte‐derived macrophages from cattle with TB (n=17) and from non‐infected, exposed cattle (in‐contacts, n=9). The disease status was established based on the tuberculin skin test and blood interferon‐gamma test responses, the presence of visible lesions at inspection on abattoirs and the histopathology and culture of M. bovis. Although macrophages from TB‐infected cattle enabled M. bovis replication, macrophages from healthy, exposed cattle had twofold lower bacterial loads, overproduced nitric oxide and had lower interleukin (IL)‐10 gene expression (P⩽0.05). Higher mRNA expression levels of inducible nitric oxide synthase, C‐C motif chemokine ligand 2 and IL‐12 were observed in macrophages from all in‐contact cattle than in macrophages from their TB‐infected counterparts, which expressed more tumour necrosis factor‐α; however, the differences were not statistically significant owing to individual variation. These results confirm that macrophage bactericidal responses have a crucial role in innate resistance to M. bovis infection in cattle.

Identification of immunodominant antigens of Mycobacterium bovis by expression library immunization

This study combines two methodologies - vector expression of a genomic library and proteomics - to identify immunogenic proteins of Mycobacterium bovis. Immunization of BALB/c mice with a plasmid DNA pool from the library, containing approximately 8000 clones, induced a humoral response that facilitated the detection of 12 antigenic proteins by Western blotting. Two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry identified four proteins (Cpn60-1, HSP70, EF-Tu, and AdoHcyase). Such genomic immunization offers the possibility of in vivo screening of potential candidate M. bovis antigens.

Mycobacterium bovis-infected macrophages from resistant and susceptible cattle exhibited a differential pro-inflammatory gene expression profile depending on strain virulence.

Mycobacterium bovis, the causative agent of bovine tuberculosis, is an intracellular bacterium that normally persists inside host macrophages. However, the influence of bacterial virulence and host resistance on the final outcome in this interaction is not well known. In this study, we infected macrophages isolated from natural disease resistant (R) and susceptible (S) cattle donors with M. bovis strains characterized as attenuated and virulent to assess pro-inflammatory cytokine (TNFα, IL-12, IL-18, IL-1β, IL-6), chemokine (MCP-1, MCP-2, MIP-1), macrophage receptor (MSR1, TLR2, TLR4, MMR) and iNOS mRNA expression levels. Our findings identified a pro-inflammatory gene expression profile as a common feature after M. bovis infection regardless of bacterial virulence, however in S macrophages a superior expression was induced by the attenuated strain, whereas in R macrophages it was accomplished by the virulent M. bovis. A macrophage pro-inflammatory profile is intended to control M. bovis intracellular growth; however the host resistant phenotype plays a determinant role in it, since R macrophages had better intracellular bacterial control than S cells.

Comparison of immune peripheral blood cells in tuberculin reactor cattle that are seropositive or seronegative for Mycobacterium bovis antigens

Bovine tuberculosis (bTB) is a major economic problem in animal husbandry and is a public health risk in nonindustrialized countries. It is generally accepted that protection against TB is generated through cell-mediated immunity. Previous investigations have shown that WC1(+) γδ, CD4(+) and CD8(+) T-cell subpopulations are important in the immune response to bTB. It is known that changes in the immune balance from a dominant T helper 1 (Th1)-type response toward a more prominent Th2 response may be observed during disease progression. In this study, we aimed to investigate immune peripheral blood cells in tuberculin reactor cattle that are seropositive or seronegative for Mycobacterium bovis antigens, using flow cytometry and hematological analysis. The evaluation of the T cell subpopulations revealed a decrease in CD8(+) T cells of the seropositive and seronegative animals compared with the control animals (p=0.0001). Moreover, the seropositive group exhibited a lower percentage of CD8(+) T cells than the seronegative group. The percentage of B cells was significantly increased in the seropositive group compared with the seronegative group and the control group (p=0.0009). No difference was observed in the percentage of WC1(+) γδ and CD4(+) T cells among the groups. Furthermore, following 24h of peripheral blood culture with bovine purified protein derivative (PPD), both apparently infected groups showed an increase in the levels of cellular activation compared with the control group (p<0.0001). The seropositive group displayed a higher level of cellular activation than the seronegative group. In both apparently infected groups, the hematological analysis showed an increase in total leukocyte (p=0.0012), lymphocyte (p=0.0057), monocyte (p=0.0010) and neutrophil (p=0.0320) counts in comparison with the healthy animals. Our results demonstrated differences in immune peripheral blood cells of tuberculin reactor cattle that are seropositive or seronegative for M. bovis antigens, probably due to different stages of bTB among the groups. The percentages of CD8(+) T cells, B cells and the T cell activation levels may represent biomarkers for the progression of the disease. However, general characteristics shared by both apparently infected groups as lymphocytosis and monocytosis may also be indicative of the disease. Further experiments are required to understand the variations between cellular and humoral immunities throughout the course of bTB infection. A detailed knowledge of the peripheral blood cells involved in all stages of the bTB immune response of naturally infected cattle is essential for the optimal exploitation of diagnosis and vaccination models.

Mycobacterium bovis infection in cattle induces differential expression of prolactin receptor isoforms in macrophages.

Prolactin receptor (PRLr) is a member of the cytokine receptor superfamily 1 showing tissue specific structural diversity. Expression of PRLr isoforms in lymphoid tissues has been associated with immunomodulatory function of prolactin. Bovine tuberculosis (bTB) is characterized by chronic inflammation caused by the persistent infection of lymphoid tissues with Mycobacterium bovis. To test the hypothesis of the influence of PRLr in the pathogenesis of bTB, the aim of this study was to identify PRLr isoforms expressed during bTB in different tissues and to analyze their association with the pathogenesis of bTB. We examined lymphoid and non-lymphoid tissues ex vivo from experimentally and naturally infected cattle, as well as from bTB-free cattle, by Western blot (WB) and immunohistochemistry (IH). In vitro, monocytes from exposed, infected, and healthy cattle were stimulated with M. bovis antigens and then analyzed by WB. To detect transcriptional levels of PRLr in macrophages (MØ) exposed to M. bovis, real time PCR was performed. WB revealed diversity of PRLr isoforms in tissues from infected cattle but not in tissues from bTB-free cattle. PRLr isoforms 100 kDa 75, 50 and 40 were found expressed in tissues of animals infected with M. bovis, while only the short isoform of 40 kDa correlated with the immunopathology and ability to infect MØ. We confirmed the synthesis of PRLr mRNA in MØ after M. bovis exposure and propose that molecular pathogen patterns of M. bovis might modulate inflammation during bTB through expression of the PRLr isoform in MØ.

Endonuclease G takes part in AIF-mediated caspase-independent apoptosis in Mycobacterium bovis-infected bovine macrophages

Mycobacterium bovis, the causative agent of bovine tuberculosis encodes different virulence mechanisms to survive inside of host cells. One of the possible outcomes in this host–pathogen interaction is cell death. Previous results from our group showed that M. bovis induces a caspase-independent apoptosis in bovine macrophages with the possible participation of apoptosis inducing factor mitochondria associated 1 (AIFM1/AIF), a flavoprotein that functions as a cell-death regulator. However, contribution of other caspase-independent cell death mediators in M. bovis-infected macrophages is not known. In this study, we aimed to further characterize M. bovis-induced apoptosis, addressing Endonuclease G (Endo G) and Poly (ADP-ribose) polymerase 1 (PARP-1). In order to accomplish our objective, we infected bovine macrophages with M. bovis AN5 (MOI 10:1). Analysis of M. bovis-infected nuclear protein extracts by immunoblot, identified a 15- and 43-fold increase in concentration of mitochondrial proteins AIF and Endo G respectively. Interestingly, pretreatment of M. bovis-infected macrophages with cyclosporine A, a mitochondrial permeability transition pore inhibitor, abolished AIF and Endo G nuclear translocation. In addition, it also decreased macrophage DNA fragmentation to baseline and caused a 26.2% increase in bacterial viability. We also demonstrated that PARP-1 protein expression in macrophages did not change during M. bovis infection. Furthermore, pretreatment of M. bovis-infected bovine macrophages with 3-aminobenzamide, a PARP-1 inhibitor, did not change the proportion of macrophage DNA fragmentation. Our results suggest participation of Endo G, but not PARP-1, in M. bovis-induced macrophage apoptosis. To the best of our knowledge this is the first report associating Endo G with caspase-independent apoptosis induced by a member of the Mycobacterium tuberculosis complex.