Necrotic Cell Death and Inflammasome NLRP3 Activity in Mycobacterium bovis-Infected Bovine Macrophages.

Mycobacterium bovis is a facultative intracellular bacterium that produces cellular necrosis in granulomatous lesions in bovines. Although M. bovis-induced inflammation actively participates in granuloma development, its role in necrotic cell death and in bovine macrophages has not been fully explored. In this study, we evaluate the effect of M. bovis AN5 and its culture filtrate protein extract (CFPE) on inflammasome activation in bovine macrophages and its consequences on cell death. Our results show that both stimuli induce necrotic cell death starting 4 h after incubation. CFPE treatment and M. bovis infection also induce the maturation of IL-1ß (>3000 pg/mL), oligomerization of ASC (apoptosis-associated speck-like protein containing CARD), and activation of caspase-1, following the canonical activation pathway of the NLRP3 inflammasome. Inhibiting the oligomerization of NLRP3 and caspase-1 decreases necrosis among the infected or CFPE-stimulated macrophages. Furthermore, histological lymph node sections of bovines naturally infected with M. bovis contained cleaved gasdermin D, mainly in macrophages and giant cells within the granulomas. Finally, the induction of cell death (apoptosis and pyroptosis) decreased the intracellular bacteria count in the infected bovine macrophages, suggesting that cell death helps to control the intracellular growth of the mycobacteria. Our results indicate that M. bovis induces pyroptosis-like cell death that is partially related to the NLRP3 inflammasome activation and that the cell death process could control bacterial growth.

NLRP3 Inflammasome Involved with Viral Replication in Cytopathic NADL BVDV Infection and IFI16 Inflammasome Connected with IL-1ß Release in Non-Cytopathic NY-1 BVDV Infection in Bovine Macrophages.

Inflammasomes are multiprotein complexes that play a role in the processing of proinflammatory cytokines such as interleukin 1 beta (IL-1ß). The secretion of IL-1ß in bovine macrophages infected with the bovine viral diarrhea virus (BVDV) cytopathic strain NADL (NADLcp-BVDV) is caspase 1-dependent. In the present study, we found that in macrophages infected with NADL, the NLRP3 inflammasome participated in the maturation of IL-1ß as the level decreased from 4629.3 pg/mL to 897.0 pg/mL after treatment with cytokine release inhibitory drug 3 (CRID3). Furthermore, NLRP3 activation has implications regarding viral replication, as there was a decrease in the viral titer until 1 log of a supernatant of macrophages that were inhibited with CRID3 remained. In the case of the non-cytopathic BVDV strain NY-1 (NY-1 ncpBVDV), IL-1ß secretion is not affected by NLRP3, but could be related to the IFI16 inflammasome; we found a colocalization of IFI16 with ASC using confocal microscopy in infected macrophages with the NY-1 ncp-BVDV biotype. To relate IFI16 activation to IL-1ß release, we used ODN TTAGGG (A151), a competitive inhibitor of IFI16; the results show a decrease in its level from 248 pg/mL to 128.3 pg/mL. Additionally, we evaluated the caspase 1 activation downstream of IFI16 and found a decrease in the IL-1ß from 252.9 pg/mL to 63.5 pg/mL when caspase 1 was inhibited with Y-VAD. Our results provide an improved understanding of the mechanisms involved in the viral replication, inflammation and pathogenesis of bovine viral diarrhea.

Brucella melitensis invA gene (BME_RS01060) transcription is promoted under acidic stress conditions.

The invA gene of Brucella melitensis codes for a NUDIX (nucleoside diphosphate linked to moiety X) hydrolase related to invasiveness. The objective of this work was to evaluate invA transcription under acidic conditions. The invA gene transcription was up regulated at pH 3 and pH 5 observed with semiquantitative real-time PCR in B. melitensis 133 strain. Results indicated that invA gene transcription at pH 3 showed a basal and decreased transcription compared to that of pH 5 incubation. Transcription levels of the dnaK gene were similar to those obtained with invA gene. The survival rates of wild type and invA mutant strains at pH 5 were above 90% in all post-incubation times. In contrast, at pH 3 there was a time-dependent reduction on both strains at 15 min (P < 0.05). These results suggest that invA gene transcription is promoted under acidic conditions in Brucella melitensis.

Association of immune responses of Zebu and Holstein-Friesian cattle and resistance to mycobacteria in a BCG challenge model.

Mycobacterium bovis is the main cause of bovine tuberculosis (BTB) in cattle and can also infect humans. Zebu cattle are considered more resistant to some infectious diseases compared with Holstein-Friesian (HF) cattle, including BTB. However, epidemiological studies may not take into account usage differences of the two types of cattle. HF cattle may suffer greater metabolic stress due to their more or less exclusive dairy use, whereas Zebu cattle are mainly used for beef production. In experiments conducted so far, the number of animals has been too small to draw statistically robust conclusions on the resistance differences between these cattle breeds. Here, we used a BCG challenge model to compare the ability of naïve and vaccinated Zebu and HF cattle to control/kill mycobacteria. Young cattle of both breeds with similar ages were housed in the same accommodation for the duration of the experiment. After correcting for multiple comparisons, we found no difference between naïve HF and Zebu (ρ = 0.862) cattle. However, there was a trend for vaccinated HF cattle to have lower cfu numbers than non-vaccinated HF cattle (ρ = 0.057); no such trend was observed between vaccinated and non-vaccinated Zebu cattle (ρ = 0.560). Evaluation of antigen-specific IFNγ secretion by PBMC indicated that Zebu and HF cattle differed in their response to mycobacteria. Thus, whilst there may be difference in immune responses, our data indicate that with the number of animals included in the study and under the conditions used in this work, we were unable to measure any differences between Zebu and HF cattle in the overall control of mycobacteria. Whilst determination of different susceptibilities between Zebu and HF cattle using the BCG challenge model will require larger numbers of animals than the number of animals used in this experiment, these data should inform future experiments.

The NADL strain of bovine viral diarrhea virus induces the secretion of IL-1ß through caspase 1 in bovine macrophages.

Bovine viral diarrhea virus (BVDV) infects different cell types including antigen-presenting cells such as macrophages. The infection induces pro-inflammatory cytokines like interleukin 1 beta (IL-1ß), which is necessary to trigger a successful inflammatory response against infections. Several authors have reported differences between IL-1ß gene expression and protein detection in BVDV-infected macrophages. These patterns may be related to inflammasome assembly, which promote the formation of active caspase 1 in order to produce mature IL-1ß molecules. Our goal was to assess BVDV ability to induce the release of IL-ß through a caspase 1 dependent pathway in bovine macrophages. We infected peripheral blood monocyte-derived macrophages using BVDV NADL strain at 0.001, 0.1, 2 and 10 multiplicities of infection (MOI) and we measured IL-1ß at different times 2, 6, 12, 24, 48, 72 h. We found an increase of 1140-2154 pg for a MOI of 10:1 and 2:1 respectively. To inhibit caspase 1, we used either carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]- fluoromethylketone (Z-VAD) or carbobenzoxy-tyr-valyl-alanyl-aspartyl-[O-methyl]- fluoromethylketone (Y-VAD). We found decreased IL-1ß secretion 2154 pg/ml to 854 pg/ml IL-1ß secretion using Y-VAD and we observed decrease from 2154 pg/ml to 22.33 pg/ml with Z-VAD, and this inhibition was followed by diminished viral replication from 2.25 × 107 to 2.1 × 105 CCID50, which suggests that caspase 1-dependent secretion of the IL-1ß active molecule is important for viral replication. This is the first report showing that BVDV infected-bovine macrophages trigger the caspase 1 dependent pathway for IL-1ß activation and that activation increases viral replication.

Genetically Related Mycobacterium bovis Strains Displayed Differential Intracellular Growth in Bovine Macrophages.

Molecular typing of bacterial isolates provides a powerful approach for distinguishing Mycobacterium bovis (M. bovis) genotypes. It is known that M. bovis strain virulence plays a role in prevalence and spread of the disease, suggesting that strain virulence and prevailing genotypes are associated. However, it is not well understood whether strain virulence correlates with particular genotypes. In this study, we assessed the in vitro intracellular growth of 18 M. bovis isolates in bovine macrophages as an indicator of bacterial virulence and sought a relationship with the genotype identified by spoligotyping. We found 14 different spoligotypes-11 were already known and three spoligotypes had never been reported before. We identified 2 clusters that were phylogenetically related, containing 10 and 6 strains, respectively, and 2 orphan strains. Intracellular growth and phagocytic rates of 18 M. bovis strains were heterogeneous. Our results suggest that M. bovis intracellular growth and phagocytosis are independent of the bacterial lineage identified by spoligotyping.

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.

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 SLC11A1 polymorphism 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.