A Common Variant in the Adaptor Mal Regulates Interferon Gamma Signaling.

Humans that are heterozygous for the common S180L polymorphism in the Toll-like receptor (TLR) adaptor Mal (encoded by TIRAP) are protected from a number of infectious diseases, including tuberculosis (TB), whereas those homozygous for the allele are at increased risk. The reason for this difference in susceptibility is not clear. We report that Mal has a TLR-independent role in interferon-gamma (IFN-γ) receptor signaling. Mal-dependent IFN-γ receptor (IFNGR) signaling led to mitogen-activated protein kinase (MAPK) p38 phosphorylation and autophagy. IFN-γ signaling via Mal was required for phagosome maturation and killing of intracellular Mycobacterium tuberculosis (Mtb). The S180L polymorphism, and its murine equivalent S200L, reduced the affinity of Mal for the IFNGR, thereby compromising IFNGR signaling in macrophages and impairing responses to TB. Our findings highlight a role for Mal outside the TLR system and imply that genetic variation in TIRAP may be linked to other IFN-γ-related diseases including autoimmunity and cancer.

Macrophage-specific responses to human- and animal-adapted tubercle bacilli reveal pathogen and host factors driving multinucleated cell formation.

The Mycobacterium tuberculosis complex (MTBC) is a group of related pathogens that cause tuberculosis (TB) in mammals. MTBC species are distinguished by their ability to sustain in distinct host populations. While Mycobacterium bovis (Mbv) sustains transmission cycles in cattle and wild animals and causes zoonotic TB, M. tuberculosis (Mtb) affects human populations and seldom causes disease in cattle. The host and pathogen determinants underlying host tropism between MTBC species are still unknown. Macrophages are the main host cell that encounters mycobacteria upon initial infection, and we hypothesised that early interactions between the macrophage and mycobacteria influence species-specific disease outcome. To identify factors that contribute to host tropism, we analysed blood-derived primary human and bovine macrophages (hMϕ or bMϕ, respectively) infected with Mbv and Mtb. We show that Mbv and Mtb reside in different cellular compartments and differentially replicate in hMϕ whereas both Mbv and Mtb efficiently replicate in bMϕ. Specifically, we show that out of the four infection combinations, only the infection of bMϕ with Mbv promoted the formation of multinucleated giant cells (MNGCs), a hallmark of tuberculous granulomas. Mechanistically, we demonstrate that both MPB70 from Mbv and extracellular vesicles released by Mbv-infected bMϕ promote macrophage multinucleation. Importantly, we extended our in vitro studies to show that granulomas from Mbv-infected but not Mtb-infected cattle contained higher numbers of MNGCs. Our findings implicate MNGC formation in the contrasting pathology between Mtb and Mbv for the bovine host and identify MPB70 from Mbv and extracellular vesicles from bMϕ as mediators of this process.

Independent genomic polymorphisms in the PknH serine threonine kinase locus during evolution of the Mycobacterium tuberculosis Complex affect virulence and host preference.

Species belonging to the Mycobacterium tuberculosis Complex (MTBC) show more than 99% genetic identity but exhibit distinct host preference and virulence. The molecular genetic changes that underly host specificity and infection phenotype within MTBC members have not been fully elucidated. Here, we analysed RD900 genomic region across MTBC members using whole genome sequences from 60 different MTBC strains so as to determine its role in the context of MTBC evolutionary history. The RD900 region comprises two homologous genes, pknH1 and pknH2, encoding a serine/threonine protein kinase PknH flanking the tbd2 gene. Our analysis revealed that RD900 has been independently lost in different MTBC lineages and different strains, resulting in the generation of a single pknH gene. Importantly, all the analysed M. bovis and M. caprae strains carry a conserved deletion within a proline rich-region of pknH, independent of the presence or absence of RD900. We hypothesized that deletion of pknH proline rich-region in M. bovis may affect PknH function, having a potential role in its virulence and evolutionary adaptation. To explore this hypothesis, we constructed two M. bovis 'knock-in' strains containing the M. tuberculosis pknH gene. Evaluation of their virulence phenotype in mice revealed a reduced virulence of both M. bovis knock-in strains compared to the wild type, suggesting that PknH plays an important role in the differential virulence phenotype of M. bovis vs M. tuberculosis.

Metabolomic changes in Mycobacterium avium subsp. paratuberculosis (MAP) challenged Holstein-Friesian cattle highlight the role of serum amino acids as indicators of immune system activation.

INTRODUCTION: Paratuberculosis, commonly known as Johne's disease, is a chronic granulomatous infection of ruminants caused by Mycobacterium avium subspecies paratuberculosis (MAP). Clinical signs, including reduced milk yields, weight loss and diarrhoea, are typically absent until 2 to 6 years post exposure. OBJECTIVES: To identify metabolomic changes profiles of MAP challenged Holstein-Friesian (HF) cattle and correlate identified metabolites to haematological and immunological parameters. METHODS: At approximately 6 weeks of age, calves (n = 9) were challenged with 3.8 × 109 cells of MAP (clinical isolate CIT003) on 2 consecutive days. Additional unchallenged calves (n = 9) formed the control group. The study used biobanked serum from cattle sampled periodically from 3- to 33-months post challenge. The assessment of sera using flow infusion electrospray high resolution mass spectrometry (FIE-HRMS) for high throughput, sensitive, non-targeted metabolite fingerprinting highlighted differences in metabolite levels between the two groups. RESULTS: In total, 25 metabolites which were differentially accumulated in MAP challenged cattle were identified, including 20 which displayed correlation to haematology parameters, particularly monocyte levels. CONCLUSION: The targeted metabolites suggest shifts in amino acid metabolism that could reflect immune system activation linked to MAP and as well as differences in phosphocholine levels which could reflect activation of the Th1 (tending towards pro-inflammatory) immune response. If verified by future work, selected metabolites could be used as biomarkers to diagnose and manage MAP infected cattle.

Integrative genomics of the mammalian alveolar macrophage response to intracellular mycobacteria.

BACKGROUND: Bovine TB (bTB), caused by infection with Mycobacterium bovis, is a major endemic disease affecting global cattle production. The key innate immune cell that first encounters the pathogen is the alveolar macrophage, previously shown to be substantially reprogrammed during intracellular infection by the pathogen. Here we use differential expression, and correlation- and interaction-based network approaches to analyse the host response to infection with M. bovis at the transcriptome level to identify core infection response pathways and gene modules. These outputs were then integrated with genome-wide association study (GWAS) data sets to enhance detection of genomic variants for susceptibility/resistance to M. bovis infection. RESULTS: The host gene expression data consisted of RNA-seq data from bovine alveolar macrophages (bAM) infected with M. bovis at 24 and 48 h post-infection (hpi) compared to non-infected control bAM. These RNA-seq data were analysed using three distinct computational pipelines to produce six separate gene sets: 1) DE genes filtered using stringent fold-change and P-value thresholds (DEG-24: 378 genes, DEG-48: 390 genes); 2) genes obtained from expression correlation networks (CON-24: 460 genes, CON-48: 416 genes); and 3) genes obtained from differential expression networks (DEN-24: 339 genes, DEN-48: 495 genes). These six gene sets were integrated with three bTB breed GWAS data sets by employing a new genomics data integration tool-gwinteR. Using GWAS summary statistics, this methodology enabled detection of 36, 102 and 921 prioritised SNPs for Charolais, Limousin and Holstein-Friesian, respectively. CONCLUSIONS: The results from the three parallel analyses showed that the three computational approaches could identify genes significantly enriched for SNPs associated with susceptibility/resistance to M. bovis infection. Results indicate distinct and significant overlap in SNP discovery, demonstrating that network-based integration of biologically relevant transcriptomics data can leverage substantial additional information from GWAS data sets. These analyses also demonstrated significant differences among breeds, with the Holstein-Friesian breed GWAS proving most useful for prioritising SNPS through data integration. Because the functional genomics data were generated using bAM from this population, this suggests that the genomic architecture of bTB resilience traits may be more breed-specific than previously assumed.

The Iron Chelator Desferrioxamine Increases the Efficacy of Bedaquiline in Primary Human Macrophages Infected with BCG.

For over 50 years, patients with drug-sensitive and drug-resistant tuberculosis have undergone long, arduous, and complex treatment processes with several antimicrobials. With the prevalence of drug-resistant strains on the rise and new therapies for tuberculosis urgently required, we assessed whether manipulating iron levels in macrophages infected with mycobacteria offered some insight into improving current antimicrobials that are used to treat drug-resistant tuberculosis. We investigated if the iron chelator, desferrioxamine, can support the function of human macrophages treated with an array of second-line antimicrobials, including moxifloxacin, bedaquiline, amikacin, clofazimine, linezolid and cycloserine. Primary human monocyte-derived macrophages were infected with Bacillus Calmette-Guérin (BCG), which is pyrazinamide-resistant, and concomitantly treated for 5 days with desferrioxamine in combination with each one of the second-line tuberculosis antimicrobials. Our data indicate that desferrioxamine used as an adjunctive treatment to bedaquiline significantly reduced the bacterial load in human macrophages infected with BCG. Our findings also reveal a link between enhanced bactericidal activity and increases in specific cytokines, as the addition of desferrioxamine increased levels of IFN-γ, IL-6, and IL-1ß in BCG-infected human monocyte-derived macrophages (hMDMs) treated with bedaquiline. These results provide insight, and an in vitro proof-of-concept, that iron chelators may prove an effective adjunctive therapy in combination with current tuberculosis antimicrobials.

Microbe Profile: Mycobacterium tuberculosis: Humanity's deadly microbial foe.

Mycobacterium tuberculosis is an expert and deadly pathogen, causing the disease tuberculosis (TB) in humans. It has several notable features: the ability to enter non-replicating states for long periods and cause latent infection; metabolic remodelling during chronic infection; a thick, waxy cell wall; slow growth rate in culture; and intrinsic drug resistance and antibiotic tolerance. As a pathogen, M. tuberculosis has a complex relationship with its host, is able to replicate inside macrophages, and expresses diverse immunomodulatory molecules. M. tuberculosis currently causes over 1.8 million deaths a year, making it the world's most deadly human pathogen.

Mycobacterium tuberculosis Complex Members Adapted to Wild and Domestic Animals.

The Mycobacterium tuberculosis complex (MTBC) is composed of several highly genetically related species that can be broadly classified into those that are human-host adapted and those that possess the ability to propagate and transmit in a variety of wild and domesticated animals. Since the initial description of the bovine tubercle bacillus, now known as Mycobacterium bovis, by Theobald Smith in the late 1800's, isolates originating from a wide range of animal hosts have been identified and characterized as M. microti, M. pinnipedii, the Dassie bacillus, M. mungi, M. caprae, M. orygis and M. suricattae. This chapter outlines the events resulting in the identification of each of these animal-adapted species, their close genetic relationships, and how genome-based phylogenetic analyses of species-specific variation amongst MTBC members is beginning to unravel the events that resulted in the evolution of the MTBC and the observed host tropism between the human- and animal-adapted member species.

Agricultural anaerobic digestion power plants in Ireland and Germany: policy and practice.

The process of anaerobic digestion (AD) is valued as a carbon-neutral energy source, while simultaneously treating organic waste, making it safer for disposal or use as a fertilizer on agricultural land. The AD process in many European nations, such as Germany, has grown from use of small, localized digesters to the operation of large-scale treatment facilities, which contribute significantly to national renewable energy quotas. However, these large AD plants are costly to run and demand intensive farming of energy crops for feedstock. Current policy in Germany has transitioned to support funding for smaller digesters, while also limiting the use of energy crops. AD within Ireland, as a new technology, is affected by ambiguous governmental policies concerning waste and energy. A clear governmental strategy supporting on-site AD processing of agricultural waste will significantly reduce Ireland's carbon footprint, improve the safety and bioavailability of agricultural waste, and provide an indigenous renewable energy source. © 2016 Society of Chemical Industry.

GOexpress: an R/Bioconductor package for the identification and visualisation of robust gene ontology signatures through supervised learning of gene expression data.

BACKGROUND: Identification of gene expression profiles that differentiate experimental groups is critical for discovery and analysis of key molecular pathways and also for selection of robust diagnostic or prognostic biomarkers. While integration of differential expression statistics has been used to refine gene set enrichment analyses, such approaches are typically limited to single gene lists resulting from simple two-group comparisons or time-series analyses. In contrast, functional class scoring and machine learning approaches provide powerful alternative methods to leverage molecular measurements for pathway analyses, and to compare continuous and multi-level categorical factors. RESULTS: We introduce GOexpress, a software package for scoring and summarising the capacity of gene ontology features to simultaneously classify samples from multiple experimental groups. GOexpress integrates normalised gene expression data (e.g., from microarray and RNA-seq experiments) and phenotypic information of individual samples with gene ontology annotations to derive a ranking of genes and gene ontology terms using a supervised learning approach. The default random forest algorithm allows interactions between all experimental factors, and competitive scoring of expressed genes to evaluate their relative importance in classifying predefined groups of samples. CONCLUSIONS: GOexpress enables rapid identification and visualisation of ontology-related gene panels that robustly classify groups of samples and supports both categorical (e.g., infection status, treatment) and continuous (e.g., time-series, drug concentrations) experimental factors. The use of standard Bioconductor extension packages and publicly available gene ontology annotations facilitates straightforward integration of GOexpress within existing computational biology pipelines.

Epitopemap: a web application for integrated whole proteome epitope prediction.

BACKGROUND: Predictions of MHC binding affinity are commonly used in immunoinformatics for T cell epitope prediction. There are multiple available methods, some of which provide web access. However there is currently no convenient way to access the results from multiple methods at the same time or to execute predictions for an entire proteome at once. RESULTS: We designed a web application that allows integration of multiple epitope prediction methods for any number of proteins in a genome. The tool is a front-end for various freely available methods. Features include visualisation of results from multiple predictors within proteins in one plot, genome-wide analysis and estimates of epitope conservation. CONCLUSIONS: We present a self contained web application, Epitopemap, for calculating and viewing epitope predictions with multiple methods. The tool is easy to use and will assist in computational screening of viral or bacterial genomes.

Investigation of the high rates of extrapulmonary tuberculosis in Ethiopia reveals no single driving factor and minimal evidence for zoonotic transmission of Mycobacterium bovis infection.

BACKGROUND: Ethiopia, a high tuberculosis (TB) burden country, reports one of the highest incidence rates of extra-pulmonary TB dominated by cervical lymphadenitis (TBLN). Infection with Mycobacterium bovis has previously been excluded as the main reason for the high rate of extrapulmonary TB in Ethiopia. METHODS: Here we examined demographic and clinical characteristics of 953 pulmonary (PTB) and 1198 TBLN patients visiting 11 health facilities in distinct geographic areas of Ethiopia. Clinical characteristics were also correlated with genotypes of the causative agent, Mycobacterium tuberculosis. RESULTS: No major patient or bacterial strain factor could be identified as being responsible for the high rate of TBLN, and there was no association with HIV infection. However, analysis of the demographic data of involved patients showed that having regular and direct contact with live animals was more associated with TBLN than with PTB, although no M. bovis was isolated from patients with TBLN. Among PTB patients, those infected with Lineage 4 reported "contact with other TB patient" more often than patients infected with Lineage 3 did (OR = 1.6, CI 95% 1.0-2.7; p = 0.064). High fever, in contrast to low and moderate fever, was significantly associated with Lineage 4 (OR = 2.3; p = 0.024). On the other hand, TBLN cases infected with Lineage 4 tended to get milder symptoms overall for the constitutional symptoms than those infected with Lineage 3. CONCLUSIONS: The study suggests a complex role for multiple interacting factors in the epidemiology of extrapulmonary TB in Ethiopia, including factors that can only be derived from population-based studies, which may prove to be significant for TB control in Ethiopia.

Iron-regulated protein HupB of Mycobacterium tuberculosis positively regulates siderophore biosynthesis and is essential for growth in macrophages.

Mycobacterium tuberculosis expresses the 28-kDa protein HupB (Rv2986c) and the Fe(3+)-specific high-affinity siderophores mycobactin and carboxymycobactin upon iron limitation. The objective of this study was to understand the functional role of HupB in iron acquisition. A hupB mutant strain of M. tuberculosis, subjected to growth in low-iron medium (0.02 µg Fe ml(-1)), showed a marked reduction of both siderophores with low transcript levels of the mbt genes encoding the MB biosynthetic machinery. Complementation of the mutant strain with hupB restored siderophore production to levels comparable to that of the wild type. We demonstrated the binding of HupB to the mbtB promoter by both electrophoretic mobility shift assays and DNA footprinting. The latter revealed the HupB binding site to be a 10-bp AT-rich region. While negative regulation of the mbt machinery by IdeR is known, this is the first report of positive regulation of the mbt operon by HupB. Interestingly, the mutant strain failed to survive inside macrophages, suggesting that HupB plays an important role in vivo.

Identification and characterization of the genetic changes responsible for the characteristic smooth-to-rough morphotype alterations of clinically persistent Mycobacterium abscessus.

Mycobacterium abscessus is an emerging pathogen that is increasingly recognized as a relevant cause of human lung infection in cystic fibrosis patients. This highly antibiotic-resistant mycobacterium is an exception within the rapidly growing mycobacteria, which are mainly saprophytic and non-pathogenic organisms. M. abscessus manifests as either a smooth (S) or a rough (R) colony morphotype, which is of clinical importance as R morphotypes are associated with more severe and persistent infections. To better understand the molecular mechanisms behind the S/R alterations, we analysed S and R variants of three isogenic M. abscessus S/R pairs using an unbiased approach involving genome and transcriptome analyses, transcriptional fusions and integrating constructs. This revealed different small insertions, deletions (indels) or single nucleotide polymorphisms within the non-ribosomal peptide synthase gene cluster mps1-mps2-gap or mmpl4b in the three R variants, consistent with the transcriptional differences identified within this genomic locus that is implicated in the synthesis and transport of Glyco-Peptido-Lipids (GPL). In contrast to previous reports, the identification of clearly defined genetic lesions responsible for the loss of GPL-production or transport makes a frequent switching back-and-forth between smooth and rough morphologies in M. abscessus highly unlikely, which is important for our understanding of persistent M. abscessus infections.

The synthesis and biological evaluation of mycobacterial p-hydroxybenzoic acid derivatives (p-HBADs).

Mycobacterium tuberculosis establishes chronic infection and causes disease through manipulation of the host's innate and adaptive immune response. The bacterial cell wall is highly complex and contains a rich variety of glycosylated compounds that are secreted during infection and have been proposed as immunomodulatory molecules. Amongst the most important of these are the p-hydroxybenzoic acid derivatives (p-HBADs). Here we report the synthesis of this important class of biomolecules and the first in vitro study of the immunomodulatory effects of these compounds in isolation from the host bacterium. The compounds do not have stimulatory properties but, in contrast, can inhibit the production of inflammatory cytokines, particularly interferon-γ (IFN-γ), by T-cells. This study offers a fundamental insight into the effect of these glycans on the immune response.

Genome-level analyses of Mycobacterium bovis lineages reveal the role of SNPs and antisense transcription in differential gene expression.

BACKGROUND: Bovine tuberculosis (bTB) is a disease with major implications for animal welfare and productivity, as well as having the potential for zoonotic transmission. In Great Britain (GB) alone, controlling bTB costs in the region of £ 100 million annually, with the current control scheme seemingly unable to stop the inexorable spread of infection. One aspect that may be driving the epidemic is evolution of the causative pathogen, Mycobacterium bovis. To understand the underlying genetic changes that may be responsible for this evolution, we performed a comprehensive genome-level analyses of 4 M. bovis strains that encompass the main molecular types of the pathogen circulating in GB. RESULTS: We have used a combination of genome sequencing, transcriptome analyses, and recombinant DNA technology to define genetic differences across the major M. bovis lineages circulating in GB that may give rise to phenotypic differences of practical importance. The genomes of three M. bovis field isolates were sequenced using Illumina sequencing technology and strain specific differences in gene expression were measured during in vitro growth and in ex vivo bovine alveolar macrophages using a whole genome amplicon microarray and a whole genome tiled oligonucleotide microarray. SNP/small base pair insertion and deletions and gene expression data were overlaid onto the genomic sequence of the fully sequenced strain of M. bovis 2122/97 to link observed strain specific genomic differences with differences in RNA expression. CONCLUSIONS: We show that while these strains show extensive similarities in their genetic make-up and gene expression profiles, they exhibit distinct expression of a subset of genes. We provide genomic, transcriptomic and functional data to show that synonymous point mutations (sSNPs) on the coding strand can lead to the expression of antisense transcripts on the opposing strand, a finding with implications for how we define a 'silent' nucleotide change. Furthermore, we show that transcriptomic data based solely on amplicon arrays can generate spurious results in terms of gene expression profiles due to hybridisation of antisense transcripts. Overall our data suggest that subtle genetic differences, such as sSNPS, may have important consequences for gene expression and subsequent phenotype.

Whole-transcriptome, high-throughput RNA sequence analysis of the bovine macrophage response to Mycobacterium bovis infection in vitro.

BACKGROUND: Mycobacterium bovis, the causative agent of bovine tuberculosis, is an intracellular pathogen that can persist inside host macrophages during infection via a diverse range of mechanisms that subvert the host immune response. In the current study, we have analysed and compared the transcriptomes of M. bovis-infected monocyte-derived macrophages (MDM) purified from six Holstein-Friesian females with the transcriptomes of non-infected control MDM from the same animals over a 24 h period using strand-specific RNA sequencing (RNA-seq). In addition, we compare gene expression profiles generated using RNA-seq with those previously generated by us using the high-density Affymetrix® GeneChip® Bovine Genome Array platform from the same MDM-extracted RNA. RESULTS: A mean of 7.2 million reads from each MDM sample mapped uniquely and unambiguously to single Bos taurus reference genome locations. Analysis of these mapped reads showed 2,584 genes (1,392 upregulated; 1,192 downregulated) and 757 putative natural antisense transcripts (558 upregulated; 119 downregulated) that were differentially expressed based on sense and antisense strand data, respectively (adjusted P-value ≤ 0.05). Of the differentially expressed genes, 694 were common to both the sense and antisense data sets, with the direction of expression (i.e. up- or downregulation) positively correlated for 693 genes and negatively correlated for the remaining gene. Gene ontology analysis of the differentially expressed genes revealed an enrichment of immune, apoptotic and cell signalling genes. Notably, the number of differentially expressed genes identified from RNA-seq sense strand analysis was greater than the number of differentially expressed genes detected from microarray analysis (2,584 genes versus 2,015 genes). Furthermore, our data reveal a greater dynamic range in the detection and quantification of gene transcripts for RNA-seq compared to microarray technology. CONCLUSIONS: This study highlights the value of RNA-seq in identifying novel immunomodulatory mechanisms that underlie host-mycobacterial pathogen interactions during infection, including possible complex post-transcriptional regulation of host gene expression involving antisense RNA.

Mycobacterial lineages causing pulmonary and extrapulmonary tuberculosis, Ethiopia.

Molecular typing of 964 specimens from patients in Ethiopia with lymph node or pulmonary tuberculosis showed a similar distribution of Mycobacterium tuberculosis strains between the 2 disease manifestations and a minimal role for M. bovis. We report a novel phylogenetic lineage of M. tuberculosis strongly associated with the Horn of Africa.

Long term dietary vitamin D3 supplementation impacts both microbicidal and inflammatory responses to ex-vivo Mycobacterium bovis BCG challenge in dairy calves.

Vitamin D deficiency (VDD) is associated with enhanced susceptibility to multiple respiratory diseases in humans, including tuberculosis. However, the consequences of VDD for disease susceptibility in calves are unknown. Previously we developed a model to drive divergent circulating 25OHD concentrations in cattle, where animals were supplemented with vitamin D3 (vit D3) from birth to 7 months of age. Calves in the control group (Ctl) received a diet containing a standard vit D3 concentration, whereas the vit D group (VitD) received a diet with the highest vit D3 concentration allowed under EU guidelines. Here, we assessed the microbicidal activity and immunoregulatory effect of divergent 25OHD circulating levels to Mycobacterium bovis BCG challenge ex-vivo. Blood samples from Ctl and VitD calves were taken at 1-, 3- and 7-months of age. 25OHD concentrations were significantly different at 7 months (but not at 1 or 3 months) with animals from the VitD group having higher serum levels. Differences in microbicidal activity followed the same pattern, with no significant differences observed at 1 and 3 months, but at 7 months a significant increase in the percentage of bacteria killed was detected. Furthermore, analysis of the reactive oxygen species (ROS) and nitric oxide (NO) in serum showed a higher production of ROS and NO in VitD-supplemented calves. In contrast, serum concentrations of IL-1ß and IL-8 were significantly lower. A similar anti-inflammatory profile was observed after gene expression analysis, with a significant downregulation of a cluster of genes including IL1B, IL1R1, CXCL1, CXCL2, CXCL5, MMP9 and COX2 and an upregulation of CXCR1, CX3CR1 and NCF1, in VitD calves after BCG challenge relative to Ctl animals. Collectively, these results suggest that dietary vit D3 boosts antimicrobial and innate immune responses and thereby could improve host anti-mycobacterial immunity.

Vitamin D induced microbicidal activity against Mycobacterium bovis BCG is dependent on the synergistic activity of bovine peripheral blood cell populations.

A growing appreciation is emerging of the beneficial role of vitamin D for health and resistance against infectious diseases, including tuberculosis. However, research has predominantly focused on murine and human species and functional data in bovines is limited. Therefore, the objective of this study was to assess the microbicidal activity and immunoregulatory effect of the vitamin D metabolite 1,25(OH)2D3 on bovine peripheral blood leukocytes (PBL) in response to Mycobacterium bovis BCG (BCG) infection using a combination of functional assays and gene expression profiling. Blood from Holstein-Friesian bull calves with low circulating levels of 25(OH)D was stimulated with 1,25(OH)2D3 for 2 h, and then infected with M. bovis BCG. Results showed that 1,25(OH)2D3 supplementation significantly increased BCG killing by on average 16 %, although responses varied between 1 % and 38 % killing. Serial cell subset depletion was then performed on PBL prior to 1,25(OH)2D3 incubation and BCG infected as before to analyse the contribution of major cell types to mycobacterial growth control. Specific antibodies and either magnetic cell separation or density gradient centrifugation of monocytes, granulocytes, CD3+, CD4+, and CD8+ T lymphocytes were used to capture each cell subset. Results showed that depletion of granulocytes had the greatest impact on BCG growth, leading to a significant enhancement of bacterial colonies. In contrast, depletion of CD4+ or CD8+ T cells individually, or in combination (CD3+), had no impact on mycobacterial growth control. In agreement with our previous data, 1,25(OH)2D3 significantly increased bacterial killing in PBL, in monocyte depleted samples, and a similar trend was observed in the granulocyte depleted subset. In addition, specific analysis of sorted neutrophils treated with 1,25(OH)2D3 showed an enhanced microbicidal activity against both BCG and a virulent strain of M. bovis. Lastly, data showed that 1,25(OH)2D3 stimulation increased reactive oxygen species (ROS) production and the expression of genes encoding host defence peptides (HDP) and pathogen recognition receptors (PRRs), factors that play an important role in the microbicidal activity against mycobacteria. In conclusion, the vitamin D metabolite 1,25(OH)2D3 improves antimycobacterial killing in bovine PBLs via the synergistic activity of monocytes and granulocytes and enhanced activation of innate immunity.