Mycobacterium tuberculosis Exploits a Molecular Off Switch of the Immune System for Intracellular Survival.

Mycobacterium tuberculosis (M. tuberculosis) survives and multiplies inside human macrophages by subversion of immune mechanisms. Although these immune evasion strategies are well characterised functionally, the underlying molecular mechanisms are poorly understood. Here we show that during infection of human whole blood with M. tuberculosis, host gene transcriptional suppression, rather than activation, is the predominant response. Spatial, temporal and functional characterisation of repressed genes revealed their involvement in pathogen sensing and phagocytosis, degradation within the phagolysosome and antigen processing and presentation. To identify mechanisms underlying suppression of multiple immune genes we undertook epigenetic analyses. We identified significantly differentially expressed microRNAs with known targets in suppressed genes. In addition, after searching regions upstream of the start of transcription of suppressed genes for common sequence motifs, we discovered novel enriched composite sequence patterns, which corresponded to Alu repeat elements, transposable elements known to have wide ranging influences on gene expression. Our findings suggest that to survive within infected cells, mycobacteria exploit a complex immune "molecular off switch" controlled by both microRNAs and Alu regulatory elements.

Innate activation of human primary epithelial cells broadens the host response to Mycobacterium tuberculosis in the airways.

Early events in the human airways determining whether exposure to Mycobacterium tuberculosis (Mtb) results in acquisition of infection are poorly understood. Epithelial cells are the dominant cell type in the lungs, but little is known about their role in tuberculosis. We hypothesised that human primary airway epithelial cells are part of the first line of defense against Mtb-infection and contribute to the protective host response in the human respiratory tract. We modelled these early airway-interactions with human primary bronchial epithelial cells (PBECs) and alveolar macrophages. By combining in vitro infection and transwell co-culture models with a global transcriptomic approach, we identified PBECs to be inert to direct Mtb-infection, yet to be potent responders within an Mtb-activated immune network, mediated by IL1ß and type I interferon (IFN). Activation of PBECs by Mtb-infected alveolar macrophages and monocytes increased expression of known and novel antimycobacterial peptides, defensins and S100-family members and epithelial-myeloid interactions further shaped the immunological environment during Mtb-infection by promoting neutrophil influx. This is the first in depth analysis of the primary epithelial response to infection and offers new insights into their emerging role in tuberculosis through complementing and amplifying responses to Mtb.

Subset-Specific Expression of Toll-Like Receptors by Bovine Afferent Lymph Dendritic Cells.

Within the ruminant system, several possibilities exist to generate dendritic cells migrating out from the tissue into the regional draining lymph nodes as afferent lymph dendritic cells (ALDCs). Here, we analyzed toll-like receptor (TLR) 1-10 mRNA expression by using quantitative real-time PCR in highly purified subsets of bovine ALDC. As TLR expression may be influenced by pathogens or vaccines and their adjuvant, it is necessary to understand what TLRs are expressed in a steady-state system to elucidate specific differences and to potentially optimize targeted vaccines. In this study, we have assessed the TLR expression profiles of the four main bovine ALDC subsets [cDC1 and cDC2 (subsets 2-4)]. We demonstrate differences in TLR expression between the four subsets that may reflect the ability of these cells to respond to different pathogens or to respond to adjuvants.

Proteomic Analysis of Kveim Reagent Identifies Targets of Cellular Immunity in Sarcoidosis.

BACKGROUND: Kveim-reagent (Kv) skin testing was a historical method of diagnosing sarcoidosis. Intradermal injection of treated sarcoidosis spleen tissue resulted in a granuloma response at injection site by 4-6 weeks. Previous work indicates proteins as the possible trigger of this reaction. We aimed to identify Kv-specific proteins and characterise the ex vivo response of Peripheral Blood Mononuclear Cells (PBMCs) from sarcoidosis, tuberculosis and healthy control patients when stimulated with both Kv and selected Kv-specific proteins. METHODS: Kv extracts were separated by 1D-SDS-PAGE and 2D-DIGE and then underwent mass spectrometric analysis for protein identification. Sarcoidosis and control PBMCs were first stimulated with Kv and then with three selected recombinant protein candidates which were identified from the proteomic analysis. PBMC secreted cytokines were subsequently measured by Multiplex Cytokine Assay. RESULTS: We observed significantly increased IFN-γ and TNF-α secretion from Kv-stimulated PBMCs of sarcoidosis patients vs. PBMCs from healthy volunteers (IFN-γ: 207.2 pg/mL vs. 3.86 pg/mL, p = 0.0018; TNF-α: 2375 pg/mL vs. 42.82 pg/mL, p = 0.0003). Through proteomic approaches we then identified 74 sarcoidosis tissue-specific proteins. Of these, 3 proteins (vimentin, tubulin and alpha-actinin-4) were identified using both 1D-SDS-PAGE and 2D-DIGE. Data are available via ProteomeXchange with identifier PXD005150. Increased cytokine secretion was subsequently observed with vimentin stimulation of sarcoidosis PBMCs vs. tuberculosis PBMCs (IFN-γ: 396.6 pg/mL vs 0.1 pg/mL, p = 0.0009; TNF-α: 1139 pg/mL vs 0.1 pg/mL, p<0.0001). This finding was also observed in vimentin stimulation of sarcoidosis PBMCs compared to PBMCs from healthy controls (IFN-γ: 396.6 pg/mL vs. 0.1 pg/mL, p = 0.014; TNF-α: 1139 pg/mL vs 42.29 pg/mL, p = 0.027). No difference was found in cytokine secretion between sarcoidosis and control PBMCs when stimulated with either tubulin or alpha-actinin-4. CONCLUSIONS: Stimulation with both Kveim reagent and vimentin induces a specific pro-inflammatory cytokine secretion from sarcoidosis PBMCs. Further investigation of cellular immune responses to Kveim-specific proteins may identify novel biomarkers to assist the diagnosis of sarcoidosis.

Differential recruitment and activation of natural killer cell sub-populations by Mycobacterium bovis-infected dendritic cells.

Through complex interplay with APCs, subsets of NK cells play an important role in shaping adaptive immune responses. Bovine tuberculosis, caused by Mycobacterium bovis, is increasing in incidence and detailed knowledge of host-pathogen interactions in the natural host is essential to facilitate disease control. We investigated the interactions of NK-cell sub-populations and M. bovis-infected DCs to determine early innate mechanisms in the response to infection. A sub-population of NK cells (NKp46(+) CD2(-) ) selectively expressing lymphoid homing and inflammatory chemokine receptors were induced to migrate towards M. bovis-infected DCs. This migration was associated with increased expression of chemokines CCL3, 4, 5, 20 and CXCL8 by M. bovis-infected DCs. Activation of NKp46(+) CD2(-) NK cells and secretion of IFN-γ was observed, a response reliant on localised IL-12 release and direct cellular interaction. In a reciprocal manner, NKp46(+) CD2(-) cells induced an increase in the intensity of cell surface MHC class II expression on DCs. In contrast, NKp46(+) CD2(+) NK cells were unable to secrete IFN-γ and did not reciprocally affect DCs. This study provides novel evidence to demonstrate distinct effector responses between bovine NK-cell subsets during mycobacterial infection.

BCG vaccination of neonatal calves: potential roles for innate immune cells in the induction of protective immunity.

Bovine tuberculosis is a disease of increasing incidence in the UK causing major economic losses and with significant impact on bovine and, potentially human health: the causative agent Mycobacterium bovis is a zoonotic pathogen. Neonatal vaccination with the attenuated M. bovis Bacille Calmette Guerin (BCG) vaccine confers a significant degree of protection in cattle, and is a widely used control strategy for human TB. The adaptive immune system is relatively immature in neonates and increased numbers of innate effector cells present in young animals and human infants may compensate for this, enabling effective immune responses to vaccination. Natural killer cells and subsets of γδ TCR+ T lymphocytes secrete high levels of interferon gamma and can interact with antigen presenting cells to promote both innate and adaptive immune responses. These cell populations may be pivotal in determining immune bias following neonatal vaccination with BCG.

The bovine chemokine receptors and their mRNA abundance in mononuclear phagocytes.

BACKGROUND: The chemokine and chemokine receptor families play critical roles in both the healthy and diseased organism mediating the migration of cells. The chemokine system is complex in that multiple chemokines can bind to one chemokine receptor and vice versa. Although chemokine receptors have been well characterised in humans, the chemokine receptor repertoire of cattle is not well characterised and many sequences are yet to be experimentally validated. RESULTS: We have identified and sequenced bovine homologs to all identified functional human chemokine receptors. The bovine chemokine receptors show high levels of similarity to their human counterparts and similar genome arrangements. We have also characterised an additional bovine chemokine receptor, not present in the available genome sequence of humans or the more closely related pigs or horses. This receptor shows the highest level of similarity to CCR1 but shows significant differences in regions of the protein that are likely to be involved in ligand binding and signalling. We have also examined the mRNA abundance levels of all identified bovine chemokine receptors in mononuclear phagocytic cells. Considerable differences were observed in the mRNA abundance levels of the receptors, and interestingly the identified novel chemokine receptor showed differing levels of mRNA abundance to its closest homolog CCR1. The chemokine receptor repertoire was shown to differ between monocytes, macrophages and dendritic cells. This may reflect the differing roles of these cells in the immune response and may have functional consequences for the trafficking of these cells in vivo. CONCLUSIONS: In summary, we have provided the first characterisation of the complete bovine chemokine receptor gene repertoire including a gene that is potentially unique to cattle. Further study of this receptor and its ligands may reveal a specific role of this receptor in cattle. The availability of the bovine chemokine receptor sequences will allow further characterisation of the function of these genes and will confer wide-reaching benefits to the study of this important aspect of the bovine immune response.