Early response of bovine alveolar macrophages to infection with live and heat-killed Mycobacterium bovis
ABSTRACT: Bovine tuberculosis, caused by Mycobacterium bovis, is a disease of considerable economic importance yet comparatively little is known about the bovine immune response to the disease. Alveolar macrophages are one of the first cells to encounter mycobacteria following infection. In this experiment we investigated the early transcriptional response of bovine alveolar macrophages following infection with M. bovis. The transcriptional response to heat-killed M. bovis was also investigated to look for genes that are only differentially transcribed in response to the live organism. Five-condition experiment, uninfected, live and heat-killed M. bovis-infected bovine alveolar macrophages from five cattle infected for two and four hours. Comparisons were within animal. Dye swaps were incorporated into the design.
Project description:Bovine tuberculosis, caused by Mycobacterium bovis, is a disease of considerable economic importance yet comparatively little is known about the bovine immune response to the disease. Alveolar macrophages are one of the first cells to encounter mycobacteria following infection. In this experiment we investigated the early transcriptional response of bovine alveolar macrophages following infection with M. bovis. The transcriptional response to heat-killed M. bovis was also investigated to look for genes that are only differentially transcribed in response to the live organism. Overall design: Five-condition experiment, uninfected, live and heat-killed M. bovis-infected bovine alveolar macrophages from five cattle infected for two and four hours. Comparisons were within animal. Dye swaps were incorporated into the design.
Project description:Innate immunity is evolutionarily conserved in multicellular organisms and was considered to lack memory until very recently. One of its more characteristic mechanisms is phagocytosis, the ability of cells to engulf, process and eventually destroy any injuring agent. We report the results of an ex vivo experiment in bovine macrophages in which improved clearance of Mycobacterium bovis (M. bovis) was induced by pre-exposure to a heat killed M. bovis preparation. The effects were independent of humoral and cellular adaptive immune responses and lasted up to six months. Specifically, our results demonstrate the existence of a training effect in the lytic phase of phagocytosis that can be activated by killed mycobacteria, thus suggesting a new mechanism of vaccine protection. These findings are compatible with the recently proposed concept of trained immunity, which was developed to explain the observation that innate immune responses provide unspecific protection against pathogens including other than those that originally triggered the immune response.
Project description:Conventional control and eradication strategies for bovine tuberculosis (BTB) face tremendous difficulties in developing countries; countries with wildlife reservoirs, a complex wildlife-livestock-human interface or a lack of veterinary and veterinary public health surveillance. Vaccination of cattle and other species might in some cases provide the only suitable control strategy for BTB, while in others it may supplement existing test-and-slaughter schemes. However, the use of live BCG has several limitations and the global rise of HIV/AIDS infections has furthermore warranted the exploration of inactivated vaccine preparations. The aim of this study was to compare the immune response profiles in response to parenteral vaccination with live BCG and two inactivated vaccine candidates in cattle. Twenty-four mixed breed calves (Bos taurus) aged 4-6 months, were allocated to one of four groups and vaccinated sub-cutaneously with live M. bovis BCG (Danish 1331), formalin-inactivated M. bovis BCG, heat-killed M. bovis or PBS/Montanide™ (control). Interferon-? responsiveness and antibody production were measured prior to vaccination and at weekly intervals thereafter for twelve weeks. At nine weeks post-priming, animals were skin tested using tuberculins and MTBC specific protein cocktails and subsequently challenged through intranodular injection of live M. bovis BCG. The animals in the heat-killed M. bovis group demonstrated strong and sustained cell-mediated and humoral immune responses, significantly higher than the control group in response to vaccination, which may indicate a protective immune profile. Animals in this group showed reactivity to the skin test reagents, confirming good vaccine take. Lastly, although not statistically significant, recovery of BCG after challenge was lowest in the heat-killed M. bovis group. In conclusion, the parenteral heat-killed M. bovis vaccine proved to be clearly immunogenic in cattle in the present study, urging further evaluation of the vaccine in challenge studies using virulent M. bovis and assessment of vaccine efficacy in field conditions.
Project description:Our goal is to discriminate specific genes in live M.leprae-infected peritoneal macrophages in comparison to heat-killed M.leprae infected peritoneal macrophages using microarray. Overall design: Two-condition experiment, Heat-killed M.leprae infected macrophage vs. Live M.leprae infected macrophage. Biological replicates: 16 mice (control), 8 heat-killed M. leprae mice (sample 1), and 8 live M. leprae infected mice (sample 2). Independently grown and harvested from isolator. One replicate per array.
Project description:Our goal is to discriminate specific genes in live M.leprae-infected peritoneal macrophages in comparison to heat-killed M.leprae infected peritoneal macrophages using microarray. Two-condition experiment, Heat-killed M.leprae infected macrophage vs. Live M.leprae infected macrophage. Biological replicates: 16 mice (control), 8 heat-killed M. leprae mice (sample 1), and 8 live M. leprae infected mice (sample 2). Independently grown and harvested from isolator. One replicate per array.
Project description:BACKGROUND:Bacterial infections are common in postpartum dairy cows. Cortisol level has been observed to increase in dairy cows during peripartum period, and is associated with the endometrial innate immunity against pathogens like E.coli. However, the mechanism underlying how cortisol regulates E.coli-induced inflammatory response in bovine endometrial epithelial cells (BEEC) remains elusive. RESULTS:Cortisol decreased the expressions of IL1?, IL6, TNF-?, IL8, and TLR4 mRNA in BEEC treated with LPS or heat-killed E.coli, but up-regulated these gene expressions in BEEC stimulated by live E.coli. CONCLUSION:Cortisol exerted the anti-inflammatory action on LPS- or heat-killed E.coli-stimulated BEEC, but the pro-inflammatory action on live E.coli-induced BEEC.
Project description:This study aimed to evaluate the capacity of Lactobacillus rhamnosus and/or its products to induce the synthesis of cytokines (TNF-α, IL-1β, IL-4, IL-6, IL-10, and IL-12) by mouse macrophages (RAW 264.7). Three microorganism preparations were used: live L. rhamnosus (LLR) suspension, heat-killed L. rhamnosus (HKLR) suspension, and the supernatant of a heat-killed L. rhamnosus (SHKLR) suspension, which were cultured with macrophages (37°C, 5% CO2) for 2 h and 30 min. After that, cells were cultured for 16 h. The supernatants were used for the quantitation of cytokines, by ELISA. The results were compared with the synthesis induced by lipopolysaccharide (LPS) and analysed, using ANOVA and Tukey test, 5%. LLR and HKLR groups were able to significantly increase the production of TNF-α, IL-6, and IL-10 (P < 0.05). SHKLR also significantly increased the production of TNF-α and IL-10 (P < 0.05) but not IL-6 (P > 0.05). All the L. rhamnosus suspensions were not able to produce detectable levels of IL-1β or significant levels of IL-4 and IL-12 (P > 0.05). In conclusion, live and heat-killed L. rhamnosus suspensions were able to induce the synthesis of different cytokines with proinflammatory (TNF-α and IL-6) or regulatory (IL-10) functions, suggesting the role of strain L. rhamnosus ATCC 7469 in the modulation or in the stimulation of immune responses.
Project description: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 beta-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 beta-10, whereas bacterial suspensions upregulated thymosin beta-10 expression, suggesting that M. bovis or mycobacterial products are essential in the process. Heat-inactivated M. bovis induced a slight increase in thymosin beta-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 beta-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 beta-10 in M. bovis-infected macrophages is linked with increased cell death due to apoptosis.
Project description:Mycobacterium bovis, the agent of bovine tuberculosis, causes an estimated $3 billion annual losses to global agriculture due, in part, to the limitations of current diagnostics. Development of next-generation diagnostics requires a greater understanding of the interaction between the pathogen and the bovine host. Therefore, to explore the early response of the alveolar macrophage to infection, we report the first application of RNA-sequencing to define, in exquisite detail, the transcriptomes of M. bovis-infected and non-infected alveolar macrophages from ten calves at 2, 6, 24 and 48?hours post-infection. Differentially expressed sense genes were detected at these time points that revealed enrichment of innate immune signalling functions, and transcriptional suppression of host defence mechanisms (e.g., lysosome maturation). We also detected differentially expressed natural antisense transcripts, which may play a role in subverting innate immune mechanisms following infection. Furthermore, we report differential expression of novel bovine genes, some of which have immune-related functions based on orthology with human proteins. This is the first in-depth transcriptomics investigation of the alveolar macrophage response to the early stages of M. bovis infection and reveals complex patterns of gene expression and regulation that underlie the immunomodulatory mechanisms used by M. bovis to evade host defence mechanisms.