Infection with Mycobacterium avium subsp. paratuberculosis results in rapid interleukin-1β release and macrophage transepithelial migration.
ABSTRACT Pathogen processing by the intestinal epithelium involves a dynamic innate immune response initiated by pathogen-epithelial cell cross talk. Interactions between epithelium and Mycobacterium avium subsp. paratuberculosis have not been intensively studied, and it is currently unknown how the bacterium-epithelial cell cross talk contributes to the course of infection. We hypothesized that M. avium subsp. paratuberculosis harnesses host responses to recruit macrophages to the site of infection to ensure its survival and dissemination. We investigated macrophage recruitment in response to M. avium subsp. paratuberculosis using a MAC-T bovine macrophage coculture system. We show that M. avium subsp. paratuberculosis infection led to phagosome acidification within bovine epithelial (MAC-T) cells as early as 10 min, which resulted in upregulation of interleukin-1β (IL-1β) at transcript and protein levels. Within 10 min of infection, macrophages were recruited to the apical side of MAC-T cells. Inhibition of phagosome acidification or IL-1β abrogated this response, while MCP-1/CCL-2 blocking had no effect. IL-1β processing was dependent upon Ca(2+) uptake from the extracellular medium and intracellular Ca(2+) oscillations, as determined by EGTA and BAPTA-AM [1,2-bis(2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid tetrakis (acetoxymethyl ester)] treatments. Thus, M. avium subsp. paratuberculosis is an opportunist that takes advantage of extracellular Ca(2+)-dependent phagosome acidification and IL-1β processing in order to efficiently transverse the epithelium and enter its niche--the macrophage.
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ABSTRACT: Vaccination is the most cost effective control measure for Johne's disease caused by Mycobacterium avium subspecies paratuberculosis (MAP) but currently available whole cell killed formulations have limited efficacy and are incompatible with the diagnosis of bovine tuberculosis by tuberculin skin test. We have evaluated the utility of a viral delivery regimen of non-replicative human Adenovirus 5 and Modified Vaccinia virus Ankara recombinant for early entry MAP specific antigens (HAV) to show protection against challenge in a calf model and extensively screened for differential immunological markers associated with protection. We have shown that HAV vaccination was well tolerated, could be detected using a differentiation of infected and vaccinated animals (DIVA) test, showed no cross-reactivity with tuberculin and provided a degree of protection against challenge evidenced by a lack of faecal shedding in vaccinated animals that persisted throughout the 7 month infection period. Calves given HAV vaccination had significant priming and boosting of MAP derived antigen (PPD-J) specific CD4+, CD8+ IFN-γ producing T-cell populations and, upon challenge, developed early specific Th17 related immune responses, enhanced IFN-γ responses and retained a high MAP killing capacity in blood. During later phases post MAP challenge, PPD-J antigen specific IFN-γ and Th17 responses in HAV vaccinated animals corresponded with improvements in peripheral bacteraemia. By contrast a lack of IFN-γ, induction of FoxP3+ T cells and increased IL-1β and IL-10 secretion were indicative of progressive infection in Sham vaccinated animals. We conclude that HAV vaccination shows excellent promise as a new tool for improving control of MAP infection in cattle.Veterinary Research 01/2014; 45(1):112. DOI:10.1186/s13567-014-0112-9 · 3.38 Impact Factor
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ABSTRACT: Thirty-one bison heifers were randomly assigned to saline or single vaccination with 10(10) CFU of B. abortus strain RB51 (RB51). Some vaccinated bison were randomly selected for booster vaccination RB51 at 11 months after initial vaccination. Mean antibody responses to RB51 were greater (P<0.05) in vaccinates after initial vaccination and booster vaccinates as compared to non-vaccinates. Proliferative responses by peripheral blood mononuclear cells (PBMC) from vaccinates were greater (P<0.05) than non-vaccinates at 16 and 24 weeks after initial vaccination, but not after booster vaccination. Relative gene expression of IFN-γ was increased (P<0.05) in RB51 vaccinates at 8, 16, and 24 weeks after initial vaccination, and at 8 weeks after booster vaccination. Vaccinated bison had greater (P<0.05) in vitro production of IFN-γ at all sampling times, IL-1β in various samplings after initial and booster vaccination, and IL-6 in one sampling time after booster vaccination. Between 170 and 180 days of gestation, bison were intraconjunctivally challenged with approximately 1 x 10(7) CFU of B. abortus strain 2308. The incidence of abortion and infection was greater (P<0.05) in non-vaccinated bison after experimental challenge as compared to either vaccination treatment. Booster vaccinates, but not single vaccinates, had reduced (P<0.05) incidence of infection in fetal tissues and maternal tissues when compared to controls. As compared to non-vaccinates, both vaccination treatments lower colonization (CFU/gm) of Brucella in all tissues except retropharyngeal and supramammary lymph nodes. Our study suggests that RB51 booster vaccination is an effective vaccination strategy for enhancing herd immunity in bison against brucellosis. Copyright © 2015, American Society for Microbiology. All Rights Reserved.Clinical and vaccine Immunology: CVI 02/2015; DOI:10.1128/CVI.00746-14 · 2.37 Impact Factor
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ABSTRACT: Johne’s disease, caused by infection with Mycobacterium avium subsp. paratuberculosis, (MAP), is a chronic intestinal disease of ruminants with serious economic consequences for cattle production in the United States and elsewhere. During infection, MAP bacilli are phagocytosed and subvert host macrophage processes, resulting in subclinical infections that can lead to immunopathology and dissemination of disease. Analysis of the host macrophage transcriptome during infection can therefore shed light on the molecular mechanisms and host-pathogen interplay associated with Johne’s disease. Here we describe results of an in vitro study of the bovine monocyte-derived macrophage (MDM) transcriptome response during MAP infection using RNA-seq. MDM were obtained from seven age- and sex-matched Holstein-Friesian cattle and were infected with MAP across a six-hour infection time course with non-infected controls. We observed 245 and 574 differentially expressed genes in MAP-infected versus non-infected control samples (adjusted P value ≤ 0.05) at 2 and 6 hours post-infection, respectively. Functional analyses of these differentially expressed genes, including biological pathway enrichment, highlighted potential functional roles for genes that have not been previously described in the host response to infection with MAP bacilli. In addition, differential expression of pro- and anti-inflammatory cytokine genes, such as those associated with the IL-10 signaling pathway, and other immune-related genes that encode proteins involved in the bovine macrophage response to MAP infection emphasize the balance between protective host immunity and bacilli survival and proliferation. Systematic comparisons of RNA-seq gene expression results with Affymetrix® microarray data generated from the same experimental samples also demonstrated that RNA-seq represents a superior technology for studying host transcriptional responses to intracellular infection.Frontiers in Immunology 02/2015; 6. DOI:10.3389/fimmu.2015.00023