bMycoplasma bovis is one of the major causative pathogens of bovine respiratory complex disease (BRD), which is characterized by enzootic pneumonia, mastitis, pleuritis, and polyarthritis. M. bovis enters and colonizes bovine respiratory epithelial cells through inhalation of aerosol from contaminated air. The nature of the interaction between M. bovis and the bovine innate immune system is not well understood. We hypothesized that M. bovis invades blood monocytes and regulates cellular function to support its persistence and systemic dissemination. We used bovine-specific peptide kinome arrays to identify cellular signaling pathways that could be relevant to M. bovis-monocyte interactions in vitro. We validated these pathways using functional, protein, and gene expression assays. Here, we show that infection of bovine blood monocytes with M. bovis delays spontaneous or tumor necrosis factor alpha (TNF-␣)/staurosporine-driven apoptosis, activates the NF-B p65 subunit, and inhibits caspase-9 activity. We also report that M. bovis-infected bovine monocytes do not produce gamma interferon (IFN-␥) and TNF-␣, although the level of production of interleukin-10 (IL-10) is elevated. Our findings suggest that M. bovis takes over the cellular machinery of bovine monocytes to prolong bacterial survival and to possibly facilitate subsequent systemic distribution.

Prebiotics are non-digestible oligosaccharides that promote the growth of beneficial gut microbes, but it is unclear whether they also have direct effects on the intestinal mucosal barrier. Here we demonstrate two commercial prebiotics, inulin and short-chain fructo-oligosaccharide (scFOS), when applied onto intestinal epithelia in the absence of microbes, directly promote barrier integrity to prevent pathogen-induced barrier disruptions. We further show that these effects involve the induction of select tight junction (TJ) proteins through a protein kinase C (PKC) δ-dependent mechanism. These results suggest that in the absence of microbiota, prebiotics can directly exert barrier protective effects by activating host cell signaling in the intestinal epithelium, which represents a novel alternative mechanism of action of prebiotics.

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BackgroundPrebiotics are non-digestible food ingredients that enhance the growth of certain microbes within the gut microbiota. Prebiotic consumption generates immune-modulatory effects that are traditionally thought to reflect microbial interactions within the gut. However, recent evidence suggests they may also impart direct microbe-independent effects on the host, though the mechanisms of which are currently unclear.MethodsKinome arrays were used to profile the host intestinal signaling responses to prebiotic exposures in the absence of microbes. Identified pathways were functionally validated in Caco-2Bbe1 intestinal cell line and in vivo model of murine endotoxemia.ResultsWe found that prebiotics directly regulate host mucosal signaling to alter response to bacterial infection. Intestinal epithelial cells (IECs) exposed to prebiotics are hyporesponsive to pathogen-induced mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NF-κB) activations, and have a kinome profile distinct from non-treated cells pertaining to multiple innate immune signaling pathways. Consistent with this finding, mice orally gavaged with prebiotics showed dampened inflammatory response to lipopolysaccharide (LPS) without alterations in the gut microbiota.ConclusionsThese findings provide molecular mechanisms of direct host-prebiotic interactions to support prebiotics as potent modulators of host inflammation.Electronic supplementary materialThe online version of this article (10.1186/s40168-017-0357-4) contains supplementary material, which is available to authorized users.

Recent investigations associate Varroa destructor (Mesostigmata: Varroidae) parasitism and its associated pathogens and agricultural pesticides with negative effects on colony health, resulting in sporadic global declines in domestic honeybee (Apis mellifera) populations. These events have motivated efforts to develop research tools that can offer insight into the causes of declining bee health as well as identify biomarkers to guide breeding programs. Here we report the development of a bee-specific peptide array for characterizing global cellular kinase activity in whole bee extracts. The arrays reveal distinct, developmentally-specific signaling profiles between bees with differential susceptibility to infestation by Varroa mites. Gene ontology analysis of the differentially phosphorylated peptides indicates that the differential susceptibility to Varroa mite infestation does not reflect compromised immunity; rather, there is evidence for mite-mediated immune suppression within the susceptible phenotype that may reduce the ability of these bees to counter secondary viral infections. This hypothesis is supported by the demonstration of more diverse viral infections in mite-infested, susceptible adult bees. The bee-specific peptide arrays are an effective tool for understanding the molecular basis of this complex phenotype as well as for the discovery and utilization of phosphorylation biomarkers for breeding programs.

A lack of appropriate disease models has limited our understanding of the pathogenesis of persistent enteric infections with Mycobacterium avium subsp. paratuberculosis. A model was developed for the controlled delivery of a defined dose of M. avium subsp. paratuberculosis to surgically isolated ileal segments in newborn calves. The stable intestinal segments enabled the characterization of host responses to persistent M. avium subsp. paratuberculosis infections after a 9-month period, including an analysis of local mucosal immune responses relative to an adjacent uninfected intestinal compartment. M. avium subsp. paratuberculosis remained localized at the initial site of intestinal infection and was not detected by PCR in the mesenteric lymph node. M. avium subsp. paratuberculosis-specific T cell proliferative responses included both CD4 and ␥␦ T cell receptor (␥␦TcR) T cell responses in the draining mesenteric lymph node. The levels of CD8 ؉ and ␥␦TcR ؉ T cells increased significantly (P < 0.05) in the lamina propria, and M. avium subsp. paratuberculosis-specific tumor necrosis factor alpha (TNF-␣) and gamma interferon secretion by lamina propria leukocytes was also significantly (P < 0.05) increased. There was a significant (P < 0.05) accumulation of macrophages and dendritic cells (DCs) in the lamina propria, but the expression of mucosal toll-like receptors 1 through 10 was not significantly changed by M. avium subsp. paratuberculosis infection. In conclusion, surgically isolated ileal segments provided a model system for the establishment of a persistent and localized enteric M. avium subsp. paratuberculosis infection in cattle and facilitated the analysis of M. avium subsp. paratuberculosis-specific changes in mucosal leukocyte phenotype and function. The accumulation of DC subpopulations in the lamina propria suggests that further investigation of mucosal DCs may provide insight into host responses to M. avium subsp. paratuberculosis infection and improve vaccine strategies to prevent M. avium subsp. paratuberculosis infection.

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Inter-individual variance in host immune responses following vaccination can result in failure to develop protective immunity leaving individuals at risk for infection in addition to compromising herd immunity. While developing more efficacious vaccines is one strategy to mitigate this problem, predicting vaccine responsiveness prior to vaccination could inform which individuals require adjunct disease management strategies. To identify biomarkers of vaccine responsiveness, a cohort of pigs (n = 120) were vaccinated and pigs representing the high (n = 6; 90th percentile) and low (n = 6; 10th percentile) responders based on vaccine-specific antibody responses following vaccination were further analyzed. Kinase-mediated phosphorylation events within peripheral blood mononuclear cells collected prior to vaccination identified 53 differentially phosphorylated peptides when comparing low responders with high responders. Functional enrichment analysis revealed pro-inflammatory cytokine signaling pathways as dysregulated, and this was further substantiated by detection of higher (p < 0.01) concentrations of interferon-gamma in plasma of low responders compared to high responders prior to vaccination. In addition, low responder pigs with high plasma interferon-gamma showed lower (p < 0.01) birth weights than high responder pigs. These associations between vaccine responsiveness, cytokine signaling within peripheral immune cells, and body weight in pigs provide both evidence and insight into potential biomarkers for identifying low responders to vaccination.