Bacterial Lipopolysaccharide Challenge Research Articles

Identification of Ovine Serum miRNAs Following Bacterial Lipopolysaccharide Challenge.

Host–pathogen interactions are complex and influenced by host genetic and epigenetic modifications. Recently, the significance of microRNAs (miRNAs) in pathogenic infection and the regulation of immune response has been highlighted. However, information on miRNAs’ role in the course of inflammation is still very limited in small ruminants. The present study was intended to identify changes in the expression of circulatory miRNAs post-lipopolysaccharide (LPS)-challenge. In this study, young ewes (n = 18) were challenged with Escherichia coli LPS (400 ng/kg i.v.) and blood samples were collected for serum miRNA isolation at two-time points; prior to challenge (T0), and 4 h (T4) post-challenge, reflecting the peak cortisol response. A total of 91 miRNAs were profiled, including 84 miRNAs on a commercial ovine miRNA-PCR array, and seven individual miRNAs. Forty five miRNAs were differentially expressed (DE) with 35 being up-regulated (Fold regulation, FR > 2) and 10 being down-regulated (FR < 1, p < 0.05) at T4. Among the up-regulated miRNAs, 14 were significantly (p < 0.05) induced, including oar-miRs: 369-3p, 495-3p, 376a-3p, 543-3p, 668-3p, 329a-3p, 655-3p, 411a-5p, and 154a-3p, which were located on ovine chromosome 18 forming four miRNA clusters within 10 kb. The elevated miRNAs belonged to different functional classes, playing roles in activating the hypothalamic-pituitary-adrenal axis; increasing cell survival and differentiation; and inducing inflammatory responses and targeted PI3K-Akt and MAPK signaling and chemokine signaling pathways. In summary, these results reveal the dynamic nature of ovine serum miRNAs during LPS-induced stress and highlight the potential role of identified miRNA-clusters on chromosome 18 to understand the regulation of the acute-phase response. Some of these identified circulating miRNAs may also serve as stress biomarkers for livestock in the future.

Potential Role of DARC-Chemokine Interaction in the Recruitment of Osteoclast Precursors in Response to Bacterial Lipopolysaccharide Challenge.

Duffy antigen receptor for chemokines (DARC) binds to a number of pro-inflammatory chemokines, and since chemokines are known to regulate trafficking of osteoclast (OC) precursors, we predicted that DARC would regulate OC recruitment to sites of inflammation by modulating chemokine activity. To test this hypothesis, we evaluated the mRNA expression of Darc and the chemokines known to bind to DARC, in endothelial cells treated with bacterial lipopolysaccharide (LPS). The mRNA expression of Mcp-1, Rantes, Darc and Ccr5 was significantly increased in endothelial cells in response to LPS treatment. Blocking the function of DARC with neutralizing antibody partially abrogated the effect of LPS on the mRNA expression of Mcp-1 and Rantes. In vivo, mice with targeted disruption of Darc gene (Darc-KO) and control wild-type (WT) mice were used to assess the role of DARC in response to single LPS application on the top of parietal bones. Five hours post-LPS injection, local expression of Cd14 mRNA (a marker of inflammatory monocytes) was significantly increased in both lines of mice. However, the magnitude of increase was greater in WT mice compared with Darc-KO mice suggesting a role for DARC in mediating the recruitment of monocytes in response to LPS. Histological staining for tartrate-resistant acid phosphatase (TRAP) in calvaria sections taken from the injection sites revealed a significant reduction in TRAP-labeled surface per bone surface in response to LPS in Darc-KO mice compared with WT mice. Based on these findings, we concluded that DARC regulates recruitment of OC precursors at the inflammation site, probably through regulation of chemokines transcytosis across endothelial cell barrier.

Characterization of chitinase‐like proteins (Cg‐Clp1 and Cg‐Clp2) involved in immune defence of the mollusc Crassostrea gigas

Chitinase-like proteins have been identified in insects and mammals as nonenzymatic members of the glycoside hydrolase family 18. Recently, the first molluscan chitinase-like protein, named Crassostrea gigas (Cg)-Clp1, was shown to control the proliferation and synthesis of extracellular matrix components of mammalian chondrocytes. However, the precise physiological roles of Cg-Clp1 in oysters remain unknown. Here, we report the cloning and the characterization of a new chitinase-like protein (Cg-Clp2) from the oyster Crassostrea gigas. Gene expression profiles monitored by quantitative RT-PCR in adult tissues and through development support its involvement in tissue growth and remodelling. Both Cg-Clp1- and Cg-Clp2-encoding genes were transcriptionally stimulated in haemocytes in response to bacterial lipopolysaccharide challenge, strongly suggesting that these two close paralogous genes play a role in oyster immunity.

Persistent Nuclear Factor-κB Activation in Ucp2-/- Mice Leads to Enhanced Nitric Oxide and Inflammatory Cytokine Production

One of the phenotypes of mice with targeted disruption of the uncoupling protein-2 gene (Ucp2-/-) is greater macrophage phagocytic activity and free radical production, resulting in a striking resistance to infectious microorganisms. In this study, the molecular mechanisms of this enhanced immune response were investigated. We found that levels of nitric oxide measured in either plasma or isolated macrophages from Ucp2-/- mice are significantly elevated in response to bacterial lipopolysaccharide challenge compared with similarly treated Ucp2+/+ mice. Likewise, expression of inducible nitric-oxide synthase and inflammatory cytokines is higher in Ucp2-/- mice in vivo and in vitro. Key steps in the activation cascade of nuclear factor (NF)-kappa B, including I kappa B kinase and nuclear translocation of NF-kappa B subunits, are all remarkably enhanced in Ucp2-/- mice, most notably even under basal conditions. The elevated basal activity of I kappa B kinase in macrophages from Ucp2-/- mice can be blocked by cell-permeable inhibitors of superoxide and hydrogen peroxide generation, but not by a specific inhibitor for inducible nitric-oxide synthase. Isolated mitochondria from Ucp2-/- cells produced more superoxide/hydrogen peroxide. We conclude that mitochrondrially derived reactive oxygen from Ucp2-/- cells constitutively activates NF-kappa B, resulting in a "primed" state to both potentiate and amplify the inflammatory response upon subsequent stimulation.

Increased Levels of LPS-Binding Protein in Bovine Blood and Milk Following Bacterial Lipopolysaccharide Challenge

Several species of gram-negative bacteria, including Escherichia coli, Klebsiella pneumoniae, and various species of Enterobacter, are common mastitis pathogens. All of these bacteria are characterized by the presence of endotoxin or lipopolysaccharide (LPS) in their outer membrane. The bovine mammary gland is highly sensitive to LPS, and LPS has been implicated, in part, in the pathogenesis of gram-negative mastitis. Recognition of LPS is a key event in the innate immune response to gram-negative infection and is mediated by the accessory molecules CD14 and LPS-binding protein (LBP). The objective of the current study was to determine whether LBP levels increased in the blood and mammary gland following LPS challenge. The left and right quarters of five midlactating Holstein cows were challenged with either saline or LPS (100μg), respectively, and milk and blood samples collected. Basal levels of plasma and milk LBP were 38 and 6μg/ml, respectively. Plasma LBP levels increased as early as 8h post-LPS challenge and reached maximal levels of 138μg/ml by 24h. Analysis of whey samples derived from LPS-treated quarters revealed an increase in milk LBP by 12h. Similar to plasma, maximal levels of milk LBP (34μg/ml) were detected 24h following the initial LPS challenge. Increments in milk LBP levels paralleled a rise in soluble CD14 (sCD14) levels and initial rises in the levels of these proteins were temporally coincident with maximal neutrophil recruitment to the inflamed gland. Because LBP and sCD14 are known to enhance LPS-induced host cell activation and to facilitate detoxification of LPS, these data are consistent with a role for these molecules in mediating mammary gland responses to LPS.

Molecular evidence for the expression of angiotensin converting enzyme in hemocytes of Locusta migratoria: stimulation by bacterial lipopolysaccharide challenge

The presence of angiotensin converting enzyme (ACE) in insects has been reported many times, but numerous questions about the functional role of this enzyme in insects remain. Here we show by RT-PCR experiments that ACE has a wide tissue distribution in Locusta migratoria, suggesting diverse roles for this enzyme in the locust. Immune challenge through injection of bacterial lipopolysaccharides resulted in a tenfold increase of ACE gene transcripts in the hemocytes and is suggestive for a role of ACE in the cellular defense of the locust. However, phenotypic knockout experiments with the ACE inhibitor captopril showed that ACE is not essential for the efficient clearance of injected E. coli bacteria.

Colonic production and expression of IL-4, IL-6, and IL-10 in neonatal suckling rats after LPS challenge.

It has been demonstrated that the neonatal suckling rat is more susceptible to endotoxin [lipopolysaccharide (LPS)]-induced colonic damage compared with weaned littermates. There is evidence to suggest that differences in the production of certain cytokines, including interleukin (IL)-4, IL-6, and IL-10, are associated with intestinal inflammation in children. We have examined the production, localization, and mRNA detection of these cytokines in suckling and weaned rat colons after bacterial LPS challenge. Suckling (10 day old) and weaned (25 day old) rats were injected with LPS (3 mg/kg ip). Colon samples were taken up to 4 h after treatment, and cytokines were measured by ELISA. LPS-induced cytokine levels were significantly different in suckling rats compared with weaned rats. Cytokine localization to the colonic mucosa was evident in suckling rats up to 4 h after LPS administration but was not consistently seen in weaned rats. The mRNA for cytokines examined were detected by RT-PCR in suckling but not in weaned rat colons after LPS treatment. Induction of neutropenia via anti-neutrophil serum (ANS) administration did not affect cytokine mRNA detection in neonates after LPS treatment. Weaned animals displayed positive detection of all cytokines examined after ANS. Therefore, we have shown that the suckling rat displays a different production and expression of colonic IL-4, IL-6, and IL-10 compared with weaned littermates after LPS challenge. Furthermore, neutrophils may be implicated in colonic cytokine expression after LPS challenge in rats.