Epstein-Barr virus (EBV) latently infected B-cells are the precursors of EBV-associated malignancies. EBV-infection induces the production of pro-survival and anti-inflammatory cytokines that may be important in the transition between latency and malignancy. One EBV protein, LMP2A, can be detected in both latently infected resting B-cells and in EBV-associated malignancies. Therefore, we tested the ability of LMP2A to influence cytokine production using both LMP2A-Tg primary B-cells and LMP2A-expressing B-cell lines. Our data demonstrate that LMP2A does not globally alter B-cell-produced cytokine levels, but specifically targets IL-10. Additional studies using ELISA and real-time-RT-PCR confirm that LMP2A utilizes PI3-kinase to increase IL-10 levels. Finally, the data demonstrate that LMP2A-expressing B-cell lines are more dependent on IL-10 for survival in comparison to LMP2A-negative B-cell lines. These data identify a novel function of LMP2A in the alteration of a cytokine that is important for both tumour survival and anti-tumour responses.
Previous data demonstrate that Epstein-Barr Virus Latent Membrane Protein 2A (LMP2A) enhances IL-10 to promote the survival of LMP2A-expressing B cell lymphomas. Since STAT3 is an important regulator of IL-10 production, we hypothesized that LMP2A activates a signal transduction cascade that increases STAT3 phosphorylation to enhance IL-10. Using LMP2A-negative and –positive B cell lines, the data indicate that LMP2A requires the early signaling molecules of the Syk/RAS/PI3K pathway to increase IL-10. Additional studies indicate that the PI3K-regulated kinase, BTK, is responsible for phosphorylating STAT3, which ultimately mediates the LMP2A-dependent increase in IL-10. These data are the first to show that LMP2A signaling results in STAT3 phosphorylation in B cells through a PI3K/BTK-dependent pathway. With the use of BTK and STAT3 inhibitors to treat B cell lymphomas in clinical trials, these findings highlight the possibility of using new pharmaceutical approaches to treat EBV-associated lymphomas that express LMP2A.
Cyanobacteria (“blue-green algae”), such as Oscillatoria sp., are a ubiquitous group of bacteria found in freshwater systems worldwide that are linked to illness and in some cases, death among humans and animals. Exposure to cyanobacteria occurs via ingestion of contaminated water or food-products. Exposure of the gut to these bacteria also exposes their toxins, such as lipopolysaccharide (LPS), to B cells in the gut associated lymphoid tissue. However, the effect of Oscillatoria sp. LPS on B cell activation is unknown. To test the hypothesis that Oscillatoria sp. LPS exposure to murine B cells would result in B cell activation, murine B cells were incubated in the absence or presence of Oscillatoria sp. LPS or E. coli LPS as a positive control. The data indicate that Oscillatoria sp. LPS induces B cells to proliferate, upregulate MHC II and CD86, enhance antigen uptake and induce IgM production at low levels. Additional studies demonstrate that this low level of stimulation may be due to incomplete TLR4 signaling induced by Oscillatoria sp. LPS, since IRF-3 is not induced in B cells after stimulation with Oscillatoria sp. LPS. These findings have important implications for the mechanisms of toxicity of cyanobacteria in both humans and animals.
Epstein-Barr virus Latent Membrane Protein 2A (LMP2A) is expressed in EBV-infected B cells in the germinal center, a site of significant apoptosis induced by engagement of Fas on activated B cells. Signals from the B cell receptor (BCR) protect germinal center B cells from Fas-mediated apoptosis, and since LMP2A is a BCR mimic, we hypothesized that LMP2A would also protect B cells from Fas-mediated apoptosis. Surprisingly, latently-infected human and murine B cell lines expressing LMP2A were more sensitive to Fas-mediated apoptosis, as determined by increases in Annexin-V staining, and cleavage of caspase-8, −3 and PARP. Additional studies show that LMP2A-expressing B cell lines demonstrate a Lyn- and Syk-dependent increase in sensitivity to Fas-mediated apoptosis, due to an LMP2A-dependent enhancement in Fas expression. These findings demonstrate the ability for LMP2A to directly increase a pro-apoptotic molecule and have implications for EBV latency as well as the treatment of EBV-associated malignancies.
The incidence of Hodgkin's lymphoma (HL) is growing due to an increase in Epstein‐Barr virus (EBV)‐associated HL in AIDS patients. The HL tumor microenvironment is vital for the survival of the malignant Hodgkin‐Reed Sternberg (HRS) cells of HL, which express the EBV protein latent membrane protein 2A (LMP2A). While previous work shows that LMP2A mimics B‐cell receptor (BCR) signaling to promote the survival of HRS cells, the ability of LMP2A to establish and maintain the tumor microenvironment through the production of chemokines remains unknown. Since BCR signaling induces the production of the chemokine macrophage inflammatory protein‐1α (MIP‐1α), and since LMP2A is a BCR mimic, we hypothesized that LMP2A increases MIP‐1α levels. A comparison of multiple LMP2A‐negative and ‐positive cell lines demonstrates that LMP2A increases MIP‐1α. Additionally, LMP2A‐mutant cell lines and pharmacologic inhibitors indicate that LMP2A activates a Syk/PI3K/NF‐κB pathway to enhance MIP‐1α. Finally, based on the finding that an NF‐κB inhibitor decreased MIP‐1α RNA/protein in LMP2A‐positive cells, we are the first to demonstrate that LMP2A increases the nuclear localization of the NF‐κB p65 subunit using DNA‐binding assays and confocal microscopy in human B cells. These findings not only have implications for the treatment of HL, but also other LMP2A‐expressing B‐cell tumors that overexpress NF‐κB.
The composition of the intestinal microbiota has been linked to the risk for developing allergic disease. Although the mechanism by which the microbiota prevents allergic sensitization is unclear, evidence suggests that it is through modulation of the immune system. 1 In this study we investigated the ability of the spore-forming gram-positive bacterium Bacillus subtilis to prevent the development of allergic disease. Previous work from our lab established B. subtilis as an immune suppressive bacterium that can prevent diarrheal disease from Citrobacter rodentium infection. 2, 3 In this study we tested the ability of B. subtilis spores to prevent house dust mite (HDM)induced eosinophilic inflammation using the treatment protocol shown in Figure 1A, that was approved by the Midwestern University Institutional Animal Care and Use Committee. C57Bl/6 mice were orally gavaged with 10 9 WT B. subtilis spores or 10 9 epsH spores that are unable to produce exopolysaccharide (EPS), due to a mutation in the eps operon 4. Our previous work found that EPS was the component of B. subtilis required for protection from C. rodentium induced inflammation 4, 5. Following the final HDM treatment, bronchial alveolar lavage (BAL) was collected from euthanized mice, by flushing the lungs with 0.8mL PBS containing 10% FCS, 1mM EDTA) and immune cell infiltration was assessed by DiffQuick (Dade Behring) staining of cytospun cells (Figure 1 BE). HDM from Dermatophagoides pteronyssinus (XPB82D3A2.5, Stallergenes Greer) alone induces a significant eosinophilia, shown as both percent and total cells/mL (Figure 1B-C). WT B. subtilis spore treatment significantly reduced the eosinophilia in HDM-treated mice. The B.
Glucocorticoid-resistant asthma, which predominates with neutrophils instead of eosinophils, is an increasing health concern. One potential source for the induction of neutrophil-predominant asthma is aerosolized lipopolysaccharide (LPS). Cyanobacteria have recently caused significant tidal blooms, and aerosolized cyanobacterial LPS has been detected near the cyanobacterial overgrowth. We hypothesized that cyanobacterial LPS contributes to lung inflammation by increasing factors that promote lung inflammation and neutrophil recruitment. To test this hypothesis, c57Bl/6 mice were exposed intranasally to LPS from the cyanobacterium member, Geitlerinema sp., in vivo to assess neutrophil infiltration and the production of pro-inflammatory cytokines and chemokines from the bronchoalveolar fluid by ELISA. Additionally, we exposed the airway epithelial cell line, A549, to Geitlerinema sp. LPS in vitro to confirm that airway epithelial cells were stimulated by this LPS to increase cytokine production and the expression of the adhesion molecule, ICAM-1. Our data demonstrate that Geitlerinema sp. LPS induces lung neutrophil infiltration, the production of pro-inflammatory cytokines such as Interleukin (IL)-6, Tumor necrosis factor-alpha, and Interferongamma as well as the chemokines IL-8 and RANTES. Additionally, we demonstrate that Geitlerinema sp. LPS directly activates airway epithelial cells to produce pro-inflammatory cytokines and the adhesion molecule, Intercellular Adhesion Molecule-1 (ICAM-1), in vitro using the airway epithelial cell line, A549. Based on our findings that use Geitlerinema sp. LPS as a model system, the data indicate that cyanobacteria LPS may contribute to the development of glucocorticoid-resistant asthma seen near water sources that contain high levels of cyanobacteria.
Cyanobacterial blooms are an increasing source of environmental toxins that affect both human and animals. After ingestion of cyanobacteria, such as Geitlerinema sp., toxins and lipopolysaccharide (LPS) from this organism induce fever, gastrointestinal illness, and even death. However, little is known regarding the effects of cyanobacterial LPS on human monocytes after exposure to LPS upon ingestion. Based on our previous data using Geitlerinema sp. LPS (which was previously named Oscillatoria sp., a genus belonging to the same order as Geitlerinema), we hypothesized that Geitlerinema sp. LPS would activate human monocytes to proliferate, phagocytose particles, and produce cytokines that are critical for promoting proinflammatory responses in the gut. Our data demonstrate that Geitlerinema sp. LPS induced monocyte proliferation and TNF-α, IL-1, and IL-6 production at high concentrations. In contrast, Geitlerinema sp. LPS is equally capable of inducing monocyte-mediated phagocytosis of FITC-latex beads when compared with Escherichia coli LPS, which was used as a positive control for our experiments. In order to understand the mechanism responsible for the difference in efficacy between Geitlerinema sp. LPS and E. coli LPS, we performed biochemical analysis and identified that Geitlerinema sp. LPS was composed of significantly different sugars and fatty acid side chains in comparison to E. coli LPS. The lipid A portion of Geitlerinema sp. LPS contained longer fatty acid side chains, such as C15:0, C16:0, and C18:0, instead of C12:0 found in E. coli LPS which may explain the decreased efficacy and toxicity of Geitlerinema sp. LPS in comparison to E. coli LPS.
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