Human Serum Albumin (HSA) Suppresses the Effects of Glycerol Monolaurate (GML) on Human T Cell Activation and Function

<div><p>Glycerol monolaurate (GML) is a monoglyceride with well characterized anti-microbial properties. Because of these properties, GML is widely used in food, cosmetics, and personal care products and currently being tested as a therapeutic for menstrual associated toxic shock syndrome, superficial wound infections, and HIV transmission. Recently, we have described that GML potently suppresses select T cell receptor (TCR)-induced signaling events, leading to reduced human T cell effector functions. However, how soluble host factors present in the blood and at sites of infection affect GML-mediated human T cell suppression is unknown. In this study, we have characterized how human serum albumin (HSA) affects GML-induced inhibition of human T cells. We found that HSA and other serum albumins bind to 12 carbon acyl side chain of GML at low micromolar affinities and restores the TCR-induced formation of LAT, PLC-γ1, and AKT microclusters at the plasma membrane. Additionally, HSA reverses GML mediated inhibition of AKT phosphorylation and partially restores cytokine production in GML treated cells. Our data reveal that HSA, one of the most abundant proteins in the human serum and at sites of infections, potently reverses the suppression of human T cells by GML. This suggests that GML-driven human T cell suppression depends upon the local tissue environment, with albumin concentration being a major determinant of GML function.</p></div

Non-catalytic functions of Pyk2 and Fyn regulate late stage adhesion in human T cells.

T cell activation drives the protective immune response against pathogens, but is also critical for the development of pathological diseases in humans. Cytoskeletal changes are required for downstream functions in T cells, including proliferation, cytokine production, migration, spreading, and adhesion. Therefore, investigating the molecular mechanism of cytoskeletal changes is crucial for understanding the induction of T cell-driven immune responses and for developing therapies to treat immune disorders related to aberrant T cell activation. In this study, we used a plate-bound adhesion assay that incorporated near-infrared imaging technology to address how TCR signaling drives human T cell adhesion. Interestingly, we observed that T cells have weak adhesion early after TCR activation and that binding to the plate was significantly enhanced 30-60 minutes after receptor activation. This late stage of adhesion was mediated by actin polymerization but was surprisingly not dependent upon Src family kinase activity. By contrast, the non-catalytic functions of the kinases Fyn and Pyk2 were required for late stage human T cell adhesion. These data reveal a novel TCR-induced signaling pathway that controls cellular adhesion independent of the canonical TCR signaling cascade driven by tyrosine kinase activity

Thermodynamics, stoichiometry, and dissociation constant of albumin with GML, lauric acid or glycerol.

<p>Thermodynamics, stoichiometry, and dissociation constant of albumin with GML, lauric acid or glycerol.</p

HSA differentially restores cytokine production in GML treated cells.

<p>APBTs were suspended in serum free RPMI or RPMI supplemented with 2%, 1% or 0.2% HSA and were treated with 0.2% ethanol vehicle control, 10 μg/ml, or 20 μg/ml of GML. Cells were plated on 2 μg/ml anti-CD3 coated plates for 24 hours. Extracellular cytokine production for <b>(A)</b> IFN-γ, <b>(B)</b> IL-2, <b>(C)</b> IL-10, or <b>(D)</b> TNFα was measured by ELISA in 3 independent experiments with different human donors. To statistically confirm that GML is suppressing cytokine production without serum, Student t’s test was done by comparing EtOH treated groups with GML treated groups under the same serum free environment, indicated by the black bars, with # denoting p<0.05. To statistically test for the effects of HSA on GML-induced cytokine production, Student t’s test was done by comparing groups with various concentrations of HSA supplemented media and serum free media with each test group (i.e. the x axis headings EtOH, 10 μg/ml GML, 20 μg/ml GML), with * denoting p<0.05, ** p<0.01, and *** p<0.005.</p

HSA allows for PLC-γ1 microcluster formation in GML treated cells.

<p>(<b>A</b>). APBTs treated with 0.1% ethanol or 10 μg/ml GML in serum free media as well as 0.1% ethanol or 10μg/ml GML in 1% HSA were stimulated by plate bound anti-CD3 (2 μg/ml) in glass covered chamber slides. They were then fixed, permeabilized, stained with antibody specific for phosphorylated PLC-γ1 Y783, and imaged using TIRF microscopy. White bar scale indicates 4 μm in length. (<b>B).</b> Pixel intensities of median axis of each cell in images obtained in (<b>A</b>) were quantified and averaged using ImageJ. Scatter plot distributions with 95% confidence intervals of 60 cells from 3 independent experiments are shown. * denotes p<0.05 in Student t’s test comparing the identified samples.</p

HSA rescues AKT microcluster formation in GML treated cells.

<p>(<b>A</b>). APBTs treated with 0.1% ethanol or 10 μg/ml GML in serum free media as well as 0.1% ethanol or 10 μg/ml GML in 1% HSA were stimulated by plate bound anti-CD3 (2 μg/ml) in glass covered chamber slides. They were then fixed, permeabilized, stained with antibody specific for total AKT, and imaged using TIRF microscopy. White bar scale indicates 4 μm in length. (<b>B</b>). Pixel intensities of median axis of each cell in images obtained in (<b>A</b>) were quantified and averaged using ImageJ. Scatter plot distributions with 95% confidence intervals of 60 cells from 3 independent experiments are shown. * denotes p<0.05 in Student t’s test comparing the identified samples.</p

HSA restores AKT phosphorylation at both threonine 308 and serine 473 residues in GML treated cells.

<p>APBTs were treated with 0.1% ethanol vehicle control in serum free media (solid black lines), 10 μg/ml GML in serum free media (solid grey lines), 0.1% ethanol vehicle control in 1% HSA (dotted black lines), or 10 μg/ml GML in 1% HSA (dotted grey lines). Cells were stimulated by crosslinking 2 μg/ml of anti-CD3 for various times. Phosphorylation of AKT at threonine 308 and serine 473 were assessed by immunoblotting. Representative blots <b>(A)</b> and immunoblot quantification <b>(B)</b> are shown from 5 independent experiments with different individual donors. # denotes p<0.05 in Student t’s test comparing ethanol and GML treated cells in serum free media. * denotes p<0.05 in Student t’s test comparing GML treated cells in serum free RPMI with GML treated cells in 1% HSA.</p

Comparison of T cell receptor-induced proximal signaling and downstream functions in immortalized and primary T cells.

BACKGROUND: Human T cells play an important role in pathogen clearance, but their aberrant activation is also linked to numerous diseases. T cells are activated by the concurrent induction of the T cell receptor (TCR) and one or more costimulatory receptors. The characterization of signaling pathways induced by TCR and/or costimulatory receptor activation is critical, since these pathways are excellent targets for novel therapies for human disease. Although studies using human T cell lines have provided substantial insight into these signaling pathways, no comprehensive, direct comparison of these cell lines to activated peripheral blood T cells (APBTs) has been performed to validate their usefulness as a model of primary T cells. METHODOLOGY/PRINCIPAL FINDINGS: We used quantitative biochemical techniques to compare the activation of two widely used human T cell lines, Jurkat E6.1 and HuT78 T cells, to APBTs. We found that HuT78 cells were similar to APBTs in proximal TCR-mediated signaling events. In contrast, Jurkat E6.1 cells had significantly increased site-specific phosphorylation of Pyk2, PLCgamma1, Vav1, and Erk1/Erk2 and substantially more Ca2+ flux compared to HuT78 cells and APBTs. In part, these effects appear to be due to an overexpression of Itk in Jurkat E6.1 cells compared to HuT78 cells and APBTs. Both cell lines differ from APBTs in the expression and function of costimulatory receptors and in the range of cytokines and chemokines released upon TCR and costimulatory receptor activation. CONCLUSIONS/SIGNIFICANCE: Both Jurkat E6.1 and HuT78 T cells had distinct similarities and differences compared to APBTs. Both cell lines have advantages and disadvantages, which must be taken into account when choosing them as a model T cell line

Acridine Orange Indicates Early Oxidation of Wood Cell Walls by Fungi

<div><p>Colonization of wood blocks by brown and white rot fungi rapidly resulted in detectable wood oxidation, as shown by a reduced phloroglucinol response, a loss of autofluorescence, and acridine orange (AO) staining. This last approach is shown to provide a novel method for identifying wood oxidation. When lignin was mildly oxidized, the association between AO and lignin was reduced such that stained wood sections emitted less green light during fluorescence microscopy. This change was detectable after less than a week, an interval that past work has shown to be too short for significant delignification of wood. Although fungal hyphae were observed in only a few wood lumina, oxidation was widespread, appearing relatively uniform over regions several hundred micrometers from the hyphae. This observation suggests that both classes of fungi release low molecular weight mild oxidants during the first few days of colonization.</p></div