Whole Proteome Analysis of Mouse Lymph Nodes in Cutaneous Anthrax

<div><p>This study aimed to characterize a soluble proteome of popliteal lymph nodes during lymphadenitis induced by intradermal injection of <i>Bacillus anthracis</i> Sterne spores in mice using tandem LC-MS/MS and reverse-phase protein microarray with antibodies specific to epitopes of phosphorylated proteins. More than 380 proteins were detected in the normal intra-nodal lymph, while the infectious process resulted in the profound changes in the protein abundances and appearance of 297 unique proteins. These proteins belong to an array of processes reflecting response to wounding, inflammation and perturbations of hemostasis, innate immune response, coagulation and fibrinolysis, regulation of body fluid levels and vascular disturbance among others. Comparison of lymph and serum revealed 83 common proteins. Also, using 71 antibodies specific to total and phosphorylated forms of proteins we carried initial characterization of circulating lymph phosphoproteome which brought additional information regarding signaling pathways operating in the lymphatics. The results demonstrate that the proteome of intra-nodal lymph serves as a sensitive sentinel of the processes occurring within the lymph nodes during infection. The acute innate response of the lymph nodes to anthrax is accompanied by cellular damage and inflammation with a large number of up- and down-regulated proteins many of which are distinct from those detected in serum. MS data are available via ProteomeXchange with identifier PXD001342.</p></div

Relative abundance of phosphoproteins and related signaling proteins in lymph of infected mice in comparison with naïve mice.

<p>*Mean protein abundance in infected relative to uninfected mice. Protein extracts from 3 individual LNs were analyzed at each time point.</p><p>**Deviation of mean (95% confidence interval) calculated across all proteins in the Table is ≤0.098. The proteins demonstrating changes of mean less than 0.098 were: SAPK/JNK, Bad, FLIP, eNOS(Ser113), IL-10, HSP90, p53 (Ser115).</p><p>Relative abundance of phosphoproteins and related signaling proteins in lymph of infected mice in comparison with naïve mice.</p

KEGG maps illustrating selected pathways relevant to the soluble protein content of LNs in <i>B. anthracis</i>-infected mice.

<p>Lists of proteins up- or down-regulated in LNs during infection (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110873#pone.0110873.s002" target="_blank">Table S2</a>) were used as an input for KEGG software, which contains a database of pathway maps reflecting different biological processes. Red stars indicate the LN proteins mapped to the corresponding pathway using the following parameters: Fisher exact <i>p</i> value for enrichment probability ≤0.05 (strongly enriched), and a minimal number of proteins mapped to the pathway ≥5.</p

Cancer Landscape (Cscape) Protein Pathway Activation map of lymph from popliteal LN of mice infected with <i>B. anthracis</i> at day 2 post infection based on RPMA data.

<p>Increase and decrease in signaling are shown in increasing shades of red or green respectively. Each balloon pin is placed over the protein measured. Magnified view of the ERK and JNK in Ras pathway (green box) and STAT in cytokine signaling pathway (red box) are shown to reveal pathway details.</p

Pie charts of the protein content (A, B) and top-scoring processes (C, D) in LNs of naïve and <i>B. anthracis</i>-infected mice.

<p>(A) LN soluble proteome of spore-challenged mice contains a large number of proteins which appeared in LNs of during infection but were undetectable in naïve mice. (B) Infection up- or down-regulated a majority of LN proteins. Only 11 proteins showed no change in abundance. (C, D) KEGG analysis revealed cellular pathways relevant to LN proteins which were up-regulated (C) or down-regulated (D) by infection. Figures in the charts indicate the number of proteins in a particular category or pathway.</p

DAVID-based analysis of enriched processes for proteins up-regulated in infection.

<p>*Count refers to a number of genes identified in a corresponding term. Terms with less than 5 genes identified were excluded from analysis.</p><p>**Enrichment value calculated by DAVID is a measure of probability (in –log scale) for members of a particular annotation cluster to be grouped together (for further explanation see Materials and Methods).</p><p>DAVID-based analysis of enriched processes for proteins up-regulated in infection.</p

Analyses of popliteal LNs in <i>B. anthracis-</i>infected mice two days post challenge <i>vs.</i> uninfected mice.

<p>(A, B) H&E-stained sections of formalin-fixed LN tissue from uninfected (A) and infected (B) animals. Top panels show global views. Bottom panels show expanded views of regions identified by squares and demonstrate changes in the histopathology of cortical LN region. Tissue edema (green arrows) and numerous pyknotic cells (black arrows) are visible. Infiltrating neutrophils immunostained brown for myeloperoxidase (red arrows) are present. (C, D) Sections of formalin-fixed LN tissue from infected (D) and uninfected (C) mice immunostaned with rabbit anti-<i>B. anthracis</i> serum followed by secondary fluorescently-labelled antibody show accumulation of a large number of green-fluorescent bacteria (arrows) in the subcapsular region of LN of infected mouse. Fluorescence was detected at 495/520 nm using Olympus BX51 microscope. No increased subcapsular staining was found in control animals. The pictures represent typical observation obtained from ≥3 infected mice. (E) Bacterial counts in LNs of infected mice after plating of the homogenized tissue onto LB agar. Error bars represent SD of mean (<i>n</i> = 3 for days 0 to 3; <i>n</i> = 1 for day 4).</p

Proteins from naïve and <i>B. anthracis-</i>infected mice common between serum and lymph ranked according to their abundance in lymph during infection.

<p>*Average from 3 mice; **Average from 4 mice; ***Average per day for LN samples collected at days 1 to 4 post challenge (4 mice per sample at days 1 to 3, one sample at day 4).</p><p>Proteins from naïve and <i>B. anthracis-</i>infected mice common between serum and lymph ranked according to their abundance in lymph during infection.</p

Activation of NF-κB complex in TC-83 infected cells.

<p>A) UV inactivation of the virus was carried out using a Stratalinker UV crosslinker (model 1800). The inactivation was achieved by delivering an energy dose equivalent to 1200 µJoules X 100 per dose five times with a 2 minute interval between dosing. UV-TC-83 and TC-83 were serially diluted and used to infect Vero cells. UV-TC-83 inactivation was confirmed by plaque assay. Plaques were photographed and counted 48 hours post-infection. Plaque counts are represented graphically. B) U87MG cells were either mock infected, treated with LPS (1 µg/mL) or infected with TC-83 or UV-TC-83 (MOI: 1). At 30 minutes, 1 and 2 hours post-infection cells were lysed and protein extracts were resolved by SDS-PAGE and subsequently immunoblotted with antibodies against phosphorylated p65 and phosphorylated IκBα. Total p65, total IκBα and β-actin served as controls. The western blot is representative of 2 independent experiments. C) U87MGs were either mock infected, treated with LPS (1 µg/mL) or UV-TC-83 or TC-83 infected (MOI: 3). One hour post-infection cells were fixed, probed with p65 antibody followed by incubation with Alexa-Fluor 568. The cells were stained with DAPI to observe the nuclei. Images were taken using Nikon Eclipse TE2000-U at 60× magnification and are representative of 2 independent experiments. ND = not detectable.</p

IKKβ inhibitors are effective in decreasing viral load of wild type VEEV.

<p>A) U87MG cells (A) or neuronal rat AP7 cells (B) were pretreated with 1 µM IKK inhibitors, BAY-11-7082, BAY-11-7085 and IKK2-IV for 2 hours. The cells were infected with the wild type strain of VEEV (TrD) at a MOI: 0.1 (A) or MOI: 1 (B) for 1 hour. The conditioned media (media containing inhibitor) was removed prior to the viral infections and replaced after the viral inoculum was removed. The cells were incubated for an additional 24 hours. The supernatants were collected from all samples and viral titers were determined by plaque assay. The graphs are representative of 2 independent experiments. Error bars (Standard deviations) for 3 replicates within the 2 independent experiments were calculated and are represented thusly. ** p≤0.01.</p