Quantitative Profiling of the Lymph Node Clearance Capacity

Abstract

Transport of tissue-derived lymphatic fluid and clearance by draining lymph nodes are pivotal for maintenance of fluid homeostasis in the body and for immune-surveillance of the self- and non-self-proteomes. Yet a quantitative analysis of nodal filtration of the tissue-derived proteome present in lymphatic fluid has not been reported. Here we quantified the efficiency of nodal clearance of the composite proteomic load using label-free and isotope-labeling proteomic analysis of pre-nodal and post-nodal samples collected by direct cannulation. These results were extended by quantitation of the filtration efficiency of fluorophore-labeled proteins, bacteria, and beads infused at physiological flow rates into pre-nodal lymphatic collectors and collected by post-nodal cannulation. We developed a linear model of nodal filtration efficiency dependent on pre-nodal protein concentrations and molecular weight, and uncovered criteria for disposing the proteome incoming from defined anatomical districts under physiological conditions. These findings are pivotal to understanding the maximal antigenic load sustainable by a draining node, and promote understanding of pathogen spreading and nodal filtration of tumor metastasis, potentially helping to improve design of vaccination protocols, immunization strategies and drug delivery.

Figure 1

Quantitative proteomic analysis of nodal clearance of afferent lymph. (A) Total protein concentration in lymph collected from afferent and efferent lymphatics. Each dot represents protein amount as detected in equal volume of pre and post-nodal lymph collected from 16 individual rats (each color represents an individual animal). Measurements were performed in triplicates for each sample and statistical analysis performed using a two-tailed t-test; average and standard deviation are reported. (b,c) Heat maps of representative biological and technical triplicates of pre- and post-nodal proteins as identified and quantified by (b) label free proteomic analysis; one representative biological sample run in technical triplicates is shown (three additional biological samples, also run in technical triplicates and a fifth sample derived from pooled lymph from 12 individual rats are shown in Fig. 1S and (c) TMT proteomic analysis on the pooled lymph from twelve individual rats. List of proteins on the heat maps and their quantification is reported in Supplement Table S1. The protein ratios from TMT quantitative analysis were used to generate the heat maps after they were rescaled using a log2 transformation, such that positive values reflect fold increases (red color) and negative values reflect fold decreases (green color). PEAKS Q significance score > 10.0 was used to assess the statistically significance of the TMT heat map. In the case of LFQ analysis, only proteins which passed a selected significance statistical threshold (ANOVA, p < 0.05 and FDR < 1% for protein and peptide expression) are represented in the heat maps. (d) Representative comparative base chromatograms (MS1/MS2) for one pair of pre and post nodal mesenteric lymph proteomes, out of the three nanoLC-MS/MS experiments run on a Q Exactive HF quadrupole orbitrap mass spectrometer with HCD ionization mode. (e,f) Volcano plot indicating statistical significance in the fold changes observed between the pre and post nodal proteome for (e) LFQ and (f) TMT proteomics. (g,h) Views of the intensity correlation are presented for different pre- and post- nodal sample pairs. (g) The Pearson’s correlation score (between 0.95–0.99) indicates the high proteomic reproducibility of all pre-nodal and post-nodal samples. (h) The Pearson’s correlation score (between 0.71–0.72) indicates the differences between pre- and post-nodal samples. Additional parameters of mass spectrometric analysis are reported in Supplement Figs S3 and S4.

Figure 2

Analysis of the protein pathways up and down-regulated following nodal filtration. (a) Quantitative pathway analysis of proteins cleared from the pre-nodal to the post-nodal lymph following nodal transit. Proteomic analysis was performed on pre- and post-nodal lymph collected from pooled lymph from twelve rats (technical quadruplicates). Numbers in parenthesis correspond to the proteins assigned to each pathway. Bars show pathways that are up (red) or down (green) regulated in the pre- vs. post-nodal lymph. (b,c) Top-scoring networks derived from the proteins with different abundance in the pre- and post-nodal lymph generated by IPA analysis. IPA identified ten significant networks (p < 0.001) with scores above 30, which are overlaid and related to carbohydrate metabolism (b) and lipids, small molecules, amino acids and proteins metabolism (c). The top networks are displayed in the Table S2. Green nodes depict proteins present in lower abundance in the post- vs. the pre-nodal lymph; red nodes depict proteins present in lower abundance in the pre- vs. the post-nodal lymph. Color intensity directly correlates with fold-changes, expressed as log2 (TMT pre-/post- ratio). (d) ELISA for total IgG and IgM pre-sent in pre- and post-nodal lymph. Each dot or square represents an individual sample, pre- or post-nodal collected from a single rat. Measurements were performed in triplicates for each sample and statistical analysis performed using a two-tailed t-test; average and standard deviation are reported. (e) Bar-graph reporting the percentage of nodal clearance of the most abundant lymph proteins. Data are reported as percentage of protein decrease in the post-nodal lymph, as compared to the pre-nodal, upon nodal transit. Measurements were collected from 4 individuals and 12 pooled lymph samples and statistical analysis performed using a two-tailed t-test; average and standard deviation are reported. (f) Proteins involved in acute phase response and coagulation pathways, as identified by IPA analysis to be statistically significant (p < 0.0001) in their fold expression calculated from the log2 (TMT ratio pre-/post-). Data were analyzed through the use of IPA (QIAGEN Inc., https://www.qiagenbioinformatics.com/products/ingenuity-pathway-analysis.

Figure 3

Anatomy of the nodal regions where the lymph percolates. (a) Schematic depicting intestinal loops with the lymphatic collectors entering and exiting the lymph node. Left Panel depicts the normal orientation of the rat ileum, mesentery and associated neurovascular bundles containing the blood (SMA) and lymphatic vessels serving the gut, the mesenteric node, and post- nodal lymphatics without the overlying adipose tissue. Right Panel depicts the arrangement of the multiple afferent pre-nodal lymphatics (A) entering the main rat mesenteric node (N), the multiple efferent post-nodal lymphatics (E) exiting the node with a complex anastomosis of the efferent lymphatics into a single main mesenteric efferent lymph trunk (T). For these experiments, we cannulated a single afferent pre-nodal lymphatic and the main mesenteric efferent lymph trunk for either collection of lymph or perfusion. (b) Pictures depicting intestinal loops with lymphatic collectors draining to and exiting the lymph node (center). (c) Picture depicting efferent lymphatic collectors entering draining lymph nodes (d) Picture depicting afferent collectors exiting the draining lymph nodes (e) Immunogold labeling with laminin (5 mm gold) and Perlecan (10 mm gold) to highlight the conduit system as the space between the central pillar of collagen and the immunogold labelled matrix proteins. (f) Immunogold labeling for MHC II of a dendritic cells connected to the conduit system.

Figure 4

Statistical Analysis of the pre- and post-nodal proteome. (a) Scatter plots of delta (pre-post- changes) with protein mass for each rat (excluding albumin). (b) Scatter plot of delta (pre-post-) and pre-nodal concentration across all samples grouped by Mass <80 kDa and Mass >80 kDa, excluding albumin. (c) Analysis of pre-nodal protein clearance by the lymph node according to the protein molecular weight. The % decrease from pre- to post-nodal protein concentration, for each range of MW was calculated using the intensities of each protein as determined by quantitative TMT proteomic. (d) Scatter plots of delta (pre-/post- changes) with pre-nodal concentration for each rat (excluding albumin).

Figure 5

Efferent Mesenteric Lymphatic Collector Cannulation and delivery of fluorochrome-labelled proteins. (a) Sequential imaging depicting lymphatic preparation and cannulation (b) still images depicting fluorochrome-labelled proteins injected into the pre-nodal lymphatic collector. (c) Fluorescent dextran beads (2 μm) and S. Aureus, as injected in the pre-nodal lymph and retrieved in the post-nodal lymph at different time points. (d) Immune cells, in the post-nodal lymph following phagocytosis of fluorophore-labelled proteins and bacteria.

Figure 6

Quantitative analysis of nodal filtration. (a) Fluorescence trace and bar graph of HEL-FITC protein as injected in the pre-nodal lymph and as detected in the post-nodal lymph, collected at different time points. (b) Graph depicting percentage of fluorochrome-labelled residual proteins in the post-nodal lymph following pre-nodal injection and nodal transit for different time-points. Measurements were collected from 4 separate rats, for each time point, and statistical analysis performed using a two-tailed t-test; average and standard deviation are reported. (c) Bar-graphs depicting the percentage of fluorochrome-labelled proteins infused, at different concentrations, in the pre-nodal lymph and collected in the post-nodal lymph. Measurements were collected from 4 separate rats and statistical analysis performed using a two-tailed t-test; average and standard deviation are reported. (d) Graph depicting percentage of clearance of exogenous (fluorochrome-labelled injected proteins) and endogenous (lymph proteomic) proteins in the post-nodal lymph following nodal transit. (e) Fluorescence trace and bar graph of S. Aureus, as injected in the pre-nodal lymph and as detected in the post-nodal lymph, collected at different time points. Measurements were collected from 3 separate rats and statistical analysis performed using a two-tailed t-test; average and standard deviation are reported. (f) Fluorescence trace and bar graph of Dextran beads, as injected in the pre-nodal lymph and as detected in the post-nodal lymph, collected at different time points. Measurements were collected from 4 separate rats and statistical analysis performed using a two-tailed t-test; average and standard deviation are reported. (g) Bar-graph depicting percentage of S. Aureus, recovered in the post-nodal lymph following injection, of different bacterial amounts, in the pre-nodal lymph. Measurements were collected from 4 separate rats and statistical analysis performed using a two-tailed t-test; average and standard deviation are reported.