Netrin-1 Augments Chemokinesis in CD4+ T Cells In Vitro and Elicits a Proinflammatory Response In Vivo

Abstract

Netrin-1 is a neuronal guidance cue that regulates cellular activation, migration, and cytoskeleton rearrangement in multiple cell types. It is a chemotropic protein that is expressed within tissues and elicits both attractive and repulsive migratory responses. Netrin-1 has recently been found to modulate the immune response via the inhibition of neutrophil and macrophage migration. However, the ability of Netrin-1 to interact with lymphocytes and its in-depth effects on leukocyte migration are poorly understood. In this study, we profiled the mRNA and protein expression of known Netrin-1 receptors on human CD4(+) T cells. Neogenin, uncoordinated-5 (UNC5)A, and UNC5B were expressed at low levels in unstimulated cells, but they increased following mitogen-dependent activation. By immunofluorescence, we observed a cytoplasmic staining pattern of neogenin and UNC5A/B that also increased following activation. Using a novel microfluidic assay, we found that Netrin-1 stimulated bidirectional migration and enhanced the size of migratory subpopulations of mitogen-activated CD4(+) T cells, but it had no demonstrable effects on the migration of purified CD4(+)CD25(+)CD127(dim) T regulatory cells. Furthermore, using a short hairpin RNA knockdown approach, we observed that the promigratory effects of Netrin-1 on T effectors is dependent on its interactions with neogenin. In the humanized SCID mouse, local injection of Netrin-1 into skin enhanced inflammation and the number of neogenin-expressing CD3(+) T cell infiltrates. Neogenin was also observed on CD3(+) T cell infiltrates within human cardiac allograft biopsies with evidence of rejection. Collectively, our findings demonstrate that Netrin-1/neogenin interactions augment CD4(+) T cell chemokinesis and promote cellular infiltration in association with acute inflammation in vivo.

Figure 1. Netrin-1 receptor expression by human CD4⁺ T lymphocytes

The expression of known Netrin-1 receptors was analyzed at the mRNA level by quantitative PCR (Panels A–C), at the protein level by Western blot analysis (Panels D–E) and by FACS after 48hrs activation (Panel F). Panels A–C show the mean fold change in mRNA expression ±SEM. In Panel D, induced expression of neogenin, UNC5A and UNC5B is illustrated and Panel E illustrates densitometric analysis of n=3 independent Western blots. The illustrated data in Panels A-F are representative of n=3 independent experiments.

Figure 2. Localization of Neogenin, UNC5A and UNC5B on CD4⁺ T cells

Expression of the Netrin-1 receptors Neogenin, UNC5A and UNC5B was analyzed on unactivated or mitogen-dependent activated (PHA 2.5μg/mL) human CD4⁺ T cells by immunofluorescent staining. Netrin-1 receptor expression (red) is illustrated on cells stained with anti-CD3 (green) and colocalization is seen in the merged image as a yellow stain. All images are representative of n=3 independent experiments.

Figure 3. Netrin-1 increases both the size, and the chemokinetic response of migrating CD4⁺ T cells

CD4⁺ T cell migration was analyzed in a microfluidic device that allows for the quantitative analysis of the fraction of migrating cells, their directionality, persistence and speed at the single cell level. Panel A illustrates the microfluidic device. CD4⁺ T cells are loaded into the central main channel and are monitored in real-time migrating through 10-micron side channels over an 8hr time period. Each cell has potential to migrate either towards (direction of reservoir) or away (direction of buffer channel) from a chemokine gradient. Panel B illustrates the quantitative analysis of bi-directional migratory patterns of 48h mitogen-activated CD4⁺ T cells (αCD3/αCD28 1µg/mL each) in response to increasing concentrations of Netrin-1, as indicated. Grey bars represent migration towards the gradient, and black bars represent migration away from the gradient (mean±SD of n=≥3 independent experiments). Panel C shows a scatter plot of migratory directional persistence of CD4⁺ cells in response to Netrin-1. The migratory patterns of individual CD4⁺ T cells were evaluated and are illustrated as either grey dots (media alone) or black dots (response to Netrin-1). The black lines represent the median and standard deviation of migratory responses under each condition. Number of cells analyzed in media alone was 449/2232 total migrating cells, and in Netrin-1 was 399/1068 total migrating cells. Illustrated data is from n=3 independent experiments. * =p< 0.05.

Figure 4. Migration characteristics of CD4⁺ T lymphocytes in response to Netrin-1

Migration patterns of individual T cells were analyzed using time-lapse videos over an 8hr time period. Panel A illustrates the time-distribution of the initial migratory response and the hourly response over the course of each experiment. The effect of Netrin-1 was prominent in the first 3 hours of migration. Black symbols represent cell migration in response to Netrin-1, whereas grey symbols represent response in media alone. In Panels B–C, the percentage of cells that migrate at each indicated speed (in μm/min) is separated into groups of 3.3 μm/min intervals, for cells migrating away from the gradient (B) and towards the gradient (C). Black bars represent cells migrating in response to Netrin-1, and grey bars represent cells migrating in response to media alone (as a control). The average migratory speed for each condition is shown in the box and whiskers plot. Error bars represent mean±SD. The number of cells analyzed in media was 449/2232 total and in Netrin-1 was 399/1069 total. Illustrated data is from n≥3 independent experiments. * =p< 0.05.

Figure 5. Effect of Netrin-1 on RANTES-induced migration

CD4⁺ T cells were loaded in the microfluidic device, and were exposed to a RANTES gradient in the absence or presence of Netrin-1 (0.1μg/ml). Panel A represents the percentage of cells migrating in the direction of the gradient over an 8 hr time period. Panel B illustrates the migratory speed of individual T cells. The black bars represent migratory speed induced by RANTES, and the grey bars represent migration speed in response to both RANTES and Netrin-1. The dashed gray line illustrates the speed distribution in media alone. Solid grey and black lines illustrate the distribution of migratory speed for RANTES with (grey) or without (black) Netrin-1. Illustrated is the combined data from n=3 independent experiments showing responses in n=377 migrating cells (RANTES alone) and n=256 migrating cells (RANTES + Netrin-1). Bars represent the mean±SD. * =p< 0.05.

Figure 6. Knockdown of neogenin attenuates the migratory response to Netrin-1

Human CD4⁺ T-cells were infected with two neogenin shRNAs, each yielding a knockdown efficiency of 50–70% (as shown in Figure S3). Control shRNA or neogenin shRNA-infected CD4⁺ T cells were loaded into microfluidic devices and the percentage of cells migrating in response to Netrin-1 was evaluated (Panel A). In Panel B, bi-directional migratory patterns were evaluated in response to Netrin-1 (0.1 μg/ml) vs. media alone using control shRNA or neogenin shRNA-infected CD4⁺ T cells. Error bars represent mean±SD. Data shown is from n=3 independent experiments with a total of n=1120 (Neogenin shRNA#1), n=1324 (Neogenin shRNA#2) and n= 1222 (Control shRNA) analyzed cells. * =p< 0.05.

Figure 7. Effect of Netrin-1 on leukocyte infiltration in vivo

Panel A: Cartoon illustrating the huSCID model; human skin is transplanted onto SCID mice, and after 6 weeks the mice are humanized by i.p. injection of 3×10⁸ human PBMC. On day 0 and day 7, Netrin-1/matrigel (5μg) or matrigel alone is injected subcutaneously into the human skin graft (n=8 mice). Panel B, H&E staining of human skin samples harvested on day 14 post humanization. Representative histology is shown in the right panels (100× mag; box insert 400× mag); the average number of infiltrates in each skin was grid counted (as described (30)) and is shown in the left bar graph (n=8 skins; **p<0.01). Panel C shows representative cryosections from day 14 skin samples immunostained with anti-CD3; the number of CD3⁺ T cells was grid counted and the average CD3⁺ T cell count (mean±SD) is illustrated in the bar graph (left panel) (n=8, **p<0.01). Panel D shows representative immunofluorescent staining of a Netrin-1-treated human skin graft using anti-neogenin (red stain) and anti-CD3 (green stain) and colocalization is seen in the merged image as yellow color. Panel E shows high magnification (400×) immunofluorescent images of Panel D, showing polarized expression of Neogenin receptors on the T cell surface as the merged yellow image. Nuclei are stained with DAPI (blue).

Figure 8. Expression of neogenin on lymphocytes within human cardiac allografts

Panel A: Immunofluorescence staining of CD3 and neogenin in a representative biopsy from a cardiac allograft with sparse CD3+ T cell infiltrates. Illustrated is coexpression of neogenin with CD3 on an isolated infiltrating lymphocyte (100× mag; box insert 200× mag). Panel B: High power photomicrograph of a representative biopsy showing colocalization of neogenin with CD3 within a focal infiltrate (200× mag; box insert 400× mag). Panel C: Confocal microscopy illustrating coexpression of CD3 and neogenin on a focal infiltrate within an allograft biopsy.