Aberrant Migratory Behavior of Immune Cells in Recurrent Autoimmune Uveitis in Horses

Abstract

The participating signals and structures that enable primary immune cells migrating within dense tissues are not completely revealed until now. Especially in autoimmune diseases, mostly unknown mechanisms facilitate autoreactive immune cells to migrate to endogenous tissues, infiltrating and harming organ-specific structures. In order to gain deeper insights into the migratory behavior of primary autoreactive immune cells, we examined peripheral blood-derived lymphocytes (PBLs) of horses with equine recurrent uveitis (ERU), a spontaneous animal model for autoimmune uveitis in humans. In this study, we used a three-dimensional collagen I hydrogel matrix and monitored live-cell migration of primary lymphocytes as a reaction to different chemoattractants such as fetal calf serum (FCS), cytokines interleukin-4 (IL-4), and interferon-γ (IFN-γ), and a specific uveitis autoantigen, cellular retinaldehyde binding protein (CRALBP). Through these experiments, we uncovered distinct differences between PBLs from ERU cases and PBLs from healthy animals, with significantly higher cell motility, cell speed, and straightness during migration of PBLs from ERU horses. Furthermore, we emphasized the significance of expression levels and cellular localization of septin 7, a membrane-interacting protein with decreased abundance in PBLs of autoimmune cases. To underline the importance of septin 7 expression changes and the possible contribution to migratory behavior in autoreactive immune cells, we used forchlorfenuron (FCF) as a reversible inhibitor of septin structures. FCF-treated cells showed more directed migration through dense tissue and revealed aberrant septin 7 and F-actin structures along with different protein distribution and translocalization of the latter, uncovered by immunochemistry. Hence, we propose that septin 7 and interacting molecules play a pivotal role in the organization and regulation of cell shaping and migration. With our findings, we contribute to gaining deeper insights into the migratory behavior and septin 7-dependent cytoskeletal reorganization of immune cells in organ-specific autoimmune diseases.

Keywords: F-actin; autoimmune uveitis; equine peripheral blood-derived lymphocytes; equine recurrent uveitis (ERU); migration; septin 7.

Figure 1

(A,B) showing cell trajectory plots of μ-slide chemotaxis live-cell experiments using fetal calf serum (FCS) as the source of chemoattractant with tracked peripheral blood-derived lymphocytes (PBLs) of 10 controls and 11 equine recurrent uveitis (ERU) cases. Starting points of each cell were placed in the center of the plots. Cells moving toward FCS were marked in blue; cells that were not attracted by the source were marked in gray. Cell numbers inside the plots represent tracked cells of either PBLs from controls or ERU cases and show amount of cells that migrated up (away from source) or down (toward the source). No differences were analyzed by comparison of values of the center of mass (C). Migration parameters of cells from ERU cases revealed increased traveled distance (D) (**p < 0.01), directness (E) (**p < 0.01), and velocity (F) (*p < 0.05). Dots or triangles within graphs D to F represent individual cells. Horizontal lines correspond to mean values.

Figure 2

Cell trajectory plots of μ-slide chemotaxis live-cell experiments of peripheral blood-derived lymphocytes (PBLs) from four healthy horses (A) and PBLs from four equine recurrent uveitis (ERU) cases (B) using interleukin-4 (IL-4) as the source of chemoattractant. Starting points of each cell were placed in the center of the plots. Tracks illustrate cells moving toward IL-4 (blue) or away from the source of chemoattractant (gray). Cell numbers inside the plots show the amount of cells that moved up (away from source) or down (toward the source of chemoattractant). No significant differences in migration parameters displacement of the center of mass (C), distance (D), directness (E), and velocity (F) were analyzed between PBLs from controls and ERU cases (not significant, p > 0.05). Dots or triangles within graphs (D–F) represent individual cells. Horizontal lines correspond to mean values.

Figure 3

Cell trajectory plots of lymphocytes from four healthy (A) and four equine recurrent uveitis (ERU) cases (B) with interferon-γ (IFN-γ) as the source of chemoattractant. Starting points of each cell were placed in the center of the plots. Blue tracks illustrate cells moving toward IFN-γ; gray tracks show cells not attracted by the source. The displacement of the center of mass (C) showed that more than 50% of control peripheral blood-derived lymphocytes (PBLs) moved in the opposite direction of IFN-γ. Analysis of migration parameter values of PBLs from controls and ERU cases revealed significantly greater traveled distances (D) (***p < 0.001), no differences in directness (E), but significant faster migration (F) (***p < 0.001) of cells from ERU cases. Dots or triangles within graphs (D–F) represent individual cells. Horizontal lines correspond to mean values.

Figure 4

Cell trajectory plots of peripheral blood-derived lymphocytes (PBLs) from healthy (A) (n = 4) and equine recurrent uveitis (ERU) cases (B) (n = 4) using retinal autoantigen cellular retinaldehyde binding protein (CRALBP) as attractant. Starting points of each cell were placed in the center of the plots. Blue tracks illustrate cells moving toward, gray tracks show cells moving away from source. Cell numbers inside the plots represent tracked cells of controls or ERU cases that migrated up (away from source) or down (toward source). Comparison of migration parameters from ERU cases and controls uncovered no differences between values of the center of mass (C), great differences in traveled distances (D) (**p < 0.01), as well as significantly more directed (E) and faster migration (F) (***p < 0.001) of cells from ERU cases. Dots or triangles within graphs (D–F) represent individual cells. Horizontal lines correspond to mean values.

Figure 5

Cell trajectory plots of untreated (A) and forchlorfenuron (FCF)-treated (B) peripheral blood-derived lymphocytes (PBLs) of healthy horses. Starting points of each cell were placed in the center of the plots. Blue tracks illustrate cells moving toward the site of attractant (FCS, fetal calf serum); gray tracks show cells moving away from source. The numbers inside the plots represent tracked cells of either PBLs of untreated or FCF-treated controls and show numbers of cells moving up (away from source) or down (toward source of chemoattractant). Analysis of migration parameters revealed that there were no significant differences between values of untreated and FCF-treated cells in the center of mass (C) and the distance (D). FCF-treated cells showed a significantly more directed movement (E) (**p < 0.01); however, there were no differences between velocity parameters (F). Dots within graphs (D–F) represent individual cells. Horizontal lines correspond to mean values.

Figure 6

Representative images of untreated and forchlorfenuron (FCF)-treated equine lymphocytes of a healthy control. First column of each row shows differential interference contrast (DIC) images of lymphocytes (A,E,I,M). F-actin is shown in magenta (B, F, J, N: single channel; D,H,L,P: overlay), septin 7 expression is displayed in green (C,G,K,O: single channel; D,H,L,P: overlay). Cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; blue). Untreated, non-migrated lymphocytes had densely packed F-actin and septin 7 structures along the cell's plasma membrane (B–D). F-actin structures of untreated, migrated cells (F) were less intense and more delicate. Septin 7 filaments were less dense and expressed at the plasma membrane and perinuclear regions (G). F-actin and septin 7 structures of non-migrated, FCF-treated cells were densely packed, mainly distributed along the cell's plasma membrane (J–L). F-actin and septin 7 structures of migrated, FCF-treated lymphocytes were distributed more evenly throughout the cell, and more expressed in perinuclear regions, with minor association to the plasma membrane (N–P). Mean intensities of F-actin were significantly reduced in untreated, migrated cells (***p < 0.001; first two magenta bar graphs), and F-actin signal was significantly reduced in FCF-treated cells that were migrated (**p < 0.01; magenta bar graphs with diagonal lines). Mean intensities of F-actin were significantly higher in migrated FCF-treated cells, compared to untreated cells that migrated (*p < 0.05; right magenta bars with and without diagonal lines). Septin 7 was significantly reduced in control peripheral blood-derived lymphocytes (PBLs) that migrated in contrast to cells that did not migrate (***p < 0.001; first green bar graphs). Septin 7 signal was also significantly reduced in FCF-treated and migrated cells compared to respective non-migrated cells (***p < 0.001; outer green bar graphs with diagonal lines).