Tumor necrosis factor superfamily member APRIL contributes to fibrotic scar formation after spinal cord injury

Abstract

Background: Fibrotic scar formation contributes to the axon growth-inhibitory environment that forms following spinal cord injury (SCI). We recently demonstrated that depletion of hematogenous macrophages led to a reduction in fibrotic scar formation and increased axon growth after SCI. These changes were associated with decreased TNFSF13 (a proliferation inducing ligand (APRIL)) expression, but the role of APRIL in fibrotic scar formation after SCI has not been directly investigated. Thus, the goal of this study was to determine the role of APRIL in fibrotic scar formation after SCI.

Methods: APRIL knockout and wild-type mice received contusive SCI and were assessed for inflammatory cytokine/chemokine expression, leukocyte infiltration, fibrotic scar formation, axon growth, and cell proliferation.

Results: Expression of APRIL and its receptor BCMA is increased following SCI, and genetic deletion of APRIL led to reduced fibrotic scar formation and increased axon growth. However, the fibrotic scar reduction in APRIL KO mice was not a result of changes in fibroblast or astrocyte proliferation. Rather, APRIL knockout mice displayed reduced TNFα and CCL2 expression and less macrophage and B cell infiltration at the injury site.

Conclusions: Our data indicate that APRIL contributes to fibrotic scar formation after SCI by mediating the inflammatory response.

Keywords: BAFF; BAFF-R; BCMA; Cell proliferation; Fibrosis; Glial scar; TACI; TNFSF13.

Fig. 1

mRNA expression of APRIL, BAFF, and their receptors at 2 weeks after contusive spinal cord injury. In the injured spinal cord, APRIL expression is significantly increased whereas its related ligand BAFF is unaltered (a–b). Of the APRIL family receptors, BCMA (c) expression is significantly increased but TACI (d) and BAFF-R (e) are not changed. n = 5 per group (biological replicates). Normalized to average of naïve for each gene. *p < 0.01 compared to naïve. Student’s t test

Fig. 2

APRIL KO mice have a reduced fibrotic scar area after SCI. Two weeks following SCI, the fibrotic scar size was assessed in APRIL KO (d–f, n = 9) and WT (a–c, n = 11) mice by evaluating the percent area of PDGFRβ signal within a 2-mm region centered around the injury site (g). The density of PDGFRβ⁺ cells was not different between WT and KO mice (h). Representative images from a WT and APRIL KO animal are shown with GFAP in red and PDGFRβ in green. n = biological replicates; *p < 0.05 compared to WT. Student’s t test. Scale bar = 250 μm

Fig. 3

APRIL KO mice have an increased number of axons in the injury site 2 weeks following SCI. Neurofilament⁺ axons (red) in the GFAP (green)-negative region were compared between WT (a–c) and APRIL KO (d–f) mice 2 weeks following injury. c, f Magnified images of dotted area (region of interest (ROI)). Quantifications are shown in g (n = 8 biological replicates). Scale bar = 250 μm (a, b, d, e), 10 μm (c, f). *p < 0.05 compared to WT. Student’s t test

Fig. 4

Proliferation of fibroblasts and astrocytes is not altered in APRIL KO mice after SCI. EdU⁺ cells (white) are widely distributed throughout the GFAP (red) and PDGFRβ (green) regions in APRIL KO (j) and WT (a–i) animals. High-magnification images of proliferating fibroblasts (b–e) and astrocytes (f–i) are shown. Quantification of proliferating fibroblasts (k) and astrocytes (l) reveals no difference between APRIL KO and WT mice following SCI. b–e From top dashed square region in (a). f–i From bottom dashed region in (a). Percent in k and l are calculated from the total number of PDGFRβ⁺ or GFAP⁺ cells within the quantified region. n = 3 per group (biological replicates). Scale bar = 250 μm (a, j), 10 μm (b–i)

Fig. 5

The number of infiltrating leukocytes is decreased in APRIL KO mice 1 week following SCI. The number of infiltrating macrophages (CD45^hiCD11b⁺) and B cells (B220⁺) were significantly reduced following SCI in APRIL KO mice (n = 7) compared to WT controls (n = 10) (a). This translated to a reduction in the overall number of infiltrating leukocytes (b), whereas the number of microglia remained similar between WT and KO mice (c). However, the percentages of each cell type, including microglia, were not different (d–f). n = biological replicates. *p < 0.05 compared to WT. Student’s t test

Fig. 6

Cytokine/chemokine expression is decreased in APRIL KO mice 1 day following SCI. Injury sites from APRIL KO mice had significantly decreased expression of TNFα (a) and CCL2 (b) compared to wild-type controls. IL6, IL-1β, CCL5, and CXCL10 expression was unchanged (c–f). n = 7–8 biological replicates per group. Normalized to average of WT for each gene. *p < 0.05 compared to WT. Student’s t test