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. 2018 Mar 20;15(1):90.
doi: 10.1186/s12974-018-1128-2.

Perturbing chondroitin sulfate proteoglycan signaling through LAR and PTPσ receptors promotes a beneficial inflammatory response following spinal cord injury

Scott Dyck  1 , Hardeep Kataria  1 , Arsalan Alizadeh  1 , Kallivalappil T Santhosh  1 , Bradley Lang  2 , Jerry Silver  2 , Soheila Karimi-Abdolrezaee  3
Affiliations

Perturbing chondroitin sulfate proteoglycan signaling through LAR and PTPσ receptors promotes a beneficial inflammatory response following spinal cord injury

Scott Dyck et al. J Neuroinflammation. .

Abstract

Background: Traumatic spinal cord injury (SCI) results in upregulation of chondroitin sulfate proteoglycans (CSPGs) by reactive glia that impedes repair and regeneration in the spinal cord. Degradation of CSPGs is known to be beneficial in promoting endogenous repair mechanisms including axonal sprouting/regeneration, oligodendrocyte replacement, and remyelination, and is associated with improvements in functional outcomes after SCI. Recent evidence suggests that CSPGs may regulate secondary injury mechanisms by modulating neuroinflammation after SCI. To date, the role of CSPGs in SCI neuroinflammation remains largely unexplored. The recent discovery of CSPG-specific receptors, leukocyte common antigen-related (LAR) and protein tyrosine phosphatase-sigma (PTPσ), allows unraveling the cellular and molecular mechanisms of CSPGs in SCI. In the present study, we have employed parallel in vivo and in vitro approaches to dissect the role of CSPGs and their receptors LAR and PTPσ in modulating the inflammatory processes in the acute and subacute phases of SCI.

Methods: In a clinically relevant model of compressive SCI in female Sprague Dawley rats, we targeted LAR and PTPσ by two intracellular functionally blocking peptides, termed ILP and ISP, respectively. We delivered ILP and ISP treatment intrathecally to the injured spinal cord in a sustainable manner by osmotic mini-pumps for various time-points post-SCI. We employed flow cytometry, Western blotting, and immunohistochemistry in rat SCI, as well as complementary in vitro studies in primary microglia cultures to address our questions.

Results: We provide novel evidence that signifies a key immunomodulatory role for LAR and PTPσ receptors in SCI. We show that blocking LAR and PTPσ reduces the population of classically activated M1 microglia/macrophages, while promoting alternatively activated M2 microglia/macrophages and T regulatory cells. This shift was associated with a remarkable elevation in pro-regenerative immune mediators, interleukin-10 (IL-10), and Arginase-1. Our parallel in vitro studies in microglia identified that while CSPGs do not induce an M1 phenotype per se, they promote a pro-inflammatory phenotype. Interestingly, inhibiting LAR and PTPσ in M1 and M2 microglia positively modulates their inflammatory response in the presence of CSPGs, and harnesses their ability for phagocytosis and mobilization. Interestingly, our findings indicate that CSPGs regulate microglia, at least in part, through the activation of the Rho/ROCK pathway downstream of LAR and PTPσ.

Conclusions: We have unveiled a novel role for LAR and PTPσ in regulating neuroinflammation in traumatic SCI. Our findings provide new insights into the mechanisms by which manipulation of CSPG signaling can promote recovery from SCI. More importantly, this work introduces the potential of ILP/ISP as a viable strategy for modulating the immune response following SCI and other neuroinflammatory conditions of the central nervous system.

Keywords: Chondroitin sulfate proteoglycans; Leukocyte common antigen-related receptor; Microglia; Neural precursor cells; Neuroinflammation; Protein tyrosine phosphatase sigma receptor; Spinal cord injury.

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Conflict of interest statement

Ethics approval

All experimental protocols in this study were approved by the Animals Care Committee of the University of Manitoba (Protocol #13-027) in accordance with the guidelines and policies established by the Canadian Council of Animal Care (CCAC).

Consent for publication

Not applicable.

Competing interests

SD, HK, AA, SKT, and SK-A declare no competing financial interests. BL and JS are inventors on the patent application (PCT/US2013/035831) for ISP.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1
Fig. 1
Summary of experimental procedures, treatment groups, and time-points for in vivo experiments
Fig. 2
Fig. 2
ILP/ISP treatment limits neutrophil infiltration but does not modulate MMP activity in acute SCI. a Myeloperoxidase (MPO) activity, a marker for neutrophils, was increased in the injured spinal cord at 1 day following SCI, which was attenuated by ILP/ISP treatment. b–d Matrix metalloproteinases (MMP)-2 and MMP-9 expression was also assessed using gel zymography. SCI-induced expression of MMP-2 and MMP-9 was observed at 1, 3, and 7 days post-SCI. However, there was no change in the levels of pro-MMP-9 (c) and pro-MMP-2 (d) under ILP/ISP treatment at any examined time-points. N = 4–6 animals/group/time-point. The data show mean ± SEM, *p < 0.05, one-way ANOVA (a–d)
Fig. 3
Fig. 3
Inhibition of LAR and PTPσ promotes an increase in the subpopulation of M2 macrophages after SCI. a Western blot analysis of Iba1 protein expression at 1, 3, 5, 7, and 14 days following SCI revealed no apparent change in the presence of microglia/macrophage within SCI lesion. b, c Similarly, flow cytometric analysis of spinal cord tissue revealed no change in the total number of infiltrated macrophages (CD45+/CD68+) at 3 and 7 days post-injury between vehicle control and ILP/ISP (10 μg/day) treated SCI animals. d, e ILP/ISP treatment resulted in a non-significant decrease in the number of CD45+/CD68+/CD86+ M1 macrophages at 3 and 7 days post-injury. f, g A significant increase in the number of CD45+/CD68+CD163+ M2 macrophages was observed at 7 days post-injury in ILP/ISP-treated animals. h Representative flow cytometry gates are depicted. N = 4–6 animals/group/time-point. The data show mean ± SEM, *p < 0.05, one-way ANOVA (a), Student t test (b–g)
Fig. 4
Fig. 4
Modulation of LAR and PTPσ attenuates pro-inflammatory cytokines while elevating anti-inflammatory mediators in SCI. a Western blot analysis of IL-1β protein expression at 1, 3, 7, and 14 days post-SCI showed a significant increase in IL-1β at 1 day post-SCI that persisted for up to 14 days after injury. ILP and ISP co-treatment attenuated this upregulation but was only statistically significant at the 3-day time-point post-SCI. b TNFα protein expression was also significantly upregulated at 1, 3, 7, and 14 days post-SCI compared to uninjured control. ILP and ISP treatment reduced TNFα levels; however, the reduction was not statistically significant. c, d Western blot analysis of IL-10 and Arginase-1 protein at various time-points showed that ILP and ISP co-treatment significantly increased both factors at 5, 7, and 14 days post-SCI compared to vehicle treatment. N = 4–6 animals/group/time-point. The data show the mean ± SEM, *p < 0.05, one-way ANOVA
Fig. 5
Fig. 5
ILP and ISP promotes a phenotypic switch in helper T cells toward a Treg phenotype. a, b Flow cytometric assessment revealed no apparent difference in the overall infiltration of helper T cells (CD45+/CD3+/CD4+) in the injured spinal cord at 3 and 7 days post-injury between vehicle and ILP/ISP-treated animals. c, d However, ILP/ISP-treated animals exhibited a significant decrease in the number of effector T cells (CD45+/CD3+/CD4+/IFNγ+) at 7 days post-injury. e, f A significant increase in the total number of regulatory T cells (CD45+/CD3+/CD4+/IL10+) was observed at 3 days post-injury in ILP/ISP-treated animals. g, h Representative flow cytometry gates are shown. i Western blot analysis showed upregulation of CD4 protein expression, at 7 and 14 days post-SCI compared to uninjured control group confirming infiltration of helper T cells in the injured spinal cord. Confirming our flow cytometry, ILP and ISP had no apparent effect on the overall protein expression of CD4. j However, ILP and ISP significantly increased FOXP3 protein expression, a marker of regulatory T cells, at both 7 and 14 days post-SCI compared to SCI vehicle control. Western blot results have been normalized to the actin loading control prior to subsequent normalization to the control values. The data show mean ± SEM, *p < 0.05, Student t test (a–f), one-way ANOVA (i, j), N = 4-6/group
Fig. 6
Fig. 6
Microglia/macrophages and T cells contribute to IL-10 expression after SCI. a–l Immunohistochemistry on spinal cord tissue confirmed an increase in IL-10 expression in ILP/ISP-treated animals at 7 days following injury compared to vehicle-treated animals. IL-10 expression was confirmed to be expressed in both CD11b + macrophages/microglia (a–f) and CD3+ T cells (g–l)
Fig. 7
Fig. 7
Polarization of primary microglia cultures to an M0, M1, or M2 phenotype. a–c Primary microglia were polarized to M1 through IFNγ and TNFα treatment or M2 through IL-10 treatment. M1 polarization was confirmed by induced expression of CD86 (d–f) and release of nitrite (j). Increased expression of mannose receptor (g–i) and IL-10 (k) were used to confirm M2 polarization. N = 5 independent experiments. The data show the mean ± SEM, *p < 0.05, one-way ANOVA
Fig. 8
Fig. 8
CSPGs modulate microglia phagocytosis, migration, and nitrite production which is partially mediated through LAR and PTPσ signaling and Rho activation. a, b Microglia phagocytosis was assessed. Success of phagocytosis was verified by intracellular detection of green fluorescent beads in microglia (Iba-1+) through Z-stack imaging. M1 microglia (TNFα + IFNγ treated) showed a reduced ability for phagocytosis. CSPGs reduced phagocytosis in microglia, which was attenuated and even promoted with ILP/ISP, inhibition of ROCK by Y-27632, or ChABC treatment but not by TAT or IMP control peptides. c Representative images of phagocytosis by M0, M1, and M2 microglia are depicted for PDL, CSPGs, and CSPGs + ILP/ISP conditions. d Nitrite production was exacerbated and significantly increased in M1 microglia when exposed to CSPG. This effect was not blocked by ILP/ISP, Y-27632, IMP, or TAT treatment but was blocked by ChABC degradation of CSPGs. IL-10 (e) and IL-1β (f) release was assessed in microglia 2 days after plating onto PDL or PDL + CSPGs substrate. CSPGs reduced IL-10 expression in M2 microglia while had no significant effect on IL-1β release. g CSPGs also significantly limited microglia migration which was overcome by ILP/ISP, Y-27632, and ChABC treatment. h RhoA activity was assessed by G-LISA in microglia demonstrating a significant increase in Rho activity when microglia were exposed to CSPGs substrate. ILP and ISP treatment significantly decreased Rho activity. The data show the mean ± SEM, *p < 0.05, one-way ANOVA, N = 3–5/group. N = 3–5 independent experiments. The data show the mean ± SEM, *p < 0.05, one-way ANOVA
Fig. 9
Fig. 9
IL-10 promotes oligodendrocyte differentiation of spinal cord NPCs in vitro. a–c Addition of recombinant IL-10 had no apparent effect on NPCs proliferation (BrdU+/DAPI+) at all doses tested. d However, IL-10 significantly reduced the percentage of GFAP+/DAPI+ astrocytes at 100 and 200 ng/ml, e while increased the percentage of Olig2+ cells at 50, 100, 200, and 400 ng/ml with the highest effect at 200 ng. This effect was significantly attenuated with IL-10 neutralizing antibody. Addition of 0.8 μg/ml of IL10 neutralizing antibody effectively blocked 100 ng/ml of IL-10 on astrocyte and oligodendrocyte differentiation of NPCs. f–k Representative images of NPC differentiation assessment are shown. N = 3–5 independent experiments. The data show the mean ± SEM, *p < 0.05, one-way ANOVA
Fig. 10
Fig. 10
M2 microglia promote oligodendrocyte differentiation of spinal cord NPCs through IL-10. a To assess the effects of microglia effects on NPC proliferation and differentiation, MCM was collected from microglia 2 days following polarization. This media was then transferred to NPC cultures to assess proliferation and differentiation. b MCM derived from M2 microglia significantly promoted NPC proliferation compared to M1 MCM. IL-10 neutralizing antibody had no effect on the overall proliferation of NPCs by M2 MCM suggesting this effect was not mediated through IL-10. c A significant decrease was observed in the percentage of GFAP+/DAPI+ astrocytes when NPCs were exposed to M2 MCM (k) compared to both M0 (g) and M1 (i) MCM. (d) M2 MCM (l) significantly increased the percentage of Olig2+/DAPI+ cells compared to both M0 (h) and M1 (j) MCM. Additionally, M1 MCM significantly reduced the percentage of Olig2+ cells compared to both M0 and M2 MCM. m, n The effect of M2 MCM was significantly reduced by IL-10 neutralizing antibody. e, f represents NPCs N = 3–5 independent experiments. The data show the mean ± SEM, *p < 0.05, one-way ANOVA
Fig. 11
Fig. 11
Blocking LAR and PTPσ receptors has no effect on formation of astrocytic scar and CSPGs following SCI. a To study scar formation under ILP and ISP treatment, we examined GFAP protein expression by Western blotting at 1, 3, 5, 7, and 14 days post-injury. As anticipated, GFAP protein expression was significantly increased following injury compared to uninjured animals. However, ILP and ISP treatment had no significant effect on the expression of GFAP at any time-points after SCI. b Using immunohistochemistry, we also studied astrocytic scar in SCI lesion. Our quantitative immunodensity analysis in the injured spinal cord tissue at the chronic 28 days post-SCI also showed no significant differences in the levels of GFAP under ILP/ISP treatment compared to vehicle SCI control group. c Slot blot analysis of CS56 expression at 1, 3, 5, 7, and 14 days post-SCI demonstrated a significant increase CSPGs following injury. Similar to GFAP, ILP and ISP treatment had no effect on the deposition of CSPGs following injury. d CS56 expression was additionally measured using immunohistochemistry at different distances to injury epicenter at 28 days post-SCI. ILP/ISP treatment showed no significant differences in the levels of CS56 compared to vehicle SCI control group. The data show the mean ± SEM, *p < 0.05, **p < 0.001, one-way ANOVA (a, c), two-way ANOVA (b–d), N = 4–6/group
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