Development of protective autoimmunity by immunization with a neural-derived peptide is ineffective in severe spinal cord injury

Abstract

Protective autoimmunity (PA) is a physiological response to central nervous system trauma that has demonstrated to promote neuroprotection after spinal cord injury (SCI). To reach its beneficial effect, PA should be boosted by immunizing with neural constituents or neural-derived peptides such as A91. Immunizing with A91 has shown to promote neuroprotection after SCI and its use has proven to be feasible in a clinical setting. The broad applications of neural-derived peptides make it important to determine the main features of this anti-A91 response. For this purpose, adult Sprague-Dawley rats were subjected to a spinal cord contusion (SCC; moderate or severe) or a spinal cord transection (SCT; complete or incomplete). Immediately after injury, animals were immunized with PBS or A91. Motor recovery, T cell-specific response against A91 and the levels of IL-4, IFN-γ and brain-derived neurotrophic factor (BDNF) released by A91-specific T (T(A91)) cells were evaluated. Rats with moderate SCC, presented a better motor recovery after A91 immunization. Animals with moderate SCC or incomplete SCT showed significant T cell proliferation against A91 that was characterized chiefly by the predominant production of IL-4 and the release of BDNF. In contrast, immunization with A91 did not promote a better motor recovery in animals with severe SCC or complete SCT. In fact, T cell proliferation against A91 was diminished in these animals. The present results suggest that the effective development of PA and, consequently, the beneficial effects of immunizing with A91 significantly depend on the severity of SCI. This could mainly be attributed to the lack of T(A91) cells which predominantly showed to have a Th2 phenotype capable of producing BDNF, further promoting neuroprotection.

Figure 1. Motor recovery of spinal cord injured rats.

Animals were subjected to a moderate (A) or severe spinal cord contusion (B). The recovery of rats subjected to a complete SCT is also shown (C). In all cases, animals were treated either with A91 or PBS. Severe injuries abolished the beneficial effect induced by immunizing with A91. ^* Different from PBS group (p<0.05, two-way ANOVA for repeated measures). Each point represents the mean ± SD of 10 rats.

Figure 2. Effect of spinal cord injury on the proliferative response to A91.

T cell proliferation against A91 was analyzed in rats with moderate (A) or severe (B) spinal cord contusion (SCC). The same evaluation was performed on rats with incomplete (C) or complete (D) spinal cord transection (SCT). Severe SCC and complete SCT inhibited the proliferative response against A91. ^* Different from PBS group (p = 0.01, Mann-Whitney U test). Bars represent the mean ± SD of 5 rats.

Figure 3. Representative histograms of A91-immunized animals.

The ability of T cells to proliferate in the presence of A91 peptide was evaluated using CFSE assays and flow cytometry. Cells stained for CFSE and CD4 were analyzed. Ten thousand events were collected for each sample.

Figure 4. IL-4/IFN-γ index in the supernatant of T cells exposed to A91.

Lymphocytes were obtained from rats with moderate spinal cord contusion (A) or incomplete spinal cord transection (B). T_A91 cells predominantly released IL-4. ^* Different from PBS (p<0.0001 (A), p = 0.001 (B) Student's t-test). Bars represent the mean ± SD of 5 rats. Data was taken from one of three experiments where the same effect was observed.

Figure 5. BDNF released by T cells from A91- or PBS-immunized rats.

In both cases, T cells were exposed to A91. The levels observed in rats with moderate spinal cord contusion (A) or incomplete spinal cord transection (B) are presented. T_A91 cells released significant amounts of BDNF. ^* Different from PBS (p = 0.01 (A), Mann-Whitney U test; p = 0.02 (B), Mann-Whitney U test). Bars represent the mean ± SD of 5 rats. Data was taken from one of three experiments where the same effect was observed.