Impact of peripheral immune status on central molecular responses to facial nerve axotomy

Abstract

When facial nerve axotomy (FNA) is performed on immunodeficient recombinase activating gene-2 knockout (RAG-2^-/-) mice, there is greater facial motoneuron (FMN) death relative to wild type (WT) mice. Reconstituting RAG-2^-/- mice with whole splenocytes rescues FMN survival after FNA, and CD4+ T cells specifically drive immune-mediated neuroprotection. Evidence suggests that immunodysregulation may contribute to motoneuron death in amyotrophic lateral sclerosis (ALS). Immunoreconstitution of RAG-2^-/- mice with lymphocytes from the mutant superoxide dismutase (mSOD1) mouse model of ALS revealed that the mSOD1 whole splenocyte environment suppresses mSOD1 CD4+ T cell-mediated neuroprotection after FNA. The objective of the current study was to characterize the effect of CD4+ T cells on the central molecular response to FNA and then identify if mSOD1 whole splenocytes blocked these regulatory pathways. Gene expression profiles of the axotomized facial motor nucleus were assessed from RAG-2^-/- mice immunoreconstituted with either CD4+ T cells or whole splenocytes from WT or mSOD1 donors. The findings indicate that immunodeficient mice have suppressed glial activation after axotomy, and cell transfer of WT CD4+ T cells rescues microenvironment responses. Additionally, mSOD1 whole splenocyte recipients exhibit an increased astrocyte activation response to FNA. In RAG-2^-/- + mSOD1 whole splenocyte mice, an elevation of motoneuron-specific Fas cell death pathways is also observed. Altogether, these findings suggest that mSOD1 whole splenocytes do not suppress mSOD1 CD4+ T cell regulation of the microenvironment, and instead, mSOD1 whole splenocytes may promote motoneuron death by either promoting a neurotoxic astrocyte phenotype or inducing Fas-mediated cell death pathways. This study demonstrates that peripheral immune status significantly affects central responses to nerve injury. Future studies will elucidate the mechanisms by which mSOD1 whole splenocytes promote cell death and if inhibiting this mechanism can preserve motoneuron survival in injury and disease.

Keywords: Amyotrophic lateral sclerosis; Axotomy; Motoneuron; Nerve injury; T cells.

Figure 1

mRNA expression of motoneuron regeneration-associated genes in the facial motor nucleus following facial nerve axotomy (Ax), relative to the control (C) facial motor nucleus. Mean percent change ± SEM was plotted across unoperated (0) and 7, 14, 28, and 56 days post operative (dpo) timepoints. Symbols used: $: p < 0.05 comparing WT to RAG-2^-/- + WT CD4+ T cells and #: p < 0.05 comparing RAG-2^-/- to RAG-2^-/- + WT CD4+ T cells.

Figure 2

mRNA expression of glial activation-associated genes comparing the axotomized (Ax) to the control (C) facial motor nucleus. Mean percent change ± SEM was plotted across uninjured (0) and 7, 14, 28, and 56 days post-operative (dpo) timepoints. Symbols used: *: p < 0.05 comparing WT to RAG-2^-/- and #: p < 0.05 comparing RAG-2- - to RAG-2^-/- + WT CD4+ T cells.

Figure 3

Mean relative gene expression ± SEM of Tnfα compared to Gapdh in the axotomized facial motor nucleus (A). mRNA expression of Tnfr1 comparing the axotomized (Ax) to the control (C) facial motor nucleus. Mean percent change ± SEM was plotted across uninjured (0) and 7, 14, 28, and 56 days post-operative (dpo) timepoints (B). Symbols used: *: p < 0.05 comparing WT to RAG-2^-/-; $: p < 0.05 comparing WT to RAG-2^-/- + WT CD4+ T cells; and #: p < 0.05 comparing RAG-2^-/- to RAG-2^-/- + WT CD4+ T cells.

Figure 4

mRNA expression of cell death-associated genes comparing the axotomized (Ax) to the control (C) facial motor nucleus. Mean percent change ± SEM was plotted across uninjured (0) and 7, 14, 28, and 56 days post-operative (dpo) timepoints. Symbols used: *: p < 0.05 comparing WT to RAG-2^-/-; $: p < 0.05 comparing WT to RAG-2^-/- + WT CD4+ T cells; and #: p < 0.05 comparing RAG-2^-/- to RAG-2^-/- + WT CD4+ T cells.

Figure 5

mRNA expression of motoneuron regeneration-associated genes in the facial motor nucleus following facial nerve axotomy (Ax), relative to the control (C) facial motor nucleus. Mean percent change ± SEM was plotted across unoperated (0) and 7, 14, 28, and 56 days post operative (dpo) timepoints. Symbols used: *: p < 0.05 comparing RAG-2^-/- + WT whole splenocytes to RAG-2^-/- + mSOD1 whole splenocytes; $: p < 0.05 comparing RAG-2^-/- + WT whole splenocytes to RAG-2^-/- + mSOD1 CD4+ T cells; and #: p < 0.05 comparing RAG-2^-/- + mSOD1 whole splenocytes to RAG-2^-/- + mSOD1 CD4+ T cells.

Figure 6

mRNA expression of glial-associated genes in the facial motor nucleus following facial nerve axotomy (Ax), relative to the control (C) facial motor nucleus. Mean percent change ± SEM was plotted across unoperated (0) and 7, 14, 28, and 56 days post operative (dpo) timepoints. Symbols used: $: p < 0.05 comparing RAG-2^-/- + WT whole splenocytes to RAG-2^-/- + mSOD1 CD4+ T cells.

Figure 7

Mean relative gene expression ± SEM of Tnfα compared to Gapdh in the axotomized facial motor nucleus (A). mRNA expression of Tnfr1 comparing the axotomized (Ax) to the control (C) facial motor nucleus. Mean percent change ± SEM was plotted across uninjured (0) and 7, 14, 28, and 56 days post-operative (dpo) timepoints (B).

Figure 8

mRNA expression of Fas/nNos cell death-associated genes in the facial motor nucleus following facial nerve axotomy (Ax), relative to the control (C) facial motor nucleus. Mean percent change ± SEM was plotted across unoperated (0) and 7, 14, 28, and 56 days post operative (dpo) timepoints. Symbols used: *: p < 0.05 comparing RAG-2^-/- + WT whole splenocytes to RAG-2^-/- + mSOD1 whole splenocytes; $: p < 0.05 comparing RAG-2^-/- + WT whole splenocytes to RAG-2^-/- + mSOD1 CD4+ T cells; and #: p < 0.05 comparing RAG-2^-/- + mSOD1 whole splenocytes to RAG-2^-/- + mSOD1 CD4+ T cells.

Figure 9

Summary of findings from this study. On the left, WT CD4+ T cells are depicted as regulators of glial responses to axotomy, resulting in a neuroprotective glial microenvironment that promotes motoneuron survival. On the right, mSOD1 whole splenocytes are shown as generating a neurotoxic glial environment by promoting an aberrant astrocyte phenotype and increasing Fas expression, thereby contributing to greater motoneuron death.