Dissecting the effects of endogenous brain IL-2 and normal versus autoreactive T lymphocytes on microglial responsiveness and T cell trafficking in response to axonal injury

Abstract

IL-2 is essential for T-helper regulatory (Treg) cell function and self-tolerance, and dysregulation of both endogenous brain and peripheral IL-2 gene expression may have important implications for neuronal injury and repair. We used an experimental approach combining mouse congenic breeding and immune reconstitution to test the hypothesis that the response of motoneurons to injury is modulated by the combined effects of IL2-mediated processes in the brain that modulate its endogenous neuroimmunological milieu, and IL2-mediated processes in the peripheral immune system that regulate T cell function (i.e., normal versus autoreactive Treg-deficient T cells). This experimental strategy enabled us to test our hypothesis by disentangling the effect of normal versus autoreactive T lymphocytes from the effect of endogenous brain IL-2 on microglial responsiveness (microglial phagocytic clusters normally associated with dead motoneurons and MHC2(+) activated microglia) and T cell trafficking, using the facial nerve axotomy model of injury. The results demonstrate that the loss of both brain and peripheral IL-2 had an additive effect on numbers of microglial phagocytic clusters at day 14 following injury, whereas the autoreactive status of peripheral T cells was the primary factor that determined the degree to which T cells entered the injured brain and contributed to increased microglial phagocytic clusters. Changes in activated MHC2(+) microglial in the injured FMN were associated with loss of endogenous brain IL-2 and/or peripheral IL-2. This model may provide greater understanding of the mechanisms involved in determining if T cells entering the injured central nervous system (CNS) have damaging or proregenerative effects.

Fig. 1

A, B and C. Comparison of CD3⁺, CD4⁺ and CD8⁺ cell counts in the axotomized FMN between subject groups. Bars represent the mean ± S.E.M for the IL-2WT/RAG2KO+WT (brainIL2WT/RAG2KO + IL2WTimmune n=8), IL-2KO/RAG2KO+WT (brainIL2KO/RAG2KO + IL2WTimmune n=8), IL-2WT/RAG2KO+KO(brainIL2WT/RAG2KO + IL2KOimmune n=7) and IL-2KO/RAG2KO+KO (brainIL2KO/RAG2KO + IL2KOimmune n=8) mice. Fig. 1. D. Assessment of lymphoproliferative autoimmunity using spleen weight for subject groups. Each bar represents mean ± S.E.M of 8 mice per group except for 7 animals/group for IL-2WT/RAG2+KO mice. Mice were immune reconstituted at age of 4 weeks and FMN T cell assessments were made at 10 weeks of age, 14 days after facial nerve axotomy. * p< .05, ** p< .01 compared to IL-2WT/RAG2KO+WT.

Fig. 2

Immunohistochemistry for CD3⁺ T cells (A–D), CD4⁺ T cells (E–H) and CD8⁺ T cells (I–L) in the FMN 14 days after facial never axotomy for the subject groups: IL-2WT/RAG2KO+WT, IL-2KO/RAG2KO+WT, IL-2WT/RAG2KO+KO and IL-2KO/RAG2KO+KO mice. High power magnification of T cells are shown in the insert of A, E and I. As viewed in the online version of the manuscript, T cells were immunostained brown and are indicated by arrows, while neuronal and glial cell bodies were counterstained with cresyl violet and are shown in blue. Scale bar = 20μm.

Fig. 3

Quantification of CD11b⁺ microglial phagocytic clusters (A) and MHC2⁺ activated microglial cells (B) in the injured FMN of subject groups at 14 days post-injury. Bars represent the mean ± S.E.M for the IL-2WT/RAG2KO+WT (brainIL2WT/RAG2KO + IL2WTimmune n=8), IL-2KO/RAG2KO+WT (brainIL2KO/RAG2KO + IL2WTimmune n=8), IL-2WT/RAG2KO+KO (brainIL2WT/RAG2KO + IL2KOimmune n=7) and IL-2KO/RAG2KO+KO (brainIL2KO/RAG2KO + IL2KOimmune n=8) mice. Mice were immune reconstituted at age of 4 weeks and, CD11b⁺ microglial phagocytic clusters and MHC2⁺ actvated microglial cells assessments were made at 10 weeks of age. * p≤ .05, ** p< .01 compared to IL-2WT/RAG2KO+WT.