Ablation of nasal-associated lymphoid tissue does not affect focal ischemic brain injury in mice

Abstract

Stroke is a devastating disease with a strong inflammatory component. It has been shown that part of this response is mediated by IL17+ γδT cells. γδT cells constitute a lymphocyte population with innate features that mainly populates epithelial surfaces including skin, intestine, and airways. We have shown that in the context of stroke, T cells migrate from the small intestine to the meninges but whether they can migrate from other epithelial surfaces is still unknown. Because of its proximity, one possible source of stroke-associated IL17+ γδT cells could be the Nasal-Associated Lymphoid Tissue (NALT) from which T cells could migrate along olfactory nerve sheaths through the cribriform plate into the brain and/or meninges. In order to study the role of NALT as a source for immune cells and/or inflammatory mediators in the context of stroke, we analyzed the effect of NALT ablation on immune cell infiltration and infarct volume after stroke. Infarct volume analysis did not show any significant difference between sham and NALT-ablated animals. In addition, no significant differences were found in immune cell infiltration in the brain or meninges of stroke animals subjected to NALT or Sham-ablation surgery. In conclusion, NALT ablation does not affect ischemic brain damage or immune cell infiltration in the meninges or brain after stroke.

Fig 1. Visualization and analysis of immune cells in the NALT.

A) Representative photomicrograph of coronal section of the nose with magnified regions at the NALT (images on the left) and the nose mucosa (images on the right). NALT γδ T cells can be visualized in green (TCR-γδ-GFP⁺) and cell nuclei in blue (DAPI). B) Flow cytometric analysis of NALT immune cells, including gating strategy and quantification of CD11b myeloid cells (CD45⁺CD11b⁺; total numbers: 266±90), B cells (CD45⁺CD11b^–CD19⁺; total numbers 8504±2193), T cells (CD45⁺CD11b^–CD19^–TCRβ⁺TCRγδ^–; total number of cells: 775±319), CD4⁺ T cells (CD45⁺CD11b^–CD19^–TCRβ⁺CD4⁺TCRγδ^–; total number of cells: 518±212), CD8⁺ T cells (CD45⁺CD11b^–CD19^–TCRβ⁺TCRγδ^–CD8⁺; total number of cells: 237±105), and γδ T cells (CD45⁺CD11b^–CD19^–TCRβ^–TCRγδ⁺; total number of cells: 14±1).

Fig 2

A) Microscopic images (Giemsa stained) of representative nasal cavities from a sham-operated mouse (left images) and a mouse subjected to NALT ablation (right images) 14 days after surgery. Arrows point to the NALT tissue (sham-surgery animal) or to the respective position (NALT-ablated animal). B) Flow cytometric gating strategy of meningeal immune cells, including total leukocytes (CD45⁺), myeloid cells (CD45⁺CD11b⁺), T cells (CD45⁺CD11b^–TCRβ⁺TCRγδ^–), γδ T cells (CD45⁺CD11b^–TCRβ^–TCRγδ⁺), Th17 cells (CD45⁺CD11b^–TCRβ⁺TCRγδ^–IL17GFP⁺), and IL17 γδT cells (CD45⁺CD11b^–TCRβ^–TCRγδ⁺IL17GFP⁺). C) Quantification of the absolute number of immune cells in the brain and meninges of mice 14 days after being subjected to NALT ablation surgery or sham surgery.

Fig 3

A) Representative images of Nissl-stained coronal brain sections (left) and quantification of infarct volumes (right) in Sham-surgery animals and NALT-ablated animals, performed three days after stroke and 17 days after NALT surgery.