Stroke affects intestinal immune cell trafficking to the central nervous system

Abstract

Stroke is an acute neurological disease with a strong inflammatory component that can be regulated by the intestinal microbiota and intestinal immune cells. Although stroke has been shown to alter immune cell populations in the gut, the dynamics of cell trafficking have not been elucidated. To study the trafficking of gut-derived immune cells after stroke, we used mice expressing the photoconvertible protein Kikume Green-Red, which turns form green to red when exposed to violet light. Mice underwent laparotomy and the small intestine was exposed to violet laser light. Immune cells were isolated from the small intestine immediately after photoconversion and 2 days later. Percentage of immune cells (CD45⁺KikR⁺) that expressed the red variant of the protein (KikR) was higher immediately after photoconversion than 2 days later, indicating cell egress from the small intestine. To investigate whether intestinal immune cells traffic to the periphery and/or the central nervous system (CNS) after stroke, we analyzed KikR⁺ immune cells (2 days after photoconversion) in peripheral lymphoid organs, meninges and brain, 3 and 14 days after transient occlusion of the middle cerebral artery (tMCAo) or sham-surgery. Although migration was observed in naïve and sham animals, stroke induced a higher mobilization of gut KikR⁺ immune cells, especially at 3 days after stroke, to all the organs analyzed. Notably, we detected a significant migration of CD45^hi immune cells from the gut to the brain and meninges at 3 days after stroke. Comparison of cell trafficking between organs revealed a significant preference of intestinal CD11c⁺ cells to migrate from the small intestine to brain and meninges after stroke. We conclude that stroke increases immune cell trafficking from the small intestine to peripheral lymphoid organs and the CNS where they might contribute to post-stroke inflammation.

Keywords: Immune cell trafficking; Intestinal immune system; Kikume Green-Red fluorescent protein; Stroke.

Figure 1.. Analysis of photoconversion and cell egress from the small intestine under homeostatic conditions.

A) Flow cytometry gating strategy for analysis of different immune populations of photoconverted cells in the small intestine. B) Analysis of photoconverted cells in the intra-epithelial leukocyte (IEL) compartment of the small intestine, immediately and two days after photoconversion; * p<0.05, ** p<0.01, unpaired t-test. C) Analysis of photoconverted cells in the lamina propria mononuclear cell (LPMC) compartment of the small intestine, immediately and two days after photoconversion; * p<0.05, ** p<0.01, unpaired t-test. D) Quantification of migration of photoconverted CD45⁺ in the IEL and LPMC compartments of the small intestine during the first two days after photoconversion. Ratio was established as (1 – fraction of CD45⁺KikR⁺ at 2 days after photoconversion/fraction of CD45⁺KikR⁺ immediately after photoconversion)*100. E) Migration of photoconverted (KikR⁺) immune cells from the small intestine to the bone marrow (BM), cervical lymph nodes (cLN), mesenteric lymph nodes (mLN), spleen (SP), brain and meninges, at 0 and 2 days after photoconversion; * p<0.05, ** p<0.01, unpaired t-test. F) Proportion of KikR⁺ intestine-derived B cells, T cells, CD11b⁺, and CD11c⁺ myeloid cells in lymphoid organs, brain, and meninges 2 days after photoconversion in naïve animals; **** p< 0.0001 for One-Way ANOVA. Cell types were defined as B cells (CD45⁺TCRβ⁻TCRγδ⁻CD19⁺), TCRβ⁺ T cells (CD45⁺TCRβ⁺TCRγδ⁻), TCRγδ⁺ T cells (CD45⁺TCRβ⁻TCRγδ⁺), CD11c⁺cells (CD45⁺CD11c⁺), CD11b⁺ cells (CD45⁺CD11b⁺), Monocytes (CD45⁺CD11c⁻CD11b⁺Ly6G⁻), Neutrophils (CD45⁺CD11c⁻CD11b⁺Ly6G⁺).

Figure 2.. Stroke-induced trafficking of immune cells from the small intestine to lymphoid organs.

Small intestine was photoconverted 1 day after stroke/sham surgery and photoconverted lymphoid (A) and myeloid subsets (B) were analyzed 2 days after photoconversion in peripheral immune organs. Ratios between photoconverted cells in stroke animals and photoconverted cells in sham-surgery animals (Migration Index) are shown. Comparisons were performed between stroke and sham animals; n=6 for sham-surgery and n=14 for stroke mice; * p<0.05, unpaired t-test. Small intestine was photoconverted 12 days after stroke/sham surgery and photoconverted lymphoid (C) and myeloid subsets (D) were analyzed 2 days after photoconversion. n=9 for sham-surgery and n=12 for stroke mice; * p<0.05, unpaired t-test.

Figure 3.. Stroke-induced trafficking of immune cells from the small intestine to the brain and meninges.

Small intestine was photoconverted 1 (A) or 12 days (B) after stroke/sham surgery, and photoconverted cells were analyzed in the brain and meninges by flow cytometry 2 days after photoconversion. Ratios between photoconverted cells in stroke animals and photoconverted cells in sham-surgery animals (Migration Index) are shown. Comparisons were performed between stroke and sham animals; n=7–8 for sham-surgery and n=9–10 for stroke mice; * p<0.05, unpaired t-test. ND, not detected.

Figure 4.. Comparison of stroke-induced migration of intestinal immune populations between the different organs.

(A-D) Small intestine was photoconverted 1 (A) or 12 days (B) after stroke/sham surgery, and photoconverted cells were analyzed in different organs by flow cytometry 2 days after photoconversion. (A-B) Comparisons were performed between the different organs, n=9 for cLN, brain and meninges and n=12 for BM, mLN and SP; * p<0.05, for one-way ANOVA with Bonferroni’s correction. (C-D) Analysis of CD11c⁺ cells in brain and meninges by flow cytometry 3 days after MCAo. (C) Gating strategy for differentiation between dendritic cells (CD45^hiCD11c⁺MHCII⁺CD64⁻ F4/80⁻) and macrophages (CD45^hiCD11c⁺MHCII⁺CD64⁺F4/80⁺; Mφ) in brain and meninges. (D Left panel) Percentage of DC and macrophages in CD45^hiCD11c⁺ cells in brain and meninges (n=4 per group; paired t-test). (D Right panel) Percentage of KikR⁺DC and KikR⁺macrophages in KikR⁺CD45^hiCD11c⁺ cells in brain and meninges (n=4 per group; paired t-test). Boxplots represent median (line) and whiskers delineate minimum and maximum values.