Mucosal-Associated Invariant T Cells Are Involved in Acute Ischemic Stroke by Regulating Neuroinflammation

Abstract

Background Mucosal-associated invariant T (MAIT) cells have been associated with inflammation in several autoimmune diseases. However, their relation to ischemic stroke remains unclear. This study attempted to elucidate the role of MAIT cells in acute ischemic stroke in mice. Methods and Results We used MR1 knockout C57BL/6 (MR1^-/-) mice and wild-type littermates (MR1^+/+). After performing a transient middle cerebral artery occlusion (tMCAO), we evaluated the association with inflammation and prognosis in the acute cerebral ischemia. Furthermore, we analyzed the tMCAO C57BL/6 mice administered with the suppressive MR1 ligand and the vehicle control. We also evaluated the infiltration of MAIT cells into the ischemic brain by flow cytometry. Results showed a reduction of infarct volume and an improvement of neurological impairment in MR1^-/- mice (n=8). There was a reduction in the number of infiltrating microglia/macrophages (n=3-5) and in their activation (n=5) in the peri-infarct area of MR1^-/- mice. The cytokine levels of interleukin-6 and interleukin-17 at 24 hours after tMCAO (n=3-5), and for interleukin-17 at 72 hours after tMCAO (n=5), were lower in the MR1^-/- mice. The administration of the suppressive MR1 ligand reduced the infarct volume and improved functional impairment (n=5). Flow cytometric analysis demonstrated there was a reduction of MAIT cells infiltrating into the ischemic brain at 24 hours after tMCAO (n=17). Conclusions Our results showed that MAIT cells play an important role in neuroinflammation after focal cerebral ischemia and the use of MAIT cell regulation has a potential role as a novel neuroprotectant for the treatment of acute ischemic stroke.

Keywords: cerebral infarction; major histocompatibility complex‐related protein 1; mucosal‐associated invariant T cells; neuroinflammation; neuroprotection.

Figure 1. Effect of the major histocompatibility complex‐related molecule 1 (MR1) deficiency on the infarct volume and neurological severity.

A, Experimental design using MR1 knockout C57BL/6 (MR1^−/−) mice and wild‐type littermate C57BL/6 (MR1^+/+) mice. B, Representative triphenyltetrazolium chloride‐stained sections of each group; MR1^+/+ mice at 24 hours (24 h) after transient middle cerebral artery occlusion (tMCAO) (MR1^+/+24), MR1^−/− mice at 24 hours after tMCAO (MR1^−/−24), MR1^+/+ mice at 72 hours (72 h) after tMCAO (MR1^+/+72), and MR1^−/− mice at 72 hours after tMCAO (MR1^−/−72). Scale bars=2 mm. C, Comparison of cerebral infarct volume between MR1^+/+ and MR1^−/− mice at 24 and 72 hours after tMCAO, respectively. Values are presented as mean±SD and were analyzed by an unpaired Student t test. *P<0.05. D, Comparison of the modified neurologic severity scores between MR1^+/+ and MR1^−/− mice at 24 and 72 hours after tMCAO, respectively (n=8 per group). Values are presented as the median and interquartile range and were analyzed by Wilcoxon’s rank‐sum test. *P<0.05.

Figure 2. Major histocompatibility complex‐related molecule 1 (MR1) deficiency and neuroinflammation after transient focal ischemia.

A, Representative images of Iba1 (ionized calcium binding adapter molecule‐1) ‐positive cells and the average cell number of Iba1‐positive cells in the ischemic boundary area in MR1^+/+ and MR1^−/− mice at 24 and 72 hours after tMCAO, respectively (MR1^+/+24 and MR1^−/−24: n=3–5, MR1^+/+72, and MR1^−/−72: n=5 per group). Scale bars=100 μm. B, A Comparison of the average cell number of activated Iba1‐positive cells between MR1^+/+ and MR1^−/− mice using morphological grading scale at each time point (MR1^+/+24 and MR1^−/−24: n=3–5, MR1^+/+72, and MR1^−/−72: n=5 per group). C, Comparison of the proinflammatory cytokine levels in ischemic hemibrain between MR1^+/+ and MR1^−/− mice at 24 hours (tMCAO24) and 72 hours (tMCAO72) after tMCAO, respectively (tMCAO24: n=3–5, tMCAO72: n=5). Sham‐operated mice without vascular occlusion at 24 hours (sham24) and 72 hours (sham72) after sham operation as controls (n=3 each). Values are presented as the mean±SD and were analyzed by an unpaired Student t test. *P<0.05. ^† P<0.01. IL indicates interleukin; tMCAO, transient middle cerebral artery occlusion; and TNF‐, tumor necrosis factor alpha.

Figure 3. Inhibition of mucosal‐associated invariant T (MAIT) cell activation and stroke outcome after transient focal ischemia.

A, Experimental design using C57BL/6 mice administered the suppressive ligand and with dimethyl sulfoxide (DMSO) as a vehicle control. B, Representative triphenyltetrazolium chloride‐stained sections of each group; DMSO‐administered mice at 24 hours after tMCAO (Vehicle24), suppressive MR1 ligand‐administered mice at 24 hours after tMCAO (Ligand24), DMSO‐administered mice at 72 hours after tMCAO (Vehicle72), and suppressive MR1 ligand‐administered mice at 72 hours after tMCAO (Ligand72). Scale bars=2 mm. C, Comparison of cerebral infarct volume between the vehicle control and the suppressive MR1 ligand‐administered mice at 24 and 72 hours after tMCAO, respectively. Values are presented as the mean±SD and were analyzed by an unpaired Student t test. *P<0.05. D, Comparison of the mNSSs between the vehicle control and the suppressive MR1 ligand‐administered mice at 24 and 72 hours after tMCAO, respectively (n=5 per group). Values are presented as the median and interquartile range and were analyzed by Wilcoxon’s rank‐sum test. *P<0.05. E, Representative images of TUNEL‐positive cells (a, Vehicle24; b, Ligand24; c, Venicle72; d, Ligand72) and the comparison of the number of TUNEL positive cells between the suppressive MR1 ligand‐administered mice and the vehicle control at 24 and 72 hours after tMCAO, respectively (e). Values are presented as the mean±SD and were analyzed by an unpaired Student t test. *P<0.05. MR1 indicates major histocompatibility complex‐related molecule 1; tMCAO, transient middle cerebral artery occlusion; and TUNEL, terminal deoxynucleotidal transferase–mediated biotin–deoxyuridine triphosphate nick‐end labeling.

Figure 4. Inhibition of MAIT cell activation and neuroinflammation after transient focal ischemia.

A, Representative images of Iba1 (ionized calcium binding adapter molecule‐1)‐positive cells and the average cell number of Iba1‐positive cells in the ischemic boundary area in tMCAO C57BL/6 mice administered with the suppressive MR1 ligand and the vehicle control at 24 and 72 hours after tMCAO, respectively (n=5 each). Scale bars=100 μm. B, A comparison of the average cell number of activated Iba1‐positive cells between the vehicle control and ligand‐administered mice using a morphological grading scale at each time point (n=5 each). C, A comparison of the proinflammatory cytokine levels in the ischemic hemibrain between the vehicle control and the ligand‐administered mice at 24 hours (tMCAO24) and 72 hours (tMCAO72) after tMCAO, respectively (n=5 each). Sham‐operated mice without vascular occlusion at 24 hours (sham24) and 72 hours (sham72) after the first injection of the ligand or DMSO in the experimental protocol as controls (n=3 each). Values are presented as the mean±SD and were analyzed by an unpaired Student t test. *P<0.05. ^† P<0.01. IL indicates interleukin; MAIT, mucosal‐associated invariant T; tMCAO, transient middle cerebral artery occlusion; and TNF‐, tumor necrosis factor alpha.

Figure 5. Administration of the suppressive MR1 ligand (isobutyryl 6‐formylpterin) and infiltrating MAIT cells in the ischemic brain.

A, Absolute MAIT cell numbers per cerebral hemisphere. B, MAIT cell percentages of the αβ T cells among tMCAO C57BL/6 mice administered with the suppressive MR1 ligand (n=6), the vehicle control mice (n=5), and the sham‐operated C57BL/6 mice (n=6). Each symbol represents data from an individual mouse. Values are presented as the median and interquartile range and were analyzed by the Kruskal‐Wallis test followed by a post hoc analysis with Dunn’s test. *P<0.05. MAIT indicates mucosal‐associated invariant T; MR1, major histocompatibility complex‐related molecule 1; and tMCAO, transient middle cerebral artery occlusion.