γδ T Cells Contribute to Injury in the Developing Brain

Abstract

Brain injury in premature infants, especially periventricular leukomalacia, is an important cause of neurologic disabilities. Inflammation contributes to perinatal brain injury development, but the essential mediators that lead to early-life brain injury remain largely unknown. Neonates have reduced capacity for mounting conventional αβT-cell responses. However, γδT cells are already functionally competent during early development and are important in early-life immunity. We investigated the potential contribution of γδT cells to preterm brain injury using postmortem brains from human preterm infants with periventricular leukomalacia and two animal models of preterm brain injury-the hypoxic-ischemic mouse model and a fetal sheep asphyxia model. Large numbers of γδT cells were observed in the brains of mice, sheep, and postmortem preterm infants after injury, and depletion of γδT cells provided protection in the mouse model. The common γδT-cell-associated cytokines interferon-γ and IL-17A were not detectable in the brain. Although there were increased mRNA levels of Il17f and Il22 in the mouse brains after injury, neither IL-17F nor IL-22 cytokines contributed to preterm brain injury. These findings highlight unique features of injury in the developing brain, where, unlike injury in the mature brain, γδT cells function as initiators of injury independently of common γδT-cell-associated cytokines. This finding will help to identify therapeutic targets for preventing or treating preterm infants with brain injury.

Figure 1

Vδ2⁺ γδT cells in the postmortem brains of preterm infants with brain injury. Paraffin-embedded cerebral hemispheres and frontal lobe sections at the level of Ammon's horn, including the lateral ventricle, were used for immunohistochemical (IHC) analysis. A–E and H–M: IHC staining of Vδ2⁺ γδT cells (arrows) in the injured brains in the periventricular white matter region adjacent to the injured area (A), the meninges tissue (B–D), and the tissue close to the cortex (E) of postmortem neonatal cases. Insets in A and B show higher magnification of Vδ2⁺ γδT cells. D is a higher magnification image of C. A–E are from case 1. F: Negative control stained with an isotype control antibody against TCRγδ. G: Thionin/acid fuchsin staining of the brain meninges area. H–M: γδT-positive staining in the meninges area of the postmortem brain outside the blood vessels, in the inner side of the blood vessels in the meninges (J), and in the cells of the blood vessel wall (K–M). I is a higher magnification image of the boxed area in H. G–L are from case 2. Arrowhead in K indicates IHC-positive staining of Vδ2 γδT cells. Scale bars: 50 μm (A, B, E, and F); 20 μm (C, I, J, L, and M); 10 µm (D); 100 μm (G, H, and K). Original magnification, ×40 (A and B, insets).

Figure 2

γδT cells are found in the mouse brain after hypoxia-ischemia (HI)–induced preterm brain injury. A–D: The mRNA expression of Trg (A), Trgv4 (B), Trgv5 (C), and Trgv7 (D) in the hemisphere ipsilateral to the injury compared with the mRNA level in the normal control mice. E–Q: Presence of γδT cells in the mouse brain after HI injury. E: γδT-cell counts from immunohistochemical staining at 6 hours after HI. F and G: Representative immunohistochemical staining of γδT cells at 6 hours after HI in the mouse brain in the meninges of the ventral part of the retrosplenial area (F) and in the meninges of the cortical amygdala area (G) in the ipsilateral hemisphere. H–K: γδT cells in the contralateral (H) and ipsilateral (I) periventricular area by the anterior pretectal nucleus (APN) and lateral posterior nucleus (LPN) of the thalamus and in the contralateral (J) and ipsilateral (K) meninges by the cortical amygdala area (COA). L–Q: In eGFP γδT-cell reporter mice, eGFP⁺ γδT-cells are shown in the meninges by the cortical amygdala area in the ipsilateral hemisphere at 6 hours after HI. L: Enhanced green fluorescent protein–positive (eGFP⁺) γδT cells. N: Nuclear staining with DAPI. P: Overlay of eGFP⁺ γδT cells and DAPI. M, O, and Q: Higher-magnification images of L, N, and P, respectively. Data are expressed as means ± SEM of the ratio of the target gene to the reference gene Rna18s1 (A–D) and means ± SEM γδT-cell counts (E). n = 6 to 8 (A–D, HI mice); n = 5 to 6 (A–D, controls); n = 8 (E, HI mice). ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001 versus control mice (n = 8); ^†P < 0.05 between the contralateral and the ipsilateral hemispheres. Scale bars = 10 μm (F and G); 50 μm (H–L, N, and P); 20 μm (M, O, and Q).

Figure 3

The activation of the IL-17/IL-22 signaling pathways in a hypoxia-ischemia (HI)–induced preterm brain injury mouse model. A and B: The gene expression profile of transcription factors, receptors, signaling adaptors, and cytokines for the IL-17 (A) and IL-22 (B) pathways in the mouse brain after HI-induced preterm brain injury. Data are expressed as means ± SEM of the ratio of the target gene to the reference gene Rna18s1. n = 5 to 6 for uninjured controls; n = 6 to 8 for HI groups. ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001.

Figure 4

Depletion of γδT cells but not IL-17A/F or IL-22 protects the mouse brain from hypoxia-ischemia (HI)–induced preterm brain injury. A–H: γδT cell depletion as seen in the subcortical white matter volume by measuring the maltose-binding protein (MBP) immunohistochemical (IHC)–positive staining area (A, C, E, and F) and in the total tissue loss using microtubule associated protein (MAP)-2 IHC staining (B, D, G, and H) in Tcrd^−/− and wild-type (WT) mice at 7 days after HI. A: Total volume of the subcortical white matter. B: Total brain tissue loss in the WT versus Tcrd^−/− mouse brain. C and D: The total subcortical white matter area (C) and total brain tissue loss at the different brain levels (D). The x axis of C and B indicates the brain levels analyzed, where level 1 refers to the frontal part of the mouse brain and level 6 refers to the posterior part of the mouse brain. E–H: Representative images of the MBP (E and F) and MAP-2 (G and H) staining from the WT (E and G) and Tcrd^−/− (F and H) mouse brains at 7 days after HI. I–L: Brain injury after blocking IL-17A/F or in Il22^−/− mice at 7 days after HI. M and N: The Il17f and Il22 mRNA levels in WT, Tcrd^−/−, and Rag1^−/− mouse brains at 6 hours and 3 days after HI and in age-matched naïve control mice at postnatal day 5 (PND5) and postnatal day 8 (PND8), respectively. Data are expressed as means ± SEM (A–D, I–L) and as means ± SEM of the ratio of the target gene to the reference gene Gapdh (M and N). n = 29 Tcrd^−/− and 29 WT mice at 7 days after HI (A–H); n = 20 IL-17, 19 anti–IL-17, 15 WT, and 20 Il22^−/− mice (I–L); n = 7 to 10 control mice and 8 to 11 HI mice (M and N). ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001. TCR, T-cell receptor.