Fig 2: Endothelial ADK deficiency prevents cerebral ischemic injury. a, b 2,3,5-tripenyltetrazolium chloride (TTC) staining (a) and quantification of infarct size (b) in ADKWT and ADKVEC-KO mice (n = 10 mice per group). c, d Representative images of TUNEL staining (c; scale bar, 50 µm) and quantification of TUNEL-positive cells (d) in the penumbra of the ipsilateral ischemic cortex (on sections at a level 0.6 mm caudal to the bregma) of ADKWT and ADKVEC-KO mice (n = 5 per group). e Neurological status of ischemic ADKWT and ADKVEC-KO mice (n = 10 mice per group). f–i Flow cytometric analysis of inflammatory cell populations in the contralateral and ipsilateral brains of ADKWT and ADKVEC-KO mice 24 h post I/R (n = 6 mice per group). f The ratio of infiltrated leukocytes (CD11b+CD45high). g The number of infiltrated neutrophils (CD11b+CD45highLy6G+). h The number of macrophages/activated microglia cells (CD11b+CD45highF4/80+). i The ratio of resident microglia cells (CD11b+CD45low). j IHC staining with Iba-1 antibody and quantification of Iba-1-positive resting microglia in contralateral brains of ADKWT and ADKVEC-KO mice 24 h post I/R. Representative images were taken in the contralateral cortex on sections at a level 0.5 mm caudal to Bregma (scale bar, 50 µm; n = 5 mice per group). k IHC staining and quantification of Mac2-positive macrophages/activated microglia cells on brain sections of ADKWT and ADKVEC-KO mice 24 h post I/R. Representative images were taken in the penumbra of the ipsilateral ischemic cortex and the corresponding contralateral cortex on sections at a level 0.4 mm caudal to the bregma (scale bar, 50 µm; n = 5 mice per group). l mRNA levels for adhesion molecules in brains of ADKWT and ADKVEC-KO mice 24 h post I/R (n = 6 mice per group). m, n Western blot detection (m) and densitometric quantification (n) of adhesion molecule expression in brains of ADKWT and ADKVEC-KO mice 24 h post I/R (n = 4 per group). For all bar graphs, data are the mean ± SEM, *P < 0.05 and **P < 0.01 (Unpaired, two-tailed Student’s t-test for b, d, and g–m; one-way ANOVA with Tukey’s post hoc test for o)

Fig 3: Immunofluorescence staining of the whole mount for immune cells. (A) control cochlea not exposed to noise, (B) Noise exposed cochlea at day 7, (C) Apical turn of the cochlea at day 7 post- noise exposure showing the immune cells (CD45 + ; red) and B cells (B220 + ; green), (D) Basal turn of the cochlea at day 7 post-noise exposure immune cells (CD45 + ; red) and B cells (B220 + ; green), (E) Apical turn of the cochlea at day 7 post-noise exposure showing the myeloid cells (CD11b + ; red) and NK cells (NK1.1 + ; green), (F) Basal turn of the cochlea at day 7 post-noise exposure showing the myeloid cells (CD11b + ; red) and NK cells (NK1.1 + ; green). C’, C”, D’, D”, E’, E”, F’, and F” show the individual images of the respective immune cell. These are the representative figures from 4 mice.

Fig 4: Ablation of meningeal lymphatics aggravates the neuroinflammatory response, the defects of exploratory behavior, and short-term working memory in SAH.a–d Impaired meningeal lymphatics worsened the microglia activation in SAH. a Representative FACS plots showing gating strategy we used in flow cytometric analysis. Cell populations in the right dot plots defined as CD11b+CD45low (microglia) were gated for further analysis. b Representative dot plots showing the ratio of CD16/32-positive subsets and CD206-positive subsets from different groups. c Quantification of CD16/32+CD206- subsets of CD11b+CD45low populations (P(SAH vs L + V + SAH) < 0.0001, P(L + SAH vs L + V + SAH) < 0.0001, P(V + SAH vs L + V + SAH) < 0.0001, P(L + V vs L + V + SAH) < 0.0001), d CD206+CD16/32- subsets of CD11b+CD45low populations (P(SAH vs L + V + SAH) = 0.0146, P(L + SAH vs L + V + SAH) < 0.0001, P(L + V vs L + V + SAH) = 0.0086), and e CD16/32+CD206+ subsets of CD11b+CD45low populations in different groups (P(V + SAH vs L +V + SAH) = 0.025). SAH/L + V + SAH; n = 14, L + SAH/V + SAH/L + V; n = 12, pooled from 2 independent experiments. f–i Ablation of meningeal lymphatics worsens the exploratory behavior and short-term working memory associated with SAH. f Percentage of time spent in the center area in the open field test (P(SAH vs L + V + SAH) = 0.0194, P(L + SAH vs L + V + SAH) = 0.0053, P(V + SAH vs L + V + SAH) = 0.009, P(L + V vs L + V + SAH) = 0.0138), g the number of entries into the center area in the open field test (P(SAH vs L + V + SAH) < 0.0001, P(L + SAH vs L + V + SAH) = 0.0001, P(V + SAH vs L + V + SAH) = 0.0018, P(L + V vs L + V + SAH) = 0.0141), h percentage of time spent in the novel arm (NA) in the Y-maze test (P(SAH vs L + V + SAH) = 0.0006, P(L + SAH vs L + V + SAH) = 0.003, P(L + V vs L + V + SAH) = 0.0198), and I the number of entries into the NA in the Y-maze test in the groups of mice indicated (P(SAH vs L + V + SAH) < 0.0001, P(L + SAH vs L + V + SAH) < 0.0001, P(V + SAH vs L + V + SAH) = 0.0338, P(L + V vs L + V + SAH) = 0.0303). SAH; n = 22, L + SAH/V + SAH/L + V + SAH; n = 19, L + V; n = 15 mice, pooled from 2 independent experiments. All data are presented as mean values ± SD, one-way ANOVA with Turkey’s multiple-comparison test, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. NS, not significant. APC allophycocyanin, PE R-phycoerythrin, FITC fluorescein isothiocyanate. Source data are provided as a Source data file.

Fig 5: PD-L1 expression is increased in Lal–/– CD11c+ cells.(A) Percentage of PD-L1+ cells in the blood of Lal+/+ and Lal–/– mice by flow cytometry analysis. (B) PD-L1 expression in blood CD11c+, MHCII+, F4/80+, CD11b+, Ly6C+, and Ly6G+ cells of Lal–/– versus Lal+/+ mice by flow cytometry analysis. (C) PD-L1 expression in CD11c- or CD11c+ double-gated myeloid cells of Lal–/– versus Lal+/+ blood by flow cytometry analysis. (D) Percentage of CD11c+ cells in the blood of Lal+/+ and Lal–/– mice by flow cytometry analysis. (E) Cytokine expression in Lal–/– versus Lal+/+ CD11c+ cells by flow cytometry analysis. Data are expressed as mean ± SD. Experiments were independently repeated, n = 6 for A–D, n = 5 for E. *P < 0.05, **P < 0.01, unpaired Student’s t test.