Fig 2: Increased Iba1+ cell infiltration at needle track of surgically wounded Dgka-/- and WT mice in acute brain injury model. (A) Representative cross sections highlighting needle track and unwounded contralateral cortex. (B) Quantification of Iba1+ cells in the needle track demonstrates an increase in cell infiltration in wounded Dgka-/- brains versus that in the contralateral, unwounded side. Each data point represents one of four brain sections cut from the site of needle injection, from each of n = 3 mice per group. Error bars: SD. *p < 0.05, **p < 0.01, ****p < 0.0001; Two-way ANOVA with Tukey post-hoc test.

Fig 3: Decreased neuronal METTL3 and m6A in the hippocampus by AAV-shMettl3. (A) Highly conserved gene sequences of shRNA targeting region in mouse and rat Mettl3 mRNA. (B) Knockdown effect of shMettl3 was confirmed in mouse N2a cells 3 days after transient transfection (Data are means±SEM from 3 independent experiments). (C) AAV-mediated gene delivery in the hippocampus was confirmed by immunostaining for GFP. WT C57BL/6 mice receiving bilaterally stereotaxic injections of AAV encoding for eGFP plus anti-Mettl3 shRNA (shMettl3) or scrambled control shRNA (shCtrl) into the hippocampus at 2 months of age were analyzed 1 month later. (D-F) Representative images of the AAV-infected areas (GFP-positive) in hippocampus for different cell markers by immunocytochemistry after fixation: MAP 2 for neurons (D), GFAP for astrocytes (E), and Iba1 for microglia (F). (G, H) Representative image of immunoreactivity of METTL3 or m6A (G) in AAV-shRNA-injected mice and quantification analysis (H) confirmed that METTL3 protein and m6A modification level were reduced in mice injected with shMettl3 virus (n = 5–7 mice per group). GFP staining has been performed in adjacent brain slices at the same time and immunoreactivities of m6A and METTL3 were measured in GFP-positive regions. (Data are means±SEM, *p < 0.05, **p < 0.01, (B, H) unpaired student’s t-test)

Fig 4: TSPO KO mice exhibited more pro-inflammatory M1 macrophages in the colon. (A) Flow cytometry analysis of the proportion of M1 type macrophages in mouse colon mononuclear cells from WT (healthy n = 3. DSS n = 3) and KO mice (healthy n = 3. DSS n = 4). (B) The statistics of the proportion of macrophages in mouse colon lymphocytes are shown in panel A. (C) Left, Iba1 immunofluorescence staining of frozen mouse colon sections from WT and KO mice was visualized by laser confocal microscopy. Red: Iba1. Blue: DAPI. The scale bar: 50 µm. Right, quantification of the expression levels of Iba1 as shown in left. (D) After DSS treatment, Iba1 expression levels in lymphocyte lysates from TSPO KO and WT mice were analyzed by Western blots. (E) Quantification of the expression levels of Iba1 as shown in Panel D. Data expressed as mean ± s.e.m., Student’s t-test, two-tailed. * p < 0.05, ** p < 0.01. n.s. means not significant.

Fig 5: Activation of microglia and innate immune response by toxic AAVs. (A) Iba1 staining of retinal sections from P30 eyes of CD-1 mice infected at P0 with the indicated AAV viruses at a dose of 3E9 vg per eye. (Scale bar: 50 µm.) (B) Quantification of displaced Iba1-positive cells by cell layer. High dose (3E9 vg per eye); low dose (8E8 vg per eye). Values are shown as mean ± SD. n = 4 per group. (C) Quantification of microglia in retinas of P20 Cx3cr1-GFP mice by flow cytometry injected with PBS or 3E8 vg per eye AAV8-CMV-TdTomato (n = 3–4 for all groups). Values are shown as mean ± SD. (D) Relative mRNA levels of TNF-a, IL-1ß, IL-6, and IFN-? by qPCR in the retinas of P30 CD-1 mice infected at P0 with the indicated AAV viruses at high dose (3E9 vg per eye). Expression level was normalized to gapdh. Values are shown as mean ± SEM. n = 4–8 per group. One-way ANOVA analysis with Tukey test, **P < 0.01; *P < 0.05; NS, not significant between the designated group and the uninjected group.