Fig 2: Vascular colonization of venules leads to efficient neutrophil recruitment and function.a Intravital imaging (maximum intensity z-projection) of neutrophil (Ly-6G, magenta) recruitment to infected venules with iRFP-expressing Neisseria meningitidis (green). The human vessels are shown in gray (UEA-1 lectin) and dashed lines. Scale bars, 30 µm. b Percentage of venules recruiting only intraluminal (white circles), only perivascular (red circles), or both intraluminal and perivascular (gray circles) neutrophils. c Numbers of intraluminal and perivascular neutrophils per square millimeter of venular endothelium during the first 6 h of the infection. Data are shown as the mean ± SEM. b, c Quantifications were performed on n = 11 vessels, in total, pooled from N = 7 infected mice imaged independently. d 3D-rendering of intravital imaging showing neutrophil (Ly-6G, magenta) migration toward and engulfing an adherent bacterial aggregate within a venule 3 h post infection with iRFP-expressing Neisseria meningitidis (green). The human venule is shown in gray (UEA-1 lectin). Scale bar, 10 µm.

Fig 3: Recruitment of neutrophils to sites of infection.a Representative time sequence (maximum intensity z-projection) of vascular colonization by iRFP-expressing Neisseria meningitidis (green). The human and murine endothelia were distinguished using UEA-1 lectin (dashed lines) and mouse-specific anti-CD31 (red) staining, respectively. Time, hh:min. Pictures shown are representatives of N = 6 mice. Scale bar, 50 µm. b Representative images (maximum intensity z-projection) of non-infected (NI) and infected human graft tissue sections at indicated times post infection (p.i.) with GFP-expressing Neisseria meningitidis (green) and stained for the human endothelium (UEA-1 lectin, gray) and neutrophils (GR-1, magenta). Pictures shown are representative of N = 3 mice per condition. Scale bar, 10 µm. c Quantification of neutrophil numbers in human xenografts of non-infected (-, gray circles) and infected (+, purple circles) mice at the indicated times post infection (p.i.). Neutrophil numbers in contralateral mouse skin at 16 h post infection is shown on the right (white circles). Two-tailed Mann–Whitney test (3 h, 6 h, 24 h) and two-tailed Kruskal–Wallis test with Dunn’s correction (16 h). n = 7 (16 h p.i.), 8 (3 h, 16 h non-infected; 6 h p.i.), 9 (3 h p.i., 6 h non-infected), 10 (mouse skin, 16 h p.i.), 18 (24 h non-infected), and 21 (24 h p.i.) mice, pooled from N = 3 (3 h, 6 h, 16 h p.i.) and 5 (24 h p.i.) independent experiments. d Movies obtained from intravital imaging were used to quantify the numbers of neutrophils per square millimeter of endothelium during the first 6 h of the infection. Quantifications were performed on n = 56 vessels, pooled from N = 7 infected mice imaged independently. c, d Data are shown as the mean ± SEM. e Intravital imaging (maximum intensity z-projection) of neutrophil (LysMGFP/+, magenta) recruitment at 16 h post infection with iRFP-expressing Neisseria meningitidis (green) in grafted Rag2-/-?c-/-LysMGFP/+ mice. Dashed lines delineate the human endothelium, stained using the UEA-1 lectin. The left panel shows an important swarm of neutrophils around the infected vessel. Time, hh:min. Scale bars, 50 µm. The right panel shows the directed migration of neutrophils towards the infected vessel as revealed by cell tracking over 30 min. Pictures shown are representative of N = 3 mice. Scale bar, 50 µm.

Fig 4: Intrinsic IFNAR in Lysozyme-expressing myeloid cells is necessary for resistance to mousepox.(A) Concatenated flow cytometry plots showing the gating strategy for cDCs (CD19-TCRß-NK1.1-CD11chighSiglecH-), pDCs (CD19-TCRß-NK1.1-CD11c+SiglecH+), eosinophils (CD19-TCRß-NK1.1-SiglecH-F4-80+SSC-Ahigh), MO (CD19-TCRß-NK1.1-SiglecH-CD11b+Gr-1+Ly6Clow/high), and neutrophils (CD19-TCRß-NK1.1-SiglecH-CD11b+Gr-1high) and concatenated histograms showing IFNAR expression in indicated cell populations from spleens of naïve Ifnar1fl/fl and Ifnar1?Lyz2 mice. (B) IFNAR1 expression in Ly6Clow, Ly6Cint, and Ly6Chigh gated MO. Histogram legend as in (A). Representative data from one out of two independent experiments (N = 6 to 7 for each group). (C) Survival of the indicated mice infected s.c. with 3000 pfu of ECTV-GFP. Data pooled from two independent experiments (N = 10 for Ifnar1?Lyz2 mice and N = 18 for Ifnar1fl/fl mice, Log-rank Mantel-Cox compared to Ifnar1fl/fl mice). (D-E) ECTV titers in the dLN (D) and the spleen (E) were quantified by plaque assay at the indicated dpi. The dashed line indicates the detection limit. Data are represented as mean ± SEM from two or three pooled independent experiments (N = 5 to 7 for each Cre+ mice and N = 11 to 24 for Ifnar1fl/fl mice; ANOVA with Tukey correction compared to Ifnar1fl/fl mice). (F-G) ECTV titers in the liver and spleen were quantified by plaque assay at 5 (F) and 7 dpi (G). The dashed line indicates the detection limit. Data are represented as mean ± SEM from two pooled independent experiments (N = 8 to 9 for each Cre+ mice and N = 18 for Ifnar1fl/fl mice, ANOVA with Tukey correction compared to Ifnar1fl/fl mice). (H) Representative flow cytometry plots showing levels of CD44 and GzmB expression and levels of Kb-TSYKFESV-specific CD8 T cells in the spleens of infected mice. (I) Total cell numbers, and percentages and total numbers of CD44+ GzmB+ CD8 T cells (NK1.1- TCRß+CD8+) in spleens of the indicated mice at 7 dpi. (J) Numbers of CD44+ Kb-TSYKFESV-specific CD8 T cells in the spleens of the indicated mice at 7 dpi. Data are represented as mean ± SEM from two pooled independent experiments (N = 8 to 9 for each Cre+ mice and N = 18 for Ifnar1fl/fl mice, ANOVA with Tukey correction compared to Ifnar1fl/fl mice).

Fig 5: Effects of single administration of AAV donor in vitro and in vivo.a Pigo-KO Neuro2a cells were transfected with pAAVCas9 and pAAV donor (pAAV-mPigoHITIgRNA) or pAAV donor only and analyzed over time by FACS for measuring the restored population. % of CD24 positive cells were shown in the right graph. b Comparison of growth in HITI-treated (AAVCas9 and AAV donor) and AAV donor treated Pigob/- mice. Data are presented as mean values + SD. c Elevation of Gr-1, a GPI-AP, levels on blood granulocytes from Pigob/- mice at 4 months after HITI-treated or AAV donor treated Pigob/- mice. n number of animals, ns not significant. (*p < 0.05) (t-test, one-sided, P-value, KIKO:KIKO + HITI, 0.035; KIKO:KIKO + Pigo, 0.035) Data are presented as mean values + SEM. d Amelioration of hyperphosphatasia. (*p < 0.05; **p < 0.01) Data are presented as mean values + SEM. (t-test, two-sided, P-value, KIKO:KIKO + HITI, 5.5 × 10-3; KIKO:KIKO + Pigo, 0.029). e Improved performance in the hanging test with 5 mm (left) and 11 mm (right) meshes of AAV donor only treated Pigob/b mice in comparison with HITI-treated Pigob/b mice. Data are presented as mean values + SEM. (t-test, two-sided). f Severity scores of tremor appeared in AAV donor only treated Pigob/b mice and HITI treated Pigob/b mice at five months (t-test, one-sided, P-value, 0.034). Data are presented as mean values + SEM. Source data are provided as a Source Data file.