Fig 2: Flow cytometry analysis of IgA and IgM positive cells in different colon immune populations of WT and Sphk2-/- mice after stress exposure. Representative dot plots showing the gating strategy used to distinguish IgA-IgM-, IgA+IgM-, IgA-IgM+, and IgA+IgM+ in each cell population. Percentage of IgA+CD45+ (a), IgA+ B cells (b), IgA+ plasmablasts (c), IgA+ plasma cells (d), IgM+CD45+ (e), IgM+ B cells (f), IgM+ plasmablasts (g), IgM+ plasma cells (h) were plotted. Data are means ± SEM of 5–8 mice per group. Two-way ANOVA considering stress (S) and Sphk2-/- as independent variables followed by Tukey's post hoc; *S p < 0.05, **S p < 0.01, ***S p < 0.001; @ (interaction) p < 0.05; +p < 0.05, ++p < 0.01 vs Control WT; &p < 0.05, &&&p < 0.001 vs Control Sphk2-/-. Sphk2: Sphingosine kinase 2; WT: wild-type; Sphk2-/-: Sphk2 knockout; Ig: immunoglobulin.

Fig 3: Generating immuno-CRISPR (iCR) mice and evaluating the CRISPR-mediated modifications by sequencing. (A) Model describing the experimental setup where CD45.1 + Lin- BM cells were modified by CRISPR targeting Zap70 and grafted into irradiated CD45.2 + recipients. (B) Flow cytometry analysis of cells in the blood of Zap70 iCR mice and WT control mice eight weeks post transplantation. Cells gated on viable, CD45.1+, single lymphocytes. (C) Quantification of B and T cells in the blood of WT and Zap70 iCR mice in (B). (D) Analysis of the level of mutations in the sgRNA targeted Zap70 region in the BM cells used for transplantation, total cells from the blood, as well as in B and T cells sorted from the spleen of Zap70 iCR mice and WT control mice 8 weeks after engraftment. (E) Model describing the experimental setup where a secondary transplantation was used to amplify the population of successfully modified mice. (F) Analysis of the level of mutations in the sgRNA targeted GFP region in blood cells of the GFP iCR mice four weeks after transplantation, in an experiment with low efficiency. One mouse showed good knockout efficiency (labeled in orange) and was used as BM donor for secondary transplantation. (G) Kinetics of the level of mutations of GFP in the secondary iCR mice. (H) As examples, representative flow cytometry plots and (I) GFP + cell population percentage of alveolar macrophages, B1 cells, neutrophils and germinal center B cells from secondary iCR mice in G. Data shown as mean and individual data points (C-D, n = 3; F, n = 4; G-I, n = 3–6). n.s. = non-significant, * = p < 0.05, ** = p < 0.01 *** = p < 0.001 by unpaired T-test (C), and one-way ANOVA and Tukey's post-test (D, G, I). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig 4: Reduced inflammatory cells in ischemic brain tissue in mice harboring female microbiota. A, B Gating strategies for monocytes and macrophages derived from microglia (CD11b + CD45inter) and macrophages derived from monocytes (CD11b + CD45high). C The infiltration of microglia and macrophages. A, D, E Gating strategies for T lymphocytes (CD3+) and B lymphocytes (B220+). F, G The infiltration of T and B lymphocytes in the infarcted hemisphere. Mean ± SD. n = 8–12/group. #p: MCAO vs. sham. #p < 0.05. ##p < 0.01. ###p < 0.001. *p: comparison between recipient mice receiving fecal microbiota from different sexes. *p < 0.05, **p < 0.01, ***p < 0.001

Fig 5: Flow cytometry analysis of B cell differentiation in the colon of WT and Sphk2-/- mice after sub-chronic stress exposure. Representative dot plots showing the gating strategy used to distinguish lymphoid lineage cells (CD45+), B cells (CD45+B220+CD138-/low), plasmablasts (CD45+B220-CD138-/low), and plasma cells (CD45+B220-CD138high). Considering the total number of CD45+ cells as 100%, percentage of B cells (a), plasmablasts (b), and plasma cells (c) were plotted. Data are means ± SEM of 5–8 mice per group. Two-way ANOVA considering stress (S) and Sphk2-/- as independent variables followed by Tukey's post hoc; *S p < 0.05, **S p < 0.01. Sphk2: Sphingosine kinase 2; WT: wild-type; Sphk2-/-: Sphk2 knockout.