Journal: Communications Biology

Article Title: Monocytes undergo multi-step differentiation in mice during oral infection by Toxoplasma gondii

doi: 10.1038/s42003-019-0718-6

Figure Lengend Snippet: Wild type (WT)/ Stat1 −/− mixed BM chimeras were infected perorally with T. gondii cysts as depicted in Fig. . a Heatmap showing, among distal regulatory regions from clusters I and II (as defined in Fig. ), which regions were more or less accessible in CD45.1 WT and CD45.2 Stat1 −/− monocytes from the BM and the spleen of infected mice. Values are represented as log 2 fold-change obtained from the median of each single region. Bars indicate the number and the relative proportion of STAT1-dependent (in red) and -independent (in blue) regions among clusters I and II. b Representative ATAC-Seq tracks from the indicated population at the loci of Il1rn , Il27a , and Il10 . STAT1-dependent and independent peaks are highlighted in blue and green, respectively. The scale represents the fraction of reads in peaks (FRiP). c Bars indicating percentage of STAT1-dependent (in red) and -independent (in blue) regions, within clusters I and II enhancer regions overlapping with ChIP-Seq peaks of resting or LPS-stimulated BMDCs and resting and IFNγ-stimulated BMMs, for the indicated transcription factors (as shown in Fig. ). ATAC samples were generated from cells isolated from a pool of seven chimeric mice.

Article Snippet: Ly6C AL-21 BV421, CD11b M1/70 AF700, CD11c HL3 PE-Cy7, CD8α 53.6.7 PerCP, CD64 a and b alloantigens X54-5/7.1 AF647 or BV785, CD40 3/23 BV711, CD80 16-10A1 PE, CD86 GL1 APC, and CD45.1 A20 Pe-Cy7 were all purchased from BD Pharmigen as well.

Techniques: Infection, ChIP-sequencing, Generated, Isolation

Journal: bioRxiv

Article Title: CD150-dependent hematopoietic stem cells sensing of Brucella instructs myeloid commitment

doi: 10.1101/2021.03.31.437872

Figure Lengend Snippet: a-d) C57BL/6J wild-type ( wt ) and CD150 -/- mice were intraperitoneally inoculated with 1×10 6 B. abortus CFUs. Eight days later, FACS analyses were performed for BM cells. Frequency of (a) MPP2-3 (lin - ,Sca + ,cKit + CD48 +, CD135 - ) (from left to right: n=22; 19; 14; 6; 11; 9; 8), (b) MPP4 (lin - ,Sca + ,cKit + CD48 + CD135 + ) (from left to right: n=22; 21; 12; 4; 9; 9; 9), (c) GMP (lin - ,Sca - ,cKit + CD34 + CD16/32 + ) (from left to right: n=16; 18; 13; 4; 8; 9; 9), in Lin - BM cells is shown for (Mock, ○) or inoculated with 1×10 6 CFU of wild-type B. abortus (Ba WT, ■), B. abortus Δ omp25 (Ba Δ omp25 ) or B. abortus Δ omp25 complemented with p:Omp25 (Ba Δomp25c □) mutants (the latter only for wt mice). d) Eight days post-infection, myeloid cells (CD45 + , CD11b + ) to lymphoid cells (CD3e + CD19 + ) ratio in blood is shown for (Mock, ○, n=11) or inoculated with 1×10 6 CFU of wild-type B. abortus (Ba WT, ■, n=9), B. abortus Δ omp25 (Ba Δ omp25 ). e) Experimental scheme: HSC from wt CD45.1 and CD150 -/- CD45.1 mice were sorted and then incubated ex vivo with B. abortus WT and B. abortus Δ omp25 for 30 minutes. After 30 minutes, cells were washed and treated for 1 hour with gentamycin to kill extra-cellular bacteria. HSC were then transplanted into lethally irradiated wt CD45.2 recipients. FACS analyses of blood samples were performed at 4, 6 and 8 weeks post-transplantation. f) myeloid cells (CD45 + , CD11b + ) to lymphoid cells (CD3e + CD19 + ) ratio in CD45.1 + blood cells is shown for hematopoietic cells provided by CD45.1 + wt mice (upper panel) or CD150 -/- mice, (lower panel), non-infected (Mock, ○) or infected with 1×10 6 CFU of wild-type B. abortus (Ba WT, ■), B. abortus Δ omp25 (Ba Δ omp25 ) as described in e (from left to right, for WT n=12; 13; 10; 10; 9; 9; 12; 8; 8 and for CD150 -/- n= 9; 4; 14; 8; 11; 7). Data were obtained from distinct samples from 4 independent experiments (a-d) or from repetitive sampling from 2 (e-f) independent experiments. Mean ± SEM is represented by horizontal bar. Significant differences from mock are shown. *** P< 0.001, ** P< 0.01, * P< 0.05. Absence of P value or ns, non-significant. Since data did not follow normal distribution, P-Value were generated using Kruskal-Wallis followed by Dunn’s test.

Article Snippet: Blood cells were stained with anti-CD11b FITC (eBioescience, clone M1/70), anti-CD19-PE-Cy7 (BioLegend, clone 6D5), anti-CD45.2-PrcpCy5.5 (BD Bioscence, clone 104), anti-CD45.1-BV421 (BD Bioscience, clone A20), anti-CD3e-APC (BD Bioscience, clone 145-2C11) and anti-Ly6G-PE (BD Bioscience, clone 1A8).

Techniques: Infection, Incubation, Ex Vivo, Irradiation, Transplantation Assay, Sampling, Generated

Journal: bioRxiv

Article Title: CD150-dependent hematopoietic stem cells sensing of Brucella instructs myeloid commitment

doi: 10.1101/2021.03.31.437872

Figure Lengend Snippet: Complete FACS gating for blood lineage output from chimeric mice. First, cells are gated based on SSC/FSC and then single cells were selected. CD150 -/- and WT cells were separated by gating on respectively CD45.1 and CD45.2 positive cells. In each, lymphoid cells were isolated by gating on CD11b negative cells and then, B cells are CD19 positive cells and T cells are CD3e positive cells. In Cd11b positive cells, granulocytes and monocytes are distinguished by gating onto respectively LY6G positive and negative cells.

Article Snippet: Blood cells were stained with anti-CD11b FITC (eBioescience, clone M1/70), anti-CD19-PE-Cy7 (BioLegend, clone 6D5), anti-CD45.2-PrcpCy5.5 (BD Bioscence, clone 104), anti-CD45.1-BV421 (BD Bioscience, clone A20), anti-CD3e-APC (BD Bioscience, clone 145-2C11) and anti-Ly6G-PE (BD Bioscience, clone 1A8).

Techniques: Isolation

Journal: bioRxiv

Article Title: CD150-dependent hematopoietic stem cells sensing of Brucella instructs myeloid commitment

doi: 10.1101/2021.03.31.437872

Figure Lengend Snippet: a) Experimental scheme: BM cells from CD150 -/- CD45.1 mice and wt CD45.2 mice were isolated from tibia and femur of mice and transplanted into lethally irradiated recipient mice. Twelve weeks after transplantation mice were intraperitoneally injected with PBSx1 (Mock, ○) or inoculated with 1×10 6 CFU of wild-type B. abortus (Ba WT), B. abortus Δ omp25 (Ba Δ omp25 ). Blood and BM were analysed 8 days after. b) Myeloid (CD45 + ,CD11b + cells) to lymphoid (CD3e + and CD19 + cells) in the blood (from left to right, n=8; 8; 8; 8; 7; 7); frequency of c) GMP (from left to right, n=6; 6; 7; 7; 9; 9), d) MPP2-3 (from left to right, n=6; 6; 6; 6; 9; 9) and e) MPP4 (from left to right, n=6; 6; 8; 8; 9; 9) in BM Lin-cells of wt (circle) and CD150 -/- (square) compartment is shown for chimeric mice intraperitoneal injected with PBS (Mock, non-filled symbols) or inoculated with 1×10 6 CFU of wild-type B. abortus (symbol filled in black) B. abortus Δ omp25 (symbol filled in grey) as described in a. e-f) CFU count per gram of organ at Day 30 post infection of WT and CD150 -/- mice for (e) BM (from left to right n=18; 9; 8; 12; 6; 6) and (f) spleen (from left to right n= 14; 18; 7; 7; 8; 6) infected with B. abortus WT (Ba WT ■), B. abortus Δomp25 (Ba Δomp25 ) or B. abortus Δomp25 complemented with p:Omp25 (Ba Δomp25c □). Mean ± SEM is represented by horizontal bar. Significant differences from mock are shown. *** P< 0.001, ** P< 0.01, * P< 0.05. Absence of P value or ns, non-significant. Since data did not follow normal distribution, P-Value were generated using Kruskal-Wallis followed by Dunn’s test.

Article Snippet: Blood cells were stained with anti-CD11b FITC (eBioescience, clone M1/70), anti-CD19-PE-Cy7 (BioLegend, clone 6D5), anti-CD45.2-PrcpCy5.5 (BD Bioscence, clone 104), anti-CD45.1-BV421 (BD Bioscience, clone A20), anti-CD3e-APC (BD Bioscience, clone 145-2C11) and anti-Ly6G-PE (BD Bioscience, clone 1A8).

Techniques: Isolation, Irradiation, Transplantation Assay, Injection, Infection, Generated

Journal: bioRxiv

Article Title: CD150-dependent hematopoietic stem cells sensing of Brucella instructs myeloid commitment

doi: 10.1101/2021.03.31.437872

Figure Lengend Snippet: a-b) wt CD45.1 and CD150 -/- CD45.1 mice were intraperitoneally inoculated with 1×10 6 CFU of B. abortus . BM cells were isolated from femur and tibia of the infected mice. a) Frequency of MEP (lin - ,Sca - ,cKit + CD34 -, CD16/32 - ), in Lin - BM cells is shown for (Mock, ○) or inoculated with 1×10 6 CFU of wild-type B. abortus (Ba WT, ■), B. abortus Δ omp25 (Ba Δ omp25 ) or B. abortus Δ omp25 complemented with p:Omp25 (Ba Δomp25c □) mutants (the latter only for wt mice) (from left to right: n=16; 16; 8; 8; 8; 7; 9). b) At eight days post-infection, the percentage of haematocrit measured in blood is shown for (Mock, ○) or inoculated with 1×10 6 CFU of wild-type B. abortus (Ba WT, ■), B. abortus Δ omp25 (Ba Δ omp25 ) (from left to right, n= 7; 7; 6; 5; 8; 8). c) Percentage of brucellosis patients that present anemia before antibiotic treatment. Men upper panel and women lower panel. Anemia was characterized by a decrease of hematocrit, hemoglobin and erythrocytes (hematocrit <40% for men and <35% for women; hemoglobin <14g/dL for men and <12 g/dL for women, erythrocyte count <4 million for men and <3.8 million/mm3 for women). Data were obtained from distinct samples from 3 independent experiments (a-b), each with at least n=4 animals per condition, are shown and mean ± SEM is represented by horizontal bar. Significant differences from mock are shown. *** P< 0.001, ** P< 0.01, * P< 0.05. Absence of P value or ns, non-significant. Since data did not follow normal distribution, P-Value were generated using Kruskal-Wallis followed by Dunn’s test.

Article Snippet: Blood cells were stained with anti-CD11b FITC (eBioescience, clone M1/70), anti-CD19-PE-Cy7 (BioLegend, clone 6D5), anti-CD45.2-PrcpCy5.5 (BD Bioscence, clone 104), anti-CD45.1-BV421 (BD Bioscience, clone A20), anti-CD3e-APC (BD Bioscience, clone 145-2C11) and anti-Ly6G-PE (BD Bioscience, clone 1A8).

Techniques: Isolation, Infection, Generated