Journal: European Journal of Immunology

Article Title: Depletion of Neonatal Neutrophilic Cells Worsens the Outcome of E. coli Sepsis in Newborn Mice

doi: 10.1002/eji.70171

Figure Lengend Snippet: Sepsis mortality after depletion of Ly6G‐positive NNC. (A) Schematic representation of the experimental procedure. (B–D) Newborn mice received either an intraperitoneal injection of an anti‐Ly6G antibody to deplete NNC or an isotype control antibody on the first postnatal day (P1). At P2 or P8 spleen cells were isolated and analyzed by flow cytometry. (B) Representative density plots showing the population of CD11b + /Ly6G + (upper right quadrant) NNC in newborn mice at P2 after administering either isotype control (left side, Isotype) or the depleting antibody (right side, anti Ly6G). Cells were pre‐gated for living CD45 + cells. (C, D) Bar graphs showing percentages of total NNC from CD45 + /CD11b + myeloid cells in spleens of newborn mice at P2 (C; 1 day after depletion; isotype: n = 7, anti Ly6G: n = 5) and P8 (D; 7 days after depletion; isotype: n = 5, anti Ly6G: n = 5). Bars show mean and standard deviation, ** p < 0.01, Mann–Whitney test. (E, F) Newborn mice received either an intraperitoneal injection of an anti‐Ly6G antibody to deplete NNC or an isotype control antibody at the first day of life (P1). At P2, mice were injected subcutaneously with either 10,000 CFU or 30,000 CFU E. coli . The pups were sacrificed when reaching the criteria for critical illness, or 48 h after sepsis induction. Probability of survival after subcutaneous injection of 10,000 CFU E. coli (E; anti Ly6G n = 8, isotype n = 8; * p < 0.05, log‐rank [Mantel–Cox] test) or 30,000 CFU E. coli (F; anti Ly6G n = 8, isotype n = 9; p:0,186: not significant, log‐rank [Mantel–Cox] test) in mice after NNC‐depletion (anti Ly6G) or injection of an isotype control (isotype). Each n is representing a single mouse. Mice for the experiments came from three litters (B–F).

Article Snippet: This was followed by a second isolation step using Ly6G biotin‐antibody and anti‐biotin microbeads (modified protocol of MDSC Isolation Kit mouse, Miltenyi Biotec, Bergisch‐Gladbach, Germany).

Techniques: Injection, Control, Isolation, Flow Cytometry, Standard Deviation, MANN-WHITNEY

Journal: European Journal of Immunology

Article Title: Depletion of Neonatal Neutrophilic Cells Worsens the Outcome of E. coli Sepsis in Newborn Mice

doi: 10.1002/eji.70171

Figure Lengend Snippet: Bacterial load and serum concentrations of IL‐6 and MCP‐1 after depletion of Ly6G‐positive NNC. Newborn mice received either an intraperitoneal injection of an anti‐Ly6G antibody (anti Ly6G) to deplete NNC or an isotype control antibody (Iso) at the first day of life (P1). At P2, mice were injected subcutaneously with either 10,000 CFU or 30,000 CFU E. coli . Injection of PBS served as negative control. The pups were sacrificed when reaching the criteria for critical illness or 48 h after sepsis induction. Spleens, lungs, livers, blood, and peritoneal lavage were collected. Organs were homogenized and samples were diluted with PBS. Suspensions were incubated for 24 h on Columbia agar plates with 5% sheep blood and colony forming units (CFU) were counted to calculate CFU per gram (g) organ weight or per milliliter (mL) fluid (blood or peritoneal lavage). Scatter diagrams with bars showing CFUs per gram organ weight (A–C) or per ml (D, E) in spleens (A), livers (B), lungs (C), blood (D), and peritoneal fluid (E) of control mice (Iso, white bars) or NNC‐depleted mice (anti‐Ly6G, striped bars) without sepsis induction (control, left side) or with sepsis induction with 10,000 CFU (middle) or 30,000 CFU (right side). Bars show mean and standard deviation. n = 5–9, * p < 0.05, ns: not significant, Mann–Whitney test. (F, G) Newborn mice received either an intraperitoneal injection of an anti‐Ly6G antibody (anti Ly6G, checked bar) to deplete NNC or an isotype control (Iso, plain bar) on the first day of life (P1). On P2, newborn mice from a litter with a size of 5–6 pups were injected subcutaneously with either 10,000 CFU (middle, n = 5–8) or 30,000 CFU (right side, n = 5–8) or with PBS as a negative control (left side, n = 6). After termination of the experiment or 48 h after beginning of the experiment, if the pups survived IL‐6 (F) and MCP‐1 concentration in serum samples was measured using enzyme‐linked immunosorbent assay (ELISA) IL‐6 (F) and MCP‐1 (G) concentration in serum samples was measured. The IL‐6 and MCP‐1 levels were normalized to the protein concentrations of each sample. Bars show mean and standard deviation. n = 3–8, * p < 0.05, ** p < 0.01; unpaired t test. Each dot (= n ) is representing a single mouse. Mice for the experiments came from three litters (A–G).We further investigated systemic inflammation during neonatal sepsis with and without depletion of Ly6G‐expressing NNC by measuring the levels of the proinflammatory cytokines IL‐6 and MCP‐1 in serum of the animals. Both, IL‐6 and MCP‐1 levels were significantly increased after Ly6G‐depletion both after sepsis induction with 10,000 (IL‐6: 187 ng/g vs. 443 ng/g; n = 7, 8; p < 0.05; MCP‐1: 3231 ng/g vs. 5737 ng/g; n = 3; ** p < 0.01) or 30.000 CFU E. coli (IL‐6: 422 ng/g vs. 552 ng/g, n = 3, p < 0.05; MCP‐1: 3808 g/g vs. 6470 g/g, n = 3; ** p < 0.01) (Figure ).

Article Snippet: This was followed by a second isolation step using Ly6G biotin‐antibody and anti‐biotin microbeads (modified protocol of MDSC Isolation Kit mouse, Miltenyi Biotec, Bergisch‐Gladbach, Germany).

Techniques: Injection, Control, Negative Control, Incubation, Standard Deviation, MANN-WHITNEY, Concentration Assay, Enzyme-linked Immunosorbent Assay, Expressing

Journal: European Journal of Immunology

Article Title: Depletion of Neonatal Neutrophilic Cells Worsens the Outcome of E. coli Sepsis in Newborn Mice

doi: 10.1002/eji.70171

Figure Lengend Snippet: Immune cell populations in spleens after depletion of Ly6G‐positive NNC. Newborn mice received either an intraperitoneal injection of an anti‐Ly6G antibody (anti Ly6G) to deplete NNC or an isotype control antibody (Isotype) at (P1). At P2 mice were injected subcutaneously with either 10,000 CFU or 30,000 CFU E. coli . Injection of PBS served as negative control. The pups were sacrificed when reaching the criteria for critical illness or 48 h after sepsis induction. Spleens were collected and homogenized to obtain single cell suspensions. Immune cell subsets were analyzed by flow cytometry. Scatter diagrams with bars showing percentages of macrophages (A), dendritic cells (B), monocytes (C), B cells (D), T cells (E), and NK cells (F) and activated T cells of leukocytes of control mice (isotype, white bars) or NNC‐depleted mice (anti‐Ly6G, striped bars) without sepsis induction (control) or with sepsis induction with 10,000 CFU or 30,000 CFU. n = 6–7, unpaired t test. Each dot (= n ) is representing a single mouse. Mice for the experiments came from three litters.

Article Snippet: This was followed by a second isolation step using Ly6G biotin‐antibody and anti‐biotin microbeads (modified protocol of MDSC Isolation Kit mouse, Miltenyi Biotec, Bergisch‐Gladbach, Germany).

Techniques: Injection, Control, Negative Control, Single Cell, Flow Cytometry