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Interleukin-3 amplifies acute inflammation and is a potential therapeutic target in sepsis

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Science  13 Mar 2015:
Vol. 347, Issue 6227, pp. 1260-1265
DOI: 10.1126/science.aaa4268
  • Fig. 1 IL-3 is detrimental in experimental sepsis.

    Comparison of Il3–/– and Balb/c (WT) mice during experimental sepsis using the CLP model. (A) Kaplan-Meier survival curve in mice not receiving antibiotics (n = 10 mice per group) and in mice receiving antibiotics (Imipenem) (n = 12 or 13 per group). d, days. (B) Clinical score and body temperature (n = 6 to 10 per group). h, hours. (C) Blood pressure. The blood pressure in WT mice was below the detection limit (n = 6 to 10 per group). (D) Bacterial titer of peritoneal cavity and blood (n = 3 to 10 per group). (E) Enumeration of neutrophils and Ly-6Chigh monocytes in 1 ml of blood at 0, 6, 12, and 24 hours after CLP (n = 3 to 12 per group). (F) Levels of IL-1β, IL-6, and TNF-α in serum 1 day after CLP (n = 8 or 9 per group). (G and H) Immunohistochemical staining and flow cytometric enumeration of monocytes (CD115) and neutrophils (Ly-6G) in entire lung (G) and liver (H) tissue 1 day after CLP (n = 6; *P < 0.05, **P < 0.01,***P < 0.001). Error bars indicate means ± SEM. Significance was assessed by log rank test (A) or Mann-Whitney test [(B) to (H)]. Data are the result of N ≥ 2 independent experiments and are grouped.

  • Fig. 2 IL-3 induces emergency hematopoiesis and potentiates the cytokine storm in sepsis.

    (A) Surface expression of IL-3Rα (CD123) on HSPCs, MEPs, CMPs, GMPs, and MDPs in bone marrow of WT and Il3–/– mice (n = 3 per group). A representative plot of n = 3 is shown. (B) Enumeration of HSPCs, CMPs/MEPs, and GMPs/MDPs in bone marrow in a steady state and 1 day after CLP in WT and Il3–/– mice (n = 3 per group). (C) Analysis and enumeration of GFP+ cells retrieved from the bone marrow 1 day after CLP from WT and Il3–/– mice that received 2 × 105 GFP+ GMP intravenously before CLP (n = 3 per group). (D) Bone marrow cells were sorted for Lin cells (i.e., enriched in HSPCs). Shown are representative CD11b versus CD115 flow cytometry plots showing cell phenotype just before placement into culture and 4 days after in vitro culture in the indicated conditions. The numbers inside the plots denote cells plated and retrieved (n = 4 per group). (E) Supernatant levels of IL-1β, IL-6, and TNF-α in the four post-culture groups shown in (D). Values are the result of technical triplicates from n = 2 experiments. (F) Enumeration of indicated cell types in (i) WT mice at a steady state; (ii) WT mice receiving rIL-3 alone; (iii) WT mice subjected to CLP; (iv) WT mice subjected to CLP and receiving antibody to CD123; (v) Il3–/– mice subjected to CLP; and (vi) Il3–/– mice subjected to CLP and receiving rIL-3 (n = 4 to 10). (G) Serum levels of IL-1β, IL-6, and TNF-α in the six groups shown in (F) (n = 4 to 10). (H) Kaplan-Meier survival curves showing the four WT mouse groups (n = 6 to 17 per group). (I) Kaplan-Meier survival curves showing the two Il3–/– mouse groups (n = 10 per group) (*P < 0.05, **P < 0.01, ***P < 0.001). Error bars indicate means ± SEM. Significance was assessed by Mann-Whitney test [(B), (C), (F), and (G)]; one-way analysis of variance (ANOVA) with Tukey’s multiple comparison test (E); Kruskal-Wallis test with Dunn’s multiple comparison test [(F) and (G)]; and log rank [(H) and (I)]. Data are the result of N = 2 independent experiments acquired in triplicates (in vitro) and n ≥ 2 independent experiments (in vivo), and are grouped.

  • Fig. 3 IRA B cells are major sources of IL-3 in sepsis.

    (A) Il3 mRNA expression in the indicated organs during a steady state and 3, 6, 12, and 24 hours after CLP (n = 6 to 8). (B) Identification of IL-3–producing cells in the spleen 4 days after CLP. (C) Enumeration of IL-3–producing B cells in spleen and thymus in a steady state and 1 and 4 days after CLP (n = 5). (D) Western blot showing IL-3 expression by B cells and non-B cells sorted from the spleen and thymus 1 day after CLP. (E) IL-3 serum levels in a steady state and 1 day after CLP with and without splenectomy (SPx) (n = 3 to 6). (F) Flow cytometric plots show the phenotype of IL-3+ and IL-3 cells retrieved from the spleen after CLP. A representative plot of n = 5 is shown. (G) Immunofluorescence microscopy of spleen tissue in the steady state and 1 day after CLP. (H) Co-staining of representative IL-3+ cells with IgM. (I) Adoptive transfer of 1.5 × 106 peritoneal B1 B cells from GFP+ mice into WT mice subjected to CLP at the time of cell transfer. Representative plots from flow cytometric analysis of n = 3 mice are shown. (J) Adoptive transfer of 3 × 106 peritoneal B1 B cells from WT or Il3–/– mice to the peritoneum of Il3–/– recipients subjected to CLP. Data show the clinical score, number of Ly-6Chigh monocytes, neutrophils, and serum cytokines 1 day after CLP (n = 5). (*P < 0.05, **P < 0.01). Error bars indicate means ± SEM. Significance was assessed by a Kruskal-Wallis test with Dunn’s multiple comparison test (E) and a Mann-Whitney test (J).

  • Fig. 4 IL-3 is an independent early predictor for outcome in human sepsis.

    (A) Total leukocyte number in nonseptic people and in septic patients at the time of sepsis onset (0) and 1, 4, 7, 14, and 28 days later. (B) Plasma levels of IL-1β, IL-6, and TNF-α in nonseptic people (n = 18) and in septic patients at the time of sepsis onset (n = 37) and 1 day later (n = 17). (C) IL-3 plasma levels in healthy people and in patients at sepsis onset and 1 day later. (D) Correlation of IL-3 plasma levels with total blood monocytes and with CD14+ and CD16+ blood monocytes in septic patients with measurable IL-3 plasma levels. (E) Kaplan-Meier analysis showing the survival of patients in the RAMMSES and SEPIL-3 studies with IL-3 at >89.4 pg/ml (top quintile, measured within 1 day after sepsis onset) versus the survival of patients with IL-3 ≤ 89.4 pg/ml. (F) Representative flow cytometry plot of n = 2 patients showing the identity of IL-3–producing human splenocytes. (G) Immunofluorescence of human spleen showing IL-3–producing B cells in high magnification (×60). A representative immunofluorescence section of n = 6 spleens is shown (*P < 0.05, ****P < 0.0001). Error bars indicate means ± SEM. Significance was assessed by one-way ANOVA with Tukey’s multiple comparison test [(A) and (C)], a Pearson correlation test (D), and log rank (E). Data in (A) to (D) are from the SEPIL-3 cohort; data in (E) are pooled from the RAMMSES and SEPIL-3 cohorts.

Supplementary Materials

  • Interleukin-3 amplifies acute inflammation and is a potential therapeutic target in sepsis

    Georg F. Weber, Benjamin G. Chousterman, Shun He, Ashley M. Fenn, Manfred Nairz, Atsushi Anzai, Thorsten Brenner, Florian Uhle, Yoshiko Iwamoto, Clinton S. Robbins, Lorette Noiret, Sarah L. Maier, Tina Zönnchen, Nuh N. Rahbari, Sebastian Schölch, Anne Klotzsche-von Ameln, Triantafyllos Chavakis, Jürgen Weitz, Stefan Hofer, Markus A. Weigand, Matthias Nahrendorf, Ralph Weissleder, Filip K. Swirski

    Materials/Methods, Supplementary Text, Tables, Figures, and/or References

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    • Materials and Methods
    • Figs. S1 to S15
    • Tables S1 to S6
    • References (34–36)

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