Research ArticleHEALTH AND MEDICINE

Treatment of severe sepsis with nanoparticulate cell-free DNA scavengers

See allHide authors and affiliations

Science Advances  29 May 2020:
Vol. 6, no. 22, eaay7148
DOI: 10.1126/sciadv.aay7148
  • Fig. 1 PAMAM-G3 reduces the cfDNA-driven proinflammatory response in vitro and in CLP-induced severe sepsis.

    (A) Serum levels of cfDNA in healthy volunteers (n = 16) and patients with sepsis (n = 15). Data are expressed as the means ± SEM, and differences were assessed with Student’s t test. (B) Activation of HEK-TLR9 reporter cells by either healthy human serum or sepsis patient serum in the absence or presence of PAMAM-G3 (10 μg/ml) for 24 hours. The corresponding embryonic alkaline phosphatase (SEAP) activity in supernatants from each group was determined with a QUANTI-Blue assay with optical density at 620 nm (OD620). (C) RAW 264.7 macrophages were stimulated with sepsis patient serum in the absence or presence of PAMAM-G3 (10 μg/ml) for 24 hours. Supernatants were assayed for TNF-α via enzyme-linked immunosorbent assay (ELISA). In (B) and (C), differences were assessed via one-way analysis of variance (ANOVA) with Tukey’s multiple comparison tests (***P < 0.001, compared with healthy serum; ###P < 0.05, compared with sepsis serum). The data are expressed as the means ± SEM. (D) The indicated BALB/c mice were subjected to CLP of different grades. Survival was monitored for 144 hours (n = 10 mice per group; *P < 0.05 and ***P < 0.001, Kaplan-Meier survival analysis). (E) High-grade CLP was performed on BALB/c mice, followed by intraperitoneal injection of PAMAM-G3 or Xuebijing (XBJ) (20 mg/kg) 12 hours before and 1 and 12 hours after surgery. Survival was monitored for 144 hours (n = 10 mice per group; *P < 0.05 and ***P < 0.001, Kaplan-Meier survival analysis). (F) Mice were monitored for 144 hours after CLP for clinical scoring. The clinical scoring of sepsis was defined according to a range from 0 (no symptoms) to 5 (loss of self-righting reflex). The data are expressed as the means ± SEM. (G to I) High-grade CLP was performed on BALB/c mice, followed by treatment as described in (E). The levels of the proinflammatory cytokines (G) TNF-α, (H) interleukin-6 (IL-6), and (I) monocyte chemoattractant protein-1 (MCP-1) were measured in the blood 24 hours after CLP. Differences were assessed via one-way ANOVA with Tukey’s multiple comparison tests (n = 6 to 8 mice per group; *P < 0.05, **P < 0.01, and ***P < 0.001). The data are expressed as the means ± SEM.

  • Fig. 2 PAMAM-G3 reverses M1 polarization of peritoneal macrophages through the TLR9-MyD88–NF-κB signaling pathway during severe sepsis.

    High-grade CLP was performed on BALB/c mice, followed by intraperitoneal injection of PAMAM-G3 or XBJ (20 mg/kg) 12 hours before and 1 and 12 hours after surgery. (A) Serum and (B) peritoneal cfDNA levels were analyzed after 24 hours after CLP. (C) The number of TLR9+ cells and (D) the percentage of M1-polarized macrophages (CD11c+F4/80+) were assessed in PLF by flow cytometry 8 hours after CLP. Differences were assessed via one-way ANOVA with Tukey’s multiple comparison tests (n = 6 to 8 mice per group; *P < 0.05, **P < 0.01, and ***P < 0.001). The data are expressed as the means ± SEM. (E to J) Peritoneal macrophages were collected 8 hours after CLP, and mRNA was extracted, converted to complementary DNA, and analyzed via real-time polymerase chain reaction (PCR) for (E) TNF-α, (F) iNOS, and (G) Arg-1 gene expression. The data are expressed as fold change relative to the saline-treated normal group and normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene expression. In parallel, macrophages were lysed in radioimmunoprecipitation assay (RIPA) buffer before analysis of (H) TLR9, (I) MyD88, and (J) p-p65 protein expression via Western blotting. The data are expressed as fold change relative to the control group and normalized to GAPDH or p65 protein expression. Differences were assessed via one-way ANOVA with Tukey’s multiple comparison tests (n = 5 mice per group; *P < 0.05, **P < 0.01, and ***P < 0.001). The data are expressed as the means ± SEM (n = 3 independent experiments in triplicate).

  • Fig. 3 PEI-functionalized biodegradable MSNs block the proinflammatory response in vitro.

    (A) The mechanism by which NABNs scavenge cfDNA to inhibit the proinflammatory response and treat severe sepsis. NABNs with a cationic corona are formed by conjugating PEI of different molecular weights onto the surface of biodegradable MSNs. After intraperitoneal injection into the CLP-induced severe sepsis models, NABNs exhibited favorable accumulation and retention in the inflamed cecum, protecting mice against death by attenuating proinflammatory effects and multiple organ injury through scavenging of cfDNA. (B to D) Transmission electron microscopy images of MSNs after incubation in buffer containing 5 × 10−3 M GSH for (B) 0 days, (C) 1 day, and (D) 3 days. Scale bar, 10 nm (B, inset). (E) ct-DNA binding efficiency of NABNs or NABPs with different mass ratios at 37°C. The data are expressed as the means ± SEM (n = 3 independent experiments in triplicate). (F) Activation of HEK-TLR9 reporter cells by either healthy human sera or sepsis patient sera in the absence or presence of NABNs or NABPs (10 μg/ml) for 24 hours. The corresponding SEAP activity in supernatants from each group was determined with a QUANTI-Blue assay at OD620. (G) RAW 264.7 macrophages were stimulated with sepsis patient sera in the absence or presence of NABNs or NABPs (10 μg/ml) for 24 hours. Supernatants were assayed for TNF-α via ELISA. In (F) and (G), differences were assessed via one-way ANOVA with Tukey’s multiple comparison tests (*P < 0.05, compared with healthy serum; #P < 0.05, compared with sepsis serum; ^P < 0.05, compared with sepsis serum + MSN-PEI 25K; &P < 0.05, compared with sepsis serum + PEI 25K). The data are expressed as the means ± SEM (n = 3 independent experiments in triplicate).

  • Fig. 4 MSN-PEI 25K protects mice against CLP-induced severe sepsis.

    High-grade CLP was performed on BALB/c mice, followed by intraperitoneal injection of MSN-PEI 25K, MSN-PEI 800, PEI 25K, PEI 800, or MSN-NH2 (20 mg/kg) 12 hours before and 1 and 12 hours after surgery. (A) Mice were monitored for 144 hours after CLP for clinical scoring. The data are expressed as the means ± SEM. (B) Survival was monitored for 144 hours (n = 10 mice per group; **P < 0.01, ***P < 0.001, Kaplan-Meier survival analysis). (C) Serum and (D) peritoneal cfDNA levels were measured at 3, 6, 12, 18, and 24 hours after CLP. (E) The number of TLR9+ cells was assessed in PLF by flow cytometry 8 hours after CLP. (F to H) 24 hours after CLP, levels of the proinflammatory cytokines (F) TNF-α, (G) IL-6, and (H) MCP-1 in serum were measured. (E to H) Differences were assessed via one-way ANOVA with Tukey’s multiple comparison tests (n = 6 to 8 mice per group; *P < 0.05, **P < 0.01, and ***P < 0.001). The data are expressed as the means ± SEM.

  • Fig. 5 MSN-PEI 25K attenuates multiple organ injury in CLP-induced severe sepsis.

    High-grade CLP was performed on BALB/c mice, followed by treatment as described in Fig. 4. (A) Twenty-four hours after CLP, the lung, kidney, heart, liver, and spleen tissues were collected, stained with H&E, and analyzed. Scale bars, 20 μm. Multiple organ injury representations, including interalveolar septum thickened in the lung, tubular epithelial cell swelling in the kidney, inflammatory cell infiltration in the liver and heart, and nuclear debris from dying cells in the spleen, are marked with arrows. The corresponding (B) liver and (E) kidney injury scores were determined and calculated according to the established criteria. In parallel, the blood serum biochemistry parameters (C) ALT, (D) AST, (F) BUN, and (G) CRE were measured. Differences were assessed via one-way ANOVA with Tukey’s multiple comparison tests (n = 6 mice per group; *P < 0.05, **P < 0.01, and ***P < 0.001). The data are expressed as the means ± SEM.

  • Fig. 6 MSN-PEI 25K shows inflammatory-specific accumulation and retention with negligible toxicity in vivo.

    (A) Merged images show intracellular localization of cationic materials and CpG in RAW 264.7 cells after 6 hours of incubation. Scale bars, 5 μm. The large white spots in NABNs-treated cells are marked with an arrow. (B to D) Ex vivo NIRF images of cecum from (B) sham and (C) CLP mice 2, 12, and 24 hours after intraperitoneal injection of Cy7-labeled NABNs or NABPs (20 mg/kg). (D) Semiquantitative analysis of ex vivo fluorescence images of the cecum in (B) and (C). Differences were assessed via one-way ANOVA with Tukey’s multiple comparison tests (n = 3 mice per group; **P < 0.01 and ***P < 0.001). The data are expressed as the means ± SEM. ROI, region of interest. (E) Quantification analysis of Si content in the lung, kidney, heart, liver, spleen, and cecum of sham or CLP mice via inductively coupled plasma optical emission spectrometry (ICP-OES) after intraperitoneal injection of MSN-PEI 25K, MSN-PEI 800, or MSN-NH2 (20 mg/kg) 12 hours before surgery and 1 and 12 hours after surgery. The data are expressed as the means ± SEM (n = 5 mice per group). % ID/g, percentage of the injected dose per gram of tissue. (F to H) Normal mice were intraperitoneally injected three times with MSN-PEI 25K, MSN-PEI 800, PEI 25K, PEI 800, or MSN-NH2 (20 mg/kg) at 0, 13, and 24 hours. (F) cfDNA and (G) TNF-α levels and (H) the white blood cell (WBC) count were measured in the blood 24 hours after the last administration. Differences were assessed via one-way ANOVA with Tukey’s multiple comparison tests (n = 5 to 6 mice per group; *P < 0.05,**P < 0.01, and ***P < 0.001). The data are expressed as the means ± SEM.

Supplementary Materials

  • Supplementary Materials

    Treatment of severe sepsis with nanoparticulate cell-free DNA scavengers

    Jianati Dawulieti, Madi Sun, Yawei Zhao, Dan Shao, Huize Yan, Yeh-Hsing Lao, Hanze Hu, Lianzhi Cui, Xiaoyan Lv, Feng Liu, Chun-Wei Chi, Yue Zhang, Mingqiang Li, Ming Zhang, Huayu Tian, Xuesi Chen, Kam W. Leong, Li Chen

    Download Supplement

    This PDF file includes:

    • Supplementary Materials and Methods
    • Figs. S1 to S20
    • Tables S1 and S2
    • References

    Files in this Data Supplement:

Stay Connected to Science Advances

Navigate This Article