In Situ Neutralization and Detoxification of LPS to Attenuate Hyperinflammation

Abstract Hyperinflammation elicited by lipopolysaccharide (LPS) that derives from multidrug‐resistant Gram‐negative pathogens, leads to a sharp increase in mortality globally. However, monotherapies aiming to neutralize LPS often fail to improve the prognosis. Here, an all‐in‐one drug delivery strategy equipped with bactericidal activity, LPS neutralization, and detoxification is shown to recognize, kill pathogens, and attenuate hyperinflammation by abolishing the activation of LPS‐triggered acute inflammatory responses. First, bactericidal colistin results in rapid bacterial killing, and the released LPS is subsequently sequestered. The neutralized LPS is further cleared by acyloxyacyl hydrolase to remove secondary fatty chains and detoxify LPS in situ. Last, such a system shows high efficacy in two mouse infection models challenged with Pseudomonas aeruginosa. This approach integrates direct antibacterial activity with in situ LPS neutralizing and detoxifying properties, shedding light on the development of alternative interventions to treat sepsis‐associated infections.


Figure S1 .
Figure S1.SLAP-S25 attenuates inflammatory response induced by LPS.A, B, C) Relative expression of NF-κB (A), TNF-α (B) and IL-6 (C) in mouse alveolar macrophages (MH-S) cells in the presence of diverse bacteria (E.coli, P. aeruginosa and K. pneumoniae) based on qRT-PCR.Experiments in A, B and C were performed as three biologically independent experiments, and the mean ± s.d. is shown, n = 3. P-values were determined using One-way ANOVA test.

Figure S2 .
Figure S2.SLAP-S25 attenuates the cell death induced by OMVs derived from K. pneumoniae.A) Cell mortality in A549 cells treated with OMVs and/or SLAP-S25 as determined by lactate dehydrogenase (LDH) assay.B) Relative expression and quantification of autophagy relative protein LC3-Ⅱ in A549 cells.Experiments in A and B were performed as three biologically independent experiments, and the mean ± s.d. is shown, n = 3. P-values were determined using One-way ANOVA test.

Figure S3 .
Figure S3.GNGs exert inhibition of bacteria growth and biofilm formation.A) Bacterial density of P. aeruginosa PAO1 in the presence of GNGs for 24 h.B) Inhibitory effects of GNGs in a dose manner on P. aeruginosa biofilm formation.The absorbance was measured at 600 nm using an Infinite M200 Microplate reader (Tecan).Experiments in A and B were performed as three biologically independent experiments, and the mean ± s.d. is shown, n = 3. P-values were determined using One-way ANOVA test.

Figure S4 .
Figure S4.GNGs attenuate the expression of either TNF-α or IL-6 in TBS, STS and TPS.A, B) Relative expression of TNF-α (A) and IL-6 (B) with the treatments added before (TBS), simultaneous (STS) or after (TPS) the stimulation of LPS in MH-S cells, respectively.Experiments in A and B were performed as three biologically independent experiments, and the mean ± s.d. is shown, n = 3.

Figure S5 .
Figure S5.Quantification of prostaglandin using HPLC.HPLC chromatograms of a prostaglandin standard.The chromatographic separation was carried out on an ODS-C18 column, which was kept at a temperature of 30 °C.The mobile phase A (acetonitrile) and mobile phase B (0.02 mol potassium dihydrogen phosphate) at a constant flow rate of 1 mL/min.A sample solution of 20 μL was injected into the HPLC system and detected at a wavelength of 196 nm.

Figure S6 .
Figure S6.Biocompatibility of GNGs in vitro.A) Cytotoxicity of RAW 264.7,MH-S, Vero, and HepG2 cells treated with GNGs were evaluated using the LDH assay.The result of staurosporine as the positive control was not shown.B) Hemolytic activity of GNGs to the red blood cells of sheep, N group as the negative control (PBS), P group as the positive control (0.2% Triton X-100).Experiments in A and B were performed as three biologically independent experiments, and the mean ± s.d. is shown, n = 3. P-values were determined using One-way ANOVA test.

Figure S7 .
Figure S7.The antibacterial and anti-inflammatory activity in the mouse lung infection model.Representative hematoxylin and eosin-stained lungs of mice in the lung infection model.Scale bar = 50 μm.B, C, D) Bacterial loads (B), the production of TNF-α (C) and IL-6 (D) in the lungs of mice in the lung infection model.E) Immunohistofluorescence analysis of TNF-α and IL-6 in the lungs of mice in the lung infection model.Scale bar = 50 μm.

Figure S8 .
Figure S8.Survival and bacterial loads of mice in the peritonitis-sepsis model.A) Survival rates of mice in the peritonitis-sepsis model.B, C, D, E, F) Bacterial loads of mice in major organs includes heart (B), liver (C), spleen (D), lung (E) and kidney (F) in the peritonitis-sepsis model.P-values were determined using One-way ANOVA test.

Figure S9 .
Figure S9.Inflammatory response of mice in the peritonitis-sepsis model.A, B, C, D) The production of TNF-α in major organs include heart (A), liver (B), lung (C) and kidney (D) in the peritonitis-sepsis model.E, F, G, H) The production of IL-6 in major organs include heart (E), liver (F), lung (G) and kidney (H) in the peritonitis-sepsis model.P-values were determined using One-way ANOVA test.

Figure S10 .
Figure S10.Histological analysis of lung in mice in the peritonitis-sepsis model.A) Representative hematoxylin and eosin-stained lung histology sections of mice in the peritonitis-sepsis model.Scale bar = 50 μm.B) Representative images of cytokines in the lungs of mice in the peritonitis-sepsis model.Blue (DAPI, nuclei), green (IL-6), red (TNF-α), and yellow (merged).Scale bar = 50 µm.

Table S1 .
Comparison of properties of different antimicrobial peptides.