SUPPLEMENT

Anti-malarial drug Artesunate restores metabolic changes in experimental allergic asthma

SUPPLEMENTARY METHODS

Toxicological and peripheral blood tests

Effect of artesunate on cell toxicity in human bronchial epithelial cells and Beas-2B cells was investigated using WST-1 assay. Beas-2B cells were incubated with 0.018% DMSO, artesunate (10 – 100 µM) or 10 mM H2O2 (positive control) for 3 h (n = 6 per treatment group). WST-1 was added 21 h later and measured 30 min later at 440 nm. Peripheral blood counts were performed independently by the National University of Singapore Laboratory Animals Centre Lab using flow cytometric analysis of peripheral blood leukocytes obtained from naïve mice treated with or without three consecutive dosages of intra-peritoneal DMSO (5%) and artesunate (30 mg/kg/d).

SUPPLEMENTARY FIGURES

Fig.S1: Artesunate does not alter BALF and serum metabolic profiles in naïve animals.OPLS-DA of global lung metabolite profiles in the BALF of naïve mice treated with (N/Arts) or without artesunate (N), detected by (A) GC-MS analysis or (B) LC-MS analysis. OPLS-DA of global metabolite profiles in the serum of naïve animals treated with (N/Arts) or without artesunate (N), detected by (C) GC/MS or (D) LC-MS analysis. In OPLS-DA analysis, the value of R2Y describes how well the data in the training set are mathematically reproduced, ranging between 0 and 1, where 1 indicates a model with a perfect fit. Models with a Q2 value greater than or equal to 0.5 are generally considered to have good predictive capability. Black open circles, N (n=12); brown asterisks, N/Arts (n=8). The x axis, t[1], and y axis, t[2], indicate the first and second principle components, respectively.

Fig.S2: Drug vehicle does not modify BALF and serum metabolic profiles in naïve animals. OPLS-DA of global lung metabolite profiles in the BALF of OVA-sensitized and -challenged mice treated with (OO/DMSO) or without DMSO (OO), detected by (A) GC-MS analysis or (B) LC-MS analysis. OPLS-DA of global serum metabolite profiles in the serum of OVA-sensitized and -challenged mice treated with (OO/DMSO) or without DMSO (OO), detected by (C) GC-MS analysis or (D) LC-MS analysis. R2Y describes the goodness of mathematical modelling of the data; while Q2 values of 0.5 or more are considered to have good predictive capability. Red filled circles, OO (n=12); Blue filled triangles, OO/DMSO (n=10). The x axis, t[1], and y axis, t[2], indicate the first and second principle components, respectively.

Fig.S3: Effects of artesunate on human epithelial cells and murine peripheral blood mononuclear cell (PBMC). Beas-2B cells were incubated with 0.018% DMSO, artesunate (10 – 100 µM) or 10 mM H2O2 (positive control) for 3 h (n = 6 per treatment group). WST-1 was added 21 h later and measured 30 min later at 440 nm. Murine plasma was collected 24 h after three consecutive days of artesunate (30mg/kg) or vehicle (DMSO) injections. Experiments of CD3 (B), CD4 (C), CD8 (D), NK1.1 (E), B220 (F) on PBMC were analyzed by FACS (n=4 mice per group). Values shown are the mean ± SEM. *Significant difference from DMSO control, p<0.05.

Fig.S4 Representative BALF metabolite changes induced by artesunate in experimental asthma.Mass abundance of BALF metabolite changes by artesunate, detected by GC/MS or LC/MS. #denotes significant change in OO as compared to OS or N; p < 0.05. *denotes significant change in OO/Arts as compared to OO; p < 0.05. n = 12 per treatment group.

Fig.S5 Representativeserum metabolite changes induced by artesunate in experimental asthma.Mass abundance of serum metabolite changes by artesunate or dexamethasone, detected by GC/MS or LC/MS.*denotes significant change in OO/Arts or OO/Dex as compared to OO; p < 0.05. n = 12 per treatment group.

TableS1 Pearson correlation coefficients (r) of key metabolites versus cytokines in BALF

IL-4 / IL-5 / IL-13 / IL-17 / IL-12 (p40) / MCP-1 / G-CSF
Acetamide / 0.449* / 0.395† / N.S. / 0.356† / N.S. / N.S. / N.S.
Urea / 0.599* / 0.656* / 0.359† / 0.611* / 0.613* / 0.433+ / 0.619*
Choline / 0.551* / 0.508* / N.S. / 0.436† / 0.662* / 0.434+ / 0.617*
Lactate / 0.628* / 0.635* / N.S. / 0.662* / 0.662* / 0.397+ / 0.652*
Arabinose / N.S. / -0.449† / N.S. / -0.476* / -0.377† / N.S. / -0.460*
Galactose / N.S. / -0.594* / N.S. / -0.399† / -0.461* / N.S. / -0.530*
Mannose / N.S. / -0.575* / N.S. / -0.382† / -0.458* / N.S. / -0.517*
Cholesterol / N.S. / -0.614* / -0.388† / N.S. / -0.448† / N.S. / -0.589*
Cholic acid / -0.385† / -0.662* / -0.450* / -0.399† / -0.539* / -0.401+ / -0.598*
Cortol / -0.368† / -0.594* / N.S. / -0.511* / -0.452* / N.S. / -0.546*
Phosphatidylcholines / N.S. / -0.583* / N.S. / -0.616* / N.S. / N.S. / -0.561*

Moderate correlations: values of -0.5 to -0.3 (negative correlation) or 0.3 to 0.5

Strong correlations: values of -1.0 to -0.5 (negative correlation) or 0.5 to 1.0

Only metabolites with significant correlations are shown.

* denotessignificant correlation, p < 0.01, n = 32

† denotes significant correlation, p < 0.05, n = 32

N.S. denotes no significant correlations, p0.05

TableS2 Pearson correlation coefficients (r) for BALF metabolites versus serum metabolites

Serum Metabolites
Urea / Homo-serine / Proline / Valine / Aminoadipic acid / 18-oxo
cortisol / 24-methylene-cholesterol
BALF Metabolites / Cholic Acid / 0.728* / -0.593* / N.S. / N.S. / -0.593* / 0.604* / -0.381†
Cholesterol / 0.599* / -0.449† / N.S. / N.S. / N.S. / 0.826* / N.S.
Cortol / 0.651* / -0.573* / -0.407† / N.S. / -0.563* / 0.636* / -0.422†
Urea / 0.800* / 0.573* / 0.508* / 0.572* / 0.537* / N.S. / N.S.
Lactate / 0.486* / 0.471† / N.S. / N.S. / 0.524* / N.S. / N.S.
Creatinine / 0.477* / N.S. / N.S. / N.S. / 0.396† / N.S. / N.S.
Arabinose / N.S. / -0.463† / N.S. / N.S. / N.S. / N.S. / N.S.
Galactose / -0.451† / N.S. / -0.406† / N.S. / N.S. / 0.557* / N.S.
Mannose / -0.444† / N.S. / -0.384† / N.S. / N.S. / 0.564* / N.S.
Acetamide / N.S / 0.471† / N.S. / N.S. / N.S. / -0.519* / N.S.
Alanine / N.S / 0.470† / N.S. / N.S. / N.S. / N.S. / N.S.

Moderate correlations: values of -0.5 to -0.3 (negative correlation) or 0.3 to 0.5

Strong correlations: values of -1.0 to -0.5 (negative correlation) or 0.5 to 1.0

Only metabolites with significant correlations are shown.

* denotessignificant correlation, p < 0.01, n=28

† denotes significant correlation, p < 0.05, n=28

N.S. denotes no significant correlations, p0.05