MOESM1 of 1-Palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol (PLAG) attenuates gemcitabine-induced neutrophil extravasation

Additional file 1. Figure S1. Analysis of Differentiation of BMDM and human neutrophil-like HL-60 cells by flow cytometry. A, FACS analysis illustrates the purity of BMDM at day 7 using a macrophage marker F4/80-PE. B, Differentiation of HL-60 cells into human neutrophil-like cells confirmed by flow cytometry and a CD11b-PE marker. Figure S2. Neutrophil counts of the blood and peritoneum in mice treated with PLAG, reparixin or NAC. Male balb/c mice of 6 to 8 weeks of age were orally administered with 250mg/kg of PLAG (in PBS), or intraperitoneally injected with 50mg/kg of reparixin (in mineral oil) or with 50mg/kg of NAC (in PBS). After 15h, blood and peritoneal fluid samples were collected for CBC analysis. The number of neutrophils from the blood and the peritoneal fluid of (A) PLAG, (B) reparixin and (C) NAC-treated mice. Each group contains five mice, and bars represent the mean ± SD. ns, not significant. Figure S3. PLAG inhibits other chemotherapeutic agents-induced neutrophil migration. Male balb/c mice of 6-8 weeks of age were orally administrated with 250mg/kg PLAG, and then intraperitoneally injected with (A) 100mg/kg 5-fluouracil or (B) AC regimen (2.5mg/kg of adriamycin and 100mg/kg of cyclophosphamide) for 24h. The number of blood neutrophils were determined by CBC analysis. Each group contains five mice, and bars represent the mean ± SD. * p<0.05, ** p<0.01, *** p<0.001. Figure S4. Gemcitabine induces a neutrophil-attracting chemokine CXCL8 production via MAPK activation in THP-1 cells. The mRNA level of CXCL8 in human monocytic THP-1 stimulated with (A) various doses and (B) different time points of gemcitabine treatment. The protein concentration of CXCL8 in THP-1 stimulated with (C) various doses and (D) different time points of gemcitabine treatment. E, Gemcitabine induces phosphorylation of ERK, p38 MAPK and JNK analyzed by western blot in THP-1 cells. The transmigration of differentiated HL-60 cells towards the conditioned medium of THP-1 stimulated with (F) various doses and (G) different time points of gemcitabine treatment. * p<0.05, ** p<0.01, *** p<0.001. Figure S5. Gemcitabine increases intracellular reactive oxygen species (ROS) levels in a time-dependent manner. A and B, THP-1 cells were treated with 10ug/ml of gemcitabine for different time points, and then the level of intracellular ROS in the cells was stained with CM-H2DCFDA and analyzed by flow cytometry. C, CM-H2DCFDA fluorescence imaging of ROS in THP-1 cells using a confocal microscope. The bars represent the mean ± SD. * p<0.05, ** p<0.01, *** p<0.001. Figure S6. PLAG does not interfere with the anti-cancer effect of gemcitabine in athymic nude mice implanted with a human myeloma cell line RPMI8226. A, The treatment design and schedule using a human RPMI8226 xenograft mice model. B, The tumor weights of the treatment groups at day 40. ns; not significant, *p < 0.05

PLAG (1-Palmitoyl-2-Linoleoyl-3-Acetyl-rac-Glycerol) Modulates Eosinophil Chemotaxis by Regulating CCL26 Expression from Epithelial Cells

<div><p>Increased number of eosinophils in the circulation and sputum is associated with the severity of asthma. The respiratory epithelium produces chemokine (C-C motif) ligands (CCL) which recruits and activates eosinophils. A chemically synthesized monoacetyl-diglyceride, PLAG (1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol) is a major constituent in the antlers of Sika deer (<i>Cervus nippon</i> Temminck) which has been used in oriental medicine. This study was aimed to investigate the molecular mechanism of PLAG effect on the alleviation of asthma phenotypes. A549, a human alveolar basal epithelial cell, and HaCaT, a human keratinocyte, were activated by the treatment of interleukin-4 (IL-4), and the expression of chemokines, known to be effective on the induction of eosinophil migration was analyzed by RT-PCR. The expression of IL-4 induced genes was modulated by the co-treatment of PLAG. Especially, CCL26 expression from the stimulated epithelial cells was significantly blocked by PLAG, which was confirmed by ELISA. The transcriptional activity of signal transducer and activator of transcription 6 (STAT6), activated by IL-4 mediated phosphorylation and nuclear translocation, was down-regulated by PLAG in a concentration-dependent manner. In ovalbumin-induced mouse model, the infiltration of immune cells into the respiratory tract was decreased by PLAG administration. Cytological analysis of the isolated bronchoalveolar lavage fluid (BALF) cells proved the infiltration of eosinophils was significantly reduced by PLAG. In addition, PLAG inhibited the migration of murine bone marrow-derived eosinophils, and human eosinophil cell line, EoL-1, which was induced by the addition of A549 culture medium.</p></div

PLAG alleviates inflammatory phenotypes in allergic asthma model.

<p>(A) Mice were sensitized and challenged by OVA, and the efficacy of PLAG and DEX was tested in the generated asthma model as depicted. (B-C) The lung tissues from the OVA-sensitized and–challenged mice were stained with H&E, and the representative images were displayed (X200 magnification, B). Infiltrated immune cells were counted, calculated and displayed as inflammation index. Eosinophils from each of the three mice per treatment group were analyzed morphometrically and mean eosinophil counts per 50 × 50 μm area ± SD were displayed. Four such areas were measured from each tissue section. PLAG was the most effective with a dose of 50 mg/Kg as showed in (B) and calculated in (C). *<i>p</i><0.001. (D) The expression level of CCL26 in BALFs of OVA-challenged mice was analyzed by ELISA. Each sample was analyzed in triplicate and displayed as mean ± SD. *<i>p</i><0.001.</p

PLAG inhibits IL-4 induced STAT6 phosphorylation.

(A) Various concentration of a STAT6 inhibitor (STAT6i, AS1517499) were treated onto A549 cells which were activated by IL-4 (10 ng/mL), and showed inhibitory effect on the secretion of CCL26 from the epithelial cells in a concentration-dependent manner. *pppppp<0.01. (D-E) Cellular extracts of IL-4 and/or PLAG treated epithelial cells were analyzed by Western blotting. PLAG decreased the phosphorylation of STAT6 with dose dependency in both A549 (D) and HaCaT (E). (F) Confocal microscopy showed that IL-4 induced nuclear localization of phosphorylated STAT6, which was inhibited by the co-treatment of PLAG.</p

IL-4 induced CCL26 expression is inhibited by PLAG.

<p>(A) A549 cells were treated with various doses of PLAG (1, 5, 10, 50 μg/mL) for 1 h and stimulated with IL-4 (10 ng/mL) for 24 h. The mRNA levels of following genes, VCAM-1, ICAM-1, MIF, CCL5, CCL7, CCL11, CCL13, CCL17, CCL24, and CCL26 were analyzed by conventional RT-PCR. Each reaction was repeated three times and representative images were displayed. GAPDH was used as an internal control. (B) The culture supernatant was harvested at 48 h, and the protein level of CCL26 was evaluated by ELISA. PLAG decreased CCL26 secretion from A549 cells in a dose-dependent manner. *<i>p</i><0.001, ***<i>p</i><0.05. (C) A549 cells were pretreated with PLAG (10 μg/mL) for 1 h and stimulated with IL-4 (10 ng/mL) for various lengths of time. PLAG decreased CCL26 secretion from A549 cells from the early time point. NC; negative control. *<i>p</i><0.001, **<i>p</i><0.01, *** <i>p</i><0.05. (D) HaCaT cells were treated with various doses of PLAG and stimulated with IL-4 (20 ng/mL) for 24 h. The mRNA levels of CCL 26 were analyzed by RT-PCR. (E) PLAG also decreased CCL26 secretion from HaCaT cells in a dose-dependent manner. *<i>p</i><0.001, ***<i>p</i><0.05.</p

PLAG inhibits eosinophil migration in vitro.

<p>(A) Murine eosinophils were differentiated from BM cells by treating SCF, FLT3 ligand and IL-5 for 2w. The differentiated cells were analyzed by FACS staining with SiglecF and CD11b. SiglecF+/CD11b+ cells were increased in the differentiated cells to ~25%. (B) CCR3 expression was increased in the differentiated murine eosinophils. (C-D) The supernatant of IL-4 and/or PLAG-treated A549 cells was prepared and used for the migration assay as described in Materials and Methods. The transmigration of eosinophil was decreased by PLAG treatment in both murine BM-derived eosinophils (C) and human eosinophil cell line, EoL-1 (D). *<i>p</i><0.001, **<i>p</i><0.01. (E) PLAG did not exhibit any cytotoxic effects on A549 cells at various concentrations.</p

PLAG decreases eosinophil infiltration into the airways of asthmatic mice.

<p>(A) Cells were harvested from the BALFs of OVA-challenged mice and analyzed by CBC. The number of infiltrated immune cells were increased in OVA-challenged mice. PLAG or DEX treatment inhibited immune cell infiltration into airways to the level of normal control. (B) After CBC, cells were collected from the BALF, and analyzed by flow cytometry. PLAG and DEX treatment induced the recovery of PMN cell populations which were increased in the allergic mice. (C) SiglecF+/CD11b+ eosinophils were increased in the OVA-challenged mice, which were decreased in PLAG- or DEX-treated mice.</p