Journal: Science signaling

Article Title: Skewing cPLA2 activity toward oxoeicosanoid production promotes neutrophil N2 polarization, wound healing, and the response to sepsis

doi: 10.1126/scisignal.add6527

Figure Lengend Snippet: (A) NETosis assays measuring neutrophil elastase activity, (B) fluorescence-based ROS assays, (C) VEGF secretion, (D) TNF secretion, and (E) phagocytosis of E. coli by cPLA2α+/+, cPLA2αKI/KI, and cPLA2α−/− primary neutrophils after 4 hours of NTEM. (F) Quantification of Syndecan-1, a marker of endothelial damage, in culture media from cPLA2α+/+, cPLA2αKI/KI, and cPLA2α−/− primary neutrophils after NTEM for the indicated durations. Data in (A) to (F) were analyzed by one-way ANOVA with Tukey post-hoc; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; n=6 biological replicates/group/time point, repeated on at least two separate occasions. (G to J) Quantification of neutrophil polarization markers in primary neutrophils from cPLA2α+/+ and cPLA2αKI/KI mice that were unchallenged (Un) or after 6 hours NTEM. N1 markers ICAM-1 (G) and CXCL10 (H) and N2 markers IL-8 (I) and CXCR2 (J) were assayed. Data in (G) to (J) were analyzed by one-way ANOVA with Tukey’s multiple comparisons test; *p < 0.05, **p < 0.01; n=4 biological replicates/group, repeated on two separate occasions. (K) Hematoxylin and eosin staining showing morphology of primary neutrophils from cPLA2α+/+ and cPLA2αKI/KI mice before challenge and after 6 hours of NTEM. Images are representative of n = 3 independent experiments. Scale bar, 10 μm.

Article Snippet: Assays for PGD, G6PD, Cytokines, Phagocytosis, ROS Production, Cell Surface Markers, and NETosis The following were utilized in this study: G6PD activity assay kit (Abcam #ab176722), PGD activity assay kit (Abcam #ab273328), NETosis assay kit (Cayman #601010), ROS detection assay kit (Cayman #601290), mouse VEGF kit (Thermo Fisher Scientific, #KHG0111 & #MMV00), mouse TNFα kit (Thermo Fisher Scientific, #BMS223-4 & #MTA00B), Phagocytosis assay kit (Cell Biolabs #CBA-222), Mouse Syndecan-1 ELISA kit (Abcam #ab273165), ICAM-1 ELISA kit (R&D Systems #MIC100), CXCL10 ELISA kit (R&D Systems #DY466-05), IL-8 ELISA kit (MyBioSource #MBS7606860), and CXCR2 ELISA kit (MyBioSource #MBS726530).

Techniques: Activity Assay, Fluorescence, Marker, Staining

Journal: Cellular and Molecular Neurobiology

Article Title: Differential Effects of Toll-Like Receptor Activation and Differential Mediation by MAP Kinases of Immune Responses in Microglial Cells

doi: 10.1007/s10571-021-01127-x

Figure Lengend Snippet: Distinct time courses of TNF and IL-6 induction after LPS and resiquimod. The microglial cells were cultured in serum-free medium overnight, stimulated by LPS (50 ng/ml), poly I:C (100 ng/ml), or resiquimod (3 μM), and RNA extracted and the culture media stored after the times indicated. A Il-6 mRNA showed meaningful changes from 8 h stimulation with LPS and resiquimod but not with poly I:C. B IL-6 release increased statistically from 8 and 24 h stimulation with LPS and resiquimod. 0.5 h stimulation data were below quantification limit. C Tnf mRNA was maximally increased at 0.5 h after stimulation with LPS or resiquimod. D TNF release was significantly increased from 8 h and this level continued to increase. The individual data points are shown along with mean ± SEM. The colour represent passages of the cells. Data were analysed by two-way ANOVA (RT-qPCR); or three-way ANOVA (ELISA), Tukey post hoc test ( n = 4–6 independent samples; * p ≤ 0.05, ** p ≤ 0.005, *** p ≤ 0.001, **** p ≤ 0.0001 vs. vehicle control at same stimulation time; $ p ≤ 0.05, $$ p ≤ 0.005, $$$ p ≤ 0.001, $$$$ p ≤ 0.0001 vs. 0.5 h stimulation within the same treatment group; # p ≤ 0.05, ## p ≤ 0.005, ### p ≤ 0.001, #### p ≤ 0.0001 overall effect Tukey comparisons; @ p ≤ 0.05, @@ p ≤ 0.005, @@@ p ≤ 0.001, @@@@ p ≤ 0.0001 LPS vs. resiquimod in a same stimulation time Tukey comparisons). ( n = 4–6/group). Details of ANOVA F values and p values and of Fisher-Pitman permutation testing are provided in Supplementary Table 2

Article Snippet: Secreted cytokines and chemokines from the stimulated cells were measured by ELISA kits (IL-6, eBioscieceTM, 12364003; TNF-a, eBioscieceTM, 155501117; CCL2, eBioscieceTM, 15561137; CCL5, R&D system, DY478-05, CXCL10, R&D system, DY466-05) per manufacturers’ instructions.

Techniques: Cell Culture, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Control

Journal: Cellular and Molecular Neurobiology

Article Title: Differential Effects of Toll-Like Receptor Activation and Differential Mediation by MAP Kinases of Immune Responses in Microglial Cells

doi: 10.1007/s10571-021-01127-x

Figure Lengend Snippet: Distinct chemokine induction after LPS and resiquimod. The microglial cells were cultured in serum-free medium overnight, stimulated by LPS (50 ng/ml), poly I:C (100 ng/ml),or resiquimod (3 μM for the times indicated. A Ccl2 mRNA levels were increased by LPS and resiquimod after 8 h stimulation but not by poly I:C. B CCL2 protein level showed meaningful changes with LPS and resiquimod stimulation at 24 h. C Ccl5 mRNA showed considerable changes after 8 h resiquimod stimulation and its level remained increased till 24 h. LPS challenge induced an even greater increase in Ccl5 mRNA levels from 8 and 24 h. D CCL5 protein levels did not change. E Cxcl10 mRNA increased from 8 h after stimulation with LPS. Resiquimod showed a lesser induction from 0.5 h and its level was no different from control levels by 24 h. F CXCL10 protein levels did not change at any timepoints with any treatments. The individual data points are shown along with mean ± SEM. The colour represent passages of the cells. The data were analysed by two-way ANOVA (RT-qPCR); three-way ANOVA (ELISA), Tukey post hoc test ( n = 4–6 independent samples; * p ≤ 0.05, ** p ≤ 0.005, *** p ≤ 0.001, **** p ≤ 0.0001 vs. vehicle control at same stimulation time; $ p ≤ 0.05, $$ p ≤ 0.005, $$$ p ≤ 0.001, $$$$ p ≤ 0.0001 vs. 0.5 h stimulation within the same treatment group; # p ≤ 0.05, ## p ≤ 0.005, ### p ≤ 0.001, #### p ≤ 0.0001 vs. overall effect Tukey comparisons; @ p ≤ 0.05, @@ p ≤ 0.005, @@@ p ≤ 0.001, @@@@ p ≤ 0.0001 LPS vs. resiquimod in a same stimulation time Tukey comparisons). ( n = 4–6/group). Details of ANOVA F values and p values and of Fisher-Pitman permutation testing are provided in Supplementary Table 2

Article Snippet: Secreted cytokines and chemokines from the stimulated cells were measured by ELISA kits (IL-6, eBioscieceTM, 12364003; TNF-a, eBioscieceTM, 155501117; CCL2, eBioscieceTM, 15561137; CCL5, R&D system, DY478-05, CXCL10, R&D system, DY466-05) per manufacturers’ instructions.

Techniques: Cell Culture, Control, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Cellular and Molecular Neurobiology

Article Title: Differential Effects of Toll-Like Receptor Activation and Differential Mediation by MAP Kinases of Immune Responses in Microglial Cells

doi: 10.1007/s10571-021-01127-x

Figure Lengend Snippet: Pro-inflammatory cytokines are selectively regulated by MAPKs in microglia after LPS and resiquimod. The microglial cells were cultured in the serum-free medium and left overnight. The cells were stimulated by LPS (50 ng/ml) or resiquimod (3 μM) for 8 h and MAPK inhibitors, JNK (JNK-IN-8, JNK inhibitor, 1 μM), ERK (PD98059, ERK inhibitor, 40 μM), or p38 (SB203580, p38 inhibitor, 5 μM), or vehicle, were added before the mimetics. A – C Il-6 mRNAs did not show any MAPKs’ inhibitor effects compare to vehicle or LPS only condition. ERK and p38 inhibition suppressed Il-6 mRNA production when it combined with resiquimod. D – F Tnf mRNA did not any statistically significance of LPS or resiquimod with MAPKs’ inhibitors . G – I TNF-a proteins were only changed by JNK inhibitor with resiquimod. Absolute quantification was performed via RT-qPCR and the data were normalised to Gapdh and Henmt1 . Total amount of protein (per 1000 cells) was presented on graphs and its amount was measured by ELISA. The individual data points are shown along with mean ± SEM. The colour represents passages of the cells. The data were analysed by three-way ANOVA, Tukey post hoc test ( n = 5–12 independent samples; * p ≤ 0.05, ** p ≤ 0.005, *** p ≤ 0.001, **** p ≤ 0.0001 vs. vehicle under the same inhibitor; $ p ≤ 0.05, $$ p ≤ 0.005, $$$ p ≤ 0.001, $$$$ p ≤ 0.0001 vs. vehicle alone within the same immune mimetic group; @ p ≤ 0.05, @@ p ≤ 0.005, @@@ p ≤ 0.001, @@@@ p ≤ 0.0001 vs. LPS+ a same inhibitor; ^ p ≤ 0.05, ^^ p ≤ 0.005, ^^^ p ≤ 0.001, ^^^^ p ≤ 0.0001 overall treatment effects; ǂ p ≤ 0.05, ǂǂ p ≤ 0.005, ǂǂǂ p ≤ 0.001, ǂǂǂǂ p ≤ 0.0001 LPS vs. LPS+PD98059 and LPS vs. SB203580 within LPS treatment group one-way ANOVA Bonferroni post hoc test). ( n = 5–12/group). Details of ANOVA F values and p values are provided in Supplementary Information

Article Snippet: Secreted cytokines and chemokines from the stimulated cells were measured by ELISA kits (IL-6, eBioscieceTM, 12364003; TNF-a, eBioscieceTM, 155501117; CCL2, eBioscieceTM, 15561137; CCL5, R&D system, DY478-05, CXCL10, R&D system, DY466-05) per manufacturers’ instructions.

Techniques: Cell Culture, Inhibition, Quantitative Proteomics, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay