Journal: The Journal of Neuroscience

Article Title: Suppression of Inflammation with Conditional Deletion of the Prostaglandin E 2 EP2 Receptor in Macrophages and Brain Microglia

doi: 10.1523/JNEUROSCI.2203-13.2013

Figure Lengend Snippet: The EP2 receptor induces expression of inflammatory enzymes and cytokines in mouse peritoneal macrophages. For all panels: *p < 0.05, **p < 0.01, ***p < 0.001, values are mean ± SEM. A, EP2 immunostaining is detected in wild-type but not EP2−/− C57BL/6 primary peritoneal macrophages costained for Cd11b. Scale bar, 100 μm. B, Peritoneal macrophages were stimulated with LPS (10 ng/ml) for 1 and 6 h. qPCR demonstrates a rapid upregulation of EP2 receptor mRNA by 1 h and subsequent downregulation by 6 h following LPS stimulation (n = 4–6 per group; two-way ANOVA for effect of time ##p < 0.01, and effect of interaction p = 0.02; Bonferroni's multiple-comparison tests comparing mean of 1 h vehicle (veh) and 1 h LPS *p < 0.05). C, Peritoneal macrophages were stimulated with LPS (10 ng/ml) +/− the EP2 agonist butaprost (1 μm) or vehicle. qPCR demonstrates an induction of proinflammatory mediators COX-2, iNOS, and gp91phox with LPS stimulation that is further enhanced by costimulation with butaprost (time points 1 h for COX-2 and 6 h for iNOS and gp91phox; n = 4–6 per group; two-way ANOVA for effect of LPS #p < 0.05, ###p < 0.001; Bonferroni's multiple-comparison tests comparing means of LPS-con and LPS-butaprost *p < 0.05, **p < 0.01). D, LPS-induced macrophage NO release is increased by EP2 receptor activation with butaprost (1 μm), whereas EP2−/− macrophages show reduced NO levels compared with EP2+/+ macrophages (n = 4 per group; Student's tests #p < 0.05, ##p < 0.01 comparing EP2+/+ to EP2−/−). E, qPCR demonstrates induction of IL-6 mRNA at 1 h and IL1β mRNA at 6 h, and a trend of decreased TNFα mRNA at 6 h in LPS-stimulated macrophages with addition of butaprost (n = 4–6 per group; two-way ANOVA for effect of LPS ##p < 0.01, ###p < 0.001; Bonferroni's multiple-comparison tests comparing means of LPS-con and LPS-butaprost **p < 0.01 for IL-6 at 1 h and IL1β at 6 h).

Article Snippet: Cells were purified with anti-mouse Cd11b Ab-conjugated magnetic beads and MACS columns (Miltenyi Biotec), as previously described ( Shi et al., 2010 ).

Techniques: Expressing, Immunostaining, Comparison, Activation Assay

Journal: The Journal of Neuroscience

Article Title: Suppression of Inflammation with Conditional Deletion of the Prostaglandin E 2 EP2 Receptor in Macrophages and Brain Microglia

doi: 10.1523/JNEUROSCI.2203-13.2013

Figure Lengend Snippet: Conditional deletion of the EP2 receptor in macrophages suppresses oxidative enzyme and cytokine gene expression. A, Genomic DNA PCR is shown for EP2+/+, EP2lox/+, and EP2lox/lox C57BL/6 mice. B, Quantitative genomic PCR was assayed for EP2+/+ wild-type, EP2+/−, EP2−/−, Cd11bCre;EP2lox/lox, Cd11bCre;EP2lox/+, and Cd11bCre;EP2+/+; values are relative to wild-type EP2+/+ control DNA. C, Peritoneal macrophages were isolated from adult Cd11bCre;EP2lox/lox and Cd11bCre;EP2+/+ mice and sorted using Cd11b antibody-conjugated magnetic beads before qPCR analysis. Basal levels of EP2 mRNA, assayed by qPCR, are reduced by 91% in Cd11bCre;EP2lox/lox compared with control macrophages (left; Cd11bCre;EP2lox/lox vs Cd11bCre;EP2+/+) and are reduced by 56% in LPS-stimulated Cd11bCre;EP2lox/lox macrophages (right; **p < 0.01; n = 5–6 per group). D, Conditional deletion of EP2 in macrophages reduces LPS-mediated increases in proinflammatory gene expression (two-way ANOVA for effect of LPS treatment is represented by ###p < 0. 001; Bonferroni's multiple-comparisons tests comparing mean of Cd11bCre;EP2+/+/LPS and Cd11bCre;EP2lox/lox/LPS were ***p < 0.001; n = 5–6 per group).

Article Snippet: Cells were purified with anti-mouse Cd11b Ab-conjugated magnetic beads and MACS columns (Miltenyi Biotec), as previously described ( Shi et al., 2010 ).

Techniques: Gene Expression, Control, Isolation, Magnetic Beads

Journal: The Journal of Neuroscience

Article Title: Suppression of Inflammation with Conditional Deletion of the Prostaglandin E 2 EP2 Receptor in Macrophages and Brain Microglia

doi: 10.1523/JNEUROSCI.2203-13.2013

Figure Lengend Snippet: Examination of levels of monocytic populations in Cd11bCre;EP2+/+ and Cd11bCre;EP2lox/lox mice. Splenocytes and peripheral blood immune cells were isolated from 3-month-old mice. A, Representative plots for CD11b+ cells gated on CD115 and Ly6C yielding four populations of cells, including CD115−/Ly6C− macrophages, CD115−/Ly6Cint-hi neutrophils, CD115int/Ly6Cint resident monocytes, and CD115hi-int/Ly6Chi inflammatory monocytes in vehicle and LPS-treated mice. B, Quantification of levels of monocytic populations, including macrophages, resident monocytes, and inflammatory monocytes does not show differences between genotypes in vehicle or LPS-treated mice. Levels of neutrophils are decreased in peripheral blood with LPS, but not vehicle stimulation (*p < 0.05; n = 4 mice per group). C, Quantification of CD11b-positive microglia derived from brains of Cd11bCre;EP2+/+ and Cd11bCre;EP2lox/lox mice does not show differences in number (n = 5–7 mice per genotype). D, Comparison of copy number of EP2/copy number of 18S is shown for adult microglia and peritoneal macrophages from Cd11bCre;EP2+/+ and Cd11bCre;EP2lox/lox mice (n = 4–6 per group; p < 0.05 unpaired t test). Macrophage expression of EP2 in Cd11bCre;EP2+/+ mice was 28-fold higher; however, the percentage reduction of expression with conditional deletion of EP2 was similar in both microglia and macrophages, and was 62.2 and 62.1%, respectively.

Article Snippet: Cells were purified with anti-mouse Cd11b Ab-conjugated magnetic beads and MACS columns (Miltenyi Biotec), as previously described ( Shi et al., 2010 ).

Techniques: Isolation, Derivative Assay, Comparison, Expressing

Journal: Function

Article Title: Calcium Signaling in Pancreatic Immune Cells In situ

doi: 10.1093/function/zqaa026

Figure Lengend Snippet: Immunostaining after Recording ATP-elicited Ca 2+ Signals in pancreatic macrophages (PMs). (A). Representative images of pancreatic lobule loaded with Fluo-4AM before (Ai) and after ATP (10 µM) application (Aii), the arrow indicates the position of the ( n = 8). A corresponding Fluo 4 trace from a PM is shown in Aiii . Corresponding immunostaining of this lobule with antibodies F4/80 Alexa Fluo 647 is shown below ( Aiv) . Hoechst 33342 staining of the same area is shown in Av . Arrow points to ear-like shape of PM nucleus. Overlay of antibody and Hoechst 33342 staining is shown in Avi . Scale bar is 10µm. (B). Immunostaining of another area in a pancreatic lobule with monoclonal F4/80 antibodies labeled with Alexa Fluor 647 (Bi) . Staining of nuclei in the same lobule with Hoechst 33342 (Bii) . Overlay of B i with Bii is shown in Biii . Scale bar is 10µm. (C). Representative images of a pancreatic lobule loaded with Fluo-4AM before (Ci) and after ATP (10 µM) application (Cii) , the arrow indicates the position of the PM. Corresponding Fluo 4 trace is shown in Ciii. Immunostaining of the same area with monoclonal CD11b antibody conjugated with Alexa Fluor 647 ( n = 8) is shown in Civ . Overlay of Cii and Civ is shown in Cv . Scale bar is 10µm.

Article Snippet: Mouse F4/80 monoclonal rat Antibody (CI-A3-1) [Alexa Fluor ® 647] and mouse CD11b/Integrin alpha M Alexa Fluor ® 647-conjugated monoclonal rat antibodies were obtained from Novus Biologicals Europe and R&D Systems Bio-techne, respectively.

Techniques: Immunostaining, Staining, Labeling

Journal: Function

Article Title: Calcium Signaling in Pancreatic Immune Cells In situ

doi: 10.1093/function/zqaa026

Figure Lengend Snippet: IgG-elicited Ca 2+ Spikes in PMs . (A). Single short Ca 2+ spike occurring after application of IgG (0.1–0.25 mg/mL) in a PM from a control pancreatic lobule. This was an infrequent observation (5 out of 29 cells tested) and is most likely not an IgG-elicited Ca 2+ signal as such single spikes have been also observed in 3 out of 15 cells in the absence of IgG stimulation. (B) . Representative trace of IgG (0.1–0.25 mg/mL)-induced Ca 2+ signals in PMs in pancreatic lobules isolated from mice with AP (FAEE-AP model—48 h). Such oscillations were observed in 9 out of 31 cells. Single short spikes have been observed in 4 out of 31 cells. No oscillations were observed in the absence of stimulation with IgG ( n = 14), while single short spikes have been observed in 2 out of 14 cells. (C). Average Ca 2+ spike frequencies in PMs displaying Ca 2+ signals under the conditions indicated. The frequencies in control PMs, both stimulated with IgG (blue bar) and unstimulated (green), as well as in unstimulated PMs from the FAEE-AP model (48 h, orange bar) were much lower than in PMs from the FAEE-AP model stimulated with IgG (red bar, P < 0.007). (D) . Average Ca 2+ spike duration in PMs displaying Ca 2+ signals under the conditions indicated. Although the average spike duration was longer in the PMs from the FAEE-AP mice stimulated with IgG than under the other conditions, the difference was not statistically different ( P > 0.2). (E). Representative images of immunostaining of PMs in lobules using antibodies F4/80 conjugated with Alexa Fluor 647. Lobules were isolated from control and FAEE-AP 3-day mice (72 h in vivo FAEE-AP model). Scale bar is 20µm. (F). Comparison of the average density of PMs in lobules from control and FAEE-AP 2-day and 3-day mice (48 h and 72 h in vivo FAEE-AP model, respectively). Control, 2.36 ± 0.6 SEM, n = 14; FAEE-AP 2 day, 9.56 ± 1.86 SEM, * P < 0.033, n = 16; FAEE-AP 3 days, 15.37 ± 1.51 SEM, * P < 0.038 as compared to FAEE-AP 2-day, n = 35. The difference between control and FAEE-AP 3-day was very highly significant (**** P < 0.0001).

Article Snippet: Mouse F4/80 monoclonal rat Antibody (CI-A3-1) [Alexa Fluor ® 647] and mouse CD11b/Integrin alpha M Alexa Fluor ® 647-conjugated monoclonal rat antibodies were obtained from Novus Biologicals Europe and R&D Systems Bio-techne, respectively.

Techniques: Isolation, Immunostaining, In Vivo, Comparison