Journal: Frontiers in Immunology

Article Title: Adenylyl Cyclase 6 Mediates Inhibition of TNF in the Inflammatory Reflex

doi: 10.3389/fimmu.2018.02648

Figure Lengend Snippet: Vagus nerve stimulation suppresses endotoxin-induced serum TNF levels for days. Animals were subjected to vagus nerve stimulation (VNS) at 10 Hz followed by intraperitoneal endotoxin (LPS) injection at a specified time after VNS and then euthanized 90 min after endotoxin administration. Serum was collected and analyzed for TNF by ELISA. (A) Rats ( n = 4–13/group) were subjected to 60 s of VNS or sham surgery. Open squares: mean TNF ± SEM in sham animals, filled diamonds: mean TNF ± SEM in vagus nerve stimulated animals. (B) Mice were subjected to 0 ( n = 9), 0.1 s ( n = 7), or 60 s ( n = 9) of VNS and rested for 24 h before endotoxin injection. Means ± SEM are plotted. (C) Wild type ( n = 7; left) and α7nAChR −/− ( n = 7; right) male mice were subjected to 60 s of VNS or sham surgery and rested for 24 h before endotoxin injection. TNF levels relative to unstimulated animals are shown as mean ± SEM. *** p < 0.001, ** p < 0.01, * p < 0.05.

Article Snippet: After 6 days of culture in the presence of 20 ng/ml M-CSF (PeproTech), cells were scraped, re-plated, and exposed to the α7nAChR selective agonist PHA568487 (Tocris) for 15 min. Wells were then washed in ice cold PBS, and cell lysis buffer (R&D systems) added on dry ice.

Techniques: Injection, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Adenylyl Cyclase 6 Mediates Inhibition of TNF in the Inflammatory Reflex

doi: 10.3389/fimmu.2018.02648

Figure Lengend Snippet: Adenylyl cyclase 6 mediates inhibition of TNF. (A) Bone-marrow derived macrophages from wild type ( n = 2) and α7nAChR −/− ( n = 3) mice were exposed to the α7nAChR selective agonist PHA568487 and cAMP analyzed in the cell lysate. Results are shown as mean cAMP levels normalized to protein concentration (pmol/μg) ± SEM. Open circles—wild type, filled circles—α7nAChR −/− cells. (B) RAW 264.7 macrophage-like cells were incubated with 10 μM acetylcholine for 1 h. The ACh was removed and 24 h post ACh, the cells were exposed to endotoxin (LPS) for 4 h. TNF was measured in the culture medium using ELISA. Plotted values are mean ± SEM. (C) RAW 264.7 cells were incubated with the adenylyl cyclase inhibitor MDL 12,330A, then exposed to choline and endotoxin. Values shown are mean fold increase of endotoxin-induced TNF mRNA ± SEM relative to cells exposed to endotoxin and choline in the absence of MDL 12,330A. (D) Adenylyl cyclase 6 (AC6) was knocked down using siRNA in RAW 264.7 cells which were then exposed to the α7nAChR selective agonist choline. Western blot shows AC6 in cells treated with siRNA targeting AC6 (si-AC6) or scrambled siRNA (si-SCR). Bars represent fold increase ± SEM of TNF mRNA compared to cells not challenged with endotoxin. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: After 6 days of culture in the presence of 20 ng/ml M-CSF (PeproTech), cells were scraped, re-plated, and exposed to the α7nAChR selective agonist PHA568487 (Tocris) for 15 min. Wells were then washed in ice cold PBS, and cell lysis buffer (R&D systems) added on dry ice.

Techniques: Inhibition, Derivative Assay, Protein Concentration, Incubation, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Frontiers in Immunology

Article Title: Adenylyl Cyclase 6 Mediates Inhibition of TNF in the Inflammatory Reflex

doi: 10.3389/fimmu.2018.02648

Figure Lengend Snippet: c-FOS mediates inhibition of TNF. (A) RAW 264.7 macrophage-like cells were exposed to endotoxin (LPS) only, or endotoxin together with the α7nAChR selective agonist choline. C-FOS was measured at 0 −6 h after endotoxin exposure by Western blot. (B) c-Fos was knocked down using siRNA in RAW 264.7 cells which were subsequently exposed to choline. Endotoxin-induced TNF mRNA levels were measured by qPCR. Bars represent fold increase ± SEM of TNF mRNA compared to cells not challenged with endotoxin. Data is representative of three independent experiments. * p < 0.05.

Article Snippet: After 6 days of culture in the presence of 20 ng/ml M-CSF (PeproTech), cells were scraped, re-plated, and exposed to the α7nAChR selective agonist PHA568487 (Tocris) for 15 min. Wells were then washed in ice cold PBS, and cell lysis buffer (R&D systems) added on dry ice.

Techniques: Inhibition, Western Blot

Journal: Frontiers in Immunology

Article Title: Adenylyl Cyclase 6 Mediates Inhibition of TNF in the Inflammatory Reflex

doi: 10.3389/fimmu.2018.02648

Figure Lengend Snippet: Functional CREB mediates persistent inhibition of TNF. (A) Human macrophages were incubated in vitro with acetylcholine (ACh), washed and rested for 24 h, and subsequently exposed to endotoxin (LPS) for 4 h. TNF was measured in the culture media using ELISA. (B) Primary human macrophages were transduced with adenovirus engineered to encode a dominant negative phosphorylation defective CREB (ACREB), or a GFP-expressing control adenovirus. After viral transduction, cells were stimulated with the selective α7nAChR agonist GTS21, washed, and cultured for 24 h before exposure to endotoxin. TNF was measured in culture media by ELISA. Mean TNF levels ± SEM in cells transduced with control virus or the phosphorylation defective ACREB-expressing virus (ACREB) are shown. * p < 0.05.

Article Snippet: After 6 days of culture in the presence of 20 ng/ml M-CSF (PeproTech), cells were scraped, re-plated, and exposed to the α7nAChR selective agonist PHA568487 (Tocris) for 15 min. Wells were then washed in ice cold PBS, and cell lysis buffer (R&D systems) added on dry ice.

Techniques: Functional Assay, Inhibition, Incubation, In Vitro, Enzyme-linked Immunosorbent Assay, Transduction, Dominant Negative Mutation, Phospho-proteomics, Expressing, Control, Cell Culture, Virus

Journal: Frontiers in Immunology

Article Title: Adenylyl Cyclase 6 Mediates Inhibition of TNF in the Inflammatory Reflex

doi: 10.3389/fimmu.2018.02648

Figure Lengend Snippet: Proposed model of α7nAChR mediated prolonged regulation of TNF in macrophages. Activation of α7nAChR promotes activity of adenylyl cyclase 6, which increases production of cAMP. Increased intracellular levels of cAMP promotes phosphorylation of CREB, which enhances expression of c-FOS, c-FOS, in turn, is involved in NFκB mediated inhibition of TNF transcription. In this way, α7nAChR activation can inhibit endotoxin-induced TNF production.

Article Snippet: After 6 days of culture in the presence of 20 ng/ml M-CSF (PeproTech), cells were scraped, re-plated, and exposed to the α7nAChR selective agonist PHA568487 (Tocris) for 15 min. Wells were then washed in ice cold PBS, and cell lysis buffer (R&D systems) added on dry ice.

Techniques: Activation Assay, Activity Assay, Phospho-proteomics, Expressing, Inhibition