Journal: Science Advances

Article Title: Lung-innervating nociceptor sensory neurons promote pneumonic sepsis during carbapenem-resistant Klebsiella pneumoniae lung infection

doi: 10.1126/sciadv.adl6162

Figure Lengend Snippet: ( A ) Levels of CGRP in BALF of PBS-treated and CRKP-infected control and Trpv1 DTA mice ( n = 8 per group). ( B ) Stained sections of lungs showing β-tubulin III (TUJ1) and CGRP fibers of control and Trpv1 DTA mice. Representative images were selected from a total of nine images of three mice in each group (three images per mouse). Scale bars, 50 μm. DAPI, 4′,6-diamidino-2-phenylindole. ( C ) Left: Schematic showing organotypic culture of VG ( n = 4 VGs per well) and their stimulation with CRKP to measure CGRP release. Right: Time course of CGRP release from VG culture after stimulation with CRKP bacteria (10 6 CFU per well and 10 8 CFU per well). Unstimulated wells are VG culture with media only. Capsaicin (1.5 μM) was used as positive control. ( D ) Heatmap showing relative mRNA levels of neuropeptide receptors in CRKP-infected monocytes and neutrophils at indicated time points of infection ( n ≥ 3 per group). Un., unstimulated cells. Data in (A), (C), and (D) are the means ± SEM. Statistical analysis: Brown-Forsythe and Welch ANOVA of two-stage liner step-up procedure of Benjamini, Krieger, and Yekutieli posttests (A), one-way ANOVA with Sidak’s multiple comparison test (C), and two-way ANOVA with Sidak’s multiple comparisons posttests (D). Level of significance: * P < 0.05, ** P < 0.01, **** P < 0.0001. BioRender.com was used to create schematic in (C).

Article Snippet: Competitive CGRP inhibitor CGRP 8–37 (GenScript) (5 μg per mouse) was administered intraperitoneally into mice at 2 hours before infection.

Techniques: Infection, Control, Staining, Bacteria, Positive Control, Comparison

Journal: Science Advances

Article Title: Lung-innervating nociceptor sensory neurons promote pneumonic sepsis during carbapenem-resistant Klebsiella pneumoniae lung infection

doi: 10.1126/sciadv.adl6162

Figure Lengend Snippet: ( A ) Schematic showing monocyte and CRKP coculture in the presence and absence of neuropeptide. ( B and C ) Intracellular viable CFUs (B) and levels of ROS (C), in CRKP-infected wild-type (WT) and Ramp − / − monocytes. ( D ) Schematic of neutrophil and CRKP coculture with and without neuropeptide. ( E ) Intracellular viable CFUs in CRKP-infected WT and Ramp − / − neutrophils. ( F ) Schematic showing BMDM isolation and coculture with CRKP in the presence and absence of neuropeptides. ( G ) Intracellular viable CFUs in WT BMDMs. Data in (B), (C), (E), and (G) involve three to eight samples per group. ( H to K ) CRKP CFUs (H), proportions of myeloid subsets (I), total BAL protein (J), and panel of 13 mouse inflammatory cytokines/chemokines (K), measured in BALF of Ramp1 + / + and Ramp1 − / − mice ( n = 7 to 8 per group) at 24 hpi. ( L ) Schematic showing administration of CGRP 8–37 (5 μg per mouse) in C57BL/6 J mice and CRKP infection. ( M to P ) CFUs in whole lung (M), blood (N), liver (O), and spleen (P) of PBS-treated and CGRP 8–37 -treated mice ( n = 8 per group) after 24 hpi. Data in (B), (C), (E), (G), (H) to (K) and (M) to (P) are the means ± SEM. Statistical analysis: one-way ANOVA with Sidak’s multiple comparisons posttests [(B), (C), (E), and (G)], Mann-Whitney test [(H), (O), and (P)], unpaired t test [(J), (M), and (N)], and two-way ANOVA of two-stage liner step-up procedure of Benjamini, Krieger, and Yekutieli posttests [(I) and (K)]. Levels of significance: * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. BioRender.com was used to create schematics in [(A), (D), (F), and (L)].

Article Snippet: Competitive CGRP inhibitor CGRP 8–37 (GenScript) (5 μg per mouse) was administered intraperitoneally into mice at 2 hours before infection.

Techniques: Infection, Isolation, MANN-WHITNEY

Journal: Science Advances

Article Title: Lung-innervating nociceptor sensory neurons promote pneumonic sepsis during carbapenem-resistant Klebsiella pneumoniae lung infection

doi: 10.1126/sciadv.adl6162

Figure Lengend Snippet: Lung-innervating nociceptor neurons suppress the recruitment of neutrophils and Ly6C hi monocytes to the airspaces of lungs during CRKP lung infection. CRKP infection induces vagal TRPV1 + neurons for releasing CGRP, which in turn acts on its receptor on Ly6C hi monocytes to dampen the ROS production. This leads to an increased CRKP survival in the lungs and enhanced dissemination of bacteria to vital organs. BioRender.com was used to create model.

Article Snippet: Competitive CGRP inhibitor CGRP 8–37 (GenScript) (5 μg per mouse) was administered intraperitoneally into mice at 2 hours before infection.

Techniques: Infection, Bacteria

Journal: Biomolecules

Article Title: Gα s -Coupled CGRP Receptor Signaling Axis from the Trigeminal Ganglion Neuron to Odontoblast Negatively Regulates Dentin Mineralization

doi: 10.3390/biom12121747

Figure Lengend Snippet: CGRP increases the intracellular cAMP level ([cAMP] i ). ( A , C ) Representative traces of the transient [cAMP] I level increases in response to 50 nM CGRP (rat) with or without 0.1 nM BIBN 4096 ( A ) or 0.1 μM SQ22536 ( C ) in the presence of extracellular Ca 2+ (2.5 mM) (white boxes at the bottom). Black boxes indicate the time periods of CGRP (rat) application to the extracellular solution. White boxes at the top indicate the time of addition of BIBN 4096 ( A ) or SQ22536 ( C ) to the extracellular solution. ( B , D ) Summary bar graphs show CGRP (rat)-induced [cAMP] i level increases in the control (upper column) or with (middle column) 0.1 nM BIBN 4096 ( B ) or 0.1 μM SQ22536 ( D ) in the presence of extracellular Ca 2+ (2.5 mM). Each recovery effect (lower column in B , D ) shows the reversible effect of BIBN 4096 ( B ) and SQ22536 ( D ). Each bar denotes the mean ± SE. Numbers in parentheses show the number of experiments. Asterisks denote statistically significant differences between columns (shown by solid lines): * p < 0.05.

Article Snippet: An AC activator, forskolin (FSK) [ , ]; an AC inhibitor, SQ22536 [ , ]; a non-selective CGRP receptor agonist, CGRP (rat) [ ]; and a non-selective CGRP receptor antagonist, BIBN 4096 [ ], were obtained from R&D Systems, Inc. (Minneapolis, MN, USA).

Techniques: Control

Journal: Biomolecules

Article Title: Gα s -Coupled CGRP Receptor Signaling Axis from the Trigeminal Ganglion Neuron to Odontoblast Negatively Regulates Dentin Mineralization

doi: 10.3390/biom12121747

Figure Lengend Snippet: Direct mechanical stimulation of TG neurons simultaneously increases [Ca 2+ ] i in neurons and [cAMP] i in the neighboring odontoblasts. ( A , B ) Representative traces showing transiently increased [Ca 2+ ] i in a mechanically stimulated TG neuron (green lines) and [cAMP] i level in neighboring odontoblasts (red lines) following focal and direct mechanical stimulation by a micropipette under standard extracellular solution. The traces shown are without ( A ) or with 0.1 nM BIBN 4096 ( B ). Horizontal dotted lines indicate the baseline ( F / F 0 = 1.0) for each response, while vertical dotted lines represent the application time of the mechanical stimulation. The black boxes at the top show the timing of the mechanical stimulation based on the displacement of a micropipette to a depth of 8 μm. The responses from the nearby odontoblasts were recorded from cells located 0–120 μm away from the stimulated TG neuron. The distance of each cell from the mechanically stimulated TG neuron is indicated on the right side of each trace ( C ). The F / F 0 values of neighboring odontoblasts located within 0–40 μm, 41–80 μm, and 81–120 μm from the stimulated TG neuron in standard extracellular solution (open columns) or standard extracellular solution with BIBN 4096 (0.1 nM) (red columns) are given. The [cAMP] i increases in the neighboring odontoblasts reduced with the increase in their distance from the mechanically stimulated TG neuron with and without BIBN 4096. Each bar denotes the mean ± SE. Asterisks denote statistically significant differences between columns or values (shown by solid lines): * p < 0.05. ( D – F ) Mechanically stimulated TG neuron showing positive immunoreactivity for CGRP ( D , F ) and negative immunoreactivity for NF-H ( E , F ). Nuclei are indicated by a gray color. Scale bar: 20 μm. Arrowheads indicate a mechanically stimulated cell. No fluorescence was detected in the negative controls (not shown). Data were obtained using a 120× magnification objective lens under a microscope. Enlarged images are shown as representative areas.

Article Snippet: An AC activator, forskolin (FSK) [ , ]; an AC inhibitor, SQ22536 [ , ]; a non-selective CGRP receptor agonist, CGRP (rat) [ ]; and a non-selective CGRP receptor antagonist, BIBN 4096 [ ], were obtained from R&D Systems, Inc. (Minneapolis, MN, USA).

Techniques: Fluorescence, Microscopy

Journal: Biomolecules

Article Title: Gα s -Coupled CGRP Receptor Signaling Axis from the Trigeminal Ganglion Neuron to Odontoblast Negatively Regulates Dentin Mineralization

doi: 10.3390/biom12121747

Figure Lengend Snippet: CGRP receptor signaling regulates the defensive reaction of odontoblasts. ( A – D ) Isolated rat dental pulp slices were cultured for 7 days in a mineralization medium without pharmacological intervention ( A ) or with 50 nM CGRP ( B ), 50 nM CGRP with 0.1 nM BIBN 4096 ( C ), or 50 nM CGRP with 0.1 μM SQ22536 ( D ) at pH 7.4 and stained using Alizarin red (red, calcium deposition). White dotted lines indicate the borderline between mineralized odontoblasts and dental pulp. Asterisks indicate the dental pulp area. Data were obtained using a 20× magnification objective lens under a microscope. Each dataset was tiled with 63 photos to acquire the constructed data. To obtain data on the mineralized area constituted by odontoblasts, the mineralized area in the total area ( I ) was divided by the mineralized area of the dental pulp ( I 0 ). ( E ) The estimated mineralization levels were 2.89 ± 0.43 I / I 0 in the absence of CGRP receptor modifiers (as controls), 1.64 ± 0.1 I / I 0 with 50 nM CGRP, 2.91 ± 0.29 I / I 0 with 50 nM CGRP and 0.1 nM BIBN 4096, and 2.82 ± 0.29 I / I 0 with 50 nM CGRP and 0.1 μM SQ22536. Each column denotes the mean ± SE of each experiment. Statistically significant differences between columns (shown by solid lines) are indicated by asterisks. * p < 0.05.

Article Snippet: An AC activator, forskolin (FSK) [ , ]; an AC inhibitor, SQ22536 [ , ]; a non-selective CGRP receptor agonist, CGRP (rat) [ ]; and a non-selective CGRP receptor antagonist, BIBN 4096 [ ], were obtained from R&D Systems, Inc. (Minneapolis, MN, USA).

Techniques: Isolation, Cell Culture, Staining, Microscopy, Construct

Journal: Cells

Article Title: Src Family Kinases Facilitate the Crosstalk between CGRP and Cytokines in Sensitizing Trigeminal Ganglion via Transmitting CGRP Receptor/PKA Pathway

doi: 10.3390/cells11213498

Figure Lengend Snippet: The protein level of phosphorylated SFKs at Y416 was increased by CGRP, which was reduced by both PKI (14-22) Amide and BIBN4096 in the mouse TG. ( A ) The representative Western blot bands of phosphorylated SFKs at Y416, SFKs, and β-actin subjected to the treatment with Kreb’s, 3 µM CGRP, 30 µM PKI (14-22) Amide or 10 µM BIBN4096 in the presence of 3 µM CGRP for 20 min. ( B – D ) Effects of Kreb’s ( n = 9), 3 μM CGRP ( n = 9), 30 μM PKI (14-22) Amide ( n = 8) or 3 µM BIBN4096 ( n = 8) in the presence of 3 μM CGRP on the protein levels of phosphorylated SFKs at Y416 and SFKs relative to that of β-actin and on the protein level of phosphorylated SFKs at Y416 relative to that of SFKs, all of which were presented in the absolute ratio. Abbreviations: PKI (14-22) Amide (PKI), BIBN4086 (BIBN), phosphorylated SFKs at Y416 (pSFKs). Two-tailed unpaired t -test was used for the comparison in the protein level of phosphorylated SFKs at Y416 between the CGRP group and either the Kreb’s group, the PKI (14-22) Amide, or the BIBN4096 in the presence of CGRP groups. Significant differences were labeled as * p < 0.05, ** p < 0.01, or *** p < 0.001. Original western blot images for the representative images in were shown in .

Article Snippet: To inhibit CGRP receptor, a CGRP receptor inhibitor, BIBN4096 (4561, Tocris, Bristol, UK), was used.

Techniques: Western Blot, Two Tailed Test, Comparison, Labeling

Journal: Cells

Article Title: Src Family Kinases Facilitate the Crosstalk between CGRP and Cytokines in Sensitizing Trigeminal Ganglion via Transmitting CGRP Receptor/PKA Pathway

doi: 10.3390/cells11213498

Figure Lengend Snippet: Model of SFKs activity facilitating the crosstalk between CGRP and cytokines by transmitting CGRP receptor signaling to potentiate TG sensitization. SFKs are activated in response to ROS to induce CGRP release in small to medium neurons; released CGRP binds to CGRP receptor (dotted line with arrow) to activate SFKs in large neurons and satellite glial cells, which causes IL-1β, CCL2, CXCL1 release and IL-1β, CCL2 gene expression, thus leading to TG sensitization.

Article Snippet: To inhibit CGRP receptor, a CGRP receptor inhibitor, BIBN4096 (4561, Tocris, Bristol, UK), was used.

Techniques: Activity Assay, Expressing