Journal: International Journal of Molecular Sciences

Article Title: Chemokinergic and Dopaminergic Signalling Collaborates through the Heteromer Formed by CCR9 and Dopamine Receptor D5 Increasing the Migratory Speed of Effector CD4 + T-Cells to Infiltrate the Colonic Mucosa

doi: 10.3390/ijms251810022

Figure Lengend Snippet: CCR9:DRD5 heteromer signalling increases the migratory speed of CD4 + T-cells in microchannels. Naïve CD4 + T-cells were isolated from the spleen of wild-type mice ( Drd5 +/+ ) and then activated with anti-CD3/anti-CD28 mAbs-coated dynabeads in the presence of IL-2 and RA for 5 d to induce gut tropism. Afterwards, cells were individually tracked in 3 μm-width confined microchannels under different conditions and the migratory speed was determined. ( A ) Migratory speed in response to increasing concentrations of CCL25 was determined; n = 52–163 cells per condition. ( B ) Migratory speed was determined in the presence of CCL25 (50 ng/mL) alone or with dopamine (1 μM), or with SKF81297 (100 nM) in the absence or in the presence of a TM-peptide irrelevant for CCR9:DRD5 heteromer assembly (TM1D) or of a TM-peptide that disrupt CCR9:DRD5 heteromer assembly (TM6D); n = 83–160 cells per condition. ( A , B ) Data correspond to the median migratory speed of lymphocytes in μm/min. In the box plots, the bars include 90% of the data points, the horizontal line in the box indicates the median and the box contains 75% of the data points. Data from two independent experiments are shown. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001 via one-way ANOVA followed by Tukey’s post hoc test. ns, non-significant.

Article Snippet: The peptide analogues to transmembrane (TM) segments derived from CCR9 and DRD5 ( ) were synthesized by GenScript (Piscataway, NJ, USA).

Techniques: Isolation

Journal: International Journal of Molecular Sciences

Article Title: Chemokinergic and Dopaminergic Signalling Collaborates through the Heteromer Formed by CCR9 and Dopamine Receptor D5 Increasing the Migratory Speed of Effector CD4 + T-Cells to Infiltrate the Colonic Mucosa

doi: 10.3390/ijms251810022

Figure Lengend Snippet: CCR9:DRD5 heteromer signalling involves the activation of the myosine light chain 2. Naïve CD4 + T-cells were isolated from the spleen of wild-type mice ( Drd5 +/+ ) and then activated with anti-CD3/anti-CD28 mAbs-coated dynabeads in the presence of IL-2 and RA for 5 d to induce gut tropism. During the last 4 h, cells were non-stimulated (vehicle) or stimulated with CCL25 (300 ng/mL) and SKF81297 (100 nM) in the presence of 4 μM of peptides TM6C or TM7C, or only DMSO as a control. Cells were stained for extracellular expression of CD4 and intracellular phosphorylation of MLC2. ( A ) Representative histograms showing the fluorescence distribution associated with the immunostaining of pMLC2 in the CD4 + live (ZAq − ) population. ( B ) Quantification of the extent of pMLC2. Values are the mean fluorescence intensity associated with the immunostaining of pMLC2. Data are represented as the mean ± SEM from three independent experiments. **, p < 0.01 via two-way ANOVA followed by the Tukey’s multiple comparisons post hoc test.

Article Snippet: The peptide analogues to transmembrane (TM) segments derived from CCR9 and DRD5 ( ) were synthesized by GenScript (Piscataway, NJ, USA).

Techniques: Activation Assay, Isolation, Control, Staining, Expressing, Phospho-proteomics, Fluorescence, Immunostaining

Journal: International Journal of Molecular Sciences

Article Title: Chemokinergic and Dopaminergic Signalling Collaborates through the Heteromer Formed by CCR9 and Dopamine Receptor D5 Increasing the Migratory Speed of Effector CD4 + T-Cells to Infiltrate the Colonic Mucosa

doi: 10.3390/ijms251810022

Figure Lengend Snippet: The disassembling of the CCR9:DRD5 heteromer reduces the recruitment of CD4+ T-cells into the colonic mucosa. Naïve CD4 + T-cells were isolated from the spleen of wild-type mice ( Drd5 +/+ ) and then activated with anti-CD3/anti-CD28 mAbs-coated dynabeads in the presence of IL-2 and RA for 5 d to induce gut tropism. During the last 4 h, cells were treated with 4 μM of peptides TM6C or TM7C, or only DMSO as a control. Afterwards, cells were i.v. transferred (6 × 10 6 cells/mouse) into Drd5 −/− mice which were previously exposed to 1.75% DSS for 3 d. Mice were exposed to 1.75% DSS for 3 more days, sacrificed, and PLA was conducted on colonic tissue. ( A ) Representative images obtained from PLA analysis. Arrows show PLA + cells. Bar, 20 μm. Images with higher magnification are shown in the right-bottom corner. ( B ) Quantification of PLA + cells. Values are the number of PLA + cells per field. Data are represented as the mean ± SEM from three independent experiments. Each symbol represents a different field. *, p < 0.05; **, p < 0.01 via one-way ANOVA followed by Sidak’s multiple comparisons post hoc test.

Article Snippet: The peptide analogues to transmembrane (TM) segments derived from CCR9 and DRD5 ( ) were synthesized by GenScript (Piscataway, NJ, USA).

Techniques: Isolation, Control

Journal: International Journal of Molecular Sciences

Article Title: Chemokinergic and Dopaminergic Signalling Collaborates through the Heteromer Formed by CCR9 and Dopamine Receptor D5 Increasing the Migratory Speed of Effector CD4 + T-Cells to Infiltrate the Colonic Mucosa

doi: 10.3390/ijms251810022

Figure Lengend Snippet: Analysis of non-equilibrium simulation during the pulling away of DRD5 TMs from the whole CCR9 protein. ( A ) Distance change during simulation at the first part of the method, before the transition point. ( B ) Simulation time when the transition point is reached. ( C ) Maximum force reached around transition point. Each symbol represents an individual simulation ( n = 6). The mean ± SEM is shown. ***, p < 0.001 via one-way ANOVA followed by Sidak’s multiple comparisons post hoc test.

Article Snippet: The peptide analogues to transmembrane (TM) segments derived from CCR9 and DRD5 ( ) were synthesized by GenScript (Piscataway, NJ, USA).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Chemokinergic and Dopaminergic Signalling Collaborates through the Heteromer Formed by CCR9 and Dopamine Receptor D5 Increasing the Migratory Speed of Effector CD4 + T-Cells to Infiltrate the Colonic Mucosa

doi: 10.3390/ijms251810022

Figure Lengend Snippet: Analysis of non-equilibrium simulation during the pulling away of CCR9 TMs from the whole DRD5 protein. ( A ) Distance change during simulation at the first part of the method, before the transition point. ( B ) Simulation time when the transition point is reached. ( C ) Maximum force reached around the transition point. Each symbol represents an individual simulation ( n = 6). The mean ± SEM is shown. ***, p < 0.001 via one-way ANOVA followed by Sidak’s multiple comparisons post hoc test.

Article Snippet: The peptide analogues to transmembrane (TM) segments derived from CCR9 and DRD5 ( ) were synthesized by GenScript (Piscataway, NJ, USA).

Techniques: