Journal: Science Advances

Article Title: Sympathetic nerve aggravates autoimmune skin disease via NE–adrenergic receptor axis: Neuroimmune cross-talk insights from vitiligo

doi: 10.1126/sciadv.aea7017

Figure Lengend Snippet: ( A and B ) Flow cytometry and statistical analysis of CD8 + CD45 + T cells in the mouse tail epidermis and dermis of vehicle, 6-OHDA, and 6-OHDA + NE groups ( n =3 per group). ( C and D ) Flow cytometry and statistical analysis of IFN-γ + CD8 + T cells in the mouse tail epidermis and dermis of vehicle, 6-OHDA, and 6-OHDA + NE groups ( n =3 per group). ( E and F ) Flow cytometry and statistical analysis of Granzyme B + CD8 + T cells in the mouse tail epidermis and dermis of vehicle, 6-OHDA, and 6-OHDA + NE groups ( n =3 per group). ( G ) The secretion levels of CXCL9, CXCL10, IL-6, and IL-15 in the tail skin of mice in the vehicle, 6-OHDA, and 6-OHDA + NE groups were detected by ELISA ( n =4 per group). All vehicle mice were only treated with 0.1% ascorbic acid in 0.9% sterile NaCl. Error bars represent mean ± SD from three independent experiments. * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: ELISA analysis on serum samples, skin samples, and cell culture supernatants were performed using the NE ELISA Kit (E-EL-0047c, Elabscience, Wuhan, China), Human CXCL9 ELISA Kit (EHC114.96, Neobioscience Technology Co, Ltd., China), Human CXCL10 ELISA Kit (EHC157.96, Neobioscience Technology Co, Ltd., China), Human IL-6 ELISA Kit (EHC007.96, Neobioscience Technology Co, Ltd., China), Human IL-15 ELISA Kit (EHC013.96, Neobioscience Technology Co, Ltd., China), Mouse CXCL9 ELISA Kit (E-EL-M3077, Elabscience, Wuhan, China), Mouse CXCL10 ELISA Kit (E-EL-M0021, Elabscience, Wuhan, China), Mouse IL-6 ELISA Kit (EMC004.96, Neobioscience Technology Co, Ltd., China), and Mouse IL-15 ELISA Kit (EMC126.96, Neobioscience Technology Co, Ltd., China) following the manufacturer’s instructions.

Techniques: Flow Cytometry, Enzyme-linked Immunosorbent Assay, Sterility

Journal: Science Advances

Article Title: Sympathetic nerve aggravates autoimmune skin disease via NE–adrenergic receptor axis: Neuroimmune cross-talk insights from vitiligo

doi: 10.1126/sciadv.aea7017

Figure Lengend Snippet: ( A ) Volcano plots illustrated the expression of adrenergic receptors on fibroblasts in human skin, including ADRA1A, ADRA1B, ADRA1D, ADRA2A, ADRA2B, ADRA2C, ADRB1, ADRB2, and ADRB3. ( B ) Bubble map illustrated the up-regulation of ADRA2A expression on fibroblasts in the lesional skin of patients with vitiligo and the normal skin of healthy controls. ( C ) Representative immunofluorescence images of fibroblasts, ADRA2A, CXCL9, CXCL10, and CD8 + T cells from the normal skin of the healthy control and the lesional skin of the patient with vitiligo ( n =3 per group). Nuclei were counterstained with DAPI (blue). Scale bars, 50 μm. ( D ) Immunofluorescence analysis of ADRA2A (red) expression in BJ cells after treatment with 5 μM NE for 48 hours. Cell nuclei were stained with DAPI (blue). Scale bar, 50 μm. ( E ) Secretion levels of CXCL9, CXCL10, IL-6, and IL-15 in BJ cells were detected by ELISA with treatment with aposcopolamine (Apos) or NE ( n =3 per group). ( F ) Secretion levels of CXCL9, CXCL10, IL-6, and IL-15 in BJ cells with ADRA2A siRNA or control siRNA were detected by ELISA ( n =3 per group). Error bars represent mean ± SD from three independent experiments. * P < 0.05, ** P < 0.01, and *** P < 0.001; ns, not significant.

Article Snippet: ELISA analysis on serum samples, skin samples, and cell culture supernatants were performed using the NE ELISA Kit (E-EL-0047c, Elabscience, Wuhan, China), Human CXCL9 ELISA Kit (EHC114.96, Neobioscience Technology Co, Ltd., China), Human CXCL10 ELISA Kit (EHC157.96, Neobioscience Technology Co, Ltd., China), Human IL-6 ELISA Kit (EHC007.96, Neobioscience Technology Co, Ltd., China), Human IL-15 ELISA Kit (EHC013.96, Neobioscience Technology Co, Ltd., China), Mouse CXCL9 ELISA Kit (E-EL-M3077, Elabscience, Wuhan, China), Mouse CXCL10 ELISA Kit (E-EL-M0021, Elabscience, Wuhan, China), Mouse IL-6 ELISA Kit (EMC004.96, Neobioscience Technology Co, Ltd., China), and Mouse IL-15 ELISA Kit (EMC126.96, Neobioscience Technology Co, Ltd., China) following the manufacturer’s instructions.

Techniques: Expressing, Immunofluorescence, Control, Staining, Enzyme-linked Immunosorbent Assay

Journal: Science Advances

Article Title: Sympathetic nerve aggravates autoimmune skin disease via NE–adrenergic receptor axis: Neuroimmune cross-talk insights from vitiligo

doi: 10.1126/sciadv.aea7017

Figure Lengend Snippet: ( A ) Volcano plots illustrated the expression of adrenergic receptors on keratinocytes in human skin, including ADRA1A, ADRA1B, ADRA1D, ADRA2A, ADRA2B, ADRA2C, ADRB1, ADRB2, and ADRB3. ( B ) Bubble map illustrated the up-regulation of ADRB2 expression on keratinocytes in the lesional skin of patients with vitiligo and the normal skin of healthy controls. ( C ) Representative immunofluorescence images of keratinocytes, ADRB2, CXCL9, CXCL10, and CD8 + T cells from the normal skin of the healthy control and the lesional skin of the patient with vitiligo ( n =3 per group). Nuclei were counterstained with DAPI (blue). Scale bars, 50 μm. ( D ) Immunofluorescence analysis of ADRB2 (red) expression in keratinocytes after treatment with 5 μM NE for 48 hours. Cell nuclei were stained with DAPI (blue). Scale bar, 50 μm. ( E ) Secretion levels of CXCL9, CXCL10, IL-6, and IL-15 in keratinocytes were detected by ELISA with treatment with NE or ICI ( n =3 per group). ( F ) Secretion levels of CXCL9, CXCL10, IL-6, and IL-15 in keratinocytes with ADRB2 siRNA or control siRNA were detected by ELISA ( n =3 per group). Error bars represent mean ± SD from three independent experiments. * P < 0.05, ** P < 0.01, and *** P < 0.001; ns, not significant.

Article Snippet: ELISA analysis on serum samples, skin samples, and cell culture supernatants were performed using the NE ELISA Kit (E-EL-0047c, Elabscience, Wuhan, China), Human CXCL9 ELISA Kit (EHC114.96, Neobioscience Technology Co, Ltd., China), Human CXCL10 ELISA Kit (EHC157.96, Neobioscience Technology Co, Ltd., China), Human IL-6 ELISA Kit (EHC007.96, Neobioscience Technology Co, Ltd., China), Human IL-15 ELISA Kit (EHC013.96, Neobioscience Technology Co, Ltd., China), Mouse CXCL9 ELISA Kit (E-EL-M3077, Elabscience, Wuhan, China), Mouse CXCL10 ELISA Kit (E-EL-M0021, Elabscience, Wuhan, China), Mouse IL-6 ELISA Kit (EMC004.96, Neobioscience Technology Co, Ltd., China), and Mouse IL-15 ELISA Kit (EMC126.96, Neobioscience Technology Co, Ltd., China) following the manufacturer’s instructions.

Techniques: Expressing, Immunofluorescence, Control, Staining, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Herpes Simplex Virus Type 2 Infection-Induced Expression of CXCR3 Ligands Promotes CD4 + T Cell Migration and Is Regulated by the Viral Immediate-Early Protein ICP4

doi: 10.3389/fimmu.2018.02932

Figure Lengend Snippet: Contribution of HSV-2 infection-induced CXCR3 ligands to CD4 + T cell infiltration into mouse vagina. Seven days prior to HSV-2 challenge, BALB/c mice were injected with progesterone in multiple sites. One day prior to HSV-2 challenge, CXCL9, CXCL10, and CXCL11 neutralizing antibodies were delivered to the vagina of mice, alone or in combination, while isotype matched control IgG was used as the control. Mice were then anesthetized with pentobarbital sodium and challenged intravaginally with 10 μL/ mouse HSV-2 at a concentration of 6 × 10 7 PFU/ml or mock- challenged. Vaginal lavage fluids and cervical-vaginal tissues were collected at day 7 after challenge. (A) HSV-2 infection induces the production of mouse CXCR3 ligands. The protein levels of CXCL9 and CXCL10 ligands in vaginal lavage fluids were measured by CBA, and the protein level of CXCL11 was detected by ELISA. (B) CXCL9 mediates the migration of CD4 + T cells to the vaginal foci of infected mice. CD4 + T cells in infection foci were detected using anti-CD4 Ab by IHC. The scale bar indicates 100 μm. Data shown are mean ± S.D. ( n = 5 mice/group) of three independent experiments (A) . *** p < 0.001. One representative out of three independent experiments is shown (B) .

Article Snippet: Abs against mouse CD4, CXCL9, CXCL10, and CXCL11 were purchased from R&D Systems (MAB554, AF-492-NA, AF-466-NA, and AF-572, USA).

Techniques: Infection, Injection, Control, Concentration Assay, Enzyme-linked Immunosorbent Assay, Migration

Journal: Frontiers in Immunology

Article Title: Herpes Simplex Virus Type 2 Infection-Induced Expression of CXCR3 Ligands Promotes CD4 + T Cell Migration and Is Regulated by the Viral Immediate-Early Protein ICP4

doi: 10.3389/fimmu.2018.02932

Figure Lengend Snippet: HSV-2 infection induces the production of CXCR3 ligands in human cervical epithelial cells. (A) HSV-2 infection activates the promoters of human CXCR3 ligands. ME180 cells in 24-well plates were co-transfected with 150 ng CXCL9-Luc, CXCL10-Luc or CXCL11-Luc, and 15 ng internal control plasmid phRL-TK. At 4 h post-transfection, cells were infected with HSV-2 or ultraviolet-inactivated HSV-2 (UV-HSV-2) at an MOI of 1 for 24 h. DLR assay was performed. Values for the samples were normalized using Renilla luciferase values and expressed as fold increase of the value induced in mock-infected samples. (B) HSV-2 infection induces the mRNA production of CXCR3 ligands. ME180 cells in 6-well plates were infected with HSV-2 at an MOI of 1 for 24 h. Cells were harvested and total RNA was extracted. The expression of CXCR3 ligands and GAPDH was evaluated by relative real-time quantitative PCR. The Ct values of GAPDH among all groups were equable and not overloaded. mRNA copies of CXCR3 ligands were normalized using GAPDH and expressed as fold increase of the value for the mock-infected control. (C) HSV-2 infection induces the production of CXCR3 ligands. As depicted in (B) , cell supernatants were collected, and the protein level of CXCR3 ligands was measured by CBA. Data shown are mean ± S.D. of three independent experiments (A, B, and C). * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Abs against mouse CD4, CXCL9, CXCL10, and CXCL11 were purchased from R&D Systems (MAB554, AF-492-NA, AF-466-NA, and AF-572, USA).

Techniques: Infection, Transfection, Control, Plasmid Preparation, Luciferase, Expressing, Real-time Polymerase Chain Reaction

Journal: Frontiers in Immunology

Article Title: Herpes Simplex Virus Type 2 Infection-Induced Expression of CXCR3 Ligands Promotes CD4 + T Cell Migration and Is Regulated by the Viral Immediate-Early Protein ICP4

doi: 10.3389/fimmu.2018.02932

Figure Lengend Snippet: HSV-2 infection-induced CXCL9 plays a predominant role in mediating CD4 + T cell migration. (A) The concentrations of CXCR3 ligands in the supernatants of ME180 cells infected with HSV-2 or mock-infected with DMEM were detected by CBA. (B,C) CXCL9 induced by HSV-2 recruits the migration of PBMCs (B) and CD4 + T cells (C) . ME180 cells in 6-well plates were infected with HSV-2 at an MOI of 1 for 24 h. Cell supernatants were collected and added to the lower chamber of transwell plates in the absence or presence of anti-CXCL9, –CXCL10, or/and –CXCL11 neutralizing Ab or control Ab for 1 h. (D) Neutralization of CXCR3 reduces the migration of CD4 + T cells induced by HSV-2 infection. ME180 cells in 6-well plates were infected with HSV-2 at an MOI of 1 for 24 h. Cell supernatants were collected and added to the lower chamber of transwell plates. The activated CD4 + T cells were incubated with RPMI 1,640 medium containing anti-CXCR3 neutralizing Ab for 1 h and placed in the upper chamber. (E) Recombinant CXCL9 significantly induces the migration of CD4 + T cells. DMEM containing recombinant CXCL9, CXCL10, or CXCL11 (48 pg/mL, 55 pg/mL and 175 pg/mL, respectively; the lowest concentration induced by HSV-2 infection) was added to the lower chamber of transwell plates. (F,G) Recombinant CXCL10 or CXCL11 mediates the migration of CD4 + T cells in a dose-dependent manner. DMEM containing recombinant CXCL10 or CXCL11 was added to the lower chamber of transwell plates. CXCL10 or CXCL11 was started from 55 pg/mL and 175 pg/mL, respectively, at a concentration gradient of two times. The activated CD4 + T cells were placed in the upper chamber. After 2 h incubation, cells migrated to lower chambers were collected and counted using an automatic cell counter. Cells migration was expressed as percentage of input. Input cells in the upper chamber were 5 × 10 5 . Data shown are mean ± S.D. of three independent experiments (A–G) . ns, not significant, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Abs against mouse CD4, CXCL9, CXCL10, and CXCL11 were purchased from R&D Systems (MAB554, AF-492-NA, AF-466-NA, and AF-572, USA).

Techniques: Infection, Migration, Control, Neutralization, Incubation, Recombinant, Concentration Assay

Journal: Frontiers in Immunology

Article Title: Herpes Simplex Virus Type 2 Infection-Induced Expression of CXCR3 Ligands Promotes CD4 + T Cell Migration and Is Regulated by the Viral Immediate-Early Protein ICP4

doi: 10.3389/fimmu.2018.02932

Figure Lengend Snippet: HSV-2 ICP4 promotes the production of human CXCR3 ligands. (A) ICP4 induces the activation of CXCR3 ligand promoters. ME180 cells in 24-well plates were transfected with 300 ng expression plasmid of HSV-2 gene or empty vector together with 150 ng CXCR3 ligand reporter and 15 ng phRL-TK. At 24 h post-transfection, DLR assay was performed. Values for the samples were normalized using Renilla luciferase values and expressed as fold increase of the value induced in cells transfected with empty vector. (B) The expression of HSV-2 genes was detected using anti-Flag Ab by Western Blot. ME180 cells were transfected with 3 μg HSV-2 gene expression plasmid for 24 h. The proteins were collected and detected using mouse anti-Flag Ab. (C) ICP4 induces the mRNA production of CXCR3 ligands. ME180 cells in 6-well plates were transfected with 3 μg ICP4 expression plasmid for 24 h. Cells were harvested and total RNA was extracted. The expression of CXCR3 ligands and GAPDH gene was evaluated by relative real-time quantitative PCR. The Ct values of GAPDH among all groups were equable and not overloaded. mRNA copies of CXCR3 ligands were normalized using GAPDH and expressed as fold increase of the value for the empty vector-transfected control. (D) ICP4 induces the production of CXCR3 ligands. As depicted in (C) , cell supernatants were collected, and the protein levels of CXCR3 ligands were measured by CBA. (E,F) CXCL9 induced by ICP4 recruits the migration of PBMCs (E) and CD4 + T cells (F) . ME180 cells in 6-well plates were transfected with 3 μg ICP4 expression plasmid for 24 h. Cell supernatants were collected and added to the lower chamber of transwell plates in the absence or presence of anti-CXCL9, –CXCL10, or/and –CXCL11 neutralizing Ab or control Ab for 1h. (G) Neutralization of CXCR3 reduces the migration of CD4 + T cells induced by ICP4. ME180 cells in 6-well plates were infected with HSV-2 at an MOI of 1 for 24 h. Cell supernatants were collected and added to the lower chamber of transwell plates. The activated CD4 + T cells were incubated with RPMI 1,640 medium containing anti-CXCR3 neutralizing Ab for 1 h and placed in the upper chamber. As depicted in Figure , cells migrated to lower chambers were counted. Cells migration was expressed as percentage of input. One representative out of three independent experiments is shown (B) . Data shown are mean ± S.D. of three independent experiments (A,C–G) . ns, not significant, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Abs against mouse CD4, CXCL9, CXCL10, and CXCL11 were purchased from R&D Systems (MAB554, AF-492-NA, AF-466-NA, and AF-572, USA).

Techniques: Activation Assay, Transfection, Expressing, Plasmid Preparation, Luciferase, Western Blot, Gene Expression, Real-time Polymerase Chain Reaction, Control, Migration, Neutralization, Infection, Incubation

Journal: Frontiers in Immunology

Article Title: Herpes Simplex Virus Type 2 Infection-Induced Expression of CXCR3 Ligands Promotes CD4 + T Cell Migration and Is Regulated by the Viral Immediate-Early Protein ICP4

doi: 10.3389/fimmu.2018.02932

Figure Lengend Snippet: HSV-2 ICP4 regulates the expression of CXCR3 ligands via the p38 MAPK signaling pathway. (A–C) HSV-2 regulates the expression of CXCL9 (A) , CXCL10 (B) , and CXCL11 (C) via p38/MAPK signaling pathway. ME180 cells in 24-well plates were co-transfected with 150 ng CXCR3 ligand reporter and 15 ng phRL-TK. At 4 h post-transfection, cells were infected with HSV-2 at an MOI of 1 and supplemented with inhibitor PD98059, SP600125, or SB203580. DLR assay was performed at 24 h post-transfection. Values for the samples were normalized using Renilla luciferase values and expressed as fold increase of the value induced in mock-infected samples. (D–F) HSV-2 ICP4 regulates the expression of CXCL9 (D) , CXCL10 (E) , and CXCL11 (F) via p38/MAPK signaling pathway. ME180 cells in 24-well plates were co-transfected with 300 ng empty vector or ICP4 expression plasmid together with 150 ng CXCR3 ligand reporter and 15 ng phRL-TK. At 4 h post-transfection, cells were cultured in complete DMEM supplemented with inhibitor PD98059, SP600125, or SB203580. DLR assay was performed at 24 h post-transfection. Values for the samples were normalized using Renilla luciferase values and expressed as fold increase of the value induced in cells transfected with empty vector. (G) ICP4 activates p38 MAPK signaling pathway. ME180 cells were transfected with 3 μg ICP4 expression plasmid. The protein level of p38, phospho-p38 (p-p38) or phospho-C/EBP-β (p-C/EBP-β) was detected by Western Blot. Data shown are mean ± S.D. of three independent experiments (A–F) . ns, not significant, *** p < 0.001. One representative out of three independent experiments is shown (G) .

Article Snippet: Abs against mouse CD4, CXCL9, CXCL10, and CXCL11 were purchased from R&D Systems (MAB554, AF-492-NA, AF-466-NA, and AF-572, USA).

Techniques: Expressing, Transfection, Infection, Luciferase, Plasmid Preparation, Cell Culture, Western Blot

Journal: eLife

Article Title: A new genetic strategy for targeting microglia in development and disease

doi: 10.7554/eLife.54590

Figure Lengend Snippet:

Article Snippet: Antibody , Anti-CXCL10 , R and D Systems , Goat polyclonal antibody. Catalog No: AF-466-NA. RRID: AB_2292487 . , Used at 1:300..

Techniques: