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: 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: Gut microbes

Article Title: Lacticaseibacillus paracasei sh2020 induced antitumor immunity and synergized with anti-programmed cell death 1 to reduce tumor burden in mice.

doi: 10.1080/19490976.2022.2046246

Figure Lengend Snippet: Figure 7. L. paracasei sh2020 promoted the expression and secretion of CXCL10 in vivo and in vitro. (a) The expression of CXCL9, CXCL10 and CXCL11 in tumor tissues from control and L. paracasei sh2020-treated mice was detected by qRT-PCR. (b-c) Representative images (b) and quantification (c) of IHC staining of CXCL10 in the tumor tissues from control and L. paracasei sh2020-treated tumors. (d) Tumor growth in each group. (e) The levels of CXCL10 in the conditioned medium. (f-g) Tumor growth in the tumor-bearing mice with intratumoral injection of L. paracasei sh2020 (n = 5–6). (h-i) Representative images (h) and quantification (i) of IHC staining of CXCL10 and CD8 in each group (n = 4–5). (j) The serum levels of CXCL10 were examined by ELISA. ns, no significant difference, *P < .05, **P < .01, ***P < .001.

Article Snippet: To neutralize Cxcl10 in vivo, 50 μg of anti-mouse Cxcl10 (134013, MAB466, R&D systems) was intraperitoneally injected into mice every 3 days for 2 weeks.

Techniques: Expressing, In Vivo, In Vitro, Control, Quantitative RT-PCR, Immunohistochemistry, Injection, Enzyme-linked Immunosorbent Assay

Journal: Gut microbes

Article Title: Lacticaseibacillus paracasei sh2020 induced antitumor immunity and synergized with anti-programmed cell death 1 to reduce tumor burden in mice.

doi: 10.1080/19490976.2022.2046246

Figure Lengend Snippet: Figure 8. CXCL10 controlled CD8+ T cell migration and the effect of L. paracasei sh2020 in vivo. (a-b) Representative images of IHC staining of CD8 and CXCL10 (a), and quantification (b) for the control and L. paracasei sh2020-treated tumors (n = 6–7). (c) IHC analysis of CD8 in tumors, which were divided into two groups according to CXCL10 high and low expression. (d) Experimental design: C57BL/6 mice were implanted subcutaneously with 5.0 × 105 MC38 cells and was treated with control vehicle or anti-CXCL10 antibody by intraperitoneal injection, every 3 days starting on D3, in total three times. The mice were given L. paracasei sh2020 with a dose of 1.0 × 109 CFU by gavage starting from D0 to D13. (e) Tumor growth in tumor-bearing mice in d. (f) Quantification of IHC staining of CXCL10 and CD8 in the tumors after neutralizing CXCL10 in vivo (n = 4–5). ns, no significant difference, *P < .05, **P < .01, ***P < .001.

Article Snippet: To neutralize Cxcl10 in vivo, 50 μg of anti-mouse Cxcl10 (134013, MAB466, R&D systems) was intraperitoneally injected into mice every 3 days for 2 weeks.

Techniques: Migration, In Vivo, Immunohistochemistry, Control, Expressing, Injection

Journal: bioRxiv

Article Title: Inflammasome mediated neuronal-microglial crosstalk: a therapeutic substrate in C9orf72 -FTD/ALS

doi: 10.1101/2022.01.27.478078

Figure Lengend Snippet: Principal component analysis (PCA) of samples. Abbreviations: Principal Component 1 (PC1); Principal Component 2 (PC2). Each point represents a single sample, with the tissue of the sample represented as a circle, triangle, or square. The treatment type is shown in red, green, or blue, for GFP and GFP-(GR) 100 (a) . Volcano plots of cortical gene transcription. X-axis represents the log2 fold change, while the Y-axis represents the negative log10 FDR-adjusted P-value. Genes with an adjusted P-value less than 0.10 are shown in blue. The 3 most highly upregulated mapped genes and 3 most highly downregulated genes are highlighted in red (b) . Inflammation related IPA pathways. Select immune system-related pathways are shown for the cortex; the p-value of each entry is represented by the size of the corresponding circle. The z-score, indicating the computed activation or repression of the given pathway, is indicated with a color gradient (c) . RT-qPCR validation of the most highly upregulated gene, Cxcl10 , in the cortex of GFP and GFP-(GR) 100 mice. For d, * p<0.05 by Two tailed Welch’s t Test.

Article Snippet: Cell supernatant and lysates were collected for assessment of LDH release and CXCL10 production (R&D Systems #DY466).

Techniques: Activation Assay, Quantitative RT-PCR, Biomarker Discovery, Two Tailed Test

Journal: bioRxiv

Article Title: Inflammasome mediated neuronal-microglial crosstalk: a therapeutic substrate in C9orf72 -FTD/ALS

doi: 10.1101/2022.01.27.478078

Figure Lengend Snippet: Evaluation of neurotoxicity of GFP-(GR) 100 (a) , Neuronal release of CXCL10 after infection with GFP-(GR) 100 (b) , CXCL10 dependent release of TNF-α (c) , Neutralization of CXCL10 and TNF-α release (d) , CXCL10 dependent release of IL-1β (e) , Neutralization of CXCL10 and IL-1β release (f) . Schematized mechanism of GFP-(GR) 100 inducing neuronal release of CXCL10, which may then bind to microglial receptors and initiate an inflammasome response (g) . The data shown correspond to average values of biological triplicates ran in technical duplicates. For a-f, * p<0.05. **p<0.01. ***p<0.001. ****p<0.0001 by one-way ANOVA.

Article Snippet: Cell supernatant and lysates were collected for assessment of LDH release and CXCL10 production (R&D Systems #DY466).

Techniques: Infection, Neutralization

Journal: eLife

Article Title: CXCL10/CXCR3 signaling contributes to an inflammatory microenvironment and its blockade enhances progression of murine pancreatic precancerous lesions

doi: 10.7554/elife.60646

Figure Lengend Snippet: Figure 1. Pre-neoplastic ADM and PanIN lesions express CXCL10. (A) ‘Normal’ acinar cells (data shown in Figure 1—figure supplement 1B), ADM areas and PanIN lesions of pancreata from KC mice were analyzed for Cxcl10 expression. Shown are representative pictures of H and E staining (overview and marked region) and FISH for Cxcl10 mRNA expression (red dots) combined with DAPI in the marked regions. Images shown represent whole-slide analysis of staining. The scale bar represents 100 mm. (B) Quantification of relative Cxcl10 expression (as determined by ISH shown in Figure 1 continued on next page

Article Snippet: DOI: https://doi.org/10.7554/eLife.60646 22 of 24 Appendix 1—key resources table continued Reagent type (species) or resource Designation Source or reference Identifiers Additional information Antibody anti-pY701-STAT1 (Rabbit monoclonal) Cell Signaling Technology Cat# 9167, RRID: AB_561284 IF (1:400) Antibody anti-pY701-STAT1 (Mouse monoclonal) Abcam Cat# ab29045, RRID:AB_778096 IF (1:200), WB (1:1000) Antibody anti-STAT1 (Rabbit polyclonal) Cell Signaling Technology Cat# 9172, RRID: AB_2198300 WB (1:1000) Antibody anti-YM1 (Rabbit polyclonal) STEMCELL Technologies Cat# 60130, RRID: AB_2868482 IF (1:200) Antibody anti-YM1/2 (Rabbit monoclonal) Abcam Cat# ab205491, RRID:AB_2891303 FC (1 mg/1 106 cells), IF (1:100) Antibody CXCL10 neutralizing antibody (NAB; Rat monoclonal) R and D Systems Cat# MAB466, RRID:AB_2292486 CXCL10 NAB Antibody Isotype control IgG2A antibody (Rat monoclonal) R and D Systems Cat# MAB006, RRID:AB_357349 CXCL10 NAB control Antibody CXCR3 neutralizing antibody (NAB; Armenian hamster monoclonal) Bio X Cell Cat# BE0249, RRID: AB_2687730 CXCR3 NAB Antibody Isotype control IgG antibody (Armenian hamster polyclonal) Bio X Cell Cat# BE0091, RRID: AB_1107773 CXCR3 NAB control Antibody anti-CD4 (Rat monoclonal) Bio X Cell Cat# BP0003-1, RRID:AB_2891358 T-cell depletion Antibody anti-CD8a (Rat monoclonal) Bio X Cell Cat# BP0061, RRID: AB_2891359 T-cell depletion Antibody Isotype control IgG2b antibody (Rat monoclonal) Bio X Cell Cat# BP0090, RRID: AB_2891360 T-cell depletion control Cell line (Mus musculus) SM3 Agbunag et al., 2006; Liou et al., 2017 Primary duct-like cells from KC mouse Chemical compound, drug NVP-BSK805 Selleckchem Cat# S2686 pan-JAK inhibitor Commercial assay or kit Mouse tumor dissociation kit Miltenyi Biotec Cat# 130-096-730 Commercial assay or kit RNAscope Assay 2.5 HD Reagent Kit- Brown Advanced Cell Diagnostics In situ hybridization (brown) Commercial assay or kit RNAscope Multiplex Fluorescent Reagent Kit v2 Advanced Cell Diagnostics In situ hybridization (fluorescent) Peptide, recombinant protein Recombinant murine CXCL10 Peprotech Cat# 250–16 Peptide, recombinant protein Recombinant murine IL-4 Peprotech Cat# 214–14 Peptide, recombinant protein Recombinant murine IFNV Peprotech Cat# 315–05 Sequence-based reagent Arg1 TaqMan (Thermo Fisher Scientific) Mm00475988_m1 qPCR probe Sequence-based reagent Chil3 TaqMan (Thermo Fisher Scientific) Mm00657889_mH qPCR probe Sequence-based reagent Cxcl10 TaqMan (Thermo Fisher Scientific) Mm00445235_m1 qPCR probe

Techniques: Expressing, Staining

Journal: eLife

Article Title: CXCL10/CXCR3 signaling contributes to an inflammatory microenvironment and its blockade enhances progression of murine pancreatic precancerous lesions

doi: 10.7554/elife.60646

Figure Lengend Snippet: Figure 2. Inflammatory macrophages are the recipients for CXCL10. (A) Primary peritoneal macrophages were isolated and either left non-polarized or were polarized to M1 and M2 macrophages. CXCR3 expression was determined by qPCR. Results shown are representative of reproducible data from three independent experiments. Statistical analysis using the Student’s t-test indicates significance (marked by an asterisk). Error bars indicate standard deviation. (B) Transwell assay. 0.5 105 non-polarized, M1- or M2-polarized peritoneal macrophages were plated into transwell inserts. 500 ng/ml Figure 2 continued on next page

Article Snippet: DOI: https://doi.org/10.7554/eLife.60646 22 of 24 Appendix 1—key resources table continued Reagent type (species) or resource Designation Source or reference Identifiers Additional information Antibody anti-pY701-STAT1 (Rabbit monoclonal) Cell Signaling Technology Cat# 9167, RRID: AB_561284 IF (1:400) Antibody anti-pY701-STAT1 (Mouse monoclonal) Abcam Cat# ab29045, RRID:AB_778096 IF (1:200), WB (1:1000) Antibody anti-STAT1 (Rabbit polyclonal) Cell Signaling Technology Cat# 9172, RRID: AB_2198300 WB (1:1000) Antibody anti-YM1 (Rabbit polyclonal) STEMCELL Technologies Cat# 60130, RRID: AB_2868482 IF (1:200) Antibody anti-YM1/2 (Rabbit monoclonal) Abcam Cat# ab205491, RRID:AB_2891303 FC (1 mg/1 106 cells), IF (1:100) Antibody CXCL10 neutralizing antibody (NAB; Rat monoclonal) R and D Systems Cat# MAB466, RRID:AB_2292486 CXCL10 NAB Antibody Isotype control IgG2A antibody (Rat monoclonal) R and D Systems Cat# MAB006, RRID:AB_357349 CXCL10 NAB control Antibody CXCR3 neutralizing antibody (NAB; Armenian hamster monoclonal) Bio X Cell Cat# BE0249, RRID: AB_2687730 CXCR3 NAB Antibody Isotype control IgG antibody (Armenian hamster polyclonal) Bio X Cell Cat# BE0091, RRID: AB_1107773 CXCR3 NAB control Antibody anti-CD4 (Rat monoclonal) Bio X Cell Cat# BP0003-1, RRID:AB_2891358 T-cell depletion Antibody anti-CD8a (Rat monoclonal) Bio X Cell Cat# BP0061, RRID: AB_2891359 T-cell depletion Antibody Isotype control IgG2b antibody (Rat monoclonal) Bio X Cell Cat# BP0090, RRID: AB_2891360 T-cell depletion control Cell line (Mus musculus) SM3 Agbunag et al., 2006; Liou et al., 2017 Primary duct-like cells from KC mouse Chemical compound, drug NVP-BSK805 Selleckchem Cat# S2686 pan-JAK inhibitor Commercial assay or kit Mouse tumor dissociation kit Miltenyi Biotec Cat# 130-096-730 Commercial assay or kit RNAscope Assay 2.5 HD Reagent Kit- Brown Advanced Cell Diagnostics In situ hybridization (brown) Commercial assay or kit RNAscope Multiplex Fluorescent Reagent Kit v2 Advanced Cell Diagnostics In situ hybridization (fluorescent) Peptide, recombinant protein Recombinant murine CXCL10 Peprotech Cat# 250–16 Peptide, recombinant protein Recombinant murine IL-4 Peprotech Cat# 214–14 Peptide, recombinant protein Recombinant murine IFNV Peprotech Cat# 315–05 Sequence-based reagent Arg1 TaqMan (Thermo Fisher Scientific) Mm00475988_m1 qPCR probe Sequence-based reagent Chil3 TaqMan (Thermo Fisher Scientific) Mm00657889_mH qPCR probe Sequence-based reagent Cxcl10 TaqMan (Thermo Fisher Scientific) Mm00445235_m1 qPCR probe

Techniques: Isolation, Expressing, Standard Deviation, Transwell Assay

Journal: eLife

Article Title: CXCL10/CXCR3 signaling contributes to an inflammatory microenvironment and its blockade enhances progression of murine pancreatic precancerous lesions

doi: 10.7554/elife.60646

Figure Lengend Snippet: Figure 3. CXCL10/CXCR3 signaling maintains the inflammatory phenotype of macrophages. (A) Peritoneal macrophages were isolated, polarized to M1, and then treated with 500 mg/ml CXCR3 NAB or isotype control IgG. After 48 hr treatment, samples were analyzed for expression of iNOS or Ym1 using immunofluorescence. Images shown are representative of three independent experiments done on peritoneal macrophages obtained from three mice (biological replicates). The scale bar indicates 100 mm. (B) Peritoneal macrophages were isolated, polarized to M1, and treated with 500 mg/ml Figure 3 continued on next page

Article Snippet: DOI: https://doi.org/10.7554/eLife.60646 22 of 24 Appendix 1—key resources table continued Reagent type (species) or resource Designation Source or reference Identifiers Additional information Antibody anti-pY701-STAT1 (Rabbit monoclonal) Cell Signaling Technology Cat# 9167, RRID: AB_561284 IF (1:400) Antibody anti-pY701-STAT1 (Mouse monoclonal) Abcam Cat# ab29045, RRID:AB_778096 IF (1:200), WB (1:1000) Antibody anti-STAT1 (Rabbit polyclonal) Cell Signaling Technology Cat# 9172, RRID: AB_2198300 WB (1:1000) Antibody anti-YM1 (Rabbit polyclonal) STEMCELL Technologies Cat# 60130, RRID: AB_2868482 IF (1:200) Antibody anti-YM1/2 (Rabbit monoclonal) Abcam Cat# ab205491, RRID:AB_2891303 FC (1 mg/1 106 cells), IF (1:100) Antibody CXCL10 neutralizing antibody (NAB; Rat monoclonal) R and D Systems Cat# MAB466, RRID:AB_2292486 CXCL10 NAB Antibody Isotype control IgG2A antibody (Rat monoclonal) R and D Systems Cat# MAB006, RRID:AB_357349 CXCL10 NAB control Antibody CXCR3 neutralizing antibody (NAB; Armenian hamster monoclonal) Bio X Cell Cat# BE0249, RRID: AB_2687730 CXCR3 NAB Antibody Isotype control IgG antibody (Armenian hamster polyclonal) Bio X Cell Cat# BE0091, RRID: AB_1107773 CXCR3 NAB control Antibody anti-CD4 (Rat monoclonal) Bio X Cell Cat# BP0003-1, RRID:AB_2891358 T-cell depletion Antibody anti-CD8a (Rat monoclonal) Bio X Cell Cat# BP0061, RRID: AB_2891359 T-cell depletion Antibody Isotype control IgG2b antibody (Rat monoclonal) Bio X Cell Cat# BP0090, RRID: AB_2891360 T-cell depletion control Cell line (Mus musculus) SM3 Agbunag et al., 2006; Liou et al., 2017 Primary duct-like cells from KC mouse Chemical compound, drug NVP-BSK805 Selleckchem Cat# S2686 pan-JAK inhibitor Commercial assay or kit Mouse tumor dissociation kit Miltenyi Biotec Cat# 130-096-730 Commercial assay or kit RNAscope Assay 2.5 HD Reagent Kit- Brown Advanced Cell Diagnostics In situ hybridization (brown) Commercial assay or kit RNAscope Multiplex Fluorescent Reagent Kit v2 Advanced Cell Diagnostics In situ hybridization (fluorescent) Peptide, recombinant protein Recombinant murine CXCL10 Peprotech Cat# 250–16 Peptide, recombinant protein Recombinant murine IL-4 Peprotech Cat# 214–14 Peptide, recombinant protein Recombinant murine IFNV Peprotech Cat# 315–05 Sequence-based reagent Arg1 TaqMan (Thermo Fisher Scientific) Mm00475988_m1 qPCR probe Sequence-based reagent Chil3 TaqMan (Thermo Fisher Scientific) Mm00657889_mH qPCR probe Sequence-based reagent Cxcl10 TaqMan (Thermo Fisher Scientific) Mm00445235_m1 qPCR probe

Techniques: Isolation, Control, Expressing, Immunofluorescence

Journal: eLife

Article Title: CXCL10/CXCR3 signaling contributes to an inflammatory microenvironment and its blockade enhances progression of murine pancreatic precancerous lesions

doi: 10.7554/elife.60646

Figure Lengend Snippet: Figure 5. Overexpression of CXCL10 in pancreatic lesions increases inflammatory macrophages and decreases lesion formation in the pancreas. (A) Athymic nude mice were orthotopically implanted with PanIN organoids obtained from lentivirally infected SM3-CXCL10 or SM3-control cells (see Figure 4—figure supplement 3G–I). Mice were euthanized 2 weeks post-surgery, and pancreatic tissue was analyzed. Shown are representative images of abnormal areas in the pancreas. The scale bar represents 500 mm. (B) Abnormal areas (lesions and stroma) were manually traced for Figure 5 continued on next page

Article Snippet: DOI: https://doi.org/10.7554/eLife.60646 22 of 24 Appendix 1—key resources table continued Reagent type (species) or resource Designation Source or reference Identifiers Additional information Antibody anti-pY701-STAT1 (Rabbit monoclonal) Cell Signaling Technology Cat# 9167, RRID: AB_561284 IF (1:400) Antibody anti-pY701-STAT1 (Mouse monoclonal) Abcam Cat# ab29045, RRID:AB_778096 IF (1:200), WB (1:1000) Antibody anti-STAT1 (Rabbit polyclonal) Cell Signaling Technology Cat# 9172, RRID: AB_2198300 WB (1:1000) Antibody anti-YM1 (Rabbit polyclonal) STEMCELL Technologies Cat# 60130, RRID: AB_2868482 IF (1:200) Antibody anti-YM1/2 (Rabbit monoclonal) Abcam Cat# ab205491, RRID:AB_2891303 FC (1 mg/1 106 cells), IF (1:100) Antibody CXCL10 neutralizing antibody (NAB; Rat monoclonal) R and D Systems Cat# MAB466, RRID:AB_2292486 CXCL10 NAB Antibody Isotype control IgG2A antibody (Rat monoclonal) R and D Systems Cat# MAB006, RRID:AB_357349 CXCL10 NAB control Antibody CXCR3 neutralizing antibody (NAB; Armenian hamster monoclonal) Bio X Cell Cat# BE0249, RRID: AB_2687730 CXCR3 NAB Antibody Isotype control IgG antibody (Armenian hamster polyclonal) Bio X Cell Cat# BE0091, RRID: AB_1107773 CXCR3 NAB control Antibody anti-CD4 (Rat monoclonal) Bio X Cell Cat# BP0003-1, RRID:AB_2891358 T-cell depletion Antibody anti-CD8a (Rat monoclonal) Bio X Cell Cat# BP0061, RRID: AB_2891359 T-cell depletion Antibody Isotype control IgG2b antibody (Rat monoclonal) Bio X Cell Cat# BP0090, RRID: AB_2891360 T-cell depletion control Cell line (Mus musculus) SM3 Agbunag et al., 2006; Liou et al., 2017 Primary duct-like cells from KC mouse Chemical compound, drug NVP-BSK805 Selleckchem Cat# S2686 pan-JAK inhibitor Commercial assay or kit Mouse tumor dissociation kit Miltenyi Biotec Cat# 130-096-730 Commercial assay or kit RNAscope Assay 2.5 HD Reagent Kit- Brown Advanced Cell Diagnostics In situ hybridization (brown) Commercial assay or kit RNAscope Multiplex Fluorescent Reagent Kit v2 Advanced Cell Diagnostics In situ hybridization (fluorescent) Peptide, recombinant protein Recombinant murine CXCL10 Peprotech Cat# 250–16 Peptide, recombinant protein Recombinant murine IL-4 Peprotech Cat# 214–14 Peptide, recombinant protein Recombinant murine IFNV Peprotech Cat# 315–05 Sequence-based reagent Arg1 TaqMan (Thermo Fisher Scientific) Mm00475988_m1 qPCR probe Sequence-based reagent Chil3 TaqMan (Thermo Fisher Scientific) Mm00657889_mH qPCR probe Sequence-based reagent Cxcl10 TaqMan (Thermo Fisher Scientific) Mm00445235_m1 qPCR probe

Techniques: Over Expression, Infection, Control