Journal: Brain

Article Title: Connexin-43 induces chemokine release from spinal cord astrocytes to maintain late-phase neuropathic pain in mice

doi: 10.1093/brain/awu140

Figure Lengend Snippet: Spinal injection of TNF-α-activated astrocytes induces mechanical allodynia via Cx43-mediated CXCL1 release. (A) Intrathecal injection of TNF-α-activated astrocytes elicited persistent mechanical allodynia for >48 h. Note this allodynia is reduced by pretreatment of astrocytes with Cx43 small interfering RNA (1 µg/ml, 18 h). *P < 0.05, compared with TNF-α or TNF-α + non-targeting control small interfering RNA treated group; n = 6 mice/group. (B) ELISA analysis shows increased CXCL1 release in the CSF at 3 h after the intrathecal injection of TNF-α-activated astrocytes. *P < 0.05, compared with vehcile group; #P < 0.05, compared with non-activated astrocytes; n = 4 mice/group. (C) Intrathecal injection of a CXCL1 neutralizing antibody (4 µg) transiently and partially reversed mechanical allodynia, induced by TNF-α-treated astrocytes. *P < 0.05, compared with control IgG group; n = 6 mice/group. (D) Intrathecal injection of the CXCR2 antagonist SB225002 (20 µg = 57 nmol) transiently and partially reversed mechanical allodynia, induced by TNF-α-activated astrocytes. *P < 0.05, compared with vehicle (PBS); n = 5–6 mice/group. All data are mean ± SEM. The differences between groups were analysed by ANOVA followed by Newman–Keuls test.

Article Snippet: The sections were then incubated overnight at 4°C with the following primary antibodies: GFAP antibody (1:1000, mouse; Millipore Bioscience Research Reagents), Cx43 antibody (1:1000, rabbit; Sigma), NeuN antibody (1:1000, mouse; Millipore Bioscience Research Reagents), CXCL1 (1: 200, rabbit; Boster), and CXCR2 antibody (1: 200, rabbit; Boster).

Techniques: Injection, Small Interfering RNA, Control, Enzyme-linked Immunosorbent Assay

Journal: Brain

Article Title: Connexin-43 induces chemokine release from spinal cord astrocytes to maintain late-phase neuropathic pain in mice

doi: 10.1093/brain/awu140

Figure Lengend Snippet: CXCL1, upregulated in spinal cord astrocytes after nerve injury, enhances excitatory synaptic transmission in spinal cord neurons and maintains neuropathic pain via CXCR2. (A) Western blotting shows CXCL1 upregulation in the spinal cord dorsal horn 21 days after CCI. Right, quantification of Cx43 levels in the dorsal horn. The western blot results are presented as a fold of sham control. *P < 0.05, compared to sham control, Student’s t-test, n = 4 mice/group. (B) Intrathecal injection of SB 225002 (20 µg), 21 days after CCI, reduced CCI-induced mechanical allodynia in the late phase. *P < 0.05, compared with vehicle (saline), Student’s t-test, n = 6 mice/group. (C) Double immunostaining of CXCL1 and GFAP in the dorsal horn 21 days after CCI. Note CXCL1 is primarily colocalized with GFAP. Arrows indicate doubled-labelled cells. Scale bar = 50 µm. (D and E) CXCL1 superfusion (100 ng/ml) increases spontaneous EPSC frequency (revealed by patch clamp recordings) in lamina IIo neurons of spinal cord slices. (E) Spontaneous EPSC frequency. *P < 0.05, Student’s t-test, n = 5 neurons/group. (F and G) CCI (21 d) increases spontaneous EPSC frequency, which is reversed by the CXCR2 antagonist SB225002 (1 µM). *P < 0.05, Student’s t-test, n = 5 neurons/group.

Article Snippet: The sections were then incubated overnight at 4°C with the following primary antibodies: GFAP antibody (1:1000, mouse; Millipore Bioscience Research Reagents), Cx43 antibody (1:1000, rabbit; Sigma), NeuN antibody (1:1000, mouse; Millipore Bioscience Research Reagents), CXCL1 (1: 200, rabbit; Boster), and CXCR2 antibody (1: 200, rabbit; Boster).

Techniques: Transmission Assay, Western Blot, Control, Injection, Saline, Double Immunostaining, Patch Clamp

Journal: Brain

Article Title: Connexin-43 induces chemokine release from spinal cord astrocytes to maintain late-phase neuropathic pain in mice

doi: 10.1093/brain/awu140

Figure Lengend Snippet: Schematic of working hypothesis for astrocytic Cx43-mediated late-phase neuropathic pain. CCI induces a persistent upregulation of Cx43 in spinal cord astrocytes. Cx43 expression and activity is also upregulated by TNF-α, secreted from microglia. Upregulation of Cx43 hemichannel activities results in CXCL1 release. Astrocytic CXCL1 secretion activates CXCR2 on neurons (central terminals of primary sensory neurons and spinal cord neurons), leading to enhanced excitatory synaptic transmission in nociceptive neurons (e.g. lamina IIo excitatory interneurons) and sustained neuropathic pain in the late-phase. Additionally, CXCL1 can also be secreted from intact or injured primary afferents in the spinal cord especially in the early phase of CCI.

Article Snippet: The sections were then incubated overnight at 4°C with the following primary antibodies: GFAP antibody (1:1000, mouse; Millipore Bioscience Research Reagents), Cx43 antibody (1:1000, rabbit; Sigma), NeuN antibody (1:1000, mouse; Millipore Bioscience Research Reagents), CXCL1 (1: 200, rabbit; Boster), and CXCR2 antibody (1: 200, rabbit; Boster).

Techniques: Expressing, Activity Assay, Transmission Assay

Journal: bioRxiv

Article Title: Mechanistic insights of radiation-induced endothelial senescence impelling glioblastoma genomic instability at relapse

doi: 10.1101/2021.12.13.472364

Figure Lengend Snippet: a. CXCR2 expression in U251 and human primary GBM cells by FACS analysis. Left panel : Representative histograms of CXCR2 staining in U251 cells. Right panel : Geomean of fluorescence compared to isotype. Geomean of CXCR2 expression in U251 cells and GBM primary cells is indicated respectively as black and white circles. b. Radio-induced MN production per cell after 5Gy in U251 cells in CM or SASP after CXCR2 blocking using monoclonal antibody MAb331(250ng/ml) or SB332235 antagonist (1nM). (mean±SD, n=3, one-way ANOVA; * p<0.05, *** p<0.001). c. Polynucleated cell frequency after 5Gy-radiation in U251 cells in CM or SASP after CXCR2 blocking using monoclonal antibody MAb331(250ng/ml) or SB332235 antagonist (1nM). (mean±SD; n=3; two-way ANOVA, * p<0.05, ** p<0.01). d. Elongation rate of U251 cells in CM (black) or SASP (blue) supplemented or not with CXCR2 monoclonal antibody (MAb331, 250ng/ml). When indicated, cells were irradiated with 5Gy-radiation. (mean±SD, n=3, two-way ANOVA; ** p<0.01, *** p<0.001).

Article Snippet: Anti-CXCR2 blocking antibody (Mab331) and antagonist drug (SB332235) were purchased from R&D and TOCRIS (Bioscience) respectively.

Techniques: Expressing, Staining, Fluorescence, Blocking Assay, Irradiation

Journal: bioRxiv

Article Title: Mechanistic insights of radiation-induced endothelial senescence impelling glioblastoma genomic instability at relapse

doi: 10.1101/2021.12.13.472364

Figure Lengend Snippet: a. Median survival (days) of tumor-bearing mice after orthotopic injections of radiation-surviving U251 cells obtained in presence of CM (R15CM), SASP (R15SASP), CM supplemented with CXCL8 and CXCL5 (R15CM -CXCL5/8 ), SASP supplemented with either CXCR2 monoclonal antibody Mab331 (R15SASP- Mab ) or CXCR2 antagonist SB3322235 (R15SASP- SB ). Log-rank p-values are indicated as compare to mice survival injected with either R15CM radioresistant cells or R15SASP radioresistant cells. b. Tumor-bearing mice survival after orthotopic injections of R15CM-CXCL (left panel), R15SASP-mAbR2 (middle panel) and R15SASP-SBR2 (right panel) (n=8/group) as compare to R15CM and R15SASP tumor-bearing mice. c. Immunofluorescence of CXCL5 and CXCL8 in primary and recurrent human GBM. d. Kaplan-Meier plots overall survival of GBM patients depending on CXCL5 and CXCL8 mRNA expression using the TCGA-glioblastoma database. (Log-rank tests analysis).

Article Snippet: Anti-CXCR2 blocking antibody (Mab331) and antagonist drug (SB332235) were purchased from R&D and TOCRIS (Bioscience) respectively.

Techniques: Injection, Immunofluorescence, Expressing

Journal: Advanced Science

Article Title: Tumor Microenvironment Responsive CD8 + T Cells and Myeloid‐Derived Suppressor Cells to Trigger CD73 Inhibitor AB680‐Based Synergistic Therapy for Pancreatic Cancer

doi: 10.1002/advs.202302498

Figure Lengend Snippet: AB680 regulates CXCL5 released by tumor cells and promotes MDSC accumulation. A) Heatmap of immune‐related differential genes in the RNA‐seq data of control and AB680‐treated tumors ( n = 3 samples per group). B,C) Over‐Representation Analysis B) and Gene Set Enrichment Analysis (C) of the above RNA‐seq data. D) Heatmap of 23 different mouse cytokine expression levels in serums obtained at the time of tumor collection, as shown in Figure using Bio‐Plex Pro Mouse Cytokine 23‐plex immunoassay ( n = 4 samples per group). E) The protein level of CXCL5 in mouse serums from the indicated treatment groups. Data presented as mean ± SD, ** p < 0.01 in Student's t ‐test. F) Dot plot of Cxcl5 expression in the identified populations in the single‐cell RNA‐sequencing dataset GSE158356 of two orthotopic KPC allografts. G,H) Quantification of CXCL5 and CXCR2% positive area in the AB680‐treated versus Control KPC tumor at the endpoint ( n = 6 samples per group). ** p < 0.01, *** p < 0.001 in the Student's t ‐test. Data presented as mean ± SD. I) Representative images of immunohistochemistry staining of CXCL5 and CXCR2 in the KPC tumors from the indicated treatment groups. J,K) The level of adenosine and CXCL5 protein in the AB680‐treated versus Control KPC tumor at the endpoint ( n = 6 samples per group). ** p < 0.01, *** p < 0.001 in Student's t ‐test. Data presented as mean ± SD. L) Schematic representation of the changes in adenosine metabolism after inhibition of CD73 activity by AB680. M) qRT‐PCR analysis of Cxcl5 mRNA expression in the KPC cells with different concentrations of AMP, adenosine, and the adenosine receptor agonist NECA at 0, 10, 20, 50, and 100 µ m ( n = 3). Data presented as mean ± SD. N) The CXCL5 protein concentration in the culture supernatant was evaluated by ELISA in the KPC cells with indicated concentrations of drugs ( n = 3). Data presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001 in Student's t ‐test.

Article Snippet: Formalin‐fixed, paraffin‐embedded sections of humans and mice were processed and incubated with primary antibodies: CD73 (Proteintech, 12231‐1‐AP), CK19 (Proteintech, 10712‐1‐AP), α‐SMA (Boster, BM0002), CD8 (Abcam, ab209775), granzyme B (Abcam, ab4059), CXCR2 (Proteintech, 19538‐1‐AP), CXCL5 (R&D Systems, AF433), LY6G (Servicebio, GB11229), Ki‐67 (CST, 9129), and cleaved caspase 3 (CST, 9664), followed by biotinylated or fluorescent secondary antibodies.

Techniques: RNA Sequencing, Control, Expressing, Immunohistochemistry, Staining, Inhibition, Activity Assay, Quantitative RT-PCR, Protein Concentration, Enzyme-linked Immunosorbent Assay

Journal: Advanced Science

Article Title: Tumor Microenvironment Responsive CD8 + T Cells and Myeloid‐Derived Suppressor Cells to Trigger CD73 Inhibitor AB680‐Based Synergistic Therapy for Pancreatic Cancer

doi: 10.1002/advs.202302498

Figure Lengend Snippet: CXCR2 inhibition further potentiates the efficacy of AB680 plus anti‐PD‐1 therapy. A) Schematic representation of the therapy schedule for AB680, anti‐PD‐1 antibody, and CXCR2 inhibitor SB225002 in the KPC orthotopic model. B,C) The image (B) and tumor weight (C) of KPC orthotopic allografts from the Control, SB225002, anti‐PD‐1+AB680, and combination groups ( n = 6/group). ** p < 0.01, *** p < 0.001 in Student's t ‐test. Data presented as mean ± SD. D) Survival of immunocompetent mice harboring KPC orthotopic allografts treated with the indicated treatment compared with the survival of those untreated mice ( n = 8/group). **p < 0.01, *** p < 0.001 in the log‐rank test. E) The percentages of total CD8 + T cells, Granzyme B+ cells among the total CD8 + T cells, and CXCR2 + MDSCs in the tumors of each group at the endpoint were determined by flow cytometry. n = 4 samples per group. Data presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001 in Student's t ‐test. F) Quantification of CD8, Granzyme B, CXCR2, and Cleaved caspase‐3% positive area in the tumors of each group at the endpoint. n = 6 samples per group. Data presented as mean ± SD. ** p < 0.01, *** p < 0.001 in Student's t ‐test (G) schematic diagram depicting the mechanism by which CXCR2 inhibitor further potentiates the efficacy of AB680 plus anti‐PD‐1 therapy. AB680 can significantly increase the infiltration of responsive CD8+ T cells by suppressing the production of immunosuppressive adenosine, but it also promotes the MDSC chemotaxis by tumor‐derived CXCL5 in an AMP manner. Targeting the CXCL5/CXCR2 pathway with the CXCR2 inhibitor SB‐225002 can disrupt the “side effect” of AB680 and potentiate the efficacy of AB680 plus anti‐PD‐1 therapy. Data presented as mean ± SD, Student's t ‐test was used unless otherwise indicated.

Article Snippet: Formalin‐fixed, paraffin‐embedded sections of humans and mice were processed and incubated with primary antibodies: CD73 (Proteintech, 12231‐1‐AP), CK19 (Proteintech, 10712‐1‐AP), α‐SMA (Boster, BM0002), CD8 (Abcam, ab209775), granzyme B (Abcam, ab4059), CXCR2 (Proteintech, 19538‐1‐AP), CXCL5 (R&D Systems, AF433), LY6G (Servicebio, GB11229), Ki‐67 (CST, 9129), and cleaved caspase 3 (CST, 9664), followed by biotinylated or fluorescent secondary antibodies.

Techniques: Inhibition, Control, Flow Cytometry, Chemotaxis Assay, Derivative Assay

Journal: eLife

Article Title: Short-range interactions between fibrocytes and CD8 + T cells in COPD bronchial inflammatory response

doi: 10.7554/eLife.85875

Figure Lengend Snippet:

Article Snippet: Antibody , Anti-CXCR2-APC-Cy7 (human recombinant monoclonal) , Miltenyi Biotec , Cat. #:130-119-571, RRID: AB_2733103 , FC (1:50).

Techniques: Blocking Assay, Recombinant

Journal: Cell reports

Article Title: Altered DNA methylation underlies monocyte dysregulation and immune exhaustion memory in sepsis

doi: 10.1016/j.celrep.2024.113894

Figure Lengend Snippet: (A) scRNA-seq UMAP feature plots for transcription factor TCF7L2 motif enrichment and gene expression. Numbers indicate different cell clusters, as outlined in . (B) RT-qPCR for key exhaustion genes in BMMCs under PBS control or repetitive LPS stimulation in the presence of different concentrations of Wnt agonist 1. Expression levels were normalized to the geometric mean of Ube2l3 , Oaz1 , and Nktr (mean expression ± SD; n = 3–6; one-way ANOVA with Sidak’s multiple comparisons test; ****p-adj < 0.0001; ***p-adj < 0.001; **p-adj < 0.01; *p-adj < 0.05; ns, not significant; differences are not significant unless otherwise specified; see for exact p values). (C) Flow cytometry mean fluorescence intensity (MFI) for exhaustion (CD38, MARCO, PD-L1, CXCR2) and macrophage (F4/80) markers relative to PBS control. Boxplots indicate median MFI values (n = 5–7; one-way ANOVA with Sidak’s multiple comparisons test). (D) Gating strategy (top) and population statistics (bottom) for non-classical (Ly6C low ), intermediate (Ly6C int ), and classical (Ly6C high ) monocyte subtypes in cultured BMMCs (n = 6–13; one-way ANOVA with Sidak’s multiple comparisons test). (E) NAD + levels in cultured BMMCs normalized to total protein levels (n = 3–6; one-way ANOVA with Sidak’s multiple comparisons test). (F) Correlation heatmap for average DNA methylation levels at key exhaustion loci in cultured BMMCs (n = 3–9 for each condition). (G) UCSC browser track views of average DNA methylation at the Plac8 promoter (chr5: 100,572,230–100,572,607), Cebpa/g distal enhancer (chr7: 35,064,805–35,065,304), and Tcf7l2 intron (chr19: 55,768,986–55,769,585). ENCODE annotated promoters (red) and enhancers (orange) are depicted in the bottom track.

Article Snippet: For Panel 2, cells were stained with antibodies against Ly6C (Pacific Blue; Biolegend #128014), CD11b (BV650; BD Biosciences #563402), Ly6g (PE-Cy5.5; Elabscience #E-AB-F1108I), CXCR2 (Alexa Fluor 488; R&D Systems # FAB2164G), CD68 (APC-Fire750; Biolegend #137041), CD172a (BV510; BD Biosciences #740159), CX3CR1 (PE-Cy7; Biolegend #149016), F4/80 (BV711; BD Biosciences #565612), CD38 (BV750; BD Biosciences #747103), PD-L1 (BV421; BD Biosciences #564716), MARCO (Alexa Fluor 647; R&D Systems #FAB29561R), FAS (BV605; Biolegend #152612), MHCII (PerCP; Biolegend #107624), CD168 (PE; Novus #NBP1–76538PE), and TREM2 (Alexa Fluor 700; R&D Systems #FAB17291N).

Techniques: Expressing, Quantitative RT-PCR, Control, Flow Cytometry, Fluorescence, Cell Culture, DNA Methylation Assay

Journal: Cell reports

Article Title: Altered DNA methylation underlies monocyte dysregulation and immune exhaustion memory in sepsis

doi: 10.1016/j.celrep.2024.113894

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: For Panel 2, cells were stained with antibodies against Ly6C (Pacific Blue; Biolegend #128014), CD11b (BV650; BD Biosciences #563402), Ly6g (PE-Cy5.5; Elabscience #E-AB-F1108I), CXCR2 (Alexa Fluor 488; R&D Systems # FAB2164G), CD68 (APC-Fire750; Biolegend #137041), CD172a (BV510; BD Biosciences #740159), CX3CR1 (PE-Cy7; Biolegend #149016), F4/80 (BV711; BD Biosciences #565612), CD38 (BV750; BD Biosciences #747103), PD-L1 (BV421; BD Biosciences #564716), MARCO (Alexa Fluor 647; R&D Systems #FAB29561R), FAS (BV605; Biolegend #152612), MHCII (PerCP; Biolegend #107624), CD168 (PE; Novus #NBP1–76538PE), and TREM2 (Alexa Fluor 700; R&D Systems #FAB17291N).

Techniques: Blocking Assay, Negative Control, Recombinant, Methylation, SYBR Green Assay, DNA Methylation Assay, Software, Isolation, Reverse Transcription, Multiplex Assay