Journal: International Journal of Biological Sciences

Article Title: CXCR4-dependent macrophage-to-fibroblast signaling contributes to cardiac diastolic dysfunction in heart failure with preserved ejection fraction

doi: 10.7150/ijbs.65802

Figure Lengend Snippet: CXCR4-deficient macrophages attenuate myofibroblast differentiation via CXCL3-CXCR2 axis. (A) Western blot analysis of FN and αSMA in CFs co-cultured in MΦ WT and MΦ MKO -CM with or without HMGB1 treatment. (B) QPCR analysis of FN and αSMA in CFs co-cultured in MΦ WT and MΦ MKO -CM with or without HMGB1 treatment. (C) Immunofluorescence staining of αSMA+ cells in CFs co-cultured in MΦ WT and MΦ MKO -CM with or without HMGB1 treatment. (αSMA. red; DAPI. blue), Scale bar = 50μm. (D) Volcano plot of RNA sequencing MΦ WT and MΦ MKO after HMGB1 treatment. (E) RNA array analysis of the indicated genes in MΦ WT and MΦ MKO after HMGB1 treatment. (F) QPCR and elisa analysis of CXCL3 MΦ WT and MΦ MKO -CM with or without HMGB1 treatment. (G) Western blot analysis of FN and αSMA in CFs co-cultured in MΦ WT -CM after HMGB1 treatment with or without CXCL3 Ab. (H) Western blot analysis of FN and αSMA in CFs co-cultured in MΦ MKO -CM after HMGB1 treatment with or without rmCXCL3 protein. (I) Western blot analysis of CXCR2 in CFs co-cultured in MΦ WT and MΦ MKO -CM with or without HMGB1 treatment. (J) Western blot analysis of CXCR2 in CFs co-cultured in MΦ WT -CM after HMGB1 treatment with or without CXCL3 Ab. (K) Western blot analysis of CXCR2 in CFs co-cultured in MΦ MKO -CM after HMGB1 treatment with or without rmCXCL3. (L) Western blot analysis of FN and αSMA in CFs co-cultured in MΦ WT -CM after HMGB1 treatment with or without SB225002. (M) Western blot analysis o FN and αSMA in CFs co-cultured in MΦ MKO -CM after HMGB1 treatment with or without rmCXCL3 or SB225002. FN, fibronectin; αSMA, a-smooth muscle actin; CFs, cardiac fibroblasts; MΦ WT macrophages derived from the bone marrow WT mice; MΦ MKO , macrophages derived from the bone marrow MKO mice. CM, conditioned medium. CXCL3, Chemokine (C‑X‑C) motif ligand 3. All data were analyzed using two-way ANOVA with Bonferroni's multiple comparisons test. *, p<0.05.

Article Snippet: The supernatants of heart tissue lysates and the conditioned medium of macrophages were collected to detect CXCL3 ( CSB-EL00624MO, CUSABIO, Wuhan, Hubei, China) and CXCL12 (MCX120, R&D Systems, Minneapolis, MN, USA) according to the enzyme-linked Immunosorbent Assay Kit protocol.

Techniques: Western Blot, Cell Culture, Immunofluorescence, Staining, RNA Sequencing, Enzyme-linked Immunosorbent Assay, Derivative Assay

Journal: Cancer Research

Article Title: Ferroptotic Neutrophils Induce Immunosuppression and Chemoresistance in Breast Cancer

doi: 10.1158/0008-5472.CAN-24-1941

Figure Lengend Snippet: Tumor neutrophil ferroptosis was related to a distinctive subset of CD4 T cells enriched in chemoresistant tumors. A, Uniform Manifold Approximation and Projection (UMAP) visualization of tumor-infiltrating CD4 T cells from five chemosensitive and five chemoresistant breast cancers by scRNA-seq. B, Three-dimensional UMAP plot of CD4 T cells colored by chemosensitivity. C, The proportions of different subpopulations of CD4 T cells in sensitive and resistant tumors. D, Heatmap displaying scaled expression of discriminating genes for each cluster of CD4 T cells in scRNA-seq data. E, Heatmap for IL1B expression in CD4 TILs from nine chemosensitive and nine chemoresistant patients by bulk RNA-seq. F, Representative flow cytometry for IL1β and CXCL3 expression in chemosensitive and chemoresistant tumor-infiltrating CD4 T cells. G, Cell death ratio of peripheral neutrophils pretreated with the inhibitors for apoptosis, necrosis, or ferroptosis and cocultured with IL1β + CXCL3 + -d or IL1β + CXCL3 + CD4 T cells. H, Representative flow cytometric images for cellular lipid peroxidation of peripheral neutrophils cocultured with different CD4 T cells. I, Proliferation of tumor-specific CTLs upon exposure to peripheral neutrophils pretreated with IL1β + CXCL3 + or IL1β + CXCL3 + -d CD4 T cells. J, Representative immunofluorescence images (left) and quantification (right) for IL1β and CXCL3 expression in CD4 T cells in chemosensitive and chemoresistant breast cancer sections. Arrows, IL1β + CXCL3 + CD4 T cells. Scale bar, 50 μm. K, Correlation between IL1β + CXCL3 + CD4 T cells and ferroptotic neutrophils in breast tumor specimens. Results are represented as mean ± SD of n = 5 ( A–D and G–I ), n = 9 ( E and F ), or n = 468 ( J ) different patients; for K , n = 468 different patients; ***, P < 0.001 by Student t test ( F ), two-sided one-way ANOVA with the Tukey test ( G , I , and J ) or two-tailed Pearson correlation coefficient test ( K ). Quantification is shown in Supplementary Figs. S4 and S5 ( F and H ).

Article Snippet: The primary antibodies used included rabbit anti-human CD4 (1:100, Abcam, cat. #ab133616, RRID: AB_2750883), rabbit anti-human CXCL3 (1:200, Biorbyt, cat. #orb13448, RRID: AB_10748863), rabbit anti-mouse CXCL3 (1:100, Abcam, cat. #ab220431, RRID: AB_2938758), rabbit anti-human/mouse IL1β (1:50, Abcam, cat. #ab254360, RRID: AB_2936299), goat anti-human/mouse MPO (10 μg/mL, R&D Systems, cat. #AF3667, RRID: AB_2250866), mouse anti-4 HNE (1:25, Abcam, cat. #ab48506, RRID: AB_867452), rabbit anti-human IL1R1 (1:100, Abcam, cat. #ab106278, RRID: AB_10865509), rabbit anti-human/mouse p65 (1:500, Cell Signaling Technology, cat. #8242S, RRID: AB_10859369), mouse anti-human/mouse cytokeratin (1 μg/mL, Abcam, cat. #ab7753, RRID: AB_306047), rabbit anti-mouse CD8 (1:100, Abcam, #ab203035), and rabbit anti-human/mouse granzyme B (1:200, Abcam, cat. #ab255598, RRID: AB_2860567).

Techniques: Expressing, RNA Sequencing, Flow Cytometry, Immunofluorescence, Two Tailed Test

Journal: Cancer Research

Article Title: Ferroptotic Neutrophils Induce Immunosuppression and Chemoresistance in Breast Cancer

doi: 10.1158/0008-5472.CAN-24-1941

Figure Lengend Snippet: Cross-talk between neutrophils and Fer-CD4 T cells maintained extensive neutrophil ferroptosis. A, Gene Ontology (GO) terms associated with upregulated genes of C2_IL1B cluster in scRNA-seq. B, Gene set enrichment analysis of bulk RNA-seq revealed enrichment of neutrophil chemotaxis genes in chemoresistant CD4 TILs. C, Scheme of chemotaxis assays with Boyden transwell chambers. D, Representative images (left) and quantification (right) of chemotaxis assays for peripheral neutrophils toward CM of different CD4 TILs. Scale bar, 100 μm. E, Migration tracks of neutrophils in μ-slide chemotaxis experiments toward CM of non–Fer-CD4 or Fer-CD4 T cells. F, Representative fluorescent images of cytoskeleton staining (left) and quantification (right) of filopodium-like protrusions (FLP) of peripheral neutrophils in the presence of CM from non–Fer-CD4 or Fer-CD4 T cells. Scale bar, 5 μm. G, Ex vivo tumor slice migration assays for the recruitment of CFSE-labeled neutrophils into breast tumor slices with high or low density of Fer-CD4 T cells. Scale bar, 100 μm. H, Dot plot for the expression of various chemokines in C2_IL1B cells in scRNA-seq. I, ELISA for CXCL3, IL8, and S100A9 release of non–Fer-CD4 or Fer-CD4 T cells. J and K, Migration ( J ) and filopodium-like protrusions ( K ) of peripheral neutrophils in the presence of CM from Fer-CD4 T cells and different antibodies. L, Cell death of peripheral neutrophils in the presence of CM of Fer-CD4 T cells and different antibodies categorized by location (top or bottom chamber of transwell system). M, CXCL3 and IL1β protein level of naïve CD4 T cells by immunoblotting after coculturing with control or ferroptotic neutrophils, which were generated by engagement with non–Fer-CD4 or Fer-CD4 T cells, respectively. Results are represented as mean ± SD of n = 7 ( D ) or n = 5 ( F and I–M ) or n = 6 ( G ). *, P < 0.05; **, P < 0.01; ***, P < 0.001 by two-sided one-way ANOVA with the Tukey test ( D , F , and J–L ) or Student t test ( G , I , and M ). Representative images are shown in Supplementary Fig. S7 ( J and K ).

Article Snippet: The primary antibodies used included rabbit anti-human CD4 (1:100, Abcam, cat. #ab133616, RRID: AB_2750883), rabbit anti-human CXCL3 (1:200, Biorbyt, cat. #orb13448, RRID: AB_10748863), rabbit anti-mouse CXCL3 (1:100, Abcam, cat. #ab220431, RRID: AB_2938758), rabbit anti-human/mouse IL1β (1:50, Abcam, cat. #ab254360, RRID: AB_2936299), goat anti-human/mouse MPO (10 μg/mL, R&D Systems, cat. #AF3667, RRID: AB_2250866), mouse anti-4 HNE (1:25, Abcam, cat. #ab48506, RRID: AB_867452), rabbit anti-human IL1R1 (1:100, Abcam, cat. #ab106278, RRID: AB_10865509), rabbit anti-human/mouse p65 (1:500, Cell Signaling Technology, cat. #8242S, RRID: AB_10859369), mouse anti-human/mouse cytokeratin (1 μg/mL, Abcam, cat. #ab7753, RRID: AB_306047), rabbit anti-mouse CD8 (1:100, Abcam, #ab203035), and rabbit anti-human/mouse granzyme B (1:200, Abcam, cat. #ab255598, RRID: AB_2860567).

Techniques: RNA Sequencing, Chemotaxis Assay, Migration, Staining, Ex Vivo, Labeling, Expressing, Enzyme-linked Immunosorbent Assay, Western Blot, Control, Generated

Journal: Frontiers in Immunology

Article Title: Pharmacological Blockade of Spinal CXCL3/CXCR2 Signaling by NVP CXCR2 20, a Selective CXCR2 Antagonist, Reduces Neuropathic Pain Following Peripheral Nerve Injury

doi: 10.3389/fimmu.2019.02198

Figure Lengend Snippet: The time course of changes in CXCR2, CXCL1, CXCL2, CXCL3 mRNAs (A–D) and proteins (E–H) in the spinal cord tissues on the 2nd, 7th, 14th, and 28th days after chronic constriction injury (CCI) in rats. The RT-qPCR and Western blot data are presented as the means ± SEM of 6–10 and 4–6 samples per group in each method, respectively. Intergroup differences were analyzed using ANOVA with Bonferroni's multiple comparisons test. * p < 0.05, *** p < 0.001 indicate differences vs. naive rats. CCI, chronic constriction injury; N, naïve.

Article Snippet: TaqMan primers and probes were used: Rn01527838_g1 (HPRT, hypoxanthine-guanine phosphoribosyltransferase); Rn02130551_s1 (CXCR2, chemokine (C-X-C motif) receptor 2); Rn00578225_m1 (CXCL1, CINC-1, chemokine (C-X-C motif) ligand 1); Rn00586403_m1 (CXCL2, CINC-3, Mip-2, chemokine (C-X-C motif) ligand 2); and Rn01414231_m1 (CXCL3, CINC-2, chemokine (C-X-C motif) ligand 3).

Techniques: Quantitative RT-PCR, Western Blot

Journal: Frontiers in Immunology

Article Title: Pharmacological Blockade of Spinal CXCL3/CXCR2 Signaling by NVP CXCR2 20, a Selective CXCR2 Antagonist, Reduces Neuropathic Pain Following Peripheral Nerve Injury

doi: 10.3389/fimmu.2019.02198

Figure Lengend Snippet: The spinal localization of CXCR2 and its ligand CXCL3 in naive and CCI-exposed rats. Immunofluorescent staining was performed on paraffin-embedded 7 μm (A,B) co-staining of CXCR2 (green) and neuronal marker NeuN (red; upper row); astroglial marker GFAP (red, middle row), and microglial marker IBA1 (red, bottom row). (C,D) co-staining of CXCL3 (green) and neuronal marker NeuN (red; upper row); astroglial marker GFAP (red, middle row), and microglial marker IBA1 (red, bottom row). White arrows indicate representative CXCL3-positive cells that co-localize with IBA1-positive cells. Scale bar for all pictures: 25 μm.

Article Snippet: TaqMan primers and probes were used: Rn01527838_g1 (HPRT, hypoxanthine-guanine phosphoribosyltransferase); Rn02130551_s1 (CXCR2, chemokine (C-X-C motif) receptor 2); Rn00578225_m1 (CXCL1, CINC-1, chemokine (C-X-C motif) ligand 1); Rn00586403_m1 (CXCL2, CINC-3, Mip-2, chemokine (C-X-C motif) ligand 2); and Rn01414231_m1 (CXCL3, CINC-2, chemokine (C-X-C motif) ligand 3).

Techniques: Staining, Marker

Journal: Frontiers in Immunology

Article Title: Pharmacological Blockade of Spinal CXCL3/CXCR2 Signaling by NVP CXCR2 20, a Selective CXCR2 Antagonist, Reduces Neuropathic Pain Following Peripheral Nerve Injury

doi: 10.3389/fimmu.2019.02198

Figure Lengend Snippet: Effects of single administrations of CXCL1, CXCL2, and CXCL3 (A–F) on nociceptive transmission in naive mice. The effects of single intrathecal administrations of CXCL1, CXCL2, CXCL3 (2, 400, or 800 ng/5 μl) on mechanical hypersensitivity (von Frey test, A–C ) and thermal hypersensitivity (cold plate test, D–F ) were measured at 1.5, 5, and 24 h after administration. Data are presented as the means ± SEM (6 mice per group). The results were evaluated using one-way ANOVA, followed by Bonferroni's test for comparisons of selected pairs measured separately at each time point. * p < 0.05, ** p < 0.01, *** p < 0.001 for the comparison of vehicle-treated naive animals with all groups at the indicated time points. Additionally, the results were evaluated using two-way ANOVA to determine the time × drug interaction (please see results in Chapter 3.3). V, vehicle.

Article Snippet: TaqMan primers and probes were used: Rn01527838_g1 (HPRT, hypoxanthine-guanine phosphoribosyltransferase); Rn02130551_s1 (CXCR2, chemokine (C-X-C motif) receptor 2); Rn00578225_m1 (CXCL1, CINC-1, chemokine (C-X-C motif) ligand 1); Rn00586403_m1 (CXCL2, CINC-3, Mip-2, chemokine (C-X-C motif) ligand 2); and Rn01414231_m1 (CXCL3, CINC-2, chemokine (C-X-C motif) ligand 3).

Techniques: Transmission Assay, Comparison

Journal: Frontiers in Immunology

Article Title: Pharmacological Blockade of Spinal CXCL3/CXCR2 Signaling by NVP CXCR2 20, a Selective CXCR2 Antagonist, Reduces Neuropathic Pain Following Peripheral Nerve Injury

doi: 10.3389/fimmu.2019.02198

Figure Lengend Snippet: Effects of the repeated administration of NVP CXCR2 20 (NVP; 10 μg/5 μl; i.t .; 16 h and 1 h before CCI and then once a day for 7 days) on the protein levels of CXCR2, IBA1, GFAP, CXCL1, CXCL2, and CXCL3 proteins (A–I) in the spinal cord (A–F) and DRG (G–I) on the 7th day after CCI in rats. The data are presented as the mean fold changes relative to the control ± SEM (5–6 samples per group). Intergroup differences were analyzed using ANOVA with Bonferroni's multiple comparisons test. * p < 0.05, ** p < 0.01, *** p < 0.001 indicate differences vs. naive rats. # p < 0.05, indicate differences between V-treated and NVP-treated rats. CCI, chronic constriction injury; N, naive; V, vehicle; NVP, NVP CXCR2 20.

Article Snippet: TaqMan primers and probes were used: Rn01527838_g1 (HPRT, hypoxanthine-guanine phosphoribosyltransferase); Rn02130551_s1 (CXCR2, chemokine (C-X-C motif) receptor 2); Rn00578225_m1 (CXCL1, CINC-1, chemokine (C-X-C motif) ligand 1); Rn00586403_m1 (CXCL2, CINC-3, Mip-2, chemokine (C-X-C motif) ligand 2); and Rn01414231_m1 (CXCL3, CINC-2, chemokine (C-X-C motif) ligand 3).

Techniques: Control

Journal: Frontiers in Immunology

Article Title: Pharmacological Blockade of Spinal CXCL3/CXCR2 Signaling by NVP CXCR2 20, a Selective CXCR2 Antagonist, Reduces Neuropathic Pain Following Peripheral Nerve Injury

doi: 10.3389/fimmu.2019.02198

Figure Lengend Snippet: Effects of single administrations of CXCL3 neutralizing antibody (A,B) on nociceptive transmission in CCI-exposed mice. The effects of single intrathecal administrations of CXCL3 neutralizing antibody (1, 4, or 8 μg/5 μl) on mechanical hypersensitivity (von Frey test, A ) and thermal hypersensitivity (cold plate test, B ) were measured at 1.5, 5, 24, and 48 h after administration at 7 days after CCI. Data are presented as the means ± SEM (6–8 mice per group). The results were evaluated using one-way ANOVA followed by Bonferroni's test for comparisons of selected pairs. * p < 0.05, ** p < 0.01, *** p < 0.001 for the comparison of CCI-exposed vehicle-treated animals with all groups at the indicated time points. Additionally, the results were evaluated using two-way ANOVA to determine the time × drug interaction (please see results in chapter 3.6). V, vehicle.

Article Snippet: TaqMan primers and probes were used: Rn01527838_g1 (HPRT, hypoxanthine-guanine phosphoribosyltransferase); Rn02130551_s1 (CXCR2, chemokine (C-X-C motif) receptor 2); Rn00578225_m1 (CXCL1, CINC-1, chemokine (C-X-C motif) ligand 1); Rn00586403_m1 (CXCL2, CINC-3, Mip-2, chemokine (C-X-C motif) ligand 2); and Rn01414231_m1 (CXCL3, CINC-2, chemokine (C-X-C motif) ligand 3).

Techniques: Transmission Assay, Comparison

Journal: Frontiers in Immunology

Article Title: Pharmacological Blockade of Spinal CXCL3/CXCR2 Signaling by NVP CXCR2 20, a Selective CXCR2 Antagonist, Reduces Neuropathic Pain Following Peripheral Nerve Injury

doi: 10.3389/fimmu.2019.02198

Figure Lengend Snippet: Effects of single NVP CXCR2 20 administration on a single CXCL3 injection and nociceptive transmission in naive mice (B,C) . Single intrathecal administrations of vehicle (V) or NVP CXCR2 20 (60 μg/5 μl) were performed 120 min before a single intrathecal administration of V or CXCL3 (2 ng/5 μl). The effects of administrations on mechanical (von Frey test; B ) and thermal (cold plate test; C ) hypersensitivity were measured 3.5, 7 and 26 h after the NVP CXCR2 20 injection (1.5, 5, and 24 h after the CXCL3) (A) . Data are presented as the means ± SEM (6–8 mice per group). The results were evaluated using one-way ANOVA followed by Bonferroni's test for comparisons of selected pairs. ** p < 0.01, *** p < 0.001 indicate differences in comparison with V+V-treated animals at the indicated time points. ## p < 0.01, ### p < 0.001 indicate differences in comparison with V+CXCL3-treated animals at the indicated time points. V, vehicle; NVP, NVP CXCR2 20.

Article Snippet: TaqMan primers and probes were used: Rn01527838_g1 (HPRT, hypoxanthine-guanine phosphoribosyltransferase); Rn02130551_s1 (CXCR2, chemokine (C-X-C motif) receptor 2); Rn00578225_m1 (CXCL1, CINC-1, chemokine (C-X-C motif) ligand 1); Rn00586403_m1 (CXCL2, CINC-3, Mip-2, chemokine (C-X-C motif) ligand 2); and Rn01414231_m1 (CXCL3, CINC-2, chemokine (C-X-C motif) ligand 3).

Techniques: Injection, Transmission Assay, Comparison

Journal: Frontiers in Immunology

Article Title: Pharmacological Blockade of Spinal CXCL3/CXCR2 Signaling by NVP CXCR2 20, a Selective CXCR2 Antagonist, Reduces Neuropathic Pain Following Peripheral Nerve Injury

doi: 10.3389/fimmu.2019.02198

Figure Lengend Snippet: Effects of on NVP CXCR2 20 levels of the CXCL1, CXCL2, and CXCL3 proteins (A–F) in primary rat microglial (A–C) and astroglial (D–F) cell cultures. Samples were analyzed 24 h after cells were stimulated with LPS. The data are presented as the fold change relative to the control and relative protein levels. Fold change relative to control : the Western blot data are presented as the means ± SEM and represent the normalized averages derived from analyses of 3–4 independent experiments. Intergroup differences were analyzed using ANOVA with Bonferroni's multiple comparisons test. * p < 0.05, ** p < 0.01, *** p < 0.001 indicate differences in comparison with the control group (vehicle-treated non-stimulated cells); # p < 0.05, ## p < 0.01, ### p < 0.001 indicate differences between vehicle-treated and NVP-treated LPS-stimulated cells. Relative protein level: Inter-group differences in relative protein level were analyzed using a t-test. ### p < 0.001 indicates differences compared to the vehicle-treated LPS-stimulated cells. LPS-, vehicle-treated non-stimulated cells; NVP-, NVP-treated non-stimulated cells, LPS+, vehicle-treated LPS-stimulated cells; NVP+, NVP-treated LPS-stimulated cells. In (D–F) the blots are cropped which was shown with a dotted line on the representation bands below the figures.

Article Snippet: TaqMan primers and probes were used: Rn01527838_g1 (HPRT, hypoxanthine-guanine phosphoribosyltransferase); Rn02130551_s1 (CXCR2, chemokine (C-X-C motif) receptor 2); Rn00578225_m1 (CXCL1, CINC-1, chemokine (C-X-C motif) ligand 1); Rn00586403_m1 (CXCL2, CINC-3, Mip-2, chemokine (C-X-C motif) ligand 2); and Rn01414231_m1 (CXCL3, CINC-2, chemokine (C-X-C motif) ligand 3).

Techniques: Control, Western Blot, Derivative Assay, Comparison