Journal: Advanced Science

Article Title: KK2DP7 Stimulates CD11b + Cell Populations in the Spleen to Elicit Trained Immunity for Anti‐Tumor Therapy

doi: 10.1002/advs.202500032

Figure Lengend Snippet: The target cells for training immunity induced by KK2DP7 are cells in the non‐lymphocyte layer of the spleen. a) Validation flowchart for KK2DP7‐induced training immunity in spleen target cell population. b) Cells in the splenic lymphocyte layer of mice trained with KK2DP7 and stimulated with LPS show no significant changes in the secretion of training‐immunity‐associated TNF‐α, IL‐1β, IL‐6, and IFN‐γ (n = 3). c) Cells in the splenic non‐lymphocyte layer of mice trained with KK2DP7 and stimulated with LPS exhibited a significant upregulation in the secretion of training‐immunity‐associated TNF‐α, IL‐1β, IL‐6, and IFN‐γ (n = 3). d) Representative flow cytometry plots of target cells in the non‐lymphocytic layer of mouse spleen are presented. e) TNF‐α production by cells in the non‐lymphocytic layer of the mouse spleen was significantly elevated after training with KK2DP7 and stimulation with LPS, whereas no changes in TNF‐α production were observed in cells in the lymphocytic layer (n = 3). f) TNF‐α production by CD45 + cells in the non‐lymphocytic layer of the mouse spleen was significantly elevated after training with KK2DP7 and stimulation with LPS, whereas no changes in TNF‐α production were observed in CD45 ‐ cells (n = 3). g) Following training with KK2DP7 and LPS stimulation, the proportion of CD45 + CD11b + cells in the non‐lymphocyte layer of the mouse spleen was found to be significantly upregulated (n = 3). h–k) Training with KK2DP7 and LPS stimulation led to a significant increase in the proportion of macrophages (CD11b + F4/80 + ), neutrophils (CD11b + Ly6G + ), monocytes (CD11b + Ly6C + ), and dendritic cells (CD11b + CD11c + ) expressing TNF‐α in the non‐lymphocytic layer of the mouse spleen (n = 3). All values presented in this figure are expressed as the mean ± S.D., unless otherwise indicated in the figure captions. Statistical analysis was performed using one‐way ANOVA with Dunnett's multiple comparison test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Two days after the last administration of KK2DP7, the CD11b + cells (1 × 10 5 ) from the non‐lymphocyte layer were isolated and cultured in a 24‐well plate, followed by tumor cells (CT26, EG7, LL2) at densities of 1 × 10 4 and 2 × 10 4 cells per well for 24 h. Subsequently, cell culture supernatants were collected and cytotoxicity was detected using a cytotoxicity LDH assay kit (MCE, US).

Techniques: Biomarker Discovery, Flow Cytometry, Expressing, Comparison

Journal: Advanced Science

Article Title: KK2DP7 Stimulates CD11b + Cell Populations in the Spleen to Elicit Trained Immunity for Anti‐Tumor Therapy

doi: 10.1002/advs.202500032

Figure Lengend Snippet: KK2DP7 induces the training of immunotherapy against tumors by activating CD11b + cells in the non‐lymphocytic layer of the spleen. a) The validation flowchart for KK2DP7‐induced training immunity in the spleen target cell population is presented below. b) CD11b + cells in the splenic non‐lymphocytic layer of mice trained with KK2DP7 and stimulated with LPS exhibited a significant upregulation of TNF‐α, IL‐1β, IL‐6, and IFN‐γ secretion, which is indicative of training immunity (n = 3). c) CD11b ‐ cells in the splenic nonlymphocytic layer of mice trained with KK2DP7 and stimulated with LPS did not exhibit any significant changes in the secretion of training‐immunity‐associated TNF‐α, IL‐1β, IL‐6, and IFN‐γ (n = 3). d,e) The CD11b + cells in the non‐lymphocyte layer of the spleen, which were trained with KK2DP7 and stimulated by tumor cells or tumor cell culture supernatant, demonstrated a significant upregulation of immune‐related TNF‐α, IL‐1β, IL‐6, and IFN‐γ secretion (n = 3). f) A flowchart of the adoptive transfer of CD11b + cells from the non‐lymphocyte layer of the spleen, following training with KK2DP7, for antitumor application is provided. g) The average tumor growth curve of mice in different groups is shown (n = 6). h) The survival rates of mice in different groups are presented (n = 6). i–k) Individual tumor growth curves from g are displayed. All values presented in this figure are expressed as the mean ± S.D., unless otherwise indicated in the figure captions. Statistical analysis was performed using one‐way ANOVA with Dunnett's multiple comparison test. Survival curves were obtained using the Kaplan–Meier method and compared by the log‐rank test. **** P < 0.0001.

Article Snippet: Two days after the last administration of KK2DP7, the CD11b + cells (1 × 10 5 ) from the non‐lymphocyte layer were isolated and cultured in a 24‐well plate, followed by tumor cells (CT26, EG7, LL2) at densities of 1 × 10 4 and 2 × 10 4 cells per well for 24 h. Subsequently, cell culture supernatants were collected and cytotoxicity was detected using a cytotoxicity LDH assay kit (MCE, US).

Techniques: Biomarker Discovery, Cell Culture, Adoptive Transfer Assay, Comparison

Journal: Advanced Science

Article Title: KK2DP7 Stimulates CD11b + Cell Populations in the Spleen to Elicit Trained Immunity for Anti‐Tumor Therapy

doi: 10.1002/advs.202500032

Figure Lengend Snippet: Significant differences were observed in the transcriptome and ATAC sequencing of CD11b + cells in the non‐lymphocyte layer of the mouse spleen following KK2DP7 treatment. a) Flowchart illustrating the target cell acquisition process for sequencing. b) Differential gene expression in CD11b + cells pretreated with KK2DP7 compared with those from PBS‐treated mice in RNA‐sequence (n=3). A volcano plot is presented, displaying the distribution of the adjusted p values (log10(padj)) and fold changes (log2 fold change). c) A heatmap of genes from KK2DP7‐treated mice as compared with PBS‐treated mice in RNA‐sequence is also included. d) An interaction network diagram of all changing genes in b is provided, as well as e) a differential peak expression in mice pre‐treated with KK2DP7 compared with from PBS‐treated mice in ATAC‐sequence. A volcano plot is presented, which shows the distribution of the adjusted p values (log10(padj)) and fold changes (log2 fold change). f) A heatmap is also provided, which shows the genes from KK2DP7‐treated mice as compared with PBS‐treated mice in ATAC sequence analysis. g) Gene ontology (GO) enrichment of immune‐related pathways in KK2DP7‐treated mice compared to PBS‐treated mice in assay for RNA‐seq analysis. h) Gene ontology (GO) enrichment of immune‐related pathways in KK2DP7‐treated mice compared to PBS‐treated mice in assay for transposase‐accessible chromatin sequencing (ATAC‐seq). i) Among the genes with shared changes in both RNA‐seq and ATAC‐seq, GO enrichment of immune‐related pathways in KK2DP7‐treated mice compared to PBS‐treated mice is analyzed. j) An expression heatmap of immune‐related genes that are commonly varied in both RNA‐seq and ATAC‐seq is provided. k) An interaction network diagram of all genes shown in j is included.

Article Snippet: Two days after the last administration of KK2DP7, the CD11b + cells (1 × 10 5 ) from the non‐lymphocyte layer were isolated and cultured in a 24‐well plate, followed by tumor cells (CT26, EG7, LL2) at densities of 1 × 10 4 and 2 × 10 4 cells per well for 24 h. Subsequently, cell culture supernatants were collected and cytotoxicity was detected using a cytotoxicity LDH assay kit (MCE, US).

Techniques: Sequencing, Gene Expression, Expressing, RNA Sequencing

Journal: Advanced Science

Article Title: KK2DP7 Stimulates CD11b + Cell Populations in the Spleen to Elicit Trained Immunity for Anti‐Tumor Therapy

doi: 10.1002/advs.202500032

Figure Lengend Snippet: The proliferation, phagocytosis and cytotoxicity of CD11b + cells in the non‐lymphocytic layer of the spleen are significantly enhanced following KK2DP7 training. a) Schematic diagram illustrating validated cell functions. b‐c) Gene Ontology (GO) enrichment analysis of cell proliferation‐related pathways in mice treated with KK2DP7 revealed a significant upregulation compared to mice treated with PBS, as evidenced by RNA and ATAC sequencing. d‐e) The expression heatmap of cell proliferation‐related genes and the interaction network diagram of all genes in the heatmap demonstrated a clear distinction between the two groups. f) The results of flow cytometry demonstrated a significant increase in the proliferation of CD11b + cells in the non‐lymphocyte layer of the spleen following KK2DP7 training and secondary stimulation. g‐h) GO enrichment of cell endocytosis‐related pathways in KK2DP7‐treated mice compared to PBS‐treated mice in RNA and ATAC sequencing. i) Expression heatmap of cell endocytosis‐related genes. j) Network diagram of all genes in i. k) GO enrichment of cell cytotoxicity‐related pathways in KK2DP7‐treated mice compared to PBS‐treated mice in RNA sequencing. l) Expression heatmap of cell cytotoxicity‐related genes. m) Network diagram of all genes in l. n) The results of flow cytometry demonstrated that following KK2DP7 training, the capacity of CD11b + cells in the non‐lymphocyte layer of the spleen to phagocytose tumor cells was significantly enhanced (n=3). o) The results of flow cytometry indicated that the capacity of CD11b + cells in the non‐lymphocyte layer of the spleen to kill tumors was significantly enhanced following KK2DP7 training (n=3). All values presented in this figure are expressed as the mean ± s.d., unless otherwise indicated in the figure captions. Statistical analysis was performed using one‐way ANOVA with Dunnett's multiple comparison test. In contrast, a student's t‐test was utilized for two‐group comparisons. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Two days after the last administration of KK2DP7, the CD11b + cells (1 × 10 5 ) from the non‐lymphocyte layer were isolated and cultured in a 24‐well plate, followed by tumor cells (CT26, EG7, LL2) at densities of 1 × 10 4 and 2 × 10 4 cells per well for 24 h. Subsequently, cell culture supernatants were collected and cytotoxicity was detected using a cytotoxicity LDH assay kit (MCE, US).

Techniques: Sequencing, Expressing, Flow Cytometry, RNA Sequencing, Comparison

Journal: Advanced Science

Article Title: KK2DP7 Stimulates CD11b + Cell Populations in the Spleen to Elicit Trained Immunity for Anti‐Tumor Therapy

doi: 10.1002/advs.202500032

Figure Lengend Snippet: Transcriptome changes and epigenetic reprogramming induced by immune training in KK2DP7. a) Pathways of KEGG enrichment in CD11b + cells of the non‐lymphocytic layer of the spleen in mice treated with KK2DP7. b) The interferon‐related pathway of Reactome enrichment in CD11b + cells of the non‐lymphocytic layer of the spleen in mice treated with KK2DP7. c‐d) Gene set enrichment analysis (GSEA) was conducted on genes related to the NOD‐ and RIG‐I‐like receptor signal pathway in CD11b + cells treated with KK2DP7 compared to those treated with PBS. The top core genes and their interactions in the NOD, RIG‐I, and interferon signaling pathways were displayed. Furthermore, a heatmap of transcription factor expression in RNA and ATAC sequences was generated. h) A genome browser track demonstrating differentially accessible regions associated with differential transcription factors. i) An analysis of GO functional networks regulated by differential transcription factors. j) An analysis of KEGG signaling pathway networks regulated by differential transcription factors.

Article Snippet: Two days after the last administration of KK2DP7, the CD11b + cells (1 × 10 5 ) from the non‐lymphocyte layer were isolated and cultured in a 24‐well plate, followed by tumor cells (CT26, EG7, LL2) at densities of 1 × 10 4 and 2 × 10 4 cells per well for 24 h. Subsequently, cell culture supernatants were collected and cytotoxicity was detected using a cytotoxicity LDH assay kit (MCE, US).

Techniques: Protein-Protein interactions, Expressing, Generated, Functional Assay

Journal: Advanced Science

Article Title: KK2DP7 Stimulates CD11b + Cell Populations in the Spleen to Elicit Trained Immunity for Anti‐Tumor Therapy

doi: 10.1002/advs.202500032

Figure Lengend Snippet: The induction of training immunity by KK2DP7 is dependent on the involvement of TLR2‐IRF7 signaling. a) Display of p‐values and expression levels of the top core genes in differential signaling pathways. b) Transcription factor regulatory gene network analysis diagram. c) Mechanistic map of KK2DP7's regulation of trained immunity. d) Up‐regulation of TLR2 expression levels in CD45 + CD11b + cells in the non‐lymphocyte layer of mouse spleen after KK2DP7 training (n=3). e‐g) The validation of TLR2, IRF7, and p‐IRF7 expression before and after immune training with KK2DP7 in wild‐type mice, TLR2 knockout mice, and IRF7 knockout mice (n=3). h‐j) The CD11b + cells in the splenic nonlymphocytic layer of TLR2 knockout mice and IRF7 knockout mice did not exhibit notable alterations in the secretion of immune‐associated cytokines following KK2DP7 induction. This was in stark contrast to the observed changes in wild‐type mice (n=3). k) Flowchart of inoculation and KK2DP7 administration in knockout mice. l) The average tumor growth curve of mice in different groups (n=6). m) The survival rates of mice in different groups. n) Individual tumor growth curves from l. o) Flow chart of KK2DP7 combined with anti‐PD‐1 antibody in the treatment of mouse tumors. p) The average tumor growth curve of mice in different groups (n=6). q) The survival rates of mice in different groups. r) Individual tumor growth curves from p. All values presented in this figure are expressed as the mean ± s.d., unless otherwise indicated in the figure captions. Statistical analysis was performed using one‐way ANOVA with Dunnett's multiple comparison test. In contrast, a student's t‐test was utilized for two‐group comparisons. Survival curves were obtained using the Kaplan–Meier method and compared by the log‐rank test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Two days after the last administration of KK2DP7, the CD11b + cells (1 × 10 5 ) from the non‐lymphocyte layer were isolated and cultured in a 24‐well plate, followed by tumor cells (CT26, EG7, LL2) at densities of 1 × 10 4 and 2 × 10 4 cells per well for 24 h. Subsequently, cell culture supernatants were collected and cytotoxicity was detected using a cytotoxicity LDH assay kit (MCE, US).

Techniques: Expressing, Protein-Protein interactions, Biomarker Discovery, Knock-Out, Comparison

Journal: Journal of translational medicine

Article Title: Cholesterol overload in macrophages drives metabolic dysfunction-associated steatohepatitis via inhibiting 7-dehydrocholesterol reductase in mice.

doi: 10.1186/s12967-024-05905-1

Figure Lengend Snippet: Fig. 1 Hepatic cholesterol accumulation and M1 macrophages infiltration in MASH. Six-week-old male C57BL/6J mice were fed either a NCD or a CDAHFD for six weeks (n = 3). (a) Body weight gain curves and (b) liver/body weight ratio was recorded. (c) Serum ALT and AST levels were detected. (d) Liver sections stained with H&E and ORO, and immunohistochemically for F4/80+ and CD11b+, 100× magnification. (e-f) Quantifies hepatic TC and TG levels. (g) NAS score. (h-j) Hepatic relative mRNA expression of Tnfa, Il1b and Il6. (k-l) Hepatic relative protein levels of CD86, ARG-1 and CD206. (m-o) Flow cytometry analysis of macrophages (live+CD45+F4/80+CD11b+) and M1 macrophages (live+CD45+F4/80+CD11b+iNOS+). (p-q) Flow cytometry analysis of TREM2 (live+CD45+F4/80+TREM2+). Data are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001 between groups

Article Snippet: Immunohistochemical analysis utilized anti-F4/80 (70076s) and anti-CD11b (17800s) antibodies from Cell Signaling Technology (CST, USA), and biotinylated goat anti-rabbit IgG (SA1022, BOSTER, China).

Techniques: Staining, Expressing, Flow Cytometry

Journal: Journal of translational medicine

Article Title: Cholesterol overload in macrophages drives metabolic dysfunction-associated steatohepatitis via inhibiting 7-dehydrocholesterol reductase in mice.

doi: 10.1186/s12967-024-05905-1

Figure Lengend Snippet: Fig. 3 Correlation between DHCR7 expression and macrophage polarization in MASH. (a) Relative expression of cholesterol biosynthesis and efflux genes in primary macrophages after ox-LDL (100 µg/ mL) or ox-LDL combined with statin treating for 24 h. (b) Expression levels of cholesterol biosyn thesis and efflux genes in primary macrophages polarized to M1 with LPS (100 ng/mL) or M2 with IL-4 (20 ng/mL) for 24 h. (c-d) Western blot analysis for DHCR7 protein in M1 and M2 macrophages, with densitometry. (e) Immunofluorescence staining for DHCR7 in the polarized-macrophages, 200× magnification. (f-g) Multiple immunofluorescence co-localization for DHCR7 and CD11B on livers of NCD or CDAHFD mice. (h-i) DHCR7 protein and mRNA levels in total liver and primary macrophages isolated from livers of mice on control and CDAHFD. (j-k) Immunofluorescence multi-labeling of liver sections from NC and MASH patients for DHCR7 and macrophage marker CD68 to determine the proportion of DHCR7+CD68+ cells among total CD68+ cells. Data are presented as mean ± SD, with n = 3 per group. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 between groups

Article Snippet: Immunohistochemical analysis utilized anti-F4/80 (70076s) and anti-CD11b (17800s) antibodies from Cell Signaling Technology (CST, USA), and biotinylated goat anti-rabbit IgG (SA1022, BOSTER, China).

Techniques: Expressing, Western Blot, Immunofluorescence, Staining, Isolation, Control, Labeling, Marker

Journal: Journal of translational medicine

Article Title: Cholesterol overload in macrophages drives metabolic dysfunction-associated steatohepatitis via inhibiting 7-dehydrocholesterol reductase in mice.

doi: 10.1186/s12967-024-05905-1

Figure Lengend Snippet: Fig. 5 Myeloid-macrophage-specific DHCR7 deficiency exacerbates MASH progression. Wildtype (WT) and myeloid-specific DHCR7 knockout (DHCR7 cKO) mice were fed with a normal control diet (CON) or a CDAHFD for 10 weeks (n = 7). (a) Body weight progression, (b) liver/body weight ratio, and (c-d) serum ALT and AST levels were detected. (e-f) Hepatic TG and TC levels were measured. (g) ELISA quantification of liver IL-1β and IL-6 levels. (h) Histological analysis of liver sections is presented through H&E, ORO staining, and immunohistochemistry for F4/80+ and CD11b+ cells (200× magnification). (i) NAS score. (j-k) Relative protein levels of CD86, ARG-1, CD206 were determined, with densitometry. (l) Flow cytometry of primary macrophage in cKO mice and WT with different diets. (m) Flow cytometry of TREM2 in macrophages. (n) Predominantly of the M1 type in macrophages, including quantification of both the percentage of M1 macrophages and M1 macrophage counts per liver weight. Data are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 between group

Article Snippet: Immunohistochemical analysis utilized anti-F4/80 (70076s) and anti-CD11b (17800s) antibodies from Cell Signaling Technology (CST, USA), and biotinylated goat anti-rabbit IgG (SA1022, BOSTER, China).

Techniques: Knock-Out, Control, Enzyme-linked Immunosorbent Assay, Staining, Immunohistochemistry, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: Crosstalk Between CD11b and Piezo1 Mediates Macrophage Responses to Mechanical Cues

doi: 10.3389/fimmu.2021.689397

Figure Lengend Snippet: CD11b is required for stretch-mediated changes in macrophage activation. (A) Representative Western blot of CD11b and GAPDH for unstimulated, IFNγ/LPS, and IL4/IL13 stimulated macrophages exposed to 0% and 20% static and cyclic stretch (left). Quantification of average across three independent experiments for CD11b expression (right). Values were normalized to GAPDH and made relative to 0% stretch and unstimulated condition. (B) Representative immunofluorescence images (top) and quantification of relative Itgam gene expression in unstimulated macrophages treated with non-target (siControl) or CD11b (siCD11b) siRNA. Data relative to siControl condition. (C) Secretion of TNFα, IL6, and MCP1 for unstimulated and IFNγ/LPS stimulated macrophages treated with siControl or siCD11b and exposed to either 0% control, 20% static, or 20% cyclic stretch. Data normalized to a siControl and IFNγ/LPS treated internal control exposed to 0% stretch within each biological replicate. (D) Relative Arg1 gene expression in IL4/IL13 stimulated and siControl or siCD11b treated BMDMs exposed to 0% control, 20% static, or 20% cyclic stretch. Data relative to 0% siControl condition. Error bars indicate standard deviation of the mean for three separate experiments and * p < 0.05 when compared to the corresponding 0% stretch condition as determined by Student’s t-test (A, C) or paired t-test (B, D) .

Article Snippet: For CD11b or Piezo1 staining, the cells were blocked in 2% BSA following fixation prior to being incubated with rat anti-CD11b (BioLegend) and rabbit anti-RFP (Rockland) primary antibodies, diluted 1:50 for CD11b and 1:400 for RFP antibodies in 2% BSA for 1 hour at room temperature.

Techniques: Activation Assay, Western Blot, Expressing, Immunofluorescence, Gene Expression, Control, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Crosstalk Between CD11b and Piezo1 Mediates Macrophage Responses to Mechanical Cues

doi: 10.3389/fimmu.2021.689397

Figure Lengend Snippet: Modulation of CD11b by adhesion time regulates stretch-mediated macrophage inflammatory responses. (A) Phase contrast images (top) of macrophages following 4 hrs (left) and 24 hrs (right) of adhesion prior to stimulation and stretch. Fluorescence images (bottom) of macrophages labelled for CD11b (red), actin (green), and nuclei (blue) following 4 hrs (left) and 24 hrs (right) of culture. (B) Averaged relative median fluorescence intensity across three independent experiments of CD11b as measured by flow cytometry. Values normalized to 4 hrs adhesion condition. (C) Secretion of TNFα for unstimulated and IFNγ/LPS stimulated macrophages exposed to 0% and 20% cyclic stretch after 4 hrs (left) and 24 hrs (right) of adhesion. Values are normalized to a 0% stretch IFNγ/LPS internal control within each biological replicate. (D) Representative Western blots (left) and corresponding quantification for ARG1 (right) for unstimulated, IFNγ/LPS, and IL4/IL13 stimulated macrophages allowed to adhere for 4 hrs prior to stimulation and stretch. Values were normalized to GAPDH and made relative to IL4/IL13 stimulated and 0% stretch conditions, respectively. Error bars indicate standard deviation of the mean for three separate experiments and * p < 0.05 when compared to the corresponding 0% stretch condition as determined by Student’s t-test (C) or paired t-test (B, D) .

Article Snippet: For CD11b or Piezo1 staining, the cells were blocked in 2% BSA following fixation prior to being incubated with rat anti-CD11b (BioLegend) and rabbit anti-RFP (Rockland) primary antibodies, diluted 1:50 for CD11b and 1:400 for RFP antibodies in 2% BSA for 1 hour at room temperature.

Techniques: Fluorescence, Flow Cytometry, Control, Western Blot, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Crosstalk Between CD11b and Piezo1 Mediates Macrophage Responses to Mechanical Cues

doi: 10.3389/fimmu.2021.689397

Figure Lengend Snippet: Crosstalk between Piezo1 and CD11b mediates macrophage response to stretch. (A) Representative immunofluorescence images (left) and quantification of mean Piezo1-tdT intensity in unstimulated, IFNγ/LPS, and IL4/IL13 treated macrophages exposed to 0% control, 20% static, or 20% cyclic stretch. Data normalized to the 0% control condition. (B) Secretion of TNFα in IFNγ/LPS stimulated macrophages treated with siControl or siPiezo1 and exposed to either 0% or 20% cyclic stretch. Data normalized to a siControl treated internal control exposed to 0% stretch within each biological replicate. (C) Secretion of TNFα in IFNγ/LPS stimulated macrophages treated with DMSO or Yoda1 and exposed to either 0% or 20% cyclic stretch. Data normalized to a DMSO treated internal control exposed to 0% stretch within each biological replicate. (D) Relative Piezo1 gene expression in unstimulated macrophages treated with non-target (siControl) or CD11b (siCD11b) siRNA. Gene expression is normalized to the siControl treated condition. (E) Relative Itgam, Itgb1, Itgb2 , and Itgb3 gene expression in unstimulated and siControl or siPiezo1 treated macrophages. Gene expression is normalized to the siControl treated condition. Error bars indicate standard deviation of the mean for three separate experiments and * p < 0.05 when compared to the corresponding 0% stretch condition as determined by Student’s t-test.

Article Snippet: For CD11b or Piezo1 staining, the cells were blocked in 2% BSA following fixation prior to being incubated with rat anti-CD11b (BioLegend) and rabbit anti-RFP (Rockland) primary antibodies, diluted 1:50 for CD11b and 1:400 for RFP antibodies in 2% BSA for 1 hour at room temperature.

Techniques: Immunofluorescence, Control, Gene Expression, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Crosstalk Between CD11b and Piezo1 Mediates Macrophage Responses to Mechanical Cues

doi: 10.3389/fimmu.2021.689397

Figure Lengend Snippet: Stretch-induced changes in macrophage activation require modulation of actin. (A) Representative images of F-actin in unstimulated, IFNγ/LPS, and IL4/IL13 stimulated macrophages exposed to 0%, 20% static, and 20% cyclic stretch. Quantification of mean F-actin fluorescence intensity across three independent experiments (right). Data normalized to the unstimulated and 0% stretch control. (B) Representative images of F-actin in unstimulated, IFNγ/LPS, and IL4/IL13 stimulated macrophages exposed to siControl or CD11b siRNA. Quantification of mean F-actin fluorescence intensity across three independent experiments (right). Values normalized to unstimulated and siControl condition. (C) Secretion of TNFα in IFNγ/LPS stimulated macrophages treated with DMSO or CytoD and exposed to 0% and 20% static or cyclic strains. Values are normalized to a DMSO, 0% stretch, and IFNγ/LPS stimulated internal control within each biological replicate. (D) Representative Western blot (left) and quantification of ARG1 expression in IL4/IL13 stimulated macrophages treated with DMSO or CytoD and exposed to 0% and 20% static or cyclic strains. Expression is relative to GAPDH. Error bars indicate standard deviation of the mean for three separate experiments and * p < 0.05 when compared to the corresponding 0% stretch condition as determined by paired t-test (A, B) and Student’s t-test (C, D) .

Article Snippet: For CD11b or Piezo1 staining, the cells were blocked in 2% BSA following fixation prior to being incubated with rat anti-CD11b (BioLegend) and rabbit anti-RFP (Rockland) primary antibodies, diluted 1:50 for CD11b and 1:400 for RFP antibodies in 2% BSA for 1 hour at room temperature.

Techniques: Activation Assay, Fluorescence, Control, Western Blot, Expressing, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Crosstalk Between CD11b and Piezo1 Mediates Macrophage Responses to Mechanical Cues

doi: 10.3389/fimmu.2021.689397

Figure Lengend Snippet: Summary of stretch mediated changes in macrophage function.

Article Snippet: For CD11b or Piezo1 staining, the cells were blocked in 2% BSA following fixation prior to being incubated with rat anti-CD11b (BioLegend) and rabbit anti-RFP (Rockland) primary antibodies, diluted 1:50 for CD11b and 1:400 for RFP antibodies in 2% BSA for 1 hour at room temperature.

Techniques: Expressing

Journal: Stem Cell Research & Therapy

Article Title: Inflammatory cytokine-primed MSC-derived extracellular vesicles ameliorate acute lung injury via enhanced immunomodulation and alveolar repair

doi: 10.1186/s13287-025-04576-z

Figure Lengend Snippet: P-MEVs alleviate lung inflammation in a mouse model of LPS-induced ARDS C57BL/6 mice were challenged with LPS (5 mg/kg, intravenously) and then received daily injections of PBS or MSC-EVs (6 × 10 9 particles, intravenously). After 72 h, the mice were sacrificed and subjected to functional analysis. ( a ) Experimental scheme for establishing an LPS-induced ALI mouse model and administration of MSC-EVs. Created using BioRender.com. ( b ) Daily monitoring of mouse body weight changes. Values are calculated as a percentage of body weight from day 0. n = 4–5. ( c ) qRT-PCR analysis of relative mRNA expression levels of TNF-α , IL-1β , and IL-6 in the lung tissues. n = 5. ( d ) ELISA analysis of TNF-α, IL-1β, and IL-6 concentrations in the lung tissues. n = 5. ( e ) Western blot analysis of lung homogenate (left, Full-length blots are presented in Supplementary Figure ). Phosphorylated bands were normalized to the respective total bands and are shown as fold changes relative to the control. n = 5. ( f ) Flow cytometric analysis of neutrophils (CD11b + Ly6G+), monocyte-derived macrophages (Siglec F- CD11b + on CD11c + F4/80+), and alveolar macrophages (Siglec F + CD11b- on CD11c + F4/80+) accumulation in lung tissue. n = 5. Con, control mice with PBS injections; LPS, mice with LPS injection only; LPS + C-MEVs, mice with LPS and control MSC-EVs injections; LPS + P-MEVs, mice with LPS and primed MSC-EVs injections. Data are presented as the mean ± SEM, analyzed by two-way ANOVA for ( b ) and analyzed with a one-way ANOVA for ( c ), ( d ), ( e ), and ( f ), followed by the Holm–Sidak multiple comparison test. Statistical differences in post hoc tests are indicated as ns = not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001

Article Snippet: All cells were fixed using 4% PFA mixed in PBS and incubated at room temperature for 10 min. After blocking, the cells were stained with the following fluorescence-conjugated antibodies: anti-CD45-APC-Cy7 antibody (103115, clone 30-F11; Biolegend, San Diego, CA, USA), anti-Ly6G-FITC antibody (127605, clone 1A8; Biolegend), anti-Siglec-F-BV421 antibody (565934, clone E50-2440; BD Life Sciences), anti-F4/80-eFluorTM 506 antibody (69-4801-80, clone BM8; Invitrogen; Thermo Fisher Scientific), anti-CD11b-PE antibody (24965; Cell Signaling Technology), and anti-CD11c-APC antibody (33293; Cell Signaling Technology).

Techniques: Functional Assay, Quantitative RT-PCR, Expressing, Enzyme-linked Immunosorbent Assay, Western Blot, Control, Derivative Assay, Injection, Comparison