Journal: Advanced Science

Article Title: Liver Kinase B1 in CD11c + Cells Inhibits Fibrosis in Chronic Pancreatitis via the Oncostatin M Signaling

doi: 10.1002/advs.75018

Figure Lengend Snippet: Lkb1 deletion in CD11c + macrophages drives fibrosis and inflammation in CP. (A) Representative images of pancreatic H&E staining. Scale bar, 100 µm. n = 8. (B) Representative images of pancreatic Masson's staining. Scale bar, 100 µm. n = 8. (C) Representative immunohistochemistry (IHC) staining images of CD11c in pancreatic tissue. Scale bar, 50 µm. n = 8. (D,E) Representative immunofluorescence images of CD11c, Lkb1, and DAPI co‐staining in CP and normal pancreatic tissues. Scale bar, 50 µm. n = 8. (F) Western blot analysis of Lkb1 expression in pancreatic CD11c + cells at multiple time points after disease induction. n = 3. (G,H) Representative immunofluorescence images of CD11c, Lkb1, and DAPI co‐staining in pancreatic tissues from control and CP model WT mice, as well as in splenic tissues from control WT mice. Scale bar, 50 µm. n = 5. (I–L) Representative images of pancreatic H&E staining and Masson's staining. Scale bar, 100 µm. From left to right: n = 8; n = 8; n = 12; n = 10. (M) Representative immunofluorescence images of co‐staining for CD45, CK19, α‐Amylase, α‐SMA, and DAPI in the pancreas with (N,O) statistical analysis. Scale bar, 50 µm. n = 5. (P) Relative pancreatic weight (pancreas/body weight). From left to right: n = 8; n = 8; n = 12; n = 10. Data are means ± SEM. Unpaired Student's t ‐tests (A, B, C, E, H, N, O) were used to evaluate statistical significance. Data were analyzed using one‐way ANOVA (J, L, P) with the Tukey test.

Article Snippet: Total RNA was extracted from the pancreatic tissue of mouse CP or CD11c magnetic bead‐sorted cells (Miltenyi Biotec, #130‐125‐835) using TRIzol reagent, following the manufacturer's guidelines.

Techniques: Staining, Immunohistochemistry, Immunofluorescence, Western Blot, Expressing, Control

Journal: Advanced Science

Article Title: Liver Kinase B1 in CD11c + Cells Inhibits Fibrosis in Chronic Pancreatitis via the Oncostatin M Signaling

doi: 10.1002/advs.75018

Figure Lengend Snippet: Lkb1 deletion in CD11c + cells modulates the inflammatory microenvironment of CP. (A) Overview of the CP mouse model and scRNA‐seq workflow. (B) UMAP plots illustrating distinct cell populations in the pancreas of CP mice. (C) UMAP plot displaying distinct cell populations in CP pancreatic tissues of Lkb1 f/f and CD11c Cre Lkb1 f/f mice. (D) Percentage of cells of different cell types in CP pancreatic tissues of Lkb1 f/f and CD11c Cre Lkb1 f/f mice. (E–G) Differences in the frequency of PSCs、T cells or macrophages as a percentage of total cells. (H) KEGG enrichment analysis of upregulated genes in PSCs. (I) UMAP plot of macrophage subclusters in CP pancreatic tissue. (J) Percentage of cells of different macrophage subclusters in macrophages of Lkb1 f/f and CD11c Cre Lkb1 f/f mice. (K) Trajectory plots display pseudo‐time trajectory analysis of various macrophage subpopulations derived from monocytes. (L) Violin plot showing the progression of cells from Lkb1 f/f and CD11c Cre Lkb1 f/f mice along the determined trajectory in pseudotime. (A–L) Lkb1 f/f ( n = 4) and CD11c Cre Lkb1 f/f ( n = 3) mice (each dot represents an animal). Data are means ± SEM. Unpaired Student's t ‐tests (E–G) were used to evaluate statistical significance.

Article Snippet: Total RNA was extracted from the pancreatic tissue of mouse CP or CD11c magnetic bead‐sorted cells (Miltenyi Biotec, #130‐125‐835) using TRIzol reagent, following the manufacturer's guidelines.

Techniques: Derivative Assay

Journal: Advanced Science

Article Title: Liver Kinase B1 in CD11c + Cells Inhibits Fibrosis in Chronic Pancreatitis via the Oncostatin M Signaling

doi: 10.1002/advs.75018

Figure Lengend Snippet: Lkb1 deletion in CD11c + cells promotes an increase in CD11c + CD206 + macrophages in CP. (A,B) Representative flow cytometry plots and the absolute number of macrophages in the pancreas. From left to right: n = 5; n = 4. (C,D) Representative flow cytometry plots and the absolute number of CD11c + CD206 + macrophages in the pancreas. From left to right: n = 5; n = 4. (E–G) Representative IHC staining images and statistical analysis of CD11c ( n = 10; n = 8), F4/80 ( n = 12; n = 9), and CD206 ( n = 12; n = 8). Scale bar, 50 µm. (H,I) Representative immunofluorescence images of co‐staining for F4/80, CD206, CD11c, α‐SMA, and DAPI in the pancreas. Scale bar, 50 µm. n = 8. (J,K) Representative immunofluorescence images of CD68, CD206, CD11c, and DAPI co‐staining in pancreatic tissues of patients with clinical CP. Scale bar, 50 µm. n = 8. Data are means ± SEM. Unpaired Student's t ‐tests were used to evaluate statistical significance.

Article Snippet: Total RNA was extracted from the pancreatic tissue of mouse CP or CD11c magnetic bead‐sorted cells (Miltenyi Biotec, #130‐125‐835) using TRIzol reagent, following the manufacturer's guidelines.

Techniques: Flow Cytometry, Immunohistochemistry, Immunofluorescence, Staining

Journal: Advanced Science

Article Title: Liver Kinase B1 in CD11c + Cells Inhibits Fibrosis in Chronic Pancreatitis via the Oncostatin M Signaling

doi: 10.1002/advs.75018

Figure Lengend Snippet: Lkb1 deletion in CD11c + cells promotes CCL2 secretion and recruitment of macrophages. (A) UMAP plots showing the gene expression and distribution of CCL2 in the subpopulation. Lkb1 f/f ( n = 4) and CD11c Cre Lkb1 f/f ( n = 3) mice. (B,C) Violin plots showing the expression levels of CCL2 in CD11c + CD206 + macrophages and PSCs. Lkb1 f/f ( n = 4) and CD11c Cre Lkb1 f/f ( n = 3) mice. (D,E) Immunofluorescence images showing co‐staining of CD11c, CD206, CCL2, α‐SMA, and DAPI in mouse pancreatic tissue. Scale bar, 50 µm. From left to right: n = 6; n = 5. (F,G) Immunofluorescence images showing co‐staining of α‐SMA, CCL2, and DAPI in mouse pancreatic tissue. Scale bar, 50 µm. From left to right: n = 6; n = 5. (H,I) Immunofluorescence images showing co‐staining of CD11c, CD206, CCL2, α‐SMA, and DAPI in human pancreatic tissue. Scale bar, 50 µm. n = 8. (J) Statistical chart of correlation between CCL2 + cells and α‐SMA + cells. (K) Schematic representation of the experimental model using CCL2 inhibitors. (L) Representative images of pancreatic tissue stained with H&E staining and Masson's staining, and (M) pathological scores indicating statistical analysis. Scale bar, 100 µm. From left to right: n = 6; n = 7; n = 6; n = 6. (N) Representative flow cytometry plots and (O) absolute numbers of total macrophages. From left to right: n = 6; n = 6; n = 5; n = 6. (P) Representative flow cytometry plots and (Q) absolute numbers of CD11c + CD206 + macrophages. From left to right: n = 6; n = 6; n = 5; n = 6. Data are means ± SEM. Unpaired Student's t ‐tests (E,F,I) were used to evaluate statistical significance. Data were analyzed using one‐way ANOVA (M,O,Q) with the Tukey test.

Article Snippet: Total RNA was extracted from the pancreatic tissue of mouse CP or CD11c magnetic bead‐sorted cells (Miltenyi Biotec, #130‐125‐835) using TRIzol reagent, following the manufacturer's guidelines.

Techniques: Gene Expression, Expressing, Immunofluorescence, Staining, Flow Cytometry

Journal: Advanced Science

Article Title: Liver Kinase B1 in CD11c + Cells Inhibits Fibrosis in Chronic Pancreatitis via the Oncostatin M Signaling

doi: 10.1002/advs.75018

Figure Lengend Snippet: Lkb1 regulates the expression of its target gene CCL2 in CD11c + CD206 + macrophages by inhibiting STAT3 phosphorylation. (A) Single‐cell regulatory network inference and clustering analysis showing the number of regulons, the number of TFs, and the number of target genes obtained by prediction. (B) Regulon activity values of subpopulations using a heatmap to demonstrate the heterogeneity and distribution characteristics of regulons in subpopulations. (C) WGCNA‐based gene co‐expression network analysis. (D) Volcano plot showing differentially expressed genes in CD11c + CD206 + macrophages. (E) KEGG enrichment analysis of upregulated differential genes in CD11c + CD206 + macrophages. (F) Schematic diagram of cell culture. (G) Western blot analysis of p‐STAT3 and t‐STAT3 in CD11c + CD206 + cells. (H) CCL2 secretion level in cell culture supernatants. (I) mRNA expression level of CCL2 . (A–E) Lkb1 f/f ( n = 4) and CD11c Cre Lkb1 f/f ( n = 3) mice, (F–I) n = 3. Data are means ± SEM. Data were analyzed using one‐way ANOVA (H,I) with the Tukey test.

Article Snippet: Total RNA was extracted from the pancreatic tissue of mouse CP or CD11c magnetic bead‐sorted cells (Miltenyi Biotec, #130‐125‐835) using TRIzol reagent, following the manufacturer's guidelines.

Techniques: Expressing, Phospho-proteomics, Single Cell, Activity Assay, Cell Culture, Western Blot

Journal: Advanced Science

Article Title: Liver Kinase B1 in CD11c + Cells Inhibits Fibrosis in Chronic Pancreatitis via the Oncostatin M Signaling

doi: 10.1002/advs.75018

Figure Lengend Snippet: Lkb1 in CD11c + cells mediates the interaction between CD11c + CD206 + macrophages and PSCs through the OSM signaling pathway. (A,B) Multi‐subpopulation network diagrams showing the number of ligand‐receptor pairs and expression abundance or probability information in cellular subpopulation pairs. (C) Bar graphs showing the proportion of CD11c + CD206 + macrophage‐PSCs interacting with signaling pathways in the two groups. (D,E) Dot plots of the cell‐cell communication between PSCs and CD11c + CD206 + macrophages. (F) Network map of cellular communication at the level of the OSM signaling pathway in the CD11c Cre Lkb1 f/f group. (G) Heatmap showing the relative importance of each cell group calculated based on four network‐centered metrics of OSM signaling in CP mice of the CD11c Cre Lkb1 f/f group. (H) Bar plots showing the contribution of each ligand‐receptor pair to the OSM signaling pathway. Characterization maps of (I) OSM and (J) OSMR gene expression. (K) Representative immunofluorescence images of co‐staining for CD11c, F4/80, OSM, and DAPI in the pancreas with (L) statistical analysis. Scale bar, 50 µm. (M) Representative immunofluorescence images of co‐staining for OSMR, α‐SMA, and DAPI in the pancreas with (N) statistical analysis. Scale bar, 50 µm. (O) Representative immunofluorescence images of co‐staining for CD11c, CD68, OSM, OSMR, α‐SMA, and DAPI in human CP and normal pancreatic tissues with (P,Q) statistical analysis. Scale bar, 50 µm. Mountain range plots showing gene expression in PSCs associated with the (R) MAPK or (S) NF‐κB signaling pathways. (A–J,R,S) Lkb1 f/f ( n = 4) and CD11c Cre Lkb1 f/f ( n = 3) mice, K‐N) n = 5, (O–Q) n = 8. Data are means ± SEM. Unpaired Student's t ‐tests (L–Q) were used to evaluate statistical significance.

Article Snippet: Total RNA was extracted from the pancreatic tissue of mouse CP or CD11c magnetic bead‐sorted cells (Miltenyi Biotec, #130‐125‐835) using TRIzol reagent, following the manufacturer's guidelines.

Techniques: Expressing, Protein-Protein interactions, Gene Expression, Immunofluorescence, Staining

Journal: Advanced Science

Article Title: Liver Kinase B1 in CD11c + Cells Inhibits Fibrosis in Chronic Pancreatitis via the Oncostatin M Signaling

doi: 10.1002/advs.75018

Figure Lengend Snippet: Mechanistic model of Lkb1 ’s role in CD11c + cells in chronic pancreatitis.

Article Snippet: Total RNA was extracted from the pancreatic tissue of mouse CP or CD11c magnetic bead‐sorted cells (Miltenyi Biotec, #130‐125‐835) using TRIzol reagent, following the manufacturer's guidelines.

Techniques: