Journal: JCI insight

Article Title: CETP inhibition enhances monocyte activation and bacterial clearance and reduces streptococcus pneumonia-associated mortality in mice.

doi: 10.1172/jci.insight.173205

Figure Lengend Snippet: Figure 4. CETPi increases macrophage infiltration into site of infection and macrophage activation while maintaining tissue homeostasis at different stages of sepsis. (A and B) Representative staining figures of M1 macrophages by marker CD86 in lung obtained 0 and 72 hours after infection by S. pneu- moniae from female APOA1.CETP mice treated with control or CETPi. Quantification was determined by random sampling spots on lungs of 3 different mice in each treatment and by measuring stained areas using ImageJ (H&E 0 hours, mean ± SD, 16.0 ± 2.6 [CETPi, n = 3] versus 2.7 ± 1.5 [Control, n = 3], unpaired t test, P = 0.002) (IHC 72 hours, mean ± SD, 6.0 ± 3.5 [CETPi, n = 3] versus 17.0 ± 7.9% [Control, n = 3], unpaired t test, P = 0.02). (C and D) Propor- tion of infiltrating inflammatory macrophages (CD11b+Ly6c+) and tissue repairing and inflammation resolving macrophages (CD11b+Ly6c–) in BAL samples obtained 24 and 72 hours after infection from female APOA1.CETP mice treated with control or CETPi (CD11b+Ly6c+ 72 hours, mean ± SD, 5.6 ± 1.8 [CETPi, n = 4] versus 14.3 ± 6.1% [Control, n = 3], unpaired t test with Bonferroni correction, P = 0.037) (CD11b+ Ly6c– 24 hours, mean ± SD, 62.1 ± 6.8 [CETPi, n = 4] ver- sus 45.6 ± 3.8 [Control, n = 3], unpaired t test with Bonferroni correction, P = 0.013; CD11b+ Ly6c– 72 hours, mean ± SD, 78.3 ± 9.1 [CETPi, n = 4] versus 34.7 ± 22.7 [Control, n = 3], unpaired t test with Bonferroni correction, P = 0.016). (E) Proportion of migrating monocytes (Ly6C++Ly6GDIM) in blood samples obtained 0 and 72 hours after infection from female APOA1.CETP mice treated with control or CETPi (mean ± SD, 12.0 ± 2.7 [CETPi, n = 5] versus 0.14 ± 0.06 [Control, n = 4], unpaired t test, P = 0.00005). Data are shown as mean ± SD. *P < 0.05, **P < 0.01, ****P < 0.0001. ApoA1, apolipoprotein A-1; CETP, cholesteryl ester transfer protein; CD86, cluster of differentiation 86; CD11b, integrin α M; Ly6-C, lymphocyte antigen 6 locus C; Ly6-G, lymphocyte antigen 6 locus G.

Article Snippet: For IHC, tissue cross-sections were incubated with a primary macrophage membrane mark antibody kit (CD86 and CD206, 97624, CST) to identify specific cell types of macrophages followed by secondary antibodies using the alkaline phosphatase system to amplify signals.

Techniques: Infection, Activation Assay, Staining, Marker, Control, Sampling

Journal: JCI insight

Article Title: CETP inhibition enhances monocyte activation and bacterial clearance and reduces streptococcus pneumonia-associated mortality in mice.

doi: 10.1172/jci.insight.173205

Figure Lengend Snippet: Figure 5. CETPi decreases caspase-1 and COX-2 protein expression in human and mouse cells. (A–C) COX-2 and Caspase-1 expression in PBMCs treated with increasing doses of CETPi. (D–E) COX-2 expression in THP1 cells treated with control or CETPi (1 μM) (mean ± SD, 1.00 ± 0.42 [Control, n = 3] versus 1.31 ± 0.38 [CETPi, n = 3], unpaired t test, P = 0.03). (F–G) Caspase-1 in THP1 cells treated with control or CETPi (1 μM) (mean ± SD, 1.00 ± 0.19 [Control, n = 3] versus 2.02 ± 0.11 [CETPi, n = 3], unpaired t test, P = 0.02). (H) Expression of ROS in THP1 cells treated with control or CETPi (2 μM), (mean ± SD, 2.45 ± 0.36 [CETPi, n = 6] versus 1.00 ± 0.19 [Control, n = 6], unpaired t test, P = 0.00005). (I–K) Expression of COX-2 and CETP in RAW264.7 cells after adenovirus transfection induced-CETP overexpression in increasing MOIs. (L–M) Expression of pro–caspase-1 and cleaved caspase-1 in RAW 264.7 cells after adeno- virus transfection–induced CETP overexpression at MOI = 1.6. Pro–caspase-1: mean ± SD, 1.00 ± 0.15 (Control, n = 3) versus 0.45 ± 0.22 (CETP overexpress, n = 3), unpaired t test, P = 0.02; cleaved caspase-1: mean ± SD, 1.00 ± 0.14 (Control, n = 3) versus 0.58 ± 0.16 (CETP overexpress, n = 3), unpaired t test, P = 0.03. Data displayed as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. Caspase-1, caspase-1/IL-1 converting enzyme; COX-2, prostaglandin-endoperoxide synthase 2; MOI, multiplicity of infection; PBMC, human peripheral blood mononuclear cell; RAW, murine macrophage cell; ROS, reactive oxygen species; THP1, human peripheral blood monocyte.

Article Snippet: For IHC, tissue cross-sections were incubated with a primary macrophage membrane mark antibody kit (CD86 and CD206, 97624, CST) to identify specific cell types of macrophages followed by secondary antibodies using the alkaline phosphatase system to amplify signals.

Techniques: Expressing, Control, Transfection, Over Expression, Virus, Infection

Journal: Frontiers in Cell and Developmental Biology

Article Title: Necroptosis in Macrophage Foam Cells Promotes Fat Graft Fibrosis in Mice

doi: 10.3389/fcell.2021.651360

Figure Lengend Snippet: Necroptosis is activated in MFCs in fat graft tissue. (A) TEM of the ultrastructure of MFCs located in fat tissue after grafting. N, nucleus; LD, lipid droplet; PS, pseudopodia; M, mitochondria; RER, rough endoplasmic reticulum; and Lys, lysosome. Red arrowheads indicated the damaged plasma membrane. (B,C) Representative pMLKL staining (B) and quantification of the pMLKL-positive area (C) in fat tissue before and after grafting. (D) Representative immunofluorescence staining of pMLKL (red) and F4/80 (green) in fat tissue after grafting. Scale bars = 50 μm. Data are presented as the mean ± SD. ## P < 0.01 compared with the pre-grafting group; ** P < 0.01.

Article Snippet: Primary antibodies against pRIPK3, RIPK3, pMLKL, and MLKL from a Mouse Reactive Necroptosis Antibody Sampler Kit (Cell Signaling Technology) were used.

Techniques: Clinical Proteomics, Membrane, Staining, Immunofluorescence

Journal: Frontiers in Cell and Developmental Biology

Article Title: Necroptosis in Macrophage Foam Cells Promotes Fat Graft Fibrosis in Mice

doi: 10.3389/fcell.2021.651360

Figure Lengend Snippet: Necroptosis is activated in MFCs in the in vitro CLS cell culture model. (A,B) Necrosis of macrophages determined by Hoechst 33342 and PI staining in the CLS cell culture group and in RAW 264.7 macrophages cultured alone at 24 and 96 h. (A) Hoechst 33342 + PI + macrophages in the pictures indicate necrotic macrophages. (B) Quantification of macrophage necrosis in both groups at 24 and 96 h. (C,D) Western blot analyses of MLKL, pMLKL, RIPK3, and pRIPK3 in the CLS cell culture group and in RAW 264.7 macrophages cultured alone at 24 and 96 h. (E) Immunofluorescence staining of pMLKL (red) and F4/80 (green) in the CLS cell culture group and in RAW 264.7 macrophages cultured alone at 24 and 96 h. Scale bars = 50 μm. A, adipocyte; M, macrophage. Data are presented as the mean ± SD. ## P < 0.01 compared with the group of RAW 264.7 macrophages cultured alone at 24 h.

Article Snippet: Primary antibodies against pRIPK3, RIPK3, pMLKL, and MLKL from a Mouse Reactive Necroptosis Antibody Sampler Kit (Cell Signaling Technology) were used.

Techniques: In Vitro, Cell Culture, Staining, Western Blot, Immunofluorescence

Journal: Frontiers in Cell and Developmental Biology

Article Title: Necroptosis in Macrophage Foam Cells Promotes Fat Graft Fibrosis in Mice

doi: 10.3389/fcell.2021.651360

Figure Lengend Snippet: Necroptosis inhibitors suppressed necroptosis of MFCs in the in vitro CLS cell culture model. (A,B) Western blot analyses of MLKL, pMLKL, RIPK3, and pRIPK3 in four groups, including RAW 264.7 macrophages cultured alone, and those in CLS cell culture incubated in the presence or absence of a necroptosis inhibitor (Nec-1 or GSK872) at 96 h. (C,D) Necrosis of macrophages determined by Hoechst 33342 and PI staining in four groups of RAW 264.7 macrophages cultured alone, and those in CLS cell culture incubated in the presence or absence of a necroptosis inhibitor (Nec-1 or GSK872) at 96 h. Scale bars = 50 μm. Data are presented as the mean ± SD. ## P < 0.01 compared with the CLS cell culture model group incubated without necroptosis inhibitors.

Article Snippet: Primary antibodies against pRIPK3, RIPK3, pMLKL, and MLKL from a Mouse Reactive Necroptosis Antibody Sampler Kit (Cell Signaling Technology) were used.

Techniques: In Vitro, Cell Culture, Western Blot, Incubation, Staining

Journal: Frontiers in Cell and Developmental Biology

Article Title: Necroptosis in Macrophage Foam Cells Promotes Fat Graft Fibrosis in Mice

doi: 10.3389/fcell.2021.651360

Figure Lengend Snippet: Necroptosis of MFCs induces collagen expression in fibroblasts via a paracrine mechanism. (A,B) MILLIPLEX MAP assays were used to detect the expression levels of cytokines/chemokines in conditioned media collected from six groups, including adipocytes (live or apoptotic), RAW 264.7 macrophages, and CLS cultured cells incubated in the presence or absence of a necroptosis inhibitor (Nec-1 or GSK872). (C) Fibroblasts were treated with conditioned media collected from the six groups. (D) Immunofluorescence staining of collagen I and collagen VI in fibroblasts to investigate the paracrine effect of the culture media from various groups on fibroblasts. (E) Quantification of the collagen I- and collagen VI-positive areas per fibroblast. Scale bars = 50 μm. Data are presented as the mean ± SD. # P < 0.05, ## P < 0.01 compared with the CLS cell culture model group incubated without necroptosis inhibitors.

Article Snippet: Primary antibodies against pRIPK3, RIPK3, pMLKL, and MLKL from a Mouse Reactive Necroptosis Antibody Sampler Kit (Cell Signaling Technology) were used.

Techniques: Expressing, Cell Culture, Incubation, Immunofluorescence, Staining

Journal: Frontiers in Cell and Developmental Biology

Article Title: Necroptosis in Macrophage Foam Cells Promotes Fat Graft Fibrosis in Mice

doi: 10.3389/fcell.2021.651360

Figure Lengend Snippet: Blockade of necroptosis alleviates fat graft fibrosis. (A) Illustration of the animal model. Fat grafting model mice were administered Nec-1 or vehicle. (B) Macroscopic views of harvested fat tissue. (C) The retention volume of the fat grafts over time. (D–F) Western blot analyses of MLKL, pMLKL, RIPK3, and pRIPK3 in vivo . (G) The gene expression level of the cytokines TNF-α and IL-6 and the chemokines MCP-1 and MIP-2 in fat grafts. (H) Representative Masson’s trichrome staining of fat tissue at week 16 after grafting in the different groups. (I) Representative Sirius red staining of fat tissue at week 16 after grafting in the different groups. (J–L) Quantification of the areas positive for Masson’s trichrome (J) , collagen I (K) , and collagen VI (L) staining in fat tissue at week 16 after grafting in the different groups. Scale bars = 50 μm. Data are presented as the mean ± SD. # P < 0.05, ## P < 0.01 compared with the control group. (M) TEM analyses of the ultrastructure of MFCs in fat tissue at week 16 after grafting in the different groups. N, nucleus; LD, lipid droplet; PS, pseudopodia; M, mitochondria; RER, rough endoplasmic reticulum; Go, golgiosome; Lys, lysosome; and Red arrowheads indicated the damaged plasma membrane.

Article Snippet: Primary antibodies against pRIPK3, RIPK3, pMLKL, and MLKL from a Mouse Reactive Necroptosis Antibody Sampler Kit (Cell Signaling Technology) were used.

Techniques: Animal Model, Western Blot, In Vivo, Gene Expression, Staining, Control, Clinical Proteomics, Membrane

Journal: Frontiers in Cell and Developmental Biology

Article Title: Necroptosis in Macrophage Foam Cells Promotes Fat Graft Fibrosis in Mice

doi: 10.3389/fcell.2021.651360

Figure Lengend Snippet: Potential role of MFC necroptosis in fat graft fibrosis. After fat grafting, lipids released from apoptotic adipocytes induce formation of MFCs and an increased level of cellular lipids mediates necroptosis of MFCs, which upregulates the expression of proinflammatory cytokines/chemokines, leading to collagen synthesis by fibroblasts. Consequently, overproduction of collagens leads to fat tissue fibrosis after grafting.

Article Snippet: Primary antibodies against pRIPK3, RIPK3, pMLKL, and MLKL from a Mouse Reactive Necroptosis Antibody Sampler Kit (Cell Signaling Technology) were used.

Techniques: Expressing