Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Transforming Growth Factor β1 Suppresses the Type I Interferon Response and Induces Mitochondrial Dysfunction in Alveolar Macrophages 1

doi: 10.4049/jimmunol.1701325

Figure Lengend Snippet: TGFβ1 decreases the Type I interferon (IFN) and proinflammatory response to respiratory syncytial virus (RSV) in CD14+ human monocyte-derived macrophages (HmMoDM). A) Gene expression profiles of M1 markers from six independent experiments performed in duplicate for each sample and reported as mean ± SEM. B) Protein expression of M1 markers measured by enzyme-linked immunosorbent assay (ELISA) from cell culture media from five independent experiments performed in duplicate for each sample and reported as mean ± SEM. C) Gene expression profiles of M2 markers from six independent experiments performed in duplicate for each sample and reported as mean ± SEM. *p ≤ 0.05 vs. control, Op ≤ 0.05 vs. RSV, #p ≤ 0.05 vs. TGFβ1.

Article Snippet: Human recombinant M-CSF was added to the monocytes according to the CellXVivo Human M2 Macrophage Differentiation Kit (R&D Systems, Minneapolis, MN).

Techniques: Virus, Derivative Assay, Gene Expression, Expressing, Enzyme-linked Immunosorbent Assay, Cell Culture, Control

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: ( A and B ) Representative staining figures of M1 macrophages by marker CD86 in lung obtained 0 and 72 hours after infection by S . pneumoniae 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 ) Proportion 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] versus 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 ++ Ly6G DIM ) 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: Staining, Marker, Infection, 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: ( 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 adenovirus 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, Infection

Journal: Aging (Albany NY)

Article Title: SIRT6 mono-ADP ribosylates KDM2A to locally increase H3K36me2 at DNA damage sites to inhibit transcription and promote repair

doi: 10.18632/aging.103567

Figure Lengend Snippet: SIRT6 mediates KDM2A displacement from chromatin to enhance NHEJ. ( A ) Schematic representation of the GFP-based NHEJ reporter, which represents an RNA Pol II-transcribed gene. DSBs are introduced by transient expression of I-SceI enzyme. The positions of the primer used in this study are indicated by small arrows below: primers for quantification of the preRNA (small black arrows); quantification of DNA DSBs (small red arrows), chromatin IP (blue arrows). ( B ) Quantification of the cutting efficiency at different time points after transfection with I-SceI vector. DSBs were quantified as a ratio between the product obtained with primers amplifying across the break (red arrows) to a PCR product from a gene away from DSB site . The experiment was repeated three times. ( C ) KDM2A dissociation from broken chromatin is SIRT6-dependent. Time-course chromatin-IP was performed using antibody against endogenous KDM2A in human skin fibroblasts harboring chromosomally-integrated NHEJ GFP reporter with I-SceI sites. Western blot shows depletion of SIRT6. ( D ) H3K36me2 accumulation post DSB induction is SIRT6-dependent. ChIP analysis of H3K36me2, at different time points post transfection with I-SceI vector using antibody specific for H3K36me2. qPCR was performed using primers positioned 50 bp downstream of TSS. ( E ) Non-ribosylatable KDM2A R1020W mutant bids to DSBs more efficiently than the wild type protein. Human skin fibroblasts carrying I-SceI reporter cassette were transfected with wild type or mutant (R1020W) KDM2A and I-SceI encoding vectors. At 12 hours post transfection cells were harvested followed by ChIP-qPCR with antibodies against FLAG. Western blot showing the levels of wild type and mutant FLAG-KDM2A proteins in cells after transfection. ( F ) Cells depleted of KDM2A have enhanced NHEJ repair efficiency. Cells were treated with siRNA to KDM2A four days before transfection with I-SceI. NHEJ efficiency was measured by reactivation of the GFP reporter normalized to transfection efficiency (DsRed) 48 h after I-SceI transfection. Western blot shows KDM2A depletion. ( G ) Downregulation of KDM2A reduces the number of 53BP1 foci. KDM2A knocked down cells were grown to confluency and fixed for IF experiment using antibody against 53BP1. On the right; quantification of the IF image obtained from 100 nuclei. All experiments were repeated at least three times. ( H ) Ku70 recruitment to DSB site requires SIRT6, and is counteracted by KDM2A. Time course of ChIP experiment using antibody against Ku70. The experiment was repeated three times. *p < 0.05; **p <0.01.

Article Snippet: The cell lysate was then used for purification by Anti-FLAG M2 Immunoprecipitation kit (SIGMA M8823) according to manufacturer protocol.

Techniques: Expressing, Chromatin Immunoprecipitation, Transfection, Plasmid Preparation, Western Blot, Mutagenesis