Journal: Molecular and Cellular Biology

Article Title: Neutrophil and Macrophage Cell Surface Colony-Stimulating Factor 1 Shed by ADAM17 Drives Mouse Macrophage Proliferation in Acute and Chronic Inflammation

doi: 10.1128/MCB.00103-18

Figure Lengend Snippet: Hematopoietic deletion of ADAM17 causes a decrease in macrophage accumulation and a decrease in transient macrophage proliferation in thioglycolate-induced acute peritonitis. (A) Number of peritoneal macrophages from Adam17+/+ or Adam17−/− hematopoietic chimeras collected 48 h after injection of thioglycolate; n = 5. The experiment was repeated 5 times. (B) Peritoneal macrophages with or without administration of BrdU 1 h before harvest were evaluated for BrdU incorporation and surface expression of different markers. The gating scheme to eliminate neutrophils and eosinophils is shown. Macrophages that were positive or negative for BrdU were further evaluated by surface markers F4/80, CD11b, CD115, Ly6C, and 7-aminoactinomycin D (7-AAD). (C) Time course of macrophage proliferation (BrdU incorporation) in elicited peritoneal macrophages. n = 8 at 24 h; n = 9 at 40 h; n = 10 at 48 h; n = 5 at 64 and 72 h. (D) Percentages of macrophages from wild-type (Adam17+/+) and Adam17−/− chimeras in S phase at 24, 40, and 64 h after thioglycolate injection. *, P < 0.01 versus wild-type controls. (E) Percentages of S phase macrophages in 50/50 mixed hematopoietic chimeras done as for panel D; n = 5. The experiment was repeated 3 times. Values are expressed as means ± SEM.

Article Snippet: To detect soluble-CSF-1 levels in mouse peritoneal fluid, a DY416 kit with both antibodies recognizing the extracellular domain of CSF-1 was purchased from R&D Systems.

Techniques: Injection, BrdU Incorporation Assay, Expressing

Journal: Molecular and Cellular Biology

Article Title: Neutrophil and Macrophage Cell Surface Colony-Stimulating Factor 1 Shed by ADAM17 Drives Mouse Macrophage Proliferation in Acute and Chronic Inflammation

doi: 10.1128/MCB.00103-18

Figure Lengend Snippet: Transient macrophage proliferation is dependent on ADAM17-mediated generation of soluble CSF-1 in acute inflammation. (A) Soluble CSF-1 in peritoneal fluid from wild-type (Adam17+/+) and Adam17−/− hematopoietic chimeras by ELISA. n = 11, n = 12, n = 6, and n = 5 for Adam17+/+ chimeras and n = 9, n = 5, n = 5, and n = 5 for Adam17−/− chimeras, evaluated at 4, 12, 24, and 48 h. *, P < 0.02; **, P = 0.0012, ***, P < 0.0001 for Adam17+/+ versus Adam17−/−. (B) CSF-1 in circulation in Adam17+/+ and Adam17−/− hematopoietic chimeras. n = 6, n = 5, n = 5, n = 5, and n = 5 for Adam17+/+ chimeras and n = 11, n = 5, n = 5, n = 10, and n = 10 for Adam17−/− chimeras evaluated at 0, 4, 12, 24, and 48 h post-thioglycolate injection. *, P < 0.02 by two-way ANOVA followed by Sidak's multiple-comparison test. (C to E) Effects of extraneous CSF-1 (10 ng/cavity 8 h post-thioglycolate i.p. injection) on Adam17+/+ and Adam17−/− hematopoietic chimeras. (C) Macrophage number (48 h; n = 6). **, P = 0.0015 versus Adam17+/+. (D) Percentages of cells in S phase (48 h). (E) Numbers of apoptotic cells (annexin V/propidium iodide at 40 h). Values are expressed as means ± SEM.

Article Snippet: To detect soluble-CSF-1 levels in mouse peritoneal fluid, a DY416 kit with both antibodies recognizing the extracellular domain of CSF-1 was purchased from R&D Systems.

Techniques: Enzyme-linked Immunosorbent Assay, Injection, Comparison

Journal: Molecular and Cellular Biology

Article Title: Neutrophil and Macrophage Cell Surface Colony-Stimulating Factor 1 Shed by ADAM17 Drives Mouse Macrophage Proliferation in Acute and Chronic Inflammation

doi: 10.1128/MCB.00103-18

Figure Lengend Snippet: CSF-1 receptor is not decreased on ADAM17-null macrophages, and another ADAM17 substrate, TNF-α, is not involved in regulating macrophage proliferation. (A) Cell surface CD115 expression on F4/80+ peritoneal macrophages was determined by flow cytometry at different times post-thioglycolate injection. n = 5 per group. *, P < 0.05; **, P < 0.01; ***, P < 0.001 versus Adam17+/+ at the same time point. The data are representative of the results of 3 experiments. MFI, mean fluorescence intensity. (B) Peritoneal fluid was collected 4 h after thioglycolate injection from Adam17+/+ and Adam17−/− hematopoietic chimeras, and TNF-α levels were determined by ELISA (n = 5). (C) Adam17+/+ and Adam17−/− hematopoietic chimeras received either PBS or TNF-α (1 ng/cavity) 4 h after thioglycolate injection, and peritoneal cells were collected at 48 h and analyzed by flow cytometry; n = 5. The experiment was repeated 2 times. *, P < 0.05 versus Adam17+/+. Values are expressed as means and SEM.

Article Snippet: To detect soluble-CSF-1 levels in mouse peritoneal fluid, a DY416 kit with both antibodies recognizing the extracellular domain of CSF-1 was purchased from R&D Systems.

Techniques: Expressing, Flow Cytometry, Injection, Fluorescence, Enzyme-linked Immunosorbent Assay

Journal: Molecular and Cellular Biology

Article Title: Neutrophil and Macrophage Cell Surface Colony-Stimulating Factor 1 Shed by ADAM17 Drives Mouse Macrophage Proliferation in Acute and Chronic Inflammation

doi: 10.1128/MCB.00103-18

Figure Lengend Snippet: Neutrophils and macrophages are major sources of csCSF-1 in acute inflammation. (A) Types of peritoneal cells (neut, neutrophils; macs, macrophages; lymphs, lymphocytes; eos, eosinophils) in wild-type mice collected 12 h after thioglycolate injection. The data are representative of the results of 5 experiments. (B) The specificity of the CSF-1 antibody used in flow cytometry was verified by the absence of staining of CSF-1-null neutrophils and macrophages. (C) Levels of csCSF-1 on peritoneal cells from wild-type mice 12 h after thioglycolate injection (IgG, goat IgG). The ratio of the mean fluorescence intensity (MFI) of csCSF-1 to IgG is shown above each cell type; n = 5. The experiment was repeated 3 times. (D) Levels of csCSF-1 on Adam17+/+ and Adam17−/− neutrophils 12 h after thioglycolate injection; n = 5. Values are expressed as means and SEM.

Article Snippet: To detect soluble-CSF-1 levels in mouse peritoneal fluid, a DY416 kit with both antibodies recognizing the extracellular domain of CSF-1 was purchased from R&D Systems.

Techniques: Injection, Flow Cytometry, Staining, Fluorescence

Journal: Molecular and Cellular Biology

Article Title: Neutrophil and Macrophage Cell Surface Colony-Stimulating Factor 1 Shed by ADAM17 Drives Mouse Macrophage Proliferation in Acute and Chronic Inflammation

doi: 10.1128/MCB.00103-18

Figure Lengend Snippet: Expression of CSF-1 in inflammatory neutrophils and macrophages is transient in the acute peritonitis model. (A and B) Representative histograms for total CSF-1 and control IgG staining in permeabilized peritoneal cells from wild-type mice 12 h after thioglycolate injection for neutrophils (A) and macrophages (B). The anti-CSF-1/control IgG MFI ratios were 10.2 for neutrophils and 5.3 for macrophages. (C and D) Surface versus total CSF-1 in peritoneal cells. An overlay of a representative histogram of surface csCSF-1 (fixed cells) and total CSF-1 (permeabilized cells) is shown for peritoneal cells 12 h after thioglycolate injection. (C) Neutrophils. (D) Macrophages. (E) Kinetics of csCSF-1 expression on peritoneal neutrophils and macrophages from wild-type mice. Shown is the anti-CSF-1/control IgG MFI ratio, with a ratio of 1.0 representing no detectable expression (dashed line). The CSF-1-to-IgG ratio in circulating blood neutrophils and monocytes is indicated as T0 blood. *, P < 0.05; **, P < 0.01 compared to time zero. The data presented are for n = 5 per group, repeated in 3 different experiments. Values are expressed as means and SEM.

Article Snippet: To detect soluble-CSF-1 levels in mouse peritoneal fluid, a DY416 kit with both antibodies recognizing the extracellular domain of CSF-1 was purchased from R&D Systems.

Techniques: Expressing, Control, Staining, Injection

Journal: Molecular and Cellular Biology

Article Title: Neutrophil and Macrophage Cell Surface Colony-Stimulating Factor 1 Shed by ADAM17 Drives Mouse Macrophage Proliferation in Acute and Chronic Inflammation

doi: 10.1128/MCB.00103-18

Figure Lengend Snippet: Neutrophil ADAM17 is responsible for the generation of soluble CSF-1 and the regulation of macrophage proliferation. (A to C) Deletion of neutrophils by anti-Ly6G antibody injection in wild-type mice (150 μg/mouse; retro-orbital administration 8 h prior to thioglycolate injection). (A) Number of peritoneal neutrophils (12 h after thioglycolate injection; 7/4 Ly6B antibody; flow analysis). (B) Soluble CSF-1 levels in peritoneal fluid (PLF) (ELISA). (C) Peritoneal macrophage proliferation (40 h after thioglycolate injection; Vybrant DyeCycle Violet stain). Values are expressed as means and SEM; n = 5 for panels A to C.

Article Snippet: To detect soluble-CSF-1 levels in mouse peritoneal fluid, a DY416 kit with both antibodies recognizing the extracellular domain of CSF-1 was purchased from R&D Systems.

Techniques: Injection, Enzyme-linked Immunosorbent Assay, Staining

Journal: Molecular and Cellular Biology

Article Title: Neutrophil and Macrophage Cell Surface Colony-Stimulating Factor 1 Shed by ADAM17 Drives Mouse Macrophage Proliferation in Acute and Chronic Inflammation

doi: 10.1128/MCB.00103-18

Figure Lengend Snippet: Absence of iRhom2 restrains ADAM17-mediated release of csCSF-1 and diminishes inflammatory macrophage proliferation. (A) Representative Western blots of iRhom2 and ADAM17 (20 μg protein/lane) from neutrophils purified from bone marrow and the peritoneum. (B) Quantification of protein levels from panel A normalized to 24-h levels (n = 3). (C) CSF-1 levels (ELISA) in lysates of isolated neutrophils presented as the fold increase compared to the bone marrow lysates. The data are representative of the results of 3 experiments. (D) Numbers of peritoneal neutrophils and macrophages from Adam17+/+ and Adam17−/− hematopoietic chimeras, along with iRhom2+/+ and iRhom2−/− hematopoietic chimeras, at 12 h post-thioglycolate injection; n = 5. The data are representative of the results of at least 3 experiments. (E) TNF-α levels in peritoneal fluid from iRhom2+/+ and iRhom2−/− chimeras 4 h after thioglycolate injection (ELISA); n = 5. (F) CSF-1 levels in peritoneal fluid from iRhom2+/+ and iRhom2−/− chimeras at 4 h and 12 h after thioglycolate injection (ELISA); n = 5. (G) Peritoneal macrophage proliferation (BrdU incorporation) in iRhom2+/+ and iRhom2−/− chimeras (40 h post-thioglycolate injection). Values are expressed as means and SEM.

Article Snippet: To detect soluble-CSF-1 levels in mouse peritoneal fluid, a DY416 kit with both antibodies recognizing the extracellular domain of CSF-1 was purchased from R&D Systems.

Techniques: Western Blot, Purification, Enzyme-linked Immunosorbent Assay, Isolation, Injection, BrdU Incorporation Assay

Journal: Molecular and Cellular Biology

Article Title: Neutrophil and Macrophage Cell Surface Colony-Stimulating Factor 1 Shed by ADAM17 Drives Mouse Macrophage Proliferation in Acute and Chronic Inflammation

doi: 10.1128/MCB.00103-18

Figure Lengend Snippet: iRhom2 is required for ADAM17 maturation and transit to the cell surface in neutrophils and macrophages. Total levels of pro-ADAM17 were evaluated in lysates without PNGase F treatment. To detect active cell surface ADAM17, peritoneal cells from iRhom2+/+ or iRhom2−/− chimeras were surface biotinylated, followed by purification of biotinylated proteins on neutravidin and PNGase F treatment. Note that active ADAM17 is absent in iRhom2−/− hematopoietic chimeras. Analysis of lysates and surface biotinylation by Western analysis are shown for peritoneal neutrophils 12 h after thioglycolate injection (A) and peritoneal macrophages 96 h after thioglycolate injection (B). Peritoneal neutrophil lysates and surface biotinylation are shown separately because of different exposure times (3 and 10 min, respectively).

Article Snippet: To detect soluble-CSF-1 levels in mouse peritoneal fluid, a DY416 kit with both antibodies recognizing the extracellular domain of CSF-1 was purchased from R&D Systems.

Techniques: Purification, Western Blot, Injection

Journal: Frontiers in Immunology

Article Title: MiR-34a deficiency enhances nucleic acid sensing and type I IFN signaling in a mouse model of Alzheimer’s disease

doi: 10.3389/fimmu.2026.1694824

Figure Lengend Snippet: Effects of miR-34a KO on expression levels of homeostatic and DAM genes. Expression levels (Heatmap) of marker genes for three different microglial stages (homeostatic, stage 1 DAM, and stage 2 DAM) are shown as fold changes miR-34a -/- to miR-34a +/+ Tg-SwDI mice, based on bulk RNAseq of the hippocampus (A) . (n=5/group, *Q < 0.05, ##Q < 0.001) Sequence alignment of the mature miR-34a with CSF1R 3’ untranslated region (3’UTR). The seed sequences and target mRNA are highlighted in red (B) . Effects of miR-34a KO on levels of CSF1R in lysates of the cerebral cortex were analyzed by western blotting using CSF1R antibody (C) and bar graph represents the results of densitometric analysis of CSF1R after normalizing with GAPDH levels. Data was analyzed by independent T-test. (n=4/group, ##P < 0.001).

Article Snippet: Membranes were incubated overnight at 4 °C with primary antibodies against IFITM3 (1:1000; Abcam Cat# ab15592, RRID: AB_2122095), CSF1R (1:1000; R and D Systems Cat# AF3818, RRID: AB_884158), and GAPDH (1:5000; Millipore Cat# MAB374, RRID: AB_2107445).

Techniques: Expressing, Marker, RNA sequencing, Sequencing, Western Blot