mouse peritoneal macrophages  (ATCC)

Journal: Journal of Advanced Research

Article Title: PEBL, a component-based Chinese medicine, reduces virus-induced acute lung injury by targeting FXR to decrease ACE2 levels

doi: 10.1016/j.jare.2025.05.003

Figure Lengend Snippet: PEBL alleviates Poly(I:C)-induced ALI in a dose-dependent manner and modulates cytokine levels in macrophage inflammation. (A) Experimental design for PEBL treatment in ALI zebrafish. (B) Dose-dependent reduction in mortality by PEBL. Survival plot of 5 dpf Tg(coro1α: GFP) larvae at 72 hpi ( n = 30). (C) Dose-dependent reduction in macrophage recruitment by PEBL. Quantitative analysis of macrophage infiltration in the swim bladder section at 4 hpi ( n = 10). (D) Fluorescence images of macrophages in the swim bladder section at 4 hpi following different concentrations of PEBL, marked by the red circle. (E-J) PEBL reduces Poly(I:C)-induced cytokine elevation in RAW264.7 cells ( n = 3). mRNA levels of IL-1β, IL-6, and TNF-α in cells were measured by qPCR (E-G), while protein concentrations of these cytokines in culture media were quantified using ELISA (H-J). ## P < 0.01, ### P < 0.001 vs. Poly(I:C); ** P < 0.01, *** P < 0.001 for group comparisons. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Mouse peritoneal mononuclear macrophage RAW264.7 cells and human embryonic kidney 293 T cells were obtained from the American Type Culture Collection (Rockville, MD, USA).

Techniques: Fluorescence, Enzyme-linked Immunosorbent Assay

Journal: Journal of Advanced Research

Article Title: PEBL, a component-based Chinese medicine, reduces virus-induced acute lung injury by targeting FXR to decrease ACE2 levels

doi: 10.1016/j.jare.2025.05.003

Figure Lengend Snippet: PEBL suppresses Poly(I:C)-induced FXR and ACE2 expression and NF-κB-p65 nuclear translocation in RAW264.7 cells. (A-E) PEBL reduces the mRNA (A-B) and protein (D-E) levels of FXR and ACE2 and diminishes NF-κB-p65 nuclear translocation (C, E). (F-H) PEBL suppresses the protein distribution of FXR and ACE2, inhibits the nuclear translocation of NF-κB-p65. Representative images show the localization of FXR (F, green), ACE2 (G, green), NF-κB-p65 (H, green), and DAPI (blue), captured by immunofluorescence at 40 × magnification using confocal microscopy. Scale bar = 10 μm. UDCA was used as a positive control. Nuc, nucleus; Cyt, cytoplasm; Mem, membrane. n = 3; * P < 0.05, ** P < 0.01, *** P < 0.001 for group comparisons. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Mouse peritoneal mononuclear macrophage RAW264.7 cells and human embryonic kidney 293 T cells were obtained from the American Type Culture Collection (Rockville, MD, USA).

Techniques: Expressing, Translocation Assay, Immunofluorescence, Confocal Microscopy, Positive Control, Membrane

Journal: Journal of Advanced Research

Article Title: PEBL, a component-based Chinese medicine, reduces virus-induced acute lung injury by targeting FXR to decrease ACE2 levels

doi: 10.1016/j.jare.2025.05.003

Figure Lengend Snippet: PEBL suppresses Poly(I:C)-induced FXR binding to ACE2 by inhibiting FXR transcription in RAW264.7 cells. (A-B) FXR overexpression reverses the effect of PEBL on the protein levels of ACE2 and NF-κB-p65. n = 3. (C-D) FXR overexpression reverses the inhibitory effect of PEBL on ACE2 distribution and NF-κB-p65 nuclear translocation. Representative images show the localization of ACE2 (C, green), NF-κB-p65 (D, green), and DAPI (blue), captured by immunofluorescence at 40 × magnification using confocal microscopy. Scale bar = 10 μm. (E-H) PEBL requires FXR to decrease ACE2 expression and mitigate Poly(I:C) infection. In FXR-KD cells (F, H), no significant change in ACE2 expression was observed following treatments with CDCA, Poly(I:C), UDCA, or PEBL, compared to WT cells (E, G). WT, wild-type RAW264.7 cells; n = 3. (I) Co-IP analysis reveals no binding between FXR and ACE2 proteins. (J-K) PEBL reduces Poly(I:C)-induced FXR binding to the ACE2 promoter, confirmed by ChIP-qPCR and agarose gel electrophoresis.Nuc, nucleus; Cyt, cytoplasm; Mem, membrane; OSTα, positive control; ACE2-NC, negative control; C, control; P, Poly(I:C). n = 6; * P < 0.05, ** P < 0.01, *** P < 0.001 for group comparisons; ns , non-significant. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Mouse peritoneal mononuclear macrophage RAW264.7 cells and human embryonic kidney 293 T cells were obtained from the American Type Culture Collection (Rockville, MD, USA).

Techniques: Binding Assay, Over Expression, Translocation Assay, Immunofluorescence, Confocal Microscopy, Expressing, Infection, Co-Immunoprecipitation Assay, ChIP-qPCR, Agarose Gel Electrophoresis, Membrane, Positive Control, Negative Control, Control

Journal: Biology Direct

Article Title: GPR43 alleviates LPS-induced acute lung injury by inhibiting NLRP3 inflammasome activation via β-arrestin 2

doi: 10.1186/s13062-025-00725-9

Figure Lengend Snippet: Activation of GPR43 inhibits NLRP3 inflammasome activation in peritoneal macrophages. LPS (100 ng/mL)-primed primary peritoneal macrophages (PM) were treated with varying concentrations of 4-CMTB (5, 10, 20 µM) and subsequently stimulated with Nigericin (Nig, 20 µM). IL-1β and caspase-1 levels in the medium supernatants (Sup) and whole cell lysates (WCL) were analyzed by WB ( A ). NLRP3 expression in cell extracts was also assessed by WB ( A ). Densitometric analysis of IL-1β in Sup ( B ), caspase-1 in Sup ( C ), NLRP3 in WCL ( D ), IL-1β in WCL ( E ), caspase-1 in WCL ( F ). ELISA analysis of IL-1β ( G ) and IL-18 ( H ) levels in the medium supernatants. PM were transfected with siRNA targeting GPR43 mRNA or an empty vector. Detection of interference efficiency of GPR43 ( I ). LPS (100 ng/mL)-primed PM were treated with 4-CMTB (20 µM) and then stimulated with Nig (20 µM). IL-1β and caspase-1 levels in Sup and WCL were analyzed by WB ( J ). Densitometric analysis of IL-1β in Sup ( K ), caspase-1 in Sup ( L ), IL-1β in WCL ( M ), caspase-1 in WCL ( N ). ELISA analysis of IL-1β ( O ) and IL-18 ( P ) levels in the medium supernatants. Data are presented as mean ± SD (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; n = 3)

Article Snippet: For transfection of mouse peritoneal macrophages, GPR43-specific small interfering RNA (siRNA) and control siRNA (Sangon Biotech, China) were utilized.

Techniques: Activation Assay, Expressing, Enzyme-linked Immunosorbent Assay, Transfection, Plasmid Preparation

Journal: Biology Direct

Article Title: GPR43 alleviates LPS-induced acute lung injury by inhibiting NLRP3 inflammasome activation via β-arrestin 2

doi: 10.1186/s13062-025-00725-9

Figure Lengend Snippet: GPR43 activation inhibits NLRP3 inflammasome activation in peritoneal macrophages induced by multiple stimuli. LPS (100 ng/mL)-primed primary PM were treated with 4-CMTB (20 µM) and then stimulated with ATP (5 mM) or MSU (200 µg/mL). IL-1β and caspase-1 levels in Sup and WCL were analyzed by WB ( A ). Under the stimulation of LPS combined with ATP, densitometric analysis of IL-1β in Sup ( B ), caspase-1 in Sup ( C ), IL-1β in WCL ( D ), caspase-1 in WCL ( E ). Under the stimulation of LPS combined with MSU, densitometric analysis of IL-1β in Sup ( F ), caspase-1 in Sup ( G ), IL-1β in WCL ( H ), caspase-1 in WCL ( I ). ELISA analysis of IL-1β ( J , L ) and IL-18 ( K , M ) levels in the medium supernatants. LPS (100 ng/mL)-primed primary PM were treated with 4-CMTB (20 µM) and then stimulated with Nig (20 µM). Cells were stained with immunofluorescence, and the green spots represent ASC specks are shown by the yellow arrows in the figure ( N ). Scale bar: 20 μm. Percentage of ASC speck-positive cells ( O ). Data are presented as mean ± SD (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; n = 3)

Article Snippet: For transfection of mouse peritoneal macrophages, GPR43-specific small interfering RNA (siRNA) and control siRNA (Sangon Biotech, China) were utilized.

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Staining, Immunofluorescence

Journal: Biology Direct

Article Title: GPR43 alleviates LPS-induced acute lung injury by inhibiting NLRP3 inflammasome activation via β-arrestin 2

doi: 10.1186/s13062-025-00725-9

Figure Lengend Snippet: GPR43 activation inhibits pyroptosis in mouse peritoneal macrophages. LPS (100 ng/mL)-primed primary PM were treated with varying concentrations of 4-CMTB (5, 10, 20 µM) and then stimulated with Nig (20 µM). GSDMD levels in cell extracts were analyzed by WB ( A ). Densitometric analysis of GSDMD-N ( B ). LDH concentration in the culture medium supernatants ( C ). Percentage of PI-positive cells ( D ) and representative images of PI-positive cells ( E ). Scale bar: 50 μm. LPS (100 ng/mL)-primed primary PM were treated with 4-CMTB (20 µM) and then stimulated with Nig (20 µM). Transmission electron microscopy (TEM) images of pyroptotic cells ( F ). Scale bars: 2 μm and 500 nm. PM were transfected with siRNA targeting GPR43 mRNA or an empty vector. LPS (100 ng/mL)-primed PM were treated with 4-CMTB (20 µM) and then stimulated with Nig (20 µM). WB analysis of GSDMD expression in cell extracts ( G ) and quantitative results ( H ). Data are presented as mean ± SD (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; n = 3)

Article Snippet: For transfection of mouse peritoneal macrophages, GPR43-specific small interfering RNA (siRNA) and control siRNA (Sangon Biotech, China) were utilized.

Techniques: Activation Assay, Concentration Assay, Transmission Assay, Electron Microscopy, Transfection, Plasmid Preparation, Expressing

Journal: Nature

Article Title: Efferocytic remodelling of pancreatic islet macrophages by limited β-cell death

doi: 10.1038/s41586-025-09560-4

Figure Lengend Snippet: a , b , NicheNet analysis of ligand–receptor interactions between intra-islet macrophages (sender cells) and CD4 + T cells (receiver cells) based on the scRNA-seq data of 5.5 week old NOD mice islets. Circos plot ( a ), and scaled expression of top-ranked ligands, their predicted target genes and the corresponding regulatory potential scores ( b ). c , Expression of Igf1r by intra-islet anergic-like CD4 + T cells, and Igf1 by e-Mac cells (scRNA-seq, 5.5-weeks-old NOD mice; see also Figs. 2g and 3f ). d , Schematic of the potential connection between efferocytosis by islet macrophages and CD4 + T cells through IGF-1 signalling. e , IGF-1 secretion by peritoneal macrophages 12 h after exposure to apoptotic β-cells (Min6), as measured by ELISA. n = 5 biologically independent samples; N = 2 experiments. f , Intracellular staining of IGF-1 among intra-islet macrophages (CD45 + F4/80 + CD11c + ), comparing the e-Mac (Gal-3 high CD9 high ) and non-e-Mac (Gal-3 low CD9 low ) subsets. Mice 1, 3 and 5 received STZ 1-low treatment; mice 2 and 4 received vehicle treatment. Islets from n = 5 individual mice; N = 2 experiments. g , Flow cytometry analysis of IGF1R staining and markers of anergic-like cells expressed by intra-islet CD4 + T cells (10 days after STZ 1-low ). Data are representative of n = 5 mice. h , i , NOD mice (4-weeks of age) were treated with recombinant IGF-1 or vehicle twice daily for 10 days before islets were collected and analysed. The representative flow cytometry plots ( h ) and quantification ( i ) show the increase in the frequency of anergic-like CD4 + T cells in the IGF-1-treated conditions. Islets from n = 6 individual mice per group; N = 2 experiments. Statistical analysis was performed using unpaired t -tests ( e and i ) and paired t -tests ( f ). s.c., subcutaneous. The diagrams in d and h were created using BioRender. Ravichandran, K. (2025) https://BioRender.com/y6jwwiv ; Ravichandran, K. (2025) https://BioRender.com/rbl2ugb .

Article Snippet: First, the macrophage population was enriched in freshly collected peritoneal lavage using the peritoneal macrophage isolation kit (Miltenyi Biotec, 130-110-434; Extended Data Fig. 9a ).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Staining, Flow Cytometry, Recombinant

Journal: Nature

Article Title: Efferocytic remodelling of pancreatic islet macrophages by limited β-cell death

doi: 10.1038/s41586-025-09560-4

Figure Lengend Snippet: a . Isolation of islet macrophages using CD11c + magnetic beads (MACS) (non-autoimmune C57BL/6 J mice). Flow cytometric analysis on dispersed islet cells before enrichment (left), and MACS-enriched Live, CD45 + F4/80 + CD11c + macrophages (right). b . Schematic of the assay when primary islet macrophages co-cultured with live or apoptotic β-cells (UV-irradiated Min6 cells). c . Flow cytometric evaluation of e-Mac frequencies in the co-culture experiment ( b ). Quantification is shown on the right. n = 4(UT), n = 2(Live), n = 8(Apo) biological replicates over N = 4 independent experiments for “UT” and “Apo” and N = 2 for “Live”. Two-tailed unpaired t -test with Welch’s correction. d . Flow cytometric analysis showing peritoneal macrophages. Peritoneal lavage cells were collected from C57BL/6 mice, plated for 2 h, washed to remove unbound cells, and analysed by flow cytometry 24 h after the beginning of the incubation. e . Flow cytometric analysis showing engulfment of apoptotic β-cells by peritoneal macrophages. Apoptotic β-cells (Min6 cell line) were labelled with pHrodo red dye and co-cultured with pMacs for 2 h; unbound apoptotic cells were then washed away, and incubation continued. The macrophage-to-β-cell ratio was 1:3. Flow cytometry was done 16 h after beginning of the incubation. f . Flow cytometric analysis of peritoneal macrophages after co-culture with live or apoptotic β-cells (Min6). Two negative controls were used: cytochalasin D (CytoD, 1 μM) – inhibitor of cytoskeletal reorganization that prevents corpse uptake; and annexin V (20 μg/ml) that binds phosphatidylserine (PtdSer) and masks it from scavenger receptors on the phagocytic cells. g . Quantification of ( f ). n = 6 (No β-cell), n = 6 (Live β-cell), n = 6 (Apo β-cell, alone), n = 3 (Apo β-cell, +CytoD), n = 3 (Apo β-cell, +Ann V) biological replicates; N = 2. Statistics - two-tailed unpaired t -test. Data are representative of N = 3 independent experiments ( a, b ). The diagrams in a and b were created in BioRender. Ravichandran, K. (2025) https://BioRender.com/oc6ssn7 ; Ravichandran, K. (2025) https://BioRender.com/laier1m .

Article Snippet: First, the macrophage population was enriched in freshly collected peritoneal lavage using the peritoneal macrophage isolation kit (Miltenyi Biotec, 130-110-434; Extended Data Fig. 9a ).

Techniques: Ex Vivo, Isolation, Magnetic Beads, Cell Culture, Irradiation, Co-Culture Assay, Two Tailed Test, Flow Cytometry, Incubation

Journal: Nature

Article Title: Efferocytic remodelling of pancreatic islet macrophages by limited β-cell death

doi: 10.1038/s41586-025-09560-4

Figure Lengend Snippet: a . Peritoneal macrophages isolated from NOD mouse using magnetic-activated cell sorting (MACS). CD11b pos macrophages are enriched into Tim-4 hi large peritoneal macrophages (LPM, expressing low level of MHC-II) and Tim-4 low small peritoneal macrophages (SPM, MHC-II hi ). Representative of N = 3 independent experiments. b . Antigen presentation assay in which naive TCR transgenic BDC2.5 CD4 + T cells were co-cultured for 3 days with SPM or LPM at indicated concentrations of antigenic peptide; N = 2. c . Gene set enrichment analysis (GSEA) plot showing enrichment of gene signature of CD4 + T cells activated by “e-Mac enriched” macrophages interrogated against publicly available transcriptional dataset comparing anergic and naive CD4 + T cells ( GSE143739 43 ). d . Summary plot of GSEA using CD4 + T cells gene signature induced by islet macrophages from STZ 1-low - or vehicle-treated mice (“e-Mac enriched” and “Vehicle” correspondingly). Differential expression comparisons between anergic cells and either naive or antigen-experienced CD4 + T cells were used as reference 43 ( GSE143739 ). The CD4 + T cells in the reference dataset were exposed to different levels of antigen. e . GSEA pathways comparing gene expression pathways upregulated in CD4 + T cells activated by islet macrophages from STZ 1-low - versus vehicle-treated mice. Gene Ontology MSigDB (GO:BP). f . Heatmap showing differentially expressed genes among CD4 + T cell subsets in the islets of NOD mice 10 days after the treatment with either STZ 1-low - or control vehicle. g . GSEA plot showing islet anergic-like CD4 + T cells gene signature interrogated against bulk transcriptional dataset 43 ( GSE143739 43 ) comparing anergic and naive cells. NES, normalized enrichment score. Statistics: weighted Kolmogorov-Smirnov test ( c, e, g ).

Article Snippet: First, the macrophage population was enriched in freshly collected peritoneal lavage using the peritoneal macrophage isolation kit (Miltenyi Biotec, 130-110-434; Extended Data Fig. 9a ).

Techniques: Activation Assay, Ex Vivo, In Vivo, Isolation, FACS, Expressing, Immunopeptidomics, Transgenic Assay, Cell Culture, Quantitative Proteomics, Gene Expression, Control

Journal: The EMBO Journal

Article Title: Macrophages form dendrite-like pseudopods to enhance bacterial ingestion

doi: 10.1038/s44318-025-00515-z

Figure Lengend Snippet: ( A ) Schematic diagram of the two-photo intravital microscopy (TIM) experimental procedure. ( B ) Illustration of imaging the abdominal pouch in live mouse with TIM. ( C ) Physical display of TIM device and 3D reconstruction of macrophages on the abdominal wall. Scale bars, 20 µm. ( D ) Representative TIM imaging of peritoneal macrophages labeled with anti-F4/80 in vivo with or without Salmonella challenge. Green cells represent macrophages with obvious pseudopods. Magnified views reveal individual macrophage examples. Scale bars, 10 µm. ( E ) Quantification of the percentage of macrophages with or without DLPs upon Salmonella infection, based on TIM data from 150 cells in 6 independent imaging views from three experiments. ( F ) Quantification of the DLPs length in adherent peritoneal macrophages with or without Salmonella challenge. n = 18 cells. ( G ) Time-lapse imaging of THP-1 macrophages revealing the process of DLPs formation with or without Salmonella infection (MOI = 20). Scale bars, 50 µm. ( H ) Pie chart quantification of the percentage of three shapes of macrophages with or without Salmonella infection (MOI = 20). Blue, round shape (I); Red, fusiform shape (II); green, deformed shape with DLPs (III). ( I ) Quantification of the search radius of shape I and III macrophages. n = 50 cells. ( J , K ) Dosage and temporal-dependent analysis of shape III macrophages at 6 h-post-infection (hpi) under varying MOIs ( J ) or at different hpi times with a fixed MOI of 20 ( K ). ( L ) Representative time-lapse imaging revealing DLPs formation in iBMDM, RAW264.7 and mouse peritoneal macrophages during Salmonella infection (MOI = 20). Scale bars, 50 µm. Pie chart quantification of the percentage of three shapes of macrophages with or without Salmonella infection (MOI = 20) for 6 h. ( M ) Immunofluorescence visualization by phalloidin staining, delineating lamellipodia, filopodia and DLPs with dashed lines. Upper panels provide definitions for the three protrusive structures. Scale bars, 10 µm. ( N ) Representative images of DLPs visualized by WGA staining in Salmonella infected THP-1 macrophages. Yellow arrows depict ‘tube’ and ‘tip’ structures. Yellow line divides the tube and tip. White line divides the cell body and DLPs. Lower panel shows the orthographic view from the white dash line in the upper panel. Scale bars, 20 µm. ( O ) Quantification of the maximum width of ‘tips’ and ‘tubes’ in ( n ). n = 75 cells. ( P ) Quantification of the percentage of macrophages with filopodia and DLPs, respectively, in the indicated MOI of Salmonella infection at 6 hpi in THP-1 cells. ( Q ) Upper panel showing the schematic diagram of Salmonella withdraw experiment. Lower panel showing the time-lapse imaging of THP-1 macrophages after washing out Salmonella . Green circles depict the radius of cell. Scale bars, 20 µm. ( R ) Quantification of search radius of shape III macrophages after washing out Salmonella . The yellow arrowheads depict the DLPs in ( D , G , L ). Data are presented as mean ± s.d. from three independent experiments. Dots in quantifications presented individual cells. P = 0.00980 ( E ). P = 0.00090 ( F ). P = 0.00090 ( H ). P = 4.26191E-10 ( I ). P values from left to right ( J ): P = 0.00700, P = 0.50150, P = 0.56538, P = 0.66530. P values from left to right ( K ): P = 0.02162, P = 0.86055, P = 0.97863. P values from left to right ( L ): P = 0.00034, P = 0.00030, P = 0.00026. P = 5.32443E-40 ( O ). ns P > 0.05; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001: unpaired two-tailed Student’s t test ( E , F , H , I , J , K , L , O ). .

Article Snippet: Mouse Peritoneal macrophages , C57BL/6J ( M. musculus ) Jackson Lab #000664; RRID: , N/A.

Techniques: Intravital Microscopy, Imaging, Labeling, In Vivo, Infection, Immunofluorescence, Staining, Two Tailed Test

Journal: The EMBO Journal

Article Title: Macrophages form dendrite-like pseudopods to enhance bacterial ingestion

doi: 10.1038/s44318-025-00515-z

Figure Lengend Snippet: ( A ) Quantification of infection rates in THP-1 macrophages with fluorescence-tagged or non-tagged Salmonella . ( B ) Pie charts show the percentage of three shapes of macrophages with Salmonella infection for 6 h in ( A ). ( C ) Quantification of the average speed of inward moving Salmonella . n = 78 cells. ( D ) Schematic diagram illustrating the CFU assay performed at distinct infection durations. ( E ) Representative images of the CFU assay at different time point post Salmonella infection of THP-1 macrophages. ( F ) Quantification of intracellular Salmonella over time in THP-1 macrophages. ( G ) Western blot analysis of vimentin in WT and VIM KO iBMDM. ( H , I ) Quantification of the bacterial load upon Salmonella infection in THP-1 macrophages treatment with TAK242 or Y-27632. ( J ) Schematic diagram of extraintestinal infection mouse model incorporating macrophage re-infusion. ( K , L ) Quantification of bacterial load indicated by colony forming unit (CFU) in peritoneal lavage fluid ( K ) and liver ( L ) infected with Salmonella . ( M – O ) Quantitative RT-qPCR analysis of the expression levels of inflammatory markers in the spleen. Data are presented as mean ± s.d. from three independent experiments. Dots in quantifications presented individual cells. P values from left to right ( F ): P = 0.65388, P = 0.00094, P = 0.00094. P = 0.00558515 ( H ). P = 0.00124 ( I ). P = 0.00805 ( K ). P = 0.00243 ( L ). P = 0.03700 ( M ). P = 0.04913 ( N ). P = 0.04496 ( O ). ns P > 0.05; * P < 0.05; ** P < 0.01; *** P < 0.001: unpaired two-tailed Student’s t test ( H , I , K – O ), one-way ANOVA followed by a Tukey’s post hoc test ( A , F ). .

Article Snippet: Mouse Peritoneal macrophages , C57BL/6J ( M. musculus ) Jackson Lab #000664; RRID: , N/A.

Techniques: Infection, Fluorescence, Colony-forming Unit Assay, Western Blot, Quantitative RT-PCR, Expressing, Two Tailed Test

Journal: The EMBO Journal

Article Title: Macrophages form dendrite-like pseudopods to enhance bacterial ingestion

doi: 10.1038/s44318-025-00515-z

Figure Lengend Snippet: ( A ) Representative images of mCherry-tagged Salmonella infected THP-1 macrophages stained with WGA. White dash line divides DLPs from the cell body. Lower panel shows the orthographic view of the upper panel image. Scale bars, 10 µm. ( B ) Time-lapse imaging of mCherry-tagged Salmonella infected THP-1 macrophages stained with WGA. White dash line depicts the cell body and DLPs. White arrowheads depict the position of an inward moving Salmonella bacterium along DLPs. Scale bars, 20 µm. ( C ) Immunofluorescence staining of endogenous LAMP1 and WGA in mCherry-tagged Salmonella infected THP-1 macrophages. The magnified views depict ROIs in cell body and in DLPs. Scale bars, 10 µm (in cell image) and 5 µm (in the magnified images). ( D , E ) Representative images of mCherry-tagged Salmonella in THP-1 macrophages with or without DLPs. Cell outlines were visualized with WGA staining. White squares depict ROIs containing ingested Salmonella . ROIs are magnified below, with ingested Salmonella . Scale bars, 10 µm. ( F ) Quantification of the number of Salmonella per infected cell in shape I and III macrophages at distinct MOIs. n = 20–30 cells. ( G ) Quantification of the ratio of bacteria numbers in shape III versus in shape I macrophages at distinct MOIs. n = 15–25 cells. ( H , I ) Quantification of the number of Salmonella per infected cell in shape I and III mouse BMDMs ( J ) and human PBMC derived macrophages ( K ). n = 20–25 cells. ( J – L ) Quantification of the bacterial load upon Salmonella infection in THP-1 WT and VIM-KO macrophages ( J ), iBMDMs WT and VIM-KO macrophages ( K ) and THP-1 macrophages with RNAi of negative control or ARHGEF3 ( L ). ( M ) Quantification of the bacterial load in P− and P+ iBMDM macrophages at distinct MOIs. ( N ) Schematic diagram of extraintestinal infection mouse model incorporating macrophage re-infusion. ( O , P ) Quantification of bacterial load indicated by colony forming unit (CFU) in peritoneal lavage fluid ( O ) and liver ( P ) infected with Salmonella . ( Q – S ) Quantitative RT-qPCR analysis of the expression levels of inflammatory markers in the spleen. Data are presented as mean ± s.d. from three independent experiments. Dots in quantifications presented individual cells. P values from left to right ( F ): P = 0.03057, P = 0.00013, P = 0.00024, P = 4.00394E-06. P = 0.016278 ( H ), P = 0.00043 ( I ), P = 0.01133 ( J ), P = 0.00855 ( K ), P = 0.00477 ( L ), P values from left to right ( M ): P = 0.79525, P = 0.16105, P = 0.06649, P = 0.00034. P = 0.00769 (N), P = 0.00362 ( O ). ns P > 0.05; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001: unpaired two-tailed Student’s t test ( H – L , O – S ), one-way ANOVA followed by a Tukey’s post hoc test ( G ), two-way ANOVA with Sidak’s analysis ( F , M ). .

Article Snippet: Mouse Peritoneal macrophages , C57BL/6J ( M. musculus ) Jackson Lab #000664; RRID: , N/A.

Techniques: Infection, Staining, Imaging, Immunofluorescence, Bacteria, Derivative Assay, Negative Control, Quantitative RT-PCR, Expressing, Two Tailed Test

Journal: bioRxiv

Article Title: Murine peritoneal macrophages undergo female-specific remodeling with aging

doi: 10.1101/2025.06.11.659200

Figure Lengend Snippet: Peritoneal macrophages are the most perturbed cell type within the peritoneal cavity immune niche as a function of age and biological sex. ( A ) Experimental scheme illustrating age-based cell profiling in mouse peritoneal lavage. ( B ) Uniform Manifold Approximation and Projection (UMAP) visualization of unsupervised clustering of peritoneal lavage cells. ( C ) UMAP representation of AUGUR area under the curve (AUC) scores for peritoneal lavage immune cell populations. ( D ) Boxplots of peritoneal cell composition quantified by scRNA-seq; n = 8 libraries per sex and age; 4 independent cohorts in 3 independent datasets. ( E ) Boxplots of peritoneal cell composition quantified by flow cytometry; n ≥ 25 per sex and age (6 independent cohorts). ( F ) Boxplots of peritoneal immune lavage cell number calculated by the proportion obtained from flow cytometry and total peritoneal lavage cell count; n ≥ 25 per sex and age (6 independent cohorts). For boxplots in panels ( D-F ), circles/squares represent NIA/JAX mice, respectively. Significance in non-parametric two-sided Wilcoxon rank-sum tests are reported in D-F . The center line of the box plots represents the sample median, the box limits consist of the 25 th and 75 th percentiles, the whiskers span 1.5x the interquartile range.

Article Snippet: Peritoneal macrophages were then isolated using Miltenyi Biotec peritoneal isolation kit (Miltenti Biotec #130-110-434), with modifications to the magnetic labeling step improve purity of macrophage isolation regardless of age, treatment, and genotype.

Techniques: Flow Cytometry, Cell Counting

Journal: bioRxiv

Article Title: Murine peritoneal macrophages undergo female-specific remodeling with aging

doi: 10.1101/2025.06.11.659200

Figure Lengend Snippet: Peritoneal macrophage transcriptome and epigenome are highly sex-dimorphic with age. (A) Experimental scheme of murine peritoneal macrophage ‘omics’ profiling. ( B ) Multidimensional scaling (MDS) plot of transcriptomic profiles, indicating distinct transcriptomic aging patterns between sexes. ( C ) MDS plot of chromatin accessibility profiles by ATAC-seq. ( D ) MDS plot of H3K4me3 profiles. ( E ) Scatterplot of age-related changes in peritoneal macrophage transcriptomes in female vs. males, reported as DESeq2 log 2 fold changes per month. 284 Male-specific (blue) and 234 female-specific (pink) aging-regulated genes (Transcriptome-wide Spearman Rho = 0.288). ( F ) Scatterplot of age-related changes in peritoneal macrophage ATAC-seq accessible regions in female vs. males, reported as DESeq2 log 2 fold changes per month. 1044 Male-specific (blue) and 2825 female-specific (pink) regions (Genome-wide Spearman Rho = 0.232). ( G ) Scatterplot of age-related changes in peritoneal macrophage H3K4me3 signal in female vs. males, reported as DESeq2 log 2 fold changes per month. 131 male-specific (blue) and 1699 female-specific (pink) marks (Genome-wide Spearman Rho = 0.329). ( H ) Heatmap of male-specific aging gene expression changes by RNA-seq. ( I ) Heatmap of female-specific aging gene expression changes by RNA-seq. ( J ) Heatmap of male-specific aging chromatin accessibility changes by ATAC-seq. ( K ) Heatmap of female-specific aging chromatin accessibility changes by ATAC-seq. ( L ) Heatmap of male-specific aging H3K4me3 peak intensity by H3K4me3 CUT&RUN. ( M ) Heatmap of female-specific H3K4me3 peak intensity by H3K4me3 CUT&RUN.

Article Snippet: Peritoneal macrophages were then isolated using Miltenyi Biotec peritoneal isolation kit (Miltenti Biotec #130-110-434), with modifications to the magnetic labeling step improve purity of macrophage isolation regardless of age, treatment, and genotype.

Techniques: Genome Wide, Gene Expression, RNA Sequencing

Journal: bioRxiv

Article Title: Murine peritoneal macrophages undergo female-specific remodeling with aging

doi: 10.1101/2025.06.11.659200

Figure Lengend Snippet: Functional enrichment analyses reveal convergent and sex-dimorphic phenotypes in aging peritoneal macrophages. ( A , B ) NetworkAnalyst putative PPI network of genes changing with age in female ( A ) and male ( B ) peritoneal macrophages using IMEx network information. ( C , D , E ) Top 5 sex-dimorphic GSEA enrichments for aging in each sex for RNA-seq ( C ), ATAC-seq ( D ), and H3K4me3 CUT&RUN ( E ). ( F ) GSEA of CRISPR-Cas9 phagocytosis regulators from screens in aging female and male peritoneal macrophages. ( G ) GSEA of metabolic gene sets from MSigDB Hallmarks in aging female and male peritoneal macrophages. ( H ) GSEA of M1/M2 polarization genes in aging female and male peritoneal macrophages. NES: Normalized Enrichment Score. FDR: False Discovery Rate.

Article Snippet: Peritoneal macrophages were then isolated using Miltenyi Biotec peritoneal isolation kit (Miltenti Biotec #130-110-434), with modifications to the magnetic labeling step improve purity of macrophage isolation regardless of age, treatment, and genotype.

Techniques: Functional Assay, RNA Sequencing, CRISPR

Journal: bioRxiv

Article Title: Murine peritoneal macrophages undergo female-specific remodeling with aging

doi: 10.1101/2025.06.11.659200

Figure Lengend Snippet: Female-specific macrophage remodeling in phagocytosis and metabolism. ( A ) Experimental scheme of murine peritoneal macrophage purification and functional analysis. ( B ) Representative microscopy image showing a phagocytosis event by young old male and female MACS-purified peritoneal macrophages engulfing Zymosan bioparticles. Blue = DAPI, Green = Zymosan bioparticles, Scale bar: 200μm. ( C ) Phagocytosis quantification of phagocytosis images from young old male and female peritoneal macrophages. The phagocytosis index is calculated by dividing phagocytosing cells by the total number of imaged cells. Due to cohort-to-cohort variation, values are normalized to the median value of each independent experiment. Animals from 5 independent cohorts (3 from NIA, 2 from JAX). Young females n = 21, old females n = 18, young males n = 22, and old males n = 20 (variation due to animal death prior to experimental day). ( D ) Flow cytometry phagocytosis quantification of peritoneal macrophage. Each point represents one animal. Animals from 6 independent cohorts (3 from NIA, 3 from JAX). Circles/squares represent NIA/JAX mice, respectively Samples with < 5000 F4/80 + events or < 75% F4/80 + purity were excluded from the analysis. ( E ) Venn diagram for CRISPR-Cas9 phagocytosis regulators and female-specific down-regulated genes according to DESeq2 at FDR < 5% and < 10%. ( F ) Lentiviral shRNA phagocytosis regulator screen experimental scheme. ( G ) Mini shRNA screen of RAW264.7 phagocytosis capacity using flow cytometry. The horizontal red line in the panel shows the median control value in the relative phagocytosis index of the macrophage. Dark green/light green for sh1/sh2 samples. Significance in non-parametric one-sided Wilcoxon rank-sum tests were reported (Hypothesis: same regulation direction as aging). ( H ) Experimental scheme of aging bone marrow-derived macrophage seahorse. ( I ) Representative normalized extracellular acidification rate (ECAR) mpH/min/DNA from 1 cohort of animals. ( J ) Glycolysis quantification using Seahorse (n = 15 for young old females and males; animals from 3 independent NIA cohorts). ( K ) Glycolysis quantification using Lactate-Glo (n = 18 for young males, n = 18 for young females, n = 17 for old males, and n = 17 for old females; animals from 5 independent cohorts (3 from NIA, 2 from JAX). For boxplots in panels ( C-D , J-H ), circles/squares represent NIA/JAX mice, respectively. Significance in non-parametric two-sided Wilcoxon rank-sum tests were reported. ( C-D , G , J-H ) The center line of the box plots represents the sample median, the box limits consist of the 25 th and 75 th percentiles, the whiskers span 1.5x the interquartile range.

Article Snippet: Peritoneal macrophages were then isolated using Miltenyi Biotec peritoneal isolation kit (Miltenti Biotec #130-110-434), with modifications to the magnetic labeling step improve purity of macrophage isolation regardless of age, treatment, and genotype.

Techniques: Purification, Functional Assay, Microscopy, Flow Cytometry, CRISPR, shRNA, Control, Derivative Assay

Journal: bioRxiv

Article Title: Murine peritoneal macrophages undergo female-specific remodeling with aging

doi: 10.1101/2025.06.11.659200

Figure Lengend Snippet: Female-specific macrophage phagocytosis declines due to decreased estrogen signaling through the Esr1 receptor. ( A ) Experimental scheme outlining the analysis of OVX mice. ( B ) Multi-dimensional scaling (MDS) analysis of gene expression profiles from Sham and OVX mice. ( C ) GSEA of OVX-regulated genes in the aging female peritoneal macrophage transcriptome. ( D ) Boxplot of phagocytosis quantification of peritoneal macrophages in Sham and OVX mice using flow cytometry (n = 11 for Sham, n = 12 for OVX; animals from 3 independent cohorts). ( E ) Boxplot of glycolysis quantification of peritoneal macrophages in Sham and OVX mice (n = 12 for Sham, n = 11 for OVX; 2 independent cohorts). ( F ) Experimental scheme describing late-life short-term 17β-estradiol (E2) supplementation in female mice. ( G ) MDS analysis of gene expression profiles in young females, old females, and E2-treated old females; n = 5 for young females, n = 5 for old females, n = 4 for E2-treated old females. ( H ) GSEA of E2-regulated genes in old female macrophages in the aging female peritoneal macrophage transcriptome. ( I ) Boxplot of phagocytosis of peritoneal macrophages in young females, old females, and E2-treated old females (n = 13 for young females, n = 13 for old females, n = 14 for E2-treated old females; animals from 3 independent cohorts). ( J ) Boxplot of glycolysis quantification of peritoneal macrophages in young females, old females, and E2-treated old females (n = 14 for young females, old females, and E2-treated old females; animals from 3 independent cohorts). ( K ) Experimental scheme for Esr1 knockout (KO) mice. ( L ) MDS analysis of gene expression profiles in WT and Esr1 KO mice (n = 5 for WT, n = 6 for Esr1 KO). ( M ) GSEA of Esr1 KO-regulated genes in the aging female peritoneal macrophage transcriptome. ( N ) Boxplot of phagocytosis of peritoneal macrophages in WT and Esr1 KO mice (n = 15 for WT, n = 13 for Esr1 KO; animals from 4 independent cohorts). ( O ) Boxplot of glycolysis quantification of peritoneal macrophages in WT and Esr1 KO mice (n = 15 for WT and Esr1 KO; animals from 4 independent cohorts). For boxplots in panels ( D , E , I , J , N , O ), circles/squares represent NIA/JAX mice, respectively. Significance in non-parametric two-sided Wilcoxon rank-sum tests were reported. The center line represents the sample median, the box limits consist of the 25 th and 75 th percentiles, and the whiskers span 1.5x the interquartile range.

Article Snippet: Peritoneal macrophages were then isolated using Miltenyi Biotec peritoneal isolation kit (Miltenti Biotec #130-110-434), with modifications to the magnetic labeling step improve purity of macrophage isolation regardless of age, treatment, and genotype.

Techniques: Gene Expression, Flow Cytometry, Knock-Out

Journal: bioRxiv

Article Title: Murine peritoneal macrophages undergo female-specific remodeling with aging

doi: 10.1101/2025.06.11.659200

Figure Lengend Snippet: A mini shRNA screen for transcription factors driving in female-specific macrophage remodeling with aging. ( A ) Flow chart outlining the steps for the selection of candidate transcription factors (TFs) for screening. ( B ) Heatmap displaying female-specific differential transcription factor gene expression with aging (FDR < 5% in aging female macrophages, FDR > 10% in aging male macrophages based on DESeq2 analysis). ( C ) Selected enriched motifs identified at female-specific differentially accessible ATAC-seq peaks with aging (HOMER; FDR < 5%). ( D ) Selected enriched motifs identified at female-specific differentially marked H3K4me3 peaks with aging (HOMER; FDR < 5%). ( E ) Experimental scheme of shRNA-mediated TF knockdown in RAW264.7 macrophages. ( F ) GSEA of TF shRNA knockdown regulated genes in the aging female peritoneal macrophage transcriptome. ( G , H , I ) Boxplots showing phagocytosis quantification using flow cytometry ( G ), glycolysis quantification using Agilent Seahorse assays ( H ), and lactate levels measured with Lactate-Glo in RAW264.7 macrophages transduced with lentiviral TF shRNA against ( I ) Irf2 , Mef2c , Meis1 , Tal1 , and Tbl1xr1 (n = 6 independent infections for each hairpin). Dark green/light green for sh1/sh2 samples. Significance in non-parametric one-sided Wilcoxon rank-sum tests were reported (Hypothesis: same direction as aging). The horizontal red line in the panel shows the median control value in the relative phagocytosis index ( G ), relative glycolysis ( H ), and relative lactate secretion ( I ) of macrophages. The center line represents the sample median, the box limits show the 25 th and 75 th percentiles, and the whiskers span 1.5 times the interquartile range.

Article Snippet: Peritoneal macrophages were then isolated using Miltenyi Biotec peritoneal isolation kit (Miltenti Biotec #130-110-434), with modifications to the magnetic labeling step improve purity of macrophage isolation regardless of age, treatment, and genotype.

Techniques: shRNA, Selection, Gene Expression, Knockdown, Flow Cytometry, Transduction, Control

Journal: bioRxiv

Article Title: Murine peritoneal macrophages undergo female-specific remodeling with aging

doi: 10.1101/2025.06.11.659200

Figure Lengend Snippet: Irf2 impacts Hk3 leading to an increase in glycolytic dependence. ( A ) Analysis of Irf2 expression at the RNA and protein levels in aging peritoneal macrophages. ( B ) Boxplot of Irf2 gene expression in mouse peritoneal macrophages using RT-qPCR (young female n = 12, old female n = 8, young male n = 12, and old male n = 9; animals from 4 independent cohorts). ( C ) Histogram of female and male Irf2 protein expression between young and old peritoneal macrophages based on intracellular immunolabelling and flow cytometry quantification. ( D ) Boxplot of Irf2 protein quantification in peritoneal macrophages between young and old female and male mice (young female n = 13, old female n = 14, young male n = 14, and old male n = 13; animals from 3 independent cohorts). ( E ) Experimental scheme for Irf2 ChIP-seq in young female BMDMs. ( F ) Top GREAT enriched terms of Irf2-bound genomic regions. (G) GSEA of direct Irf2 target genes in the aging female peritoneal macrophage transcriptome. Peaks were assigned to target genes based on their absolute distance to transcriptional start sites (500bp-5kb). ( H ) Integrated Genomics Viewer (IGV) snapshot of Irf2 ChIP-seq signal at the Hk3 gene locus on chromosome 13. ( I ) Quantitative RT-qPCR analysis of Hk3 mRNA levels in young and old female peritoneal macrophages samples (n = 12 young, n = 8 old female; animals from 4 independent cohorts). ( J ) Experimental design for lentiviral knockdown of Irf2 in J774A.1 cells. ( K ) Boxplot of Hk3 expression from RNA-seq after knockdown of Irf2 in macrophage cells. Circles/squares for RAW264.7/J774A.1 samples; shLuciferase (goldenrod), non-targeting control (light goldenrod), sh1 (dark green), and sh2 (light green); n = 3 infections per hairpin. Significance as FDR from DESeq2. ( L ) Boxplot of glycolysis quantification using Seahorse assays on lentiviral knockdown of Irf2 RAW264.7 and J774A.1 cell line (n = 6 infections per hairpin). Note that the RAW264.7 data is repeated from for comparison convenience. ( M ) Boxplot of glycolysis quantification using Lactate-Glo on lentiviral knockdown Irf2 in RAW264.7 and J774A.1 cells (n = 6 infections per hairpin). Note that the RAW264.7 data is repeated from for comparison convenience. ( N ) Proposed mechanism for Irf2-driven age-related glycolytic shift in aging female peritoneal macrophages. For boxplots in panels ( B , D , I) , circles/squares represent NIA/JAX mice, respectively. Significance in non-parametric two-sided Wilcoxon rank-sum tests is reported. For boxplots in panels ( L , M ) circles/squares for RAW264.7/J774A.1 samples; dark green/light green for sh1/sh2 samples. Significance in non-parametric one-sided Wilcoxon rank-sum tests were reported (Hypothesis: same regulation direction as aging). The horizontal red line in the panel shows the median control value of the normalized glycolysis ( L ) and normalized lactate ( M ).

Article Snippet: Peritoneal macrophages were then isolated using Miltenyi Biotec peritoneal isolation kit (Miltenti Biotec #130-110-434), with modifications to the magnetic labeling step improve purity of macrophage isolation regardless of age, treatment, and genotype.

Techniques: Expressing, Gene Expression, Quantitative RT-PCR, Flow Cytometry, ChIP-sequencing, Knockdown, RNA Sequencing, Control, Comparison