ccr2 inhibitor  (MedChemExpress)

Journal: Journal of Advanced Research

Article Title: Novel function of macrophage migration inhibitory factor in regulating post-infarct inflammation and the therapeutic significance

doi: 10.1016/j.jare.2025.05.030

Figure Lengend Snippet: Macrophage migration inhibitory factor (MIF) promotes inflammatory cell migration and infiltration through C – C motif chemokine receptor 2 (CCR2) and C-X-C motif chemokine receptor (CXCR) 4 and promotes splenic monocyte mobilization via interaction with angiotensin Ⅱ type 1 receptor (AT-1R) after myocardial infarction (MI). A, peripheral blood mononuclear cells (PBMCs) chemotaxis in response to homogenized sham or infarct heart tissue from wild type (WT) mice at day 3 post-MI, or MI homogenate together with inhibition of C–C motif chemokine receptor 2 (CCR2), C-X-C motif chemokine receptor (CXCR) 2 and CXCR4, respectively. Cells/HPF, cells/high power field. PBMCs harvested from WT mice at 3 days post-MI. CCR2i, CCR2 inhibition. CXCR2i, CXCR2 inhibition. CXCR4i, CXCR4 inhibition. n = 4–8 per group. *** P < 0.001 vs. sham . B, PBMCs chemotaxis in response to recombinant human MIF (rMIF) or rMIF with inhibition of CCR2, CXCR2 and CXCR4, respectively. PBMCs harvested from WT mice at 3 days post-MI. n = 4–8 per group. *** P < 0.001 vs. control . C, Representative immunoblotting images for monocyte chemoattractant protein-1 (MCP-1), CXCR4 and internal reference protein (heat shock protein 60, HSP-60) in hearts from WT and global MIF deficient (MIFKO) mice with sham-operation or 72 h MI. D-E, Quantitative analysis of MCP-1 and CXCR4 expression. n = 6–9 per group. ** P < 0.01 vs. sham in the same genotype. *** P < 0.001 vs. sham in the same genotype. F, Splenic monocyte (from normal WT mice) chemotaxis in response to plasma from normal mice or mice with 1.5 h MI, or MI plasma together with losartan. Lor, losartan, AT-1R inhibitor. n = 6–8 per group. *** P < 0.001 vs. normal. G, Splenic monocytes (from normal WT mice) chemotaxis in response to rMIF and angiotensin II (Ang II), or together with losartan addition, respectively. n = 6–8 per group. ** P < 0.01 vs. Control. *** P < 0.001 vs. Control. H, Representative immunoblotting images for AT-1R and internal reference protein (HSP-60) in spleens from WT and global MIF deficient (MIFKO) mice with sham-operation and 1.5 h MI. I, Quantitative analysis of AT-1R expression. n = 8 per group. *** P < 0.001 vs. sham in the same genotype . J. Co-immunoprecipitation (Co-IP) assay using an anti-AT-1R antibody or IgG demonstrated the interaction between AT-1R and MIF, as well as between AT-1R and CD74 in splenic monocytes and spleen tissue. Input, input sample. IgG, negative control sample. IP, Co-IP sample.

Article Snippet: For both groups 1 and 2, PBMCs were pre-treated for 1 h using DMEM with or without CCR2 inhibitor (CCR2 antagonist 4 hydrochloride, 100 mM), CXCR2 inhibitor (SB225002, 800 nM) or CXCR4 inhibitor (Plerixafor, AMD3100, 10 μM) (all from MedChemExpress, USA).

Techniques: Migration, Chemotaxis Assay, Inhibition, Recombinant, Control, Western Blot, Expressing, Clinical Proteomics, Co-Immunoprecipitation Assay, Negative Control

Journal: Signal Transduction and Targeted Therapy

Article Title: Piezo1 activation suppresses bone marrow adipogenesis to prevent osteoporosis by inhibiting a mechanoinflammatory autocrine loop

doi: 10.1038/s41392-025-02455-w

Figure Lengend Snippet: Piezo1 suppresses Lcn2 expression and secretion via the inhibition of Ccl2-evoked NF-κB activation. BMMSCs were isolated from femurs and tibias of 10-week-old male PDGFRα-Piezo1 KO mice and WT controls. a Gene expression of Ccr2 in BMMSCs, as determined by real-time PCR. b–g Recombinant mouse Ccl2 protein (rmCcl2, 100 ng/mL) or the CCR2 antagonist INCB3344 (10 nM) was added to the adipogenic and osteogenic induction medium during BMMSC differentiation. The expression levels of osteogenic genes ( b–d ) and adipogenic genes ( e–g ) were determined via real-time qPCR. h–n WT and KO BMMSCs were treated with rmCcl2 (100 ng/mL) or the CCR2 antagonist INCB3344 (10 nM) for 24 h. h Sonicated BMMSCs were subjected to chromatin immunoprecipitation (ChIP) using an anti-p65 antibody. The ChIP DNA samples were then subjected to qPCR amplification using specific primers against the promoter region of Lcn2, as shown in supplementary Fig. . Representative gel images of end-point ChIP-qPCR products are shown. i Quantitative results are shown as the percentage of input DNA: % of Input = 2^(CT Input −CT IP )/dilution factor × 100%. j Luciferase reporter assay was performed in isolated WT and KO BMMSCs transfected with pGL3 constructs containing the mouse wild-type Lcn2 promoter (Lcn2-WT) or the mutated Lcn2 promoter with mutations in the NF-κB binding motif (Lcn2-Mut, sequence in supplementary Fig. ). The firefly luciferase activity was measured and normalized to Renilla luciferase activity. k Schematic diagram illustrating the actions of rmCcl2, the CCR2 antagonist, and the NF-κB-specific inhibitor QNZ on NF-κB translocation and Lcn2 expression; created with BioRender ( https://BioRender.com ). l ELISA analysis of total p65 in the nucleus and cytoplasm of BMMSCs. m Representative immunofluorescence images of intracellular p65 localization. Nuclei were stained with DAPI (blue). Scale bar, 50 μm. n P65 nuclear translocation was quantified by ImageJ software via Pearson’s correlation coefficient (PCC) between the p65 immunofluorescence signal and DAPI staining across segmented nuclear regions. o , p WT and KO BMMSCs were treated with the NF-κB-specific inhibitor QNZ (10 nM) for another 2 h after 24-h treatment of rmCcl2 (100 ng/mL) and INCB3344 (10 nM). o The mRNA level of Lcn2 . p Lcn2 levels in the culture medium were determined by ELISA. q Illustration of how the AP-1 inhibitor modulates Ccl2 gene expression; created with BioRender ( https://BioRender.com ). r–u WT and KO BMMSCs were treated with the AP-1-specific inhibitor T-5224 (10 μM) for 24 h. r Sonicated BMMSCs were subjected to ChIP assay using an anti-c-Jun antibody. Representative gel images of end-point ChIP-qPCR products are shown. s Quantitative results are shown as the percentage of input DNA. t The mRNA level of Ccl2 . u Ccl2 levels in the culture medium were determined by ELISA. v Luciferase reporter assay was performed in isolated WT and KO BMMSCs transfected with pGL3 constructs containing the mouse wild-type Ccl2 promoter (Ccl2-WT) or the mutated Ccl2 promoter with mutations in the c-Jun binding motif (Ccl2-Mut, sequence in supplementary Fig. ). The firefly luciferase activity was measured and normalized to Renilla luciferase activity. n = 5 for each group. The data are presented as the means ± SEMs; * p < 0.05, ** p < 0.01, *** p < 0.001. See also supplementary Figs. –

Article Snippet: To investigate the regulatory effects of AP-1 inhibitor on Ccl2, 10 μM AP-1 inhibitor T-5224 (MCE, #HY-12270) was supplemented into the medium of WT or Piezo1 KO BMMSCs for 24 h. To investigate the regulatory role of NF-κB on Lcn2 expression, WT and PDGFRα-Piezo1 KO BMMSCs were first treated with recombinant mouse Ccl2 protein (rmCcl2, 100 ng/mL) and CCR2 antagonist INCB3344 (10 nM) for 24 h, followed by supplementation with the NF-κB-specific inhibitor QNZ (10 nM, MCE, #HY-13812) for additional 2 h. To investigate the effects of Klf2 on BMMSC differentiation, 40 MOI of lentivirus encoding eGFP or eGFP together with Klf2 (WZ Biosciences Inc) was used to infect WT or Piezo1 KO BMMSCs for 72 h with the presence of 5 μg/mL polybrene before differentiation.

Techniques: Expressing, Inhibition, Activation Assay, Isolation, Gene Expression, Real-time Polymerase Chain Reaction, Recombinant, Sonication, Chromatin Immunoprecipitation, Amplification, ChIP-qPCR, Luciferase, Reporter Assay, Transfection, Construct, Binding Assay, Sequencing, Activity Assay, Translocation Assay, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining, Software

Journal: Advanced Science

Article Title: Deciphering the Role and Mechanism of Decidual Monocyte‐Derived Macrophage Infiltration in Obstetric Antiphospholipid Syndrome at Single‐Cell Resolution

doi: 10.1002/advs.202503480

Figure Lengend Snippet: Abnormal infiltration of monocyte‐derived CCR2 + macrophages in OAPS decidua and their pro‐inflammatory roles. A) Bar plot showing the proportions of MDMs in decidual immune cells from OAPS patients and HCs in scRNA‐seq data. B) FCM analysis of the proportion of CCR2 + macrophages in the decidua from OAPS patients (n = 11) compared with HCs (n = 18). C) Representative images for CCR2 + and CCR2 − macrophages from the decidua of OAPS patients and HCs under the transmission electron microscope. Scale bar: 2 µm. D) Bubble plot showing the functional gene expressions in different types of decidual myeloid cells based on scRNA‐seq data. E) Bar plot showing the GO terms of marker genes in decidual MDMs. F) Bar plot showing the KEGG terms of marker genes in decidual MDMs. G) GSEA indicates the term of inflammatory response is significantly enriched in decidual CCR2 + macrophages. H) Bar plot showing the relative expression levels of genes related to inflammatory factors in decidual CCR2 + and CCR2 − macrophages, as detected using RT‐qPCR (n = 3). I) Representative immunoblots and semi‐quantified results of Arginase 1, NF‐κB P65, and STAT3 expressions in decidual CCR2 + and CCR2 − macrophages from OAPS patients (n = 3). J) Representative immunofluorescence images for CD14 (green), CCR2 (red), IGFBP1 (croci), HLA‐G (cyan), and DAPI (blue) co‐staining in decidua from HCs. Scale bar: 1000 µm. K) Representative immunofluorescence images for CD14 (green), CCR2 (red), IGFBP1 (croci), HLA‐G (cyan), and DAPI (blue) co‐staining in decidua from OAPS patients. Scale bar: 2000 µm. L) Line chart showing the effects of medium supernatants from CCR2 + and CCR2 − macrophages on HTR‐8/SVneo proliferation, as measured by CCK‐8 (n = 3). M) Bar plot showing relative expression levels of CASP3 , MMP2 , and MMP9 genes in HTR‐8/SVneo treated with supernatants from CCR2 + and CCR2 − macrophages, as detected by RT‐qPCR (n = 3). Data in (A) and (B) are presented as mean ± SD and analyzed using Student's t ‐test. Data in (H) and (I) are presented as mean ± SD and analyzed using a paired t ‐test. Data in (L) and (M) are presented as mean ± SD and analyzed using one‐way ANOVA with Dunnett's multiple comparisons test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. OAPS, obstetric antiphospholipid syndrome; HCs, healthy controls; MDM, monocyte‐derived macrophage; FCM, flow cytometry; scRNA‐seq, single‐cell RNA sequencing; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; GSEA, Gene Set Enrichment Analysis; RT‐qPCR, real‐time quantitative polymerase chain reaction; SD, standard deviation; ANOVA, analysis of variance.

Article Snippet: To investigate the effects of CCR2 on chemotaxis, THP‐1 cells or M0 macrophages were pre‐treated with the CCR2 antagonist RS 504 393 (MCE, USA, 500 nM) for 3 h before the experiment.

Techniques: Derivative Assay, Transmission Assay, Microscopy, Functional Assay, Marker, Expressing, Quantitative RT-PCR, Western Blot, Immunofluorescence, Staining, CCK-8 Assay, Flow Cytometry, RNA Sequencing, Real-time Polymerase Chain Reaction, Standard Deviation

Journal: Advanced Science

Article Title: Deciphering the Role and Mechanism of Decidual Monocyte‐Derived Macrophage Infiltration in Obstetric Antiphospholipid Syndrome at Single‐Cell Resolution

doi: 10.1002/advs.202503480

Figure Lengend Snippet: Elevated CCL2 levels in the decidua and serum of OAPS patients and OAPS mice compared with HCs and control mice. A) Representative immunohistochemistry images for CCL2 in decidua from HCs (n = 6) and OAPS patients (n = 8). Three random fields of each sample were used for semiquantitative analyses. Scale bars: 50 µm. B) Scatter plot showing the average OD of CCL2 in decidua. A point represents a random field. C) The expression levels of CCL2 in medium supernatant of primary decidual cells from HCs (n = 6) and OAPS patients (n = 6). D) The expression levels of CCL2 in medium supernatant of primary decidual cells from HCs (n = 4) after stimulation of PBS, HC‐IgG/β2GPI complex, and aPL‐IgG/β2GPI complex. E) Flow chart depicting the methods of establishing OAPS mouse model by intravenous injection of aPL‐IgG. F) Representative figures for the uterus from D15.5 pregnant mice after injection of 0.9% NaCI, HC‐IgG, and aPL‐IgG. G) The embryo absorption rates of D15.5 pregnant mice after injection of 0.9% NaCI, HC‐IgG, and aPL‐IgG (n = 6). H) Representative HE images for decidua and placenta from D15.5 pregnant mice after injection of 0.9% NaCI, HC‐IgG, and aPL‐IgG. The decidua and placenta were detected for sites where embryos were live; for sites where embryos were absorbed, the whole uterus was detected. Scale bars: 1 mm. I) The levels of serum CCL2 in mice of different groups (n = 6). J) The expression of the Ccl2 gene was detected by RT‐qPCR in the decidua from mice in different groups (n = 6). K) Representative immunohistochemistry images for CCL2 in the decidua from mice of different groups. Three random fields of each sample were used for semiquantitative analyses. Scale bars: 5 µm. L) Bar plot showing the average OD of CCL2 in decidua. A point represents a sample. M) The proportion of macrophages in the decidua of mice from different groups, as detected by FCM (n = 6). N) Representative immunofluorescence images for CCR2 + macrophage in decidua from mice of different groups. Scale bars = 100 µm. O) The Tnf and Ifng gene expression in decidua from mice in different groups, detected by RT‐qPCR (n = 6). Data in (B) and (C) are presented as mean ± SD and analyzed using Student's t ‐test. Data in (D), (G), (J), (L), (M), and (O) are presented as mean ± SD and analyzed using one‐way ANOVA with Dunnett's multiple comparisons test. For (I), boxplot features: minimum whisker, 25th percentile – 1.5 × IQR or the lowest value within; minimum box, 25th percentile; center, median; maximum box, 75th percentile; maximum whisker, 75th percentile + 1.5 × IQR or the greatest value within. * p < 0.05, ** p < 0.001, *** p < 0.001, **** p < 0.0001. OAPS, obstetric antiphospholipid syndrome; HCs, healthy controls; OD, optical density; aPL, antiphospholipid antibody; Ig, immunoglobin; β2GPI, β2 glycoprotein I; RT‐qPCR, real time‐quantitative reverse transcription polymerase chain reaction; FCM, flow cytometry; IQR, interquartile range; SD, standard deviation; ANOVA, analysis of variance.

Article Snippet: To investigate the effects of CCR2 on chemotaxis, THP‐1 cells or M0 macrophages were pre‐treated with the CCR2 antagonist RS 504 393 (MCE, USA, 500 nM) for 3 h before the experiment.

Techniques: Control, Immunohistochemistry, Expressing, Injection, Quantitative RT-PCR, Immunofluorescence, Gene Expression, Whisker Assay, Reverse Transcription, Polymerase Chain Reaction, Flow Cytometry, Standard Deviation

Journal: Advanced Science

Article Title: Deciphering the Role and Mechanism of Decidual Monocyte‐Derived Macrophage Infiltration in Obstetric Antiphospholipid Syndrome at Single‐Cell Resolution

doi: 10.1002/advs.202503480

Figure Lengend Snippet: CCL2 promotes CCR2 + macrophage infiltration in OAPS decidua. A) Schematic diagram of an in vitro chemotaxis system where primary decidual cells chemoattract THP‐1 or THP‐1‐derived M0 macrophages. Briefly, the culture supernatant of primary decidual cells was added to the lower chamber of a 3.0 µm Transwell insert, and THP‐1 or THP‐1‐derived M0 macrophages, resuspended in FBS‐free medium, were added to the upper chamber. B) Bar plot showing the number of THP‐1 cells in the lower chamber after chemotaxis induced by decidual cells from OAPS patients and HCs (n = 6). C) Representative images and statistical results showing the number of THP‐1 M0 macrophages that migrated through the Transwell membrane (n = 5). Each sample was analyzed in three fields for quantification. D) Bar plot showing the number of THP‐1 cells in the lower chamber after chemotaxis induced by decidual cells from OAPS patients with or without Bindarit treatment (n = 4). E) Representative images and statistical results showing the number of THP‐1 M0 macrophages that migrated through the Transwell membrane (n = 5). Each sample was analyzed in three fields for quantification. F) Bar plot showing the number of THP‐1 cells in the lower chamber after chemotaxis induced by decidual cells from OAPS patients, with or without RS504393 treatment (n = 6). G) Representative images and statistical results showing the number of THP‐1 M0 macrophages that migrated through the Transwell membrane (n = 5). Each sample was analyzed in three fields for quantification. H) Flow chart depicting the methods of treating the OAPS mice model by intravenous injection of PF‐4136309. I) Representative images of uteri from D15.5 pregnant mice after injection of solvent or PF‐4136309. J) Embryo resorption rates in D15.5 pregnant mice following injection of solvent or PF‐4136309 (n = 6). K) Representative HE images for decidua and placenta from D15.5 pregnant mice after injection of solvent and PF‐4136309. L) The levels of serum CCL2 in mice of different groups (n = 6). M) The expression of the Ccl2 gene was detected by RT‐qPCR in the decidua from mice (n = 5 and 6, respectively). N) Representative immunohistochemistry images for CCL2 in decidua from mice of different groups (n = 6). Three random fields of each sample were used for semiquantitative analyses. Scale bars: 50 µm. O) Bar plot showing the average OD of CCL2 in decidua. A point represents a sample. P) The proportion of macrophage in the decidua of mice that received different treatments was detected by FCM (n = 6). Q) Representative immunofluorescence images for CCR2 + macrophage in the decidua from mice that received different treatments. Scale bars: 50 µm. Data in (B), (C), (D), (E), (F), (G), (J), (K), (M), and (P) are presented as mean ± SD and analyzed using Student's t ‐test. For (K), boxplot features: minimum whisker, 25th percentile – 1.5 × IQR or the lowest value within; minimum box, 25th percentile; center, median; maximum box, 75th percentile; maximum whisker, 75th percentile + 1.5 × IQR or the greatest value within. Data in (O) are presented as mean ± SD and analyzed using one‐way ANOVA with Dunnett's multiple comparisons test. ns: not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. OAPS, obstetric antiphospholipid syndrome; HCs, healthy controls; RT‐qPCR, real time‐quantitative reverse transcription polymerase chain reaction; ELISA, enzyme‐linked immunosorbent assay; OD, optical density; FCM, flow cytometry; SD, standard deviation; IQR, interquartile range; ANOVA, analysis of variance.

Article Snippet: To investigate the effects of CCR2 on chemotaxis, THP‐1 cells or M0 macrophages were pre‐treated with the CCR2 antagonist RS 504 393 (MCE, USA, 500 nM) for 3 h before the experiment.

Techniques: In Vitro, Chemotaxis Assay, Derivative Assay, Membrane, Injection, Solvent, Expressing, Quantitative RT-PCR, Immunohistochemistry, Immunofluorescence, Whisker Assay, Reverse Transcription, Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Standard Deviation

Journal: Advanced Science

Article Title: Deciphering the Role and Mechanism of Decidual Monocyte‐Derived Macrophage Infiltration in Obstetric Antiphospholipid Syndrome at Single‐Cell Resolution

doi: 10.1002/advs.202503480

Figure Lengend Snippet: The aPL‐IgG/β2GPI complex promotes macrophages to express more CCL2 via the NF‐κB pathway in vitro. (A) UMAP presenting the expression of CCL2 gene in the integrated single‐cell landscape. (B) FCM analysis of CCL2 secretion in major decidual immune cells after 18 h LPS stimulation (n = 6). (C) FCM analysis of CCL2 secretion in CCR2 + and CCR2 − macrophages after 18 h LPS stimulation (n = 6). (D) Violin plot showing CCL2 gene expression levels in decidual macrophages from OAPS patients compared with HCs. (E) Representative immunofluorescence images of CD14 (red), CCL2 (green), and DAPI (blue) co‐staining in the decidua from OAPS patients and HCs. Scale bar: 100 µm. (F) Bar plot showing the proportion of CCL2 + macrophages, as determined by semi‐quantitative immunofluorescence staining (n = 4 for HCs, n = 5 for OAPS). (G) FCM analysis of CCL2 secretion in macrophages from the decidua of HCs after stimulation with HC‐IgG/β2GPI and aPL‐IgG/β2GPI complexes (n = 6). (H) Expression levels of CCL2 , TNF, IL1B , and IL6 genes in THP‐1 M0 macrophages, as detected by RT‐qPCR following stimulation with PBS, HC‐IgG/β2GPI, and aPL‐IgG/β2GPI complexes (n = 3). (I) Levels of CCL2 and TNF‐α in the culture supernatant of THP‐1 M0 macrophages, as detected by ELISA following stimulation with PBS, HC‐IgG/β2GPI, and aPL‐IgG/β2GPI complexes (n = 3). (J) Volcano plot of DEGs in THP‐1 M0 macrophages under the stimulation of HC‐IgG/β2GPI and aPL‐IgG/β2GPI complex. (K) GO terms of upregulated DEGs in (J). (L) KEGG terms of upregulated DEGs in (J). (M) Representative immunoblot showing the expression of TLR4, NF‐κB p65, IκBα, and IKKα in THP‐1 M0 macrophages stimulated with PBS, HC‐IgG/β2GPI, and aPL‐IgG/β2GPI complexes. (N) Bar plot showing the levels of TLR4 in THP‐1 M0 macrophages after different stimulations (n = 3). (O) Bar plot showing the phosphorylation levels of NF‐κB P65, IκBα, and IKKα in THP‐1 M0 macrophages with different stimulations (n = 3). (P) Representative immunofluorescence images for NF‐κB P65 (green) and DAPI (blue) in THP‐1 M0 macrophages after different stimulations. Scale bar: 50 µm. Data in (C) and (G) are presented as mean ± SD and analyzed using a paired t ‐test. Data in (D) are presented as mean ± SD and analyzed using Student's t ‐test. Data in (B), (H), (I), (N), and (O) are presented as mean ± SD and analyzed using one‐way ANOVA with Dunnett's multiple comparisons test. * p < 0.05, ** p < 0.001, *** p < 0.001, **** p < 0.0001. OAPS, obstetric antiphospholipid syndrome; HCs, healthy controls; DEGs, differentially expressed genes; aPL, antiphospholipid antibody; Ig, immunoglobin; β2GPI, β2 glycoprotein I; RT‐qPCR, real time‐quantitative reverse transcription polymerase chain reaction; ELISA, enzyme‐linked immunosorbent assay; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; FCM, flow cytometry; ANOVA, analysis of variance; SD, standard deviation.

Article Snippet: To investigate the effects of CCR2 on chemotaxis, THP‐1 cells or M0 macrophages were pre‐treated with the CCR2 antagonist RS 504 393 (MCE, USA, 500 nM) for 3 h before the experiment.

Techniques: In Vitro, Expressing, Gene Expression, Immunofluorescence, Staining, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Western Blot, Phospho-proteomics, Reverse Transcription, Polymerase Chain Reaction, Flow Cytometry, Standard Deviation

Journal: Advanced Science

Article Title: Deciphering the Role and Mechanism of Decidual Monocyte‐Derived Macrophage Infiltration in Obstetric Antiphospholipid Syndrome at Single‐Cell Resolution

doi: 10.1002/advs.202503480

Figure Lengend Snippet: Inhibition of TLR4 decreases CCL2 expression in macrophages in vitro and reduces decidual CCR2 + macrophage infiltration and abortion in vivo. (A) Expression levels of CCL2, TNF, IL1B, and IL6 genes in THP‐1 M0 macrophages stimulated with aPL‐IgG/β2GPI complex and inhibited by TAK‐242, as detected by RT‐qPCR (n = 3). (B) Levels of CCL2 and TNF‐α in THP‐1 M0 macrophage culture supernatant stimulated with aPL‐IgG/β2GPI complex and inhibited by TAK‐242, detected by ELISA (n = 3). (C) Representative immunoblot visualizing the expressions of NF‐κB P65, IκBα, and IKKα in THP‐1 M0 macrophages of different groups (n = 3). (D) Bar plot showing the phosphorylation levels of NF‐κB P65, IκBα, and IKKα in THP‐1 M0 macrophages from different groups (n = 3). (E) Representative immunofluorescence images for NF‐κB P65 (green) and DAPI (blue) in THP‐1 M0 macrophages from different groups. Scale bar: 50 µm. (F) Flow chart depicting the methods of treating the OAPS mice model by intraperitoneal injection of TAK‐242. (G) Representative images of uteri from D15.5 pregnant mice after injection of solvent or TAK‐242. (H) Embryo resorption rates in D15.5 pregnant mice following injection of solvent or TAK‐242 (n = 6). (I) Representative HE‐stained images of decidua and placenta from D15.5 pregnant mice in different groups. (J) The levels of serum CCL2 in mice of different groups (n = 6). (K) Expression levels of Ccl2 gene in the decidua of mice, as detected by RT‐qPCR (n = 6). (L) Representative immunohistochemistry images showing CCL2 expression in the decidua of mice from different groups. Three random fields per sample were used for semiquantitative analysis. Scale bars: 50 µm. (M) Bar plot showing the average OD of CCL2 in decidua. A point represents a sample. (N) Proportion of macrophages in the decidua of mice with different treatments, as detected by FCM (n = 6). (O) Representative immunofluorescence images showing CCR2 + macrophages in the decidua of mice from different groups. Scale bars: 50 µm. (P) Schematic diagram of the OAPS mouse model following treatment with PF‐4136309 and TAK‐242. Data in (H), (I), (K), and (N) are presented as mean ± SD and analyzed using Student's t ‐test. For (I), boxplot features: minimum whisker, 25th percentile – 1.5 × IQR or the lowest value within; minimum box, 25th percentile; center, median; maximum box, 75th percentile; maximum whisker, 75th percentile + 1.5 × IQR or the greatest value within. Data in (A), (B), (D), and (M) are presented as mean ± SD and analyzed using one‐way ANOVA with Dunnett's multiple comparisons test. * p < 0.05, ** p < 0.001, *** p < 0.001, **** p < 0.0001. OAPS, obstetric antiphospholipid syndrome; HCs, healthy controls; aPL, antiphospholipid antibody; Ig, immunoglobin; β2GPI, β2 glycoprotein I; RT‐qPCR, real time‐quantitative reverse transcription polymerase chain reaction; ELISA, enzyme‐linked immunosorbent assay; TNF, tumor mecrosis factor; FCM, flow cytometry; IQR, interquartile range; SD, standard deviation; ANOVA, analysis of variance.

Article Snippet: To investigate the effects of CCR2 on chemotaxis, THP‐1 cells or M0 macrophages were pre‐treated with the CCR2 antagonist RS 504 393 (MCE, USA, 500 nM) for 3 h before the experiment.

Techniques: Inhibition, Expressing, In Vitro, In Vivo, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Western Blot, Phospho-proteomics, Immunofluorescence, Injection, Solvent, Staining, Immunohistochemistry, Whisker Assay, Reverse Transcription, Polymerase Chain Reaction, Flow Cytometry, Standard Deviation

Journal: Advanced Science

Article Title: Deciphering the Role and Mechanism of Decidual Monocyte‐Derived Macrophage Infiltration in Obstetric Antiphospholipid Syndrome at Single‐Cell Resolution

doi: 10.1002/advs.202503480

Figure Lengend Snippet: Vascular ECs express higher levels of ACKR1 to aggregate CCL2. (A) Representative immunofluorescence images for vWF (red), CCL2 (green), and DAPI (blue) co‐staining in decidua from OAPS patients and HCs. Scale bar: 100 µm. (B) UMAP plot of EC subclusters at single‐cell resolution. (C) Bar plot showing the proportions of EC subclusters in the decidua of OAPS patients and HCs. (D) Violin plot showing the marker genes of EC subclusters. (E) Bubble plot showing the intercellular communications mediated by ACKR1 and CCL2 between macrophages and vascular ECs in decidua from OAPS patients and HCs. (F) Violin plot of the expression of the ACKR1 gene in vascular ECs of OAPS patients compared with those of HCs decidua. (G) Representative immunofluorescence images for vWF (red), ACKR1 (green), and DAPI (blue) co‐staining in decidua from OAPS patients and HCs. Scale bar: 100 µm. (H) Volcano plot of DEGs for vascular ECs from OAPS patients’ decidua and those from HCs decidua. (I) GO terms of the upregulated DEGs in (H). (J) Heatmap showing the functional scores of vascular ECs in the OAPS and HC groups. (K) Representative immunohistochemistry images for IFN‐γ and TNF‐α in decidua from HCs (n = 6) and OAPS patients (n = 8). Three random fields of each sample were used for semiquantitative analyses. Scale bars: 50 µm. (L) Scatter plot showing the average OD of IFN‐γ and TNF‐α in the decidua. Each point represents a random field. (M) Expression levels of IFN‐γ and TNF‐α in the supernatant of primary decidual cells from HCs (n = 6) and OAPS patients (n = 6). (N) Expression levels of IFN‐γ and TNF‐α in the supernatant of primary decidual cells from HCs following stimulation with PBS, HC‐IgG/β2GPI complex, and aPL‐IgG/β2GPI complex (n = 4). (O) Line chart showing ACKR1 gene expression in EA.hy926 cells under different concentrations of TNF‐α (n = 3). (P) Line chart showing ACKR1 gene expression in EA.hy926 cells under different concentrations of IFN‐γ (n = 3). (Q, S) Representative immunoblot and semi‐quantified results showing ACKR1 protein expression in EA.hy926 cells under different concentrations of TNF‐α (n = 3). (R, T) Representative immunoblot and semi‐quantified results showing ACKR1 protein expression in EA.hy926 cells under different concentrations of IFN‐γ (n = 3). (U) Schematic diagram summarizing the findings of this study. In brief, the aPL‐IgG/β2GPI complex promotes decidual macrophages to express CCL2 via the TLR4‐NF‐κB pathway. CCL2 accumulates on the surface of vascular ECs through ACKR1, recruiting more monocytes into the decidua. Infiltrated CCR2 + macrophages exacerbate decidual inflammatory injury in OAPS patients. ACKR1 overexpression in vascular ECs is driven by increased IFN‐γ and TNF‐α levels in the decidual environment of OAPS patients. Data in (L) and (M) are presented as mean ± SD and analyzed using Student's t ‐test. Data in (N), (O), (P), (S), and (T) are presented as mean ± SD and analyzed using one‐way ANOVA with Dunnett's multiple comparisons test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. OAPS, obstetric antiphospholipid syndrome; HCs, healthy controls; aPL, antiphospholipid antibody; Ig, immunoglobin; β2GPI, β2 glycoprotein I; EC, endothelial cell; OD, optical density; TNF, tumor necrosis factor; IFN, interferon; ELISA, enzyme‐linked immunosorbent assay; DEGs, differential expressed genes; SD, standard deviation; ANOVA, analysis of variance.

Article Snippet: To investigate the effects of CCR2 on chemotaxis, THP‐1 cells or M0 macrophages were pre‐treated with the CCR2 antagonist RS 504 393 (MCE, USA, 500 nM) for 3 h before the experiment.

Techniques: Immunofluorescence, Staining, Marker, Expressing, Functional Assay, Immunohistochemistry, Gene Expression, Western Blot, Over Expression, Enzyme-linked Immunosorbent Assay, Standard Deviation