Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Japanese encephalitis virus-associated human microglia induce cell death of human microvascular endothelial cells in receptor-independent infection

doi: 10.3389/fcimb.2025.1580958

Figure Lengend Snippet: Impact of CX 3 CR1-CX 3 CL1 inhibition in receptor-independent infection. (a) Representative micrographs of CX 3 CL1 expression in the grey matter (left panel) and the white matter (right panel) in the frontal lobe of the human brain. In vascular areas, red arrows highlight CX 3 CL1 expression in endothelial cells of blood vessels. In cerebral matter, green arrows CX 3 CL1 expression out of cellular bodies, black arrows CX 3 CL1 non-expressing cells, and solid and open blue arrows show CX 3 CL1 expressing cells with high and low intensity, respectively. Scale is indicated. (b) Representative histogram plot of flow cytometry analysis showing frequencies of CX 3 CL1 + microvascular endothelial cells at steady state including the isotype control (filled grey), after single cell selection. (c) Scatter dot plot representing virus titres in supernatants of receptor-independent infection of microvascular endothelial cells with mock and JEV-associated microglia, with a MOI of 10 TCID 50 /cell for 6 days, in presence of DMSO and indicated concentration of CX 3 CR1 antagonist. (d) Scatter dot plot representing the frequencies of Annexin-V + single microvascular endothelial cells in a receptor-independent infection of microvascular endothelial cells mock and JEV-associated microglia, with a MOI of 10 TCID 50 /cell for 6 days, in presence of DMSO and indicated concentration of CX 3 CR1 antagonist, as gated in Figure 2c . (c, d) Data are of (c) 2 and (d) 3 independent experiments (# of blood donors) with each condition performed in triplicate, the solid line is the mean value and (d) the dashed line is the baseline. Statistics are calculated with (d) the unpaired t-test (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001).

Article Snippet: For flow cytometry, cells were characterized using fluorescently labelled anti-human CD11b (mouse IgG1κ, clone ICRF44, FITC, BD Biosciences, Franklin Lakes, NJ), anti-human CX 3 CR1 (rat IgG2b, clone 2A9-1, PE, Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) and anti-human CD31 (mouse IgG1κ, clone WM59, AF647, BD Biosciences) antibodies as well as primary anti-human CX 3 CL1 antibody (rabbit polyclonal IgG, clone PA5-23062, Thermofischer Scientific, Waltham, MA) followed by secondary fluorescent-labelled anti-rabbit IgG antibody (donkey, AF647 Abcam, Cambridge, UK).

Techniques: Inhibition, Infection, Expressing, Flow Cytometry, Control, Selection, Virus, Concentration Assay

Journal: npj Viruses

Article Title: Respiratory syncytial virus glycoprotein G impedes CX 3 CR1-activation by CX 3 CL1 and monocyte function

doi: 10.1038/s44298-024-00075-9

Figure Lengend Snippet: A Dose-response curve showing CX 3 CR1 activation in Chem-4 cells stimulated with increasing concentrations of CX 3 CL1 (0, 1, 5, 10, 50, 100, 500 nM). Cells pretreated with 10 µM JMS-17-2 inhibitor served as controls, with subsequent stimulation using the same concentrations of CX 3 CL1. Fluorescence intensity, indicative of calcium mobilization, was measured over 180 s. Data represent mean values ± SD ( n = 6 from three independent experiments). B CX 3 CR1 activation in Chem-4 cells stimulated with recombinant RSV sG (WT or CX 3 C Mut ) at concentrations equivalent to those in ( A ). The red dotted line indicates the mean relative fluorescence units (RFU) in response to 500 nM CX 3 CL1 (as shown in ( A )). Data represent mean values ± SD (n = 3 from three independent experiments). p values were determined by Welsh t-tests compared as indicated. C Receptor competition assay with 150 nM CX 3 CL1 and varying molar ratios of RSV sG WT or RSV sG CX 3 C Mut (5:1, 1:1, 1:2, 1:10, 1:20 CX 3 CL1) compared to untreated cells. Fluorescence intensity was measured to assess calcium flux and receptor activation. Mean values ± SD are shown ( n = 6 from three independent experiments). p values were determined by Dunnett’s One-way ANOVA compared to untreated cells. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001.

Article Snippet: To assess the assay specificity, Chem-4 cells were additionally treated with 10 μM of the CX 3 CR1 inhibitor JMS-17-2 (MedChemExpress) or the corresponding concentration of DMSO for the generation of the dose response curve.

Techniques: Activation Assay, Fluorescence, Recombinant, Competitive Binding Assay

Journal: npj Viruses

Article Title: Respiratory syncytial virus glycoprotein G impedes CX 3 CR1-activation by CX 3 CL1 and monocyte function

doi: 10.1038/s44298-024-00075-9

Figure Lengend Snippet: A Flow cytometry analysis of CX 3 CR1 expression on THP-1 cells compared to HEK293 cells (negative control). B Quantification of THP-1 cell migration in response to 300 nM rBSA, 50 nM, 150 nM, and 300 nM RSV A rsG WT, 50 nM, 150 nM, and 300 nM RSV A rsG CX 3 C Mut , 50 nM, 150 nM, and 300 nM CX 3 CL1. Data represent mean values ± SD ( n = 6 from three independent experiments). Statistical significance was determined using One-Way ANOVA and Kruskal-Wallis tests compared to untreated cells. C THP-1 cell migration in response to 150 nM CX 3 CL1 in cells pretreated with 10 μM AZD8797, or equimolar ratios of rBSA, RSV A rsG WT, RSV A rsG CX 3 C Mut . Data represent mean values ± SD ( n = 6 from three independent experiments). Statistical significance was determined using One-Way ANOVA and Kruskal-Wallis tests compared to 150 nM CX 3 CL1 stimulation on untreated THP-1 cells. D Migration of THP-1 cells induced by supernatants from A549 cells treated with 300 nM or 150 nM RSV A rsG WT, 300 nM or 150 nM RSV A rsG CX 3 C Mut , 150 nM rBSA, or infected with RSV A 0594 at MOI 0.1, 1, or 3. Data represent mean values ± SD ( n = 6 from three independent experiments). Statistical significance was determined using One-Way ANOVA and Kruskal-Wallis tests compared to untreated cells. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001.

Article Snippet: To assess the assay specificity, Chem-4 cells were additionally treated with 10 μM of the CX 3 CR1 inhibitor JMS-17-2 (MedChemExpress) or the corresponding concentration of DMSO for the generation of the dose response curve.

Techniques: Flow Cytometry, Expressing, Negative Control, Migration, Infection

Journal: npj Viruses

Article Title: Respiratory syncytial virus glycoprotein G impedes CX 3 CR1-activation by CX 3 CL1 and monocyte function

doi: 10.1038/s44298-024-00075-9

Figure Lengend Snippet: A Immunofluorescence staining of A549 cells for CX 3 CL1 expression after 12-h treatment with 30 µg/mL LTA, 10 µg/mL LPS, 150 nM RSV A sG WT, 150 nM RSV A sG CX 3 C Mut , and infection with RSV A 0594 at MOI 1. CX 3 CL1-FITC antibody staining shows the level of CX 3 CL1 expression under each condition. B Quantification of monocyte attachment to A549 cells pretreated with 10 µg/mL LPS and subsequently exposed 7.5 µM AZD8797, 150 nM or 300 nM RSV A sG WT, 150 nM or 300 nM RSV A sG CX 3 C Mut , 150 nM or 300 nM rBSA. Monocyte attachment was assessed by counting calcein-positive THP-1 cells using an ImageJ automated cell counting macro adopted from ref. . Data represent mean values ± SD ( n = 27 from three independent experiments). Statistical significance was determined using Dunnett’s One-way ANOVA compared to untreated cells. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001.

Article Snippet: To assess the assay specificity, Chem-4 cells were additionally treated with 10 μM of the CX 3 CR1 inhibitor JMS-17-2 (MedChemExpress) or the corresponding concentration of DMSO for the generation of the dose response curve.

Techniques: Immunofluorescence, Staining, Expressing, Infection, Cell Counting

Journal: Frontiers in Immunology

Article Title: CX 3 CR1 fate mapping in vivo distinguishes cochlear resident and recruited macrophages after acoustic trauma

doi: 10.3389/fimmu.2025.1678176

Figure Lengend Snippet: Cre recombination efficiency in CX 3 CR1-lineage leukocytes in adult mouse cochlea and blood in CX 3 CR1 YFP−CreER/wt :R26 RFP mice. (A) Experimental regime. (B–I) Representative confocal images of cochlear whole mounts immunostained to label for (B, F) CX 3 CR1-expressing macrophages (YFP/GFP; green), (C, G) Cre recombination (RFP/tdTomato; red), (D, H) sensory hair cells (Myosin 7A; white), and (E, I) merged (yellow) at 60 days post-vehicle (corn oil) (B–E) or tamoxifen (F–I) injections. Scale bar = 130 µm. (J) Percentage of Cre recombination (RFP+) in YFP+ CX 3 CR1-expressing cochlear macrophages at 60 days after vehicle injections or at 2 and 60 days after tamoxifen injections. (K) Percentage of CD45+ leukocytes in the cochleae of vehicle- and tamoxifen-injected mice at 60 days post-injections. (L, M) Blood flow cytometry gating strategy (L) and quantification (M) of CX 3 CR1 lineage (CD45+, CD11b+, Ly6G−) (black box in panel L ). N = 3 mice per condition in (J, K, M) . Data in (J, K, M) are presented as mean ± SD. ***p < 0.001, ****p < 0.0001; ns, not significant; one-way ANOVA (J, M) or two-way ANOVA (K) .

Article Snippet: CX 3 CR1 YFP−CreER/YFP−CreER (stock no. 021160) and the Cre reporter mouse line, Rosa-lsl-tdTomato (R26 RFP ) (stock no. 007914) were purchased from Jackson Laboratories (Bar Harbor, ME, USA).

Techniques: Expressing, Injection, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: CX 3 CR1 fate mapping in vivo distinguishes cochlear resident and recruited macrophages after acoustic trauma

doi: 10.3389/fimmu.2025.1678176

Figure Lengend Snippet: Fate mapping in CX 3 CR1 YFP−CreER/wt :R26 RFP mice distinguishes CX 3 CR1-expressing resident macrophages (RM) and monocytes and monocyte-derived macrophages (Mo/Mo-M) in the neuronal region of the cochlea after acoustic trauma. (A) Experimental regime. (B) Representative confocal images of the spiral ganglion from tamoxifen-injected Cre mice after sham exposure (top) and at 7 days after acoustic trauma (bottom) shows the presence of only resident macrophages (yellow overlay) after sham exposure and resident macrophages (YFP+ RFP+; yellow overlay, white asterisks) and recruited monocyte-derived macrophages (YFP+ RFP; green overlay, white arrows) after acoustic trauma. Scale bar = 50 µm. (C, D) Quantification of resident and recruited macrophages in the apical, middle, and basal spiral ganglia (C) and osseous spiral lamina (D) on different days after exposure. Data in (C, D) are presented as mean ± SD from N = 3 mice per time point. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; one-way ANOVA comparing macrophage numbers at different days post-noise exposure with those of the sham group. Asterisks are color-coded based on resident (magenta) or recruited (green) macrophages, as shown in the graph legends.

Article Snippet: CX 3 CR1 YFP−CreER/YFP−CreER (stock no. 021160) and the Cre reporter mouse line, Rosa-lsl-tdTomato (R26 RFP ) (stock no. 007914) were purchased from Jackson Laboratories (Bar Harbor, ME, USA).

Techniques: Expressing, Derivative Assay, Injection

Journal: Frontiers in Immunology

Article Title: CX 3 CR1 fate mapping in vivo distinguishes cochlear resident and recruited macrophages after acoustic trauma

doi: 10.3389/fimmu.2025.1678176

Figure Lengend Snippet: CX 3 CR1-expressing resident macrophages (RM) and monocytes and monocyte-derived macrophages (Mo/Mo-M) in the spiral ligament, stria vascularis, and sensory epithelium of the cochlea after acoustic trauma. Representative confocal images of the spiral ligament (A) , stria vascularis (B) , and sensory epithelium (C, D) from tamoxifen-injected Cre mice after sham exposure (top) and at 7 days after acoustic trauma (bottom). White asterisks show YFP+ RFP+ resident macrophages (yellow overlay), and white arrows show YFP+ RFP− recruited monocyte/monocyte-derived macrophages (green overlay). Mo/Mo-M (green) are present in the lower spiral ligament as the primary site for macrophage accumulation (A) and around the sensory epithelium (D) , but not in the stria vascularis (B) of the noise-injured cochlea. Scale bars = 30 µm (A) , 5 µm (B) , 50 µm (C) , and 10 µm (D) .

Article Snippet: CX 3 CR1 YFP−CreER/YFP−CreER (stock no. 021160) and the Cre reporter mouse line, Rosa-lsl-tdTomato (R26 RFP ) (stock no. 007914) were purchased from Jackson Laboratories (Bar Harbor, ME, USA).

Techniques: Expressing, Derivative Assay, Injection

Journal: Frontiers in Immunology

Article Title: CX 3 CR1 fate mapping in vivo distinguishes cochlear resident and recruited macrophages after acoustic trauma

doi: 10.3389/fimmu.2025.1678176

Figure Lengend Snippet: Morphology of CX 3 CR1-expressing resident macrophages (RM) and monocytes and monocyte-derived macrophages (Mo/Mo-M). Representative confocal micrographs showing indistinguishable morphology of CX 3 CR1-expressing resident (YFP+ RFP+) and recruited (YFP+ RFP−, white arrows) macrophages in the spiral ganglion at 1 (A) and 7 (B) days post-noise exposure. The recruited circulating Mo were transformed from being round or less ramified at 1 DPNE to more ramified by 7 DPNE (white arrows). This suggests that the infiltrated circulating Mo may differentiate into cochlear RM. Scale bars = 17 µm (top panel) and 13 µm (bottom panel) in (A) and 20 µm in (B) .

Article Snippet: CX 3 CR1 YFP−CreER/YFP−CreER (stock no. 021160) and the Cre reporter mouse line, Rosa-lsl-tdTomato (R26 RFP ) (stock no. 007914) were purchased from Jackson Laboratories (Bar Harbor, ME, USA).

Techniques: Expressing, Derivative Assay, Transformation Assay

Journal: Frontiers in Immunology

Article Title: CX 3 CR1 fate mapping in vivo distinguishes cochlear resident and recruited macrophages after acoustic trauma

doi: 10.3389/fimmu.2025.1678176

Figure Lengend Snippet: Fate of CX 3 CR1-expressing resident macrophages (RM) and monocytes and monocyte-derived macrophages (Mo/Mo-M). Number of (A) cleaved caspase 3 (CC3) and (B) Ki67-positive macrophages in the apical, middle, and basal spiral ganglia on different days after noise exposure. Data are plotted as mean ± SD. N = 3–4 mice per recovery time. (C) Representative confocal micrographs showing Ki67-positive resident (YFP+ RFP+; white asterisks) and recruited (YFP+ RFP−; white arrows) macrophages in the spiral ganglion of the middle turn at 1 and 3 days post-noise exposure. Scale bars = 31 µm (top panel) and 70 µm (bottom panel) in (C) .

Article Snippet: CX 3 CR1 YFP−CreER/YFP−CreER (stock no. 021160) and the Cre reporter mouse line, Rosa-lsl-tdTomato (R26 RFP ) (stock no. 007914) were purchased from Jackson Laboratories (Bar Harbor, ME, USA).

Techniques: Expressing, Derivative Assay

Journal: Frontiers in Immunology

Article Title: CX 3 CR1 fate mapping in vivo distinguishes cochlear resident and recruited macrophages after acoustic trauma

doi: 10.3389/fimmu.2025.1678176

Figure Lengend Snippet: Working model of the ontogeny and dynamics of resident and recruited macrophages in the injured cochlea. Schematic illustrating disruption of the blood–labyrinth barrier (BLB), fibrinogen glycoprotein extravasation from blood, and subsequent recruitment of circulating monocytes (green) into the injured cochlea following an acute (2 hours) acoustic trauma imparting permanent hearing loss. The short-lived recruited monocytes may ultimately differentiate into macrophages [monocyte-derived macrophages (Mo-M)]. The long-lived cochlear resident macrophages (RM; magenta) undergo self-renewal and expansion in the injured cochlea. The overall increase in the number of macrophages in the acute noise-injured cochlea is attributed to the recruitment of blood-circulating monocytes, their prospective differentiation into macrophages, and the in situ proliferation of resident (local) macrophages. Defining the dynamics and the differential neuroprotective functions of CX 3 CR1-expressing cochlear resident and recruited macrophages in acute and chronic pathological cochleae is underway. The illustration is created in BioRender .

Article Snippet: CX 3 CR1 YFP−CreER/YFP−CreER (stock no. 021160) and the Cre reporter mouse line, Rosa-lsl-tdTomato (R26 RFP ) (stock no. 007914) were purchased from Jackson Laboratories (Bar Harbor, ME, USA).

Techniques: Disruption, Derivative Assay, In Situ, Expressing