Journal: Frontiers in Immunology

Article Title: Patrolling monocytes mediate virus neutralizing IgG effector functions: beyond neutralization capacity

doi: 10.3389/fimmu.2025.1600056

Figure Lengend Snippet: Macrophages and inflammatory monocytes are dispensable for nAbs-mediated LCMV control. (A) Flow cytometric analysis of monocytes subsets, CD45 + Lin - (CD45R + CD8 + CD4 + NK + ), CD11b + Ly6G - , in the peripheral blood of MC21 or isotype (rat IgG2a)-treated mice 3 days pot infection (d.p.i). Mice were infected with LCMV-WE (2x10 5 PFU) on day 0, preceded by treatment with 25 µg MC21 or isotype (rat IgG2b) on day -1 and again on day 1. See ( Supplementary Figure S2 ) for gating strategy of the monocyte subsets. (B) Numbers of patrolling monocytes (-Mo), inflammatory monocytes (-Mo), neutrophils, and lymphocytes in the peripheral blood of MC21 or isotype (rat IgG2a)-treated mice 3 d.p.i. Results show the pooled data from two independent experiments with similar results. (n=3–5 mice/group/experiment). (C) Virus titres (3 d.p.i) in spleen of WT mice infected with LCMV-WE (2x10 5 PFU) on day 0 and treated on day 1 with Wen3 (350 µg) or isotype (IgG2a), On day -1 and day 1 mice were treated with 25 µg MC21 or isotype (rat IgG2b). Results show the pooled data from two independent experiments with similar results. (n=3–5 mice/group/experiment). (D) Schematic presentation of the experiments in (E-G) . (E) Numbers of patrolling monocytes (-Mo), inflammatory monocytes (-Mo), neutrophils, and lymphocytes in the peripheral blood and spleen (3 d.p.i.) of mice treated with 10 µL/g bodyweight control or clodronate liposomes one day before LCMV infection (2x10 5 PFU). Results show the pooled data from two independent experiments with similar results. Experiment was repeated three times with similar results (n=4–5 mice/group/experiment). (F) Shown are the virus titres (3 d.p.i.) in spleen of WT mice infected with LCMV-WE (2x10 5 PFU) on day 0 and treated on day 1 with Wen3 (350 µg) or isotype (IgG2a). On day -1 mice were treated with 10µL/g bodyweight control or clodronate liposomes. Results show the pooled data from two independent experiments with similar results. Experiment was repeated three time with similar results (n=3–5 mice/group/experiment). (G) Spleen sections collected 3 d.p.i. were stained for F4/80 (blue), CD169 (green), and LCMV nucleoprotein (−NP) (red). Scale bar 300 µm. Shown are representative pictures from three independent experiments (n=2–4 mice/group/experiment). Statistical analysis was performed by using the One-way ANOVA test (C, F) or Student’s t-test (B, E) . *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.001, ns, non-significant. Horizontal dotted lines indicating the detection limit.

Article Snippet: Depletion antibodies utilized were CD115 (Clone: AFS98), Ly6G (Clone: 1A8), NK (Clone: PK136), all procured from BioXcell, and each was administered at a dosage of 200 μg per mouse (except CD115 100 μg).

Techniques: Control, Infection, Virus, Liposomes, Staining

Journal: Frontiers in Immunology

Article Title: Patrolling monocytes mediate virus neutralizing IgG effector functions: beyond neutralization capacity

doi: 10.3389/fimmu.2025.1600056

Figure Lengend Snippet: Contribution of patrolling monocytes for antiviral activity of Wen3. (A) Schematic presentation of the experiments in (B-E) . (B) Flow cytometric analysis of monocyte subsets in the peripheral blood and spleen of control or clodronate liposomes-treated mice 3 days post infection (d.p.i.). Animals were treated with 0.5 µL/g control or clodronate liposomes (Clo. (low) ) on day -1 and 1. Mice were infected with LCMV-WE (2x10 5 PFU) on day 0. See ( Supplementary Figure S2 ) for gating strategy of the monocyte subsets. (C) Numbers of patrolling monocytes (-Mo), inflammatory monocytes (-Mo), neutrophils, and lymphocytes/macrophages in the peripheral blood and spleen of control or clodronate liposome-treated mice 3 d.p.i. Animals were treated with 0.5µL/g control or clodronate liposomes (Clo. (low) ) on day -1 and 1. mice were infected with LCMV-WE (2x10 5 PFU) on day 0. Results show the pooled data from two independent experiments with similar results. (n=3–5 mice/group/experiment). (D) Virus titres (3 d.p.i.) in spleen of WT mice infected with LCMV-WE (2x10 5 PFU) on day 0 and treated on day 1 with Wen3 (350 µg) or isotype (IgG2a), On day -1 and day 1 mice were treated with 0.5µL/g bodyweight control or clodronate liposomes (Clo. (low) ). Results show the pooled data from three independent experiments with similar results (n=3–4 mice/group/experiment). (E) Spleen sections collected 3 d.p.i. were stained for CD169 (green) and LCMV nucleoprotein (−NP) (red). Shown are the representative merged images from three independent experiments. Scale bar= 300 µm. (F) Virus titres (3 d.p.i.) in spleen of WT mice infected with LCMV-WE (2x10 5 PFU) on day 0 and treated on day 1 with neutralizing serum (Neutralizing S.) or naïve serum (see methods). On day -1 and day 1 mice were treated with 0.5µL/g control or clodronate liposomes (Clo. (low) ). Results show the pooled data from two independent experiments with similar results (n=3–5 mice/group/experiment). (G) Numbers of patrolling monocytes (-Mo), inflammatory monocytes (-Mo), neutrophils, along with lymphocytes in the peripheral blood or macrophages in spleen (3 d.p.i.) of mice treated with anti-CD115 or Isotype. Mice were infected with LCMV-WE (2x10 5 PFU) on day 0, preceded by treatment with 100 µg anti-CD115 or isotype (rat IgG2b) on day -1 and again on day 1. Results show the pooled data from two independent experiments with similar results (n=3 mice/group/experiment). (H) Shown are the virus titres (3 d.p.i.) in spleen of WT mice infected with LCMV-WE (2x10 5 PFU) on day 0 and treated on day 1 with Wen3 (350 µg) or isotype (IgG2a). On day -1 and day 1 mice were treated with 100 µg anti-CD115 or isotype. Results show the pooled data from two independent experiments with similar results (n=3 mice/group/experiment). (I) Shown are the virus titres (3 d.p.i.) in spleen of CD11c-cre +/+ /iDTR and CD11c-cre tg/+ /iDTR mice infected with LCMV-WE (2x10 5 PFU) on day 0 and treated on day 1 with Wen3 (350 µg) or isotype (IgG2a), on day -1 and day 1 mice were treated with diphtheria toxin 12ng/g bodyweight. Results show the pooled data from two independent experiments with similar results (n=3–4 mice/group/experiment). (J) Shown are the virus titres (3 d.p.i.) in spleen of WT mice infected with LCMV-WE (2x10 5 PFU) on day 0 and treated on day 1 with Wen3 (350 µg) or isotype (IgG2a), mice were treated with 200 µg 9E9 or isotype for blocking FcγRIV. Results show the pooled data from three independent experiments with similar results (n=3–4 mice/group/experiment). (K) Heat map showing the expression of FcγRIV on CD11b + Ly6G - population gated from CD45 + Lin - (CD45R + CD8 + CD4 + ), 24 hour following infection with LCMV. Statistical analysis was performed by using the Student’s t-test (C, G) or One-way ANOVA test (D, F, H–J) . *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.001, ns, non-significant. Horizontal dotted lines indicating the detection limit.

Article Snippet: Depletion antibodies utilized were CD115 (Clone: AFS98), Ly6G (Clone: 1A8), NK (Clone: PK136), all procured from BioXcell, and each was administered at a dosage of 200 μg per mouse (except CD115 100 μg).

Techniques: Activity Assay, Control, Liposomes, Infection, Virus, Staining, Blocking Assay, Expressing

Journal: JCI Insight

Article Title: Combination treatment with anti-RANKL and antibiotics for preventing joint destruction in septic arthritis

doi: 10.1172/jci.insight.184954

Figure Lengend Snippet: ( A ) The protective effect of neutrophils was examined by depleting mice of neutrophils ( n = 10/group) using the anti-Ly6G antibody (Neutrophil depletion) or an isotype control (Controls). Knee swelling was measured in millimeters over a period of 7 days after intra-articular (i.a.) injection of 20 μL of PBS containing S . aureus LS-1 strain (4 × 10 3 CFU/knee) into the knee joints of NMRI mice. ( B ) Bacterial counts in the mouse knee joints ( n = 5/group) were assessed. ( C ) Bone erosion scores of the knee joints ( n = 5/group) were determined after micro-CT (μCT) scan. ( E ) Cumulative survival was monitored daily. ( F ) The pathogenic role of monocytes was investigated by depleting mice of monocytes ( n = 22/group) using clodronate liposomes (Monocyte depletion) or PBS control liposomes (Controls), and knee swelling was followed for 10 days after infection using the same strategy. ( G ) Bacterial counts in mouse knee joints in the monocyte depletion group ( n = 5) and controls ( n = 6). ( H ) Bone erosion scores for the monocyte depletion group ( n = 7) and controls ( n = 16) and ( I ) cumulative survival. ( D ) Representative μCT images of knee joints from the control, neutrophil depletion, and monocyte depletion groups. Arrows indicate bone erosion. The data were pooled from 2 independent experiments. Statistical evaluations were performed using the Mann-Whitney test ( A – C and F – H ) or log-rank (Mantel-Cox) test ( E and I ). Data are presented as mean with SEM. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: Selective depletion of murine blood neutrophils was accomplished using the anti-Ly6G monoclonal antibody (clone 1A8; BioXCell), as previously documented ( ).

Techniques: Control, Injection, Micro-CT, Liposomes, Infection, MANN-WHITNEY

Journal: JCI Insight

Article Title: Combination treatment with anti-RANKL and antibiotics for preventing joint destruction in septic arthritis

doi: 10.1172/jci.insight.184954

Figure Lengend Snippet: Knee joints from NMRI mice, collected on day 3 after intra-articular (i.a.) injection of 20 μL of PBS or Lpl1(+sp) (4 μg/knee) or PBS containing S . aureus LS-1 strain (4 × 10 3 CFU/knee), underwent anti-CD68 immunohistochemistry examination ( A – C ). Scale bar: 100 μm. Lpl1(+sp), S . aureus intact Lpp. Knee synovial tissues from mice that were injected i.a. with 20 μL of PBS ( n = 4) or PBS containing S . aureus LS-1 strain (4 × 10 3 CFU/knee, n = 6) were analyzed on day 3 after injection using flow cytometry. t-Distributed stochastic neighbor embedding (tSNE) analysis of monocyte subsets based on Ly6C expression with gating on CD11b + CD45 + Ly6G – population was conducted ( D ). Representative mean fluorescence intensity (MFI) histograms of monocyte subsets for RANK, c-Fms, and CCR2 are shown ( E ). Statistical analyses of RANK, c-Fms, and CCR2 levels are presented ( F ). Statistical evaluations were performed using 1-way ANOVA with Holm-Šídák multiple-comparison test, with data presented as mean with SEM. * P < 0.05; ** P < 0.01; **** P < 0.0001.

Article Snippet: Selective depletion of murine blood neutrophils was accomplished using the anti-Ly6G monoclonal antibody (clone 1A8; BioXCell), as previously documented ( ).

Techniques: Injection, Immunohistochemistry, Flow Cytometry, Expressing, Fluorescence, Comparison

Journal: Cell Genomics

Article Title: Spatial transcriptomics identifies novel Pseudomonas aeruginosa virulence factors

doi: 10.1016/j.xgen.2025.100805

Figure Lengend Snippet: Non-biased whole-mouse transcriptome analysis of P. aeruginosa -infected tissues reveals differential enrichment for host transcripts depending on the anatomical site (A) A schematic of the workflow. Ocular tissues were harvested 24 h post challenge with 1 × 10 6 CFUs of PAO1. Tissues were paraffin embedded, sectioned, stained for morphological markers, and hybridized with the whole host transcriptome probe library. ROIs were selected to encompass the epithelial layer, stromal layer, and AC of the eye. Probes were cleaved post hybridization, collected with GeoMx, and amplified, and the generated libraries were sequenced. The image was generated with BioRender. (B) Representative immunohistochemical images of noninfected (contralateral control eyes) and P. aeruginosa -infected ocular tissues stained for cytokeratin (green), Ly6G (red), DNA (blue), and c-KIT (yellow) were visualized with GeoMx (scale bars, 300 μm; n = 2 biological replicas). Robust neutrophil infiltration (red) in the corneal stroma and epithelium is readily observed in the infected site. (C) Volcano plots illustrating differentially expressed transcripts in epithelium and stroma tissue layers of the eye following infection compared to tissues of the non-infected contralateral eye 24 h post challenge. Data are presented cumulatively and derived from biological replicates. The analyzed tissue segments include 16 epithelial ROIs and 16 stromal ROIs collected from 2 biological replicas with 2 technical replicas each. The control contralateral ocular tissue analysis included 8 epithelial ROIs and 8 stromal ROIs from 2 technical replicas. The x axis indicates fold change (log2 scale), and the y axis shows the statistical significance (−log10 false discovery rate [FDR]-adjusted p values). Sample metadata are included. (D) Bar plot of GO (Gene Ontology) functional enrichment analysis, highlighting tissue site-specific gene transcript clustering at level 7. The x axis displays the FDR-adjusted p values (−log10 scale), indicating the statistical significance of the enrichment. The y axis lists the level 7 GO categories with enriched transcripts. This analysis incorporates data from ROI profiles comprising 16 epithelial and 16 stromal samples from ocular tissues harvested 24 h post challenge ( n = 2 biological replicas, n = 2 technical replicas per biological sample) and 8 epithelial and 8 stromal samples from control samples ( n = 2, technical replicas). Data are presented cumulatively and are derived from biological replicates. GO enrichment analysis is listed in Table S1 .

Article Snippet: Ly6G , Novus Biologicals , Clone: 1A8 NB253131AFD PRID:AB2572720.

Techniques: Infection, Staining, Hybridization, Amplification, Generated, Immunohistochemical staining, Control, Derivative Assay, Functional Assay

Journal: Cell Genomics

Article Title: Spatial transcriptomics identifies novel Pseudomonas aeruginosa virulence factors

doi: 10.1016/j.xgen.2025.100805

Figure Lengend Snippet: Bacterial transcript profiling of P. aeruginosa -infected corneas reveal differential tissue enrichment (A) Heatmap showing the distribution of 100 bacterial transcripts in corneas infected with the P. aeruginosa ExoS-expressing strain PAO1. Each column represents data from individual ROIs. Data are presented cumulatively and derived from measurements of 2 biological and 2 technical replicas. Surface epithelial ROIs ( n = 14 ROIs, N = 2 biological replicas, n = 2 technical replicas per biological sample) (green), stromal ROIs ( n = 16 ROIs, n = 2 biological replicas, n = 2 technical replica per biological sample) (yellow), and anterior chamber (AC) ROIs ( n = 16, n = 2 biological replicas, n = 2 technical replicas per biological sample) (purple) are grouped together. The curved lines at the top of the heatmap connect juxtaposing ROIs. (B) Immunohistochemistry analysis of infected corneal sections stained for cytokeratin (green) and Ly6G+ neutrophils (red). Scale bar, 0.5 mm. Also shown is an overlay plot of host and bacterial transcript levels in PAO1-infected tissues. Logarithmic transcript abundances (log2(count + 1)) of host TNF-α and bacterial PA2590, F fpvB , rplL , and a AlgE are represented as circles, where the diameter reflects transcript numbers in the individual ROIs. (C) Bacterial enrichment was calculated as relative transcript abundance in the surface segments compared to the stromal segments. Bacterial transcript levels were normalized to housekeeping bacterial rRplL transcripts. FDR values and fold change (FC) are annotated per gene. Representative bacterial enrichment data depict (1) transcripts that were enriched in the stroma fpvB , PA2590, and femA ; (2) transcripts showing no enrichment; and (3) transcripts showing decreased abundance, such as algE and piv . Complete enrichment analysis is provided in Table S4 .

Article Snippet: Ly6G , Novus Biologicals , Clone: 1A8 NB253131AFD PRID:AB2572720.

Techniques: Infection, Expressing, Derivative Assay, Immunohistochemistry, Staining

Journal: Cell Genomics

Article Title: Spatial transcriptomics identifies novel Pseudomonas aeruginosa virulence factors

doi: 10.1016/j.xgen.2025.100805

Figure Lengend Snippet: Bacterial transcript profiling of PA14-infected corneas (A) Heatmap showing the distribution of 100 bacterial transcripts in uninfected and PA14-infected eyes. The sham (uninfected) and infected tissues were harvested and processed 48 h post challenge. Each column represents data from an individual ROI. Transcript abundance was compared between surface epithelial sham ROIs ( n = 6 ROIs and n = 2 technical replicas) (green), stromal uninfected ROIs ( N = 4 and N = 2 technical replicas) (yellow), and infected surface (epithelial) ROIs ( N = 11 ROIs consisting of 2 or 3 surface ROIs per ocular section, n = 3 biological replicas and up to 2 technical replicas per biological sample) (green), infected stromal ROIs ( n = 11 ROIs comprising 2 or 3 surface ROIs per ocular section, n = 3 biological replicas and up to 2 technical replicas per biological sample) (yellow), and infected AC ROIs ( n = 8 consisting of 2 AC ROIs per ocular section and n = 2 biological replicas with 2 technical replicas per biological sample) (purple) are grouped together. (B) Bacterial enrichment was calculated as relative transcript abundance in the surface (epithelial) segments compared to the stromal segments. Bacterial transcript levels were normalized to the housekeeping bacterial rplL transcripts. FDR values and FC are annotated for fpvB , PA2590, and femA . Complete enrichment analysis is provided in Table S5 . (C) Immunohistochemistry analysis of uninfected and PA14-infected corneal sections stained for OprI (yellow), cytokeratin (green), Ly6G+ neutrophils (red), and DNA (blue). Scale bar, 0.1 mm. Arrows indicate bacterial invasion from the surface lesion into the corneal stroma. Data are representative from sham ( n = 3) and infected tissue ( n = 11) biological replicates. (D) Venn diagram depicting differentially present and shared bacterial transcripts that are enriched within the PAO1- and PA14-infected tissues. FDR values ≤ 0.1.

Article Snippet: Ly6G , Novus Biologicals , Clone: 1A8 NB253131AFD PRID:AB2572720.

Techniques: Infection, Immunohistochemistry, Staining

Journal: Cell Genomics

Article Title: Spatial transcriptomics identifies novel Pseudomonas aeruginosa virulence factors

doi: 10.1016/j.xgen.2025.100805

Figure Lengend Snippet:

Article Snippet: Ly6G , Novus Biologicals , Clone: 1A8 NB253131AFD PRID:AB2572720.

Techniques: Virus, Recombinant, Software