Journal: bioRxiv

Article Title: Neutrophil-microglia interaction drives reversible motor dysfunction in neuromyelitis optica model induced by subarachnoid AQP4-IgG

doi: 10.1101/2025.08.22.671883

Figure Lengend Snippet: A and B. In the spinal grey matter of AQP4-IgG-recipient mice, C5a-immunoreactivity was not seen in GFAP + astrocytes (A) and rarely seen in IBA1 + microglia/macrophage (B). The C5 proenzyme was seen only in luminal plasma, not in any CNS parenchymal cells . C. Triple immunostaining of lumbar cord of AQP4-IgG recipient mice revealed C5a in neutrophils (MPO+, left) and monocytes (CCR2-GFP + , enhanced by anti-GFP-IgG, right). D. The percentage of spinal cord cells expressing C5a immunoreactivity in AQP4-IgG-infused mice (determined by Image J) ( n =4 mice in each group). E. Colocalization analysis of C5a and MPO signals in C using Zen software (LSM980). F. After transcardiac washout of vasculature, lumbar cords from IgG-infused mice were dissociated enzymatically and subjected to high parametric flow cytometric analysis (Cytek). G. tSNE maps identifying CD11b + , Ly6G + , Cx3cr1 + and C5aR1 + cells and their expression levels among CD45 + immune cells in the spinal cord, revealed that C5aR1 is expressed predominantly in microglial and neutrophil subsets. H. Public database ( Brain RNA-Seq ) documents that C5aR1 mRNA in normal mouse brain is predominantly expressed in microglia and macrophages. I. Flow cytometric plot shows that, after 3 days of AQP4-IgG infusion, greater numbers of neutrophils (Ly6G + cells) infiltrate the lumbar parenchyma in WT mice than in C5aR1 deficient mice. Percentages are quantified in the bar graph ( n = 4 mice in each group). J. Quantification of the percentage of infiltrating neutrophils (CD45 + CD11b + Ly6G + Ly6C − cells among CD45 + CD11b + cells) in spinal cords of wild-type and C5aR1 −/− mice at day 3 of AQP4-IgG infusion. K. Motor function, assessed by Rotarod performance (latency to fall), in WT and C5aR1 −/− mice infused with AQP4-IgG or (only C5aR1 −/− mice) normal mouse IgG (0.1 μg/μL, n = 6 mice per group). L. Motor function of mice assessed as fall latency from Rotarod; (time: F (8, 48) = 1.543, p =0.1675; treatment: F (1, 6) = 14.90, p = 0.0084; interaction: F (8, 39) = 2.070, p =0.0629; n = 6-7 mice per group). In all graphs, data represent means ± SEM and all statistical tests are two-sided. One-way ANOVA, followed by Tukey’s post hoc multiple comparisons test in D. Unpaired Student t -test in J. Two-way repeated measures ANOVA with Sidak post hoc test in K. p < 0.05 was considered a significant difference.

Article Snippet: Trifluoroacetate was removed from the cyclic hexapeptide C5aR1 antagonist PMX 205 (HY-110136 A; MedChemExpress, NJ, USA) by repeated hydrochloric acid exchange followed by lyophilization .

Techniques: Clinical Proteomics, Triple Immunostaining, Expressing, Software, RNA Sequencing

Journal: bioRxiv

Article Title: Neutrophil-microglia interaction drives reversible motor dysfunction in neuromyelitis optica model induced by subarachnoid AQP4-IgG

doi: 10.1101/2025.08.22.671883

Figure Lengend Snippet: A. Representative confocal images from lumbar cord of WT and C5aR1 -deficient mice infused with normal IgG or AQP4-IgG. Lysosomal CD68-immunoreactivity (red) is more abundant in microglia (green) of WT recipients of AQP4-IgG than in C5aR1 −/− recipients. Imaris 3D rendering images illustrate the magnitude of lysosomal expansion. B. Image J analysis of the percentage area occupied by lysosome inside microglia in the lumbar cord of different experimental groups (Treatment: F (1, 20) = 145.8, p < 0.0001; genotype: F (1, 20) = 1.144, p = 0.2975; n = 5-6 mice per group). C. Sholl analysis of microglial branching revealed by Imaris AI-powered filament tracing which counts the number of microglial filaments intersected by 1 μm spherical steps (Treatment: F (77, 3730) = 39.35, p < 0.0001; Radius: F (3, 95) = 17.02, p < 0.0001; n = 19-32 microglia from 5 mice per group). In all graphs, data represent means ± SEM and all statistical tests are two-sided. Two-way (treatment × genotyping) ANO VA with Sidak post hoc multiple comparisons test in B. Two-way repeated measures ANOVA with Sidak post hoc test in C. p < 0.05 was considered significant difference.

Article Snippet: Trifluoroacetate was removed from the cyclic hexapeptide C5aR1 antagonist PMX 205 (HY-110136 A; MedChemExpress, NJ, USA) by repeated hydrochloric acid exchange followed by lyophilization .

Techniques:

Journal: bioRxiv

Article Title: Neutrophil-microglia interaction drives reversible motor dysfunction in neuromyelitis optica model induced by subarachnoid AQP4-IgG

doi: 10.1101/2025.08.22.671883

Figure Lengend Snippet: A. Experimental design: WT mice were infused continuously with AQP4-IgG using osmotic pumps from day 0 through day 7; infusion was stopped on day 8. B-D. Quantification of Nissl bodies (B), Nissl area (C) and percentage area occupied by ChAT + motor neurons in grey matter of lumbar cord of Ctrl-IgG-infused mice at day 3, AQP4-IgG-infused mice at days 3, 14 and 28. Ctrl: (Nissl body count 251.5± 85.7/mm 2 , Ctrl Nissl body area: 4.928± 0.6 mm 2 , Ctrl ChAT signal area: 14.83 ± 1.8 mm 2 ); Day 3: (Nissl body count 165.8 ± 21.6/mm 2 , Nissl body area 3.0 ± 0.4 mm 2 , ChAT signal area: 4.1 ± 0.8 mm 2 ); Day 28: (Nissl body count: 278.8 ± 33.08/mm 2 , Nissl body area: 4.786 ± 0.6 mm 2 , ChAT signal area: 9.517 ± 1.8 mm 2 ). E-H. Representative immunostained images of Nissl bodies (red) in ventral gray matter (vGM) neurons of wild-type and C5aR1 −/− mice after 3 days’ Ctrl-IgG or AQP4-IgG infusion. Boxed areas are enlarged on the right. I, J. Numbers and sizes of Nissl body-positive neurons in ventral gray matter ( n = 8 mice per group). K. Representative motor neuron confocal images and quantification in ventral gray matter of WT mice infused with Ctrl-IgG or AQP4-IgG, and C5aR1 −/− mice infused with AQP4-IgG or control IgG (not shown). ANNA1[HuD] + (green); ChAT + (magenta). L, M. Numbers of ChAT + motor neurons (MNs) and their percentage among total neurons (ANNA1 [HuD] + in vGM. ( n = 4 mice per group). N. Representative images of BODIPY + lipid droplets (green) in ChAT + motor neurons (grey) and NeuN + neurons (grey) in ventral gray matter of AQP4-IgG-infused WT and C5aR1 −/− mice. 3D reconstruction of BODIPY + lipid and NeuN + neurons using Imaris (right). O. Quantification of lipid droplet numbers in the cytoplasm of NeuN + neurons in ventral gray matter of WT and C5aR1 deficient mice infused for 3 days with AQP4-IgG ( n = 20 neurons from 4 individual mice per group). P. Representative confocal images of 4HNE (peroxidative stress marker) in ChAT + motor neurons of WT and C5aR1 −/− mice infused with AQP4-IgG. Q and R. Q, Quantification of 4HNE and ChAT immunoreactivity intensities across 30 μm neuronal diameter; R, presented as mean ±SEM ( n =20 neurons from 4 individual mice per group). All data represent means ± SEM, all statistical tests are two-sided. Two-way (treatment × genotyping) ANOVA with Sidak post hoc test multiple comparisons test in I and J. One-way ANOVA with Tukey post hoc in B-D, L and M. Unpaired Student t -test in O and R. p < 0.05 was considered significant difference.

Article Snippet: Trifluoroacetate was removed from the cyclic hexapeptide C5aR1 antagonist PMX 205 (HY-110136 A; MedChemExpress, NJ, USA) by repeated hydrochloric acid exchange followed by lyophilization .

Techniques: Control, Marker

Journal: Bone Research

Article Title: Transcriptional reprogramming during human osteoclast differentiation identifies regulators of osteoclast activity

doi: 10.1038/s41413-023-00312-6

Figure Lengend Snippet: C5AR1, SSTR2 and GPR120/FFAR4 activation modulates osteoclastogenesis and/or resorptive activity of mature osteoclasts. a Box plot of RNA-seq-based gene expression levels of complement 5A receptor 1 ( C5AR1 ). b UMAP plot of C5AR1 gene expression in differentiated human osteoclasts at the single-cell level. c Box plot quantifying osteoclast numbers and nuclei per osteoclast after 9 days of osteoclast differentiation in the presence of the C5AR1 agonist BM221 (1 µg/mL) and/or antagonist PMX205 (5 µg/mL) ( n = 6). d Box plot quantifying osteoclast resorptive activity as the percentage of eroded surface per bone surface for mature osteoclasts (Day 9) after 72 h on bovine bone slices in the presence of the C5AR1 agonist BM221 (1 µg/mL) and/or antagonist PMX205 (5 µg/mL) ( n = 6). e Box plot of the RNA-seq-based gene expression levels of free fatty acid receptor 4 ( FFAR4 ). f Box plot quantifying IP-1 levels in an HTRF assay, showing that the specific FFAR4 agonist TUG-891 increases IP-1 levels in mature osteoclasts (Day 9) after 30 min ( n = 6). g Box plot quantifying osteoclast numbers and nuclei per osteoclast after 9 days of osteoclast differentiation in the presence of the FFAR4 agonist TUG-891 (10 nmol/L) ( n = 4). h Box plot quantifying osteoclast resorptive activity as the percentage of eroded surface per bone surface for mature osteoclasts (Day 9) after 72 h on bovine bone slices in the presence of the FFAR4 agonist TUG-891 (10 nmol/L) ( n = 5). i Box plot of RNA-seq-based gene expression levels of somatostatin receptor 2 ( SSTR2 ). The inlet highlights the expression at Days 5 and 9. Lines represent individual donors. j Box plot quantifying the cAMP-Glo assay. k Box plot quantifying osteoclast numbers and nuclei per osteoclast after 9 days of osteoclast differentiation in the presence of the SSTR2 agonist somatostatin (100 nmol/L) and octreotide (10 nmol/L) and antagonism by BIM-23627 (100 nmol/L) ( n = 4). l Box plot quantifying osteoclast resorptive activity as a percentage of eroded surface area per bone surface for mature osteoclasts (Day 9) after 72 h on bovine bone slices in the presence of the SSTR2 agonists somatostatin (100 nmol/L) and octreotide (10 nmol/L) and antagonism by BIM-23627 (100 nmol/L) ( n = 6). m Box plot of real-time PCR-based gene expression levels of SSTR2 relative to TBP in osteoclasts on Day 9 of differentiation ( n = 6). n Box plot quantifying osteoclast resorptive activity after 72 h on bovine bone slices in the presence of the SSTR2 agonist somatostatin (100 nmol/L) as a percentage of the eroded surface area per bone surface ( n = 6). o Scatter plot quantifying the percentage change in bone resorption following SSTR2 knockdown (vehicle values from Fig. : (siCTR – siSSTR2)/siCTR) relative to changes in SSTR2 expression (values from Fig. : siCTR – siSSTR2) ( n = 6). p Scatter plot quantifying the percentage change in bone resorption in response to somatostatin (values from Fig. : (Veh – somatostatin)/Veh) over relative SSTR2 expression (values from Fig. ) ( n = 6). The small scatterplot was used to quantify the delta of the x and y values of siCTR and si SSTR2 , and the big scatterplot P value was calculated based on a linear regression model

Article Snippet: The following compounds were used at the following concentrations according to the literature and purchased from the following companies: BM221 (ref. ) (0.2 µg/mL, 1 µg/mL and 10 µg/mL; Product No. GLXC-24851; Glixx Laboratories, Inc., MA, USA); PMX205 (ref. ) (5 µg/mL and 15 µg/mL; Product No. 5196; Bio-Techne Tocris, MN, USA); somatostatin-14 (ref. ) (1, 10 and 100 nmol/L; Product No. S9129; Merck, London, UK); octreotide (1 and 10 nmol/L; Product No. O1014; Merck, London, UK); BIM-23627 (ref. ) (1, 10 and 100 nmol/L; Product No. 4041642; Bachem, Bubendorf, CH); and TUG-891 (ref. , ) (0.1, 1 and 10 μmol/L; Product No. SML1914; Merck, London, UK).

Techniques: Activation Assay, Activity Assay, RNA Sequencing Assay, Expressing, HTRF Assay, Glo Assay, Real-time Polymerase Chain Reaction, Knockdown

Journal: Medical Microbiology and Immunology

Article Title: Exploration of compounds to inhibit the Panton-Valentine leukocidin of Staphylococcus aureus

doi: 10.1007/s00430-024-00803-1

Figure Lengend Snippet: Half-maximal effective concentrations (EC 50 ) of Panton-Valentine leukocidin (PVL) at different concentrations of potential competitors

Article Snippet: These compounds were avacopan/CCX168 (C5aR antagonist, Thermo Fisher Scientific, Waltham, MA, USA), BM213 (C5aR agonist, MedChemExpress, Monmouth Junction, NJ, USA), DF2593A (C5aR antagonist, Sigma-Aldrich, St. Louis, MO, USA), JPE-1375 (C5aR antagonist, MedChemExpress), PMX205 (C5aR antagonist, Hycultec, Beutelsbach, Germany), W-54,011 (C5aR antagonist, MedChemExpress) as well as NQ301 (CD45 inhibitor, MedChemExpress).

Techniques: Concentration Assay, Control