Journal: bioRxiv

Article Title: SARM1 is required for macrophage immunophenotype switching that is essential for nerve repair

doi: 10.64898/2026.04.07.716973

Figure Lengend Snippet: ( A ) Representative still images from live cell imaging sequences following a 2D scratch assay of WT and Sarm1-/- stimulated Mɸ. t=0 is immediately after the scratch and t=48 hours is 48 hours post-scratch. Scratch boundaries are indicated by yellow dashed lines. Scale bar = 300µm. ( B,C ) Quantification of the relative wound density of WT ( B ) or Sarm1-/- ( C ) Mɸ cultures after the scratch assay in A represented as a percentage of t=0. N=3 biological replicates. Error = SEM. *p<0.05; **p<0.01; ***p<0.005; letters (a,b,c) p<0.05. by two-way repeated measures ANOVA with Dunnett post-hoc test. Blue asterisk = mCSF compared to t=0. Green asterisk = IL-4 compared to t=0. Orange asterisk = LPS compared to t=0. a = mCSF compared to IL-4; b = mCSF compared to LPS; c = mCSF compared to IFNɣ conditions at indicated timepoints. ( D ) Representative still images from live cell imaging sequences following a 3D invasion assay in Matrigel of WT and Sarm1-/- stimulated Mɸ. t=0 immediately after the scratch and t=48 hours is 48 hours post-scratch. Scratch boundaries are indicated by yellow dashed lines. Scale bar = 300µm. ( E ) Quantification of the relative wound density of the invasion assay without C5a added (top) and with C5a added as a chemoattractant (bottom) represented as a percentage of t=0. N=2-4 biological replicates. Error = SEM. Letters (b,c) p<0.05; **p<0.01; ***p<0.005; ****p<0.001 by two-way ANOVA, main effects model with Tukey post-hoc test after simple linear regression model indicated slopes were not different. Wildtype Mɸ + C5a main effects could not be calculated because slopes were significantly different. Orange asterisk = LPS compared to t=0. b = mCSF compared to LPS; c = mCSF compared to IFNɣ conditions at indicated timepoints. ( F ) Representative images of F4/80 (green) positive Mɸ stained for Oil Red O lipids (magenta) following a myelin challenge assay. No clearance indicates a 24 hour myelin treatment and clearance indicates 24 hours of myelin treatment followed by myelin removal for 24 hours. Scale bar = 100µm. ( G ) Quantification of OilRedO intensity per Mɸ (normalized to Mɸ cell area). N = 3 biological replicates and each data point = 1 Mɸ. Mean +/- SEM is shown. Outliers were removed with ROUT Q=1. **p<0.01; ***p<0.005; ****p<0.001 by Kruskal-Wallis test with Dunn’s correction for multiple comparisons. Black asterisk = WT to Sarm1-/- comparisons. Purple asterisk = Sarm1-/- to Sarm1-/- comparisons. Blue asterisk = WT to WT comparisons.

Article Snippet: Additional media with or without recombinant mouse complement component C5a (R&D system, #2150-C5/CF) was added after incubation.

Techniques: Live Cell Imaging, Wound Healing Assay, Invasion Assay, Staining

Journal: The Journal of Clinical Investigation

Article Title: Urine proteins reveal distinct coagulation and complement cascades underlying acute versus chronic lupus nephritis

doi: 10.1172/JCI186143

Figure Lengend Snippet: ( A ) Shown are representative fields from 3 independent experiments. Complement proteins C3a and C5a increased the expression of ECM proteins in THP1 macrophages, BMDMs, and HK2 proximal tubule cells after 72 hours of treatment in serum-free medium. Scale bars: 50 μm. ( B – D ) The scatter plots show the mean staining intensity per THP-1 macrophage ( B ), BMDM ( C ), and HK2 proximal tubule cell ( D ), normalized to expression levels in their respective vehicle-treated groups. Each data point corresponds to quantified fluorescence intensity in a single field of view (FOV) from the microscope, and the larger dots represent the average of FOVs in biological replicates, each of which is color coded. RT-qPCR analysis of ECM protein coding genes were measured in BMDMs ( E ) and in HK2 proximal tubule cells ( F ). Gene expression was normalized to the expression of 18S ribosomal RNA in the same sample and then normalized to the expression level of vehicle-treated group ( n = 4). * P < 0.05, ** P < 0.01, and *** P < 0.001 by 2-tailed Student’s t test.

Article Snippet: After 3 days of differentiation, the medium was replaced with serum-free medium for 24 hours, after which the cells were treated for 72 hours with either vehicle or 10 ng/mL C3a (R&D Systems 3677-C3-025) or 10 ng/mL of C5a (R&D Systems 2037-C5-025/CF).

Techniques: Expressing, Staining, Fluorescence, Microscopy, Quantitative RT-PCR, Gene Expression

Journal: The Journal of Clinical Investigation

Article Title: Urine proteins reveal distinct coagulation and complement cascades underlying acute versus chronic lupus nephritis

doi: 10.1172/JCI186143

Figure Lengend Snippet: Circulating immune complexes and Abs planted directly within glomerular and tubulo-interstitial regions of the kidneys may fix complement, resulting in complement activation. The alternative pathway may further amplify complement activation within the kidneys. The products of C3 and C5 convertases, including the anaphylatoxins C3a and C5a, engage cognate receptors on a wide spectrum of immune cells, leading to immune cell activation, release of cytokines and chemokines, and acute inflammation, leading to high AI, as depicted on the left. Long-standing, unresolved complement activation and eventual formation of MAC may additionally engage and activate more immune and renal-resident cells, leading to tissue damage and repair, ECM deposition, and renal fibrosis, leading to high CI, as depicted on the right.

Article Snippet: After 3 days of differentiation, the medium was replaced with serum-free medium for 24 hours, after which the cells were treated for 72 hours with either vehicle or 10 ng/mL C3a (R&D Systems 3677-C3-025) or 10 ng/mL of C5a (R&D Systems 2037-C5-025/CF).

Techniques: Activation Assay

Journal: Frontiers in Immunology

Article Title: P-Rex1 controls phagocytosis and the killing of bacteria by murine neutrophils independently of its catalytic activity

doi: 10.3389/fimmu.2025.1591006

Figure Lengend Snippet: P-Rex1 mediates the killing of S. aureus by neutrophils independently of its catalytic Rac-GEF activity, whereas chemotaxis, ROS, and NETs require its Rac-GEF activity. (A) Bactericidal activity. Purified neutrophils from Prex1 –/– (red squares), Prex1 GD (green triangles), and wild type mice (grey circles) were primed with 20 ng/ml TNFα and 50 ng/ml GM-CSF for 45 min before incubation with serum-opsonized S. aureus for 90 min at a ratio of 1.5 bacteria per neutrophil. Heat-killed neutrophils were used as negative controls. Surviving bacteria were grown overnight and CFU enumerated. The % killing of bacteria by live neutrophils compared to heat-killed controls is plotted. Data are mean ± SEM of 3–4 independent experiments; each symbol represents the mean of one experiment. Statistics are one-way ANOVA with Tukey’s multiple comparisons test on log-transformed raw data; black p-values are significant, grey p-values non-significant. (B) Chemotaxis. Bone marrow cells from Prex1 –/– , Prex1 GD , and wild type mice were primed with 20 ng/ml TNFα and 50 ng/ml GM-CSF for 45 min before being stimulated with 3 nM C5a in transwell filters for 40 min, or mock stimulated. Transmigrated cells were analyzed by flow cytometry in parallel to control cells, using Ly6G hi /Mac1 hi staining to identify neutrophils. Data are mean ± SEM of 3–4 independent experiments; each symbol represents the mean of one experiment. Statistics are two-way ANOVA with Sidak’s multiple comparisons test on raw data. (C) fMLP-stimulated ROS production. Purified neutrophils as in (A) were primed with 1 μg/ml LPS for 90 min and then stimulated with 3 µM fMLP (filled symbols), or mock-stimulated (open symbols). ROS production was measured by real-time chemiluminescence assay with luminol and HRP for extra- and intracellular ROS. Left-hand panel shows luminometer traces from one representative experiment; right-hand panel shows the quantification as AUC over 2 min. Data are mean ± SEM of 3–5 independent experiments; each symbol represents the mean AUC from one experiment. Statistics are two-way ANOVA with Sidak’s multiple comparisons tests on log-transformed raw data. (D) S. aureus -stimulated intracellular ROS. Neutrophils were primed with 20 ng/ml TNFα and 50 ng/ml GM-CSF for 45 min in the presence of 50 units/ml SOD and 2000 units/ml catalase to scavenge extracellular ROS and were then stimulated with S. aureus at a ratio of 10 bacteria per neutrophil (filled symbols), or mock-stimulated (open symbols). ROS production was measured as in (C) except without HRP and in the presence of SOD and catalase, and quantification was done over 60 min. Data are mean ± SEM of 4 independent experiments; statistics are two-way ANOVA with Sidak’s multiple comparisons tests. (E) Formation of NETs. Neutrophils were seeded onto glass slides and allowed to adhere for 30 min before stimulation with serum-opsonised S. aureus at a ratio of 10 bacteria per neutrophil (closed symbols), or mock stimulation (open symbols). Non-cell permeable Sytox Green and cell-permeable Hoechst 33342 DNA dyes were added to samples 15 min before the end of the incubation, and cells were live-imaged by wide-field microscopy. Left-hand panel shows representative images from one experiment after 120 min stimulation or mock stimulation. Insets are magnifications of the indicated areas. Red arrows highlight NETs, white arrows dead cells without NETs. Right-hand panel shows quantification of NETs by ImageJ. Data are mean ± SEM of 3–4 independent experiments. Statistics are two-way ANOVA with Sidak’s multiple comparisons tests on raw data; significant p-values between and Prex1 –/– and wild type are indicated in red, and between Prex1 GD and wild type in green. For all panels, closed symbols show stimulated cells, open symbols mock-treated cells.

Article Snippet: Cells were primed with 20 ng/ml TNFα and 50 ng/ml GM-CSF for 45 min at 37°C, pipetted into transwell filters (400 μl/filter) in a 24-well plate containing HBSS ++++ (600 μl/well) in the presence or absence of 3 nM complement factor C5a anaphylatoxin (C5a, R&D Systems, 2037-C5-025), and incubated for 40 min at 37°C.

Techniques: Activity Assay, Chemotaxis Assay, Purification, Incubation, Bacteria, Transformation Assay, Flow Cytometry, Control, Staining, Chemiluminescence Immunoassay, Microscopy