Journal: medRxiv

Article Title: Differential virulence potential of different clades of multidrug-resistant Klebsiella pneumoniae ST258

doi: 10.64898/2026.03.28.26349612

Figure Lengend Snippet: (A and B) Bacteria recovered after 30-minute exposure of 10 8 CFU/mL to 85% human serum. Limit of detection (LOD) is indicated with a dashed line. (A) 13 Clade 1 isolates and 36 Clade 2 isolates were tested, each with at least two biological replicates. (B) 6 biological replicates per strain were tested. (C and D) C3 binding to bacteria was measured using an indirect ELISA. (C) 13 Clade 1 isolates and 36 Clade 2 isolates were tested, each with at least two biological replicates. (D) 6 biological replicates were tested per strain, each with at least 5 technical replicates. (E) Correlation between C3 binding and serum survival of Clade 2 isolates was assessed using Spearman correlation. (F) Uronic acid was quantified from a selection of Clade 1 (n=6) and Clade 2 (n=10) isolates with 3 biological replicates per isolate. Significance was assessed with Welch’s t-test. (G) Correlation between uronic acid quantity and serum survival of 10 Clade 2 isolates was assessed using Spearman correlation. (H) OD-normalized bacteria were reacted with 5% human serum in PBS for 30 minutes. C5a from the supernatant of the reaction was quantified using a sandwich ELISA. At least two biological replicates per isolate were performed. For panels A-D and H, P ≤ 0.05: *, P ≤ 0.01: **, P ≤ 0.001: *** by Mann-Whitney test.

Article Snippet: C5a quantification was performed using the Human Complement Component C5a DuoSet ELISA from R&D systems.

Techniques: Bacteria, Binding Assay, Indirect ELISA, Selection, Sandwich ELISA, MANN-WHITNEY

Journal: Nature Communications

Article Title: Orphan G protein-coupled receptor GPRC5B controls macrophage function by facilitating prostaglandin E receptor 2 signaling

doi: 10.1038/s41467-025-56713-0

Figure Lengend Snippet: A Library size-normalized counts detected by RNA sequencing in mouse RPM and in M0-differentiated BMDM (3 mice each; only GPCRs with average count >15 displayed). B Gprc5b expression in RPM and lymph node-derived lymphocytes was determined by NanoString RNA analysis (cells pooled from two mice per data point). C Knockout efficiency in RPM from control mice (white) and M-G5b-KOs (gray) was analyzed by qRT-PCR (C, n = 4, data normalized to Gapdh and controls set to 1). D Knockout efficiency in RPM was analyzed by immunoblotting (unspecific band around 38 kDa; the higher of the two specific bands probably represents glycosylated GPRC5B ; GAPDH as loading control). E, F Expression of Nos2 (E) and Tnf (F) was determined in RPM by qRT-PCR under basal conditions and after 6 h of stimulation with 1 µg/ml LPS ( n = 11/12/12/12 in E, 12/10/11/12 in F), data normalized to Gapdh and control set to 1). G Basal and C5a (20 ng/ml)-induced transwell migration in RPM (all cells pretreated with LPS 1 µg/ml for 3 h to facilitate migration) ( n = 3). H The distance travelled by individual RPM in response to different chemotactic factors (C5a, 20 nM; CCL5, 10 ng/ml; fMLP, 10 nM) was determined by live cell imaging ( n = 512 cells from 2 mice per group; cells pretreated with LPS 1 µg/ml for 3 h to facilitate migration, arb. units: arbitrary units). Phagocytic activity of LPS (1 µg/ml, 6 h)-stimulated RPM was determined by uptake of pHrodo E. coli bioparticles ( I , J , n = 5) or pHrodo-labeled apoptotic thymocytes ( K , L n = 10); I + K show original traces, J + L statistical evaluation of areas under the curve (AUC). Body weight change ( M ) and bacterial colony-forming units in peritoneal lavage fluid harvested 24 h after injection of fecal bacteria ( N ) ( n = 10). O, P Numbers of CD11b + , F4/80 + , MHCII - , Tim4 + RPM and CD11b + , F4/80 lo , MHCII + , CCR2 + BMDM before and 3, 24, and 54 h after i.p. injection of fecal bacteria ( n = 3/3/3/3/11/12/5/5). Data are means ± SEM; comparisons between genotypes were performed using unpaired two-sided Student’s t -test (C, J, L, N), two-way ANOVA (E-H) or two-way RM-ANOVA (M) with Sidak’s multiple comparison test, unpaired two-sided t-test corrected for multiple testing by two-stage step-up method Benjamini, Krieger and Yekutieli (O, P). * P < 0.05; *** P < 0.001; **** P < 0.0001; n , number of individual mice. Source data are provided as a Source Data file.

Article Snippet: Complement component C5a (2150-C5-025) was from R&D systems.

Techniques: RNA Sequencing, Expressing, Derivative Assay, Knock-Out, Control, Quantitative RT-PCR, Western Blot, Migration, Live Cell Imaging, Activity Assay, Labeling, Injection, Bacteria, Comparison

Journal: Nature Communications

Article Title: Orphan G protein-coupled receptor GPRC5B controls macrophage function by facilitating prostaglandin E receptor 2 signaling

doi: 10.1038/s41467-025-56713-0

Figure Lengend Snippet: A Knockout efficiency was determined by qRT-PCR in RPM and M0 BMDM (data normalized to Gapdh and RPM controls set to 1) ( n = 12). Analyses in resting and LPS (1 μg/ml, 6 h)-stimulated M0 BMDM: Expression of inflammatory genes ( B , C ; n = 15/14/15/15 in B, 15/14/15/15 in C), production of NOx ( D ; n = 3) or release of cytokines ( E , n = 3). F Transwell migration of M1 BMDM in response to different chemotactic factors ( n = 6) (CCL5: 75 ng/ml, CCL2: 10 ng/ml, SDF-1β: 100 ng/ml, C5a: 20 ng/ml, fMLP: 10 nM). Uptake of pHrodo E.coli fragments by M0 BMDM: G , exemplary curves; H , statistical analysis of AUC ( n = 6). I Flow cytometric analysis of CD11b-positive cells in the combined infarct and border zones of hearts harvested 4 days after infarction ( n = 5). J Echocardiographic analysis of ejection fraction (EF%) before and after infarction (8 controls, 4 KOs). K Histological analysis of scar size in hearts harvested 21 days after infarction ( n = 7 controls, 4 KOs), left ventricle (LV). L, M DSS colitis: Disease activity index integrating body weight change, stool consistency, intestinal bleeding (L) and colon length on day 6 (M) ( n = 7(L), 7/7/10/11 in M). Data are means ± SEM; comparisons between genotypes were performed using unpaired two-sided Student’s t- test (A, H, K), two-way ANOVA with Sidak’s multiple comparisons test (B-F, J, M), unpaired two-sided t -test corrected for multiple testing by two-stage step-up method Benjamini, Krieger and Yekutieli (I), two-way repeated measures ANOVA with Sidak’s multiple comparisons test (L). n, number of mice per group; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. Source data are provided as a Source Data file.

Article Snippet: Complement component C5a (2150-C5-025) was from R&D systems.

Techniques: Knock-Out, Quantitative RT-PCR, Expressing, Migration, Activity Assay

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: Life

Article Title: Macrophage-Induced Pro-Fibrotic Gene Expression in Tubular Cells after Ischemia/Reperfusion Is Paralleled but Not Directly Mediated by C5a/C5aR1 Signaling

doi: 10.3390/life14081031

Figure Lengend Snippet: Time course of the C5a/C5aR1 axis, expression in rat kidney sections, C5a concentration in rat plasma and numbers of renal macrophages. The left kidneys were harvested after I/R at the following time points: 10 min, 6 h, 24 h, 3 d, 5 d and 8 w. The healthy right kidney was nephrectomized one week before and served as a control. mRNA was isolated from fresh-frozen rat kidney tissue. C5a was measured in rat plasma using an ELISA ( A ). Plasma C5a concentrations were correlated with serum urea ( B ). Expression of C5ar1 ( C ) and C5 ( D ). mRNA was analyzed at different time points post-perfusion. C5ar1 mRNA expression correlated with the expression of the macrophage marker Cd68 ( E ), while no correlation was found between Cd68 expression and plasma C5a levels ( F ). Immunohistochemistry was used for the evaluation of CD163-positive ( G ) and CD68-positive macrophages in kidney sections ( H ) (ns = not significant; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

Article Snippet: Rat plasma samples collected at 10 min, 6 h, 24 h, 3 days, 5 days and 8 weeks after I/R were analyzed for C5a using a rat complement component C5a ELISA kit (NBP2-82137, Novus Biologicals, Centennial, CO, USA) following the manufacturer’s instructions and using a Synergy microplate reader and Gen5 software (BioTek Instruments GmbH, Friedrichshall, Germany).

Techniques: Expressing, Concentration Assay, Clinical Proteomics, Control, Isolation, Enzyme-linked Immunosorbent Assay, Marker, Immunohistochemistry

Journal: Life

Article Title: Macrophage-Induced Pro-Fibrotic Gene Expression in Tubular Cells after Ischemia/Reperfusion Is Paralleled but Not Directly Mediated by C5a/C5aR1 Signaling

doi: 10.3390/life14081031

Figure Lengend Snippet: Expression of pro-fibrotic genes in human tubular cells and supernatant concentration of TGF-ß after co-cultivation under normoxic or hypoxic conditions with or without C5a stimulation. mRNA isolated from human proximal tubular cells (HPTC) after a 24 h incubation (21% O 2 or 1% O 2 ) in co-culture with THP-1 monocytes differentiated to macrophages and subsequent stimulation with 50 nM C5a for 24 h. Expression of FGF2 ( A ), SNAI1 ( B ), ACTA2 ( C ), VIM ( D ) and TGFB1 ( F ). Supernatant was collected and TGF-ß concentration was determined by an ELISA ( E ) (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; § p < 0.05 vs. Ctrl.).

Article Snippet: Rat plasma samples collected at 10 min, 6 h, 24 h, 3 days, 5 days and 8 weeks after I/R were analyzed for C5a using a rat complement component C5a ELISA kit (NBP2-82137, Novus Biologicals, Centennial, CO, USA) following the manufacturer’s instructions and using a Synergy microplate reader and Gen5 software (BioTek Instruments GmbH, Friedrichshall, Germany).

Techniques: Expressing, Concentration Assay, Isolation, Incubation, Co-Culture Assay, Enzyme-linked Immunosorbent Assay

Journal: PloS one

Article Title: Sphingosine kinase 1 mediation of expression of the anaphylatoxin receptor C5L2 dampens the inflammatory response to endotoxin.

doi: 10.1371/journal.pone.0030742

Figure Lengend Snippet: Figure 2. Sphingosine-1-phosphate (S1P) and anaphylatoxin C5a concentrations in plasma and lung tissue. S1P concentrations in plasma (A) or lung tissue lysate (B) or C5a concentrations in plasma (C) or lung tissue lysates (D) from Sphk1+/+ or Sphk12/2 mice were determined before or after i.p. LPS challenge (0.5 mg/kg), using the LC-MS/MS (46) or ELISA techniques. Error bars represent s.d. *p,0.005, **p,0.001, by Student’s t-test. No significant differences in (B, C, D). n = 10 for each genotype and time point. doi:10.1371/journal.pone.0030742.g002

Article Snippet: For the C5a ELISA, purified rat anti-mouse C5a (BD Pharmingen, 558027) was used as capturing Ab and rat biotinylated anti-mouse C5a (BD Pharmingen, 558028) was used as detection Ab and a standurd curve for C5a protein measurment was established using mouse recombinant C5a (R&D Systems) (see Figure S1).

Techniques: Clinical Proteomics, Liquid Chromatography with Mass Spectroscopy, Enzyme-linked Immunosorbent Assay

Journal: PloS one

Article Title: Sphingosine kinase 1 mediation of expression of the anaphylatoxin receptor C5L2 dampens the inflammatory response to endotoxin.

doi: 10.1371/journal.pone.0030742

Figure Lengend Snippet: Figure 4. Anaphylatoxin C5a-mediated reduction in cytokine and chemokine production depends on Sphk1. BMDMs from Sphk1+/+ or Sphk12/2 mice were stimulated with LPS (500 ng/ml), with LPS concomitant with C5a (1 nM), or without C5a. Tissue culture supernatants were harvested 2 h (TNF-a) or 8 h (IL-6 and KC) after the addition of stimuli. (A) TNF-a; (B); IL-6; (C) KC. Error bars represent s.d. *p,0.05 by Student’s t-test; n = 5 for each genotype; representative for at least three independent experiments. (D) Reduced C5a-induced ERK1/2 and activation. BMDMs from Sphk1+/+ or Sphk12/2 mice were stimulated with C5a (10 nM), LPS (500 ng/ml), LPS and C5a, or saline for 5 min. Cell lysates were processed for immunoblotting with indicated antibodies. Representative of 3 independent experiments showing similar results. UD, undetected. (E) Model: Reduction of inflammatory cytokine production by phagocytes stimulated with C5a requires Sphk1. The model links Sphk1 activity to the cell surface expression of the anaphylatoxin receptor C5L2. LPS, C5a and inflammatory cytokines activate Sphk1 which is required to maintain S1P during inflammation. S1P regulates C5L2 cell surface expression on phagocytes. C5a, via C5L2 expressed on the cell surface, reduces neutrophil inflammation and inflammatory cytokine production by macrophages. doi:10.1371/journal.pone.0030742.g004

Article Snippet: For the C5a ELISA, purified rat anti-mouse C5a (BD Pharmingen, 558027) was used as capturing Ab and rat biotinylated anti-mouse C5a (BD Pharmingen, 558028) was used as detection Ab and a standurd curve for C5a protein measurment was established using mouse recombinant C5a (R&D Systems) (see Figure S1).

Techniques: Activation Assay, Saline, Western Blot, Activity Assay, Expressing

Journal: Theranostics

Article Title: C5aR1 is a master regulator in Colorectal Tumorigenesis via Immune modulation

doi: 10.7150/thno.45058

Figure Lengend Snippet: The strong correlation between complement activation and CRC. ( A-D ) The associations between the mRNA levels of C3 (A), C5 (B), C5AR1 (C), and C5AR2 (D) and the overall survival of CRC patients (n=364). ( E, F ) The effect of C3 , C5 , C5ar1 or C5ar2 deficiency on AOM/DSS-induced colorectal tumorigenesis compared to WT control. Images of the colorectum, where CRC often developed at distal (rectum) sites (E), and quantitative analysis of tumor number and mass (F). The experiment was duplicated. Scale bar in (E), 1 cm. ( G ) Pathological analysis of control or tumor tissues, including H&E, Ki-67 and C3d staining. Scale bar, 100 µm for H&E and 50 µm for IHC. ( H ) C5a concentration in colon tissue homogenates (CTHs) measured by ELISA. n≥ 7 in each group of WT, C3 -KO, C5 -KO, C5ar1 -KO, or C5ar2 -KO mice. Data are represented as mean ± SEM; ns, not significant; * P <0.05; and *** P <0.001. NC, negative control.

Article Snippet: The level of mouse C5a in colon tissue homogenates (CTHs) was measured with Mouse Complement Component C5a DuoSet ELISA kit according to the manufacturer's instructions (R&D Systems, Minneapolis, MN).

Techniques: Activation Assay, Control, Staining, Concentration Assay, Enzyme-linked Immunosorbent Assay, Negative Control

Journal: Theranostics

Article Title: C5aR1 is a master regulator in Colorectal Tumorigenesis via Immune modulation

doi: 10.7150/thno.45058

Figure Lengend Snippet: Schematic for the mechanisms by which C5a/C5aR1 signaling initiates colorectal tumorigenesis by modulating versatile immune responses. Complement C5a/C5aR1 signaling, independent of C3 activation, recruits MDSCs into the inflamed colorectal tissues to impair CD8 + T cells, and modulates the production of a variety of cytokines/chemokines (IL-1, IL-6, IL-11, IL-17A, TNF-α, IL-9, IL-10, IL-23, IL-27, CCL2, CCL17, CXCL1/5), thus fostering AOM/DSS-induced colorectal tumorigenesis. C5aR1 inhibition by PMX205 impedes CRC tumorigenesis. ACF, aberrant crypt foci.

Article Snippet: The level of mouse C5a in colon tissue homogenates (CTHs) was measured with Mouse Complement Component C5a DuoSet ELISA kit according to the manufacturer's instructions (R&D Systems, Minneapolis, MN).

Techniques: Activation Assay, Inhibition

Journal: Cells

Article Title: Extracellular Vesicles Derived from Endothelial Progenitor Cells Protect Human Glomerular Endothelial Cells and Podocytes from Complement- and Cytokine-Mediated Injury

doi: 10.3390/cells10071675

Figure Lengend Snippet: Time- and dose-dependent cytotoxicity and apoptosis assays on Glomerular Endothelial Cells (GECs) and Podocytes. GECs. Time-dependent XTT citotoxity assay on GECs ( A ) and podocytes ( B ) incubated with a medium containing CK (20 ng/mL TNF-α, 2.5 ng/mL IL-6, and 50 ng/mL human recombinant C5a protein) or vehicle alone at different time-points (12, 18, 24, 36, 48 h) Significant differences in viability were observed on both GECs and podocytes at 18, 24, 36, and 48 h (* p < 0.05, CK vs. vehicle). At 12 h, only the GECs (A) had different viability between treated (CK) and control cells (vehicle, * p < 0.05, CK vs. vehicle). Cytotoxicity tests by XTT ( C ) and apoptosis by TUNEL assay ( D ), evaluating the protective dose of EPC-derived EVs on GECs (black columns) and podocytes (white columns) after 24 h. Incubation with CK alone was effective in inducing cytotoxic (C) and apoptotic damage (D) in both cell types (* p < 0.05, CK vs. vehicle). Adding 1 or 10 μg/mL EPC-derived EVs to cells incubated with CK did not significantly reduce cell damage (* p < 0.05, CK + 1 μg/mL EV and CK + 10 μg/mL EV vs. vehicle), except with GEC incubated with 10 μg/mL EV in TUNEL assay (§ CK + 10 μg/mL EV vs. CK). In contrast, the adding of 25 or 50 μg/mL EPC-derived EVs to cells incubated with CK significantly reduced cell damage at the level of GECs and podocytes (§ p < 0.05, CK + 25 μg/mL EV and CK + 50 μg/mL EV vs. CK). XTT assays are reported as average optical density (O.D.) intensity ± 1SD. For TUNEL assays, data are expressed as the average number of green fluorescent apoptotic cells ± 1 SD. We performed three experiments for each test and the statistical analysis by ANOVA with Newmann–Keuls multiple comparison test.

Article Snippet: In selected experiments, we incubated cells in an appropriate medium containing 20 ng/mL tumor necrosis factor (TNF)-α (Sigma Aldrich), 2.5 ng/mL IL-6, and 50 ng/mL human recombinant C5a protein (R&D Systems, Minneapolis, MN, USA) in the presence or absence of different concentrations of EPC-derived EVs assessed by Nanosight analysis.

Techniques: Incubation, Recombinant, Control, TUNEL Assay, Derivative Assay, Comparison

Journal: Cells

Article Title: Extracellular Vesicles Derived from Endothelial Progenitor Cells Protect Human Glomerular Endothelial Cells and Podocytes from Complement- and Cytokine-Mediated Injury

doi: 10.3390/cells10071675

Figure Lengend Snippet: EPC-derived EVs protected GECs from complement- and cytokine-induced damage ( A , B ). Graphs showing GEC cytotoxicity by XTT assay ( A ) and apoptosis by TUNEL assay ( B ). For XTT assays, data are expressed as average OD intensity ± 1 SD, whereas we expressed TUNEL assays data as the average number of green fluorescent apoptotic cells ± 1 SD. We performed three experiments with similar results for all the assays and the statistical analysis by ANOVA with Newmann–Keuls multiple comparison test and Student’s t -test. ( C , D ) FACS analysis ( C ) and representative micrographs ( D ) of ROS expression of GEC (green staining) by confocal microscopy studies (magnification ×400, scale bar 50 μm). Nuclei were counterstained in blue by 2.5 μg/mL Hoechst. We performed three experiments with similar results for all the assays, and we performed the statistical analysis by ANOVA with Newmann–Keuls multiple comparison test and the Kolmogorov–Smirnov test. Incubation with cytokines 20 ng/mL TNF -α, 2.5 ng/mL IL-6, plus 50 ng/mL human recombinant C5a protein CKs significantly increased GEC vitality ( A ), inhibited resistance to apoptosis ( B ), and increased oxidative stress (C) in comparison to treatment with vehicle alone (vehicle, * p < 0.05 CK vs. vehicle). EV stimulation significantly inhibited these effects (§ p < 0.05 CK + EV vs. CK), but not EV were pre-treated with 1 U/mL RNase (# p < 0.05 CK + EV RNase vs. CK + EV).

Article Snippet: In selected experiments, we incubated cells in an appropriate medium containing 20 ng/mL tumor necrosis factor (TNF)-α (Sigma Aldrich), 2.5 ng/mL IL-6, and 50 ng/mL human recombinant C5a protein (R&D Systems, Minneapolis, MN, USA) in the presence or absence of different concentrations of EPC-derived EVs assessed by Nanosight analysis.

Techniques: Derivative Assay, XTT Assay, TUNEL Assay, Comparison, Expressing, Staining, Confocal Microscopy, Incubation, Recombinant