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: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: FGF19-Induced Inflammatory CAF Promoted Neutrophil Extracellular Trap Formation in the Liver Metastasis of Colorectal Cancer.

doi: 10.1002/advs.202302613

Figure Lengend Snippet: Figure 5. FGF19 induced NET formation by facilitating complement C5a and IL-1𝛽production in iCAFs. A) Cytokine array of the media of LX-2 cells pretreated with rhFGF19 (50 ng mL−1) for 24 h. Seven factors were upregulated in the supernatant of LX-2 cells stimulated with rhFGF19. B) Quantification of NETs formed by neutrophils stimulated with complement C5a, CXCL11, IL-1𝛼, IL-1𝛽, IL-18, MIF, or PAI-1 (n = 18 RMFs from 6 experimental replicates per group). C) Intracellular and D) extracellular expression of complement C5a and IL-1𝛽in LX-2 cells treated with rhFGF19 or FGF19-containing CM (n = 3 for WB, or 6 for ELISA). E) Quantification of NETs formed by neutrophils cocultured with LX-2 cells that had been pretreated with FGF19-containing CM in the presence of C5a neutralizing antibody (50 ng mL−1) or IL-1𝛽neutralizing antibody (1 μg mL−1) (n = 18 RMFs from 6 experimental replicates per group). F) Representative IHC staining of C5a or IL-1𝛽expression in paired primary CRC and CRLM tissues. Scale bar: 50 μm. G) Expression of complement C5a and IL-1𝛽were higher in CRLM tissues (n = 30) than that in paired primary CRC tissues (n = 30). H) Correlation between IHC scores of FGF19 and C5a or IL-1𝛽in human CRLM tissues (n = 30). I) ChIP‒qPCR assays showed the recruitment of STAT3 to the promoter regions of C5 and IL1B (n = 3). J,K) Intracellular and L) extracellular expression of C5a and IL-1𝛽in LX-2 cells stimulated with rhFGF19 or FGF19-containing CM and treated with or without fisogatinib (100 nм), fedratinib (10 μм), C188-9 (5 μg mL−1), and anakinra (20 mg mL−1) (n = 3 for WB, or 6 for ELISA). M) Extracellular expression of complement C5a and IL-1𝛽in LX-2 cells treated with FGF19-containing CM for 24 h and then subjected to regular medium, regular medium with rhIL-1𝛼(1 ng mL−1), FGF19-free CM, fresh FGF19-containing CM, or anakinra for 24 h (n = 6). ** or ##p < 0.01; *** or ###p < 0.001; n.s., nonsignificant. In (B), (D), (I), (L), and (M), data were subjected to Student’s t-test. In (E), data were subjected to Mann–Whitney test. In (G), data were subjected to paired Student’s t-test. See also related Figure S6, Supporting Information.

Article Snippet: Human FGF19 neutralizing antibody (AF969, R&D Systems), human IL-1β neutralizing antibody (AF-201-NA, R&D Systems), and human complement C5a neutralizing antibody (MAB2037, R&D Systems) were used in this study.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Immunohistochemistry, MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: Epithelial C5aR1 Signaling Enhances Uropathogenic Escherichia coli Adhesion to Human Renal Tubular Epithelial Cells

doi: 10.3389/fimmu.2018.00949

Figure Lengend Snippet: Effect of C5a stimulating renal tubular epithelial cell (RTEC) on bacterial adhesion to the RTEC. RTEC were pre-treated with C5a (as indicated or otherwise 10 nM) and/or LPS (800 ng/mL) for 24 h and subjected to assessment of bacteria binding to RTEC. (A,B) Bacterial adhesion to RTEC, evaluated by bacterial plate count assay. (C,D) Bacterial adhesion to RTEC, evaluated by fluorescence microscopy analysis. (C) Representative fluorescence images of tri-iodothyronine and tetramethylrhodamine-labeled J96 adhesion to RTEC, J96 (red), and 4’,6-diamidino-2-phenylindole (blue). Scale bars, 50 µm. (D) Quantification of bacteria adhesion to RTEC corresponding to the images in (C) . Results were expressed as number of bacteria per 10 3 RTEC. (A,B,D) data were analyzed by one-way ANOVA with Tukey’s multiple comparisons test [ (A) n = 12 individual wells per group, (B) n = 8 individual wells per group, (C) n = 6 individual images (×200 magnification) from two coverslips per group]. All results shown are representative of three independent experiments. * P < 0.05, *** P < 0.001, **** P < 0.0001.

Article Snippet: Co., USA); human recombinant C5a (with an endotoxin level of ≤0.1 EU per 1 μg of protein) (R&D systems).

Techniques: Bacteria, Binding Assay, Fluorescence, Microscopy, Labeling

Journal: Frontiers in Immunology

Article Title: Epithelial C5aR1 Signaling Enhances Uropathogenic Escherichia coli Adhesion to Human Renal Tubular Epithelial Cells

doi: 10.3389/fimmu.2018.00949

Figure Lengend Snippet: Effect of C5a stimulating renal tubular epithelial cell (RTEC) on bacterial internalization into the RTEC. (A) Bacterial uptake by RTEC, evaluated by bacterial plate count assay. RTEC were pre-treated with C5a (10 nM) and/or LPS (800 ng/mL) for 24 h and subjected to the assay. Data were analyzed by one-way ANOVA with Tukey’s multiple comparisons test ( n = 8 individual wells) and representative of three independent experiments. (B,C) Bacterial uptake by RTEC, evaluated by fluorescence microscopy analysis. RTEC were pre-treated with or without C5a (10 nM) for 24 h and subjected to the assay. (B) Representative fluorescence images of (tri-iodothyronine and tetramethylrhodamine-labeled) bacteria internalization into RTEC. Bacteria (red), F-actin (green), and 4’,6-diamidino-2-phenylindole (blue) are shown. Scale bars, 25 µm. Lower panel image corresponding to the boxed region in the top-right image shows the cross-sectional views in z stack (bottom and side panel) of RTEC, F-actin, and bacteria, demonstrating J96 within the cytoplasm of RTEC. A representative of three experiments is shown. (C) Quantification of bacteria uptake by RTEC corresponding to the images in (B) . Results were expressed as number of bacteria per 10 3 RTEC. Data were analyzed by unpaired two-tailed Student’s t -test ( n = 15 individual images per group). * P < 0.05, *** P < 0.001, **** P < 0.0001.

Article Snippet: Co., USA); human recombinant C5a (with an endotoxin level of ≤0.1 EU per 1 μg of protein) (R&D systems).

Techniques: Fluorescence, Microscopy, Labeling, Bacteria, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Epithelial C5aR1 Signaling Enhances Uropathogenic Escherichia coli Adhesion to Human Renal Tubular Epithelial Cells

doi: 10.3389/fimmu.2018.00949

Figure Lengend Snippet: Effect of C5a stimulating renal tubular epithelial cell (RTEC) on bacterial transmigration through the monolayer of RTEC. Bacterial transmigration in RTEC that had been pre-treated with or without C5a (10 nM) for 24 h, then incubated with or without bacteria up to 3 h. (A) Transmigrated bacteria recovered from the lower chamber of RTEC cultures, evaluated by plate count assay. Data were analyzed by two-way ANOVA with multiple comparisons ( n = 4 individual wells per group). (B–D) Epithelial barrier damage, evaluated at 2 h after incubation with bacteria by reduction of tight junction marker ZO-1 expression. (B) Western blot analysis for ZO-1 in RTEC. Data were analyzed by unpaired two-tailed Student’s t -test ( n = 3 individual images per group). (C) Representative fluorescence images of ZO-1 (green) staining in RTEC. Scale bars, 10 µm. (D) Quantification of ZO-1-positive cells, shown as C5a treatment relative to control, corresponding to the RTEC shown in (C) . Data were analyzed by unpaired two-tailed Student’s t -test [ n = 4 individual images (×200 magnification) per group]. * P < 0.05, **** P < 0.0001. All results shown are representative of three independent experiments.

Article Snippet: Co., USA); human recombinant C5a (with an endotoxin level of ≤0.1 EU per 1 μg of protein) (R&D systems).

Techniques: Transmigration Assay, Incubation, Bacteria, Marker, Expressing, Western Blot, Two Tailed Test, Fluorescence, Staining, Control

Journal: Frontiers in Immunology

Article Title: Epithelial C5aR1 Signaling Enhances Uropathogenic Escherichia coli Adhesion to Human Renal Tubular Epithelial Cells

doi: 10.3389/fimmu.2018.00949

Figure Lengend Snippet: C5a stimulation upregulates Man expression in renal tubular epithelial cell (RTEC). RTEC were pre-treated with C5a (10 nM) and/or LPS (800 ng/mL) or with C5a, in the presence of C5aR1 antagonist (PMX53, 5 µM) or vehicle (Ctrl) for 24 h and subjected to assessment of Man expression and bacteria binding to RTEC. (A,B) Fluorescence microscopy analysis. (A) Representative fluorescence images of Man expression in RTEC (non-permeabilized). Man (green) detected by fluorescein-labeled Galanthus nivalis lectin (GNL) and 4’,6-diamidino-2-phenylindole (blue) are shown. Scale bars, 25 µm. (B) Quantification of Man expression, shown as relative fluorescence intensity in GNL-positively stained area corresponding to the images shown in (A) . (C,D) Fluorescence intensity-based microplate assay. (C) Man expression in RTEC that had been treated with C5a and/or LPS. (D) Man expression in RTEC that had been treated with C5a and PMX53. (E) Bacterial adhesion to RTEC, evaluated by bacterial plate count assay. (B–D) Data were analyzed by one-way ANOVA with Tukey’s multiple comparisons [ (B) n = 6 coverslips per group, under ×100 magnification, (C) n = 9 individual wells per group, (D,E) n = 6–8 individual wells per group]. All results shown are representative of three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Abbreviations: Ctrl, control; RFI, relative fluorescence intensity; RFU, relative fluorescence units.

Article Snippet: Co., USA); human recombinant C5a (with an endotoxin level of ≤0.1 EU per 1 μg of protein) (R&D systems).

Techniques: Expressing, Bacteria, Binding Assay, Fluorescence, Microscopy, Labeling, Staining, Control

Journal: Frontiers in Immunology

Article Title: Epithelial C5aR1 Signaling Enhances Uropathogenic Escherichia coli Adhesion to Human Renal Tubular Epithelial Cells

doi: 10.3389/fimmu.2018.00949

Figure Lengend Snippet: Uropathogenic Escherichia coli adhesion to renal tubular epithelial cell (RTEC) through binding to Man on the cell surface of the RTEC. (A) Confocal microscopic images of bound bacteria in RTEC (non-permeabilized) that had been incubated with labeled J96 for 1 h. Bacteria (red), Man (green) detected by fluorescein-labeled Galanthus nivalis lectin and 4’,6-diamidino-2-phenylindole (blue) are shown. Left image: compressed image. Scale bars, 10 µm. Right image: corresponding to the boxed region in the left image show the cross-sectional views in z stack (bottom and side panel) of RTEC, Man, and bacteria, demonstrating association of Man and J96 at cell surface of RTEC. (B) Bacteria binding to RTEC, evacuated by bacterial plate count assay. RTEC were incubated with d -mannose or glucose (1 or 5%) for 30 min before the addition of bacteria. Data were analyzed by two-way ANOVA with multiple comparisons ( n = 8 individual wells per group). (C,E) Man expression in RTCE that had been pre-treated without C5a (C) or with C5a (E) for 24 h and then treated with buffer alone (PBS) or containing α-mannosidase (5 mM) or control enzyme (β-galactosidase) for 1 h, evacuated by fluorescence intensity-based microplate assay. Data were analyzed by one-way ANOVA with Tukey’s multiple comparisons ( n = 6 individual wells per group). (D,F) Bacterial binding to RTEC that had undergone the same treatments as described in (C,E) , evacuated by bacterial plate count assay. (D) Without C5a pre-treatment. (F) With C5a pre-treatment. Data were analyzed by one-way ANOVA with Tukey’s multiple comparisons ( n = 8 individual wells per group). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. All results shown are representative of three independent experiments.

Article Snippet: Co., USA); human recombinant C5a (with an endotoxin level of ≤0.1 EU per 1 μg of protein) (R&D systems).

Techniques: Binding Assay, Bacteria, Incubation, Labeling, Expressing, Control, Fluorescence

Journal: Frontiers in Immunology

Article Title: Epithelial C5aR1 Signaling Enhances Uropathogenic Escherichia coli Adhesion to Human Renal Tubular Epithelial Cells

doi: 10.3389/fimmu.2018.00949

Figure Lengend Snippet: Intracellular signaling/molecules responsible for the action of C5a on Mannosyl residue expression. (A,B) Western blot analysis for ERK1/2 and IκB phosphorylation in renal tubular epithelial cell (RTEC) after C5a (10 nM) stimulation for up to 60 min. In each set of blots, the top row of bands corresponds to incubating membrane with appropriate anti-phospho-antibody and the bottom row of bands corresponds to incubating membrane with appropriate total antibody. Relative amounts of protein phosphorylation are shown in the lower panel of each set of blots. Data were analyzed by one-way ANOVA ( n = 3/group, resulting from three independent experiments). (C) Effect of inhibition of ERK1/2 pathway on Man expression in RTECs assessed by fluorescence intensity-based microplate assay. RTEC monolayers were pre-incubated with C5a for 24 h in the presence of ERK1/2 pathway inhibitor (U0126, 10 μM) and the vehicle control (DMSO) then were used for quantification of Man expression. Data were analyzed by unpaired two-tailed Student’s t -test ( n = 9–12 individual wells per group). A representative result of three experiments is shown. (D,E) Production of TNF-α in RTEC that had been treated with C5a (10 nM) and/or LPS (800 ng/mL) for 24 h and subjected to RT-qPCR (D) and ELISA (E) . Data were analyzed by one-way ANOVA with Tukey’s multiple comparisons ( n = 3/group, resulting from three independent experiments). (F) Man expression in RTEC that had been pre-treated with recombinant human TNF-α (10 ng/mL) or vehicle control (BSA) for 24 h, evaluated by fluorescence intensity-based microplate assay. Data were analyzed by unpaired two-tailed Student’s t -test ( n = 9 replicate wells/group). A representative result of three experiments is shown. (G,H) Bacterial adhesion to RTEC. (G) Representative microscopic images of bacteria adhesion to RTEC that had been pre-treated with TNF-α for 24 h, then incubated with tri-iodothyronine and tetramethylrhodamine-labeled J96 for 1 h. Bacteria (red), Man (green) detected by fluorescein-labeled Galanthus nivalis lectin, and 4’,6-diamidino-2-phenylindole (blue) are shown. Scale bars, 25 µm. (H) Quantification of bound bacteria corresponding to the images shown in (G) . Data were analyzed by unpaired two-tailed Student’s t -test [ n = 6 individual images (×200 magnification) from two coverslips per group]. A representative result of three experiments is shown. * P < 0.05, ** P < 0.005, *** P < 0.001, **** P < 0.0001.

Article Snippet: Co., USA); human recombinant C5a (with an endotoxin level of ≤0.1 EU per 1 μg of protein) (R&D systems).

Techniques: Residue, Expressing, Western Blot, Phospho-proteomics, Membrane, Inhibition, Fluorescence, Incubation, Control, Two Tailed Test, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Recombinant, Bacteria, Labeling

Journal: Frontiers in Immunology

Article Title: Epithelial C5aR1 Signaling Enhances Uropathogenic Escherichia coli Adhesion to Human Renal Tubular Epithelial Cells

doi: 10.3389/fimmu.2018.00949

Figure Lengend Snippet: Detection of C5a and C5aR1 in human urinary tract. (A–C) C5a and creatinine concentrations in urine from urinary tract infection (UTI) patients and healthy controls. (A) Urinary C5a concentrations. (B) Urinary creatinine concentrations. (C) Ratios of urinary C5a/creatinine concentration. Data were analyzed by Mann–Whitney U two-tailed test. ** P < 0.01, n.s. no significant. (D) C5aR1 expression in human kidney tissue determined by immunochemical staining. Control: negative control, tissue stained with non-specific mouse IgG2a. Scale bar, 50 µm.

Article Snippet: Co., USA); human recombinant C5a (with an endotoxin level of ≤0.1 EU per 1 μg of protein) (R&D systems).

Techniques: Infection, Concentration Assay, MANN-WHITNEY, Two Tailed Test, Expressing, Staining, Control, Negative Control