Journal: bioRxiv

Article Title: ERK5 is required for neutrophil-mediated ROS release and essential in epidermolysis bullosa acquisita

doi: 10.1101/2025.11.12.688045

Figure Lengend Snippet: (A) Lesional and healthy skin obtained from immunization-induced EBA and healthy control mice was immunohistochemically stained for phosphorylated ERK5 (pERK5). Scale bar represents 100 µM. The dermal-epidermal junction is indicated by the black lines. Arrows indicate stained cells. (B) Enhanced staining in both dermis and epidermis in lesional treated mice was observed in a semi-quantitative measurement. (C) Immortalized keratinocytes (HaCaT) were stimulated with anti-COL7 C IgG and C5a concentration was measured using ELISA, which showed no significant changes upon ERK5 inhibition. (D-F) Antibody transfer–induced EBA was induced by injection of rabbit anti-mCOL7 C antibodies in C57BL/6J mice, and prophylactic treatment with XMD8-92 twice daily significantly reduced clinical disease scores, affected ear surface area (AESA), and delta ear thickness (referring to day 0) compared to vehicle control. (G,H) In skin samples obtained on the final day of the antibody transfer–induced EBA model, histological analyses revealed no significant effects of ERK5 inhibition on leukocyte infiltration (asterisk) or dermal–epidermal split formation (arrows). IgG or C3 deposition remained unchanged (arrows). (I–J) Cryosections were stained for IgG and C3 deposition, which were both unaffected by ERK5 inhibition. B : n = 5, Tukey boxplot Mann-Whitney test, *p ≤ 0.05; C : n = 3,, mean ± SEM; E : n = 6 (control)/5 (XMD8-92), mean ± SEM, mixed effect analysis with Šidák’s multiple comparisons test, **p ≤ 0.01, ***p ≤0.001, ****p ≤ 0.0001; G-J : n = 6 (control)/5 (XMD8-92), Tukey boxplot, Mann-Whitney test

Article Snippet: C5a concentration in supernatants was determined using the Human Complement Component C5a DuoSet ELISA (R&D Systems GmbH, Wiesbaden, Germany) in accordance with manufacturer instructions and measured in a GloMax® Discover microplate reader (Promega, Walldorf, Germany).

Techniques: Control, Staining, Concentration Assay, Enzyme-linked Immunosorbent Assay, Inhibition, Injection, MANN-WHITNEY

Journal: PLoS ONE

Article Title: Complement Activation and STAT4 Expression Are Associated with Early Inflammation in Diabetic Wounds

doi: 10.1371/journal.pone.0170500

Figure Lengend Snippet: (A) C5a concentration in wound beds of diabetic and control mice absorbed by filter paper and assayed by ELISA. Left and right wounds averaged for n = 3 mice in each group and time point: 10 min (P = 0.05) 2h (P = 0.002), 4h (P = 0.001), 24h (P = 0.05). * P ≤0.05 vs. hetero (control). (B) C3-fragment deposition (C3 opsonization) in the subcutaneous tissue at the edges of the wound beds of diabetic and control mice assayed by immunofluorescence (n = 2). (C) Nucleated cell infiltration into the subcutaneous tissue at the edges of the wound beds of diabetic and control mice assayed by DAPI fluorescence (n = 2). Data are means ± SEM.

Article Snippet: The liquid samples were then measured using the mouse Complement C5a ELISA kit (R&D Systems, MN).

Techniques: Concentration Assay, Control, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Fluorescence

Journal: PLoS ONE

Article Title: Complement Activation and STAT4 Expression Are Associated with Early Inflammation in Diabetic Wounds

doi: 10.1371/journal.pone.0170500

Figure Lengend Snippet: (A) C5a concentration in the wound beds of diabetic and heterozygous control mice absorbed by filter paper and assayed by ELISA. Left and right wounds averaged for n = 3 mice in each group and time point: 4H (P = 0.002), 8H (P = 0.007) and 48H (P = 0.05). (B) C5a concentration in the wound beds of diabetic and heterozygous control mice treated with vehicle control gel or PIC1 gel (combined time points). db/db ± PIC1 (P = 0.05). Heterozygous ± PIC1 (P = 0.01); data are means ± SEM. (C) C3-fragment deposition (C3 opsonization) in the subcutaneous tissue at the edges of the wound beds of diabetic and control mice treated with vehicle control gel or PIC1 gel (combined time points). db/db ± PIC1 (P = 0.09). Heterozygous ± PIC1 (P = 0.12), Data are means ± SEM. * P ≤0.05 vs. saline control.

Article Snippet: The liquid samples were then measured using the mouse Complement C5a ELISA kit (R&D Systems, MN).

Techniques: Concentration Assay, Control, Enzyme-linked Immunosorbent Assay, Saline

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: Journal of neuroinflammation

Article Title: Modulation of C5a-C5aR1 signaling alters the dynamics of AD progression.

doi: 10.1186/s12974-022-02539-2

Figure Lengend Snippet: Fig. 1 Overproduction of C5a accelerates memory decline in Arctic mice at 7 months. A Overview of experimental design for object location memory (OLM) test. B Discrimination index (%) was calculated for the OLM test. Data shown as mean ± SEM. *p < 0.05. Two-way ANOVA with Tukey’s post hoc test. N = 17 (WT), 10 (C5a+), 11 (Arc), 8 (ArcC5a+)

Article Snippet: C5a concentration was determined with the R&D Systems Mouse Complement Component C5a DuoSet ELISA (DY2150) according to the manufacturer’s instructions, in triplicate.

Techniques:

Journal: Journal of neuroinflammation

Article Title: Modulation of C5a-C5aR1 signaling alters the dynamics of AD progression.

doi: 10.1186/s12974-022-02539-2

Figure Lengend Snippet: Fig. 5 C5a overexpression leads to increase in expression of genes associated with synapse transmission and assembly in the hippocampus. A Heatmap of selected genes in the hippocampus present in RNA clusters 3 and 4. Genes were clustered based on biological processes. RNA-seq data (TPM) is row-mean normalized. B Gene ontology (GO) and pathway enrichment analyses of genes present in Rc6. C Representative diagram of the GABAergic synapse pathway with select genes present in the panel A heatmap highlighted in red. N = 4–10 mice/genotype/age

Article Snippet: C5a concentration was determined with the R&D Systems Mouse Complement Component C5a DuoSet ELISA (DY2150) according to the manufacturer’s instructions, in triplicate.

Techniques: Over Expression, Expressing, Transmission Assay, RNA Sequencing

Journal: Journal of neuroinflammation

Article Title: Modulation of C5a-C5aR1 signaling alters the dynamics of AD progression.

doi: 10.1186/s12974-022-02539-2

Figure Lengend Snippet: Fig. 6 CD11c expression changes with C5a overexpression or C5aR1 ablation. A Representative images from 7 months of CD11b positive microglia in WT, Arctic, ArcticC5aR1KO, and ArcticC5a+ in the hippocampus. B–D Quantification of gene expression (TPM) of Itgam in the hippocampus (B) and percent field area of CD11b in hippocampus (C) and cortex (D) at 5, 7, and 10 months. E Representative images from 7 months of CD11c-positive microglia in WT, Arctic, ArcticC5aR1KO, and ArcticC5a+ in the hippocampus. F–H Quantification of gene expression (TPM) of Itgax in the hippocampus (F) and percent field area of CD11c in hippocampus (G) and cortex (H) at 5, 7, and 10 months. C5aR1KO and C5a+ cohorts were stained and analyzed separately. Four sections were stained per mouse and the percent field area was averaged over the 4 data points. Data shown as mean ± SEM. *p < 0.05, #0.1 > p > 0.05. Two-way ANOVA with Tukey’s post hoc test. N = 2–6 mice/genotype/age. Scale bar 500 µm

Article Snippet: C5a concentration was determined with the R&D Systems Mouse Complement Component C5a DuoSet ELISA (DY2150) according to the manufacturer’s instructions, in triplicate.

Techniques: Expressing, Over Expression, Gene Expression, Staining

Journal: BMC Oral Health

Article Title: Different concentrations of C5a affect human dental pulp mesenchymal stem cells differentiation

doi: 10.1186/s12903-021-01833-4

Figure Lengend Snippet: Morphological appearance of DPSCs cultured with different concentration of C5a for 2 weeks. A 50, B 100, C 200, D 300, E 400 ng/ml C5a groups and F Control group. DPSCs, dental pulp mesenchymal stem cells; C5a, complement component 5a

Article Snippet: Cells were cultured in a 37 °C, 5% CO 2 incubator and divided into six groups (n = 1 × 10 5 cells/group, using Corning® 25cm 2 Rectangular Canted Neck Cell Culture Flask with Vent Cap): (i) cells treated with basic cell culture medium containing 10 nmol/l dexamethasone, 5 mmol/l β-glycerophosphate and 50 mg/ml vitamin-C-phosphate as control; (ii) DPSCs treated with 50 ng/ml recombinant human complement component C5a protein (C5a; R&D Systems, Inc.); (iii) DPSCs treated with 100 ng/ml C5a; (iv) DPSCs treated with 200 ng/ml C5a; (v) DPSCs treated with 300 ng/ml C5a; and (vi) DPSCs treated with 400 ng/ml C5a.

Techniques: Cell Culture, Concentration Assay, Control

Journal: BMC Oral Health

Article Title: Different concentrations of C5a affect human dental pulp mesenchymal stem cells differentiation

doi: 10.1186/s12903-021-01833-4

Figure Lengend Snippet: Effect of different concentration of C5a stimulation on DSPP protein expression in DPSC. All 6 groups were cultured in dentinogenic medium. DSPP expression was determined by Western blot after 28 days of culture time. Results are expressed as relative expression to Actin. Data are presented as mean ± SEM of 3 independent experiments. *P < 0.05, *showed 100 ng/ml and 200 ng/ml groups expressed significantly lower DSP protein compared with other groups.. △ P < 0.05, △showed 400 ng/ml group expressed significantly higher DSP protein. DSPP, dentin sialophosphoprotein; DPSC, dental pulp mesenchymal stem cell

Article Snippet: Cells were cultured in a 37 °C, 5% CO 2 incubator and divided into six groups (n = 1 × 10 5 cells/group, using Corning® 25cm 2 Rectangular Canted Neck Cell Culture Flask with Vent Cap): (i) cells treated with basic cell culture medium containing 10 nmol/l dexamethasone, 5 mmol/l β-glycerophosphate and 50 mg/ml vitamin-C-phosphate as control; (ii) DPSCs treated with 50 ng/ml recombinant human complement component C5a protein (C5a; R&D Systems, Inc.); (iii) DPSCs treated with 100 ng/ml C5a; (iv) DPSCs treated with 200 ng/ml C5a; (v) DPSCs treated with 300 ng/ml C5a; and (vi) DPSCs treated with 400 ng/ml C5a.

Techniques: Concentration Assay, Expressing, Cell Culture, Western Blot

Journal: BMC Oral Health

Article Title: Different concentrations of C5a affect human dental pulp mesenchymal stem cells differentiation

doi: 10.1186/s12903-021-01833-4

Figure Lengend Snippet: Cell morphology and mineralized nodules of hDPSCs cultured with C5a and mineralized medium for 28 days. A 50 ng/ml C5a culture hDPSCs (arrows nodules). B 100 ng/ml C5a culture hDPSCs displayed smaller and less mineralized nodules (arrows nodules). C 200 ng/ml C5a culture hDPSCs, displayed smaller and less mineralized nodules (arrows nodules). D 300 ng/ml C5a culture hDPSCs (arrows nodules). E 400 ng/ml C5a culture hDPSCs displayed bigger and more mineralized nodules (arrows nodules). F Control group. C5a, complement component 5a, hDPSCs, human dental pulp mesenchymal stem cell (arrows nodules). G Quantification of mineralized nodule formation. Data are shown as mean OD/µg of total protein ± SD (n = 6). *Indicates significant differences compared to conrol group (P < 0.05)

Article Snippet: Cells were cultured in a 37 °C, 5% CO 2 incubator and divided into six groups (n = 1 × 10 5 cells/group, using Corning® 25cm 2 Rectangular Canted Neck Cell Culture Flask with Vent Cap): (i) cells treated with basic cell culture medium containing 10 nmol/l dexamethasone, 5 mmol/l β-glycerophosphate and 50 mg/ml vitamin-C-phosphate as control; (ii) DPSCs treated with 50 ng/ml recombinant human complement component C5a protein (C5a; R&D Systems, Inc.); (iii) DPSCs treated with 100 ng/ml C5a; (iv) DPSCs treated with 200 ng/ml C5a; (v) DPSCs treated with 300 ng/ml C5a; and (vi) DPSCs treated with 400 ng/ml C5a.

Techniques: Cell Culture, Control

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