Journal: bioRxiv

Article Title: Complement activation at injury sites drives the phagocytosis of necrotic cell debris and resolution of liver injury

doi: 10.1101/2024.08.23.609344

Figure Lengend Snippet: (A-B) Representative immunofluorescence images of liver cryosections from control mice or mice challenged with acetaminophen (APAP; 600 mg/kg) for 24h. Green: (i)C3b; red: fibrin(ogen); Orange: IgM; Cyan: C1q. Scale bar represents 50 µm. (C-F) Quantification area percentage stained with fibrin(ogen) (C), IgM (D), C1q (E) and (i)C3b (F) in liver cryosections of control mice and mice receiving an APAP overdose for 24, 48 or 72h. (G) Pearson’s correlation coefficient of (i)C3b and nuclei (Hoechst) or fibrin(ogen) in the injured livers 24, 48 and 72h after APAP overdose. (H) Pearson’s correlation coefficient between (i)C3b, IgM and C1q in the injured livers 24h after APAP overdose. (I) C3 levels in the serum of control mice or challenged with APAP for 24h. (J) C3a levels in the serum of control mice or challenged with APAP for 24h. Image quantifications were pooled from 8 fields of view. Each dot represents a single mouse. Data are represented as mean ± SEM. *p≤0.05 compared to control; #p≤0.05 between indicated groups. APAP = acetaminophen.

Article Snippet: Sections were incubated overnight at 4°C with 10 µg/ml polyclonal rabbit anti-human/mouse fibrin(ogen) (Dako), 5 µg/ml rat anti-mouse C3b/iC3b (clone 3/26, Hycult Biotec) and 5 µg/ml rabbit anti-mouse C1q (clone 4.8, Abcam).

Techniques: Immunofluorescence, Control, Staining

Journal: bioRxiv

Article Title: Complement activation at injury sites drives the phagocytosis of necrotic cell debris and resolution of liver injury

doi: 10.1101/2024.08.23.609344

Figure Lengend Snippet: (A) Representative immunofluorescence images of liver cryosections from WT and Rag2 -/- mice 24h after an overdose of acetaminophen (APAP; 600 mg/kg). Red: IgM; Cyan: C1q; Green: (i)C3b. Scale bar represents 100 µm. (B) Quantification of area percentage stained with IgM in liver cryosections of WT and Rag2 -/- mice 24h after APAP overdose. (C) Mean fluorescence intensity (MFI) of IgM staining at the necrotic injury sites in liver cryosections. (D) Quantification of area percentage stained with fibrin(ogen) in liver cryosections of WT and Rag2 -/- mice 24h APAP overdose. (E) Quantification of C1q deposition in the liver of WT and Rag2 -/- mice 24h after APAP overdose, normalized to the degree of fibrin(ogen) staining. (F) Mean fluorescence intensity of C1q staining at the necrotic injury sites in liver cryosections. (G) Quantification of (i)C3b deposition in the liver of WT and Rag2 -/- mice 24h after APAP overdose, normalized to the degree of fibrin(ogen) staining. (H) Mean fluorescence intensity of (i)C3b staining at the necrotic injury site of liver cryosections. Image quantifications were pooled from 8 fields of view. Each dot represents a single mouse. Data are represented as mean ± SEM. *p≤0.05 compared to WT mice. APAP= acetaminophen, MFI = Mean fluorescence intensity.

Article Snippet: Sections were incubated overnight at 4°C with 10 µg/ml polyclonal rabbit anti-human/mouse fibrin(ogen) (Dako), 5 µg/ml rat anti-mouse C3b/iC3b (clone 3/26, Hycult Biotec) and 5 µg/ml rabbit anti-mouse C1q (clone 4.8, Abcam).

Techniques: Immunofluorescence, Staining, Fluorescence

Journal: bioRxiv

Article Title: Complement activation at injury sites drives the phagocytosis of necrotic cell debris and resolution of liver injury

doi: 10.1101/2024.08.23.609344

Figure Lengend Snippet: (A) Representative image of neutrophils (green) and necrotic debris (pHRodo; red) in the liver 6h after FTI. pHRodo-SE was applied on top of the lesion 6h before imaging. Scale bar represents 50 µm, scale bar zoomed image represents 20 µm. (B) Quantification of the percentage neutrophils carrying pHRodo labeled debris at different areas of the injury. (C) Representative immunofluorescence stainings of liver cryosections 6h post burn injury. Green: (i)-C3b; Cyan: C1q. Scale bar represents 200 µm. (D-G) Flow cytometry of non-parenchymal cells isolated from liver FTI sites showing the percentage (D) pHrodo neutrophils (Ly6G), (E) pHrodo non-classical monocytes (Ly6C / CX 3 CR1), (F) pHrodo classical monocytes (Ly6C / CX 3 CR1 / CCR2) and macrophages (F4/80). Data are represented as mean ± SEM. Each dot represents a single mouse. *p≤0.05 compared to WT.

Article Snippet: Sections were incubated overnight at 4°C with 10 µg/ml polyclonal rabbit anti-human/mouse fibrin(ogen) (Dako), 5 µg/ml rat anti-mouse C3b/iC3b (clone 3/26, Hycult Biotec) and 5 µg/ml rabbit anti-mouse C1q (clone 4.8, Abcam).

Techniques: Imaging, Labeling, Immunofluorescence, Flow Cytometry, Isolation

Journal: bioRxiv

Article Title: Complement activation at injury sites drives the phagocytosis of necrotic cell debris and resolution of liver injury

doi: 10.1101/2024.08.23.609344

Figure Lengend Snippet: (A) 3D reconstruction image of human neutrophils stained with Calcein (green) phagocytosing pHrodo-labeled necrotic debris (red). Scale bar represents 10 µm. (B) Percentage of bone-marrow derived mouse neutrophils phagocytosing necrotic debris opsonized with serum, C3-depleted serum (from C3 -/- mice) or antibody-depleted serum (from Rag2 -/- mice). (C) Volume of necrotic debris phagocytosed by bone-marrow derived mouse neutrophils which was opsonized with serum, C3 serum or C1q serum. (D) Percentage of human neutrophils phagocytosing necrotic debris opsonized with serum, C1q-depleted serum or C3-depleted serum. (E) Volume of necrotic debris phagocytosed by human neutrophils which was opsonized with serum, C1q-depleted serum or C3-depleted serum. (F-J) Gene expression of human neutrophils incubated with unopsonized, serum-opsonized or C3-depleted serum-opsonized human necrotic debris. Data are normalized to the average expression of 3 housekeeping genes (GAPDH, 18s and CDKN1A) and represented as 2 −ΔΔCt relative to the unopsonized group. 10 µM latrunculin is used as a negative control. Data are represented as mean ± SEM. Each dot represents an independent experiment. *p≤0.05 compared to serum group or between indicated groups.

Article Snippet: Sections were incubated overnight at 4°C with 10 µg/ml polyclonal rabbit anti-human/mouse fibrin(ogen) (Dako), 5 µg/ml rat anti-mouse C3b/iC3b (clone 3/26, Hycult Biotec) and 5 µg/ml rabbit anti-mouse C1q (clone 4.8, Abcam).

Techniques: Staining, Labeling, Derivative Assay, Expressing, Incubation, Negative Control

Journal: The Journal of Immunology Author Choice

Article Title: Engineering Agonistic Bispecifics to Investigate the Influence of Distance on Surface-Mediated Complement Activation

doi: 10.4049/jimmunol.2400091

Figure Lengend Snippet: C1q-targeting bispecific Nb activates complement on lipid membranes. (A) Schematic of the C1 complex bound to a bispecific Nb (left). The domain organization of C1 is also shown (right). The bispecific Nb comprises two Nb domains that bind gC1q (green Nb, PDB:6Z6V) and the membrane-bound alfaTag (brown Nb, light pink alfaTag, PDB:6I2G). (B) ELISAs showing binding of the bispecific Nb to both the alfaTag (via detection of the His6-tag on the bispecific Nb) and C1q in the presence of human serum. (C) C1s activation assay; cleavage of a short peptide substrate induces fluorescence. Fluorescence units converted to μM AMC converted per minute. (D) Liposomes synthesized to display a cholesterol-functionalized alfaTag on the membrane surface. Liposomes were incubated with BC and human serum (10%, v/v). BC16 recruits C1 and activates the complement pathway leading to MAC-mediated lysis of the liposome, SRB dilution, and fluorescence increase. Complement activation was measured for a range of bispecific concentrations (technical triplicate). Negative controls where either BC16 or serum was omitted are represented in black and orange, respectively.

Article Snippet: Primary mouse anti–6× His-tag (1:2,000) (Thermo Fisher Scientific), rabbit anti-C1q (1:2,000) (Dako), goat anti-human C4 (1:150,000) (Complement Technology), or mouse anti-human 5b–9 (Dako) was added to PBS, 0.05% Tween 20, 1% BSA (PBS-BT).

Techniques: Membrane, Binding Assay, Activation Assay, Fluorescence, Liposomes, Synthesized, Incubation, Lysis

Journal: The Journal of Immunology Author Choice

Article Title: Engineering Agonistic Bispecifics to Investigate the Influence of Distance on Surface-Mediated Complement Activation

doi: 10.4049/jimmunol.2400091

Figure Lengend Snippet: Increased spacer length inhibits C4 deposition. (A) Liposomes were functionalized with cholesterol and alfaTag-modified DNA linkers, incubated in 1.5% (v/v) human serum in PBS with or without BC16. Western blots were used to detect C4 with a polyclonal anti-human C4 Ab (n = 2 separate experiments). DNA and BC concentrations were the same as in Fig. 2. (B–D) DNA constructs were functionalized with biotin, bound to an ELISA plate coated with streptavidin, and subsequently incubated with 1% human serum. Concentrations of DNA with BC16 were covaried to measure C1q (B), C4 (C), and 5b–9 deposition (D). White-filled symbols indicate that no bispecific was present, but DNA-alfaTag construct was (n = 2, separate experiments). The boxes in the graphs indicate deposition at 6.25 nM BC.

Article Snippet: Primary mouse anti–6× His-tag (1:2,000) (Thermo Fisher Scientific), rabbit anti-C1q (1:2,000) (Dako), goat anti-human C4 (1:150,000) (Complement Technology), or mouse anti-human 5b–9 (Dako) was added to PBS, 0.05% Tween 20, 1% BSA (PBS-BT).

Techniques: Liposomes, Modification, Incubation, Western Blot, Construct, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Immunology Author Choice

Article Title: Engineering Agonistic Bispecifics to Investigate the Influence of Distance on Surface-Mediated Complement Activation

doi: 10.4049/jimmunol.2400091

Figure Lengend Snippet: Positioning of C1qNB75 via protein linkers displays a similar effect on complement activation to DNA-based linkers. (A) Schematic depictions of the BC variants, which are differentiated by their linker. Estimated distancing that the Nbs should be able to achieve is depicted. The derivation of these distances is clarified in the main text. (B) ELISAs detecting C1q binding to alfaTag-bound BCs (left), and the capacity of BCs to bind to alfaTag peptides (right). (C) Liposome lysis assays of BCs on surfaces functionalized with cholesterol-alfaTag (0.5%). The averages (n = 3, technical replicates) are shown of the fluorescence intensity values after 20 min of measuring over a range of BC concentrations. The controls with no BC are shown in gray. (D) Liposome lysis assay of the BC variants with increasing DNA spacer lengths. Either cholesterol-modified alfaTag or cholesterol-modified DNA was used. In the latter case, 300 nM cholesterol-DNA-alfaTag was added and unbound DNA was purified. Next, 250 nM BC and 10% serum was added to lyse the liposomes (n = 3, technical replicates).

Article Snippet: Primary mouse anti–6× His-tag (1:2,000) (Thermo Fisher Scientific), rabbit anti-C1q (1:2,000) (Dako), goat anti-human C4 (1:150,000) (Complement Technology), or mouse anti-human 5b–9 (Dako) was added to PBS, 0.05% Tween 20, 1% BSA (PBS-BT).

Techniques: Activation Assay, Binding Assay, Lysis, Fluorescence, Modification, Purification, Liposomes

Journal: RSC Chemical Biology

Article Title: Selection and characterization of a peptide-based complement modulator targeting C1 of the innate immune system †

doi: 10.1039/d4cb00081a

Figure Lengend Snippet: RaPID selection, sequence analysis and peptide screening. (a) qPCR quantifying DNA recovery after each selection round. cDNA conjugated peptides were either incubated with SCgC1q-presenting beads (positive) or beads without protein (negative). (b) Phylogenetic tree representation of the top 100 sequences. (c) Representative sequences obtained from each cluster in (b). Lines connecting N-terminal tyrosine and cysteine denote the position of cyclization, and (bio) denotes the C-terminal lysine analogue that was modified with biotin. (d) and (e) ELISAs where peptides were incubated with either purified C1q or human serum (1% in RPMI medium). Single datapoints are shown. Peptides were incubated at two concentrations 50 μM and 500 nM, either with purified C1q or 1% human serum. (f) Structure of cL3, which was modified with either a biotin (cL3-Bt) or azide (cL3-Az) moiety.

Article Snippet: Primary Rabbit anti-C1q or Mouse anti C5b-9 (Dako, Denmark) was added to PBS, 0.05% Tween-20, 0.1% BSA (PBS-BT) in 1 : 2000 and 1 : 333 dilution, respectively, then incubated and washed.

Techniques: Selection, Sequencing, Incubation, Modification, Purification

Journal: RSC Chemical Biology

Article Title: Selection and characterization of a peptide-based complement modulator targeting C1 of the innate immune system †

doi: 10.1039/d4cb00081a

Figure Lengend Snippet: Activation of complement via C1q binding to cL3. (a) Representative ELISA detecting C1q was performed to evaluate the ability of cL3-Bt to bind C1 from 1% human serum in RPMI medium (two technical replicates). See Fig. S2 (ESI ) for fitted models of the data and additional technical replicates collected on different days. The control consists of unbound cL3, but no streptavidin, indicating the necessity of the streptavidin monolayer to recruit C1q. (b) ELISA detecting C5b-9 deposition to evaluate complement activation by cL3-Bt (two technical replicates). In both (a) and (b), streptavidin-coated plates are used and cL3-Bt is bound directly the biotin moiety. (c) As in (a), but this time the cL3-Az variant was immobilized on the plate using a DBCO-biotin linker that was co-incubated with the cL3-Az peptide on the plate for 1 hour at 37 °C. Thereafter, excess peptide was washed away and peptides were incubated with 1% human serum in RPMI medium before C1q was detected. Control is the condition with only biotin-DBCO linker present. See Fig. S4 (ESI ) for fitted model of the data. (d) As in (c), but this time with detection of C5b-9. (e) Deletion mutants of cL3 as in c: binding was assessed to determine recruitment of C1q from 1% human serum in PRMI. Lys(N3) denotes the lysine residue with the sidechain amine replaced by an azide, and * denotes that the cysteine has been cyclized via the N-terminal chloroacetyl modification. (c) and (d) display the data of one titration of cL3-Az. Further replicates of cL3-Az are contained in (e), which displays technical duplicates.

Article Snippet: Primary Rabbit anti-C1q or Mouse anti C5b-9 (Dako, Denmark) was added to PBS, 0.05% Tween-20, 0.1% BSA (PBS-BT) in 1 : 2000 and 1 : 333 dilution, respectively, then incubated and washed.

Techniques: Activation Assay, Binding Assay, Enzyme-linked Immunosorbent Assay, Control, Variant Assay, Incubation, Residue, Modification, Titration

Journal: RSC Chemical Biology

Article Title: Selection and characterization of a peptide-based complement modulator targeting C1 of the innate immune system †

doi: 10.1039/d4cb00081a

Figure Lengend Snippet: Inhibition of antibody-dependent complement activation by cL3. (a) The ability of cL3 to inhibit complement activation by IgG antibody platforms was assessed. Human serum, 10%, was incubated with 100 μM of cL3 10 min, and fluorescence increase caused by liposome lysis was monitored. (one representative experiment of data shown in (b)). (b) A titration of cL3 was performed under the same conditions as (a) ( n = 4 technical replicates). A one-way ANOVA, followed by a Tuckey test was performed comparing the fluorescent data after 20 minutes to the IgG condition ( p < 0.05 *, p < 0.01 **, p < 0.001 ***). (c) Competition ELISAs, performed in 1% human serum, determining cL3 mediated inhibition of C1q and C5b-9 deposition by pooled IgG, using cL3-Az. Samples are normalized to the DMSO control (maximal signal, 100%) and no serum samples (minimal signal, 0%) (two technical replicates). (d) Competition ELISAs as in (c) determining inhibition of C5b-9 deposition by cL3-deletion mutants (two technical replicates).

Article Snippet: Primary Rabbit anti-C1q or Mouse anti C5b-9 (Dako, Denmark) was added to PBS, 0.05% Tween-20, 0.1% BSA (PBS-BT) in 1 : 2000 and 1 : 333 dilution, respectively, then incubated and washed.

Techniques: Inhibition, Activation Assay, Incubation, Fluorescence, Lysis, Titration, Control

Journal: RSC Chemical Biology

Article Title: Selection and characterization of a peptide-based complement modulator targeting C1 of the innate immune system †

doi: 10.1039/d4cb00081a

Figure Lengend Snippet: cL3 variants effect on complement modulation. (a) ELISA determining the activation of complement in 1% human serum by azide modified inv-cL3 detecting C1q and C5b-9 (technical singlet). (b) Competition ELISA showing inv-cL3 inhibiting C1q and C5b-9 deposition by blocking binding to pooled IgG (two technical replicates). (c) Competition ELISA showing lin-cL3 inhibiting C1q and C5b-9 deposition by blocking binding to pooled IgG (two technical replicates). (d) The ability of lin-L3 to inhibit complement activation by IgG was assessed in liposome lysis assays (single experiment shown, experiment was repeated twice, once at 10 min pre-incubation, once at 1 hour).

Article Snippet: Primary Rabbit anti-C1q or Mouse anti C5b-9 (Dako, Denmark) was added to PBS, 0.05% Tween-20, 0.1% BSA (PBS-BT) in 1 : 2000 and 1 : 333 dilution, respectively, then incubated and washed.

Techniques: Enzyme-linked Immunosorbent Assay, Activation Assay, Modification, Blocking Assay, Binding Assay, Lysis, Incubation

Journal: RSC Chemical Biology

Article Title: Selection and characterization of a peptide-based complement modulator targeting C1 of the innate immune system †

doi: 10.1039/d4cb00081a

Figure Lengend Snippet: Competition assays between cL3 and C1q binders. (a) Competition ELISAs where either cL3-Bt (left) or C1qNB75 (right) are immobilised and competed against solution phase C1qNB75 (100 nM) or cL3 (20 μM), respectively, in the presence of 1% human serum. Binding of complement component C1q and deposition of C5b-9 was measured. (Three technical replicates) (b) ELISAs determining cL3-mediated inhibition of C1q binding and C5b-9 deposition by CRP. 1% human serum was incubated with cL3-Az before adding to the wells containing CRP. Samples are normalized to the DMSO control (maximal signal, 100%) and no serum samples (minimal signal, 0%). (Two technical replicates) (c) binding locations of CRP (orange) and C1qNB75 (green) on gC1q (blue). (d) C1 complex showing potential cL3 binding locations (orange).

Article Snippet: Primary Rabbit anti-C1q or Mouse anti C5b-9 (Dako, Denmark) was added to PBS, 0.05% Tween-20, 0.1% BSA (PBS-BT) in 1 : 2000 and 1 : 333 dilution, respectively, then incubated and washed.

Techniques: Binding Assay, Inhibition, Incubation, Control

Journal: bioRxiv

Article Title: CD11c-expressing microglia are transient, driven by interactions with apoptotic cells

doi: 10.1101/2024.06.24.600082

Figure Lengend Snippet: (A) Left – schematic of retinal flat mount depicting where analysis was conducted, with dorsal leaf outlined in dashed yellow box. Right – max projected confocal images of CD11c-GFP+ cells in the dorsal leaf of retinal flat mounts at postnatal days (P) 0, 7, and 30. Optic nerve head outlined in dashed white line. Scale bar, 100μm. (B) Densities of CD11c-GFP+ cells from retinal flat mount across embryonic and postnatal ages. Data are presented as means ± SEM ( - animals age). One-way ANOVA F(9,17) =18.87 P<.0001. (C) Representative Max projected confocal image of C1q+ (red) and CD11c-GFP+ (green) microglia at P5. Scale bar 50μm. (D) Proportion of CD11c-GFP+ retinal microglia from retinal flat mounts at embryonic day (e) 16.6, P0, P5, P12, and P30. Data are presented as mean ± SEM ( - animals/age. One-way ANOVA F(4, 5) =15.76 P=.0049 and Tukey’s multiple comparisons. * P ≤.05, NS =no significance. (E) Max projected confocal images of individual CD11c-GFP+ microglia in retinal flat mounts from embryonic day 16.5 (e16.5) through postnatal day 30 (P30). Scale bar at 10μm. (F) Confocal images of retinal cross sections from CD11c-DTR/GFP transgenic mice at P0, P3, P7, and P30. CD11c-GFP (Magenta); Hoechst (blue). For P0/3, brackets segment retinal layers, gray – nerve fiber layer and ganglion cell layer (NFL/GCL), purple – inner plexiform layer (IPL), and teal – neuroblastic layer (NbL). P7/P30, gray – nerve fiber layer and ganglion cell layer (NFL/GCL), purple – inner plexiform layer and inner nuclear layer (IPL/INL), and blue – outer plexiform layer (OPL) for P7 and P30. Scale bar, 100μm. (G) Percentage of CD11c-GFP+ microglia across all retinal layers at P0, P3, P7, and P30 (n = 2 animals). Data are presented as means ± SEM.

Article Snippet: Antibodies used include Goat polyclonal anti-GFP ( :2000, Abcam ab5450, RRID: AB_304897), Rabbit monoclonal anti-C1q ( :1500, Abcam an182451, RRID: AB_2732849), Rabbit polyclonal anti-IBA1 ( :1000, Wako 019-19741, RRID: AB_839504), Rabbit monoclonal anti-CC3 ( :500, BD Biosciences 559565, RRID: AB_397274), Guinea pig polyclonal anti-RBPMS ( :750, Millipore Sigma ABN1376, RRID: AB_2687403), Rat monoclonal anti-CD68 ( :250, Bio-Rad MCA1957, RRID: AB_322219), and Goat polyclonal anti-Osteopontin ( :100, Thermo Fischer PA5-34579, RRID: AB_2551931).

Techniques: Transgenic Assay

Journal: bioRxiv

Article Title: CD11c-expressing microglia are transient, driven by interactions with apoptotic cells

doi: 10.1101/2024.06.24.600082

Figure Lengend Snippet: (A) Confocal image of P3 retinal flat mount (Arrowhead and magnified boxes illustrate interactions) in NFL/GCL. CD11c-GFP+ microglia (green); cleaved caspase 3, CC3 (pink); and RBPMS (blue). Scale bar, 50μm. (B) Percent CD11c-GFP+ microglia contacting apoptotic RGCs (RBPMS+CC3+) at P3 out of total CD11c-GFP+ microglia within GCL. (n=5 animals). (C) Confocal images of retinal flat mounts of P3 CD11c-GFP animals in GCL showing a range of microglial GFP expression from none to high. Scale bar, 10μm. Dashed line demarcates distinction between CD11c-GFP negative and positive. (D) Cell area immunostained for CD68 ( Top ) and C1q ( Bottom ) in CD11c-GFP- and CD11c-GFP+ microglia (n=4 animals, 352 GFP-cells and 422 GFP+ cells). Mann-Whitney test Top **P =. 0011 and Bottom ****P <. 0001. (E) Scatter plot of CD68 area/cell compared to CD11c-GFP area/cell at P3 in GCL. Pearson r= 0.4145 **** P < .0001. (F) Flow cytometry analysis showing the percent CD11c Hi of total CD45+CD11b+ or CD45+CX3CR1-GFP+ microglia from retinas across all genotypes. CX3CR1-GFP/+ (n=10), CX3CR1-KO (n=6), CR3 KO (n=6), MerTK KO (n=9), Axl KO (n=8), and MerTK/Axl dKO (n=6), ≥ 2 litters collected for each genotype, ± SEM. One-way ANOVA F(5, 39) = 34.64 P <.0001 and Tukey’s multiple comparisons. **** P <.0001, *** P =.0003, ** P =.0056, * P =.02, NS, not significant. (G) Max projected confocal image of P5 retinal flat mount from WT and AXL KO, Spp1+ (green) and total microglia (IBA1+; red). Scale bar, 100μm. (H) Density of Spp1+IBA1+ microglia in AXL WT and KO (n=5, n=4 animals) ± SEM. Unpaired t test * P = .0139. (I) Percent Spp1+IBA1+ of total IBA1+ microglia in AXL WT and KO (n=5, n=4 animals) ± SEM. Unpaired t test ***P <.001

Article Snippet: Antibodies used include Goat polyclonal anti-GFP ( :2000, Abcam ab5450, RRID: AB_304897), Rabbit monoclonal anti-C1q ( :1500, Abcam an182451, RRID: AB_2732849), Rabbit polyclonal anti-IBA1 ( :1000, Wako 019-19741, RRID: AB_839504), Rabbit monoclonal anti-CC3 ( :500, BD Biosciences 559565, RRID: AB_397274), Guinea pig polyclonal anti-RBPMS ( :750, Millipore Sigma ABN1376, RRID: AB_2687403), Rat monoclonal anti-CD68 ( :250, Bio-Rad MCA1957, RRID: AB_322219), and Goat polyclonal anti-Osteopontin ( :100, Thermo Fischer PA5-34579, RRID: AB_2551931).

Techniques: Expressing, MANN-WHITNEY, Flow Cytometry

Journal: bioRxiv

Article Title: CD11c-expressing microglia are transient, driven by interactions with apoptotic cells

doi: 10.1101/2024.06.24.600082

Figure Lengend Snippet: (A) Depiction of two depletion strategies, Diphtheria toxin (DT) targeted ablation of CD11c-DTR/GFP+ microglia (Top) and PLX3397 (PLX)-mediated depletion to target more homeostatic microglia (Bottom). (B) Confocal images of CD11c-DTR/GFP+ microglia (green) in retinal flat mounts at P5 of Vehicle and DT-treated CD11c-DTR/GFP mice. Scale bar, 50μm. (C) Density of CD11c-DTR/GFP+ microglia in vehicle and DT-treated CD11c-DTR/GFP (n=6, n=7 animals) ± SEM. Unpaired t test **** P <.0001. (D) Confocal images of C1q+ microglia (white) in immunostained retinal flat mounts at P5 of vehicle and DT-treated CD11c-DTR/GFP mice. Scale bar, 50μm. (E) Densities of C1q+ cells in retinas of vehicle and DT-treated CD11c-DTR/GFP mice (n=6, n=7 animals) ± SEM. Mann Whitney test *P = .0140. (F) Max projected confocal images of C1q+ microglia in retinal flat mounts at P5 of vehicle and PLX-treated Cx3CR1/GFP+ mice. C1q (white). Scale bar, 50μm. (G) Density of C1q+ cells in retinas of naïve and PLX-treated CX3CR1-GFP (n=6, n=8) ± SEM. Unpaired t test *P = .0239. (H) Max projected confocal images of apoptotic RGCs in retinal flat mounts at P5 of all conditions/genotypes. CC3 (magenta); RBPMS (green). Scale bar, 50μm. (I) Density of CC3+RBPMS+ cells in retinas from vehicle and DT-treated CD11c-DTR/GFP mice and DT-treated wildtype mice (WT) (n=8, n=10, n=10 animals respectively) ± SEM. Ordinary one-way ANOVA **P = .0018 and Tukey’s multiple comparisons test: Veh vs DT ** P =.006, WT+DT vs DT **P = .0043, NS , not significant. (J) Density of CC3+RBPMS+ cells in naïve and PLX-treated CX3CR1-GFP/+ (n=8, n=8 animals) ± SEM. Unpaired t test **** P <.0001 (K) Scatter plot of dying RGC density (CC3+RBPMS+/mm 2 ) compared to microglial density (C1q+/mm 2 ) of same animal (n=4 CD11c + vehicle, n=5 CD11c + DT, n=10 PLX animals). Pearson r= -0.6350 ** P =.0035.

Article Snippet: Antibodies used include Goat polyclonal anti-GFP ( :2000, Abcam ab5450, RRID: AB_304897), Rabbit monoclonal anti-C1q ( :1500, Abcam an182451, RRID: AB_2732849), Rabbit polyclonal anti-IBA1 ( :1000, Wako 019-19741, RRID: AB_839504), Rabbit monoclonal anti-CC3 ( :500, BD Biosciences 559565, RRID: AB_397274), Guinea pig polyclonal anti-RBPMS ( :750, Millipore Sigma ABN1376, RRID: AB_2687403), Rat monoclonal anti-CD68 ( :250, Bio-Rad MCA1957, RRID: AB_322219), and Goat polyclonal anti-Osteopontin ( :100, Thermo Fischer PA5-34579, RRID: AB_2551931).

Techniques: MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: Proangiogenic properties of complement protein C1q can contribute to endometriosis

doi: 10.3389/fimmu.2024.1405597

Figure Lengend Snippet: Gene expression analysis of C1q in EM lesions based on EndometDB. (A) Histograms representing C1QA , C1QB , and C1QC mRNA expression in control endometrium (CE), patient endometrium (PE), and in different EM lesions (peritoneal; deep; and ovarian, OMA). Gene expression profiling (GEP) analysis, based on data extracted from GEO (GSE141549), revealed a significantly higher expression of all three C1q genes in EM lesions as compared to CE. (B) Analysis of C1q gene expression in EM patients clustered into different disease stages (stage I-IV). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (Mann-Whitney U Test).

Article Snippet: Bound C1q was probed by polyclonal rabbit anti-human C1q (1:300; Dako) and alkaline phosphatase-conjugated secondary antibody (1:20,000; Sigma–Aldrich).

Techniques: Expressing, Control, MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: Proangiogenic properties of complement protein C1q can contribute to endometriosis

doi: 10.3389/fimmu.2024.1405597

Figure Lengend Snippet: C1q is abundantly present in endometriotic lesions and in healthy ovary. Representative microphotographs showing the presence of C1q in ovarian endometriotic lesions (A, B, E) , patients’ eutopic endometrium (C) , and healthy ovary (D) . AEC (red) chromogen was used to visualize the binding of rabbit anti-human C1q antibody. Red arrows indicate vessels (A) , while yellow arrows indicate isolated cells scattered in EM stroma which resulted due to positive staining for C1q (B) . (F, G) Representative microphotographs showing the presence of C4d (F) or C1q (G) in serial sections of endometriotic lesions. C1q is present in the endometriotic lesion; however, the classical pathway is feebly activated. AEC (red) chromogen was used to visualize the binding of secondary antibodies. Nuclei were counterstained in blue with Harris Hematoxylin. Magnification, 10x (A, C-E) ; 20x (B) . Scale bars, 50 µm (A, B) ; 100 µm (C-G) .

Article Snippet: Bound C1q was probed by polyclonal rabbit anti-human C1q (1:300; Dako) and alkaline phosphatase-conjugated secondary antibody (1:20,000; Sigma–Aldrich).

Techniques: Binding Assay, Isolation, Staining

Journal: Frontiers in Immunology

Article Title: Proangiogenic properties of complement protein C1q can contribute to endometriosis

doi: 10.3389/fimmu.2024.1405597

Figure Lengend Snippet: Double immunofluorescence microscopy for C1q in EM lesions. Representative images showing double staining for C1q (red) and vWF (A, D, E) , CD68 (B) , or CD34 (C) (green) in endometriotic lesions. After deparaffinization, tissue sections were incubated overnight with anti-human C1q and anti-human vWF, CD68, or CD34 primary antibodies, followed by incubation with anti-rabbit Cy3 and anti-mouse Alexa Fluor™ 488 secondary antibodies. Cell nuclei were stained with DAPI. Scale bars, 50 µm.

Article Snippet: Bound C1q was probed by polyclonal rabbit anti-human C1q (1:300; Dako) and alkaline phosphatase-conjugated secondary antibody (1:20,000; Sigma–Aldrich).

Techniques: Immunofluorescence, Microscopy, Double Staining, Incubation, Staining

Journal: Frontiers in Immunology

Article Title: Proangiogenic properties of complement protein C1q can contribute to endometriosis

doi: 10.3389/fimmu.2024.1405597

Figure Lengend Snippet: C1QA , C1QB , and C1QC gene expression in endometriotic lesions and primary isolated endometriotic cells. (A) Graphical representation of primary EM cell isolation procedure. (B) Characterization of endometriotic cells (EMCs) isolated from EM ovary cysts by immunofluorescence. Around 10% of EMCs resulted positively stained for cytokeratin (CK)8/18, and 100% were positive for mucin-1 (MUC-1) and vimentin. Cell nuclei were stained with DAPI. (C) After total RNA extraction and retro-transcription, C1q gene expression was analyzed by performing RT-qPCR. Peripheral blood mononuclear cells (PBMCs) were used as positive control. GAPDH was used as the housekeeping gene. Scatter plots were generated with the software GraphPad Prism 8.4.3. (D, E) The clustering of EM patients in stages (I-III or IV) or for adenomyosis presence respectively highlighted a significant difference in terms of C1q expression, with higher C1q levels in the most severe group (D) , and in adenomyosis-positive EM patients (E) . C1q expression was evaluated by examining the collective mean of individual C1QA , C1QB , and C1QC genes. Data are expressed as box-plots (median, interquartile range). *p < 0.05 (unpaired two-tailed t-test).

Article Snippet: Bound C1q was probed by polyclonal rabbit anti-human C1q (1:300; Dako) and alkaline phosphatase-conjugated secondary antibody (1:20,000; Sigma–Aldrich).

Techniques: Expressing, Isolation, Cell Isolation, Immunofluorescence, Staining, RNA Extraction, Quantitative RT-PCR, Positive Control, Generated, Software, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Proangiogenic properties of complement protein C1q can contribute to endometriosis

doi: 10.3389/fimmu.2024.1405597

Figure Lengend Snippet: Binding of C1q to EECs, OVECs, or HUVECs. (A) Different endothelial cells [ECs; i.e., ECs isolated from healthy ovary (OVECs), n = 3; human umbilical vein ECs (HUVECs), n = 3; and endometriotic ECs (EECs)] grown to confluence on 96-well tissue culture plates were incubated with 10 µg/mL of purified C1q at different time points (0, 15, 30, 60, or 120 minutes) at room temperature. The binding of C1q was revealed by whole-cell ELISA assay. The data are presented as mean ± SD of three separate experiments. (B) Schematic representation of functional assays for the evaluation of C1q proangiogenic properties by migration, scratch, proliferation, and tube formation assays. Image created with BioRender.com , as an adaptation from Laschke et al. . * p <0.05; ** p <0.01; **** p <0.0001.

Article Snippet: Bound C1q was probed by polyclonal rabbit anti-human C1q (1:300; Dako) and alkaline phosphatase-conjugated secondary antibody (1:20,000; Sigma–Aldrich).

Techniques: Binding Assay, Isolation, Incubation, Purification, Enzyme-linked Immunosorbent Assay, Functional Assay, Migration

Journal: Frontiers in Immunology

Article Title: Proangiogenic properties of complement protein C1q can contribute to endometriosis

doi: 10.3389/fimmu.2024.1405597

Figure Lengend Snippet: C1q promotes angiogenesis in EECs and OVECs. (A) Migration assays were performed in a trans-well system using endothelial cells (ECs) isolated from endometriotic cysts (EECs), healthy ovary (OVECs), or human umbilical vein (HUVECs). Cells were stained with FAST DiI™, seeded in FluoroBlok™ Inserts (1.5x10 5 cells/insert), and the lower chamber was loaded with C1q (10 μg/mL) or VEGF (20 ng/mL), as chemoattractant stimuli. After 24h, fluorescence was read via INFINITE 200 Fluorescence Plate Reader. Data are expressed as mean ± standard deviation (SD). *p < 0.05; **p < 0.01. (B) Wound healing assays were performed using EECs, OVECs, and HUVECs. Cells (5x10 4 /well) were grown until 60–70% of confluence in a 24-well plate. After scratching the middle of endothelial monolayer, cells were stimulated with C1q (10 μg/mL) or VEGF (20 ng/mL). Images of the wound fields were captured after 18h, allowing calculation of percentage wound closure. Data are expressed as mean ± SD. *p < 0.05; **p < 0.01. (C) Tube formation assays were performed in EECs, OVECs, and HUVECs. Cells (5x10 4 ) were seeded onto Matrigel ® in CultureSlides, and stimulated with C1q (10 μg/mL) or VEGF (20 ng/mL). After 18h, using TiEsseLab BDS 600 microscope, the capillary-like structures formed by ECs were manually counted, comparing the different conditions. Data are expressed as mean ± SD. *p < 0.05. (D) Proliferation assays were performed using EECs, OVECs, and HUVECs. Cells (7x10 3 /well) were seeded in a 96-well plate, and stimulated with C1q (10 μg/mL) or VEGF (20 ng/mL) for 24h. MTS was then added in each well, and cell proliferation was measured using PowerWave Select X Microplate Reader. Data are expressed as mean ± SD. REST, resting cells.

Article Snippet: Bound C1q was probed by polyclonal rabbit anti-human C1q (1:300; Dako) and alkaline phosphatase-conjugated secondary antibody (1:20,000; Sigma–Aldrich).

Techniques: Migration, Isolation, Staining, Fluorescence, Standard Deviation, Microscopy

Journal: Frontiers in Immunology

Article Title: Proangiogenic properties of complement protein C1q can contribute to endometriosis

doi: 10.3389/fimmu.2024.1405597

Figure Lengend Snippet: gC1qR is expressed in EECs and OVECs and can modulate C1q-induced proangiogenic behaviour. (A, B) Representative images displaying gC1qR positive staining (green) in EECs and OVECs, comparing permeabilized and not permeabilized cells. Cell nuclei were stained with DAPI (blue). (C, D) Surface biotinylation assay for the detection of gC1qR fraction present on the cell surface of EECs ( n = 3) and OVECs ( n = 3). Cells were treated with Sulfo-NHS-biotin reagent, biotinylated cell surface proteins were isolated upon binding to a Streptavidin-coated resin, and separated on a 10% SDS-PAGE. Membrane was then probed with anti-gC1qR antibody and anti-rabbit IRDye 800CW secondary antibody by Western blot analysis. Signal intensity was detected using an Odyssey CLx near-infrared scanner (LI-COR Biosciences, Lincoln, NE, USA). Image acquisition, processing, and data analysis were performed with Image Studio 5.2 (LI-COR Biosciences). β-actin was used to normalize the results. Data are expressed as mean ± standard deviation (SD). EECs displayed a higher amount of gC1qR compared to OVECs, considering both total protein and cell surface fraction; *p < 0.05. B, biotinylated; MW, molecular weights; NB, not biotinylated; PD, pull down. (E) Wound healing assay was performed using HUVECs ( n = 3) after transfection with siC1QBP (gC1qR gene) or siCTRL for 48h. After scratching the middle of endothelial monolayer, cells were stimulated with C1q (10 μg/mL) or VEGF (20 ng/mL). Images of the wound fields were captured at 0 and 8h, percentage wound closure was calculated. The percentage of wound healing was considered as relative to siCTRL in resting conditions (100% of wound healing). Data are expressed as mean ± SD; *p < 0.05.

Article Snippet: Bound C1q was probed by polyclonal rabbit anti-human C1q (1:300; Dako) and alkaline phosphatase-conjugated secondary antibody (1:20,000; Sigma–Aldrich).

Techniques: Staining, Surface Biotinylation Assay, Isolation, Binding Assay, SDS Page, Membrane, Western Blot, Standard Deviation, Wound Healing Assay, Transfection