Journal: Theranostics

Article Title: Microglia exacerbate white matter injury via complement C3/C3aR pathway after hypoperfusion

doi: 10.7150/thno.35841

Figure Lengend Snippet: Activation of complement C3-C3aR/ITGAM pathway in BCCAO rats. A Quantitative RT-PCR analysis of the expression of C1qa, C1qb, C4b, C3 , C3ar , and Itgam in the striatum of control and BCCAO rats at day 7, 14, and 28 after BCCAO surgery. The values are normalized to those of the control group. n = 3-7 in each group. B Western blots and quantification for C3, C3aR, ITGAM, and β-actin in the striatum of control and BCCAO rats at day 7, 14, and 28 after surgery. C Representative images and quantification of complement C3 puncta (red) deposition on myelin (MBP + , green) in the striatum of BCCAO and control rats. n = 3-4 animals in each group at day 7, 14, and 28 after surgery. Scale bar=10 μm. The data are shown as the mean ± SD. ***, p < 0.001; **, p < 0.01; *, p < 0.05; NS, not significant; the BCCAO group vs. the control group.

Article Snippet: Rat anti-MBP (Abcam, ab7349, 1:400), mouse anti-SMI32 (Biolegend, San Diego, CA, 801701, 1:100), rabbit anti-APC (Abcam, ab72040, 1:50), rabbit anti-Iba-1 (Wako, Richmond, VA, 019-19741, 1:300), rabbit anti-CD86 (Abcam, ab112490, 1:200), mouse anti-CD68 (AbD Serotec, MCA341, 1:200), rabbit anti-C3 (Abcam, ab200999, 1:200), mouse anti-ITGAM (AbD Serotec, Oxford, UK, MCA618, 1:100), rat anti-C3aR (Hycult Biotech, Uden, Netherlands, HM1123, 1:100) antibodies were used.

Techniques: Activation Assay, Quantitative RT-PCR, Expressing, Western Blot

Journal: Theranostics

Article Title: Microglia exacerbate white matter injury via complement C3/C3aR pathway after hypoperfusion

doi: 10.7150/thno.35841

Figure Lengend Snippet: Genetic deletion of C3ar1 attenuates microglia activation and reverses white matter injury in BCAS mice. A Representative images and quantification of C3aR (green) and Iba-1 (red) double-positive cells and microglia cells (Iba-1 + cells, red) in the striatum of WT, C3aR-KO, BCAS, and BCAS/C3aR-KO mice at day 28 after surgery. Scale bar=25 μm. B Western blots and quantification for CD86, iNOS, MBP and β-actin in the striatum of WT, C3aR-KO, BCAS, and BCAS/C3aR-KO mice at day 28 after surgery. C Representative images and quantification of damaged axon (SMI32 + , red) relative to myelin (MBP + , green) and mature oligodendrocyte (APC + cells, red) in the striatum of WT, C3aR-KO, BCAS, and BCAS/C3aR-KO mice at day 28 after surgery. Scale bar=50 μm. D Western blots of total- and phospho-STAT3 (pSTAT3) and β-actin in the striatum of WT, C3aR-KO, BCAS, and BCAS/C3aR-KO mice at day 28 after surgery. E and F Quantification of total STAT3/ β-actin ( E ) and phospho-STAT3/STAT3 ( F ) levels in the striatum of WT, C3aR-KO, BCAS, and BCAS/C3aR-KO mice at day 28 after surgery. The data are shown as the mean ± SD. n = 3-4 animals in each genotype group. ***, p < 0.001; **, p < 0.01; *, p < 0.05; NS, not significant.

Article Snippet: Rat anti-MBP (Abcam, ab7349, 1:400), mouse anti-SMI32 (Biolegend, San Diego, CA, 801701, 1:100), rabbit anti-APC (Abcam, ab72040, 1:50), rabbit anti-Iba-1 (Wako, Richmond, VA, 019-19741, 1:300), rabbit anti-CD86 (Abcam, ab112490, 1:200), mouse anti-CD68 (AbD Serotec, MCA341, 1:200), rabbit anti-C3 (Abcam, ab200999, 1:200), mouse anti-ITGAM (AbD Serotec, Oxford, UK, MCA618, 1:100), rat anti-C3aR (Hycult Biotech, Uden, Netherlands, HM1123, 1:100) antibodies were used.

Techniques: Activation Assay, Western Blot

Journal: Theranostics

Article Title: Microglia exacerbate white matter injury via complement C3/C3aR pathway after hypoperfusion

doi: 10.7150/thno.35841

Figure Lengend Snippet: C3aR inhibition suppresses microglial activation and microglia redistribution to myelin in BCCAO rats. A Western blots and quantification for CD86, iNOS, and β-actin in the striatum of the sham+vehicle, sham+C3aR antagonist, BCCAO+vehicle, and BCCAO+C3aR antagonist groups. n = 3-6 in each group. B Representative images of microglia cells (Iba-1 + cells, red) contacting with myelin (MBP + , green) in the striatum of sham+vehicle, sham+C3aR antagonist, BCCAO+vehicle, and BCCAO+C3aR antagonist groups. Scare bar=50 μm. n= 3-4 in each group. C-D Quantification of the proportion of microglia cells adhered to myelin relative to the number of myelin fibers ( C ) and the number of microglia cells ( D ). The data are shown as the mean ± SD. ***, p < 0.001; **, p < 0.01; *, p < 0.05; NS, not significant; the BCCAO group vs. control group. C3aRA: C3aR antagonist.

Article Snippet: Rat anti-MBP (Abcam, ab7349, 1:400), mouse anti-SMI32 (Biolegend, San Diego, CA, 801701, 1:100), rabbit anti-APC (Abcam, ab72040, 1:50), rabbit anti-Iba-1 (Wako, Richmond, VA, 019-19741, 1:300), rabbit anti-CD86 (Abcam, ab112490, 1:200), mouse anti-CD68 (AbD Serotec, MCA341, 1:200), rabbit anti-C3 (Abcam, ab200999, 1:200), mouse anti-ITGAM (AbD Serotec, Oxford, UK, MCA618, 1:100), rat anti-C3aR (Hycult Biotech, Uden, Netherlands, HM1123, 1:100) antibodies were used.

Techniques: Inhibition, Activation Assay, Western Blot

Journal: Theranostics

Article Title: Microglia exacerbate white matter injury via complement C3/C3aR pathway after hypoperfusion

doi: 10.7150/thno.35841

Figure Lengend Snippet: C3aR inhibition prevents behavioral deficits and white matter injury in BCCAO rats. A Five-day spatial learning performance measured as the latency to reach the platform in the Morris water maze test in sham+vehicle, sham+C3aR antagonist, BCCAO+vehicle, and BCCAO+C3aR antagonist groups. n= 12-15 animals for each group. B Spatial memory performance measured as the number of entries into the platform quadrant and the percentage of time spent in the platform quadrant in the Morris water maze test in the sham+vehicle, sham+C3aR antagonist, BCCAO+vehicle, and BCCAO+C3aR antagonist groups, n = 12-15 animals in each group. C Spatial memory performance measured as discrimination time in the new object recognition test in the sham+vehicle, sham+C3aR antagonist, BCCAO+vehicle, and BCCAO+C3aR antagonist groups. n = 4-6 animals for each group. D Western blots and quantification for myelin basic protein (MBP) and β-actin in the striatum of sham+vehicle, sham+C3aR antagonist, BCCAO+vehicle, and BCCAO+C3aR antagonist groups. E - F Representative images and quantification of damaged axon (SMI32 + , red) relative to myelin (MBP + , green) ( E ) and mature oligodendrocyte (APC + cells, red) ( F ) in the striatum of sham+vehicle, sham+C3aR antagonist, BCCAO+vehicle, and BCCAO+C3aR antagonist groups. n = 3-4 animals in each group. Scale bar=50 μm. The data are shown as the mean ± SD. **, p < 0.01; *, p < 0.05; the BCCAO group vs. control group. C3aRA: C3aR antagonist.

Article Snippet: Rat anti-MBP (Abcam, ab7349, 1:400), mouse anti-SMI32 (Biolegend, San Diego, CA, 801701, 1:100), rabbit anti-APC (Abcam, ab72040, 1:50), rabbit anti-Iba-1 (Wako, Richmond, VA, 019-19741, 1:300), rabbit anti-CD86 (Abcam, ab112490, 1:200), mouse anti-CD68 (AbD Serotec, MCA341, 1:200), rabbit anti-C3 (Abcam, ab200999, 1:200), mouse anti-ITGAM (AbD Serotec, Oxford, UK, MCA618, 1:100), rat anti-C3aR (Hycult Biotech, Uden, Netherlands, HM1123, 1:100) antibodies were used.

Techniques: Inhibition, Western Blot

Journal: Frontiers in Cellular Neuroscience

Article Title: Complement activation and increased anaphylatoxin receptor expression are associated with cortical grey matter lesions and the compartmentalised inflammatory response of multiple sclerosis

doi: 10.3389/fncel.2023.1094106

Figure Lengend Snippet: Primary antibodies used in this study.

Article Snippet: C3aR1 , Mouse , hC3aRZ8 , Hycult Biotech.

Techniques: Binding Assay

Journal: Frontiers in Cellular Neuroscience

Article Title: Complement activation and increased anaphylatoxin receptor expression are associated with cortical grey matter lesions and the compartmentalised inflammatory response of multiple sclerosis

doi: 10.3389/fncel.2023.1094106

Figure Lengend Snippet: Anaphylatoxin receptor expression at sites of complement activation. Complement C3b (blue reaction product) and C5aR1 + cells (brown reaction product) and subpial and perivascular sites of the cortical GM (A–C) . TMEM119 + immunopositive microglia (blue) in close contact with large neuron-like cells expressing C3 transcript (D) and an example of a C5aR1 + cell contacting a C3b immunoreactive cortical neuron (E–E”) . Immunostaining revealed an elevated number and more darkly stained C3aR1 and C5aR1 + cells with a microglia-like morphology in the demyelinated cortical GM (GM lesion; F,H ) in comparison to adjacent normal appearing GM (NAGM; G,I ). Scale bars; panels (A,F–I) = 50 μm and panels (B–E”) = 20 μm.

Article Snippet: C3aR1 , Mouse , hC3aRZ8 , Hycult Biotech.

Techniques: Expressing, Activation Assay, Immunostaining, Staining

Journal: Frontiers in Cellular Neuroscience

Article Title: Complement activation and increased anaphylatoxin receptor expression are associated with cortical grey matter lesions and the compartmentalised inflammatory response of multiple sclerosis

doi: 10.3389/fncel.2023.1094106

Figure Lengend Snippet: Microglia/macrophages express complement anaphylatoxin C3a and C5a receptors. C5aR1 microglia/macrophages were seen in the cortical GM in association with TMEM119 (A–A”) and HLA-D (B–B”) , and an example of C3aR1 + /TMEM119 + (C–C”) . Dual C3a/C5aR1 immunostaining showing co-positive C3aR1/C5aR1 cells (D–D”) . Human monocyte-derived microglia-like cells (i-microglia) were cultured for 10 days in vitro and expressed TMEM119 (E) , TREM2 (F), and C3aR1 and C5aR1 (G,H) . Multiplex immunocytochemistry of primary cultures from neonatal mouse brains revealed C5aR1 expression was associated with IBA-1 + microglia but not astrocytes, oligodendrocyte precursor cells (GFAP+, Olig2+, respectively, I–J” ) or neurons (MAP2 + ; J”’ , arrows represent cells shown in inserts) in day 5 cultures. Scale bars = 20 μm.

Article Snippet: C3aR1 , Mouse , hC3aRZ8 , Hycult Biotech.

Techniques: Immunostaining, Derivative Assay, Cell Culture, In Vitro, Multiplex Assay, Immunocytochemistry, Expressing

Journal: Frontiers in Cellular Neuroscience

Article Title: Complement activation and increased anaphylatoxin receptor expression are associated with cortical grey matter lesions and the compartmentalised inflammatory response of multiple sclerosis

doi: 10.3389/fncel.2023.1094106

Figure Lengend Snippet: C5aR1 + microglia/macrophages are increased in density at the expanding grey matter lesion edge. Microglia/macrophages immunopositive for activation markers HLA-D and CD68 were quantified in leukocortical and subpial GM lesions (A,B) . The density of immunostained cells were significantly increased at the centre and edge, and at the edge of GM lesions, respectively, for both markers compared to non-neurological control GM at matched anatomical cortical level (A,B) . Examples of HLA-D (C,E) and CD68 (D,F) immunostaining in GM lesion (GML), normal appearing GM (NAGM), and control (ctrl). Quantification of C3aR1 + microglia/macrophages revealed a significant increase in quantity at the centre of deep cortical GM lesions compared to controls and NAGM and there were no significant changes in subpial GM lesions (G,I,K) . C5aR1 + microglia/macrophages were significantly elevated at the GM lesion centre and edge of leukocortical GM lesions compared to control GM, and at the GM lesion edge of subpial lesions compared to NAGM (H,J,L) . Blue arrows represent the expanding lesion edge of leukocortical and subpial lesions. Black arrows show the pial surface and grey lines represent the grey/white matter border. Data compared by Kruskal–Wallis and Dunn’s multiple comparison post-test. Scale bars = 100 μm.

Article Snippet: C3aR1 , Mouse , hC3aRZ8 , Hycult Biotech.

Techniques: Activation Assay, Immunostaining

Journal: Antioxidants

Article Title: Effect of Heme Oxygenase-1 Depletion on Complement Regulatory Proteins Expression in the Rat

doi: 10.3390/antiox12010061

Figure Lengend Snippet: CRP and C3aR expression in WT and Hmox1 -/- tissue samples and C3b deposition.

Article Snippet: Sections were incubated with either of anti-rat DAF (clone: RDIII-7), Crry (clone: TLD-1C11), CD59 (clone:TH9), C3/C3b (clone: 2B10B9B2) (Hycult, Uden, Netherlands) and C3aR (clone: D-12), (DTX, Santa Cruz, CA, USA) primary antibodies, followed by incubation with species-specific secondary antibodies, using established techniques.

Techniques: Expressing

Journal: Antioxidants

Article Title: Effect of Heme Oxygenase-1 Depletion on Complement Regulatory Proteins Expression in the Rat

doi: 10.3390/antiox12010061

Figure Lengend Snippet: C3aR staining of WT and Hmox1 −/− rat tissue sections. Staining of C3b was detected in all tissue sections examined. Magnification ×200.

Article Snippet: Sections were incubated with either of anti-rat DAF (clone: RDIII-7), Crry (clone: TLD-1C11), CD59 (clone:TH9), C3/C3b (clone: 2B10B9B2) (Hycult, Uden, Netherlands) and C3aR (clone: D-12), (DTX, Santa Cruz, CA, USA) primary antibodies, followed by incubation with species-specific secondary antibodies, using established techniques.

Techniques: Staining

Journal: Acta Neuropathologica Communications

Article Title: Protective effects of decay-accelerating factor on blast-induced neurotrauma in rats

doi: 10.1186/2051-5960-1-52

Figure Lengend Snippet: DAF treatment reduces interaction of C3a-C3aR in the rat brain tissue after blast exposure. Representative photomicrographs of C3a-C3aR interaction in frontal grey matter ( a ) and hippocampus (DG) ( b ) of frozen sections stained with anti-C3a (red) and anti-C3aR (green) antibodies. Original magnification of × 200 (grey matter) and × 400 (DG). Scale bars, 200 μm (grey matter) and 100 μm (DG). n = 8 for control, 3 and 24 h experimental groups. n = 5 for 48 h experimental groups.

Article Snippet: Mouse anti-rat C3a receptor (C3aR) and mouse anti-rat C5b-9 antibodies were acquired from Hycult Biotech Inc (Plymouth Meeting, PA).

Techniques: Staining

Journal: PLoS Pathogens

Article Title: Genome-scale CRISPR screening reveals that C3aR signaling is critical for rapid capture of fungi by macrophages

doi: 10.1371/journal.ppat.1010237

Figure Lengend Snippet: A. WT and C3ar-/- BMDMs were infected with live and PFA-killed mCherry-expressing Hc yeast (MOI2), and the phagocytosis rate was monitored over-time using flow-cytometry (n = 3 biological replicates). B. WT and C3ar-/- BMDMs were infected with FITC-labelled zymosan or mCherry-expressing Hc (MOI2) and the phagocytosis rate infected cells was monitored using flow cytometry (n = 3 biological replicates). C. BMDMs were infected with Candida albicans ( Ca ) (MOI3). Cells were imaged using confocal microscopy to quantify phagocytosis (n = 2 biological replicates, >350 cells/replicate). CFW staining was used to exclude extracellular Ca . D. BMDMs were infected with FITC-labelled Coccidioides posadasii ( Cp ) arthroconidia (MOI1), and extracellular conidia were labelled with calcofluor white. BMDM infection rates were determined using confocal microscopy (n = 3 biological replicates, 200–400 cells/rep). E. BMDMs were infected with FITC-labelled E . coli bioparticles (MOI4) and the E . coli -association with BMDMs was monitored via flow cytometry (n = 2 biological replicates). F. BMDMs were infected with 2 μm or 0.5 μm red fluorescent latex beads (MOI2), and the rate of BMDM association with the beads was measured using flow cytometry (n = 3 biological replicates). G. BMDMs were treated with a C3aR antagonist (1 μM SB290157) and infected with Hc yeast (MOI2). Phagocytosis was measured using flow cytometry (n = 3 biological replicates). H. BMDMs were pre-treated for 2 h with 1 μg/mL pertussis toxin (Ptx), which inhibits Gαi, and infected with Hc (MOI5, n = 3 biological replicates). I. BMDMs were pre-treated for 90 min with 10 μg/mL CD18 blocking antibody (GAME-46) and infected with Hc yeast (MOI5, n = 3 biological replicates) Phagocytosis was measured using flow cytometry. Emc1 is required for C3aR expression in BMDMs (J-L). J. Emc1 CRISPRKO BMDMs and control sgRNA transduced BMDMs, and C3aR levels were measured via flow cytometry following C3aR surface staining (n = 2 biological replicates). K. Histogram of C3aR levels in control and Emc1 CRISPRKO BMDMs. L. Frequency of C3aR+ cells in the indicated BMDMs. M. The mean fluorescence intensity (MFI) of the C3aR signal in the indicated BMDMs.

Article Snippet: Coverslips were blocked with PBS + 5% FBS for 1 h at RT, and stained with an anti-mouse C3aR antibody (Clone 14D4, Hycult, 1:1000) overnight at 4°C in 5% FBS.

Techniques: Infection, Expressing, Flow Cytometry, Confocal Microscopy, Staining, Blocking Assay, Fluorescence

Journal: PLoS Pathogens

Article Title: Genome-scale CRISPR screening reveals that C3aR signaling is critical for rapid capture of fungi by macrophages

doi: 10.1371/journal.ppat.1010237

Figure Lengend Snippet: A. FBS stimulates macrophage phagocytosis of fungi in a C3aR-dependent manner. BMDMs were infected with mCherry-expressing Hc or FITC-labelled zymosan (30 min, MOI5) in the presence or absence of 20% heat-treated FBS (FBS). Phagocytosis was assessed via flow cytometry (n = 3 biological replicates). B. FBS does not promote macrophage phagocytosis of Hc via opsonization. Hc and zymosan particles were pre-incubated with 10% heat-treated FBS for 30 min at 37°C, washed, and used to infect BMDMs (2h, MOI2). Phagocytosis was measured using flow cytometry (n = 2 biological replicates). C-D. Prolonged or intense heat-treatment and zymosan treatment eliminates the phagocytosis-stimulating properties of serum. C. Macrophage phagocytosis of Hc (MOI5, 45 min, n = 3 biological replicates) was assessed in media supplemented with 10% FBS that had been subjected to heat treatment (C) at 56°C for up to 2h, at 65°C for 30 min, or that had been pre-treated with zymosan (D) (1X10 8 particles/mL, 60 min at 37°C). Phagocytosis was measured by flow cytometry. E. Normal mouse serum (NMS) stimulates macrophage phagocytosis of Hc in a C3-dependenent manner. BMDMs were infected with Hc yeast (MOI = 5, 60min) in serum-free media or media supplemented with 5% FBS, 5% NMS from WT mice, 5% NMS from C3-/- mice, or 5% heat-inactivated NMS (hiNMS) from WT mice and phagocytosis was measured by flow cytometry (n = 3 biological replicates). F. BMDMs in serum-free media were infected with Hc opsonized with 10% WT or C3-/- NMS. Phagocytosis was measured by flow cytometry (n = 3 biological replicates). G. C5-deficient serum promotes macrophage phagocytosis of Hc in a C3aR-dependant manner. BMDMs were infected with Hc yeast (MOI5) in media supplemented with 5% NMS from C57BL/6 mice or DBA2 (C5-deficient) mice. Phagocytosis was measured by flow cytometry (n = 2 biological replicates). H-J. Normal human serum (NHS) stimulates macrophage phagocytosis of Hc yeast. H. BMDMs were infected with Hc (MOI5, 60 min) in media supplemented with 5% untreated, heat-inactivated, or C3-depleted (C3d) NHS, and phagocytosis was monitored by flow cytometry (n = 3 biological replicates). I. Hc was opsonized with 10% untreated or C3d NHS, used to infect BMDMs in serum-free media (MOI5, 60 min), and phagocytosis was monitored by flow cytometry (n = 3 biological replicates) J. BMDMs were infected with Hc (MOI5) in media supplemented with 5% untreated or C5-depleted (C5d) NHS, and phagocytosis was monitored by flow cytometry (n = 3 biological replicates). K-L. Mouse serum promotes complement opsonization of yeast and release of C3a via multiple pathways. Hc was incubated in 10% serum from WT, C3-/- , or DBA2 mice for 30 min at 37°C. 10 mM EGTA or EDTA were added to the reactions to chelate Ca 2+ or Mg 2+ , respectively. K. Supernatants were harvested following incubation, and mouse C3a levels were measured by ELISA (n = 3 biological replicates). L. Yeast were stained with a FITC conjugated anti-mouse C3, and imaged using confocal microscopy (representative slices are shown from 2 biological replicates).

Article Snippet: Coverslips were blocked with PBS + 5% FBS for 1 h at RT, and stained with an anti-mouse C3aR antibody (Clone 14D4, Hycult, 1:1000) overnight at 4°C in 5% FBS.

Techniques: Infection, Expressing, Flow Cytometry, Incubation, Enzyme-linked Immunosorbent Assay, Staining, Confocal Microscopy

Journal: PLoS Pathogens

Article Title: Genome-scale CRISPR screening reveals that C3aR signaling is critical for rapid capture of fungi by macrophages

doi: 10.1371/journal.ppat.1010237

Figure Lengend Snippet: C3aR localizes to Hc -containing phagosomes (A) to a greater extent than latex bead-containing phagosomes (B). BMDMs were infected with the indicated particles (MOI = 5, n = 2 biological replicates per time point). Cells were then stained with a C3aR-specific antibody and imaged using optical sectioning with a confocal microscope. Representative images from a single slice are shown. C. Enlarged views of insets outlined in panels A and B by a white box. Scale bar = 20 μm. D. The mean fluorescence intensity of C3aR in the particle-containing phagosomes was quantified using ImageJ (N>91 phagosomes, **** p<0.0001, **p<0.01 by two-tailed Wilcoxon rank-sum test). The line represents the median phagosomal C3aR intensity.

Article Snippet: Coverslips were blocked with PBS + 5% FBS for 1 h at RT, and stained with an anti-mouse C3aR antibody (Clone 14D4, Hycult, 1:1000) overnight at 4°C in 5% FBS.

Techniques: Infection, Staining, Microscopy, Fluorescence, Two Tailed Test

Journal: PLoS Pathogens

Article Title: Genome-scale CRISPR screening reveals that C3aR signaling is critical for rapid capture of fungi by macrophages

doi: 10.1371/journal.ppat.1010237

Figure Lengend Snippet: J774A.1 cells were engineered to express Lifeact-mEGFP to label F-actin, co-cultured with mCherry-expressing Hc yeast, and subjected to live-cell confocal microscopy in a temperature-and-CO 2 controlled chamber in media supplemented with 10% FBS. Cells were treated with a C3aR antagonist (10 μM SB290157) or a vehicle control. A. Representative images from a confocal time series showing a macrophage extending an F-actin-rich protrusion towards an mCherry expressing Hc yeast, followed by phagocytosis and formation of an actin-rich phagosome. The corresponding DIC images are shown below. B. A similar time series of macrophages treated with SB290157 showing a failure to initiate formation of a membrane protrusion and much slower capture of Hc yeast. Scale bar = 20 μm. The movement of membrane structures that successfully caputured yeast were analyzed using MtrackJ to quantify the behaviors of these structures (C-E) (n = 2 biological replicates, >50 tracks per replicate), including the phagocytosis rate, quantified as the time required for the macrophage to successfully engulf the yeast divided by the distance of the yeast to the macrophage at the start of the series (C), the mean velocity of the membrane structure closest to the yeast (D), and the outreach ratio quantified as the max displacement of the track divided by the length of the track (E) (**** p<0.0001 by two-tailed Wilcoxon rank sum test). The line represents the median measurement.

Article Snippet: Coverslips were blocked with PBS + 5% FBS for 1 h at RT, and stained with an anti-mouse C3aR antibody (Clone 14D4, Hycult, 1:1000) overnight at 4°C in 5% FBS.

Techniques: Cell Culture, Expressing, Confocal Microscopy, Two Tailed Test

Journal: PLoS Pathogens

Article Title: Genome-scale CRISPR screening reveals that C3aR signaling is critical for rapid capture of fungi by macrophages

doi: 10.1371/journal.ppat.1010237

Figure Lengend Snippet: A-B C3ar-/- mice (n≥10) and age-matched WT C57BL/6 mice (n≥10) were infected intranasally with varying doses of Hc yeast to initiate either a sub-lethal (A) or lethal (B) infection. D. C3-/- mice and age-matched WT mice were infected intranasally with a sub-lethal dose of Hc yeast. Susceptibility is illustrated by a Kaplan-Meier survival curve. ns = not significant, *p < 0.05 by logrank test. C,E. The indicated mice were infected with a sub-lethal dose of Hc . The fungal burden in lung and spleen homogenates was determined by enumeration of colony forming units (CFUs) at the indicated time points (n≥5). X-axis label for C is the same as that indicated for E.

Article Snippet: Coverslips were blocked with PBS + 5% FBS for 1 h at RT, and stained with an anti-mouse C3aR antibody (Clone 14D4, Hycult, 1:1000) overnight at 4°C in 5% FBS.

Techniques: Infection

Journal: PLoS Pathogens

Article Title: Genome-scale CRISPR screening reveals that C3aR signaling is critical for rapid capture of fungi by macrophages

doi: 10.1371/journal.ppat.1010237

Figure Lengend Snippet: We propose the following model for the role of complement and C3aR in macrophage recognition of Hc : C3, derived from serum, reacts with the Hc cell-wall, leading to C3b/iC3b deposition on the cell-wall, and release of C3a, which diffuses away from the yeast surface leading to a concentration gradient emanating from the yeast cell-wall. C3a activates C3aR, which signals through Gαi and Gβ2 to promote the formation and directional movement of actin-rich membrane protrusions, and possibly to promote local activation or increased motility of the integrin receptor CR3. Active CR3 can then recognize C3b/iC3b or other features of the Hc cell-wall. C3aR and/or CR3 activation then coordinates actin polymerization and phagocytic cup formation by regulating the activity of actin polymerization regulators Arp2/3 and SCAR/WAVE. In the presence of C5-containing serum, the C5 convertase can similarly catalyze the cleavage of C5 at the fungal surface, leading to release of C5a and activation of C5aR, which may also drive local chemotaxis and activation of phagocytic integrins to promote phagocytosis.

Article Snippet: Coverslips were blocked with PBS + 5% FBS for 1 h at RT, and stained with an anti-mouse C3aR antibody (Clone 14D4, Hycult, 1:1000) overnight at 4°C in 5% FBS.

Techniques: Derivative Assay, Concentration Assay, Activation Assay, Activity Assay, Chemotaxis Assay

Journal: PLoS Pathogens

Article Title: Genome-scale CRISPR screening reveals that C3aR signaling is critical for rapid capture of fungi by macrophages

doi: 10.1371/journal.ppat.1010237

Figure Lengend Snippet: A. WT and C3ar-/- BMDMs were infected with live and PFA-killed mCherry-expressing Hc yeast (MOI2), and the phagocytosis rate was monitored over-time using flow-cytometry (n = 3 biological replicates). B. WT and C3ar-/- BMDMs were infected with FITC-labelled zymosan or mCherry-expressing Hc (MOI2) and the phagocytosis rate infected cells was monitored using flow cytometry (n = 3 biological replicates). C. BMDMs were infected with Candida albicans ( Ca ) (MOI3). Cells were imaged using confocal microscopy to quantify phagocytosis (n = 2 biological replicates, >350 cells/replicate). CFW staining was used to exclude extracellular Ca . D. BMDMs were infected with FITC-labelled Coccidioides posadasii ( Cp ) arthroconidia (MOI1), and extracellular conidia were labelled with calcofluor white. BMDM infection rates were determined using confocal microscopy (n = 3 biological replicates, 200–400 cells/rep). E. BMDMs were infected with FITC-labelled E . coli bioparticles (MOI4) and the E . coli -association with BMDMs was monitored via flow cytometry (n = 2 biological replicates). F. BMDMs were infected with 2 μm or 0.5 μm red fluorescent latex beads (MOI2), and the rate of BMDM association with the beads was measured using flow cytometry (n = 3 biological replicates). G. BMDMs were treated with a C3aR antagonist (1 μM SB290157) and infected with Hc yeast (MOI2). Phagocytosis was measured using flow cytometry (n = 3 biological replicates). H. BMDMs were pre-treated for 2 h with 1 μg/mL pertussis toxin (Ptx), which inhibits Gαi, and infected with Hc (MOI5, n = 3 biological replicates). I. BMDMs were pre-treated for 90 min with 10 μg/mL CD18 blocking antibody (GAME-46) and infected with Hc yeast (MOI5, n = 3 biological replicates) Phagocytosis was measured using flow cytometry. Emc1 is required for C3aR expression in BMDMs (J-L). J. Emc1 CRISPRKO BMDMs and control sgRNA transduced BMDMs, and C3aR levels were measured via flow cytometry following C3aR surface staining (n = 2 biological replicates). K. Histogram of C3aR levels in control and Emc1 CRISPRKO BMDMs. L. Frequency of C3aR+ cells in the indicated BMDMs. M. The mean fluorescence intensity (MFI) of the C3aR signal in the indicated BMDMs.

Article Snippet: Cells were blocked for 20min with PBS5 (PBS+5% FBS) and stained with a C3aR antibody (Clone 14D4, Hycult, 1:500) in PBS5 for 20min on ice.

Techniques: Infection, Expressing, Flow Cytometry, Confocal Microscopy, Staining, Blocking Assay, Fluorescence

Journal: PLoS Pathogens

Article Title: Genome-scale CRISPR screening reveals that C3aR signaling is critical for rapid capture of fungi by macrophages

doi: 10.1371/journal.ppat.1010237

Figure Lengend Snippet: A. FBS stimulates macrophage phagocytosis of fungi in a C3aR-dependent manner. BMDMs were infected with mCherry-expressing Hc or FITC-labelled zymosan (30 min, MOI5) in the presence or absence of 20% heat-treated FBS (FBS). Phagocytosis was assessed via flow cytometry (n = 3 biological replicates). B. FBS does not promote macrophage phagocytosis of Hc via opsonization. Hc and zymosan particles were pre-incubated with 10% heat-treated FBS for 30 min at 37°C, washed, and used to infect BMDMs (2h, MOI2). Phagocytosis was measured using flow cytometry (n = 2 biological replicates). C-D. Prolonged or intense heat-treatment and zymosan treatment eliminates the phagocytosis-stimulating properties of serum. C. Macrophage phagocytosis of Hc (MOI5, 45 min, n = 3 biological replicates) was assessed in media supplemented with 10% FBS that had been subjected to heat treatment (C) at 56°C for up to 2h, at 65°C for 30 min, or that had been pre-treated with zymosan (D) (1X10 8 particles/mL, 60 min at 37°C). Phagocytosis was measured by flow cytometry. E. Normal mouse serum (NMS) stimulates macrophage phagocytosis of Hc in a C3-dependenent manner. BMDMs were infected with Hc yeast (MOI = 5, 60min) in serum-free media or media supplemented with 5% FBS, 5% NMS from WT mice, 5% NMS from C3-/- mice, or 5% heat-inactivated NMS (hiNMS) from WT mice and phagocytosis was measured by flow cytometry (n = 3 biological replicates). F. BMDMs in serum-free media were infected with Hc opsonized with 10% WT or C3-/- NMS. Phagocytosis was measured by flow cytometry (n = 3 biological replicates). G. C5-deficient serum promotes macrophage phagocytosis of Hc in a C3aR-dependant manner. BMDMs were infected with Hc yeast (MOI5) in media supplemented with 5% NMS from C57BL/6 mice or DBA2 (C5-deficient) mice. Phagocytosis was measured by flow cytometry (n = 2 biological replicates). H-J. Normal human serum (NHS) stimulates macrophage phagocytosis of Hc yeast. H. BMDMs were infected with Hc (MOI5, 60 min) in media supplemented with 5% untreated, heat-inactivated, or C3-depleted (C3d) NHS, and phagocytosis was monitored by flow cytometry (n = 3 biological replicates). I. Hc was opsonized with 10% untreated or C3d NHS, used to infect BMDMs in serum-free media (MOI5, 60 min), and phagocytosis was monitored by flow cytometry (n = 3 biological replicates) J. BMDMs were infected with Hc (MOI5) in media supplemented with 5% untreated or C5-depleted (C5d) NHS, and phagocytosis was monitored by flow cytometry (n = 3 biological replicates). K-L. Mouse serum promotes complement opsonization of yeast and release of C3a via multiple pathways. Hc was incubated in 10% serum from WT, C3-/- , or DBA2 mice for 30 min at 37°C. 10 mM EGTA or EDTA were added to the reactions to chelate Ca 2+ or Mg 2+ , respectively. K. Supernatants were harvested following incubation, and mouse C3a levels were measured by ELISA (n = 3 biological replicates). L. Yeast were stained with a FITC conjugated anti-mouse C3, and imaged using confocal microscopy (representative slices are shown from 2 biological replicates).

Article Snippet: Cells were blocked for 20min with PBS5 (PBS+5% FBS) and stained with a C3aR antibody (Clone 14D4, Hycult, 1:500) in PBS5 for 20min on ice.

Techniques: Infection, Expressing, Flow Cytometry, Incubation, Enzyme-linked Immunosorbent Assay, Staining, Confocal Microscopy

Journal: PLoS Pathogens

Article Title: Genome-scale CRISPR screening reveals that C3aR signaling is critical for rapid capture of fungi by macrophages

doi: 10.1371/journal.ppat.1010237

Figure Lengend Snippet: C3aR localizes to Hc -containing phagosomes (A) to a greater extent than latex bead-containing phagosomes (B). BMDMs were infected with the indicated particles (MOI = 5, n = 2 biological replicates per time point). Cells were then stained with a C3aR-specific antibody and imaged using optical sectioning with a confocal microscope. Representative images from a single slice are shown. C. Enlarged views of insets outlined in panels A and B by a white box. Scale bar = 20 μm. D. The mean fluorescence intensity of C3aR in the particle-containing phagosomes was quantified using ImageJ (N>91 phagosomes, **** p<0.0001, **p<0.01 by two-tailed Wilcoxon rank-sum test). The line represents the median phagosomal C3aR intensity.

Article Snippet: Cells were blocked for 20min with PBS5 (PBS+5% FBS) and stained with a C3aR antibody (Clone 14D4, Hycult, 1:500) in PBS5 for 20min on ice.

Techniques: Infection, Staining, Microscopy, Fluorescence, Two Tailed Test

Journal: PLoS Pathogens

Article Title: Genome-scale CRISPR screening reveals that C3aR signaling is critical for rapid capture of fungi by macrophages

doi: 10.1371/journal.ppat.1010237

Figure Lengend Snippet: J774A.1 cells were engineered to express Lifeact-mEGFP to label F-actin, co-cultured with mCherry-expressing Hc yeast, and subjected to live-cell confocal microscopy in a temperature-and-CO 2 controlled chamber in media supplemented with 10% FBS. Cells were treated with a C3aR antagonist (10 μM SB290157) or a vehicle control. A. Representative images from a confocal time series showing a macrophage extending an F-actin-rich protrusion towards an mCherry expressing Hc yeast, followed by phagocytosis and formation of an actin-rich phagosome. The corresponding DIC images are shown below. B. A similar time series of macrophages treated with SB290157 showing a failure to initiate formation of a membrane protrusion and much slower capture of Hc yeast. Scale bar = 20 μm. The movement of membrane structures that successfully caputured yeast were analyzed using MtrackJ to quantify the behaviors of these structures (C-E) (n = 2 biological replicates, >50 tracks per replicate), including the phagocytosis rate, quantified as the time required for the macrophage to successfully engulf the yeast divided by the distance of the yeast to the macrophage at the start of the series (C), the mean velocity of the membrane structure closest to the yeast (D), and the outreach ratio quantified as the max displacement of the track divided by the length of the track (E) (**** p<0.0001 by two-tailed Wilcoxon rank sum test). The line represents the median measurement.

Article Snippet: Cells were blocked for 20min with PBS5 (PBS+5% FBS) and stained with a C3aR antibody (Clone 14D4, Hycult, 1:500) in PBS5 for 20min on ice.

Techniques: Cell Culture, Expressing, Confocal Microscopy, Two Tailed Test

Journal: PLoS Pathogens

Article Title: Genome-scale CRISPR screening reveals that C3aR signaling is critical for rapid capture of fungi by macrophages

doi: 10.1371/journal.ppat.1010237

Figure Lengend Snippet: A-B C3ar-/- mice (n≥10) and age-matched WT C57BL/6 mice (n≥10) were infected intranasally with varying doses of Hc yeast to initiate either a sub-lethal (A) or lethal (B) infection. D. C3-/- mice and age-matched WT mice were infected intranasally with a sub-lethal dose of Hc yeast. Susceptibility is illustrated by a Kaplan-Meier survival curve. ns = not significant, *p < 0.05 by logrank test. C,E. The indicated mice were infected with a sub-lethal dose of Hc . The fungal burden in lung and spleen homogenates was determined by enumeration of colony forming units (CFUs) at the indicated time points (n≥5). X-axis label for C is the same as that indicated for E.

Article Snippet: Cells were blocked for 20min with PBS5 (PBS+5% FBS) and stained with a C3aR antibody (Clone 14D4, Hycult, 1:500) in PBS5 for 20min on ice.

Techniques: Infection

Journal: PLoS Pathogens

Article Title: Genome-scale CRISPR screening reveals that C3aR signaling is critical for rapid capture of fungi by macrophages

doi: 10.1371/journal.ppat.1010237

Figure Lengend Snippet: We propose the following model for the role of complement and C3aR in macrophage recognition of Hc : C3, derived from serum, reacts with the Hc cell-wall, leading to C3b/iC3b deposition on the cell-wall, and release of C3a, which diffuses away from the yeast surface leading to a concentration gradient emanating from the yeast cell-wall. C3a activates C3aR, which signals through Gαi and Gβ2 to promote the formation and directional movement of actin-rich membrane protrusions, and possibly to promote local activation or increased motility of the integrin receptor CR3. Active CR3 can then recognize C3b/iC3b or other features of the Hc cell-wall. C3aR and/or CR3 activation then coordinates actin polymerization and phagocytic cup formation by regulating the activity of actin polymerization regulators Arp2/3 and SCAR/WAVE. In the presence of C5-containing serum, the C5 convertase can similarly catalyze the cleavage of C5 at the fungal surface, leading to release of C5a and activation of C5aR, which may also drive local chemotaxis and activation of phagocytic integrins to promote phagocytosis.

Article Snippet: Cells were blocked for 20min with PBS5 (PBS+5% FBS) and stained with a C3aR antibody (Clone 14D4, Hycult, 1:500) in PBS5 for 20min on ice.

Techniques: Derivative Assay, Concentration Assay, Activation Assay, Activity Assay, Chemotaxis Assay