Journal: Acta Neuropathologica Communications

Article Title: Mass cytometric analysis of the immune cell landscape after traumatic brain injury elucidates the role of complement and complement receptors in neurologic outcomes

doi: 10.1186/s40478-023-01583-0

Figure Lengend Snippet: List of CyTOF antibodies and their conjugated heavy metals

Article Snippet: 4 , C3aR , Hycult , 14D4 , HM1123 , Dy , 161.

Techniques:

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: 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: bioRxiv

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

doi: 10.1101/2021.12.30.474615

Figure Lengend Snippet: A. Characterization of Hc- mediated lysis in J774A.1 macrophage-like cells. J774A.1 cells were infected with WT Hc, or Hc with a disruption in a gene, CBP1 , that is required for Hc to lyse macrophages. Lysis over time was measured using the LDH release assay. B. Validation and clonal expansion of Cas9-expressing J774A.1 cells. Cells were transduced with an Ef1a-Cas9-Blast expression vector and grown under blasticidin selection to generate a population of Cas9-expressing cells. These were subjected to single-cell sorting and clonal expansion to generate Cas9-expressing J774A.1 clones with high Cas9 activity. Cas9 activity was measured by transducing J774A.1 cells with a guide RNA vector that co-expressed EGFP with a sgRNA targeting EGFP. Cas9 activity leads to silencing of the GFP following puromycin selection. Cas9 clone 9 was chosen for the large-scale CRISPR screens due to its high-efficiency GFP silencing. C-D. Characterizing lysis and recovery from infection with uracil pulses during infection with a Ura5-deficient Hc . J774A.1 macrophages were infected with ura5 mutant Hc in the presence or absence of exogenous uracil (0.4ug/mL). Uracil-containing cells were washed and media was replaced with uracil-poor media after 2d of lysis, which allowed the macrophages to recover. Recovery was assessed using LDH release quantification to assess lysis, and the confluency of viable cells in the wells was estimated using the pico-green dsDNA assay kit following lysis of macrophages with water. E. macrophages that had been recovered from lysis by removal of uracil from culture media were passaged for several days, and uracil was added to selected wells. Macrophage lysis over time was monitored by assessing LDH release over time to determine whether dormant yeast would be able to re-activate upon introduction of uracil. F. Reproducibility of the casTLE score across two replicates of the screens. G. Histograms comparing the distribution of negative control sgRNAs and sgRNAs targeting Gnb2 or C3ar in the H. capsulatum infected pool compared to the uninfected pool. H. Analysis of essential gene behavior during J7 library growth. Scatter plot showing the gene effect resulting from passaging of J7s, either going from the plasmid pool to the T0 pool, or the T0 pool to the uninfected pool. Genes annotated as “essential” or “non-essential” were plotted to determine whether essential genes appeared more likely to drop out of the uninfected pools.

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: Lysis, Infection, Lactate Dehydrogenase Assay, Expressing, Transduction, Plasmid Preparation, Selection, FACS, Clone Assay, Activity Assay, CRISPR, Mutagenesis, dsDNA Assay, Negative Control, Passaging

Journal: bioRxiv

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

doi: 10.1101/2021.12.30.474615

Figure Lengend Snippet: A. WT and C3ar-/- BMDMs were infected with live and PFA-killed mCherry-expressing Hc yeast, 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 and the phagocytosis rate infected cells was monitored using flow cytometry (n=3 biological replicates). C. BMDMs were infected with Candida albicans ( Ca ). 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, 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 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, 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. 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 (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 (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 were stained with an anti-C3aR antibody, and C3aR levels were measured via flow cytometry (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: bioRxiv

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

doi: 10.1101/2021.12.30.474615

Figure Lengend Snippet: BMDMs from C3ar-/- and WT mice, in addition to BMDMs expressing Cas9 and control or Emc1- targeting sgRNAs, were stained with anti-CD18 and anti-CD11b antibodies and analyzed by flow cytometry (n=2 biological replicates). A. Representative histograms showing CD11b and CD18 levels in control, C3ar-/- , and Emc1 CRISPRKO BMDMs. The percentage of CD11b ( B ) and CD18 ( D ) positive macrophages was analyzed. The mean fluorescence intensity of CD11b ( C ) and CD18 ( D ) were also measured.

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: Expressing, Staining, Flow Cytometry, Fluorescence

Journal: bioRxiv

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

doi: 10.1101/2021.12.30.474615

Figure Lengend Snippet: WT and C3ar-/- BMDMs were infected with Hc (MOI=5 for 6 h), and TNFα levels in macrophage supernatants were measured using the BD Cytometric Bead Array kit (n=3 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

Journal: bioRxiv

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

doi: 10.1101/2021.12.30.474615

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 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. Phagocytosis was measured using flow cytometry (n=2 biological replicates). C. Normal mouse serum (NMS) stimulates BMDM phagocytosis of fungi in a C3-dependenent manner. BMDMs were infected with Hc yeast 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. BMDMs in serum-free media were also infected with Hc opsonized with 10% WT or C3-/- NMS. Phagocytosis was measured as described above (n=3 biological replicates). D. C5-deficient serum promotes macrophage phagocytosis of Hc in a C3aR-dependant manner. BMDMs were infected with Hc yeast in media supplemented with 5% NMS from C57BL/6 mice or DBA2 (C5-deficient) mice. Phagocytosis was measured as described above (n=2 biological replicates). E-F . Mouse serum promotes complement opsonization of yeast via multiple pathways. Hc ( E ) or Zymosan ( F ) were incubated in PBS with 10% sera from WT or C3-/- mice. 10 mM EGTA or EDTA were added to the reactions to chelate Ca 2+ or Mg 2+ , respectively. Yeast were stained with FITC conjugated anti-mouse C3, and imaged using confocal microscopy (representative slices are shown from 2 biological replicates). G. Incubation of Hc with mouse serum leads to C3a release. Supernatants were harvested following incubation of mouse serum with Hc or zymosan, and mouse C3a levels were measured by ELISA.

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, Staining, Confocal Microscopy, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

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

doi: 10.1101/2021.12.30.474615

Figure Lengend Snippet: ( A-C ) BMDMs from WT and C3ar-/- mice were infected with Hc in the presence of 20% FBS from three different lots from 2 separate suppliers. In addition, WT BMDMs differentiated in different lots of serum were treated with 10 µM of the C3aR antagonist and infected with Hc ( B ). Phagocytosis of Hc was measured by flow cytometry as described previously (n=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, Flow Cytometry

Journal: bioRxiv

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

doi: 10.1101/2021.12.30.474615

Figure Lengend Snippet: Uninfected WT and C3ar-/- BMDMs were stained with a C3aR-specific antibody and imaged using confocal microscopy and optical sectioning. Representative slices of 2 biological replicates are shown. The antibody specifically detects C3aR, as staining was not observed in C3ar-/- BMDMs. C3aR exhibits punctate localization near the plasma membrane in WT 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: Staining, Confocal Microscopy

Journal: bioRxiv

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

doi: 10.1101/2021.12.30.474615

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. The mean fluorescence intensity of C3aR in the particle-containing phagosomes was quantified using ImageJ (N>91 phagosomes).

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

Journal: bioRxiv

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

doi: 10.1101/2021.12.30.474615

Figure Lengend Snippet: A. Hc stimulates chemotaxis of J774A.1 macrophage-like cells. H. capsulatum yeast were seeded into multiple-well plates at varying concentrations, and WT J774A.1 cells were seeded onto transwell permeable supports with 5 μm pores. Serum-free media supplemented with 0.25% BSA was used as the diluent in both the chamber and well unless otherwise indicated. After 3 h of migration, transwells were stained with crystal violet, and non-migratory cells were wiped off of the upper side of the transwell using a Q-tip. The number of migratory cells in each condition was quantified by microscopy (n=2 biogical replicates, 3 fields/biological replicate). B. Migration towards Hc is Gai-dependent. J774A.1 cells with or without pre-treatment with 1 µg/mL pertussis toxin (PTX) for 2 h were seeded into transwell permeable supports and migration towards 1e5 Hc/uL was quantified as described above. The number of migrating cells was quantified as described. C. The C3aR antagonist does not inhibit macrophage migration towards Hc. J774A.1 macrophages were treated with 1 µM SB290157, a C3aR antagonist, and migration towards H. capsulatum was assessed as described. D. C3aR-deficiency moderately impacts migration of J774A.1 cells towards Hc. Cas9-expressing J774A.1 macrophages transduced with non-targeting or C3aR-targeting sgRNAs were assessed for their ability to migrate towards Hc as described previously. E. Hc- dependent migration is abolished in the presence of FBS. The transwell migration assay was performed with media supplemented with BSA or 10% FBS to determine whether FBS affected the migration of macrophage-like cells towards Hc yeast.

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: Chemotaxis Assay, Migration, Staining, Microscopy, Expressing, Transduction, Transwell Migration Assay

Journal: bioRxiv

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

doi: 10.1101/2021.12.30.474615

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. The movement of membrane structures that successfully caputured yeast were analyzed using MtrackJ to quantify the behaviors of these structures ( C-E ), 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 ). These metrics demonstrate that macrophages treated with the C3aR antagonist are defective at the extension of membrane protrusions in the direction of Hc yeast that facilitate 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: Cell Culture, Expressing, Confocal Microscopy

Journal: bioRxiv

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

doi: 10.1101/2021.12.30.474615

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 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.

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