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Identification of phagocytosis regulators using magnetic genome-wide CRISPR screens

Abstract

Phagocytosis is required for a broad range of physiological functions, from pathogen defense to tissue homeostasis, but the mechanisms required for phagocytosis of diverse substrates remain incompletely understood. Here, we developed a rapid magnet-based phenotypic screening strategy, and performed eight genome-wide CRISPR screens in human cells to identify genes regulating phagocytosis of distinct substrates. After validating select hits in focused miniscreens, orthogonal assays and primary human macrophages, we show that (1) the previously uncharacterized gene NHLRC2 is a central player in phagocytosis, regulating RhoA-Rac1 signaling cascades that control actin polymerization and filopodia formation, (2) very-long-chain fatty acids are essential for efficient phagocytosis of certain substrates and (3) the previously uncharacterized Alzheimer’s disease–associated gene TM2D3 can preferentially influence uptake of amyloid-β aggregates. These findings illuminate new regulators and core principles of phagocytosis, and more generally establish an efficient method for unbiased identification of cellular uptake mechanisms across diverse physiological and pathological contexts.

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Fig. 1: Genome-wide CRISPR screening for genetic regulators of phagocytosis.
Fig. 2: Putative modifiers of phagocytosis of diverse substrates from eight genome-wide screens.
Fig. 3: Orthogonal measurements of phagocytosis validate magnetic screen results.
Fig. 4: Loss of NHLRC2 inhibits phagocytosis through Rho/Rac1 signaling.
Fig. 5: Role for VLCFAs in phagocytosis.
Fig. 6: Identification of substrate specific regulators of phagocytosis.

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Data availability

The RNA-seq FASTQ files are available at GEO under accession GSE107566.

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Acknowledgements

We thank the members of the Bassik and Barres laboratories for feedback and support throughout, and S. Grinstein, J. Pritchard, J. Pluvinage, and T. Wyss-Coray for helpful suggestions. We thank T. Flores, J. Mulholland and J. Perrino of the Stanford Electron Microscopy facility for excellent assistance with SEM images, and D. Lysko and W. Talbot for experimental advice. This work was supported by the Christopher and Dana Reeve Foundation International Research Consortium on Spinal Cord Injury, the Dr. Miriam and Sheldon G. Adelson Medical Research Foundation, the JPB Foundation, the Novartis Institute of Basic Research, a National Institutes of Health (NIH) grant to B.A.B. (R01 DA015043), a Jane Coffin Childs Fellowship to R.A.K., the Howard Hughes Medical Institute (J.A.T. and D.T.) and the Stanford Medical Scientist Training Program NIH T32-GM007365 (for L.L.), a Kimmel Scholar Award, an NIH Director’s New Innovator Award (DP2 HD094656) to S.R.C., an NIH Director’s New Innovator Award (1DP2HD084069-01) and Stanford Neuroscience Institute Brain Rejuvenation Project Award to M.C.B., and generous contributions from Vincent and Stella Coates. C.J.B. was supported by the Damon Runyon Cancer Research Foundation (DRG-2125-12).

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Authors and Affiliations

Authors

Contributions

C.J.B., M.S.H., B.A.B., S.R.C. and M.C.B. conceptualized the study. C.J.B., M.S.H. and D.W.M. generated and analyzed screen data. A.L. assisted in the cloning of sgRNA libraries. K.T. performed FACS-based phagocytosis screening. J.A.O. and B.E. assisted in generating sgRNA-expressing U937 and RAW 264.7 cell lines and clonally derived knockout lines. H.C. and A.T. assisted in performing phagocytosis microscopy assays. J.A.O. made BirA-NHLRC2 construct and prepared samples for mass spectrometry with advice from E.C.; J.C. and L.J. ran mass spectrometry samples and assisted with analysis; E.R. performed neutrophil migration assays with S.R.C.; J.T., D.V., L.L. and R.L. advised on microscopy experiments. R.L. advised on and performed phalloidin microscopy for NHLRC2 knockouts and frustrated phagocytosis assay.

Corresponding authors

Correspondence to Christopher J. Bohlen or Michael C. Bassik.

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Integrated supplementary information

Supplementary Figure 1 Differentiated U937 cells are highly phagocytic.

a,b, Staining of undifferentiated (a) or PMA-differentiated (b) U937 cells with a CD11b antibody (green) and DAPI (blue). Representative of two independent experiments. c, Flow cytometry of CD11b levels in undifferentiated or PMA-differentiated U937 cells. Representative of two independent experiments. d, Phagocytic index of differentiated (red), undifferentiated (green), or differentiated U937 cells treated with the actin polymerization inhibitor cytochalasin D (blue). Phagocytic index is measured by live-cell microscopy using 1.3-μm beads labeled with the pH-sensitive dye pHrodo, which becomes fluorescent upon reaching lysosomal pH. Phagocytic index was calculated at each time point by measuring the total area of pHrodo signal normalized by the total area of live cells (indicated by calcein AM), and the index is presented relative to the average value of the control condition at the time point at 5 h. Values represent mean ± s.e.m. of n = 4 replicate wells. Representative of three independent experiments. e, Phagocytic index for pHrodo-labeled zymosan in differentiated U937 cells (red) and differentiated U937 cells treated with the actin polymerization inhibitor cytochalasin D (blue). Values represent the means ± s.e.m. of n = 4 replicate wells. Representative of three independent experiments. f, Proportion of WT differentiated U937 cells in the bound fraction of a magnetic column after the addition 1.3-μm magnetic beads (red) and proportion of WT differentiated U937 cells treated with the actin polymerization inhibitor cytochalasin D in the bound fraction of a magnetic column after the addition of 1.3-μm magnetic beads. g, Schematic of manually categorized genes passing 10% FDR (by casTLE) according to cellular process or compartment.

Supplementary Figure 2 Generalization of the magnetic based phagocytosis screening method to diverse substrates.

a, Myelin phagocytosis by differentiated U937 cells (red) is inhibited by addition of the actin polymerization inhibitor cytochalasin D (blue). Phagocytic index was calculated at each time point by measuring the total area of pHrodo signal normalized by the total area of live cells (indicated by calcein AM), and the index is presented relative to the average value of the control condition at the time point at 5 h. Values represent mean ± s.e.m. of n = 4 replicate wells. Representative of three independent experiments. b, Titration of IONP concentration applied to myelin shows an increase in the proportion of differentiated U937 cells bound to the magnetic column after phagocytosis (red), while titration of IONPs alone shows minimal increase in differentiated U937 cells bound to a magnetic column (blue). Magnetic labeling density chosen for genome-wide screens is indicated with the gray dotted line. c, Schematic of the pilot experiment to demonstrate the method of separating differentiated U937 cells based on the amount of IONP-labeled material phagocytosed. Myelin labeled with pHrodo-red and IONPs was applied to unlabeled U937 cells, allowing for 6 h of phagocytosis. Alternatively, myelin labeled with pHrodo-red and IONPs was applied to a second population of U937 cells labeled with calcein AM (green), allowing for 2 h of phagocytosis. These two populations were then mixed 1:1 and separated by a magnetic column. d, Microscopy shows an even distribution of calcein AM–labeled U937 cells (2 h of phagocytosis) and U937 cells with pHrodo-red myelin (6 h of phagocytosis) before magnetic separation, but an enrichment of pHrodo-red-labeled myelin in the bound column (without calcein AM) and a corresponding enrichment of green-labeled cells in the unbound column. Representative of three independent experiments. e, Proportion of differentiated U937 cells bound to the magnetic column after IONP-labeled myelin was applied for 2 or 6 h. Values represent mean ± s.e.m. of n = 4 replicate wells. Representative of three independent experiments. f, Proportion of a 1:1 mixture of green and unlabeled differentiated U937 cells in each fraction before and after separation on a magnetic column. g, Microscopy demonstrating enrichment of pHrodo- (red) and IONP-labeled red blood cells opsonized with IgG in the bound fraction of a magnetic column compared to the unbound fraction. Live cells were labeled with calcein AM dye (green). Representative of two independent experiments. h, Microscopy demonstrating enrichment of pHrodo- (red) and IONP-labeled zymosan in the bound fraction of a magnetic column compared to the unbound fraction. Live cells were labeled with calcein AM dye (green). Representative of two independent experiments. i, Demonstration that red blood cells labeled with IONPs but not opsonized with IgG (gray) are not phagocytosed at the same rate as red blood cells labeled with IONPs and opsonized with IgG (red). This phagocytosis was eliminated with the actin polymerization inhibitor cytochalasin D (blue). Values represent the means ± s.e.m. of n = 4 replicate wells. Representative of three independent experiments. j, Demonstration that red blood cells labeled with IONPs but not opsonized with C3b (gray) are not phagocytosed at the same rate as red blood cells labeled with IONPs and opsonized with C3b (red). Phagocytosis was eliminated with the actin polymerization inhibitor cytochalasin D (blue). Values represent mean ± s.e.m. of n = 4 replicate wells. Representative of three independent experiments.

Supplementary Figure 3 mRNA expression in myeloid cells of genes discovered to be phagocytosis screen hits and correlation of replicate screens.

a, mRNA expression levels of genes passing 10% FDR for each substrate in differentiated U937 cells and primary human microglia. Plots illustrate mean (bar), minimum, and maximum values, with each gene’s mean FPKM value derived from n = 4 sequenced replicates. b, Correlation of replicate genome-wide screens. Signed confidence score (casTLE score) of each genome-wide screen plotted against a biological replicate screen. The signed confidence score is negative if gene knockout inhibits phagocytosis and positive if it promotes phagocytosis. Each casTLE score is derived from n = 2 replicate screens. c, Correlation of replicate-focused batch retest screens. The signed confidence score (casTLE score) of each focused batch retest screen is plotted against a biological replicate screen. The signed confidence score is negative if gene knockout inhibits phagocytosis and positive if it promotes phagocytosis. Each casTLE score is derived from n = 2 replicate screens.

Supplementary Figure 4 Validation of genome-wide screen results using focused sublibraries and individual gene KO cell lines.

a, Comparison of genome-wide and focused sublibrary screens. Scatterplots of confidence scores for genes in the focused sublibrary batch retest screens and original genome-wide results across various substrates. Confidence scores are calculated by casTLE and are negative if the gene KO inhibits phagocytosis and positive if the gene KO promotes phagocytosis. Hits discovered in the genome-wide screen for a given substrate are highlighted in red. Genes that were present in the batch library but were not hits for the given substrate in the genome-wide screen are in gray. Note that the focused sublibrary screen reveals phenotypes for many genes that were missed in the genome-wide screens (false negatives). b, Representation of the number of hits for each focused sublibrary screen. Hits are defined as the gene KO having a nonzero effect on phagocytosis within a 95% credible interval. c, Summary of all microscopy-based zymosan validations. U937 cells containing the indicated guide were treated with pHrodo-labeled zymosan and phagocytosis was monitored over time using automated microscopy. The phagocytic index of the time point at 5 h is presented. Data are the average of four independent replicate wells, and error bars represent the s.e.m. d, Summary of all microscopy-based positive bead validations. U937 cells containing the indicated guide were treated with pHrodo-labeled positively charged beads and phagocytosis was monitored over time using automated microscopy. The phagocytic index of the time point at 5 h is presented. Data are the average of four independent replicate wells, and error bars represent s.e.m. e, Validation by magnetic separation of magnetized zymosan and positively charged beads for predicted phagocytosis-promoting hits ICAM1 and CCNC. U937 cells containing the indicated guide were treated with magnetized zymosan or magnetic positively charge beads. Cells were separated by magnetic column as conducted in genome-wide screens. Cells in the unbound and bound fractions were counted and the fold enrichment of the bound:unbound cell ratio was calculated. f, Validation by magnetic separation of magnetized zymosan and positively charged beads for the predicted substrate-specific hits PLEK and TLN1. U937 cells containing the indicated guide were treated with magnetized zymosan or magnetic positively charge beads. Cells were separated by magnetic column as conducted in genome-wide screens. Cells in the unbound and bound fractions were counted and the fold enrichment of the bound:unbound cell ratio was calculated. g, Validation of sgRNAs editing target genes in U937 cells. Genomic DNA from U937 cells containing the indicated guide was submitted to Sanger sequencing at the location of sgRNA homology. The percentage of edited cells at the sgRNA target location was calculated using the ICE software package. Sanger sequencing data are available upon request. h, Validation of sgRNAs editing target genes in clonal U937 and RAW 264.7 cells. Genomic DNA from U937 and RAW 264.7 pooled cell populations containing the indicated guide was single-cell sorted, clonally expanded and submitted to Sanger sequencing at the location of sgRNA homology. The percentage of edited cells at the sgRNA target location was calculated using the ICE software package. Sanger sequencing data are available upon request. i, Validation of sgRNAs editing target genes in primary human macrophage cells. Genomic DNA from primary cells containing the indicated guide was submitted to Sanger sequencing at the location of sgRNA homology. The percentage of edited cells at the sgRNA target location was calculated using the ICE software package. Sanger sequencing data are available upon request.

Supplementary Figure 5 NHLRC2 knockout inhibits phagocytosis.

a, Multiple independent sgRNAs reduce phagocytosis in pools of RAW 264.7 cells expressing sgRNAs targeting Nhlrc2 (blue lines) or Nckap1l (red, orange lines) when compared to a control sgRNA (gray). Values represent the means ± s.e.m. of n = 4 replicate wells. Representative of three independent experiments. b, Validation of phagocytic impairment in the RAW 264.7 Nhlrc2 homozygous clonal knockout line (blue) by measuring the phagocytic index of pHrodo-labeled zymosan, compared to RAW 264.7 cells expressing a control sgRNA (gray) or the same line with addition of the actin polymerization inhibitor cytochalasin D (red). Values represent the means ± s.e.m. of n = 4 replicate wells. Representative of three independent experiments. c, Amount of active (GTP-bound) RAC1 in RAW 264.7 cells expressing a control sgRNA (red) or confirmed Nhlrc2 clonal knockout RAW 264.7 cells (blue) as determined by ELISA with or without addition of a phagocytic substrate (beads). ELISA signal was normalized against recombinant GTP-bound RAC1. Values represent mean ± s.e.m. of n = 3 replicate wells. Representative of two independent experiments. d, Additional images of F-actin staining of RAW 264.7 cells expressing a control sgRNA (left) and a confirmed Nhlrc2 KO cell line (right) imaged on a spinning-disk confocal microscope. Images were generated from extended-focus projections of confocal sections acquired at 0.4-μm intervals of cells stained with AlexaFluor-488 phalloidin. Representative of four independent experiments. Scale bar, 10 μm. e, Additional SEM images of RAW 264.7 cells expressing a control sgRNA (top), a confirmed Nckap1l KO cell line (middle) and a confirmed Nhlrc2 KO cell line (bottom). Cells were either exposed to IgG-opsonized 7-μm beads for 10 min prior to fixation or fixed without exposure to beads. Representative of two independent experiments.

Supplementary Figure 6 Phagocytic and motility defects in ELOVL1 knockout and knockdown cells.

a, Additional confocal microscopy of RAW 264.7 cells expressing a control sgRNA (left) or ELOVL1 sgRNA (right). Cells were incubated with IgG-coated 7-µm beads for 10 min and fixed. Donkey anti-rabbit IgG signal (red) marks exposed bead surfaces that are not obstructed by contact with a cell. Cells were stained with phalloidin (green) and Hoechst nuclear stain (blue). A white asterisk marks a fully internalized bead. Representative of four independent experiments. b, Additional SEM of RAW 264.7 cells expressing a control sgRNA (right) or ELOVL1 sgRNA (left) that were incubated with IgG-coated 7-μm beads for 10 min and fixed. A white asterisk marks a fully internalized bead. Representative of two independent experiments. c, Differentiated PLB-985 cells were imaged in an under-agarose chemotaxis assay both before and after generation of a chemoattractant gradient by photo-uncaging of a caged derivative of the chemoattractant fMLF. Each experimental well contained both mCherry-labeled experimental cells expressing dCas9 and an sgRNA targeting the indicated gene, and mTurquoise-labeled control cells expressing dCas9 without an sgRNA. RAC2 and ACTR2 were included as positive controls for migration defects, and FPR1 (formyl peptide receptor) was included as a positive control for response to attractant stimulation. Mean cell speed was measured by tracking cell movement without chemoattractant gradient generation (basal migration speed). Shown are the mean cell speeds for the experimental cells, normalized according to the speed of the control cells in the same well and then normalized by the mean of the no-sgRNA wells on each day. A total of at least n = 39 replicate wells (technical replicates) were measured for each sgRNA over a total of five independent days. Error bars indicate the s.e.m. for normalized well-average speed measurements. d, Mean cell speed was measured by tracking cell movement without chemoattractant gradient generation (post-stimulation). Shown are the mean cell speeds for the experimental cells, normalized according to the speed of the control cells in the same well and then normalized by the mean of the no-sgRNA wells on each day. A total of at least n = 39 replicate wells (technical replicates) were measured for each sgRNA over a total of five independent days. Error bars indicate the s.e.m. for normalized well-average speed measurements. e, qPCR was used to measure the transcript abundance of ELOVL1 for cells expressing dCas9 and the indicated sgRNA.

Supplementary Figure 7 Substrate-specific modifiers of phagocytosis.

Assay of U937 cell lines expressing sgRNAs that are simultaneously challenged with two phagocytic substrates within the same well, one substrate labeled with pHrodo green and the other with pHrodo red. Phagocytosis of both substrates is measured over time using live-cell microscopy. a, Ratio of the total red area (indicating zymosan phagocytosis) to the total green area (indicating 1.3-μm bead phagocytosis) at the time point at 5 h of the phagocytosis assay in cells expressing control sgRNAs (gray) or sgRNAs targeting ITGB2, TLN1, FERMT3 and PLEK (blue). Values represent mean ± s.e.m. of n = 4 replicate wells. Representative of three independent experiments. b, Ratio of the total red area (indicating C3b-opsonized RBC phagocytosis) to the total green area (1.3-μm bead phagocytosis) at the time point at 5 h of the phagocytosis assay in cells expressing control sgRNAs (gray) or sgRNAs targeting ITGB2, TLN1, FERMT3, and PLEK (blue). Values represent mean ± s.e.m. of n = 4 replicate wells. Representative of two independent experiments. c, Phagocytosis of pHrodo-labeled amyloid-β (Aβ) aggregates over time in differentiated U937 cells expressing control sgRNAs (black and gray), a clonal NCKAP1L KO U937 line (light blue), a clonal NHLRC2 KO U937s line (dark blue), and a U937 line expressing a control sgRNA with the actin polymerization inhibitor cytochalasin D (red). Values represent mean ± s.e.m. of n = 4 replicate wells. Representative of three independent experiments.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–7 and Supplementary Note

Reporting Summary

Supplementary Table 1

Results from genome-wide KO phagocytosis screens for eight phagocytic substrates

Supplementary Table 2

Sequencing counts from genome-wide screens

Supplementary Table 3

Labeling densities of IONPs and pHrodo for each substrate tested

Supplementary Table 4

RNA-seq results of differentiated and undifferentiated U937 cells

Supplementary Table 5

Results from targeted (batch library) KO phagocytosis FACS and magnetic-based validation screens

Supplementary Table 6

Sequencing counts from batch library screens

Supplementary Table 7

Single-gene validation data of phagocytosis phenotypes using time-lapse fluorescence microscopy

Supplementary Table 8

Results from BioID followed by mass spectrometry

Supplementary Video 1

Frustrated phagocytosis assay of control-sgRNA-expressing RAW 264.7 cells. Representative of four independent experiments

Supplementary Video 2

Frustrated phagocytosis assay of control of NHLRC2 KO RAW 264.7 cells. Representative of four independent experiments

Supplementary Video 3

z stack of control-sgRNA-expressing RAW 264.7 cells. Representative of two independent experiments

Supplementary Video 4

z stack of ELOVL1 KO RAW 264.7 cells. Representative of two independent experiments

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Haney, M.S., Bohlen, C.J., Morgens, D.W. et al. Identification of phagocytosis regulators using magnetic genome-wide CRISPR screens. Nat Genet 50, 1716–1727 (2018). https://doi.org/10.1038/s41588-018-0254-1

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