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A noncanonical role for the engulfment gene ELMO1 in neutrophils that promotes inflammatory arthritis

Nature Immunology volume 20, pages 141–151 (2019)Cite this article

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Abstract

Rheumatoid arthritis is characterized by progressive joint inflammation and affects ~1% of the human population. We noted single-nucleotide polymorphisms (SNPs) in the apoptotic cell–engulfment genes ELMO1, DOCK2, and RAC1 linked to rheumatoid arthritis. As ELMO1 promotes cytoskeletal reorganization during engulfment, we hypothesized that ELMO1 loss would worsen inflammatory arthritis. Surprisingly, Elmo1-deficient mice showed reduced joint inflammation in acute and chronic arthritis models. Genetic and cell-biology studies revealed that ELMO1 associates with receptors linked to neutrophil function in arthritis and regulates activation and early neutrophil recruitment to the joints, without general inhibition of inflammatory responses. Further, neutrophils from the peripheral blood of human donors that carry the SNP in ELMO1 associated with arthritis display increased migratory capacity, whereas ELMO1 knockdown reduces human neutrophil migration to chemokines linked to arthritis. These data identify ‘noncanonical’ roles for ELMO1 as an important cytoplasmic regulator of specific neutrophil receptors and promoter of arthritis.

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Fig. 1: Engulfment protein ELMO1 contributes to inflammatory arthritis.
Fig. 2: ELMO1 expression in neutrophils regulates disease severity in arthritis.
Fig. 3: The neutrophil-specific ELMO1 protein interactome includes regulators of chemotaxis linked to human arthritis.
Fig. 4: ELMO1 promotes neutrophil migration to inflamed joints.
Fig. 5: Elmo1 function regulates cell type-specific transcriptional programs.
Fig. 6: ELMO1 SNP rs11984075 promotes migration of human neutrophils.

Data availability

The data that support the findings from this study, including the R code used for bioinformatics analysis and heatmap generation, are available from the corresponding authors upon reasonable request. Sequencing data have been deposited in the GEO database under accession number GSE122412. Reagents used in this study are also listed in the Nature Research Reporting Summary.

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Acknowledgements

The authors thank members of the Ravichandran laboratory for discussions and critical reading of the manuscript, A. Criss and A. Smirnov for human neutrophils, S. T. Fleury for assistance with neutrophil purification, and K. Koster for assistance with bone marrow preparations. This work is supported by grants to K.S.R. from NIGMS R35GM122542, NIMH (MH096484), NHLBI (P01HL120840), NICHD (HD07498), and the Center for Cell Clearance/University of Virginia School of Medicine, and the Odysseus Award from the FWO, Belgium, to M.R.E. from NIAID (AI114554), to M.K. from NIAID (P01 AI102851), and to A.G. (NS083542 and NS101281). Additional support was provided by the Philip S. Magaram, Esq. Research Award from the Arthritis Foundation to S.A.. J.S.A.P. is supported by a Mark Foundation award from the Cancer Research Institute and a postdoctoral enrichment award from Burroughs Wellcome and was previously supported by a postdoctoral fellowship through a T32 Cancer Training Grant. C.D.L. is supported by an award from The Wellcome Trust (206566/Z/17/Z). L.S.S. is supported by the Roaring Fork Valley Postdoctoral Research Fellowship (PF-17-098-01-CSM) from the American Cancer Society, and K.K.P. is supported by an NHLBI F30 award (F30 HL126385) and was previously supported by an NIH T32 Immunology Training Grant (T32 AI007496).

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Authors and Affiliations

  1. Center for Cell Clearance, Department of Microbiology, Immunology and Cancer Biology, University of Virginia, Charlottesville, VA, USA

    Sanja Arandjelovic, Justin S. A. Perry, Christopher D. Lucas, Kristen K. Penberthy, Alexandra M. Bettina, Laura S. Shankman, Amanda H. Cohen & Kodi S. Ravichandran

  2. Centre for Inflammation Research, University of Edinburgh, Edinburgh, Scotland, UK

    Christopher D. Lucas

  3. David H. Smith Center for Vaccine Biology and Immunology, Department of Microbiology and Immunology, University of Rochester, Rochester, NY, USA

    Tae-Hyoun Kim, Minsoo Kim & Michael R. Elliott

  4. Inova Center for Personalized Health, Inova Schar Cancer Institute, Fairfax, VA, USA

    Ming Zhou & Thomas P. Conrads

  5. Center for Brain Immunology and Glia, Department of Neuroscience, University of Virginia, Charlottesville, VA, USA

    Dorian A Rosen, Tzu-Ying Chuang & Alban Gaultier

  6. Inflammation Research Centre, VIB, Ghent, Belgium

    Kodi S. Ravichandran

  7. Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium

    Kodi S. Ravichandran

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  1. Sanja Arandjelovic
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Conceptualization, S.A. and K.S.R.; methodology, S.A. and K.S.R.; software, J.S.A.P.; investigation, S.A., J.S.A.P., C.D.L., K.K.P., T.-H.K., M.Z., D.A R., T.-Y.C., A.M.B., L.S.S., A.H.C., and A.G.; data curation, J.S.A.P.; writing, S.A. and K.S.R.; resources, A.G., T.P.C., M.K., M.R.E. and K.S.R.; funding acquisition, K.S.R.

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Correspondence to Sanja Arandjelovic or Kodi S. Ravichandran.

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Integrated supplementary information

Supplementary Figure 1 Loss of ELMO1, but not BAI1, reduces disease severity in arthritis.

a) Mice with spontaneous arthritis (K/BxN) or their healthy littermates were used to analyze engulfment machinery expression in isolated paws. b) Expression of apoptotic cell clearance components genes in total paw extracts from K/BxN mice by qRT-PCR. Each data point represents one mouse. c) Incidence of CIA in male Elmo1+/+-DBA (n = 3, white symbols), Elmo1+/– -DBA (n = 12, black symbols) and Elmo1–/–-DBA (n = 6, green symbols). d) Swelling of the paws (left panel) and clinical scores (right panel) were measured in male Elmo1+/+-DBA (n = 3, white symbols), Elmo1+/– -DBA (n = 12, black symbols) and Elmo1–/–-DBA (n = 5, green symbols). e) Incidence of CIA in female Elmo1+/+-DBA (n = 5, white symbols), Elmo1+/– -DBA (n = 8, black symbols) and Elmo1–/–-DBA (n = 7, green symbols). f) Swelling of the paws (left panel) and clinical scores (right panel) were measured in female Elmo1+/+-DBA (n = 5, white symbols), Elmo1+/–-DBA (n = 8, black symbols) and Elmo1–/–-DBA (n = 5, green symbols). g) Elmo1, Elmo2 and Elmo3 expression was analyzed by quantitative RT-PCR in the total paw extracts of Elmo1+/– (black symbols, n = 7) and Elmo1–/– (green symbols, n = 7) mice on day 10 after K/BxN serum injection. Each symbol represents an individual animal. h) Schematic of BAI1 binding to ELMO1-DOCK2, leading to the activation of GTPase RAC1. i) Paw swelling and clinical scores of BAI1+/+ (n = 4, black symbols) and BAI1–/– mice (n = 11, blue symbols) injected with 150 μl of K/BxN serum on day 0.

Supplementary Figure 2 ELMO1 expression in neutrophils regulates disease severity in arthritis.

a) Immunoblot analysis of ELMO1 protein expression in resident peritoneal macrophages from indicated mice. The blot was cropped to show relevant bands. b)Paw swelling and clinical scores of (n = 4, black symbols) and Lyz2-Cre (n = 3, blue symbols) mice injected with 150 μl of K/BxN serum on day 0 and 2. c) Paw swelling and clinical scores of Elmo1fl/fl (Control, n = 7) or Elmo1fl/flLyz2-Cre (n = 5) mice injected with 150 μl of K/BxN serum on day 0 and 2. d) Flow cytometry analysis of paws from of Elmo1fl/fl (n = 3) or Elmo1fl/flLyz2-Cre (n = 3) mice on day 10 after K/BxN serum injection. Cells in the singlet gate are shown. e) Paw swelling and clinical scores of Elmo1fl/fl (Control, n = 5) or Elmo1fl/flCx3cr1-Cre (n = 5) mice injected with 150 μl of K/BxN serum on day 0 and 2. f) Representative hind paw ankle sections stained with hematoxylin and eosin on day 10 after K/BxN serum injection (left panels). Areas of inflammatory cell infiltration are indicated with yellow asterisks. Scale bar = 1 mm. Quantification of inflammation (right panel) in Elmo1fl/fl (Control, n = 5) or Elmo1fl/flCx3cr1-Cre (n = 5). g) Paw swelling and clinical scores of Elmo1fl/fl (Control, n = 6) or Elmo1fl/flMrp8-Cre (n = 3) mice injected with 150 μl of K/BxN serum on day 0 and 2. h) Immunoblot analysis of ELMO1 protein expression in the Ly6G+ cells purified from the bone marrow of indicated mouse strains, as described in the Methods. The blot was cropped to show relevant bands. i) Paw swelling and clinical scores of (n = 8, black symbols) and Mrp8-Cre (n = 3, magenta symbols) mice were injected with 150 μl of K/BxN serum on day 0 and 2. j) Immunoblotting of ELMO1 protein expression in neutrophils and fibroblast like synoviocytes (FLS) from the indicated strains of mice. Each lane represents an individual animal. The blot was cropped to show relevant bands.

Supplementary Figure 3 Bacterial-challenge response and EAE are not changed in Elmo1–/–mice.

a) Mice were challenged with LPS administered intranasally as described in the Methods, and mobilization of neutrophils was analyzed 8 hours later by flow cytometry of cells in the BAL. b) Quantification of 7-AAD-CD11b+Ly6C+Ly6G+ neutrophils in the BAL of Elmo1+/+, Elmo1–/–, Elmo1fl/fl and Elmo1fl/flMrp8-Cre mice. Each symbol represents an animal. c) Elmo1+/+ (n = 18) and Elmo1–/– (n = 19) mice were injected with fecal contents (1.5 mg/g) intraperitoneally to induce fecal-induced peritonitis (FIP) and disease parameters were monitored as described in Methods. d) Number of Ly6G+ neutrophils in the peritoneum 4 hours post FIP induction. Each symbol represents an animal. e) Temperature of mice in c). f) Clinical scores of mice in c) were measured as described in Methods. g) Survival curves of mice in c). All mice alive at 72 hours post FIP induction exhibited complete recovery. h) Purified neutrophils from Elmo1+/+ (n = 4) and Elmo1–/– (n = 4) mice were incubated with Klebsiella pneumoniae at a 1:2 ratio for 1 hour, as described in the Methods, and bacterial killing was analyzed. Each symbol represents neutrophils from an individual animal. i) Elmo1+/– (n = 8, black symbols) and Elmo1–/– (n = 8, green symbols) mice were immunized to induce EAE and disease was scored over the indicated time as described in the Methods. j) Luxol Fast Blue staining of the spinal cords on day 26 after EAE induction (left panel). Demyelination was scored at four different levels of the spinal cord and composite result is shown (right panel). Each symbol represents an individual animal.

Supplementary Figure 4 Elmo1 deletion in neutrophils inhibits neutrophil migration.

a) Total bone marrow cells were purified as described in the Methods, and cell migration toward the indicated concentrations of LTB4 or CXCL1 was evaluated after 3 hours at 37 °C by flow cytometry. Each symbol represents an individual animal. b) Transcript levels of the LTB4 receptor Blt1 are analyzed by qPCR. Each symbol represents an individual animal. c) Flow cytometry analysis of cell surface levels of CXCR2 in Elmo1+/+ and Elmo1–/– mice on Ly6G+ neutrophils in the blood (top panels, n = 9, 11) and the bone marrow (bottom panels, n = 3, 3). MFI, mean fluorescence index. FMO, fluorescence minus one. Each symbol represents an individual animal. d) Flow cytometry analysis of cell surface levels of FcγRIII and FcγRIV on CD11b+Ly6G+ neutrophils in the blood (top panels) and the bone marrow (bottom panels) of Elmo1+/+ and Elmo1–/– mice. e) Immunoblotting of FcγRI protein expression in Ly6G+ neutrophils purified from the bone marrow of Elmo1+/+ and Elmo1–/– mice. The blot was cropped to show relevant bands in non-adjacent lanes. f) Immunoblotting of Syk protein expression in Ly6G+ neutrophils purified from the bone marrow of Elmo1+/+ and Elmo1–/– mice. The blot was cropped to show relevant bands in non-adjacent lanes.

Supplementary Figure 5 Inducible deletion of Elmo1 in ongoing arthritis.

(a) Paw swelling of male and female Elmo1fl/flUbc-CreERt2 (n = 9 for males, n = 7 for females, green symbols) and littermate control (n = 12 for males, n = 10 for females, black symbols) mice with K/BxN serum transfer induced arthritis and tamoxifen administration (arrows). b) Paw swelling (left panel) and clinical scores (right panel) of Elmo1fl/fl (control, n = 5) and Ubc-CreERt2/Elmo1fl/fl (n = 3) mice with K/BxN serum transfer induced arthritis without tamoxifen administration. c) Schematic of the K/BxN serum transfer arthritis induction and treatment with tamoxifen to induce deletion of Elmo1 during ongoing arthritis. d) Paw swelling of male Ubc-CreERt2/Elmo1fl/fl (n = 3, green symbols) and littermate control (n = 6, black symbols) mice with K/BxN serum transfer induced arthritis and tamoxifen administration (arrows). e) ELMO1 protein expression in human peripheral blood neutrophils (duplicates are shown) and buffy coat cells from two different donors (1 and 2). ERK protein expression is used as a loading control. The blot was cropped to show relevant bands.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–5 and Supplementary Tables 1–3

Reporting Summary

Supplementary Video 1 (Elmo1KO): Reduced migration of Elmo1–/– neutrophils toward the site of laser induced injury.

Representative time-lapse videos showing movement of neutrophils in response to laser-induced injury, as described in the Methods. Damaged area is indicated by the yellow dotted line. Second harmonic generation is shown in gray, neutrophils are shown in green. Time-scale, min:sec, Scale bar = 20µm.

Supplementary Video 2 (Elmo1WT): Reduced migration of Elmo1–/– neutrophils toward the site of laser induced injury.

Representative time-lapse videos showing movement of neutrophils in response to laser-induced injury, as described in the Methods. Damaged area is indicated by the yellow dotted line. Second harmonic generation is shown in gray, neutrophils are shown in green. Time-scale, min:sec, Scale bar = 20µm.

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Arandjelovic, S., Perry, J.S.A., Lucas, C.D. et al. A noncanonical role for the engulfment gene ELMO1 in neutrophils that promotes inflammatory arthritis. Nat Immunol 20, 141–151 (2019). https://doi.org/10.1038/s41590-018-0293-x

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