Defining the role of CD177 in neutrophil biology Ricardo Grieshaber - - PowerPoint PPT Presentation

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Defining the role of CD177 in neutrophil biology Ricardo Grieshaber Brigham and Women's Hospital Division of Rheumatology, Immunology and Allergy Medical Student Heidelberg University Peter Nigrovics Lab Agenda Neutrophils and Ly6G in

SLIDE 1

Defining the role of CD177 in neutrophil biology

Ricardo Grieshaber

Medical Student Heidelberg University Brigham and Women's Hospital Division of Rheumatology, Immunology and Allergy Peter Nigrovic’s Lab

SLIDE 2

Agenda

Neutrophils and Ly6G in murine arthritis
CD177 as human homolog
Characterization of CD177pos/CD177neg neutrophils

→ Key neutrophil functions  → CyTOF  → RNAseq

Outlook: CD177 genetics

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Neutrophils are indispensable for K/BxN serum transfer arthritis

Wipke and Allen J Immunol (2001)

αGR-1 depletes neutrophils … and blocks arthritis

(αGR-1)

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Ly6G ligation blocks arthritis in sub-depleting doses

Wang … Nigrovic Blood (2012)

1 2 3 4 1 2 3 4 5 6 7 8 2 5 0 µg 1 0 0 µg 5 0 µg 1 0 µg 5 µg P B S A n ti-G r-1 (a n ti-L y 6 C /G ) o r P B S K /B xN se ru m (D a y ) C lin ic a l S c o re

Normal PBS Anti-Gr-1 10 g 0.00 0.25 0.50 0.75 1.00

* *

n.s. Number of neutrophils in blood (x 10-3/ L) µ

Clinical arthritis score

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Is there a human Ly6G?

Lee … Nigrovic J Leukoc Biol (2013)

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CD177 expression is bimodal

Specific to neutrophils
Interacts with PECAM-1
Associates with β2 integrins
Tethers ANCA-autoantigen PR3

to neutrophil surface

102 103 104 105 0

56.4 43.6

CD15 CD177

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Hypothesis independent analysis of neutrophil subsets

1. High dimensional analysis

→ Clustering by CD177

2. Exclusion of CD177

→ No clustering

CD11b CD177

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Similar expression pattern in CD177pos and CD177neg neutrophils

BL SF

Pos Neg Pos Neg

CD177 CD11a CD11b (a) CD11b CD11c CD15 CD16 CD181 CD182 CD183 CD184 CD18 (a ) CD18 CD273 CD274 CD279 CD32 CD44 CD45 CD59 CD62L CD64 CD66b HLADR 150 300

CD177

Marked differences between  blood and synovial fluid No difference in surface protein MFI  between CD177pos and CD177neg

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Functional characterization of CD177

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CD177 signals via β2 integrins to promote migratory arrest

Bai … Nigrovic (in preparation)

αCD177 triggers  downstream signaling

None Iso αCD177 200 400 600 800 Relative migration (% of unstim ctrl) LTB4 *

αCD177 attenuates migration αCD177  increases spreading → →

iso αCD177 50 100 % of CD177pos neutorphils attached spreading

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No difference in key neutrophil functions

unstim PMA 5000 10000 15000 MFU SytoxOrange

ns ns

CD177neg CD177pos

unstim PMA 2000 4000 6000 8000 MFI (DCFDA) ns

In vivo migration ROS NETs Also tested: Integrin and activation marker expression, cell death

PB SF 20 40 60 80 100 %CD177pos

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“And a step backward, after making a wrong turn, is a step in the right direction.” ― Kurt Vonnegut (Player Piano)

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Pipeline for RNAseq

Patient recruitment BCH joint tap clinic (Lauren) BWH Rheumatology Attendings Successful joint tap Synovial fluid +  paired peripheral blood Processing Blood: EasySep Isolation Fluid: RBC lysis Staining  CD15, CD66b, CD177 Sorting  Human Immunology  Flow Core  (Adam, Mike)

CD177 CD66b CD66b CD15 FSC/SSC FSC Doublets SSC Doublets

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RNAseq on sorted CD177pos/CD177neg neutrophils

6x inflammatory arthritis (paired blood + fluid)
7x healthy controls (2 donors twice, 5 donors once)
2x CD177null (healthy)
2x unstim/LPS/LTB4 stimulated neutrophils

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Promoting collaborations in the JBC

Ricardo Pui Lauren

→ Low input SmartSeq2 at

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Future directions: CD177 genetics

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CD177: Striking genotype-phenotype correlation

Wu PLOS Gen (2016)

A T A T A T T T T T T T A T A T A T T T T T T T P s e u d

e n e

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CD177 and CD177P1 are highly homologous

CD177 CTTTCCCTCTCACCCTCAGGACTCACATCAACCCTGGTGGGGACAAAAGGCTGCAGCACT |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| CD177P1 CTTTCCCTCTCACCCTCAGGACTCACATCAACCCTGGTGGCGACCTAAAGCTGCAGCGCT | || | |

5 SNPs in complete linkage disequilibrium Variant mapped to human genome build? hg19 hg38

Lysine

Stop

→ incorrect/missing annotation in genetic databases → poor representation in Illumina MEGA chips (e.g. Partners Biobank)

TT genotype Wu et al. (535 healthy donors, deep sequencing) 2.6 % (in HWE) 1000 Genomes (4x coverage) 10.7 % (not in HWE)

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Genetic linkage of CD177 with RA

Harm-Jan Westra,  Soumya Raychaudhuri

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Acknowledgements

BWH Peter Nigrovic Marta Martinez-Bonet  Margaret Chang  Darren Chiu Pierre Cunin  Olha Halyabar  Minah Iqbal Pui Lee  Anaïs Levescot Gang Li  Nate Nelson-Maney  Yu Yang Former: Ming Bai, Junxia Wang Soumya Raychaudhuri  Harm-Jan Westra  Susan Hannes Jim Lederer, Anu Seshadri  Michael B. Brenner Adam Chicoine  Michael Gurish  Helena Johnnson  Edy Kim  Deepak Rao  Kevin Wei Children’s Lauren Henderson, Kacie Hoyt MGH Roy Soberman, Angela Schmider Broad Chad Nusbaum, Jim Bochicchio Funding Boehringer Ingelheim Fonds MD Fellowship German Academic Scholarship Foundation JBC Microgrant

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Appendix

SLIDE 22

αCD177 causes migratory arrest

Liu Nat Rev Immunol (2007)

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