SUPPLEMENTARY INFORMATION Articles DOI:./s-7-- In the format provided y the authors and unedited. Asian Zika virus strains target CD + lood monocytes and induce M-skewed immunosuppression during pregnancy Suan-Sin Foo, Weiqiang Chen, Yen Chan, James W. Bowman, Lin-Chun Chang, Younho Choi, Ji Seung Yoo, Jianning Ge, Genhong Cheng, Alexandre Bonnin, Karin Nielsen-Saines, Patrícia Brasil and Jae U. Jung Department of Molecular Microiology and Immunology, University of Southern California, Keck School of Medical, Zilkha Neurogenetic Institute, San Palo Street, Los Angeles, CA 9, USA. Division of Maternal-Fetal Medicine, Department of Ostetrics and Gynecology, University of Southern California, Keck School of Medicine, Zilkha Neurogenetic Institute, San Palo Street, Los Angeles, CA 9, USA. Department of Microiology, Immunology and Molecular Genetics, University of California, Los Angeles, CA 99, USA. Department of Cell and Neuroiology, University of Southern California, Keck School of Medicine, Zilkha Neurogenetic Institute, San Palo Street, Los Angeles, CA 9, USA. Division of Pediatric Infectious Diseases, David Geffen School of Medicine, University of California, Los Angeles, Marion Davies Children s Health Center, LeConte Avenue, Los Angeles, CA 99, USA. Laoratório de Pesquisa Clínica em Doenças Feris Agudas, Instituto Nacional de Infectologia Evandro Chagas, FIOCRUZ, Avenida Brasil, Rio de Janeiro, RJ -, Brazil. e-mail: jaeujung@med.usc.edu Nature Microiology www.nature.com/naturemicroiology 7 Macmillan Pulishers Limited, part of Springer Nature. All rights reserved.
NK#cells Mock ZIKV CD NK#cells CD Lym phocytes Myeloid Monocytes Monocytes Total# SSC leukocytes CD CD FSC SSC CD T#cells T# cells B#cells CD B cells CD9 Unstained FMO#control# (minus#cd) FMO#control# (minus#cd) Fully#stained CD lo# CD hi#cd+ CD lo# CD hi# CD + CD lo# CD hi#cd+ CD lo# Intermediate# # CD hi#cd+ CD +# CD +# CD +# CD +# CD CD hi#cde CD hi#cde CD hi#cde CD hi#cde CD Supplementary Figure. Gating strategy for each immune suset within PBMCs. PBMCs were isolated from whole lood of healthy donors for FACS surface staining. a, Major lood immune susets which include CD + CD9 - T cells, CD - CD9 + B cells and CD - CD + NK cells were gated from CD hi SSC lo lymphocyte population, while CD hi/lo CD +/- monocytes were gated from CD hi SSC hi myeloid cells. Gating of were ased on total population of gated monocytes/nk cells/t cells/b cells., Specific gating strategy for monocyte susets with the use of fluorescence minus one (FMO) controls.
IFN-α........ MR7 H/PF/ IL-β MR7 H/PF/ IFN-λ/ MR7 H/PF/ IFN-β MR7 H/PF/ IL-..... MR7 H/PF/ IL-7A.... MR7 H/PF/ MyD.... MR7 H/PF/ Type,I,IFN,signaling,genes Inflammatory,genes PTX MR7 H/PF/ IL-.... MR7 H/PF/ IRF7 IRF9 NF-kB p MR7 H/PF/ MR7 H/PF/ MR7 H/PF/ IFN-γ..... MR7 H/PF/ CD.... MR7 H/PF/ AXL MR7 H/PF/ CD IFN-λ MR7 H/PF/ NF-kB p MR7 H/PF/... MR7 H/PF/ c M,macrophage:related,genes d M,macrophage:related,genes.... IL- p MR7 H/PF/... inos MR7 H/PF/ CCL MR7 H/PF/ YKL- MR7 H/PF/ Supplementary Figure. Differential gene induction profiles of PBMCs following infection with different ZIKV lineages. Gene expression profiling of PBMCs isolated from mock- or ZIKVinfected (MOI ) whole lood of healthy donors (n = ) at hpi. Gene expression of a, type I IFN-signaling genes,, inflammatory genes, c, M macrophage-related genes and d, M macrophage-related genes were normalized to GAPDH and expressed as fold change relative to mock controls. Data (mean ± SEM) were presented in ox plot showing upper (7%) and lower (%) quartiles, with horizontal line as median and whiskers as maximum and minimum values oserved. P <., P <., Mann-Whitney U test.
hpi: PBMCs Monocyte susets shift c CXCL IL- ZIKV NS viral load (Log RNA copies/ ng) MR7 PRVABC9 % change relative to mock MR7 PRVABC9 Intermediate Non- MR7 PRVABC9 MR7 PRVABC9 d hpi: PBMCs e Monocyte susets shift f CXCL IL- ZIKV NS viral load (Log RNA copies/ ng) IH H/PF/ % change relative to mock IH H/PF/ Intermediate Non- IH H/PF/. IH H/PF/ Supplementary Figure. Different virological and immunologic traits etween African and Asian lineage ZIKV strains. Whole lood from healthy donors (n = ) were infected with different ZIKV strains for h prior to the purification of PBMCs. Infection with MR7 (African ZIKV) and PRVABC9 (Asian ZIKV) was performed at MOI, while IH (African ZIKV) and H/PF/ (Asian ZIKV) was performed at MOI.. a,d, Viral urdens of total PBMCs were detected using viral load qrt-pcr with the specific proes and primers against the ZIKV NS RNA.,e, FACS analyses were performed to determine (CD + CD - ), intermediate (CD hi CD + ) and non- (CD lo CD + ) monocyte susets of mock- and ZIKV-infected PBMCs. c,f, Expression of CXCL and IL- genes were normalized to GAPDH and expressed as fold changes relative to mock controls. Data (mean ± SEM) were presented in ox plot showing upper (7%) and lower (%) quartiles, with horizontal line as median and whiskers as maximum and minimum values oserved. P <., P <., Mann-Whitney U test in (a, c, d,f) or two-way ANOVA, Bonferroni post-test in (,e).
Mock-infected$PBMCs IL- Arg- CDR CCL...... YKL-....... CD..... VEGFα...... CCL................ Mock-infected$PBMCs CXCL IL-A CD CCR7 IDO......................... IL- CD SOCS IL- p................... c M$macrophage-related$genes d M$macrophage-related$genes CD IL-A IL- p IL- CD VEGFα CCL 9 MR7 H/PF/..... MR7 H/PF/..... Supplementary Figure. Pregnancy primes lood immunity towards M-skewed immunosuppression, which was further amplified upon Asian ZIKV infection. Gene expression profiling of PBMCs isolated from mock- or ZIKV-infected (MOI ) whole lood of nonpregnant (n = ) and pregnant women (n = per trimester) at hpi. Basal expressions of a,
M macrophage-related genes, and, M macrophage-related genes, were determined in mockinfected specimens. Gene expressions were normalized to GAPDH and expressed as relative expression. Additional c, M macrophage-related genes and d, M macrophage-related genes expressions were determined. Gene expressions were normalized to GAPDH and expressed as fold change relative to mock controls. Data (mean ± SEM) were presented in ox plot showing upper (7%) and lower (%) quartiles, with horizontal line as median and whiskers as maximum and minimum values oserved. P <., P <., Mann-Whitney U test.
; Mock H/PF/ MR7 IFNA FCGRA PTAFR VCAM IFNG CD STAT 7 9 MX ISG IRF MX IFIT USP IRF IRF HLA7DQB IFNGR PSMB IRF MR7 H/PF/ SOCS SOCS ICAM FLNB IRF Supplementary Figure. Nanostring analysis of non-pregnant women s monocyte specimens. Pan monocytes which include (CD + CD - ), intermediate (CD hi CD + ) and non- (CD lo CD + ) monocyte susets were isolated from mock- and ZIKV-infected (MOI ) PBMCs harvested at hpi. Whole cell lysates of ~, cells/specimen were sujected to NanoString multiplex gene analysis using Human Myeloid panel consisting of ~ genes. a, Venn diagram representation of the numers of differentially induced genes etween MR7 (African ZIKV) and H/PF/ (Asian ZIKV) of non-pregnant women s monocytes specimens., Heat map representation of IFN-signaling pathway. 7