1. Immunology and Inflammation
  2. Microbiology and Infectious Disease
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Virus infection is controlled by hematopoietic and stromal cell sensing of murine cytomegalovirus through STING

  1. Sytse J Piersma  Is a corresponding author
  2. Jennifer Poursine-Laurent
  3. Liping Yang
  4. Glen N Barber
  5. Bijal A Parikh
  6. Wayne M Yokoyama
  1. Division of Rheumatology, Department of Medicine, Washington University School of Medicine, United States
  2. Department of Cell Biology and Sylvester Comprehensive Cancer Center, University of Miami School of Medicine, United States
  3. Department of Pathology and Immunology, Washington University School of Medicine, United States
Research Article
Cite this article as: eLife 2020;9:e56882 doi: 10.7554/eLife.56882
6 figures, 1 table and 1 additional file

Figures

MyD88 and STING control morbidity and mortality during MCMV infection.

Mice were infected with 50,000 PFU MCMV WT-1, weight loss and survival was monitored over time. (A) Weight loss over time in wildtype (n = 12), STING-deficient (STING GT, n = 21), MyD88-deficient (MyD88 KO, n = 9) and mice deficient in both STING and MyD88 (DKO; n = 14). The numbers indicate the number of mice at the start of the experiment, weight loss of surviving mice at each timepoint is plotted. (B) Survival curves of wildtype (n = 17), STING GT (n = 18), MyD88 KO (n = 17) and DKO mice (n = 20). Cumulative data of 3 independent experiments. Error bars indicate SEM; *p<0.05, **p<0.01, ****p<0.0001.

Figure 1—source data 1

MyD88 and STING control morbidity and mortality during MCMV infection.

https://cdn.elifesciences.org/articles/56882/elife-56882-fig1-data1-v2.xlsx
Figure 2 with 1 supplement
STING contributes to control of MCMV in the hematological and stromal compartment, whereas MyD88 in the hematological compartment potently controls infection.

Mice were infected with 50,000 PFU (A) and (B) or 20,000 PFU (C) MCMV. Viral load was quantified 2 days (A) or 5 days (B) and (C) p.i. (C) Indicated bone marrow was adoptively transferred into irradiated wildtype (WT) or STING-deficient (STING GT) hosts. Bone marrow chimeras were infected 6 weeks post transfer and viral load was analyzed 5 days p.i. Each panel shows cumulative data of 2 independent experiments. Error bars indicate SEM; ns, not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Figure 2—source data 1

STING contributes to control of MCMV in the hematological and stromal compartment, whereas MyD88 in the hematological compartment potently controls infection.

https://cdn.elifesciences.org/articles/56882/elife-56882-fig2-data1-v2.xlsx
Figure 2—figure supplement 1
Viral load for 20,000 PFU infection 5 days p.i. and extended statistical analysis for bone marrow chimeras.

(A) Mice were infected with 20,000 PFU and viral load was quantified 5 p.i. (B) Extended statistical analysis for bone marrow chimeras presented in Figure 2C. Each panel shows cumulative data of 2 independent experiments. Error bars indicate SEM; ns, not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Figure 3 with 1 supplement
MCMV-infected cells specifically produce IFNβ upon infection.

WT mice were infected with 100,000 PFU MCMV IE1-GFP reporter virus. (A) Analysis of GFP expression in CD45- stromal cells and CD45+CD11c+ DC at 8 hr and 36 hours p.i. (B) GFP+ and GFP- stromal cells and DC were FACS-sorted 36 hours p.i. and Ifnb1 and pan-Ifna transcript levels were quantified by real-time PCR. Both panels show representative experiments from two independent experiments. Error bars indicate SD; ns, not significant, *p<0.05, **p<0.01, ***p<0.001.

Figure 3—source data 1

MCMV-infected cells specifically produce IFNβ upon infection.

https://cdn.elifesciences.org/articles/56882/elife-56882-fig3-data1-v2.xlsx
Figure 3—figure supplement 1
Gating strategy and purity of sorted cell populations.

WT mice were infected with 100,000 PFU MCMV IE1-GFP reporter virus. GFP+ and GFP- stromal cells and DC were FACS-sorted 36 hours p.i. Representative gating strategy and purity of sorted cells that were used in Figure 3BC are shown.

Figure 4 with 1 supplement
pDCs produce IFNβ in a MyD88-dependent but STING-independent manner in IFNβ-YFP reporter mice.

IFNβ-YFP reporter mice were backcrossed to MyD88- (MyD88 KO), STING- (STING GT) and double-deficient (DKO) mice. Animals were infected with 200,000 PFU WT1 MCMV and analyzed 48 hr post infection. Spleens were digested to a single cell suspension, stained and analyzed by flow cytometry. Error bars indicate SD; **p<0.01.

Figure 4—source data 1

pDCs produce IFNβ in a MyD88-dependent but STING-independent manner in IFNβ-YFP reporter mice.

https://cdn.elifesciences.org/articles/56882/elife-56882-fig4-data1-v2.xlsx
Figure 4—figure supplement 1
Gating strategy for analysis of IFNβ-YFP reporter mice.

IFNβ-YFP reporter mice were backcrossed to MyD88- (MyD88 KO), STING- (STING GT) and double-deficient (DKO) mice. Animals were infected with 200,000 PFU WT1 MCMV and analyzed 48 hr post infection. Gating strategy for samples presented in Figure 4 are shown.

MCMV-induced fibroblast IFNβ is triggered by cGAS-STING-dependent but MyD88-Trif-MAVS-independent mechanisms.

(A) IFNB1 mRNA levels of primary splenic fibroblasts infected with WT1 MCMV (MOI = 5) 8 hr post-infection. (B) IFNB1 mRNA levels of murine embryonic fibroblasts (MEF) from wildtype (WT) or indicated deficient mice were infected and analyzed as in (A). (C) IFNB1 mRNA levels in infected WT or cGAS-deficient primary splenic fibroblasts, analyzed as in (A). (D) Secreted IFNβ by WT or indicated gene deficient MEF, infected with MCMV (MOI = 0.5); supernatant was analyzed 48 hours p.i. by ELISA. Panels show representative experiments from two independent experiments performed in duplicate. WT, STING GT, and TBK1-, MAVS-, ZBP1-, DNA-PK-, and cGAS-deficient MEF represent data from two independent MEF preparations. Error bars indicate SEM; ns, not significant, *p<0.05, **p<0.01, ***p<0.001.

Figure 5—source data 1

MCMV-induced fibroblast IFNβ is triggered by cGAS-STING-dependent but MyD88-Trif-MAVS-independent mechanisms.

https://cdn.elifesciences.org/articles/56882/elife-56882-fig5-data1-v2.xlsx
MyD88 and STING are required for NK cell cytolytic capacity during MCMV infection.

(A and B) Mice deficient in MyD88 and/or STING were infected with MCMV and 2 days later splenocytes were harvested and analyzed for GzmB, Prf1, and IFNγ expression by FACS. Representative contour plots of individual mice are shown in (A) and quantification for multiple mice is shown in (B). (C) Differentially labelled WT, m157-Tg and MHC-I deficient splenocytes were adoptively transferred into indicated day 3-infected mice. Specific rejection was analyzed 3 hr post-transfer in the spleen. Representative experiments from two independent experiments per panel are shown. Error bars indicate SEM; ns, not significant, *p<0.05, **p<0.01, ***p<0.001.

Figure 6—source data 1

MyD88 and STING are required for NK cell cytolytic capacity during MCMV infection.

https://cdn.elifesciences.org/articles/56882/elife-56882-fig6-data1-v2.xlsx

Tables

Key resources table
Reagent type
(species) or
resource
DesignationSource or
reference
IdentifiersAdditional
information
Strain, C57BL/6 background (Mus musculus)C57BL/6Charles River Laboratories556; RRID:MGI:2160593
Strain, BALB/c background (Mus musculus)BALB/cCharles River Laboratories555; RRID:MGI:2160915
Strain, C57BL/6 background (Mus musculus)STING golden ticketJackson Laboratories017537; RRID:IMSR_JAX:017537
Strain, C57BL/6 background (Mus musculus)IFNβ-YFP reporter miceJackson Laboratories010818; RRID:IMSR_JAX:010818
Strain, C57BL/6 background (Mus musculus)DNA-PK SCIDJackson Laboratories001913; RRID:IMSR_JAX:001913
Strain, C57BL/6 background (Mus musculus)Β2m KOJackson Laboratories002087; RRID:IMSR_JAX:002087
Strain, C57BL/6 background (Mus musculus)M157-TgTripathy et al., 2008
Strain, C57BL/6 background (Mus musculus)H-2Kb x H-2Db KOTaconic4215; RRID:IMSR_TAC:4215
Strain, C57BL/6 background (Mus musculus)MyD88 KOS. AkiraRRID:MGI:3577712through the JCRB Laboratory Animal Resource Bank of the National Institute of Biomedical Innovation
Strain, C57BL/6 background (Mus musculus)TBK1 KOS. Akiranbio156; RRID:MGI:3053427through the JCRB Laboratory Animal Resource Bank of the National Institute of Biomedical Innovation
Strain, C57BL/6 background (Mus musculus)ZBP1 KOS. Akiranbio155; RRID:MGI:3776852through the JCRB Laboratory Animal Resource Bank of the National Institute of Biomedical Innovation
Strain, C57BL/6 background (Mus musculus)IPS1 KOMichael Gale
Strain, C57BL/6 background (Mus musculus)cGAS KOHerbert Virgin
OtherIRF3/7 KO MEFMichael DiamondPrimary murine embryonic fibroblasts.
OtherSTING KO MEFGlen BarberPrimary murine embryonic fibroblasts.
OtherMyD88xTRIF KO MEFThis paperPrimary murine embryonic fibroblasts. See Materials and methods, Section 2
OtherTBK1 KO MEFThis paperPrimary murine embryonic fibroblasts. See Materials and methods, Section 2
OtherTBK1 HET MEFThis paperPrimary murine embryonic fibroblasts. See Materials and methods, Section 2
OtherSTING GT MEFThis paperPrimary murine embryonic fibroblasts. See Materials and methods, Section 2
OtherMAVS (IPS1) KO MEFThis paperPrimary murine embryonic fibroblasts. See Materials and methods, Section 2
OtherMAVS (IPS1) KO MEFThis paperPrimary murine embryonic fibroblasts. See Materials and methods, Section 2
OtherMAVS (IPS1) KO MEFThis paperPrimary murine embryonic fibroblasts. See Materials and methods, Section 2
OtherZBP1 KO MEFThis paperPrimary murine embryonic fibroblasts. See Materials and methods, Section 2
OtherDNA-PKSCID MEFThis paperPrimary murine embryonic fibroblasts. See Materials and methods, Section 2
OthercGAS KO MEFThis paperPrimary murine embryonic fibroblasts. See Materials and methods, Section 2
Virus (murine cytomegalovirus)MCMV WT1Cheng et al., 2010
Virus (murine cytomegalovirus)MCMV GFPHenry et al., 2000
Sequence-based reagentMCMV IE1IDT DNATAQman assayForward: 5’-CCCTCTCCTAACTCTCCCTTT-3’; Reverse: 5’-TGGTGCTCTTTTCCCGTG −3’; Probe: 5’-
TCTCTTGCCCCGTCCTGAAAACC-3’
Sequence-based reagentACTBIDT DNATAQman assayForward: 5’-AGCTCATTGTAGAAGGTGTGG-3’; Reverse: 5’- GGTGGGAATGGGTCAGAAG-3’; Probe: 5’-TTCAGGGTCAGGATACCTCTCTTGCT-3’
Sequence-based reagentIFNB1Thermo Fisher ScientificTAQman assayMm00439546_s1
Sequence-based reagent(pan)IfnaIDT DNATAQman assayForward: 5’-CTTCCACAGGATCACTGTGTACCT-3’; Reverse: 5’-TTCTGCTC TGACCACCTCCC-3’; Probe: 5’-AGAGAGAAGAAACACAGCCC CTGTGCC-3’
Sequence-based reagentGAPDHThermo Fisher ScientificTAQman assayMm99999915_g1
AntibodyAnti-mouse NK1.1 PE-Cy7 (Mouse monoclonal)Thermo Fisher ScientificCat#: 25-5941-82; RRID:AB_469665FACS (1:100)
AntibodyAnti-mouse NKp46 PerCP-eFluor710 (Rat monoclonal)Thermo Fisher ScientificCat#: 46-3351-82; RRID:AB_1834441FACS (1:100)
AntibodyAnti-mouse CD3 APC-eFluor780 (Armenian hamster monoclonal)Thermo Fisher Scientificcat# 47-0031-82, RRID:AB_11149861FACS (1:100)
AntibodyAnti-mouse CD19 APC-eFluor780 (Rat monoclonal)Thermo Fisher ScientificCat# 47-0193-82, RRID:AB_10853189FACS (1:100)
AntibodyLy49H FITC (Mouse monoclonal)Made in-houseFACS (1:200)
AntibodyAnti-mouse CD31 PE (Rat monoclonal)Thermo Fisher ScientificCat# 12-0311-83, RRID:AB_465633FACS (1:100)
AntibodyAnti-mouse PDCA1 PE (Mouse monoclonal)Thermo Fisher ScientificCat# 12-3171-81, RRID:AB_763427FACS (1:50)
AntibodyAnti-mouse gp38 PE-Cy7 (Syrian hamster monoclonal)Thermo Fisher ScientificCat# 25-5381-82, RRID:AB_2573460)FACS (1:100)
AntibodyAnti-mouse CD45 APC (Rat monoclonal)Thermo Fisher ScientificCat# 17-0451-83, RRID:AB_469393)FACS (1:50)
AntibodyAnti-mouse CD11c APC-eFluor780 (Armenian hamster monoclonal)Thermo Fisher ScientificCat# 47-0114-82, RRID:AB_1548652)FACS (1:50)
AntibodyAnti-mouse Ly49H APC (Mouse monoclonal)Thermo Fisher ScientificCat# 17-5886-82, RRID:AB_10598809FACS (1:100)
AntibodyAnti-mouse Perforin PE (Rat monoclonal)Thermo Fisher ScientificCat# 12-9392-82, RRID:AB_466243FACS (1:50)
AntibodyAnti-mouse Granzyme B APC (Mouse monoclonal)Thermo Fisher ScientificCat# MHGB05, RRID:AB_10373420FACS (1:100)
AntibodyAnti-mouse IFNg eFluor450 (Rat monoclonal)Thermo Fisher ScientificCat# 48-7311-82, RRID:AB_1834366FACS (1:100)
Commercial assay or kitMouse
IFNB ELISA
Biolegend439407
Commercial assay or kitCytofix/Cytoperm kitBD Biosciences554714
Software, algorithmPrismGraphpadRRID: SCR_002798
Software, algorithmFlowjoTreestar IncRRID:SCR_008520
OtherViability stain eFluor 506Thermofisher Scientific65-0866-14FACS (1:1000)

Data availability

All data generated or analysed during this study are included in the manuscript and supporting files. Source data files have been provided for all figures.

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