Comparative analysis of signature genes in PRRSV-infected porcine monocyte-derived dendritic cells at

differential activation statuses.

Laura Miller, USDA-ARS (Co-PD)Yongming Sang, KSU (PD)

Raymond R. R. Rowland, KSU (Co-PD)Frank Blecha, KSU (Co-PD)

College of Veterinary Medicine

Kansas State University, Manhattan, KS 66506, USA

Activation statuses of monocytic cells including monocytes, macrophages and dendritic cells (DCs) are critically important for antiviral immunity. In particular, some devastating viruses, including porcine reproductive and respiratory syndrome virus (PRRSV), are capable of directly infecting these cells to subvert host immunity.

Introduction

Table 1. Monocytotropic viruses and pathogenic effect of macrophage manipulation/infection

Virus*[genome, family]

Macrophage-related primary infection cells/sites Effect of manipulation/infection in monocytes, MΦs and DCs

DENV[(+)ssRNA, Flaviviridae]

Monocytes, MΦs and DCs in multiple tissues of IFN-αβγR KO mice

MΦ-depletion: Tenfold increase in systemic viral titer, and massive infiltration of monocytes

RSV[(-)ssRNA, Paramyxoviridae]

Blood monocytes, DCs, lung epithelial cells and MΦs in mice/humans

MΦ-depletion: Abolished local inflammatory cytokine peak at 1 dpi, and enhanced viral load in the lung at 4 dpi

HIV1[(+)ssRNA, Retroviridae]

Macrophages and T cells in humans Deficiency of CCR5, a co-receptor that mediates HIV macrophage-tropism, showed resistance to HIV-1infection

WNV[(+)ssRNA, Flaviviridae]

Murine keratinocytes and skin-resident DCs, and probable peripheral MΦs and DCs mediating neuroinvasion

MΦ-depletion: Higher and extended viremia, and accelerated encephalitis and death. Inhibition of NOS activity of infiltrating MΦs relieved encephalitis and prolonged survival

SARS-Cov[(+)ssRNA, Coronaviridae]

Human respiratory epithelial cells, and antibody-enhanced infection of macrophages and immune cells

Depletion of alveolar MΦs 1-2 day before infection, (but not at 2 dpi), prevented lethal disease, and enhanced viral clearance

IAV[Segmented (-)RNA, Orthomyxoviridae]

Airway and lung epithelial cells, DCs, and MΦs of mice/humans/pigs

MΦ-depletion: Strain-dependent exacerbation of viral replication, increased airway inflammation and viral pneumonia

CSFV[(+)ssRNA, Flaviviridae]

Porcine blood monocytes/macrophages Viral infection stimulated arginase-1 (ARG-1) but suppressed nitric oxide synthase (iNOS) expression, i.e., induced M1-M2 repolarization

PrV[dsRNA, Herpesviridae]

Porcine lung epithelial cells and MΦs and spread via infected blood monocytes

Acute IFN-α response is important in diminishing the spread of PrV in the connective tissue but not in epithelial cells (IFN cell preferences)

ASFV[dsRNA, Asfarviridae]

Primarily and persistently infected tissuemonocytes/ MΦs and fibroblasts in multiple tissues

Massive M1 polarization served as a modulator of the viral pathogenesis including pulmonary edema, hemorrhage, and lymphoid depletion that characterize the disease

PCV2[ssDNA, Circoviridae]

Monocyte/MΦ lineage cells, including alveolar MΦs, are the major target cells

Acute infection reduced alveolar MΦs phagocytosis and microbicidal capability; and persistence increased inflammatory and pro-apoptotic responses, which led to lymphopenia and immunosuppression

FMDV[(+)ssRNA, Picornaviridae]

Early infection of porcine T and B cells caused viremia; immunocomplex promoted productive infection and killing of mDCs

Increase IL-10 production in infected DCs, loss of pDC cell function coincides with lymphopenia in FMDV-infected pigs; macrophage depletion in vaccinated mice severely decreased vaccine protection

PRRSV[(+)ssRNA, Arteriviridae]

Tissue macrophages, monocytes and mDCs especially those in reproductive and respiratory tracts.

Massive cell death of infected monocytic cells; increase of IL-10 and reduction of phagocytic, microbicidal, pro-inflammatory, and antigen-presentation activity in MΦs and DCs. Pathogenicity-related suppression of IFN-α production in pDCs.

Sang Y, Miller LC, Blecha F. Macrophage Polarization in Virus-Host Interactions . J Clin Cell Immunol. 2015 Apr;6(2). pii: 311.

Objectives

Our long-term goal is two-fold: 1) to integrate activation status with antiviral responses in monocytic cells2) to functionally modulate them for a prototypic cellular adjuvant/vaccine that is ideal for potentiating antiviral immunity.

Methods

• To study how PRRSV infection alters cell activation, we have systematically characterized the activation status and determined, genome-wide, signature genes regulating the activation status in porcine monocytic innate immune cells with PRRSV pathogenicity in ex-vivo stimulated cells using our established RNA-seq procedure.

• Porcine monocyte-derived dendritic cells (mDCs) were polarized with mediators (PBS, IFN-γ, IL-4, LPS, IL-10, IFN-α) for 30 hours, then mock-infected, or infected with PRRSV strain VR-2332, or highly pathogenic PRRSV strain HP-PRRSV rJXwn06, for 5 h.

• Each sample represents a pooled RNA from four replicates.

Methods

Comparisons were made within each treatment group of activation status (mediator vs. PBS control) and between treatment group for each mediator.

CTRL Grp I:Polarization mediators

PRRSV (moi: 0.1)

1 PBS -2 IFNγ -3 IL4 -4 LPS -5 IL10 -6 IFNα -

VR Grp II: Polarization mediators

PRRSV (moi: 0.1)

1V PBS VR23322V IFNγ VR23323V IL4 VR23324V LPS VR23325V IL10 VR23326V IFNα VR2332

HP Grp III: Polarization mediators

PRRSV (moi: 0.1)

1H PBS HP-JX2H IFNγ HP-JX3H IL4 HP-JX4H LPS HP-JX5H IL10 HP-JX6H IFNα HP-JX

Table 1. Sample organization table

Results and discussionCorrelation of cell activation status with PRRSV pathogenicity in ex-vivo stimulated cells.

Results and discussion

PCA by mediator PCA by virus

Correlation of cell activation status with PRRSV pathogenicity in ex-vivo stimulated cells.

Results and discussionVisualization of the DESeq2 dispersion estimates.

.

Results and discussionHeat map of the top 35 most variable genes in the dataset.

mediators

Results and discussionviruses

U2U2U4Rnase_MRPU1novel geneSNORA487SKRnaseP_nucnovel genenovel genessc-mir-4332ELOVL5IRG6DOK6CXCL9POSTNCEP55TAGLNCOL3A1COL1A2COL5A2novel geneCAV1novel geneCOL6A3DCNtenascinCALD1COL1A1novel genenovel geneGLMNtenascinSFRP2

Polarization mediators Control vs PBS VR2332 vs PBS HP-PRRSV vs PBS

IFN-γ

RNA ALDH POSTNACT ACAN ACTRNA TNN COLCXCL COL CXCLSAA CXCL SAAUBD POSTN UBDIDO1 COL SAA

IL-4

COL THBS1 RNACOL CYTB RNAACT MMP RNasePCXCL POSTN CXCLIL-17 SFRP2 CDH2CDH2 CXCL IL17RB

LPS

COL TNN RNasePACT COL U splicesomal RNARNA GJA1 RNASAA IL1B SAAIDO1 SAA MMPIL1B CXCL IL1B

IL-10

RNA DEFB133 RNAIFIT1 IL2RA RNasePRNA S100A1 U splicesomal RNA

MMP CELSR1 MMPSAA GTSE1 IL7IL7 TTC38 MMP

IFN-α

RNA THBS1 RNAACT CYTB RNaseP

RNaseP IL1B U splicesomal RNAISG12 ISG12 ISG12TNF IRG6 XAF1IFI IFIT1/IFIT2 IFIT1

CTRL Grp I:Polarization mediators

PRRSV (moiI: 0.1)

1 PBS -2 IFNg -3 IL4 -4 LPS -5 IL10 -6 IFNa -

VR Grp II: 1V PBS VR23322V IFNg VR23323V IL4 VR23324V LPS VR23325V IL10 VR23326V IFNa VR2332

HP Grp III: 1H PBS HP-JX2H IFNg HP-JX3H IL4 HP-JX4H LPS HP-JX5H IL10 HP-JX6H IFNa HP-JX

2 vs 13 vs 14 vs 15 vs 16 vs 1

2V vs 1V3V vs 1V4V vs 1V5V vs 1V6V vs 1V

2H vs 1H3H vs 1H4H vs 1H5H vs 1H6H vs 1H

Results and discussion

DE Polarization mediators VR-2332 HP-PRRS

é

PBS

RNA CXCL10RNaseP RNAACT ISG12

êMORC3 AREGTNN U splicesomal RNATNFSF10 CXCL2

é

IFN-γ

ETNPPL IRG6CYP1A1 COLCOL FAM111C

êPOSTN RNACCL11 U splicesomal RNACAV2 RNaseP

é

IL-4

RNA RNARNaseP RNaseP

U spliceosomal RNA U splicesomal RNA

êPOSTN POSTNIRG6 COLIFIT1/IFIT2 SFRP2

é

LPS

LAG3 CXCL10MARCO IRG6TRBV19 IFIT1/IFIT2/IFIT3

êTGM2 COL1A2MMP COL3A1IL1A POSTN

é

IL-10

ACT RNACOL RNasePCOL RNA

êUBCH5B CCNL1IL1A SEPINB2CXCL2 CXCL2

é

IFN-α

COL1A2 CXCL10COL1A2 IRG6COL6A3 IFIT3/IFIT5/IFIT2

êPMAIP1 WNT5ARNA AREGCXCL10 MMP

1V vs 12V vs 23V vs 34V vs 45V vs 56V vs 6

1H vs 12H vs 23H vs 34H vs 45H vs 56H vs 6

DE Polarization mediators VR2332 vs HP-PRRS

éPBS

CXCL10IRG6IFIT1

êCOL3A1RNACOL1A2

éIFN-γ

POSTNMXRA5IRG6

êRNARNasePU splicesomal RNA

éIL-4

CXCL10IRG6IFIT1/IFIT2/IFIT3

êACTA2CAV1COL3A1/CXCL2

éLPS

IRG6CXCL10IFIT1/IFIT2/IFIT3

êCOL1A2COL3A1SFRP2

éIL-10

CXCL10IRG6IFIT1

êCOL3A1ACTA2COL1A2

éIFN-α

CXCL10IRG6IFIT3

êCOL1A2COL3A1COL5A2

1H vs 1V2H vs 2V3H vs 3V4H vs 4V5H vs 5V6H vs 6V

Results and discussion

• Clustering of samples was consistent with virus strain and then by mediator.

• Many of the genes showing the most variability were related to cellular structure and innate immune response.

• The magnitude of differentially expressed gene profiles detected in HP-PRRSV rJXwn06 infected mDCs as compared to VR-2332 infected mDCs was consistent with the increased pathogenicity of the HP-PRRSV in vivo.

Conclusions

Cytokine and signature gene phenotyping to correlate cell activation statuses with PRRSV pathogenicity.Blood –Whole-blood and PBMCs for isolation of monocytes, cDC and pDCsBroncheo-alveolar lug fluid (BALF)

Correlate cell activation status with PRRSV pathogenicity in primary cells isolated from virus-infected pigs.

Current/future work

Real-time RT-PCRComparisons:1) treatments of control, vaccinated and infected; 2) pigs within a treatment;3) groups of the monocytic cells; and 4) subsets of MФs and DCs.

Flow (FSC)PBMCPanel #1 CD4+ CD8+ CD172a+

Panel #2 CD80+ MHCIIHi MHCIIlo

Panel #3 CD16+ CD163+ MHCIIHi MHCIIlo

Panel #4 CD163+ SDOW17+

BALFPanel #2 CD80+ MHCIIHi MHCIIlo

Panel #3 CD16+ CD163+ MHCIIHi MHCIIlo

Correlate cell activation status with PRRSV pathogenicity in primary cells isolated from virus-infected pigs.

Current/future work

Acknowledgements

Technicians: Sarah Anderson USDA ARS NADCSequencing: ISU DNA facilityBioinformatics: Dr Darrell Bayles, USDA ARS NADCAnimal study: Dr Vikas Kulshrestha, Dr Albert Van Geelen, Dr Alexa Buckley, Dr Nestor Montiel, Dr Kelly Lager, Sarah Anderson, NADC animal caretakersFlow cytometry: Sam Humphrey USDA ARS NADCFlow cytometry analyses: Dr Nestor Montiel, Sam Humphrey USDA ARS NADCFunding: This work was supported by USDA NIFA AFRI grant 2013-67015-21236

CTRL

Fluorescent

ToFA(2.5 μg/ml)

ToFA(5 μg/ml)

mDCs infected with DsRed-PRRSV for 48 h

Merged

ToFA (5-(Tetradecyloxy)-2-furoic acid), a competitive inhibitor of acetyl-CoA carboxylase (ACC)

FL1 (PRRSV)

99.42 0.58

100 101 102 103 104

MФsMARC-145 mDCs

6.09 93.91

83.16 16.84

98.52 1.48

43.26 56.74

62.55 37.45

74.51 25.49

86.97 13.02

98.96 1.04

100 101 102 103 104 100 101 102 103 104

FSC-H

1000800600400200

01000800600400200

01000800600400200

0

Mock

PRRSVToFA+PRRSV

Sang et al. Animal Health Review 2011, 12:149-67. PLoS One. 2014, 9:e87613. and J. Virol. 2014, Oct;88(19):11395-410.

Antiviral regulation via AMPK pathway and lipid metabolism

Develop a prototypic adjuvant/vaccine system based on functional modulation of activation statuses in porcine monocytic innate immune cells

ORF1aORF1b

ORF2-7

Afl II

Mlu I

IFN ORF6X Histidine tag

12-AA protease cleavage site

Vector CMV promoter

Viral RNA cDNA

Sang et al., Viruses. 2012, 4:102-16; J. Virol. 2014, Oct;88(19):11395-410.

Anti-PRRSV N

Anti-IFNα Merged

Virus-replication competent IFN expression: acts against viral suppression of IFN production in situ

Validation for Therapeutic Designs

Sang et al. Animal Health Review 2011, 12:149-67, and J. Virol. 2014, 88(19):11395-410. Brockmeier et al., Clin Vaccine Immunol. 2012, 19:508-14.

3. Synthetic/natural lipids

2. Metabolic mediators,

such as ToFA

1. Virus-replication competent IFN expression

Regulatory lipid nano-particle

(LNP)

Validation for Therapeutic Designs

Cytokine and signature gene phenotyping to correlate cell activation statuses with PRRSV pathogenicity.

Blood –Whole-blood and PBMCs for isolation of monocytes, cDC and pDCs (LM)BALF –LMSamples prepped for flow cytometry/sorting

RT-PCR (RNAlater) and Searchlight (cytokine buffer.

The 2015 North American PRRS Symposium wishes to thank the following sponsors for their generous

support: