Download Innate Immune Response to Ebolavirus Infection

2012 Research Grant Program Winning Abstract
Innate Immune Response to Ebolavirus Infection
By Elizabeth Fritz
Ebolavirus (EBOV) and Marburgvirus (MARV) are single-stranded, negative-sense RNA
viruses of the Filoviridae family recognized for their impressive lethality. Filoviruses are
causative agents of viral hemorrhagic fever (VHF) characterized by hypotension,
inflammation, lymphopenia, thrombocytopenia, coagulation disorders, hemorrhage, and
a rampant cytokine-storm response resulting in multi-organ failure and death. There is
one species of MARV, Lake Victoria marburgvirus, whereas there are five distinct EBOV
species: Sudan ebolavirus (SEBOV), Zaire ebolavirus (ZEBOV), Ivory Coast ebolavirus
(ICEBOV), Bundibugyo ebolavirus (BEBOV), and Reston ebolavirus (REBOV). Mortality
rates are approximately 40 to 90%, depending on the virus, with ZEBOV and MARVAngola being the most virulent. There are no FDA-approved vaccines or therapeutics to
combat EBOV or MARV infection, and we still lack an understanding of the host’s innate
immune response to these Category A Priority Pathogens.
Antiviral host-response elements are affected by filovirus infection: the interferon (IFN)
response, natural killer (NK cells), macrophages, and dendritic cells (DCs).
Macrophages and DCs are primary targets of filovirus infection. DCs are potent antigenpresenting cells that capture foreign antigens for uptake and processing to target
secondary lymphoid tissues for stimulation of T and B cells. The latter process occurs
by antigen presentation, co-stimulation via cell surface expression of CD-40, CD86, and
CD80, and DC cytokines (i.e. IL-2, IL-12, IL-10, and TNF-alpha). Immature DCs display
low MHC class II and co-stimulatory molecule surface expression, whereas high
expression of MHC II, CD80, CD86, CD40, DC83, and IL-12 categorizes mature DCs.
MARV may affect DC maturation and MHC class II antigen presentation, as we
observed low surface expression of HLA-DR in MARV-positive cells of infected nonhuman primates (NHPs). Plasmacytoid DCs in the spleens of these animals showed low
HLA-DR surface staining. Filovirus envelope glycoproteins bind to DC-SIGN, and
diminished DC-SIGN expression was noted in the spleens and lymph nodes of MARVinfected animals. This suggests the virus causes a loss of DCs in lymphoid tissues
and/or affects DC-SIGN surface expression and maturation. A report found that the
EBOV VP35 viral protein impedes the maturation and function of EBOV-stimulated DCs,
with decreased CD4+ cell activation.
DCs and NK cells are in close proximity in secondary lymphoid tissues. NK cells are
activated by DC cytokines (IL-2, IL-12, and IL-18), and DC IL-15 is crucial for NK cell
development, priming, function, and survival. NK cells co-stimulate T and B cells by
surface expression of CD40L, OX40, CD86, and CD70. Two activating killer
immunoglobulin-like receptor (KIR) genes, KIR2DS1 and KIR2DS3, were correlated with
lethal EBOV infection. KIRs are NK cell receptors that control NK function following
specific interactions with MHC class I EBOV-infected NHPs showed a depletion of NK
cells and CD8+ T cells soon after infection. NKp30 up-regulation was linked to potent
NK cytolytic activity against DCs infected with MARV virus-like particles (VLPs).
We will study the DC and NK cell response to EBOV infection in vitro with the objective
of elucidating the innate immune response to EBOV. We hypothesize that filoviruses
immediately target DCs and NK cells allowing rampant viral replication/spread resulting
in an immunosuppressive state incapable of battling infection. These studies will be
conducted in a biosafety level-4 (BSL-4) laboratory equipped with a BD FACSCanto™ II
instrument with BD FACSDiva™ software.
Aim 1: Determine DC and NK cell responses to EBOV. We identified changes in DCs
(HLA-DRlo and DC-SIGNlo) and depletion of NK cells following filovirus infection. To
determine the effect EBOV has on cellular maturation, activation, and function, DCs and
NK cells will be purified from peripheral blood mononuclear cells (PBMCs), checked for
purity using BD fluorochrome-conjugated antibodies (lineage cocktail, CD123, CD11c,
HLA-DR, CD3, CD8, CD16, CD56) and cultured following standard techniques. DCs will
be infected with ZEBOV, a highly pathogenic virus in humans, or REBOV, a
nonpathogenic virus in humans, and stained for cellular markers by using BD
fluorochrome-conjugated antibodies (CD123, CD11c, HLA-DR, CD209, CD86, CD80,
CD40) following a time course of infection (8, 24, 48, and 72 hours). Standard protocols
will apply for using BD Pharmingen™ stain and BD Cytofix™ fixation buffers. In parallel,
DC cytokines (IL-2, IL-10, IL-12, IL-6, IL-18, IL-15, TNF-alpha, IFN-alpha/beta, IFNgamma) will be measured by intracellular cytokine staining by using standard protocols
(BD GolgiPlug™ and BD Cytofix/Cytoperm™ buffer) and standard ELISAs for secreted
protein. EBOV-infected DCs will be co-cultured with homologous donor NK cells to
determine altered activation and function by using BD fluorochrome-conjugated
antibodies against cell surface and intracellular targets (NKG2D, NKp46, NKp30, CD16,
CD3, CD56, CD8, Granzyme B, IFN-gamma) and following standard protocols as above.
Aim 2: Determine DC and NK cell signaling pathways affected by EBOV. EBOV and
MARV are known to interfere with host cell signal transduction pathways, especially that
of STAT phosphorylation. We will examine the effect of ZEBOV and REBOV on DCs
and NK cell signal transduction and apoptosis, and correlate with Aim 1 findings. We will
use BD Phosflow™ and apoptosis reagents (ERK, p38, MEK1, TBK1, STATs, Annexin
V, APO-BRDU kit) to examine signal transduction and apoptosis in DCs and co-cultured
NK cells, following a time course of viral infection (0.5, 1.5, 4.0, 8.0, 12.0, and 24 hours).
Western blot analysis will be used for additional targets and confirm flow cytometry
findings.
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