Project 2: Cellular Mechanisms and Interaction of Neurokinin1 Receptor NK1R Antagonists and HIV CoRe

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Project 2 Cellular Mechanisms and Interaction of
Neurokinin-1 Receptor (NK-1R) Antagonists and HIV
Co-Receptors CCR5 and CXCR4
Principal Investigator - S.D. Douglas, M.D.
Co-Investigators -L.E. Kilpatrick, Ph.D
J.-P. Lai, M.D.
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SP and NK-1R in Macrophages

• SP is a mediator of neuroimmuno-regulation
• Macrophages synthesize SP and express SP
  specific receptors (NK-1R)- autocrine loop
• SP enhances HIV infection of macrophages
• NK-1R antagonists inhibit HIV infection of
  macrophages

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Role of CCR5 in HIV Infection of Macrophages

• The chemokine receptors CCR5 and CXCR4 are the
  primary co-receptors for HIV infection
• NK-1R antagonists selectively inhibit HIV strains
  which use CCR5 as a co-receptor
• Thus, there is a critical relationship between
  NK-1R and CCR5 receptors

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NK-1RCCR5 Interaction in Macrophages
SP
Hetero- dimerization?
NK-1R Antagonists
HIV
CCR5
NK-1R
CD4
Signal Transduction?
CXCR4
Protein Synthesis?
Cytokines, Chemokines, CCR5, CXCR4
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Goal of Project 2 To determine the mechanism by
which SPNK-1R regulates CCR5 function
1) GPCR crosstalk at the level of the receptor or
between signaling pathways 2) Direct effect on
CCR5 expression through alterations in receptor
synthesis 3) Indirect interactions through
cytokines and chemokines
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• The Specific Aims of this Project are designed to
  examine
• Selectivity of NK-1R Antagonists for CCR5
• Mechanism of Interaction between CCR5 and NK-1R
• Level of Receptor (heterodimer complexes)
• Signaling Pathways (crosstalk)
• Regulation of CCR5 expression
• Regulation of cytokines and chemokines
• Specificity of NK-1R Antagonists (siRNA)

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Specific Aim 1
We will characterize the antiviral and
immunomodulating effects of Aprepitant and other
candidate NK-1R antagonists on CCR5 function. We
will ascertain whether the interaction between
NK-1R and CCR5 is unique to the CCR5 receptor or
a common feature of chemokine receptors,
chemotactic receptors, or GPCRs.
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In vitro Inhibition of Pseudotyped HIV infection
of Macrophages by NK-1R Antagonists
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Aim 1 Characterization of the effect of NK-1R
Antagonists on CCR5 receptor-mediated functions
NK-1R antagonists
Untreated controls
Macrophages

• 1. Antiviral Infection by pseudotype HIV (ADA)
• Immunomodulatory Chemotaxis to ligands
• of CCR5, CXCR4, FPR, CSF-1
• Immunomodulatory and Antiviral Expression
  (protein
• and mRNA) of CCR5, CXCR4, chemokines, and
  cytokines

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Test the hypothesis that NK-1R antagonists
alter CCR5 mediated physiological responses
through G-protein coupled receptor (GPCR)
crosstalk.
Specific Aim 2
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Enhancement of RANTES mediated Ca2 Mobilization
by SP
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Aim 2 NK-1R antagonists alter CCR5-mediated
physiological responses through receptor crosstalk
SP and/or NK-1R antagonists
Untreated controls
Macrophages
1. Heterodimers of NK-1R and CCR5
(Constitutive and agonist-mediated) 2.
Receptor phosphorylation and G-protein
coupling 3. Receptor internalization and
desensitization
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Specific Aim 3
Test the hypothesis that activation of NK-1R
mediated signaling amplifies CCR5 mediated
intracellular signaling resulting in synergistic
crosstalk between NK-1R and CCR5 signal
transduction pathways.
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Aim 3 NK-1R antagonists interrupt positive
crosstalk Between NK-1R and CCR5 receptors
SP and/or NK-1R antagonists
Untreated controls
Macrophages
1. CCR5-mediated Ca2 mobilization and PKC
activation 2. CCR5-mediated activation of MAP
kinases and NFkB 3. Specificity of NK-1R for
CCR5 vs. CXCR4, FPR, CSF-1
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Specific Aim 4
We will further define the specificity of
Aprepitant and other NK-1R antagonists for the
NK-1R receptor through knockout studies using
NK-1R siRNA in the macrophage cell line THP-1.
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Aim 4 The relationship between NK-1R and CCR5
(as determined by NK-1R Knockout)
Untreated controls (NK-1R, CCR5, CXCR4)
NK-1R Knockout cells (CCR5 and CXCR4)
1. Infectivity of THP-1 cells with R5 or X4 HIV
strains 2. CCR5/CXCR4 expression 3. CCR5/CXCR4
signaling and chemotaxis
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Interaction with other Projects and Cores
Effects of Chronic Antagonist Exposure
Data analysis and PKA
Project 2 (Douglas, Kilpatrick)
Core B (BBI)
Core C (Biostatistics)
NK-1R-CCR5 interaction
Immuno- modulatory effect
Immuno- modulatory effect
Project 3 (Lackner)
Project 1 (Ho)
Project 4 (Tebas)