david carratu dtc presentation
TRANSCRIPT
MHV-68 infection increases IL-1β in HBCs and in HBC culture media
David CarratuDr. Seth GullerZhonghua Tang
Background
Viral infections and Pregnancy
• Viral infections have severe pregnancy complications due to activation of the inflammatory response: – Miscarriage – Preterm delivery – Passing the virus to the fetus• Severe infections• Brain, lung, and liver damage
– Sensitize the pregnant mother to bacterial infections
Herpes Virus
• Herpes viruses are a common cause of viral-related perinatal neurologic injury
• Murine γ-herpesvirus 68 (MHV-68) – Murine herpes virus that shares significant
genomic collinearity with human herpes viruses– Used in earlier studies • Mor et al. AJRI 2010
Hofbauer Cells (HBCs)
• M2 Placental macrophage– Macrophage: white blood cell that engulfs and
digests cellular debris, microbes, and foreign substances
– M2 phenotype: decrease inflammation and encourage tissue repair
• Role in microbial driven placental/fetal inflammation
Interleukin-1beta (IL-1β)
• A cytokine protein• Produced by activated macrophages in pro
form• Processed to mature form by Caspase-1• Important mediator of the inflammatory
response– Regulates immune responses &
inflammatory reactions
NLRP3 Inflammasome
• A multiprotein oligomer consisting of Caspase-1, ASC, and NLRP3 – ASC and NLRP3 are proteins that are crucial to
inflammasome activation• Responsible for activation of inflammatory
responses – Converts Pro-caspase-1 to (active) Caspase-1
Mechanisms of NLRP3 Inflammasome activation
NF-κB Activation
• NF-κB is a protein complex that plays a key role in regulating the immune response to infection and promoting gene expression
• IκBα, when attached to NF-κB, deactivates NF-κB
• Following phosphorylation of IκBα by IKK, IκBα will dissociate from NF-κB and then NF-κB is translocated to the nucleus and promotes activation of the genes for Pro IL-1β
Extracellular signal-regulated kinases (ERKs)
• Protein kinase intracellular signaling molecules
• Pathway activated by virus infections • Known to activate many transcription factors
IL-1β Production PathwayVirus
Transcription Pro IL-1B
ASC
Pro-Caspase
1
NLRP3
Caspase1
Mature IL-1B
ERKsIKK
p50
IkBα
p65 ?
IκBαp50 p65
NLRP3 Inflammasome
Inactive NF-κB
Active NF-κB
Nucleus
Objective #1
• Analyze the effect of MHV-68 infection on levels of cell associated (pro) IL-1β and secreted (mature) IL-1β in HBCs and in their conditioned media, respectively
Methods
Isolation of HBCs and MHV-68 Infection
• Isolated HBCs from term placental tissue (reference: Tang et al. AJRI 2011)
• Incubated with MHV-68 for 1hr, removed media, and added fresh media
• Collected samples from cell material and media at the following times:– T0, 2hr, 4hr, 8hr, 24hr
Western Blotting• Gel electrophoresis• Transfer to membrane• Primary antibody• Secondary antibody• Scan membrane
qPCR
• Exponential amplification of DNA• Non-specific fluorescent dyes intercalate with
double-stranded DNA• Machine measures fluorescents after each
cycle
ELISA
Methods • Western blotting– Measures protein levels in cells and media– Measured Pro IL-1β, mature IL-1β, ASC, NLRP3,
IkBα, and p-ERK• qPCR– Measured IL-1β mRNA (converted IL-1β mRNA to
cDNA • ELISA– Measures concentration of protein in cell media– Measured secreted IL-1β
Results
MHV infection increases pro IL-1β in HBCs
IL-1β
HSP-90
- + - + - + - + - +T0 2hr 4hr 8hr 24hr
MHV 37 kDa
100 kDa
75 kDa
4h 8h 24h0.00
100.00
200.00
300.00
400.00
500.00
600.00
700.00
800.00
MHV infection increases IL-1β mRNA in HBCs by qPCR
-MHV +MHV
Time(hr)
Fold
Incr
ease
MHV infection increases IL-1β in HBCCulture Media
37 kDa
25 kDa
15 kDa
20 kDa
- + - + - + - +
2hr 4hr 8hr 24hr
MHV
IL-1β(mature)
IL-1β(pro)
2 4 8 240.00
500.00
1000.00
1500.00
2000.00
2500.00
MHV infection increases Secreted IL-1β in HBC
Culture Media by ELISA
MHV-MHV+
Time (hr)
Conc
entr
ation
(pg/
ml)
Conclusion #1
• MHV-68 infection increases IL-1β pro form and mature form in HBCs and in HBC culture media
Objective #2
• Understand the mechanisms behind increased IL-1β production following MHV infection– NLRP3 inflammasome and NF-κB activation– ERK Pathway
MHV infection increases levels of components of NLRP3 inflammasome?
100 kDa
75 kDa
100 kDa
75 kDa
25 kDa
- + - + - + - + - +T0 2hr 4hr 8hr 24hr
HSP-90
NLRP3
ASC
MHV
MHV Infection effects IκBαthrough IKK phosphorylation
100 kDa75 kDa
HSP-90
IκBα
- + - + - + - + - +T0 2hr 4hr 8hr 24hr
MHV
37kDa
pIKK 100kDa75kDa
MHV infection affects ERK pathway
- + - + - + - + - +T0 2hr 4hr 8hr 24hr
MHV
P-ERK50kDa
37kDa
100 kDa75 kDa
HSP-90
Conclusions #2
• MHV-68 infection :• Increases NLRP3 levels in HBCs (potentially
increases inflammasome activity)
• Increases IKK phosphorylation• Decreases IκBα Levels • Increased NF-kB action• Increases ERK pathway
Increased IL-1β mRNA production }
Future Directions
• Analyze the effect of other viral infections on IL-1β levels in HBCs
• Further understand the role that Caspase-1 plays in increased IL-1β levels
• Further study Erk and NF-κB pathways and their effect on pro IL-1β transcription