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