Forward genetic analysis of Toll-like receptor induced necroptosis and RIP1 kinase driven inflammation in wild-derived mice.
Abstract: The innate
immune system, including the Toll-like receptor family, recognizes invading pathogens
and activates the immune response through the induction of cytokines and other
antimicrobial factors. The outcomes of TLR ligation and signaling extend beyond
activation of gene expression to include programmed cell death. When virus-encoded
caspase inhibitors block caspase activity or ... read morewhen caspase expression in tissues is low,
cells activate a necrotic cell death pathway through RIP1 and RIP3 known as necroptosis.
In addition to established signaling pathways to trigger necroptosis such as TNF-R1,
TLRs activate necroptosis when caspases are inhibited. Peritoneal macrophages from the
wild-derived mouse strain MOLF are resistant to necroptosis when compared with the
classical inbred strain C57BL/6. Forward genetic analysis using an N2 backcross panel
identified two loci in which harboring a MOLF allele conferred resistance to TLR-induced
necroptosis. One locus exhibited differential regulation of CYLD, a deubiquitinase that
cleaves K63 and linear polyubiquitin chains from RIP1 and is known to promote
necroptosis downstream of TNF-R1. CYLD was shown here to promote necroptosis driven by
TRIF-dependent TLRs in the absence of TNF-R1. In MOLF peritoneal macrophages, TLR
stimulation reduced Cyld mRNA expression and diminished protein levels. Decreased
recruitment of RNA polymerase II to a promoter proximal site in MOLF Cyld suggested that
a defect in promoter pausing or elongation may underlie CYLD downregulation. With low
levels of CYLD, MOLF peritoneal macrophages are resistant to TLR-induced necroptosis
when compared to B6, which express higher levels of CYLD. RIP1 kinase activity can also
contribute to activation of gene transcription and cytokine production in the absence of
caspase activity. While a scaffolding role for RIP1 is well documented for TNF-R1 and
TRIF-mediated signaling pathways, RIP1 kinase activity augmented the induction of TNF
and IFNβ after TLR4 ligation when caspases were inhibited by the pan-caspase
inhibitor ZVAD. MOLF macrophages generated more cytokine mRNA through the RIP1 kinase
dependent pathway than B6. MOLF but not B6 macrophages activated p38 in a RIP1
kinase-dependent manner through a mechanism involving IRAK2 and the activation of this
arm of the MAPK pathway likely contributed to the more robust response of MOLF
Thesis (Ph.D.)--Tufts University, 2016.
Submitted to the Dept. of Immunology.
Advisor: Alexander Poltorak.
Committee: Peter Brodeur, Stephen Bunnell, Miguel Stadecker, and Michelle Kelliher.
Keyword: Immunology.read less