Rip Proteins in Innate Immune Signaling

NIH Pandemic-Era Grants

Pandemic Era Grants

2024

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Principal Investigator: MICHELLE ALICE KELLIHER
Organization: UNIV OF MASSACHUSETTS MED SCH WORCESTER
Fiscal Year: 2024
Award: $502,500
Funding agency: National Institute of Allergy and Infectious Diseases

PROJECT SUMMARY/ABSTRACT
The increased incidence of autoimmune and inflammatory disorders highlights the need for improved
therapeutics to treat these diseases. Our work places RIPK1 kinase at the apex of inflammatory pathways
where it regulates a form of inflammatory cell death called necroptosis mediated by RIPK1, RIPK3 and
MLKL. In the work proposed, we will delineate novel necroptosis pathways important in autoimmunity and
immune-mediated liver injury operative in autoimmune and viral hepatitis.
We demonstrate that mice with a dendritic cell RIPK1-deficiency (Ripk1DC KO) develop inflammation and
autoimmunity; unexpectedly revealing that RIPK1 restrains necroptosis in dendritic cells (DC) in contrast to
it well established role as an initiator of necroptosis. During development, RIPK1 prevents necroptosis
mediated by the nucleic acid sensor Z-DNA binding protein (ZBP1) leading us to hypothesize that ZBP1-
mediated dendritic cell necroptosis breaks tolerance and induces autoimmunity. We will test this hypothesis
in Aim 1 by determining whether a ZBP1-deficiency prevents inflammation and autoimmunity in Ripk1DC KO
mice. We will identify the nucleic acid species bound to ZBP1 and will establish whether nucleic acid binding
is required to induce inflammation and/or autoimmunity. Nucleic acids in the Z-form are known to be highly
immunogenic and anti-Z-DNA autoantibodies occur in lupus patients; therefore, we will test genetically
whether inhibiting ZBP1 or necroptosis ameliorates disease in other autoimmune prone mice. In Aim 2, we
will investigate whether RIPK1-kinase-dependent necroptosis contributes to a mouse model of autoimmune
hepatitis (AIH) induced by the plant lectin Concanavalin A (Con A). Based on our preliminary data in
reciprocal hematopoietic chimeras we hypothesize that AIH is mediated by canonical necroptosis in
hematopoietic cells and noncanonical necroptosis in hepatocytes. Consistent with increased RIPK1 and
MLKL expression in the livers of AIH patients, our preliminary data implicate RIPK1 and MLKL but not
RIPK3 in hepatocyte cell death, leading us to hypothesize that a novel RIPK3 independent necroptosis
pathway is induced in hepatocytes. Going forward we will test this hypothesis rigorously by generating mice
that express kinase inactive RIPK1D138N or delete Ripk3 or Mlkl specifically in liver parenchymal cells. An
additional goal will be to delineate this non-canonical necroptosis pathway, by identifying the RIPK1-
interacting kinase responsible for MLKL activation in hepatocytes and to assess its contribution to AIH.
Successful completion of these Aims will elucidate the contribution(s) of these new necroptosis pathways
to autoimmunity and acute liver inflammation with the long-term goal to identify those
autoinflammatory/immune diseases that might benefit from RIPK1 kinase targeted therapy

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