Inflammasomes alert the mammalian immune system to the presence of infection and tissue damage. These sensory complexes activate caspase-1 in the "canonical" pathway to cleave IL-1 family cytokines for secretion and eliminate infected host cells via a form of lytic cell death termed pyroptosis. In addition, a new "non- canonical" inflammasome has emerged that enlists caspase-11 specifically in response to Gram-negative bacteria. How each pathway discriminates microbial from danger signals and the host proteins responsible for this functional dichotomy remain unknown. Both questions are central to understanding the biology of inflammasome-mediated host defense. Here, we tackle this issue by focusing on a new family of 65-73kDa immune GTPases termed the Guanylate binding Proteins (GBPs) that uncouple these two activities. GBP5 specifically promotes canonical NLRP3 inflammasome activation to microbial but not danger signals, the first such protein to exhibit this profile. Subsequent work has found GBP1 and GBP2 confer the same selectivity. Thus the GBPs offer a unique opportunity to understand how one side of this bimodal response operates.
In Aim 1 we will test whether GBP1, GBP2 and GBP5 promotes not only the canonical inflammasome pathway but also the non-canonical inflammasome triggered by Gram-negative bacterial stimuli. This will be examined using genetically-deficient human and mouse macrophages as well as newly-created Gbp1-/-, Gbp2-/- and Gbp5-/- mice infected with Salmonella typhimurium (Stm) variants that stimulate either inflammasome pathway. Our powerful genetic approach will reveal which GBP governs what inflammasome activities in vitro and in vivo. Thereafter, we will dissect the molecular mechanisms used by the inflammasome-related GBPs to confer their intracellular functions as part of Aim 2. Here gene-deficient macrophages complemented with GBP mutants with distinct biochemical lesions will test whether they are involved in cytosolic ligand detection or spatial assembly of the inflammasome complex during infection. "Reconstitution" studies will also reveal if the core inflammasome machinery is physically assembled by the GBPs as part of this macromolecular complex. Collectively, our proposal examines a new set of host factors that partition the inflammasome response with important implications for human therapies designed to promote antimicrobial activities while avoiding tissue damage.

Public Health Relevance

Inflammasomes are protein assemblies inside host cells that detect the presence of infection as well as sterile damage during inflammation. This proposal focuses on a new group of proteins - the Guanylate Binding Proteins (GBPs) - that have recently been discovered to orchestrate inflammasome responses specifically against infection by human pathogens such as Salmonella that are a major cause of food-borne gastroenteritis and blood-borne sepsis in U.S. intensive care units. Our efforts will uncover the different type of inflammasomes regulated by the GBPs to protect against bacterial infection as well as the mechanisms they employ to regulate these protective complexes inside host cells. Understanding the molecular basis for how inflammasomes recognize different microbial signatures is critical to the intense research effort aimed at generating inflammsome-based therapeutic approaches to treat a variety of infectious and inflammatory diseases.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
1R01AI108834-01A1
Application #
8816302
Study Section
Innate Immunity and Inflammation Study Section (III)
Program Officer
Palker, Thomas J
Project Start
2014-09-19
Project End
2019-08-31
Budget Start
2014-09-19
Budget End
2015-08-31
Support Year
1
Fiscal Year
2014
Total Cost
$416,250
Indirect Cost
$166,250
Name
Yale University
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
043207562
City
New Haven
State
CT
Country
United States
Zip Code
06520