During the past decade, major efforts using genomic and genetic approaches have identified two major classes of innate immune receptors for sensing microbial nucleic acids: Toll-like receptors (TLR: TLR3, 7, 8, 9) and retinoic acid-inducible gene I-like helicases (RLH: RIG-I, LGP2, MDA-5). However, genomic and genetic approaches have left a major gap in our understanding on how these receptors bind nucleic acids and whether additional receptors or coreceptors exist. We decided to open this "Black Box" by taking a biochemical approach to directly isolate and characterize microbial nucleic acid-binding proteins in human plasmacytoid dendritic cells (pDCs) by CpG-DNA pull down and mass spectrometric peptide sequencing experiments. We identified two novel members of the DExD/H-box helicase family DHX36 and DHX9 that specifically bind CpG- A and CpG-B, respectively, in human pDCs. Knocking down DHX36 expression by siRNA in a pDC cell line was associated with over 50% reduction in type 1 IFN responses and abolished IRF7 nuclear translocation induced by CpG-A and knocking down DHX9 expression was associated with a diminished TNF and IL-6 response and blocking of NF-kB p50 nuclear translocation induced by to CpG-B. The DExD/H helicase family has over 50 members. Based on these preliminary data and the fact that the other viral sensors, including RIG- I-like helicases (RIG-I, LGP2, MDA-5) and Dicer, all belong to the DExD/H helicase family, this proposal will test our central hypotheses: 1) the DExD/H-box helicases DHX36 and DHX9 may represent novel TLR9- independent DNA sensors in pDCs;and 2) the DExD/H helicase family (59 members) may play a much broader role in anti-viral innate immunity than previously thought. )

Public Health Relevance

By CpG-DNA pull down and mass spectrometric peptide sequencing, we identified two novel DExD/H- box helicase family members, DHX36 and DHX9, that specifically bind and respond to CpG-A and CpG-B, respectively, in human pDCs. Because many other viral sensors, including RIG-I like helicases (RIG-I, LGP2, MDA-5) and Dicer, all belong to the DExD/H helicase family, this proposal will test our central hypotheses: 1) the DExD/H-box helicases DHX36 and DHX9 may represent novel TLR9-independent DNA sensors in pDCs;and 2) DExD/H helicase family (59 members) may play a much broader role in anti-viral innate immunity than previously thought. )

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37AI091947-04
Application #
8459503
Study Section
Cellular and Molecular Immunology - A Study Section (CMIA)
Program Officer
Palker, Thomas J
Project Start
2011-05-01
Project End
2016-04-30
Budget Start
2013-05-01
Budget End
2014-04-30
Support Year
4
Fiscal Year
2013
Total Cost
$368,480
Indirect Cost
$133,480
Name
Baylor Research Institute
Department
Type
DUNS #
145745022
City
Dallas
State
TX
Country
United States
Zip Code
75204
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