A Novel Pathway for Inducible Nitric-oxide Synthase Activation through Inflammasomes

Julia C. Campopiano,Silvia L. Lage,Gustavo P. Amarante-Mendes,Dario S. Zamboni,Momtchilo Russo,Rafael Leme-Souza,Larissa D. Cunha,Liliana M. Massis,Karina R. Bortoluci,Alexandra A. Cassado,Carina L. Buzzo

doi:10.1074/jbc.m110.124297

Abstract

Innate immune recognition of flagellin is shared by transmembrane TLR5 and cytosolic Nlrc4 (NOD-like receptor family CARD (caspase activation recruitment domain) domain containing 4)/Naip5 (neuronal apoptosis inhibitory protein 5). TLR5 activates inflammatory genes through MYD88 pathway, whereas Nlrc4 and Naip5 assemble multiprotein complexes called inflammasomes, culminating in caspase-1 activation, IL-1β/IL-18 secretion, and pyroptosis. Although both TLR5 and Naip5/Nlrc4 pathways cooperate to clear infections, little is known about the relative anti-pathogen effector mechanisms operating through each of them. Here we show that the cytosolic flagellin (FLA-BSDot) was able to activate iNOS, an enzyme previously associated with TLR5 pathway. Using Nlrc4- or Naip5-deficient macrophages, we found that both receptors are involved in iNOS activation by FLA-BSDot. Moreover, distinct from extracellular flagellin (FLA-BS), iNOS activation by intracellular flagellin is completely abrogated in the absence of caspase-1. Interestingly, IL-1β and IL-18 do not seem to be important for FLA-BSDot-mediated iNOS production. Together, our data defined an additional anti-pathogen effector mechanism operated through Naip5 and Nlrc4 inflammasomes and illustrated a novel signaling transduction pathway that activates iNOS.

Highlights

Toll-like receptors (TLRs) and NOD-like receptors (NLRs) have been described as central elements in triggering innate immune responses [1, 2]
Extracellular flagellin is recognized by transmembrane TLR5 [8], whereas cytosolic sensing of flagellin requires Naip5 and Nlrc4 (NLR family CARD domain-containing 4), called Ipaf (ICE-(IL-1converting enzyme) protease activating factor), both members of the NLR family (9 –13)
It was recently demonstrated that both Nlrc4 and Naip5 are required for signaling in response to the 35-amino acid of a peptide located on the C-terminal D0 domains of Legionella pneumophila and Salmonella typhimurium flagellins and that this region of flagel

Summary

EXPERIMENTAL PROCEDURES

Mice— 6 – 8-Week-old wild-type (WT) C57BL/6 and Myd88deficient female mice were bred in our animal facilities at the University of Sao Paulo. Naip functions of the F7 mice were monitored by measuring restriction of L. pneumophila multiplication in macrophages (supplemental Fig. 1). These mice were bred and constructed in the animal facilities at the University of Sao Paulo-Ribeirao Preto. Macrophage Infection with L. pneumophila—BMDMs from C57BL/6- and Myd88-deficient mice (2 ϫ 105 cells seeded in 24-well plates) were infected with wild-type or flagellin-deficient mutant FlaA L. pneumophila at a multiplicity of infection of 0.015 for 24, 48, and 72 h in the presence or absence of 0.1 mM aminoguanidine (selective inhibitor of iNOS [23]; SIGMA). Differences between experimental groups were considered significant for p Ͻ 0.05 (*), p Ͻ 0.01 (**), and p Ͻ 0.001 (***)

RESULTS

Macrophages were cultured with

DISCUSSION