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. 2010 Oct 15;285(42):32087-95.
doi: 10.1074/jbc.M110.124297. Epub 2010 Aug 11.

A novel pathway for inducible nitric-oxide synthase activation through inflammasomes

Carina L Buzzo  1 , Julia C Campopiano, Liliana M Massis, Silvia L Lage, Alexandra A Cassado, Rafael Leme-Souza, Larissa D Cunha, Momtchilo Russo, Dario S Zamboni, Gustavo P Amarante-Mendes, Karina R Bortoluci
Affiliations

A novel pathway for inducible nitric-oxide synthase activation through inflammasomes

Carina L Buzzo et al. J Biol Chem. .

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.

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Figures

FIGURE 1.
FIGURE 1.
Differential activation of macrophages by extracellular and cytosolic B. subtilis. PM from C57BL/6 mice (3 × 105/200 μl) were stimulated with 1, 3, and 6 μg/ml LPS or purified flagellin from B. subtilis in its free form (FLA-BS) or inserted into DOTAP, a cationic lipid vesicules that permits its delivery to cell cytosol (FLA-BSDot). The release of IL-1β (A), IL-6 (C), and macrophage lysis (B) were determined in culture supernatants at 24 h by ELISA and at 6 h by LHD release, respectively. Numbers represent the mean ± S.D. of triplicate samples. Data are representative of three independent experiments.
FIGURE 2.
FIGURE 2.
Cytosolic flagellin induces iNOS activation. PM (A) or BMDM (B) from C57BL/6 mice (3 × 105/200 μl) were stimulated with 3 μg/ml purified flagellin from B. subtilis in its free form (FLA-BS) or inserted into DOTAP, a cationic lipid vesicules that permits its delivery to cell cytosol (FLA-BSDot). After 24 h iNOS expression was evaluated in cell extracts by Western blot, and nitrite concentrations in culture supernatants were determined by Griess method. Numbers represent the mean ± S.D. of triplicate samples. *, p < 0.05; **, p < 0.01 when compared with control group. Data are representative of three independent experiments.
FIGURE 3.
FIGURE 3.
Nlrc4 and Naip5 are required for cytosolic flagellin-induced iNOS expression. BMDM from wild-type and Nlrc4−/− C57BL/6 mice (A and C) or Naip5-competent (Lgn1B6/AJ) and Naip5-deficient (Lgn1AJ/AJ) mice (B and D) were stimulated with 3 μg/ml purified flagellin from B. subtilis inserted into DOTAP, a cationic lipid vesicule that permits its delivery to cell cytosol (FLA-BSDot). After 24 or 5 h, iNOS expression was evaluated in cell extracts by Western blot (A and B) or at transcriptional levels by real-time RT-PCR (C and D), respectively. Data are representative of three independent experiments. Numbers represent the mean ± S.D. of triplicate samples. ***, p < 0.001 when compared with control group. #, p < 0.05; ###, p < 0.001 when compared with FLA-BSDot group from deficient mice. Ctrl, control.
FIGURE 4.
FIGURE 4.
Caspase-1 is required for cytosolic flagellin-induced iNOS expression. PMs or BMDM from C57BL/6 or caspase-1−/− mice were stimulated with 3 μg/ml purified free (FLA-BS) or cytosolic (FLA-BSDot) flagellin from B. subtilis (A, B, and E) or flagellin from S. typhimurium (FliC or FliCDot, respectively) (C and D) in the presence or absence of z-YVAD-fmk (2 μm). After 24 or 5 h, iNOS expression was evaluated in cell extracts by Western blot (B, C, and D) or at transcriptional levels by real-time RT-PCR (E), respectively. IL-1β secretion was determined in culture supernatants by ELISA (A). Numbers represent the mean ± S.D. of triplicate samples. ***, p < 0.001 when compared with control group. ###, p < 0.001 when compared with FLA-BSDot group from caspase-1−/− BMDM. Data are representative of three independent experiments.
FIGURE 5.
FIGURE 5.
Induction of iNOS expression by cytosolic flagellin is independent of IL-1β and IL-18. PMs (A, B) and BMDM (C) from C57BL/6 were stimulated with 3 μg/ml purified free (FLA-BS) or cytosolic (FLA-BSDot) flagellin from B. subtilis in the presence or absence of neutralizing anti-IL-1β (1 μg/ml) or anti-IL-18 (5 μg/ml). After 24 h, iNOS expression was evaluated in cell extracts by Western blot, and IL-1β secretion was determined in culture supernatants by ELISA. Numbers represent the mean ± S.D. of triplicate samples. ***, p < 0.001 when compared with control group. ###, p < 0.001 when compared with FLA-BSDot in the presence of neutralizing anti-IL-1β. Data are representative of three independent experiments.
FIGURE 6.
FIGURE 6.
MYD88 is not required for cytosolic flagellin-induced macrophage responses. PMs from Myd88−/− mice were stimulated with 3 μg/ml purified cytosolic flagellin from B. subtilis (FLA-BSDot). After 6 and 24 h macrophage lysis was monitored by LDH release in culture supernatants (B), and iNOS expression was evaluated in cell extracts by Western blot (A), respectively. Data are representative of three independent experiments.
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