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

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. 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. IntroductionInnate immune system employs intracellular and extracellular pattern recognition receptors (PRRs) 2The abbreviations used are: PRRpattern recognition receptorNLRNOD-like receptorTLRToll-like receptorIRFIFN-responsive factorsNaip5neuronal apoptosis inhibitory protein 5CARDcaspase activation recruitment domainiNOSinducible nitric-oxide synthaseBMDMbone marrow-derived macrophagePMperitoneal macrophagez-YVAD-fmkbenzyloxycarbonyl-YVAD-fluoromethyl ketonePAMPpathogen-associated molecular patternDOTDOTAP (lipidic vesicles). to detect microbial products through the identification of pathogen-associated molecular patterns (PAMPs). Among many families of PRRs, Toll-like receptors (TLRs) and NOD-like receptors (NLRs) have been described as central elements in triggering innate immune responses (1Franchi L. Eigenbrod T. Muñoz-Planillo R. Nuñez G. Nat. Immunol. 2009; 10: 241-247Crossref PubMed Scopus (1259) Google Scholar, 2Medzhitov R. Nature. 2007; 449: 819-826Crossref PubMed Scopus (1962) Google Scholar).TLRs and NLRs have been shown to activate distinct biochemical pathways, resulting in diverse mechanisms of resistance to pathogens. These mechanisms may act independently or may cooperate to the resolution of infections. TLRs primarily activate transcriptional factors such as NF-κB and IFN-responsive factors (IRF) (3Akira S. Takeda K. Nat. Rev. Immunol. 2004; 4: 499-511Crossref PubMed Scopus (6573) Google Scholar, 4Iwasaki A. Medzhitov R. Nat. Immunol. 2004; 5: 987-995Crossref PubMed Scopus (3274) Google Scholar), culminating in the production of effector molecules such as inflammatory cytokines and type I interferons. The contribution of these pathways to resistance to infection is manifold: recruitment of other inflammatory cells, production of cytotoxic and cytostatic molecules, and activation of adaptive immune response. NLRs, on the other hand, although also capable of activating NF-κB, have a peculiar ability of initiating an inflammatory response through caspase-1 activation via the assembly of a multiprotein complex called inflammasome (5Inohara N. Ogura Y. Fontalba A. Gutierrez O. Pons F. Crespo J. Fukase K. Inamura S. Kusumoto S. Hashimoto M. Foster S.J. Moran A.P. Fernandez-Luna J.L. Nuñez G. J. Biol. Chem. 2003; 278: 5509-5512Abstract Full Text Full Text PDF PubMed Scopus (1386) Google Scholar, 6Inohara Chamaillard McDonald C. Nuñez G. Annu. Rev. Biochem. 2005; 74: 355-383Crossref PubMed Scopus (802) Google Scholar, 7Bortoluci K.R. Medzhitov R. Cell. Mol. Life Sci. 2010; 67: 1643-1651Crossref PubMed Scopus (109) Google Scholar). Besides the well known role of caspase-1 on IL-1β and IL-18 maturation and secretion, this protease has also been described to induce macrophages death by a process known as pyroptosis (6Inohara Chamaillard McDonald C. Nuñez G. Annu. Rev. Biochem. 2005; 74: 355-383Crossref PubMed Scopus (802) Google Scholar, 7Bortoluci K.R. Medzhitov R. Cell. Mol. Life Sci. 2010; 67: 1643-1651Crossref PubMed Scopus (109) Google Scholar). Importantly, pyroptosis has been ascribed as a mechanism of host immune response against certain pathogens, such as Salmonella, Legionella, Pseudomonas, and Shigella, although it is still not clear how pyroptosis effectively contributes for pathogen clearance (7Bortoluci K.R. Medzhitov R. Cell. Mol. Life Sci. 2010; 67: 1643-1651Crossref PubMed Scopus (109) Google Scholar).The recognition of PAMPs by TLRs and NLRs occur independently. However, there are cases where a certain PAMP can be recognized by both TLRs and NLRs. Flagellin, the monomeric subunit of flagella present in several Gram-negative and Gram-positive bacteria is a good example of this redundancy. Extracellular flagellin is recognized by transmembrane TLR5 (8Hayashi F. Smith K.D. Ozinsky A. Hawn T.R. Yi E.C. Goodlett D.R. Eng J.K. Akira S. Underhill D.M. Aderem A. Nature. 2001; 410: 1099-1103Crossref PubMed Scopus (2762) Google Scholar), whereas cytosolic sensing of flagellin requires Naip5 (neuronal apoptosis inhibitory protein 5) and Nlrc4 (NLR family CARD (caspase activation recruitment domain) domain-containing 4), also called Ipaf (ICE-(IL-1-converting enzyme) protease activating factor), both members of the NLR family (9Amer A. Franchi L. Kanneganti T.D. Body-Malapel M. Ozören N. Brady G. Meshinchi S. Jagirdar R. Gewirtz A. Akira S. Núñez G. J. Biol. Chem. 2006; 281: 35217-35223Abstract Full Text Full Text PDF PubMed Scopus (380) Google Scholar, 10Franchi L. Amer A. Body-Malapel M. Kanneganti T.D. Ozören N. Jagirdar R. Inohara N. Vandenabeele P. Bertin J. Coyle A. Grant E.P. Núñez G. Nat. Immunol. 2006; 7: 576-582Crossref PubMed Scopus (903) Google Scholar, 11Lightfield K.L. Persson J. Brubaker S.W. Witte C.E. von Moltke J. Dunipace E.A. Henry T. Sun Y.H. Cado D. Dietrich W.F. Monack D.M. Tsolis R.M. Vance R.E. Nat. Immunol. 2008; 9: 1171-1178Crossref PubMed Scopus (371) Google Scholar, 12Mariathasan S. Newton K. Monack D.M. Vucic D. French D.M. Lee W.P. Roose-Girma M. Erickson S. Dixit V.M. Nature. 2004; 430: 213-218Crossref PubMed Scopus (1382) Google Scholar, 13Zamboni D.S. Kobayashi K.S. Kohlsdorf T. Ogura Y. Long E.M. Vance R.E. Kuida K. Mariathasan S. Dixit V.M. Flavell R.A. Dietrich W.F. Roy C.R. Nat. Immunol. 2006; 7: 318-325Crossref PubMed Scopus (419) Google Scholar). 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 flagellin is distinct from the D1 domain sensed by TLR5 (11Lightfield K.L. Persson J. Brubaker S.W. Witte C.E. von Moltke J. Dunipace E.A. Henry T. Sun Y.H. Cado D. Dietrich W.F. Monack D.M. Tsolis R.M. Vance R.E. Nat. Immunol. 2008; 9: 1171-1178Crossref PubMed Scopus (371) Google Scholar). Importantly, TLR5 engagement by flagellin leads to NF-κB activation, whereas the recognition of flagellin by Naip5 and Nlrc4 induces the assembly of inflammasomes with the recruitment and activation of pro-caspase-1 (7Bortoluci K.R. Medzhitov R. Cell. Mol. Life Sci. 2010; 67: 1643-1651Crossref PubMed Scopus (109) Google Scholar).Regardless the particularities between intracellular and extracellular receptor pathways, the relative contribution of TLR5 and Naip5/Nlrc4 inflammasomes to host resistance to infection is still not clear. Here, by using a purified flagellin, we defined an additional anti-pathogen effector mechanism operating through Naip5 and Nlrc4 inflammasomes that was previously associated with TLR pathway (14Brightbill H.D. Libraty D.H. Krutzik S.R. Yang R.B. Belisle J.T. Bleharski J.R. Maitland M. Norgard M.V. Plevy S.E. Smale S.T. Brennan P.J. Bloom B.R. Godowski P.J. Modlin R.L. Science. 1999; 285: 732-736Crossref PubMed Scopus (1399) Google Scholar, 15Thoma-Uszynski S. Stenger S. Takeuchi O. Ochoa M.T. Engele M. Sieling P.A. Barnes P.F. Rollinghoff M. Bolcskei P.L. Wagner M. Akira S. Norgard M.V. Belisle J.T. Godowski P.J. Bloom B.R. Modlin R.L. Science. 2001; 291: 1544-1547Crossref PubMed Scopus (586) Google Scholar), namely the activation of inducible nitric-oxide synthase (iNOS), the enzyme responsible for nitric oxide (NO) production (16Bogdan C. Trends Cell Biol. 2001; 11: 66-75Abstract Full Text Full Text PDF PubMed Scopus (454) Google Scholar). Interestingly, distinct from extracellular flagellin, iNOS activation by intracellular flagellin is completely abrogated in the absence of caspase-1 but not with neutralization of IL-1β and/or IL-18. Importantly, this effect is preserved in Myd88-deficient macrophage, ruling out the participation of Together, our data that by using distinct pathways, TLR and NLR can with a certain of the host resistance against is of the protein sensed by the innate immune system and can activate both TLR and NLR Extracellular flagellin is recognized by transmembrane TLR5 (8Hayashi F. Smith K.D. Ozinsky A. Hawn T.R. Yi E.C. Goodlett D.R. Eng J.K. Akira S. Underhill D.M. Aderem A. Nature. 2001; 410: 1099-1103Crossref PubMed Scopus (2762) Google Scholar), whereas cytosolic flagellin is sensed by Naip5 and Nlrc4 (9Amer A. Franchi L. Kanneganti T.D. Body-Malapel M. Ozören N. Brady G. Meshinchi S. Jagirdar R. Gewirtz A. Akira S. Núñez G. J. Biol. Chem. 2006; 281: 35217-35223Abstract Full Text Full Text PDF PubMed Scopus (380) Google Scholar, 10Franchi L. Amer A. Body-Malapel M. Kanneganti T.D. Ozören N. Jagirdar R. Inohara N. Vandenabeele P. Bertin J. Coyle A. Grant E.P. Núñez G. Nat. Immunol. 2006; 7: 576-582Crossref PubMed Scopus (903) Google Scholar, 11Lightfield K.L. Persson J. Brubaker S.W. Witte C.E. von Moltke J. Dunipace E.A. Henry T. Sun Y.H. Cado D. Dietrich W.F. Monack D.M. Tsolis R.M. Vance R.E. Nat. Immunol. 2008; 9: 1171-1178Crossref PubMed Scopus (371) Google Scholar, 12Mariathasan S. Newton K. Monack D.M. Vucic D. French D.M. Lee W.P. Roose-Girma M. Erickson S. Dixit V.M. Nature. 2004; 430: 213-218Crossref PubMed Scopus (1382) Google Scholar, 13Zamboni D.S. Kobayashi K.S. Kohlsdorf T. Ogura Y. Long E.M. Vance R.E. Kuida K. Mariathasan S. Dixit V.M. Flavell R.A. Dietrich W.F. Roy C.R. Nat. Immunol. 2006; 7: 318-325Crossref PubMed Scopus (419) Google Scholar, T. D.S. Roy C.R. Dietrich W.F. Vance R.E. 2006; PubMed Scopus Google Scholar, E.A. M. Aderem A. Nat. Immunol. 2006; 7: PubMed Scopus Google Scholar). Interestingly, TLR5 to whereas sensing of Nlrc4 and Naip5 on present in such as the type of Salmonella L. Amer A. Body-Malapel M. Kanneganti T.D. Ozören N. Jagirdar R. Inohara N. Vandenabeele P. Bertin J. Coyle A. Grant E.P. Núñez G. Nat. Immunol. 2006; 7: 576-582Crossref PubMed Scopus (903) Google Scholar, E.A. M. Aderem A. Nat. Immunol. 2006; 7: PubMed Scopus Google and the type of Legionella (9Amer A. Franchi L. Kanneganti T.D. Body-Malapel M. Ozören N. Brady G. Meshinchi S. Jagirdar R. Gewirtz A. Akira S. Núñez G. J. Biol. Chem. 2006; 281: 35217-35223Abstract Full Text Full Text PDF PubMed Scopus (380) Google Scholar, 13Zamboni D.S. Kobayashi K.S. Kohlsdorf T. Ogura Y. Long E.M. Vance R.E. Kuida K. Mariathasan S. Dixit V.M. Flavell R.A. Dietrich W.F. Roy C.R. Nat. Immunol. 2006; 7: 318-325Crossref PubMed Scopus (419) Google Scholar). These the of products the host Nlrc4- and caspase-1 activation can be by purified flagellin the host by of or by with L. Amer A. Body-Malapel M. Kanneganti T.D. Ozören N. Jagirdar R. Inohara N. Vandenabeele P. Bertin J. Coyle A. Grant E.P. Núñez G. Nat. Immunol. 2006; 7: 576-582Crossref PubMed Scopus (903) Google Scholar, E.A. M. Aderem A. Nat. Immunol. 2006; 7: PubMed Scopus Google Scholar, D.S. 2010; PubMed Scopus Google Scholar). with this TLR5 primarily leads to the activation of effector mechanisms that are in a effector mechanism of a inflammatory death is in with bacteria (7Bortoluci K.R. Medzhitov R. Cell. Mol. Life Sci. 2010; 67: 1643-1651Crossref PubMed Scopus (109) Google the of capable of sensing the or present on the pathogen is to the The of responses to a against pathogens. Extracellular and intracellular recognition of flagellin induces such distinct of with whereas IL-1β and pyroptosis with and However, as the of is not not host are to and pathogens, it be for the host to have family activation by TLRs can be of the effector mechanism for intracellular C. Nat. Immunol. 2001; PubMed Scopus Google Scholar). TLRs are able to NF-κB and both to induce iNOS and activation R. T. M. J. D. J. T. S.J. Science. PubMed Scopus Google Scholar, P. S.W. Sci. PubMed Scopus Google Scholar). and are the NLR members known to activate NF-κB S.E. 2007; PubMed Scopus Google Scholar), resulting in the activation of a of iNOS S. M. A. D. C. Underhill D. P. D. C. J. Immunol. 2006; PubMed Scopus Google Scholar). and Nlrc4 inflammasomes, on the other hand, caspase-1 activation and the IL-1β and IL-18 and/or a death called pyroptosis (1Franchi L. Eigenbrod T. Muñoz-Planillo R. Nuñez G. Nat. Immunol. 2009; 10: 241-247Crossref PubMed Scopus (1259) Google Scholar, 7Bortoluci K.R. Medzhitov R. Cell. Mol. Life Sci. 2010; 67: 1643-1651Crossref PubMed Scopus (109) Google Scholar, Nat. Rev. Immunol. 2008; PubMed Scopus Google Scholar). activation by cytosolic flagellin independently of TLR5 L. Amer A. Body-Malapel M. Kanneganti T.D. Ozören N. Jagirdar R. Inohara N. Vandenabeele P. Bertin J. Coyle A. Grant E.P. Núñez G. Nat. Immunol. 2006; 7: 576-582Crossref PubMed Scopus (903) Google Scholar, E.A. M. Aderem A. Nat. Immunol. 2006; 7: PubMed Scopus Google Scholar), our data iNOS by the of innate recognition a role for on the that both Nlrc4 and Naip5 can induce caspase-1 activation, IL-1β secretion, and pyroptosis in response to L. pneumophila and S. typhimurium flagellin, it is still how Nlrc4 and Naip5 in the recognition of flagellin and in the assembly of the Nlrc4 can to pro-caspase-1 via a but the molecular of Naip5 to be Naip5 do not in of the Nlrc4 has been shown to be in host for a of such as Salmonella, Legionella, Pseudomonas, and Shigella, and be by a Naip5 the other hand, Naip5 is for the resistance to L. pneumophila infection D.S. Kobayashi K.S. Kohlsdorf T. Ogura Y. Long E.M. Vance R.E. Kuida K. Mariathasan S. Dixit V.M. Flavell R.A. Dietrich W.F. Roy C.R. Nat. Immunol. 2006; 7: 318-325Crossref PubMed Scopus (419) Google Scholar). Interestingly, iNOS was in macrophages and completely in the absence of a Naip5 using such as we able to detect a transcriptional of iNOS in the a Naip5 and still with The of Naip5 from (11Lightfield K.L. Persson J. Brubaker S.W. Witte C.E. von Moltke J. Dunipace E.A. Henry T. Sun Y.H. Cado D. Dietrich W.F. Monack D.M. Tsolis R.M. Vance R.E. Nat. Immunol. 2008; 9: 1171-1178Crossref PubMed Scopus (371) Google for the iNOS an effect that not be in for these is that Naip5 an additional role in the of we show for the distinct from flagellin, the activation of iNOS by cytosolic flagellin via is on caspase-1 The or of caspase-1 in the of iNOS protein and in response to FLA-BSDot. the of IL-1β and in can activate iNOS Annu. Rev. Immunol. 2009; PubMed Scopus Google Scholar). it was recently shown that and iNOS activation in and is on IL-1β M. Mol. Cell. 2007; PubMed Scopus Google Scholar). However, our data show that neutralization of IL-1β and/or IL-18 with in a iNOS by ruling out the for these cytokines in iNOS 5). These are by the that macrophages are able to iNOS in response to cytosolic flagellin in both and IL-18 signaling the of iNOS be to activation as and not able to activate iNOS in the absence of flagellin Moreover, and have effect on in response to on iNOS These data that IL-1β and IL-18 are not required for iNOS activation in response to but do not participation in of iNOS the iNOS for NF-κB and R. T. M. J. D. J. T. S.J. Science. PubMed Scopus Google Scholar, P. S.W. Sci. PubMed Scopus Google Scholar), additional is required to the of iNOS by caspase-1 the activation of or both it was demonstrated that and factors seem to be Naip5 and sensing of L. pneumophila A. K. M. S. P. 2009; PubMed Scopus Google Scholar). Moreover, these in the absence of flagellin, there is a activation of and iNOS, the and transcriptional the clear of Nlrc4 and caspase-1 and activation, it is not clear these are although Nlrc4 can and activate caspase-1 via CARD domain J.L. M. M. T. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, F. J. Cell 2007; PubMed Scopus Google Scholar), the activation of caspase-1 by Naip5 requires with other Naip5 a for other of apoptosis family members are known to with S. Cell 2010; PubMed Scopus Google Scholar). of the between Naip5 and Nlrc4 D.S. Kobayashi K.S. Kohlsdorf T. Ogura Y. Long E.M. Vance R.E. Kuida K. Mariathasan S. Dixit V.M. Flavell R.A. Dietrich W.F. Roy C.R. Nat. Immunol. 2006; 7: 318-325Crossref PubMed Scopus (419) Google Scholar), these NLR do not (11Lightfield K.L. Persson J. Brubaker S.W. Witte C.E. von Moltke J. Dunipace E.A. Henry T. Sun Y.H. Cado D. Dietrich W.F. Monack D.M. Tsolis R.M. Vance R.E. Nat. Immunol. 2008; 9: 1171-1178Crossref PubMed Scopus (371) Google Scholar), the of Naip5 and other complexes containing NLR can be found the Nat. Rev. Immunol. 2008; PubMed Scopus Google Scholar, T. 2009; 7: Scholar), the sensing of cytosolic flagellin to multiprotein complexes or to other NLR members and involved in the activation of there are still in the Nlrc4 and Naip5 inflammasome role in clearance of is clear. on Nlrc4 or caspase-1 macrophages to such as S. typhimurium L. Amer A. Body-Malapel M. Kanneganti T.D. Ozören N. Jagirdar R. Inohara N. Vandenabeele P. Bertin J. Coyle A. Grant E.P. Núñez G. Nat. Immunol. 2006; 7: 576-582Crossref PubMed Scopus (903) Google Scholar, E.A. M. Aderem A. Nat. Immunol. 2006; 7: PubMed Scopus Google Scholar), E.A. M. Aderem A. Sci. 2008; PubMed Scopus Google Scholar, L. Ogura Y. Flavell R.A. J. 2007; PubMed Scopus Google and T. Franchi L. C. M. Y. Inohara N. C. Nuñez G. 2007; PubMed Scopus Google Scholar), whereas Naip5 to against L. pneumophila infection D.S. Kobayashi K.S. Kohlsdorf T. Ogura Y. Long E.M. Vance R.E. Kuida K. Mariathasan S. Dixit V.M. Flavell R.A. Dietrich W.F. Roy C.R. Nat. Immunol. 2006; 7: 318-325Crossref PubMed Scopus (419) Google Scholar). the also to resistance to pyroptosis to be the effector mechanism to these pathogens. However, the of death an infection and the of pyroptosis in are still of (7Bortoluci K.R. Medzhitov R. Cell. Mol. Life Sci. 2010; 67: 1643-1651Crossref PubMed Scopus (109) Google Scholar). this a activation and maturation responsible for the of bacteria A. L. S. C. D. C. A. C. J. S. Kanneganti T.D. Amer 2009; 5: PubMed Scopus Google was recently we are a novel pathway for iNOS activation by the Naip5 and Nlrc4 and operated through a effector mechanism by Importantly, this pathway cooperate with production for the of L. pneumophila of iNOS of both and macrophages to L. pneumophila but not to bacteria The role of iNOS to the resistance against L. pneumophila was previously demonstrated J.T. J. Biol. PubMed Scopus Google Scholar, S.J. T.R. PubMed Google Scholar). However, iNOS seem not to be involved in the of L. pneumophila by macrophages from Naip5-deficient S.J. Y. Newton C. PubMed Google Scholar), our data that the iNOS activation is in the of It to be how such as cytokines secretion, and iNOS activation cooperate to the of infections. IntroductionInnate immune system employs intracellular and extracellular pattern recognition receptors (PRRs) 2The abbreviations used are: PRRpattern recognition receptorNLRNOD-like receptorTLRToll-like receptorIRFIFN-responsive factorsNaip5neuronal apoptosis inhibitory protein 5CARDcaspase activation recruitment domainiNOSinducible nitric-oxide synthaseBMDMbone marrow-derived macrophagePMperitoneal macrophagez-YVAD-fmkbenzyloxycarbonyl-YVAD-fluoromethyl ketonePAMPpathogen-associated molecular patternDOTDOTAP (lipidic vesicles). to detect microbial products through the identification of pathogen-associated molecular patterns (PAMPs). Among many families of PRRs, Toll-like receptors (TLRs) and NOD-like receptors (NLRs) have been described as central elements in triggering innate immune responses (1Franchi L. Eigenbrod T. Muñoz-Planillo R. Nuñez G. Nat. Immunol. 2009; 10: 241-247Crossref PubMed Scopus (1259) Google Scholar, 2Medzhitov R. Nature. 2007; 449: 819-826Crossref PubMed Scopus (1962) Google Scholar).TLRs and NLRs have been shown to activate distinct biochemical pathways, resulting in diverse mechanisms of resistance to pathogens. These mechanisms may act independently or may cooperate to the resolution of infections. TLRs primarily activate transcriptional factors such as NF-κB and IFN-responsive factors (IRF) (3Akira S. Takeda K. Nat. Rev. Immunol. 2004; 4: 499-511Crossref PubMed Scopus (6573) Google Scholar, 4Iwasaki A. Medzhitov R. Nat. Immunol. 2004; 5: 987-995Crossref PubMed Scopus (3274) Google Scholar), culminating in the production of effector molecules such as inflammatory cytokines and type I interferons. The contribution of these pathways to resistance to infection is manifold: recruitment of other inflammatory cells, production of cytotoxic and cytostatic molecules, and activation of adaptive immune response. NLRs, on the other hand, although also capable of activating NF-κB, have a peculiar ability of initiating an inflammatory response through caspase-1 activation via the assembly of a multiprotein complex called inflammasome (5Inohara N. Ogura Y. Fontalba A. Gutierrez O. Pons F. Crespo J. Fukase K. Inamura S. Kusumoto S. Hashimoto M. Foster S.J. Moran A.P. Fernandez-Luna J.L. Nuñez G. J. Biol. Chem. 2003; 278: 5509-5512Abstract Full Text Full Text PDF PubMed Scopus (1386) Google Scholar, 6Inohara Chamaillard McDonald C. Nuñez G. Annu. Rev. Biochem. 2005; 74: 355-383Crossref PubMed Scopus (802) Google Scholar, 7Bortoluci K.R. Medzhitov R. Cell. Mol. Life Sci. 2010; 67: 1643-1651Crossref PubMed Scopus (109) Google Scholar). Besides the well known role of caspase-1 on IL-1β and IL-18 maturation and secretion, this protease has also been described to induce macrophages death by a process known as pyroptosis (6Inohara Chamaillard McDonald C. Nuñez G. Annu. Rev. Biochem. 2005; 74: 355-383Crossref PubMed Scopus (802) Google Scholar, 7Bortoluci K.R. Medzhitov R. Cell. Mol. Life Sci. 2010; 67: 1643-1651Crossref PubMed Scopus (109) Google Scholar). Importantly, pyroptosis has been ascribed as a mechanism of host immune response against certain pathogens, such as Salmonella, Legionella, Pseudomonas, and Shigella, although it is still not clear how pyroptosis effectively contributes for pathogen clearance (7Bortoluci K.R. Medzhitov R. Cell. Mol. Life Sci. 2010; 67: 1643-1651Crossref PubMed Scopus (109) Google Scholar).The recognition of PAMPs by TLRs and NLRs occur independently. However, there are cases where a certain PAMP can be recognized by both TLRs and NLRs. Flagellin, the monomeric subunit of flagella present in several Gram-negative and Gram-positive bacteria is a good example of this redundancy. Extracellular flagellin is recognized by transmembrane TLR5 (8Hayashi F. Smith K.D. Ozinsky A. Hawn T.R. Yi E.C. Goodlett D.R. Eng J.K. Akira S. Underhill D.M. Aderem A. Nature. 2001; 410: 1099-1103Crossref PubMed Scopus (2762) Google Scholar), whereas cytosolic sensing of flagellin requires Naip5 (neuronal apoptosis inhibitory protein 5) and Nlrc4 (NLR family CARD (caspase activation recruitment domain) domain-containing 4), also called Ipaf (ICE-(IL-1-converting enzyme) protease activating factor), both members of the NLR family (9Amer A. Franchi L. Kanneganti T.D. Body-Malapel M. Ozören N. Brady G. Meshinchi S. Jagirdar R. Gewirtz A. Akira S. Núñez G. J. Biol. Chem. 2006; 281: 35217-35223Abstract Full Text Full Text PDF PubMed Scopus (380) Google Scholar, 10Franchi L. Amer A. Body-Malapel M. Kanneganti T.D. Ozören N. Jagirdar R. Inohara N. Vandenabeele P. Bertin J. Coyle A. Grant E.P. Núñez G. Nat. Immunol. 2006; 7: 576-582Crossref PubMed Scopus (903) Google Scholar, 11Lightfield K.L. Persson J. Brubaker S.W. Witte C.E. von Moltke J. Dunipace E.A. Henry T. Sun Y.H. Cado D. Dietrich W.F. Monack D.M. Tsolis R.M. Vance R.E. Nat. Immunol. 2008; 9: 1171-1178Crossref PubMed Scopus (371) Google Scholar, 12Mariathasan S. Newton K. Monack D.M. Vucic D. French D.M. Lee W.P. Roose-Girma M. Erickson S. Dixit V.M. Nature. 2004; 430: 213-218Crossref PubMed Scopus (1382) Google Scholar, 13Zamboni D.S. Kobayashi K.S. Kohlsdorf T. Ogura Y. Long E.M. Vance R.E. Kuida K. Mariathasan S. Dixit V.M. Flavell R.A. Dietrich W.F. Roy C.R. Nat. Immunol. 2006; 7: 318-325Crossref PubMed Scopus (419) Google Scholar). 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 flagellin is distinct from the D1 domain sensed by TLR5 (11Lightfield K.L. Persson J. Brubaker S.W. Witte C.E. von Moltke J. Dunipace E.A. Henry T. Sun Y.H. Cado D. Dietrich W.F. Monack D.M. Tsolis R.M. Vance R.E. Nat. 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Importantly, this effect is preserved in Myd88-deficient macrophage, ruling out the participation of Together, our data that by using distinct pathways, TLR and NLR can with a certain of the host resistance against infections.