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• W2020458505 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 TLR5. Together, our data indicated that by using distinct pathways, TLR and NLR systems can operate with a certain level of redundancy, which might improve the host resistance against infections.DISCUSSIONFlagellin is one of the rare protein structures sensed by the innate immune system and can activate both TLR and NLR receptors. 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, 26Ren T. Zamboni D.S. Roy C.R. Dietrich W.F. Vance R.E. PLoS Pathog. 2006; 2: e18Crossref PubMed Scopus (415) Google Scholar, 27Miao E.A. Alpuche-Aranda C.M. Dors M. Clark A.E. Bader M.W. Miller S.I. Aderem A. Nat. Immunol. 2006; 7: 569-575Crossref PubMed Scopus (890) Google Scholar). Interestingly, TLR5 seems to broadly recognize flagellated bacteria, whereas sensing of Nlrc4 and Naip5 depends on specialized transport systems present in virulent bacteria, such as the type III secretion systems of Salmonella (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, 27Miao E.A. Alpuche-Aranda C.M. Dors M. Clark A.E. Bader M.W. Miller S.I. Aderem A. Nat. Immunol. 2006; 7: 569-575Crossref PubMed Scopus (890) Google Scholar) and the type IV (T4SS) 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 secretion systems allow the delivery of bacterial products directly into the host cell cytoplasm. In experimental models, Nlrc4- and Naip5-induced caspase-1 activation can be achieved by delivering purified flagellin into the host cell cytosol by means of cationic liposomes or by transducing cells with appropriated gene-expression systems (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, 27Miao E.A. Alpuche-Aranda C.M. Dors M. Clark A.E. Bader M.W. Miller S.I. Aderem A. Nat. Immunol. 2006; 7: 569-575Crossref PubMed Scopus (890) Google Scholar, 28Silveira T.N. Zamboni D.S. Infect. Immun. 2010; 78: 1403-1413Crossref PubMed Scopus (88) Google Scholar). Consistent with this view, TLR5 stimulation primarily leads to the activation of microbicidal effector mechanisms that are supposedly effective in controlling less aggressive, non-pathogenic bacterial infection. In contrast, a more dramatic effector mechanism consisting of a Nlrc4/Naip5-caspase-1-induce inflammatory cell death is observed only in infections with virulent bacteria (7Bortoluci K.R. Medzhitov R. Cell. Mol. Life Sci. 2010; 67: 1643-1651Crossref PubMed Scopus (109) Google Scholar).Therefore, the existence of different PRRs capable of sensing the same agonist or different agonists present on the same pathogen is obviously beneficial to the host. The combined action of distinct, non-redundant, specialized responses should lead to a synergistic, more robust immunity against potential pathogens. Extracellular and intracellular recognition of flagellin induces such distinct macrophage responses. High levels of IL-6 were observed only with FLA-BS, whereas IL-1β secretion and pyroptosis were detected only with FLA-BSDot (Fig. 1 and supplemental Fig. 2). However, as the expression of PRRs is not ubiquitous and, therefore, not all host cells are equally prepared to properly recognize and handle potential pathogens, it should be interesting for the host immunity to have different PRR family sharing at least some microbicidal mechanisms.iNOS activation by TLRs can be considered one of the major effector mechanism for controlling intracellular pathogens (29Bogdan C. Nat. Immunol. 2001; 2: 907-916Crossref PubMed Scopus (2530) Google Scholar). TLRs are able to recruit NF-κB and IRF transcription factors, both necessary to induce iNOS expression and activation (30Kamijo R. Harada H. Matsuyama T. Bosland M. Gerecitano J. Shapiro D. Le J. Koh S.I. Kimura T. Green S.J. Science. 1994; 263: 1612-1615Crossref PubMed Scopus (782) Google Scholar, 31Lowenstein C.J. Alley E.W. Raval P. Snowman A.M. Snyder S.H. Russell S.W. Murphy W.J. Proc. Natl. Acad. Sci. U.S.A. 1993; 90: 9730-9734Crossref PubMed Scopus (1002) Google Scholar). In contrast, Nod1 and Nod2 are the only NLR members known to activate NF-κB (32Tattoli I. Travassos L.H. Carneiro L.A. Magalhaes J.G. Girardin S.E. Semin. Immunopathol. 2007; 29: 289-301Crossref PubMed Scopus (93) Google Scholar), resulting in the activation of a large repertoire of genes, including iNOS (33Tötemeyer S. Sheppard M. Lloyd A. Roper D. Dowson C. Underhill D. Murray P. Maskell D. Bryant C. J. Immunol. 2006; 176: 4804-4810Crossref PubMed Scopus (61) Google Scholar). Nlrps, Naip5, and Nlrc4 inflammasomes, on the other hand, mediate caspase-1 activation and the consequent IL-1β and IL-18 secretion and/or a programmed cell 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, 34Ting J.P. Willingham S.B. Bergstralh D.T. Nat. Rev. Immunol. 2008; 8: 372-379Crossref PubMed Scopus (286) Google Scholar). As macrophage activation by cytosolic flagellin occurs independently of TLR5 (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, 27Miao E.A. Alpuche-Aranda C.M. Dors M. Clark A.E. Bader M.W. Miller S.I. Aderem A. Nat. Immunol. 2006; 7: 569-575Crossref PubMed Scopus (890) Google Scholar), our data demonstrating iNOS up-regulation by FLA-BSDot (Fig. 2) highlights the plasticity of innate immunity recognition strategies, adding a role for NO on inflammasome-mediated microbicidal pathways.Despite the fact 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 obscure how exactly Nlrc4 and Naip5 individually participate in the recognition of flagellin and in the assembly of the inflammasomes. Nlrc4 can bind directly to pro-caspase-1 via a CARD-CARD homotypic interaction, but the molecular partner(s) of Naip5 remains to be solved. Interesting, Naip5 do not participate in every function of the Nlrc4-inflammasome. Nlrc4 has been shown to be crucial in host defense for a number of pathogens such as Salmonella, Legionella, Pseudomonas, and Shigella, and its genetic defect cannot be substituted by a functional Naip5 (9–12, 17, 35–37). On the other hand, Naip5 is indispensable for the resistance to L. pneumophila infection (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). Interestingly, FLA-BSDot-induced iNOS expression was significantly reduced in Nlrc4-deficient macrophages and completely abolished in the absence of a functional Naip5 (Fig. 3). By using highly sensitive techniques such as real-time RT-PCR, we were able to detect a slight transcriptional induction of iNOS in the mice harboring a partially mutant A/J Naip5 allele and still significantly less when compared with Nlrc4-deficient macrophages. The partial functionality of Naip5 from A/J mice (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) could account for the observed flagellin-dependent iNOS mRNA induction, an effect that possibly would not be observed in Naip5−/− mice. Another possible explanation for these results is that Naip5 plays an additional Nlrc4-independent role in the post-transcriptional regulation of iNOS.Importantly, we show here for the first time that, distinct from free flagellin, the activation of iNOS by cytosolic flagellin via Nlrc4/Naip5 is dependent on caspase-1 (Fig. 4). The genetic or pharmacological inactivation of caspase-1 resulted in the abrogation of iNOS protein and mRNA expression in response to FLA-BSDot. Caspase-1 promotes the release of IL-1β and IL-18, which in turn can activate iNOS (25Dinarello C.A. Annu. Rev. Immunol. 2009; 27: 519-550Crossref PubMed Scopus (2428) Google Scholar). In fact, it was recently shown that IFN-γ- and TNF-α-induced iNOS activation in astrocytes and cerebral endothelial cells is dependent on caspase-1-mediated IL-1β secretion (38Jüttler E. Bonmann E. Spranger M. Kolb-Bachofen V. Suschek C.V. Mol. Cell. Neurosci. 2007; 34: 612-620Crossref PubMed Scopus (14) Google Scholar). However, our data show that neutralization of IL-1β and/or IL-18 with specific antibodies resulted in a rather increased iNOS expression by FLA-BSDot, ruling out the requirement for these cytokines in flagellin-mediated iNOS up-regulation (Fig. 5). These observations are reinforced by the fact that myd88−/− macrophages are able to express iNOS in response to cytosolic flagellin (Fig. 6) besides their deficiency in both IL-1 and IL-18 receptor, signaling pathways. Also, the observed up-regulation of iNOS cannot be attributed to unspecific macrophage activation as anti-IL-1β- and anti-IL-18-neutralizing antibodies were not able to activate iNOS in the absence of flagellin (Fig. 5C). Moreover, anti-IL-1β and anti-IL-18 have no effect on IL-6 produced in response to FLA-BSDot, showing their particular involvement on iNOS modulation (supplemental Fig. 3). These data indicate that IL-1β and IL-18 are not required for iNOS activation in response to FLA-BSDot but do not discard their participation in some aspects of iNOS regulation.Because the iNOS promoter contains binding sites for NF-κB and IRF-1 (30Kamijo R. Harada H. Matsuyama T. Bosland M. Gerecitano J. Shapiro D. Le J. Koh S.I. Kimura T. Green S.J. Science. 1994; 263: 1612-1615Crossref PubMed Scopus (782) Google Scholar, 31Lowenstein C.J. Alley E.W. Raval P. Snowman A.M. Snyder S.H. Russell S.W. Murphy W.J. Proc. Natl. Acad. Sci. U.S.A. 1993; 90: 9730-9734Crossref PubMed Scopus (1002) Google Scholar), additional investigation is required to elucidate whether the up-regulation of iNOS by caspase-1 involves the activation of either or both transcription factors. Recently, it was demonstrated that IRF-1 and IRF-8 transcription factors seem to be regulated during Naip5 and Nlrc4-mediated sensing of L. pneumophila (39Fortier A. Doiron K. Saleh M. Grinstein S. Gros P. Infect. Immun. 2009; 77: 4794-4805Crossref PubMed Scopus (35) Google Scholar). Moreover, these authors observed that, in the absence of flagellin, there is a defective activation of IL-12p40 and iNOS, the IRF-1 and IRF-8 transcriptional targets. Despite the clear evidences of Nlrc4 and Naip5-mediated caspase-1 and IRF-1/IRF-8 activation, it is not clear whether these events are correlated.Additionally, although Nlrc4 can directly interact and activate caspase-1 via its N-terminal CARD domain (40Poyet J.L. Srinivasula S.M. Tnani M. Razmara M. Fernandes-Alnemri T. Alnemri E.S. J. Biol. Chem. 2001; 276: 28309-28313Abstract Full Text Full Text PDF PubMed Scopus (340) Google Scholar, 41Martinon F. Tschopp J. Cell Death Differ. 2007; 14: 10-22Crossref PubMed Scopus (652) Google Scholar), the activation of caspase-1 by Naip5 possibly requires interaction with other proteins since Naip5 possesses a BIR domains, which for other inhibitor of apoptosis proteins family members are known to interact with different proteins (42Mace P.D. Shirley S. Day C.L. Cell Death Differ. 2010; 17: 46-53Crossref PubMed Scopus (70) Google Scholar). Regardless of the possible interaction between Naip5 and Nlrc4 (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 NLR do not share all caspase-1-mediated functions (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), opening the possibility of interactions among Naip5 and other unknown proteins. Because multiple complexes containing more than one NLR member can be found within the same cell (34Ting J.P. Willingham S.B. Bergstralh D.T. Nat. Rev. Immunol. 2008; 8: 372-379Crossref PubMed Scopus (286) Google Scholar, 43Bergsbaken T. Fink S.L. Cookson B.T. Nature Reviews. 2009; 7: 99-109Google Scholar), the sensing of cytosolic flagellin could lead to multiprotein complexes formation including Nlrc4, Naip5, caspase-1, IRF-1, IRF-8, or alternatively, to other NLR members and proteins involved in the activation of iNOS.Although there are still missing pieces in the Nlrc4 and Naip5 inflammasome complexes, their role in macrophage clearance of bacterial pathogens is clear. Defects on Nlrc4 or caspase-1 render macrophages susceptible to infections such as S. typhimurium (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, 27Miao E.A. Alpuche-Aranda C.M. Dors M. Clark A.E. Bader M.W. Miller S.I. Aderem A. Nat. Immunol. 2006; 7: 569-575Crossref PubMed Scopus (890) Google Scholar), Pseudomonas aeruginosa (35Miao E.A. Ernst R.K. Dors M. Mao D.P. Aderem A. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 2562-2567Crossref PubMed Scopus (235) Google Scholar, 36Sutterwala F.S. Mijares L.A. Li L. Ogura Y. Kazmierczak B.I. Flavell R.A. J. Exp. Med. 2007; 204: 3235-3245Crossref PubMed Scopus (404) Google Scholar) and Shigella flexneri (37Suzuki T. Franchi L. Toma C. Ashida H. Ogawa M. Yoshikawa Y. Mimuro H. Inohara N. Sasakawa C. Nuñez G. PLoS Pathog. 2007; 3: e111Crossref PubMed Scopus (414) Google Scholar), whereas Naip5 deficiency lead to susceptibility against L. pneumophila infection (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). Because the same defects also lead to resistance to caspase-1-mediated cell death, pyroptosis seems to be the major effector mechanism to control these pathogens. However, the requirement of macrophage death during an infection and the relevance of pyroptosis in vivo are still matters of debate (7Bortoluci K.R. Medzhitov R. Cell. Mol. Life Sci. 2010; 67: 1643-1651Crossref PubMed Scopus (109) Google Scholar). In this regard, a Naip5/Nlrc4-caspase-1-dependent caspase-7 activation and phagosome maturation responsible for the inhibition of bacteria replication (44Akhter A. Gavrilin M.A. Frantz L. Washington S. Ditty C. Limoli D. Day C. Sarkar A. Newland C. Butchar J. Marsh C.B. Wewers M.D. Tridandapani S. Kanneganti T.D. Amer A.O. PLoS Pathog. 2009; 5: e1000361Crossref PubMed Scopus (144) Google Scholar) was recently described.Here, we are describing a novel pathway for iNOS activation orchestrated by the Naip5 and Nlrc4 and operated through caspase-1, adding a new effector mechanism mediated by inflammasomes. Importantly, this pathway cooperate with TLR-induced NO production for the control of L. pneumophila since pharmacological inhibition of iNOS renders higher susceptibility of both WT C57BL/6 and myd88−/− macrophages to flagellated L. pneumophila but not to flagellin-mutant bacteria (supplemental Fig. 4). The role of iNOS to the resistance against L. pneumophila was previously demonstrated (45Summersgill J.T. Powell L.A. Buster B.L. Miller R.D. Ramirez J.A. J. Leukoc. Biol. 1992; 52: 625-629Crossref PubMed Scopus (66) Google Scholar, 46Skerrett S.J. Martin T.R. Infect. Immun. 1996; 64: 3236-3243Crossref PubMed Google Scholar). However, iNOS seem not to be involved in the control of L. pneumophila by macrophages from Naip5-deficient A/J mice (47Gebran S.J. Yamamoto Y. Newton C. Klein T.W. Friedman H. Infect. Immun. 1994; 62: 3197-3205Crossref PubMed Google Scholar), corroborating our data showing that the TLR-independent iNOS activation is observed only in the presence of functional flagellin. It remains to be determined how caspase-1-mediated cellular events, such as pyroptosis, pro-inflammatory cytokines secretion, caspase-7, and iNOS activation cooperate to the control 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. 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 TLR5. Together, our data indicated that by using distinct pathways, TLR and NLR systems can operate with a certain level of redundancy, which might improve the host resistance against infections." @default.
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