Nitric oxide synthase in innate and adaptive immunity: an update.

TREIMM-1157; No. of Pages 18

Feature review

Nitric oxide synthase in innate and adaptive immunity: an update Christian Bogdan Mikrobiologisches Institut – Klinische Mikrobiologie, Immunologie, und Hygiene, Friedrich-Alexander-Universita¨t (FAU) ErlangenNu¨rnberg, Universita¨tsklinikum Erlangen, Wasserturmstraße 3/5, 91054 Erlangen, Germany

Thirty years after the discovery of its production by activated macrophages, our appreciation of the diverse roles of nitric oxide (NO) continues to grow. Recent findings have not only expanded our understanding of the mechanisms controlling the expression of NO synthases (NOS) in innate and adaptive immune cells, but have also revealed new functions and modes of action of NO in the control and escape of infectious pathogens, in T and B cell differentiation, and in tumor defense. I discuss these findings, in the context of a comprehensive overview of the various sources and multiple reaction partners of NO, and of the regulation of NOS2 by micromilieu factors, antisense RNAs, and ‘unexpected’ cytokines. NO and the immune system: no end (yet) to new paradigms The production of large amounts of nitrite (NO2) and nitrate (NO3) by mouse macrophages stimulated with lipopolysaccharide (LPS) and interferon (IFN)-g [1,2], and the L-arginine-dependency of the NO2 generation and of the tumor cytotoxic activity of activated macrophages [3,4], were the pioneering observations that ignited the interest of immunologists in the small inorganic radical of nitric oxide (NO). With the subsequent purification and cloning of the enzyme NOS2 [5,6], the production of polyclonal and monoclonal anti-NOS2-peptide antibodies, the development of NOS2-selective inhibitors [7,8], and the generation of mice with a functionally complete [9,10] or partial deletion of the Nos2 gene [11,12], the necessary tools became available to study in detail the expression, regulation, and function of NOS2 in the immune system. While at the beginning of NOS2 research NO was mainly viewed as an antimicrobial, tumoricidal, and tissue-damaging effector molecule operating in the innate arm of the immune system, it soon became clear that it strongly affects adaptive immune responses and also exerts cytoprotective effects due to its pleiotropic and versatile signaling functions (reviewed in [13–19]). In recent years, our understanding of the role of NO in the immune system has been enriched by fascinating discoveries which include the exquisite regulation of Corresponding author: Bogdan, C. ([email protected]). Keywords: nitric oxide synthases (NOS1/nNOS; NOS2/iNOS; NOS3/eNOS); myeloid cells; microenvironment; antimicrobial activity; Th17 cells; B cells. 1471-4906/ ß 2015 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.it.2015.01.003

NOS2 expression by micromilieu factors, the function of bacterial pathogen-derived NO in modulating and evading the host defense, and the NO-dependent control of pathogens by depletion of micronutrients. In addition, recent studies have provided novel insights into the effect of exogenous or endogenous NO on the differentiation of T helper cell subsets and into the NOS2-dependent communication between mesenchymal cells and T cells. This review aims to integrate these new findings into previously existing concepts. Sources of NO Mammalian NO synthases NOS2 is a homodimeric enzyme that, like all other NOS isoforms, converts L-arginine and oxygen into L-citrulline and NO in a complex oxidoreductase reaction (Box 1 and Figure 1A). A characteristic feature of NOS2 is its lack of expression in strictly resting cells. Instead, it is induced by immunological stimuli in a calcium-independent manner, which led to its original designation as inducible NO synthase (iNOS) [13]. The host cell localization of NOS2 has been mainly investigated in macrophages and neutrophils, where enzymatically active NOS2 can be detected in the cytosol, in small vesicles (still awaiting further characterization), in primary and tertiary granules, in the vicinity of phagosomes bearing inert particles or avirulent pathogens, attached to the submembranal actin cytoskeleton, or in mitochondria [19–26]. There is evidence from non-myeloid cells for sequestration of inactive NOS2 in cytoplasmic, perinuclear inclusion-like bodies (aggresomes) [27]. NOS2 has also been reported to localize to the nucleus, but the biological role of nuclear NOS2 (and of other NOS isoforms) is currently unclear ([25,28,29] and references therein). In addition to NOS2 there are two other isoforms of NOS: neuronal NOS (nNOS, NOS1) and endothelial NOS (eNOS, NOS3), originally named according to their predominant tissue distribution. These two isoforms have also been termed cNOS, because, unlike NOS2, they are constitutively expressed and their activity is dependent on prior calcium fluxes that enable the binding of calmodulin [13]. However, NOS1 and NOS3 are now recognized to have a wider cell and tissue distribution, and to be also regulated by cytokines, microbial products, hormones, and micromilieu factors [30–33]. NOS1 and NOS3 affect the differentiation and function of immune cells in vitro and modulate immune responses and inflammatory processes in vivo [29,34–45] (for further references see also [14,17]). Trends in Immunology xx (2015) 1–18

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Feature review Box 1. Type 2 nitric oxide synthase Type 2 nitric oxide synthase (NOS2) is a heme-containing flavoprotein and oxidoreductase which requires the presence of molecular oxygen, NADPH, calmodulin, and four redox-active prosthetic groups [i.e., FAD, FMN, iron protoporphyrin IX (heme), and tetrahydrobiopterin] to catalyze the conversion of the semi-essential amino acid L-arginine into L-citrulline, water, and the NO radical (in the following abbreviated as NO), with Nv-hydroxy-L-arginine being formed as transient, enzyme-bound intermediate [246]. Owing to failure to crystallize the holoenzyme, the structure of NOS2 has so far remained speculative. Recently, high-throughput single-particle electron microscopy revealed that in the dimeric NOS2 holoenzyme the two oxidase domains are dimerized in an antiparallel fashion and are flanked by the two separate reductase domains [247], thus visually confirming earlier mechanistic models [246]. Three flexible linker domains account for the high conformational mobility of the enzyme. This allows one of the reductase domains to flip over and closely interact with the oxidase domain, which is essential for electron transfer and catalysis [247].

Trends in Immunology xxx xxxx, Vol. xxx, No. x

(A)

Eukaryotes L-arginine

Asl

+ O2

Argininosuccinate

NOHA NOS

Ass1

Aspartate

Arg

L-citrulline + NO

pH↓ XO heme UV

NO targets and signaling NO radical, the primary product released from the Fe2+– heme complex within NOS2, is a labile compound. Its multiple reaction partners, which explain both the toxic and the regulatory effects of NO, include (i) the thiol groups of cysteines within peptides or proteins, leading 2

ADC

Urea + Ornithin

AGAT

CO2 + Agmane

Ornithine + Guanidinoacetate

NO2 –

Pyrimidine nucleodes NO3 –

(B)

Other sources of NO Under normoxia, NO derived from mammalian NOS is rapidly oxidized to nitrite and nitrate, which are in principle stable products. Nitrite, however, can easily become a source of NO, either non-enzymatically under acidic conditions (e.g., in the stomach or in inflamed tissues) or via the nitrite reductase activities of xanthine oxidoreductase and iron porphyrin-containing proteins during hypoxia (e.g., deoxyhemoglobin, desoxymyoglobin, cytochrome c) [46–48] (Figure 1A). The Fe3+–porphyrin-catalyzed reduction of nitrite can be efficiently assisted by thiols (e.g., glutathione) and sulfides (e.g., H2S), which then, in addition to NO, leads to the generation of nitroxyl (HNO) and thionitrous acid (HSNO), a potent S-nitrosating agent [49]. Irradiation of human skin with ultraviolet A light (315– 400 nm) or with blue light (420–453 nm) led to enzymeindependent formation of NO and other reactive nitrogen species (RNS) due to decomposition of S-nitrosothiol and nitrite stores in the epidermis and dermis of the skin [50,51]. While beneficial effects of UVA irradiation on blood pressure and blood flow have recently been reported [52], the immunological consequences and potential tissuedamaging effects of this pathway still need to be established. Finally, it has long been known that many environmental, commensal, and pathogenic bacteria reduce NOS2derived or dietary nitrate or nitrite to NO [or even further to nitrous oxide (N2O) or nitrogen] under anaerobic conditions, via a process termed nitrate respiration, nitrate dissimilation, or denitrification [53] (Figure 1B). More recently, shortened NOS-like proteins (bNOS) with high sequence similarity to the mammalian NOS-oxygenase domain, but without reductase domain, were discovered in Gram-positive bacteria [54]. Surprisingly, these bacterial NOS seem to play an important role in host–pathogen interactions, as discussed further below.

+ glycine

NOS

Creane

Polyamines Proline collagen

Prokaryotes

NO3 – NAR

NO2 – NIR

NO NOR

L-arginine bNOS

L-arginine

L-arginine

Arg

ADI

+ O2

NOHA

H2 O NH3 + H+

bNOS

L-citrulline + NO

Urea + Ornithine

Citrulline PO4 – + H+ OCT

N2 O N 2 OR

N2

Ornithine Carbamoylphosphate ADP + 2H+ CK ATP + NH3 CO2 TRENDS in Immunology

Figure 1. Arginine metabolism and the generation of NO in host cells and bacteria. (A) In mammalian cells four enzymatic pathways account for arginine metabolism –, the NO synthases (NOS), the arginases (Arg), L-arginine decarboxylase (ADC), and L-arginine:glycine amidinotransferase (AGAT). NO also arises NOS-independently by enzymatic (xanthine oxidase – XO) or nonenzymatic reduction of nitrite (i.e., by acidic pH, UV irradiation, or reaction with Fe3+ porphyrins such as heme). The arginase product ornithine and the ADC product agmatine are precursors for the synthesis of polyamines; ornithine is also a precursor for the generation of proline and collagens. In macrophages the NOS product L-citrulline can be recycled to L-arginine via argininosuccinate synthase 1 (Ass1) and argininosuccinate lyase (Asl). Arginine can also be generated from proteins or peptides carrying N- or C-terminal arginine residues via the activity of carboxypeptidases (e.g., CPD, CPM) or aminopeptidases (e.g., ERAP1). (B) Several bacterial species (e.g., E. coli, S. enterica Typhimurium, M. tuberculosis, M. catarrhalis) use nitrate and nitrite as energy sources under anaerobic conditions (nitrate respiration or dissimilation), which leads to the generation of NO, nitrous oxide (dinitrogen monoxide, N2O), or nitrogen via the activities of respiratory nitrate reductase (NAR), nitrite reductase (NIR), nitric oxide reductase (NOR), or nitrous oxide reductase (N2OR). In some prokaryotes (e.g., S. aureus), NO synthesis results from the presence of a bacterial NO synthase (bNOS). Some bacterial pathogens are able to degrade (host-derived) arginine by expressing arginase (e.g., Helicobacter pylori) or the enzymes of the ADI pathway (ADI, arginine deiminase or arginine dihydrolase; OCT, ornithine carbamoyl transferase; CK, carbamate kinase) (e.g., S. pyogenes, S. enterica). NOHA, Nv-hydroxy-L-arginine.

to the formation of S-nitrosothiols [55]; (ii) superoxide anions (O2), which gives rise to peroxynitrous acid/peroxynitrite (ONOO), capable of modifying proteins (e.g., by tyrosine nitration [56]); (iii) divalent cations such as


Feature review Fe2+ (in heme groups or iron–sulfur clusters) or Zn2+ (in zinc–sulfur clusters), which regulate the function of many transcription factors and enzymes, including the mammalian NO sensor sGC (soluble guanylate cyclase) that generates cGMP after activation by NO [57,58]; (iv) nucleic acids, where NO affords deamination and leads to mutagenesis [59]; and (v) unsaturated lipids, where, for example, NO-derived NO2 can attack alkene double bonds leading to nitrolipids [60]. The post-translational cysteine modification by S-nitrosation and its reversal (Box 2) is a key mechanism for the activation or inactivation of protein functions, and underlies many of the known signaling effects of NOS2-derived NO in the immune system (reviewed in [61–63]) (for examples see also text below). Based on a recent analysis using a 16, 368 protein microarray chip, the human S-nitrosocysteine proteome comprises at least 834 proteins [64]. In an earlier study, mass spectrometry of mouse tissues (e.g., lung, liver, thymus) identified more than 1000 S-nitrosylated cysteine residues in 647 proteins, many of which belong to key metabolic pathways. Depending on the organ, 56–90% of Snitrosylation events resulted from NOS3 activity [65]. A whole new avenue of research has been opened up by the possible interactions of RNS (e.g., NO, S-nitrosothiols, ONOO) with hydrogen sulfide (also termed sulfane; H2S) or its anion (HS) [66,67]. H2S/HS, which similarly to NO has been recognized as an inorganic signaling molecule in mammals, appears to modulate multiple physiological processes including immune and inflammatory responses

Box 2. S-nitrosation and denitrosation S-nitrosothiols (R-S-NO) are formed, when a nitroso (NO)-group is covalently added to the thiol (syn. sulfhydryl) group of the amino acid cysteine. The chemically correct name for this process and its reversal is S-nitrosation and S-denitrosation, but, by analogy to the nomenclature of other post-translational modifications (e.g., glycosylation), the terms S-nitrosylation or S-denitrosylation are frequently used [62,248,249]. As described in detail elsewhere [248], S-nitrosothiols can emerge by the reaction of: (i) NO radical with NO 2 (e.g., derived from the decay of peroxynitrite), leading to the formation of N2O3, which then reacts with thiolates (RS) to give rise to R-S-NO (NO autooxidation pathway), where R signifies any cysteine residue within small peptides (e.g., glutathione) or proteins; (ii) iron-nitrosyl-complexes (heme-Fe3+-NO , heme-Fe2+-+NO) with R-S to form R-S-NO and Fe2+-heme (transition metalcatalyzed pathway); (iii) a thiol (R-S-H) with another radical (X) leading to X-H and a thiyl radical (R-S) which then pairs with NO to form R-S-NO (thiyl radical recombination pathway); (iv) an already formed S-nitrosothiol (R1-S-NO) with a thiolate (R2S) leading to an R1-S-(N=O)-S-R2 intermediate with subsequent transfer of the NO-group and the generation of R1-S and R2-S-NO (the transnitrosation or exchange pathway). S-nitrosated proteins can be denitrosated by the catalytic action of proteins (‘denitrosylases’) such as thioredoxin or S-nitrosoglutathione-reductase (GSNO-R). This might relieve nitrosative stress and provide the basis for a redox-switch in protein function. GSNOR is crucial for the development and function of the immune system, as has been demonstrated with GSNO-R deficient mice [55,204] (see main text). Recently, S-nitrosylated proteins were also detected in NOexposed bacteria, where they conferred protection against further nitrosative stress [189] (see main text for further details).


[68]. It will be a major challenge in the future to unravel the full chemistry and the functional consequences of the crosstalk between H2S and RNS. Regulation of NOS2 expression Cytokines and microbial products In the mouse system interferons [IFN-g and type I IFNs (IFN-a/b)] and microbial pathogens or products (e.g., LPS) are prototypic transcriptional inducers of NOS2 which effectively stimulate macrophages for the release of high amounts of NO. IFNs and LPS elicit the dimerization of STAT1, the expression of interferon-regulatory factor (IRF)-1, and the formation of the interferon-stimulated gamma factor (ISGF) 3-complex (consisting of STAT1, STAT2, and IRF9), or the activation of NF-kB, respectively, all of which interact with binding sites in the Nos2 promoter (reviewed in [69]). Using mouse bone marrowderived macrophages infected with Listeria (L.) monocytogenes and chromatin immunoprecipitation, Farlik et al. unraveled the molecular basis for the previously described [70,71] synergistic induction of NOS2 gene transcription by type I IFN and microbial components [72]. As a first step, bacterial components interacting with membrane-bound or cytosolic pattern recognition receptors (PRR) trigger the activation of NF-kB and the production of endogenous IFNab, respectively. The binding of NF-kB to the Nos2 promoter led to the subsequent recruitment of the transcription factor (TF) IIH and its associated cyclin-dependent kinase (CDK) 7 subunit. In a second step, IFN-a/b feedback signaling caused the formation and promoter binding of the ISGF3 complex, thus enabling the co-recruitment and promoter-binding of RNA polymerase II and its phosphorylation (C-terminal domain at serine position 7) by TFIIH– CDK7 [72]. This latter process also required the binding of members of the bromodomain and extra terminal domain (BET) protein family to the Nos2 promoter [73]. Thus, NFkB and ISGF3 act sequentially and cooperatively at the Nos2 promoter. Research on NOS2 has been dominated by mouse studies. The molecular analysis of NOS2 expression in other species, however, is highly relevant to settle discussions on the extent and biological relevance of species-specific differences. Today, there is no doubt that human cells (e.g., hepatocytes, monocyte-derived macrophages or dendritic cells, tissue macrophages) are able to express NOS2 protein and activity in vitro and in vivo (reviewed in [14,15,74– 80]). Characterization of NOS2 expression in human cells has confirmed the crucial role of NF-kB and STAT1a, but also revealed differences in the promoter structure (explaining the hyporesponsiveness to IFN-g and LPS), the importance of post-transcriptional mechanisms, and the impact of cell origin, culture conditions, and stimuli [77,81,82]. In human alveolar macrophages stimulated with IFN-g plus LPS, the lack of NOS2 mRNA and protein expression was recently attributed to epigenetic gene silencing by CpG methylation, histone modifications, and chromatin compaction [83]. While these results further corroborate the hyporesponsiveness of human macrophages to IFN-g/LPS, they contrast starkly with the presence of NOS2 protein in alveolar and granulomatous macrophages of patients infected with, for example, M. 3


Feature review tuberculosis ([79] and references therein) and by no means question the expression of NOS2 by human cells. In hamster macrophages the low levels or expression of NOS2 in response to IFN-g and LPS could be partially explained by the absence of the NF-IL6 binding element in the proximal basal promoter [84]. In recent years, several unexpected cytokines and growth factors have been identified as (co)inducers of NOS2 in macrophages. Mouse macrophages infected with Leishmania (L.) amazonensis parasites and stimulated with interleukin (IL)-1b released remarkable amounts of NO. Roughly 40% of the NO production was dependent on inflammasome (caspase 1) activation and consecutive synthesis of endogenous IFN-g [85]. This suggests that IL-1b itself can confer NOS2-inducing signals, confirming previous findings obtained with other cell types of different species [14,86,87]. In keeping with this in vitro observation, L. amazonensis-infected mice deficient for caspase 1, the respective inflammasome components (i.e., NLRP3 and ASC), or the IL-1 receptor (IL1R) developed significantly more severe skin lesions than wild type controls [85]. IL-33, another member of the IL-1 family and best known for its potent induction of type 2 T helper (Th2) cell responses, triggered the expression of NOS2 mRNA, protein and activity in mouse macrophages. The effect of IL-33 required the ST2 ligand-binding component of the IL-33R, protein kinase B (Akt) and b-catenin activity, and partially also the IL-1R (raising the possibility of secondary induction of IL-1 by IL-33). The biological relevance of the findings was demonstrated in a Staphylococcus (S.) aureus infection model, where IL-33 inhibited the growth of the bacteria in a NOS2-dependent manner in vitro and in vivo [88]. Erythropoietin (EPO) is the key growth factor for red blood cell development and differentiation and has been successfully used for the treatment of some forms of anemia. Because non-erythroid cells and tissues can also respond to EPO, based on their expression of tissue-protective EPO heteroreceptors, several groups have investigated the function of EPO in the immune system [89]. This led to the discovery that EPO inhibited the activation and promoter-binding of NF-kB p65, and thereby blocked the induction of NOS2 and proinflammatory cytokines by microbial Toll-like receptor (TLR) ligands in macrophages, an effect which EPO shares with classical macrophage-deactivating cytokines (IL-4, IL-10, IL-13, TGF-b). As expected, EPO impaired the control of Salmonella (S.) enterica Typhimurium in vivo, but improved the outcome of cytokine-driven pathologies such as chemically (TNBS – 2,4,6trinitrobenzenesulfonic acid)-induced colitis [90] or LPSmediated endotoxin shock [91]. These results are relevant for the application of EPO in patients with anemia due to malignancies or chronic inflammatory diseases. Non-coding RNAs MicroRNAs (miRNAs) have been reported to affect the level of NOS2 mRNA and protein expression in various cell types including mouse macrophages [92–95], splenic lymphocytes [96], mouse mesenchymal stem cells (MSCs) [97], human endothelial cells [98], and hepatocytes [86]. However, in most cases the identified miRNAs (i.e., 4


miR-125a-5p, miR-146a, miR-149, miR-155, miR-301a) acted indirectly by blocking the expression of signaling or transcription factors (e.g., SOCS-1, IRAK-1, NF-kBrepressing factor, Kru¨ppel-like factor 13, Rheb) that positively or negatively regulated the expression of NOS2 mRNA. Direct interactions of miRNAs with the 30 -untranslated region (UTR) of NOS2 mRNA, with subsequent downregulation of NOS2 protein levels and NO production, have been convincingly documented for miR-939 (in human hepatocytes) [86] and miR-26a (in human T cell lymphoma cells) [99], and possibly also exist for miR146a as shown in mouse renal carcinoma cells [100]. A related form of NOS regulation has emerged from the discovery of natural non-coding antisense transcripts (NATs) that are derived from the opposite DNA strand (i.e., the strand complementary to the coding strand). NATs are generated by a large proportion of the mammalian genome [101]. In the case of the NOS genes, antisense transcripts were detected that either originated from the same gene locus as their sense counterparts (cis-NATs) [102,103] or from a different locus (trans-NATs) [104]. Depending on whether the NATs interacted with untranslated or translated regions of the mRNA, they stabilized or downregulated NOS2 mRNA or inhibited NOS3 mRNA translation ([102–104] and references therein). Micromilieu factors and metabolites Low oxygen tension is a characteristic both of sites of inflammation and of tumor tissues. Hypoxia causes stabilization of the transcription factor hypoxia-inducible factor (HIF)-1a, which then switches on genes that ultimately help to improve tissue oxygenation and resolution of inflammation [22,105,106]. Hypoxia has three major effects on NOS2. First, it impairs the synthesis of NO by NOS2 because of the lack of the substrate oxygen ([107] and references therein). Second, hypoxia disrupts the binding between NOS2 and the adaptor protein a-actinin 4, and thereby prevents the attachment of NOS2 to the actin cytoskeleton, which is a prerequisite for NOS2 activity [22]. Third, NOS2 belongs to those genes that are transcriptionally coinduced by HIF-1a in myeloid cells such as macrophages [108]. As shown in mouse dendritic cells (DC), hypoxia alone was a weak inducer of NOS2 mRNA. However, it strongly synergized with TLR3, TLR4, and TLR9 agonists for the upregulation of NOS2 mRNA and protein. Unexpectedly, under normoxic conditions TLRinduced NOS2 expression did not only require NF-kB and Myd88 signaling, but was partially also dependent on HIF-1a [109]. Using an innovative transcutaneous sensor technology, a direct correlation between tissue oxygenation, NO production, and antimicrobial activity was recently documented in mouse L. major skin infections [107]. In a Mycobacterium (M.) marinum zebrafish model, stabilization of HIF-1a was paralleled by an increased NOS2 activity and an improved pathogen control [110]. Another micromilieu factor that is poorly reflected by the standard tissue culture conditions is tonicity (osmolarity). In two pioneering studies, mouse lymphoid tissues were discovered to be hyperosmolar relative to serum [111], and macrophages were found to respond to the


Feature review interstitial accumulation of sodium chloride in the skin [112]. In vitro, the TLR-dependent production of proinflammatory cytokines and NOS2-derived NO was enhanced under hypertonic conditions [113,114], which offers an explanation for the local or systemic hyperosmolarity seen in particular inflammatory disease states [115], including bacterial skin infections [114]. A highsalt diet of mice was associated with an increased expression of NOS2 mRNA and protein, and improved control of L. major parasites, which was dependent on the central osmosensor, the transcription factor tonicityresponsive enhancer-binding protein (TonEBP, also known as NFAT5) [114]. Notably, NFAT5 was also a major inducer of proinflammatory cytokines and NOS2 in macrophages in the absence of hyperosmotic stress [116]. Thus, similarly to HIF-1a NFAT5 not only senses the micromilieu, but also participates in TLR-dependent NOS2 regulation. NO itself or its derivatives (e.g., S-nitrosothiols) also contribute to the cellular micromilieu and the regulation of NOS2 expression. The feedback activation or inhibition by NO involves transcriptional (e.g., S-nitrolysation or nitration of transcription factors and signaling molecules), translational (e.g., inhibition of NOS2 dimerization), or post-translational effects (e.g., S-nitrosylation of actin and disruption of its interaction with NOS2 protein) [14,19,23,117,118]. H2S, which is synthesized in mammalian tissues from homocysteine or cysteine in a pathway involving four different enzymes (cystathionine b-synthase, cystathionine g-lyase, 3-mercaptopyruvate sulfurtransferase, and cysteine aminotransferase) [68], is another microenvironmental factor with the potential to regulate NOS. Although it is currently unknown whether (and to what extent) H2S affects the expression or activity of NO synthases under physiological or inflammatory conditions, in vitro studies reported both stimulatory and inhibitory effects of H2S on NOS1, NOS2, or NOS3 [119]. In mouse endothelial cells H2S/HS caused S-sulfhydration of NOS3 at Cys 443 (forming a hydrodisulfide residue, R-S-SH), which prevented NOS3 S-nitrosylation and promoted NOS3 phosphorylation, dimerization, and activity [120]. In a mouse macrophage cell line exogenous or endogenous H2S/HS downregulated LPS-mediated NF-kB activation, NOS2 protein expression, and NO production via induction of heme oxygenase 1 (HO-1) [121,122]. HO-1, which converts heme into biliverdin, carbon monoxide (CO), and ferrous iron (Fe2+), is induced in response to reactive oxygen species (ROS) and RNS that inactivate the repressor BACH and at the same time release the transcription factor Nrf2 (also known as NFE2L2) from its negative regulator KEAP1 in a S-nitrosylation-dependent process [123,124]. Expression of HO-1 and exposure of mouse or human cells to Fe2+ or CO have been described to suppress cytokine- or LPS-induced NOS2 expression [125–127]. However, depending on the cell type, organ, and model system studied, upregulation of NOS2 by CO [125] and oxidative preprogramming of the inflammatory response by HO-1 have also been observed [123,128,129]. Thus, the HO-1 pathway should not be viewed as a general antagonist of NOS2.


Protein–NOS2 interactions and substrate availability In addition to transcriptional, post-transcriptional (mRNA stability), and translational levels of control (reviewed in [15,19,69,82]), the activity of NOS2 in many cell types is strongly influenced by post-translational events, notably by its interaction with other proteins and by the availability of the substrate L-arginine. Several proteins have been identified that either enhance or block the activity of NOS2 by acting as adaptors, scaffolds [e.g., a-actinin-4, ezrin/ radizin/moesin-binding phosphoprotein 50 (EBP50), kinase suppressor of Ras-1 (Ksr1)], allosteric activators (e.g., Hsp90, Rac2) or dimerization inhibitors (e.g., kalirin), or that direct NOS2 towards proteasomal degradation (e.g., Rpn13/ARDM1/NAP110, UCH37) [15,25,26,130– 134]. The availability and import of extracellular L-arginine is essential for NOS2 (and NOS3) activity, despite a sufficient intracellular arginine pool (the so-called arginine paradox). Three enzymatic pathways are known to affect the L-arginine supply for NOS2 in the immune system. First, specific metallo-carboxypeptidases (i.e., the membrane-bound CPD and CPM) and the secreted endoplasmic reticulum (ER)-associated aminopeptidase 1 (ERAP1) generate L-arginine from peptides with C- or N-terminal arginine residues, and thereby support the NOS2-dependent NO production by macrophages or microvascular endothelial cells [135,136] (Figure 1A). Second, cytosolic arginase (Arg) 1, a characteristic marker of macrophages or DCs stimulated with IL-4, IL-13, or TGF-b, but which is also found in NOS2-positive macrophages, cleaves L-arginine into urea and ornithine (the latter being a precursor of and collagen synthesis) [15,137,138] polyamine (Figure 1A). Arg1 depletes macrophages of L-arginine, and thereby not only impedes their generation of NO and antimicrobial activity [139], but also their expression of NOS2 protein [140]. Arg1-mediated arginine depletion also helps to restrain T cell proliferation and T cell-mediated immunopathology, especially in the absence of NOS2 activity [141]. At least in endothelial cells, the intracellular localization of Arg1 (or Arg2) relative to NOS3 appeared to be irrelevant for the degree of L-arginine depletion and inhibition of NO production [142]. Third, whereas macrophages are unable to convert ornithine into L-arginine, they can regenerate L-arginine from reimported L-citrulline via the activity of argininosuccinate synthase 1 (Ass1) and argininosuccinate lyase (Asl) (Figure 1A). The relevance of this citrulline recycling pathway under argininerestricted conditions was recently demonstrated in macrophages and mice deficient for Ass1, which presented a reduced capacity for sustained NO production and for controlling M. bovis BCG or M. tuberculosis in vitro and in vivo [143]. There is also evidence that citrulline supplementation helps to restore arginine availability and NO production in endotoxemic mice [144]. By contrast, the enhanced pathogen replication observed in herpes simplex virus-1 infected human fibroblasts after RNAi of Ass1 was not due to the lack of L-arginine and NO, but resulted from the accumulation of aspartate and an improved synthesis of pyrimidine nucleotides [145] (Figure 1A). In addition to NOS2 and the arginases, there are two other L-arginine degrading pathways in mammalian 5


Feature review


organisms. These are initiated by the mitochondrial enzymes L-arginine decarboxylase (ADC; leading to the formation of agmatine and consecutively to polyamines) and L-arginine:glycine amidinotransferase (AGAT; giving rise to guanidinoacetate and subsequently to creatine) [146] (Figure 1A). However, with the exception of an inhibitory effect of agmatine on macrophage NOS2 activity, and an expression analysis of ADC and AGAT during the interaction of the intestinal protozoan parasite Giardia lamblia with human epithelial cells [147–149], the role of these pathways in the immune system is largely unexplored. Regulation at the post-activity level The denitrosylation of S-nitrosothiols (see above) can reverse the (signaling) effects of NOS2-derived NO, and therefore must be viewed as an additional level of NOS2 regulation. There is now intriguing evidence that the nitration of tyrosine residues, another biochemical footprint of NOS2 activity, can also be reversed in a controlled enzymatic process. A denitrase activity capable of specifically denitrating tyrosine-nitrated cyclooxygenase (Cox)-1 was discovered in mouse macrophages, human endothelial cells, and various mouse and rat tissues (e.g., spleen, liver, lung, brain). Upon partial purification, glutathione-S-transferase and copper/zinc-superoxide dismutase were identified as two enzymes with denitrase activity ([150] and references therein). Understanding the regulation of denitrosylation and denitration will be of great importance to judge the relevance of these processes during immune responses.

Taken together, the induction of NOS2 transcription and mRNA stability by interferons and microbial ligands remains the hallmark of NOS2 expression. However, microenvironmental factors (hypoxia, salt, substrate availability), other gasotransmitters (e.g., H2S, CO), new cytokine regulators of NOS2 (e.g., IL-33, EPO), and additional levels of control (post-translational, post-activity) have emerged as regulators of NO production. Antimicrobial and antiviral activity of NOS2 The antimicrobial and antiviral activity of NOS2 has been mostly studied with macrophages [14,15,19,151,152], but other myeloid (e.g., dendritic cells, neutrophils, and eosinophils) as well as non-myeloid cells (e.g., hepatocytes) can also exert NOS2-dependent effector functions as shown by in vitro killing assays, in vivo expression analyses, application of NOS2 inhibitors, the use of NOS2-deficient mice, or cell transfer studies [14,25,153–157]. It is important to emphasize that the expression of NOS2 and certain cytokines by myeloid cells, which led to eponymous designations (e.g., TNF/iNOS-producing dendritic cells – TIPDCs), defines a reversible activation state with the potential to kill infectious pathogens rather than a new subset of myeloid cells. Direct effects against infectious pathogens NO and other RNS can react with structural elements, components of the replication machinery, nucleic acids, metabolic enzymes, or with virulence-associated molecules

Indirect anmicrobial effects of NO, e.g., Host cell apoptosis Autophagosomal degradaon of bacteria Fpn1-mediated iron deprivaon Phagolysosomal fusion Inhibion of the expression of bacterial effectors/toxins/adhesins Dispersion of bacterial biofilms Immunoregulatory effects

Macrophage

Nrf2 Fe2+ Phagolysosome

NO

NOS2

L-arg

Direct anmicrobial effects of NO, e.g., Inhibion of pathogen proliferaon DNA mutagenesis Disrupon of [FeS] clusters Metabolic blockade (e.g., Krebs cycle) Inacvaon of virulence factors

Fpn1

Fe2+

Key: Indirect anmicrobial effects of NO

Direct anmicrobial effects of NO

Intracellular pathogen (e.g., S. enterica Typhimurium) TRENDS in Immunology

Figure 2. Examples for direct and indirect antimicrobial activities of NO. NOS2-derived NO exerts numerous direct and indirect antimicrobial effects on intracellular bacterial and protozoan pathogens. Amongst these, NO activates the transcription factor Nrf2, which leads to upregulation of the iron exporter ferroportin-1 (Fpn1) and subsequent iron deprivation of intracellular bacteria such as S. enterica Typhimurium. For further details see text.

6


Feature review of infectious pathogens, which is the basis for their direct antiviral or antimicrobial effects [19,152] (Figure 2). Although NO is likely to attack multiple structures of an infectious pathogen at the same time, the disruption of a single microbial target structure (e.g., [Fe–S] clusters) can be sufficient for a strong antimicrobial effect [158]. New microbial targets of RNS continue to be discovered. In S. enterica serovar Typhimurium, NO inactivated the dihydrolipoyl (or lipoamide) dehydrogenase component (LpdA) of the a-ketoglutarate dehydrogenase (a-KDH), an enzymatic complex of the tricarboxylic acid cycle (Krebs cycle). This led to reduced synthesis of methionine and lysine, whose synthesis is dependent on succinyl-CoA, the product of the a-KDH reaction [159]. In Clostridium difficile infections, NO derived from NOS2 (or NOS3) S-nitrosylated cysteine residues of the two major exotoxins of the bacterium (TcdA and TcdB), which blocked their cysteine protease activity and self-cleavage by catalytic site-inhibition and by displacement of the allosteric activator inositol hexakisphosphate [160]. Control of pathogens by NOS2-derived NO does not necessarily imply their killing and elimination. In fact, NO-mediated reduction of microbial metabolic activity caused sufficient impairment of pathogen proliferation in vivo and allowed clinical resolution of the disease by the immune response, as was shown for the protozoan parasite L. major [161]. Although an enhanced translocation of NOS2 towards a pathogen-containing vacuole has been associated with improved infection control in vitro [19,162], localization of NOS2 in the immediate vicinity to an intracellular microbe is not a conditio sine qua non for executing its antimicrobial activity. Transcellular diffusion of NO from uninfected NOS2-expressing macrophages to Leishmania-infected NOS2-negative host cells was also effective in parasite killing in vitro, and might even represent the prevailing scenario in vivo during acute and latent infections [163,164]. The detection of NOS2 in granulomas of humans and macaques infected with M. tuberculosis is also indicative for an antimicrobial role of NO during acute and latent tuberculosis [79]. Indirect effects against infectious pathogens Whereas the direct toxicity of RNS, especially against extracellular infectious pathogens, is well documented, the activity of NOS2 against intracellular microbes appears to be far more complex and can also entail indirect effects (Figure 2). In mouse bone marrow-derived macrophages, NO generated by NOS2 was found to elicit host cell apoptosis and thereby restricted the growth of intracellular M. tuberculosis [165], whereas in L. monocytogenesinfected macrophages NOS2-derived NO caused host cell necrosis without affecting bacterial survival [166]. NOS2derived NO also led to the formation of 8-nitro-cGMP, which caused S-guanylation and subsequent autophagosomal degradation of cytosolic bacteria such as group A streptococci [167]. Autophagy, in turn, might support NOS2 expression, as was seen in Pseudomonas aeruginosa-infected macrophages [168]. In S. typhimurium infection NOS2 expressed by macrophages was required for the Nrf2-dependent transcriptional induction and upregulation of ferroportin-1 (Fpn1), an iron exporter expressed in


the phagosomal and the cell membrane. The resulting iron deprivation partially accounted for the NOS2-mediated control of intracellular Salmonella by macrophages in vitro and in vivo [169]. Other indirect antimicrobial activities of NOS2-derived NO include (i) the stimulation of phagosomal maturation and phagolysosomal fusion [170]; (ii) the inhibition of the expression of bacterial secretion systems, effector molecules, toxins, or adhesins [171,172]; (iii) the dispersion of bacterial biofilms [173]; and (iv) various immunomodulatory effects on different types of immune cells that help to convey protection against infectious pathogens (reviewed in [14,15,17,19]). In Anopheles mosquitoes, tyrosine nitration of Plasmodium falciparum parasites, which resulted from the cooperation of the invertebrate NOS2 with heme peroxidase 2 and NADPH oxidase 5, was reported to facilitate the recognition of the pathogen by the complement-like innate defense system in the hemolymph of the vector ([174] and references therein). Future research will need to tackle the question how these multiple direct and indirect antimicrobial activities of NOS2 interact and cooperate with NOS2-independent effector mechanisms (e.g., NADPH oxidase, myeloperoxidase) in vivo to achieve pathogen control in various cell types and organs during the different phases of infection. Single cell and in situ kinetic analyses have provided the first insights [175]. NO and microbial escape of host defenses Microorganisms have developed various constitutive or inducible mechanisms to resist oxidative and nitrosative stress, and also to evade killing by activated phagocytes. Classically, these include (i) the production of scavenger molecules (e.g., thiols), (ii) detoxifying enzymes (e.g., mycobacterial peroxiredoxins; truncated hemoglobin HbN of M. tuberculosis, which converts host-derived NO into nitrite; flavohemoglobin Hmp of e.g., Salmonella or Escherichia coli, which oxidizes – ‘denitrosylates’ – host-derived NO to nitrate; nitric oxide reductase NorB of Neisseria meningitidis, which deprives the host cells of S-nitrosothiols; assimilatory reductases of Salmonella which reduce nitrite to nitrogen or ammonium), (iii) mechanisms of repair (e.g., degradation of nitrosylated proteins by mycobacterial proteasomes), and (iv) strategies of avoidance (e.g., inhibition of phagosomal recruitment of NOS2 by M. tuberculosis or Salmonella effector proteins) (reviewed in [19,152]; [133,176]). Several recent discoveries illustrate that this list is far from being complete. Host- or pathogen-derived RNS support pathogen survival and contribute to disease pathogenesis The observation that NOS2-derived NO itself might facilitate the survival of intracellular pathogens has added an unexpected facet to the scenario of microbial escape of the antimicrobial machinery of phagocytes. In infections with the food-borne pathogen Listeria (L.) monocytogenes, Cole et al. showed that host cell derived NO promoted the listeriolysin-dependent escape of the bacterium from the phagosomal vacuole into the cytosol by inhibiting the proton-pumping activity of V-ATPase and delaying phagolysosomal fusion [177,178]. An even more intricate case of 7


Feature review NO function was observed with bacterial pathogens that express their own bacterial NO synthase (bNOS). In Bacillus (B.) anthracis, bNOS-derived NO activated the bacterial expression of catalase and antagonized the generation of hydroxyl radicals in the Fenton reaction [179]. bNOS of S. aureus conferred resistance to ROS, cathelicidin, and particular antibiotics, and caused enhanced virulence in vivo, presumably also due to the induction of antioxidative mechanisms (e.g., superoxide dismutase – SOD) [180]. These results need to be reconciled with previous studies in which NO generated by NO donors or host cells strongly inhibited catalase activity and exerted synergistic antibacterial effects together with ROS [19,152,181,182]. Presumably, the amount of NO generated by bNOS is sufficient to elicit signaling events, but too low to inactivate catalase or to damage the bacteria. Bacteria such as S. enterica Typhimurium, which are devoid of bNOS, express ROS- and/or RNS-sensitive transcription factors (e.g., OxyR, SoxR) or transcriptional repressors (e.g., NsrR, Fur), which become activated or deactivated, respectively, during oxidative or nitrosative stress, and which regulate the expression of detoxification mechanisms (e.g., Hmp, NorB) [152,183,184]. S. aureus lacks the NO-sensor NsrR, but instead uses the SrrAB twocomponent system to control the expression its NO resistance genes [185]. A special form of evasion and adaptation to NO is seen in bacteria that are capable of utilizing nitrate for respiration, especially in hypoxic tissues such as the gut or intracellularly in granulomata when oxygen is limiting. In mouse models of colitis, NOS2-derived NO3 promoted the outgrowth of facultative anaerobic S. enterica Typhimurium or Escherichia (E.) coli (which express nitrate, nitrite, and nitric oxide reductases) in the inflamed colon [186–188]. This raises the possibility that the expression of NOS2 during intestinal inflammations catalyzes the dysbiosis frequently seen in colitis. Importantly, anaerobic respiration of E. coli on nitrate was accompanied by extensive S-nitrosylation of bacterial proteins – including the transcription factor OxyR, which switched on a specific set of genes that protected E. coli from endogenous nitrosative stress [189]. Similarly, NO produced by human macrophages supported the intracellular growth of M. tuberculosis, a phenotype that required bacterial nitrate reductase [190]. The mycobacterial respiration of nitrate and the generation of nitrite protected the bacteria against various forms of stress typically encountered in infected tissues (acidic pH, NO, and hypoxia) [191,192]. The pro-survival effect of nitrite resulted from bacterial growth retardation, reduced consumption of ATP, and the expression of resistance-mediating genes [193]. Another respiratory tract pathogen, Moraxella (M.) catarrhalis, generated NO by reducing nitrite. The nitrite-derived NO not only activated bronchial epithelial cells for the production of proinflammatory cytokines (TNF and IL-1a), but also inhibited host cell division and caused host cell apoptosis by caspasedependent and -independent mechanisms [194]. Depletion of host cell arginine The consumption of arginine by extra- and intracellular microorganisms can deprive host cell NOS2 of its substrate. 8


Metabolic pathways that were found to impair the NOS2dependent production of NO by myeloid cells include the arginase activity (e.g., Helicobacter pylori; reviewed in [195]) and the arginine deiminase activity of bacteria (e.g., Streptococcus pyogenes, S. enterica Typhimurium) or protozoa (e.g., Giardia intestinalis) [147,196,197] (Figure 1B). Mutant bacteria lacking the arginine deiminase gene showed attenuated virulence in wild type, but not in NOS2-deficient mice [196]. Depletion of host cell arginine can presumably also result from upregulation of microbial arginine transporters and subsequent sequestration of arginine within the pathogen [198]. Evasion mechanisms that enable microbes to at least partially or temporarily circumvent the antimicrobial effects of NOS2 reflect the coevolution of host organisms and infectious pathogens. It is important to bear in mind that pathogen killing and escape is frequently not a blackand-white situation. Evasion of host defenses can lead to pathogen persistence while still allowing the control and clinical resolution of the infection [199]. As outlined above, NOS2 activity is one of the players in this scenario that can serve both the host (pathogen killing, immunoregulation) and the pathogen (growth on NOS2 metabolites; transcriptional induction of antioxidant defenses by NO). NOS and cell differentiation, function, and survival NOS and myeloid cells The expression of NOS2 by myeloid cells is associated with two major functional consequences: the acquisition and/or alteration of cell-intrinsic capabilities and phenotypes, and regulatory effects on neighboring immune cells. The first category is reflected not only by the NOS2-mediated antimicrobial activity of macrophages and other myeloid cells (as discussed above), but also by their cell-autonomous modulation of phagocytosis, expression of MHC class II and costimulatory molecules, antigen presentation, cytokine production, survival, and apoptosis of myeloid cells [15,200]. The second category is best exemplified by the NOS2-dependent inhibition of T cell responses (see below). Myeloid cells specialized in this latter function have been described as ‘suppressor macrophages’, ‘myeloid-derived suppressor cells’ (MDSC), ‘regulatory macrophages’, or ‘immunosuppressive monocytes’. In recent years, several new facets of NO function in myeloid cells have emerged. First, NO was shown to drive the differentiation of myeloid cells. In experimental myocarditis, macrophage colony-stimulating factor (M-CSF) converted CD133+ progenitor cells into CD133+F4/80+ macrophages and prevented the development of myofibroblasts, provided that the mice and macrophages expressed NOS2 [201]. In human DCs, inflammatory cytokines induced NOS1 (but not NOS2) which was required for DC maturation [38]. Second, the cNOS isoforms (NOS1 and NOS3) were found to contribute to the inflammatory functions of macrophages. In mouse bone marrow-derived macrophages, LPS-induced NOS3 supported the expression of NOS2 in vitro and in vivo [35]; engagement of Fcgreceptors triggered NOS1 (and NOS3) activity and lowoutput NO production, which facilitated autocrine and paracrine phagocytosis [44]. Third, tissue regeneration is dependent on NOS2-expression by myeloid cells even after


Feature review sterile injury. In a muscle-injury mouse model, NO released by infiltrating NOS2-positive macrophages was necessary for the proliferation and differentiation of myogenic precursor cells [202]. Finally, NO, derived from any of the three NOS isoforms, can either upregulate or inhibit autophagy in distinct contexts, thereby supporting antimicrobial defense (see above) or contributing to the development of neurodegenerative or malignant diseases [167,203]. NOS and T cells The response of T lymphocytes to NO generated by NOS2positive myeloid cells was one of its first discovered immunomodulatory activities. Exogenous NO (i.e., NO not generated by T cells themselves) inhibited the proliferation or even caused the death of T cells (reviewed in [15,17]). Mice lacking an important antioxidant mechanism (i.e., GSNOR) exhibited a significant deficiency of T and B cells in the periphery as a result of excessive S-nitrosylation and lymphocyte apoptosis [204]. On the other hand, smaller quantities of NO supported the survival and differentiation of T cell subpopulations, notably Th1 cells and a FoxP3-negative regulatory T cell population termed NOTreg [15,17], which potently inhibited Th17 cell differentiation [205]. Furthermore, recent studies have shown that exogenous NO also regulates Th9 and Th17 cells. Under in vitro Th9 stimulatory conditions (i.e., antiCD3 plus anti-CD28 plus IL-4 and TGF-b), the presence of a NO donor (50 mM) promoted the generation of mouse and human Th9 cells. NOS2-deficient mice showed a reduced frequency of Th9 cells during allergic airway inflammation using the ovalbumin asthma model. The mechanism underlying the induction of IL-9 by NO was identified by Niedbala et al. as enhanced expression of p53 (presumably via S-nitrosylation and inactivation of its negative regulator MDM2) followed by increased IL-2 production, STAT5phosphorylation, and IRF4 activity, and also by upregulation of IL-4Ra and TGF-bR2. Evidence for Th9 cell-intrinsic expression and function of NOS2 was not obtained [206]. The expression of NOS isoforms by T cells has been a matter of debate. The important methodological issues of cell purity, cell origin (primary T cells vs cell lines), and NOS detection method (mRNA vs protein vs enzyme activity; analysis at a population vs single cell level), as well as the results of previous studies on NOS1, NOS2, or NOS3 in T cells, have been discussed in detail elsewhere [17]. Two groups recently provided convincing evidence for the expression of NOS2 in mouse or human Th17 cells, but reached different conclusions as to its functional role. Yang et al. analyzed mouse CD4+ T cells at the single cell level and found that stimulation with plate-bound anti-CD3 plus anti-CD28 under neutral (Th0) or Th17-polarizing conditions (IL-6 plus TGF-b) induced NOS2 protein. In the absence of NOS2, the differentiation of Th0 cells into Th17 cells was strikingly enhanced, whereas the development of Th1 and Th2 cells was unimpaired; NOS1- or NOS3-deficiency had no detectable effect [207]. Exogenous NO (10–200 mM) inhibited Th17 differentiation, confirming earlier data by others [208]. The negative regulatory effect of T cell NOS2 resulted from the nitration of defined


tyrosine residues within the transcription factor RORgt, and this strongly impaired its ability to activate the IL-17 promoter [207]. The in vivo relevance of the NOS2-dependent downregulation of Th17 responses was demonstrated in T cell transfer colitis and in experimental allergic encephalomyelitis (elicited by immunization with myelin oligodendrocyte glycoprotein), where the absence of NOS2 led to more severe disease driven by higher rates of Th17 cells [207]. Obermajer et al. observed that low doses of exogenous NO (10–25 mM), delivered by NO donors or myeloid-derived suppressor cells (MDSC), strongly supported the development of RORgt+IL-23R+IL-17+ Th17 cells from naive human CD4+ T cells stimulated with anti-CD3/ CD28 beads in the presence of a Th17-inducing cytokine cocktail (IL-1b, IL-6, IL-23, TGF-b). More importantly, human Th17 cells, especially memory Th cells, expressed NOS2 mRNA and protein, and this was confirmed at the single cell level. Endogenous NOS2 and NO-induced cGMP signaling were required for maintaining the Th17 cell phenotype (IL-17- and IL23R-expression) [209]. To date, there is no molecular explanation for the differential effects of NOS2-derived NO in mouse versus human Th17 cells. The effects of exogenous or endogenous NO on T cell differentiation are illustrated in Figure 3. NOS, B cells, and plasma cells Mouse or human peritoneal B1 cells, IgD+ B cells, and B220lowCD138+ plasma cells were found to express NOS2 or NOS3 [14,15,210,211]. However, to date, we do not have a coherent picture of the function of NOS2 in B cells. The serum of influenza A virus-infected NOS2-deficient mice contained higher levels of virus-specific IgG2a compared to wild type controls [212]. These findings contrasted with another report where naive Nos2/ mice exhibited a striking lack of mucosal IgA and IgG2b in the serum. In naive IgD+ B cells from the spleen, mesenteric lymph node, or intestinal lamina propria of NOS2-deficient mice, both T cell dependent (simulated by anti-CD40 plus TGF-b) and T cell independent (mediated by the B cell stimulatory cytokines BAFF and APRIL) IgA class-switch recombination and IgA production were severely impaired. Mechanistic analyses revealed that the expression of TGF-bRII on B cells and TLR-induced production of APRIL and BAFF by dendritic cells strictly required NOS2-derived NO [213]. In a recent study, the antibody responses towards a type 2 Tindependent (TI-2) antigen (NP-Ficoll) were enhanced in NOS2-deficient mice, which correlated with a raised production of BAFF by monocytes and DCs in the absence of NOS2 [214]. Thus, NOS2-derived NO, either expressed by B cells themselves or delivered by myeloid cells, appeared to be essential for homeostatic mucosal IgA production, but was inhibitory to antibody responses driven by viral pathogens or type 2 TI-2 B cell antigens. In a study by Saini et al., the yield of antibody-secreting B220lowCD138+ plasma cells [following polyclonal B cell stimulation with LPS or after immunization of mice with a TI-2 antigen (NP-Ficoll) or a T cell dependent antigen (ovalbumin)] was strikingly lower in the absence of NOS2. Unlike to their wild type counterparts, Nos2/ plasma cells failed to show prolonged survival or increased 9


Feature review


Mouse (A)

Exogenous NO

NO donor

NO donor

Th1↑ (B)

Human

Endogenous NO

Th0

–/–

Nos2

NO

NO

Th0

Th0

Th9↑

Treg↑ Th17↓

IL-17↓

Th0

Th9↑

Th17↑

IL- 23R↑

RORγt ↓

Th1

++

++

Th2

++

++

NO

++

cGMP ↑ NO

Th17 NOS2

Th17

IL-17↑

+/+

Nos2

Th17 NOS2

(+) TRENDS in Immunology

Figure 3. Effect of exogenous or endogenous NOS2-derived NO on mouse and human T cell differentiation. (A) Under defined cytokine conditions (not shown), exogenous NO (derived from chemical compounds donating NO or from NOS2-expressing myeloid cells) facilitated the differentiation of mouse Th0 cells into Th1 cells [12], Th9 cells [206], or into a subset of regulatory T cells (termed NO-Tregs) [205], but inhibited the development of Th17 cells [207,208]. NO-Tregs also blocked Th17 cell differentiation [205]. In human T cells, exogenous NO supported the development of Th9 [206] and Th17 cells [209]. (B) Expression of endogenous NOS2 in mouse Th0 cells had no effect on Th1 or Th2 development, but hindered the differentiation of Th17 cells by inactivating the transcription factor RORgt [207]. In human (memory) Th17 cells expression of NOS2 and production of NO activated guanylate cyclase. The resulting cGMP helped to sustain the expression of IL-17 and of the IL-23 receptor which signify the Th17 phenotype [209].

antibody secretion in response to IL-6 or APRIL. In addition, Nos2/ plasma cells exhibited reduced activity of the NO/cGMP-dependent protein kinase G, an impaired ER stress response, and enhanced activation of initiator and executioner caspases [211]. Notably, NOS2 did not affect the activation (expression of CD69, MHC class II, and CD44) or proliferation of B cells in response to LPS (a TI-1 antigen) or anti-IgM stimulation [211]. These data strongly argue for a cell-intrinsic prosurvival effect of NOS2 specifically in plasma cells. NOS2 and mesenchymal cells Non-hematopoietic mesenchyme, such as connective, adipose, or muscle tissue, is derived from mesenchymal stem cells (MSCs), also termed multipotent mesenchymal stroma cells. MSCs prove to be important modulators of immune responses. In response to supernatants from activated T cells or recombinant proinflammatory cytokines (IFN-g together with TNF, IL-1a or IL-1b) mouse MSCs not only strongly expressed a plethora of chemokines, but also produced high amounts of NO derived from NOS2. Several of these chemokines (notably CXCL-9, -10, and -11 acting via the CXCR3 receptor) attracted T cells towards the MSCs, whereas the released NO potently suppressed T cell proliferation [215]. Human MSCs, by contrast, caused T cell suppression mainly by producing prostaglandin E2 and by upregulating indoleamine 2,3dioxygenase (IDO), which led to local tryptophan depletion 10

(reviewed in [216]). Similarly to MSCs, differentiated stromal cells in mouse lymphoid organs [i.e., fibroblastic reticular cells (FRCs) and lymphoid endothelial cells (LECs)] also inhibited T cell proliferation in a NOS2-dependent manner [217,218]. The findings summarized above support the concept that cell-intrinsic NO or low doses of exogenous NO (10– 50 mM) primarily regulate the differentiation and survival of lymphocytes, whereas high amounts of exogenous NO (>100 mM) delivered by neighboring myeloid or mesenchymal stromal cells exert an anti-proliferative effect. NOS-mediated induction versus resolution of inflammation Owing to its diverse sources, complex regulation, and multiple cellular and molecular targets and interaction partners, it is not surprising that NO has both stimulatory and suppressive properties in the immune system (see above and [15,17]). Which of these two categories will dominate the outcome of an immune response in a given in vivo situation, is hard to predict and difficult to analyze, especially because transgenic mice for cell type-specific and inducible NOS2 or NOS3 deletion are still unavailable. Nevertheless, available in vitro systems and mouse models have revealed fascinating new insights into the pro- and anti-inflammatory activities of NO. Although the available data are not yet consistent, NOS2 and NOS3 (expressed by endothelial and myeloid cells) are clearly


Feature review involved in the homeostatic and inflammatory regulation of the frequency and strength of lymphatic vessel contractions, which are essential for the transport of dendritic cells and antigen into the draining lymph node [40,219]. During infection with herpes simplex virus type 2, NOS3 was indispensable for the remodeling (caliper increase) of arterioles feeding the draining lymph node [45]. NOS3 was also required for the adhesion and transendothelial migration of neutrophils [43]. Several findings support the concept that NOS2, and to some extent also the other NOS isoforms, can help to limit immune responses and thereby contribute to the resolution of inflammation. These include (i) the downregulation of leukocyte recruitment by NOS2-dependent functional inactivation of chemokines via nitration of crucial tyrosine residues or suppression of chemokine production [202,220,221]; (ii) the NOS1-, NOS2-, or NOS3-dependent inhibition of leukocyte adhesion and transendothelial migration [15,23,29,39,222]; (iii) the blockade of T cell expansion in lymph nodes by NOS2-expressing FRCs and LECs [217,218]; (iv) the restriction of Th1 responses by NOS2positive Ly6C+CD86+PDCA1+ DCs, which express less T cell stimulatory cytokines compared to their NOS2-deficient counterparts [223], confirming earlier data obtained with macrophages [15]; (v) the nitration of STAT1 by NOS2, which switched off an IFN-g or IFN-a/b response [118]; (vi) the S-nitrosylation of NLRP3 by IFN-g-, IFN-a/ b-, or LPS-induced NOS2, which led to the suppression of caspase 1 activation and IL-1b production, and thereby protected tuberculous or endotoxemic mice from excessive immunopathology [224–226]; (vii) the shedding of the TNF receptor 1 by the protease TACE (TNF converting enzyme), which became activated via the NOS2/NO/cGMP pathway [257]; and (viii) the induction of apototic cell death in myeloid cells and lymphoid cells (e.g., effector memory T cells) [15,200,227]. NO in disease and treatment The striking antimicrobial and immunoregulatory effects of NO in vitro, and the expression of NOS2 and other NOS isoforms during infectious, autoimmune, chronic inflammatory, malignant, and degenerative diseases in humans and in animal models [14,15,42,152,228,229], have stimulated immunologists and clinicians to investigate the functional role of NOS2 in vivo (e.g., by using transgenic mouse models) and to test the therapeutic application of NOS inhibitors or NO donors [230]. Because comprehensive discussion of this topic is beyond the scope of this review, I will briefly highlight some results recently obtained in three disease models (sepsis, tumor, neurodegeneration). In endotoxemia the earlier use of NOS2 inhibitors for treatment has yielded disappointing results in preclinical models [231]. Retrospectively, this is not surprising given the potent anti-inflammatory effects of NOS2-derived NO in addition to its sepsis-promoting proinflammatory activities. A recent study by Fletcher et al. now documents an unexpected therapeutic potency of the transfer of ex vivo expanded FRCs into mice with endotoxemia or cecal-ligation-and-puncture sepsis, which was completely dependent on the NOS2 activity expressed by the FRCs [232]. In a similar sepsis model, NOS2-dependent upregulation of


Box 3. Overview of the principal effects of NOS2-derived NO in tumor biology and antitumor defensea Tumor-promoting effects Increased growth and metastasis of tumors due to (i) enhanced blood flow (vasodilatation), (ii) improved vascular permeability, (iii) increased tumor vessel formation [expression of angiogenic growth factors such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF)], (iv) activation of tumorigenic signaling cascades by S-nitrosylation [e.g., Wnt/bcatenin, epidermal growth factor (EGF) receptor, inhibitor of differentiation 4 (ID4)–JAGGED–NOTCH signaling, Ets-1 transcription factor), (v) suppression of anti-tumorigenic signaling cascades (e.g., cell division autoantigen-1 – CDA1), and (vi) invasion of host cell tissue [e.g., upregulation of metalloproteinases (MMPs) and, downregulation of tissue inhibitor of metalloproteinase-1 (TIMP-1)]. Inhibition of immune cell chemotaxis, adhesion, and infiltration (downregulation of VCAM-1, ICAM-1, or P-selectin; inactivation of T cell chemokines). Direct inhibition of T cell proliferation, induction of T cell death. Indirect blockade of T cell proliferation via increased infiltration of MDSC. Anti-tumor effects Killing of tumor cells by induction of apoptosis. Normalization of tumor vasculature (improved expression of adhesion molecules on tumor endothelial cells). Suppression of angiogenic and tumor growth factors. Upregulation of chemokines with subsequent improved recruitment of tumor-specific cytotoxic T cells. Inhibition of metastasis by downregulation of matrix metalloproteases and upregulation of TIMP-1 and E-cadherin. a Further details can be found in [14,15,221,229,234–236,250–255].

cGMP and subsequent activation of TACE protected against organ injury [257]. The role of NOS2 in tumor immunology is controversial because both beneficial (i.e., tumoricidal and tumor immunity-inducing) and detrimental (i.e., immunosuppressive and tumor growth- and metastasis-promoting) effects have been described (Box 3). Accordingly, NOS2 expression in human tumors has been positively or negatively correlated with tumor progression [229]. In addition to the use of different in vitro and in vivo models, key factors for the discrepancies between different published results appear to be (i) the spatiotemporal and quantitative relationship between NOS2-positive tumor cells, NOS2-expressing infiltrating myeloid cells, and anti-tumor effector T cells [233], (ii) the balance between the actual effects exerted by tumor-expressed NOS2 or by myeloid cell-expressed NOS2, and (iii) the amount of NO generated and available within the tumor tissue. Although high amounts of NO (e.g., produced by tumor cells expressing NOS2 or by NOS2-positive tumor-infiltrating myeloid cells) have been associated with tumor cell death [234], smaller amounts of NO produced by hypoxic tumor tissue or by infiltrating myeloid cells were found to have regulatory effects on angiogenesis, metastasis, or on the adhesion and infiltration of immune cells with either suppressive (e.g., MDSC) or (in-)direct tumoricidal activities (e.g., NOS2+Arg1 macrophages, cytotoxic T cells) [229,235]. The expression of NOS2 by tumor cells is subject to regulation by cytokines, miRNAs, hypoxia, and the availability of arginine [99,100,236–238], which in addition to its role as a NOS2 substrate is thought to function as a tumor-promoting amino acid. In tumors that lack Ass1, arginine-degrading 11


Feature review


Table 1. Selection of recent studies demonstrating the effects of immune cell- or tumor cell-derived NOS2 on tumor cell survival and/or anti-tumor immune responsesa Tumor model

Source of NOS2

Induction of NOS2 protein by

Effect of NOS2-derived NO

Refs

Tumor-infiltrating macrophage

Low-dose local g- irradiation

[252]

Mouse renal adenocarcinoma (RENCA)

Tumor cell

Mouse renal adenocarcinoma (RENCA)

Tumor-infiltrating macrophage

IFN-g/LPS plus miR-146a inhibitor IL-2/anti-CD40 immunotherapy; IFN-g

Suppression of angiogenic VEGF and MDSCrecruiting GM-CSF Endothelial cell activation (VCAM-1") Induction of T cell-recruiting chemokines (RANTES) and improved infiltration of T cells Promotes macrophage-mediated tumor cell death in vitro and in vivo Inhibition of metastasis by downregulation of matrix metalloproteinases and upregulation of TIMP-1

Tumor cell

N.a.

Anti-tumorigenic effects of NO Spontaneous mouse pancreatic island carcinoma (RIP1-Tag5-mice); tumor control by low-dose g-irradiation and adoptive T cell transfer

Protumorigenic effects of NO Human glioma stem cells

NPM-ALK+ human T cell lymphoma

Tumor cell

Mouse melanoma MT-RET-1

Tumor cell

Mouse colon adenocarcinoma C26

Gr1+ myeloid cells (MDSC) Tumor cells and myeloid cells (MDSC)

Mouse colon adenocarcinoma C26; mouse thymoma EG7-OVA; mouse prostate carcinoma (TRAMP mouse); human prostate, colon, or nasopharyngeal carcinoma Mouse xenograft model with human breast cancer cells (e.g., MDA-MB-231)

Tumor cells (and myeloid cells?)

Lack of miR-26a expression N.a.

N.a. N.a.

IFN-g, hypoxia, serum withdrawal

Downregulation of cell cycle inhibitor cell division autoantigen-1 (CDA1) Upregulation of inhibitor of differentiation 4 (ID4) Tumor cell survival, adhesion, and migration Secretion of VEGF by tumor cells, which in turn causes recruitment, maturation, and accumulation of MDSC in the tumor and T cell suppression Inhibition of splenocyte IFN-a and IFN-g response due to nitration of STAT1 Nitration of tyrosine- and tryptophan-residues in mouse and human CCL2; nitrated CCL2 still attracts myeloid cells, but no longer effector T cells Upregulation of proinflammatory markers associated with poor tumor survival (e.g., S100A8, IL-6, IL-8) Increased tumor cell migration and brain metastasis

[100] [254]

[253] [256] [99] [251]

[118] [221]

[236]

a

GM-CSF, granulocyte-macrophage colony stimulating factor; MDSC, myeloid-derived suppressor cells; N.a., not analyzed; NPM-ALK, nucleophosmin-anaplastic lymphoma kinase; RANTES, regulated on activation, normal T cell expressed and secreted; TIMP-1, tissue inhibitor of metalloproteinase 1; VCAM-1, vascular cellular adhesion molecule-1; VEGF, vascular endothelial growth factor.

enzymes (e.g., pegylated forms of bacterial ADI or human arginase) are currently being evaluated as novel therapeutics [239,240]. Table 1 summarizes the results of some recent mouse and human studies, which illustrate the complexity of the role of NOS2 in tumor biology and anti-tumor immune defenses. Novel tumor treatment strategies are likely to arise from the molecular targets of NOS2-derived NO. In mouse models of Alzheimer’s dementia, NOS3 activity protected against loss of spatial learning and memory [241]. By contrast, the expression of NOS2 was associated with the deposition of extracellular amyloid b and cognitive dysfunction [242]. The disease-promoting effect of NOS2, which is upregulated by the NLRP3 inflammasome, appeared to result from the NO-dependent inhibition of amyloid b-degrading enzymes and from the accelerated aggregation and plaque formation of amyloid b following nitration of defined tyrosine residues ([243,244] and references therein). In humans, progressive Alzheimer’s disease was paralleled by loss of NOS1 and NOS3 expression and activity in various regions of the brain versus normal agematched controls, whereas conflicting results were obtained for NOS2 ([245] and references therein). These data argue for an isoform-dependent effect of NOS in 12

Alzheimer’s dementia, which might reflect the canonical cellular distribution and function of NOS1, NOS2, and NOS3. Concluding remarks In recent years our understanding of the role of NO in immunity has significantly changed. Originally conceived as a key effector mechanism of myeloid cells and the innate immune response, NOS2 must now also be considered as an integral component of the development, differentiation, and function of B and T lymphocytes, and also of non-hematopoietic cells. In addition to NOS2, NOS1 and NOS3 have to be taken into account as sources of immunomodulatory NO. Whereas in the past NO was selectively recognized as a product of host cells for defense against pathogens, novel research has revealed that NO generated by bacterial NOS or nitrite reductases clearly helps bacterial pathogens to resist the antimicrobial activities of myeloid cells or even modulates host cell immune responses. All these insights into the activities of NOS and NO provide the basis for novel therapeutic approaches of infectious, malignant, autoimmune, and chronic inflammatory or neurodegenerative diseases. On the other hand, the increasing knowledge of the multiple functions and targets of NO also raises our


Feature review

Box 4. Outstanding questions What is the chemical basis for stimulatory versus inhibitory effects of H2S/HS on the expression of NOS isoforms? How does CO, the product of the heme oxygenase-1, induce or inhibit NOS2 expression? What is the phenotype of mice with a cell type (e.g., macrophage, dendritic cells, CD4+ T cell, B cell)-specific deletion of NOS2 in models of infection, autoimmunity, or tumor? What are the inducers of human NOS2 in vivo? Are microenvironmental factors (e.g., hypoxia, tonicity, microbial ligands) key signals in humans? Does NOS2 during infection of humans fulfill an antimicrobial (direct? indirect?) or rather a T cell regulatory function? Does the tissue microenvironment determine whether NOmediated pathogen killing out-competes the NO-triggered antioxidant defense mechanisms? Do molecular mechanisms other than impaired protein synthesis underlie the suppression of T cell proliferation following arginine depletion? How can NO generated by NOS2 or NOS3 support or inhibit the endothelial adhesion and transmigration of immune cells? Is the putatively therapeutic effect of arginine degradation in particular malignancies due to nutrient deprivation of the tumor cells or to impaired generation of pro-tumorigenic NO? Can NO donors be designed that are only taken up by specific cell types and only release NO inside a defined compartment of the target cell?

awareness of the complexity of events that might ensue from the activation or inhibition of NOS isoforms. Outstanding questions are listed in Box 4. Clearly, without tissue- or cellspecific strategies for the deletion of NOS or for the delivery of NO it currently appears unlikely that systemically applied NOS-based therapies will readily become clinical practice. However, this skepticism continues to be dispelled by the truly encouraging results from preclinical mouse models as outlined above. Acknowledgements The preparation of this manuscript and some of the work reviewed was supported by the Deutsche Forschungsgemeinschaft (SFB 643, project grant A6; GRK 1660), the Interdisciplinary Center for Clinical Research (IZKF) of the Universita¨tsklinikum Erlangen (project grants A49 and A61), the Emerging Field Initiative of FAU Erlangen-Nu¨rnberg (project grant within the ‘Metal Redox Inorganic Chemistry’ consortium) and by the Dr Robert Pfleger Stiftung. I apologize to all authors, whose work could only be acknowledged by referring to previous review articles because of space limitations.

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