The role of interleukin 18 and interleukin 18 binding protein in autoinflammatory diseases
L'interleukine (IL) 18 est une cytokine pro-inflammatoire naturellement inhibée par l'IL-18 binding protein (IL-18BP). Cette dernière est présente en grande quantité dans la circulation et lie l'IL-18 avec une forte affinité, si bien que la majorité de l'IL-18 circule sous une forme inactive non seulement à l'état basal, mais aussi dans de nombreuses situations pathologiques. Nous avons montré que la fraction libre, bioactive, de l'IL-18 était spécifiquement élevée dans des maladies autoinflammatoires incluant la maladie de Still de l'adulte, le syndrome d'activation macrophagique (SAM), et le syndrome autoinflammatoire de l'enfant avec entérocolite lié à une mutation de l'inflammasome NLRC4, faisant de l'IL-18BP un outil thérapeutique intéressant dans ces maladies. Nous avons par ailleurs confirmé le rôle pathogène de l'IL-18 dans un modèle murin de SAM en utilisant des souris déficientes en IL-18BP. Elucider les mécanismes de production et de régulation de l'IL-18 et l'IL-18BP reste essentiel pour mieux appréhender ces maladies.
GIRARD, Charlotte. The role of interleukin 18 and interleukin 18 binding protein in autoinflammatory diseases. Thèse de doctorat : Univ. Genève, 2018, no. Sc. Méd. 33
URN : urn:nbn:ch:unige-1149817
DOI : 10.13097/archive-ouverte/unige:114981
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Faculty of Medicine
Department of Internal Medicine Specialties Division of Rheumatology
The role of interleukin 18 and interleukin 18 binding protein in autoinflammatory diseases
Presented at the Faculty of Medicine of University of Geneva for the MD-PhD Doctorate in Medical Sciences
Prof. Cem Gabay, Thesis supervisor Prof. Beat Imhof, Thesis committee member Prof. Walter Reith, Thesis committee member Prof. Francesco Negro, President of the scientific committee
Prof. Patrick Matthys, Expert
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Table of contents
Acknowledgements ... 1
Summary ... 3
Résumé ... 5
Abbreviations ... 7
I. Introduction ... 10
I.1. Interleukin (IL)-18 and IL-18 binding protein (IL-18BP) ... 10
I.1.a. IL-18: Characterization, production, signaling, function... 10
Characterization ... 10
Production and regulation ... 11
IL-18 gene and its regulation ... 11
Post-translational processing ... 13
Membrane-bound IL-18 ... 15
IL-18 receptor (IL-18R) and signaling pathways ... 15
IL-18R ... 16
Intracellular signaling pathways ... 17
Soluble IL-18R ... 18
IL-37 ... 19
IL-18 functions ... 20
Induction of IFN-γ production ... 20
Other functions ... 21
IL-18 genetically modified mice ... 22
I.1.b. IL-18BP: characterization, function, production ... 24
Isolation and characterization ... 24
Function ... 24
IL-18/IL-18BP interaction ... 26
IL-18BP production and regulation ... 26
I.1.c. IL-18 and IL-18BP in human diseases and mouse models ... 28
Role of IL-18/IL-18BP in inflammatory diseases ... 28
Role of IL-18/IL-18BP in Crohn’s disease ... 30
Role of IL-18/IL-18BP in rheumatoid arthritis ... 32
Role in other inflammatory diseases ... 35
Role of IL-18 in cancer ... 39
Role of IL-18 in infectious diseases ... 40
Role in other diseases ... 41
I.2. Autoinflammatory diseases and Macrophage Activation Syndrome ... 41
I.2.a. Autoinflammatory diseases and inflammasomes... 41
The inflammasomes ... 42
Monogenic vs polygenic AIDs ... 45
Monogenic AIDs ... 45
Polygenic AIDs ... 47
I.2.b. AOSD and sJIA: clinical and laboratory features, epidemiology, pathophysiology47 AOSD ... 47
sJIA ... 48
I.2.c. Macrophage Activation Syndrome ... 50
Clinical and biological features ... 50
Classification of haemophagocytic syndromes ... 51
Pathogenesis ... 53
I.3. IL-18 in autoinflammatory diseases ... 57
I.3.a. IL-18 in AOSD and sJIA ... 57
I.3.b. IL-18 in the NLRC4/MAS syndrome ... 58
I.3.c. IL-18 in HLH ... 60
II. Hypotheses and objectives of the thesis ... 63
III. Publications ... 64
III.1. Elevated serum levels of free interleukin-18 in adult-onset Still's disease ... 65
III.2. Unopposed IL-18 signaling leads to severe TLR9-induced macrophage activation syndrome in mice ... 87
III.3. Interleukin-18 diagnostically distinguishes and pathogenically promotes human and murine macrophage activation syndrome ... 113
IV. Discussion ... 141
V. Conclusion ... 146
References ... 147
Annex ... 167
I sincerely want to thank:
Prof. Cem Gabay for proposing me to work on this exciting project, for his wise, thoughtful and kind supervision, and for inviting me to become a Swiss rheumatologist!
The members of my jury for doing me the honor to evaluate my work,
Sylvette Bas for teaching me with rigor and precision during my first year in Geneva, Gaby Palmer for her availability, her intelligence and her valuable advice,
All the members of the lab for their very warm welcome, their friendship, their personal and technical support,
Deshire for her valued friendship, her joie de vivre, and our spiritual discussions, Aleksandra for our daily young mum and more scientific exchanges!
All the people of PATIM and Rheumatology departments and of Faculty of Medicine who made these 3 years more fun and easy,
Andrew, Eduardo, Greg and the whole AB2 Bio team for their trust, cordial meetings and fruitful discussions,
Our collaborators, especially Prof. Scott Canna, for their efficiency and responsiveness and for including me in their outstanding projects,
My dear parents for their everlasting love and support,
Pierre-Marie, for attracting me to Geneva (!) and, above all, for opening my mind and making my life more meaningful every day,
2 My parents in law for their generosity and affection,
My sweet Raphaël, for the daily happiness he brought in our life.
I would also like to thank AB2 Bio, Institut Servier and Fondation Jean Tua for financial support.
Interleukin (IL)-18 is a pro-inflammatory cytokine of IL-1 family, which was first known as a potent interferon-γ (IFN-γ) inducer and which maturation and release is dependent on the inflammasome-caspase-1 pathway activation. Its biological activity is further regulated by IL-18 binding protein, which naturally inhibits most of circulating IL-18 by forming high affinity complexes. IL-18 has been involved in the pathogenesis of many disorders, especially inflammatory diseases, but the results of clinical trials testing IL-18BP in rheumatoid arthritis and psoriasis proved to be disappointing. We participated to the development of an ELISA that allowed us to dose specifically bioactive free IL-18 and found that free IL-18 serum levels were elevated in patients with adult onset Still’s disease (AOSD), a rare systemic autoinflammatory disease (AID) of unknown origin, and correlated with disease activity. We identified additional AIDs where free IL-18 levels were increased, including systemic-onset juvenile idiopathic arthritis (sJIA), macrophage activation syndrome (MAS) (a life-threatening complication of both AOSD and sJIA characterized by overwhelming inflammation and multi-organ failure), and the recently described autoinflammation with infantile enterocolitis (AIFEC), related to a gain-of-function mutation of the inflammasome component NLRC4. We and others showed that IL-18 inhibition by recombinant IL-18BP was efficient in the treatment of AOSD and AIFEC.
In a mouse model of MAS induced by repeated Toll-like receptor (TLR)9 stimulation, we demonstrated that excessive IL-18 signaling, as observed in IL-18BP-deficient or IL-18- transgenic mice, worsened the disease and that IL-18 blockade could reverse this phenotype, further arguing for a pathogenic role of IL-18 in MAS. IL-18 acted through IFN-γ in this model since worsened phenotype was associated with enhanced IFN-γ signature and IFN-γ blockade recapitulated the effects of IL-18 inhibition. Mice bearing the Nlrc4 T337S gain-of-function mutation, identical to that found in patients with AIFEC, displayed chronically elevated total IL-18 levels but were not more susceptible to the TLR9-induced MAS model than wild type
4 animals. Serum free IL-18 levels were also not elevated in these mice, probably because of the high levels of IL-18BP present in the circulation. Of note, intestinal epithelial cells were the source of overproduced IL-18 in these mutated mice. Deciphering the mechanisms of IL-18 and IL-18BP production is critical to understand the IL-18/IL-18BP imbalance seen in diseases where free IL-18 levels are elevated, including AIDs.
L’interleukine (IL)-18 est une cytokine pro-inflammatoire de la famille de l’IL-1, initialement décrite comme un puissant inducteur d’interféron-γ (IFN-γ), et dont la maturation et la sécrétion sont sous la dépendance de l’activation de la voie de l’inflammasome et de la caspase-1. Son activité biologique est par ailleurs régulée par l’IL-18 binding protein (IL- 18BP), qui inhibe naturellement la grande majorité de l’IL-18 circulante en formant des complexes de haute affinité. L’IL-18 a été impliquée dans de nombreuses pathologies, parmi lesquelles les maladies inflammatoires. Toutefois, les résultats de deux essais cliniques étudiant l’effet de l’IL-18BP dans la polyarthrite rhumatoïde et le psoriasis se sont révélés décevants.
Nous avons participé à la mise au point d’un test ELISA permettant de doser spécifiquement la fraction libre et active de l’IL-18 et avons montré que les concentrations d’IL-18 libre étaient élevés dans la circulation des patients atteints de maladie de Still de l’adulte (MSA), une maladie autoinflammatoire (MAI) systémique rare, et qu’ils corrélaient avec l’activité de la maladie. Nous avons identifié d’autres MAI avec des taux élevés d’IL-18 libre, parmi lesquels la forme systémique d’arthrite juvénile idiopathique (sAJI), le syndrome d’activation macrophagique (SAM) (une complication grave de la MSA et sAJI qui se caractérise par un syndrome inflammatoire intense et des défaillances d’organe), et le syndrome autoinflammatoire de l’enfant avec entérocolite (AIFEC) récemment décrit et lié à une mutation avec gain de fonction de l’inflammasome NLRC4. En collaboration avec d’autres équipes, nous avons montré que l’inhibition de l’IL-18 par l’IL-18BP permettait de traiter efficacement des cas de MSA et une patiente avec AIFEC. Par ailleurs, nous avons démontré que, dans un modèle murin de SAM induit par la stimulation du récepteur Toll-like (TLR)9 de manière répétée, des quantités excessives d’IL-18 circulantes, telles qu’observées dans des souris déficientes en IL- 18BP ou surexprimant l’IL-18, aggravaient la maladie, et que le blocage de l’IL-18 permettait de corriger ce phénotype. L’IL-18 agissait par le biais de l’IFN-γ dans ce modèle, comme
6 attesté par la signature moléculaire IFN-γ associée à la sévérité du phénotype, de même que par l’efficacité du blocage de l’IFN-γ, reproduisant les effets de l’inhibition de l’IL-18. Des souris porteuses de la mutation T337S de Nlrc4, identique à celle retrouvée chez les sujets avec AIFEC, présentaient des taux élevés d’IL-18 totale de manière chronique mais ne présentaient aucun phénotype spontané et n’étaient pas davantage susceptibles au modèle de SAM induit par TLR9 que les souris sauvages. Les taux d’IL-18 libre n’étaient pas élevés chez ces souris mutées, probablement en raison des grandes quantités d’IL-18BP présentes dans la circulation.
Par ailleurs, nous avons démontré que c’étaient les cellules de l’épithélium intestinal qui étaient responsables de la production accrue d’IL-18 chez ses souris mutées. Nous pensons qu’il est essentiel de mieux définir les sources et mécanismes régulant la production de l’IL-18 et de l’IL-18BP pour comprendre le déséquilibre entre IL-18 et IL-18BP observé dans les maladies à taux élevé d’IL-18 libre, telles que les MAI.
AcP AID AIFEC AIM2 AOSD AP-1 ASC BMDM CANDLE CAPS CAR CARD CD CIA CINCA COPD CXCL DAMPS DAS DC DMARD DNBS DSS EAE ERK FasL FCAS fHLH FMF GAS GM-CSF hIL-18BP HLH ICAM ICSBP IEC IFI16 IFN Ig
Autoinflammation with infantile enterocolitis Absent in melanoma 2
Adult onset Still’s disease Activator protein 1
Adaptor protein apoptosis-associated speck-like protein containing caspase-recruitment domain
Bone marrow derived monocyte
Chronic atypical neutrophilic dermatosis, lipodystrophy, elevated temperature syndrome
Cryopyrin-associated periodic syndromes Chimeric antigen receptor
Caspase-recruitment domain Crohn’s disease
Chronic infantile neurological cutaneous articular syndrome Chronic obstructive pulmonary disease
(C-X-C motif) ligand
Danger-associated molecular patterns Disease activity score
Disease-modifying anti-rheumatic drug Dinitrobenzene sulfonic acid
Dextran sulfate sodium
Experimental autoimmune encephalomyelitis Extra cellular-regulated kinase
Familial cold autoinflammatory syndrome Familial haemophagocytic lymphohistiocytosis Familial Mediterranean fever
Granulocyte-macrophage colony-stimulating factor Human interleukin-18 binding protein
Haemophagocytic lymphohistiocytosis Intercellular adhesion molecule
Interferon consensus sequence-binding protein Intestinal epithelial cell
Interferon-γ-inducible protein 16 Interferon
IκB IKK IL IL-1R IL-18BP IL-18R ILC IRAK IRF JNK LCMV LPS LRR MAPK MAS MCMV MCV MCP MIF mIL-18 mIL-18BP MOG
M. tuberculosis MyD
MWS NAIP NFATc4 NF-κB NIK NK NLR NLRC NLRP NO NOD NOMID OA P. acnes PAMPs PBMC
Interferon-γ inducing factor Inhibitor of κB
Inhibitor of κB kinase Interleukin
Interleukin-18 binding protein Interleukin-18 receptor
Innate lymphoid cell
Interleukin-1 receptor associated kinase Interferon regulatory factor
c-Jun N-terminal kinase
Lymphocytic choriomeningitis virus Lipopolysaccharide
Leucine rich repeat
p38 mitogen activated protein kinase Macrophage activation syndrome Murine cytomegalovirus
Molluscum contagiosum virus Monocyte chemoattractant protein Macrophage migration inhibitory factor
Membrane-bound form of mature interleukin-18 Mouse interleukin-18 binding protein
Myelin oligodendrocyte glycoprotein Mycobacterium tuberculosis
Myeloid differentiation factor Muckle-Wells syndrome
Neuronal apoptosis inhibitory protein
Nuclear factor of activated T-cells, cytoplasmic 4 Nuclear factor-κB
Nuclear factor-κB-inducing kinase Natural killer
Nucleotide-binding oligomerization domain-like receptor Nucleotide-binding oligomerization domain-like receptor - and caspase-recruitment domain-containing protein
Nucleotide-binding oligomerization domain-like receptor family, pyrin domain containing
Neonatal-onset multisystem inflammatory disorder Osteoarthritis
Pathogen-associated molecular patterns Peripheral blood mononuclear cell
PRR PYD PYHIN RA RIG-1 SIGIRR sIL-18R sJIA SLE STAT Th TIR TLR TNBS TNF TRAF TRAPS Treg Tyk VCAM VEGF WT XIAP
Phosphoinositide 3-kinase Pattern recognition receptor Pyrin domain
Pyrin and HIN domains containing protein Rheumatoid arthritis
Retinoic acid-inducible gene 1
Single immunoglobulin interleukin-1-related receptor Soluble form of interleukin-18 receptor
Systemic-onset juvenile idiopathic arthritis Systemic Lupus Erythematosus
Signal transducer and activator of transcription factor T helper
Toll/interleukin-1 receptor Toll-like receptor
Trinitrobenzene sulfonic acid Tumor necrosis factor
Tumor necrosis factor receptor-associated factor
Tumor necrosis factor receptor-associated periodic syndrome Regulatory T cell
Janus kinase tyrosine protein kinase Vascular cell adhesion molecule Vascular endothelial growth factor Wild type
X-linked inhibitor of apoptosis
10 I. Introduction
I.1. Interleukin (IL)-18 and IL-18 binding protein (IL-18BP) I.1.a. IL-18: Characterization, production, signaling, function Characterization
IL-18 is a pro-inflammatory cytokine that was first identified as an interferon (IFN)-γ inducer in the serum of BCG-infected mice challenged with lipopolysaccharide (LPS) (1). It was then purified and cloned from mice infected with heat-killed Propionibacterium acnes (P.
acnes) where toxic shock was subsequently induced by LPS injection (2, 3).
IL-18, which was originally termed IGIF (for IFN-γ inducing factor), showed no similarities to known sequences in protein or nucleotide databases. Amino acid sequence of human IL-18 is 65% homologous with that of mouse IL-18 (4). IGIF was first suggested to be part of the IL-1 family of cytokines by Bazan et al. who matched its β-pleated structure to the one of IL-1β, using fold recognition methods (5). They identified what could be a pro-domain and a cleavage site for the IL-1β convertase-like enzyme (now termed caspase-1) between amino acids Asp35 and Asn36. Murine IGIF is indeed known to be produced as a 192 amino acids precursor (193 in human (4)) devoid of a conventional signal sequence before being cleaved in its N-terminal region, after Asp35, as a biologically active 18.3-kDa protein (3). Gu et al. and Ghayur et al. separately demonstrated that the cysteine protease caspase-1 was responsible for the cleavage of the 24-kDa IGIF precursor and its release out of the cell in its active form (6, 7).
11 Production and regulation
IL-18 gene and its regulation
The gene encoding IL-18 maps to chromosome 9 in mouse and 11 in human (8, 9). It is composed of 7 exons in mouse (10), 6 in human (11) (Figure 1), of which the last 5 are translated. The first two exons in mouse are non-coding whereas the human IL18 gene contains only one non-coding exon. In mouse, the promoter region is shared between the two 5’flanking fragments of exons 1 and 2. The promoter region upstream of exon 1 contains a binding site for the IFN consensus sequence-binding protein (ICSBP), which is a member of the IFN regulatory factors (IRF). The second promoter, located upstream of exon 2, includes a PU.1 binding site and is constitutively active (10). PU.1 is a transcription factor that is activated by IFN-γ and the Toll-like receptor (TLR)4-agonist LPS. The IL18 human gene comports only one promoter region which includes 3 signal transducer and activator of transcription factor (STAT)-binding sites (11), a PU.1-binding site and a GC-rich region (12). Indeed, STAT5 was found to specifically bind and activate the IL18 gene promoter (11). Moreover, in human myeloid cells, PU.1 or GC-binding proteins alone were not sufficient for optimal promoter activity and the formation of a PU.1/GC-binding protein complex was required (12). Both mouse and human promoters of the IL-18 gene are TATA-less.
Indeed, LPS has been shown to induce IL-18 mRNA expression in human peripheral blood mononuclear cells (PBMCs) (13) as well as in unstimulated mouse bone marrow derived monocytes (BMDMs) (14). In mouse BMDMs, the TLR3-agonist poly(I:C) and IFN-β also upregulated Il18 mRNA expression. Finally, CpG oligo deoxy nucleotide induced IL18 mRNA expression in fresh human macrophages and human monocyte-derived dendritic cells (15, 16).
Figure 1. The IL-18 gene. Mouse (A, from (17)) and human (B, from (11)) IL-18 genes are represented.
Moreover, one potential nuclear factor-κB (NF-κB) recognition sequence has been identified upstream of exon 1 in mouse Il18 gene, suggesting that NF-κB may be involved in upregulation of Il18 gene expression (10). Indeed, tumor necrosis factor (TNF)-α was able to induce IL-18 mRNA expression via NF-κB activation, in rat cardiomyocytes (18).
13 Besides, unlike many cytokines, which mRNA display a short half-life, Il18 gene has no, either only one RNA-destabilizing element, so that Il18 mRNA is remarkably stable (10).
As mentioned above, IL-18 is translated as a pro-peptide that requires cleavage by caspase-1 to become biologically active and released out of the cell. Constitutive expression of pro-IL-18 in many cells and tissues highlights the significance of its enzymatic processing in the regulation of its production as a functional cytokine. Of note, caspase-1 is also first produced as an inactive proenzyme which cleavage occurs upon inflammasome activation (see below for further details). Alternative pathways to caspase-1-dependent activation have been reported (Figure 2). Proteinase 3 and granzyme B were also shown to cleave pro-IL-18 in its 18-kDa mature form in vitro(19, 20). Cleavage of pro-IL-18 between Asn51 and Asp52 into biologically active 17-kDa protein occurred after treatment with the β subunit of the metalloproteinase meprin (21). In the human monocytic cell line THP-1, caspase-3 cleavage of pro and mature IL-18 after Asp71 and Asp76 generated biologically inactive products (22). Nonetheless, caspase-3 inhibitors substantially reduced the release of mature IL-18 in influenza A and Sendai virus-infected macrophages, which makes ambiguous the role of caspase-3 in pro-IL-18 processing (23). It has also been reported that P. acnes-primed caspase-1-deficient mice could produce biologically active IL-18 upon Fas stimulation by membrane-associated or soluble Fas ligand (FasL) (24). Bossaller et al. demonstrated that Fas-mediated activation of IL-18 was a caspase-8-dependent mechanism (25). Finally, in human mast cells, the serine protease chymase was responsible for the cleavage of pro-IL-18 into a 16-kDa biologically active fragment (26).
Figure 2. Protein sequence of human pro-IL-18 is represented. Sites of cleavage for various
proteolytic enzymes are shown. In green are the proteases that give rise to biologically active product, in orange those which lead to protein degradation. From (27)
Unlike IL-1β, IL-18 is constitutively expressed in many cells and tissues. Puren et al.
demonstrated for the first time the presence of IL18 mRNA and 24-kDa IL-18 precursor in unstimulated freshly isolated human PBMCs, as well as in snap frozen mouse spleen (28). IGIF mRNA was also highly expressed in Kupffer cells from untreated or P. acnes-infected mice (3).
Low but detectable levels of Il18 mRNA had also been found in mouse keratinocytes (29). In human, IGIF mRNA could be detected in pancreas, kidney, skeletal muscle, and, to a lesser extent, in liver and lung (4). To date, monocytes, macrophages, dendritic cells, T cells, B cells, but also non-immune cells including intestinal and airway epithelial cells, keratinocytes, osteoblasts, chondrocytes, astrocytes and microglial cells, and pancreatic islet β-cells are known to constitutively express IL-18 (reviewed in (17, 27, 30)).
15 It is hypothesized that mature IL-18 would be produced primarily by monocytes/macrophages and dendritic cells and that epithelial and mesenchymal cells would release immature IL-18 when dying, which would then be processed extracellularly, e.g. by proteinase 3, into a bioactive cytokine (31).
Once secreted, mature IL-18 biological activity is further regulated by its natural inhibitor IL-18BP.
Intriguingly, Bellora et al. identified, by flow cytometry and confocal microscopy analyses, a membrane-bound form of mature IL-18 (mIL-18) at the cell surface of M0 and M2 macrophages (32). Treatment with LPS or BCG infection induced shedding of mIL-18 that became detectable in the supernatant and promoted IFN-γ production and C-C chemokine receptor type 7 expression by natural killer (NK) cells. It was suggested that IL-18 expression at the cell surface of macrophages was dependent on caspase-1 and LPS-induced proteases.
Nonetheless, proteases involved in the release of mIL-18 have not yet been identified, neither have been the molecular events leading to cell surface retention of IL-18. Moreover, this membrane-bound form of IL-18 has not been reported by others so far.
IL-18 receptor (IL-18R) and signaling pathways
IL-18 is again very similar to IL-1 regarding its receptors and signaling pathways.
Like IL-1 receptor (IL-1R), IL-18R is a heterodimer composed of one binding chain, IL-18Rα, and one signaling chain, IL-18Rβ. IL-18Rα (also known as IL-1R-related protein) is a receptor of the IL-1 family, which had been first considered an orphan receptor. It binds to IL-18 with low affinity (33). IL-18Rβ, which shows similarities with IL-1R accessory protein (AcP) and was thus also named AcP-like (AcPL), is essential for IL-18 signaling (34). The two sub-units of the receptor and IL-18 form a high affinity complex that is able to transduce pro- inflammatory signals inside the cells. Experiments using IL-18Rα and IL-18Rβ gene-deficient mice have shown that both chains are essential for IL-18-mediated signaling (35, 36).
IL-18Rα is constitutively expressed on many cell types: immune cells (naïve T cells and mature T helper (Th)1 lymphocytes, NK cells, monocytes, macrophages, dendritic cells, B cells, neutrophils, basophils, mast cells) but also non-immune cells (endothelial cells, smooth muscle cells, synovial fibroblasts, chondrocytes, epithelial cells) (reviewed in (37, 38)). On the other hand, IL-18Rβ expression seems to be rather modulated by various cytokines (IL-2, IL-12, IL- 15, IL-21, IL-23) (reviewed in (37)). IL-18Rβ has been detected on the cell surface of T, NK and dendritic cells, macrophages, and non-immune cells (endothelial and smooth muscle cells) (reviewed in (38)). IL-18Rα expression is further upregulated by IL-12 (39-42), which participates in the synergy with IL-18 in the induction of IFN-γ (see below). IL-15 is also able to enhance IL-18Rα expression (40). On the other hand, T cell stimulation in the presence of IL-4 resulted in a downregulation of IL-18Rα expression (41). More recently, both sub-units of IL-18R were found to be expressed by type 3 innate lymphoid cells (ILC) (43).
17 Intracellular signaling pathways
IL-18 signaling pathways are, at least in part, shared with IL-1 and TLRs. Like receptors of the IL-1R family and TLRs, the intracellular region of IL-18Rβ contains a Toll/IL-1 receptor (TIR) domain that, upon IL-18/IL-18R complex formation, binds to the adaptor molecule myeloid differentiation factor (MyD)88 (44) to stimulate an intracellular signaling cascade.
MyD88 serves as a platform for IL-1R associated kinases (IRAK)4, 1 and 2, that subsequently autophosphorylate, leading to the recruitment of TNF receptor-associated factor (TRAF)6.
TRAF6 is then thought to phosphorylate NF-κB-inducing kinase (NIK). In turn, NIK phosphorylates the inhibitor of κB (IκB)-kinases (IKK) which results in the phosphorylation and degradation of IκB and the release of free NF-κB into the cytoplasm that will translocate to the nucleus where it will act as a transcription factor for many pro-inflammatory molecules (reviewed in (27), Figure 3).
Similar to IL-1, IL-18 signaling pathway seems mainly NF-κB pathway-dependent.
Nonetheless, as for IL-1, additional pathways are stimulated by IL-18 signaling. Functional c- Jun N-terminal Kinase (JNK), activator protein 1 (AP-1), p38 mitogen activated protein kinase (MAPK), extra cellular-regulated kinases (ERK), nuclear factor of activated T-cells, cytoplasmic 4 (NFATc4), phosphoinositide 3-kinase (Pi3K), Akt, Janus kinase Tyrosine protein kinase (Tyk)2 and STAT3 were required for full IL-18 biological activity, depending on the cell type, the species, and the experiment conditions ((45-48), reviewed in (27, 49), Figure 3).
18 Figure 3. IL-18 signaling pathways. Adapted from (27)
Soluble forms of IL-18Rα (sIL-18Rα) and IL-18Rβ (sIL-18Rβ) have been identified.
sIL-18Rβ stems from differential IL-18Rβ mRNA splicing (50) and was reported to exacerbate clinical symptoms and articular damage in the collagen-induced arthritis (CIA) mouse model, by affecting regulatory T cell (Treg) levels and supporting Th17 responses (51). A complex
19 made of sIL-18Rα, IL-18 and sIL-18Rβ, was isolated in human blood serum and inhibited IL- 18 biological activity in vitro (52). Moreover, sIL-18Rα serum levels were significantly higher in patients with rheumatoid arthritis (RA) and adult onset Still’s disease (AOSD) as compared to patients with systemic lupus erythematosus (SLE), osteoarthritis (OA) and healthy controls (52), and in patients with allergic asthma as compared to non-asthmatic allergic patients and healthy subjects (53). sIL-18Rα was presented as a useful diagnostic biomarker for allergic asthma and RA. Whether sIL-18Rα results from alternative splicing events and/or intramembrane proteolysis of IL-18Rα is not known to date (52).
IL-37 (previously known as IL-1F7) is another member of the IL-1 family of cytokines that mainly exerts an inhibitory effect on immune responses and does not exist in mouse (54).
Of note, it was shown to bind IL-18Rα but was unable to recruit IL-18Rβ nor to induce IFN-γ production by target cells (55). Nonetheless, it is not believed that IL-37 is an IL-18R antagonist because its binding affinity to IL-18Rα is low, and no competition occurs between IL-18 and IL-37 in vitro (56). It was shown later that, once bound to IL-18Rα, IL-37 could recruit IL-1R8 (single immunoglobulin IL-1-related receptor, SIGIRR) that bears a mutated TIR domain which is able to sequester MyD88, without inducing any downstream phosphorylation cascade ((57), reviewed in (58)). Moreover, IL-37 can bind IL-18BP, and the complex is able to recruit IL- 18Rβ, which prevents the formation of an active IL-18/IL-18R complex (56).
20 IL-18 functions
Induction of IFN-γ production
The first and most characterized function of IL-18 is the induction of IFN-γ production by its target cells. First reports demonstrated that mouse IGIF could stimulate IFN-γ production by mouse splenocytes, including established Th1 cells, in the presence of IL-2 or anti-CD3 antibodies (1-3, 59). Similar effects of IGIF/IL-18 were described in human mitogen-stimulated PBMCs (4, 60) and in anti-CD3-activated enriched human T cells (61).
IL-12, a pro-inflammatory cytokine identified in 1992, was also shown to induce IFN-γ production and to share other biological functions but has no structural similarity with IL-18 (62). It was early discovered that IGIF and IL-12 acted independently but synergistically in inducing IFN-γ secretion by mouse and human T cells (3, 61). The combination of IL-18 and IL-12 was also reported to strongly enhance IFN-γ production by mouse BMDMs and peritoneal macrophages (63, 64), human NK cells (40), anti-CD40-activated human B cells (65) and type 1 ILCs (66). The synergy between IL-18 and IL-12 is supported by their distinct signaling pathways. Indeed, while IL-18 activates NF-κB and AP-1, IL-12 signaling involves STAT4 which is also a well-known transcription factor binding to the IFN-γ promoter (67).
Moreover, it has been shown that IL-12 and IL-18 reciprocally induced the cell surface expression of their receptors. Indeed, IL-12 was first reported to upregulate IL-18Rα expression by the IL-12 responding-T cell clone 2D6 (39), then on mouse T and B cells and human NK cells (40, 42). IL-12 also had a positive effect on the expression of the signaling chain IL-18Rβ in mouse thymocytes (68). Reciprocally, IL-18 induced IL-12Rβ2 expression on mouse Th1 lymphocytes (69).
IL-15 and IL-21 have also been shown to act synergistically with IL-18 in IFN-γ production by human NK and T cells (70). Of note, both cytokines also upregulated IL-18Rα
21 and IL-18Rβ gene expression in human NK and T cells (71). Moreover, IL-15 was recently reported to act in synergy with IL-18 in promoting type 3 ILCs expansion and IL-22 production (43).
Surprisingly, IL-18 alone has marginal effect on IFN-γ production (39). Further, while it is considered, in collaboration with IL-12, as a major player in Th1 cell development, IL-18 was demonstrated to induce Th2 immune responses in the absence of IL-12. Indeed, in the presence of IL-2, IL-18 strongly induced IL-13 mRNA expression in mouse NK and T cells (72). It also enhanced production of IL-4 by basophils in vivo and, in vitro, mouse basophils released large amounts of IL-4 and IL-13 in response to stimulation with IL-3 and IL-18 (73).
Whereas IL-12 and IL-18 inhibited immunoglobulin (Ig)E production by activated B cells(65), IL-18 alone was shown to induce IgE production in BALB/c mice, in an IL-4-dependent manner (74).
Apart from IFN-γ induction, IL-18 has been widely involved in the regulation of innate and adaptive immunity. IL-18 promotes proliferation of mouse and human T cells (3, 59, 61) and induces IL-2 secretion by T cells (61). IL-18 enhances the development of CD8+ effector T cells (75) and upregulates their cytotoxic functions by increasing FasL expression (76). IL- 18 has further been shown to prevent CD8+ T cells from activation-induced cell death (77). IL- 18 could also play a role in Th17 responses by promoting Th17 cell differentiation (78, 79) and inducing the secretion of IL-17 by γδ-T cells, together with IL-23 (80). IL-18 is a major player in NK cell activation, not only by enhancing proliferation (81-83) but also by increasing cytolytic activities (4, 59) and inhibiting NK cell death (84). NK cytotoxic functions were shown to be enhanced via upregulation of FasL expression (85) or induction of perforin-
22 mediated pathway (86). CD25 expression was upregulated on mouse NK cells stimulated with IL-18 (87). IL-18 also promoted recruitment and activation of neutrophils (88). Notably, it has been demonstrated to induce secretion of the chemokine IL-8 by human PBMCs (60). IL-18 also promotes the release of TNF-α, IL-1β, monocyte chemoattractant protein (MCP)-1 and granulocyte-macrophage colony-stimulating factor (GM-CSF) by human PBMCs (4, 60) but inhibits IL-10 production (4). Bone marrow-derived basophils released IL-4 and histamine after treatment with IL-18 (73). IL-18 was also reported to induce nitric oxide (NO) synthesis in synovial membrane cultures (89). IL-18 is thought to have a critical role in anti-microbial peptides production by intestinal mucosa (90). It also induced anti-microbial peptides expression in macrophages infected by Mycobacterium tuberculosis (M. tuberculosis) (48).
Another potent function of IL-18 is its ability to foster tumor expansion and metastases. Indeed, IL-18 induced the expression of the vascular cell adhesion molecule (VCAM)-1 on hepatic sinusoidal endothelium (91, 92) and the production of matrix metallopeptidase-9 by the human myeloid leukemia cell line HL-60 (93). Moreover, IL-18 has been involved in the vascular endothelial growth factor (VEGF)-regulated migration of human gastric cancer cells, where IL- 18 blockade markedly reduced the level of VEGF-enhanced migration (94).
IL-18 genetically modified mice
First evidence of the in vivo pro-inflammatory role of IL-18 was demonstrated in P.
acnes-primed, LPS-challenged IL-18-deficient mice (95). In comparison to wild type (WT) mice, IL-18-deficient mice displayed decreased IFN-γ production, significant impairment of NK cells activity and reduction of Th1 response. In contrast, IL-18-transgenic mice, which overexpressed murine recombinant IL-18, showed an increased number of CD8+ T cells and macrophages, a decreased number of B cells and a significant raise of serum IgE, IgG1, IL-4
23 and IFN-γ levels (96). Moreover, splenic T cells of IL-18-transgenic mice produced higher levels of IFN-γ, IL-4, IL-5 and IL-13 as compared to WT splenic T cells.
To summarize, IL-18 exerts mainly pro-inflammatory effects by strongly promoting Th1 and NK cell responses as well as by stimulating the production of IFN-γ and other pro- inflammatory cytokines (Figure 4). However, according to the cytokine milieu, for instance in the absence of IL-12, IL-18 alone can drive rather Th2 cytokines or even Th17 responses in the presence of IL-23. IL-18 is thought to play a noteworthy role in cancer progression by a pro- angiogenic effect and the activation of matrix metalloproteases.
Figure 4. Main IL-18 biological functions. From (49)
24 I.1.b. IL-18BP: characterization, function, production
Isolation and characterization
With the aim to identify a soluble receptor for IL-18, IL-18BP was discovered and purified from normal human urine and serum of endotoxin-treated mice by two independent teams in 1999 (97, 98). Human IL-18BP molecular weight was estimated at around 40 kDa on SDS-PAGE electrophoresis, and it was thought to be highly glycosylated, given an approximate molecular weight of 20 kDa after treatment with N-glycanase (97). Cloning in Jurkat cells, human PBMCs and human and mouse spleen cells libraries brought out 4 different human IL- 18BP isoforms (hIL-18BPa, b, c and d) and 2 distinct mouse isoforms (mL-18BPc and d) that differ in their C-terminal region and are generated by alternative mRNA splicing (97, 99). In human cells, IL-18BPa is the most abundantly expressed isoform. All isoforms include a signal peptide and 4 potential N-glycosylation sites (Gln residues). No transmembrane domain could be identified. Importantly, IL-18BP is not a soluble form of IL-18R. All isoforms except hIL- 18BPd share an Ig domain that is homologous to the third Ig domain of the decoy receptor IL- 1R type II and is thought to be the binding site for IL-18. Furthermore, IL-18BP display significant homology to a family of putative proteins encoded by several poxviruses, including Molluscum contagiosum virus (MCV) (97, 100). Human IL18BP gene is located on chromosome 11q13, mouse Il18bp gene on chromosome 7.
Human IL-18BP obtained from urine was shown to prevent IL-18-induced IFN-γ production by murine splenocytes and human KG-1 cells and PBMCs in vitro, in a dose- dependent manner (97). Recombinant human IL-18BPa, expressed in monkey COS7 cells,
25 inhibited IL-18 bioactivity in vitro, similarly to urine-purified IL-18BP, but also in vivo, in LPS- treated mice (97). Aizawa et al. also demonstrated that recombinant IL-18BP prevented IL-18 binding to IL-18R-expressing cells (98). Thus, IL-18BP can be considered as a decoy receptor for IL-18.
Interestingly, recombinant MCV-encoded proteins MC53L and MC54L inhibited recombinant human IL-18-induced IFN-γ production by KG-1 cells similarly to human and mouse recombinant IL-18BP (101). In human, MCV infection is common and responsible for multiple small skin lesions characterized by high viral load but limited infiltration by inflammatory cells. The ability of IL-18BP-like MCV-encoded proteins to bind and inhibit human IL-18 may explain the capacity of MCV to evade the immune system.
As mentioned above, IL-18BP is able to bind not only to IL-18, but also to the anti- inflammatory cytokine IL-37 (56). At low IL-18BP concentrations, the IL-18BP/IL-37 complex has been shown to recruit IL-18Rβ, which could participate in IL-18 antagonism. It has been hypothesized that, in contrast, high concentrations of IL-18BP, especially in the case of clinical therapy by recombinant IL18BP, may counteract IL-37 anti-inflammatory activity (102).
Fantuzzi et al. reported that human IL-18BPa-transgenic mice had a normal phenotype at steady state but displayed lower serum levels of IFN-γ and macrophage inflammatory protein-2 after P. acnes infection followed by LPS treatment (103). Splenocytes of these mice produced less IFN-γ in response to LPS. IL-18BP-transgenic mice were also protected against concanavalin A-induced hepatitis. More recently, Harms et al. described a normal weight gain of IL-18BP-deficient mice as compared to WT, but disrupted NK cells phenotype characterized by decreased number and immature profile (104). Moreover, NK cells from IL-18BP-deficient mice were polarized to TNF-α rather than IFN-γ production. Finally, high circulating levels of IFN-γ were measured in IL-18BP-deficient mice following LPS treatment.
26 IL-18/IL-18BP interaction
Chemical cross-linking reactions demonstrated that recombinant human IL-18BP could bind recombinant human and murine IL-18, as well as recombinant murine IL-18BP bound recombinant murine and human IL-18 (98). Moreover, these bindings were considered as specific, since IL-18BP could not bind IL-1β.
At equimolar concentrations, hIL-18BPa and c and mIL-18BPd inhibited 50% of human IL-18-induced IFN-γ production by the NKO cell line. Full inhibition of IL-18 bioactivity occurred at 2-fold molar excess of IL-18BP over IL-18 (99). Human isoforms IL-18BPb and d and mouse IL-18BPc had no effect on human IL-18 inhibition.
BIAcore analyses showed that IL-18BP was unable to bind pro-IL-18. Human IL-18BPa bound human mature IL-18 with the highest affinity (dissociation constant of 400 pM) (99).
This exceptionally strong affinity suggests that, in opposition to other known cytokine soluble receptors (IL-1R type I and II, TNF-receptor p55 and p75, IL-6 receptor α), IL-18BP does serve rather as an inhibitor than a carrier protein for IL-18.
IL-18BP production and regulation
First reports explored the expression of IL-18BP at the mRNA level in different tissues and showed that IL-18BP was constitutively expressed in mouse and human spleen cells as well as in resting human PBMCs, normal human colon, heart and lung (97, 98). IL-18BP levels measured in the serum of healthy individuals were 0.5 to 7 ng/mL (2.15 ± 0.15 ng/mL), which represents a 20-fold molar excess compared to IL-18 (64 ± 17 pg/mL assessed by electrochemiluminescence assay) (105). According the law of mass action, 85% of IL-18 was
27 thought to circulate in its free, IL-18BP-unbound, form. Serum levels of IL-18BP increased up to 21.9 ± 1.44 ng/mL in patients with sepsis (105).
IL-18BP expression is highly controlled at transcriptional level. In constitutively non- expressing cells, IL-18BPa was shown to be potently upregulated by IFN-γ. Indeed, IFN-γ treatment induced IL-18BPa gene expression in human keratinocyte cell line HaCaT, colon carcinoma/epithelial cell line DLD-1 and primary renal glomerular mesangial cells from two distinct donors (106). Moreover, at the protein level, IFN-γ enhanced IL-18BPa secretion by DLD-1 but also LoVo, Caco-2 and HCT116 human colon carcinoma cell lines, HaCat, and cultures of colonic biopsy specimen (107). IFN-γ was thereafter shown to robustly induce IL- 18BP in other cell types including the human liver cancer cell line HepG2 and fibroblast-like synoviocytes (108, 109). The proximal γ-activated site (GAS) at the IL-18BP promoter appeared to be involved in this regulation process. Depending on the cell type, it was activated by the transcription factor STAT1 (in DLD-1 (110)) or a complex IRF-1/CCAAT/enhancer binding protein β (in HepG2 cells (108)). Thus, IFN-γ triggers a negative feedback loop in the IL-18/IL-18BP balance.
Remarkably, IFN-γ-induced IL-18BP gene expression was weaker in PBMCs and monocytic THP-1 cells as compared to DLD-1 (110). It appeared that a specific CpG motif (coined CpG2) located in the IL-18BP promoter, next to the proximal GAS site, was consistently methylated in THP-1 and primary monocytes, but not in DLD-1, HaCat and primary keratinocytes. This suggests that epigenetic silencing determines differential IL-18BP inducibility in monocytic vs epithelial cells, supporting cell type-specific tasks in host defense (102).
Besides, IL-27, a member of the IL-12 family of cytokines strongly upregulates IL- 18BPa expression in HaCat but also human primary keratinocytes and dermal fibroblasts (111).
28 IL-27 has also been involved in IL-18BP secretion by human epithelial ovarian cancer cells (112). Again, the proximal GAS site at the IL-18BP promoter and the transcription factor STAT1 were critical for IL-27-induced IL-18BP production (111, 112).
Finally, it has been reported that IFN-α and β2-adrenergic activation could induce IL- 18BP respectively in chronic hepatitis C patients (113) and cardiomyocytes (114).
I.1.c. IL-18 and IL-18BP in human diseases and mouse models
Being a major IFN-γ inducer, IL-18 was first known as a key player in host defense against intracellular microbial and viral infections. Its pathogenic role in inflammatory and autoimmune diseases, as well as its involvement in cancer, has been gradually deciphered.
Role of IL-18/IL-18BP in inflammatory diseases
Increased circulating levels of IL-18 and/or IL-18BP have been reported in many inflammatory diseases (Table 1).
A few authors pointed out the necessity to assess the levels of circulating free IL-18 (the IL-18 fraction that is not bound to IL-18BP), which is the biologically active form of the cytokine. So far, this could be done only indirectly by calculation, using the law of mass action (105). Indeed, in some diseases, free IL-18 is present despite markedly increased levels of IL- 18BP, indicating that the buffering capacities of IL-18BP are overwhelmed by excessive production of mature IL-18 (105, 115-117).
29 Disease References IL-18BP levels Free IL-18 levels
(118) - -
(119) - -
(105) ↗ ↗
(120) - -
(121) - -
(115) ↗ ↗
(122) ↗ ↗
(123) - -
(124) ↗ -
(125) - -
(126) ↘ -
Systemic lupus erythematosus
(127) - -
(128) ↗ ↗
Multiple sclerosis (129) - -
Myasthenia gravis (130) - -
Graves’ disease (131) - -
Dermato/polymyositis (132) - -
Sjögren’s syndrome (133) - -
Type 1 diabetes (134) = ↗
Chronic liver disease (116) ↗ -
Cutaneous psoriasis (135) - -
Psoriatic arthritis (136) - -
(137) - -
(138) - -
Chronic spontaneous urticaria
(117) ↗ ↗
(139) - -
(140) - -
(141) - -
(142) ↗ -
(143) - -
(144) - -
Inflammatory lung diseases
Chronic obstructive pulmonary disease
(145) - -
(146) - -
(147) - -
Idiopathic pulmonary fibrosis
(148) - -
Pulmonary sarcoidosis (149) - -
Table 1. Inflammatory diseases with elevated levels of circulating IL-18. If assessed, levels of circulating IL-18BP and/or calculated free IL-18 are also indicated (=: comparable to healthy controls; ↗ or ↘ : increased or decreased in comparison to healthy controls)
Besides high circulating levels of IL-18 and/or IL-18BP, other findings, both in patients and animal models, argue for a role of IL-18 and IL-18BP in the pathogenesis of various inflammatory diseases such as in Crohn’s disease (CD) and RA.
Role of IL-18/IL-18BP in Crohn’s disease
Inflammatory bowel diseases include CD and ulcerative colitis and are among the most frequent chronic inflammatory diseases. CD is a prototypic Th1-mediated condition, in which the role of IL-18 has been early and extensively studied. In addition to circulate at high levels in the blood of CD patients, where it has also been detected in its free bioactive form despite
31 elevated levels of IL-18BP (115, 122), IL-18 was reported to be upregulated at the mRNA and protein level in inflamed colonic tissue from CD patients as well as in a variety of experimental models of colitis in mice such as dextran sulfate sodium (DSS), trinitrobenzene sulfonic acid (TNBS) or dinitrobenzene sulfonic acid (DNBS)-induced colitis (120, 150-157). Importantly, not only pro- but also mature IL-18 were overexpressed at the site of inflammation, as compared to normal tissues from healthy subjects or naive mice (151, 154, 157). IL-18 neutralization using anti-IL-18 antiserum (155), anti-IL-18 antibodies (154) or recombinant IL-18BP (157, 158) remarkably attenuated clinically and histologically the severity of experimental colitis. IL- 18-deficient mice were protected against DSS (159), TNBS (154) and DNBS-colitis (160) whereas IL-18-overexpressing transgenic mice (161), IL-18BP-deficient mice or mice receiving daily IL-18 injections (159) exhibited exacerbated DSS-induced colitis. However, conflicting results were observed regarding the role of the inflammasome-caspase-IL-18 pathway in the severity of experimental colitis. Mice deficient for the nucleotide-binding oligomerization domain-like receptor (NLR) family, pyrin domain containing (NLRP) 3 inflammasome or caspase-1 developed less severe DSS-induced colitis than WT animals (162, 163). Other reports described a higher susceptibility to DSS-induced colitis in IL-18 and IL- 18Rα-deficient mice(164). Mice deficient in caspase-11 (165) or NLRP6 (166), which display defective intestinal IL-18 production, developed more severe DSS-induced colitis. Moreover, exogenous IL-18 administration was able to attenuate the severity of colitis in caspase-11- deficient mice (165).
Intestinal epithelial cells (IECs) appear to be the main source of IL-18 at steady state and during the acute phase of colitis (155). As chronic inflammation settles, a dramatic shift of IL-18 expression occurs from the IECs to the infiltrating macrophages and dendritic cells (150).
It has been proposed that IECs-produced IL-18 would have a protective effect against colitis, by promoting the integrity of the epithelial barrier, at the acute phase of the disease. Notably, it
32 has been shown that IL-18 controls the outgrowth of colitogenic bacteria, which could be dependent on anti-microbial peptides production (90, 166). Regulatory properties of epithelial- derived IL-18 also act by suppressing colonic Th17 cell differentiation and enhancing the expression of key regulatory T cell molecules (167). At the chronic phase of colitis, where IL- 18 is massively secreted by immune cells infiltrating the lamina propria, the pro-inflammatory effects of IL-18 predominate (168).
Another argument for IL-18 involvement in the pathogenesis of CD is the significant association of a polymorphism in the IL18R1-IL18RAP locus, encoding both chains of IL-18R, with early-onset CD, in a large cohort of European and North-American patients (169).
The production of IL-18BP is enhanced in colon samples from CD patients and DSS- treated mice (156, 159). In CD patients, its level of expression correlated with that of IL-18.
The submucosal endothelial cells and macrophages were identified as major sources of IL- 18BP (156).
Role of IL-18/IL-18BP in rheumatoid arthritis
RA is a chronic autoimmune and inflammatory disease primarily targeting the joints leading to structural damage associated with significant disability and increased morbidity and mortality.
Many reports have involved IL-18 in the pathogenesis of RA. First, IL-18 is highly expressed in affected joints of RA patients. IL18 mRNA and protein have been detected in the synovial tissue and more precisely in the lining layer within both macrophages and synovial fibroblasts (89, 123, 170). The levels of IL-18 were increased in RA in comparison to OA patients (126, 170). Remarkably, the mature/pro-IL-18 ratio was higher in RA than in OA
33 synovial tissue (170). The synovial fluid levels of IL-18 were higher in RA than in OA patients (123, 125).
Similarly, serum IL-18 levels were higher in patients with RA as compared to OA (123, 125, 126) but also to psoriatic arthritis (124). Of note, IL-18 levels in serum were lower than in synovial fluid, suggesting a local IL-18 production (89, 125). Surprisingly, Bokarewa et al. did not find a significant difference regarding serum levels of IL-18 between RA patients and healthy controls (171). Moreover, some studies did not find a correlation between serum IL-18 levels and disease activity or biomarkers of inflammation (124, 171). Yet, Yamamura et al.
reported that serum IL-18 correlated with C-reactive protein levels in RA patients (123).
Petrovic-Rackov et al. showed that both serum and synovial fluid levels of IL-18 correlated with the disease activity score (DAS) 28, a valid composite index used to evaluate disease activity in RA patients (125). This finding was however not observed in another report (124).
Bokarewa et al. measured higher serum IL-18 levels in erosive or rheumatoid factor-positive RA patients than in non-erosive or rheumatoid factor-negative RA patients, respectively, but did not find any correlation between IL-18 levels and any marker of inflammation (171).
A further argument supporting the role of IL-18 in RA pathogenesis is the identification of RA-associated polymorphism in the promoter region of the IL18 gene. According to a recent meta-analysis, the single nucleotide polymorphism 607A/C is associated with the risk of developing RA (172).
Importantly, IL-18R mRNA and protein have been detected in synovial tissue from RA patients (89, 126, 170). Moreover, IL-18Rα and β mRNA and IL-18Rα protein levels were increased in RA patients as compared to OA and healthy subjects (126, 170). Synovial tissue- infiltrating leukocytes were the first IL-18R-expressing cells to be identified (89) but it is increasingly clear that synovial fibroblasts also respond to IL-18 (173).
34 IL-18BP may be rather downregulated in RA joint, where its levels were decreased in RA synovial fluid and tissue as compared to OA and healthy subjects (126, 174). Consequently, Marotte et al. calculated increased free IL-18 levels in the synovial fluid from RA patients. Of note, IL-18BP serum levels were also lower in RA than in OA patients and healthy subjects (126) but raised in RA as compared to psoriatic arthritis (124).
IL-18 was also reported to be upregulated in experimental mouse models of arthritis.
Joosten et al. detected IL-18 in patella washouts and increased mRNA levels in synovial biopsies of mice subjected to streptococcal cell wall arthritis, and Plater-Zyberk et al. reported raised serum IL-18 levels in the CIA model (175, 176). More importantly, IL-18 blockade using anti-IL-18 antibodies reduced disease incidence and activity, cartilage destruction, bone erosion and articular inflammation in both models (175, 176). IL-18BP administered as recombinant protein or overexpressed as a transgene attenuated the severity of CIA (176-178). In line with these results, IL-18 deficiency prevented the development of arthritis in mice subjected to collagen- or zymosan-induced arthritis (179, 180). The reduced incidence and severity of CIA in IL-18KO mice was reversed by the administration of recombinant murine IL-18 (179). The concomitant administration of recombinant murine IL-18 and type II collagen + incomplete Freund’s adjuvant, during CIA priming and challenge, reproduced the effects of usual type II collagen + complete Freund’s adjuvant treatment (89, 181), indicating that IL-18 acts as an adjuvant.
In vitro studies with RA synovial explants showed increased production of NO, TNF-α, GM-CSF, IFN-γ and IL-6 under direct stimulation by IL-18 (89, 123). In addition, IL-18 induced the expression of the intercellular adhesion molecule (ICAM)-1 and VCAM-1, and the secretion of various chemokines, including IL-8 by RA synovial fibroblasts. IL-18 stimulation of RA synovial fibroblast also led to the production of angiogenic mediators such as VEGF. In addition, IL-18, itself, may exert pro-angiogenic activities (reviewed in (173)). To summarize,
35 IL-18 not only promotes Th1 responses but also recruits leukocytes in the synovial tissue and contribute to the formation of new blood vessels, which is critical for the development of the pannus, consisting in proliferating synovial tissue.
Role in other inflammatory diseases
Other examples of inflammatory diseases where IL-18 has been explored and involved in pathogenesis are numerous and, for some of them, detailed in Table 2.
As active IL-18 production is the result of inflammasome activation, inflammasome- dependent disorders and other autoinflammatory diseases (AID)s, including AOSD, its children counterpart systemic-onset juvenile idiopathic arthritis (sJIA) and macrophage activation syndrome (MAS) have been linked to IL-18. This will be discussed in detail in a following section.
Altogether the results described above suggest that IL-18 is an interesting therapeutic target in many inflammatory diseases. A first Phase I clinical trial assessed the pharmacokinetics, pharmacodynamics and safety of recombinant human IL-18BPa (tadekinig α) as an IL-18 blocking agent in moderate-to-severe RA and in plaque psoriasis. Tadekinig α had a favorable safety profile but was devoid of any therapeutic effect in neither RA nor in plaque psoriasis (182).
Disease References Data from patients Data from animal models
Systemic lupus erythematosus
Lymph node cells from MRL lpr/lpr mice are hyperresponsive to IL-18 and highly express the IL- 18Rβ chain
Endogenous anti-IL-18 antibody production secondary to IL-18 cDNA vaccination protects MRL lpr/lpr mice from renal damage and mortality
(185) Higher IL-18 serum levels in patient with lupus nephritis ; positive IL-18 staining in renal biopsies
(186) CD4+ T cells from patients secrete more IL-18
upon in vitro stimulation -
- IL-18Rα-deficient mice are resistant to MOG peptide- induced EAE
(188) IL18R1 mRNA is increased in the cerebrospinal
fluid and PBMCs from patients -
- Administration of IL-18BP-expressing adenoviral
vector reduces EAE incidence and severity (80)
Dendritic cells stimulated with M. tuberculosis and MOG promote IL-17 production by T cells and induce EAE following transfer to naive mice, in a caspase-1- dependant manner. IL-18 administration reverses EAE suppression by a caspase-1 inhibitor in these mice
Type I diabetes
IL-18 blockade using IL-18BP-Fc fusion molecule delays the development of adoptive transfer and cyclophosphamide-induced diabetes in NOD mice (191)
- IL-18-deficient mice have reduced islet reactive T cells compared to WT NOD mice
Chronic obstructive pulmonary disease
(192) IL-18 is highly expressed in alveolar
macrophage and CD8+ T cells, as well as in epithelial cells from patient lungs
In the smoke-induced COPD mouse model, IL-18 is increased in lung biopsies and the bronchiolo-alveolar lavage fluid; IL-18Rα-deficient mice develop less severe disease after smoke exposure
- Lung-specific IL-18-transgenic mice demonstrate
typical COPD lesions (145) Serum IL-18 concentrations correlate with the
severity of COPD -
(194) IL-18Rα polymorphism is associated with
allergic asthma -
(195) IL-18 polymorphism is associated with asthma
severity in adults -
(141) IL-18 expression is increased in lung epithelial
and smooth muscle cells from patient biopsies -
Ovalbumin sensitization/inhalation in lung-specific IL- 18-transgenic mice induces airway hyperresponsiveness and severe inflammation
(197) Plasma levels of IL-18 correlate with disease
severity in psoriatic patients -
(198) IL-18 expression is increased in early active and
progressive plaque-type psoriatic lesions -
(199) Increased levels of expression of IL-18 in keratinocytes from psoriatic patients correlate with disease severity
Cooperatively with IL-23, IL-18 enhances psoriasis-like dermal hyperplasia in the IL-23-induced psoriasis-like skin inflammation model
38 Atopic dermatitis
(137) IL-18 serum levels are elevated in children and adults with atopic dermatitis
IL-18 serum levels are elevated in NC/Nga mice, an inbred mouse model that spontaneously develop atopic dermatitis-like lesions
- Skin-specific IL-18-transgenic mice spontaneously develop atopic dermatitis-like skin inflammation
(202) IL-18 expression is increased in the horny layer of skin lesions from patients and correlates with the severity of atopic dermatitis
(203) IL-18 serum concentrations in atopic dermatitis
patients correlate with disease severity -
Table 2. Examples of other inflammatory diseases which pathogenesis may involve IL-18 and main data supporting this hypothesis. MOG = myelin oligodendrocyte glycoprotein; EAE = experimental autoimmune encephalomyelitis; NOD = non-obese diabetic; COPD = chronic obstructive pulmonary disease.