Marcel F. Nold

Differential requirement for the activation of the inflammasome for processing and release of IL-1beta in monocytes and macrophages.

The processing of pro-interleukin-1beta depends on activation of caspase-1. Controversy has arisen whether Toll-like receptor (TLR) ligands alone can activate caspase-1 for release of interleukin-1beta (IL-1beta). Here we demonstrate that human blood monocytes release processed IL-1beta after a one-time stimulation with either TLR2 or TLR4 ligands, resulting from constitutively activated caspase-1 and release of endogenous adenosine triphosphate. The constitutive activation of caspase-1 depends on the inflammasome components, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), and NALP3, but in monocytes caspase-1 activation is uncoupled from pathogen-associated molecular pattern recognition. In contrast, macrophages are unable to process and release IL-1beta solely by TLR ligands and require a second adenosine triphosphate stimulation. We conclude that IL-1beta production is differentially regulated in monocytes and macrophages, and this reflects their separate functions in host defense and inflammation.

IL-37 is a fundamental inhibitor of innate immunity

The function of interleukin 37 (IL-37; formerly IL-1 family member 7) has remained elusive. Expression of IL-37 in macrophages or epithelial cells almost completely suppressed production of pro-inflammatory cytokines, whereas the abundance of these cytokines increased with silencing of endogenous IL-37 in human blood cells. Anti-inflammatory cytokines were unaffected. Mice with transgenic expression of IL-37 were protected from lipopolysaccharide-induced shock, and showed markedly improved lung and kidney function and reduced liver damage after treatment with lipopolysaccharide. Transgenic mice had lower concentrations of circulating and tissue cytokines (72–95% less) than wild-type mice and showed less dendritic cell activation. IL-37 interacted intracellularly with Smad3 and IL-37-expressing cells and transgenic mice showed less cytokine suppression when endogenous Smad3 was depleted. IL-37 thus emerges as a natural suppressor of innate inflammatory and immune responses.

IL-1 family nomenclature

To the Editor: n nNewly cloned interleukin 1 (IL-1) family members1–3 were originally given an IL-1 family (IL-1F) designation4, but as functions have now been elucidated for several of these5,6, we propose that each now be assigned an individual interleukin designation. IL-1F6, IL-1F8 and IL-1F9 are encoded by distinct genes but use the same receptor complex (IL-1Rrp2 and AcP), are proinflammatory and deliver nearly identical signals7–12. We propose these be designated IL-36α, IL-36β and IL-36γ, respectively. IL-1F5 also binds to IL-1Rrp2 but antagonizes those cytokines in a manner analogous to that used by IL-1Ra to antagonize IL-1α and IL-1β7–9. We propose that IL-1F5 be renamed IL-36Ra (for ‘receptor antagonist’). In the IL-1 nomenclature, IL-1Ra is used for the natural product, whereas IL-1ra is used for the recombinant product; therefore, IL-36Ra is appropriate for natural IL-1F5. n nIL-1F7 produces anti-inflammatory effects by suppressing innate immune responses; it does this by decreasing the production of inflammatory cytokines induced by Toll-like receptor agonists as well as that of IL-1 and tumor necrosis factor13,14. We propose this IL-1 family member be renamed IL-37. IL-1F7 has various splice forms1,2,15,16, of which IL-1F7b is the most studied. We propose that IL-1F7a, IL-1F7b and so on be renamed IL-37a, IL-37b and so on. The one remaining IL-1 family member, for which no function has yet been demonstrated, is IL-1F10; however, as evidence of its properties remains limited, we suggest that it retain its IL-1F designation until a function is clearly identified, although it might be prudent to reserve the designation IL-38 for this eventuality.

Interleukin 37 expression protects mice from colitis

IL-37, a newly described member of the IL-1 family, functions as a fundamental inhibitor of innate inflammation and immunity. In the present study, we examined a role for IL-37 during experimental colitis. A transgenic mouse strain was generated to express human IL-37 (hIL-37tg), and these mice were subjected to dextran sulfate sodium (DSS)-induced colitis. Despite the presence of a CMV promoter to drive expression of IL-37, mRNA transcripts were not present in colons at the resting state. Expression was observed only upon disruption of the epithelial barrier, with a six- to sevenfold increase (P = 0.02) on days 3 and 5 after continuous exposure to DSS. During the development of colitis, clinical disease scores were reduced by 50% (P < 0.001), and histological indices of colitis were one-third less in hIL-37tg mice compared with WT counterparts (P < 0.001). Reduced inflammation was associated with decreased leukocyte recruitment into the colonic lamina propria. In addition, release of IL-1β and TNFα from ex vivo colonic explant tissue was decreased 5- and 13-fold, respectively, compared with WT (P ≤ 0.005), whereas IL-10 was increased sixfold (P < 0.001). However, IL-10 was not required for the anti-inflammatory effects of IL-37 because IL-10-receptor antibody blockade did not reverse IL-37-mediated protection. Mechanistically, IL-37 originating from hematopoietic cells was sufficient to exert anti-inflammatory effects because WT mice reconstituted with hIL-37tg bone marrow were protected from colitis. Thus, IL-37 emerges as key modulator of intestinal inflammation.

IL-37: a new anti-inflammatory cytokine of the IL-1 family

The IL-1 family of cytokines encompasses eleven proteins that each share a similar β-barrel structure and bind to Ig-like receptors. Some of the IL-1-like cytokines have been well characterised, and play key roles in the development and regulation of inflammation. Indeed, IL-1α (IL-1F1), IL-1β (IL-1F2), and IL-18 (IL-1F4) are well-known inflammatory cytokines active in the initiation of the inflammatory reaction and in driving Th1 and Th17 inflammatory responses. In contrast, IL-1 receptor antagonist (IL-1Ra, IL-1F3) and the receptor antagonist binding to IL-1Rrp2 (IL-36Ra, IL-1F5) reduce inflammation by blocking the binding of the agonist receptor ligands. In the case of IL-37 (IL-1F7), of which five different splice variants have been described, less is known of its function, and identification of the components of a heterodimeric receptor complex remains unclear. Some studies suggest that IL-37 binds to the α chain of the IL-18 receptor in a non-competitive fashion, and this may explain some of the disparate biological effects that have been reported for mice deficient in the IL-18R. The biological properties of IL-37 are mainly those of down-regulating inflammation, as assessed in models where human IL-37 is expressed in mice. In this review, an overview of the role of IL-37 in the regulation of inflammation is presented. The finding that IL-37 also locates to the nucleus, as do IL-1α and IL-33, for receptor-independent organ/tissue-specific regulation of inflammation is also reviewed.

The IL-1 Family Member 7b Translocates to the Nucleus and Down-Regulates Proinflammatory Cytokines

The IL-1 family member 7b (IL-1F7b) is a novel homolog of the IL-1 cytokine family discovered by computational cloning. We have reported that IL-1F7b shares critical amino acid residues with IL-18 and binds the IL-18-binding protein; in doing so, IL-1F7b augments the inhibition of IFN-γ by the IL-18-binding protein. IL-1F7b also binds IL-18Rα but neither induces signal nor acts as a receptor antagonist. Hence, the function of IL-1F7b remains unknown. In the present study, we analyzed the intracellular expression pattern of IL-1F7b. Using two variants of GFP fusion constructs of human IL-1F7b stably expressed in RAW macrophages, only the postcleavage mature form of the IL-1F7b precursor—but not the N-terminal propiece—specifically translocates to the nucleus following LPS stimulation. IL-1F7b, like IL-1β, IL-18, and IL-33, is processed by caspase-1 to generate the mature cytokines. Therefore, we tested whether caspase-1-mediated cleavage of the IL-1F7b precursor is required for mature IL-1F7b to translocate actively into the nucleus. Indeed, a specific caspase-1 inhibitor markedly reduced nuclear entry of IL-1F7b. In stable transfectants of human IL-1F7b in RAW macrophages stimulated with LPS, levels of TNF-α, IL-1α, IL-6, as well as the chemokine MIP-2, were substantially reduced (72–98%) compared with LPS-stimulated cells transfected with the empty plasmid. These results demonstrate that IL-1F7b translocates to the nucleus after caspase-1 processing and may act as a transcriptional modulator reducing the production of LPS-stimulated proinflammatory cytokines, consistent with IL-1F7b being an anti-inflammatory member of the IL-1 family.

Expression and Release of IL-18 Binding Protein in Response to IFN-γ

IL-18 and IL-18 binding protein (IL-18BP) are two newly described opponents in the cytokine network. Local concentrations of these two players may determine biological functions of IL-18 in the context of inflammation, infection, and cancer. As IL-18 appears to be involved in the pathogenesis of Crohn’s disease and may modulate tumor growth, we investigated the IL-18/IL-18BPa system in the human colon carcinoma/epithelial cell line DLD-1. In this study, we report that IFN-γ induces expression and release of IL-18BPa from DLD-1 cells. mRNA induction and secretion of IL-18BPa immunoreactivity were associated with an activity that significantly impaired release of IFN-γ by IL-12/IL-18-stimulated PBMC. Inducibility of IL-18BPa by IFN-γ was also observed in LoVo, Caco-2, and HCT116 human colon carcinoma cell lines and in the human keratinocyte cell line HaCaT. Induction of IL-18BPa in colon carcinoma/epithelial cell lines was suppressed by coincubation with sodium butyrate. IFN-γ-mediated IL-18BPa and its suppression by sodium butyrate were confirmed in organ cultures of intestinal colonic biopsy specimens. In contrast, sodium butyrate did not modulate expression of IL-18. The present data suggest that IFN-γ may limit biological functions of IL-18 at sites of colonic immune activation by inducing IL-18BPa production. Down-regulation of IL-18BPa by sodium butyrate suggests that reinforcement of local IL-18 activity may contribute to actions of this short-chain fatty acid in the colonic microenvironment.

IL-37 requires the receptors IL-18Rα and IL-1R8 (SIGIRR) to carry out its multifaceted anti-inflammatory program upon innate signal transduction

Interleukin 37 (IL-37) and IL-1R8 (SIGIRR or TIR8) are anti-inflammatory orphan members of the IL-1 ligand family and IL-1 receptor family, respectively. Here we demonstrate formation and function of the endogenous ligand-receptor complex IL-37–IL-1R8–IL-18Rα. The tripartite complex assembled rapidly on the surface of peripheral blood mononuclear cells upon stimulation with lipopolysaccharide. Silencing of IL-1R8 or IL-18Rα impaired the anti-inflammatory activity of IL-37. Whereas mice with transgenic expression of IL-37 (IL-37tg mice) with intact IL-1R8 were protected from endotoxemia, IL-1R8-deficient IL-37tg mice were not. Proteomic and transcriptomic investigations revealed that IL-37 used IL-1R8 to harness the anti-inflammatory properties of the signaling molecules Mer, PTEN, STAT3 and p62(dok) and to inhibit the kinases Fyn and TAK1 and the transcription factor NF-κB, as well as mitogen-activated protein kinases. Furthermore, IL-37–IL-1R8 exerted a pseudo-starvational effect on the metabolic checkpoint kinase mTOR. IL-37 thus bound to IL-18Rα and exploited IL-1R8 to activate a multifaceted intracellular anti-inflammatory program.

Endogenous IL-32 Controls Cytokine and HIV-1 Production

IL-32, a proinflammatory cytokine that activates the p38MAPK and NF-κB pathways, induces other cytokines, for example, IL-1β, IL-6, and TNF-α. This study investigated the role of endogenous IL-32 in HIV-1 infection by reducing IL-32 with small interfering (si)RNA in freshly infected PBMC and in the latently infected U1 macrophage cell line. When PBMC were pretreated with siRNA to IL-32 (siIL-32), IL-6, IFN-γ, and TNF-α were reduced by 57, 51, and 36%, respectively, compared with scrambled siRNA. Cotransfection of NF-κB and AP-1 reporter constructs with siIL-32 decreased DNA binding of these transcription factors by 42 and 46%, respectively. Cytokine protein array analysis revealed that the inhibitory activity of siIL-32 primarily targeted Th1 and proinflammatory cytokines and chemokines, e.g., MIP-1α/β. Unexpectedly, HIV-1 production (as measured by p24) increased 4-fold in these same PBMC when endogenous IL-32 was reduced. Because IFN-γ was lower in siIL-32-treated PBMC, we blocked IFN-γ bioactivity, which enhanced the augmentation of p24 by siIL-32. Furthermore, siIL-32 reduced the natural ligands of the HIV-1 coreceptors CCR5 (MIP-1α/β and RANTES) and CXCR4 (SDF-1). Inhibition of endogenous IL-32 in U1 macrophages also increased HIV-1. When rhIL-32γ was added to these cells, p24 levels fell by 72%; however, in the same cultures IFN-α increased 4-fold. Blockade of IFN-α/β bioactivity in IL-32γ-stimulated U1 cells revealed that IFN-α conveys the anti-HIV-1 effect of rhIL-32γ. In summary, depletion of endogenous IL-32 reduced the levels of Th1 and proinflammatory cytokines but paradoxically increased p24, proposing IL-32 as a natural inhibitor of HIV-1.

Role of caspase-1 in nuclear translocation of IL-37, release of the cytokine, and IL-37 inhibition of innate immune responses

Significance IL-37 exerts broad inhibitory properties on the innate inflammatory and acquired immune responses. We mutated the caspase-1 site in IL-37 and show that caspase-1 processing is required for maturation of the intracellular IL-37 precursor for its translocation to the nucleus. Because nuclear translocation of IL-37 is required for the suppression of LPS-induced IL-6, the data define a unique consequence for caspase-1 inhibition, that is, reversal of the anti-inflammatory activities of endogenous IL-37. In addition, neutralizing antibodies reverse the suppression of LPS-induced IL-6 in IL-37 transgenic mice, supporting a role for extracellular signaling by IL-37. Thus, similar to IL-1α and IL-33, IL-37 now emerges as a dual-function cytokine with both intra- and extracellular mechanisms of action. IL-37 is a fundamental inhibitor of innate immunity. Human IL-37 has a caspase-1 cleavage site and translocates to the nucleus upon LPS stimulation. Here, we investigated whether caspase-1 processing affects IL-37–mediated suppression of LPS-induced cytokines and the release from cells by analyzing a caspase-1 cleavage site mutant IL-37 (IL-37D20A). Nuclear translocation of IL-37D20A is significantly impaired compared with WT IL-37 in transfected cells. LPS-induced IL-6 was decreased in cells expressing WT IL-37 but not IL-37D20A. The function of IL-37 in transfected bone marrow-derived macrophages is nucleotide-binding oligomerization domain-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome-dependent, because IL-37 transfection in apoptosis-associated speck-like protein containing a carboxyl-terminal caspase recruitment domain- and NLRP3-deficient cells does not reduce levels of IL-6 and IL-1β upon LPS stimulation. IL-37–expressing macrophages release both precursor and mature IL-37, but only the externalization of mature IL-37 was dependent on ATP. Precursor and mature IL-37 was also secreted from human dendritic cells and peripheral blood mononuclear cells. To determine whether IL-37 is active in the extracellular compartment, we pretreated IL-37 transgenic mice with IL-37–neutralizing antibodies before LPS challenge. In IL-37–expressing mice, neutralizing IL-37 antibodies reversed the suppression of LPS-induced serum IL-6. In contrast, the addition of neutralizing antibody did not reverse suppression of LPS-induced IL-6 in mouse macrophages transfected with IL-37. Although caspase-1 is required for nuclear translocation of intracellular IL-37 and for secretion of mature IL-37, the release of the IL-37 precursor is independent of caspase-1 activation. IL-37 now emerges as a dual-function cytokine with intra- and extracellular properties for suppressing innate inflammation.