Structural transitions enable interleukin-18 maturation and signaling.

Several interleukin-1 (IL-1) family members, including IL-1ß and IL-18, require processing by inflammasome-associated caspases to unleash their activities. Here, we unveil, by cryoelectron microscopy (cryo-EM), two major conformations of the complex between caspase-1 and pro-IL-18. One conformation is similar to the complex of caspase-4 and pro-IL-18, with interactions at both the active site and an exosite (closed conformation), and the other only contains interactions at the active site (open conformation). Thus, pro-IL-18 recruitment and processing by caspase-1 is less dependent on the exosite than the active site, unlike caspase-4. Structure determination by nuclear magnetic resonance uncovers a compact fold of apo pro-IL-18, which is similar to caspase-1-bound pro-IL-18 but distinct from cleaved IL-18. Binding sites for IL-18 receptor and IL-18 binding protein are only formed upon conformational changes after pro-IL-18 cleavage. These studies show how pro-IL-18 is selected as a caspase-1 substrate, and why cleavage is necessary for its inflammatory activity.

IL-18BP is a secreted immune checkpoint and barrier to IL-18 immunotherapy.

Cytokines were the first modern immunotherapies to produce durable responses in patients with advanced cancer, but they have only modest efficacy and limited tolerability1,2. In an effort to identify alternative cytokine pathways for immunotherapy, we found that components of the interleukin-18 (IL-18) pathway are upregulated on tumour-infiltrating lymphocytes, suggesting that IL-18 therapy could enhance anti-tumour immunity. However, recombinant IL-18 previously did not demonstrate efficacy in clinical trials3. Here we show that IL-18BP, a high-affinity IL-18 decoy receptor, is frequently upregulated in diverse human and mouse tumours and limits the anti-tumour activity of IL-18 in mice. Using directed evolution, we engineered a 'decoy-resistant' IL-18 (DR-18) that maintains signalling potential but is impervious to inhibition by IL-18BP. Unlike wild-type IL-18, DR-18 exerted potent anti-tumour effects in mouse tumour models by promoting the development of poly-functional effector CD8+ T cells, decreasing the prevalence of exhausted CD8+ T cells that express the transcriptional regulator of exhaustion TOX, and expanding the pool of stem-like TCF1+ precursor CD8+ T cells. DR-18 also enhanced the activity and maturation of natural killer cells to effectively treat anti-PD-1 resistant tumours that have lost surface expression of major histocompatibility complex class I molecules. These results highlight the potential of the IL-18 pathway for immunotherapeutic intervention and implicate IL-18BP as a major therapeutic barrier.

IL-18 receptor-α signalling pathway contributes to autoantibody-induced arthritis via neutrophil recruitment and mast cell activation.

OBJECTIVES: The interleukin (IL)-18 signalling pathway is involved in animal models of collagen-induced arthritis, but the role of this pathway in autoantibody-induced arthritis is poorly understood. An autoantibody-induced arthritis model, K/BxN serum transfer arthritis, reflects the effector phase of arthritis and is important in innate immunity including neutrophils and mast cells. This study aimed to investigate the role of the IL-18 signalling pathway in autoantibody-induced arthritis using IL-18 receptor (IL-18R) α-deficient mice. METHODS: K/BxN serum transfer arthritis was induced in IL-18Rα-/- and wild-type B6 (controls) mice. The severity of arthritis was graded, and histological and immunohistochemical examinations were performed on paraffin-embedded ankle sections. Total Ribonucleic acid (RNA) isolated from mouse ankle joints was analysed by real-time reverse transcriptase-polymerase chain reaction. RESULTS: IL-18 Rα-/- mice had significantly lower arthritis clinical scores, neutrophil infiltration, and numbers of activated, degranulated mast cells in the arthritic synovium than in controls. IL-1ß, which is indispensable for the progression of arthritis, was significantly downregulated in inflamed ankle tissue in IL-18 Rα-/- mice. CONCLUSIONS: IL-18/IL-18Rα signalling contributes to the development of autoantibody-induced arthritis by enhancing synovial tissue expression of IL-1ß and inducing neutrophil recruitment and mast cell activation. Therefore, inhibition of the IL-18Rα signalling pathway might be a new therapeutic strategy for rheumatoid arthritis.

Interleukin-18: Biological properties and role in disease pathogenesis.

Initially described as an interferon (IFN)γ-inducing factor, interleukin (IL)-18 is indeed involved in Th1 and NK cell activation, but also in Th2, IL-17-producing γδ T cells and macrophage activation. IL-18, a member of the IL-1 family, is similar to IL-1ß for being processed by caspase 1 to an 18 kDa-biologically active mature form. IL-18 binds to its specific receptor (IL-18Rα, also known as IL-1R7) forming a low affinity ligand chain. This is followed by recruitment of the IL-18Rß chain. IL-18 then uses the same signaling pathway as IL-1 to activate NF-kB and induce inflammatory mediators such as adhesion molecules, chemokines and Fas ligand. IL-18 also binds to the circulating high affinity IL-18 binding protein (BP), such as only unbound free IL-18 is active. IL-18Rα may also bind IL-37, another member of the IL-1 family, but in association with the negative signaling chain termed IL-1R8, which transduces an anti-inflammatory signal. IL-18BP also binds IL-37 and this acts as a sink for the anti-inflammatory properties of IL-37. There is now ample evidence for a role of IL-18 in various infectious, metabolic or inflammatory diseases such as influenza virus infection, atheroma, myocardial infarction, chronic obstructive pulmonary disease, or Crohn's disease. However, IL-18 plays a very specific role in the pathogenesis of hemophagocytic syndromes (HS) also termed Macrophage Activation Syndrome. In children affected by NLRC4 gain-of-function mutations, IL-18 circulates in the range of tens of nanograms/mL. HS is treated with the IL-1 Receptor antagonist (anakinra) but also specifically with IL-18BP. Systemic juvenile idiopathic arthritis or adult-onset Still's disease are also characterized by high serum IL-18 concentrations and are treated by IL-18BP.

Effects of IL-18 on the proliferation and steroidogenesis of bovine theca cells: Possible roles in the pathogenesis of polycystic ovary syndrome.

Interleukin 18 (IL-18) is a pleiotropic pro-inflammatory cytokine and is associated with arrested follicle development and anovulation which are the typical pathological changes of PCOS. Theca cells (TCs) have a key role in follicular growth and atresia. But whether IL-18 can directly affect ovarian TCs function is unknown. Therefore, the objective of this study was to determine the effect of IL-18 on proliferation and steroidogenesis of bovine TCs and to explore the biological effect of IL-18 on folliculogenesis. This work revealed that at 300-1000 pg/mL, IL-18 led to a time- and dose-dependently increase in cell proliferation (P < .05). IL-18 increased 17-hydroxyprogesterone (17OHP4) and androstenedione (A2) secretion with up-regulation of key steroidogenesis-related genes CYP11A1 and CYP17A1 (P < .05). Furthermore, our data demonstrated that the IL-18R protein is predominantly expressed in small-follicle (3-6 mm) TCs than large follicles (8-22 mm) by immunohistochemistry. We also found that the stimulation effects of IL-18 on TCs can be reversed with the addition of IL-18BP as early as at 4 hours of culture and reached the peak at 16 hours. We conclude that IL-18 appears to target TCs in bovine, and suggest an important role for this cytokine in ovarian function. Present findings further validate potential effects of IL-18 in the conditions associated with follicular dysplasia and excessive growth of ovarian TCs (such as PCOS). But additional research is needed to further understand the mechanism of action of IL-18 in theca cells as well as its precise role in folliculogenesis.

IL-18R-dependent and independent pathways account for IL-18-enhanced antitumor ability of CAR-T cells.

Interleukin-18 (IL-18) has been demonstrated to augment the antitumor capacity of chimeric antigen receptor-T cells (CAR-T) but the underlying mechanisms are largely unknown. Here we explored the effects and mechanisms of exogenous IL-18 on the antitumor response of CAR-T cells. IL-18 boosted the cytotoxicity of human epidermal growth factor receptor-2 (HER2)-specific CAR-T cells ex vivo and enhanced the antitumor efficacy of the CAR-T cells in immunodeficient mice, moreover, IL-18 improved the antitumor capacity of OVA-specific T cells in immunocompetent mice, indicating the universal enhancing function of IL-18 for adoptive cell therapy. To address the roles of IL-18 receptor (IL-18R) in the enhancing function, we evaluated the effects of IL-18R knockout (IL-18R-/-) condition in immunocompetent host and CAR-T cells on the IL-18-enhanced antitumor activities. Interestingly, IL-18 persisted to improve the antitumor ability of IL-18R intact CAR-T cells in IL-18R-/- mice. For IL-18R-/- CAR-T cells, however, IL-18 still holds the enhancing ability to boost the antitumor efficacy in IL-18R-/- mice, albeit the ex vivo tumor-killing ability was lower than that of IL-18R intact CAR-T cells, indicating that IL-18R-independent pathway is involved in the enhancement. Furthermore, tagged IL-18 binded to the membrane of IL-18R-/- splenic and lymph node cells and IL-18R intact and IL-18R-/- CAR-T cells showed distinct transcriptomic profiles when stimulated by IL-18. These data demonstrate that IL-18R-independent pathways contribute to functions of IL-18.

IL-18 but Not IL-1 Signaling Is Pivotal for the Initiation of Liver Injury in Murine Non-Alcoholic Fatty Liver Disease.

Non-alcoholic fatty liver disease (NAFLD) is rising in prevalence, and a better pathophysiologic understanding of the transition to its inflammatory phenotype (NASH) is key to the development of effective therapies. To evaluate the contribution of the NLRP3 inflammasome and its downstream effectors IL-1 and IL-18 in this process, we applied the true-to-life "American lifestyle-induced obesity syndrome" (ALiOS) diet mouse model. Development of obesity, fatty liver and liver damage was investigated in mice fed for 24 weeks according to the ALiOS protocol. Lipidomic changes in mouse livers were compared to human NAFLD samples. Receptor knockout mice for IL-1 and IL-18 were used to dissect the impact of downstream signals of inflammasome activity on the development of NAFLD. The ALiOS diet induced obesity and liver steatosis. The lipidomic changes closely mimicked changes in human NAFLD. A pro-inflammatory gene expression pattern in liver tissue and increased serum liver transaminases indicated early liver damage in the absence of histological evidence of NASH. Mechanistically, Il-18r-/-- but not Il-1r-/- mice were protected from early liver damage, possibly due to silencing of the pro-inflammatory gene expression pattern. Our study identified NLRP3 activation and IL-18R-dependent signaling as potential modulators of early liver damage in NAFLD, preceding development of histologic NASH.

Interleukin-18 in Health and Disease.

Interleukin (IL)-18 was originally discovered as a factor that enhanced IFN-γ production from anti-CD3-stimulated Th1 cells, especially in the presence of IL-12. Upon stimulation with Ag plus IL-12, naïve T cells develop into IL-18 receptor (IL-18R) expressing Th1 cells, which increase IFN-γ production in response to IL-18 stimulation. Therefore, IL-12 is a commitment factor that induces the development of Th1 cells. In contrast, IL-18 is a proinflammatory cytokine that facilitates type 1 responses. However, IL-18 without IL-12 but with IL-2, stimulates NK cells, CD4⁺ NKT cells, and established Th1 cells, to produce IL-3, IL-9, and IL-13. Furthermore, together with IL-3, IL-18 stimulates mast cells and basophils to produce IL-4, IL-13, and chemical mediators such as histamine. Therefore, IL-18 is a cytokine that stimulates various cell types and has pleiotropic functions. IL-18 is a member of the IL-1 family of cytokines. IL-18 demonstrates a unique function by binding to a specific receptor expressed on various types of cells. In this review article, we will focus on the unique features of IL-18 in health and disease in experimental animals and humans.

Expression of mRNA and protein of IL-18 and its receptor in human follicular granulosa cells.

PURPOSE: There is no information available about the IL-18 receptor in ovarian follicles, so the present study attempts to demonstrate the expression of IL-18 and its receptor in human granulosa cells (GCs). METHODS: To evaluate the concentration of IL-18 in serum and follicular fluid (FF), we collected serum and FF from 102 women undergoing oocyte retrieval. Also, to detect expression of IL-18 and its receptor by luteinized GCs, these cells were pooled six times from a total of twenty individual patients with 5-16 follicles each. The IL-18 concentration was determined by ELISA and the expression of IL-18 and its receptor by immunocytochemistry and reverse transcription polymerase chain reaction. RESULTS: Our results showed that the median IL-18 concentration in serum, 159.27 pg/ml (IQR 121.41-210.1), was significantly higher than in FF, 142.1 pg/ml (IQR 95.7-176.5), p < 0.001. Moreover, we found that IL-18 and its receptor are expressed by GCs. CONCLUSION: The presence of IL-18 in FF and the expression of IL-18 and its receptor by GCs suggest an important role for this cytokine in ovarian function.

Blockade of IL-18 signaling diminished neuropathic pain and enhanced the efficacy of morphine and buprenorphine.

Currently, the low efficacy of antinociceptive drugs for the treatment of neuropathic pain is a major therapeutic problem. Here, we show the potential role of interleukin (IL)-18 signaling in this phenomenon. IL-18 is an important molecule that performs various crucial functions, including the alteration of nociceptive transmission in response to neuropathic pain. We have studied the changes in the mRNA and protein levels (qRT-PCR and Western blot analysis, respectively) of IL-18, IL-18-binding protein (IL-18BP) and the IL-18 receptor (IL-18R) over time in rats following chronic constriction injury (CCI) of the sciatic nerve. Our study demonstrated that the spinal levels of IL-18BP were slightly downregulated at days 7 and 14 in the rats subjected to CCI. In contrast, the IL-18 and IL-18R mRNA expression and protein levels were elevated in the ipsilateral spinal cord on days 2, 7 and 14. Moreover, in rats exposed to a single intrathecal administration of IL-18BP (50 and 100 ng) 7 or 14 days following CCI, symptoms of neuropathic pain were attenuated, and the analgesia pursuant to morphine and buprenorphine (0.5 and 2.5 µg) was enhanced. In summary, the restoration of the analgesic activity of morphine and buprenorphine via the blockade of IL-18 signaling suggests that increased IL-18 pathway may account for the decreased analgesic efficacy of opioids for neuropathic pain.

The role of IL-18 in type 1 diabetic nephropathy: The problem and future treatment.

Diabetic vascular complication is a leading cause of diabetic nephropathy, a progressive increase in urinary albumin excretion coupled with elevated blood pressure leading to declined glomerular filtration and eventually end stage renal failure. There is growing evidence that activated inflammation is contributing factor to the pathogenesis of diabetic nephropathy. Meanwhile, IL-18, a member of the IL-1 family of inflammatory cytokines, is involved in the development and progression of diabetic nephropathy. However, the benefits derived from the current therapeutics for diabetic nephropathy strategies still provide imperfect protection against renal progression. This imperfection points to the need for newer therapeutic agents that have potential to affect primary mechanisms contributing to the pathogenesis of diabetic nephropathy. Therefore, the recognition of IL-18 as significant pathogenic mediators in diabetic nephropathy leaves open the possibility of new potential therapeutic targets.

Flagellin induces antibody responses through a TLR5- and inflammasome-independent pathway.

Flagellin is a potent immunogen that activates the innate immune system via TLR5 and Naip5/6, and generates strong T and B cell responses. The adaptor protein MyD88 is critical for signaling by TLR5, as well as IL-1Rs and IL-18Rs, major downstream mediators of the Naip5/6 Nlrc4-inflammasome. In this study, we define roles of known flagellin receptors and MyD88 in Ab responses generated toward flagellin. We used mice genetically deficient in flagellin recognition pathways to characterize innate immune components that regulate isotype-specific Ab responses. Using purified flagellin from Salmonella, we dissected the contribution of innate flagellin recognition pathways to promote Ab responses toward flagellin and coadministered OVA in C57BL/6 mice. We demonstrate IgG2c responses toward flagellin were TLR5 and inflammasome dependent; IgG1 was the dominant isotype and partially TLR5 and inflammasome dependent. Our data indicate a substantial flagellin-specific IgG1 response was induced through a TLR5-, inflammasome-, and MyD88-independent pathway. IgA anti-FliC responses were TLR5 and MyD88 dependent and caspase-1 independent. Unlike C57BL/6 mice, flagellin-immunized A/J mice induced codominant IgG1 and IgG2a responses. Furthermore, MyD88-independent, flagellin-induced Ab responses were even more pronounced in A/J MyD88(-/-) mice, and IgA anti-FliC responses were suppressed by MyD88. Flagellin also worked as an adjuvant toward coadministered OVA, but it only promoted IgG1 anti-OVA responses. Our results demonstrate that a novel pathway for flagellin recognition contributes to Ab production. Characterization of this pathway will be useful for understanding immunity to flagellin and the rationale design of flagellin-based vaccines.

Increased Expression of Interleukin-18 in Lenses of Ovariectomized Rats.

Previous studies showed an increased prevalence of cataracts in postmenopausal women. In this study, we investigated changes in the levels of calcium ion (Ca(2+)) and interleukin (IL)-18, which are factors in cataract development, in the lenses of ovariectomized (OVX) rats, a model of postmenopausal woman. Although the Ca(2+) content in the blood of OVX rats increased 1 month after ovariectomy and subsequently decreased, the Ca(2+) content in the lenses was unchanged in OVX rats 1-3 months after ovariectomy. The Ca(2+)-ATPase activity in the lenses of OVX rats peaked 1 month after ovariectomy, and the behavior of Ca(2+)-ATPase activity in lenses of OVX rats was similar to that of the Ca(2+) concentration in the blood. It is possible that hypercalcemia increases the Ca(2+) inflow into the lens; however, the enhanced Ca(2+)-ATPase activity prevents the Ca(2+) level from rising. On the other hand, we found that the levels of both IL-18 and interferon (IFN)-γ in the lenses of OVX rats were significantly increased as compared with the lenses of sham (control) rats during the period 1-3 months after surgery. These results suggest that the expression of IFN-γ via IL-18 in the lenses of OVX rats is induced by ovariectomy, and that excessive IL-18 and IFN-γ production in the lenses may be related to cataract development in postmenopausal women. These findings support those of previous studies that assessed lens opacification in postmenopausal women.

Correlation of IL-18 with Tryptase in Atopic Asthma and Induction of Mast Cell Accumulation by IL-18.

Interleukin- (IL-) 18 and tryptase were previously reported to relate to asthma, but the correlation between these two potent proinflammatory molecules in asthma and their roles in mast cell accumulation remain uninvestigated. Using flow cytometric analysis technique and ovalbumin- (OVA-) sensitized mouse model, it was found that IL-18 and tryptase levels in the plasma of moderate and severe asthma were elevated, and they correlated well with each other. Tryptase and agonist peptides of protease activated receptor- (PAR-) 2 induced substantial quantity of IL-18 release. IL-18 and tryptase provoked mast cell accumulation in peritoneum of OVA-sensitized mice. OVA-sensitization increased number of IL-18 receptor (R)(+) mast cells. IL-18 and tryptase induced dramatic increase in IL-18R(+) mast cells and mean fluorescence intensity (MFI) of IL-18R on mast cells. Moreover, while IL-18 induced an increase in PAR-2(+) mast cells in nonsensitized mice, IL-18 and tryptase provoked increases in IL-4 and thymic stromal lymphopoietin (TSLP) in the peritoneum of OVA-sensitized mice. In summary, the correlation between IL-18 and tryptase in plasma of patients with asthma indicates close interactions between them, which should be considered for development of anti-IL-18 and antitryptase therapies. Interactions between IL-18 and tryptase may contribute to mast cell recruitment in asthma.

CD161++ CD8+ T cells, including the MAIT cell subset, are specifically activated by IL-12+IL-18 in a TCR-independent manner.

CD161(++) CD8(+) T cells represent a novel subset that is dominated in adult peripheral blood by mucosal-associated invariant T (MAIT) cells, as defined by the expression of a variable-α chain 7.2 (Vα7.2)-Jα33 TCR, and IL-18Rα. Stimulation with IL-18+IL-12 is known to induce IFN-γ by both NK cells and, to a more limited extent, T cells. Here, we show the CD161(++) CD8(+) T-cell population is the primary T-cell population triggered by this mechanism. Both CD161(++) Vα7.2(+) and CD161(++) Vα7.2(-) T-cell subsets responded to IL-12+IL-18 stimulation, demonstrating this response was not restricted to the MAIT cells, but to the CD161(++) phenotype. Bacteria and TLR agonists also indirectly triggered IFN-γ expression via IL-12 and IL-18. These data show that CD161(++) T cells are the predominant T-cell population that responds directly to IL-12+IL-18 stimulation. Furthermore, our findings broaden the potential role of MAIT cells beyond bacterial responsiveness to potentially include viral infections and other inflammatory stimuli.

Allergen-induced interleukin-18 promotes experimental eosinophilic oesophagitis in mice.

Elevated levels of interleukin (IL)-18 have been reported in a number of allergic diseases. We recently reported that IL-18 in the blood and IL-18Rα mRNA in the oesophagus are induced during human eosinophilic oesophagitis (EoE). Additionally, we earlier showed that invariant natural killer T (iNKT) cells are critical to EoE pathogenesis; however, the mechanism of iNKT cell activation in EoE is not well understood. Therefore, the current study focused on the hypothesis that allergen-induced IL-18 may have an important role in iNKT cell-mediated EoE pathogenesis. We first validated the human EoE findings of IL-18 in experimental EoE by examining blood levels of IL-18 and oesophageal IL-18Rα mRNA levels in aeroallergen- and food allergen-induced experimental mouse models of EoE. We demonstrate that blood IL-18 protein and oesophageal IL-18Rα mRNA are induced in the mouse model of EoE and that IL-18Rα is expressed by iNKT cells in the oesophagus. Intranasal delivery of rIL-18 induced both mast cells and eosinophilic inflammation in the oesophagus in a time- and dose-dependent manner. To establish the significance of IL-18 in EoE pathogenesis, we examined DOX-inducible rtTA-CC10-IL-18 bitransgenic mice that induce IL-18 protein expression in the oesophagus. Our analysis indicated that induction of IL-18 in these mice resulted in the development of many of the characteristics of EoE, including oesophageal intraepithelial eosinophilia, increased mast cells, oesophageal remodelling and fibrosis. The current study provides evidence that IL-18 may induce iNKT cell activation to release the eosinophil-activating cytokine IL-5, as IL-5-deficient mice and iNKT cell-deficient (CD1d null) mice do not induce EoE in response to intranasal IL-18 challenge. Taken together, these findings provide evidence that allergen-induced IL-18 has a significant role in promoting IL-5- and iNKT-dependent EoE pathogenesis.

Cord factor and peptidoglycan recapitulate the Th17-promoting adjuvant activity of mycobacteria through mincle/CARD9 signaling and the inflammasome.

Although adjuvants are critical vaccine components, their modes of action are poorly understood. In this study, we investigated the mechanisms by which the heat-killed mycobacteria in CFA promote Th17 CD4(+) T cell responses. We found that IL-17 secretion by CD4(+) T cells following CFA immunization requires MyD88 and IL-1ß/IL-1R signaling. Through measurement of Ag-specific responses after adoptive transfer of OTII cells, we confirmed that MyD88-dependent signaling controls Th17 differentiation rather than simply production of IL-17. Additional experiments showed that CFA-induced Th17 differentiation involves IL-1ß processing by the inflammasome, as mice lacking caspase-1, ASC, or NLRP3 exhibit partially defective responses after immunization. Biochemical fractionation studies further revealed that peptidoglycan is the major component of heat-killed mycobacteria responsible for inflammasome activation. By assaying Il1b transcripts in the injection site skin of CFA-immunized mice, we found that signaling through the adaptor molecule caspase activation and recruitment domain 9 (CARD9) plays a major role in triggering pro-IL-1ß expression. Moreover, we demonstrated that recognition of the mycobacterial glycolipid trehalose dimycolate (cord factor) by the C-type lectin receptor mincle partially explains this CARD9 requirement. Importantly, purified peptidoglycan and cord factor administered in mineral oil synergized to recapitulate the Th17-promoting activity of CFA, and, as expected, this response was diminished in caspase-1- and CARD9-deficient mice. Taken together, these findings suggest a general strategy for the rational design of Th17-skewing adjuvants by combining agonists of the CARD9 pathway with inflammasome activators.

Pressure overload induces IL-18 and IL-18R expression, but markedly suppresses IL-18BP expression in a rabbit model. IL-18 potentiates TNF-α-induced cardiomyocyte death.

Recurrent or sustained inflammation plays a causal role in the development and progression of left ventricular hypertrophy (LVH) and its transition to failure. Interleukin (IL)-18 is a potent pro-hypertrophic inflammatory cytokine. We report that induction of pressure overload in the rabbit, by constriction of the descending thoracic aorta induces compensatory hypertrophy at 4weeks (mass/volume ratio: 1.7±0.11) and ventricular dilatation indicative of heart failure at 6weeks (mass/volume ratio: 0.7±0.04). In concordance with this, fractional shortening was preserved at 4weeks, but markedly attenuated at 6weeks. We cloned rabbit IL-18, IL-18Rα, IL-18Rß, and IL-18 binding protein (IL-18BP) cDNA, and show that pressure overload, while enhancing IL-18 and IL-18R expression in hypertrophied and failing hearts, markedly attenuated the level of expression of the endogenous IL-18 antagonist IL-18BP. Cyclical mechanical stretch (10% cyclic equibiaxial stretch, 1Hz) induced hypertrophy of primary rabbit cardiomyocytes in vitro and enhanced ANP, IL-18, and IL-18Rα expression. Further, treatment with rhIL-18 induced its own expression and that of IL-18Rα via AP-1 activation, and induced cardiomyocyte hypertrophy in part via PI3K/Akt/GATA4 signaling. In contrast, IL-18 potentiated TNF-α-induced cardiomyocyte death, and by itself induced cardiac endothelial cell death. These results demonstrate that pressure overload is associated with enhanced IL-18 and its receptor expression in hypertrophied and failingrabbit hearts. Since IL-18BP expression is markedly inhibited, our results indicate a positive amplification in IL-18 proinflammatory signaling during pressure overload, and suggest IL-18 as a potential therapeutic target in pathological hypertrophy and cardiac failure.

Cytokine activation induces human memory-like NK cells.

Natural killer (NK) cells are lymphocytes that play an important role in the immune response to infection and malignancy. Recent studies in mice have shown that stimulation of NK cells with cytokines or in the context of a viral infection results in memory-like properties. We hypothesized that human NK cells exhibit such memory-like properties with an enhanced recall response after cytokine preactivation. In the present study, we show that human NK cells preactivated briefly with cytokine combinations including IL-12, IL-15, and IL-18 followed by a 7- to 21-day rest have enhanced IFN-γ production after restimulation with IL-12 + IL-15, IL-12 + IL-18, or K562 leukemia cells. This memory-like phenotype was retained in proliferating NK cells. In CD56(dim) NK cells, the memory-like IFN-γ response was correlated with the expression of CD94, NKG2A, NKG2C, and CD69 and a lack of CD57 and KIR. Therefore, human NK cells have functional memory-like properties after cytokine activation, which provides a novel rationale for integrating preactivation with combinations of IL-12, IL-15, and IL-18 into NK cell immunotherapy strategies.

Up-regulation of interleukin-23 induces persistent allodynia via CX3CL1 and interleukin-18 signaling in the rat spinal cord after tetanic sciatic stimulation.

Tetanic stimulation of the sciatic nerve (TSS) induces sciatic nerve injury and long-lasting pain hypersensitivity in rats, and spinal glial activation and proinflammatory cytokines releases are involved. In the present study, we showed that spinal interleukin (IL)-23 and its receptor, IL-23R, are crucial for the development of mechanical allodynia after TSS. In the spinal dorsal horn, both IL-23 and IL-23R are expressed in astrocytes, and this expression is substantially increased after TSS. Inhibition of IL-23 signaling attenuated TSS-induced allodynia and decreased the induction of glial fibrillary acidic protein (GFAP, an astrocytic marker). Conversely, intrathecally delivered IL-23 induced a persistent allodynia. Similar to IL-23 signaling, an increase in IL-18 and its receptor, IL-18R, as well as CX3CL1 and its receptor, CX3CR1, was simultaneously observed in the spinal dorsal horn after TSS. Interestingly, IL-18 and CX3CR1 were exclusively expressed in microglia, while IL-18R was mainly localized in astrocytes. In contrast, CX3CL1 was predominately expressed in neurons and secondarily in astrocytes. The functional inhibition of CX3CL1 and IL-18 signaling attenuated TSS-induced allodynia and suppressed IL-23 and IL-23R upregulation. Activation of CX3CR1 and IL-18R induced similar behavioral and biochemical changes to those observed after TSS. These results indicate that the interaction among CX3CL1, IL-18 and IL-23 signaling in the spinal cord plays a critical role in the development of allodynia. Thus, interrupting this chemokine-cytokine network might provide a novel therapeutic strategy for neuropathic pain.