Eli C. Lewis

Deficiency of interleukin-18 in mice leads to hyperphagia, obesity and insulin resistance

Here we report the presence of hyperphagia, obesity and insulin resistance in knockout mice deficient in IL-18 or IL-18 receptor, and in mice transgenic for expression of IL-18 binding protein. Obesity of Il18−/− mice resulted from accumulation of fat tissue based on increased food intake. Il18−/− mice also had hyperinsulinemia, consistent with insulin resistance and hyperglycemia. Insulin resistance was secondary to obesity induced by increased food intake and occurred at the liver level as well as at the muscle and fat-tissue level. The molecular mechanisms responsible for the hepatic insulin resistance in the Il18−/− mice involved an enhanced expression of genes associated with gluconeogenesis in the liver of Il18−/− mice, resulting from defective phosphorylation of STAT3. Recombinant IL-18 (rIL-18) administered intracerebrally inhibited food intake. In addition, rIL-18 reversed hyperglycemia in Il18−/− mice through activation of STAT3 phosphorylation. These findings indicate a new role of IL-18 in the homeostasis of energy intake and insulin sensitivity.

Cisplatin-induced acute renal failure is associated with an increase in the cytokines interleukin (IL)-1beta, IL-18, IL-6, and neutrophil infiltration in the kidney.

We have demonstrated that caspase-1-deficient (caspase-1–/–) mice are functionally and histologically protected against cisplatin-induced acute renal failure (ARF). Caspase-1 exerts proinflammatory effects via the cytokines interleukin (IL)-1β, IL-18, IL-6, and neutrophil recruitment. We sought to determine the role of the cytokines IL-1β, IL-18, and IL-6 and neutrophil recruitment in cisplatin-induced ARF. We first examined IL-1β; renal IL-1β increased nearly 2-fold in cisplatin-induced ARF and was reduced in the caspase-1–/– mice. However, inhibition with IL-1 receptor antagonist (IL-1Ra) did not attenuate cisplatin-induced ARF. Renal IL-18 increased 2.5-fold; however, methods to inhibit IL-18 using IL-18 antiserum and transgenic mice that overproduce IL-18-binding protein (a natural inhibitor of IL-18) did not protect. Renal IL-6 increased 3-fold; however, IL-6-deficient (IL-6–/–) mice still developed cisplatin-induced ARF. We next examined neutrophils; blood neutrophils increased dramatically after cisplatin injection; however, prevention of peripheral neutrophilia and renal neutrophil infiltration with the neutrophil-depleting antibody RB6-8C5 did not protect against cisplatin-induced ARF. In summary, our data demonstrated that cisplatin-induced ARF is associated with increases in the cytokines IL-1β, IL-18, and IL-6 and neutrophil infiltration in the kidney. However, inhibition of IL-1β, IL-18, and IL-6 or neutrophil infiltration in the kidney is not sufficient to prevent cisplatin-induced ARF.

IL-1 family nomenclature

To the Editor: Newly 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. IL-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.

Mycobacterium tuberculosis Induces Interleukin-32 Production through a Caspase- 1/IL-18/Interferon-γ-Dependent Mechanism

Background Interleukin (IL)–32 is a newly described proinflammatory cytokine that seems likely to play a role in inflammation and host defense. Little is known about the regulation of IL-32 production by primary cells of the immune system. Methods and Findings In the present study, freshly obtained human peripheral blood mononuclear cells were stimulated with different Toll-like receptor (TLR) agonists, and gene expression and synthesis of IL-32 was determined. We demonstrate that the TLR4 agonist lipopolysaccharide induces moderate (4-fold) production of IL-32, whereas agonists of TLR2, TLR3, TLR5, or TLR9, each of which strongly induced tumor necrosis factor α and IL-6, did not stimulate IL-32 production. However, the greatest amount of IL-32 was induced by the mycobacteria Mycobacterium tuberculosis and M. bovis BCG (20-fold over unstimulated cells). IL-32-induced synthesis by either lipopolysaccharide or mycobacteria remains entirely cell-associated in monocytes; moreover, steady-state mRNA levels are present in unstimulated monocytes without translation into IL-32 protein, similar to other cytokines lacking a signal peptide. IL-32 production induced by M. tuberculosis is dependent on endogenous interferon-γ (IFNγ); endogenous IFNγ is, in turn, dependent on M. tuberculosis–induced IL-18 via caspase-1. Conclusions In conclusion, IL-32 is a cell-associated proinflammatory cytokine, which is specifically stimulated by mycobacteria through a caspase-1- and IL-18-dependent production of IFNγ.

α1-Antitrypsin monotherapy induces immune tolerance during islet allograft transplantation in mice

Human pancreatic islet transplantation offers diabetic patients tight glucose control but has low graft survival rates. The immunosuppressive drugs that are administered to graft recipients lack the antiinflammatory benefits of corticosteroids because of their diabetogenic effects. The serum protease inhibitor α1-antitrypsin (AAT) possesses antiinflammatory properties and reduces cytokine-mediated islet damage. In the present study, diabetic mice were grafted with allogeneic islets and treated with AAT monotherapy (n = 24). After 14 days of treatment, mice remained normoglycemic and islet allografts were functional for up to 120 treatment-free days. After graft removal and retransplantation, mice accepted same-strain islets but rejected third-strain islets, thus confirming that specific immune tolerance had been induced. Explanted grafts exhibited a population of T regulatory cells in transplant sites. According to RT-PCR, grafts contained high levels of mRNA for foxp3, cytotoxic T lymphocyte antigen-4, TGF-β, IL-10, and IL-1 receptor antagonist; expression of proinflammatory mediators was low or absent. After implantation of skin allografts, AAT-treated mice had greater numbers of foxp3-positive cells in draining lymph nodes (DLNs) compared with control treatment mice. Moreover, dendritic cells in DLNs exhibited an immature phenotype with decreased CD86 activation marker. Although the number of CD3 transcripts decreased in the DLNs, AAT did not affect IL-2 activity in vitro. Thus, AAT monotherapy provides allografts with antiinflammatory conditions that favor development of antigen-specific T regulatory cells. Because AAT treatment in humans is safe, its use during human islet transplantation may be considered.

Interleukin-32 induces the differentiation of monocytes into macrophage-like cells

After emigration from the bone marrow to the peripheral blood, monocytes enter tissues and differentiate into macrophages, the prototype scavenger of the immune system. By ingesting and killing microorganisms and removing cellular debris, macrophages also process antigens as a first step in mounting a specific immune response. IL-32 is a cytokine inducing proinflammatory cytokines and chemokines via p38-MAPK and NF-κB. In the present study, we demonstrate that IL-32 induces differentiation of human blood monocytes as well as THP-1 leukemic cells into macrophage-like cells with functional phagocytic activity for live bacteria. Muramyl dipepide (MDP), the ligand for the intracellular nuclear oligomerization domain (NOD) 2 receptor, has no effect on differentiation alone but augments the monocyte-to-macrophage differentiation by IL-32. Unexpectedly, IL-32 reversed GM-CSF/IL-4-induced dendritic cell differentiation to macrophage-like cells. Whereas the induction of TNFα, IL-1β, and IL-6 by IL-32 is mediated by p38-MAPK, IL-32-induced monocyte-to-macrophage differentiation is mediated through nonapoptotic, caspase-3-dependent mechanisms. Thus, IL-32 not only contributes to host responses through the induction of proinflammatory cytokines but also directly affects specific immunity by differentiating monocytes into macrophage-like cells.

Anti-inflammatory and immunomodulatory properties of α1-antitrypsin without inhibition of elastase

The rationale of α1-antitrypsin (AAT) augmentation therapy to treat progressive emphysema in AAT-deficient patients is based on inhibition of neutrophil elastase; however, the benefit of this treatment remains unclear. Here we show that clinical grade AAT (with elastase inhibitory activity) and a recombinant form of AAT (rAAT) without anti-elastase activity reduces lung inflammatory responses to LPS in elastase-deficient mice. WT and elastase-deficient mice treated with either native AAT or rAAT exhibited significant reductions in infiltrating neutrophils (23% and 68%), lavage fluid levels of TNF-α (70% and 80%), and the neutrophil chemokine KC (CXCL1) (64% and 90%), respectively. Lung parenchyma TNF-α, DNA damage-inducible transcript 3 and X-box binding protein-1 mRNA levels were reduced in both mouse strains treated with AAT; significantly lower levels of these genes, as well as IL-1β gene expression, were observed in lungs of AAT-deficient patients treated with AAT therapy compared with untreated patients. In vitro, LPS-induced cytokines from WT and elastase-deficient mouse neutrophils, as well as neutrophils of healthy humans, were similarly reduced by AAT or rAAT; human neutrophils adhering to endothelial cells were decreased by 60–80% (P < 0.001) with either AAT or rAAT. In mouse pancreatic islet macrophages, LPS-induced surface expression of MHC II, Toll-like receptor-2 and -4 were markedly lower (80%, P < 0.001) when exposed to either AAT or rAAT. Consistently, in vivo and in vitro, rAAT reduced inflammatory responses at concentrations 40- to 100-fold lower than native plasma-derived AAT. These data provide evidence that the anti-inflammatory and immunomodulatory properties of AAT can be independent of elastase inhibition.

The unique hypusine modification of eIF5A promotes islet β cell inflammation and dysfunction in mice

In both type 1 and type 2 diabetes, pancreatic islet dysfunction results in part from cytokine-mediated inflammation. The ubiquitous eukaryotic translation initiation factor 5A (eIF5A), which is the only protein to contain the amino acid hypusine, contributes to the production of proinflammatory cytokines. We therefore investigated whether eIF5A participates in the inflammatory cascade leading to islet dysfunction during the development of diabetes. As described herein, we found that eIF5A regulates iNOS levels and that eIF5A depletion as well as the inhibition of hypusination protects against glucose intolerance in inflammatory mouse models of diabetes. We observed that following knockdown of eIF5A expression, mice were resistant to beta cell loss and the development of hyperglycemia in the low-dose streptozotocin model of diabetes. The depletion of eIF5A led to impaired translation of iNOS-encoding mRNA within the islet. A role for the hypusine residue of eIF5A in islet inflammatory responses was suggested by the observation that inhibition of hypusine synthesis reduced translation of iNOS-encoding mRNA in rodent beta cells and human islets and protected mice against the development of glucose intolerance the low-dose streptozotocin model of diabetes. Further analysis revealed that hypusine is required in part for nuclear export of iNOS-encoding mRNA, a process that involved the export protein exportin1. These observations identify the hypusine modification of eIF5A as a potential therapeutic target for preserving islet function under inflammatory conditions.

Interleukin-1β regulates fat-liver crosstalk in obesity by auto-paracrine modulation of adipose tissue inflammation and expandability.

The inflammasome has been recently implicated in obesity-associated dys-metabolism. However, of its products, the specific role of IL-1β was clinically demonstrated to mediate only the pancreatic beta-cell demise, and in mice mainly the intra-hepatic manifestations of obesity. Yet, it remains largely unknown if IL-1β, a cytokine believed to mainly function locally, could regulate dysfunctional inter-organ crosstalk in obesity. Here we show that High-fat-fed (HFF) mice exhibited a preferential increase of IL-1β in portal compared to systemic blood. Moreover, portally-drained mesenteric fat transplantation from IL-1βKO donors resulted in lower pyruvate-glucose flux compared to mice receiving wild-type (WT) transplant. These results raised a putative endocrine function for visceral fat-derived IL-1β in regulating hepatic gluconeogenic flux. IL-1βKO mice on HFF exhibited only a minor or no increase in adipose expression of pro-inflammatory genes (including macrophage M1 markers), Mac2-positive crown-like structures and CD11b-F4/80-double-positive macrophages, all of which were markedly increased in WT-HFF mice. Further consistent with autocrine/paracrine functions of IL-1β within adipose tissue, adipose tissue macrophage lipid content was increased in WT-HFF mice, but significantly less in IL-1βKO mice. Ex-vivo, adipose explants co-cultured with primary hepatocytes from WT or IL-1-receptor (IL-1RI)-KO mice suggested only a minor direct effect of adipose-derived IL-1β on hepatocyte insulin resistance. Importantly, although IL-1βKOs gained weight similarly to WT-HFF, they had larger fat depots with similar degree of adipocyte hypertrophy. Furthermore, adipogenesis genes and markers (pparg, cepba, fabp4, glut4) that were decreased by HFF in WT, were paradoxically elevated in IL-1βKO-HFF mice. These local alterations in adipose tissue inflammation and expansion correlated with a lower liver size, less hepatic steatosis, and preserved insulin sensitivity. Collectively, we demonstrate that by promoting adipose inflammation and limiting fat tissue expandability, IL-1β supports ectopic fat accumulation in hepatocytes and adipose-tissue macrophages, contributing to impaired fat-liver crosstalk in nutritional obesity.

IL-37 protects against obesity-induced inflammation and insulin resistance

Cytokines of the IL-1 family are important modulators of obesity-induced inflammation and the development of systemic insulin resistance. Here we show that IL-1 family member IL-37, recently characterized as an anti-inflammatory cytokine, ameliorates obesity-induced inflammation and insulin resistance. Mice transgenic for human IL-37 (IL-37tg) exhibit reduced numbers of adipose tissue macrophages, increased circulating levels of adiponectin and preserved glucose tolerance and insulin sensitivity after 16 weeks of HFD. In vitro treatment of adipocytes with recombinant IL-37 reduces adipogenesis and activates AMPK signalling. In humans, elevated steady-state IL-37 adipose tissue mRNA levels are positively correlated with insulin sensitivity and a lower inflammatory status of the adipose tissue. These findings reveal IL-37 as an important anti-inflammatory modulator during obesity-induced inflammation and insulin resistance in both mice and humans, and suggest that IL-37 is a potential target for the treatment of obesity-induced insulin resistance and type 2 diabetes.