Foo Y. Liew

Negative regulation of Toll-like receptor-mediated immune responses

Toll-like receptors (TLRs) are involved in host defence against invading pathogens, functioning as primary sensors of microbial products and activating signalling pathways that induce the expression of immune and pro-inflammatory genes. However, TLRs have also been implicated in several immune-mediated and inflammatory diseases. As the immune system needs to constantly strike a balance between activation and inhibition to avoid detrimental and inappropriate inflammatory responses, TLR signalling must be tightly regulated. Here, we discuss the various negative regulatory mechanisms that have evolved to attenuate TLR signalling to maintain this immunological balance.

Disease-associated functions of IL-33: the new kid in the IL-1 family

Interleukin-33 (IL-33), a newly described member of the IL-1 family, is expressed by many cell types following pro-inflammatory stimulation and is thought to be released on cell lysis. The IL-33 receptor, consisting of ST2 and IL-1 receptor accessory protein, is also widely expressed, particularly by T helper 2 (TH2) cells and mast cells. IL-33 is host-protective against helminth infection and reduces atherosclerosis by promoting TH2-type immune responses. However, IL-33 can also promote the pathogenesis of asthma by expanding TH2 cells and mediate joint inflammation, atopic dermatitis and anaphylaxis by mast cell activation. Thus IL-33 could be a new target for therapeutic intervention across a range of diseases.

A proinflammatory role for IL-18 in rheumatoid arthritis

IL-18 is a novel cytokine with pleiotropic activities critical to the development of T-helper 1 (Th1) responses. We detected IL-18 mRNA and protein within rheumatoid arthritis (RA) synovial tissues in significantly higher levels than in osteoarthritis controls. Similarly, IL-18 receptor expression was detected on synovial lymphocytes and macrophages. Together with IL-12 or IL-15, IL-18 induced significant IFN-gamma production by synovial tissues in vitro. IL-18 independently promoted GM-CSF and nitric oxide production, and it induced significant TNF-alpha synthesis by CD14(+) macrophages in synovial cultures; the latter effect was potentiated by IL-12 or IL-15. TNF-alpha and IFN-gamma synthesis was suppressed by IL-10 and TGF-beta. IL-18 production in primary synovial cultures and purified synovial fibroblasts was, in turn, upregulated by TNF-alpha and IL-1beta, suggesting that monokine expression can feed back to promote Th1 cell development in synovial membrane. Finally, IL-18 administration to collagen/incomplete Freunds adjuvant-immunized DBA/1 mice facilitated the development of an erosive, inflammatory arthritis, suggesting that IL-18 can be proinflammatory in vivo. Together, these data indicate that synergistic combinations of IL-18, IL-12, and IL-15 may be of importance in sustaining both Th1 responses and monokine production in RA.

Coordinated and distinct roles for IFN-alpha beta, IL-12, and IL-15 regulation of NK cell responses to viral infection

NK cell cytotoxicity, IFN-γ expression, proliferation, and accumulation are rapidly induced after murine CMV infections. Under these conditions, the responses were shown to be elicited in overlapping populations. Nevertheless, there were distinct signaling molecule requirements for induction of functions within the subsets. IL-12/STAT4 was critical for NK cell IFN-γ expression, whereas IFN-αβ/STAT1 were required for induction of cytotoxicity. The accumulation/survival of proliferating NK cells was STAT4-independent but required IFN-αβ/STAT1 induction of IL-15. Taken together, the results define the coordinated interactions between the cytokines IFN-αβ, IL-12, and IL-15 for activation of protective NK cell responses during viral infections, and emphasize these factors’ nonredundant functions under in vivo physiological conditions.

ST2 is an inhibitor of interleukin 1 receptor and Toll-like receptor 4 signaling and maintains endotoxin tolerance

The Toll–interleukin 1 receptor (TIR) superfamily, defined by the presence of an intracellular TIR domain, initiates innate immunity through activation of the transcription factor NF-κB, leading to the production of proinflammatory cytokines. ST2 is a member of the TIR family that does not activate NF-κB and has been suggested as an important effector molecule of T helper type 2 (TH2) responses. We show here that the membrane-bound form of ST2 negatively regulated type I interleukin 1 receptor (IL-1RI) and Toll-like receptor 4 (TLR4) but not TLR3 signaling by sequestrating the adaptors MyD88 and Mal. In contrast to wild-type mice, ST2-deficient mice failed to develop endotoxin tolerance. Thus, these results provide a molecular explanation for the function of ST2 in TH2 responses, as inhibition of TLRs promotes a TH2 response, and also identify ST2 as a key regulator of endotoxin tolerance.

IL-33 reduces the development of atherosclerosis

Atherosclerosis is a chronic inflammatory disease of the vasculature commonly leading to myocardial infarction and stroke. We show that IL-33, which is a novel IL-1–like cytokine that signals via ST2, can reduce atherosclerosis development in ApoE−/− mice on a high-fat diet. IL-33 and ST2 are present in the normal and atherosclerotic vasculature of mice and humans. Although control PBS-treated mice developed severe and inflamed atherosclerotic plaques in the aortic sinus, lesion development was profoundly reduced in IL-33–treated animals. IL-33 also markedly increased levels of IL-4, -5, and -13, but decreased levels of IFNγ in serum and lymph node cells. IL-33 treatment also elevated levels of total serum IgA, IgE, and IgG1, but decreased IgG2a, which is consistent with a Th1-to-Th2 switch. IL-33–treated mice also produced significantly elevated antioxidized low-density lipoprotein (ox-LDL) antibodies. Conversely, mice treated with soluble ST2, a decoy receptor that neutralizes IL-33, developed significantly larger atherosclerotic plaques in the aortic sinus of the ApoE−/− mice compared with control IgG-treated mice. Furthermore, coadministration of an anti–IL-5 mAb with IL-33 prevented the reduction in plaque size and reduced the amount of ox-LDL antibodies induced by IL-33. In conclusion, IL-33 may play a protective role in the development of atherosclerosis via the induction of IL-5 and ox-LDL antibodies.

IL-33 Amplifies the Polarization of Alternatively Activated Macrophages That Contribute to Airway Inflammation

Alternatively activated macrophages (AAM) play a crucial role in type 2 immunity. Mice deficient in ST2, a receptor for the latest member of the IL-1 family, IL-33, have impaired type 2 immune responses. We therefore reasoned that IL-33/ST2 signaling may be involved in the differentiation and activation of AAM during airway inflammation. We report here that IL-33 changed the quiescent phenotype of alveolar macrophages toward an AAM phenotype that expressed mannose receptor, IL-4Rα, and produced high levels of CCL24 and CCL17 in an IL-13-dependent manner during IL-33-induced airway inflammation. Neutralization of AAM-derived CCL24 led to an amelioration of IL-33-induced eosinophilia in the lungs. Moreover, depletion of alveolar macrophages reduced IL-33-induced airway inflammation. Additionally, the attenuated OVA-induced airway inflammation in ST2−/− mice was associated with a decrease in AAM differentiation. In vitro, IL-33 amplified IL-13-induced polarization of alveolar- and bone marrow-derived macrophage toward an AAM phenotype by increasing the expression of arginase I, Ym1, as well as the production of CCL24 and CCL17. IL-13/IL-4Rα signaling was crucial for IL-33-driven AAM amplification by inducing the expression of ST2L. Finally, we showed that IL-33 was more abundantly expressed in the lung epithelial cells of asthma patients than those from healthy controls, suggesting that IL-33 may be involved in lung macrophage activation in clinical asthma. Taken together, we demonstrate here that IL-33/ST2 plays a significant role in the amplification of AAM polarization and chemokine production which contribute to innate and Ag-induced airway inflammation.

A Novel Anti-Inflammatory Role for Simvastatin in Inflammatory Arthritis

3-Hydroxy-3-methylglutaryl-CoA reductase inhibitors (statins) exert favorable effects on lipoprotein metabolism, but may also possess anti-inflammatory properties. Therefore, we explored the activities of simvastatin, a lipophilic statin, in a Th1-driven model of murine inflammatory arthritis. We report in this study that simvastatin markedly inhibited not only developing but also clinically evident collagen-induced arthritis in doses that were unable to significantly alter cholesterol concentrations in vivo. Ex vivo analysis demonstrated significant suppression of collagen-specific Th1 humoral and cellular immune responses. Moreover, simvastatin reduced anti-CD3/anti-CD28 proliferation and IFN-γ release from mononuclear cells derived from peripheral blood and synovial fluid. Proinflammatory cytokine production in vitro by T cell contact-activated macrophages was suppressed by simvastatin, suggesting that such observations have direct clinical relevance. These data clearly illustrate the therapeutic potential of statin-sensitive pathways in inflammatory arthritis.

IL-35 is a novel cytokine with therapeutic effects against collagen-induced arthritis through the expansion of regulatory T cells and suppression of Th17 cells

Epstein‐Barr virus‐induced gene 3 (EBI3) and the p35 subunit of IL‐12 have been reported to form a heterodimeric hematopoietin in human and mouse. We have constructed a heterodimeric protein covalently linking EBI3 and p35, to form a novel cytokine which we now call IL‐35. The Fc fusion protein of IL‐35 induced proliferation of murine CD4+CD25+ and CD4+CD25– T cells when stimulated with immobilized anti‐CD3 and anti‐CD28 antibodies in vitro. The IL‐35‐expanded CD4+CD25+ T cell population expressed Foxp3 and produced elevated levels of IL‐10, whereas the IL‐35‐induced CD4+CD25– T cells produced IFN‐γ but not IL‐4. The in vitro expanded CD4+CD25+ T cells retained their suppressive functions against CD4+CD25– effector cells. Furthermore, when cultured with soluble anti‐CD3 antibody and antigen‐presenting cells, IL‐35 suppressed the proliferation of CD4+CD25– effector cells. Moreover, IL‐35 inhibited the differentiation of Th17 cells in vitro. In vivo, IL‐35 effectively attenuated established collagen‐induced arthritis in mice, with concomitant suppression of IL‐17 production but enhanced IFN‐γ synthesis. Thus, IL‐35 is a novel anti‐inflammatory cytokine suppressing the immune response through the expansion of regulatory T cells and suppression of Th17 cell development.

Feedback inhibition of nitric oxide synthase activity by nitric oxide

1 A murine macrophage cell line, J774, expressed nitric oxide (NO) synthase activity in response to interferon‐gamma (IFN‐γ, 10 u ml−1) plus lipopolysaccharide (LPS, 10 ng ml−1). The enzyme activity was first detectable 6 h after incubation, peaked at 12 h and became undetectable after 48 h. 2 The decline in the NO synthase activity was not due to inhibition by stable substances secreted by the cells into the culture supernatant. 3 The decline in the NO synthase activity was significantly slowed down in cells cultured in a low l‐arginine medium or with added haemoglobin, suggesting that NO may be involved in a feedback inhibitory mechanism. 4 The addition of NO generators, S‐nitroso‐acetyl‐penicillamine (SNAP) or S‐nitroso‐glutathione (GSNO) markedly inhibited the NO synthase activity in a dose‐dependent manner. The effect of NO on the enzyme was not due to the inhibition of de novo protein synthesis. 5 SNAP directly inhibited the inducible NO synthase extracted from activated J774 cells, as well as the constitutive NO synthase extracted from the rat brain. 6 The enzyme activity of J774 cells was not restored after the removal of SNAP by gel filtration, suggesting that NO inhibits NO synthase irreversibly.