Reiko Horai

IL-17 production from activated T cells is required for the spontaneous development of destructive arthritis in mice deficient in IL-1 receptor antagonist

IL-17 is a T cell-derived, proinflammatory cytokine that is suspected to be involved in the development of various inflammatory diseases. Although there are elevated levels of IL-17 in synovial fluid of patients with rheumatoid arthritis, the pathogenic role of IL-17 in the development of rheumatoid arthritis remains to be elucidated. In this report, the effects of IL-17 deficiency were examined in IL-1 receptor antagonist-deficient (IL-1Ra−/−) mice that spontaneously develop an inflammatory and destructive arthritis due to unopposed excess IL-1 signaling. IL-17 expression is greatly enhanced in IL-1Ra−/− mice, suggesting that IL-17 activity is involved in the pathogenesis of arthritis in these mice. Indeed, the spontaneous development of arthritis did not occur in IL-1Ra−/− mice also deficient in IL-17. The proliferative response of ovalbumin-specific T cells from DO11.10 mice against ovalbumin cocultured with antigen-presenting cells from either IL-1Ra−/− mice or wild-type mice was reduced by IL-17 deficiency, indicating insufficient T cell activation. Cross-linking OX40, a cosignaling molecule on CD4+ T cells that plays an important role in T cell antigen-presenting cell interaction, with anti-OX40 Ab accelerated the production of IL-17 induced by CD3 stimulation. Because OX40 is induced by IL-1 signaling, IL-17 induction is likely to be downstream of IL-1 through activation of OX40. These observations suggest that IL-17 plays a crucial role in T cell activation, downstream of IL-1, causing the development of autoimmune arthritis.

Caspase 1-independent IL-1β release and inflammation induced by the apoptosis inducer Fas ligand

Fas ligand is a well-characterized apoptosis inducer. Here we demonstrate that Fas ligand induces the processing and secretion of interleukin-1β (IL-1β) in peritoneal exudate cells. This IL-1β secretion is independent of IL-1β converting enzyme (caspase 1), yet it is inhibited by caspase inhibitors, indicating that a caspase(s) in addition to IL-1β converting enzyme can process IL-1β. Inoculation of tumor cells expressing Fas ligand into wild-type mice induces a massive neutrophil infiltration that is, in contrast, suppressed in IL-1α/β knockout mice. These results demonstrate a newly discovered role for Fas ligand in inflammation, and challenge the dogma that apoptosis does not induce inflammation.

Cutting Edge: NKT Cells Constitutively Express IL-23 Receptor and RORγt and Rapidly Produce IL-17 upon Receptor Ligation in an IL-6-Independent Fashion

Th17 cells require IL-6 and TGFβ for lineage commitment and IL-23 for maintenance. Unexpectedly, naive IL-6−/− splenocytes stimulated with anti-CD3 and IL-23 produced normal amounts of IL-17 during the first 24 h of culture. These rapid IL-6-independent IL-17 producers were identified as predominantly DX5+ TCRβ+ NKT cells, and a comparable response could be found using the invariant NKT-specific ligand α-galactosylceramide. Human NKT cells also produced IL-17. NKT cells constitutively expressed IL-23R and RORγt. Ligation of either TCR or IL-23R triggered IL-17 production and both together had a synergistic effect, suggesting independent but convergent pathways. IL-17 production was not restricted to a particular subset of NKT cells but they were NK1.1 negative. Importantly, in vivo administration of α-galactosylceramide triggered a rapid IL-17 response in the spleen. These data suggest an important biological role for innate IL-17 production by NKT cells that is rapid and precedes the adaptive IL-17 response.

Six1 controls patterning of the mouse otic vesicle

Six1 is a member of the Six family homeobox genes, which function as components of the Pax-Six-Eya-Dach gene network to control organ development. Six1 is expressed in otic vesicles, nasal epithelia, branchial arches/pouches, nephrogenic cords, somites and a limited set of ganglia. In this study, we established Six1-deficient mice and found that development of the inner ear, nose, thymus, kidney and skeletal muscle was severely affected. Six1-deficient embryos were devoid of inner ear structures, including cochlea and vestibule, while their endolymphatic sac was enlarged. The inner ear anomaly began at around E10.5 and Six1 was expressed in the ventral region of the otic vesicle in the wild-type embryos at this stage. In the otic vesicle of Six1-deficient embryos, expressions of Otx1, Otx2, Lfng and Fgf3, which were expressed ventrally in the wild-type otic vesicles, were abolished, while the expression domains of Dlx5, Hmx3, Dach1 and Dach2, which were expressed dorsally in the wild-type otic vesicles, expanded ventrally. Our results indicate that Six1 functions as a key regulator of otic vesicle patterning at early embryogenesis and controls the expression domains of downstream otic genes responsible for respective inner ear structures. In addition, cell proliferation was reduced and apoptotic cell death was enhanced in the ventral region of the otic vesicle, suggesting the involvement of Six1 in cell proliferation and survival. In spite of the similarity of otic phenotypes of Six1- and Shh-deficient mice, expressions of Six1 and Shh were mutually independent.

IL-1 plays an important role in lipid metabolism by regulating insulin levels under physiological conditions.

IL-1 is a proinflammatory cytokine that plays important roles in inflammation. However, the role of this cytokine under physiological conditions is not known completely. In this paper, we analyzed the role of IL-1 in maintaining body weight because IL-1 receptor antagonist–deficient (IL-1Ra−/−) mice, in which excess IL-1 signaling may be induced, show a lean phenotype. Body fat accumulation was impaired in IL-1Ra−/− mice, but feeding behavior, expression of hypothalamic factors involved in feeding control, energy expenditure, and heat production were normal. When IL-1Ra−/− mice were treated with monosodium glutamate (MSG), which causes obesity in wild-type mice by ablating cells in the hypothalamic arcuate nucleus, they were resistant to obesity, indicating that excess IL-1 signaling antagonizes the effect of MSG-sensitive neuron deficiency. IL-1Ra−/− mice showed decreased weight gain when they were fed the same amount of food as wild-type mice, and lipid accumulation remained impaired even when they were fed a high-fat diet. Interestingly, serum insulin levels and lipase activity were low in IL-1Ra−/− mice, and the insulin levels were low in contrast to wild-type mice after MSG treatment. These observations suggest that IL-1 plays an important role in lipid metabolism by regulating insulin levels and lipase activity under physiological conditions.

Protective role of interleukin-1 in mycobacterial infection in IL-1 α/β double-knockout mice

To understand the role of the proinflammatory cytokine interleukin-1 (IL-1) in mycobacterial inflammation, IL-1 α/β double-knockout (KO) mice were produced. These mice were infected with either Mycobacterium tuberculosis H37Rv by the airborne route using an airborne infection apparatus, and their capacities to control mycobacterial growth, granuloma formation, cytokine, and nitric oxide (NO) production were examined. The IL-1 α/β mice developed significantly larger (p < 0.01) granulomatous, but not necrotic, lesions in their lungs than wild-type (WT) mice after infection with H37Rv. Inflammatory lesions, but not granulomas, were observed in spleen and liver tissues from both IL-1 α/β KO and wild-type mice. Granulomatous lesion development in IL-1 α/β KO mice was not significantly inhibited by treatment with exogenous recombinant IL-1α/β. Compared with wild-type mice, splenic IFN-γ and IL-12 levels were within the normal range. NO production by cultured alveolar macrophages from IL-1 α/β KO mice was lower than in wild-type mice but were increased by the addition of recombinant IL-1 α/β. Our data clearly indicate that IL-1 is important for the generation of early-phase protective immunity against mycobacterial infection.

SAP regulates T cell–mediated help for humoral immunity by a mechanism distinct from cytokine regulation

X-linked lymphoproliferative disease is caused by mutations affecting SH2D1A/SAP, an adaptor that recruits Fyn to signal lymphocyte activation molecule (SLAM)-related receptors. After infection, SLAM-associated protein (SAP)−/− mice show increased T cell activation and impaired humoral responses. Although SAP−/− mice can respond to T-independent immunization, we find impaired primary and secondary T-dependent responses, with defective B cell proliferation, germinal center formation, and antibody production. Nonetheless, transfer of wild-type but not SAP-deficient CD4 cells rescued humoral responses in reconstituted recombination activating gene 2−/− and SAP−/− mice. To investigate these T cell defects, we examined CD4 cell function in vitro and in vivo. Although SAP-deficient CD4 cells have impaired T cell receptor–mediated T helper (Th)2 cytokine production in vitro, we demonstrate that the humoral defects can be uncoupled from cytokine expression defects in vivo. Instead, SAP-deficient T cells exhibit decreased and delayed inducible costimulator (ICOS) induction and heightened CD40L expression. Notably, in contrast to Th2 cytokine defects, humoral responses, ICOS expression, and CD40L down-regulation were rescued by retroviral reconstitution with SAP-R78A, a SAP mutant that impairs Fyn binding. We further demonstrate a role for SLAM/SAP signaling in the regulation of early surface CD40L expression. Thus, SAP affects expression of key molecules required for T–B cell collaboration by mechanisms that are distinct from its role in cytokine regulation.

NR2B tyrosine phosphorylation modulates fear learning as well as amygdaloid synaptic plasticity

Phosphorylation of neural proteins in response to a diverse array of external stimuli is one of the main mechanisms underlying dynamic changes in neural circuitry. The NR2B subunit of the NMDA receptor is tyrosine‐phosphorylated in the brain, with Tyr‐1472 its major phosphorylation site. Here, we generate mice with a knockin mutation of the Tyr‐1472 site to phenylalanine (Y1472F) and show that Tyr‐1472 phosphorylation is essential for fear learning and amygdaloid synaptic plasticity. The knockin mice show impaired fear‐related learning and reduced amygdaloid long‐term potentiation. NMDA receptor‐mediated CaMKII signaling is impaired in YF/YF mice. Electron microscopic analyses reveal that the Y1472F mutant of the NR2B subunit shows improper localization at synapses in the amygdala. We thus identify Tyr‐1472 phosphorylation as a key mediator of fear learning and amygdaloid synaptic plasticity.

Alteration of behavioural phenotype in mice by targeted disruption of the progranulin gene.

Sexual differentiation of the brain in rodents is achieved by estrogens, which are converted from androgens in the brain, during the perinatal period. We have identified the progranulin (PGRN) gene as one of the sex steroid-inducible genes that may be involved in masculinization of the rat brain. In the present study, we generated a line of mice with targeted disruption of the PGRN gene, and investigated male sexual behaviour, aggression and anxiety. PGRN-deficient mice exhibited a decrease in ejaculation incidence, while the latency and frequency of both mount and intromission were unchanged. For the aggressive behaviour test, the resident-intruder paradigm was used, and PGRN-deficient mice exhibited enhanced aggressiveness. In wild-type mice, males exhibited lower levels of anxiety than females by the open field test, while male PGRN-deficient mice exhibited an elevated level of anxiety and sex difference in anxiety was not observed. In addition, mRNA expression of the serotonergic receptor 5-HT1A, which could be related to the inhibition of aggression and anxiety, was significantly reduced in the hippocampus of PGRN-deficient mice after aggressive encounters. On the other hand, deficiency of the PGRN gene did not affect serum testosterone concentrations. These results suggest that PGRN gene plays a role in establishing sexual dimorphic behaviours at least partially by modulating the brain serotonergic system.

Interleukin-1β, but not interleukin-1α, is required for T-cell-dependent antibody production

Interleukin‐1 (IL‐1) consists of two molecules, IL‐1α and IL‐1β, and IL‐1 receptor antagonist (IL‐1Ra) is a natural inhibitor of these molecules. Although the adjuvant effects of exogenously administered IL‐1 in the humoral immune response are well known, the roles of endogenous IL‐1 and the functional discrimination between IL‐1α and IL‐1β have not been elucidated completely. In this report, we investigated the role of IL‐1 in the humoral immune response using gene‐targeted mice. Both primary and secondary antibody production against T‐dependent antigen, sheep red blood cells (SRBC), was significantly reduced in IL‐1α/β−/− mice, and was enhanced in IL‐1Ra−/− mice. The intrinsic functions of B cells, such as antibody production against type 1 T‐independent antigen, trinitrophenyl–lipopolysaccharide and proliferative responses against mitogenic stimuli, were normal in IL‐1α/β−/− mice. The proliferative response of T cells and cytokine production upon stimulation with anti‐CD3 monoclonal antibody were also normal, as was the phagocytotic ability of antigen‐presenting cells (APCs). However, SRBC‐specific proliferative response and cytokine production of T cells through the interaction with APCs were markedly impaired in IL‐1α/β−/− mice, and enhanced in IL‐1Ra−/− mice. Moreover, we show that SRBC‐specific antibody production was reduced in IL‐1β−/− mice, but not in IL‐1α−/− mice. These results show that endogenous IL‐1β, but not IL‐1α, is involved in T‐cell‐dependent antibody production, and IL‐1 promotes the antigen‐specific T‐cell helper function through the T‐cell–APC interaction.