室井 正志

Inducible nitric oxidase (iNOS) encoded by Nos2 is a representative IFNγ-inducible effector molecule that plays an important role in both innate and adaptive immunity. In the present study, we demonstrated that full-length NF-κB p105 (p105), which is a precursor of NF-κB p50 (p50), is required for full activation of IFNγ-induced iNOS expression in the RAW264.7 mouse macrophage cell line. In comparison to wild-type (WT) RAW264.7 cells, p105 KO RAW264.7 (p105 KO) cells completely lost IFNγ-induced iNOS expression. Despite the limited effect of exogenous expression of p50 in p105 KO cells on IFNγ-induced Nos2 promoter activity, p105 expression fully restored IFNγ-induced Nos2 promoter activity to a level comparable to that of WT cells, suggesting an important role for full-length p105 in IFNγ-induced iNOS expression. While the expression and phosphorylation of JAK1 and STAT1 were rather enhanced in p105 KO cells, the phosphorylation of c-Jun downstream of MAPK signaling was decreased. IFNγ-induced phosphorylation of ERK, a kinase for IFNγ-induced c-Jun phosphorylation, was not significantly reduced in p105 KO cells, although the nuclear activity of ERK was significantly decreased due to its reduced translocation to the nucleus. Expression of iNOS, nuclear translocation of ERK, and phosphorylation of c-Jun were restored by stable supplementation of p105 in p105 KO cells. These results suggest that p105 is required for the nuclear translocation of ERK and the subsequent phosphorylation of c-Jun, which are necessary for full activation of IFNγ-induced iNOS expression. Reduced nuclear translocation of ERK in p105 KO cells was also observed in the activation of ERK following serum starvation, further suggesting that the involvement of p105 in ERK nuclear translocation is not limited to IFNγ-stimulated cells but is a more general function of p105.

Abstract Eosinophils are typical effector cells associated with type 2 immune responses and play key roles in the pathogenesis of allergic diseases. These cells are activated by various stimuli, such as cytokines, chemokines, and growth factors, but the regulatory mechanisms of eosinophil effector functions remain unclear. Glucocorticoid-induced TNF receptor family-related protein (GITR), a transmembrane protein belonging to the TNF receptor superfamily, is a well-known regulatory molecule for T cell activation. Here, we show that GITR is also constitutively expressed on eosinophils and functions as a co-stimulatory molecule for these cells. Although degranulation was unaffected by GITR engagement of murine bone marrow-derived eosinophils (bmEos), secretion of inflammatory cytokines such as interleukin (IL)-4, IL-6, and IL-13 from IL-33-activated bmEos were augmented by anti-mouse GITR agonistic antibody (DTA-1). In conclusion, our results provide a new regulatory pathway of cytokine secretion from eosinophils where GITR functions as a co-stimulatory molecule.

Abstract LPS interacts with TLR4, which play important roles in host-against-pathogen immune responses, by binding to MD-2 and inducing an inflammatory response. In this study, to our knowledge, we found a novel function of lipoteichoic acid (LTA), a TLR2 ligand, that involves suppression of TLR4-mediated signaling independently of TLR2 under serum-free conditions. LTA inhibited NF-κB activation induced by LPS or a synthetic lipid A in a noncompetitive manner in human embryonic kidney 293 cells expressing CD14, TLR4, and MD-2. This inhibition was abrogated by addition of serum or albumin. LTAs from different bacterial sources also inhibited NF-κB activation, although LTA from Enterococcus hirae had essentially no TLR2-mediated NF-κB activation. The TLR2 ligands tripalmitoyl-Cys-Ser-Lys-Lys-Lys-Lys (Pam3CSK4) and macrophage-activating lipopeptide-2 (MALP-2) did not affect the TLR4-mediated NF-κB activation. In bone marrow–derived macrophages from TLR2−/− mice, LTA inhibited LPS-induced IκB-α phosphorylation and production of TNF, CXCL1/KC, RANTES, and IFN-β without affecting cell surface expression of TLR4. LTA did not suppress IL-1β–induced NF-κB activation mediated through signaling pathways shared with TLRs. LTAs including E. hirae LTA, but not LPS, induced association of TLR4/MD-2 complexes, which was suppressed by serum. LTA also increased association of MD-2, but not TLR4 molecules. These results demonstrate that, under serum-free conditions, LTA induces association of MD-2 molecules to promote formation of an inactive TLR4/MD-2 complex dimer that in turn prevents TLR4-mediated signaling. The presence of LTA that poorly induces TLR2-mediated activation but inhibits TLR4 signaling provides insight into the role of Gram-positive bacteria in suppressing inflammation induced by Gram-negative bacteria in organs such as the intestines where serum is absent.

Excessive activation of Toll-like receptor (TLR) leads to sepsis. Inflammatory responses to various microbiological components are initiated via different TLR proteins, but all TLR signals are transmitted by TRAF6. We reported that TRAF6 associated with ubiquitinated IRAK-1 undergoes proteasome-mediated degradation, suggesting that IRAK-1 has a negative regulatory role in TLR signaling. Here, we investigated the minimal structural region of IRAK-1 needed for degradation of TRAF6. The IRAK-1 protein contains an N-terminal death domain (DD; amino acids 1-102), a serine/proline/threonine-rich ProST domain (amino acids 103-197), a central kinase domain with an activation loop (amino acids 198-522), and the C-terminal C1 and C2 domains (amino acids 523-712), which contain two and one putative TRAF6-binding (TB) sites, respectively. TRAF6 degradation was severely impaired by deletion of the DD or C1 domain, and a mutant (DC1) containing only the DD and C1 domains could induce TRAF6 degradation. IRAK-1 mutants lacking the N- or C-terminal amino acids of DD induced little degradation. Deletion or mutation of TB2 (amino acids 585-591) in the C1 domain also inhibited TRAF6 degradation. An IRAK-1 mutant possessing only DD and TB2 did not induce TRAF6 degradation, although a mutant in which a short spacer was inserted between DD and TB2 induced TRAF6 degradation, which and DC1-induced degradation were inhibited by proteasome inhibitors. All IRAK-1 mutants that induced TRAF6 degradation could be immunoprecipitated with TRAF6. Meanwhile, NF-κB activation was observed for all IRAK-1 mutants-including those that failed to induce degradation and was severely impaired only for a mutant carrying mutations in both TBs of C1. These results demonstrate that only DD and TB2 separated by an appropriate distance can induce TRAF6 degradation. Conformational analysis of this minimal structural unit may aid development of low molecular compounds that negatively regulate TLR signaling.

BACKGROUND: Type 2 innate lymphoid cells (ILC2s) are one of the sources of IL-5 and IL-13 in allergic airway inflammation. Innate immune receptors such as Toll-like receptors (TLRs) expressed on epithelial cells could contribute to ILC2 activation through IL-33 production, but a direct effect of TLRs on ILC2s remains to be elucidated. OBJECTIVES: We hypothesized that TLRs can directly activate lung ILC2s and participate in the pathogenesis of asthma. METHODS: After intranasal administration of IL-33 to wild-type (WT), TLR2KO and TLR4KO female mice, ILC2s were isolated from harvested lungs. ILC2s were incubated with IL-2 and TLR stimulants (pam3csk4 (PAM), house dust mite extract (HDM)). In some experiments, TLR2 or dectin-1 signalling inhibitors were used. As an in vivo model, the mice were treated with IL-33 and rested until lung recruitment of eosinophils regressed. Then they were treated intranasally with PAM + HDM or vehicle and analysed. RESULTS: In vitro stimulation of isolated ILC2s showed that PAM could induce IL-13 and IL-5 production, and HDM had a synergistic effect on this stimulation. Both effects were dependent on TLR2 and NF-κB signalling. PAM + HDM stimulation of WT mice led to increased ILC2s, airway hyperresponsiveness and increased levels of both neutrophils and eosinophils in bronchoalveolar lavage fluid. These observations were dependent on TLR2. CONCLUSIONS & CLINICAL RELEVANCE: TLR2 can directly activate lung ILC2s, an effect that is augmented by HDM. Asthmatic characteristics mediated through the TLR2 pathway were evident in the in vivo mice model. These data implicate a new pathway of ILC2 activation in the pathogenesis of asthma.

Assays using lysate reagents prepared from horseshoe crab hemocyte extract (limulus amoebocyte lysate, LAL) are commonly and widely used to detect and measure endotoxin in parenteral drugs and medical devices. However, lysate reagents suffer from lot-to-lot variations leading to possible fluctuations in testing. Also, this continued usage of lysate reagents leads to the possible decline of the horseshoe crab population. Recently, a new recombinant chromogenic reagent, PyroSmart, consisting of three recombinant factors was introduced to the market. There are now three recombinant products; two with recombinant factor C reagents and PyroSmart with the complete recombinant LAL system. We evaluated the applicability of the reagent to the harmonized bacterial endotoxins test in the United States, European and Japanese pharmacopeias. The recombinant product showed equivalent potency of thirteen endotoxins from different bacterial strains to conventional chromogenic lysate reagents as long as their assay modes are identical. All analytical characteristics or assay parameters of the reagent satisfied the acceptance criteria which are set for the use for the bacterial endotoxins test filed in the pharmacopeias. All of 109 parenteral drugs tested can be measured with PyroSmart within respective maximum allowable dilutions. The lot-to-lot variation in recovery of endotoxin added in the parenteral drugs for PyroSmart was equal to or less than those of six limulus lysate reagents. In conclusion, the present study suggests that the recombinant reagent, PyroSmart, provide a good alternative to the LAL reagents with better lot-to-lot variation.

House dust mites (HDMs) are a common source of allergens that trigger both allergen-specific and innate immune responses in humans. Here, we examined the effect of allergen concentration and the involvement of Toll-like receptor 4 (TLR4) in the process of sensitization to house dust mite allergens in an HDM extract-induced asthma mouse model. Intranasal administration of HDM extract induced an immunoglobulin E response and eosinophilic inflammation in a dose-dependent manner from 2.5 to 30 μg/dose. In TLR4-knockout mice, the infiltration of eosinophils and neutrophils into the lung was decreased compared with that in wild-type mice in the early phase of inflammation (total of three doses). However, in the late phase of inflammation (total of seven doses), eosinophil infiltration was significantly greater in TLR4-knockout mice than in wild-type mice. This suggests that the roles of TLR4 signaling are different between the early phase and the later phase of HDM allergen-induced inflammation. Thus, innate immune response through TLR4 regulated the response to HDM allergens, and the regulation was altered during the phase of inflammation.

近年、酸化チタンやシリカなどの種々ナノマテリアルが香粧品・医薬品成分として汎用されつつあり、その適用範囲は飛躍的に拡大している。一方で、ナノマテリアル暴露が炎症や感染症リスクを増加させるなど、ナノマテリアルの免疫攪乱作用が危惧されているものの、その免疫毒性をはじめとする生体影響に関する基礎情報は極めて乏しい。我々は、100 nm以下のナノマテリアルが経皮的に体外から体内に移行し得ることを見いだしており、全身性・皮膚局所での免疫系への影響を精査する必要を認めている。以上の観点から、本発表では、香粧品に汎用される非結晶性ナノシリカの自然免疫応答に及ぼす影響に関して検討した。まず、粒子径70 nmの非結晶性ナノシリカ (nSP)を用い、マウスマクロファージ細胞株RAW264.7に対するサイトカイン産生誘導能を指標に起炎性を評価した。その結果、nSP作用において、炎症性サイトカインTNFa、IL-6産生は全く誘導されないことが判明した。一方で、TLR4リガンドであるLPSとnSPを共作用させた場合、nSPはLPS刺激によるTNFaの産生を顕著に増大させた一方で、IL-6の産生は有意に抑制した。そこで次に、nSP がTLRの下流シグナル伝達に与える影響を検討した。その結果、nSPはLPS刺激によるNF-kB活性化を有意に抑制するとともに、MyD88依存的経路の活性化を抑制し得ることが判明した。以上の結果より、nSPはマクロファージの自然免疫応答を抑制し得る可能性が示唆された。本成果は、ナノマテリアルの安全性確保を目指した免疫毒性研究に新たな知見を与えるものである。

日本薬局方で使用が指定されている菌株の継代・培地等の差による特性の変化を検討した。

日本薬局方で使用が指定されている菌株の特性と保存管理法を検討した。