Koubun Yasuda

The Jmjd3- Irf4 axis regulates M2 macrophage polarization and host responses against helminth infection

Polarization of macrophages to M1 or M2 cells is important for mounting responses against bacterial and helminth infections, respectively. Jumonji domain containing-3 (Jmjd3), a histone 3 Lys27 (H3K27) demethylase, has been implicated in the activation of macrophages. Here we show that Jmjd3 is essential for M2 macrophage polarization in response to helminth infection and chitin, though Jmjd3 is dispensable for M1 responses. Furthermore, Jmjd3 (also known as Kdm6b) is essential for proper bone marrow macrophage differentiation, and this function depends on demethylase activity of Jmjd3. Jmjd3 deficiency affected trimethylation of H3K27 in only a limited number of genes. Among them, we identified Irf4 as encoding a key transcription factor that controls M2 macrophage polarization. Collectively, these results show that Jmjd3-mediated H3K27 demethylation is crucial for regulating M2 macrophage development leading to anti-helminth host responses.

Administration of IL-33 induces airway hyperresponsiveness and goblet cell hyperplasia in the lungs in the absence of adaptive immune system

Systemic administration of IL-18 induces polyclonal IgE responses by causing NKT cells to express CD40 ligand and to produce IL-4. Administration of IL-33 also induces IgE response, although the mechanism underlying IgE response is unclear. Here, we compared the effects of IL-18 and IL-33 on bone marrow-derived mast cells and basophils as well as non-polarized and T(h)2-polarized CD4(+) T cells in vitro. Basophils, comprising IL-18Ralpha(+) cells (14.2%) and IL-33Ralpha(+) cells (34.6%), and mast cells, comprising IL-18Ralpha(+) cells (2.0%) and IL-33Ralpha(+) cells (95.6%), produce IL-4, IL-6, IL-13, granulocyte macrophage colony-stimulating factor (GM-CSF) and chemokines (RANTES, MIP-1alpha, MIP-1beta and MCP-1), upon stimulation with IL-18 and/or IL-33 in the presence of IL-3. Only basophils strongly produce IL-4. Furthermore, compared with mast cells, basophils produce larger amounts of the above cytokines and chemokines in response to IL-33. Level of IL-33Rbeta-mRNA expression in basophils is higher than that in mast cells. Effect of IL-33 is dependent on ST2 binding, and its signal is transduced via MyD88 in vitro. We also found that IL-2 plus IL-18 or IL-33 alone stimulates non-polarized or T(h)2-polarized CD4(+) T cells to produce IL-4 and IL-13 or IL-5 and IL-13, respectively. We finally showed that administration of IL-33 into mice ST2/MyD88 dependently induces airway hyperresponsiveness (AHR) and goblet cell hyperplasia by induction of IL-4, IL-5 and IL-13 in the lungs. Furthermore, same treatment of RAG-2(-/-) mice, lacking T and B cells, more strikingly induced AHR with marked goblet cell hyperplasia and eosinophilic infiltration in the lungs. Thus, IL-33 induces asthma-like symptom entirely independent of acquired immune system.

Contribution of IL-33–activated type II innate lymphoid cells to pulmonary eosinophilia in intestinal nematode-infected mice

When animals are infected with helminthic parasites, resistant hosts show type II helper T immune responses to expel worms. Recently, natural helper (NH) cells or nuocytes, newly identified type II innate lymphoid cells, are shown to express ST2 (IL-33 receptor) and produce IL-5 and IL-13 when stimulated with IL-33. Here we show the relevant roles of endogenous IL-33 for Strongyloides venezuelensis infection-induced lung eosinophilic inflammation by using Il33−/− mice. Alveolar epithelial type II cells (ATII) express IL-33 in their nucleus. Infection with S. venezuelensis or intranasal administration of chitin increases in the number of ATII cells and the level of IL-33. S. venezuelensis infection induces pulmonary accumulation of NH cells, which, after being stimulated with IL-33, proliferate and produce IL-5 and IL-13. Furthermore, S. venezuelensis infected Rag2−/− mice increase the number of ATII cells, NH cells, and eosinophils and the expression of IL-33 in their lungs. Finally, IL-33–stimulated NH cells induce lung eosinophilic inflammation and might aid to expel infected worms in the lungs.

Skin-specific expression of IL-33 activates group 2 innate lymphoid cells and elicits atopic dermatitis-like inflammation in mice

Transgenic mice expressing the mouse interleukin 33 (IL-33) gene driven by a keratin 14 promoter were generated. The skin-selective expression of the IL-33 gene was enhanced, and intense immunofluorescence for IL-33 was evident in the nuclei of the epidermis. Spontaneous itchy dermatitis developed in those mice at 6–8 wk of age in specific pathogen-free conditions. In the lesional skin, the epidermis was thickened and the eosinophils were infiltrated with increased expression of the eosinophil peroxidase and major basic protein genes. Mast cells were also abundant there, and blood histamine and total IgE levels were high. Those phenotypes closely resemble the features of atopic dermatitis. In peripheral blood and lesional skin, IL-5, IL-13, regulated upon activation, normally T-expressed, and presumably secreted (RANTES)/CCL5, and Eotaxin 1/CCL11 were increased, whereas TNF-α, IFN-γ, and thymic stromal lymphopoietin (TSLP) were unaltered. Furthermore, the proportion of group 2 innate lymphoid cells (ILC2s), which produce IL-5, were significantly increased in the lesional skin, peripheral blood, and regional lymph nodes. The dermatitis with eosinophil infiltration was improved by the administration of an anti-IL-5 antibody. These results suggest that the expression of IL-33 in the skin activates an immune response involving ILC2 and that this process might play a crucial role in the pathogenesis of allergic inflammation that is characteristic of atopic dermatitis.

ASC is essential for LPS‐induced activation of procaspase‐1 independently of TLR‐associated signal adaptor molecules

Toll‐like receptors (TLRs) initiate a signalling cascade via association with an adaptor molecule, myeloid differentiation factor 88 (MyD88) and/or TIR domain‐containing adaptor inducing‐IFN‐β (Trif), to induce various pro‐inflammatory cytokines for microbial eradication. After stimulation of TLR4 with lipopolysaccharide (LPS), both IL‐1β and IL‐18 are processed, depending on the activation of caspase‐1, although its mechanism remains unclear. ASC is an adapter protein possibly involved in the activation of procaspase‐1. To unravel the requirement of ASC, we generated Asc−/– mice. Upon stimulation with LPS, Asc−/– macrophages failed in the processing of procaspase‐1 and maturation of pro‐IL‐1β and pro‐IL‐18, but normally produced other pro‐inflammatory cytokines including TNF‐α and IL‐6. MyD88−/– and Trif−/– macrophages showed normal activation of caspase‐1, demonstrating a dispensable role for MyD88 and Trif. After, LPS‐challenged Asc−/– mice lacked serum elevation of IL‐1β and IL‐18. Moreover, the Asc−/– mice exhibited neither acute liver injury nor lethal shock. These results demonstrate critical roles for ASC in the release of IL‐1β/IL‐18 via activation of caspase‐1 and provide new insights into the inflammatory responses for host defence and diseases.

IL-27 suppresses Th2 cell development and Th2 cytokines production from polarized Th2 cells: a novel therapeutic way for Th2-mediated allergic inflammation.

IL-27 up-regulates Th1 but down-regulates Th2 responses. However, its molecular mechanism and regulatory effects on polarized Th2 cells remain unclear. In this study, we have revealed that IL-27 inhibits Th2 cell development as well as Th2 cytokines production from already polarized Th2 cells by down-regulation of GATA-3 and up-regulation of T-bet expression simultaneously. In vivo daily IL-27 treatment for 1 wk after Leishmania major infection protects BALB/c mice from footpad swelling by diminishing parasite burden via reciprocal regulation of Th1 and Th2 responses. Furthermore, IL-27 stimulation causes marked reduction in the capacity of host mouse to mount a Th2 response against Strongyloides venezuelensis infection. Thus, IL-27-treated mice failed to develop intestinal mastocytosis after S. venezuelensis infection and exhibited a marked delay in parasite expulsion. Finally, intranasal administration of IL-27 inhibits OVA-induced airway hyperresponsiveness and inflammation in OVA-sensitized animals. Thus, IL-27 could provide us with a novel therapeutic way for treating Th2-associated diseases such as bronchial asthma.

Cutting Edge: Fyn Is Essential for Tyrosine Phosphorylation of Csk-Binding Protein/Phosphoprotein Associated with Glycolipid-Enriched Microdomains in Lipid Rafts in Resting T Cells

In resting T cells, Csk is constitutively localized in lipid rafts by virtue of interaction with a phosphorylated adaptor protein, Csk-binding protein (Cbp)/phosphoprotein associated with glycolipid-enriched microdomains, and sets an activation threshold in TCR signaling. In this study, we examined a kinase responsible for Cbp phosphorylation in T cell membrane rafts. By analyzing T cells from Fyn−/− mice, we clearly demonstrated that Fyn, but not Lck, has its kinase activity in membrane rafts, and plays a critical role in Cbp phosphorylation, Cbp-Csk interaction, and Csk kinase activity. Naive CD44lowCD62 ligandhigh T cells were substantially reduced in Fyn−/− mice, presumably due to the inhibition of Cbp phosphorylation. Thus, Fyn mediates Cbp-Csk interaction and recruits Csk to rafts by phosphorylating Cbp. Csk recruited to rafts would then be activated and inhibit the kinase activity of Lck to keep resting T cells in a quiescent state. Our results elucidate a negative regulatory role for Fyn in proximal TCR signaling in lipid rafts.

Contribution of IL-33 to induction and augmentation of experimental allergic conjunctivitis

IL-33, a member of the IL-1 family of cytokines, is the ligand for ST2 (IL-33Ralpha chain). IL-33 has the capacity to induce T(h)2 cytokine production from T(h)2 cells, mast cells and basophils, indicating that IL-33 has the potential to induce T(h)2 cytokine-mediated allergic inflammation of the eye. Thus, we tested the pathological role of IL-33 in allergic conjunctivitis (AC). As reported elsewhere, animals immunized with ragweed pollen (RW)/alum and boosted with RW/PBS developed AC promptly (within 15 min) and conjunctival eosinophilic inflammation after a delay (within 24 h) in response to eye drop challenge with RW. Furthermore, RW-immunized mice, when topically challenged with both RW and IL-33, developed more striking eosinophilia in their conjunctiva without exacerbation of the clinical AC score. This in vivo IL-33 treatment significantly increased the capacity of T cells in the cervical lymph nodes of RW-immunized mice to produce IL-4, IL-5 and IL-13 upon challenge with anti-CD3 and anti-CD28 antibodies in vitro. Furthermore, the infiltrating cells were largely eosinophils and a small proportion of CD4(+) T cells, both of which express ST2. We also found that even splenic eosinophils express ST2 and show increased expression in response to IL-5, granulocyte-macrophage colony-stimulating factor (GM-CSF) or IL-33. Eosinophils, stimulated with IL-5 and/or GM-CSF, are responsive to IL-33, which induces production of IL-4 and chemokines. Finally, we showed that conjunctival tissues constitutively express biologically active IL-33, suggesting that IL-33 might play a crucial role in the induction and augmentation of AC.

Serine 6 of Lck Tyrosine Kinase: A Critical Site for Lck Myristoylation, Membrane Localization, and Function in T Lymphocytes

Lck is a member of the Src family kinases expressed predominantly in T cells, and plays a pivotal role in TCR-mediated signal transduction. Myristoylation of glysine 2 in the N-terminal Src homology 4 (SH4) domain of Lck is essential for membrane localization and function. In this study, we examined a site within the SH4 domain of Lck regulating myristoylation, membrane localization, and function of Lck. A Lck mutant in which serine 6 (Ser6) was substituted by an alanine was almost completely cytosolic in COS-7 cells, and this change of localization was associated with a drastic inhibition of myristoylation in this mutant. To assess the role of Ser6 of Lck in T cell function, we established stable transfectants expressing various Lck mutants using Lck-negative JCaM1 cells. The Lck mutant of Ser6 to alanine, most of which did not target to the plasma membrane, was not able to reconstitute TCR-mediated signaling events in JCaM1 cells, as analyzed by tyrosine phosphorylation of intracellular proteins and CD69 expression. These results demonstrate that Ser6 is a critical factor for Lck myristoylation, membrane localization, and function in T cells, presumably because the residue is important for N-myristoyl transferase recognition.

Contribution of TIR domain-containing adapter inducing IFN-β-mediated IL-18 release to LPS-induced liver injury in mice

BACKGROUND/AIMS After treatment with heat-killed Propionibacterium acnes mice show dense hepatic granuloma formation. Such mice develop liver injury in an interleukin (IL)-18-dependent manner after challenge with a sublethal dose LPS. As previously shown, LPS-stimulated Kupffer cells secrete IL-18 depending on caspase-1 and Toll-like receptor (TLR)-4 but independently of its signal adaptor myeloid differentiation factor 88 (MyD88), suggesting importance of another signal adaptor TIR domain-containing adapter inducing IFN-beta (TRIF). Nalp3 inflammasome reportedly controls caspase-1 activation. Here we investigated the roles of MyD88 and TRIF in P. acnes-induced hepatic granuloma formation and LPS-induced caspase-1 activation for IL-18 release. METHODS Mice were sequentially treated with P. acnes and LPS, and their serum IL-18 levels and liver injuries were determined by ELISA and ALT/AST measurement, respectively. Active caspase-1 in LPS-stimulated Kupffer cells was determined by Western blotting. RESULTS Macrophage-ablated mice lacked P. acnes-induced hepatic granuloma formation and LPS-induced serum IL-18 elevation and liver injury. Myd88(-/-) Kupffer cells, but not Trif(-/-) cells, exhibited normal caspase-1 activation upon TLR4 engagement in vitro. Myd88(-/-) mice failed to develop hepatic granulomas after P. acnes treatment and liver injury induced by LPS challenge. In contrast, Trif(-/-) mice normally formed the hepatic granulomas, but could not release IL-18 or develop the liver injury. Nalp3(-/-) mice showed the same phenotypes of Trif(-/-) mice. CONCLUSIONS Propionibacterium acnes treatment MyD88-dependently induced hepatic granuloma formation. Subsequent LPS TRIF-dependently activated caspase-1 via Nalp3 inflammasome and induced IL-18 release, eventually leading to the liver injury.