Koji Kawata

β-Glucan derived from Aureobasidium pullulans is effective for the prevention of influenza in mice.

β-(1→3)-D-glucans with β-(1→6)-glycosidic linked branches produced by mushrooms, yeast and fungi are known to be an immune activation agent, and are used in anti-cancer drugs or health-promoting foods. In this report, we demonstrate that oral administration of Aureobasidium pullulans-cultured fluid (AP-CF) enriched with the β-(1→3),(1→6)-D-glucan exhibits efficacy to protect mice infected with a lethal titer of the A/Puerto Rico/8/34 (PR8; H1N1) strain of influenza virus. The survival rate of the mice significantly increased by AP-CF administration after sublethal infection of PR8 virus. The virus titer in the mouse lung homogenates was significantly decreased by AP-CF administration. No significant difference in the mRNA expression of inflammatory cytokines, and in the population of lymphocytes was observed in the lungs of mice administered with AP-CF. Interestingly, expression level for the mRNA of virus sensors, RIG-I (retinoic acid-inducible gene-I) and MDA5 (melanoma differentiation-associated protein 5) strongly increased at 5 hours after the stimulation of A. pullulans-produced purified β-(1→3),(1→6)-D-glucan (AP-BG) in murine macrophage-derived RAW264.7 cells. Furthermore, the replication of PR8 virus was significantly repressed by pre-treatment of AP-BG. These findings suggest the increased expression of virus sensors is effective for the prevention of influenza by the inhibition of viral replication with the administration of AP-CF.

Alteration of circulating type 2 follicular helper T cells and regulatory B cells underlies the comorbid association of allergic rhinitis with bronchial asthma.

Allergic rhinitis (AR), the most common allergic disorder of the airway, is often accompanied by bronchial asthma. However, little is known about the mechanism by which AR advances to AR comorbid with bronchial asthma (AR+Asthma). To determine the pathophysiologic features of AR and AR+Asthma, we examined subsets of follicular helper T (Tfh) cells and regulatory B (Breg) cells in peripheral blood from AR and AR+Asthma patients. The results showed polarization of Tfh2 cells within Tfh cell subsets in both AR and AR+Asthma cases. Interestingly, the %Breg cells in total B cells were decreased in AR cases and, more extensively, in AR+Asthma cases. Moreover, we found significant correlations of fractional exhaled nitric oxide and blood eosinophil levels with the index %Tfh2 cells per %Breg cells. Our findings indicate that relative decrease in Breg cells under the condition of Tfh2 cell skewing is a putative exaggerating factor of AR to bronchial asthma.

ΔNp63 Controls a TLR3-Mediated Mechanism That Abundantly Provides Thymic Stromal Lymphopoietin in Atopic Dermatitis

In the skin lesions of atopic dermatitis (AD), keratinocytes release large quantities of thymic stromal lymphopoietin (TSLP), causing unfavorable inflammation along with skin damage. Nevertheless, how TSLP influences keratinocytes themselves is still unknown. In this study, we showed that ΔNp63, a p53-homologue, predominantly expressed in keratinocytes regulated the receptor complex of TSLP, which determines susceptibility to self-derived TSLP. Expression of TSLP receptors in skin tissues and keratinocytes was assessed by immunohistochemistry and quantitative RT-PCR, and in vitro studies were also performed to examine the functional relevance of ΔNp63 in the expression of TSLP receptors and the constituting autocrine and/or paracrine pathway of TSLP under the condition of stimuli to innate receptors sensing cell damage. The results showed that normal keratinocytes in the upper epidermis preferentially expressed TSLP receptors and conversely lacked ΔNp63, which has an inhibitory effect on the expression of TSLP receptors. Interestingly, the epidermis of AD lesions was found to abundantly contain keratinocytes with low or undetectable levels of ΔNp63 (ΔNp63lo/-). Moreover, in the absence of ΔNp63, keratinocytes readily presented TSLP and other cytokines by stimuli through Toll-like receptor 3 (TLR3). Together with the evidence that extrinsic TSLP itself augments TSLP production by keratinocytes without ΔNp63, the results indicate that ΔNp63lo/- keratinocytes generate TSLP through a putative autocrine and/or paracrine pathway upon TLR3 stimulation within AD lesions, since moieties of damaged cells and pathogens stimulate TLR3.

Cutting Edge: A Critical Role of Lesional T Follicular Helper Cells in the Pathogenesis of IgG4-Related Disease

IgG4-related disease (IgG4-RD) is a newly recognized systemic chronic fibroinflammatory disease. However, the pathogenesis of IgG4-RD remains unknown. To determine the pathophysiologic features of IgG4-RD, we examined T follicular helper (Tfh) cells in lesions and blood from patients with IgG4-RD. Patients with IgG4-related dacryoadenitis and sialadenitis (IgG4-DS) showed increased infiltration of Tfh cells highly expressing programmed death 1 and ICOS in submandibular glands. Tfh cells from IgG4-DS submandibular glands had higher expression of B cell lymphoma 6 and a greater capacity to help B cells produce IgG4 than did tonsillar Tfh cells. We also found that the percentage of programmed death 1hi circulating Tfh cells in IgG4-DS patients was higher than that in healthy volunteers and was well correlated with clinical parameters. Our findings indicate that anomalous Tfh cells in tissue lesions of IgG4-RD have features distinct from those in lymphoid counterparts or blood and potentially regulate local IgG4 production in IgG4-RD.

Stimulation with the Aureobasidium pullulans -produced β-glucan effectively induces interferon stimulated genes in macrophage-like cell lines

A β-(1,3),(1,6)-D-glucan produced by A. pullulans (AP-PG) is known to be an immune stimulating agent. In this study, we demonstrate that the stimulation with AP-PG effectively induces the interferon (IFN) stimulated genes (ISGs) in macrophage-like cell lines. The ISGs, Mx1, ISG15, and viperin mRNAs were significantly increased in RAW264.7 cells after stimulation with AP-PG. The stimulation with AP-PG transiently induced IFN-β mRNA. However, the expression of viperin mRNA was also increased after stimulation with AP-PG even when new protein synthesis was completely blocked by treatment with cycloheximide. Further, in IFN-α receptor knockdown RAW264.7 cells, AP-PG stimulation more effectively induced viperin mRNA compared with that of IFN-α stimulation. The phosphorylation of Ser 727 in STAT1 involved in the enhancement of STAT1 activation was immediately increased after stimulation with AP-PG. In addition, viperin mRNA expression induced after stimulation with IFN-α was significantly increased by combined stimulation with AP-PG. These results suggest that stimulation with AP-PG effectively induces the ISGs through the induction of IFN and the enhancement of STAT1-mediated transcriptional activation.

Lipid mediators foster the differentiation of T follicular helper cells

Lipid mediators such as leukotrienes and lipoxines broadly regulate innate and acquired immunity, and their dysfunction causes various immune-mediated disorders. We previously reported a salient feature of arachidonate 5-lipoxyganase (Alox5), which is responsible for the production of such lipid mediators, in the regulation of high affinity antibodies in vivo. The aim of this study was to determine the functional significance of Alox5-related lipid mediators during the processes of acquired humoral responses. The results of in vitro experiments using lymphocytes in tonsils and blood specimens showed that lipoxin A4 (LXA4) and leukotriene B4 (LTB4) have the capacity to differentiate naïve CD4+ T cells into T follicular helper (Tfh) cells, which activate naïve B cells to form germinal centers. Such a function of LXA4 was further supported by results of in vitro studies using BML-111 and BOC-2, which are an agonist and an antagonist, respectively, of FPR2 of an LXA4-specific cell-surface receptor. The results suggest that such lipid mediators have a potential role in the development of lymphoid follicles through the regulation of Tfh cell differentiation.

Novel Mechanisms of Compromised Lymphatic Endothelial Cell Homeostasis in Obesity: The Role of Leptin in Lymphatic Endothelial Cell Tube Formation and Proliferation

Leptin is a hormone produced by adipose tissue that regulates various physiological processes. Recent studies have shown that the level of circulating leptin is elevated in obese patients and have suggested a relationship between obesity and postoperative lymphedema. However, the mechanisms by which postoperative lymphedema develops in obese patients and the mechanisms by which leptin regulates lymphatic endothelial cell homeostasis such as tube formation and cell proliferation remain unknown. Here we report that leptin regulates tube formation and cell proliferation in human dermal lymphatic endothelial cells (HDLECs) by activation of the signal transducer and activator of transcription 3 pathway, which is downstream signaling of the leptin receptor. Additionally, we found that upregulation of suppressor of cytokine signaling 3 underlies the mechanisms by which a high dose of leptin inhibits cell proliferation and tube formation. Leptin also enhanced expression of the proinflammatory cytokine IL-6 in HDLECs. Interestingly, IL-6 rescues the compromised cell proliferation and tube formation caused by treatment with a high dose of leptin in an autocrine or paracrine manner. Taken together, our findings reveal a novel mechanism by which compromised HDLECs maintain their homeostasis during inflammation mediated by leptin and IL-6. Thus, regulating the level of leptin or IL-6 may be a viable strategy to reduce the incidence of postoperative lymphedema.

Oral administration of the Aureobasidium pullulans -derived β-glucan effectively prevents the development of high fat diet-induced fatty liver in mice

Aureobasidium pullulans-derived β-glucan (AP-PG) consisting of a β-(1,3)-linked glucose main chain and β-(1,6)-linked glucose branches is taken as a supplement to improve health. This study demonstrates that oral administration of AP-PG is effective to prevent the development of high-fat diet (HFD)-induced fatty liver in mice. Here, C57BL/6N mice were fed with a normal diet or HFD, and AP-PG diluted in drinking water was administered orally. After 16 weeks, the serological analysis showed that HFD-induced high blood cholesterol and triglyceride levels were reduced by the oral administration of AP-PG. Further, HFD induced-fatty liver was significantly reduced by the oral administration of AP-PG. The triglyceride accumulation in the liver was also significantly reduced in mice administered AP-PG. Liver injury as indicated by an increase in serum alanine aminotransferase (ALT) in the HFD-fed mice was significantly reduced in the mice administered AP-PG orally, and the gene expression of cholesterol 7 alpha-hydroxylase (CYP7A1) which is known to be involved in cholesterol degradation in the liver was significantly increased in the AP-PG administered mice. These results suggest the possibility that the oral administration of AP-PG is effective to prevent the development of non-alcoholic fatty liver disease (NAFLD).

Bob1 limits cellular frequency of T‐follicular helper cells

T follicular helper (Tfh) cells are involved in specific humoral immunity at initial and recall phases. The fact that the transcription repressors B‐cell lymphoma‐6 and Blimp‐1 determine lineages of Tfh cells and other types of effector CD4+ T cells, respectively, suggests that there are unique mechanisms to establish Tfh‐cell identity. In this study, we found that Tfh cells preferentially express the transcriptional coactivator Bob1. Bob1 of Tfh cells was dispensable for the expression of B‐cell lymphoma‐6 and the functional property of the cells for B cell help. However, upon initial immunization of foreign antigens, the percentages of Tfh cells in Bob1−/− mice were much higher than those in wild‐type (WT) mice. In addition, expansion of Tfh cells within Bob1−/−CD4+ T cells transferred into WT mice revealed that the high frequency of Tfh cells was caused by a T‐cell‐intrinsic mechanism. These findings were further supported by the results of in vitro studies demonstrating that Bob1−/− Tfh cells had greater proliferative activity in response to stimuli by CD3/CD28 monoclonal antibody and were also refractory to CD3‐induced cell death in comparison to WT Tfh cells. These results suggest that Tfh cells harbor a Bob1‐related mechanism to restrict numerical frequency against stimulation of TCRs.

Stimulation of Macrophages with the β-Glucan Produced by Aureobasidium pullulans Promotes the Secretion of Tumor Necrosis Factor-Related Apoptosis Inducing Ligand (TRAIL)

A β-glucan produced by Aureobasidium pullulans (AP-PG) is consisting of a β-(1,3)-linked main chain with β-(1,6)-linked glucose side residues. Various β-glucans consisting of β-(1,3)-linked main chain including AP-PG are believed to exhibit anti-tumor activities, and actually, anti-tumor activities of AP-PG in mice have been demonstrated. In this study, we demonstrate that stimulation with AP-PG induces TRAIL expression in mouse and human macrophage-like cell lines. TRAIL is known to be a cytokine which specifically induces apoptosis in transformed cells, but not in untransformed cells. The expression of TRAIL mRNA after stimulation with AP-PG was increased in RAW264.7 cells, Mono Mac 6 cells, and macrophage-differentiated THP-1 cells. The mRNA expression of TNF-α and FasL is only weakly increased after stimulation with AP-PG. The induction activity of TRAIL by curdlan, a bacterial β-glucan, was very similar to that by AP-PG in RAW264.7 cells, but weaker in macrophage-differentiated THP-1 cells. Activation of caspases was found in HeLa cells after treatment with the supernatant of cultured medium from AP-PG-stimulated Mono Mac 6 cells, and was inhibited by the anti-TRAIL neutralizing antibody. These findings suggest that the stimulation with AP-PG effectively induces TRAIL in macrophages, and that it may be related to apoptosis induction of tumor cells.