Knock-Out of ACY-1 Like Gene in Spodoptera litura Supports the Notion that FACs Improve Nitrogen Metabolism.

Volicitin [N-(17-hydroxylinolenoyl)-L-glutamine] and N-linolenoyl-L-glutamine were originally identified in the regurgitant of Spodoptera exigua larvae. These fatty acid amino acid conjugates (FACs) are known to be elicitors that induce plants to release volatile compounds which in turn attract natural enemies of the larvae such as parasitic wasps. FAC concentrations are regulated by enzymatic biosynthesis and hydrolysis in the intestine of Lepidoptera larvae. It has been proposed that FAC metabolism activates glutamine synthetase and plays an important role in nitrogen metabolism in larvae. In this study, we identified candidate genes encoding a FACs hydrolase in Spodoptera litura using genomic information of various related lepidopteran species in which FACs hydrolases have been reported. We analyzed the importance of FAC hydrolysis on caterpillar performance with CRISPR/Cas9 knock outs. Larvae of strains with an inactive FACs hydrolase excreted FACs in their feces. They absorbed 30% less nitrogen from the diet compared to WT caterpillars resulting in a reduction of their body weight of up to 40% compared to wild type caterpillars. These results suggest that the hydrolysis of FACs is an important metabolism for insects and that FACs are important for larval growth.

MyD88 associated ROS generation is crucial for Lactobacillus induced IL-12 production in macrophage.

It is well known that some strains of lactic acid bacteria (LAB) can induce IL-12 which plays an important role in modulating immune responses. However, the mechanisms by which LAB induce IL-12 production remain unclear. Here, we examine the role of toll-like receptors (TLR's) and reactive oxygen species (ROS) in IL-12 production by LAB stimulated peritoneal macrophages. Our results indicate that a TLR is not necessary for IL-12 induction by LAB, whilst the universal adaptor protein, MyD88, is essential. Specific strains of LAB induced ROS that correlated with both the frequency of phagocytosis and IL-12 production. Reduction in IL-12 production by NADPH oxidase inhibitors or ROS scavengers demonstrates the crucial role of ROS in IL-12 induction. Interestingly, deficiency of TLR2, 4, 9 or MyD88 did not affect the phagocytosis of LAB strain KW3110, a potent IL-12 inducer, and ROS production was significantly reduced only in MyD88 deficient macrophages. These results suggest the existence of TLR-MyD88 independent LAB recognition and MyD88 related ROS induction mechanisms. We show here the importance of ROS for IL-12 induction and provide new insights into IL-12 induction by LAB.

Orally administered Lactobacillus paracasei KW3110 induces in vivo IL-12 production.

Lactic acid bacteria (LAB) are popularly used as probiotics, and some strains of LAB have anti-allergic functions in vivo. Although in vitro studies show that LAB modulate the T helper type (Th) 1/Th2 balance and inhibit IgE secretion by inducing IL-12, it is not known how LAB regulates allergies in vivo. In this study, we evaluated in vivo IL-12 production after oral administration of Lactobacillus paracasei KW3110, a strain reported to improve allergies, to mice. Orally administered KW3110 interacted with CD11b positive cells and induced IL-12 mRNA expression at Peyer's patch. In addition, blood IL-12 levels increased transiently 10 h after administration of KW3110. Based on these results, we found that oral administration of KW3110 induces IL-12 in vivo. Our findings should contribute to understanding of the in vivo function of LAB.

MyD88 but not TLR2, 4 or 9 is essential for IL-12 induction by lactic acid bacteria.

Although lactic acid bacteria (LAB) affect the immune system, for example, having an anti-allergic effect, little is known about the actual mechanisms of immune modulation. Toll-like receptors (TLRs) recognize conserved microbial molecular patterns, and are presumed to be involved in the recognition of LAB. However, there are few detailed reports examining the relationships between TLR and LAB. We measured here production of IL-12, a cytokine considered to play an important role in anti-allergic effects, induced by Lactobacillus paracasei strain KW3110 and other typical LAB by cells from TLR2-, TLR4-, TLR9- and myeloid differentiation factor 88 (MyD88)-deficient mice. Unexpectedly, similar cytokine production from wild-type and TLR2-, 4- and 9-deficient mice was observed. In contrast, cells from MyD88-deficient mice failed to respond to stimulation with LAB. It is therefore concluded that although LAB, including strain KW3110, are not likely to be recognized by TLR2, 4 or 9, MyD88 is essential for the response to these bacteria.

Ameliorating effect of saporin-conjugated anti-CD11b monoclonal antibody in a murine T-cell-mediated chronic colitis.

BACKGROUND: Crohn's disease (CD) is an inflammatory bowel disease that is associated with several changes in the immune system, including an increased number of infiltrating macrophages. These macrophages release a variety of pro-inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-alpha) which are critically involved in the onset and the development of CD. The present study was performed to explore the initial involvement of macrophages in the development of T-cell-mediated chronic colitis. METHODS: The effects were evaluated of saporin-conjugated anti-CD11b monoclonal antibody (mAb) on the development of chronic colitis in severe combined immunodeficiency (SCID) mice induced by adoptive transfer of CD4(+)CD45RB(high) T cells as an animal model of CD. RESULTS: Significantly increased CD11b-expressing macrophages as well as CD4(+) T cells were found in inflamed colon from colitic mice. Administration of saporin-conjugated anti-CD11b mAb markedly ameliorated the clinical and histopathological disease. In vivo treatment with saporin-conjugated anti-CD11b mAb decreased CD4(+) T-cell infiltration in the colon and suppressed interferon-gamma (IFN-gamma) and TNF-alpha production by lamina propria CD4(+) T cells. CONCLUSIONS: Collectively, the present results suggest an initial role of macrophages in the pathogenesis of T-cell-mediated chronic colitis. Furthermore, the macrophage-specific targeting may be a promising strategy for therapeutic intervention in CD.

Synthetic alpha-mannosyl ceramide as a potent stimulant for an NKT cell repertoire bearing the invariant Valpha19-Jalpha26 TCR alpha chain.

A NKT cell repertoire is characterized by the expression of the Valpha19-Jalpha26 invariant TCR alpha chain (Valpha19 NKT cell). This repertoire, as well as a well-established Valpha14-Jalpha281 invariant TCR alpha(+) NKT cell subset (Valpha14 NKT cell), has been suggested to have important roles in the regulation of the immune system and, thus, is a major therapeutic target. Here, we attempted to find specific antigens for Valpha19 NKT cells. Valpha19 as well as Valpha14 NKT cells exhibited reactivity to alpha-galactosyl ceramide (alpha-GalCer). Thus, a series of monoglycosyl ceramides with an axially oriented glycosidic linkage between the sugar and ceramide moiety were synthesized and their antigenicity to Valpha19 NKT cells was determined by measuring their immune responses in culture with glycolipids. Comprehensive examinations revealed substantial antigenic activity for Valpha19 NKT cells by alpha-mannosyl ceramide.

Regulation of murine chronic colitis by CD4+CD25- programmed death-1+ T cells.

Naturally arising CD4(+)CD25(+) regulatory T (T(R)) cells are engaged in the maintenance of self tolerance and prevention of autoimmune diseases. However, accumulating evidence suggests that a fraction of peripheral CD4(+)CD25(-) T cells also possesses regulatory activity. Programmed death-1 (PD-1) is a new member of the CD28/CTLA-4 family, which has been implicated in the maintenance of peripheral self tolerance. Here, we identified a subpopulation of CD4(+)CD25(-)PD-1(+) T cells in the spleen of naive mice that constitutively expressed CTLA-4 and FoxP3 and was hypoproliferative in response to anti-CD3 antibody stimulation in vitro. However, the CD4(+)CD25(-)PD-1(+) T cells uniquely produced large amounts of IL-4 and IL-10 in response to anti-CD3 and anti-CD28 mAb stimulation, unlike the CD4(+)CD25(+) T(R) cells. The CD4(+)CD25(-)PD-1(+) T cells exhibited a suppressor activity against the proliferation of anti-CD3 antibody-stimulated CD4(+)CD25(-)PD-1(-) T cells in vitro, which was partially abrogated by anti-CTLA-4 mAb, but not by anti-IL-10 or anti-PD-1 mAb. Remarkably, the CD4(+)CD25(-)PD-1(+) T cells inhibited the development of colitis induced by adoptive transfer of CD4(+)CD45RB(high) T cells into C.B17-scid/scid mice, albeit to a lesser extent than CD4(+)CD25(+) T(R) cells, in a CTLA-4-dependent manner. These results indicate that the CD4(+)CD25(-)PD-1(+) T cells contain substantial amounts of T(R) cells that are involved in the maintenance of peripheral tolerance.