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Intestinal inflammation can be accompanied by osteoporosis, but their relationship, mediated by immune responses, remains unclear. Here, we investigated a non-IgE-mediated food-allergic enteropathy model of ovalbumin (OVA) 23-3 mice expressing OVA-specific T-cell-receptor transgenes. Mesenteric lymph nodes (MLNs) and their pathogenic CD4+T cells were important to enteropathy occurrence and exacerbation when the mice were fed an egg-white (EW) diet. EW-fed OVA23-3 mice also developed bone loss and increased CD44hiCD62LloCD4+T cells in the MLNs and bone marrow (BM); these changes were attenuated by MLN, but not spleen, resection. We fed an EW diet to F1 cross offspring from OVA23-3 mice and a mouse line expressing the photoconvertible protein KikGR to track MLN CD4+T cells. Photoconverted MLN CD44hiCD62LloCD4+T cells migrated predominantly to the BM; pit formation assay proved their ability to promote bone damage via osteoclasts. Significantly greater expression of IL-4 mRNA in MLN CD44hiCD62LloCD4+T cells and bone was observed in EW-fed OVA23-3 mice. Anti-IL-4 monoclonal antibody injection canceled bone loss in the primary inflammation phase in EW-fed mice, but less so in the chronic phase. This novel report shows the specific inflammatory relationship, via Th2-dominant-OVA-specific T cells and IL-4 production, between MLNs and bone, a distant organ, in food-allergic enteropathy.

Intestinal inflammation can be accompanied by osteoporosis, but their relationship, mediated by immune responses, remains unclear. Here, we investigated a non-IgE-mediated food-allergic enteropathy model of ovalbumin (OVA) 23-3 mice expressing OVA-specific T-cell-receptor transgenes. Mesenteric lymph nodes (MLNs) and their pathogenic CD4+T cells were important to enteropathy occurrence and exacerbation when the mice were fed an egg-white (EW) diet. EW-fed OVA23-3 mice also developed bone loss and increased CD44hiCD62LloCD4+T cells in the MLNs and bone marrow (BM); these changes were attenuated by MLN, but not spleen, resection. We fed an EW diet to F1 cross offspring from OVA23-3 mice and a mouse line expressing the photoconvertible protein KikGR to track MLN CD4+T cells. Photoconverted MLN CD44hiCD62LloCD4+T cells migrated predominantly to the BM; pit formation assay proved their ability to promote bone damage via osteoclasts. Significantly greater expression of IL-4 mRNA in MLN CD44hiCD62LloCD4+T cells and bone was observed in EW-fed OVA23-3 mice. Anti-IL-4 monoclonal antibody injection canceled bone loss in the primary inflammation phase in EW-fed mice, but less so in the chronic phase. This novel report shows the specific inflammatory relationship, via Th2-dominant-OVA-specific T cells and IL-4 production, between MLNs and bone, a distant organ, in food-allergic enteropathy.

Gastric adenocarcinoma cells secrete sulfomucins, but their role in gastric tumorigenesis remains unclear. To address that question, we generated A4gnt/Chst4double-knockout (DKO) mice by crossing A4gntknockout (KO) mice, which spontaneously develop gastric adenocarcinoma, with Chst4KO mice, which are deficient in the sulfotransferase GlcNAc6ST-2. A4gnt/Chst4DKO mice lack gastric sulfomucins but developed gastric adenocarcinoma. Unexpectedly, severe gastric erosion occurred in A4gnt/Chst4DKO mice at as early as 3 weeks of age, and with aging these lesions were accompanied by gastritis cystica profunda (GCP). Cxcl1, Cxcl5, Ccl2, and Cxcr2transcripts in gastric mucosa of 5-week-old A4gnt/Chst4DKO mice exhibiting both hyperplasia and severe erosion were significantly upregulated relative to age-matched A4gntKO mice, which showed hyperplasia alone. However, upregulation of these genes disappeared in 50-week-old A4gnt/Chst4DKO mice exhibiting high-grade dysplasia/adenocarcinoma and GCP. Moreover, Cxcl1and Cxcr2were downregulated in A4gnt/Chst4DKO mice relative to age-matched A4gntKO mice exhibiting adenocarcinoma alone. These combined results indicate that the presence of sulfomucins prevents severe gastric erosion followed by GCP in A4gntKO mice by transiently regulating a set of inflammation-related genes, Cxcl1, Cxcl5, Ccl2, and Cxcr2at 5 weeks of age, although sulfomucins were not directly associated with gastric cancer development:

The cytokines IL-17A and IL-17F have 50% amino-acid identity and bind the same receptor; however, their functional differences have remained obscure. Here we found that Il17f–/–mice resisted chemically induced colitis, but Il17a–/–mice did not, and that Il17f−/−CD45RBhiCD4+T cells induced milder colitis in lymphocyte-deficient Rag2–/–mice, accompanied by an increase in intestinal regulatory T cells (Tregcells). Clostridiumcluster XIVa in colonic microbiota capable of inducing Tregcells was increased in both Il17f−/−mice and mice given transfer Il17f−/−T cells, due to decreased expression of a group of antimicrobial proteins. There was substantial production of IL-17F, but not of IL-17A, not only by naive T cells but also by various colon-resident cells under physiological conditions. Furthermore, antibody to IL-17F suppressed the development of colitis, but antibody to IL-17A did not. These observations suggest that IL-17F is an effective target for the treatment of colitis. The cytokines IL-17A and IL-17F bind via same receptor. Iwakura and colleagues demonstrate different functions for IL-17A and IL-17F in the gut, with the latter altering the production of antimicrobial peptides with consequent effects on the microbiota, the induction of Tregcells and immune-system homeostasis.

Hematopoietic stem cell (HSC) transplantation (HSCT) for malignancy requires toxic pre‐conditioning to maximize anti‐tumor effects and donor‐HSC engraftment. While this induces bone marrow (BM)‐localized inflammation, how this BM environmental change affects transplanted HSCs in vivo remains largely unknown. We here report that, depending on interval between irradiation and HSCT, residence within lethally irradiated recipient BM compromises donor‐HSC reconstitution ability. Both in vivo and in vitro we demonstrate that, among inflammatory cytokines, TNF‐α plays a role in HSC damage: TNF‐α stimulation leads to accumulation of reactive oxygen species (ROS) in highly purified hematopoietic stem/progenitor cells (HSCs/HSPCs). Transplantation of flow‐cytometry—sorted murine HSCs reveals damaging effects of accumulated ROS on HSCs. Short‐term incubation either with an specific inhibitor of tumor necrosis factor receptor 1 signaling or an antioxidant N‐acetyl‐L‐cysteine (NAC) prevents TNF‐α‐mediated ROS accumulation in HSCs. Importantly, pre‐transplantation exposure to NAC successfully demonstrats protective effects in inflammatory BM on graft‐HSCs, exhibiting better reconstitution capability than that of nonprotected control grafts. We thus suggest that in vivo protection of graft‐HSCs from BM inflammation is a feasible and attractive approach, which may lead to improved hematopoietic reconstitution kinetics in transplantation with myeloablative conditioning that inevitably causes inflammation in recipient BM. StemCells2017;35:989–1002 Schematic illustration of the “HSC protection in transplantation” concept that we proposed in this study. Following harsh pre‐conditioning, a certain level of inflammation occurs within the bone marrow environment. At the site of injury, the presence of TNF‐α will lead to an accumulation of reactive oxygen species within graft‐hematopoietic stem cells (HSCs) resulting in perturbed hematopoiesis. Prevention of these negative outcomes may be achieved by pre‐incubating HSCs with N‐acetylcysteine. In this scenario, graft‐HSCs are considered to be “protected” within an inflammation‐prone microenvironment thus improving hematopoiesis.

Hematopoietic cell transplantation has proven beneficial for various intractable diseases, but it remains unclear how hematopoietic stem/progenitor cells (HSPCs) home to the bone marrow (BM) microenvironment, initiate hematopoietic reconstitution, and maintain life‐long hematopoiesis. The use of newly elucidated molecular determinants for overall HSPC engraftment should benefit patients. Here, we report that modification of C‐X‐C chemokine receptor type 4 (Cxcr4) signaling in murine HSPCs does not significantly affect initial homing/lodging events, but leads to alteration in subsequent BM repopulation kinetics, with observations confirmed by both gain‐ and loss‐of‐function approaches. By using C‐terminal truncated Cxcr4 as a gain‐of‐function effector, we demonstrated that signal augmentation likely led to favorable in vivo repopulation of primitive cell populations in BM. These improved features were correlated with enhanced seeding efficiencies in stromal cell cocultures and altered ligand‐mediated phosphorylation kinetics of extracellular signal‐regulated kinases observed in Cxcr4 signal‐augmented HSPCs in vitro. Unexpectedly, however, sustained signal enhancement even with wild‐type Cxcr4 overexpression resulted in impaired peripheral blood (PB) reconstitution, most likely by preventing release of donor hematopoietic cells from the marrow environment. We thus conclude that timely regulation of Cxcr4/CXCR4 signaling is key in providing donor HSPCs with enhanced repopulation potential following transplantation, whilst preserving the ability to release HSPC progeny into PB for improved transplantation outcomes. StemCells2014;32:1929–1942

The mechanism by which thrombotic vessel occlusion occurs independently of plaque development or endothelial cell (EC) disruption remains unclear, largely because of an inability to visualize the formation of thrombus, especially at the single-platelet level in real time. Here we demonstrate that rapidly developing thrombi composed of discoid platelets can be induced in the mesenteric capillaries, arterioles, and large-sized arteries of living mice, enabling characterization of the kinetics of thrombosis initiation and the multicellular interrelationships during thrombus development. Platelet aggregation without EC disruption was triggered by reactive oxygen species (ROS) photochemically induced by moderate power laser irradiation. The inflammatory cytokines TNF-α and IL-1 could be key components of the EC response, acting through regulation of VWF mobilization to the cell surface. Thrombus formation was then initiated by the binding of platelet GPIbα to endothelial VWF in our model, and this effect was inhibited by the ROS scavenger N-acetylcysteine. Actin linker talin-dependent activation of alphaIIb-beta3 integrin or Rac1 in platelets was required for late-phase thrombus stability. Our novel imaging technology illustrates the molecular mechanism underlying inflammation-based thrombus formation by discoid platelets on undisrupted ECs and suggests control of ROS could be a useful therapeutic target for the prevention of thrombotic diseases.

The mechanism by which thrombotic vessel occlusion occurs independently of plaque development or endothelial cell (EC) disruption remains unclear, largely because of an inability to visualize the formation of thrombus, especially at the single-platelet level in real time. Here we demonstrate that rapidly developing thrombi composed of discoid platelets can be induced in the mesenteric capillaries, arterioles, and large-sized arteries of living mice, enabling characterization of the kinetics of thrombosis initiation and the multicellular interrelationships during thrombus development. Platelet aggregation without EC disruption was triggered by reactive oxygen species (ROS) photochemically induced by moderate power laser irradiation. The inflammatory cytokines TNF-α and IL-1 could be key components of the EC response, acting through regulation of VWF mobilization to the cell surface. Thrombus formation was then initiated by the binding of platelet GPIbα to endothelial VWF in our model, and this effect was inhibited by the ROS scavenger N-acetylcysteine. Actin linker talin-dependent activation of alphaIIb-beta3 integrin or Rac1 in platelets was required for late-phase thrombus stability. Our novel imaging technology illustrates the molecular mechanism underlying inflammation-based thrombus formation by discoid platelets on undisrupted ECs and suggests control of ROS could be a useful therapeutic target for the prevention of thrombotic diseases.

Kjellmaniella crassifoliaMiyabe (gagome) is a brown alga. Oral gagome administration (oral gagome) resulted in significant upregulation of IL-10 and IFNγ production by Peyer’s patch cells. To assess the adjuvant activity of oral gagome, treated mice were subcutaneously injected with ovalbumin (OVA). The production of cytokines from antigen (Ag)-specific T cells in draining lymph nodes (dLN) and their proliferative response were significantly increased as compared with the control group. These enhancements were associated with increased CD44hiCD62L−activated/memory T cells in dLN as well as upregulation of Ag-specific IgA level in luminal contents. No upregulation of cytokine production by dLN T cells was observed in dectin-1-deficient mice, suggesting that the effect of gagome on cytokine production is largely dependent on the dectin-1 pathway despite its composite constituents. Our findings indicate that gagome is an effective immunomodulator and a potent adjuvant for both the intestinal and the systemic immune response.

2′,5′-Oligoadenylate synthetase (2-5OAS) is one of the interferon (IFN)-induced proteins and mediates the antiviral action of IFN. In human, three classes of 2-5OAS genes (OAS1, OAS2, and OAS3) and one OAS-like gene (OASL) are reported. In mice, however, OAS genes corresponding to human OAS2 and OAS3 have not been identified. In this report, we identified six novel OAS family genes in mice by screening mouse genomic library and expressed sequence tag (EST) database. These genes include three homologs of the human OAS1 and each homologous gene of the human OAS2, OAS3, and OASL, respectively. Each gene displays 52%-65% amino acid identity to the corresponding human homologs. Nine 2-5OAS genes, except for two OASL genes, locate within the 210-kb genomic region and form a cluster. Each novel 2-5OAS gene showed a characteristic expression pattern among different tissues, and all of them were induced by polyinosinic-polycytidylic acid. Biochemical analyses using recombinant proteins produced in Escherichia coli showed that all the novel mouse 2-5OAS molecules have double-stranded RNA (dsRNA) binding ability, but they do not have 2-5OAS activity except for the OAS2 and OAS3 mouse homologs. These results show that there are at least 11 OAS genes, which are classified into four groups, in the mouse.

The glycolipid α -galactosylceramide (α -GalCer), which is presented by CD1d and specifically activates Vα 14 NKT cells, exerts a potent anti-metastatic effect when administered in vivo. In this study, we demonstrated that α -GalCer administration led to rapid elimination of NKT cells by apoptosis in the liver and spleen, after they produced IFN-γ  and IL-4. In contrast, a more prolonged secretion of IFN-γ  was observed by liver and splenic NK cells after α -GalCer administration. Cytotoxic activity of liver mononuclear cells was not augmented 3h after α -GalCer administration, but was increased at 24 h when NKT cells were mostly depleted. The α -GalCer-induced cytotoxic activity was abolished in IFN-γ -deficient and NK cell-depleted mice as well as CD1-deficient mice, suggesting that the α -Galcer-induced cytotoxicity was mainly mediated by IFN-γ -activated NK cells. While the α -GalCer-induced cytotoxicity in vitro was mostly perforin dependent, anti-metastatic effect of α -GalCer was impaired in NK cell-depleted or IFN-γ -deficient mice but not in perforin-deficient mice. Collectively, these results indicated that the anti-metastatic effect of α -GalCer is mainly mediated by NK cells, which are activated secondarily by IFN-γ  produced by α -GalCer-activated NKT cells, in aperforin-independent manner.

Concanavalin A (Con A)-induced hepatitis is an experimental hepatitis model in which hepatic injury is caused by the action of cytokines produced by T cells. Using IFN-γ-deficient mice, we previously demonstrated that IFN-γ plays a central role in Con A-induced hepatitis. Here, we show that development of the disease is completely suppressed in gld/gld mice, in which Fas ligand is defective. In contrast, suppression of the disease in lpr/lpr mice was incomplete, since a small amount of the fas mRNA was produced in these mice. The data indicate that activation of the Fas/Fas ligand system is a necessary step in the development of Con A-induced hepatitis. Furthermore, we found that not only fas but also caspase-1 expression was reduced in IFN-γ-deficient mice. Since caspase-1 is an integral component of Fas signal transduction, these observations suggest that IFN-γ-induced activation of both fas and caspase-1 expression causes enhancement of hepatocyte apoptosis resulting in the development of hepatitis.

Abstract 1888

No relevant conflicts of interest to declare.

A very-high-frequency (VHF) plasma at 100 MHz is experimentally investigated in parallel-plate configuration in H2using spatiotemporal optical emission spectroscopy and current-voltage-power waveforms. The observed optical emission intensity and net excitation rate profiles have strong temporal and spatial variations. The VHF plasma is still capacitive, and is maintained by reflected electrons in the oscillating sheath next to the instantaneous cathode. The characteristics of VHF plasmas, that the excitation rate increases in proportion to the square of driving frequency, and that the minimum sustaining voltage and pressure decrease with increasing frequency, are experimentally confirmed at pressures, ranging from 0.01 to 2 Torr and at the frequency of 100 MHz .