Murine model identifies tropomyosin as IgE cross-reactive protein between house dust mite and coho salmon that possibly contributes to the development of salmon allergy.

Background: Recently, we have developed a method to identify IgE cross-reactive allergens. However, the mechanism by which IgE cross-reactive allergens cause food allergy is not yet fully understood how. In this study, we aimed to understand the underlying pathogenesis by identifying food allergens that cross-react with house dust mite allergens in a murine model. Material and methods: Allergenic protein microarray analysis was conducted using serum from mice intraperitoneally injected with Dermatophagoides pteronyssinus (Der p) extract plus alum or alum alone as controls. Der p, Dermatophagoides farinae (Der f), coho salmon extract-sensitized and control mice were analyzed. Serum levels of IgE against Der p, Der f, coho salmon extract, protein fractions of coho salmon extract separated by ammonium sulfate precipitation and anion exchange chromatography, and recombinant coho salmon tropomyosin or actin were measured by an enzyme-linked immunosorbent assay. A murine model of cutaneous anaphylaxis or oral allergy syndrome (OAS) was established in Der p extract-sensitized mice stimulated with coho salmon extract, tropomyosin, or actin. Results: Protein microarray analysis showed that coho salmon-derived proteins were highly bound to serum IgE in Der p extract-sensitized mice. Serum IgE from Der p or Der f extract-sensitized mice was bound to coho salmon extract, whereas serum IgE from coho salmon extract-sensitized mice was bound to Der p or Der f extract. Analysis of the murine model showed that cutaneous anaphylaxis and oral allergic reaction were evident in Der p extract-sensitized mice stimulated by coho salmon extract. Serum IgE from Der p or Der f extract-sensitized mice was bound strongly to protein fractions separated by anion exchange chromatography of coho salmon proteins precipitated with 50% ammonium sulfate, which massively contained the approximately 38 kDa protein. We found that serum IgE from Der p extract-sensitized mice was bound to recombinant coho salmon tropomyosin. Der p extract-sensitized mice exhibited cutaneous anaphylaxis in response to coho salmon tropomyosin. Conclusion: Our results showed IgE cross-reactivity of tropomyosin between Dermatophagoides and coho salmon which illustrates salmon allergy following sensitization with the house dust mite Dermatophagoides. Our method for identifying IgE cross-reactive allergens will help understand the underlying mechanisms of food allergies.

Dll1 Can Function as a Ligand of Notch1 and Notch2 in the Thymic Epithelium.

T-cell development in the thymus is dependent on Notch signaling induced by the interaction of Notch1, present on immigrant cells, with a Notch ligand, delta-like (Dll) 4, on the thymic epithelial cells. Phylogenetic analysis characterizing the properties of the Dll4 molecule suggests that Dll4 emerged from the common ancestor of lobe- and ray-finned fishes and diverged into bony fishes and terrestrial organisms, including mammals. The thymus evolved in cartilaginous fishes before Dll4, suggesting that T-cell development in cartilaginous fishes is dependent on Dll1 instead of Dll4. In this study, we compared the function of both Dll molecules in the thymic epithelium using Foxn1-cre and Dll4-floxed mice with conditional transgenic alleles in which the Dll1 or Dll4 gene is transcribed after the cre-mediated excision of the stop codon. The expression of Dll1 in the thymic epithelium completely restored the defect in the Dll4-deficient condition, suggesting that Dll1 can trigger Notch signaling that is indispensable for T-cell development in the thymus. Moreover, using bone marrow chimeras with Notch1- or Notch2-deficient hematopoietic cells, we showed that Dll1 is able to activate Notch signaling, which is sufficient to induce T-cell development, with both the receptors, in contrast to Dll4, which works only with Notch1, in the thymic environment. These results strongly support the hypothesis that Dll1 regulates T-cell development via Notch1 and/or Notch2 in the thymus of cartilaginous fishes and that Dll4 has replaced Dll1 in inducing thymic Notch signaling via Notch1 during evolution.

Anti-CD80/86 antibodies inhibit inflammatory reaction and improve graft survival in a high-risk murine corneal transplantation rejection model.

We investigated the effects of anti-CD80/86 antibodies in a murine high-risk corneal transplantation rejection model. A mixed lymphocyte reaction (MLR) assay was conducted with anti-CD80/86 antibodies. Inflammatory cytokine levels in the culture supernatant were measured using an enzyme-linked immunosorbent assay. Interferon (IFN)-γ-producing CD4+ T cell frequencies in the MLR were assessed using flow cytometry. In vivo, high-risk corneal allograft survival and IFN-γ-producing CD4+ T cell frequencies in corneal grafts were assessed with intraperitoneal injection of anti-CD80/86 antibodies compared to phosphate-buffered saline (PBS). RNA-sequencing was performed on corneal grafts 2 weeks post-transplantation. Anti-CD80/86 antibodies significantly decreased T-cell proliferation, IFN-γ+-producing CD4+ T cell frequencies, and IFN-γ, interleukin (IL)-1ß, IL-2, IL-10, and tumor necrosis factor-α production in the MLR compared to PBS injection. Intraperitoneal injection of anti-CD80/86 antibodies significantly prolonged corneal graft survival and decreased IFN-γ+-producing CD4+ T cell frequencies compared to PBS injection. Gene set enrichment analysis showed that the gene sets mainly enriched in the control group were related to allograft rejection and inflammatory response compared to PBS injection. Anti-CD80/86 antibodies significantly prolonged corneal graft survival by inhibiting T-cell proliferation and inflammatory response.

Ex Vivo-Induced Bone Marrow-Derived Myeloid Suppressor Cells Prevent Corneal Allograft Rejection in Mice.

Purpose: To investigate the effects of ex vivo-induced bone marrow myeloid-derived suppressor cells (BM-MDSCs) on allogeneic immune responses in corneal transplantation. Methods: Bone marrow cells from C57BL/6J (B6) mice were cultured with IL-6 and GM-CSF for four days. The ex vivo induction of the BM-MDSCs was assessed using flow cytometry, inducible nitric oxide synthase (iNOS) mRNA expression using reverse transcription-quantitative polymerase chain reaction, and nitric oxide (NO) production in allogeneic stimulation. T-cell proliferation and regulatory T-cell (Treg) expansion were investigated on allogeneic stimulation in the presence of ex vivo-induced BM-MDSCs. IFN-γ, IL-2, IL-10, and TGF-ß1 protein levels were measured using enzyme-linked immunosorbent assays. After subconjunctival injection of ex vivo-induced BM-MDSCs, the migration of the BM-MDSCs into corneal grafts, allogeneic corneal graft survival, neovascularization, and lymphangiogenesis were assessed using flow cytometry, slit-lamp microscopy, and immunohistochemistry. Results: The combination of GM-CSF and IL-6 significantly induced BM-MDSCs with increased iNos mRNA expression. The ex vivo-induced BM-MDSCs promoted NO release in allogeneic stimulation in vitro. The ex vivo-induced BM-MDSCs inhibited T-cell proliferation and promoted Treg expansion. Decreased IFN-γ and increased IL-2, IL-10, and TGF-ß1 production was observed in coculture of ex vivo-induced BM-MDSCs. Injected ex vivo-induced BM-MDSCs were confirmed to migrate into the grafts. The injected BM-MDSCs also prolonged corneal graft survival and prevented angiogenesis and lymphangiogenesis. Conclusions: The ex vivo-induced BM-MDSCs have suppressive effects on allogeneic immune responses and prolong corneal allograft survival via the iNOS pathway, indicating that they may be a potential therapeutic tool for corneal transplantation.

Novel immunotherapeutic effects of topically administered ripasudil (K-115) on corneal allograft survival.

Corneal allograft survival is mediated by the variety of immunological reactions and wound healing process. Our aim was to explore the effects of topical administration of ripasudil, a selective Rho-associated coiled-coil protein kinase inhibitor, on corneal allograft survival. Ripasudil was administered to mice thrice a day after allogeneic corneal transplantation. Corneal graft survival, opacity, neovascularization, re-epithelization, immune cell infiltration, and mRNA levels of angiogenic and pro-inflammatory factors in the grafted cornea and draining lymph nodes (dLNs) were evaluated with slit-lamp microscopy, immunohistochemistry, flow cytometry, and polymerase chain reaction. Graft survival was significantly prolonged with lower graft opacity and neovascularization scores in 0.4% and 2.0% ripasudil-treated groups, and mRNA levels of angiogenic and pro-inflammatory factors in ripasudil-treated grafted corneas were reduced. Moreover, 0.4% and 2.0% ripasudil reduced CD45+-infiltrated leukocyte frequency, Cd11b and Cd11c mRNA levels, and the frequencies of mature dendritic cells, IFNγ-, and IL-17- producing CD4+T cells in the dLNs of recipients. Re-epithelization rate of the grafted cornea was significantly higher in the 0.4% and 2.0% ripasudil groups than in the control. Topically applied ripasudil prolonged graft survival by downregulating neovascularization and inflammation factors, while promoting corneal re-epithelization, suggesting that ripasudil may be useful for suppressing immunological rejection in corneal transplantation.

CD155-Transducing Signaling through TIGIT Plays an Important Role in Transmission of Tolerant State and Suppression Capacity.

The precise mechanism of how the regulatory T cell population elicits and maintains tolerant state in activated T cells is poorly understood. To address this issue, we established an in vitro coculture system using mouse T cells and showed that tolerant state is serially passed from preinduced-tolerant T cells into new TCR-stimulated T cells across generations in a dendritic cell-independent manner. In this successive induction process of tolerant state, TIGIT was found to play an important role: TIGIT expression on induced-tolerant T cells was promoted in stimulated T cells cocultured with the tolerant cells. In addition, these stimulated T cells in the coculture also expressed high B lymphocyte-induced maturation protein 1 accompanied by IL-2 suppression. Because CD155, a partner of TIGIT, is known to transduce signaling inside by trans-interaction with its ligands, these phenotypical changes in TCR-stimulated naive T cells were reproduced when naive T cells were double cross-linked by CD3 and CD155. These results indicate that TIGIT enhanced on tolerant T cells may function as a ligand of its paired receptor CD155 to transduce signaling into its expressing naive T cells to accelerate new TIGIT expressions as well as IL-2 suppression via B lymphocyte-induced maturation protein 1 enhancement. In consideration of these results, we propose a novel process in which tolerant state in T cell population is maintained by successive generation of new tolerant T cells from naive T cells as one of the regulating mechanisms in immune responses.

Allogeneic Neural Stem/Progenitor Cells Derived From Embryonic Stem Cells Promote Functional Recovery After Transplantation Into Injured Spinal Cord of Nonhuman Primates.

UNLABELLED: : Previous studies have demonstrated that neural stem/progenitor cells (NS/PCs) promote functional recovery in rodent animal models of spinal cord injury (SCI). Because distinct differences exist in the neuroanatomy and immunological responses between rodents and primates, it is critical to determine the effectiveness and safety of allografted embryonic stem cell (ESC)-derived NS/PCs (ESC-NS/PCs) in a nonhuman primate SCI model. In the present study, common marmoset ESC-NS/PCs were grafted into the lesion epicenter 14 days after contusive SCI in adult marmosets (transplantation group). In the control group, phosphate-buffered saline was injected instead of cells. In the presence of a low-dose of tacrolimus, several grafted cells survived without tumorigenicity and differentiated into neurons, astrocytes, or oligodendrocytes. Significant differences were found in the transverse areas of luxol fast blue-positive myelin sheaths, neurofilament-positive axons, corticospinal tract fibers, and platelet endothelial cell adhesion molecule-1-positive vessels at the lesion epicenter between the transplantation and control groups. Immunoelectron microscopic examination demonstrated that the grafted ESC-NS/PC-derived oligodendrocytes contributed to the remyelination of demyelinated axons. In addition, some grafted neurons formed synaptic connections with host cells, and some transplanted neurons were myelinated by host cells. Eventually, motor functional recovery significantly improved in the transplantation group compared with the control group. In addition, a mixed lymphocyte reaction assay indicated that ESC-NS/PCs modulated the allogeneic immune rejection. Taken together, our results indicate that allogeneic transplantation of ESC-NS/PCs from a nonhuman primate promoted functional recovery after SCI without tumorigenicity. SIGNIFICANCE: This study demonstrates that allogeneic embryonic stem cell (ESC)-derived neural stem/progenitor cells (NS/PCs) promoted functional recovery after transplantation into the injured spinal cord in nonhuman primates. ESC-NS/PCs were chosen because ESC-NS/PCs are one of the controls for induced pluripotent stem cell-derived NS/PCs and because ESC derivatives are possible candidates for clinical use. This translational research using an allograft model of a nonhuman primate is critical for clinical application of grafting NS/PCs derived from various allogeneic pluripotent stem cells, especially induced pluripotent stem cells, into injured spinal cord at the subacute phase.

Delta-like 4-mediated Notch signaling is required for early T-cell development in a three-dimensional thymic structure.

Delta-like 4 (Dll4)-mediated Notch signaling is critical for specifying T-cell fate, but how Dll4-mediated Notch signaling actually contributes to T-cell development in the thymus remains unclear. To explore this mechanism in the thymic three-dimensional structure, we performed fetal thymus organ culture using Dll4-deficient mice. DN1a/b+DN2mt cells, which had not yet committed to either the αß T or γδ T/NK cell lineage, did not differentiate into the αß T-cell lineage in Dll4-deficient thymus despite the lack of cell fate conversion into other lineages. However, DN3 cells efficiently differentiated into a later developmental stage of αß T cells, the double-positive (DP) stage, although the proliferation was significantly impaired during the differentiation process. These findings suggest that the requirement for Notch signaling differs between the earliest and pre-TCR-bearing precursors and that continued Notch signaling is required for proper differentiation with active proliferation of αß T lineage cells. Furthermore, we showed that Notch signaling increased the c-Myc expression in DN3 cells in the thymus and that its overexpression rescued the proliferation and differentiation of DN3 cells in the Dll4-null thymus. Therefore, c-Myc plays a central role in the transition from stage DN3 to DP as a downstream target of Notch signaling.

Disappearance of centroacinar cells in the Notch ligand-deficient pancreas.

Notch signaling has been shown to contribute to murine pancreatic development at various stages. Delta-like 1 (Dll1) or Jagged1 (Jag1) are the Notch ligands that solely function to trigger this signaling during the pancreatic bud stage (~e9.5) or after birth, respectively. However, it has not been elucidated whether these Notch ligands are required at the later stage (e10.5-18.5) when the particular pancreas structures form. Here, we detected the dual expression of Dll1 and Jag1 in the epithelium after e10.5, which was restricted to the ductal cell lineage, including centroacinar cells expressing Sox9, CD133 and Hes1 but not the ductal cell markers Hnf1ß and DBA, at e18.5. To evaluate the significance of the Notch ligands during this period, we established double-floxed mice of Dll1 and Jag1 genes with Ptf1a-Cre knock-in allele and examined the effects on development. The abrogation of both ligands but not a single one led to the loss of centroacinar cells, which was due to the decrease in cell proliferation and the increase in cell death, as well as to the reduction of Sox9. These results suggested that Dll1 and Jag1 function redundantly and are necessary to maintain the centroacinar cells as an environmental niche in the developing pancreas.

Commensal bacteria regulate thymic Aire expression.

Commensal bacteria in gastrointestinal tracts are reported to function as an environmental factor to regulate intestinal inflammation and immune responses. However, it remains largely unknown whether such bacterial function exerts any effect on other immune organs distant from the intestine. In this study, the influence of commensal bacteria in the thymus, where T cell lineages develop into mature type to form proper repertoires, was investigated using germ-free (GF) mice and Nod1-deficient mice lacking an intracellular recognition receptor for certain bacterial components, in which a commensal bacterial effect is predicted to be less. In both mice, there was no significant difference in the numbers and subset ratios of thymocytes. Interestingly, however, autoimmune regulator (Aire) expression in thymic epithelial cells (TECs), main components of the thymic microenvironment, was decreased in comparison to specific pathogen-free (SPF) mice and Nod1 wild-type (WT) mice, respectively. In vitro analysis using a fetal thymus organ culture (FTOC) system showed that Aire expression in TECs was increased in the presence of a bacterial component or a bacterial product. These results suggest that through their products, commensal bacteria have the potential to have some effect on epithelial cells of the thymus in tissues distant from the intestine where they are originally harbored.

Effective expansion of engrafted human hematopoietic stem cells in bone marrow of mice expressing human Jagged1.

The human immune system can be reconstituted in experimental animals by transplanting human hematopoietic stem cells (hHSCs) into immunodeficient mice. To generate such humanized mice, further improvements are required, particularly to ensure that transplanted hHSCs are maintained in mice and proliferate long enough to follow prolonged immune responses to chronic diseases or monitor therapeutic effects. To prepare the relatively human bone marrow environment in mice, we generated nonobese diabetic/severe combined immunodeficiency/interleukin-2 receptor gamma chain null (NOG) mice expressing human Jagged1 (hJ1) in an osteoblast-specific manner (hJ1-NOG mice) to examine whether Notch signaling induced by hJ1 mediates hHSC proliferation and/or maintenance in mice. The established hJ1-NOG mice possess relatively larger bone marrow space and thinner cortical bone compared with nontransgenic littermates, but the number of c-kit(+) Sca-1(+) lineage(-) cells was not significantly different between hJ1-NOG and nontransgenic littermates. In the transplantation experiments of CD34(+) cells obtained from human cord blood, CD34(+)CD38(-) cells (hHSCs) were more increased in hJ1-NOG recipient mice than in nontransgenic littermates in mouse bone marrow environment. In contrast, the transplanted mouse c-kit(+) Sca-1(+) lineage(-) cells did not show significant increase in the same hJ1-NOG mice. These results suggest that hJ1-NOG mice could contribute to the growth of transplanted human CD34(+) cells in a human-specific manner and be useful to study the in vivo behavior and/or development of human stem cells, including cancer stem cells and immune cells.

Osteosclerosis and inhibition of human hematopoiesis in NOG mice expressing human Delta-like 1 in osteoblasts.

NOD/Shi-scid IL2rγnull (NOG) mice with severe immunodeficiency are excellent recipients to generate "humanized" mice by the transplantation of human CD34(+) hematopoietic stem cells (HSCs). In this study, we developed NOG mice carrying a human Delta-like1 (DLL1) gene, which is a ligand of the Notch receptor and is known to be important in HSC maintenance and self-renewal. We also analyzed the effect of DLL1 signaling on human hematopoiesis and HSC maintenance using humanized DLL1 transgenic NOG mice. To develop DLL1 transgenic NOG (NOG-D1-Tg) mice, a transgenic vector consisting of a human DLL1 complementary DNA fragment placed downstream of the α1(I) collagen (Col1a1) promoter for expression specifically in osteoblasts was constructed. Human CD34(+) HSCs were transplanted into NOG-D1-Tg mice, and differentiation of lymphoid or myeloid lineage cells from human HSCs and maintenance of HSCs in bone marrow were analyzed. Severe osteosclerosis accompanied by increased bone mass and a decreased number of bone marrow cells were observed in NOG-D1-Tg mice. After human HSC transplantation, development of human B lymphocytes, but not T lymphocytes, was significantly suppressed in both bone marrow and the periphery of NOG-D1-Tg mice. Contrary to the initial expectation, retention of human CD34(+) HSCs was inhibited in the bone marrow of NOG-D1-Tg mice. In conclusion, our data suggest that the development of human B lymphocytes and HSC maintenance in osteosclerotic bone may be suppressed by introducing DLL1. These unique humanized mice with sclerotic bone reconstituted by human HSCs are useful models of hematopoiesis in patients with osteosclerosis, such as osteopetrosis, and for investigation of osteogenesis via Notch signaling.

Delta-like 4 is indispensable in thymic environment specific for T cell development.

The thymic microenvironment is required for T cell development in vivo. However, in vitro studies have shown that when hematopoietic progenitors acquire Notch signaling via Delta-like (Dll)1 or Dll4, they differentiate into the T cell lineage in the absence of a thymic microenvironment. It is not clear, however, whether the thymus supports T cell development specifically by providing Notch signaling. To address this issue, we generated mice with a loxP-flanked allele of Dll4 and induced gene deletion specifically in thymic epithelial cells (TECs). In the thymus of mutant mice, the expression of Dll4 was abrogated on the epithelium, and the proportion of hematopoietic cells bearing the intracellular fragment of Notch1 (ICN1) was markedly decreased. Corresponding to this, CD4 CD8 double-positive or single-positive T cells were not detected in the thymus. Further analysis showed that the double-negative cell fraction was lacking T cell progenitors. The enforced expression of ICN1 in hematopoietic progenitors restored thymic T cell differentiation, even when the TECs were deficient in Dll4. These results indicate that the thymus-specific environment for determining T cell fate indispensably requires Dll4 expression to induce Notch signaling in the thymic immigrant cells.

Expression of Delta-like 1 in the splenic non-hematopoietic cells is essential for marginal zone B cell development.

The Notch ligand Delta-like 1 (Dll1) is critical for the generation of marginal zone (MZ) B cells in the spleen. However, the precise mechanism underlying the differentiation of MZB cells is unclear. To determine whether hematopoietic cells or non-hematopoietic cells provides the Dll1-mediated signals to primitive hematopoietic cells, we transplanted lineage(-)c-kit(+)Sca-1(+) (KSL) bone marrow cells derived from wild-type (Dll1(+/+)) GFP-transgenic mice into lethally irradiated Dll1 conditional knockout (cKO) mice. After transplantation, we examined the kinetics of hematopoietic reconstitution and found that although the frequency of stem/progenitor subsets and of more mature lymphoid, myeloid, and erythroid lineages were normal, the donor-derived hematopoietic cells failed to differentiate into MZB cells. We further demonstrated that while the splenic stromal cells of wild-type mice expressed Dll1 molecule, the splenic stromal cells of recipient Dll1 cKO mice deleted the expression of Dll1. These results suggesting that the expression of Dll1 in splenic non-hematopoietic stromal cells, but not hematopoietic cells, is essential for the development of MZB cells.

Notch signaling is necessary for GATA3 function in the initiation of T cell development.

GATA3 and Notch1 are essential for T cell development at the earliest stage, but their mutual roles in this process remain to be clarified. In this study, we demonstrated that impairment of T lymphopoiesis in hematopoietic progenitor cells (HPC) of GATA3-deficient fetal liver (FL) on day 11.5 of gestation (E11.5) was rescued only by introduction of both GATA3 and the intracellular region of Notch1 but not by either alone. However, the introduction of GATA3 only was sufficient for T cell induction in GATA3-deficient FL cells at the advanced stage, where Notch signaling is well detectable. This indicates that Notch signaling is necessary for GATA3 to function for T cell fate specification but is not sufficient without GATA3. On the other hand, Notch signaling is sufficient for blockage of B cell development without GATA3, suggesting that T cell fate specification at the branching point does not result simply from the developmental arrest of B cell lineage by Notch signaling.

Delta-like 1 is necessary for the generation of marginal zone B cells but not T cells in vivo.

Notch receptors and their ligands contribute to many developmental systems, but it is not apparent how they function after birth, as their null mutants develop severe defects during embryogenesis. Here we used the Cre-loxP system to delete the Delta-like 1 gene (Dll1) after birth and demonstrated the complete disappearance of splenic marginal zone B cells in Dll1-null mice. In contrast, T cell development was unaffected. These results demonstrated that Dll1 was dispensable as a ligand for Notch1 at the branch point of T cell-B cell development but was essential for the generation of marginal zone B cells. Thus, Notch signaling is essential for lymphocyte development in vivo, but there is a redundancy of Notch-Notch ligand signaling that can drive T cell development within the thymus.