Mucin degrader Akkermansia muciniphila accelerates intestinal stem cell-mediated epithelial development.

Mucin-degrading bacteria are densely populated in the intestinal epithelium; however, their interaction with intestinal stem cells (ISCs) and their progeny have not been elucidated. To determine whether mucin-degrading bacteria play a role in gut homeostasis, mice were treated with Akkermansia muciniphila, a specialized species that degrades mucin. Administration of A. muciniphila for 4 weeks accelerated the proliferation of Lgr5+ ISCs and promoted the differentiation of Paneth cells and goblet cells in the small intestine (SI). We found similar effects of A. muciniphila in the colon. The levels of acetic and propionic acids were higher in the cecal contents of A. muciniphila-treated mice than in PBS-treated mice. SI organoids treated with cecal contents obtained from A. muciniphila-treated mice were larger and could be diminished by treatment with G protein-coupled receptor (Gpr) 41/43 antagonists. Pre-treatment of mice with A. muciniphila reduced gut damage caused by radiation and methotrexate. Further, a novel isotype of the A. muciniphila strain was isolated from heathy human feces that showed enhanced function in intestinal epithelial regeneration. These findings suggest that mucin-degrading bacteria (e.g., A. muciniphila) may play a crucial role in promoting ISC-mediated epithelial development and contribute to intestinal homeostasis maintenance.

Food antigens drive spontaneous IgE elevation in the absence of commensal microbiota.

Immunoglobulin E (IgE), a key mediator in allergic diseases, is spontaneously elevated in mice with disrupted commensal microbiota such as germ-free (GF) and antibiotics-treated mice. However, the underlying mechanisms for aberrant IgE elevation are still unclear. Here, we demonstrate that food antigens drive spontaneous IgE elevation in GF and antibiotics-treated mice by generating T helper 2 (TH2)-skewed T follicular helper (TFH) cells in gut-associated lymphoid tissues (GALTs). In these mice, depriving contact with food antigens results in defective IgE elevation as well as impaired generation of TFH cells and IgE-producing cells in GALT. Food antigen-driven TFH cells in GF mice are mostly generated in early life, especially during the weaning period. We also reveal that food antigen-driven TFH cells in GF mice are actively depleted by colonization with commensal microbiota. Thus, our findings provide a possible explanation for why the perturbation of commensal microbiota in early life increases the occurrence of allergic diseases.

Interleukin-2/antibody complex expanding Foxp3＋ regulatory T cells exacerbates Th2-mediated allergic airway inflammation.

Foxp3＋ regulatory CD4＋ T (Treg) cells play an essential role in preventing overt immune responses against self and innocuous foreign antigens. Selective expansion of endogenous Treg cells in response to the administration of interleukin (IL)-2/antibody complex, such as the IL-2/JES6-1 complex (IL-2C) in mice, is considered an attractive therapeutic approach to various immune disorders. Here, we investigated the therapeutic potential of IL-2C in allergic airway inflammation models. IL-2C treatment ameliorated Th17-mediated airway inflammation; however, unexpectedly, IL-2C treatment exacerbated Th2-mediated allergic airway inflammation by inducing the selective expansion of Th2 cells and type-2 innate lymphoid cells. We also found that IL-2 signaling is required for the expansion of Th2 cells in lymphoproliferative disease caused by Treg cell depletion. Our data suggest that IL-2C is selectively applicable to the treatment of allergic airway diseases depending on the characteristics of airway inflammation. [BMB Reports 2019; 52(4): 283-288].

Roles of major histocompatibility complex class II in inducing protective immune responses to influenza vaccination.

Major histocompatibility complex class II-deficient (MHC-II KO; Aß(-/-)) mice were used to assess the roles of MHC-II molecules in inducing protective immune responses to vaccination. After vaccination with influenza A/PR8 virus-like particle (VLP) vaccine, in vivo and in vitro vaccine antigen-specific IgG isotype antibodies were not detected in MHC-II KO mice, which is quite different from CD4 T cell-deficient mice that induced vaccine-specific IgG antibodies. The deficiency in MHC-II did not significantly affect the induction of antigen-specific IgM antibody in sera. MHC-II KO mice that were vaccinated with influenza VLP, whole inactivated influenza virus, or live attenuated influenza virus vaccines were not protected against lethal infection with influenza A/PR8 virus. Adoptive transfer of fractionated spleen cells from wild-type mice to MHC-II KO mice indicated that CD43(+) cell populations with MHC-II contributed more significantly to producing vaccine-specific IgG antibodies than CD43(-) B220(+) conventional B cell or CD4 T cell populations, as well as conferring protection against lethal infection. Bone marrow-derived dendritic cells from MHC-II KO mice showed a significant defect in producing interleukin-6 and tumor necrosis factor alpha cytokines. Thus, results indicate that MHC-II molecules play multiple roles in inducing protective immunity to influenza vaccination. Importance: Major histocompatibility complex class II (MHC-II) has been known to activate CD4 T helper immune cells. A deficiency in MHC-II was considered to be equivalent to the lack of CD4 T cells in developing host immune responses to pathogens. However, the roles of MHC-II in inducing protective immune responses to vaccination have not been well understood. In the present study, we demonstrate that MHC-II-deficient mice showed much more significant defects in inducing protective antibody responses to influenza vaccination than CD4 T cell-deficient mice. Further analysis showed that CD43 marker-positive immune cells with MHC-II, as well as an innate immunity-simulating adjuvant, could rescue some defects in inducing protective immune responses in MHC-II-deficient mice. These results have important implications for our understanding of host immunity-inducing mechanisms to vaccination, as well as in developing effective vaccines and adjuvants.

Distinct B-cell populations contribute to vaccine antigen-specific antibody production in a transgenic mouse model.

The generation of memory B cells by vaccination plays a critical role in maintaining antigen-specific antibodies and producing antibody responses upon re-exposure to a pathogen. B-cell populations contributing to antibody production and protection by vaccination remain poorly defined. We used influenza virus-like particle (VLP) vaccine in a transgenic mouse model that would identify germinal centre-derived memory B cells with the expression of yellow fluorescent protein (YFP(+) cells). Immunization with influenza VLP vaccine did not induce significant increases in YFP(+) cells although vaccine antigen-specific antibodies in sera were found to confer protection against a lethal dose of influenza A virus (A/PR8). In addition, CD43(+)  B220(-) populations with low YFP(+) cells mainly contributed to the production of vaccine antigen-specific IgG isotype-switched antibodies whereas CD43(-)  B220(+) populations with high YFP(+) cells were able to produce vaccine antigen-specific IgM antibodies. Challenge infection of immunized transgenic mice with live influenza A virus resulted in significant increases in YFP(+) cells in the B220(-) populations of spleen and bone marrow cells. These results suggest that CD43(+)  B220(-) B cells generated by vaccination are important for producing influenza vaccine antigen-specific antibodies and conferring protection.

Proangiogenic features of Wharton's jelly-derived mesenchymal stromal/stem cells and their ability to form functional vessels.

Mesenchymal stromal/stem cells derived from human Wharton's jelly (WJ-MSC) have emerged as a favorable source for autologous and allogenic cell therapy. Here, we characterized the proangiogenic features of WJ-MSCs and examined their ability to form functional vessels in in vivo models. First, we examined whether WJ-MSCs express endothelial and smooth muscle cell specific markers after culture in endothelial growth media. WJ-MSCs expressed an endothelial specific marker, VEGFR1, at mRNA and protein levels, but did not express other specific markers (VEGFR2, Tie2, vWF, CD31, and VE-cadherin). Rather, WJ-MSCs expressed smooth muscle cell specific markers, α-SMA, PDGFR-ß and calponin, and were unable to form tube-like structures with lumen on Matrigel. WJ-MSCs secreted growth factors including angiogenin, IGFBP-3, MCP-1, and IL-8, which stimulated endothelial proliferation, migration, and tube formation. When WJ-MSCs suspended in Matrigel were implanted into nude mice, it led to formation of functional vessels containing erythrocytes after 7 days. However, implantation of endothelial cell-suspended Matrigel resulted in no perfused vessels. The implanted WJ-MSCs were stained positively for calponin or PDGFR-ß and were located adjacent to the lining of mouse endothelial cells that were stained with labeled BS-lectin B4. In a murine hindlimb ischemia model, the transplantation of MSCs (5×10(5)cells) into the ischemic limbs improved perfusion recovery and neovascularization of the limbs compared to control group. Therefore, the results suggest that WJ-MSCs promote neovascularization and perfusion by secreting paracrine factors and by functioning as perivascular precursor cells, and that WJ-MSCs can be used efficiently for cell therapy of ischemic disease.

The Rho kinase inhibitor fasudil augments the number of functional endothelial progenitor cells in ex vivo cultures.

Rho kinase (ROCK) has been implicated in the regulation of vascular tone, endothelial dysfunction, inflammation and remodeling. Endothelial progenitor cells (EPC) have been proven to have the efficacy of therapeutic neovascularization in ischemia. However, the scarcity of EPCs limits cell therapy. Using an in vitro EPC culture assay, Y27632 was found to increase the number of adherent EPCs. In this study, we investigated the effect of fasudil, another ROCK inhibitor being used in the clinic, on EPC number and examined whether EPCs expanded by fasudil are functional in vitro and in vivo. In ex vivo cultures of EPCs, fasudil effectively increased the number of ac-LDL/UEA-1 positive cells as well as adherent cells, in contrast to H89, a less selective ROCK inhibitor. Fasudil also increased EPC numbers in culture up to 10 µM, in a dose-dependent manner. When EPCs expanded with fasudil were examined for the migratory activity toward stromal cell-derived factor-1 and vascular endothelial growth factor, these cells retained functional properties in migration, albeit with some decrease. Fasudil-cultured EPCs labeled with PKH26 showed an activity similar to non-treated EPCs for cellular adhesion into an endothelial cell (EC) monolayer and incorporation into capillary-like structures formed by ECs. Finally, when EPCs cultured with fasudil (106 cells/mouse) were injected into ischemic limbs, these cells showed a blood flow recovery at a level comparable to non-treated control EPCs and increased neovascularization. Therefore, these data suggest that the ROCK inhibitor fasudil can provide a beneficial effect in the treatment of ischemic diseases by increasing EPC numbers.

Efficient nonadhesive ex vivo expansion of early endothelial progenitor cells derived from CD34+ human cord blood fraction for effective therapeutic vascularization.

Endothelial progenitor cells (EPCs) have been shown to have therapeutic potential in ischemic disease. However, the number of EPCs for cell therapy is limited. In this study, instead of the typical adherent culture method, we investigated a more efficient, clinically applicable nonadhesive expansion method for early EPCs using cord blood-derived cells to overcome rapid cellular senescence. After a suspension culture of isolated CD34(+) cells in serum-free medium containing each cytokine combination was maintained for 9 d, the number of expanded functional EPCs was assessed by an adherent culture assay. Compared to mononuclear cells, the CD34(+) fraction was superior in its expansion of functional EPCs that could differentiate into acLDL/UEA-1(+) cells without significant cellular senescence, whereas the CD34(-) fraction showed no EPC expansion. Among the cytokine combinations tested for the CD34(+) fraction, a combination (SFIb) consisting of stem cell factor (SCF), FMS-like tyrosine kinase 3 ligand, interleukin-3, and basic fibroblast growth factor resulted in a reproducible 64- to 1468-fold EPC expansion from various cord blood origins. Interestingly, the SFIb combination displayed markedly increased EPC expansion (2.43-fold), with a higher percentage of CD34(+) cells (2.17-fold), undifferentiated blasts (2.38-fold) and CXCR4(+) cells (1.68-fold) compared to another cytokine combination (SCF, thrombopoietin, and granulocyte colony-stimulating factor), although the two cytokine combinations had a similar level of total mononucleated cell expansion (∼ 10% difference). Accordingly, the cells expanded in the SFIb combination were more effective in recovery of blood flow and neovascularization in hind-limb ischemia in vivo. Taken together, these results suggest that the nonadhesive serum-free culture conditions of the CD34(+) fraction provide an effective EPC expansion method for cell therapy, and an expansion condition leading to high percentages of CD34(+) cells and blasts is likely important in EPC expansion.

Enhancement of anti-tumor activity by low-dose combination of the recombinant urokinase kringle domain and celecoxib in a glioma model.

The kringle domain of urokinase-type plasminogen activator (UK1) has anti-angiogenic and anti-tumor effects. Celecoxib, an inhibitor of cyclooxygenase type 2, also suppresses angiogenesis and tumor growth. To look for potential additive effects in their activities, we examined the anti-angiogenic and anti-tumor effects of the combination of UK1 and celecoxib for malignant gliomas. In vitro, the combination of UK1 and celecoxib enhanced inhibition of proliferation, migration, and tube formation of endothelial cells, although showing no enhancement of inhibition of U87 cell growth. However, in vivo models, combination treatment of intracerebral U87 malignant glioma xenografts in nude mice with UK1 (10mg/kg/day) and celecoxib (10mg/kg/day) at lower doses resulted in even more potent inhibition of tumor growth than each monotherapy (by 81% compared to untreated tumors), with drastic decrease of the expression of angiogenesis-related factors and increase of apoptosis in the tumor tissues. Interestingly, UK1 inhibited VEGF or bFGF-induced phosphorylation of ERK1/2 in ECs, whereas celecoxib showed no such effects. However, celecoxib inhibited U87 cell growth and directly suppressed their VEGF production. Therefore, our data suggest that combined use at low doses of UK1 and celecoxib with different anti-angiogenic mechanisms provides a desirable strategy for anti-glioma therapy.

Integrin alphavbeta3 is not significantly implicated in the anti-migratory effect of anti-angiogenic urokinase kringle domain.

The recombinant kringle domain (UK1) of urokinase-type plasminogen activator (uPA) has been shown to possess anti-angiogenic activity in vitro and in vivo. It has also been found to inhibit in vivo malignant glioma growth. In contrast, direct interaction of the kringle domain of uPA and integrin alphavbeta3 has been reported to be involved in plasminogen and leukocyte activation by uPA. Since integrin alphavbeta3 is involved in tumor angiogenesis, we investigated whether integrin alphavbeta3 is involved in the inhibitory function of UK1 in angiogenesis, by examining its anti-migratory activity. In a modified Boyden chamber assay, the Pichia-expressed UK1 dose-dependently inhibited the VEGF-induced migration of human umbilical vein endothelial cells (HUVECs). However, in the absence of growth factor stimulation, soluble UK1 alone did not induce or inhibit HUVEC migration. In cell adhesion, immobilized UK1 promoted HUVEC adhesion and spreading which were compared to BSA. Pretreatment of the anti-alphavbeta3 integrin antibody, significantly inhibited HUVEC binding to immobilized UK1, whereas neither anti-alpha2beta1 nor anti-alpha5beta1 integrin antibody had any effect, although pre-treatment of the soluble UK1 showed no marked alteration of the binding level of anti-alphavbeta3 antibody to HUVECs in FACS analysis. In a modified Boyden chamber assay, the function blocking antibodies against integrins alphavbeta3, alpha2beta1 and alpha5beta1 did not completely prevent the inhibitory effect of UK1 in HUVEC migration. These results suggest that UK1 interacts with integrin alphavbeta3, but its anti-migratory activity on endothelial cells is not significantly mediated by integrin alphavbeta3.

Tumor angiogenesis promoted by ex vivo differentiated endothelial progenitor cells is effectively inhibited by an angiogenesis inhibitor, TK1-2.

Neovascularization plays a critical role in the growth and metastatic spread of tumors and involves recruitment of circulating endothelial progenitor cells (EPC) from bone marrow as well as sprouting of preexisting endothelial cells. In this study, we examined if EPCs could promote tumor angiogenesis and would be an effective cellular target for an angiogenesis inhibitor, the recombinant kringle domain of tissue-type plasminogen activator (TK1-2). When TK1-2 was applied in the ex vivo culture of EPCs isolated from human cord blood, TK1-2 inhibited adhesive differentiation of mononuclear EPCs into endothelial-like cells. In addition, it inhibited the migration of ex vivo cultivated EPCs and also inhibited their adhesion to fibronectin matrix or endothelial cell monolayer. When A549 cancer cells were coimplanted along with ex vivo cultivated EPCs s.c. in nude mice, the tumor growth was increased. However, the tumor growth and the vascular density of tumor tissues increased by coimplanted EPCs were decreased upon TK1-2 treatment. Accordingly, TK1-2 treatment reduced the remaining number of EPCs in tumor tissues and their incorporation into the host vascular channels. In addition, overall expression levels of vascular endothelial growth factor (VEGF) and von Willebrand factor in tumor tissues were decreased upon TK1-2 treatment. Interestingly, strong VEGF expression by implanted EPCs was decreased by TK1-2. Finally, we confirmed in vitro that TK1-2 inhibited VEGF secretion of EPCs. TK1-2 also inhibited endothelial cell proliferation and migration induced by the conditioned medium of EPCs. Therefore, we concluded that EPCs, as well as mature endothelial cells, could be an important target of TK1-2.