Concurrent exposure to a dectin-1 agonist suppresses the Th2 response to epicutaneously introduced antigen in mice

Background

Epicutaneous sensitization with protein allergen that induces predominant Th2 responses is an important sensitization route in atopic dermatitis. Fungal components have been shown to modulate Th cell differentiation. However, the effects of fungal components on epicutaneous sensitization are unclear.

Results

In this study, we showed that co-administration of curdlan, a dectin-1 agonist, during epicutaneous ovalbumin sensitization of BALB/c mice decreased the IL-5 and IL-13 levels in supernatants of lymph node cell ovalbumin reactivation cultures. Mechanistically, curdlan co-administration decreased IL-4 and IL-1β expressions in draining lymph nodes. Curdlan co-administration also lower the migration of langerin⁺ CD103^- epidermal Langerhans cells into draining lymph nodes at 96 hours post-sensitization which might be attributed to decreased expressions of IL-18 and IL-1β in patched skin. Moreover, adoptive transfer of CFSE-labeled transgenic CD4 T cells confirmed that curdlan co-administration decreased the proliferation and IL-4-production of ovalbumin -specific T cells primed by epidermal Langerhans cells.

Conclusions

These results indicated that concurrent exposure to a dectin-1 agonist suppresses the epicutaneously induced Th2 response by modulating the cytokine expression profiles in draining LNs and the migration of epidermal Langerhans cells. These results highlight the effects of fungal components on epicutaneous allergen sensitization in atopic diseases.     

Cutting edge: Identification of a motile IL-17-producing gammadelta T cell population in the dermis

Dendritic epidermal T cells (DETCs) are a well-studied population of γδ T cells that play important roles in wound repair. In this study, we characterize a second major population of γδ T cells in the skin that is present in the dermis. In contrast to DETCs, these Vγ5-negative cells are IL-7R(hi)CCR6(hi) retinoic acid-related orphan receptor γt(+) and are precommitted to IL-17 production. Dermal γδ T cells fail to reconstitute following irradiation and bone marrow transplantation unless the mice also receive a transfer of neonatal thymocytes. Real-time intravital imaging of CXCR6(GFP/+) mouse skin reveals dermal γδ T cells migrate at ～4 μm/min, whereas DETCs are immobile. Like their counterparts in peripheral lymph nodes, dermal γδ T cells rapidly produce IL-17 following exposure to IL-1β plus IL-23. We have characterized a major population of skin γδ T cells and propose that these cells are a key source of IL-17 in the early hours after skin infection.     

OX40 ligand regulates splenic CD8 dendritic cell-induced Th2 responses in vivo

In mice, splenic conventional dendritic cells (cDCs) can be separated, based on their expression of CD8α into CD8⁻ and CD8⁺ cDCs. Although previous experiments demonstrated that injection of antigen (Ag)-pulsed CD8⁻ cDCs into mice induced CD4 T cell differentiation toward Th2 cells, the mechanism involved is unclear. In the current study, we investigated whether OX40 ligand (OX40L) on CD8⁻ cDCs contributes to the induction of Th2 responses by Ag-pulsed CD8⁻ cDCs in vivo, because OX40–OX40L interactions may play a preferential role in Th2 cell development. When unseparated Ag-pulsed OX40L-deficient cDCs were injected into syngeneic BALB/c mice, Th2 cytokine (IL-4, IL-5, and IL-10) production in lymph node cells was significantly reduced. Splenic cDCs were separated to CD8⁻ and CD8⁺ cDCs. OX40L expression was not observed on freshly isolated CD8⁻ cDCs, but was induced by anti-CD40 mAb stimulation for 24 h. Administration of neutralizing anti-OX40L mAb significantly inhibited IL-4, IL-5, and IL-10 production induced by Ag-pulsed CD8⁻ cDC injection. Moreover, administration of anti-OX40L mAb with Ag-pulsed CD8⁻ cDCs during a secondary response also significantly inhibited Th2 cytokine production. Thus, OX40L on CD8⁻ cDCs physiologically contributes to the development of Th2 cells and secondary Th2 responses induced by Ag-pulsed CD8⁻ cDCs in vivo.     

Probiotic Bifidobacterium breve Induces IL-10-Producing Tr1 Cells in the Colon

Specific intestinal microbiota has been shown to induce Foxp3⁺ regulatory T cell development. However, it remains unclear how development of another regulatory T cell subset, Tr1 cells, is regulated in the intestine. Here, we analyzed the role of two probiotic strains of intestinal bacteria, Lactobacillus casei and Bifidobacterium breve in T cell development in the intestine. B. breve, but not L. casei, induced development of IL-10-producing Tr1 cells that express cMaf, IL-21, and Ahr in the large intestine. Intestinal CD103⁺ dendritic cells (DCs) mediated B. breve-induced development of IL-10-producing T cells. CD103⁺ DCs from Il10 ^−/−, Tlr2 ^−/−, and Myd88 ^−/− mice showed defective B. breve-induced Tr1 cell development. B. breve-treated CD103⁺ DCs failed to induce IL-10 production from co-cultured Il27ra ^−/− T cells. B. breve treatment of Tlr2 ^−/− mice did not increase IL-10-producing T cells in the colonic lamina propria. Thus, B. breve activates intestinal CD103⁺ DCs to produce IL-10 and IL-27 via the TLR2/MyD88 pathway thereby inducing IL-10-producing Tr1 cells in the large intestine. Oral B. breve administration ameliorated colitis in immunocompromised mice given naïve CD4⁺ T cells from wild-type mice, but not Il10 ^−/− mice. These findings demonstrate that B. breve prevents intestinal inflammation through the induction of intestinal IL-10-producing Tr1 cells.     

Amygdalin mediates relieved atherosclerosis in apolipoprotein E deficient mice through the induction of regulatory T cells

Objective: Regulatory T cells (Tregs) play a critical role in the regulation of T cell-mediated immune responses in atherosclerosis, a chronic autoimmune-like disease. Therefore, in this study, we aimed to investigate the therapeutic effect of amygdalin on atherosclerosis of apolipoprotein E deficient (ApoE^−/−) mice, and to explore its immune regulatory function by stimulation of Tregs. Methods and results: To evaluate the anti-atherosclerotic effect of amygdalin and for in vivo Treg expansion/activation analysis, ApoE^−/− mice received intraperitoneal injections of amygdalin, and this therapy resulted in a comparatively 2-fold decrease in triglyceride (TG), 1.5-fold decrease in total cholesterol (TC) and low density lipoprotein (LDL). By comparing the vessel areas, lumen areas, plaque areas, and aortic plaque coverage percentage, the effects of amygdalin on pre-existing lesions were assessed. Studies on IL-10 and TGF-β indicated that mice treated with amygdalin had increased expression of Treg-related cytokines. Meanwhile, flow cytometry and real-time PCR data showed that mice treated with amygdalin had higher percentage of CD4⁺CD25⁺Foxp3⁺ T cells than untreated mice and increased expression of forkhead box P3 (FOXP3) gene. Conclusion: Our data showed amygdalin could attenuate the development of atherosclerosis by suppressing inflammatory responses and promoting the immunomodulation function of Tregs. The effects of amygdalin ultimately resulted in the enlarged lumen area and the loss of atherosclerotic plaque. All these data indicated the therapeutic potential of amygdalin in preventing and/or treating of atherosclerosis.     

Reduced atherosclerosis and inflammatory cytokines in apolipoprotein-E-deficient mice lacking bone marrow-derived interleukin-1α

Objective

Interleukin (IL)-1α and IL-1β are products of macrophages, endothelial cells and vascular smooth muscle cells; moreover, each of these cell types is affected by the pro-inflammatory properties of both IL-1’s. Whereas several studies demonstrate the proatherogenic properties of IL-1β, the role of IL-1α in atherogenesis remains unclear. We assessed whether IL-1α and IL-1β from tissue resident vascular cells or emigrating bone marrow-derived cells promote the development of atherosclerosis in apoE−/− mice and determined the effect of selective macrophage IL-1α or IL-1β deficiency on degradation of LDL and cytokine production.

Methods

We generated strains of double knock-out (KO) mice (apoE−/−/IL-1α−/− and apoE−/−/IL-1β−/−) and created chimeras consisting of apoE−/− mice reconstituted with bone marrow-derived cells from apoE−/−/IL-1+/+, apoE−/−/IL-1α−/− and apoE−/−/IL-1β−/−.

Results

The areas of aortic sinus lesions were lower in either double KO mice compared to solely apoE−/− mice, despite higher non-HDL cholesterol levels. Importantly, selective deficiency of IL-1α or IL-1β in bone marrow-derived cells inhibited atherogenesis to the same extent as in double KO mice without affecting plasma lipids. Aortic sinus lesions in apoE−/− mice transplanted with IL-1β−/− or IL-1α−/− cells were 32% and 52% lower, respectively, than in IL-1+/+ transplanted mice. Ex vivo, isolated IL-1α−/− macrophages from atherosclerotic mice degraded LDL and secreted IL-6, TNFα and IL-12 similarly to IL-1+/+ macrophages; however, IL-1α deficient macrophages secreted reduced levels of IL-1β (−50%) and 2–3-fold higher levels of the anti-inflammatory cytokine IL-10.

Conclusion

We show for the first time that it is IL-1α from bone marrow-derived cells that accelerates atherogenesis in apoE-deficient mice rather than constitutive IL-1α in vascular cells, possibly by increasing the inflammatory cytokine profile of macrophages.     

Participation of NK1.1+ T Cells in the Rejection of lpr αβT Cells When Bone Marrow Cells of Ipr Mice Are Transplanted into B6 Mice

When C57BL/6 (B6) mice were irradiated (9 Gy) and received bone marrow (BM) cells of B6-lpr/lpr mouse origin (i.e., lpr→B6), all mice died within 6 days. In the irradiated B6 mice, radioresistant CD3^? IL-2Rβ⁺ NK cells and IL-2Rβ CD3^int cells (i.e., CD3^int cells of extrathymic origin) remained, especially in the liver. There were two subsets, NK1.1⁺ and NK1.1^?, among the IL-2Rβ⁺ CD3^int cells. However, the NK1.1⁺ subset (i.e., NK1.1⁺ T cells) was much more radioresistant, and the majority of CD3^int cells belonged to this subset in irradiated mice. The expansion of lymphocytes from injected BM cells did not occur in the irradiated B6 mice. However, such expansion did take place in irradiated B6-lpr/lpr mice injected with both BM cells of B6-lpr/lpr and B6 origin. As a result, the mice subjected to BM cells survived. Irradiated B6 mice were treated in vivo with anti-NK1.1 mAb or anti-asialoGM₁ antibody to eliminate NK cells alone or both NK cells and NK1.1⁺ T cells. When irradiated B6 mice were pretreated with anti-NK1.1 mAb, the mice could survive. These results suggest that intact NK1.1⁺ T cells of extrathymic origin may recognize abnormal BM cells with the lpr gene and inhibit the expansion of lymphocytes, including abnormal double-negative CD4^?8^? cells, in B6-lpr/lpr mice. To inhibit the expansion of lymphocytes, mechanisms other than Fas ligand/Fas molecules on extrathymic T cells may be responsible.     

Interleukin-36 potently stimulates human M2 macrophages,Langerhans cells and keratinocytes to produce pro-inflammatory cytokines

Interleukin (IL)-36 cytokines belong to the IL-1 family and include three agonists, IL-36 α, β and γ and one inhibitor, IL-36 receptor antagonist (IL-36Ra). IL-36 and IL-1 (α and β) activate similar intracellular pathways via their related heterodimeric receptors, IL-36R/IL-1RAcP and IL-1R1/IL-1RAcP, respectively. However, excessive IL-36 versus IL-1 signaling induces different phenotypes in humans, which may be related to differential expression of their respective receptors.We examined the expression of IL-36R, IL-1R1 and IL-1RAcP mRNA in human peripheral blood, tonsil and skin immune cells by RT-qPCR. Monocyte-derived dendritic cells (MDDC), M0, M1 or M2-polarized macrophages, primary keratinocytes, dermal macrophages and Langerhans cells (LC) were stimulated with IL-1β or IL-36β. Cytokine production was assessed by RT-qPCR and immunoassays.The highest levels of IL-36R mRNA were found in skin-derived keratinocytes, LC, dermal macrophages and dermal CD1a⁺ DC. In the blood and in tonsils, IL-36R mRNA was predominantly found in myeloid cells. By contrast, IL-1R1 mRNA was detected in almost all cell types with higher levels in tonsil and skin compared to peripheral blood immune cells. IL-36β was as potent as IL-1β in stimulating M2 macrophages, keratinocytes and LC, less potent than IL-1β in stimulating M0 macrophages and MDDC, and exerted no effects in M1 and dermal macrophages. Levels of IL-1Ra diminished the ability of M2 macrophages to respond to IL-1.Taken together, these data are consistent with the association of excessive IL-36 signaling with an inflammatory skin phenotype and identify human LC and M2 macrophages as new IL-36 target cells.     

CD103+CD11b+ Dendritic Cells Induce Th17 T Cells in Muc2-Deficient Mice with Extensively Spread Colitis

Mucus alterations are a feature of ulcerative colitis (UC) and can drive inflammation by compromising the mucosal barrier to luminal bacteria. The exact pathogenesis of UC remains unclear, but CD4⁺ T cells reacting to commensal antigens appear to contribute to pathology. Given the unique capacity of dendritic cells (DCs) to activate naive T cells, colon DCs may activate pathogenic T cells and contribute to disease. Using Muc2^-/- mice, which lack a functional mucus barrier and develop spontaneous colitis, we show that colitic animals have reduced colon CD103⁺CD11b^- DCs and increased CD103^-CD11b⁺ phagocytes. Moreover, changes in colonic DC subsets and distinct cytokine patterns distinguish mice with distally localized colitis from mice with colitis spread proximally. Specifically, mice with proximally spread, but not distally contained, colitis have increased IL-1β, IL-6, IL-17, TNFα, and IFNγ combined with decreased IL-10 in the distal colon. These individuals also have increased numbers of CD103⁺CD11b⁺ DCs in the distal colon. CD103⁺CD11b⁺ DCs isolated from colitic but not noncolitic mice induced robust differentiation of Th17 cells but not Th1 cells ex vivo. In contrast, CD103^-CD11b⁺ DCs from colitic Muc2^-/- mice induced Th17 as well as Th1 differentiation. Thus, the local environment influences the capacity of intestinal DC subsets to induce T cell proliferation and differentiation, with CD103⁺CD11b⁺ DCs inducing IL-17-producing T cells being a key feature of extensively spread colitis.     

CD98hc regulates the development of experimental colitis by controlling effector and regulatory CD4 T cells

CD4⁺ T cell activation is controlled by signaling through the T cell receptor in addition to various co-receptors, and is also affected by their interactions with effector and regulatory T cells in the microenvironment. Inflammatory bowel diseases (IBD) are caused by the persistent activation and expansion of auto-aggressive CD4⁺ T cells that attack intestinal epithelial cells. However, the molecular basis for the persistent activation of CD4⁺ T cells in IBD remains unclear. In this study, we investigated how the CD98 heavy chain (CD98hc, Slc3a2) affected the development of colitis in an experimental animal model. Transferring CD98hc-deficient CD4⁺CD25⁻ T cells into Rag2^−/− mice did not cause colitis accompanied by increasing Foxp3⁺ inducible regulatory T cells. By comparison, CD98hc-deficient naturally occurring regulatory T cells (nTregs) had a decreased capability to suppress colitis induced by CD4⁺CD25⁻ T cells, although CD98hc-deficient mice did not have a defect in the development of nTregs. Blocking CD98hc with an anti-CD98 blocking antibody prevented the development of colitis. Our results indicate that CD98hc regulates the expansion of autoimmune CD4⁺ T cells in addition to controlling nTregs functions, which suggests the CD98hc as an important target molecule for establishing strategies for treating colitis.     

Differential expression of co-signal molecules and migratory properties in four distinct subsets of migratory dendritic cells from the oral mucosa

Variations in co-signal ligand expression and cytokine production greatly influence the antigen-presenting properties of migrating DCs in regional lymph nodes (RLNs). Here we investigated DCs migrating from the oral mucosa using CD326 and CD103 antigens for discriminate CD207⁺ Langerhans cells (LCs) from CD207⁺ submucosal DCs (SMDCs). Similar to DCs migrating from the skin, we identified four distinct oral mucosal DC (OMDC) subsets, CD11c^hiCD207⁻CD103⁻CD326^intCD11b^hi (F1; resident CD11b^hi SMDCs), CD11c^int/loCD207^-CD103^-CD326^loCD11b^int/hi (F2; newly recruited blood-derived SMDCs), CD11c^int/loCD207⁺CD103⁺CD326^int/hiCD11b^lo (CD103⁺ F3; resident CD207⁺ SMDCs), and CD11c^int/loCD207⁺CD103^-CD326^int/hiCD11b^lo (CD103^- F3; resident LCs). F1 DCs migrated rapidly after fluorescein isothiocyanate (FITC) painting and expressed notably high levels of CD86, CD273, and CD274 at an earlier time point. In contrast, CD103⁻ LCs expressing the highest levels of the epithelial cell adhesion molecule CD326 accounted for a minor subset at the earlier time point, but increased slowly with CD103⁺CD207⁺ SMDCs. However, their expression of CD86, CD273, and CD274 was very limited. The delayed migration and limited induction of co-signal ligands suggest that roles of OMLCs are distinct from those of the other three DC subsets. The identification of distinct subsets of OMDCs in RLNs may benefit efforts to determine the functional specialization of each subset in T cell responses against orally administrated antigens.     

Dendritic cell-derived TNF-α is responsible for development of IL-10-producing CD4 T cells

Immature dendritic cells (DCs) appear to be involved in peripheral immune tolerance via induction of IL-10-producing CD4⁺ T cells. We examined the role of TNF-α in generation of the IL-10-producing CD4⁺ T cells by immature DCs. Immature bone marrow-derived DCs from wild type (WT) or TNF-α^−/− mice were cocultured with CD4⁺ T cells from OVA specific TCR transgenic mice (OT-II) in the presence of OVA_323-339 peptide. The WT DCs efficiently induced the antigen-specific IL-10-producing CD4⁺ T cells, while the ability of the TNF-α^−/− DCs to induce these CD4⁺ T cells was considerably depressed. Addition of exogenous TNF-α recovered the impaired ability of the TNF-α^−/− DCs to induce IL-10-producing T cells. However, no difference in this ability was observed between TNF-α^−/− and WT DCs after their maturation by LPS. Thus, TNF-α appears to be critical for the generation of IL-10-producing CD4⁺ T cells during the antigen presentation by immature DCs.     

Potentiation of Th17 cytokines in aging process contributes to the development of colitis

Th17 cells, which produce IL-17 and IL-22, promote autoimmunity in mice and have been implicated in the pathogenesis of autoimmune/inflammatory diseases in humans. However, the Th17 immune response in the aging process is still not clear. In the present study, we found that the induction of IL-17-produing CD4⁺ T cells was significantly increased in aged individuals compared with young healthy ones. The mRNA expression of IL-17, IL-17F, IL-22, and RORC2 was also significantly increased in aged people. Similar to humans, Th17 cells as well as mRNAs encoding IL-17, IL-22 and RORγt were dramatically elevated in naïve T cells from aged mouse compared to young ones. In addition, CD44 positive IL-17-producing CD4⁺ T cells were significantly higher in aged mice, suggesting that memory T cells are an important source of IL-17 production. Furthermore, the percentage of IL-17-produing CD4⁺ T cells generated in co-culture with dendritic cells from either aged or young mice did not show significant differences, suggesting that dendritic cells do not play a primary role in the elevation of Th17 cytokines in aged mouse cells. Importantly, transfer of CD4⁺CD45Rb^hi cells from aged mice induced more severe colitis in RAG^−/− mice compared to cells from young mice, Taken together, these results suggest that Th17 immune responses are elevated in aging humans and mice and may contribute to the increased development of inflammatory disorders in the elderly.     

Differential Capacity of Human Skin Dendritic Cells to Polarize CD4+T Cells into IL-17, IL-21 and IL-22 Producing Cells

Accumulating evidence suggests a contribution of T cell-derived IL-17, IL-21 and IL-22 cytokines in skin immune homeostasis as well as inflammatory disorders. Here, we analyzed whether the cytokine-producing T lymphocytes could be induced by the different subsets of human skin dendritic cells (DCs), i.e., epidermal Langerhans cells (LCs), dermal CD1c⁺CD14⁻ and CD14⁺ DCs (DDCs). DCs were purified following a 2-day migration from separated epidermal and dermal sheets and co-cultured with allogeneic T cells before cytokine secretion was explored. Results showed that no skin DCs could induce substantial IL-17 production by naïve CD4⁺ or CD8⁺T lymphocytes whereas all of them could induce IL-17 production by memory T cells. In contrast, LCs and CD1c⁺CD14⁻DDCs were able to differentiate naïve CD4⁺T lymphocytes into IL-22 and IL-21-secreting cells, LCs being the most efficient in this process. Intracellular cytokine staining showed that the majority of IL-21 or IL-22 secreting CD4⁺T lymphocytes did not co-synthesized IFN-γ, IL-4 or IL-17. IL-21 and IL-22 production were dependent on the B7/CD28 co-stimulatory pathway and ICOS-L expression on skin LCs significantly reduced IL-21 level. Finally, we found that TGF-β strongly down-regulates both IL-21 and IL-22 secretion by allogeneic CD4⁺ T cells. These results add new knowledge on the functional specialization of human skin DCs and might suggest new targets in the treatment of inflammatory skin disorders.     

Downregulation of immune response by the human cytokines Interleukin-32α and β in cell-mediated rejection

Xenotransplantation of porcine organs has the potential to help overcome the severe shortage of human tissues and organs available for human transplantation. However, numerous hurdles such as immune-mediated xenograft rejection remain before clinical xenotransplantation.In this study, we elucidated the role of human TNF-α-inducing factor, Interleukin-32 (IL-32), in porcine kidney cells (PK-15) during cell-mediated rejection by examining host cell responses. CD8⁺ and CD4⁺ T cells numbers were reduced in the lymph nodes of PK-15/IL-32β injected mice. CD3⁺ Tcells were in mice injected with control cells but PK-15/IL-32α- and PK-15/IL-32β-injected cell numbers were lower in lymphnodes than un transfected controls. In Mixed lymphocyte reaction cultures, the rates of cell proliferation were increased in both PK-15/IL-32α- and PK-15/IL-32β-injected groups compared to the untransfected control groups. The Stable porcine PK-15 cells expression IL-32α and IL-32β inhibited cytotoxic T lymphocyte (CTLs) after cellular xenograft. Our results suggest that human IL-32α and IL-32β regulates on xenograft rejection in cellular xenotransplantation.     

Salmon cartilage proteoglycan suppresses mouse experimental colitis through induction of Foxp3+ regulatory T cells

Proteoglycans (PGs) are complex glycohydrates which are widely distributed in extracellular matrix (ECM). PGs are involved in the construction of ECM, cell proliferation and differentiation. ECM components are involved in transduction of proinflammatory responses, but it is still unknown whether PGs are involved in inflammatory response. In this study, we investigated the effect of PG extracted from salmon cartilage on the progression of experimental colitis-induced in severe combined immunodeficiency mice by cell transfer from interleukin-10 (IL-10)^−/− mice. IL-10^−/− cell-transferred mice showed weight loss, colon shortening and histological appearance of mild colitis. Daily oral administration of PG attenuated the clinical progression of colitis in a dose-dependent manner. Colitis-induced mice showed the elevated expression of IFN-γ, IL-12, TNF-α, IL-21, IL-23p19, IL-6, IL-17A and retinoic acid-related orphan receptor γt (RORγt) in lamina propria mononuclear cells (LPMCs) and oral administration of PG suppressed the expression of these factors. Conversely, expression of Foxp3 that induces CD4⁺CD25⁺ regulatory T cells in LPMCs was enhanced by PG administration. These findings suggested that salmon PG attenuated the progression of colitis due to suppression of inflammatory response by enhancement of regulatory T cell induction.