Bacillus‐derived poly‐γ‐glutamic acid attenuates allergic airway inflammation through a Toll‐like receptor‐4‐dependent pathway in a murine model of asthma

Background Asthma is an inflammatory disease of the airways that is mediated by Th2 responses. Poly‐γ‐glutamic acid (γ‐PGA) is an extracellular polymeric compound that is synthesized by Bacillus cells. Previously, we found that γ‐PGA promoted Th1 cell development in a manner dependent on antigen‐presenting cells, but inhibited Th2 cell development. Objective To investigate the effect of γ‐PGA on dendritic cells (DCs), and its potential for treating Th2‐mediated allergic asthma. Methods Wild‐type, Toll‐like receptor (TLR)‐2 deficient, and TLR‐4‐defective mice were used. DCs derived from the bone marrow and extracted from the lung were stimulated with γ‐PGA and assayed for the expression of signalling molecules, costimulatory molecules, and cytokines. Mice were sensitized and challenged with ovalbumin (OVA) to induce asthma. They were repeatedly injected intranasally with γ‐PGA before and during the challenge period, and inflammation and structural remodelling of the airways were examined. Results γ‐PGA selectively signalled conventional DCs to activate NF‐κB and mitogen‐activated protein kinase, leading to the up‐regulation of CD86, CD40, and IL‐12, but not IL‐10 and IL‐6. These effects of γ‐PGA were dependent on TLR‐4 and independent of TLR‐2. Importantly, the intranasal administration of γ‐PGA to OVA‐sensitized/challenged mice reduced the airway hyperresponsiveness and allergic inflammation such as leucocyte influx, goblet cell hyperplasia, eosinophilia, and Th2 cytokine production. In addition to lowered IgE titres, the treatment of mice with γ‐PGA significantly reduced the multiplication and Th2 polarization of mediastinal lymph node T cells upon allergen‐specific restimulation. These anti‐asthmatic effects of γ‐PGA were also abolished in TLR‐4‐defective mice. Conclusions and Clinical Relevance Our data indicate that γ‐PGA activates DCs to favour Th1 cell induction through a TLR‐4‐dependent pathway and alleviates pathologic symptoms in a Th2‐biased asthmatic model. These findings highlight the potential of γ‐PGA for the treatment of asthma and other allergic disease in which Th2 polarization plays an important role. Cite this as: K. Lee, S.‐H. Kim, H. J. Yoon, D. J. Paik, J. M. Kim and J. Youn, Clinical & Experimental Allergy, 2011 (41) 1143–1156.     

Toll‐like receptor 4 agonists adsorbed to aluminium hydroxide adjuvant attenuate ovalbumin‐specific allergic airway disease: role of MyD88 adaptor molecule and interleukin‐12/interferon‐γ axis

Background Epidemiological and experimental data suggest that bacterial lipopolysaccharides (LPS) can either protect from or exacerbate allergic asthma. Lipopolysaccharides trigger immune responses through toll‐like receptor 4 (TLR4) that in turn activates two major signalling pathways via either MyD88 or TRIF adaptor proteins. The LPS is a pro‐Type 1 T helper cells (Th1) adjuvant while aluminium hydroxide (alum) is a strong Type 2 T helper cells (Th2) adjuvant, but the effect of the mixing of both adjuvants on the development of lung allergy has not been investigated. Objective We determined whether natural (LPS) or synthetic (ER‐803022) TLR4 agonists adsorbed onto alum adjuvant affect allergen sensitization and development of airway allergic disease. To dissect LPS‐induced molecular pathways, we used TLR4‐, MyD88‐, TRIF‐, or IL‐12/IFN‐γ‐deficient mice. Methods Mice were sensitized with subcutaneous injections of ovalbumin (OVA) with or without TLR4 agonists co‐adsorbed onto alum and challenged with intranasally with OVA. The development of allergic lung disease was evaluated 24 h after last OVA challenge. Results Sensitization with OVA plus LPS co‐adsorbed onto alum impaired in dose‐dependent manner OVA‐induced Th2‐mediated allergic responses such as airway eosinophilia, type‐2 cytokines secretion, airway hyper‐reactivity, mucus hyper production and serum levels of IgE or IgG1 anaphylactic antibodies. Although the levels of IgG2a, Th1‐affiliated isotype increased, investigation into the lung‐specific effects revealed that LPS did not induce a Th1 pattern of inflammation. Lipopolysaccharides impaired the development of Th2 immunity, signaling via TLR4 and MyD88 molecules and via the IL‐12/IFN‐γ axis, but not through TRIF pathway. Moreover, the synthetic TLR4 agonists that proved to have a less systemic inflammatory response than LPS also protected against allergic asthma development. Conclusion Toll‐like receptor 4 agonists co‐adsorbed with allergen onto alum down‐modulate allergic lung disease and prevent the development of polarized T cell‐mediated airway inflammation.     

Antigen‐specific airway IL‐33 production depends on FcγR‐mediated incorporation of the antigen by alveolar macrophages in sensitized mice

Although interleukin (IL)‐33 is a candidate for the aggravation of asthma, the mechanisms underlying antigen‐specific IL‐33 production in the lung are unclear. Therefore, we analysed the mechanisms in mice. Intra‐tracheal administration of ovalbumin (OVA) evoked increases in IL‐33 and IL‐33 mRNA in the lungs of both non‐sensitized and OVA‐sensitized mice, and the increases in the sensitized mice were significantly higher than in the non‐sensitized mice. However, intra‐tracheal administration of bovine serum albumin did not increase the IL‐33 level in the OVA‐sensitized mice. Depletion of neither mast cells/basophils nor CD4⁺ cells abolished the OVA‐induced IL‐33 production in sensitized mice, suggesting that the antigen recognition leading to the IL‐33 production was not related with either antigen‐specific IgE‐bearing mast cells/basophils or memory CD4⁺ Th2 cells. When a fluorogenic substrate‐labelled OVA (DQ‐OVA) was intra‐tracheally administered, the lung cells of sensitized mice incorporated more DQ‐OVA than those of non‐sensitized mice. The lung cells incorporating DQ‐OVA included B‐cells and alveolar macrophages. The allergic IL‐33 production was significantly reduced by treatment with anti‐FcγRII/III mAb. Depletion of alveolar macrophages by clodronate liposomes significantly suppressed the allergic IL‐33 production, whereas depletion of B‐cells by anti‐CD20 mAb did not. These results suggest that the administered OVA in the lung bound antigen‐specific IgG Ab, and then alveolar macrophages incorporated the immune complex through FcγRII/III on the cell surface, resulting in IL‐33 production in sensitized mice. The mechanisms underlying the antigen‐specific IL‐33 production may aid in development of new pharmacotherapies.     

Allergic sensitization is enhanced in early life through toll‐like receptor 7 activation

Background Prospective cohort studies suggest that children hospitalized in early life with severe infections are significantly more likely to develop recurrent wheezing and asthma. Objective Using an inhalational mouse model of allergic airways inflammation, we sought to determine the effect of viral and bacterial‐associated molecular patterns on the magnitude of the allergic inflammatory response and whether this effect was age dependent. Methods BALB/c mice were sensitized by intranasal administration of endotoxin^low ovalbumin (OVA) in the absence or presence of viral single‐stranded (ss)RNA, lipoteichoic acid or flagellin as neonates (within the first 24 h of life) or as weanlings (4 weeks of age). Mice were challenged four times with OVA at 6 weeks of age and end‐points (bronchoalveolar lavage cytology, histology, antigen‐specific T and B cell responses) determined at 7 weeks of age. Results Inhalational sensitization (<24 h or 4 weeks of age) and challenge with OVA induced a mild allergic inflammatory response in the airways as indicated by increased numbers of eosinophils and mucus cells, elevated serum OVA‐specific IgG1, and production of T helper 2 (Th2) cytokines. Mice sensitized to endotoxin^low OVA at birth in the presence of ssRNA or lipoteichoic acid, but not flagellin, showed an increase in the numbers of airway and tissue eosinophils, mucus producing cells and antigen‐specific production of IL‐13 as compared with mice exposed only to endotoxin^low OVA. By contrast, all three TLR ligands failed to increase the magnitude of OVA‐induced allergic inflammation in mice sensitized as weanlings. Conclusions Recognition of distinct microbial‐associated patterns in early life may preferentially promote the de novo differentiation of bystander, antigen‐specific CD4⁺ T cells toward a Th2 phenotype, and promote an asthma‐like phenotype upon cognate antigen exposure in later life.     

Myeloid leukemia cells with a B7‐2+ subpopulation provoke Th‐cell responses and become immuno‐suppressive through the modulation of B7 ligands

Expression of the B7 family molecules in acute myeloid leukemia (AML) has been demonstrated by independent clinical studies. Intriguingly, the expression of the most potent costimulatory molecules B7‐2 (CD86) and B7‐H2 (ICOS Ligand) on AML cells has been associated with poor prognosis and disease severity. Here, this phenomenon was modeled in vitro with the myeloid leukemia cell line HL‐60, which is capable of differentiating through the FAB M2/M3 and M4/M5 immunophenotypes. These derivatives of HL‐60 harbored a B7‐2⁺ subpopulation and recapitulated the distribution of B7 ligands previously reported in primary AML cases. B7‐2⁺ AML cells significantly contributed to T‐cell responses. This costimulatory activity enabled helper (Th)‐cell activation, proliferation, and production of Th1‐associated cytokines. Conversely, even a short‐term incubation with stimulated T cells resulted in upregulation of inhibitory B7‐H1 (PD‐L1) and B7‐DC (PD‐L2), and downregulation of stimulatory B7‐H2 molecules on leukemia cells. Purified from iHL‐60‐T‐cell co‐cultures, these myeloid leukemia cells severely suppressed Th‐cell responses specifically through the PD‐1 pathway. In conclusion, Th‐cell responses can be directly supported by B7‐2⁺ leukemia subpopulations. However, this interaction can facilitate the acquisition of a suppressive character that may contribute to immune evasion in myeloid leukemia.     

IL‐34‐ and M‐CSF‐induced macrophages switch memory T cells into Th17 cells via membrane IL‐1α

Macrophages orchestrate the immune response via the polarization of CD4⁺ T helper (Th) cells. Different subsets of macrophages with distinct phenotypes, and sometimes opposite functions, have been described. M‐CSF and IL‐34 induce the differentiation of monocytes into IL‐10^high IL‐12^low immunoregulatory macrophages, which are similar to tumor‐associated macrophages (TAMs) in ovarian cancer. In this study, we evaluated the capacity of human macrophages induced in the presence of M‐CSF (M‐CSF macrophages) or IL‐34 (IL‐34 macrophages) and ovarian cancer TAMs to modulate the phenotype of human CD4⁺ T cells. Taken together, our results show that M‐CSF‐, IL‐34 macrophages, and TAMs switch non‐Th17 committed memory CD4⁺ T cells into conventional CCR4⁺ CCR6⁺ CD161⁺ Th17 cells, expressing or not IFN‐gamma. Contrary, the pro‐inflammatory GM‐CSF macrophages promote Th1 cells. The polarization of memory T cells into Th17 cells is mediated via membrane IL‐1α (mIL‐1α), which is constitutively expressed by M‐CSF‐, IL‐34 macrophages, and TAMs. This study elucidates a new mechanism that allows macrophages to maintain locally restrained and smoldering inflammation, which is required in angiogenesis and metastasis.     

IFN‐γ stimulates CpG‐induced IL‐10 production in B cells via p38 and JNK signalling pathways

The production of IL‐10, a potent immunosuppressive cytokine, must be strictly regulated to ensure a balanced immune response. IFN‐γ, a key cytokine in multiple immune processes and pathologies, is known as an inhibitor of IL‐10 production by monocytes and macrophages, but also has some regulatory functions. In the present study, we explored the role of IFN‐γ on Toll‐like receptor (TLR)‐induced IL‐10 production in murine peritoneal and spleen cells and in human peripheral blood mononuclear cells. IFN‐γ inhibited IL‐10 production induced by TLR2, TLR3, TLR4 and TLR7/8 agonists, but stimulated IL‐10 production when cells were triggered with CpG oligodeoxynucleotides, a specific TLR9 agonist. The stimulatory effect of IFN‐γ on TLR9‐induced IL‐10 was restricted to B cells. In line with the increased IL‐10, B cells stimulated with CpG and IFN‐γ profoundly inhibited CD4 T cell proliferation. Further research into the mechanisms involved, revealed that the mitogen‐activated protein kinases p38 and JNK are essential players in this stimulatory effect, and that the phosphatase MKP1 – an inhibitor of p38 and JNK activity – is downregulated after combined stimulation with IFN‐γ and CpG. Our data may represent a novel immunoregulatory role of IFN‐γ in B cells after triggering of TLR9, by stimulating IL‐10 production.     

Environmental pollutant tributyltin promotes Th2 polarization and exacerbates airway inflammation

It has been shown that a relatively high dose of tributyltin (TBT), which is recognized as a particularly notable environmental pollutant, exerts immunotoxic effects such as thymic atrophy via induction of T cell apoptosis. However, the effect of low doses of TBT on the immune responses remains unknown. Here we show that environmentally relevant doses of TBT promoted strong Th2 polarization via suppression and augmentation of Th1 and Th2 development, respectively, from naive CD4(+) T cells primed with anti-CD3 and splenic antigen-presenting cells (APC). TBT-induced Th2 polarization was indirect, working through APC via suppression of IL-12 production by macrophages/DC and the augmentation of IL-10 production by B cells. Th2 polarization was also induced in mice treated with TBT and immunized with OVA or infected with Nippostrongylus brasiliensis. Furthermore, airway inflammation in mice sensitized and challenged with OVA was exacerbated by the administration of TBT with concomitant augmentation of Th2-type immunity. Our results highlight the fact that an important environmental pollutant TBT may present significant risk for the induction of allergic diseases via promotion of Th2 polarization.     

Expression of toll‐like receptors in T lymphocytes stimulated with N‐(3‐oxododecanoyl)‐L‐homoserine lactone from Pseudomonas aeruginosa

The establishment of chronic Pseudomonas aeruginosa infections is correlated with the disturbance of the host immune system. The P. aeruginosa quorum‐sensing molecule N‐3‐(oxododecanoyl)‐L‐homoserine lactone (3‐O‐C12‐HSL) has the potential to modulate the host immune system. The immune system recognizes pathogens via toll‐like receptors (TLRs). We found that 3‐O‐C12‐HSL induced TLR changes in monocytes. However, the role of T cells in P. aeruginosa infection has not been delineated. In order to understand this activity, we examined whether 3‐O‐C12‐HSL has an effect on the immune function and the expression of TLRs in T lymphocytes. Human peripheral blood mononuclear cells (PBMCs) cells were cultured with 0, 1, 10, 50, or 100 μM 3‐O‐C12‐HSL for 12 h. TLR2/TLR4 expression and T‐lymphocyte proliferation were increased in a dose‐dependent manner, and 100 μM 3‐O‐C12‐HSL significantly increased TLR2 expression. Moreover, tumor necrosis factor‐α production of these PBMCs was inhibited. To conclude, 3‐O‐C12‐HSL can induce lymphocyte cell proliferation. These findings provide a new perspective on our understanding of the persistence of the chronic inflammation that accompanies P. aeruginosa infection.     

Impaired antigen‐specific lymphocyte priming in mice after Toll‐like receptor 4 activation via induction of monocytic myeloid‐derived suppressor cells

In sepsis, the pathology involves a shift from a proinflammatory state toward an immunosuppressive phase. We previously showed that an agonistic anti‐TLR4 antibody induced long‐term endotoxin tolerance and suppressed antigen‐specific secondary IgG production when primed prior to immunization with antigen. These findings led us to speculate that TLR4‐induced innate tolerance due to primary infection causes an immunosuppressive pathology in sepsis. Therefore, the mechanism underlying impaired antigen‐specific humoral immunity by the TLR4 antibody was investigated. We showed, in a mouse model, that primary antigen‐specific IgG responses were impaired in TLR4 antibody‐induced tolerized mice, which was the result of reduced numbers of antigen‐specific GC B cells and plasma cells. Ovalbumin‐specific CD4 and CD8 T‐cell responses were impaired in TLR4 antibody‐injected OT‐I and ‐II transgenic mice ex vivo. Adoptive transfer studies demonstrated suppression of OVA‐specific CD4 and CD8 T‐cell responses by the TLR4 antibody in vivo. The TLR4 antibody induced Gr1⁺CD11b⁺ myeloid‐derived suppressor cell (MDSC) expansion with suppression of T‐cell activation. Monocytic MDSCs were more suppressive and exhibited higher expression of PD‐L1 and inducible nitric oxidase compared with granulocytic MDSCs. In conclusion, immune tolerance conferred by TLR4 activation induces the expansion of monocytic MDSCs, which impairs antigen‐specific T‐cell priming and IgG production.     

Mycobacterium tuberculosis RpfE promotes simultaneous Th1‐ and Th17‐type T‐cell immunity via TLR4‐dependent maturation of dendritic cells

Reciprocal induction of the Th1 and Th17 immune responses is essential for optimal protection against Mycobacterium tuberculosis (Mtb); however, only a few Mtb antigens are known to fulfill this task. A functional role for resuscitation‐promoting factor (Rpf) E, a latency‐associated member of the Rpf family, in promoting naïve CD4⁺ T‐cell differentiation toward both Th1 and Th17 cell fates through interaction with dendritic cells (DCs) was identified in this study. RpfE induces DC maturation by increasing expression of surface molecules and the production of IL‐6, IL‐1β, IL‐23p19, IL‐12p70, and TNF‐α but not IL‐10. This induction is mediated through TLR4 binding and subsequent activation of ERK, p38 MAPKs, and NF‐κB signaling. RpfE‐treated DCs effectively caused naïve CD4⁺ T cells to secrete IFN‐γ, IL‐2, and IL‐17A, which resulted in reciprocal expansions of the Th1 and Th17 cell response along with activation of T‐bet and RORγt but not GATA‐3. Furthermore, lung and spleen cells from Mtb‐infected WT mice but not from TLR4^?/? mice exhibited Th1 and Th17 polarization upon RpfE stimulation. Taken together, our data suggest that RpfE has the potential to be an effective Mtb vaccine because of its ability to activate DCs that simultaneously induce both Th1‐ and Th17‐polarized T‐cell expansion.     

BMP‐7 ameliorates cobalt alloy particle‐induced inflammation by suppressing Th17 responses

Metal wear debris has been shown to activate an aseptic osteolytic process that causes failure in total joint arthroplasty (TJA). This osteolysis is characterized by a proinflammatory, self‐propagating immune response involving primarily macrophages, dendritic cells, and activated osteoclasts, as well as T cells and B cells. The human bone morphogenic protein (BMP)‐7, on the other hand, was shown to promote osteoblast survival, and reversed the downregulation of anabolic Smad proteins and Runx2 following cobalt injury. Therefore, we investigated the effect and mechanism of BMP‐7 on the proinflammatory immune responses in osteoarthritis patients with previous TJA. Cobalt‐treated monocytes/macrophages presented significantly elevated levels of interleukin 6 (IL‐6) and tumor necrosis factor (TNF), both of which were suppressed by the addition of exogenous BMP‐7. In patients with TJA, the serum BMP‐7 level was inversely associated with the level of IL‐6 and TNF secreted by monocytes/macrophages. Cobalt‐treated monocytes/macrophages effectively supported Th17 inflammation, by an IL‐6‐dependent but not TNF‐dependent mechanism. BMP‐7, however, significantly suppressed cobalt‐induced Th17 inflammation. In patients with TJA, the risk of osteolysis development was positively associated with the frequency of Th17 cells and negatively associated with the level of BMP‐7. Together, these results demonstrated that BMP‐7 could serve as a therapeutic agent in treating patients with metal wear debris‐induced inflammation.     

Marked enhancement of antigen‐specific T‐cell responses by IL‐7‐fused nonlytic,but not lytic,Fc as a genetic adjuvant

IL‐7 plays a crucial role in the homeostatic proliferation, differentiation and survival of T cells, as well as in the survival and proliferation of precursor B cells. Here, we demonstrated that utilizing nonlytic Fc‐fused IL‐7 (IL‐7‐Fc^m) as a genetic adjuvant significantly enhanced not only CD4⁺ but also CD8⁺ T‐cell responses by E7 DNA immunization, in addition to improving protection against TC‐1‐induced tumors in comparison to IL‐7 alone. Similar results were obtained in OT‐1 adoptive transfer experiments with OVA DNA injection, suggesting independence from antigenic nature and experimental conditions. In particular, the increased frequency of CD8⁺ T cells was mainly due to enhanced T‐cell proliferation in T‐cell priming, and not to decreased cellular apoptosis. Interestingly, the enhanced adjuvant effect was not seen in the co‐delivery of lytic Fc‐fused IL‐7 (IL‐7‐Fc) which increases T‐cell apoptosis as well as T‐cell proliferation, suggesting that the T‐cell proliferative effect may be neutralized by T‐cell apoptosis. Thus, our findings suggest that nonlytic Fc, in contrast to lytic Fc, fusion to cytokines may provide an insight in designing a potent genetic adjuvant for inducing CD4⁺ and CD8⁺ T‐cell responses.     

Japanese encephalitis virus expands regulatory T cells by increasing the expression of PD‐L1 on dendritic cells

The mechanisms underlying Japanese encephalitis virus (JEV) pathogenesis need to be thoroughly explored to delineate therapeutic approaches. It is believed that JEV manipulates the innate and adaptive compartments of the host's immune system to evade immune response and cross the blood–brain barrier. The present study was thus designed to investigate the functional modulation of DCs after exposure to JEV and to assess the consequences on CD4⁺ T‐lymphocyte functions. Human monocyte‐derived DCs were either infected with 1 MOI of live virus, UV‐inactivated virus, or were mock‐infected. Replication‐competent JEV induced a significant increase in the expression of maturation markers 48 h postinfection, along with that of programmed cell death 1 ligand 1 (PD‐L1; also called B7‐H1 and CD274). JEV‐infected DCs expanded the Treg cells in allogenic mixed lymphocyte reactions. The expansion of Treg cells by JEV‐infected DCs was significantly reduced upon blocking PD‐L1 using an antagonist. In addition, JEV‐infected DCs significantly altered the proliferation and reduced the polarization of Th cells toward the Th1‐cell phenotype. The results, for the first time, suggest that JEV evades the host's immune system by modulating the crosstalk between DCs and T lymphocytes via the PD‐L1 axis.     

Fcγ receptor‐mediated antigen uptake by lung DC contributes to allergic airway hyper‐responsiveness and inflammation

During asthma, lung DC capture and process antigens to initiate and maintain allergic Th2 cell responses to inhaled allergens. The aim of the study was to investigate whether allergen‐specific IgG, generated during sensitization, can potentiate the acute airway inflammation through Fcγ receptor (FcγR)‐mediated antigen uptake and enhance antigen presentation resulting in augmented T‐cell proliferation. We examined the impact of antigen presentation and T‐cell stimulation on allergic airway hyperresponsiveness and inflammation using transgenic and gene‐deficient mice. Both airway inflammation and eosinophilia in bronchoalveolar lavage fluid were markedly reduced in sensitized and challenged FcγR‐deficient mice. Lung DC of WT, but not FcγR‐deficient mice, induced increased antigen‐specific CD4⁺ T‐cell proliferation when pulsed with anti‐OVA IgG immune complexes. Intranasal application of anti‐OVA IgG immune complexes resulted in enhanced airway inflammation, eosinophilia and Th2 cytokine release, mediated through enhanced antigen‐specific T‐cell proliferation in vivo. Finally, antigen‐specific IgG in the serum of sensitized mice led to a significant increase of antigen‐specific CD4⁺ T‐cell proliferation induced by WT, but not FcγR‐deficient, lung DC. We conclude that FcγR‐mediated enhanced antigen presentation and T‐cell stimulation by lung DC has a significant impact on inflammatory responses following allergen challenge in asthma.     

Toll‐like receptor 2 agonist Pam3CSK4 enhances the induction of antigen‐specific tolerance via the sublingual route

Background Sublingual immunotherapy (SLIT) has been established in humans as a safe and efficacious treatment for type I respiratory allergies. Objective In this study, we compared three Toll‐like receptor (TLR) 2 ligands (Pam3CSK4, Porphyromonas gingivalis lipopolysaccharide and lipoteichoic acid) as potential adjuvants for sublingual allergy vaccines. Methods These molecules were tested in co‐cultures of adjuvant‐pre‐treated dendritic cells (DCs) with murine naïve CD4⁺ T lymphocytes. Patterns of cytokine production, phenotype, proliferation and gene expression were analysed by ELISA, cytofluorometry and quantitative PCR, respectively. TLR2 ligands were subsequently tested in a model of SLIT in BALB/c mice sensitized with ovalbumin (OVA). Results Among the three TLR2 ligands tested, the synthetic lipopeptide Pam3CSK4 is the most potent inducer of IL‐12p35 and IL‐10 gene expression in murine bone marrow‐derived DCs, as well as in purified oral myeloid DCs. Only Pam3CSK4‐treated DCs induce IFN‐γ and IL‐10 secretion by naïve CD4⁺ T cells. Sublingual administration of Pam3CSK4 together with the antigen in BALB/c mice sensitized to OVA decreases airway hyperresponsiveness as well as OVA‐specific T‐helper type 2 (Th2) responses in cervical lymph nodes dramatically. Conclusion Pam3CSK4 induces Th1/regulatory T cell responses, and as such, is a valid candidate adjuvant for sublingual allergy vaccines.     

15‐lipoxygenase metabolites play an important role in the development of a T‐helper type 1 allergic inflammation induced by double‐stranded RNA

Background We recently demonstrated that the T‐helper type 1 (Th1) immune response plays an important role in the development of non‐eosinophilic inflammation induced by airway exposure of an allergen plus double‐stranded RNA (dsRNA). However, the role of lipoxygenase (LO) metabolites in the development of Th1 inflammation is poorly understood. Objective To evaluate the role of LO metabolites in the development of Th1 inflammation induced by sensitization with an allergen plus dsRNA. Methods A Th2‐allergic inflammation mouse model was created by an intraperitoneal injection of lipopolysaccharide‐depleted ovalbumin (OVA, 75 μg) and alum (2 mg) twice, and the Th1 model was created by intranasal application of OVA (75 μg) and synthetic dsRNA [10 μg of poly(I : C)] four times, followed by an intranasal challenge with 50 μg of OVA four times. The role of LO metabolites was evaluated using two approaches: a transgenic approach using 5‐LO^?/? and 15‐LO^?/? mice, and a pharmacological approach using inhibitors of cysteinyl leucotriene receptor‐1 (cysLTR1), LTB4 receptor (BLT1), and 15‐LO. Results We found that the Th1‐allergic inflammation induced by OVA+dsRNA sensitization was similar between 5‐LO^?/? and wild‐type (WT) control mice, although Th2 inflammation induced by sensitization with OVA+alum was reduced in the former group. In addition, dsRNA‐induced Th1 allergic inflammation, which is associated with down‐regulation of 15‐hydroxyeicosateraenoic acids production, was not affected by treatment with cysLTR1 or BLT1 inhibitors, whereas it was significantly lower in 12/15‐LO^?/? mice compared with WT control mice. Moreover, dsRNA‐induced allergic inflammation and the recruitment of T cells following an allergen challenge were significantly inhibited by treatment with a specific 15‐LO inhibitor (PD146176). Conclusion 15‐LO metabolites appear to be important mediators in the development of Th1‐allergic inflammation induced by sensitization with an allergen plus dsRNA. Our findings suggest that the 15‐LO pathway is a novel therapeutic target for the treatment of virus‐associated asthma characterized by Th1 inflammation.     

TLR8‐mediated activation of human monocytes inhibits TL1A expression

TLR play important roles in inflammation and innate immune response to pathogens. TLR8 recognizes ssRNA and induces NF‐κB via MyD88 signaling. TL1A is a member of the TNF superfamily that markedly enhances IFN‐γ production by IL‐12/IL‐18‐stimulated peripheral and mucosal CD4⁺ T cells. TL1A expression is increased in the mucosa of patients with inflammatory bowel disease and is considered a key mediator of Crohn's disease (CD). We have previously shown that TL1A is strongly induced by immune complexes (IC) but not TLR ligands in antigen‐presenting cells. However, a potential interaction between these pro‐inflammatory signaling pathways has not been investigated. IC‐induced TL1A expression of monocytes was potently inhibited by a TLR8 or TLR7/8 ligand (R848) in a dose‐dependent manner. Furthermore, when co‐cultured with CD4⁺ T cells, TLR8 ligands inhibited TL1A production, resulting in almost complete inhibition of IFN‐γ production by the CD4⁺ T cells. Furthermore, we demonstrate that IFN‐α is not required for this suppressive effect by TLR8 signaling. Our data demonstrate for the first time a direct interaction between TLR and TL1A signaling pathways. TLR8 activation may be an important, novel pathway for targeted treatment of Th1‐mediated diseases, such as CD.     

The engagement of oral‐associated lymphoid tissues during oral versus gastric antigen administration

The role of oral‐associated lymphoid tissues during induction of oral tolerance still remains elusive. Therefore, the aim was to compare T‐cell activation and induction of tolerance to ovalbumin (OVA) presented through either of two routes; deposited into the oral cavity, or the stomach, thereby bypassing the oral cavity. OVA was administered by the oral or gastric route to BALB/c mice that had received OVA‐specific DO11.10+ CD4⁺ T cells, stained with CellTrace^? Violet dye, through intravenous injection. Proliferating OVA‐specific T cells were detected in the nose‐associated lymphoid tissues (NALT) and the cervical, mesenteric and peripheral lymph nodes at different time‐points following OVA exposure. OVA‐specific T‐cell proliferation was initially observed in the NALT 1 hr after oral, but not gastric, administration. However, at day 1, proliferation at this site was also detected after gastric administration and profound proliferation was observed at all sites by day 4. For the oral route the degree of proliferation observed was lower in the peripheral lymph nodes by day 4 compared with the other sites. These results demonstrate a similar activation pattern achieved by the two routes. However, the NALT distinguishes itself as a site of rapid T‐cell activation towards fed antigens irrespective of feeding regimen. To evaluate induction of tolerance a semi‐effective OVA dose was used, to detect differences in the degree of tolerance achieved. This was performed in a model of OVA‐induced airway hypersensitivity. No differences in tolerance induction were observed between the two administration routes.