TIM-3 is not essential for development of airway inflammation induced by house dust mite antigens.

BACKGROUND: T cell immunoglobulin domain and mucin domain-containing molecule 3 (TIM-3), which is preferentially expressed on Th1 cells rather than Th2 cells, is considered to be a negative regulator of Th1 cell function. This suggests that TIM-3 indirectly enhances Th2-type immune responses by suppressing Th1 cell function. METHODS: To investigate TIM-3's possible involvement in Th2-type acute and chronic airway inflammation, wild-type and TIM-3-deficient (TIM-3-/-) mice were sensitized and challenged with a house dust mite (HDM) extract. Airway inflammation and the number of inflammatory cells in bronchoalveolar lavage fluids (BALFs) in the mice were determined by histological analysis and with a hemocytometer, respectively. Expression of mRNA in the lungs was determined by quantitative PCR, while the levels of cytokines in the BALFs and IgE in sera were determined by ELISA. RESULTS: Despite constitutive expression of TIM-3 mRNA in the lungs, the number of eosinophils in bronchoalveolar lavage fluids (BALFs) and the score of pulmonary inflammation were comparable between wild-type and TIM-3-/- mice during both acute and chronic HDM-induced airway inflammation. On the other hand, the number of lymphocytes in the BALFs of TIM-3-/- mice was significantly increased compared with wild-type mice during HDM-induced chronic, but not acute, airway inflammation, while the levels of Th2 cytokines in the BALFs and HDM-specific IgG1 and IgG2a and total IgE in the sera were comparable in both groups. CONCLUSIONS: Our findings indicate that, in mice, TIM-3 is not essential for development of HDM-induced acute or chronic allergic airway inflammation, although it appears to be involved in reduced lymphocyte recruitment during HDM-induced chronic allergic airway inflammation.

Potential role of myeloid cell/eosinophil-derived IL-17 in LPS-induced endotoxin shock.

IL-17RA is a shared receptor subunit for several cytokines of the IL-17 family, including IL-17A, IL-17C, IL-17E (also called IL-25) and IL-17F. It has been shown that mice deficient in IL-17RA are more susceptible to sepsis than wild-type mice, suggesting that IL-17RA is important for host defense against sepsis. However, it is unclear which ligands for IL-17RA, such as IL-17A, IL-17C, IL-17E/IL-25 and/or IL-17F, are involved in the pathogenesis of sepsis. Therefore, we examined IL-17A, IL-17E/IL-25 and IL-17F for possible involvement in LPS-induced endotoxin shock. IL-17A-deficient mice, but not IL-25- or IL-17F-deficient mice, were resistant to LPS-induced endotoxin shock, as compared with wild-type mice. Nevertheless, studies using IL-6-deficient, IL-21Rα-deficient and Rag-2-deficient mice, revealed that neither IL-6 and IL-21, both of which are important for Th17 cell differentiation, nor Th17 cells were essential for the development of LPS-induced endotoxin shock, suggesting that IL-17A-producing cells other than Th17 cells were important in the setting. In this connection, IL-17A was produced by macrophages, DCs and eosinophils after LPS injection. Taken together, these findings indicate that IL-17A, but not IL-17F or IL-25, is crucial for LPS-induced endotoxin shock. In addition, macrophages, DCs and eosinophils, but not Th17 cells or γδ T cells, may be sources of IL-17A during LPS-induced endotoxin shock.

Epithelial cell-derived IL-25, but not Th17 cell-derived IL-17 or IL-17F, is crucial for murine asthma.

IL-17A, IL-17F, and IL-25 are ligands for IL-17RA. In the current study, we demonstrated that IL-25-deficient mice-but not IL-17A-, IL-17F-, IL-17A/F-, IL-23p19-, or retinoic acid-related orphan receptor (ROR)-γt-deficient mice-showed significant suppression of 1) the number of eosinophils and the levels of proinflammatory mediators in bronchoalveolar lavage fluids, 2) airway hyperresponsiveness to methacholine, and 3) OVA-specific IgG1 and IgE levels in the serum during OVA-induced Th2-type/eosinophilic airway inflammation. The IL-25 deficiency did not affect lung dendritic cell migration or Ag-specific memory-Th2 cell expansion during Ag sensitization. Adoptive transfer of T cells, mast cells, or bone marrow cells from IL-25-deficient mice revealed that induction of Th2-type/eosinophilic airway inflammation was dependent on activation of lung epithelial cells and eosinophils by IL-25 produced by airway structural cells such as epithelial cells but not by such hematopoietic stem-cell-origin immune cells as T cells and mast cells. Therefore, airway structural cell-derived IL-25-rather than Th17 cell-derived IL-17A and IL-17F-is responsible for induction of local inflammation by promoting activation of lung epithelial cells and eosinophils in the elicitation phase of Th2-type/eosinophilic airway inflammation. It is not required for Ag-specific Th2 cell differentiation in the sensitization phase.

Leptin enhances survival and induces migration, degranulation, and cytokine synthesis of human basophils.

Basophils are the rarest leukocytes in human blood, but they are now recognized as one of the most important immunomodulatory as well as effector cells in allergic inflammation. Leptin, a member of the IL-6 cytokine family, has metabolic effects as an adipokine, and it is also known to participate in the pathogenesis of inflammatory reactions. Because there is an epidemiologic relationship between obesity and allergy, we examined whether basophil functions are modified by leptin. We found that human basophils express leptin receptor (LepR) at both the mRNA and surface protein levels, which were upregulated by IL-33. Leptin exerted strong effects on multiple basophil functions. It induced a strong migratory response in human basophils, similar in potency to that of basophil-active chemokines. Also, leptin enhanced survival of human basophils, although its potency was less than that of IL-3. Additionally, CD63, a basophil activation marker expressed on the cell surface, was upregulated by leptin, an effect that was neutralized by blocking of LepR. Assessments of basophil degranulation and cytokine synthesis found that leptin showed a strong priming effect on human basophil degranulation in response to FcεRI aggregation and induced Th2, but not Th1, cytokine production by the cells. In summary, the present findings indicate that leptin may be a key molecule mediating the effects of adipocytes on inflammatory cells such as basophils by binding to LepR and activating the cellular functions, presumably exacerbating allergic inflammation.

Screening of novel malaria DNA vaccine candidates using full-length cDNA library.

No licensed malaria vaccine exists, in spite of intensive development efforts. We have been investigating development of a DNA vaccine to prevent malaria infection. To date, we have established a full-length cDNA expression library from the erythrocytic-stage murine malaria parasite, Plasmodium berghei. We found that immunization of mice with combined 2000 clones significantly prolonged survival after challenge infection and that splenocytes from the immunized mice showed parasite-specific cytokine production. We determined the 5'-end one-pass sequence of these clones and mapped a draft genomic sequence for P. berghei for use in screening vaccine candidates for efficacy. In this study, we annotated these cDNA clones by comparing them with the genomic sequence of Plasmodium falciparum. We then divided them into several subsets based on their characteristics and examined their protective effects against malaria infection. Consequently, we selected 104 clones that strongly induced specific IgG production and decreased the mortality rate in the early phase. Most of these 104 clones coded for unknown proteins. The results suggest that these clones represent potential novel malaria vaccine candidates.

Th17 cell-derived IL-17 is dispensable for B cell antibody production.

IL-17, which is preferentially produced by Th17 cells, is important for host defense against pathogens and is also involved in the development of autoimmune and allergic disorders. Antibody (Ab) production was shown to be impaired in IL-17-deficient mice, suggesting that IL-17 may promote B cell activation and direct secretion of Ab. However, the precise role of IL-17 in Ab production by B cells remains unclear. In the present study, we found constitutive expression of IL-17R in murine splenic B cells. Nevertheless, IL-17, IL-17F or IL-25 alone could not induce Ab production by B cells even in the presence of agonistic anti-CD40 Ab. IL-17 also could not affect IFN-γ-, IL-4- or TGF-ß1-mediated Ig class-switching. Furthermore, in co-cultures of B cells and IL-17(-/-) CD4(+) T cells or IL-17(-/-) Th17 cells, IL-17 deficiency did not influence Ab production by B cells in vitro, suggesting that Th17 cell-derived IL-17 was not required for B cell Ab production through T cell-B cell interaction in vitro. Thus, in vivo, IL-17 may be indirectly involved in Ab production by enhancing production of B cell activator(s) by other immune cells.

N-acetylglucosaminyltransferase V-deficiency increases susceptibility to murine malaria.

It is considered that several glycoproteins on erythrocytes in mammalian species are involved in malaria parasite infection. To elucidate the role of N-glycans on malaria parasite infection, we induced experimental murine malaria infection (using Plasmodium berghei ANKA) in mice deficient in N-acetylglucosaminyltransferase V (Mgat5), which is one of the enzymes involved in ß1,6-GlcNAc N-glycan biosynthesis. After infection, Mgat5(-/-) mice showed severe body weight loss and parasitemia compared with wild-type mice. The Mgat5(-/-) mice, but not wild-type mice, also showed severe pathology accompanied by marked infiltration of plasma cells into the lungs and liver. These results suggest that ß1,6-GlcNAc N-glycans on/in host erythrocytes may interfere with invasion of the parasites and progression to severe malaria.

Alteration of immune responses by N-acetylglucosaminyltransferase V during allergic airway inflammation.

BACKGROUND: ß-1,6-N-acetylglucosaminyltransferase V (Mgat5 or GlcNac-TV), which is involved in the glycosylation of proteins, is known to be important for down-regulation of TCR-mediated T-cell activation and negatively regulates induction of contact dermatitis and experimental autoimmune encephalomyelitis. However, the role of Mgat5 in the induction of allergic airway inflammation remains unclear. METHODS: To elucidate the role of Mgat5 in the pathogenesis of allergic airway inflammation, ovalbumin (OVA)-induced airway inflammation was induced in Mgat5-deficient mice. The OVA-specific lymphocyte proliferation and cytokine production levels, OVA-specific IgG1, IgG2a and IgE levels in the serum, and the number of leukocytes and cytokine levels in the bronchoalveolar lavage (BAL) fluid were compared between wild-type and Mgat5-deficient mice. RESULTS: OVA-specific lymphocyte proliferation and production of IFN-γ and IL-10, but not IL-4, were increased in Mgat5-deficient mice, suggesting that Th2-type immune responses are seemed to be suppressed by increased IFN-γ and IL-10 production in these mice. However, Th2-type responses such as OVA-specific IgG1, but not IgE, and IL-5 levels in BAL fluids were increased in Mgat5-deficient mice. Meanwhile, the number of eosinophils was normal, but the numbers of neutrophils, macrophages and lymphocytes were reduced, in these mutant mice during OVA-induced airway inflammation. CONCLUSIONS: Mgat5-dependent glycosylation of proteins can modulate acquired immune responses, but it is not essential for the development of OVA-induced eosinophilic airway inflammation.

CD4(+) T cell response in early erythrocytic stage malaria: Plasmodium berghei infection in BALB/c and C57BL/6 mice.

Plasmodium berghei ANKA causes lethal malaria in mice. It is well established that C57BL/6 mice die early with fulminant symptoms including convulsion, whereas BALB/c mice survive this phase and die later of anemia and prostration. Early death in C57BL/6 mice has been considered to result from the adverse effects of inflammatory cytokines. To elucidate the CD4(+) T cell responses in early death due to severe malaria, the kinetics of CD4(+) T cells were compared by analyzing cell surface markers and the production of cytokines and transcription factors. The results revealed that cytokine production by CD4(+) T cells was induced as early as 5 days after infection and the maintenance of higher levels of IL-4 and IL-10 may be associated with the protection of BALB/c mice from early death. These results suggest that parasite control in the early phase of infection may be important for the development of an effective vaccine.

IL-25, IL-33 and TSLP receptor are not critical for development of experimental murine malaria.

IL-25, IL-33 and TSLP, which are produced predominantly by epithelial cells, can induce production of Th2-type cytokines such as IL-4, IL-5 and/or IL-13 by various types of cells, suggesting their involvement in induction of Th2-type cytokine-associated immune responses. It is known that Th2-type cytokines contribute to host defense against malaria parasite infection in mice. However, the roles of IL-25, IL-33 and TSLP in malaria parasite infection remain unclear. Thus, to elucidate this, we infected wild-type, IL-25-/-, IL-33-/- and TSLP receptor (TSLPR)-/- mice with Plasmodium berghei (P. berghei) ANKA, a murine malaria strain. The expression levels of IL-25, IL-33 and TSLP mRNA were changed in the brain, liver, lung and spleen of wild-type mice after infection, suggesting that these cytokines are involved in host defense against P. berghei ANKA. However, the incidence of parasitemia and survival in the mutant mice were comparable to in the wild-type mice. These findings indicate that IL-25, IL-33 and TSLP are not critical for host defense against P. berghei ANKA.

Impaired recognition by Toll-like receptor 4 is responsible for exacerbated murine Pneumocystis pneumonia.

We investigated the effect of Toll-like receptor 4 (TLR4) on the progression of murine Pneumocystis pneumonia. TLR4-mutant C3H/HeJ and wild-type C3H/HeN mice were infected with Pneumocystis after depletion of CD4 T cells. Mutant mice lost body weight more quickly and showed exacerbated pulmonary injury even though there was no difference in Pneumocystis organism burden in the lung. Mutant mice showed reduced levels of IL-10, IL-12p40 and MIP-2 accompanied by elevated levels of TNF-alpha and IL-6 in the bronchoalveolar lavage fluid compared with those of wild-type mice 8 weeks after the infection. In response to stimulation with Pneumocystis antigen, the production of IL-10, IL-12p40 and MIP-2 by alveolar macrophages was partially impaired in mutant mice, while that in wild-type mice was suppressed by the anti-TLR4/MD-2 mAb, MTS510. Unlike the response to lipopolysaccharide stimulation, TLR4-reconstituted HEK293 cells showed no elevated NF-kappaB activation after stimulation with Pneumocystis antigen. Taken together, these findings suggest that recognition of Pneumocystis by TLR4 helps to regulate the host inflammatory responses through cytokine and chemokine production by alveolar macrophages.

Fundamental study of expression-library immunization against Taenia taeniaeformis in rats.

Expression-library immunization has been proposed as an effective means to screen a large number of genes of the pathogen as candidate protective molecules. In this study, we examined the efficacy of expression-library immunization using a T. taeniaeformis rat model system. Total RNAs were isolated from the last 15 segments of adult T. taeniaeformis and poly A RNA was purified. cDNA library was produced using SuperScript Plasmid System, which contains a mammalian expression vector, pCMV*SPORT6. From about 3,500 clones examined, more than 800 clones were found to contain DNA fragments. About 200 clones were sequenced and the homology search was carried out. The blast search revealed that 29% of the expression genes were mitochondrial genes (rRNA; 17%, protein; 12%). Nuclear rRNA genes (10%), nuclear protein (9%) and genes from Escherichia coli were also detected. Forty-two percent of sequences did not show a significant similarity to any genes deposited in the public database. Rats were immunized with expression-library and injected orally with 1,000 T. taeniaeformis eggs. However the protective effect of expression-library vaccine was not confirmed.

IL-33, but not IL-25, is crucial for the development of house dust mite antigen-induced allergic rhinitis.

Both interleukin (IL)-33 and IL-25 induce Th2 cytokine production by various cell types, suggesting that they contribute to development of allergic disorders. However, the precise roles of IL-33 and IL-25 in house dust mite (HDM)-induced allergic rhinitis (AR) remain unclear. Both IL-33 and IL-25 were produced mainly by nasal epithelial cells during HDM-induced AR. Eosinophil and goblet cell counts in the nose and IL-5 levels in lymph node cell culture supernatants were significantly decreased in IL-33-deficient, but not IL-25-deficient, mice compared with wild-type mice during HDM-induced AR, but the serum IgE and IgG1 levels did not differ. On the other hand, HDM-induced AR developed similarly in wild-type mice transferred with either IL-33-deficient BM cells or wild-type BM cells. IL-33, but not IL-25, produced by nasal epithelial cells was crucial for the development of murine HDM-induced AR. These observations suggest that IL-33 neutralization may be a potential approach for treatment of HDM-induced AR in humans.

A novel method for development of malaria vaccines using full-length cDNA libraries.

We describe a novel method to screen malaria DNA vaccine candidates using a full-length cDNA library and a murine malaria infection model. For the development of effective malaria vaccines, much effort has been made with meager success. The completion of genome sequencing of Plasmodium falciparum has provided invaluable information for achieving this goal. We have been studying full-length cDNA libraries of malaria parasites as a part of genome analysis. Mice vaccinated with a DNA vaccine consisting of 2000 pooled clones showed significantly prolonged survival after challenge infection. In addition, spleen cells of vaccinated mice produced augmented levels of IL-2 and IFN-gamma when incubated with the crude parasite antigens, indicating that cellular immunity plays an important role in the protection. This approach will not only form the basis for development of malaria vaccines but will also be applicable to other parasites and pathogenic microorganisms.