Type II Collagen Induces Peripheral Tolerance in BALB/c Mice via the Generation of CD8+ T Regulatory Cells

Antigens introduced into the anterior chamber (AC) of the eye induce a potent form of antigen-specific peripheral immune tolerance termed AC-associated immune deviation (ACAID), which prevents inflammatory immune responses and is characterized by impaired delayed-type hypersensitivity (DTH) responses. Type-II collagen (CII) is a fibrillar protein expressed exclusively in cartilage tissues. Although of its clinical relevance to Rheumatoid arthritis, aging, and osteoarthritis, there have been no studies to date to test if CII has the ability to induce ACAID. We hypothesized that ACAID could be generated via AC injection of CII in BALB/c mice. Using a DTH assay, the hypothesis was supported and led to another hypothesis that CII is capable of inducing specific immune tolerance via CD8⁺ T regulatory cells (Tregs). Thus, we performed functional local adoptive transfer (LAT) assays to examine the regulatory roles of spleen cells, T cells, and CD8⁺ T cells in the specific immune regulation induced by CII injection into the AC. Results indicated that CII induced ACAID when injected into the AC. Spleen cells of mice injected with CII in the AC significantly suppressed DTH responses. The T cell compartment of the spleen was capable of expressing this suppression. CD8⁺ Tregs could solely express this CII-driven suppression and even exerted more noticeable suppression than spleen cells or splenic T cells. This study suggests a crucial role for CD8⁺ Tregs in mediating CII-driven ACAID-mediated immune tolerance. This could have therapeutic implications in Rheumatoid arthritis, aging, osteoarthritis, and other diseases in which CII is involved.     

T cells that are naturally tolerant to cartilage-derived type II collagen are involved in the development of collagen-induced arthritis

The immunodominant T-cell epitope that is involved in collagen-induced arthritis (CIA) is the glycosylated type II collagen (CII) peptide 256-270. In CII transgenic mice, which express the immunodominant CII 256-270 epitope in cartilage, the CII-specific T cells are characterized by a partially tolerant state with low proliferative activity in vitro, but with maintained effector functions, such as IFN-γ secretion and ability to provide B cell help. These mice were still susceptible to CIA. The response was mainly directed to the glycosylated form of the CII 256-270 peptide, rather than to the nonglycosylated peptide. Tolerance induction was rapid; transferred T cells encountered CII within a few days. CII immunization several weeks after thymectomy of the mice did not change their susceptibility to arthritis or the induction of partial T-cell tolerance, excluding a role for recent thymic emigrants. Thus, partially tolerant CII autoreactive T cells are maintained and are crucial for the development of CIA.     

Feeding transgenic plants that express a tolerogenic fusion protein effectively protects against arthritis

Although much explored, oral tolerance for treatment of autoimmune diseases still awaits the establishment of novel and effective vectors. We investigated whether the tolerogenic CTA1(R7K)‐COL‐DD fusion protein can be expressed in edible plants, to induce oral tolerance and protect against arthritis. The fusion protein was recombinantly expressed in Arabidopsis thaliana plants, which were fed to H‐2^q‐restricted DBA/1 mice to assess the preventive effect on collagen‐induced arthritis (CIA). The treatment resulted in fewer mice exhibiting disease and arthritis scores were significantly reduced. Immune suppression was evident in treated mice, and serum biomarkers for inflammation as well as anticollagen IgG responses were reduced. In spleen and draining lymph nodes, CD4⁺ T‐cell responses were reduced. Concomitant with a reduced effector T‐cell activity with lower IFNγ, IL‐13 and IL‐17A production, we observed an increase in IL‐10 production to recall antigen stimulation in vitro, suggesting reduced Th1, Th2 and Th17 activity subsequent to up‐regulated IL‐10 and regulatory T‐cell (Treg) functions. This study shows that edible plants expressing a tolerogen were effective at stimulating CD4 T‐cell tolerance and in protecting against CIA disease. Our study conveys optimism as to the potential of using edible plants for oral treatment of rheumatoid arthritis.     

Natural killer T cells promote collagen-induced arthritis in DBA/1 mice

The role of NKT cells in the pathogenesis of collagen-induced arthritis (CIA) remains unclear since most studies have used C57BL/6 (B6) mice, which are less susceptible to CIA than mice with a DBA/1 background. To clarify the immunological functions of NKT cells in CIA, it is necessary to analyze in detail the effects of NKT cell deficiency on CIA development in DBA/1 mice. The incidence and severity of CIA were significantly exacerbated in DBA/1CD1d^+/− mice as compared to DBA/1CD1d^−/− mice. In DBA/1CD1d^+/− mice, antigen-specific responses of B and T cells against CII were remarkably increased and inflammatory cytokine levels were also increased in vivo and in vitro. The number of IL-17-producing NKT cells significantly increased in DBA/1CD1d^+/− mice as the disease progressed. Our results clearly show that NKT cells are involved not only in accelerating the severity and incidence of CIA but also in perpetuating the disease progression.     

Diet‐Induced Changes in Stearoyl‐CoA Desaturase 1 Expression in Obesity‐Prone and ‐Resistant Mice

Objective: To investigate stearoyl‐coenzyme A desaturase (SCD) 1 expression in obesity‐prone C57BL/6 mice and in obesity‐resistant FVB mice to explore the relationship of SCD1 expression and susceptibility to diet‐induced obesity. Research Methods and Procedures: Nine‐week‐old C57BL/6 and FVB mice were fed either a high‐ or low‐fat diet for 8 weeks. Body weight and body composition were measured before and at weeks 4 and 8 of the study. Energy expenditure was measured at weeks 1 and 5 of the study. Hepatic SCD1 mRNA was measured at 72 hours and at the end of study. Plasma leptin and insulin concentrations were measured at the end of study. Results: When C57BL/6 mice were switched to a calorie‐dense high‐fat diet, animals gained significantly more body weight than those maintained on a low‐calorie density diet primarily due to increased fat mass accretion. Fat mass continued to accrue throughout 8 weeks of study. Increased calorie intake did not account for all weight gain. On the high‐fat diet, C57BL/6 mice decreased their energy expenditure when compared with mice fed a low‐fat diet. In response to 8 weeks of a high‐fat diet, SCD1 gene expression in liver increased >2‐fold. In contrast, feeding a high‐fat diet did not change body weight, energy expenditure, or SCD1 expression in FVB mice. Discussion: Our study showed that a high‐fat hypercaloric diet increased body adiposity first by producing hyperphagia and then by decreasing energy expenditure of mice susceptible to diet‐induced obesity. Consumption of a high‐fat diet in species predisposed to obesity selectively increased SCD1 gene expression in liver.     

Suppression of collagen‐induced arthritis by oral administration of transgenic rice seeds expressing altered peptide ligands of type II collagen

Rheumatoid arthritis (RA) is an autoimmune disease associated with the recognition of self proteins secluded in arthritic joints. We previously reported that altered peptide ligands (APLs) of type II collagen (CII256‐271) suppress the development of collagen‐induced arthritis (CIA). In this study, we generated transgenic rice expressing CII256‐271 and APL6 contained in fusion proteins with the rice storage protein glutelin in the seed endosperm. These transgene products successfully and stably accumulated at high levels (7–24 mg/g seeds) in protein storage vacuoles (PB‐II) of mature seeds. We examined the efficacy of these transgenic rice seeds by performing oral administration of the seeds to CIA model mice that had been immunized with CII. Treatment with APL6 transgenic rice for 14 days significantly inhibited the development of arthritis (based on clinical score) and delayed disease onset during the early phase of arthritis. These effects were mediated by the induction of IL‐10 from CD4⁺ CD25^? T cells against CII antigen in splenocytes and inguinal lymph nodes (iLNs), and treatment of APL had no effect on the production of IFN‐γ, IL‐17, IL‐2 or Foxp3⁺ Treg cells. These findings suggest that abnormal immune suppressive mechanisms are involved in the therapeutic effect of rice‐based oral vaccine expressing high levels of APLs of type II collagen on the autoimmune disease CIA, suggesting that the seed‐based mucosal vaccine against CIA functions via a unique mechanism.     

Type IX collagen deficiency enhances the binding of cartilage-specific antibodies and arthritis severity

Joint cartilage is attacked in both autoimmune inflammatory and osteoarthritic processes. Type IX collagen (CIX) is a protein of importance for cartilage integrity and stability. In this study we have backcrossed a transgenic disruption of the col9a1 gene, which leads to an absence of CIX, into two different inbred mouse strains, DBA/1 and B10.Q. None of the CIX-deficient mice developed observable clinical or microscopic osteoarthritis, but DBA/1 male mice had more pronounced enthesopathic arthritis, the so-called stress-induced arthritis. Both DBA/1 and B10.Q strains are susceptible to the induction of collagen-induced arthritis, and CIX deficiency in both strains led to the development of a more severe arthritis than in the controls. Induction of arthritis with monoclonal antibodies against type II collagen (CII) led to an earlier arthritis in the paws that also involved the knee joints. The antibodies used, which were specific for the J1 and the C1^I epitopes of CII, initiate their arthritogenic attack by binding to cartilage. The C1^I-specific antibodies bound to cartilage better in CIX-deficient mice than in wild-type animals, demonstrating that the lack of CIX in cartilage leads to an increased accessibility of structures for antibody binding and thus making the joints more vulnerable to inflammatory attack. These findings accentuate the importance of cartilage stability; cartilage disrupted as a result of genetic disorders could be more accessible and vulnerable to an autoimmune attack by pathogenic antibodies.     

A cross-reactive idiotype on anti-collagen antibodies in collagen-induced arthritis: identification and relevance to disease

Immunization of mice with type II collagen (CII) leads to the production of anti-CII antibodies and, in susceptible strains, to the induction of arthritis. Specifically purified anti-CII antibodies from arthritic DBA/1 mice were used to prepare a rabbit anti-idiotypic antiserum. This antiserum recognizes a cross-reactive idiotype (CRI) present on 20-25% of anti-CII antibodies from DBA/1 mice immunized with bovine CII. The CRI is not present on DBA/1 anti-trinitrophenyl, undetectable in normal Ig and not Igh allotype linked. The presence of this CRI was examined after antigen specific suppression of the anti-CII antibody response by intravenous administration of chick or bovine CII. While intravenous injection of bovine CII, prior to immunization with chick CII, greatly reduces both the incidence of arthritis and the anti-CII response, the fraction of anti-bovine CII which expresses the CRI is increased by this treatment. These findings suggest that the CRI characterizes a disease-unrelated fraction of anti-CII which recognizes bovine and chick CII, but probably not mouse CII. In addition, attempts at idiotypic regulation of arthritis incidence and antibody response by in vivo administration of anti-idiotypic serum also indicate that the CRI-bearing antibody is not important for the induction of arthritis.     

Helminth Antigens Enable CpG-Activated Dendritic Cells to Inhibit the Symptoms of Collagen-induced Arthritis through Foxp3+ Regulatory T Cells

Dendritic cells (DC) have the potential to control the outcome of autoimmunity by modulating the immune response. In this study, we tested the ability of Fasciola hepatica total extract (TE) to induce tolerogenic properties in CpG-ODN (CpG) maturated DC, to then evaluate the therapeutic potential of these cells to diminish the inflammatory response in collagen induced arthritis (CIA). DBA/1J mice were injected with TE plus CpG treated DC (T/C-DC) pulsed with bovine collagen II (CII) between two immunizations with CII and clinical scores CIA were determined. The levels of CII-specific IgG2 and IgG1 in sera, the histological analyses in the joints, the cytokine profile in the draining lymph node (DLN) cells and in the joints, and the number, and functionality of CD4+CD25+Foxp3+ T cells (Treg) were evaluated. Vaccination of mice with CII pulsed T/C-DC diminished the severity and incidence of CIA symptoms and the production of the inflammatory cytokine, while induced the production of anti-inflammatory cytokines. The therapeutic effect was mediated by Treg cells, since the adoptive transfer of CD4+CD25+ T cells, inhibited the inflammatory symptoms in CIA. The in vitro blockage of TGF-β in cultures of DLN cells plus CII pulsed T/C-DC inhibited the expansion of Treg cells. Vaccination with CII pulsed T/C-DC seems to be a very efficient approach to diminish exacerbated immune response in CIA, by inducing the development of Treg cells, and it is therefore an interesting candidate for a cell-based therapy for rheumatoid arthritis (RA).     

BID‐dependent release of mitochondrial SMAC dampens XIAP‐mediated immunity against Shigella

The X‐linked inhibitor of apoptosis protein (XIAP) is a potent caspase inhibitor, best known for its anti‐apoptotic function in cancer. During apoptosis, XIAP is antagonized by SMAC, which is released from the mitochondria upon caspase‐mediated activation of BID. Recent studies suggest that XIAP is involved in immune signaling. Here, we explore XIAP as an important mediator of an immune response against the enteroinvasive bacterium Shigella flexneri, both in vitro and in vivo. Our data demonstrate for the first time that Shigella evades the XIAP‐mediated immune response by inducing the BID‐dependent release of SMAC from the mitochondria. Unlike apoptotic stimuli, Shigella activates the calpain‐dependent cleavage of BID to trigger the release of SMAC, which antagonizes the inflammatory action of XIAP without inducing apoptosis. Our results demonstrate how the cellular death machinery can be subverted by an invasive pathogen to ensure bacterial colonization.     

Aging is associated with increased regulatory T‐cell function

Regulatory T‐cell (Treg, CD4⁺CD25⁺) dysfunction is suspected to play a key role in immune senescence and contributes to increased susceptibility to diseases with age by suppressing T‐cell responses. FoxP3 is a master regulator of Treg function, and its expression is under control of several epigenetically labile promoters and enhancers. Demethylation of CpG sites within these regions is associated with increased FoxP3 expression and development of a suppressive phenotype. We examined differences in FoxP3 expression between young (3–4 months) and aged (18–20 months) C57BL/6 mice. DNA from CD4⁺ T cells is hypomethylated in aged mice, which also exhibit increased Treg numbers and FoxP3 expression. Additionally, Treg from aged mice also have greater ability to suppress effector T‐cell (Teff) proliferation in vitro than Tregs from young mice. Tregs from aged mice exhibit greater redox remodeling–mediated suppression of Teff proliferation during coculture with DCs by decreasing extracellular cysteine availability to a greater extent than Tregs from young mice, creating an adverse environment for Teff proliferation. Tregs from aged mice produce higher IL‐10 levels and suppress CD86 expression on DCs more strongly than Tregs from young mice, suggesting decreased T‐cell activity. Taken together, these results reveal a potential mechanism of higher Treg‐mediated activity that may contribute to increased immune suppression with age.     

Relevance of posttranslational modifications for the arthritogenicity of type II collagen

To establish the role of posttranslational modification in modulating the immune response to collagen, recombinant human type II collagen (rCII) was produced using a yeast expression system (rCII(pic)) and a baculovirus expression system (rCII(bac)). The biosynthesis of CII requires extensive posttranslational modification including the hydroxylation of prolyl and lysyl residues and glycosylation of selected hydroxylysyl residues. Amino acid analyses indicated that the rCII(bac) was adequately hydroxylated at prolyl residues but underhydroxylated at lysyl residues and underglycosylated compared with tissue-derived CII, whereas rCII(pic) was adequately hydroxylated at prolyl residues but unhydroxylated at lysyl residues and had no glycosylation. When DBA/1 mice were immunized with rCII, rCII(pic) induced a lower incidence of arthritis than tissue-derived CII, whereas rCII(bac) induced an intermediate level of arthritis. The severity of the arthritis was significantly lower in mice immunized with rCII(pic) compared with mice immunized with tissue-derived CII, whereas that of rCII(bac) was intermediate. These data indicate that the degree of lysine hydroxylation and glycosylation plays a role in the induction of arthritis. The recombinant collagens were then compared with tissue-derived CII when given as i.v. or oral tolerogens to suppress arthritis. Both recombinant collagens were less potent than tissue-derived CII, and this decrease in arthritis was associated with a decrease in Ab response to CII. These data suggest that the degree of glysosylation affects the immune response to CII, so that underglycosylated CII is less effective in the induction of arthritis and in its ability to suppress collagen-induced arthritis.     

Injection of soluble antigen into the anterior chamber of the eye induces expansion and functional unresponsiveness of antigen-specific CD8+ T cells

The injection of soluble Ag into the anterior chamber (a.c.) of the eye induces systemic tolerance, termed a.c.-associated immune deviation (ACAID), characterized by Ag-specific inhibition of delayed-type hypersensitivity responses and a reduction in complement-fixing Abs. Recently, we have shown that CD8(+) CTL responses are also inhibited in ACAID. In this study, we have used an adoptive transfer approach to follow the fate of Ag-specific CD8(+) TCR transgenic (OT-I) T cells in vivo during the induction and expression of ACAID. C57BL/6 (B6) recipients of OT-I splenocytes that were injected with chicken OVA in the a.c. displayed reduced OVA-specific delayed-type hypersensitivity and CTL responses, compared with those of mice given OVA in the subconjunctiva or an irrelevant Ag human IgG in the a.c. OT-I T cells increased 9-fold in the submandibular lymph nodes and 3-fold in the spleen following an a.c. injection with OVA, indicating that expansion rather than deletion of Ag-specific CD8(+) T cells was induced by this treatment. OT-I T cells expanded equivalently upon administration of OVA in CFA to mice previously given OVA in the a.c. or subconjunctiva. However, the lytic activity attributed to OT-I T cells was reduced on a per-cell basis in mice previously given OVA in the a.c. We conclude that tolerance of CTL responses in mice given Ag via the a.c. results from unresponsiveness of Ag-specific CD8(+) T cells.     

Thymus‐specific deletion of insulin induces autoimmune diabetes

Insulin expression in the thymus has been implicated in regulating the negative selection of autoreactive T cells and in mediating the central immune tolerance towards pancreatic β‐cells. To further explore the function of this ectopic insulin expression, we knocked out the mouse Ins2 gene specifically in the Aire‐expressing medullary thymic epithelial cells (mTECs), without affecting its expression in the β‐cells. When further crossed to the Ins1 knockout background, both male and female pups (designated as ID‐TEC mice for insulin‐deleted mTEC) developed diabetes spontaneously around 3 weeks after birth. β‐cell‐specific autoimmune destruction was observed, as well as islet‐specific T cell infiltration. The presence of insulin‐specific effector T cells was shown using ELISPOT assays and adoptive T cell transfer experiments. Results from thymus transplantation experiments proved further that depletion of Ins2 expression in mTECs was sufficient to break central tolerance and induce anti‐insulin autoimmunity. Our observations may explain the rare cases of type 1 diabetes onset in very young children carrying diabetes‐resistant HLA class II alleles. ID‐TEC mice could serve as a new model for studying this pathology.     

Suppressive effect of epigallocatechin‐3‐O‐gallate on endoglin molecular regulation in myocardial fibrosis in vitro and in vivo

Epigallocatechin‐3‐O‐gallate (EGCG), derived from green tea, has been studied extensively because of its diverse physiological and pharmacological properties. This study evaluates the protective effect of EGCG on angiotensin II (Ang II)‐induced endoglin expression in vitro and in vivo. Cardiac fibroblasts (CFs) from the thoracic aorta of adult Wistar rats were cultured and induced with Ang II. Western blotting, Northern blotting, real‐time PCR and promoter activity assay were performed. Ang II increased endoglin expression significantly as compared with control cells. The specific extracellular signal‐regulated kinase inhibitor SP600125 (JNK inhibitor), EGCG (100 μM) and c‐Jun N‐terminal kinase (JNK) siRNA attenuated endoglin proteins following Ang II induction. In addition, pre‐treated Ang II‐induced endoglin with EGCG diminished the binding activity of AP‐1 by electrophoretic mobility shift assay. Moreover, the luciferase assay results revealed that EGCG suppressed the endoglin promoter activity in Ang II‐induced CFs by AP‐1 binding. Finally, EGCG and the JNK inhibitor (SP600125) were found to have attenuated endoglin expression significantly in Ang II‐induced CFs, as determined through confocal microscopy. Following in vivo acute myocardial infarction (AMI)‐related myocardial fibrosis study, as well as immunohistochemical and confocal analyses, after treatment with endoglin siRNA and EGCG (50 mg/kg), the area of myocardial fibrosis reduced by 53.4% and 64.5% and attenuated the left ventricular end‐diastolic and systolic dimensions, and friction shortening in hemodynamic monitor. In conclusion, epigallocatechin‐3‐O‐gallate (EGCG) attenuated the endoglin expression and myocardial fibrosis by anti‐inflammatory effect in vitro and in vivo, the novel suppressive effect was mediated through JNK/AP‐1 pathway.     

NKT cell-derived urokinase-type plasminogen activator promotes peripheral tolerance associated with eye

In a model of peripheral tolerance called anterior chamber-associated immune deviation (ACAID), the differentiation of the T regulatory cells depends on NKT cells and occurs in the spleen. In this study, we show that NKT cells that express the invariant (i) TCR and are the CD1d-reactive NKT cells (required for development of peripheral tolerance) actually produced urokinase-type plasminogen activator (uPA) during tolerance induction. The RT-PCR and in vitro plasmin assay showed that splenic iNKT cells derived uPA-converted plasminogen to plasmin. Moreover, uPA was required for tolerance induction because uPA knockout (KO) mice did not develop peripheral tolerance or develop CD8(+) T regulatory cells after Ag inoculation into the anterior chamber. In contrast, other aspects of ACAID-induced tolerance, including recruitment of iNKT cells to the spleen and production of IL-10 by iNKT cells, were unchanged in uPA-deficient mice. The adoptive transfer of splenic NKT cells from wild-type mice restored ACAID in Jalpha18 KO mice (iNKT cell deficient), but NKT cells from uPA KO mice did not. We postulate that the mechanism of action of uPA is through its binding to the uPAR receptor, and enzymatic cleavage of plasminogen to plasmin, which in turn activates latent TGFbeta. In conclusion, uPA derived from iNKT cells is required to induce peripheral tolerance via the eye.     

Isolation of T cell line capable of protecting mice against collagen-induced arthritis

A T cell line specific to human type II collagen (CII) was selected and propagated from DBA/1J mice immunized with human CII. The line cells were not reactive to type I or type III collagen of human origin, but they were cross-reactive to bovine, rat, and rabbit CII and they recognized both native and heat-denatured human CII. The cells were reactive to an N-terminal three-quarters fragment of human CII, produced by tadpole collagenase digestion of human CII, but not to a C-terminal one-quarter fragment of human CII. The cells showed Thy-1+, Lyt-1+, Lyt-2-, and L3T4+ phenotypes characteristic of T helper cells or delayed-type hypersensitive cells, determined by the immunofluorescence method. To clarify the role of T cells in the pathogenesis of collagen-induced arthritis, we inoculated this cell line into DBA/1J mice and found that they developed clinical arthritis, albeit at a low incidence. The cells attenuated by x-ray were capable of inducing resistance to the subsequent induction of collagen-induced arthritis of DBA/1J mice. The sera from mice protected by inoculation of the cell line exhibited anti-idiotypic antibody response against conventional and monoclonal anti-CII antibodies. Anti-T cell receptor response may be involved in the mechanism for the protective effect of the cell line against autoimmune murine arthritis.     

Diet restriction‐induced healthy aging is mediated through the immune signaling component ZIP‐2 in Caenorhabditis elegans

Dietary restriction (DR) robustly delays the aging process in all animals tested so far. DR slows aging by negatively regulating the target of rapamycin (TOR) and S6 kinase (S6K) signaling pathway and thus inhibiting translation. Translation inhibition in C. elegans is known to activate the innate immune signal ZIP‐2. Here, we show that ZIP‐2 is activated in response to DR and in feeding‐defective eat‐2 mutants. Importantly, ZIP‐2 contributes to the improvements in longevity and healthy aging, including mitochondrial integrity and physical ability, mediated by DR in C. elegans. We further show that ZIP‐2 is activated upon inhibition of TOR/S6K signaling. However, DR‐mediated activation of ZIP‐2 does not require the TOR/S6K effector PHA‐4/FOXA. Furthermore, zip‐2 was not activated or required for longevity in daf‐2 mutants, which mimic a low nutrition status. Thus, DR appears to activate ZIP‐2 independently of PHA‐4/FOXA and DAF‐2. The link between DR, aging, and immune activation provides practical insight into the DR‐induced benefits on health span and longevity.     

Bifidobacterium CECT 7765 improves metabolic and immunological alterations associated with obesity in high‐fat diet‐fed mice

Objectives: To evaluate the effects of administration of Bifidobacterium pseudocatenulatum CECT 7765 on metabolic and immune alterations in obese mice. Design and Methods: Adult male wild‐type C57BL‐6 mice were fed a standard diet or high‐fat diet (HFD), supplemented or not with B. pseudocatenulatum CECT 7765 for 7 weeks. The assessments included biochemical and immunological parameters, insulin resistance, glucose tolerance, histology of liver, white‐adipose and intestinal tissues, immunocompetent cell functions, and microbiota‐related features. Results: B. pseudocatenulatum CECT 7765 reduced serum cholesterol, triglyceride, and glucose levels and decreased insulin resistance and improved glucose tolerance in obese mice. This strain reduced serum levels of leptin, interleukin (IL)‐6 and monocyte chemotactic protein‐1, while increased those of IL‐4 in HFD‐fed mice. B. pseudocatenulatum CECT7765 reduced liver steatosis and the number of larger adipocytes and number of fat micelles in enterocytes of obese mice. The strain also improved the function of macrophages and dendritic cells in relation to phagocytosis, cytokine production, and induction of T‐lymphocyte proliferation. The strain administration increased bifidobacteria and reduced enterobacteria and the inflammatory properties of the gut content in HFD‐fed mice. Conclusion: B. pseudocatenulatum CECT 7765 was shown to ameliorate both metabolic and immunological dysfunctions related to obesity in HFD‐fed mice.     

Transgenic expression of n‐3 fatty acid desaturase (fat‐1) in C57/BL6 mice: Effects on glucose homeostasis and body weight

The fat‐1 gene, derived from Caenorhabditis elegans, encodes for a fatty acid n‐3 desaturase. In order to study the potential metabolic benefits of n‐3 fatty acids, independent of dietary fatty acids, we developed seven lines of fat‐1 transgenic mice (C57/BL6) controlled by the regulatory sequences of the adipocyte protein‐2 (aP2) gene for adipocyte‐specific expression (AP‐lines). We were unable to obtain homozygous fat‐1 transgenic offspring from the two highest expressing lines, suggesting that excessive expression of this enzyme may be lethal during gestation. Serum fatty acid analysis of fat‐1 transgenic mice (AP‐3) fed a high n‐6 unsaturated fat (HUSF) diet had an n‐6/n‐3 fatty acid ratio reduced by 23% (P < 0.025) and the n‐3 fatty acid eicosapentaenoic acid (EPA) concentration increased by 61% (P < 0.020). Docosahexaenoic acid (DHA) was increased by 19% (P < 0.015) in white adipose tissue. Male AP‐3‐fat‐1 line of mice had improved glucose tolerance and reduced body weight with no change in insulin sensitivity when challenged with a high‐carbohydrate (HC) diet. In contrast, the female AP‐3 mice had reduced glucose tolerance and no change in insulin sensitivity or body weight. These findings indicate that male transgenic fat‐1 mice have improved glucose tolerance likely due to increased insulin secretion while female fat‐1 mice have reduced glucose tolerance compared to wild‐type mice. Finally the inability of fat‐1 transgenic mice to generate homozygous offspring suggests that prolonged exposure to increased concentrations of n‐3 fatty acids may be detrimental to reproduction. J. Cell. Biochem. 107: 809–817, 2009. © 2009 Wiley‐Liss, Inc.