Influence of high-fat feeding on both naive and antigen-experienced T-cell immune response in DO10.11 mice.

Obesity is becoming one of the most serious public health problems in industrialized societies, due to the profound changes in lifestyle, and notably in nutrition. Beside diabetes, cardiovascular diseases or hypertension, increased susceptibility to infection is one of the pathological consequences of being overweight. In this paper, we have assessed the influence of a high-fat diet (HFD) rich in saturated fatty acids on the immune system of DO11.10 mice, which are transgenic for a T-cell receptor specifically recognizing a peptide of ovalbumin. We showed that the specific T-cell immune response was impaired by high-fat feeding, and that the expression of this defect is different depending on whether T cells are naive or Ag experienced. Indeed, on in vitro ovalbumin stimulation, spleen T cells from naive HFD-fed transgenic mice showed proliferation similar to that of cells from standard diet (SD)-fed mice, but exhibited a strong inflammatory profile as shown by the markedly increased IFN-gamma/IL-4 ratio. Inversely, spleen T cells from ovalbumin-immunized HFD mice were impaired in their Ag-dependent proliferation compared to cells from SD mice. By co-culture experiments, we showed that both T cells and antigen-presenting cells were involved in this impairment. Moreover, in ovalbumin-immunized HFD animals, a trend towards Th2 response was noted, compared to immunized SD mice. This data implies that naive T cells could participate actively in the low-grade systemic inflammation observed in overweight patients. Moreover, the impaired activity of Ag-experienced T cells could have major consequences both in defence against infection and/or in vaccination protocols.

Genes involved in obesity: Adipocytes, brain and microflora.

The incidence of obesity and related metabolic disorders such as cardiovascular diseases and type 2 diabetes, are reaching worldwide epidemic proportions. It results from an imbalance between caloric intake and energy expenditure leading to excess energy storage, mostly due to genetic and environmental factors such as diet, food components and/or way of life. It is known since long that this balance is maintained to equilibrium by multiple mechanisms allowing the brain to sense the nutritional status of the body and adapt behavioral and metabolic responses to changes in fuel availability. In this review, we summarize selected aspects of the regulation of energy homeostasis, prevalently highlighting the complex relationships existing between the white adipose tissue, the central nervous system, the endogenous microbiota, and nutrition. We first describe how both the formation and functionality of adipose cells are strongly modulated by the diet before summarizing where and how the central nervous system integrates peripheral signals from the adipose tissue and/or the gastro-intestinal tract. Finally, after a short description of the intestinal commensal flora, rangingfrom its composition to its importance in immune surveillance, we enlarge the discussion on how nutrition modified this perfectly well-balanced ecosystem.

Ocular transfer of retinal glial cells transduced ex vivo with adenovirus expressing viral IL-10 or CTLA4-Ig inhibits experimental autoimmune uveoretinitis.

Gene transfer using immunomodulatory molecules is a promising tool for in vivo regulation of immune responses. Experimental autoimmune uveitis (EAU), which serves as a model for human ocular inflammation, is induced by systemic immunization with autoantigens, but its expression is restricted to the eye. Previously, we reported protection of rodents against EAU by intravenous or/and periocular injection of vIL-10-expressing adenovirus. Here, the expression of vIL-10 was targeted into the rat Lewis eye, by intravitreal injection of either the free virus or ex vivo transfected retinal Müller glial cells (RMG-vIL-10). As shown using GFP-expressing adenovirus, a longer expression of transgene was observed in the eye after transfer of transfected syngeneic RMG cells than was seen after injection of free virus. Intravitreal injection of RMG-vIL-10 led to significant decrease in ocular pathological manifestations, compared to control RMG cells. This was observed when cells were injected simultaneously with autoantigen, but also after a delayed administration of transfected cells. Finally, injection of RMG cells transfected with adenovirus expressing CTLA4 had a strongly protective effect. In conclusion, inhibition of antigen presentation at the site of expression of the autoimmune disorders represents an attractive alternative to treat ocular inflammation, and the transfer of ex vivo genetically modified cells provides a promising method to target the factor of interest into the eye.

Inhibition of experimental autoimmune uveoretinitis by systemic and subconjunctival adenovirus-mediated transfer of the viral IL-10 gene.

Pathological ocular manifestations result from a dysregulation in the balance between proinflammatory type 1 cytokines and regulatory type 2 cytokines. Interleukin-10 (IL-10) is an anti-inflammatory cytokine with potent immunosuppressive effects. We have examined the efficiency of viral IL-10 adenovirus (Ad-vIL-10)-mediated gene transfer on experimental autoimmune uveoretinitis (EAU) induced in mice and rats by purified retinal autoantigens, respectively, interphotoreceptor binding protein (IRBP) and S-antigen (S-Ag). B10-A mice that received a single unilateral injection of Ad-vIL-10 in the retro-orbital sinus venosus performed 1 day before immunization with IRBP in the footpads showed high levels of circulating vIL-10 in their sera and a significant reduction in pathological ocular manifestations. Lower levels of IFN-gamma and IL-2 were found in cellular supernatants from IRBP-stimulated splenic cells in these treated mice. The local effect on ocular disease of vIL-10 was neutralized completely by injection of a monoclonal anti-vIL-10 antibody, demonstrating the specificity of the treatment. To determine whether the transfer of the vIL-10 gene within the periocular tissues of the eye could prevent acute EAU, a subconjunctival injection of Ad-vIL-10 was performed in Lewis rats simultaneously with S-antigen in the footpads. This injection determined in situ vIL-10 expression with very low circulating vIL-10 and led to a significant reduction of EAU without affecting the systemic immune response. The present results suggest that Ad-mediated gene transfer resulting in systemic and local expression of vIL-10 provide a promising approach for the treatment of uveitis.

Direct evidence of cytoplasmic delivery of PKC-alpha, -epsilon and -zeta pseudosubstrate lipopeptides: study of their implication in the induction of apoptosis.

Protein kinases C (PKC) are serine/threonine kinase enzymes involved in the mechanism of cell survival. Their pseudosubstrate sequences are autoinhibitory domains, which maintain the enzyme in an inactive state in the absence of allosteric activators, thus representing an attractive tool for the modulation of different PKC isoforms. Here, we report the use of palmitoylated modified PKC-alpha, -epsilon, and -zeta pseudosubstrate peptides, and determine their intracellular distribution together with their respective PKC isoenzymes. Finally, we propose that the differential distribution of the peptides is correlated with a selective induction of apoptosis and therefore argues for different involvement of PKC isoforms in the anti-apoptotic program.

IFN-gamma-derived lipopeptides: influence of lipid modification on the conformation and the ability to induce MHC class II expression on murine and human cells.

Two truncated analogues of a previously identified lipopeptide agonist toward the IFN-gamma receptor were synthesized in an attempt to determine the minimal compound able to induce expression of MHC class II molecules on murine and human cells and to study the role of the lipid tail. Circular dichroism studies were used to probe the induced conformationnal changes. Our results indicate at least a double role for the lipid modification that contributes to the stabilization of helical organization of the associated peptide and to its passive delivery into the cytoplasm. The persistence of biological activity in a truncated peptide of half of the residues present in the lead compound suggests that the lipid tail could also contribute to the stabilization of the peptide-receptor binding through additional hydrophobic interactions. This study allowed to readjust the minimal requirements for intracellular IFN-gamma receptor stimulation. More generally, we suggest that lipidated analogues of functional peptides could be utilized for intracellular target validation in the drug discovery process.

Systemic delivery of an adenovirus expressing EBV-derived vIL-10 in mice infected with Schistosoma mansoni or Leishmania amazonensis: controversial effects on the development of pathological parameters.

Within the context of microorganism/host interactions, those which last over weeks are expected to be sensitive to more or less sustained and targeted immuno-intervention, such as delivery of cytokines known to operate as down-regulators of acute inflammatory processes. IL-10 has received growing attention as a potential tool in immunotherapy, due to its anti-inflammatory and immunosuppressive properties. Therefore, using two experimental models of long-term interactions between parasites and laboratory mice, we monitored some effects of the systemic delivery of an adenovirus (Ad) expressing EBV-derived IL-10 (vIL-10) designated Ad-vIL-10. We first monitored the vIL-10 serum level following intranasal, intraperitoneal, intramuscular and intravenous administration. The i. p. and i.v. delivery of Ad-vIL-10 allowed a high serum level of vIL-10 (= 100 ng/ml), the i.v. route leading to a more sustained expression (up to 3 weeks). As a first model of parasite/mouse interaction, Schistosoma mansoni/C57Bl/6 mouse was selected. Ad-vIL-10 delivery was performed 4 weeks after S. mansoni infection i.e. at the time of egg-laying, and several parameters were monitored: (i) number of adult worms in the mesenteric vein, (ii) number of eggs trapped in the liver and intestine, (iii) liver fibrosis, (iv) serum levels of egg-reactive antibody subclasses, (v) serum content of cytokines, and (vi) cytokine production in the supernatant of antigen-stimulated mesenteric lymph node cells. No apparent effect was observed, either on the different parasitological parameters or on fibrosis development at day 70 of infection. Surprisingly, a marked increase in both Th1 and Th2 type cytokines was observed in the sera of the Ad-vIL-10 injected animals, as well as in the supernatants of their Ag-stimulated mesenteric lymph node cells. Nevertheless, polarization of the humoral response towards a Th2 profile was demonstrated by an increase in the IgE level in the Ad-vIL-10-injected animals. As far as the second model is concerned, namely the Leishmania amazonensis /C57Bl6 mouse interactions, Ad-vIL-10 was delivered intravenously one day before subcutaneous injection of stationary promastigotes and footpad swelling was monitored over 110 days. Under these conditions, vIL-10 exhibited a biphasic effect, decreasing the lesion size at the early stages of infection, but leading to a more pronounced lesion size during the chronic phase. This observation suggests a deactivation of the macrophage host cells under the influence of vIL-10. The results are discussed in the context of immunotherapy and the paradoxical effects observed in immunointervention with vIL-10.

Systemic viral interleukin-10 gene delivery prevents cartilage invasion by human rheumatoid synovial tissue engrafted in SCID mice.

OBJECTIVE: To assess the effects of viral interleukin-10 (vIL-10) gene delivery on human rheumatoid synovial tissue. METHODS: SCID mice were engrafted subcutaneously with human rheumatoid synovial tissue and homologous cartilage before systemic injection of 10(9) plaque-forming units of type 5 E1a Elb-deficient non-replicative adenovirus vector containing the vIL-10 gene under control of the cytomegalovirus promoter (AdvIL-10; n = 10) or a control gene (AdvIL-10mut; n = 7). Three weeks later, the graft was removed for histologic analysis of cartilage invasion by synovial tissue. The number of CD3-positive mononuclear cells was assessed in the synovial tissue by immunohistology. Messenger RNA (mRNA) expression of matrix metalloproteinase 3 (MMP-3), tissue inhibitor of metalloproteinases 1 (TIMP-1), and proinflammatory cytokines was determined by polymerase chain reaction. RESULTS: Systemic vIL-10 gene transfer resulted in high sustained production of vIL-10 protein in SCID mouse sera (mean +/- SD 25 +/- 5 ng/ml on day 40 post vector injection). Moreover, vIL-10 mRNA expression was detected in the synovial tissue 3 weeks after intravenous injection of AdvIL-10, reflecting the gene transfer in the human graft. In animals treated with AdvIL-10, cartilage invasion by rheumatoid synovial tissue was significantly inhibited compared with the control vector (mean +/- SD histologic score 2.5 +/- 0.52 versus 0.75 +/-0.8; P < 0.0001). The number of T cells infiltrating the synovium and perichondral resorption in the animals treated with AdvIL-10 gene were not significantly modified relative to the control vector. In animals treated with AdvIL-10, the MMP-3-TIMP-1 balance was partially restored, independent of the effect on mRNA expression of tumor necrosis factor a, IL-1, IL-6, or IL-8. CONCLUSION: Systemic vIL-10 gene transfer prevented cartilage invasion by synovial tissue engrafted in SCID mice. This model offers the opportunity to study the biologic effects of gene transfer in vivo in rheumatoid synovium.

Adenovirus-mediated transfer of viral IL-10 gene inhibits murine collagen-induced arthritis.

IL-10 is a potent anti-inflammatory cytokine that has received growing attention for its therapeutic potential. We examined the efficiency of adenoviral-mediated gene transfer of IL-10 on the incidence and severity of murine collagen-induced arthritis (CIA). Male DBA1 mice immunized with collagen II were treated by systemic administration of 10(9) plaque-forming units of replication-defective adenoviral vector expressing viral IL-10 (vIL-10) on day 30, when clinical symptoms of arthritis start. The transgene was shown to inhibit the onset of CIA, to decrease severity, and profoundly suppress the overall joint histopathology of the experimental arthritis. Significant IL-10 concentrations were obtained in the serum of injected animals for 7 days. Inhibition of arthritis was enhanced by administration of increasing doses of adenovirus-vIL-10. In addition, the local immunosuppressive effect of gene-delivered vIL-10 could be neutralized by a monoclonal anti-vIL-10 Ab. The CIA symptoms in the group treated with the same construct expressing inactive vIL-10 (vIL-10 mut) were similar to those in untreated animals. Our data indicate that a single systemic administration of an adenoviral vector encoding vIL-10 may be a good candidate to suppress arthritis.

Unrestricted agonist activity on murine and human cells of a lipopeptide derived from IFN-gamma.

We describe the isolation of a synthetic agonist of IFN-gamma (L-mIFN-gamma-CT) active on mouse and human cells. Its biological activity is the result of the ability of the C-terminal extremity of murine IFN-gamma to interact with the intracellular part of IFNgamma-R and the observation that the modification of peptides by a palmitic acid enables their cytoplasmic delivery. L-mIFN-gamma-CT stimulated murine cells exhibited an increase in MHC class II molecules and FcgammaRII/III expression and conferred protection against viral lysis. Unresponsiveness to L-mIFN-gamma-CT of cells recovered from IFNgamma-R alpha-chain knockout mice indicated the involvement of IFNgamma-R in the biological activities observed. Induction of VCAM-1, ICAM-1, and HLA-DR expression on human cells stimulated with L-mIFN-gamma-CT demonstrated an abrogation of species specificity. These results describe the development of a new synthetic agonist of IFN-gamma, which substitutes for the native cytokine in any IFN-gamma responsive cells, by acting intracellularly on IFN-gammaR.

Cellular immune response and pathology in schistosomiasis.

In the present review, some aspects of the cellular response following the murine Schistosoma mansoni infection are described. Due to the peculiar route used by the schistosome to infect its definitive host, the skin appears as a critical site in which the initial events of the host/parasite relationship occur and where the immune response is initiated. Moreover, the induction and the modulation of the granuloma formation, which represent the main aspect of the pathology of this parasitic disease, is under the control of several cellular populations n which CD4 and CD8 T cells play a key role. The cytokines produced in response to the parasite, such as IL7 in the skin and IFN gamma in the liver, seem to influence the further development of immunity against Schistosoma mansoni.

Trypanosoma cruzi glutathione-binding proteins (TcGBP): protection induced by native proteins in an experimental model and analysis of the antibody response.

Three Trypanosoma cruzi glutathione-binding proteins (TcGBP) of 45, 30 and 25 kDa presenting glutathione S transferase activity were characterized from T. cruzi epimastigotes. We show here that immunization of mice using TcGBP and complete Freund's adjuvant (CFA) did not protect the animals against a challenge with bloodstream trypomastigotes. In contrast, immunization of mice using TcGBP in association with Bordetella pertussis plus alum (BpAI) resulted in greatly diminished parasitaemia and significantly protected the animals from lethal infection. Using TcGBP mixed with BpAI and a lower challenge dose, we obtained strongly diminished parasitaemia and 100% protection in terms of survival. Only sera from mice immunized with TcGBP plus BpAI were able to kill trypomastigotes by complement-mediated lysis, whereas sera from mice immunized with TcGBP plus CFA did not. Interestingly, sera from mice immunized with TcGBP plus BpAI showed significant levels of specific IgE, IgG2a and IgG2b antibodies, whereas these isotypes were not detected in sera from mice immunized with TcGBP in CFA. All these levels were increased in sera of protected animals. These results demonstrate that TcGBP antigens can confer resistance to T. cruzi acute infection in mice, and suggest a possible functional role for both IgE and IgG2 isotypes in the induction of protective immunity.

Schistosoma mansoni 28-kDa glutathione S-transferase and immunity against parasite fecundity and egg viability. Role of the amino- and carboxyl-terminal domains.

We have previously shown that a mAb that inhibits the enzymatic activity of the Schistosoma mansoni 28-kDa glutathione S-transferase (Sm28 GST) also reduces female worm fecundity and egg viability in vivo and in vitro. By peptidic epitope mapping and an activity reconstitution assay, the carboxyl terminus (CT) amino acid residues 190-211 and to a lesser extent the truncated amino terminus (NT) residues 10-43 of the enzyme were identified as mAb recognition sites. Sera from rats immunized with the NT (10-43) and CT (190-211) peptides showed a partial inhibitory effect on Sm28 GST activity in a late phase (6 to 7 wk) but not in an early phase (2 to 4 wk) after immunization. Passive transfer of Sm28 GST-inhibiting anti-N- and C-terminal sera, but not of the noninhibitory sera, protected the infected mice by reducing tissue egg deposition and the ability of eggs to hatch. In active immunization experiments, the CT peptide significantly decreased the worm burden (37 to 40%) in mice as did the rSm28 GST (28 to 52%). In terms of tissue egg deposition and egg-hatching ability, immunization with both the NT and CT peptides reproduced the reduction observed after immunization with rSm28 GST. A constant reduction in egg numbers was noted in the small intestines and the livers of the immunized mice. A clear reduction in the ability of intestinal or hepatic eggs to hatch was observed. The results are discussed in terms of the conformational participation of the NT and CT of Sm28 in the expression of GST activity.

Trypanosoma cruzi glutathione-binding proteins: immunogenicity during human and experimental Chagas' disease.

Following purification by affinity chromatography, three glutathione-binding proteins (TcGBP) of 45, 30, and 25 kDa were co-purified from Trypanosoma cruzi epimastigotes. Using 1-chloro-2,4 dinitrobenzene as substrate, a glutathione S-transferase activity of 70 nmol/min/mg of proteins was detected in the GSH binding fraction. An increased expression of TcGBP and total GST activity was observed upon incubation of parasites with phenobarbital, which is an inducer of GST synthesis. Immunofluorescence and electron microscopic experiments demonstrated that TcGBP were expressed by all developmental stages of the parasite, including infective forms. The expression of these proteins by intracellular dividing amastigotes could be in favour of a potential defensive role of these molecules against host attack. Results obtained by immunoprecipitation of in vitro translation products using anti-TcGBP antisera suggested that these three polypeptides are not glycosylated. In addition, antibodies directed against the TcGBP were found in a high proportion of T. cruzi-infected chronic chagasic patients' sera and in sera of chronically infected BALB/c mice. In contrast, acute chagasic patients' sera and acute-phase mouse sera were found to be poorly reactive with these proteins. Our results identify a new class of potential target antigens, which may be essential for the development of T. cruzi in its host. Their protective role in experimental models deserves to be investigated.

T-cell-dependent immunity and thrombocytopenia in rats infected with Plasmodium chabaudi.

Normal, splenectomized, and athymic Fischer rats were infected with Plasmodium chabaudi. In normal rat infections, acute-phase infection resolved rapidly and completely. In splenectomized rats, infection resulted in high parasitemia and ultimately death. In nude rats, parasite growth was reduced compared with normal rats, and a persistent parasitemia (between 20 and 45%) was observed for several months. Complete resolution of the infection was achieved after adoptive transfer of T lymphocytes, even when transfer occurred during the course of infection. These results indicated that an acquired, T-lymphocyte-dependent immunity was necessary for the complete recovery observed in normal rats. In normal rats, thrombocytopenia and splenomegaly occurred during infection. By contrast, in nude rats, both of these pathological manifestations were only observed after thymus grafting. Thrombocytopenia was also absent in the splenectomized animals. Despite an increase in platelet-associated immunoglobulin levels during the infection, thrombocytopenia was not transferred by injection of infected rat serum to normal recipients. It has been concluded that the nude rat infection can be regarded as a novel and useful model for studying the T-cell-dependent effector and pathological mechanisms and to investigate the anti-P. chabaudi immune response.

A monoclonal antibody blocking the Schistosoma mansoni 28-kDa glutathione S-transferase activity reduces female worm fecundity and egg viability.

The protective effects of two different monoclonal antibodies (mAb) raised against the Schistosoma mansoni 28-kDa glutathione S-transferase (Sm 28 GST) were investigated. Two mAb of the same isotype (IgM) have been selected according to the blocking effect on Sm 28 GST enzymatic activity (S13) or the lack of blockade (H12). When passively transferred into Fischer rats, both S13 and H12 significantly reduced the worm burden. In BALB/c mice clear effects on female worm fecundity and egg viability were observed when the S13 mAb was transferred; these effects included significantly reduced loads of intestinal eggs, reduced egg hatching rates and an increased proportion of non-living eggs. No effect on egg production and egg hatching was observed in H12-treated mice. In addition, worm pairs recovered from S13-but not H12-treated mice laid significantly fewer eggs in vitro, and normal worm pairs incubated in vitro with the S13 mAb produced significantly fewer eggs than those incubated with H12 mAb. The impairment of egg hatching ability was also reproduced in vitro by the S13 mAb. These data suggest the existence of two different effector mechanisms induced by immunization with Sm 28 GST. The effect on the schistosome worm burden appears to be independent of GST activity whereas the effect on S. mansoni female fecundity and egg viability seems to be significantly linked to the inactivation of the enzymatic site.

Molecular cloning and tissue distribution of a 26-kilodalton Schistosoma mansoni glutathione S-transferase.

A Schistosoma mansoni cDNA library was constructed from the mRNA of adult worms in the expression vector lambda gt11 and screened with a rabbit antiserum raised against the 26-kDa S. mansoni glutathione S-transferase isoforms (Sm GST 26). Two clones were selected and the nucleotide sequences deduced. The predicted amino acid sequence, specified by these cDNAs, shows strong homology with a Schistosoma japonicum 26 kDa glutathione S-transferase and a lower level of homology with mammalian glutathione S-transferase class mu isoenzymes (EC 2.5.1.18). No significant homology score was found with a 28-kDa S. mansoni glutathione S-transferase (Sm GST 28). A study of the tissue distribution of the cloned Sm GST 26 by immunoelectron microscopy shows similarities to Sm GST 28 in that they are present in the tegument and in subtegumentary parenchymal cells. However, a major difference exists in the protonephridial region in which Sm GST 26 is present in the cytoplasmic digitations localized in the apical chamber delineated by the flame cell body, suggesting that Sm GST 26 may be actively excreted by adult worms.

Protective effects of anti-antiidiotypic IgE antibodies obtained from an IgE monoclonal antibody specific for a 26-kilodalton Schistosoma mansoni antigen.

A rat IgE mAb specific for larval Ag (26 kDa, 56 kDa) has been shown to protect rats against Schistosoma mansoni infection. Immunizations of Lou/M rats performed with this IgE (Ab1) induced the production of antiidiotypic antibodies (Ab2). Moreover, after this Ab2 production, anti-antiidiotypic antibodies (Ab3) were revealed. The screening of Ab3 isotypes showed the presence of IgG Ab3 and more interestingly of IgE Ab3, i.e., the same isotype as Ab1. These IgE and IgG antibodies recognized predominantly the 26-kDa Ag and were cytotoxic for schistosomula in the presence of platelets for IgE Ab3 and eosinophils for IgG Ab3. Both IgE and IgG Ab3 conferred by passive transfer protective immunity to infected rats (up to 50%). Thus the immunization with an IgE mAb led in part to the production of Ab3 of the same isotype as Ab1. In conclusion, these results suggest that the isotype selection of the antibodies of the third generation (Ab3) might be influenced by the Ab1. The respective role of the idiotope and isotype of Ab1 in isotype regulation is discussed.