Suppression of experimental myasthenia gravis by a B-cell epitope-free recombinant acetylcholine receptor.

Myasthenia gravis (MG) and experimental autoimmune MG (EAMG) are antibody-mediated autoimmune diseases in which the nicotinic acetylcholine receptor (AChR) is the major autoantigen. Previously we have revealed that oral treatment with the less native recombinant fragment of the extracellular domain of the human AChR (Halpha1-205) suppressed ongoing EAMG, whereas the more native recombinant Trx-Halpha1-210 exacerbated EAMG. In this study, we speculated on the role of B-cell epitopes in oral tolerogens for the induction of oral tolerance in EAMG. We developed a B-cell epitope-free AChR fragment (BF-AChR) by removing two major B-cell epitopes (67-76 and 129-145) from Trx-Halpha1-210. BF-AChR exhibited a poor response to EAMG sera and to AChR-specific B- and T-cells while its parent fragment, Trx-Halpha1-210, showed much higher reactivity. Oral administration of BF-AChR ameliorated the symptoms in ongoing myasthenic rats accompanied by a significant decrease in AChR-specific humoral and Th1 cellular responses. The underlying mechanism for BF-AChR-induced oral tolerance was mediated by a shift from Th1 to regulatory T-cell (IL-10(+), CD4(+) TGF-beta(+) or Foxp3(+)) responses. This shift was assessed by changes in the cytokine profile and a deviation in the anti-AChR IgG isotypes from IgG2a/IgG2b to IgG1. Our results suggest that the removal of pathogenic B-cell epitopes from AChR fragments increases tolerogenicity by reducing the activation and proliferation of autoreactive B- and T-cells. Collectively, careful consideration of the immunogenicity of a tolerogen is necessary to induce successful oral tolerance in autoimmune disorders.

Lactobacillus casei suppresses experimental arthritis by down-regulating T helper 1 effector functions.

Although the beneficial effects of probiotics on wide variety of diseases have been shown, little is known about how probiotics modulate the immune system. In this study we elucidated the underlying mechanisms how Lactobacillus casei (L. casei) protects against rheumatoid arthritis (RA) progression by investigating the effector functions of CD4(+) T cells. Oral administration of L. casei suppressed collagen-induced arthritis (CIA) and reduced paw swelling, lymphocyte infiltration and destruction of cartilage tissue. L. casei administration reduced type II collagen (CII)-reactive proinflammatory molecules (IL-1beta, IL-2, IL-6, IL-12, IL-17, IFN-gamma, TNF-alpha and Cox-2) by CD4(+) T cells. L. casei administration also reduced translocation of NF-kappaB into nucleus and CII-reactive Th1-type IgG isotypes IgG2a and IgG2b, while up-regulating immunoregulatory IL-10 levels. Our results suggest that oral administration of L. casei suppresses the type II collagen-reactive effector function of Th1-type cellular and humoral immune responses in arthritic inflammation.

Lactobacillus casei potentiates induction of oral tolerance in experimental arthritis.

Probiotics have been shown to exert beneficial effects on modulation of diverse diseases. However, no information is available for the effect of probiotics in the induction of oral tolerance in autoimmune diseases. The main purpose of this study was to elucidate whether Lactobacillus casei (L. casei) affect the induction of oral tolerance in experimental rheumatoid arthritis (RA). Type II collagen (CII) alone or together with L. casei was orally administered into collagen-induced arthritis (CIA) rats, and its effects on the clinical and histopathological aspects of RA were investigated. Co-administration of L. casei with CII more effectively suppressed clinical symptoms, paw swelling, lymphocyte infiltration and destruction of cartilage tissues of experimental arthritis than the rats treated with CII alone. The enhanced therapeutic efficacy was associated with an increase in anti-inflammatory cytokines (IL-10 and TGF-beta) while decreasing pro-inflammatory cytokines (IL-1beta, IL-2, IL-6, IL-12, IL-17, IFN-gamma and TNF-alpha). Co-administration of L. casei with CII more effectively suppressed CII-reactive T cell proliferation and the levels of Th1-type IgG isotypes (IgG2a and IgG2b), while up-regulating Foxp3 expression levels and the population of Foxp3(+) CD4(+) T cells. Our study provides evidence that L. casei could potentiate antigen-specific oral tolerance and suppress Th1-type immune responses of arthritic inflammation.

Defect in TCR-CD3zeta signaling mediates T cell hypo-responsiveness in mesenteric lymph node.

Mesenteric lymph node (MLN) in gut-associated lymphoid tissue plays obligatory roles in the induction of oral tolerance and ignorance to commensals. However, little is known about its immunological characteristics. In this study, we investigated the hypo-responsiveness of MLN CD4(+) T cells, comparing them with spleen CD4(+) T cells. MLN CD4(+) T cells were hypo-proliferative and expressed low levels of Th1-type cytokines in response to antigen or CD3/T cell receptor (TCR) stimulation. The hypo-responsiveness of MLN CD4(+) T cells is linked neither with changes in the regulatory T cell population (CD4(+)CD25(+), CD4(+)Foxp3(+)) nor the apoptotic population. Rather, MLN CD4(+) T cells showed deformity of T cell:APC conjugation and reduced expression of TCR signaling molecules such as CD3zeta, PLC-gamma1, PKC-theta, Zap70, with reduced phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs). Among the alterations in TCR signaling molecules, defective CD3zeta expression is the most evident, and reversal of the anergic state by CD3/CD28 costimulation restored CD3zeta expression levels. Collectively, we suggest that reduced CD3zeta expression and defects in TCR signaling mediate the anergy state of MLN CD4(+) T cells, which play a critical role in maintenance of mucosal tolerance in gut-associated lymphoid tissue.

A novel splicing variant of mouse interleukin (IL)-24 antagonizes IL-24-induced apoptosis.

Alternative splicing of mRNA enables functionally diverse protein isoforms to be expressed from a single gene, allowing transcriptome diversification. Interleukin (IL)-24/MDA-7 is a member of the IL-10 gene family, and FISP (IL-4-induced secreted protein), its murine homologue, is selectively expressed and secreted by T helper 2 lymphocytes. A novel splice variant of mouse IL-24/FISP, designated FISP-sp, lacks 29 nucleotides from the 5'-end of exon 4 of FISP. The level of FISP-sp expression is 10% of the level of total primary FISP transcription. Unlike FISP, FISP-sp does not induce growth inhibition and apoptosis. FISP-sp is exclusively localized in endoplasmic reticulum, and its expression is up-regulated by endoplasmic reticulum stress. Our results suggest that the novel splicing variant FISP-sp dimerizes with FISP and blocks its secretion and inhibits FISP-induced apoptosis in vivo.

Structural analysis of immunotherapeutic peptides for autoimmune Myasthenia gravis.

Myasthenia gravis (MG) and its animal model, experimental MG (EAMG), are autoimmune disorders in which major pathogenic antibodies are directed against the main immunogenic region (MIR) of the nicotinic acetylcholine receptor (nAChR). In an earlier attempt to develop peptide mimotopes capable of preventing the anti-MIR-mediated pathogenicity, the peptide Pep.1 was initially identified from phage display, and subsequently, Cyclic extended Pep.1 (Cyc.ext.Pep.1), which incorporates eight additional residues into the Pep.1 sequence and has an affinity for the anti-MIR antibody mAb198 3 orders of magnitude greater than that of Pep.1, was developed. In an animal model, Pep.1 shows no ability to inhibit mAb198-induced EAMG, whereas Cyc.ext.Pep.1 successfully blocks anti-MIR antibody 198 (mAb198)-induced EAMG. Our aim in this study was to identify the structural characteristics related to the different affinities for mAb198 of Pep.1 and Cyc.ext.Pep.1 using NMR spectroscopy and alanine scanning analysis. The NMR structural analysis revealed that Pep.1 is very flexible in solution, whereas Cyc.ext.Pep.1 is highly rigid within a region containing several turn structures. Interestingly, TRNOE experiments revealed that mAb198-bound Pep.1, particularly in the region between Asn7 and Glu11, shows significant structural similarity to the region between Asn10 and Glu14 of Cyc.ext.Pep.1, which is critical for interaction with mAb198. We therefore conclude the higher affinity of Cyc.ext.Pep.1 for mAb198 reflects the fact that incorporation of additional residues producing a single disulfide bond endows Pep.1 with a conformational rigidity that mimics the structure of mAb198-bound Pep.1. Furthermore, our results suggest that cyclic extended peptides could be utilized generally as useful tools to optimize the affinity of phage library-derived peptide antigens.