Preferential production of IgG1, IL-4 and IL-10 in MuSK-immunized mice.

Myasthenia gravis (MG) is an autoimmune disease characterized by muscle weakness associated with acetylcholine receptor (AChR), muscle-specific receptor kinase (MuSK) or low-density lipoprotein receptor-related protein 4 (LRP4)-antibodies. MuSK-antibodies are predominantly of the non-complement fixing IgG4 isotype. The MuSK associated experimental autoimmune myasthenia gravis (EAMG) model was established in mice to investigate immunoglobulin (Ig) and cytokine responses related with MuSK immunity. C57BL/6 (B6) mice immunized with 30µg of recombinant human MuSK in incomplete or complete Freund's adjuvant (CFA) showed significant EAMG susceptibility (>80% incidence). Although mice immunized with 10µg of MuSK had lower EAMG incidence (14.3%), serum MuSK-antibody levels were comparable to mice immunized with 30µg MuSK. While MuSK immunization stimulated production of all antibody isotypes, non-complement fixing IgG1 was the dominant anti-MuSK Ig isotype in both sera and neuromuscular junctions. Moreover, MuSK immunized IgG1 knockout mice showed very low serum MuSK-antibody levels. Sera and MuSK-stimulated lymph node cell supernatants of MuSK immunized mice showed significantly higher levels of IL-4 and IL-10 (but not IFN-γ and IL-12), than those of CFA immunized mice. Our results suggest that through activation of Th2-type cells, anti-MuSK immunity promotes production of IL-4, which in turn activates anti-MuSK IgG1, the mouse analog of human IgG4. These findings might provide clues for the pathogenesis of other IgG4-related diseases as well as development of disease specific treatment methods (e.g. specific IgG4 inhibitors) for MuSK-related MG.

Targeting complement system to treat myasthenia gravis.

While the complement system is desired for protective immunity, antibody- and complement-mediated neuromuscular junction (NMJ) destruction, a hallmark of myasthenia gravis (MG) or experimental autoimmune MG (EAMG), is a significant concern. Evidence suggests that the binding of complement factors to the pathogenic anti-acetylcholine receptor (AChR) autoantibody induces the formation of membrane attack complexes (MAC), which ultimately lead to NMJ destruction and muscle weakness. Studies corroborating the evidence show that the complement (C3-C6)-deficient or complement inhibitor (anti-C1q, soluble CR1, anti-C6, and C5 inhibiting peptide)-treated animals are highly resistant to EAMG induction, whereas the deficiency of the naturally occurring complement inhibitors, such as the decay-accelerating factor (DAF), increases EAMG susceptibility. Notably, the complement-inhibited animals do not exhibit significant immunosuppression but only a marginal reduction in the production of certain cytokines and immunoglobulin isotypes. A preliminary clinical trial using antibody-based C5 inhibitor eculizumab has been shown to be of potential use for MG treatment. The inhibition of the classic complement pathway (CCP) alone appears to be enough to suppress EAMG, suggesting that the complement inhibitors targeting specifically the classic pathway could effectively treat MG without causing immunosuppressive and other side effects. For instance, a recent non-antibody-based therapeutic approach selectively targeting the CCP component C2 by small interfering RNA (siRNA) has proven useful in EAMG treatment. The treatment strategies developed for MG might also be beneficial for other complement-mediated autoimmune diseases.

Complement C2 siRNA mediated therapy of myasthenia gravis in mice.

Activation of complement components is crucial in the progression and severity of myasthenia gravis and experimental autoimmune myasthenia gravis (EAMG). Mice deficient in complement component C4 or treated with monoclonal antibody to C1q are resistant to EAMG. In this study, we show that inhibition of complement cascade activation by suppressing the expression of a critical low-abundant protein, C2, in the classical complement pathway, significantly improved clinical and immunopathological manifestations of EAMG. Two weeks after a second booster immunization with acetylcholine receptor, when mice exhibit muscle weakness, i.p. injection of C2 siRNA significantly suppressed C2 mRNA in the blood cells and liver of EAMG mice. Treatment of EAMG mice with C2 siRNA, once a week for 5 weeks, significantly improved muscle strength, which was further evidenced by functional AChR preservation in muscle, reduction in number of C3 and membrane-attack complexes at neuro-muscular junctions in forelimb muscle sections, and a transient decrease in serum IgG2b levels. Our study shows for the first time that siRNA-mediated suppression of C2, a component of the classical complement system, following established disease, can effectively contribute to the remission of EAMG. Therefore, C2 siRNA mediated therapy can be applied in all complement mediated autoimmune diseases.

Ocular and generalized myasthenia gravis induced by human acetylcholine receptor γ subunit immunization.

INTRODUCTION: HLA-DQ8 transgenic mice develop ocular myasthenia gravis (oMG), which then progresses to generalized MG (gMG) when immunized with the human acetylcholine receptor (H-AChR) α subunit. Because the fetal AChR γ subunit is expressed in adult extraocular muscles, we anticipated that γ subunit immunization would generate an immune response to mouse AChR and induce MG in mice. RESULTS: H-AChR γ subunit immunization in HLA-DQ8 mice induced an autoimmune response to mouse AChR and led to the destruction of AChR in the neuromuscular junction (NMJ) by anti-AChR antibody and complement activation, and it triggered upregulation of AChR gene transcription. CONCLUSION: Our findings indicate that oMG may be induced by immunity to the AChR γ subunit.

Characterization of peripheral blood acetylcholine receptor-binding B cells in experimental myasthenia gravis.

In myasthenia gravis (MG), the neuromuscular transmission is impaired by antibodies (Abs) specific for muscle acetylcholine receptor (AChR). Anti-AChR Abs can be detected in the serum of MG patients, although their levels do not correlate with disease severity. In this study, we developed a flow cytometric assay for the detection of peripheral blood AChR-specific B cells to characterize B cell phenotypes associated with experimental autoimmune myasthenia gravis (EAMG). Alexa-conjugated AChR was used as a probe for AChR-specific B cells (B220+Ig+). Mice with EAMG had significantly elevated frequencies of AChR-specific IgG2+ and IgM+ B cells. While the frequencies of IgG2+ B cells and plasma anti-AChR IgG2 levels significantly correlated with the clinical grades of EAMG, the frequencies of IgM+ B cells and plasma anti-AChR IgM levels did not. These results indicate that the frequency of AChR-specific and IgG1+ (mouse IgG2 equivalent) peripheral blood B cells and anti-AChR IgG1 levels could be potential biomarkers for MG disease severity.

Complement and cytokine based therapeutic strategies in myasthenia gravis.

Myasthenia gravis (MG) is a T cell-dependent and antibody-mediated disease in which the target antigen is the skeletal muscle acetylcholine receptor (AChR). In the last few decades, several immunological factors involved in MG pathogenesis have been discovered mostly by studies utilizing the experimental autoimmune myasthenia gravis (EAMG) model. Nevertheless, MG patients are still treated with non-specific global immunosuppression that is associated with severe chronic side effects. Due to the high heterogeneity of AChR epitopes and antibody responses involved in MG pathogenesis, the specific treatment of MG symptoms have to be achieved by inhibiting the complement factors and cytokines involved in anti-AChR immunity. EAMG studies have clearly shown that inhibition of the classical and common complement pathways effectively and specifically diminish the neuromuscular junction destruction induced by anti-AChR antibodies. The inborn or acquired deficiencies of IL-6, TNF-α and TNF receptor functions are associated with the lowest EAMG incidences. Th17-type immunity has recently emerged as an important contributor of EAMG pathogenesis. Overall, these results suggest that inhibition of the complement cascade and the cytokine networks alone or in combination might aid in development of future treatment models that would reduce MG symptoms with highest efficacy and lowest side effect profile.

Suppression of ongoing experimental autoimmune myasthenia gravis by transfer of RelB-silenced bone marrow dentritic cells is associated with a change from a T helper Th17/Th1 to a Th2 and FoxP3+ regulatory T-cell profile.

OBJECTIVE: To observe the therapeutic effect of RelB-silenced dendritic cells (DCs) in experimental autoimmune myasthenia gravis (EAMG), and further to investigate the mechanism of this immune system therapeutic. METHODS: (1) RelB-silenced DCs and control DCs were prepared and the supernatants were collected for IL-12p70, IL-6, and IL-23 measurement by ELISA. (2) RelB-silenced DCs and control DCs were co-cultured with AChR-specific T cells, and the supernatant was collected to observe the IL-17, IFN-gamma, IL-4 production. (3) EAMG mice with clinical scores of 1 to 2 were collected and enrolled randomly into the RelB-silenced DC group or the control DC group. RelB-silenced DCs or an equal amount of control DCs were injected intravenously on days 0, 7, and 14 after enrollment. Clinical scores were evaluated every other day. Twenty days after allotment, serum from individual mice was collected to detect serum concentrations of anti-mouse AChR IgG, IgG1, IgG2b, and IgG2c. The splenocytes were isolated for analysis of lymphocyte proliferative responses, cytokine (IL-17, IFN-gamma, IL-4) production, and protein levels of RORgammat, T-bet, GATA-3, and FoxP3 (the special transcription factors of Th17, Th1, Th2, and Treg, respectively). RESULTS: (1) RelB-silenced DCs produced significantly reduced amounts of IL-12p70, IL-6, and IL-23, as compared with control DCs. (2) RelB-silenced DCs spurred on the CD4(+) T cells from Th1/Th17 to the Th2 cell subset in the co-culture system. (3) Treatment with RelB-silenced DCs effectively reduced myasthenic symptoms and levels of serum anti-acetylcholine receptor autoantibody in mice with ongoing EAMG. Th17-related markers (RORgammat, IL-17) and Th1-related markers (T-bet, IFN-gamma) were downregulated, whereas Th2 markers (IL-4 and GATA3) and Treg marker (FoxP3) were upregulated. CONCLUSIONS: RelB-silenced DCs were able to create a particular cytokine environment that was absent of inflammatory cytokines. RelB-silenced DCs provide a practical means to normalize the differentiation of the four T-cell subsets (Th17, Th1, Th2, and Treg) in vivo, and thus possess therapeutic potential in Th1/Th17-dominant autoimmune disorders such as myasthenia gravis.

Dendritic cells transduced with lentiviral-mediated RelB-specific ShRNAs inhibit the development of experimental autoimmune myasthenia gravis.

Dendritic cells (DC) are professional APC that are able to modulate immune response in either a positive or negative manner depending upon their lineage and state of maturation. RelB is a NF-kappaB family member which plays a key role in the differentiation and maturation of DC. In this study, we constructed lentiviral vector expressing RelB-specific short hairpin RNAs (ShRNAs) that efficiently silenced the RelB gene in bone marrow-derived dendritic cells (BMDCs). These RelB-silenced BMDCs were maturation resistant and could functionally decrease antigen-specific T cells proliferation. We tested the therapeutic effect of RelB-silenced BMDCs in C57BL/6 mice with experimental autoimmune myasthenia gravis (EAMG). Injection i.v. with RelB-silenced BMDCs plused with Torpedo acetylcholine receptor (TAChR) dominant peptide Talpha(146-162) on days 3, 33, and 63 after first immunization decreased the incidence and severity of clinical EAMG with suppressed IFN-gamma production and increased IL-10 and IL-4 production in vitro and in vivo, and also leads to a decreased serum anti-AChR IgG, IgG1, IgG2b Ab levels. Furthermore, RelB-silenced BMDCs promoted regulatory T cell profiles as indicated by a marked increase of FoxP3 in splenocyte. Our data suggested that lentiviral-mediated RNAi targeting RelB was effective methods to inhibit the maturation of BMDCs, thus possess therapeutic potential to prevent autoimmune disorders such as EAMG or human MG.

Inhibitory IgG receptor FcgammaRIIB fails to inhibit experimental autoimmune myasthenia gravis pathogenesis.

Deficiency of the inhibitory FcgammaRIIB renders mice susceptible to autoimmune disorders characterized with cellular infiltration of target tissue. To analyze the role of FcgammaRIIB in an antibody-mediated autoimmune disease, experimental autoimmune myasthenia gravis (EAMG), FcgammaRIIB knockout (KO) and wild-type mice were immunized with acetylcholine receptor (AChR). In contrast with previous reports, FcgammaRIIB KO mice were mildly resistant to EAMG despite preserved anti-AChR antibody production and neuromuscular junction complement deposition capacity. EAMG resistance was associated with reduced lymph node cell IL-6 and IL-10 production and increased CD4(+)CD25(+) cell ratios in lymph nodes. Our data suggest that FcgammaRIIB promotes antibody-mediated autoimmunity.

C5a is not involved in experimental autoimmune myasthenia gravis pathogenesis.

C5 deficient mice are highly resistant to experimental autoimmune myasthenia gravis (EAMG) despite intact immune response to acetylcholine receptor (AChR), validating the pivotal role played by membrane attack complex (MAC, C5b-9) in neuromuscular junction destruction. To distinguish the significance of C5a from that of C5b in EAMG pathogenesis, C5a receptor (C5aR) knockout (KO) and wild-type (WT) mice were immunized with AChR to induce pathogenic anti-AChR antibodies. In contrast with C5 deficient mice, C5aR KO mice were equally susceptible to EAMG as WT mice and exhibited comparable antibody and lymphocyte proliferation response to AChR implicating that C5a is not involved in EAMG development.

Classical complement pathway in experimental autoimmune myasthenia gravis pathogenesis.

Mice deficient for complement factors C3, C4, or C5 are resistant to experimental autoimmune myasthenia gravis (EAMG). Acetylcholine receptor (AChR) immune lymph node cells (LNC) of C3 deficient mice produce less interleukin 6 (IL-6), and EAMG-resistant IL-6 deficient mice have less serum C3. Increased serum C1q-circulating immune complex (CIC) levels correlated with EAMG disease severity in RIIIS/J mice. The CIC promotes EAMG severity by stimulating the production of LNC IL-6, serum C1q, and C3 via FCgammaR interaction. Therefore, EAMG/MG could be treated by blocking the activation of classical complement pathway (CCP) and/or IL-6. Anti-C1q antibody administration before and following AChR immunization suppressed EAMG by reducing LNC IL-6 production and neuromuscular junction deposits of IgG, C3, and C5b-C9 complexes. Treatment with low dose (10 microg) of anti-C1q antibody twice a week for 4 weeks in mice with ongoing clinical EAMG reduced the clinical severity of disease and LNC IL-6 production. Therefore, inhibitors of CCP factors C1q, C2, or C4 could treat MG and would preserve the alternate complement pathway activation. Our goal is to tailor MG therapy using anti-C2/C4 reagents in combination, with or without anti-cytokine (e.g., anti-IL-6) reagents.

Targeting classical complement pathway to treat complement mediated autoimmune diseases.

Mice deficient for classical complement pathway (CCP) factor C4 are resistant to antibody and complement mediated experimental autoimmune myasthenia gravis (EAMG). Anti-C1q antibody administration before or following acetylcholine receptor immunization suppresses EAMG development by reducing lymph node cell IL-6 production and neuromuscular junction IgG, C3 and C5b-C9 deposition. This effect is achieved by treating mice with 10 microg of anti-C1q antibody, twice weekly for 4 weeks. Treatment with a higher amount of anti-C1q antibody gives rise to increased serum anti-acetylcholine receptor antibody, immune complex and C3 levels, facilitates kidney C3 and IgG deposits and thus reduces the treatment efficacy. C4 KO and anti-C1q antibody treated mice display normal immune system functions and intact antibody production capacity. Furthermore, CCP inhibition preserves alternative complement pathway activation, which is required for host defense against microorganisms. Therefore, CCP inhibition might constitute a specific treatment approach for not only myasthenia gravis but also other complement mediated autoimmune diseases.

Mapping myasthenia gravis-associated T cell epitopes on human acetylcholine receptors in HLA transgenic mice.

Susceptibility to myasthenia gravis (MG) is positively linked to expression of HLA-DQ8 and DR3 molecules and negatively linked to expression of the DQ6 molecule. To elucidate the molecular basis of this association, we have induced experimental autoimmune MG (EAMG) in mice transgenic for HLA-DQ8, DQ6, and DR3, and in DQ8xDQ6 and DQ8xDR3 F(1) transgenic mice, by immunization with human acetylcholine receptor (H-AChR) in CFA. Mice expressing transgenes for one or both of the HLA class II molecules positively associated with MG (DQ8 and DR3) developed EAMG. T cells from DQ8 transgenic mice responded well to three cytoplasmic peptide sequences of H-AChR (alpha320-337, alpha304-322, and alpha419-437), of which the response to alpha320-337 was the most intense. DR3 transgenic mice also responded to this sequence very strongly. H-AChR- and alpha320-337 peptide-specific lymphocyte responses were restricted by HLA class II molecules. Disease resistance in DQ6 transgenic mice was associated with reduced synthesis of anti-AChR IgG, IgG(2b), and IgG(2c) Ab's and reduced IL-2 and IFN-gamma secretion by H-AChR- and peptide alpha320-337-specific lymphocytes. Finally, we show that DQ8 imparts susceptibility to EAMG and responsiveness to an epitope within the sequence alpha320-337 as a dominant trait.

Pros and cons of treating murine myasthenia gravis with anti-C1q antibody.

To test the feasibility of classical complement pathway manipulation in experimental autoimmune myasthenia gravis (EAMG) treatment, C57BL/6 (B6) and RIIIS/J mice with EAMG were treated with 10 microg or 100 microg of anti-C1q Ab or isotype Ab. Treatment with 10 microg anti-C1q Ab significantly reduced the clinical severity, decreased lymph node cell IL-6 production and T cell populations. Conversely, administration of 100 microg anti-C1q Ab caused harmful side effects such as increased serum anti-acetylcholine receptor antibody, immune complex, C3 and lymph node B cell levels and kidney C3 and IgG deposits, which reduced the treatment efficacy.

A new mouse model of autoimmune ocular myasthenia gravis.

PURPOSE: To establish a novel model of autoimmune ocular myasthenia gravis (oMG) in mice and study the pathogenic mechanisms of oMG. METHODS: oMG was induced in HLA-DQ8 transgenic, HLA-DR3 transgenic, major histocompatibility complex (MHC) class II-deficient, C57BL/6, and C57BL/10 mice by immunization with an Escherichia coli plasmid expressing the recombinant human acetylcholine receptor (AChR) alpha subunit. RESULTS: All strains of immunized mice developed ocular myasthenia gravis with varying disease incidence and severity. HLA-DQ8 transgenic mice were highly susceptible to oMG. Mice with oMG had serum autoantibodies to the mouse extraocular AChR, pathologic deposits of IgG, C3, and C5b-C9 in their extraocular and limb neuromuscular junctions, and droopiness of eyelids. HLA-DR3 transgenic and MHC class II-deficient mice were relatively resistant to oMG induced by AChR alpha subunit immunization and had minimal ocular abnormalities. CONCLUSIONS: These findings suggest that oMG pathogenesis could be triggered by immunity to the human AChR alpha subunit and that MHC class II molecule is required for human AChR alpha subunit presentation and CD4 cell-mediated anti-AChR antibody class switching. Differential oMG susceptibility observed in DQ8 and DR3 transgenic mice correlated with the intensity of lymphocytes to respond to the human AChR alpha subunit. This new model of oMG will be a valuable tool for studying the mechanism of oMG and gMG pathogenesis in humans and for preclinical therapeutic analysis.

Major Histocompatibility Complex Class II Alleles Influence Induction of Pathogenic Antiphospholipid Antibodies in a Mouse Model of Thrombosis.

OBJECTIVE: Both environmental and genetic factors are important in the development of antiphospholipid antibodies (aPL) in patients with antiphospholipid syndrome (APS). Currently, the only available data on predisposing genetic factors have been obtained from epidemiologic studies, without mechanistic evidence. Therefore, we studied the influence of major histocompatibility complex (MHC) class II alleles on the production of aPL in a mouse model of APS. METHODS: Three groups of mice, MHC class II-deficient (MHCII-/- ) mice, MHCII-/- mice transgenic for human HLA-DQ6 (DQ6), DQ8, or DR4 alleles, and the corresponding wild-type (WT) mouse strains were immunized; half were immunized with human ß2 -glycoprotein I (ß2 GPI), and the other half were immunized with control ovalbumin (OVA) protein. Thrombus formation in vivo, tissue factor activity in carotid and peritoneal macrophages, and serum levels of tumor necrosis factor (TNF), IgG anticardiolipin (aCL), antibodies, and anti-OVA antibodies were determined. RESULTS: Immunization with ß2 GPI induced significant production of aCL and anti-ß2 GPI in WT mice compared with control mice immunized with OVA (P < 0.001) but diminished aCL (P < 0.001) and anti-ß2 GPI (P = 0.016) production in MHCII-/- mice. Anti-ß2 GPI production was fully restored in DQ6 and DQ8 mice, while levels of anti-ß2 GPI in DR4 mice and aCL in all transgenic lines were only partially restored (P < 0.001 to P < 0.046). Thrombus size in WT mice was twice that in MHCII-/- mice (P < 0.001) but similar to that in all transgenic lines. Carotid and peritoneal macrophage tissue factor levels decreased by >50% in MHCII-/- mice compared with wild-type B6 mice and were restored in DQ8 mice but not DR4 mice or DQ6 mice. TNF levels decreased 4-fold in MHCII-/- mice (P < 0.001) and were not restored in transgenic mice. CONCLUSION: Our mechanistic study is the first to show that MHC class II alleles influence not only quantitative aPL production but also the pathogenic capacity of induced aPL.

Predictive value of serum anti-C1q antibody levels in experimental autoimmune myasthenia gravis.

Components of the complement cascade and circulating immune complexes play important roles in both experimental autoimmune myasthenia gravis and myasthenia gravis in humans. Thus far, no serological factor has been identified to predict the clinical severity of either myasthenia gravis. Upon immunization with acetylcholine receptor, levels of complement factors C1q, C3 and CIC increase with time in sera from C57BL/6 (B6) mice. Both these and plasma samples from myasthenia gravis patients also contain anti-C1q antibodies. The serum levels of anti-C1q antibodies but not C1q, C3 and CIC are significantly correlated with the clinical severity in the experimental myasthenia mice. However, this correlation is not observed in myasthenia gravis patients.

Complement regulator CD59 deficiency fails to augment susceptibility to actively induced experimental autoimmune myasthenia gravis.

Complement deficient mice are resistant to experimental autoimmune myasthenia gravis (EAMG), suggesting a pivotal role for the membrane attack complex (MAC) in EAMG pathogenesis. To test the significance of MAC regulation in EAMG pathogenesis, CD59 KO and wild type mice were immunized with acetylcholine receptor (AChR). Interestingly, deletion of CD59, the regulator of MAC assembly, failed to augment EAMG susceptibility. The CD59 KO mice had reduced serum anti-AChR IgG1, IgG2b and complement levels. Their lymph node cell IL-2 production and lymphocyte proliferation response to AChR were reduced. The data challenge the current paradigm that CD59 is solely involved in MAC regulation and suggest a role for this molecule in antigen-driven T cell and B cell activation.

Genetic evidence for the involvement of Fcgamma receptor III in experimental autoimmune myasthenia gravis pathogenesis.

Immune complexes and classical complement pathway play vital roles in experimental autoimmune myasthenia gravis (EAMG). To analyze the role of immune complex receptors in EAMG, FcgammaRIII knockout (KO) mice were immunized with AChR and were found out to be resistant to EAMG induction. This was associated with reduced neuromuscular junction deposits, lymph node cell (LNC) IL-6 production and serum complement levels. EAMG resistance of anti-C1q Ab-administered mice was also associated with reduced LNC IL-6 production and neuromuscular junction deposits, indicating C1q involvement in EAMG resistance. The data provide the first direct genetic evidence for Fcgamma receptor involvement in EAMG pathogenesis.

MuSK induced experimental autoimmune myasthenia gravis does not require IgG1 antibody to MuSK.

Sera of myasthenia gravis (MG) patients with muscle-specific receptor kinase-antibody (MuSK-Ab) predominantly display the non-complement fixing IgG4 isotype. Similarly, mouse IgG1, which is the analog of human IgG4, is the predominant isotype in mice with experimental autoimmune myasthenia gravis (EAMG) induced by MuSK immunization. The present study was performed to determine whether IgG1 anti-MuSK antibody is required for immunized mice to develop EAMG. Results demonstrated a significant correlation between clinical severity of EAMG and levels of MuSK-binding IgG1+, IgG2+ and IgG3+ peripheral blood B cells in MuSK-immunized wild-type (WT) mice. Moreover, MuSK-immunized IgG1 knockout (KO) and WT mice showed similar EAMG severity, serum MuSK-Ab levels, muscle acetylcholine receptor concentrations, neuromuscular junction immunoglobulin and complement deposit ratios. IgG1 and IgG3 were the predominant anti-MuSK isotypes in WT and IgG1 KO mice, respectively. These observations demonstrate that non-IgG1 isotypes can mediate MuSK-EAMG pathogenesis.