Use of Toll-Like Receptor Agonists to Induce Ectopic Lymphoid Structures in Myasthenia Gravis Mouse Models

Myasthenia gravis (MG) is an autoimmune disease mediated by autoantibodies against the acetylcholine receptor (AChR) at the neuromuscular junction. MG symptoms are characterized by muscle weaknesses. The thymus of MG patients is very often abnormal and possesses all the characteristics of tertiary lymphoid organs such as neoangiogenesis processes, overexpression of inflammatory cytokines and chemokines, and infiltration of B lymphocytes leading to ectopic germinal center (GC) development. We previously demonstrated that injections of mice with polyinosinic–polycytidylic acid [Poly(I:C)], a synthetic double-stranded RNA mimicking viral infection, induce thymic changes and trigger MG symptoms. Upon Poly(I:C) injections, we observed increased thymic expressions of α-AChR, interferon-β and chemokines such as CXCL13 and CCL21 leading to B-cell recruitment. However, these changes were only transient. In order to develop an experimental MG model associated with thymic GCs, we used Poly(I:C) in the classical experimental autoimmune MG model induced by immunizations with purified AChR emulsified in complete Freund’s adjuvant. We observed that Poly(I:C) strongly favored the development of MG as almost all mice displayed MG symptoms. Nevertheless, we did not observe any ectopic GC development. We next challenged mice with Poly(I:C) together with other toll-like receptor (TLR) agonists known to be involved in GC development and that are overexpressed in MG thymuses. Imiquimod and CpG oligodeoxynucleotides that activate TLR7 and TLR9, respectively, did not induce thymic changes. In contrast, lipopolysaccharide that activates TLR4 potentiated Poly(I:C) effects and induced a significant expression of CXCL13 mRNA in the thymus associated with a higher recruitment of B cells that induced over time thymic B-lymphoid structures. Altogether, these data suggest that tertiary lymphoid genesis in MG thymus could result from a combined activation of TLR signaling pathways

Role of IL-1β in experimental cystic fibrosis upon P. aeruginosa Infection

Cystic fibrosis is associated with increased inflammatory responses to pathogen challenge. Here we revisited the role of IL-1β in lung pathology using the experimental F508del-CFTR murine model on C57BL/6 genetic background (Cftrtm1eur or d/d), on double deficient for d/d and type 1 interleukin-1 receptor (d/d X IL-1R1-/-), and antibody neutralization. At steady state, young adult d/d mice did not show any signs of spontaneous lung inflammation. However, IL-1R1 deficiency conferred partial protection to repeated P. aeruginosa endotoxins/LPS lung instillation in d/d mice, as 50% of d/d mice succumbed to inflammation, whereas all d/d x IL-1R1-/- double mutants survived with lower initial weight loss and less pulmonary collagen and mucus production, suggesting that the absence of IL-1R1 signaling is protective in d/d mice in LPS-induced lung damage. Using P. aeruginosa acute lung infection we found heightened neutrophil recruitment in d/d mice with higher epithelial damage, increased bacterial load in BALF, and augmented IL-1β and TNF-α in parenchyma as compared to WT mice. Thus, F508del-CFTR mice show enhanced IL-1β signaling in response to P. aeruginosa. IL-1β antibody neutralization had no effect on lung homeostasis in either d/d or WT mice, however P. aeruginosa induced lung inflammation and bacterial load were diminished by IL-1β antibody neutralization. In conclusion, enhanced susceptibility to P. aeruginosa in d/d mice correlates with an excessive inflammation and with increased IL-1β production and reduced bacterial clearance. Further, we show that neutralization of IL-1β in d/d mice through the double mutation d/d x IL-1R1-/- and in WT via antibody neutralization attenuates inflammation. This supports the notion that intervention in the IL-1R1/IL-1β pathway may be detrimental in CF patients

Review on Toll-Like Receptor Activation in Myasthenia Gravis: Application to the Development of New Experimental Models

International audienceAbnormal toll-like receptor (TLR) activation and uncontrolled resolution of inflammation are suspected to play a key role in the development of autoimmune diseases. Acquired myasthenia gravis (MG) is an invalidating neuromuscular disease leading to muscle weaknesses. MG is mainly mediated by anti-acetylcholine receptor (AChR) autoantibodies, and thymic hyperplasia characterized by ectopic germinal centers is a common feature in MG. An abnormal expression of certain TLRs is observed in the thymus of MG patients associated with the overexpression of interferon (IFN)-β, the orchestrator of thymic changes in MG. Experimental models have been developed for numerous autoimmune diseases. These models are induced by animal immunization with a purified antigen solubilized in complete Freund’s adjuvant (CFA) containing heat-inactivated mycobacterium tuberculosis (MTB). Sensitization against the antigen is mainly due to the activation of TLR signaling pathways by the pathogen motifs displayed by MTB, and attempts have been made to substitute the use of CFA by TLR agonists. AChR emulsified in CFA is used to induce the classical experimental autoimmune MG model (EAMG). However, the TLR4 activator lipopolysaccharide (LPS) has proved to be efficient to replace MTB and induce a sensitization against purified AChR. Poly(I:C), the well-known TLR3 agonist, is also able by itself to induce MG symptoms in mice associated with early thymic changes as observed in human MG. In this review, we discuss the abnormal expression of TLRs in MG patients and we describe the use of TLR agonists to induce EAMG in comparison with other autoimmune experimental models

Novel CXCL13 transgenic mouse: inflammation drives pathogenic effect of CXCL13 in experimental myasthenia gravis

International audienceAbnormal overexpression of CXCL13 is observed in many inflamed tissues and in particular in autoimmune diseases. Myasthenia gravis (MG) is a neuromuscular disease mainly mediated by anti-acetylcholine receptor autoantibodies. Thymic hyperplasia characterized by ectopic germinal centers (GCs) is a common feature in MG and is correlated with high levels of anti-AChR antibodies. We previously showed that the B-cell chemoattractant, CXCL13 is overexpressed by thymic epithelial cells in MG patients. We hypothesized that abnormal CXCL13 expression by the thymic epithelium triggered B-cell recruitment in MG. We therefore created a novel transgenic (Tg) mouse with a keratin 5 driven CXCL13 expression. The thymus of Tg mice overexpressed CXCL13 but did not trigger B-cell recruitment. However, in inflammatory conditions, induced by Poly(I:C), B cells strongly migrated to the thymus. Tg mice were also more susceptible to experimental autoimmune MG (EAMG) with stronger clinical signs, higher titers of anti-AChR antibodies, increased thymic B cells, and the development of germinal center-like structures. Consequently, this mouse model finally mimics the thymic pathology observed in human MG. Our data also demonstrated that inflammation is mandatory to reveal CXCL13 ability to recruit B cells and to induce tertiary lymphoid organ development

Preconditioned mesenchymal stem cells treat myasthenia gravis in a humanized preclinical model

International audienceMyasthenia gravis (MG) with anti–acetylcholine receptor (AChR) Abs is an autoimmune disease characterized by severe defects in immune regulation and thymic inflammation. Because mesenchymal stem cells (MSCs) display immunomodulatory features, we investigated whether and how in vitro–preconditioned human MSCs (cMSCs) could treat MG disease. We developed a new humanized preclinical model by subcutaneously grafting thymic MG fragments into immunodeficient NSG mice (NSG-MG model). Ninety percent of the animals displayed human anti-AChR Abs in the serum, and 50% of the animals displayed MG-like symptoms that correlated with the loss of AChR at the muscle endplates. Interestingly, each mouse experiment recapitulated the MG features of each patient. We next demonstrated that cMSCs markedly improved MG, reducing the level of anti-AChR Abs in the serum and restoring AChR expression at the muscle endplate. Resting MSCs had a smaller effect. Finally, we showed that the underlying mechanisms involved (a) the inhibition of cell proliferation, (b) the inhibition of B cell–related and costimulatory molecules, and (c) the activation of the complement regulator DAF/CD55. In conclusion, this study shows that a preconditioning step promotes the therapeutic effects of MSCs via combined mechanisms, making cMSCs a promising strategy for treating MG and potentially other autoimmune diseases

Anti-IL-1β antibody treatment has no significant effect on lung ultrastructure, nor on IL-1β, IL-6 and CCL2 mRNA production in lungs.

<p>Wild type (WT) and F508del CFTR mutation homozygote (d/d) mice (14-week-old females and males) were treated intra-peritoneally, once a week for 8 weeks, an anti-IL-1β antibody (10 mg/kg), or PBS. Mice were euthanatized 1 week after the last treatment and lung structure was observed on H&E stained slides observed at ×20 magnification (A). Cellular infiltration (B) was quantified on these H&E slides and mucus production (C) was analyzed on CAB stained lung slides. Lung injury score was recorded in anti-IL-1β antibody treated mice compared with PBS treated mice. (D and E) mRNA production of IL-1β, IL-6 and CCL2 was measured, and normalized with 2 housekeeping gene expression (<i>Hprt1</i> and <i>Gapdh</i>). The effect of anti-IL-1β antibody treatment compared to PBS control in WT and d/d animals is shown in (D), and the effect of F508del CFTR mutation compared to WT is shown in (E). (n = 6–7)</p

IL-1β participates in <i>P. aeruginosa</i>-induced inflammation at 20 h in WT mice.

<p>WT mice received 10⁶ CFU of <i>P. aeruginosa</i> PA011 intranasally, and WT mice treated with NaCl 0.9% were used as control. A group of WT mice was also treated intraperitoneally with anti-IL-1β antibody, 200 µg/mice, 15 h and 1 h before infection. Body weight variation 20 h after infection is shown in (A). Bacterial load was determined in BALF and in lung homogenate (B), myeloperoxidase activity was quantified (C) and absolute numbers of cells, (macrophages and neutrophils) were measured in BALF (D). Protein concentration was evaluated in BALF (E) and IL-1β (F), KC (G), TNF-α (H) and IL-6 (I), were measured in BALF and in lung homogenate. Cell infiltration was observed on H&E stained slide (J) and scored (K). (n = 5–6) Values are in mean +/− SEM; * for p<0.05; ** for p<0.01 and *** for p<0.001. ns for non-significant.</p

IL-1β participates in pathologic inflammation in F508del CFTR mutants, in response to <i>P. aeruginosa</i> LPS.

<p>d/d and double KO d/d x IL-1R1^−/− mice were treated intranasally with 80 µg of <i>P. aeruginosa</i> endotoxins/LPS in 40 µL PBS, once a week for 5 weeks. Untreated WT mice (UT) were used as control. Survival is presented in (A) and body weight variation 24 h after the last LPS challenge in (B). Absolute numbers of cells, (macrophages, lymphocytes and neutrophils) were measured in BALF 24 h after the last LPS challenge (C). CCL2 (D), IL-6 (E) and IL-1β (F) were measured in lung homogenate. Cell infiltration was observed on H&E staining (G), mucus production on PAS staining (H) and collagen deposition on CAB staining (I). Histopathological scores are shown in (J). n = 6–7 initially, n = 3 for d/d+LPS mice 24 h after last challenge instillation. Values are in mean +/− SEM; * for p<0.05 and ns for non-significant.</p