Helicobacter pylori Impairs Murine Dendritic Cell Responses to Infection

International audienceBACKGROUND: Helicobacter pylori, a human pathogen associated with chronic gastritis, peptic ulcer and gastric malignancies, is generally viewed as an extracellular microorganism. Here, we show that H. pylori replicates in murine bone marrow derived-dendritic cells (BMDCs) within autophagosomes. METHODOLOGY/PRINCIPAL FINDINGS: A 10-fold increase of CFU is found between 2 h and 6 h p.i. in H. pylori-infected BMDCs. Autophagy is induced around the bacterium and participates at late time points of infection for the clearance of intracellular H. pylori. As a consequence of infection, LC3, LAMP1 and MHC class II molecules are retained within the H. pylori-containing vacuoles and export of MHC class II molecules to cell surface is blocked. However, formalin-fixed H. pylori still maintain this inhibitory activity in BMDC derived from wild type mice, but not in from either TLR4 or TLR2-deficient mice, suggesting the involvement of H. pylori-LPS in this process. TNF-alpha, IL-6 and IL-10 expression was also modulated upon infection showing a TLR2-specific dependent IL-10 secretion. No IL-12 was detected favoring the hypothesis of a down modulation of DC functions during H. pylori infection. Furthermore, antigen-specific T cells proliferation was also impaired upon infection. CONCLUSIONS/SIGNIFICANCE: H. pylori can infect and replicate in BMDCs and thereby affects DC-mediated immune responses. The implication of this new finding is discussed for the biological life cycle of H. pylori in the host

Cholesterol-Dependent Anaplasma phagocytophilum Exploits the Low-Density Lipoprotein Uptake Pathway

In eukaryotes, intracellular cholesterol homeostasis and trafficking are tightly regulated. Certain bacteria, such as Anaplasma phagocytophilum, also require cholesterol; it is unknown, however, how this cholesterol-dependent obligatory intracellular bacterium of granulocytes interacts with the host cell cholesterol regulatory pathway to acquire cholesterol. Here, we report that total host cell cholesterol increased >2-fold during A. phagocytophilum infection in a human promyelocytic leukemia cell line. Cellular free cholesterol was enriched in A. phagocytophilum inclusions as detected by filipin staining. We determined that A. phagocytophilum requires cholesterol derived from low-density lipoprotein (LDL), because its replication was significantly inhibited by depleting the growth medium of cholesterol-containing lipoproteins, by blocking LDL uptake with a monoclonal antibody against LDL receptor (LDLR), or by treating the host cells with inhibitors that block LDL-derived cholesterol egress from late endosomes or lysosomes. However, de novo cholesterol biosynthesis is not required, since inhibition of the biosynthesis pathway did not inhibit A. phagocytophilum infection. The uptake of fluorescence-labeled LDL was enhanced in infected cells, and LDLR expression was up-regulated at both the mRNA and protein levels. A. phagocytophilum infection stabilized LDLR mRNA through the 3′ UTR region, but not through activation of the sterol regulatory element binding proteins. Extracellular signal–regulated kinase (ERK) was up-regulated by A. phagocytophilum infection, and inhibition of its upstream kinase, MEK, by a specific inhibitor or siRNA knockdown, reduced A. phagocytophilum infection. Up-regulation of LDLR mRNA by A. phagocytophilum was also inhibited by the MEK inhibitor; however, it was unclear whether ERK activation is required for LDLR mRNA up-regulation by A. phagocytophilum. These data reveal that A. phagocytophilum exploits the host LDL uptake pathway and LDLR mRNA regulatory system to accumulate cholesterol in inclusions to facilitate its replication

Major histocompatibility complex class II gene disruption prevents experimental autoimmune myasthenia gravis.

Abstract To analyze the impact of lack of MHC class II gene expression, and to demonstrate the direct genetic evidence for the involvement of the MHC class II gene product in the development of experimental autoimmune myasthenia gravis (EAMG), MHC class II gene-disrupted C57BL6 mutant (-/-) and EAMG-susceptible MHC class II wild-type C57BL6 mice (+/+) were evaluated for the clinical and immunopathologic manifestations of EAMG. The deficiency of MHC class II, and therefore, CD4+ T cells, completely prevented the C57BL6 MHC class II mutant (-/-) mice from mounting an autoimmune response to the nicotinic acetylcholine receptor. Further, the mutant (-/-) mice failed to show any immunopathologic and clinical manifestations of EAMG. The data unequivocally provide direct genetic evidence for the essential role of MHC class II molecules in the induction of EAMG, and rule out any pathogenic effector role for MHC class I-restricted CD8+ T cells, gamma delta TCR-bearing cells, or NK cells, which are intact in the MHC class II mutant mice in the induction of EAMG.</jats:p

Tolerance to a dominant T cell epitope in the acetylcholine receptor molecule induces epitope spread and suppresses murine myasthenia gravis.

Abstract Myasthenia gravis (MG) is a T cell-dependent, Ab-mediated autoimmune disease. T cells reactive to a dominant peptide alpha 146-162 of acetylcholine receptor (AChR) alpha subunit participate in murine MG pathogenesis. To suppress the autoimmune response to AChR, a high dose of alpha146-162 peptide in IFA was administered parenterally as a tolerogen, after the development of a primary T cell immune response to AChR. This form of AChR T cell peptide tolerance suppressed the in vitro T cell proliferative response to AChR and its dominant alpha146-162 and subdominant alpha182-198 peptides through epitope spread. Administration of alpha146-162 peptide in IFA after the primary immune response to AChR also significantly suppressed the serum anti-AChR Ab of the IgG2b isotype and clinical incidence of MG in C57BL/6 mice. Furthermore, the production of IFN-gamma, IL-2, and IL-10 cytokines by AChR, alpha146-162, and alpha182-198 peptide-reactive cells was suppressed by alpha146-162 peptide tolerance, and the epitope spread observed could be attributed to the reduction in the above cytokine production. Therefore, AChR T cell-dominant peptide tolerance could be adapted in the Ag-specific therapy of MG.</jats:p

Genetic evidence for involvement of classical complement pathway in induction of experimental autoimmune myasthenia gravis

Abs to acetylcholine receptor (AChR) and complement are the major constituents of pathogenic events causing neuromuscular junction destruction in both myasthenia gravis (MG) and experimental autoimmune MG (EAMG). To analyze the differential roles of the classical vs alternative complement pathways in EAMG induction, we immunized C3(-/-), C4(-/-), C3(+/-), and C4(+/-) mice and their control littermates (C3(+/+) and C4(+/+) mice) with AChR in CFA. C3(-/-) and C4(-/-) mice were resistant to disease, whereas mice heterozygous for C3 or C4 displayed intermediate susceptibility. Although C3(-/-) and C4(-/-) mice had anti-AChR Abs in their sera, anti-AChR IgG production by C3(-/-) mice was significantly suppressed. Both C3(-/-) and C4(-/-) mice had reduced levels of B cells and increased expression of apoptotis inducers (Fas ligand, CD69) and apoptotic cells in lymph nodes. Immunofluorescence studies showed that the neuromuscular junction of C3(-/-) and C4(-/-) mice lacked C3 or membrane attack complex deposits, despite having IgG deposits, thus providing in vivo evidence for the incapacity of anti-AChR IgGs to induce full-blown EAMG without the aid of complements. The data provide the first direct genetic evidence for the classical complement pathway in the induction of EAMG induced by AChR immunization. Accordingly, severe MG and other Ab- and complement-mediated diseases could be effectively treated by inhibiting C4, thus leaving the alternative complement pathway intact

IFN-alpha treatment suppresses the development of experimental autoimmune myasthenia gravis.

Abstract Myasthenia gravis (MG) is an Ab-mediated autoimmune neuromuscular disease and is linked to MHC class II beta-chain polymorphism. Corticosteroids and azathioprine are the primary immunosuppressive drugs used in the treatment of MG. These drugs have significant side effects and have limited efficacy. Therefore, drugs with fewer side effects and greater efficacy are being sought. IFN-alpha is a potent immunomodulator and has been shown to down-regulate MHC class II expression on lymphoid cells. MHC class II expression is critical for the development of experimental autoimmune myasthenia gravis (EAMG). Because of the immunomodulating effects of IFN-alpha and its effect on the MHC class II expression, we tested the therapeutic efficacy of IFN-alpha on EAMG induced by immunization with acetylcholine receptor (AChR) in CFA. IFN-alpha (10(5) IU three times weekly for 5 wk) treatment started 1 wk after the second immunization with AChR in CFA, when autoimmunity to AChR is well established, reduced the incidence of clinical EAMG by more than 50% in two separate experiments (p = 0.04 and 0.008). Therefore, IFN-alpha could be a potential agent for the control of MG, and other Ab-mediated autoimmune diseases.</jats:p

Effect of MHC class I and CD8 cell deficiency on experimental autoimmune myasthenia gravis pathogenesis.

Abstract MHC class I and CD8+ cell deficiency have either prevented systemic lupus erythematosus-like disease in mice or enhanced type I diabetes in nonobese diabetic mice. To study the involvement of MHC class I and class I-restricted CD8+ T cells in the induction of a classical Ab-mediated disease, experimental autoimmune myasthenia gravis (EAMG), we immunized beta 2 microglobulin (beta 2-m) gene-disrupted (beta 2 m-/-) C57BL10 (B10) mice, deficient in class I gene expression and CD8+ cells, and heterozygous (beta 2-m+/-) B10 mice with normal expression of class I molecules and sufficient CD8+ cells with Torpedo acetylcholine receptor in CFA, and assessed them for clinical and immunopathologic manifestations of EAMG. Despite MHC class I and CD8+ cell deficiency, beta 2-m-/- mice developed EAMG. Moreover, the incidence of EAMG in the beta 2-m-/- mice was higher than that of beta 2-m+/- heterozygous mice with normal class I expression and frequency of CD8+ cells. The finding provided direct genetic evidence against a pathogenic effector role in C57BL10 mice for MHC class I molecule and class I-restricted CD8+ T cells in EAMG pathogenesis.</jats:p