Isolation of F. novicida-Containing Phagosome from Infected Human Monocyte Derived Macrophages

Francisella is a gram-negative bacterial pathogen, which causes tularemia in humans and animals. A crucial step of Francisella infection is its invasion of macrophage cells. Biogenesis of the Francisella-containing phagosome (FCP) is arrested for ∼15min at the endosomal stage, followed by gradual bacterial escape into the cytosol, where the microbe proliferates. The crucial step in pathogenesis of tularemia is short and transient presence of the bacterium within phagosome. Isolation of FCPs for further studies has been challenging due to the short period of time of bacterial residence in it and the characteristics of the FCP. Here, we will for the first time present the method for isolation of the FCPs from infected human monocytes-derived macrophages (hMDMs). For elimination of lysosomal compartment these organelles were pre-loaded with dextran coated colloidal iron particles prior infection and eliminated by magnetic separation of the post-nuclear supernatant (PNS). We encountered the challenge that mitochondria has similar density to the FCP. To separate the FCP in the PNS from mitochondria, we utilized iodophenylnitrophenyltetrazolium, which is converted by the mitochondrial succinate dehydrogenase into formazan, leading to increased density of the mitochondria and allowing separation by the discontinuous sucrose density gradient ultracentrifugation. The purity of the FCP preparation and its acquisition of early endosomal markers was confirmed by Western blots, confocal and transmission electron microscopy. Our strategy to isolate highly pure FCPs from macrophages should facilitate studies on the FCP and its biogenesis.1

Toll-Like Receptor 2, Toll-Like Receptor 4, Myeloid Differentiation Response Gene 88, and Toll-IL-1 Receptor Domain-Containing Adaptor-Inducing Interferon-gamma (TRIF) Selectively Regulate Susceptibility of PO106-125-Induced Murine Experimental Autoimmune Neuritis

The functional relevance of the innate immune system has not yet been dissected in PO106-125-induced murine experimental autoimmune neuritis. Therefore, the role of Toll-like receptor (TLR) 2, TLR4, myeloid differentiation response gene 88, and ToLL IL-1 receptor domain-containing adaptor-inducing interferon-i (TRIF), factors critically involved in the TLR signaling pathway, was studied in experimental autoimmune neuritis. In the absence of TLR2, TLR4, myeloid differentiation response gene 88, or TRIF, the clinical course was significantly attenuated compared to wild-type mice. This could be attributed to impaired NE-kappa B activation, as shown by the absence of nuclear translocation of ReIA with a decreased expression of IL-6, IL 12p40, and IL-17A. Remarkably, PO106-125-immunized TLR2(0/0) mice exhibited a delayed recovery as compared to TLR4(0/0) mice, which was because of an impaired T helper cell 2 polarization. Immunized TLR2(0/0) mice' were unable to induce OX40 and OX4OL by matrix metalloproteinase-2 on spLenic dendritic cells. Subsequently, M2 polarization was impaired and macrophages were unable to sufficiently induce T regulatory cells (T-regs). Thus, in the recovery phase, Tregs were significantly increased in TLR4(0/0) mice as compared to wild-type mice, whereas T-regs in immunized TLR2(0/0) mice were only slightly increased. Our data highlight the relevance of innate immunity and, especially, the tight interaction between the innate and the adaptive immune system, which should be considered for therapeutic approaches of autoimmune diseases

IL-10, IL-4, and STAT6 Promote an M2 Milieu Required for Termination of PO106-125-Induced Murine Experimental Autoimmune Neuritis

The role of the type 2 helper T cell (Th2) polarizing cytokines IL-4 and IL-10 has not yet been studied in PO105-125-induced murine experimental autoimmune neuritis (EAN). We, therefore, addressed the functional relevance of these cytokines and signaling via the IL-4 associated transcription factor STAT6. The clinical course of PO106-125-induced EAN in mice deficient for IL-10(0/0), IL-4(0/0), or STAT6(0/0) was significantly aggravated compared with that of wild-type control mice. In addition, treatment of PO106-125-immunized C57BL/6 mice at the onset of clinical symptoms with a monoclonal IL-10 neutralizing antibody aggravated symptoms and prolonged disease to a similar degree as in IL-10(0/0) mice. This exacerbated course was attributed to a more prominent Th1 immune response associated with a persistent M1 milieu in the sciatic nerve and in the regional and systemic Lymphatic system. These data suggest a Th2-polarized milieu being required to prevent axonal damage of the sciatic nerve and to terminate the PO106-125-Specific immune response in EAN. Beyond the already known role of macrophages as pathogenic effector cells in EAN, these data suggest that M2-differentiated macrophages do not damage and may even protect neural tissues in EAN. Thus, these data highlight the pathogenetic relevance of the macrophage polarization status in EAN. Therapeutic modulation of immune responses from an M1 toward an M2 milieu may be a promising novel strategy in peripheral nervous system neuritis

Costimulatory Molecule CD40 Is Essential for Myelin Protein 0 Peptide 106Y125YInduced Experimental Autoimmune Neuritis in Mice

Myelin protein 0 peptide 106-125-induced murine experimental autoimmune neuritis (EAN) is a CD4-positive T cell-mediated monophasic axonal inflammatory neuropathy; interferon- is the key proinflammatory mediator. Experimental autoimmune neuritis is well suited for elucidating pathogenetic mechanisms underlying human acute axonal Guillain-Barre syndrome. Here, the functional role of the costimulatory molecule CD40 was defined by characterization of EAN in CD40-deficient mice. In contrast to immunized C57BL/6 mice, CD40-deficient mice were resistant to EAN owing to impaired priming of CD4-positive T-effector cells. To determine whether CD40 is a suitable candidate for the treatment of EAN, we administered monoclonal anti-CD40 antibody either before immunization or upon onset of neurologic signs. Prophylactic anti-CD40 treatment completely abolished CD4-positive T-cell priming. Therapeutic application of anti-CD40 prevented full activation of CD4-positive T cells that were in the process of priming and suppressed production of interferon- in peripheral lymph nodes, spleen, and serum, and of interleukin-6, interleukin-12p40, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1, which are associated with activation of the nuclear factor-B signaling pathway. This resulted in enhanced recovery by early generation of CD25-positive, Foxp3-positive, CD4-positive regulatory T cells. Thus, these experiments highlight the crucial role of CD40 as an important costimulatory molecule in EAN and suggest that it has potential as a therapeutic target in human neuritis

Costimulatory Molecule CD40 Is Essential for Myelin Protein 0 Peptide 106–125–Induced Experimental Autoimmune Neuritis in Mice

Myelin protein 0 peptide 106-125-induced murine experimental autoimmune neuritis (EAN) is a CD4-positive T cell-mediated monophasic axonal inflammatory neuropathy; interferon- is the key proinflammatory mediator. Experimental autoimmune neuritis is well suited for elucidating pathogenetic mechanisms underlying human acute axonal Guillain-Barre syndrome. Here, the functional role of the costimulatory molecule CD40 was defined by characterization of EAN in CD40-deficient mice. In contrast to immunized C57BL/6 mice, CD40-deficient mice were resistant to EAN owing to impaired priming of CD4-positive T-effector cells. To determine whether CD40 is a suitable candidate for the treatment of EAN, we administered monoclonal anti-CD40 antibody either before immunization or upon onset of neurologic signs. Prophylactic anti-CD40 treatment completely abolished CD4-positive T-cell priming. Therapeutic application of anti-CD40 prevented full activation of CD4-positive T cells that were in the process of priming and suppressed production of interferon- in peripheral lymph nodes, spleen, and serum, and of interleukin-6, interleukin-12p40, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1, which are associated with activation of the nuclear factor-B signaling pathway. This resulted in enhanced recovery by early generation of CD25-positive, Foxp3-positive, CD4-positive regulatory T cells. Thus, these experiments highlight the crucial role of CD40 as an important costimulatory molecule in EAN and suggest that it has potential as a therapeutic target in human neuritis

IL-10, IL-4, and STAT6 Promote an M2 Milieu Required for Termination of P0106-125-Induced Murine Experimental Autoimmune Neuritis

The role of the type 2 helper T cell (Th2) polarizing cytokines IL-4 and IL-10 has not yet been studied in PO105-125-induced murine experimental autoimmune neuritis (EAN). We, therefore, addressed the functional relevance of these cytokines and signaling via the IL-4 associated transcription factor STAT6. The clinical course of PO106-125-induced EAN in mice deficient for IL-10(0/0), IL-4(0/0), or STAT6(0/0) was significantly aggravated compared with that of wild-type control mice. In addition, treatment of PO106-125-immunized C57BL/6 mice at the onset of clinical symptoms with a monoclonal IL-10 neutralizing antibody aggravated symptoms and prolonged disease to a similar degree as in IL-10(0/0) mice. This exacerbated course was attributed to a more prominent Th1 immune response associated with a persistent M1 milieu in the sciatic nerve and in the regional and systemic Lymphatic system. These data suggest a Th2-polarized milieu being required to prevent axonal damage of the sciatic nerve and to terminate the PO106-125-Specific immune response in EAN. Beyond the already known role of macrophages as pathogenic effector cells in EAN, these data suggest that M2-differentiated macrophages do not damage and may even protect neural tissues in EAN. Thus, these data highlight the pathogenetic relevance of the macrophage polarization status in EAN. Therapeutic modulation of immune responses from an M1 toward an M2 milieu may be a promising novel strategy in peripheral nervous system neuritis