Rosanne Seguin

Type 2 Monocyte and Microglia Differentiation Mediated by Glatiramer Acetate Therapy in Patients with Multiple Sclerosis

Glatiramer acetate (GA) therapy of patients with multiple sclerosis (MS) represents a unique setting in which in vivo Th2 deviation of T cells is consistently observed and associated with clinical benefit in a human autoimmune disease. We postulated that APCs are important targets of GA therapy and demonstrate that treatment of MS patients with GA reciprocally regulates the IL-10/IL-12 cytokine network of monocytes in vivo. We further show that Th1- or Th2-polarized GA-reactive T cells isolated from untreated or treated MS patients mediate type 1 and 2 APC differentiation of human monocytes, based on their ability to efficiently induce subsequent Th1 and Th2 deviation of naive T cells, respectively. These observations are extended to human microglia, providing the first demonstration of type 2 differentiation of CNS-derived APCs. Finally, we confirm that the fundamental capacity of polarized T cells to reciprocally modulate APC function is not restricted to GA-reactive T cells, thereby defining a novel and dynamic positive feedback loop between human T cell and APC responses. In the context of MS, we propose that GA therapy results in the generation of type 2 APCs, contributing to Th2 deviation both in the periphery and in the CNS of MS patients. In addition to extending insights into the therapeutic mode of action of GA, our findings revisit the concept of bystander suppression and underscore the potential of APCs as attractive targets for therapeutic immune modulation.

Differential infectivity and division of Toxoplasma gondii in human peripheral blood leukocytes.

ABSTRACT When tachyzoites were incubated with human peripheral blood leukocytes in vitro, more monocytes and dendritic cells than neutrophils or lymphocytes were infected. Although tachyzoites were able to divide in each of these cell types, monocytes and dendritic cells were more permissive to rapid tachyzoite division than neutrophils or lymphocytes.

Phagocytosis of Apoptotic Inflammatory Cells by Microglia and Its Therapeutic Implications: Termination of CNS Autoimmune Inflammation and Modulation by Interferon-Beta

Apoptosis of autoaggressive T‐cells in the CNS is an effective, noninflammatory mechanism for the resolution of T‐cell infiltrates, contributing to clinical recovery in T‐cell‐mediated neuroinflammatory diseases. The clearance of apoptotic leukocytes by tissue‐specific phagocytes is critical in the resolution of the inflammatory infiltrate and leads to a profound downregulation of phagocyte immune functions. Adult human microglia from surgically removed normal brain tissue was used in a standardized, light‐microscopic in vitro phagocytosis assay of apoptotic autologous peripheral blood‐derived mononuclear cells (MNCs). Microglia from five different patients had a high capacity for the uptake of apoptotic MNCs in contrast to nonapoptotic target cells with the phagocytosis rate for nonapoptotic MNCs amounting to only 61.6% of the apoptotic MNCs. A newly described phosphatidylserine receptor, critical in the phagocytosis of apoptotic cells by macrophages, is also expressed at similar levels on human microglia. The effects of the therapeutically used immunomodulatory agent interferon‐beta (IFNβ) were investigated using Lewis rat microglia and apoptotic, encephalitogenic, myelin basic protein‐specific autologous T‐cells. Also, rat microglia had a high capacity to phagocytose apoptotic T‐cells specifically. IFNβ increased the phagocytosis of apoptotic T‐cells to 36.8% above the untreated controls. The enhanced phagocytic activity was selective for apoptotic T‐cells and was not mediated by increased IL‐10 secretion. Apoptotic inflammatory cells may be efficiently and rapidly removed by microglial cells in the autoimmune‐inflamed human CNS. The in vitro increase of phagocytosis by IFNβ merits further investigations whether this mechanism could also be therapeutically exploited.

Cryptogenic Epilepsy: An Infectious Etiology?

Summary:  Purpose: Cryptogenic epilepsy, the group of epilepsy syndromes for which an etiology is unknown, comprises ∼20% of all epilepsy syndromes. We selected patients in this subgroup of epilepsy and tested them for evidence of Toxoplasma gondii IgG antibodies by the enzyme‐linked immunosorbent assay. T. gondii is found in up to 20% of the U.S. population forming dormant brain cysts in the latent bradyzoite form. We investigated the hypothesis that dormant T. gondii infection might be associated with cryptogenic epilepsy.

NK cell-mediated lysis of autologous human oligodendrocytes.

Although considered an autoimmune disease, the mechanisms underlying oligodendrocyte (OL)/myelin injury in multiple sclerosis (MS) remain to be established. We utilized in vitro assays to demonstrate that human OLs, as well as other glial elements (astrocytes, microglia), were susceptible to injury mediated by peripheral blood-derived mononuclear cell preparations (MNCs) enriched for natural killer (NK cells) by depleting CD3(+) +/- CD19(+) cells through use of either magnetic beads or cell sorting. Cytotoxic effects of the NK cell-enriched effectors were dependent on pre-exposure of these cells to IL-2. Furthermore, we found that autologous OLs were as susceptible to injury mediated by IL-2 activated NK cells as were heterologous OLs. In context of the tissue injury that occurs in MS, our results suggest that the inflammatory milieu in MS lesions could provide conditions required for NK cell activation and that such effector cells can bypass the putative protective effects of self-MHC class I molecules that may be expressed on OLs.

Differential effects of Th1 and Th2 lymphocyte supernatants on human microglia

We assessed the effects of soluble molecules (supernatants) produced by pro‐ (Th1) and anti‐ (Th2) inflammatory T‐cell lines on the capacity of adult human CNS‐derived microglia to express or produce selected cell surface and soluble molecules that regulate immune reactivity or impact on tissue protection/repair within the CNS. Treatment of microglia with supernatants from allo‐antigen and myelin basic protein‐specific Th1 cell lines augmented expression of cell surface molecules MHC class II, CD80, CD86, CD40, and CD54, enhanced the functional antigen‐presenting cell capacity of microglia in a mixed lymphocyte reaction, and increased cytokine/chemokine secretion (TNFα, IL‐6, and CXCL10/IP‐10). These Th1‐induced effects were not reproduced by interferon‐γ (IFNγ) alone and were only incompletely blocked by anti‐IFNγ antibody. Th2 cell supernatant treatments did not alter costimulatory/adhesion molecule expression or induce cytokine/chemokine production by microglia. Th2 treatment, furthermore, failed to reduce the induction observed in response to Th1 supernatants. Neither Th1 nor Th2 supernatants induced production of the neurotrophin molecules, nerve growth factor, or brain‐derived neurotrophic factor. Our results suggest that soluble molecules released by Th1 and not Th2 cells that infiltrate the CNS can stimulate resident microglia to acquire enhanced effector and accessory cell functions; the Th1‐induced effects were not downregulated by Th2 supernatant‐mediated bystander suppression. GLIA 42:36–45, 2003.

Identification and Role of Thiols in Toxoplasma gondii Egress

The nucleoside triphosphate hydrolase of Toxoplasma gondii is a potent apyrase that is secreted into the parasitophorous vacuole where it appears to be essentially Inactive in an oxidized form. Recent evidence shows that nucleoside triphosphate hydrolase can be activated by dithiothreitol in vivo. On reduction of the enzyme, there is a rapid depletion of host cell ATP. Previous results also demonstrate a dithiothreitol induced egress of parasites from the host cell with a concurrent Ca2+ flux, postulated to be a consequence of the release of ATP-dependent Ca2+ stores within the tubulovesicular network of the parasitophorous vacuole. Reduction of the nucleoside triphosphate hydrolase appears crucial for Its activation; however, the exact mechanism of reduction/activation has not been determined. Using a variety of techniques, we show here that glutathione promoters activate a Ca2+ flux and decrease ATP levels in Infected human fibroblasts. We further show the in vitro activation of nucleoside triphosphate hydrolase by endogenous reducing agents, one of which we postulate might be secreted into the PV by T. gondii. Our findings suggest that the reduction of the parasite nucleoside triphosphate hydrolase, and ultimately parasite egress, is under the control of the parasites themselves.

Human brain endothelial cells supply support for monocyte immunoregulatory functions

Blood-derived monocytic cells comprise a significant component of most inflammatory responses that occur in the CNS. We utilized human brain-derived endothelial cells (HBECs) coated membranes in Boyden chambers to assess immune function related properties of human blood-derived monocytes following interaction with HBECs. Monocytes in contact with HBECs maintained functional antigen-presenting capacity and chemokine/cytokine production in contrast to monocytes that migrated through the HBEC barrier. These results indicate that HBECs, although themselves incapable of serving as competent antigen-presenting cells during the course of inflammatory CNS disorders, supply support needed for infiltrating perivascular monocytes to maintain their functions. Monocyte migration across HBECs was inhibited by interferon-beta.

In situ microRNA profiles of astrocytes in the context of ischemic brain injury and multiple sclerosis

expression of Ly6C and Ly6G, very important in infectious, autoimmune and tumor models. The present work will further characterize the potential role of miR-223 in the EAE model and MS. First we found an upregulation of miR-233 in the Peripheral Blood Mononuclear Cell (PBMC) of 20 MS samples vs. 20 controls (fold change over controls 1.64 ± 1.25 vs. 1.20 ± 0.95, P = 0.018). This result was confirmed in a different cohort of subjects, including 15 untreated MS subjects (population from Italy: 11 RRMS, 4 PPMS) and 12 healthy controls. In this cohort, miR-233 was upregulated in MS vs. control subjects (fold change over controls 0.81 ± 0.65 vs. 0.40 ± 0.26, P = 0.010). We also performed several active EAE experiments in miR-223 knockout (miR-223 KO) mice and littermate control mice. MiR-223 KO mice developed a significantly less severe disease (P b 0.0001 by two-way ANOVA) with a significantly higher percentage of PMN-MDSC (CD11b/Ly6G positive cells) and MO-MDSC (CD11b/Ly6C positive cells) in the spleens and spinal cords compared to control mice. We found also that MO-MDSC from miR-223 KO mice had greater immune-suppressive effects on CD4 T cell proliferation than controls in antigen T cell stimulatory conditions. It is established that MO-MDSCs inhibit CD4 and CD8 T cell proliferation mostly via ARG1 action. ARG1 was promptly upregulated in MO-MDSC from miR-223 KO cells corresponding to their high immunosuppressive function. These results demonstrate altered levels of miR 223 in the PBMC of MS patients and suggest that miR-223 plays a role in EAE. This may lead to the identification of new disease biomarkers of therapeutic targets.

Regular ArticlesCerebral Malaria in Mice: Interleukin-2 Treatment Induces Accumulation of γδ T Cells in the Brain and Alters Resistant Mice to Susceptible-Like Phenotype

In this study, we report that infection with Plasmodium yoelii 17XL, a lethal strain of rodent malaria, does not result in death in the DBA/2 strain of mice. In contrast to BALB/c mice, DBA/2 mice developed significantly less parasitemia and never manifested symptoms of cerebral malaria (CM) on infection with this parasite. Moreover, the histological changes evident in the brain of susceptible BALB/c were absent in DBA/2 mice. Interestingly, the resistant DBA/2 mice when treated with recombinant interleukin (IL)-2, were found to develop CM symptoms and the infection became fatal by 6 to 8 days after infection. This condition was associated with an augmented interferon-gamma and nitric oxide production. Unexpectedly, IL-10 levels were also elevated in IL-2-treated DBA/2 mice during late stage of infection (at day 6 of infection) whereas the inverse relationship between IL-10 and interferon-gamma or nitric oxide was maintained in the early stage of infection (at day 3 after infection). The level of tumor necrosis factor-alpha production was moderately increased in the late phase of infection in these mice. Histology of brain from IL-2-treated mice demonstrated the presence of parasitized erythrocytes and infiltration of lymphocytes in cerebral vessels, and also displayed some signs of endothelial degeneration. Confocal microscopical studies demonstrated preferential accumulation of gammadelta T cells in the cerebral vessels of IL-2-treated and -infected mice but not in mice treated with IL-2 alone. The cells recruited in the brain were activated because they demonstrated expression of CD25 (IL-2R) and CD54 (intercellular adhesion molecule 1) molecules. Administration of anti-gammadelta mAb prevented development of CM in IL-2-treated mice until day 18 after infection whereas mice treated with control antibody showed CM symptoms by day 6 after infection. The information concerning creating pathological sequelae and death in an otherwise resistant mouse strain provides an interesting focus for the burden of pathological attributes on death in an infectious disease.