Brett T. Lund

Mycobacteria-induced Gr-1+ subsets from distinct myeloid lineages have opposite effects on T cell expansion

Similar to the regulation of vasodilation, the balance between NO and superoxide (O2–) regulates expansion of activated T cells in mice. Reduction of suppressive NO levels by O2– is essential for T cell expansion and development of autoimmunity. In mice primed with heat‐killed Mycobacterium, a splenocyte population positive for Gr‐1 (Ly‐6G/C) is the exclusive source of both immunoregulatory free radicals. Distinct Gr‐1+ cell subpopulations were separated according to Ly‐6G expression. In culture with activated T cells, predominantly monocytic Ly‐6G− Gr‐1+ cells produced T cell‐inhibitory NO but no O2–. However, mostly granulocytic Ly‐6G+ cells produced O2– simultaneously but had no measurable effect on proliferation. Recombination of the two purified Gr‐1+ subpopulations restored controlled regulation of T cell proliferation through NO and O2– interaction. Coculture of p47phox−/− and inducible NO synthase−/− Gr‐1+ cells confirmed this intercellular interaction. These data suggest that bacterial products induce development of distinct Gr‐1+ myeloid lineages, which upon stimulation by activated T cells, interact via their respective free radical products to modulate T cell expansion.

T cell vaccination in secondary progressive multiple sclerosis

Four secondary progressive MS patients were vaccinated with bovine myelin-reactive irradiated T cell lines from their peripheral blood. Patients were followed for 30-39 months, and monitored for immunological responses toward the vaccine, and for their clinical characteristics. Two patients showed stable EDSS score over time, one patient showed improvement by one EDSS step, and in the remaining patient her EDSS advanced over time. After the second inoculation there was a progressive decline of circulating whole myelin-reactive T cells, MBP143-168, PLP104-117, and MOG43-55-peptide-reactive T cells. In contrast the frequency of tetanus toxoid-reactive T cells remained unchanged. T cell vaccination (TCV) was also associated with a decline of myelin-specific IL-2- and IFN-gamma-secreting T cells. Twelve T cell lines (TCL) that recognize the inoculates were isolated from the peripheral blood of two patients. Ten of these TCL were CD8(+) and lysed the inoculates in a MHC Class I restricted manner. The remaining two TCL were CD4(+), and lysed the inoculates by MHC Class II restricted cytolytic activity. All T cell lines lysed not only myelin-reactive T cells, but also TCL specific for MBP143-168, PLP104-117 and MOG43-55 peptides. Control TCL specific for tetanus toxoid were not lysed. Neutralizing anti-Fas mAb did not influence the killing. Moreover, culture supernatants from two TCL which produce IL-10, were able to block the proliferation of myelin protein-specific TCL. This effect was abrogated using mAbs specific for IL-10. The data obtained indicated that TCV using autologous irradiated bovine myelin-reactive T cells promotes an effective depletion of T cells reactive against different myelin antigens.

Maternal Inflammation Disrupts Fetal Neurodevelopment via Increased Placental Output of Serotonin to the Fetal Brain

Maternal inflammation during pregnancy affects placental function and is associated with increased risk of neurodevelopmental disorders in the offspring. The molecular mechanisms linking placental dysfunction to abnormal fetal neurodevelopment remain unclear. During typical development, serotonin (5-HT) synthesized in the placenta from maternal l-tryptophan (TRP) reaches the fetal brain. There, 5-HT modulates critical neurodevelopmental processes. We investigated the effects of maternal inflammation triggered in midpregnancy in mice by the immunostimulant polyriboinosinic-polyribocytidylic acid [poly(I:C)] on TRP metabolism in the placenta and its impact on fetal neurodevelopment. We show that a moderate maternal immune challenge upregulates placental TRP conversion rapidly to 5-HT through successively transient increases in substrate availability and TRP hydroxylase (TPH) enzymatic activity, leading to accumulation of exogenous 5-HT and blunting of endogenous 5-HT axonal outgrowth specifically within the fetal forebrain. The pharmacological inhibition of TPH activity blocked these effects. These results establish altered placental TRP conversion to 5-HT as a new mechanism by which maternal inflammation disrupts 5-HT-dependent neurogenic processes during fetal neurodevelopment. SIGNIFICANCE STATEMENT The mechanisms linking maternal inflammation during pregnancy with increased risk of neurodevelopmental disorders in the offspring are poorly understood. In this study, we show that maternal inflammation in midpregnancy results in an upregulation of tryptophan conversion to serotonin (5-HT) within the placenta. Remarkably, this leads to exposure of the fetal forebrain to increased concentrations of this biogenic amine and to specific alterations of crucially important 5-HT-dependent neurogenic processes. More specifically, we found altered serotonergic axon growth resulting from increased 5-HT in the fetal forebrain. The data provide a new understanding of placental function playing a key role in fetal brain development and how this process is altered by adverse prenatal events such as maternal inflammation. The results uncover important future directions for understanding the early developmental origins of mental disorders.

Resistance to glucocorticoid-induced apoptosis in PLP peptide-specific T cell clones from patients with progressive MS

Glucocorticoids (GC) are commonly used to treat inflammatory disorders such as multiple sclerosis (MS) and may exert their immunosuppressive activity by inducing apoptosis in activated lymphocytes. However, unlike relapsing-remitting MS patients, those with progressive disease respond poorly to GC treatment. The data in this communication indicate that PLP peptide-specific T cell clones from progressive, but not relapsing-remitting MS patients are resistant to GC-induced apoptosis in vitro, in a fashion associated with expression of B-7 co-stimulatory molecules. Thus, failure to respond to GC treatment may reflect defect in apoptosis that develop during the progressive stages of chronic inflammatory disease.

Association of MBP peptides with Hsp70 in normal appearing human white matter.

Multiple Sclerosis is an autoimmune disease directed against myelin proteins. The etiology of MS is poorly defined though, with no definitive causative agent yet identified. It has been hypothesized that MS may be a multifactorial disease resulting in the same end product: the destruction of myelin by the immune system. In this report we describe a potential role for heat shock proteins in the pathogenesis of MS. We isolated Hsp70 from the normal appearing white matter of both MS and normal human brain and found this was actively associated with, among other things, immunodominant MBP peptides. Hsp70-MBP peptide complexes prepared in vitro were shown to be highly immunogenic, with adjuvant-like effects stimulating MBP peptide-specific T cell lines to respond to normally sub-optimal concentrations of peptide. This demonstration of a specific interaction between Hsp70 and different MBP peptides, coupled with the adjuvanticity of this association is suggestive of a possible role for Hsp70 in the immunopathology associated with MS.

The dual role of CXCL8 in human CNS stem cell function: Multipotent neural stem cell death and oligodendrocyte progenitor cell chemotaxis

Research into multiple sclerosis (MS) has shown that cells purportedly important to myelin repair within the CNS, namely neural stem cells (NSC) and oligodendrocyte progenitor cells (OPC), are recruited to active lesion sites during the course of the disease. However, over time these cells appear to become depleted or functionally blocked in and around lesions, accompanied by a failure of repair mechanisms. We have previously demonstrated elevated CXCL8 in patients with MS, and hypothesized that this chemokine may play a role in the pathology of this disease. Using NSC and OPC derived in vitro from human embryonic stem cells (hESC) we demonstrate here that CXCL8 has a dual role on stem cell biology in vitro. CXCL8 caused CXCR1‐mediated death of NSC, but not OPC, whilst also acting as a potent chemoattractant for both cell types. These data support a context‐dependent role for CXCL8 expression in the CNS in which it may drive recruitment of NSC and OPC to sites of inflammation, but as a side‐effect could also contribute to the failure of myelin repair in MS.

Assessment of changes in immune measures of multiple sclerosis patients treated with laquinimod

Laquinimod is a novel orally active agent with immunomodulatory properties that was shown to be effective in suppressing disease activity in relapsing-remitting multiple sclerosis patients. Though many mechanisms of action of laquinimod have been described, little is known about the in vivo effects of laquinimod on the functionality of circulating human peripheral blood mononuclear cell populations. We assessed both phenotypical and functional measures of PBMC in a prospective longitudinal analysis comparing laquinimod and placebo treated cohorts. We determined that there were no significant changes in the relative proportion of T-cells, B-cells, monocytes & macrophages, NK-cells, dendritic cells or FoxP3(+) CD25(hi) T-regs in laquinimod treated patients. There were also no significant differences in the proliferative response to PHA or tetanus antigen, or in the inflammatory cytokine bias of these responses. These data demonstrated that there were no significant changes in immune function of PBMC in patients receiving two years of continuous laquinimod therapy who retained a full complement of the major populations of circulating PBMC and retained their capacity to respond to immunologic stimuli.

Gene regulation networks related to neural differentiation of hESC.

With the unique property of self-renewal and developmental pluripotency, human embryonic stem cells (hESC) provide an opportunity to study molecular aspects of developmental biology. Understanding gene regulation of hESC pluripotency is a critical step toward directing hESC differentiation for regenerative medicine. However, currently little is known about hESC gene regulation of hESC pluripotency. Applying network analysis to microarray gene expression profiling data, we compared gene expression profiles from pluripotent hESC to hESC-derived astrocytes and identified potential gene regulation networks. These gene regulation networks suggest that hECS has stringent control of cell cycle and apoptosis. Our data reveal several potential hESC differentiation biomarkers and suggest that IGF2 and A2M could play a role in hESC pluripotency by altering the availability of cytokines at the local environment of hECS. These findings underscore the importance of network analysis among differentially expressed genes, and should facilitate future study for understanding the gene regulation of hESC pluripotency.

Measuring apoptosis in neural stem cells.

In trauma to, and diseases of, the central nervous system (CNS), apoptotic events are frequently observed in and around areas of damage. Human embryonic stem cells (hESCs) and their progeny have been suggested as possible therapeutic agents in the treatment of CNS diseases. The success of stem cell transplantation not only depends on the capacity of these cells to retain their functionality after transplant into the CNS but also on their ability to resist the in situ environmental cues that may lead to apoptosis. Although there are many methods used to detect apoptosis, the assessment of apoptosis in adherent cultures of primary stem cells and their progeny is more limited. We describe a series of protocols we have used to assess apoptosis in these cells.

In vitro assessment of the direct effect of laquinimod on basic functions of human neural stem cells and oligodendrocyte progenitor cells

Laquinimod is an orally active immunomodulatory small molecule that has shown clear clinical benefit in trials for relapsing-remitting multiple sclerosis and in experimental rodent models that emulate multiple sclerosis (MS). Studies in healthy mice, and in mice with experimental autoimmune encephalomyelitis, have demonstrated that laquinimod is capable of entering the central nervous system. It is therefore important to determine if laquinimod is capable of a direct influence on basic functions of neural stem cells (NSC) or oligodendrocyte progenitor cells (OPC)--cells critical for myelin repair in MS. In order to address this question, a series of experiments was conducted to determine the effect of exogenous laquinimod on viability, proliferation, migration and differentiation of human NSC and OPC in vitro. These data show, for the first time in cells of human origin, that direct, short-term interaction between laquinimod and NSC or OPC, in an isolated in vitro setting, is not detrimental to the basic cellular function of these cells.