Ingrid E. Gienapp

Oral Tolerance in Experimental Autoimmune Encephalomyelitis

In work performed by a number of laboratories, it has become quite clear that the oral administration of autoantigens exerts a profoundly suppressive effect on the development and long-term clinical course of autoimmune disease. Specific peptide sequences derived from the autoantigens are similarly suppressive. An interesting sidelight to emerge from specificity studies is that oral administration of a self-protein or peptide sequence (i.e., rat MBP peptide administered to a rat) is markedly less tolerogenic than oral administration of a non-self or even closely related sequence (guinea pig MBP peptide administered to a rat). The dose of oral antigen is now known to play a critical role in determination of the mechanism of oral tolerance, with low doses of antigen causing active suppression with concomitant release of TGFbeta1. Studies outlined here suggest that oral administration of higher antigen doses (e.g., 20 mg MBP to rats or mice) results in deletion of specific antigen-reactive T lymphocytes. This conclusion stems from the fact that injections of IL-2 could not reverse high-dose tolerance while reversing low-dose oral tolerance. Moreover, feeding MBP to MBP-TCR transgenic mice caused trafficking of transgenic cells to the intestine followed by a profound depletion of transgene-positive cells and reduction in proliferative function in all peripheral lymphoid organs. Oral tolerance has proven to be of therapeutic benefit in other animal models of autoimmune disease as well, including uveitis, collagen-induced arthritis, adjuvant arthritis, thyroiditis, myasthenia gravis, and diabetes. Initial human trials in multiple sclerosis, rheumatoid arthritis, and uveitis show promising results.

Sex differences in experimental autoimmune encephalomyelitis in multiple murine strains

Multiple sclerosis (MS) is more prevalent in women than men. We evaluated seven different mouse strains commonly used in the study of autoimmune diseases, for sex differences in the disease course of experimental autoimmune encephalomyelitis (EAE). Greater severity of EAE was observed in the female SJL immunized with two different peptides of myelin proteolipid protein (PLP) and myelin oligodendrocyte glycoprotein (MOG) as well as in the female ASW relative to males. Female NZW mice showed a greater incidence of EAE than males. However, male B10.PL and PL/J mice showed more severe disease than females. No sex differences were noted in the C57BL/6 or NOD strains.

Estriol Generates Tolerogenic Dendritic Cells In Vivo That Protect against Autoimmunity

Chronic inflammation contributes to numerous diseases, and regulation of inflammation is crucial for disease control and resolution. Sex hormones have potent immunoregulatory abilities. Specifically, estrogen influences immune cells and inflammation, which contributes to the sexual dimorphism of autoimmunity and protection against disease seen during pregnancy in multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). Although long thought to act primarily on T cells, recent evidence demonstrated that myeloid cells, such as dendritic cells (DCs), are essential in mediating estrogen’s protective effects. Estriol (E3), a pregnancy-specific estrogen, has therapeutic efficacy in MS and EAE, and we evaluated whether E3 could act exclusively through DCs to protect against the inflammatory autoimmune disease EAE. Levels of activation markers (CD80 and CD86) and inhibitory costimulatory markers (PD-L1, PD-L2, B7-H3, and B7-H4) were increased in E3 DCs. E3 DCs had decreased proinflammatory IL-12, IL-23, and IL-6 mRNA expression, increased immunoregulatory IL-10 and TGF-β mRNA expression, and a decreased ratio of IL-12/IL-10 protein production. Importantly, transfer of E3 DCs to mice prior to active induction of EAE protected them from developing EAE through immune deviation to a Th2 response. This protection was apparent, even in the face of in vitro and in vivo inflammatory challenge. In summary, our results showed that E3 generates tolerogenic DCs, which protect against the inflammatory autoimmune disease EAE. Targeted generation of tolerogenic DCs with immunomodulatory therapeutics, such as E3, has potential applications in the treatment of numerous autoimmune and chronic inflammatory diseases.

T-cell activation and receptor downmodulation precede deletion induced by mucosally administered antigen

The fate of antigen-specific T cells was characterized in myelin basic protein (MBP) T-cell receptor (TCR) transgenic (Tg) mice after oral administration of MBP. Peripheral Th cells are immediately activated in vivo, as indicated by upregulation of CD69 and increased cytokine responses (Th1 and Th2). Concurrently, surface TCR expression diminishes and internal TCR levels increase. When challenged for experimental autoimmune encephalomyelitis during TCR downmodulation, Tg mice are protected from disease. To characterize Th cells at later times after antigen feeding, it was necessary to prevent thymic release of naive Tg cells. Therefore, adult Tg mice were thymectomized before treatment. TCR expression returns in thymectomized Tg mice 3 days after MBP feeding and then ultimately declines in conjunction with MBP-specific proliferation and cytokine responses (Th1-type and Th2-type). The decline correlates with an increase in apoptosis. Collectively, these results demonstrate that a high dose of fed antigen induces early T-cell activation and TCR downmodulation, followed by an intermediate stage of anergy and subsequent deletion.

Pregnancy Suppresses Experimental Autoimmune Encephalomyelitis through Immunoregulatory Cytokine Production

Women with multiple sclerosis (MS) often experience a decrease in relapse rate during pregnancy, most notably during the third trimester, with a flare of disease activity 3–6 mo postpartum. Studies in experimental autoimmune encephalomyelitis (EAE), an animal model for MS, have shown that pregnancy delays the onset and decreases the incidence of disease. We investigated the effect of pregnancy and the postpartum period in a remitting-relapsing model of murine EAE. When immunization occurs during pregnancy, mice show a reduction in the incidence of EAE as well as a decrease in clinical severity, while mice immunized during the postpartum period exhibit more severe disease. No differences in lymphocyte proliferation or expression of activation markers were noted when immunization occurred during pregnancy as compared with the nonpregnant controls. Mice immunized during pregnancy produced less TNF-α and IL-17, and showed an increased number of IL-10-secreting cells within the CD11b+, CD11c+, CD19+, and CD4+/CD25+ populations. No differences were noted in the production of IFN-γ, IL-2, IL-4, and IL-5. These results suggest that when an Ag is introduced during pregnancy, an immunoregulatory rather than an immunosuppressive or Th2 environment predominates.

Cutting edge: macrophage migration inhibitory factor is necessary for progression of experimental autoimmune encephalomyelitis.

Macrophage migration inhibitory factor (MIF) has been implicated in the pathogenesis of inflammatory and autoimmune diseases. The role of MIF in the progression of experimental autoimmune encephalomyelitis (EAE) was explored using MIF−/− mice. Wild-type mice showed a progressive disease course, whereas MIF−/− mice exhibited acute signs but no further progression of clinical disease. MIF−/− mice displayed markedly elevated corticosterone levels and significant decreases in the inflammatory cytokines TNF-α, IFN-γ, IL-2, and IL-6 before, during, and after EAE onset. Taken together, these findings support that MIF is an important mediator of EAE progression through glucocorticoid antagonism and up-regulation of the inflammatory response.

Alterations in Immune Cell Phenotype and Function after Experimental Spinal Cord Injury

Traumatic injury to the spinal cord initiates a cascade of inflammatory-mediated injury and repair processes within the nervous system. In parallel, spinal injury could influence peripheral mechanisms of host defense (e.g., wound healing, antibody production) by altering lymphocyte phenotype and function. The goal of this study was to evaluate the physiological impact of spinal contusion injury on phenotypic and functional indices of lymphocyte activation. A flow cytometric time-course analysis of lymphocytes isolated from lymph node and spleen revealed an increase in CD4+ and a decrease in CD8+ lymphocytes during the first week post injury. The functional potential of lymphocytes was also evaluated based on their ability to proliferate in the presence of a biologically relevant antigen (myelin basic protein, MBP) or a lymphocyte mitogen. The data revealed increased proliferation to MBP by 3 days postinjury in lymphocytes isolated from lymph node but not spleen. By 1 week postinjury, increased proliferation to mitogen was noted in both the lymph node and the spleen suggesting a general increase in lymphocyte reactivity during this time interval. Circulating corticosterone (CORT), an endogenous glucocorticoid with significant effects on lymphocyte phenotype and function, was elevated within 24 h after spinal cord injury (SCI) and remained above control levels throughout the duration of our studies (up to 1 month postinjury). The present data suggest injury-associated changes in immune cell phenotype and function paralleled by the activation of the hypothalamic-pituitary-adrenal (HPA) axis.

Suppression of Experimental Autoimmune Encephalomyelitis Using Peptide Mimics of CD28

The B7:CD28/CTLA-4 costimulatory pathway plays a critical role in regulating the immune response and thus provides an ideal target for therapeutic manipulation of autoimmune disease. Previous studies have shown that blockade of CD28 signaling by mAbs can both prevent and exacerbate experimental autoimmune encephalomyelitis (EAE). In this study, we have designed two CD28 peptide mimics that selectively block B7:CD28 interactions. By surface plasmon resonance, both the end group-blocked CD28 peptide (EL-CD28) and its retro-inverso isomer (RI-CD28) compete effectively with the extracellular domain of CD28 for binding to B7-1. Both the CD28 peptide mimics inhibited expansion of encephalitogenic T cells in vitro. A single administration of EL-CD28 or RI-CD28 peptide significantly reduced disease severity in EAE. Importantly, we show that either CD28 peptide mimic administered during acute disease dramatically improved clinical signs of EAE, suppressing ongoing disease. The ratio of CD80:CD86 expression was significantly lower on CD4+ and F4/80+ spleen cells in CD28 peptide-treated mice. Peripheral deletion of Ag-specific CD4+ T cells occurs following in vivo blockade of CD28 with synthetic CD28 peptides.

Neuroendocrine modulation of chronic relapsing experimental autoimmune encephalomyelitis: A critical role for the hypothalamic-pituitary-adrenal axis

Murine relapsing EAE can be profoundly suppressed by restraint stress (RST) administered beginning prior to neuroantigen immunization. This study determined what hormone pathway(s) mediate disease suppression. Our results showed that nadolol (NAD), a beta2-adrenergic antagonist, did not reverse the RST-induced suppression of EAE. However, administration of either RU486 or aminoglutethimide, which block the action of peripheral glucocorticoids, resulted in a partial reversal of EAE suppression. Administration of exogenous corticosterone mimicked the effects of RST, in terms of suppression of EAE, decrease in lymphoid cell numbers and decrease in Thl cytokine production. Therefore, the HPA axis plays a more profound role in the RST-induced suppression of EAE than does the sympathetic nervous system.

A small-molecule inhibitor of macrophage migration inhibitory factor for the treatment of inflammatory disease.

Multiple sclerosis (MS) is a chronic, debilitating disease of the central nervous system (CNS) characterized by demyelination and axon loss. The proinflammatory cytokine macrophage migration inhibitory factor (MIF) has been shown to be elevated in the cerebrospinal fluid of patients during relapses. The purpose of this study was to evaluate a new small‐molecule inhibitor of MIF and its ability to reduce the severity of an animal model of MS, experimental autoimmune encephalomyelitis (EAE). We utilized 2 structurally related isoxazolines, which show in vitro inhibition of MIF tautomerase activity. We found that administration of an inhibitor of MIF to mice with established EAE immediately reduced the severity of clinical signs and expanded a population of regulatory T lymphocytes. We also noted that the inhibitor reduced relapses of disease in a relapsing/remitting model of EAE. An analysis of leukocyte migration into the brain revealed that administration of inhibitor reduced entry of these cells. No effects on inflammatory cytokine production or T‐cell activation in the periphery were noted. From these studies, we conclude that a small‐molecule inhibitor of MIF reduces the severity of EAE and prevents access of immune cells into the CNS, which could be of therapeutic relevance to MS.—Kithcart, A. P., Cox, G. M., Sielecki, T., Short, A., Pruitt, J., Papenfuss, T., Shawler, T., Gienapp, I., Satoskar, A. R., Whitacre, C. C. A small‐molecule inhibitor of macrophage migration inhibitory factor for the treatment of inflammatory disease. FASEB J. 24, 4459–4466 (2010). www.fasebj.org