Melissa A. Brown, Ph.D. - US grants

Much current research has focused on the role of polypeptide growth factors in normal and neoplastic cell growth. It has been hypothesized that some cells become malignant by virtue of the endogenous production of polypeptide growth factors acting on the producer cells through functional external receptors. Such a phenomenon is known as autocrine growth. One growth factor, Interleukin-4 (IL-4), a product of activated T cells, has multiple biological activities that affect cells of most hematopoietic lineages. Among these is the ability to co-stimulate the growth of B cells, T cells and mast cells. Recently, it was found that infection of interleukin-3 dependent mast lines with Abelson murine leukemia virus resulted in loss of growth factor dependence and a transformed phenotype. These cells did not produce autocrine IL-3 but a majority of cells expressed large amounts of IL-4 mRNA and IL-4 biological activity compared to non-transformed mast cells. This project proposes to 1) determine whether IL-4 acts as an autocrine growth factor for transformed mast cells and thus plays a role in the malignant phenotype of these cells and 2) identify cis and trans acting regulatory elements responsible for high constitutive expression of the IL-4 in transformed cells as well as in physiological activation of the gene. Eucaryotic expression vectors containing the IL-4 gene will be introduced into non-transformed mast cells and the consequences of high constitutive expression of factor dependence and tumorigenicity will be examined. Anti-sense oligonucleotides corresponding to regions critical for the initiation of translation of the IL-4 mRNA will be employed during culture of transformed mast cells in an attempt to block protein production. The effect of such treatment on in vitro growth will be examined. In addition, the elements regulating the transcriptional activity of the IL-4 gene in both constitutive high IL-4 expressors and inducible cells will be studied using DNAse I hypersensitivity, DNA methylation, transient expression systems using reporter fusion gene constructs and DNA protein binding assays.

Interleukin 4 (IL-4) is a potent immunoregulatory cytokine with extremely diverse effects on a number of target cells. IL-4 plays an important physiologic role in hematopoietic cell growth and differentiation, tumor surveillance and local protective inflammatory responses. However, dysregulated IL-4 production has been shown to have extremely detrimental effects. Pathological conditions such as atopic disease and inability to resolve certain parasitic infections are clearly associated with overproduction of IL-4. IL-4 was originally defined as a T cell cytokine, but it is now clear that mast cells are also important IL-4 producing cells. Mast cells are widely distributed in vascularized tissues in the proximity of many IL-4 target cells. This location provides the opportunity for mast cell-derived IL-4 to easily access such cells and effectively modulate a wide range of local inflammatory responses. Because of the unique tissue distribution of mast cells and thus potentially distinct functions of mast cell-derived IL-4 on local inflammatory responses, it was predicted that mechanisms which regulate IL-4 gene expression in mast cells would differ from those in T cells. Recent studies have confirmed this prediction. It was demonstrated that there are unique cis acting sequences which regulate IL-4 gene transcription in T and mast cells. A major component of tissue-specific transcriptional activity in mast cells appears to be contained in a sequence in the second intron of the IL-4 gene. This sequence exhibits prototypic enhancer activity in mast cell lines and its activity is dependent on the interaction of both positive and negative regulatory elements. The specific aims of this proposal are: 1. To identify essential positive and negative acting-sequences that comprise the IL-4 intronic enhancer. 2. To identify DNA binding proteins which specifically govern the function of the IL-4 intronic enhancer and determine their expression and binding dynamics in mast cells and cells of other lineages. 3. To examine populations of freshly isolated mast cells for their ability to express IL-4 upon stimulation and analyze the expression of mast cell specific transcription factors in these cells. 4. To determine how the IL-4 intronic enhancer interacts with other IL-4 regulatory sequences to modulate IL-4 expression in mast cells. DNA- protein binding assays and response element-reporter gene constructs in concert with transient transfection assays will be used to accomplish these aims. Understanding the molecular basis for tissue specific regulation in mast cells is an important first step in ultimately devising therapeutic strategies for selectively limiting or augmenting mast cell or T cell-derived IL-4 activity in allergic disease or immunotherapy of tumors such as melanoma.

DESCRIPTION (Adapted from the applicant's abstract): Interleukin 4 is a pleiotropic cytokine that exerts its diverse effects on a wide variety of target cells of hematopoietic and non-hematopoietic lineage. IL-4 plays an important physiologic role in hematopoietic cell growth and differentiation both directly by influencing differentiation processes and indirectly by modulating the production of other hematopoietic growth factors such as IL-1 and TNF-alpha. It is a critical immunoregulatory molecule and modulates the inflammatory response, in part, due to its ability to affect adhesion molecule expression and cytokine production by endothelial cells and its ability to affect growth and/or activation state of neutrophils, mast cells, T cells and eosinophils. Dysregulated IL-4 production has been shown to result in pathological conditions such as atopic disease. The physiologic production of IL-4 is tightly regulated: it is expressed only by a subset of activated T cells and mast cells/basophils. Based on their different tissue distribution and access to distinct target cells, T and mast cell derived IL-4 may have quite different effects on physiologic/immunologic processes. Recently, information regarding regulatory elements that dictate IL-4 gene transcription in T cells has been reported. However, very little is known about regulation of IL-4 in mast cells. The long term goals of this proposal are to define the molecular basis of the cell specific-regulation of IL-4. With this information, strategies to selectively modulate its expression in a cell specific manner can ultimately be devised and used therapeutically. Dr. Brown has defined a key IL-4 regulatory element (ARE) that is a target for activation signals leading to IL-4 transcription in both T and mast cells. There are clear differences in the T and mast cell transcription factors that interact with this element. Clearly, the next step in the analysis of this region is identification and comparison of these component proteins. The experiments in this proposal are designed to assess the cell specific differences in IL-4 regulation mediated by the ARE and other IL-4 regulatory regions. Specifically, the following is proposed: (1) To compare the transcription factors that directly associate with an IL-4 activation responsive element in T and mast cells: T cell ARE binding proteins will be purified by affinity chromatography and microsequence information will be used to design oligonucleotides to screen an activated T cell cDNA library. The cDNA(s) identified will be characterized and used as probes to assess their expression in mast cells. (2) To compare T and mast cell ARE transcription factors that require cooperative binding or interact solely through protein-protein interactions for ARE association: A two hybrid cloning system that is specifically designed to detect interacting proteins will be used to clone those proteins that do not directly bind the ARE. The interacting factors in T and mast cells can be compared. (3) To examine the potential of other IL-4 regulatory elements, defined in T cells, to modulate mast cell IL-4 gene transcription: Mutations in defined T cell IL-4 regulatory elements will be introduced into 5' IL-4 sequences and their effect on the expression of linked CAT reporter gene will be assessed in mast cells.

DESCRIPTION: (Adapted from the applicant's abstract) Interleukin-4 (IL-4) is an important immunoregulatory cytokine with diverse effects on immune system maturation and function. Two major cell types are known to produce IL-4, T cells and mast cells. The regulation of IL-4 gene expression in these cell types has been shown to be different and a regulatory element in the second intron of the gene is mast cell specific, contributing to the different mechanisms of regulation in the two cell types. This project is based on a report that mast cells express a truncated IL-4 mRNA, encompassing only exons 3 and 4. This truncated mRNA was only detected by RNAse protection assays as Northern blot analysis was not sufficiently sensitive to distinguish the truncated mRNA from the full length mRNA. Analysis of intronic DNA has revealed the presence of a "TATA-less" promoter elements, suggesting that transcription of this truncated mRNA may initiate in this intronic regulatory element. This project is focused on further characterizing this truncated transcript and assessing the possible biological consequences of its expression. The aims are: 1) to assess the expression profile of the truncated transcript; 2) to assess the function of the transcript and 3) to define the regulatory elements within the intron that regulate transcription of the truncated mRNA. As it is possible that this novel transcript may represent an important mechanism for regulating IL-4 gene expression and for understanding the mechanisms behind its expression and by which it may regulate IL-4 biological activity.

We have previously characterized a DNA regulatory element in the second intron of the murine IL-4 gene. This element exhibits mast cell- specific enhancer activity in transient transfection assays. Analysis of the components of this element revealed that the cell specific activity is due to critical sites within the enhancer that serve as binding sites for transcription factors expressed in mast cells but no T cells. Although this element works well with a heterologous promoter in reporter gene assays, it has only marginal activity with the 5"IL-4 promoter, suggesting its physiological role is not involved in enhancing 5"IL-4 promoter-mediated transcription. It has recently become apparent that a TATA-less promoter element (a consensus Inr sequence) is located just downstream of the previously defined enhancer elements. The Inr- containing promoter element is located just downstream of the previously defined enhancer elements. The Inr-containing promoter element is active and appears to drive transcription of a unique, truncated IL-4 transcript. This transcript contains sequences that initiate immediately 3' of the Inr sequence that are spliced in exons II and IV. Studies of the expression profile of this "truncated" mRNA indicate it is expressed at relatively high levels in unstimulated mast cells and splenic cultures. This expression of this transcript is inversely correlated with that of the full length transcript: cell stimulation results in the disappearance of this mRNA species and an increase in the full length IL-4 element. Based on these data, we hypothesize that the truncated IL-4 mRNA is involved in the regulation of IL-4 production and/or activity, and its transcription is controlled by the intronic enhancer. This proposal describes experiments aimed at defining the function of the transcript and exploring the details of its expression and regulation.

DESCRIPTION (adapted from investigator's abstract): IL-4 is a pleiotropic cytokine that plays a central role in immunoregulation. Although both T and mast cells express IL-4, each cell type utilizes distinct cis- and trans-acting factors to regulate transcription. A number of both promoter-proximal and intronic DNA elements are targets of lineage-specific factors. These factors include proteins belonging to the NF-AT, STAT, GATA, Ets, and c-maf families. Such differences likely reflect the distinct extracellular signals that elicit IL-4 in these cell types and the unique role each cell type has in the immune response. In addition to transcriptional activation of the IL-4 gene in differentiated cells, there is a clearly an additional level of regulation. Recent studies of T cells as well as those in mast cells by Dr. Brown's laboratory have established a role for developmentally regulated chromatin accessibility in IL-4-gene expression. DNAseI hypersensitivity and demethylation within the IL-4 chromosomal gene locus are observed at an early stage in the commitment to a Th2 or mast cell lineage. Importantly, these hallmarks of "open" chromatin evident in both cell types are independent of active IL-4 transcription. Dr. Brown proposes that these observations demonstrate that two temporally distinct events regulate the acquisition of an IL-4-producing phenotype: 1) developmentally-determined signals confer locus opening and 2) in differentiated cells, cell activation signals promote the association of transcription factors with accessible regulatory elements within the IL-4 gene. The studies described in this proposal will continue Dr. Brown's ongoing investigation on the cell-type specific regulatory elements that dictate IL-4 gene expression in T and mast cells. Her recent data suggests that some of these regulatory elements have striking parallels to the u and k Ig gene intron enhancers. Although initially defined as regulatory elements based on their ability to enhance transcription in conventional reporter assays, Eu and Ek also function to regulate lineage and cell-stage specific accessibility of the Ig locus. Dr. Brown hypothesizes that 1) the IL-4 gene is regulated in a similar manner; some sequences defined as enhancers have dual roles as accessibility determinants; 2) lineage specific factors regulate chromatin accessibility at the IL-4 gene locus in T and mast cells; and 3) differences in the general locus accessibility of the IL-4/IL-13 gene cluster contribute to strain-specific differences in IL-4 gene expression levels. The specific aims of this proposal are: 1. To determine the mechanisms that underlie the ability of the intron "enhancer" to mediate chromatin accessibility in mast cells and determine its potential role in T cells. 2. To characterize the cis- and trans-acting elements that comprise a defined enhancer located in the IL-4/IL-13 intergenic region and determine its in vivo role. 3. To identify other sequences that influence transcription of IL-4 and/or IL-13 in both T and mast cells. 4. To examine the molecular basis of strain-specific differences in murine IL-4 gene expression.

[unreadable] DESCRIPTION (provided by applicant): Mast cells are potent inflammatory cells that have been best studied in the realm of allergy research. Recent evidence has established a role for mast cells in murine models of autoimmune diseases such as multiple sclerosis and arthritis. Mast cells are normally present in the pancreas and increase in numbers in inflammatory conditions of the pancreas such as pancreatitis. In addition, they are important producers of mediators that are known to regulate T cell responses that initiate the inflammation that contributes to diabetes. It is hypothesized that mast cells influence inflammatory events that lead to pancreatic islet beta cell destruction. To test this, we will utilize a mast cell-deficient mouse model. Genes that give rise to a mast cell-deficient phenotype (two distinct ckit mutations) will be bred onto the NOD/Lt mouse background and the effects on indices of pre-diabetes and diabetes will be evaluated. The specific aims are: 1. To develop and characterize a line of mast cell-deficient NOD mice 2. To examine the consequencess of mast cell-deficiency on the development of spontaneous insulitis and diabetes. 3. To determine the sites of mast cell influence on diabetes development. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): It is well established that CD4 + T cells are of central importance in initiating the autoimmune destruction associated with multiple sclerosis (MS) and the rodent model of MS, experimental allergic encephalomyelitis (EAE). However, a variety of other inflammatory cells, including B cells and macrophages, contribute to the events that lead to the varying degrees of myelin and axonal damage observed in this disease syndrome. Mast cells, best known for their role in allergic responses in the skin and respiratory tract, exhibit widespread distribution in many tissues throughout the body. Relevant to this application, these cells are prevalent within sites of initial T cell activation such as the spleen and lymph node. In addition, their intimate association with blood vessels and nerves and the plethora of immunoregulatory mediators that can be produced by mast cells make them viable candidates for profoundly influencing immune function. Using the myelin oligodendrocyte glycoprotein (MOG)-induced model of EAE, we recently demonstrated that mast cell-deficient mice exhibit delayed onset and less severe disease than their wild type littermates. Reconstitution of the mast cell population with wild type bone marrow mast cells, a procedure that does not correct other hematological abnormalities in these animals, restores the susceptibility to severe disease. Surprisingly, this restoration of disease susceptibility occurs without detectable reconstitution of mast cells in the CNS, revealing a role for mast cells in the periphery. These findings do not rule out the possibility that mast cells directly influence inflammatory events in the CNS in mast cell competent animals. However, they do illuminate a model for examining how mast cells can influence the generation and character of the autoimmune T cell response, independent of any CNS effects. In this application, we propose experiments to determine the sites of mast cell influence on disease course and to explore the mechanism through which mast cells exert their influence. The specific aims are as follows: 1) To determine the modes of mast cell activation in disease; 2) To characterize specific mast cell-regulated lymphoctye immune responses in EAE; 3) To determine the mediators that confer mast cell influence on disease [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Although mast cells are best known for their role in the pro-inflammatory processes that mediate allergic responses, recent data from several laboratories including ours have implicated mast cells as critical players in a variety of other pathologic and protective immune responses. For example, mast cells are required for maximal disease in murine models of multiple sclerosis (Experimental allergic encephalomyelitis - EAE) and arthritis and for resistance to many bacterial infections. In EAE, mast cells exert their effects on both CD4+ and CD8+ autoreactive T cell responses. T cells derived from immunized mast cell-deficient mice (W/Wv) exhibit reduced antigen-specific IFN?, IL-17 production, attenuated alterations in activation markers including CD44, CD11a, CD69 and CD62L as well as an inability to efficiently traffic to the target tissues in the CNS when compared with cells isolated from their wild type littermates. In this setting, the sub-optimal CD8+ T cell response was most striking and may be due to inefficient mast cell-dependent CD4+ T cell help. It may reflect a more direct influence of mast cells on CD8+ T cells. Alternatively, mast cells may indirectly alter T cell responses through effects on dendritic cell function and migration. Experiments in this application will utilize mast cell-deficient mice to test the hypothesis that mast cells contribute to a microenvironment that shapes events governing primary and memory CD4+ and CD8+ T cell function using a well characterized infection model that elicits robust CD4+ and CD8+ T cell responses to lymphocytic choriomeningitis (LCMV) virus epitopes that are necessary for long-term protective immunity to this virus. Understanding all of the factors that lead to a strong memory response is essential for effective vaccine development. The specific aims are: 1) To define the roles of mast cells in LCMV-specific CD4+ and CD8+ T responses. 2) To examine the influence of mast cells on dendritic cell maturation, migration and T cell stimulatory function in LCMV-infected mice. [unreadable] [unreadable]

Mast cells have a well-established role in the inflammatory processes that regulate allergic responses. Recent data have implicated mast cells in a variety of other pathologic and protective immune responses. These include exacerbation of symptoms in murine models of multiple sclerosis (Experimental allergic encephalomyelitis - EAE) and arthritis and resistance to bacterial infections. In addition to mediators that are known to affect the recruitment and activation state of cells of the innate immune system, mast cells also express many molecules that have documented effects on the regulation of adaptive immune responses. These include CD40L, IL-2, IL-4, IL-7, IL-15, IL-23, LTB4 and histamine. We recently compared the T cell responses of mast cell-deficient mice (W/Wv) with their wild type littermates after immunization with a myelin peptide in CFA, a protocol used to induce EAE. Although there is no inherent deficit in W/Wv-derived T cells activated in a mast cell-sufficient environment, several indices of activation including stimulation-dependent changes in the expression of CD44, CD11a, CD69 and CD62L are attenuated in T cells primed in a mast cell-deficient setting as is the ability to express IFN gamma. Notably, both the CD4+ and CD8+ T cell responses were consistently and significantly affected. The sub-optimal CDS response may be due to inefficient mast cell-dependent T cell help. Alternatively, it may reflect the more direct influence of mast cells on CD8+ T cells. Using a well-defined Listeria monocytogenes infection model, experiments in this application will test the hypothesis that mast cells contribute to a microenvironment that shapes several events governing CD4+ and CD8+ T cell function. The specific aims are as follows: 1. To determine the effect of mast cell-deficiency on CD4+ and CD8+ T responses after infection with a recombinant L. monocytogenes expressing gp33-41, the class I LCMV epitope (rLM33). 2. To examine the influence of mast cells on dendritic cell maturation, migration and T cell stimulatory function in rl_M33 infected mice. 3. 3. To assess the changes in mast cell phenotype during rLM33 infection and determine their relationship to alterations in T cell responses

DESCRIPTION (provided by applicant): Until recently, mast cells and basophils were considered to be rather obscure cells of the immune system whose main role is to induce allergy. However, increasing evidence over the last few years has illuminated their critical role in the initiation and regulation of the immune system. It has also become evident that improper functioning of these cells is central to the pathogenesis of chronic inflammatory and autoimmune diseases such as multiple sclerosis and rheumatoid arthritis, and even cancer. Hence, understanding the ontogeny and development of these cells has become imperative. Considering the fact that mast cells and basophils are similar in several aspects including the relatively unique expression of the high affinity IgE receptor and share common modes of activation, degranulation and mediator release, it is not surprising that they have parallel developmental pathways as well. It has been reported that mast cells and basophils arise from a common hematopoietic precursor - the basophil-mast cell precursor (BMCP). However, the molecular mechanisms directing lineage choice between mast cell vs. basophil are unclear. The granulocyte-related transcription factor CCAAT/enhancer-binding protein 1 (C/EBP1) is reported to steer the common precursor towards the basophil lineage. We have recently demonstrated that the transcription factor Ikaros regulates mast cell IL-4 gene expression. Furthermore, preliminary data from our laboratory suggests that Ikaros is involved in mast cell development and without Ikaros, cells default to the basophil lineage. In this proposal we hypothesize that the transcription factor Ikaros drives the lineage choice of the BMCP towards mast cell lineage. As set forth in these aims, we will first determine whether Ikaros regulates lineage choice towards mast cells at the expense of basophils, in a cell intrinsic manner, using Ikaros-null mice. Secondly, we will determine whether Ikaros suppresses basophil lineage promoting transcription factors such as C/EBP1. Finally, we will assess the in vivo relevance of loss of Ikaros in mast vs. basophil development by analyzing basophil specific responses in Ikaros-null mice. Results obtained from these experiments will further our understanding of lineage choice decisions between mast cells and basophils and elucidate potential therapeutic targets for inflammatory and autoimmune disorders. PUBLIC HEALTH RELEVANCE: All cells of the hematopoietic lineage, including red blood cells, platelets and white blood cells, can arise from the same single precursor stem cell. This stem cell must go through unique development steps (lineage choices) that involve the activation or suppression of specific genes in order to develop correctly into the various cell types of our blood system. Our studies focus on the development of mast cells and basophils, highly related cells that not only are the major cause of allergic symptoms, but are also critical for some protective functions of our immune system: Here we propose to study the mechanisms by which a common precursor cell chooses to become a mast cell or basophil.

Acute; Antigens; Bacteria; base; Biological Assay; CD4 Positive T Lymphocytes; Cell Line; Cell Lineage; cell type; Cells; Chromatin; Chromatin Remodeling Factor; cytokine; Cytokine Gene; Deoxyribonuclease I; Development; DNA Methylation; Dominant-Negative Mutation; Ectopic Expression; Event; extracellular; fungus; Gene Expression; Gene Targeting; Genes; Genetic Transcription; Helper-Inducer T-Lymphocyte; Hematopoietic; Histone Acetylation; Histone H3; histone modification; Hypersensitivity; IL4 gene; Immune; Immunity; In Vitro; indexing; Interleukin-10; Interleukin-12; Interleukin-17; Interleukin-4; Interleukin-6; Laboratories; Lymphoid; Molecular; Molecular Conformation; Nucleic Acid Regulatory Sequences; Organ; pathogen; Pathologic; Pathology; Pathway interactions; Peripheral; Phenotype; Play; Production; Protein Isoforms; Proteins; Regulatory T-Lymphocyte; response; Role; Sequence-Specific DNA Binding Protein; Signal Pathway; T-Lymphocyte; Th1 Cells; Th2 Cells; Time; transcription factor; Virus

DESCRIPTION (provided by applicant): Multiple sclerosis (MS), the most common inflammatory disease of the central nervous system (CNS), affects more than 2.5 million people worldwide. MS is characterized by perivascular inflammation in the CNS, demyelination of nerve fibers as well as axonal damage. The resulting interruption of motor and sensory impulses as they pass through demyelinated regions of the brain or spinal cord often leads to visual disturbances, incontinence as well as sensory and motor disturbances. The prevalence of this disease is estimated to be at least three times greater in females than males and while genetic, hormonal and immune response differences have been implicated, the basis for this gender bias is still not fully understood. MS is considered to be autoimmune in nature and myelin-specific CD4+ Th1 and Th17 cells are major orchestrators of the CNS inflammation. It is assumed that these cells are initially activated in peripheral lymphoid organs but must cross the relatively impermeable blood-brain barrier (BBB) to become reactivated in the CNS. Other innate immune cells that infiltrate or reside in the CNS contribute to inflammation-mediated neurological damage. Many of these events have been initially defined by studying the similar, albeit imperfect rodent model of MS, experimental allergic/autoimmune encephalomyelitis (EAE). Mast cells are granulocytes that reside in most tissues and are among the innate cells that exert an important amplifying effect on disease severity in females. Mice bearing mutations in Kit, which encodes the SCF receptor, ckit, fail to develop mast cells and have been used to study the role of mast cells in a relapsing-remitting EAE model. We previously demonstrated that female SJLW/Wv mice exhibit attenuated EAE, a phenotype dependent on mast cells. However, male SJLW/Wv mice have exacerbated disease, indicating either mast cells or other c-kit related defects are pathologic in males. This is not due to disparate serum testosterone levels between wild type and ckitW/Wv males, which show no significant differences. There are at least two explanations for these observations: i) Male mast cells, under the influence of sex hormones such as testosterone, show distinct responses in the context of disease compared to female mast cells; and/or ii) SCF, the ligand for c-kit, exerts a neuroprotective effect in concert with testosterone that acts to minimize immune-mediated damage in the CNS. The specific aims of this study are: 1) Compare the events (e.g. T cell priming in the periphery, inflammatory cell entry to the CNS, local CNS inflammatory responses) that lead to development of EAE in wild type versus ckitW/Wv male mice to determine where c-kit signals exert their protective influence 2) Identify whether and how mast cells or other c-kit related factors alter disease susceptibility in males.

Proteasome inhibitors are novel biologically targeted anti-cancer drugs. The first-in-class proteasome inhibitor Bortezomib (BZ, also called Velcade) was approved for use in multiple myeloma (MM) patients in US in 2003, and in Europe in 2004 (1). During last five years, the use of BZ in cancer treatment has been significantly extended. It is currently used for both the initial therapy and salvage therapy of recurrent/relapsed MM as well as mantle cell lymphoma (MCL), two commonly diagnosed hematological malignancies that have a combined prevalence of more than 85,000 cases in the United States alone (1). BZ has been also extensively tested in clinical trials for the treatment of large variety of solid tumors that showed promising results. BZ is a reversible inhibitor of the 26S proteasome. After a single treatment, there is inhibition of proteasome activity followed by the recovery. Thus, BZ must be delivered to cancer patients by i.v. injection every 72 hrs., a procedure that allows sustained targeting of the 26S proteasome activity. (2-4). However, there are dose-limiting toxicities in patients that include neurotoxicity, fatigue, diarrhea, and cutaneous toxicity. Relevant to this proposal are the severe adverse cutaneous effects including rash, cutaneous vasculitis, and dermatitis that affect 10-15 % of BZ treated patients (5-8). Yet, BZ treatment effects on the skin are poorly characterized and mechanism(s) of these adverse effects are unknown. Intravenous BZ delivery has also been used successfully for experimental anti-cancer therapy in mice (3, 4). We asked if we could recapitulate the cutaneous effects of BZ in a mouse model. In our pilot experiment, we treated (C57BL/6 x DBA) Fl female mice intravenously with a therapeutic dose of BZ (MTD, 1 mg/kg; Ref. 3). We observed extensive recruitment of mast cells to the skin accompanied by modest inflammatory infiltration and pronounced epidermal hyperplasia 24 hrs. after treatment (Figure 1). Most of these mast cells showed typical indices of cell activation as measured by the appearance of multiple granules that are released from the cells (Figure 1). Such granules are characteristic of mast cells. They are present in the cytoplasm of an intact, unactivated cell and contain a number of pre-formed pro-inflammatory mediators, including histamine, proteases and cytokines. Release of these cytokines can strongly impact the inflammatory response. These experiments were repeated using mice from different strains (C57BL/6, FVB, SENCAR) as well as using alternative routes of treatment including topical application of high doses of BZ (5-10 ug/animal in acetone). We obtained similar results in these repeated experiments (data not shown). Thus, this BZ-induced mast cell recruitment and epidermal hyperplasia is not strain-specific and is observed under multiple delivery modes. These are novel observations and further study of this phenomenon could ultimately lead to information that will impact our ability to prevent/treat BZ induced cutaneous toxicity

Abstract Females are more to susceptible many autoimmune diseases. In multiple sclerosis (MS), not only is there a 3-4 fold increase in disease incidence, but there are sex-determined differences in the average age of onset and clinical course. Yet the cellular and molecular underpinnings of this sex-dimorphism have remained undefined. The SJL mouse model of MS recapitulates these differences in that female mice exhibit higher incidence, more severe disease, and a more consistent relapsing-remitting pattern than their male counterparts. This difference in disease susceptibility corresponds to qualitatively distinct anti-myelin Th cell cytokine responses. Whereas females generate pro-inflammatory Th1/Th17-dominant responses, the response in males is Th2-skewed and non-pathogenic. In this application we provide evidence that type 2 innate lymphoid cells (ILC2s) exert a male-specific protective influence. Best studied in allergic airway models, ILC2s are c-kit+ and are essential for inducing Th2 immunity through production of IL-13. We propose that mast cell activation in immunized in male mice elicits production of ILC2 activating factors such as IL-33 that promote ILC2 functionality. The inability to generate a robust IL-33 response in females leads to a functional deficit in ILC2 activity. Mast cells (c-kit+ Fc?R1+) are one important source of IL-33 in vivo and testosterone directly induces Il33 exclusively in male-derived cells, despite equivalent androgen receptor expression by female-derived mast cells. These data suggest a cellular and molecular target of testosterone and identify a potential mechanism of action for testosterone-mediated protection in CNS autoimmune disease. Specific Aim 1: Determine the IL-33 expression kinetics, cellular source (s) and its requirement for protection in immunized male mice. Using IL-33- reporter mice (Il33Cit/+) or IL-33-deficient mice (Il33Cit/Cit (Il33-deficient) these experiments will define the IL-33-expressing cells, when and where it is produced. Specific Aim 2: Determine how testosterone influences the expression of IL-33 and other factors that regulate sex-dimorphic EAE protection. IL-33 gene expression will be evaluated in mice treated with testosterone or androgen receptor (AR) antagonists. We will ask if testosterone induces epigenetic changes at the Il33 locus conferring changes in chromatin accessibility. Other AR target genes will also be examined. Specific Aim 3: Explore the contributions of sex determining genes on ILC2 and mast cell function in EAE. The potential effect of sex chromosomes independent of hormonal influences on ILC2 and mast cell gene expression and function will be examined using four core genotype mice.