Thomas G. Forsthuber - US grants

DESCRIPTION (Adapted from the Investigator's abstract): One of the major objectives of vaccine development is the establishment of immunization protocols that permit the selective induction of either Th1 or Th2 type immunity, as differentially required for the immune system to successfully overcome different infectious agents. For example, Th1 immunity is though to be required for controlling Leishmania major infection, while a Th2 response to this agent has pathological effects. The reverse might also be true, e.g. in the case of Helicobacter pylori infection. Recently the investigator has shown that immunization with a prototypic antigen, hen egg white lysozyme (HEL) injected in complete Freunds adjuvant (CFA), induces an immune response that is essentially pure Th1, while injection of HEL in incomplete Freunds adjuvant (IFA) results in an apparently pure Th2 response to HEL. Can adjuvants be used selectively to engage the desired class of response as needed for vaccinations against different infectious agents? Moreover, do the rules for adjuvant guided immune responses established in young adult mice also apply for aged mice? Specific Aim 1 proposes to characterize more closely the cytokine profile and immunoglobulin isotype of the anti-HEL response induced in Th2 biased BALB/c and Th1 biased C57BL/6 mice following immunizations with CFA, IFA or aluminum hydroxide, the primary adjuvant used in humans. Specific Aim 2 proposes to test whether Th1 or Th2 type immunity can be selectively induced against Leishmania antigens by use of different adjuvants (preliminary data suggest that indeed this may be the case). Finally, Specific Aim 3 will test whether the anti-Leishmania Th1 response induced by CFA immunization protects otherwise susceptible, Th2 biased mice from Leishmania major infection. In the reverse experiments, it will be tested whether induction of Th2 immunity to Leishmania antigens renders naturally resistant C57 BL/6 mice susceptible to this infection. These studies will compared the immune responses of young adult and aged mice.

Rheumatoid arthritis (RA) is thought to be an autoimmune disease in which T cells erroneously attack hyaline cartilage matrix proteins like, for example type II collagen. Amongst the autoantigens implicated in RA, a novel candidate target antigen, human cartilage gp-39 (HC-gp-39) has been described. Defining the parameters of the autoimmune response may facilitate diagnosis and formulation of novel therapeutic strategies. For example, if the determinant(s) of HC gp-39 or other cartilage antigens recognized in the context of human MHC alleles were known, specific strategies for immune intervention could be designed which would interfere specifically with the autoimmune response. For the most part, determinant mapping studies of patients with T cell-mediated autoimmune diseases like RA and Multiple Sclerosis (MS) have failed to provide clear results and studies on inbred mouse strains have so far usually focused on determinant utilization in the context of murine MHC haplotypes and so may not pertain to humans. I propose to overcome this limitation for the experiments described in this application by using mice that have been engineered to express human MHC class II alleles including the RA susceptibility alleles HLA-DR4 (HLA-DRB1 0401) and HLA-DRW 14 (HLA-DRB1 0404), which will thus create a murine immune system with humanized antigen presentation properties. Our experiments will test the hypothesis that T cell recognition of human collagen type II and HC gp-39 epitopes are of importance in induction and/or propagation of polyarthritis in HLA-DR4 and HLA-DR14 transgenic mice, and whether intra-and intermolecular spreading of T cell responses are of significance in the propagation of polyarthritis. Based on this information, we will then be in a position to monitor responses in patients and design strategies for specific immune intervention. This essential information cannot be obtained by testing conventional mouse strains, nor through human studies, but it can readily be obtained through the use of humanized mice as proposed in this study.

[unreadable] DESCRIPTION (provided by applicant): Multiple sclerosis (MS) is a chronic inflammatory and demyelinating disease of the central nervous system (CNS), which is thought to be mediated by an erroneous attack of T cells on myelin autoantigens present in the central nervous system (CNS). Characterization of the T cell epitopes targeted in MS patients has remained technically challenging, and the functional characteristics of the neuroantigen-specific T cells, particularly in the CNS, have remained unresolved. We have obtained preliminary results showing vigorous T cell responses to MOG peptides in PBL of MS patients by cytokine ELISPOT assay. Based on these results, we want to examine the MOG- specific T cell response in individual patients longitudinally over the course of MS, and test the epitope specificity and functional avidity of these cells. To overcome the difficulties that arise when examining M0G responses directly ex vivo from the brain of MS patients, we propose to study T cell responses in the CNS of "humanized" HLA-DR2 and -DR4 transgenic mice. Our preliminary studies have indicated that T cell responses in HLA-DR transgenic mice are directed against similar MOG epitopes as T cell responses in MS patients with this HLA-DR haplotype. We will test the hypothesis that MOG-specific T cells undergo avidity maturation over the course of MS, and high-avidity T cells accumulate in the peripheral blood of patients prior to relapses or exacerbation of disease. Furthermore, we will test the function of MOG-specific T cells by studying the fine specificity and functional avidity of these cells in the CNS. We will test this hypothesis in the fol1owing aims: In Aim 1 we will examine the MOG epitope-specific T cell response over time in MS patients by cytokine ELISPOT. In Aim 2 we will test the avidity maturation of MOG-specific T cells over the course of disease in MS patients. In Aim 3 we will test the MOG-specific T cell repertoire in the CNS of HLA-DR2 and -DR4 transgenic mice over the course of EAE. In Aim 4 we will test the avidity maturation of MOG-specific T cells in the CNS and blood of the transgenic mice over the course of EAE. At the end of this project, we will have learned whether MOG-specific T cells undergo avidity maturation in MS patients and how this relates to relapses/remissions. The experiments in the HLA-DR transgenic mice will complement the human studies and define the specificity and function of MOG-reactive T cells in the CNS.

[unreadable] DESCRIPTION (provided by applicant): Glucocorticoids (GC) remain the mainstay of therapy for acute MS episodes. However, it is also clear that GC fail to prevent MS relapses and disease progression, and appear to have little effect on long-term T cell responses to myelin antigens in patients. Similarly, GC treatment efficiently down-regulates EAE in rodents, but fails to prevent subsequent relapses. Importantly, GC fail to inhibit proliferation and cytokine production by T cells at inflammatory sites, which could be particularly relevant in the CMS where expression of MIF in a number of cell types including neurons could contribute to this observation. Our own preliminary data and a recent report by Powell and colleagues strongly support a link between MIF and glucocorticoids in EAE. We found that Wt mice show exacerbation of EAE and relapsing disease upon termination of Dexamethasone treatment, but not MIF-/- mice. Furthermore, EAE is substantially exacerbated in MIF-/- mice treated with the glucocorticoid receptor inhibitor Mifepristone (RU-486). Furthermore, the frequencies and functional avidity of myelin-specific T cells were decreased when MIF was neutralized in vivo with anti-MIF antibodies. Based on these results and the literature it is highly likely that MIF interferes with GC-mediated immunosuppression, induction of apoptosis, and/or migration of inflammatory cells to the CMS. This proposal will test the hypothesis that: MIF promotes EAE by counter-regulating the immunosuppressive effects of glucocorticoids on T cell and macrophage apoptosis, effector functions, and CMS migration. This hypothesis will be tested with the following specific aims: Aim 1. To determine whether MIF interferes with glucocorticoid-mediated apoptosis of encephalitogenic T cells and APCs in EAE. Aim 2. To determine whether MIF counter-regulates glucocorticoid-mediated immunosuppression of T cell effector functions in EAE. Aim 3. To determine the mechanism of MIF interference with apoptosis. [unreadable] [unreadable] [unreadable]

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Currently there is no cure for multiple sclerosis (MS). Immunosuppressive and immunomodulatory treatments including glucocorticoids (GC) are used to treat acute attacks of MS and slow relapses. However, no adequate biomarkers exist to show treatment efficacy and/or development of treatment resistance. Therefore, the goal of this project is to identify biomarkers to monitor whether immunomodulatory treatments in MS are effective and/or whether patients develop treatment resistance. We will study glucocorticoid (GC) treatment of mice with experimental autoimmune encephalomyelitis (EAE) as a generally accepted model of MS. Combining immunological studies with proteomics methods we expect to discover proteins (biomarkers) that indicate when the GC treatment is effective and furthermore provide us information on the mechanisms of GC resistance.

DESCRIPTION (provided by applicant): Myocarditis is an important cause of sudden death in up to 20% of young adults and frequently progresses to dilated cardiomyopathy, which accounts for up to 1 in 25 cases of heart failure; yet, there is no specific and effective treatment for thi devastating condition. Strong evidence supports that progression of acute (e.g. viral) myocarditis to chronic myocarditis and ensuing dilated cardiomyopathy are most frequently mediated by an autoimmune assault against heart tissue; however, for unknown reasons immunosuppressive treatments are not very effective and current treatment focuses on managing clinical symptoms and providing supportive care. In our preliminary studies we found that immunosuppressive glucocorticoid (GC) hormone treatment of macrophage migration inhibitory factor (MIF) knockout mice completely prevented the progression of experimental autoimmune myocarditis (EAM), an animal model of myocarditis, to dilated cardiomyopathy (DCM). Therefore, we will test in the present application inhibitors of MIF in combination with GC treatment as novel therapeutic approach for this disease. The rational for these studies is that MIF has been shown to promote EAM. Furthermore, MIF stimulates IL-17 production by pathogenic T cells, which is a critical cytokine for induction of EAM. Importantly, MIF is unique i that it is the only known proinflammatory cytokine induced by GCs and to subsequently counter-regulate GC- mediated immunosuppression. Since treatment of MIF knockout (MIF-/-) mice with Dexamethasone (Dex) completely prevented progression of EAM to DCM (our preliminary results), we hypothesize that MIF plays a critical role in resistance to immunosuppressive treatment and that inhibiting this cytokine in combination with GCs in myocarditis patients could represent a novel treatment approach and major therapeutic advance. We will test our central hypothesis with the following specific aims: Aim 1. To determine the efficacy of MIF inhibitors in preventing resistance to GC treatment in EAM and progression to dilated cardiomyopathy. We will test the hypothesis that inhibition of MIF with anti-MIF mAb or small molecule MIF inhibitors will prevent DCM in Dex treated BALB/c wild-type (wt) mice analogous to MIF-/- mice. Aim 2. To determine the key mechanism(s) how MIF promotes resistance to Dex in EAM and progression to DCM. We will test the hypothesis that MIF inhibits GC treatment effects in EAM and progression to DCM by modulating chemokines/chemokine receptors and homing molecules guiding recruitment of inflammatory cells and Prominin-1/CD133+ myeloid precursor cells to the heart and their differentiation into fibroblasts and DCM. We expect that the proposed studies will provide important information that is currently not available and which will have significant and lasting impact on research and treatment of myocarditis and DCM.

DESCRIPTION (provided by applicant): Approximately 1/1000 people develop multiple sclerosis (MS), but even among people that express the MS- associated MHC II allele HLA-DR2 we cannot predict who will develop disease and only 1/200 HLA-DR2+ people ever will. Surprisingly, even in studies of the murine experimental autoimmune encephalomyelitis (EAE) model of MS, where active or passive disease is induced under well-defined conditions, not all of the genetically-identical mice develop disease with the same trajectory or severity and we cannot accurately predict who will develop disease. In our own preliminary studies we have identified several protein biomarker candidates that correlate with onset, peak, and remission of EAE using a novel quantitative proteomics method that we recently reported (Raphael, I. Electrophoresis, 2012). Furthermore, we have developed a method to induce EAE of predetermined severity using adoptive transfer EAE in combination with pre-transfer cytokine ELISPOT analysis. Importantly, we have preliminary results that predicted candidate protein biomarkers can be detected in serum of mice with EAE by ELISA. By combining these approaches for inducing EAE and correlating protein expression to disease progression we will provide proof-of-principle in a unique system to identifying predictive markers of disease incidence and severity. The objective of this proposal is to provide proof-of-principle of predictive biomarkers using the EAE model. Our central hypothesis is that the clinical onset of EAE symptoms is preceded by release of CNS disease- specific proteins into blood (serum) that can be used to predict which animal will develop disease and how severe. This hypothesis is based on our own preliminary results and supported by published studies. The rationale for the proposed research is that proof-of-principle in the EAE model will provide the basis for developing homologous predictive biomarkers for MS patients, which will revolutionize treatment and drug development for MS. We will test our central hypothesis with the following specific aims: Aim 1. To determine key CNS disease-related protein isoforms with altered expression in CNS tissue prior to the onset of EAE and as a function of disease severity. Aim 2. To determine the CNS disease-related proteins isoforms released into blood (serum) most predictive of EAE incidence and severity. We expect that the proposed studies will provide important information that is currently not available and that will have significant and lasting impact on research and treatment of MS.

A pathogenic role for autoantibodies in neuroinflammatory diseases has been established in neuromyelitis optica (NMO), where autoantibodies directed against aquaporin-4 (AQP4) are present in the majority of patients and are believed to be pathogenic. The vast majority of autoantibodies is produced by long-lived plasma cells (LLPCs) in bone marrow, but current MS treatments, such as anti-CD20 antibody treatment or interferons do not specifically target these cells and therefore lack therapeutic efficacy and show significant side effects. Novel approaches to eliminating autoantibody-producing LLPCs are therefore urgently needed. Plasma cells (PCs) are terminally differentiated from germinal center (GC) B cells and they are the main source for antibodies found in serum and other body fluids. GC B cells can give rise to short-lived plasmablasts, which enter the blood stream and recirculate for a short period until they find a survival niche in the bone marrow (BM), where they develop into LLPCs. The molecular players that guide the differentiation of GC B cells towards the plasmablast fate and further direct their differentiation into PCs and promote their survival and antibody-production are only partially understood. PCs are also found in the CNS in MS lesions, and data from the experimental autoimmune encephalomyelitis (EAE) model suggest that they contribute to disease severity. The extracellular signal-regulated kinases 1 (ERK1) and 2 (ERK2) are the best-studied members of the mitogen-activated protein kinase (MAPK) family and are located downstream of many critical signaling pathways in plasmablasts and PCs. Experimental evidence suggests that they may play a key role in the formation, survival, and/or antibody-producing function of PCs. However, the data concerning the requirements of ERK1 versus ERK2 in PCs are controversial, and the role of these MAPKs in generating and/or sustaining autoantibody-producing LLPCs, and their antibody-producing function in BM or CNS in neuroinflammatory disease has not been investigated. We have developed a novel Mx1creYFP+/-Erk2fl/fl mouse model in which the deletion of Erk2 is accompanied by the expression of the fluorescent reporter protein, eYFP. This model allows us to follow the fate of antigen-specific Erk2- deficient (Erk2? eYFP+) B cells and B cell subsets, their identification and isolation using flow cytometry cell sorting, and functional studies to investigate key molecules for cell proliferation, function and survival. Our preliminary studies show that Erk2? B cells fail to develop into LLPCs and are virtually absent from BM. Our data strongly suggest that ERK2 signaling is critical for the survival of PCs and that targeting this pathway may be a viable strategy to eliminate autoantibody- producing LLPCs from BM and CNS. We propose that exploring the underlying mechanisms could lead to novel therapies. We will test the central hypothesis that ERK2-signaling is critical for generation and survival of autoantibody- producing LLPCs in bone marrow and CNS niches. We will determine the requirement for ERK2 for generating autoantibody-producing long-lived plasma cells in bone marrow and CNS in neuroinflammation. We will determine the role of ERK2 for sustaining already established autoantibody-producing LLPCs in BM and CNS in neuroinflammation.

The mechanisms driving progressive multiple sclerosis (MS) remain enigmatic. Progression of MS correlates with the expression of the cytokine tumor necrosis factor (TNF) in cerebrospinal fluid of patients, suggesting that TNF plays a key role in the disease process. Thus, targeting TNF was suggested as an attractive therapy for MS. Surprisingly, however, TNF-blocking drugs with proven efficacy in other autoimmune diseases triggered onset and exacerbations of MS, which provided evidence that TNF can also contribute to CNS protection and repair. Subsequent work showed that pathogenic TNF effects in EAE are mediated via TNF receptor (TNFR) 1, whereas TNFR2 signaling ameliorates disease and reduces demyelination. TNFR2 is expressed by different cell types in the CNS, including microglia and oligodendrocytes, and its expression by these cells can promote repair and remyelination. Likewise, astrocytes can express TNFR2, but the role of TNFR2 expressed by astrocytes for MS/EAE progression remains unresolved. Astrocytes with pathogenic (A1) or neuroprotective/anti-inflammatory properties (A2) have been described; however, the mechanisms orchestrating detrimental versus beneficial astrocyte functions are still not fully understood. We have obtained exciting preliminary results supporting a central role for astrocyte TNFR2 in curtailing EAE progression by investigating the role of TNFR2 in a ?humanized? transgenic mouse model expressing the human MS-associated MHC II allele HLA-DR2b and lacking the expression of TNFR2 molecules, herein called DR2b?R2 mice. This model provided evidence that the HLA- DR2b molecule favored Th17 development, while impairing Foxp3+ Treg cell formation. Importantly, we observed that DR2b?R2 mice developed progressive EAE and astrogliosis when CNS resident cells were TNFR2 deficient. Moreover, in DR2b?R2 animals with EAE, astrocytes showed increased expression of pro-inflammatory cytokines and increased expression of CXCR5. Additionally, CXCL13 was upregulated in the CNS of these mice in line with previous reports that CNS expression of CXCL13 aggravates EAE and promotes chronic white matter lesions that is not dependent on recruitment of CXCR5+ leukocytes from the periphery, and that CXCL13 expressed by damaged neurons can activate astrocytes. Moreover, we observed a striking dichotomy in the expression of TNFR2 between astrocyte populations in DR2b mice with EAE, suggesting that TNFR2 expression may favor astrocyte A2 versus A1 subpopulations. Thus, our proposal will test the central hypothesis that TNFR2 signaling in astrocytes curtails progression of neuroinflammation by restraining pathogenic and promoting protective astrocyte functions. We will test our hypothesis by (1) determining the role of TNFR2 for curtailing pathogenic astrocyte effector functions and preventing EAE progression; and (2) determining the role of TNFR2-regulated CXCL13/CXCR5 signaling for chronic astrocyte activation and function in progressive EAE in DR2b?R2 mice. We will accomplish the objectives of this proposal by applying innovative new approaches including our progressive EAE model, developing astrocyte-specific TNFR2 and CXCR5 knockout mice, and innovative immunological and molecular biological methods, including single-cell RNA-seq and RNAScope.