Center of Biomedical Research Excellence
Role of the SHP-2 phosphatase during NK cell response to MCMV infection
The mouse model of cytomegalovirus infection has been extensively studied. Accumulating data have demonstrated the critical roles of Natural Killer (NK) cells in the control of the infection. However, despite the presence of NK cells in the salivary glands, MCMV persists in this organ for several weeks to months eventually becoming latent for the life of the host. Lymphocyte inhibitory receptors such as Ly49 molecules associate predominantly with the tyrosine phosphatase SHP-1 and SHP-2 to exert downstream functions. We hypothesized that the hyporesponsive phenotype of salivary gland NK cells is due in part to SHP-2 mediated inhibition. This enzyme is well expressed by NK cells. In Specific Aim 1, we will determine if mice conditionally deficient for SHP-2 in the NK cell lineage control MCMV infection. In Specific Aim 2, we will examine the effector functions of SHP-2 deficient NK cells. The work proposed in this application may lead to the development of new approaches to reverse the hyporesponsive phenotype of salivary glands NK cells and perhaps latency. We believe our studies on this relatively unexplored research area will expand our current knowledge of NK cells and provide insights into strategies to restore adequate immune functions during latency.
Role of vitamin A metabolism in immunity against intestinal bacteria
Dietary derived substances and commensal bacteria are critical regulators of intestinal immunity. During homeostasis and response to infection in the GI tract, environmental signals derived from commensal bacteria as well as dietary sources are integrated to regulate mucosal immunity. A key factor in orchestrating mucosal immune responses in the intestine is retinoic acid (RA), a metabolite of vitamin A. Currently not much is known about how RA production and consequently retinoic acid receptor (RAR) signaling is regulated in the intestine to generate paradoxically opposite immune responses that range from immunoprotective to immunosuppressive in nature. The overall aim of this proposal is to define the molecular mechanism by which gut associated dendritic cells produce RA and how this regulates RAR signaling dependent immune function in the mucosal tissues. In order to understand how intestinal RA levels are regulated at cellular and molecular level we have identified a key component of the vitamin A metabolic machinery in the gut associated DCs, called retinol dehydrogenase 7 (Rdh7). We have shown by employing lineage deletion strategy that mice lacking DC specific Rdh7 show reduced lymphocyte homing to the gut thereby uncovering a crucial role for Rdh7 in generating mucosal immune responses. We will use the DC Rdh7 loss of function mouse models along with RA receptor reporter mouse models to map regions of RA synthesis and RA signaling within the gut mucosa. The proposed work will provide novel molecular insight into the mechanisms of RA generation in the intestine. In particular, the studies delineating the role of Rdh7 will be the first to explore a role of vitamin A metabolizing enzyme in generating intestinal immunity. By unraveling the molecular mechanisms of RA generation and signaling in the intestine, we anticipate identifying novel target for regulating mucosal immunity through pharmacologic and/or dietary manipulation, with the goal of protecting against infectious diseases of the intestine.
Immunoglobulin A as a Novel Biomarker of Intestinal Dysbiosis in Patients with Inflammatory Bowel Disease
Inflammatory bowel disease (IBD) is a chronic, debilitating condition characterized by relapsing and remitting episodes of gastrointestinal inflammation that affects over 1.4 million Americans. Pathogenesis has been attributed to a combination of genetic predisposition, alterations in the gut microbiome, defects in the mucosal immune system and various environmental exposures. Disease phenotype is highly variable and there are currently no reliable biomarkers to predict which patients will go on to have a more complicated clinical course. While alterations in the microbiome have been implicated in disease susceptibility, the clinical utility of these observations is yet to be demonstrated. Recent work suggests that pro-inflammatory bacteria may be identified based on the degree to which they are coated with immunoglobulin A (IgA). This research proposal evaluates translational, high-throughput microbial datasets from the Ocean State Crohn’s and Colitis Area Registry (OSCCAR) in an effort to identify and validate novel biomarkers of disease-specific outcomes. An inception cohort of patients with newly diagnosed IBD in the state of Rhode Island, OSCCAR is an ideal substrate from which to conduct this work. The objectives of this pilot project are to: 1) Evaluate differential patterns of IgA-coated bacteria in adult and pediatric patients with IBD and; 2) Explore the stability of these findings over time relative to disease phenotype, clinical course and immune-modulating treatment exposures. Future work will focus on how these findings can be translated into novel, microbialdirected therapeutic approaches for this devastating chronic disease.
Past Pilot Projects
Do human-like T cell epitopes contained in Env protein help HIV achieve Immune Camouflage?
Human immunodeficiency virus type 1 (HIV-1) has adopted several strategies for evading host immunity over the course of its evolution. As a consequence, developing a vaccine against HIV-1 has been a difficult task. In previous work, we have identified a novel mechanism by which viruses that cause chronic infection like HIV might escape the human immune response by mutating their epitopes to present “human-like” amino acid sequences to the T cell receptor (TCR) when displayed on antigen-presenting cells. As T cells that bear TCR that recognize autologous epitopes with high affinity are either deleted in the thymus or converted to regulatory T cells (Tregs), viruses that incorporate human-like epitopes may exploit host tolerance to avoid or suppress effector responses.
To search more rapidly for such epitopes, we developed an immunoinformatics tool, JanusMatrix, which identifies pathogen-derived epitopes whose TCR-facing amino acids are identical to those of numerous peptides from the human proteome, and published our initial observation that viruses causing chronic infections in humans contain higher numbers of these JanusMatrix-discoverable highly cross-reactive T cell epitopes. We have also identified T cell epitopes in H7N9 influenza HA protein and hepatitis C p7 protein that are highly conserved with human genome epitopes. T cells that are exposed to these epitopes in vitro are phenotypically and functionally regulatory (Treg). We hypothesized that the presence of such Treg-activating epitopes would reduce the efficacy of vaccines.
Naturally we turned our attention to HIV-1 and found several such epitopes in the envelope (Env) protein of HIV-1, one of which was included in both the HIV-1 E and HIV-1 B Env antigens that were used in the ‘moderately effective’ HIV RV144 trial in Thailand. Therefore, we propose to characterize the phenotype and function of T cells that expand or react when stimulated by human-like epitopes contained in HIV-1 (Aim 1), and to determine whether the phenotype of dendritic cells exposed to the epitopes in vitro is modified towards a tolerogenic phenotype (Aim 2). Validation of our hypothesis may provide one explanation for the limited efficacy of Env-based HIV-1 vaccines, and point a way forward to improving the efficacy of vaccines for HIV-1 and other important human pathogens.
Immunoregulatory effects of H. pylori’s cytotoxin-associated protein A (CagA)
Abstract: Helicobacter pylori (H. pylori) is a stomach-adapted global pathogen that infects over half the world’s population. H. pylori’s cytotoxic-associated gene A (cagA) encoded CagA is its most important virulence factor, possessing both immunodominant and oncogenic properties. H. pylori strains carrying cagA gene are associated with severe clinical outcomes including gastric adenocarcinoma. Moreover, previous studies suggest that Human Leukocyte Antigen (HLA) genotypes are linked to susceptibility to H. pylori chronic infection and HLA DRB1 and DQB1 gene loci are associated with gastric cancer development. Therefore, functional discrepancies in immune responses against H. pylori’s CagA may due to genetic differences derived from HLA haplotypes. Interestingly, recent epidemiological and animal studies indicate a potentially beneficial immunoregulatory role of H. pylori’s CagA, which suggests it may have both immunostimulatory and immunoregulatory functions. Thus, a deeper understanding of CagA’s dual immune function is critical for gastric cancer prevention through immune-based H. pylori interventions. Based on these new findings and our ongoing preliminary work, we hypothesize that there will be a variability in immune responses to CagA according to an individual’s HLA class II haplotype and certain HLA class II alleles may predispose the host to a distinct immunoregulatory response. To examine these, the proposed work has two specific aims:
Aim 1: Identify and characterize immunogenic H. pylori’s CagA peptides that elicit distinct T regulatory responses.
To accomplish this goal, human peripheral mononuclear cells (PBMCs) from healthy blood donors will be co-cultured with 32 individual immunogenic peptides derived from H. pylori’s cagA sequences. IL-10 EliSpot, flow cytometric analysis as well as multi-analyte ELISArrays will be conducted.
Aim 2: Evaluate the association of significant immunoregulatory responses to H. pylori’s CagA peptides with human HLA class II haplotypes.
The results from Aim 1 studies will be correlated with an individual blood donor’s haplotype. We will perform HLA Class II genetic typing at high resolution by using PCR sequence specific oligonucleotide probes (SSOP). Data will be analyzed under the help of Statistics and Data Management core in the Center of Biomedical Research Excellence (COBRE).
Successfully completing this project will lead to a better understanding of variant inter-personal immunoregulatory functions of H. pylori’s CagA. It may help to explain the differences in H. pylori persistent colonization and severity of clinical outcomes. Furthermore, identifying HLA alleles that increase or decrease an individual’s specific immunoregulatory response to H. pylori's CagA may also provide a novel preventive strategy to immune-based precision medicine in the future.
Galectin-3 in neonatal host defense against disseminated candidiasis
Joseph Bliss is a neonatologist with a special interest in fungal infections in premature infants. He obtained his BA in Biology from Ithaca College in 1990, and his M.D. and Ph.D. degrees from the University of Rochester in 1998. After completing his residency and fellowship training, he joined the Brown faculty in 2003. In addition to his clinical work, he studies host-defense mechanisms against infection with the fungus, C. albicans with special emphasis on the premature infant.
Education & Training University of Rochester University of Rochester School of Medicine and Dentistry
Certifications & Licensure RI State Medical License 2003 - 2016 MA State Medical License MA State Medical License 2003 - 2016 American Board of Pediatrics Neonatal-Perinatal Medicine
Overcoming HIV-1 latency by regulating stem-loop binding protein (SLBP)
Although current antiretroviral therapy can effectively manage HIV-1, there is always a small reservoir of latent HIV-1 virus that cannot be targeted by antiretroviral medications. HIV-1 multiplies once treatment stops. In order to achieve an HIV-1 cure (completely clearing the virus from the body), ways to stimulate and eliminate inactive HIV-1 must be developed. Based on our recent laboratory research, a human protein, called stem-loop binding protein (SLBP), can dramatically stimulate HIV-1 RNA expression. Although there are a few classes of drugs, such as histone deacetylase inhibitors (SAHA), NF-κB activators (Prostrain) and histone methyltransferase inhibitors (Chaetocin), available at different research and clinical stages to target HIV-1 latency, SLBP provides a brand new angle to purge HIV-1 provirus by affecting histone (chromatin) metabolism and the accessibility of transcription factors to the HIV-1 promoter.
This pilot study will focus on: 1) defining the relationship between SLBP expression and HIV-1 transcription in primary human CD4+ T cells; 2) confirming the association between SLBP expression and HIV-1 latency in purified memory CD4+ T cells isolated from HIV-1 infected patients under active antiretroviral therapy (ART); 3)exploring new effective ways of purging latent HIV-1 by combining SLBP depletion with other, such as SAHA, treatments.
This study will not only expand our understanding of how SLBP can impact HIV-1 expression but also broadens the scope of designing therapeutic means to activate latent HIV-1.
Inhibition of T. brucei PLK as a novel strategy for treating trypanosomaisis.
The protist parasite Trypanosoma brucei is the causative agent of African trypanosomiasis (sleeping sickness) in humans and nagana in cattle, which cause debilitating illness and economic hardship in sub-Saharan Africa. T. brucei is an obligate extracellular pathogen that is constantly exposed to the immune system of its mammalian and insect hosts. To survive, T. brucei has developed a variety of strategies to avoid detection and disseminate itself. The parasite has a distinct awl-like shape and a single flagellum that nucleates at its posterior end and is adhered along the length of the cell body. This positioning of the flagellum is essential for the proper motility and viability of the parasite. During cell division, the new flagellum must be correctly positioned within the cell body of the daughter cell for cytokinesis to proceed normally. We have shown that the single polo like kinase homolog in T. brucei (TbPLK) is essential for the proper inheritance of the new flagellum. The kinase controls the duplication and segregation of a suite of cytoskeletal organelles that are tasked with positioning the new flagellum. Inhibition of TbPLK blocks the assembly of these organelles, which leads to detachment of the new flagellum and profound cytokinetic defects.