Browsing by Subject "Immunity, Innate"
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Item Enhancement of the AN1 Population by BAFF: Potential Role in Autoimmune Disease(2010-01-12T19:01:56Z) Sadat, Eva Lasmin; Scheuermann, Richard H.The cytokine BAFF is a TNF family member found to be essential for the homeostasis of B cells. Numerous studies have shown BAFF to be involved in the pathogenesis of autoimmune diseases, including Systemic Lupus Erythematosus (SLE) and Rheumatoid Arthritis. In an effort to understand the role of BAFF in the pathogenesis of autoimmune diseases, extensive in vitro studies on primary B cells have been performed. A recent study has described the existence of an anergic or silent autoreactive B cell population called An1, present within the wild-type repertoire of B cells in the periphery. Our differentiation studies using primary murine splenic B cells indicate an increase in this An1 population in response to BAFF stimulation. In order to ascertain whether this population is truly anergic, calcium flux assays have been performed. Interestingly, these assays show a decrease in response to BCR signaling in BAFF-treated cultures compared to untreated controls. Additional studies with sorted populations of cells indicate that these An1 cells are emerging mainly from the mature and the T2 populations. Survival studies using Annexin V as an apoptosis marker show that BAFF increases the survival of B cells in each of their different stages affirming the role of BAFF in promoting survival of the An1 cells. Microarray gene expression studies done on splenic B cells treated with or without BAFF show higher expression of a set of genes that have been reported to be upregulated in anergic B cells. Purified B cells from mice injected with BAFF also showed an increase in AA4.1 hi cells, which include the An1 cells. B cells from these mice show a lowered calcium flux upon BCR stimulation indicating anergic properties. These data suggest that BAFF stimulation results in the induction of B cell anergy, both in vitro and in vivo. The induction of anergy in B cells in response to stimulation through the antigen receptor may provide a mechanism by which autoreactive cells can evade deletion. Ultimately, the presence of these anergic B cells in the periphery poses a risk of activation and reversion to autoreactivity thus leading to automimmune disease.Item Hepatitis C Virus Entry into Hepatocytes and Engagement of Innate Immune Defenses(2011-08-26T17:34:10Z) Owen, David Matthew; Gale, Michael Jr.Hepatitis C virus (HCV) infection is a major cause of liver disease and a global health problem with inadequate treatment options. An improved understanding of how HCV exploits and subverts host factors to establish infection should yield potential targets for therapy. This study uses a recently developed cell culture model of HCV infection to examine HCV entry and engagement of innate immune defenses. HCV associates with host apolipoproteins and enters hepatocytes through complex processes involving some combination of CD81, claudin-I, occludin, and scavenger receptor BI. Here I show that HCV forms a complex with very low density lipoprotein (VLDL) within infected hepatocytes and uses this association to support infection through the low density lipoprotein receptor (LDL-R). Blocking experiments demonstrate that beta-VLDL and apolipoprotein E (apoE) can compete with HCV for entry. Knockdown of the LDL-R by treatment with 25-hydroxycholesterol or siRNA ablated ligand uptake and reduced HCV infection of cells, whereas infection was rescued upon cell ectopic LDL-R expression. Analyses of gradient-fractionated HCV demonstrate that apoE is associated with HCV virions exhibiting peak infectivity and dependence upon the LDL-R for cell entry. These results define the LDL-R as a cooperative HCV co-receptor that supports viral entry and infectivity through interaction with apoE ligand present in an infectious HCV/lipoprotein complex comprising the virion. Furthermore, upon entry HCV induces an initial transient activation of interferon regulatory factor-3 (IRF3) which is dependent on retinoic acid inducible gene I (RIG-I) and interferon-beta promoter stimulator-1 (IPS-1). This activation produces an antiviral activity which inhibits HCV entry and replication. HCV NS3/4A protease activity blocks this activation within 48 hours. At later time points post infection HCV activates NF-kappaB in a RIG-I independent manner leading to inflammatory cytokine production. These studies identify 3 potential targets for future HCV therapy: 1) alteration of HCV-lipoprotein interaction to disrupt entry, 2) blockade of NS3/4A protease activity to restore innate antiviral response, and 3) modulation of HCV induced NF-kappaB signaling to downregulate chronic inflammation.Item Host Modulagtors of the Death Response to Influenza a Infection(2012-07-17) Ward, Samuel Enoch; White, Michael A.Influenza A virus infects 5-20% of the population annually, resulting in ~35,000 deaths and significant morbidity. Current treatments include vaccines and drugs that target viral proteins. However, both of these approaches have limitations, as vaccines require yearly development and the rapid evolution of viral proteins gives rise to drug resistance. In consequence additional intervention strategies, that target host factors required for the viral life cycle, are under investigation. Here I employed arrayed whole-genome siRNA screening strategies to identify cell-autonomous molecular components that are subverted to support H1N1 influenza A virus infection of human mucosal epithelial cells. Integration across relevant public data sets exposed druggable gene products required for epithelial cell infection or required for viral proteins to deflect host cell suicide checkpoint activation. Pharmacological inhibition of representative targets, RGGT and CHEK1, resulted in significant protection against infection of human epithelial cells by the A/WS/33 virus. In addition, chemical inhibition of RGGT partially protected against H5N1 and the 2009 H1N1 pandemic strain. The observations reported here thus contribute to decoding vulnerabilities in the command and control networks specified by influenza virulence factors. [Keywords: Influenza A, genomewide screen, RNAi, innate immunity, CHEK1, IFITM3, virus]Item Innate Immune Responses and Viral Changes after Oral Transmission of SIV in Rhesus Macaques(2010-01-12T18:51:12Z) Durudas, Andre; Sodora, Donald L.Recent HIV vaccine trial failures indicated the need to increase our knowledge with regard to mucosal immune responses after exposure to HIV/SIV. The studies presented here utilized oral transmission of simian immunodeficiency virus (SIV) in Rhesus macaques with the goals of (1) determining associations between innate immune responses at different tissue sites and disease progression and (2) assessing the differences in immune and viral changes that occur after oral administrations with low or high SIV doses. During previous work in the Sodora laboratory, mRNA levels of innate immune responses were assessed at mucosal sites of orally SIV-infected macaques. The studies presented here build upon this work by assessing innate immune modulator transcripts within lymph nodes and peripheral blood. Dividing the SIV+ macaques with regard to their rate of disease progression, rapidly progressing macaques exhibited elevated expression of IFNα, OAS, CXCL9 and CXCL10 mRNA at lymph nodes. In peripheral blood, only expression of OAS and CXCL10 mRNA was associated with faster disease progression. Assessment of macaques orally infected by high or low doses of SIV revealed that high doses, as expected, resulted in transmission of more virions, and viral replication exhibited slightly faster kinetics early after transmission. Furthermore, high dose macaques exhibited higher levels of the anti-viral factors IFNα and OAS in tissues. However, other innate and adaptive immune responses were comparable between macaques infected by high or low doses. Also, mRNA expression of immune modulators in peripheral blood was similar between the two groups, with only expression of OAS and CXCL10 transcripts being upregulated. These findings indicate that expression of these two immune modulators is preferentially upregulated during faster disease progression and is independent of viral dose. This suggests that OAS and CXCL10 transcript levels could potentially be used as diagnostic markers of AIDS progression. Taken together, these studies indicate that multiple tissue compartments need to be assessed to obtain a complete understanding of immune and viral factors in SIV/HIV disease. A more complete knowledge of the mucosal and systemic immune responses prior to, during and following HIV transmission is likely to lead to new approaches for the development of novel vaccines and therapies to combat HIV infection/disease. [Keywords: HIV; AIDS; SIV; oral transmission; Rhesus Macaques; innate immune responses; low dose]Item The Ligand and Function of the RegIII Family of Bactericidal C-Type Lectins(2006-08-11) Cash, Heather Lynn; Hooper, LoraBeginning at birth, the intestines of humans and other mammals are colonized with a diverse society of resident bacteria that play a crucial role in host nutrient metabolism. To maintain this commensal relationship, resident microbes must be prevented from crossing the intestinal epithelium into host tissues where they can cause inflammation and sepsis. The innate immune system plays a crucial role in preventing bacterial incursions across gut epithelial surfaces. Mucosal epithelial cells produce a variety of secreted antimicrobial proteins that help to prevent bacterial attachment and encroachment at epithelial surfaces. Among these, Paneth cells are specialized small intestinal epithelial cells that have been shown to produce and secrete antimicrobial proteins and peptides. To gain new insights into the adaptation of mucosal surfaces to microbial challenges, the Hooper lab has used DNA microarrays to screen for Paneth cell genes whose expression is modulated by intestinal microbes. This screen revealed that expression of two C-type lectins, RegIIIbeta and RegIIIgamma , is strongly induced following intestinal colonization with resident microbes. Two features suggested that members of the RegIII family may have microbicidal functions. First, they are C-type lectin family members. Other C-type lectins, including the mannose binding lectin, have well-characterized innate immune functions and play critical roles in microbial killing by recruiting complement. Second, I have shown that the murine RegIII lectins localize to intestinal crypt cells, including Paneth cell secretory granules, and that they bind to luminal bacteria harvested from intestinal conditions. Based on these observations, we hypothesized that this family of proteins may perform an innate immune function, specifically antimicrobial defense. The studies reported in this thesis characterize a family of C-type lectins. Specifically, we determined that these proteins interact with peptidoglycan by binding with high affinity to its glycan structure, representing a unique blend of peptidoglycan recognition and lectin function. Additionally, we have demonstrated that this binding results in the specific disruption of the Gram positive bacterial cell wall, where peptidoglycan is exposed, which is the first example of a family of directly bactericidal C-type lectins. We also present evidence for the regulation of these bactericidal proteins by colonization with an intestinal microflora. Therefore, the research presented in this thesis elucidates the function of three members of the RegIII family, in both mice and humans.Item The Pathogenic Cascade of Acanthamoeba Keratitis(2006-05-15) Clarke, Daniel William; Niederkorn, JerryAcanthamoeba keratitis is a blinding infection of the cornea caused by the ubiquitous, free-living amoeba, Acanthamoeba. The pathogenic cascade of Acanthamoeba keratitis is a sequential process that begins with adherence of trophozoites to the corneal epithelium and culminates in the destruction of the corneal epithelium and the dissolution of the corneal stroma. This work examined the pathophysiology and immunobiology of Acanthamoeba keratitis. First, we explored possible mechanisms to explain why A. castellanii remains restricted to the cornea and rarely produces intraocular infections. One hypothesis proposed that trophozoites cannot penetrate Descemet's membrane and the corneal endothelium to enter the anterior chamber. However, amoebae utilized a mannose-induced serine protease to penetrate Descemet's membrane within 24 hr of in vitro culture. The second hypothesis proposed that the trophozoites can enter the anterior chamber; however, the aqueous humor contains factors that either induce encystment or kill the amoebae. Injection of amoebae into the anterior chamber induced a robust neutrophil infiltrate, which was associated with complete clearance by day 15 post anterior chamber injection. This indicates that neutrophils of the innate immune apparatus are important in preventing Acanthamoeba keratitis from progressing to become an intraocular infection. Previous reports have shown that intracorneal instillation of sterile latex beads results in resistance to Acanthamoeba keratitis and mitigation of corneal inflammation. This study examined the mechanisms that could be responsible for the latex bead protective effect. Latex bead treatment induced a significant increase in the infiltration of macrophages into the corneas that peaked at day 4 of infection. Additionally, depletion of conjunctival macrophages with the macrophagicidal drug, clodronate, eliminated the latex bead protective effect, providing further evidence that macrophages are crucial in resistance to Acanthamoeba keratitis. With the exception of mucosal IgA antibody, the adaptive immune apparatus is not typically effective against Acanthamoeba keratitis. The results presented here provide evidence that neutrophils and macrophages of the innate immune response are crucial in resistance to Acanthamoeba keratitis. Collectively, these results suggest that recruitment and/or activation of the innate immune apparatus may lead to resolution of disease in Acanthamoeba keratitis patients.Item Systems Biology of Staphylococcus Aureus Infection Ex Vivo and in Vitro(2012-07-09) Banchereau, Romain; Ramilo, OctavioStaphylococcus aureus has emerged as one of the most common community-acquired bacterial infections, with significant morbidity and mortality. Emergence of multidrug resistant strains worldwide, combined with limited treatment options demand novel approaches to further elucidate host-pathogen interactions, and especially host responses to infection. To this end, we leveraged systems biology approaches to better characterize the status of the host immune system during S. aureus infection ex vivo and in vitro. The transcriptional profiles of PBMC and whole blood from patients with community-acquired S. aureus infection were characterized by microarray analysis, and leukocyte population frequencies were measured by polychromatic flow cytometry. To refine our understanding of inflammatory networks involved, an in vitro system of antigen-presenting cell stimulation with various pathogens, including S. aureus as well as other bacteria and viruses, and their components, was used to identify early inflammatory programs induced in innate immune cells. To reduce the dimension and complexity of the data generated, we developed modular frameworks to analyze and interpret the fingerprints obtained from both the ex vivo and in vitro studies. // Overall, the blood transcriptional response to S. aureus infection was characterized by over-expression of innate immunity and hematopoiesis transcriptional programs, and under-expression of adaptive immunity programs. Flow cytometry and standard cell blood count (CBC) revealed an increase in absolute numbers of circulating monocytes, neutrophils and antigen-presenting cells, including dendritic cells and B cells, combined with a decrease in central memory T cells. To identify transcriptional correlates of clinical heterogeneity, we obtained individual fingerprints and derived the molecular distance to health, a numerical score of transcriptional perturbation for each patient. Patient-by-patient analysis without a priori knowledge of clinical diagnoses identified four major transcriptional clusters based on inflammation, erythropoiesis and interferon-induced profiles. Clinical presentation, bacterial dissemination and time between hospitalization and blood sampling were identified as major factors influencing the signature. The framework obtained from in vitro stimulation of monocyte-derived DC helped us refine the characterization of inflammatory programs activated during S. aureus infection. In addition to inflammatory antibacterial programs, S. aureus induced a subset of interferon response modules, also observed in viral infections and autoimmunity, as well as a specific set of modules linked to cell compartmentalization and lipid biosynthesis. Systems biology approaches provide a global and comprehensive assessment of host responses to acute bacterial infections, bringing a new understanding of disease pathogenesis and underlying patient heterogeneity. [Keywords; Staphylococus aureus, microarray, systems biology, module framework, transcription]