Human Granulocytic Anaplasmosis and Anaplasma phagocytophilum

Understanding how Anaplasma phagocytophilum alters neutrophils will improve diagnosis, treatment, and prevention of this severe illness

Epigenetic Silencing of Host Cell Defense Genes Enhances Intracellular Survival of the Rickettsial Pathogen Anaplasma phagocytophilum

Intracellular bacteria have evolved mechanisms that promote survival within hostile host environments, often resulting in functional dysregulation and disease. Using the Anaplasma phagocytophilum–infected granulocyte model, we establish a link between host chromatin modifications, defense gene transcription and intracellular bacterial infection. Infection of THP-1 cells with A. phagocytophilum led to silencing of host defense gene expression. Histone deacetylase 1 (HDAC1) expression, activity and binding to the defense gene promoters significantly increased during infection, which resulted in decreased histone H3 acetylation in infected cells. HDAC1 overexpression enhanced infection, whereas pharmacologic and siRNA HDAC1 inhibition significantly decreased bacterial load. HDAC2 does not seem to be involved, since HDAC2 silencing by siRNA had no effect on A. phagocytophilum intracellular propagation. These data indicate that HDAC up-regulation and epigenetic silencing of host cell defense genes is required for A. phagocytophilum infection. Bacterial epigenetic regulation of host cell gene transcription could be a general mechanism that enhances intracellular pathogen survival while altering cell function and promoting disease

RAGE Expression in Human T Cells: A Link between Environmental Factors and Adaptive Immune Responses

The Receptor for Advanced Glycation Endproducts (RAGE) is a scavenger ligand that binds glycated endproducts as well as molecules released during cell death such as S100b and HMGB1. RAGE is expressed on antigen presenting cells where it may participate in activation of innate immune responses but its role in adaptive human immune responses has not been described. We have found that RAGE is expressed intracellularly in human T cells following TCR activation but constitutively on T cells from patients with diabetes. The levels of RAGE on T cells from patients with diabetes are not related to the level of glucose control. It co-localizes to the endosomes. Its expression increases in activated T cells from healthy control subjects but bystander cells also express RAGE after stimulation of the antigen specific T cells. RAGE ligands enhance RAGE expression. In patients with T1D, the level of RAGE expression decreases with T cell activation. RAGE+ T cells express higher levels of IL-17A, CD107a, and IL-5 than RAGE− cells from the same individual with T1D. Our studies have identified the expression of RAGE on adaptive immune cells and a role for this receptor and its ligands in modulating human immune responses

A Structural Study of the Potential Inter- and Intramolecular Interactions Involving the Unique 5′ Region of the HIV-1 5′-UTR

The most conserved region of the HIV-1 genome, the 5’ - Untranslated Region (5’-UTR), is comprised of several structural elements that regulate various activities during the retroviral life cycle. During the late phase of the HIV-1 replication cycle, assembling retroviruses selectively packaged two copies of their genomic RNA into viral particles. Recent phylogenetic analysis, secondary structure probing, and free-energy based secondary calculations suggest a novel long-range interaction between residues of the unique 5’ element and residues near the gag coding region. Utilizing Fluorescence Energy Resonance Transfer (FRET), Isothermal Titration Calorimetry (ITC), native polyacrylamide gel electrophoresis (PAGE), and Nuclear Magnetic Resonance (NMR), we show that an 11-oligoribonucleotide spanning residues 105-115 of the 5’ - UTR (U5) binds to oligoribonucleotides containing the gag start codon (AUG), disrupts the pre-existing GNRA tetraloop, and forms a heterodimer (Kd = 0.47 +/- 0.16 μM) stabilized by Watson-Crick base pairs, indicating that the long-range U5:AUG interaction is feasible. Recent studies suggest that the U5: AUG long-range interaction may play a significant role in controlling the monomer-dimer equilibrium of the HIV-1 5’ - UTR. Mutations in the 5’ - UTR that inhibit the proposed U5: AUG interaction, either by deletion of the AUG hairpin or stabilization of the AUG hairpin, strongly block dimerization. Furthermore, the addition of the AUG oligoribonucleotides in trans promotes dimerization by the GC-rich kissing loop in the DIS hairpin. These finding imply that the DIS is sequestered in the monomeric RNA via a possible interaction with U5 and the formation of the U5: AUG long-range interaction exposes the DIS to promote dimerization. Using mutagenesis and multi-dimensional NMR detection, we have devised an approach to directly probe for the novel U5: DIS interaction in the monomeric RNA. Moreover, our studies provide preliminary evidence that the novel long-range interaction between U5 and DIS is possible

A Structural Study of the Potential Inter- and Intramolecular Interactions Involving the Unique 5′ Region of the HIV-1 5′-UTR

The most conserved region of the HIV-1 genome, the 5’ - Untranslated Region (5’-UTR), is comprised of several structural elements that regulate various activities during the retroviral life cycle. During the late phase of the HIV-1 replication cycle, assembling retroviruses selectively packaged two copies of their genomic RNA into viral particles. Recent phylogenetic analysis, secondary structure probing, and free-energy based secondary calculations suggest a novel long-range interaction between residues of the unique 5’ element and residues near the gag coding region. Utilizing Fluorescence Energy Resonance Transfer (FRET), Isothermal Titration Calorimetry (ITC), native polyacrylamide gel electrophoresis (PAGE), and Nuclear Magnetic Resonance (NMR), we show that an 11-oligoribonucleotide spanning residues 105-115 of the 5’ - UTR (U5) binds to oligoribonucleotides containing the gag start codon (AUG), disrupts the pre-existing GNRA tetraloop, and forms a heterodimer (Kd = 0.47 +/- 0.16 μM) stabilized by Watson-Crick base pairs, indicating that the long-range U5:AUG interaction is feasible. Recent studies suggest that the U5: AUG long-range interaction may play a significant role in controlling the monomer-dimer equilibrium of the HIV-1 5’ - UTR. Mutations in the 5’ - UTR that inhibit the proposed U5: AUG interaction, either by deletion of the AUG hairpin or stabilization of the AUG hairpin, strongly block dimerization. Furthermore, the addition of the AUG oligoribonucleotides in trans promotes dimerization by the GC-rich kissing loop in the DIS hairpin. These finding imply that the DIS is sequestered in the monomeric RNA via a possible interaction with U5 and the formation of the U5: AUG long-range interaction exposes the DIS to promote dimerization. Using mutagenesis and multi-dimensional NMR detection, we have devised an approach to directly probe for the novel U5: DIS interaction in the monomeric RNA. Moreover, our studies provide preliminary evidence that the novel long-range interaction between U5 and DIS is possible

Diminished Adhesion of Anaplasma phagocytophilum-Infected Neutrophils to Endothelial Cells Is Associated with Reduced Expression of Leukocyte Surface Selectin

Anaplasma phagocytophilum propagates within neutrophils and causes a disease marked by inflammatory tissue injury or complicated by opportunistic infections. We hypothesized that infection with A. phagocytophilum modifies the binding of neutrophils to endothelial cells and the expression of neutrophil adhesion molecules and studied these changes in vitro. Infected dimethyl sulfoxide-differentiated HL-60 cells and neutrophils showed reduced binding to cultured brain and systemic endothelial cells and lost expression of P-selectin glycoprotein ligand 1 (PSGL-1, CD162) and L-selectin (CD62L) (to 33 and 5% of control values, respectively), at a time when the levels of β(2) integrin and immunoglobulin superfamily adhesion molecules and activation markers Mac-1 and intercellular adhesion molecule 1 increased (5 to 10 times that of the control). The loss of CD162 and CD62L expression was inhibited by EDTA, which suggests that neutrophil activation and sheddase cleavage occurred. The loss of selectin expression and the retained viability of the neutrophils persisted for at least 18 h with A. phagocytophilum infection, whereas Escherichia coli and Staphylococcus aureus rapidly killed neutrophils. The adhesion defect might increase the numbers of infected cells and their persistence in the blood prior to tick bites. However, decreased CD162 expression and poor endothelial cell binding may partly explain impaired host defenses, while simultaneous neutrophil activation may aggravate inflammation. These observations may help us to understand the modified biological responses, host inflammation, and immune response that occur with A. phagocytophilum infections