HIV Infection Dysregulates MicroRNA Expression by  Human Macrophages Aim ée Joanne Luers Laboratory of Dr. Joan W. Berman
HIV/AIDS: A Global Health Crisis Total: 33.4 million (31.1 – 35.8 million) 2009   http://www.unaids.org/en/KnowledgeCentre...
HIV/AIDS <ul><li>Acute HIV infection </li></ul><ul><ul><li>High blood viremia </li></ul></ul><ul><ul><li>Drop of in the nu...
HIV/AIDS <ul><li>Combinatorial Anti-Retroviral Therapy (cART)   does not cure AIDS   </li></ul><ul><ul><li>Virus may be is...
Macrophages (M ɸ) <ul><li>Among the first cells to become infected during the course of HIV infection </li></ul><ul><li>In...
Macrophages (Mɸ) <ul><li>Key cells of both innate and adaptive immunity </li></ul><ul><li>Monocytes differentiate into Mac...
Macrophages (Mɸ)
Macrophages (M ɸ) <ul><li>The M ɸ  response to infection and inflammation requires regulation to fight disease while avoid...
MicroRNAs <ul><li>~22 nucleotide-long non-coding RNAs repressing protein production in plants and animals </li></ul><ul><l...
MicroRNA  Maturation  and  Machinery
MicroRNAs and Macrophages   <ul><li>The examination of the miRNAs of immune cells as they pertain to infectious disease is...
Hypothesis:  Macrophages express MicroRNA and their required machinery.  MicroRNA expression is altered during HIV infecti...
Hypothesis:  Macrophages express MicroRNA and their required machinery.   MicroRNA expression is altered during HIV infect...
Hypothesis:  Macrophages express MicroRNA and their required machinery.  MicroRNA expression is altered during HIV infecti...
Hypothesis:  Macrophages express MicroRNA and their required machinery.  MicroRNA expression is altered during HIV infecti...
Whole Blood or Leukopak Separate by Ficoll Gradient PBMC Incubate with Magnetized CD14 Immuno-  Beads Magnet CD14 - CD14 +...
Mɸ Produce the Proteins Required for  MicroRNA Machinery Function Luers et al,  Cellular Immunology,  2010 <ul><li>Variabi...
Aims: Specific Aim 1:   To determine whether human macrophages produce the proteins of the MicroRNA machinery and profile ...
MicroRNA Microarray Profiling of  Human Primary Macrophages: 2 Methods <ul><li>  Method 1: </li></ul><ul><li>1.  Trizol ex...
Fractionation, Purification, and Analysis of Macrophage RNA Total RNA Small RNA Fraction < 40 nt Fractionation Purificatio...
<ul><li>Electrophoresis separates RNA species by size </li></ul><ul><li>Breast cancer tissue control indicates RNA fractio...
MicroRNA Microarray Profiling of  Human Primary Macrophages: 2 Methods <ul><li>  Method 1: </li></ul><ul><li>1.   Trizol e...
<ul><li>  Method 2: </li></ul><ul><li>Trizol extraction of total RNA </li></ul><ul><li>Sent to LC Sciences for microarray ...
119 MicroRNAs Expressed by  Human Primary M acrophages Luers et al,  Cellular Immunology,  2010 MicroRNA Microarray
qRT-PCR Verification of MicroRNA Expression Profile <ul><li>Cycle Threshold:  point at which amplification is detectable a...
qRT-PCR Verification of MicroRNA Expression Profile <ul><li>Previous literature showed that the HL-60 promyelocytic monocy...
qRT-PCR Verification of Profiling: Consistent Subset <ul><li>Examination of miRNAs that had high, moderate, and low expres...
<ul><li>There can be a large degree of  variability  among donors in the expression of some miRNAs  </li></ul><ul><ul><li>...
<ul><li>qRT-PCR of miRNAs not identified by array  </li></ul><ul><li>Showed that they are not expressed in Mɸ thereby vali...
Aim 1 Summary: Do Macrophages express  miRNA machinery proteins? Yes. Do Macrophages express miRNAs? Yes.
Hypothesis:  Macrophages express MicroRNA and their required machinery.  MicroRNA expression is altered during HIV infecti...
Connections between HIV Transcription and the MicroRNA Machinery <ul><li>When RNA Pol II transcribes the HIV genome, the e...
<ul><li>TAR RNA Binding Protein  (TRBP) complements Tat in its binding of TAR </li></ul><ul><li>Dicer may bind here as wel...
MicroRNA Expression in the Context of Viral Infection <ul><li>Viruses can produce their own miRNAs to support their replic...
MicroRNA Activity in HIV-Infected Monocyte Differentiation into Macrophages <ul><li>MicroRNA-28, -150, -223, and -382 </li...
Specific Aim 2:   To identify MicroRNA expression alterations in HIV-infected human primary macrophages.   Does HIV-1 infe...
LC Sciences Array Results <ul><li>Total RNA from HIV-infected and uninfected human primary macrophages was profiled </li><...
Macrophage Cultures Do Not  Become HIV-Infected Uniformly <ul><li>Inter-culture variability </li></ul><ul><ul><li>Human pr...
Using the HL-60/OM10.1 Model System to Verify MicroRNAs of Interest <ul><li>HL-60 promyelocytic monocytes (Gallo, 1980) </...
PMA Causes OM10.1 Cells to Produce High Levels of HIV and Differentiate to a Macrophage Phenotype <ul><li>p24 is a structu...
<ul><li>miR-99b – increased in HIV infection </li></ul><ul><ul><li>Putatively down-regulates  C/EBP β </li></ul></ul><ul><...
qRT-PCR Indicates that Expression of MicroRNA-99b Is Increased during HIV Production
qRT-PCR Indicates that Expression of MicroRNA-125a Is Increased during HIV Production
qRT-PCR Indicates that Expression of MicroRNA-146a Is Decreased during HIV Production
Hypothesis:  Macrophages express MicroRNA and their required machinery.  MicroRNA expression is altered during HIV infecti...
Hypothesis:  Macrophages express MicroRNA and their required machinery.  MicroRNA expression is altered during HIV infecti...
Are the MicroRNAs that are differentially expressed during HIV infection of macrophages affecting  viral production ? <ul>...
Are the MicroRNAs that are differentially expressed during HIV infection of macrophages affecting  viral production ? <ul>...
<ul><li>MicroRNA-146a is decreased during production of HIV </li></ul><ul><li>The Mimic to -146a restores  miR-146a activi...
<ul><ul><li>Transfected OM10.1 cells for 24h with miR-146a Mimic </li></ul></ul><ul><ul><li>Treated with PMA </li></ul></u...
OM10.1 p24 Production is Increased by Mimic-146a
<ul><ul><li>CCL3 is a putative target of miR-146a </li></ul></ul><ul><ul><li>Transfected miR-146a Mimic  </li></ul></ul><u...
Over-Expression of MicroRNA-146a  Does Not  Affect CCL-3 Production <ul><li>PMA increases CCL-3 production as expected </l...
<ul><ul><li>Other miR-146a targets will need to be tested to determine the effect of its decrease during HIV production </...
<ul><ul><li>Transfected OM10.1 cells for 24h with miR-99b AntagomiR </li></ul></ul><ul><ul><li>Treated with PMA </li></ul>...
The miR-99b AntagomiR Decreases the Activity of miR-99b <ul><li>MicroRNA-99b is increased during production of HIV </li></...
OM10.1 p24 Production is Increased by  AntagomiR-99b
<ul><li>During HIV infection, miR-99b increases </li></ul><ul><li>Data suggests that if miR-99b is decreased with an Antag...
 
<ul><li>During HIV infection, miR-99b increases </li></ul><ul><li>Data suggests that if miR-99b is decreased with an Antag...
Chromatin  Immunoprecipitation (ChIP) Performed in collaboration with Loreto Carvallo C/EBP β
AntagomiR to miR-99b Increases the Presence of C/EBP β   at the HIV LTR in Transfected OM10.1 Cells
AntagomiR to miR-99b May Increase and/or Stabilize the  Occupancy of C/EBP β  at the Viral Promoter
 
Summary of Aims <ul><li>Hypothesis:   Macrophages express MicroRNA and their required machinery.  MicroRNA expression is a...
Future Directions <ul><li>Determine target for miR-146a </li></ul><ul><ul><li>What could be mediating increase in viral pr...
Conclusions <ul><li>There is variability in the production of the miRNA machinery and miRNA expression </li></ul><ul><ul><...
Conclusions <ul><li>These data underscore the importance of miRNA study in HIV-macrophages, as their alteration affects th...
Acknowledgements <ul><li>Berman Lab   </li></ul><ul><li>Dr. Joan W. Berman </li></ul><ul><li>Dr. Tina Calderon </li></ul><...
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HIV Infection Dysregulates MicroRNA Expression by Human Macrophages. This seminar was presented in partial fulfillment of the requirements of the State of NY for obtaining my Ph.D. in the Biomedical Sciences.

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  • 2 different projects/perspectives – ChIP and microRNA
  • I then FACS the cells on Days 3 and 6 for the following monocytic markers.
  • Thesis for resume presentation

    1. 1. HIV Infection Dysregulates MicroRNA Expression by Human Macrophages Aim ée Joanne Luers Laboratory of Dr. Joan W. Berman
    2. 2. HIV/AIDS: A Global Health Crisis Total: 33.4 million (31.1 – 35.8 million) 2009 http://www.unaids.org/en/KnowledgeCentre/HIVData/EpiUpdate/EpiUpdArchive/2009/default.asp Western & Central Europe 850,000 [710 000 – 970 000] Middle East & North Africa 310,000 [250 000 – 380 000] Sub-Saharan Africa 22.4 million [20.8 – 24.1 million] Eastern Europe & Central Asia 1.5 million [1.4 – 1.7 million] South & South-East Asia 3.8 million [3.4 – 4.3 million] Oceania 59,000 [51 000 – 68 000] North America 1.4 million [1.2 – 1.6 million] Latin America 2.0 million [1.8 – 2.2 million] East Asia 850,000 [700 000 – 1.0 million] Caribbean 240,000 [220 000 – 260 000]
    3. 3. HIV/AIDS <ul><li>Acute HIV infection </li></ul><ul><ul><li>High blood viremia </li></ul></ul><ul><ul><li>Drop of in the number of CD4+ T-cells circulating in the periphery </li></ul></ul><ul><li>Most but not all individuals who become infected will succumb to progressive immunodeficiency, AIDS </li></ul>
    4. 4. HIV/AIDS <ul><li>Combinatorial Anti-Retroviral Therapy (cART) does not cure AIDS </li></ul><ul><ul><li>Virus may be isolated from cells throughout the body in individuals receiving cART </li></ul></ul><ul><li>This underscores the need for </li></ul><ul><ul><li>Improved therapies </li></ul></ul><ul><ul><li>Effective vaccine strategies </li></ul></ul><ul><ul><li>Transmission prevention alternatives </li></ul></ul>
    5. 5. Macrophages (M ɸ) <ul><li>Among the first cells to become infected during the course of HIV infection </li></ul><ul><li>Involved in the pathogenesis of many diseases including AIDS </li></ul><ul><li>Continue to be an important reservoir of the virus even during cART </li></ul><ul><ul><li>HIV-infected macrophages are a reservoir for the virus in almost every organ </li></ul></ul><ul><ul><li>HIV-infected macrophage precursor cells can be isolated from the peripheral blood of HIV+ individuals on cART </li></ul></ul>
    6. 6. Macrophages (Mɸ) <ul><li>Key cells of both innate and adaptive immunity </li></ul><ul><li>Monocytes differentiate into Macrophages </li></ul><ul><li>In the periphery, monocytes migrate towards sites of damage and infection by responding to chemotactic signals </li></ul><ul><li>At the target site, monocytes extravasate and differentiate into macrophages, which are capable of </li></ul><ul><ul><li>Chemotaxis within the affected tissue </li></ul></ul><ul><ul><li>Phagocytosis of cellular debris and pathogens </li></ul></ul><ul><ul><li>Stimulation of lymphocytes to destroy affected cells through antigen presentation </li></ul></ul><ul><ul><li>Secretion of inflammatory mediators </li></ul></ul><ul><ul><li>Participation in tissue repair </li></ul></ul>
    7. 7. Macrophages (Mɸ)
    8. 8. Macrophages (M ɸ) <ul><li>The M ɸ response to infection and inflammation requires regulation to fight disease while avoiding disease pathogenesis </li></ul><ul><ul><li>One method of self-regulation is the expression of MicroRNAs </li></ul></ul><ul><ul><li>MicroRNAs (miRNAs) limit the expression of target proteins </li></ul></ul><ul><ul><li>Examination of this regulatory process during HIV infection of macrophages </li></ul></ul>
    9. 9. MicroRNAs <ul><li>~22 nucleotide-long non-coding RNAs repressing protein production in plants and animals </li></ul><ul><li>Diverse roles in many cellular processes: </li></ul><ul><ul><li>Expression patterning, development, cell proliferation, and differentiation </li></ul></ul><ul><li>Computer algorithms indicate that some miRNAs target over a thousand proteins </li></ul><ul><ul><li>Few of the targets of the almost 1000 identified human miRNAs have been verified experimentally </li></ul></ul>
    10. 10. MicroRNA Maturation and Machinery
    11. 11. MicroRNAs and Macrophages <ul><li>The examination of the miRNAs of immune cells as they pertain to infectious disease is an expanding field </li></ul><ul><li>The miRNA expression profile of and miRNA machinery production in macrophages has not been well studied, especially in the context of HIV infection </li></ul>
    12. 12. Hypothesis: Macrophages express MicroRNA and their required machinery. MicroRNA expression is altered during HIV infection, thereby affecting viral production in and proteins produced by macrophages. Aims: Specific Aim 1: To determine whether human macrophages produce the proteins of the MicroRNA machinery and profile macrophage expression of MicroRNAs.
    13. 13. Hypothesis: Macrophages express MicroRNA and their required machinery. MicroRNA expression is altered during HIV infection, thereby affecting viral production in and proteins produced by macrophages. Aims: Specific Aim 1: To determine whether human macrophages produce the proteins of the MicroRNA machinery and profile macrophage expression of MicroRNAs. Specific Aim 2: To identify MicroRNA expression alterations in HIV-infected human primary macrophages.
    14. 14. Hypothesis: Macrophages express MicroRNA and their required machinery. MicroRNA expression is altered during HIV infection, thereby affecting viral production in and proteins produced by macrophages. Aims: Specific Aim 1: To determine whether human macrophages produce the proteins of the MicroRNA machinery and profile macrophage expression of MicroRNAs. Specific Aim 2: To identify MicroRNA expression alterations in HIV-infected human primary macrophages. Specific Aim 3: To determine whether differentially expressed MicroRNAs have an effect on HIV production and/or host cell protein production in HIV-infected human macrophages.
    15. 15. Hypothesis: Macrophages express MicroRNA and their required machinery. MicroRNA expression is altered during HIV infection, thereby affecting viral production in and proteins produced by macrophages. Aims: Specific Aim 1: To determine whether human macrophages produce the proteins of the MicroRNA machinery and profile macrophage expression of MicroRNAs.
    16. 16. Whole Blood or Leukopak Separate by Ficoll Gradient PBMC Incubate with Magnetized CD14 Immuno- Beads Magnet CD14 - CD14 + Column Culture CD14 + Cells with MCSF for 6 days Apply to Magnetized Column Isolation of CD14+ Monocytes and Culture of Monocyte-Derived Macrophages CD14 + , CD68 + , CD11b + Migrate, Phagocytic Y Y Y Y
    17. 17. Mɸ Produce the Proteins Required for MicroRNA Machinery Function Luers et al, Cellular Immunology, 2010 <ul><li>Variability of miRNA machinery </li></ul><ul><li>protein expression may explain </li></ul><ul><li>earlier finding by Klase et al, 2007, who were unable to find Dicer expression in human primary macrophages using </li></ul><ul><li>Only two donors </li></ul><ul><li>Less lysate </li></ul>
    18. 18. Aims: Specific Aim 1: To determine whether human macrophages produce the proteins of the MicroRNA machinery and profile macrophage expression of MicroRNAs. Do macrophages express miRNA machinery proteins? Yes. Do macrophages express miRNAs?
    19. 19. MicroRNA Microarray Profiling of Human Primary Macrophages: 2 Methods <ul><li> Method 1: </li></ul><ul><li>1. Trizol extraction of total RNA </li></ul><ul><li>2. Fractionation and Purification of miRNA </li></ul>
    20. 20. Fractionation, Purification, and Analysis of Macrophage RNA Total RNA Small RNA Fraction < 40 nt Fractionation Purification Bioanalyzer Analysis
    21. 21. <ul><li>Electrophoresis separates RNA species by size </li></ul><ul><li>Breast cancer tissue control indicates RNA fraction that contains miRNAs </li></ul><ul><li>Fractionated RNA electropherogram indicates enrichment for small RNA in macrophages </li></ul><ul><li>Control and macrophage samples have differing RNA profiles </li></ul>Analysis of RNA Using Agilent Small RNA Chip Luers et al, Cellular Immunology, 2010
    22. 22. MicroRNA Microarray Profiling of Human Primary Macrophages: 2 Methods <ul><li> Method 1: </li></ul><ul><li>1. Trizol extraction of total RNA </li></ul><ul><li>2. Fractionation and Purification of miRNA </li></ul><ul><li>Cy3/Cy5 Labeling </li></ul><ul><li>Hybridization to a chip containing DNA that complements known miRNAs </li></ul><ul><li>Scan the chip/Analyze data </li></ul><ul><ul><li>MicroRNAs with signals of less than than 1000 in 3 spots were excluded </li></ul></ul>
    23. 23. <ul><li> Method 2: </li></ul><ul><li>Trizol extraction of total RNA </li></ul><ul><li>Sent to LC Sciences for microarray profiling </li></ul><ul><ul><li>MicroRNAs with a detectable signal and present in an aggregate of 6 or more out of 8 spots were included </li></ul></ul>MicroRNA Microarray Profiling of Human Primary Macrophages: 2 Methods
    24. 24. 119 MicroRNAs Expressed by Human Primary M acrophages Luers et al, Cellular Immunology, 2010 MicroRNA Microarray
    25. 25. qRT-PCR Verification of MicroRNA Expression Profile <ul><li>Cycle Threshold: point at which amplification is detectable above background </li></ul><ul><li>Amplification above a C T of 32 is background noise due to assay sensitivity </li></ul><ul><li>Positive Control: Endogenous Target </li></ul><ul><ul><li>Rnu44: small RNA molecular consistently expressed in macrophages </li></ul></ul><ul><li>Negative Control: Two kinds </li></ul><ul><ul><li>No Template Control (NTC) – no RNA added to reaction </li></ul></ul><ul><ul><li>Non-endogenous Target – known not to be expressed in the sample </li></ul></ul>Cycle Threshold C T Positive Control Negative Controls
    26. 26. qRT-PCR Verification of MicroRNA Expression Profile <ul><li>Previous literature showed that the HL-60 promyelocytic monocyte cell line expresses miR-146a when differentiated to a macrophage phenotype </li></ul><ul><li>Our miR-146a primary macrophage microarray and qRT-PCR data corroborated what was demonstrated in the HL-60 cell line </li></ul>Luers et al, Cellular Immunology, 2010
    27. 27. qRT-PCR Verification of Profiling: Consistent Subset <ul><li>Examination of miRNAs that had high, moderate, and low expression on arrays </li></ul><ul><li>There is a subset of miRNAs that are consistently expressed among Mɸ donors </li></ul><ul><ul><li>miR-30b, miR-23a, and </li></ul></ul><ul><ul><li>miR-212 </li></ul></ul>Luers et al, Cellular Immunology, 2010
    28. 28. <ul><li>There can be a large degree of variability among donors in the expression of some miRNAs </li></ul><ul><ul><li>hsa-let-7a, miR-16, miR-103, and miR-378 </li></ul></ul>qRT-PCR Verification of Profiling: Variable Subset Luers et al, Cellular Immunology, 2010
    29. 29. <ul><li>qRT-PCR of miRNAs not identified by array </li></ul><ul><li>Showed that they are not expressed in Mɸ thereby validating the cut-off values used </li></ul>qRT-PCR Verification of Background Cut-Offs Luers et al, Cellular Immunology, 2010
    30. 30. Aim 1 Summary: Do Macrophages express miRNA machinery proteins? Yes. Do Macrophages express miRNAs? Yes.
    31. 31. Hypothesis: Macrophages express MicroRNA and their required machinery. MicroRNA expression is altered during HIV infection, thereby affecting viral production in and proteins produced by macrophages. Aims: Specific Aim 1: To determine whether human macrophages produce the proteins of the MicroRNA machinery and profile macrophage expression of MicroRNAs. Specific Aim 2: To identify MicroRNA expression alterations in HIV-infected human primary macrophages. Specific Aim 3: To determine whether differentially expressed MicroRNAs have an effect on HIV production and/or host cell protein production in HIV-infected human macrophages.
    32. 32. Connections between HIV Transcription and the MicroRNA Machinery <ul><li>When RNA Pol II transcribes the HIV genome, the enzyme stalls and creates a double-stranded stem-loop RNA structure that similar to a miRNA </li></ul><ul><li>Tat binds this stem-loop and facilitates transcription of the viral genome in cooperation with many host cell proteins </li></ul>Tat CyclinT Cdk9 p300 Ac RNAPol II P P P P SWI/SNF
    33. 33. <ul><li>TAR RNA Binding Protein (TRBP) complements Tat in its binding of TAR </li></ul><ul><li>Dicer may bind here as well </li></ul><ul><li>Tat may interfere with Dicer activity which may facilitate TRBP activity at the viral promoter </li></ul><ul><li>MicroRNA-like TAR RNA released from non-processive RNA Pol II may interfere with Tat and TRBP binding at the viral promoter and explain viral latency in T-cells </li></ul>Connections between HIV Transcription and the MicroRNA Machinery Tat TRBP
    34. 34. MicroRNA Expression in the Context of Viral Infection <ul><li>Viruses can produce their own miRNAs to support their replication cycle </li></ul><ul><li>Infected host cells can be induced by viruses to produce miRNAs that support the viral replication cycle </li></ul><ul><li>Host cells can respond to infection by producing anti-viral miRNAs to counteract viral infection </li></ul>
    35. 35. MicroRNA Activity in HIV-Infected Monocyte Differentiation into Macrophages <ul><li>MicroRNA-28, -150, -223, and -382 </li></ul><ul><ul><li>Monocytes express anti-HIV miRNA </li></ul></ul><ul><ul><li>As Monocytes differentiate into Macrophages, the expression of these miRNA decreases enabling M ɸ infection </li></ul></ul><ul><li>Decrease of miR-198 during Macrophage differentiation increases Cyclin-T1, allowing Macrophages to be infected </li></ul><ul><li>Dicer expression is suppressed when HIV-infected monocytes are differentiated into macrophages </li></ul><ul><ul><li>Dicer is suppressed by Vpr during HIV infection during macrophage differentiation </li></ul></ul><ul><ul><li>Found an overall decrease in miRNA expression in infected macrophages </li></ul></ul>Wang et al, 2008 Blood ; Rice and Sung, 2009 PLOS Pathogens; Coley et al, 2010 JBC
    36. 36. Specific Aim 2: To identify MicroRNA expression alterations in HIV-infected human primary macrophages. Does HIV-1 infection alter the production of miRNAs? Yes, some are increased, some are decreased.
    37. 37. LC Sciences Array Results <ul><li>Total RNA from HIV-infected and uninfected human primary macrophages was profiled </li></ul><ul><li>Identified 13 cellular miRNAs with significantly different expression during HIV infection (p < 0.01) </li></ul>Two Macrophage Donors LC Control Ratio miRNA Custom LC Control Ratio miRNA Custom
    38. 38. Macrophage Cultures Do Not Become HIV-Infected Uniformly <ul><li>Inter-culture variability </li></ul><ul><ul><li>Human primary Mɸ cultures exhibit high variability in infection from donor to donor </li></ul></ul><ul><li>Intra-culture variability </li></ul><ul><ul><li>Not every cell in a given culture is infected </li></ul></ul><ul><ul><li>Not every cell in a given culture produces the same quantity of virus </li></ul></ul><ul><li>Utilization of cell lines to identify miRNAs of interest </li></ul>
    39. 39. Using the HL-60/OM10.1 Model System to Verify MicroRNAs of Interest <ul><li>HL-60 promyelocytic monocytes (Gallo, 1980) </li></ul><ul><li>PMA treatment differentiates HL-60 to a Mɸ phenotype </li></ul><ul><li>HL-60s were Infected with HIV LAV (Butera, 1991) and expanded to </li></ul><ul><li>make OM10.1 cells </li></ul><ul><li>Each OM10.1 cell contains latent integrated virus </li></ul><ul><li>PMA treatment differentiates OM10.1 cells to a Mɸ phenotype </li></ul><ul><li>PMA Treatment expands OM10.1 virus production </li></ul>
    40. 40. PMA Causes OM10.1 Cells to Produce High Levels of HIV and Differentiate to a Macrophage Phenotype <ul><li>p24 is a structural protein of the virus core </li></ul><ul><li>The presence of p24 in the supernatant of infected cells indicates production of HIV </li></ul><ul><li>PMA treatment of OM10.1 cells causes differentiation into a macrophage phenotype </li></ul><ul><li>Characterized by adherence and projections </li></ul>
    41. 41. <ul><li>miR-99b – increased in HIV infection </li></ul><ul><ul><li>Putatively down-regulates C/EBP β </li></ul></ul><ul><ul><li>C/EBP β required by HIV for viral production </li></ul></ul><ul><li>miR-125a – increased in HIV infection </li></ul><ul><ul><li>Regulates apoptosis in hematopoietic stem cells and cancer </li></ul></ul><ul><ul><li>HIV-infected macrophages are resistant to apoptosis </li></ul></ul><ul><li>miR-146a – decreased in HIV infection </li></ul><ul><ul><li>Putatively down-regulates CCL-3 (MIP-1 α ) </li></ul></ul><ul><ul><ul><li>CCL-3 is a macrophage chemoattractant that binds to CCR5, a macrophage co-receptor for HIV </li></ul></ul></ul><ul><ul><ul><li>Secretion of CCL-3 is increased during HIV infection </li></ul></ul></ul><ul><ul><li>Regulates numerous proteins of the NF- κ B pathway </li></ul></ul>MicroRNAs of Interest
    42. 42. qRT-PCR Indicates that Expression of MicroRNA-99b Is Increased during HIV Production
    43. 43. qRT-PCR Indicates that Expression of MicroRNA-125a Is Increased during HIV Production
    44. 44. qRT-PCR Indicates that Expression of MicroRNA-146a Is Decreased during HIV Production
    45. 45. Hypothesis: Macrophages express MicroRNA and their required machinery. MicroRNA expression is altered during HIV infection, thereby affecting viral production in and proteins produced by macrophages. Aim 2 Summary: Specific Aim 2: To identify MicroRNA expression alterations in HIV-infected human primary macrophages. MicroRNA-99b and -125a are increased and MicroRNA-146a is decreased during HIV production in OM10.1 cells
    46. 46. Hypothesis: Macrophages express MicroRNA and their required machinery. MicroRNA expression is altered during HIV infection, thereby affecting viral production in and proteins produced by macrophages. Aims: Specific Aim 1: To determine whether human macrophages produce the proteins of the MicroRNA machinery and profile macrophage expression of MicroRNAs. Specific Aim 2: To identify MicroRNA expression alterations in HIV-infected human primary macrophages. Specific Aim 3: To determine whether differentially expressed MicroRNAs have an effect on HIV production and/or host cell protein production in HIV-infected human macrophages.
    47. 47. Are the MicroRNAs that are differentially expressed during HIV infection of macrophages affecting viral production ? <ul><li>Study affect on HIV using p24 ELISA </li></ul><ul><li>AntagomiR to knock down miR-99b activity </li></ul><ul><li>Mimic to increase miR-146a activity </li></ul><ul><li>AntagomiR and Mimic are made of Locked Nucleic Acids, for binding stability </li></ul>
    48. 48. Are the MicroRNAs that are differentially expressed during HIV infection of macrophages affecting viral production ? <ul><li>Experimental controls include: </li></ul><ul><ul><li>Locked Nucleic Acid controls </li></ul></ul><ul><ul><li>Unrelated dsRNA controls </li></ul></ul><ul><ul><li>Transfection Reagent controls </li></ul></ul>
    49. 49. <ul><li>MicroRNA-146a is decreased during production of HIV </li></ul><ul><li>The Mimic to -146a restores miR-146a activity in these cells </li></ul><ul><li>Transfection of the Mimic is expected to decrease the presence of -146a target proteins </li></ul><ul><li>Scramble is a dsRNA structure of similar properties to the 146a mimic but does not produce a mature miRNA </li></ul>The miR-146a Mimic Restores the Activity of miR-146a
    50. 50. <ul><ul><li>Transfected OM10.1 cells for 24h with miR-146a Mimic </li></ul></ul><ul><ul><li>Treated with PMA </li></ul></ul><ul><ul><li>Supernatants collected Day 1, 2, 3 post PMA-Tx </li></ul></ul>Transfection of OM10.1 Cells with Mimic
    51. 51. OM10.1 p24 Production is Increased by Mimic-146a
    52. 52. <ul><ul><li>CCL3 is a putative target of miR-146a </li></ul></ul><ul><ul><li>Transfected miR-146a Mimic </li></ul></ul><ul><ul><li>Collected supernatants for CCL-3 ELISA </li></ul></ul>Tested Effect of MicroRNA-146a on CCL3 Expression in OM10.1 Cells
    53. 53. Over-Expression of MicroRNA-146a Does Not Affect CCL-3 Production <ul><li>PMA increases CCL-3 production as expected </li></ul><ul><li>Transfection alone appears to decrease CCL-3 production </li></ul>Fold Change in pg/ml of CCL-3
    54. 54. <ul><ul><li>Other miR-146a targets will need to be tested to determine the effect of its decrease during HIV production </li></ul></ul>MicroRNA-146a Does Not Affect CCL3 Expression by OM10.1 Cells
    55. 55. <ul><ul><li>Transfected OM10.1 cells for 24h with miR-99b AntagomiR </li></ul></ul><ul><ul><li>Treated with PMA </li></ul></ul><ul><ul><li>Supernatants collected Day 1, 2, 3 post PMA-Tx </li></ul></ul>Transfection of OM10.1 Cells with AntagomiR
    56. 56. The miR-99b AntagomiR Decreases the Activity of miR-99b <ul><li>MicroRNA-99b is increased during production of HIV </li></ul><ul><li>The AntagomiR interferes with miR-99b activity in these cells </li></ul><ul><li>Transfection of the AntagomiR is expected to increase the presence of miR-99b target proteins </li></ul><ul><li>Scramble is a dsRNA structure of similar properties to the 99b AntagomiR but does not interfere with any miRNAs </li></ul>
    57. 57. OM10.1 p24 Production is Increased by AntagomiR-99b
    58. 58. <ul><li>During HIV infection, miR-99b increases </li></ul><ul><li>Data suggests that if miR-99b is decreased with an AntagomiR, viral production increases </li></ul><ul><li>C/EBP β is a putative target of miR-99b </li></ul><ul><li>C/EBP β binding at the LTR increases viral production </li></ul>What Is the Mechanism of AntagomiR-99b Increase in HIV Production?
    59. 60. <ul><li>During HIV infection, miR-99b increases </li></ul><ul><li>Data suggests that if miR-99b is decreased with an AntagomiR, viral production increases </li></ul><ul><li>C/EBP β is a putative target of miR-99b </li></ul><ul><li>C/EBP β binding at the LTR increases viral production </li></ul><ul><li>If miR-99b is decreased, C/EBP β should increase at the viral LTR, which would explain increase in the production of HIV during miR-99b knockdown </li></ul>OM10.1 p24 Production is Increased by AntagomiR-99b
    60. 61. Chromatin Immunoprecipitation (ChIP) Performed in collaboration with Loreto Carvallo C/EBP β
    61. 62. AntagomiR to miR-99b Increases the Presence of C/EBP β at the HIV LTR in Transfected OM10.1 Cells
    62. 63. AntagomiR to miR-99b May Increase and/or Stabilize the Occupancy of C/EBP β at the Viral Promoter
    63. 65. Summary of Aims <ul><li>Hypothesis: Macrophages express MicroRNA and their required machinery. MicroRNA expression is altered during HIV infection, thereby affecting viral production in and proteins produced by macrophages. </li></ul><ul><li>Macrophages produce the miRNA machinery and express 119 miRNAs </li></ul><ul><li>There is variability in the production of the miRNA machinery and miRNA expression </li></ul><ul><li>Macrophage expression is altered by HIV infection </li></ul><ul><ul><li>miR-99b: increased </li></ul></ul><ul><ul><li>miR-125a: increased </li></ul></ul><ul><ul><li>miR-146a:decresed </li></ul></ul><ul><li>Knockdown of miR-99b and over-expression of miR-146a increase viral production </li></ul><ul><li>Knockdown of miR-99b increases the presence of C/EBP β at the viral LTR </li></ul>
    64. 66. Future Directions <ul><li>Determine target for miR-146a </li></ul><ul><ul><li>What could be mediating increase in viral production during mimic transfection? </li></ul></ul><ul><ul><li>Begin by evaluating the NF- κ B pathway </li></ul></ul><ul><li>Examine putative miR-99b regulation of C/EBP β </li></ul><ul><ul><li>Is the increase in viral production a direct effect? </li></ul></ul><ul><ul><li>Reporter constructs, site mutagenesis </li></ul></ul><ul><li>Examine role of miR-125a in apoptosis </li></ul><ul><ul><li>It is known that macrophages can survive for long periods of time even when HIV-infected </li></ul></ul><ul><ul><li>Is miR-125a responsible for macrophage survival during infection with HIV? </li></ul></ul><ul><ul><li>Examination of the miR-125a~let-7e~99b cluster </li></ul></ul>
    65. 67. Conclusions <ul><li>There is variability in the production of the miRNA machinery and miRNA expression </li></ul><ul><ul><li>May be an important consideration in choosing new therapeutic targets, especially in RNA-based therapies </li></ul></ul><ul><li>Macrophage expression is altered by HIV infection </li></ul><ul><ul><li>miR-99b and miR-125a are increased, miR-146a is decreased </li></ul></ul><ul><ul><li>May be due to macrophage response to viral infection </li></ul></ul><ul><ul><li>May be due to viral activity to promote its replication cycle </li></ul></ul><ul><ul><li>These miRNAs may become an important part of strategies to alter therapeutically macrophage responses to HIV infection </li></ul></ul>
    66. 68. Conclusions <ul><li>These data underscore the importance of miRNA study in HIV-macrophages, as their alteration affects the production of macrophage proteins as well as the virus </li></ul><ul><li>Macrophage miRNA expression is an important and new field of study for the examination of gene expression alterations during HIV infection which will contribute to our understanding of the macrophage response during HIV infection </li></ul>
    67. 69. Acknowledgements <ul><li>Berman Lab </li></ul><ul><li>Dr. Joan W. Berman </li></ul><ul><li>Dr. Tina Calderon </li></ul><ul><li>Dr. Eliseo Eugenin </li></ul><ul><li>Dr. PJ Gaskill </li></ul><ul><li>Dr. Loreto Carvallo Torres </li></ul><ul><li>Lillie Lopez </li></ul><ul><li>Joy Hazleton </li></ul><ul><li>Jackie Coley </li></ul><ul><li>Dionna Williams </li></ul><ul><li>Dr. Jessie King </li></ul><ul><li>Dr. Clarisa Buckner </li></ul><ul><li>Dr. Toni Roberts </li></ul><ul><li>Funding </li></ul><ul><li>NIMH, NIDA </li></ul><ul><li>In Collaboration with </li></ul><ul><li>Dr. Olivier Loudig </li></ul><ul><li>Christina Liu </li></ul><ul><li>Advisory Committee </li></ul><ul><li>Dr. Fernando Macian </li></ul><ul><li>Dr. Ganjam Kalpana </li></ul><ul><li>Dr. Olivier Loudig </li></ul><ul><li>Dr. Moshe Sadofsky </li></ul><ul><li>Dr. Janice Clements </li></ul><ul><li>(Johns Hopkins University) </li></ul><ul><li>Thesis Committee </li></ul><ul><li>Dr. Fernando Macian </li></ul><ul><li>Dr. Moshe Sadofsky </li></ul><ul><li>Dr. Herbert Tanowitz </li></ul><ul><li>Dr. Louis Weiss </li></ul><ul><li>Dr. Kami Kim </li></ul><ul><li>Dr. Janice Clements </li></ul><ul><li>(Johns Hopkins University) </li></ul><ul><li>Sue Golding Graduate Divison </li></ul><ul><li>Dr. Victoria Freedman </li></ul><ul><li>Sheila Cleeton and Vera Rico </li></ul>Resources and Reagents NIH AIDS Research and Reference Reagent Program (HIV ADA and OM10.1 cells) AECOM Center for AIDS Research Dr. Harris Goldstein Monica Dutta Dr. Ganjam Kalpana Aldo Massimi Department of Pathology The laboratories of Drs. Sadofsky, Belbin, and Childs Dr. Michael Prystowsky Annie D’Alauro, Althea Fenton-Humphreys, Krystle Agard, Barbara Groszewski, Trina Abbate, Karen Goldman, Camilla Davis, Charissa Monteczuma, and Christine Hubertus

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