Damage Associated Molecular Pattern Molecule Laboratory: Role of immune and inflammatory cells in the response to unscheduled cell death: implications for immunotherapy of chronic inflammatory diseases including cancer

Description of projects available to graduate students:
It is becoming increasingly clear that the development of invasive cancer is not simply the result of genetic changes in the transformed cell but is critically dependent upon the coordinate function of infiltrating immune and stromal cells. A growing number of studies suggest that development of chronic inflammation may be responsible for the disordered tumor microenvironment that favors tumor progression, based on release by the tumors of so called Damage Associated Molecular Pattern Molecules [DAMPs]. HMGB1 is the best characterized DAMP A rapidly growing body of literature supports the dual function of the DNA binding protein HMGB1 as regulator of intracellular transcription and as an extracellular cytokine/inflammatory mediator. HMGB1 can be released into the extracellular space under two conditions: 1) HMGB1 is acetylated, translocated into the cytosol, and actively secreted from macrophages that have been exposed to either LPS, TNF α, or IL-1. 2) HMGB1 is also released by cells dying nonapoptotic (unscheduled) cell death. HMGB1 is loosely associated with chromatin in normal cells but in cells undergoing apoptosis chemical alteration of the DNA results in tight binding that prevents its release into the extracellular space. Cells dying alternative deaths (necrotic) do not modify their DNA to bind HMGB1, this results in it being liberated into the extra-cellular milieu. Once liberated into the extracellular space HMGB1 mediates 1) endothelial cell activation 2) angiogenesis, 3) stem cell migration and perhaps most importantly 4) activation of the innate immune effectors. HMGB1 results in activation of multiple important innate immune cell effectors including macrophages, neturophils, and myeloid and plasmacytoid dendritic cells. Treatment of human neutrophils with HMGB1 results in release of inflammatory cytokines IL-6, IL-8,IL-1β and TNFα. HMGB1 matures myeloid dendritic cells alone and synergistically with TNF-α or ATP. The 60 amino acid B box domain of HMGB1 upregulates CD40, CD54, CD80, CD83, and MHC Class II molecules on the surface of immature myeloid DC. HMGB1 also increases production of the pro-inflammatory cytokines/dendrikines IL-12, IL-6, IL-1α, IL-8. When released from necrotic cells, HMGB1 serves as an endogenous adjuvant enhancing cellular and humoral immune responses. Treatment with recombinant HMGB1 converts poorly immunogenic apoptotic lymphomas into efficient vaccines. In our hands rHMGB1 suppresses alpha interferon release from plasmacytoid DCs.
Recent work from David Baltimore`s lab has demonstrated that the monocyte derived cell line THP-1 stimulated with LPS (a pathogen-associated molecular pattern molecule [PAMP)) upregulates hsa-miR-146a. An NF-κB-regulated gene, one of the functional targets of hsa-miR-146 was shown to be TRAF6, an intermediate in TLR4 signaling. Several other important transcription factors known to be important for myeloid differention, including PU.1/Spi-1 and IRF-8 are also activated through specific changes in miRNA when monocytes are stimulated by a PAMP, such as LPS. Like the PAMP, LPS, we believe that the DAMP, HMGB1, will lead to a specific and unique pattern of microRNAs up regulation in monocytes
The currently active projects include:

- CELLULAR INTERACTIONS BETWEEN NK, MACROPHAGES, EOSINOPHILS, AND DC POLARIZATION IN RESPONSE TO DAMPs
- USE OF NK POLARIZED DC TO INDUCE THERAPEUTIC RESPONSES AGAINST CANCER

- FUNCTIONAL MODULATION OF PDC AND MDC BY DAMPs
- IDENTIFICATION AND VALIDATION OF miRNAs IN INFLAMMATORY CELLS
- DEMONSTRATION OF DAMP INHIBITION STRATEGIES TO AFFECT MURINE CANCERS

Techniques graduate student will learn:
Techniques graduate student will learn:
• Isolation of immune cells from peripheral blood and bone marrow (human, mouse)
• Cell culture techniques (generation of dendritic cells, macrophages and eosinophils from blood in vitro induction of primary responses of CD8 and CD4 T cells; generation of Ag-specific CD4+ T cell lines, Th1 and Th2 clones; generation of NK cells from bone marrow precursors)
• In vitro analysis of the interactions between different immune cell types (DC, CD8 T cells, CD4 T cells, NK cells, eosinophils, tumor cells)
• FACSArray and Imaging Cytometric-based analysis of cell surface phenotype.
• Cytokine production tests: intracellular staining, ELISA, ELISPOT, RT-PCR.
• Antigen presentation assays.
• miRNA isolation and identification; siRNA knockdown of gene expression
• Imaging and flow-based apoptosis, autophagy, and necrosis assays