PhD SCIENCE REPORT RICARDO RAJSBAUM

RICARDO RAJSBAUM

My PhD project relates to the Tripartite motif (TRIM) proteins, which have been shown to be involved in many cellular functions including cell differentiation, apoptosis, cytokine signalling and some have antiviral activity. I have performed a comprehensive analysis of expression of TRIM molecules in cells of the innate and adaptive immune system. The cells used for my study include macrophages, myeloid and plasmacytoid dendritic cells (DC), and a panel of CD4+T cells (naïve T cells, Th1, Th2, and CD25+T regulatory cells and IL-10 T regulatory cells). My work up to date has revealed that TRIM molecules can be grouped in clusters based on their patterns of mRNA expression in the different cell populations. My main work shows that a group of TRIMs that share similar structural features, namely the COS-FN3 domains important in protein-protein interactions and localization to the microtubules, are preferentially expressed in the CD4+ T cell subsets, suggesting they may share a common function in these cells. On the other hand, a different cluster of TRIMs is constitutively expressed at high levels in plasmacytoid dendritic cells (pDC). Conversely, I found that a different group containing a large number of TRIM genes are induced in primary mouse macrophages, myeloid DC (mDC) and pDC upon influenza virus infection in a type-1 IFN dependent manner, suggesting an anti-viral function. However, stimulation of macrophages and mDC with LPS and double stranded RNA also led to type-1 IFN dependent up-regulation of these TRIM genes, demonstrating that their expression is not directly regulated by the virus, and that they may have broader functions in innate immune responses. A subgroup of the TRIMs induced in macrophages and DC by viruses in a type-I IFN dependent manner, mapped to mouse chromosome 7, which is syntenic to human chromosome 11, where TRIMs with known anti-viral activity are localised, suggesting that they may have co-evolved to combat viruses. Our study highlights the importance of TRIM molecules as effectors of the innate immune response.
During this project I was able to analyse the large and complex data obtained as a result of my training during the Mugen Course for microarray analysis run by Ricardi-Castagnoli, and forms the basis for analysis of the data obtained in the following microarray study. As part of this work, I have also contributed in work done in our lab showing that certain MAP kinases are involved in the regulation of IL-10 and IL-12 as well as IFN?. In support of this we have performed some microarray analysis of macrophages and DC from different genetic backgrounds of mice, stimulated with CpG, in the presence or absence of a MEK inhibitor, to identify downstream transcription factors involved in the regulation of IL-10, IL-12, and IFN?, in collaboration with P. Ricciardi-Castagnoli.
The following work has been done so far:
a)Microarray analysis of unstimulated and anti-CD3/CD28 stimulated T cells (naïve CD4+ T cells, Th1, Th2, IL-10Treg, CD25+Treg)(done by a previous PhD student in the lab: John Shoemaker. Analysis of TRIM proteins done by me in this study). Validation by quantitative RT-PCR (TaqMan). Analysis of cytokine protein expression by ELISA and FACS (intracellular staining), and mRNA by TaqMan.

b)Preparation of antigen presenting cells (macrophages, myeloid dendritic cells (mDC) and plasmacytoid dendritic cells (pDC). Stimulation of these cells with LPS, CpG, Poly I:C and infection with influenza virus. Analysis of TRIM mRNA expression by TaqMan and cytokine analysis by ELISA and TaqMan.

c)Analysis of TRIM mRNA expression and cytokine protein in TRIF -/-, and wild type macrophages.

d)Analysis of TRIM mRNA expression and cytokine protein in IFNR1 -/- and wild type bone marrow macrophages, myeloid DC (BM-mDC) and pDC.

e)Data analysis and data presentation by generating heat maps of TRIM mRNA expression to identify clusters or groups of TRIMs co-expressed in each condition.

f)Phylogenetic analysis of TRIMs used in this study and correlation with the expression patterns obtained for each group of TRIMs.

g) Microarray analysis of macrophages from different genetic backgrounds of mice, stimulated with CpG, in the presence or absence of a MEK inhibitor.

I have Finished the writing of the Thesis and it has been submitted to the examiners.
My PhD examination will take place on January 15th, 2009.
After my PhD I intend to continue my research training as a post-doc and work related to the collaboration in DC-THERA.

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