Nir Hacohen

Center for Immunology and Inflammatory Diseases
Massachusetts General Hospital
149 13th St., Room 8410
Charlestown, MA 02129
Tel: 617-724-3768
Lab: 617-724-3781; 617-724-0916
Fax: 617-726-5651

Broad Institute of Harvard and MIT
7 Cambridge Center
Cambridge MA 02142
617-324-4543

e-mail: nhacohen@partners.org

Web pages: http://www.broad.mit.edu/genome_bio/trc
http://www.massgeneral.org/rai/index.asp?page=research&subpage=hacohen
3 Postdoctoral Fellows, 3 Graduate Students, 1 Undergraduate Student

Pathogen-sensing by the innate immune system. The immune system can normally distinguish and respond appropriately to a broad diversity of pathogens and antigens that it encounters during a lifetime – including the products of bacteria, fungi, viruses and mammalian cells. Dendritic cells (DCs) play an important role in this process through their highly developed machinery to sense and engulf pathogens and to process and present digested peptides to T cells. We have previously shown that dendritic cells exhibit unique gene expression signatures, including specific cytokine, chemokine and costimulator expression, in response to distinct pathogens. We are interested in addressing the following kinds of questions: (1) What are the mechanisms by which each innate sensor induces specific and common signatures of gene expression? (2) What is the role of innate sensory pathways in the induction of protective immunity to infections and tumors? (3) How are these pathways dysregulated under some conditions, leading to inflammatory disorders or autoimmune disease? To address these questions, we utilize genetic, biochemical and cell biological approaches to systematically dissect the genetic circuitry of pathogen-sensing pathways and their role in initiating and guiding immune responses.

RNAi library for loss-of-function genetics in mammalian cells. Genetic screening in lower organisms has been the basis of critical discoveries across many fields. To develop a method for systematic genetic screens in mammals, we have: (a) generated genome-wide lentiviral shRNA libraries targeting human and mouse genes, in collaboration with several laboratories at the Broad Institute (Moffat et al., 2006); (b) developed high-throughput protocols to generate viral particles and infect cells in order to enable large-scale loss-of-function screens in mammalian cells; (c) demonstrated that these viruses can infect primary dendritic cells (and many other immune and non-immune cell types) and silence genes that control DC functions. As the Broad Institute RNAi Consortium and Platform continues to refine this technology and develop applications to many biological systems, we have begun to apply this powerful methodology to dissect the circuitry of the innate immune system.

What are the genes that control dendritic cell maturation and antigen presentation in response to microbes and their products? Dendritic cells exhibit an ordered cascade of processes during maturation including: pathogen recognition, engulfment and destruction; antigen processing and presentation; cytokine, chemokine and costimulator production; migration to draining lymph nodes; and finally, CD4 and CD8 T cell engagement. How are these processes regulated by TLR and other pathogen sensors upon exposure of DCs to microbes or their products? What are the pathway branchpoints that confer specificity of output? How are these pathways self-limiting? (1) To identify genes and pathways required in these processes, we have infected mouse bone marrow-derived DCs with thousands of lentiviruses from the RNAi library and assayed several cell biological processes quantitatively, seeking to identify pathways that mediate induction of DC cytokines and costimulators in response to Gram-negative bacteria and their components. A screen for genes that control IL-12/23 production has recovered known genes (including IkB, NF-kB subunits, IRAK and MAPK family members, PU.1 and others) and identified novel genes that regulate these processes. The development of an effective system to reveal gene function in primary DCs now provides us a unique approach to address several important questions in the field. (1) Our first screen is aimed at identifying mediators that account for the specificity and quantitative regulation of responses to innate immune stimulators, such as LPS, dsRNA, CpG DNA and other ligands that engage members of the Toll-like receptor family of sensors. (2) In a second screen in DCs, we have been searching for genes that mediate or regulate cross-presentation of microbial antigens on MHC Class I, a process that is not understood at a mechanistic level. (3) In a third screen, we are dissecting the pathways that mediate the sensing of viral and self nucleic acids. This system is important for protecting the host from viral infections but may also be switched on inappropriately under some conditions to induce autoimmunity. (4) Finally, to complement the RNAi screening approach, we are collecting genome-wide measurements of cell states in response to innate immune ligands to help us reconstruct the underlying genetic networks.

What are the host factors involved in detection and replication of RNA viruses? While most viral genomes encode for proteins that mediate viral entry, replication and assembly, viruses are still dependent on host factors for their life cycle within cells. The genome of RNA viruses, for example, is synthesized, processed, transported and packaged within a host cell. For many of these processes, the identity and function of the host factors are not known. In addition, RNA viruses are detected by a system of RNA sensors that trigger host defenses. We are using an RNAi-based screen to comprehensively identify host gene products that play a role in: (1) innate immune sensing of RNA and RNA viruses and (2) the life cycle of RNA viruses. We are currently focusing our efforts on the segmented negative-stranded influenza A virus and have identified several essential host factors that confer susceptibility or resistance to influenza infection in human lung epithelial cells.

Papers & Publications:

Huang Q, Liu D, Majewski P, Schulte LC, Korn J, Young RA, Lander E, Hacohen N. The plasticity of dendritic cell responses to pathogens and their components. Science 2001; 294:870-875

Alcamo E*, Hacohen N*, Schulte LC, Rennert P, Hynes R, Baltimore D. Requirement for the NF-kB family member RelA in the Development of Secondary Lymphoid Organs. J. Exp. Med. 2002; 195:233-244.

Moffat J, Grueneberg D, Yang X, Kim SY, Kloepfer AM, Hinkle G, Piqani B, Eisenhaure TM, Luo B, Grenier JK, Carpenter AE, Foo SY, Stewart SA, Stockwell BR, Hacohen N*, Hahn WC*, Lander ES*, Sabatini* DM, Root DE*. A lentiviral RNAi library for human and mouse genes applied to an arrayed viral high-content screen. Cell. 2006; 124:1283-98

Root DE, Hacohen N, Hahn WC, Lander ES, Sabatini DM. Genome-scale loss-of-function screening with a lentiviral RNAi library. Nat Methods. 2006; 3:715-19