Our work identified a protein of hitherto unknown function, TRAF3, as part of the Toll-like receptor (TLR) signaling pathway. It investigates the function of TRAF3 during immune cell activation and shows its specific role in regulation of type I interferons (IFN-I) and Interleukin-10 (IL-10). Both IFN-I and IL-10 play important roles during immune responses. IFN-I is critical for the immune defense against viruses, while anti-inflammatory IL-10 protects us from potentially harmful pro-inflammatory activities of activated immune cells. The functional characterization of TRAF3 as a signaling molecule in this pathway contributes important information to the question of how TLRs control specific, yet diverse, effector functions of the immune system.

“Our work identified a molecule of hitherto unknown functions, TRAF3, as part of this intracellular signaling pathway.”

The paper describes two new methodologies. First, it uses a novel approach to purify signaling complexes as they are marshaled by adaptor proteins, such as the TLR-signaling molecule MyD88. This approach took advantage of former work by M. Farrar and colleagues, who established a system to induce artificial dimerization of molecules inside of living cells. When hooked up to MyD88 and provided with an additional epitope tag, this system allowed us to purify the TLR signaling complex and identify novel components, such as TRAF3 by mass spectrometry.

Second, it uses a new method, very recently described by M. Kamps and colleagues, which can be used to conditionally immortalize myeloid progenitor cells. This allowed us to generate sufficient amounts of immune cells (macrophages) from perinatally lethal, TRAF3-deficient mice.

Humans are constantly exposed to and threatened by various infectious agents, such as viruses and bacteria. The major function of the immune system is to recognize these pathogens and orchestrate an immune response, which ultimately resolves the infection.

There is a considerable amount of knowledge on how cells of the immune system recognize pathogens. Several cell membrane receptors, e.g., the so-called "Toll-like" receptors, which directly recognize parts of pathogens, have been characterized. However, it is still largely unknown how these receptors transduce and translate the signal from the cell surface into effector functions, such as production of cytokines, which are important regulators of the immune response.

Our work identified a molecule of hitherto unknown functions, TRAF3, as part of this intracellular signaling pathway. Moreover, we found that TRAF3 is specifically involved in regulation of cytokines called type I interferons (IFN-I) and Interleukin-10 (IL-10). IFN-I is critically involved in our immune defense against viruses, while IL-10’s main function is to limit ongoing immune responses in order to protect us from potentially harmful inflammatory processes as they occur during immune cell activation.

Notably, the same pathways used for regulation of immune responses during natural infections are also important for generation of immune responses during vaccination and are, most likely, also involved in pathological immune responses, e.g., autoimmune diseases or sepsis. To understand these signaling pathways in more detail will be the basis in designing strategies for the therapeutic manipulation of immune cell functions, either as part of a prevention plan (vaccination) or therapeutic intervention during infectious disease (sepsis).

Our work covers a period of about five years. It originates from my previous work with pathogen-derived, unmethylated DNA, a strong activator of innate immune cells and our finding that DNA activates cells through a molecule called MyD88, which later was found to be an important component of the signaling pathway engaged by TLRs—receptors that are used to recognize pathogens.

To determine its molecular mechanism of action and identify downstream mediators, we adopted the above-mentioned method to initiate dimerization of molecules inside of living cells and were soon able not only to imitate receptor-mediated cell activation, but also to purify the (artificially) activated signaling complex.

Still working in the labs of H. Wagner and G. Häcker at the Technical University Munich, Germany, I sought to test whether it might be possible to use such an approach to purify signaling complexes in a systematic way. This project led me to join the lab of M. Karin at UCSD, La Jolla, California, where we first developed the biochemical aspect of the project.

The mass spectrometry analysis of purified protein samples was performed by B. Blagoev and I. Kratchmarova in M. Mann’s lab at the CEBI, Odense, Denmark. One protein identified was TRAF3, which was then functionally characterized and compared to TRAF6, a protein already known to be involved in this pathway.

For our functional analyses we took advantage of already established TRAF3-deficient mice and cells, which were provided by G. Cheng and his colleagues G. Oganesyan and S.K. Saha, as well as TRAF6-deficient mice, which were provided by J. Inoue.

Although these cells could already be used for many immunological assays, which were performed in collaboration with V. Redecke in E. Raz’s lab at UCSD, some of the biochemical assays required larger amounts of cells. Coincidentally, M.P. Kamps and his colleague G.G. Wang, also at UCSD, had just developed a technique to generate conditionally immortalized, myeloid progenitor cells, which can inducibly be differentiated into macrophages. This allowed us to generate sufficient amounts of TRAF3- and TRAF6-deficient immune cells, which could then be used for our assays.

The project was based on a novel technique and, expectedly, we did encounter many problems, primarily of a biochemical nature. However, most of these problems could indeed be resolved and, as detailed above, often in collaboration.

I don’t think that our research has any immediate social or political implications beyond the one any medical research has, i.e., to further human life and human society by contributing to the prevention or cure of diseases.