Macrophages And Natural Killer cells INteractions During intracellular bacterial infections – MANKIND

Infectious diseases are responsible for ˜30% deaths worldwide. Interactions between phagocytes and lymphocytes are key to orchestrate efficient antimicrobial responses. Here, we focus on the cross-talk between infected macrophages and Natural Killer (NK) cells and on the central role of Interferon-gamma (IFN-g) in controlling intracellular bacterial infections. Thanks to innovative vectorology strategies, we will perform two Clustered Regularly-Interspaced Short Palindromic Repeats (CRISPR)/Cas9 genome-wide screens in primary human cells to decipher 1) the antimicrobial role of IFN-g in macrophages against cytosolic bacteria 2) the regulatory network controlling IFN-? production in NK cells in response to cytokines (IL-12 and IL-18) produced by infected macrophages.
Francisella novicida, a close relative of Francisella tularensis, the agent of tularemia is a bacterial pathogen replicating in the macrophage cytosol. This bacterium elicits AIM2 inflammasome activation and secretion of IL-12 and IL-18. NK cells are among the first cells responding in a synergistic manner to these two cytokines to produce large amount of IFN-g. Surprisingly, the signalling downstream of the IL-12 and the IL-18 receptors and the synergy of these two cytokines are still poorly understood. IFN-g, in turn, activates the macrophage antimicrobial response leading to the restriction of the bacterial growth. While the IFN-inducible mechanisms restricting the growth of intracellular bacteria replicating in a membrane-bound compartment are well understood, the mechanisms restricting bacterial growth in the host cytosol are still largely unknown. Guanylate binding proteins (GBPs) have been recently shown to kill cytosolic bacteria. The mechanism by which GBPs are targeted to cytosolic bacteria and their bacteriolytic mechanism remain unclear. Of note, the lack of knowledge on the IL-12/IL-18 signalling and the cytosolic antimicrobial activity is particularly striking in human cells.
The CRISPR/Cas9 is an extraordinary tool to investigate in an unbiased manner these questions. Yet, there are a number of technological challenges associated with genome-wide CRISPR/Cas9 screens especially in primary human cells. Innovative vectorology strategies will be developed to ensure an efficient delivery of Cas9 and its associated guide RNA (gRNA) to primary human cells. Then two screens will be performed to understand i) how IL-12 and IL-18 synergize in NK cells to induce IFN-g ii) how IFN-g kills cytosolic bacteria in F. novicida-infected macrophages. The most interesting hits from these screens will be further characterized in vitro in human cells and in a mouse model of tularemia where the cross-talk between NK cells and infected macrophages can be assessed in a physiological context. These screens will identify the bacteriolytic mechanisms taking place in the host cytosol and will reveal the complex signalling network controlling IFN-? production. In addition to developing safe and efficient means to vectorize the Cas9 enzyme and its gRNA, the obtained results should pave the way for the development of novel therapies targeting intracellular bacteria and auto-inflammatory syndromes associated with deregulated IFN-? production.