Sridharan Ganesan

Zinc is one of the most important trace elements for life and its deficiency, like its excess, can be fatal. In the bacterial opportunistic pathogen Pseudomonas aeruginosa, Zn homeostasis is not only required for survival, but also for virulence and antibiotic resistance. Thus, the bacterium possesses multiple Zn import/export/storage systems. In this work, we determine the expression dynamics of the entire P. aeruginosa Zn homeostasis network at both transcript and protein levels. Precisely, we followed the switch from a Zn-deficient environment, mimicking the initial immune strategy to bacterial infections, to a Zn-rich environment, representing the phagocyte metal boost used to eliminate an engulfed pathogen. Thanks to the use of the NanoString technology, we timed the global silencing of Zn import systems and the orchestrated induction of Zn export systems. We show that the induction of Zn export systems is hierarchically organized as a function of their impact on Zn homeostasis. Moreover, we identify PA2807 as a novel Zn resistance component in P. aeruginosa and highlight new regulatory links among Zn-homeostasis systems. Altogether, this work unveils a sophisticated and adaptative homeostasis network, which complexity is key in determining a pathogen spread in the environment and during host-colonization.

Natural killer (NK) cell function is regulated by a balance between activating and inhibitory receptors, but the details of this receptor interplay are not extensively understood. We developed a flow cytometry-based assay system in which Ca²⁺ flux downstream of antibody-mediated activating receptor triggering was studied in the presence or absence of inhibitory receptor co-crosslinking. We show that the inhibitory influence on activating receptor-induced Ca²⁺ flux is quantitatively regulated, both on murine and human NK cells. Furthermore, both activating and inhibitory receptors operate in an additive way, suggesting that a fine-tuned balance between activating and inhibitory receptors regulate proximal NK cell signaling. We also demonstrate that murine NK cell expression of H2D^d lowered the capacity of Ly49A to deliver inhibitory signals after antibody crosslinking, suggesting that the cis interaction between H2D^d and Ly49A reduce the signaling capacity of Ly49A in this setting. Finally, we show that priming of NK cells by IL-15 rapidly augments Ca²⁺ flux after activating receptor signaling without attenuating the potential of inhibitory receptors to reduce Ca²⁺ flux. Our data shed new light on NK cell inhibition and raises new questions for further studies.

Natural killer (NK) cell responsiveness in the mouse is determined in an education process guided by inhibitory Ly49 and NKG2A receptors binding to MHC class I molecules. It has been proposed that inhibitory signalling in human NK cells involves Abl‐1 (c‐Abl)‐mediated phosphorylation of Crk, lowering NK cell function via disruption of a signalling complex including C3G and c‐Cbl, suggesting that NK cell education might involve c‐Abl. Mice deficient in c‐Abl expression specifically in murine NK cells displayed normal inhibitory and activating receptor repertoires. Furthermore, c‐Abl‐deficient NK cells fluxed Ca²⁺ normally after triggering of ITAM receptors, killed YAC‐1 tumour cells efficiently and showed normal, or even slightly elevated, capacity to produce IFN‐γ after activating receptor stimulation. Consistent with these results, c‐Abl deficiency in NK cells did not affect NK cell inhibition via the receptors Ly49G2, Ly49A and NKG2A. We conclude that signalling downstream of murine inhibitory receptors does not involve c‐Abl and that c‐Abl plays no major role in NK cell education in the mouse.

During infection and inflammation, dendritic cells (DC) provide priming signals for natural killer (NK) cells via mechanisms distinct from their antigen processing and presentation functions. The influence of DC on resting NK cells, i.e. at steady-state, is less well studied. We here demonstrate that as early as 1 day after DC depletion, NK cells in naïve mice downregulated the NKG2D receptor and showed decreased constitutive phosphorylation of AKT and mTOR. Subsequently, apoptotic NK cells appeared in the spleen concomitant with reduced NK cell numbers. At 4 days after the onset of DC depletion, increased NK cell proliferation was seen in the spleen resulting in an accumulation of Ly49 receptor-negative NK cells. In parallel, NK cell responsiveness to ITAM-mediated triggering and cytokine stimulation dropped across maturation stages, suggestive of a functional deficiency independent from the homeostatic effect. A role for IL-15 in maintaining NK cell function was supported by a gene signature analysis of NK cell from DC-depleted mice as well as by in vivo DC transfer experiments. We propose that DC, by means of IL-15 transpresentation, are required to maintain not only homeostasis, but also function, at steady-state. These processes appear to be regulated independently from each other.

Natural killer (NK) cells deliver cytotoxic granules and immunomodulatory cytokines in response to transformed and virally infected cells. NK cell functions are under the control of a large number of germline-encoded receptors that recognize various ligands on target cells, but NK cells also respond to cytokines in the surrounding environment. The interaction between NK cell receptors and their ligands delivers either inhibitory or activating signals. The cytokine milieu further shapes NK cell responses, either directly or by influencing the way inhibitory or activating signals are perceived by NK cells. In this review, we discuss how NK cell function is controlled by inhibitory receptors and MHC-I molecules, how activating receptors contribute to NK cell education, and finally, how cytokines secreted by the surrounding cells affect NK cell responsiveness. Inputs at these three levels involve different cell types and are seamlessly integrated to form a functional NK cell population.