How does the interferon-induced protein CALHM6 facilitate cell-cell cross-talk to boost an innate immune response?

1. IMPORTANCE: Macrophages instruct the functions of other innate cells to coordinate the type and timing of inflammatory responses. We recently identified CALHM6 as an interferon- and TLR-inducible membrane protein on macrophages, important for macrophage-mediated instruction of early innate responses to infections. Proteins of the CALHM family have only been characterised in the neuronal system, where they act as ATP-permeable ion channels. They promote cell-cell crosstalk at neuronal synapses by releasing a small molecule ATP, which acts as a neurotransmitter. CALHM6 is the only CALHM family member highly expressed in immune cells. It has been linked to the induction of natural killer (NK) cell anti-tumour activity. The mechanism of action and broader functions of CALHM6 in the immune system were unclear. We generated CALHM6-deficient mice and found that CALHM6 controlled infection-induced innate cell cross-talk at the immunological synapse, and was required for the early control of Listeria monocytogenes infection in vivo. CALHM6 expression was highest on activated macrophages in the presence of pathogen-derived signals or interferons. CALHM6 expression was transient and transcriptionally terminated by anti-inflammatory cytokines. Mechanistically, our patch clamp studies in Xenopus oocytes revealed that CALHM6 can form a membrane ion channel, in which a conserved acidic residue E119 controlled channel opening. When overexpressed in Xenopus oocytes, CALHM6 was constitutively open and toxic to cells. In mammalian cells, however, CALHM6 localized to the inside of the cell and, hence, no channel activity or toxicity was detected or reported. We found that only upon macrophage activation does CALHM6 rapidly re-localise from the intracellular compartment to the immune synapse, where it controls the early kinetics of natural killer cell activation in vivo and innate defences from infection. How is CALHM6 recruited to the synapse? Does CALHM6 form a synaptic ATP channel in activated macrophages? What functional programmes does CALHM6 regulate in NK cells? These key questions form the focus of this proposal. 2. HYPOTHESIS AND PROJECT AIMS: Hypothesis: In activated macrophages, CALHM6 translocates to the immune synapse to form a metabolite-permeable membrane channel important for natural killer cell activation. We will use bone marrow-derived macrophages from our WT and Calhm6-/- mice reconstituted WT or E119R channel-dead mutant control to address the following Aims: Objective 1: Generate a time-resolved map of CALHM6 trafficking in resting and activated macrophages as they form immune synapses with natural killer cells. Objective 2: Identify CALHM6 interactome in the resting state and upon synapse formation, to understand the molecular basis for intracellular retention and activation-induced relocalisation. Objective 3: Test whether membrane-targeted CALHM6 can form a metabolite-permeable synaptic channel and define CALHM6-dependent functional programmes.