Non-cytotoxic Cardiac Innate Lymphoid Cells Are a Resident and Quiescent Type 2-Commited Population

Innate lymphoid cells (ILC) are a subset of leukocytes with lymphoid properties that lack antigen specific receptors. They can be stimulated by and exert their effect via specific cytokine axes, whereas Natural Killers (NK) cells are the only known cytotoxic member of this family. ILCs are considered key in linking the innate and adaptive response in physiologic and pathologic environments. In this study, we investigated the properties of non-cytotoxic cardiac ILCs in physiologic, inflammatory, and ischemic conditions. We found that in healthy humans and mice, non-cytotoxic cardiac ILCs are predominantly a type 2-committed population with progenitor-like features, such as an absence of type-specific immunophenotype, intermediate GATA3 expression, and capacity to transiently express Pro-myelocytic Leukemia Zinc Finger protein (PLZF) upon activation. During myocarditis and ischemia, in both human and mice, cardiac ILCs differentiated into conventional ILC2s. We found that cardiac ILCs lack IL-25 receptor and cannot become inflammatory ILC2s. We found a strong correlation between IL-33 production in the heart and the ability of cardiac ILCs to become conventional ILC2s. The main producer of IL-33 was a subset of CD29+Sca-1+ cardiac fibroblasts. ILC2 expansion and fibroblast-derived IL-33 production were significantly increased in the heart in mouse models of infarction and myocarditis. Despite its progenitor-like status in healthy hearts, cardiac ILCs were unable to become ILC1 or ILC3 in vivo and in vitro. Using adoptive transfer and parabiosis, we demonstrated that the heart, unlike other organs such as lung, cannot be infiltrated by circulating ILCs in adulthood even during cardiac inflammation or ischemia. Thus, the ILC2s present during inflammatory conditions are derived from the heart-resident and quiescent steady-state population. Non-cytotoxic cardiac ILCs are a resident population of ILC2-commited cells, with undifferentiated progenitor-like features in steady-state conditions and an ability to expand and develop pro-inflammatory type 2 features during inflammation or ischemia

Mechanisms of phagosome formation, maturation and acidification, implications for intracellular infection

grantor: University of TorontoTuberculosis is the leading infectious cause of death worldwide, and is characterized by the intracellular survival 'Mycobacterium tuberculosis ' (MTB) within host phagocytes. Internalized pathogens become sequestered into vacuoles called phagosomes, which normally mature rapidly to become acidic, microbicidal organelles. MTB thwarts phagosomal maturation and therefore survives within the phagosome. To understand and treat infection with MTB, it is essential to understand the mechanisms governing phagocytosis, phagosomal acidification, and phagosomal maturation. Two mechanisms essential for the process of Fc receptor-mediated phagocytosis are described. In the first, it was determined that the cytoskeletal rearrangements and intracellular signalling cascade that occur in response to Fc g -receptor ligation require the small GTPase Rho. Inhibition of Rho by microinjection of macrophages with C3 exotoxin altered cell morphology, and impaired the ability of Fc g receptors to cluster within the membrane, thereby inhibiting the signalling in response to particle adherence. Secondly, we determined that contrary to current dogma, phagocytosis was accompanied by an increase in cell surface area, suggesting the concomitant occurrence of exocytosis. Selective cleavage of components of the secretory machinery profoundly inhibited phagocytosis, indicating that exocytosis of endomembranes is essential for particle internalization. To determine the mechanisms governing phagosomal acidification, we investigated whether proton transporters active at the cell surface were present and functional in phagosomal membranes. We found that both the sodium-proton exchanger and the vacuolar type H+-ATPase were expressed and functional on phagosomes, although the V-ATPase predominated in acidifying phagosomes containing inert and mycobacterial particles. Finally, two protein family members with potential roles in phagosomal maturation were studied. First, we identified that three isoforms of syntaxins, which are general regulators of vesicular transport, were expressed in macrophages and recruited to the phagosome. Second, we examined the effects of the Natural Resistance Associated Macrophage Protein (Nramp), which confers resistance to murine mycobacterial infection. We found that expression of Nramp in phagosomes containing live mycobacteria resulted in increased phagosomal acidification compared to Nramp-deficient macrophages. This effect was associated with increased phagosome-lysosome fusion and increased delivery of V-ATPases to the mycobacterial phagosome, demonstrating that Nramp plays a central role in phagosomal maturation.Ph.D