Final Report Summary - INNATE_CROSSTALK (Innate immunity crosstalk in immunoregulation)
INNATE CROSSTALK endeavoured to investigate different aspects of the often neglected innate interactions between natural killer (NK) cells and neutrophils, which are likely to occur in the early phase of an immune response. We investigated the functional consequences of these interactions: firstly, how this crosstalk modulates NK cell function and NK interactions with dendritic cells (DC) and, in this way, affects the development of adaptive immune responses; secondly, how NK - neutrophil crosstalk influences the resolution of inflammation. We also aimed to identify the ligands for the two innate immunity receptors, IRp60, which is expressed by NK cells and neutrophils, and NKp30, an activating NK cell receptor that is of importance in the NK-DC crosstalk.
To achieve our goals, we assessed the production by neutrophils of different chemokines in response to stimulation with different Toll-like receptor ligands. Supernatants from these neutrophil cultures were used in NK cell chemotaxis experiments to determine the fraction of migrating NK cells. We further preformed co-culture experiments with neutrophils and NK cells to evaluate the neutrophil-mediated changes of NK cell surface expression of different receptors. Moreover, NK cytotoxicity against different tumour target cells was assessed with or without co-incubation with stimulated neutrophils. We also addressed other aspects of NK cell function co-culture with neutrophils, such as cytokine production, responsiveness to subsequent stimulation etc. Since NK cells have been described to specifically kill mononuclear myeloid cells under certain circumstances, we developed assays to determine whether NK cells also exert cytotoxicity against neutrophils.
It is known that NK cells display specific cytotoxicity against immature DCs and LPS-activated macrophages. Within the project we have discovered that NK cells also exert cytotoxicity against normal neutrophils. Surprisingly, neutrophils were only inefficiently lysed by NK cells, and thus cytotoxicity assays dependent on loss of plasma membrane integrity (51Cr release or cell-impermant nuclear stains) failed to detect significant NK cytotoxicity. Instead, NK cell-induced cytotoxicity against neutrophils could be monitored using traditional apoptosis assays. We found that human NK cells triggered caspase-dependent neutrophil apoptosis in vitro. Neutrophil apoptosis was dependent on cell-cell contact and mediated through the NK cell receptor NKp46. Mixed lymphocyte populations had minimal effects on neutrophil survival, suggesting that the ability to induce neutrophil apoptosis was specific for NK cells. Our findings implicate that NK cells may accelerate neutrophil apoptosis through cell-cell interactions, and that such interactions could play an important role in the resolution of inflammation.
The findings raised multiple questions that will be addressed beyond the project itself as part of a long-term collaboration between the Italian hosting laboratory and the researcher, Fredrik Bergh Thoren. In ongoing studies initiated six months into the project, we are investigating NK-neutrophil interactions in a human in vivo skin chamber model: using multicolor flow cytometry, NK cells present in the aseptic inflammatory exudate of induced blisters will be phenotypically compared with those in peripheral blood, and it is anticipated that these studies to shed some light on the relevance of NK - neutrophil interactions in the resolution of inflammation. Finally, additional investigations of the elevated responsiveness of NK cells to DC-derived signals will be pursued by collaborating laboratories to clarify the potential role of NK - neutrophil interactions during the early phase of the immune response for the development of the adaptive immune response.
To achieve our goals, we assessed the production by neutrophils of different chemokines in response to stimulation with different Toll-like receptor ligands. Supernatants from these neutrophil cultures were used in NK cell chemotaxis experiments to determine the fraction of migrating NK cells. We further preformed co-culture experiments with neutrophils and NK cells to evaluate the neutrophil-mediated changes of NK cell surface expression of different receptors. Moreover, NK cytotoxicity against different tumour target cells was assessed with or without co-incubation with stimulated neutrophils. We also addressed other aspects of NK cell function co-culture with neutrophils, such as cytokine production, responsiveness to subsequent stimulation etc. Since NK cells have been described to specifically kill mononuclear myeloid cells under certain circumstances, we developed assays to determine whether NK cells also exert cytotoxicity against neutrophils.
It is known that NK cells display specific cytotoxicity against immature DCs and LPS-activated macrophages. Within the project we have discovered that NK cells also exert cytotoxicity against normal neutrophils. Surprisingly, neutrophils were only inefficiently lysed by NK cells, and thus cytotoxicity assays dependent on loss of plasma membrane integrity (51Cr release or cell-impermant nuclear stains) failed to detect significant NK cytotoxicity. Instead, NK cell-induced cytotoxicity against neutrophils could be monitored using traditional apoptosis assays. We found that human NK cells triggered caspase-dependent neutrophil apoptosis in vitro. Neutrophil apoptosis was dependent on cell-cell contact and mediated through the NK cell receptor NKp46. Mixed lymphocyte populations had minimal effects on neutrophil survival, suggesting that the ability to induce neutrophil apoptosis was specific for NK cells. Our findings implicate that NK cells may accelerate neutrophil apoptosis through cell-cell interactions, and that such interactions could play an important role in the resolution of inflammation.
The findings raised multiple questions that will be addressed beyond the project itself as part of a long-term collaboration between the Italian hosting laboratory and the researcher, Fredrik Bergh Thoren. In ongoing studies initiated six months into the project, we are investigating NK-neutrophil interactions in a human in vivo skin chamber model: using multicolor flow cytometry, NK cells present in the aseptic inflammatory exudate of induced blisters will be phenotypically compared with those in peripheral blood, and it is anticipated that these studies to shed some light on the relevance of NK - neutrophil interactions in the resolution of inflammation. Finally, additional investigations of the elevated responsiveness of NK cells to DC-derived signals will be pursued by collaborating laboratories to clarify the potential role of NK - neutrophil interactions during the early phase of the immune response for the development of the adaptive immune response.