Neutrophils as social network cells

Neutrophils have long been viewed as the final effector cells of an acute inflammatory response only, exerting a primary role in the killing and clearance of extracellular pathogens. However, more recent observations have provided solid evidence that neutrophils can exert a wide variety of functions. For instance, the newly discovered repertoire of effector molecules that neutrophils possess in their armamentarium allows them to play a key role in the activation and regulation of innate and adaptive immune cells. Accordingly, it has been shown that neutrophils are part of the networks that form the foundations of immunity, in this manner dictating instructions to many immune cells. Although an increasing body of literature corroborates the neutrophil capacity to interact with immune cells (e.g., monocytes, macrophages, dendritic cells (DCs), etc.), our knowledge of cross-talks involving neutrophils remains not completely characterized. For such a reason, during my doctoral studies, I focused on how human neutrophils establish novel interactions with: different classes and subsets of T lymphocytes (TASK 1); natural killer cells (TASK 2). TASK 1. In the first part of my project, I have described the interactions that neutrophils engage with cytotoxic CD8+ T lymphocytes, CD4+ T helper (Th) lymphocytes and the Th17 subpopulation. In particular, I show how anti-CD3-activated CD4+ T cells and, more potently, anti-CD3-activated CD8+ T cells, positively modulate neutrophil survival and expression of activation markers, such as CD11b, CD64 and CD62L. The effects mediated by activated T cells occur independently from direct cellular interactions, as they are mainly attributable to their capacity to release tumor necrosis factor-alpha (TNF-a), interferon-gamma (IFN-g) and granulocyte-macrophage colony-stimulating factor (GM-CSF). Moreover, I describe that Th17 cells favor the survival and functionality of neutrophils, effects that are, again, attributable to Th17-derived GM-CSF, IFN-g and TNF-a cytokines and not to interleukin-17 (IL-17), as initially presumed. A further important observation is that neutrophils and Th17 cells, once appropriately activated, secrete chemokines that induce their reciprocal recruitment. Consistent with the results obtained in vitro, neutrophils and Th17 cells were found to co-localize in gut tissue from Crohn’s disease and in the synovial fluid (SF) of rheumatoid arthritis (RA) patients. TASK 2. The second part of my thesis describes the reciprocal interactions that human neutrophils establish with NK cells. Indeed, I show how activated NK cells strongly protect the survival of neutrophils as well as potently up-regulate the expression of membrane molecules (CD64, CD11b and CD69). Analysis of the molecular events occurring in neutrophil/NK co-cultures has revealed that: a) neutrophil survival is positively affected by NK-derived GM-CSF; b) NK-derived IFN-g is almost entirely responsible for the induction of CD64; c) NK-derived IFN-g and GM-CSF, as well as direct neutrophil-NK cell-cell contact, up-regulate CD11b and CD69 in neutrophils. Finally, I also report the characterization of a complex network that neutrophil establish with both NK cells and a recently described subtype of circulating myeloid DC, namely the 6-Sulfo LacNac+ DCs (slanDCs). Specifically, I show that stimulated neutrophils directly interact with, and potentiate the activity of, both slanDCs and NK cells. As a result, neutrophils potentiate the production of IL-12p70 by slanDCs via a CD18/ICAM-1 interaction, which, in turn, stimulates activated NK cells to produce IFN-g. IFN-g further reinforces the interaction between neutrophils and slanDCs and the release of slanDC-derived IL-12p70, thus creating a positive feedback loop. On the other hand, neutrophils directly co-stimulate NK cells via CD18-CD11d/ICAM-3 interaction, leading to an increased production of IFN-g. All these in vitro data are also supported by additional results showing that neutrophils, NK cells, and slanDCs co-localize in inflamed tissues of both Crohn’s disease and psoriasis patients. In summary, the data collected in my thesis help to extend our knowledge on the complexity of all the possible neutrophil-leukocyte interactions that might regulate the immune response under physiological and/or pathological conditions.