Neutrophil extracellular traps : function in infectious and non-Infectious conditions

Neutrophil extracellular traps (NETs) are composed of a backbone of chromatin, decorated with microbicidal peptides and proteolytic enzymes, and are used by neutrophils as a weapon against pathogens. Since their initial discovery in 2004, NETs have challenged scientists in terms of the nature of the triggers and the mechanisms of neutrophil release of NETs, as well as the implication of these extracellular structures in infectious and non-infectious human diseases. The work presented in this thesis, performed in an interdisciplinary perspective using an array of different methodologies, aims to deepen the knowledge on the functionality and dysfunctionality of NETs, including the mechanism of NET disposal and its immunological consequences. Once NETs have fulfilled their anti-microbial function, the timely clearance of these structures is needed in order to avoid a misguided autoinflammatory response.

In Paper I, we showed that primary human macrophages employ TREX1, while dendritic cells use DNase1L3 to digest NETs purified from activated neutrophils. In addition, on the basis of cytokine profiling, we showed that NETs have immunomodulatory effects on phagocytes. In Paper II, we showed that JAGN1 is required for efficient fungal killing in NETs. Patients suffering from severe congenital neutropenia (SCN) present homozygous mutations in the gene encoding JAGN1 and are susceptible to bacterial and fungal infections. We found that JAGN1-deficient neutrophils isolated from an SCN patient, as well as neutrophil-like HL-60 cells with silenced JAGN1 expression, released NETs, but the expression of myeloperoxidase (MPO) was altered. In Paper III and IV we explored the interactions of NETs with graphene oxide (GO), a carbon-based 2-D material. Using ToF-SIMS, a mass spectrometry-based surface analytical method, we could show in Paper III that GO interacts with the plasma membrane of neutrophils, promoting cholesterol oxidation. In addition, we could define a size-dependent mechanism of GO induced release of NETs. Furthermore, we showed in Paper IV that GO undergoes efficient extracellular degradation through neutrophil degranulation or in NETs in a process dependent on MPO. Moreover, intermediate degradation products of GO did not cause DNA damage in lung cells.

Overall, the work presented in this thesis has shed light on the clearance and degradation of NETs by phagocytic cells and the involvement of several endonucleases in a cell type-specific manner, and has confirmed the importance of NETs in fungal killing, with new evidence for a role of JAGN1 in this process. In addition, we have shown for the first time that GO triggers NETs and reported that GO may also undergo degradation in NETs in a similar manner as pathogens. Our studies have thus revealed that MPO expressed in NETs is a key element in eliminating infectious as well as non-infectious agents.

List of scientific papers

I. Lazzaretto, B. and Fadeel, B. Intra- and extracellular degradation of neutrophil extracellular traps by macrophages and dendritic cells. 2018. [Manuscript]

II. Khandagale, A., Lazzaretto, B., Carlsson, G., Sundin, M., Shafeeq, S., Römling, U. and Fadeel, B. JAGN1 is required for myeloperoxidase-mediated fungal killing in neutrophil extracellular traps. 2018. [Manuscript]

III. Mukherjee, S.P., Lazzaretto, B., Hultenby, K., Newman, L., Rodrigues, A.F., Lozano, N., Kostarelos, K., Malmberg, P. and Fadeel, B. Graphene oxide elicits membrane lipid changes and neutrophil extracellular trap formation. 2018. Chem. 4: 334-358.
https://doi.org/10.1016/j.chempr.2017.12.017

IV. Mukherjee, S.P., Gliga, A.R., Lazzaretto, B., Brandner, B., Fielden, M., Vogt, C., Newman, L., Rodrigues, A.F., Shao, W., Fournier, P.M., Toprak, M.S., Star, A., Kostarelos, K., Bhattacharya, K. and Fadeel, B. Graphene oxide is degraded by neutrophils and the degradation products are non-genotoxic. 2018. Nanoscale. 10: 1180-1188.
https://doi.org/10.1039/c7nr03552g