Investigating the role of neutrophils in Leishmania mexicana infection

Neutrophil NETs have been a keen area of research in the few years since they were initially described. As well as apoptosis and necrosis, it was discovered that neutrophils also undergo a process known as NETosis by which DNA, histones and granule proteins are expelled from within the cell to form traps capable of killing fungi and bacteria. Here, we investigated the role of neutrophil NETs in response to infection with Leishmania mexicana. Using murine bone marrow derived neutrophils and dendritic cells, we used immunofluorescent staining of known NET markers and were able to identify that neutrophil NETs were capable of the capture of L. mexicana promastigotes following stimulation of neutrophils with PMA. Using flowcytometry and FITC-conjugated anti-H3cit antibodies, we were able todevelop a novel, cost effective and high throughout method of quantifyingNET release, which allowed us to determine that L. mexicana promastigotesdid not induce a significant increase in NET release by neutrophils. Using flow cytometry to determine parasite uptake, we were also able to determine that phagocytosis by dendritic cells was significantly downregulated in the presence of PMA-activated neutrophil NETs, suggesting that NETs may play a role in the activation of dendritic cells. Further investigation into the activation of dendritic cells using flow cytometry, however, showed that there was significant decrease of activation which may be due to parasite manipulation of dendritic cell activation. We were also able to determine that L. mexicana promastigotes were capable of downregulating glucose uptake in neutrophils potentially due to the metabolic activity of the parasite itself in growing and carrying out replication. Together, these findings suggest an involvement of neutrophil NETs in the immune response to infection with Leishmania mexicana, with more research necessary to determine the precise role in which they play.Neutrophil NETs have been a keen area of research in the few years since they were initially described. As well as apoptosis and necrosis, it was discovered that neutrophils also undergo a process known as NETosis by which DNA, histones and granule proteins are expelled from within the cell to form traps capable of killing fungi and bacteria. Here, we investigated the role of neutrophil NETs in response to infection with Leishmania mexicana. Using murine bone marrow derived neutrophils and dendritic cells, we used immunofluorescent staining of known NET markers and were able to identify that neutrophil NETs were capable of the capture of L. mexicana promastigotes following stimulation of neutrophils with PMA. Using flowcytometry and FITC-conjugated anti-H3cit antibodies, we were able todevelop a novel, cost effective and high throughout method of quantifyingNET release, which allowed us to determine that L. mexicana promastigotesdid not induce a significant increase in NET release by neutrophils. Using flow cytometry to determine parasite uptake, we were also able to determine that phagocytosis by dendritic cells was significantly downregulated in the presence of PMA-activated neutrophil NETs, suggesting that NETs may play a role in the activation of dendritic cells. Further investigation into the activation of dendritic cells using flow cytometry, however, showed that there was significant decrease of activation which may be due to parasite manipulation of dendritic cell activation. We were also able to determine that L. mexicana promastigotes were capable of downregulating glucose uptake in neutrophils potentially due to the metabolic activity of the parasite itself in growing and carrying out replication. Together, these findings suggest an involvement of neutrophil NETs in the immune response to infection with Leishmania mexicana, with more research necessary to determine the precise role in which they play