Extracellular Trap Formation in Response to Trypanosoma cruzi Infection in Granulocytes Isolated From Dogs and Common Opossums, Natural Reservoir Hosts

Abstract

Granulocytes mediate the first line of defense against infectious diseases in humans as well as animals and they are well known as multitasking cells. They can mediate antimicrobial activity by different strategies depending on the pathogen they encounter. Besides phagocytosis, a key strategy against extracellular pathogens is the formation of extracellular traps (ETs). Those ETs mainly consist of DNA decorated with antimicrobial components and mediate entrapment of various pathogens. In the last years, various studies described ET formation as response to bacteria, viruses and parasites e.g., Trypanosma (T.) cruzi. Nevertheless, it is not fully understood, if ET formation helps the immune system to eliminate intracellular parasites. The goal of this study was to analyze ET formation in response to the intracellular parasite Trypanosma (T.) cruzi by granulocytes derived from animals that serve as natural reservoir. Thus, we investigated the ET formation in two T. cruzi reservoirs, namely dogs as domestic animal and common opossums (Didelphis marsupialis) as wild animal. Granulocytes were harvested from fresh blood by density gradient centrifugation and afterwards incubated with T. cruzi. We conducted the analysis by determination of free DNA and immunofluorescence microscopy. Using both methods, we show that T. cruzi efficiently induces ET formation in granulocytes derived from common opossum as well as dog blood. Most ETs from both animal species as response to T. cruzi are decorated with the protease neutrophil elastase. Since T. cruzi is well known to circulate over years in both analyzed animals as reservoirs, it may be assumed that T. cruzi efficiently evades ET-mediated killing in those animals. Therefore, ETs may not play a major role in efficient elimination of the pathogen from the blood of dogs or common opossums as T. cruzi survives in niches of their body. The characterization of granulocytes in various animals and humans may be helpful to understand the anti-pathogenic capacity and overall role of ETs against zoonotic pathogens like T. cruzi.

Trypanosoma brucei brucei Induces Polymorphonuclear Neutrophil Activation and Neutrophil Extracellular Traps Release

“…In this context, local pro-inflammatory responses in skin lesions in AAT, resulting in focal edema, were associated with PMN recruitment and granuloma formation surrounding T. b. brucei replication sites (10). PMN reacts against protozoan and metazoan parasites by different effector mechanisms which include the release of immunomodulatory molecules [e.g., cytokines, chemokines (CXCL1, CXCL8, CXCL10) (11,12)], phagocytosis, production of reactive oxygen species (ROS), and release of neutrophil extracellular traps (NETs) (11,13,14). So far, different parasite species were identified to induce either nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX)-dependent or NOX-independent NETs formation (15,16).…”

Extracellular Traps: An Ancient Weapon of Multiple Kingdoms

“…Analysis of the cells revealed a high percentage of eosinophils in the granulocyte fraction. Free extracellular DNA was detected after didelphine granulocytes were stimulated by T. cruzi [77]. The addition of cholesterol-depleting methyl-β-cyclodextrin (CD) also induced the release of extracellular traps, as has been previously shown in human [31] and bovine neutrophils [126], indicating a conserved role of lipid membrane composition in NET formation.…”

“…The study showed that ERK1/2, MAPK, and SOCE pathways were involved in the phenotype, and the parasites were entrapped within the NET structures [74]. Recently, two studies showed that canine PMNs also release extracellular traps in response to Trypanosoma cruzi [77] and Dirofilaria immitis microfilariae, and their L3 stage larvae [78]. Finally, also an overexposure to nickel in the form of nickel nitrate, a commonly used heavy metal material in battery manufacturing, triggered the release of extracellular traps from canine neutrophils [143].…”

“…Belonging to the mammalian class, marsupials, however, split from the phylogenetic tree further down, developing a front pouch for carrying their offspring. In a study analysing extracellular traps from granulocytes derived from domesticated dogs, granulocytes from wild opossums Didelphis (D.) marsupialis were used as a comparison [77]. Analysis of the cells revealed a high percentage of eosinophils in the granulocyte fraction.…”

“…Neutrophil elastase was found within the fibres, after stimulation with both T. cruzi and CD. Three-dimensional (3D) imaging further revealed an accumulation of T. cruzi within the trap formation [77].…”