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Parasites release extracellular vesicles (EVs) which, in some cases, modulate the host's immune response contributing to the establishment of the infection. In this work we have isolated and characterized the EVs released by trophozoites of the human protozoan parasite Entamoeba histolytica, the causal agent of amoebiasis, when alone or in coculture with human neutrophils, and determined their effect on neutrophil NETs and ROS production. Nanoparticle tracking analysis showed that amoebic EVs are variable in size, ranging from less than 50 nm to nearly 600 nm in diameter (average of 167 nm), whereas neutrophil EVs are more uniform in size, with an average of 136 nm. In cocultures amoeba:neutrophil (1:100) most EVs are 98 nm in size, which is the typical size of exosomes. EVs from amoebae and neutrophils showed almost equal levels of ROS, which were considerably increased in EVs from cocultures. Uptake of amoebic EVs by neutrophils was demonstrated by fluorescence and resulted in a significant reduction in the oxidative burst and NET release triggered by PMA, ionophore A23187, or the amoebae itself used as stimuli. Interestingly, uptake of EVs from cocultures did not affect ROS production, but instead caused a greater delay in the onset of NETs release and in their quantity. A comparative proteomic analysis between the EVs of amoebae and neutrophils separately vs the cocultures showed a similar distribution of protein categories in the GO analysis, but differences in the expression and abundance of proteins such as the N-acetyl-D-galactosamine (GalNAc) inhibitable surface lectin and calreticulin in amoeba EVs, and various antimicrobial molecules in neutrophil EVs, such as lactoferrin and myeloperoxidase. These results highlight the importance of EVs in the immunomodulatory effects exerted by amoeba on human neutrophils. [ABSTRACT FROM AUTHOR]

Parasites release extracellular vesicles (EVs) which, in some cases, modulate the host’s immune response contributing to the establishment of the infection. In this work we have isolated and characterized the EVs released by trophozoites of the human protozoan parasite Entamoeba histolytica, the causal agent of amoebiasis, when alone or in coculture with human neutrophils, and determined their effect on neutrophil NETs and ROS production. Nanoparticle tracking analysis showed that amoebic EVs are variable in size, ranging from less than 50 nm to nearly 600 nm in diameter (average of 167 nm), whereas neutrophil EVs are more uniform in size, with an average of 136 nm. In cocultures amoeba:neutrophil (1:100) most EVs are 98 nm in size, which is the typical size of exosomes. EVs from amoebae and neutrophils showed almost equal levels of ROS, which were considerably increased in EVs from cocultures. Uptake of amoebic EVs by neutrophils was demonstrated by fluorescence and resulted in a significant reduction in the oxidative burst and NET release triggered by PMA, ionophore A23187, or the amoebae itself used as stimuli. Interestingly, uptake of EVs from cocultures did not affect ROS production, but instead caused a greater delay in the onset of NETs release and in their quantity. A comparative proteomic analysis between the EVs of amoebae and neutrophils separately vs the cocultures showed a similar distribution of protein categories in the GO analysis, but differences in the expression and abundance of proteins such as the N-acetyl-D-galactosamine (GalNAc) inhibitable surface lectin and calreticulin in amoeba EVs, and various antimicrobial molecules in neutrophil EVs, such as lactoferrin and myeloperoxidase. These results highlight the importance of EVs in the immunomodulatory effects exerted by amoeba on human neutrophils.

The ability of motile immune cells to detect and follow gradients of chemoattractant is critical to numerous vital functions, including their recruitment to sites of infection and-in emerging immunotherapeutic applications-to malignant tumors. Facilitated by a multitude of chemotactic receptors, the cells navigate a maze of stimuli to home in on their target. Distinct chemotactic processes direct this navigation at particular times and cell-target distances. The expedient coordination of this spatiotemporal hierarchy of chemotactic stages is the central element of a key paradigm of immunotaxis. Understanding this hierarchy is an enormous interdisciplinary challenge that requires, among others, quantitative insight into the shape, range, and dynamics of the profiles of chemoattractants around their sources. We here present a closed-form solution to a diffusion-reaction problem that describes the evolution of the concentration gradient of chemoattractant under various conditions. Our ready-to-use mathematical prescription captures many biological situations reasonably well and can be explored with standard graphing software, making it a valuable resource for every researcher studying chemotaxis. We here apply this mathematical model to characterize the chemoattractant cloud of anaphylatoxins that forms around bacterial and fungal pathogens in the presence of host serum. We analyze the spatial reach, rate of formation, and rate of dispersal of this locator cloud under realistic physiological conditions. Our analysis predicts that simply being small is an effective protective strategy of pathogens against complement-mediated discovery by host immune cells over moderate-to-large distances. Leveraging our predictions against single-cell, pure-chemotaxis experiments that use human immune cells as biosensors, we are able to explain the limited distance over which the cells recognize microbes. We conclude that complement-mediated chemotaxis is a universal, but short-range, homing mechanism by which chemotaxing immune cells can implement a last-minute course correction toward pathogenic microbes. Thus, the integration of theory and experiments provides a sound mechanistic explanation of the primary role of complement-mediated chemotaxis within the hierarchy of immunotaxis, and why other chemotactic processes are required for the successful recruitment of immune cells over large distances.

The ability of motile immune cells to detect and follow gradients of chemoattractant is critical to numerous vital functions, including their recruitment to sites of infection and—in emerging immunotherapeutic applications—to malignant tumors. Facilitated by a multitude of chemotactic receptors, the cells navigate a maze of stimuli to home in on their target. Distinct chemotactic processes direct this navigation at particular times and cell-target distances. The expedient coordination of this spatiotemporal hierarchy of chemotactic stages is the central element of a key paradigm of immunotaxis. Understanding this hierarchy is an enormous interdisciplinary challenge that requires, among others, quantitative insight into the shape, range, and dynamics of the profiles of chemoattractants around their sources. We here present a closed-form solution to a diffusion–reaction problem that describes the evolution of the concentration gradient of chemoattractant under various conditions. Our ready-to-use mathematical prescription captures many biological situations reasonably well and can be explored with standard graphing software, making it a valuable resource for every researcher studying chemotaxis. We here apply this mathematical model to characterize the chemoattractant cloud of anaphylatoxins that forms around bacterial and fungal pathogens in the presence of host serum. We analyze the spatial reach, rate of formation, and rate of dispersal of this locator cloud under realistic physiological conditions. Our analysis predicts that simply being small is an effective protective strategy of pathogens against complement-mediated discovery by host immune cells over moderate-to-large distances. Leveraging our predictions against single-cell, pure-chemotaxis experiments that use human immune cells as biosensors, we are able to explain the limited distance over which the cells recognize microbes. We conclude that complement-mediated chemotaxis is a universal, but short-range, homing mechanism by which chemotaxing immune cells can implement a last-minute course correction toward pathogenic microbes. Thus, the integration of theory and experiments provides a sound mechanistic explanation of the primary role of complement-mediated chemotaxis within the hierarchy of immunotaxis, and why other chemotactic processes are required for the successful recruitment of immune cells over large distances.