Beschreibung
Cryptosporidium parvum, a zoonotic protozoan parasite, is found globally and has the ability to infect both humans and livestock. The main reservoir of this parasitic infection are neonatal calves. The occurrence of life-threatening cryptosporidiosis in HIV patients infected with C. parvum highlights the importance of a strong adaptive immune response in effectively combating this parasite. Currently, there is limited knowledge regarding the activation of innate and adaptive immune responses against C. parvum in humans and bovines. Neutrophil extracellular traps (NETs), also referred to as suicidal NETosis, are a highly effective and long-standing innate defense mechanism used by polymorphonuclear neutrophils (PMN) to combat parasitic organisms such as protozoa and helminths. Mammalian monocytes are a type of myeloid leukocytes that originate from the bone marrow. They play a crucial role in the early innate immune response of the host due to their various defense mechanisms. These include the presence of ATP purinergic-, CD14- and CD16-receptors, as well as their ability to adhere to surfaces, migrate, and phagocytose pathogens. Monocytes also possess inflammatory and anti-parasitic properties, further contributing to their important role in the immune system. Recently, there have been reports on the formation of monocyte extracellular traps (METs) as an additional mechanism to combat apicomplexan parasites. However, there is currently no information available in the literature regarding the extrusion of METs in relation to C. parvum-oocysts or sporozoites. In this study, we investigated the ATP purinergic receptor P2X1, glycolysis, Notch signaling, and lactate monocarboxylate transporters (MCT) in bovine monocytes and PMN exposed to C. parvum under two different oxygen conditions: intestinal physioxia (5% O2) and hyperoxia (21% O2, which is commonly used in laboratory settings). The confirmation of C. parvum-triggered suicidal NETs/METs were achieved through several methods. Firstly, the complete rupture of exposed monocytes and PMN were observed. Additionally, the co-localization of extracellular DNA with myeloperoxidase (MPO) and histones (H1-H4) was detected using immunofluorescence- and confocal microscopy analyses. According to scanning electron microscopy (SEM) analyses, suicidal NETs/METs induced by C. parvum not only led to oocyst entrapment but also hindered the egress of sporozoites from the oocysts. Using live cell 3D-holotomographic microscopy analysis, we were able to uncover the early activation of bovine PMN/monocytes induced by parasites. This activation was characterized by the formation of membrane protrusions towards C. parvum-oocysts/sporozoites.
The analysis using live cell 3D-holotomographic microscopy visualized early morphological changes in PMN induced by C. parvum. These changes included the formation of membrane protrusions towards C. parvum while undergoing NETosis. A significant decrease in C. parvum-induced suicidal NETosis was observed when PMN were treated with the purinergic receptor P2X1 inhibitor NF449, regardless of the oxygen conditions. This finding suggests that the P2X1 receptor plays a crucial role in the formation of NETs, highlighting its significance. In a similar way, when PMN glycolysis was inhibited through treatments with 2-deoxy glucose, there was a slight decrease in C. parvum-triggered suicidal NETosis, although not to a significant extent. According to measurements of PMN energetic state, the exposure to C. parvum did not result in an increase in extracellular acidification rates (ECAR) or oxygen consumption rates (OCR) in the cells. The use of inhibitors targeting plasma membrane monocarboxylate transporters (MCTs) of lactate resulted in a significant reduction of C. parvum-induced NET extrusion. No significant reduction in Notch signaling was observed following treatments with two specific Notch inhibitors, namely DAPT and compound E, in relation to PMN. In this study, we present the first description of the important role played by the ATP purinergic receptor P2X1 in C. parvum-induced suicidal NETosis under physioxia (5% O2). Additionally, we highlight the anti-cryptosporidial properties of this receptor. When monocytes were exposed to different levels of oxygen, the administration of NF449, an inhibitor of the ATP purinergic receptor P2X1, did not result in a significant reduction in C. parvum-induced METosis. This implies that the process of cell death does not rely on P2X1. Furthermore, when monocytes were treated with 2-deoxy glucose (2-DG) to inhibit glycolysis, there was a reduction in C. parvum-induced METosis, although the decrease was not statistically significant. Based on measurements of monocyte energetic state, it was found that cells exposed to C. parvum did not show any increase in extracellular acidification rates (ECAR) or oxygen consumption rates (OCR). The treatment with an inhibitor of lactate monocarboxylate transporters (MCT), such as AR-C 141990, significantly reduced the extrusion of METs induced by C. parvum under physioxic conditions (5% O2). In the same vein, the administration of either DAPT or compound E, which are two selective Notch inhibitors, did not show any notable inhibitory effects on the production of bovine MET. In this study, we present the first evidence of C. parvum-mediated METosis as a defense mechanism. We found that this process is independent of P2X1, but relies on MCT. Moreover, we observed that these effects occur specifically under intestinal physioxia conditions with 5% CO2. The findings from METs suggest that there are anti-cryptosporidial effects achieved by trapping the parasites and inhibiting the process of sporozoite excystation.
The interaction between dendritic cells (DC) and pathogenic microorganisms is a crucial first step in any adaptive immune response. DC detect molecules derived from pathogens and alarm signals, and respond by activating and maturing. We generated primary human DC from monocytes (MO-DC) obtained from healthy blood donors. These MO-DC were then exposed to C. parvum oocysts and sporozoites in a controlled laboratory environment. In general, exposure to parasites significantly increased the production of the pro-inflammatory cytokines/chemokines IL-6 and IL-8 in DC. This increase was comparable to the level observed with lipopolysaccharide (LPS) stimulation, which was used as a positive control. In addition, there was an upregulation of maturation markers and costimulatory molecules necessary for T cell stimulation, such as CD83, CD40, and CD86. Antigen-presenting molecules like HLA-DR and CD1a, as well as adhesion molecules like CD11b and CD58 were also upregulated. Furthermore, human DCs exposed to parasites exhibited improved cell adherence, increased mobility, and a heightened, albeit temporary, ability to engulf C. parvum oocysts and sporozoites. This enhanced phagocytosis serves as an additional requirement for effective antigen presentation. In contrast to other microbial stimuli, exposure to C. parvum resulted in increased oxidative consumption rates (OCR) rather than extracellular acidification rates (ECAR) in DC. This suggests that different metabolic pathways were utilized to generate energy for DC activation. When human DC were exposed to C. parvum, they exhibited all the characteristics of successful maturation, which allowed them to effectively initiate an adaptive immune response in the host.
