AMPK and CAMKK activation participate in early events of Toxoplasma gondii-triggered NET formation in bovine polymorphonuclear neutrophils

Abstract

Toxoplasma gondii is an obligate intracellular apicomplexan parasite that infects humans, eventually causing severe diseases like prenatal or ocular toxoplasmosis. T. gondii also infects cattle but rarely induces clinical signs in this intermediate host type. So far, the innate immune mechanisms behind the potential resistance of bovines to clinical T. gondii infections remain unclear. Here, we present evidence on sustained activation of bovine polymorphonuclear neutrophils PMN by T. gondii tachyzoites, which is linked to a rise in cytoplasmic calcium concentrations, an enhancement of calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK) and AMP-activated protein kinase (AMPK). NETosis is a specific form of programmed cell death, characterized by the release chromatin from the nucleus to the extracellular space resulting in formation of neutrophil extracellular traps (NETs). NETs can kill and entrap pathogens. In our experiments, NETosis was triggered by T. gondii, and this effector mechanism was enhanced by pre-treatments with the AMPK activator AICAR. Moreover, tachyzoite-mediated bovine neutrophil DNA release depended on MAPK- and store operated calcium entry- (SOCE) pathways since it was diminished by the inhibitors UO126 and 2-APB, respectively. Overall, we here provide new insights into early polymorphonuclear neutrophils responses against T. gondii for the bovine system.

Keywords: AMPK; CAMKK; NET; PMN; Toxoplasma gondii; bovine; cattle; innate immunity.

Figure 1

Toxoplasma gondii exposure induces cytoplasmatic calcium increase, AMPK and CAMKK phosphorylation in bovine PMN. Fluo-4 AM-loaded bovine PMN (A) (n = 3) were confronted with T. gondii or stimulated with the calcium ionophore A23187 for positive control. Fluo 4-AM derived fluorescence was measured by a flow cytometer and the mean of the fluorescence intensity after 5 min of co-incubation was represented as bar graph (A). Bovine PMN isolated from peripheral blood from 6 different animals (n = 6) were exposed to T. gondii at 1:4 PMN: T. gondii ratio. After 30 min of co-incubation, protein extracts were generated from PMN and tested for AMPK, p-AMPK, CAMKK and p-CAMKK expression by Western blotting. The expression of vinculin was used as internal reference protein. (B) Representative Western blot and (C) densitometric analysis of protein bands for AMPK, p-AMPK. (D) Densitometric analysis for CAMKK and p-CAMKK. Bars in the graph represent the mean ± SD. p values were calculated by applying a Mann–Whitney test.

Figure 2

Studies on autophagy-related proteins on T. gondii-exposed PMN. Bovine PMN isolated from peripheral blood from four different animals (n = 4) were exposed to T. gondii at 1:4 PMN: T. gondii ratio. After 0–30 min of incubation, total protein extracts were generated from PMN and tested for Beclin-1, p-Beclin-1 and ULK1 expression by Western blotting. The expression of vinculin was used as internal reference protein. (A) Representative Western blot and densitometric analysis of protein bands for Beclin-1 (B), p-Beclin-1 (C,D) ULK1 at 0, 5, 15 and 30 min of co-incubation. Bars in the graph represents mean ± SD. p values were calculated by unpaired two-tailed t-tests comparing control PMN vs. PMN incubated with T. gondii at each time point.

Figure 3

AICAR treatments enhance T. gondii-induced NET formation in bovine PMN. (A) Immunofluorescence images showing DNA (blue, DAPI), neutrophil elastase (NE, green) and DNA-histone complexes (magenta) in PMN (negative control), PMN-T. gondii -cocultures and AICAR-pretreated bovine PMN (30 min before T. gondii exposure). (B) The percentage of NET-releasing PMN was calculated by a semi-automated quantification method via image analysis (Image J, Fiji version) and is represented as bar graph, mean ± SD. p-values were calculated using a ANOVA test followed by a Tukey multiple comparison test.

Figure 4

Toxoplasma gondii-induced DNA release in bovine PMN is dependent on ERK and SOCE signalling. Bovine PMN (n = 3) were pre-treated for 30 min with UO126 (50 μM), LY294002 (1 μM) or 2-APB (50 μM) before the addition of T. gondii (1:4 PMN: tachyzoites ratio). After 4 h of co-incubation, the Picogreen-derived fluorescence, corresponding to extracellular DNA amount, was determined in a plate reader. DNAse I (90 U) was added after the 4 h of incubation in the corresponding experiments to confirm the DNA-nature of the emitted fluorescence. Bars represent the mean ± SD. p-values were calculated by applying an ANOVA test followed by a Dunnet multiple comparison test.