Fig 1.
Experimental design showing that macrophages were pre-incubated with apoptotic Jurkat cells, previously infected or not with E. cuniculi.
After efferocytosis, they were challenged with infection by E. cuniculi in a total of four groups: Macrophages with E. cuniculi; macrophages with ACs; macrophages with ACs and E. cuniculi; macrophages with IACs and E. cuniculi.
Fig 2.
Evaluation of the phagocytic activity of pre-incubated (+) or not (–) macrophages with apoptotic cells (ACs) or infected apoptotic cells (IACs) and challenged with E. cuniculi.
(A) Percentage of macrophages phagocyting the spores during 1 h and 24 h of incubation. (B) Average spores inside the macrophages in 1 h and 24 h of incubation. (C) Image of macrophages with spores of E. cuniculi stained by Calcofluor after pre-incubation with ACs or IACs. One-way analysis of variance (ANOVA) with Tukey’s post-test revealed significance between the groups. *p < 0.05, ***p < 0.001.
Fig 3.
Infection of Jurkat cells (ACs) by E. cuniculi before inducing apoptosis.
(A) Jurkat cells (ACs) stained with PKH; E. cuniculi spores marked with CFSE; core fluorescence stained with DAPI; phase-contrast cultures-DIC; overlay of images showing internalized and intact spores inside the Jurkat cells, confirmed in the 3D image (arrow). (Images) (B) Ultramicrographic images of a Jurkat cell fragment infected with E. cuniculi (arrow).
Fig 4.
Macrophages pre-incubated with apoptotic cells and challenged with E. cuniculi.
(A) Image showing clusters of E. cuniculi spores in a lighter area of the cytoplasm, suggesting a parasitophorous vacuole (arrows). Spores inside the macrophage with preserved characteristics (arrowhead) stained with toluidine blue. (B) Ultramicrography showing clusters of E. cuniculi spores in the macrophage cytoplasm with ruptured cell membrane following the multiplication of the pathogen (arrow). (C) Ultramicrography of E. cuniculi spores in the macrophage cytoplasm showing signs of polar tubule extrusion (arrow), as a sign of viability. Note that the rupture of the resin next to the spores is a characteristic finding of microsporidia.
Fig 5.
Analysis of the viability of pre-incubated (+) or not (–) macrophages with apoptotic cells (ACs) or infected apoptotic cells (IACs) and challenged (+) or not (–) with E. cuniculi, after 1 h or 24 h of observation.
Percentages of dead, apoptotic, necrotic, and live cells (A) after 1 h of infection with E. cuniculi and (B) after 24 h of infection with E. cuniculi.
Fig 6.
Ultramicrography image of macrophages pre-incubated with E. cuniculi in infected and apoptotic cells.
(A) Apoptotic bodies phagocyted (arrowhead) by macrophages containing intact E. cuniculi spores inside (insert) (B) necrotic cells (Ne) with the spores of pathogen close to macrophages. (C) A macrophage in apoptosis (Ac).
Fig 7.
Ultramicrography image of pre-incubation of macrophages with infected and apoptotic Jurkat cells after challenge with E. cuniculi.
(A) Parasitophore vacuole (Vp) with E. cuniculi spores (arrows) and other forms of development. (B) Detail of meronte (me) and spores (e) with rupture of resin, characteristic of microsporidia. (C) Detail of immature spores (e) showing an enameled polar tubule. (D) Detail of spores (e) with rupture of resin, characteristic of microsporidia and meronte (me) next to the Vp membrane.
Fig 8.
Expression of polarization and activation surface markers in pre-incubated (+) or not (–) macrophages with apoptotic cells (ACs) or infected apoptotic cells (IACs) and challenged (+) or not (–) with E. cuniculi, after an hour or 24 h of observation.
(A) Median fluorescence (MFI) for CD40 in macrophages. (B) MFI for CD206 in macrophages. (C) The ratio between MFI CD40/MFI CD206. (D) MFI for CD80/86 and MHC II in macrophages. One-way analysis of variance (ANOVA) with Tukey’s post-test revealed significance between the groups. *p < 0.05, **p < 0.01, ***p < 0.001.
Fig 9.
Quantification of inflammatory (TNF-α, MCP-1, and IL-6) and anti-inflammatory (IL-10) cytokines released pre-incubated (+) or not (–) macrophages with apoptotic cells (ACs) or infected apoptotic cells (IACs) and challenged (infected E. cuniculi) or not (infected) in one hour, 12 and 24 h of observation.
One-way analysis of variance (ANOVA) with Tukey’s post-test revealed significance between the groups. *p < 0.05, **p < 0.01, ***p < 0.001.