Figure 1.
Labelling live cercariae with the amine reactive tracer CFDA-SE.
A; Typical bright field / fluorescent confocal images of CFDA-SE-labelled cercaria with unlabelled cercaria shown insert (arrows represent, i: Tail, ii: Head, iii: post-acetabular glands, iv: pre-acetabular glands, v: acetabular ducts). B; 3D image of a CFDA-SE-labelled cercaria constructed using Volocity image software. Video S1: 3D image of CFDA-SE-labelled cercaria.
Figure 2.
Transformation of cercariae into schistosomula results in the release of CFDA-SE labelled ES gland material.
A; The RFU of CFDA-SE labelled untransformed cercariae and transformed schistosomula was compared after 3 hrs of culture (Unl cerc = unlabelled cercariae, CFDA-SE cerc = CFDA-SE labelled cercariae, Medium CF cerc = Medium from untransformed CFDA-SE labelled cercariae, CFDA-SE Sch = CFDA-SE labelled schistosomula, Medium CF Sch = medium from labelled transformed schistosomula). Results are mean±SEM from 5 independent experiments; significant difference of transformed schistosomula versus untransformed cercariae P<0.01. B; Representative confocal images of a transforming CFDA-SE-labelled cercaria showing the release of gland material (C) as discrete vesicles.
Figure 3.
Material released by CFDA-SE-labelled cercariae is phagocytosed by adherent MHC-II+ cells.
A; Uptake of the labelled material in adherent cells is significantly greater than non-adherent cells; (p<0.001). B; Representative fluorescent, and merged with brightfield, images of adherent PECs exposed to unlabelled or CFDA-SE labelled cercariae, or directly to free CFDA-SE. C; Cells that take up ES material expressed elevated levels of MHC-II and 40.4% of these were also positive for CFDA-SE. All results for PEC are representative of 5 independent experiments. D; Fluorescence of BMMФ stimulated with unlabelled or CFDA-SE labelled cercariae, or unlabelled or CFDA-SE labelled 0-3hRP with flow cytometry histogram plots displaying MFI (Unl Cerc = unlabelled cercariae, Unt CFDA-SE Cerc = untransformed labelled cercariae, Tr CFDA-SE Cerc = transformed labelled cercariae, RPMIc = concentrated RPMI, 0-3hRP = 0-3 hour released preparation). E; Addition of EGTA (5 mg/ml) or Cytochalasin D (10 ug/ml) significantly decreased uptake of CFDA-SE 0-3hRP. Representative plots are shown, bars are means±SEM where n = 6 mice.
Figure 4.
Prolonged translocation of CFDA-SE labelled 0-3hRP to the LAMP-1+ phagosome compared to Alexa Fluor488 E. coli bioparticles.
A & B; Confocal images of BMMФ cultured with CFDA-SE-labelled 0-3hRP (green) or Alexa Fluor488 E. coli bioparticles (green) co-localised with EEA-1+ early endosomes (purple) and LAMP-1+ late phagosomes (red) after 15 mins (A) and 30 mins (B), scale bar = 10 µm. C; Co-localisation of CFDA-SE 0-3hRP with EEA-1 endosomes is prolonged compared to Alexa Fluor488 E. coli bioparticles and (D) is slower to be translocated to LAMP-1+ phagosome. The co-localisation coefficients (Mx) were determined over 18 hr using the imaging software Volocity. Results are mean±SEM of 3 separate experiments. Video S2: co-localisation of 0-3hRP with EEA-1 (purple) and LAMP-1 (red) within BMMΦ after 30 mins. Video S3: Co-localisation of Alexa Fluor488 E. coli bioparticles with EEA-1 (purple) and LAMP-1 (red) after 30 mins.
Figure 5.
In vivo infection with CFDA-SE-labelled cercariae results in the release of labelled material in skin and its uptake by cells in the skin and sdLN.
A; Stills from a time-lapse video (see supplementary Video S4) showing confocal images of a CFDA-SE-labelled infective cercaria penetrating and migrating through a mouse pinna, (scale bar = 100 µM). Attachment of the cercaria to the stratum corneum at 00:01:00, the loss of its tail by 00:18:00, penetration through outer layers of epidermis and deposition of gland material through 00:10:00 and onward migration up to 2 hours post-infection 02:00:00. B; Pinnae from mice infected with CFDA-SE labelled cercariae were digested and skin cell suspensions enumerated for CFDA-SE+ cells by flow cytometry (significant difference compared to 3 hrs). C; Phenotype of CFDA-SE+ cells from digested skin of pinnae exposed to CFDA-SE labelled cercariae showing uptake by GR-1+ MHC-II−, F4/80+ MHC-II+, and CD11c+ MHC-II+ cells. D; Phenotype of CFDA-SE+ cells from the sdLN of the same mice above showing uptake by F4/80+MHC-IIl+, and CD11c+MHC-II+ cells with a significant increased level of CFDA-SE+ cells present at 48 hrs (** denotes p = <0.01). Data in B–D is represents data from 6 different mice.
Figure 6.
Comparison of the relative ability of BMMФ and BMDC to internalise and be activated by CFDA-SE labelled material released from cercariae, or CFDA-SE-labelled 0-3hRP.
A; BMDC internalise greater quantities of released material from cercariae or 0-3hRP compared to BMMФ and B; express higher MFI±SEM. C; BMDC internalise 0-3hRP at a faster rate than BMMΦ. D; Expression of CD40, CD86 and MHC II of BMDC (red) and BMMΦ (blue) after stimulation with cercariae (dotted line) and 0-3hRP (solid line), values shown in insert are MFI±SEM. E; Levels of IL-12p40, TNF-α, IL-6 and IL-10 in BMMФ (open bars) and BMDC (hatched bars). F; Levels of mRNA measured by qPCR for arginase 1 and iNOS (relative to GAPDH) in BMMΦ and BMDC. Cultures of BMMФ and BMDC were derived from the same mouse, and data shows the mean±SEM for separate animals. Data represent cells obtained from 6 different mice.
Figure 7.
Phagosome maturation of 0-3hRP in BMMΦ is prolonged compared to BMDC.
A; Co-localisation coefficients (Mx) of CFDA-SE labelled 0-3hRP and B; AF594 E. coli bioparticles with EEA-1+ and LAMP-1+ compartments in BMMΦ and BMDC, data represent cells obtained from 6 different mice. C; Confocal images of CFDA-SE 0-3hRP (green) and E. coli (red) present within different LAMP-1 compartments (turquoise) of the same BMDC and BMMΦ, scale bar = 10 µm.