Fig 1.
Summary of experimental procedures.
C57BL/6J female mice were infected with T. regenti cercariae and examined at desired time points (indicated by filled red dots). Uninfected mice were used as controls; particular age-matching is shown for each analysis (filled blue dots). Empty dot means that the analysis was not performed at the respective time point.
Fig 2.
General features of T. regenti infection in C57BL/6J mice.
(A) Infected mice exhibited a lower gain in body weight than age-matched uninfected controls. (B) The levels of T. regenti-specific IgG1 and IgG2a detected in mouse sera rose steadily throughout the infection. Th2-associated IgG1 dominated over Th1-associated IgG2a, especially 28 dpi. (C) Blood eosinophil counts have increased in infected mice 7–21 dpi. (D) Spleen enlargement was observed in infected mice 7–21 dpi. (E) Within the CNS, most viable schistosomula were found 7 dpi being localized predominantly in the spinal cord. The amount of released schistosomula remarkably decreased at later time points. (F) Contrary to those from hemispheres, T. regenti DNA was detected in most of the spinal cord tissue samples 7–21 dpi. In each graph, points show data from individual mice. Pooled data from 2–3 independent experiments are shown. Data were evaluated by 2-way ANOVA for repeated measures and Šidák’s test (A), ordinary 2-way ANOVA and Šidák’s test (B, F), Kruskal-Wallis and Dunn’s test (C), or ordinary 1-way ANOVA and Dunnett’s test (D); *p<0.05, **p<0.01, ***p<0.001.
Fig 3.
Effects of T. regenti infection on mouse behavior 7 dpi.
(A) Beam walking on wide and narrow beams, impaired motor coordination in infected mice. (B) Grid test, worse endurance, and strength of the forelimbs and hindlimbs of infected mice. (C) Footprint analysis of forelimbs and hindlimbs, altered posture and gait in infected mice. (D) Bar holding, normal endurance, and strength of the forelimbs in both groups. (E) Open field, unaffected locomotor activity, and anxiety in both groups. (F) Novelty-induced hypophagia, higher amount of eaten oat flakes caused by stronger appetitive motivation, rather than increased anxiety of infected mice. In each graph, points show data from individual mice. Pooled data from 3 independent experiments are presented. Data were evaluated by Mann-Whitney test (A, B, F—eating) or unpaired t-test (C, D, E, F—feces); *p<0.05, **p<0.01, ***p<0.001.
Fig 4.
Histological examination of the T. regenti-infected spinal cord.
(A) In the uninfected spinal cord, no leukocyte infiltration was apparent. (B) The intact schistosomulum in the white matter 7 dpi. No inflammatory cells surrounded the schistosomulum, but eosinophil extravasation was recorded in the adjacent areas (inset, eosinophils marked by white arrowheads). (C) The “rocket-tail” leukocyte cluster behind the schistosomulum 14 dpi. Eosinophils and mononuclear cells prevailed in the cluster (gray arrowhead and inset). (D) The inflammatory lesion around the destroyed schistosomulum 14 dpi. The adjoining leptomeningeal spaces (gray asterisks) were thickened and heavily inflamed. (E) The schistosomulum remnants within the fading inflammatory lesion 21 dpi. (F) The spinal cord tissue 28 dpi, no schistosomula residues or inflammatory foci were evident. Representative images from 2 independent experiments (each with 2–3 mice) per time point are shown. Scale bar (large image) = 50 μm, scale bar (inset) = 10 μm.
Fig 5.
Dynamics of major immune cell populations in the mouse CNS during T. regenti infection.
(A–D) Microglia, neutrophils, eosinophils, macrophages/monocyte, and lymphoid cells were analyzed by flow cytometry in the spinal cord (A), the brain stem (B), the cerebellum (C), and the hemisphere (D). In all examined segments, the most prominent increase was observed in eosinophil counts 14 dpi. Gating strategies are shown in S2 Text. Data (n = 5 per each time point) were evaluated by ordinary 1-way or Welch’s ANOVA followed by Dunnett’s test; *p<0.05, **p<0.01, ***p<0.001. (E–H) The relative proportion of the examined immune cell populations in the spinal cord (E), the brain stem (F), the cerebellum (G), and the hemisphere (H). Massive infiltration of peripheral leukocytes caused an apparent decrease in the proportion of microglia. Representative data from 1 out of 2 independent experiments are shown.
Fig 6.
Transcriptomic analysis of the spinal cord of T. regenti-infected mice.
(A) The number of transcripts identified in the spinal cords of infected or age-matched uninfected (control) mice 7, 14, and 21 dpi. The transcriptomic analysis was performed with a group of mice (n = 4 per each time point) infected independently from other experiments presented herein. Data were evaluated by ordinary 2-way ANOVA and Šidák’s test; *p<0.05. (B) The number of differentially expressed transcripts 7, 14, and 21 dpi based on log2fold change (FC; upregulated >2, downregulated <-2). (C) Differentially expressed transcripts with Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation and their exclusive/shared occurrence in the infected spinal cord 7, 14, and 21 dpi. (D) Enriched immune system pathways of upregulated transcripts according to KEGG annotation. (E) Enriched pathways of downregulated transcripts linked with the nervous system and development and regeneration according to KEGG annotation. (F) Top 20 up-/downregulated transcripts (based on log2FC) 7, 14, and 21 dpi. Abbreviated gene names are shown, gray background marks genes found among the “top 20” at all time points.
Fig 7.
Expression of immune and neural markers in the spinal cord of T. regenti-infected mice.
(A–F) The heatmaps show log2fold change (FC) in the expression of chemokines (A), chemokine receptors (B), Toll-like receptors (C), neurofilaments (D), myelin-associated markers (E), and pathology-associated markers (F). Genes/transcripts with log2FC >2 or <–2 were considered as upregulated or downregulated, respectively. Only genes with a significant log2FC are shown unless indicated otherwise (crossed cells). Spinal cords of 4 infected and 4 uninfected age-matched mice were analyzed at each time point.
Fig 8.
Proteomic analysis of the spinal cord tissue around T. regenti schistosomula.
(A) A schematic representation of the region of interest. The area within 100 μm from the schistosomulum was microdissected as well as the schistosomulum digestive tract (marked by an asterisk; for intestinal results see the main text). (B) A real image from the microscope, labeling is consistent with (A)–i.e., the already microdissected area is outlined in red. Scale bar = 100 μm. (C) A summary of the proteomic analysis of the microdissected nervous tissue. Proteins exclusively found (thick line) or more abundant (dashed line) in uninfected (blue) or T. regenti-infected (red) spinal cords are shown. The up/down arrows indicate a log2fold change (FC) in the expression of the protein mRNA as revealed by the transcriptomic analysis (see Fig 6). More arrows per one protein mean that more isoforms were detected in the transcriptome. If no arrow is shown, the log2FC was negligible (–1; +1) or not significant. The proteomic analysis was performed with samples obtained from a group of mice (n = 3–4, see Materials and Methods for details) infected independently from other experiments presented herein.
Fig 9.
M1/M2 polarization in the spinal cord of T. regenti-infected mice.
(A–C) The heatmaps show log2fold change (FC) in the expression of markers associated with microglia (A), M1 (B), and M2 (C) polarization. Genes/transcripts with log2FC >2 were considered upregulated, and only genes with a significant log2FC are shown unless indicated otherwise (crossed cells). Spinal cords of 4 infected and 4 uninfected age-matched mice were analyzed at each time point. (D–E) Immunolocalization of IL-4+Iba-1 (D), and Arg-1 (E). Representative images are shown, the white line indicates the space occupied by the schistosomulum. Scale bar = 50 μm.
Fig 10.
Major histocompatibility complex (MHC) II in the spinal cord of T. regenti-infected mice.
(A) The heatmaps show log2fold change (FC) in the expression of markers associated with the MHC II pathway. Genes/transcripts with log2FC >2 were considered upregulated, and only genes with a significant log2FC are shown unless indicated otherwise (crossed cells). Spinal cords of 4 infected and 4 uninfected age-matched mice were analyzed at each time point. (B) Immunolocalization of MHC II and Iba-1. Representative images are shown, the white line indicates the space occupied by the schistosomulum. Scale bar = 50 μm. (C) 3D images obtained by light-sheet fluorescence microscopy (LSFM) showing MHC II+ cells in the spinal cord. Schistosomula are marked with grey arrowheads. (D) Quantification of MHC II+ volume around schistosomula. It was computed in the virtual box around the schistosomula (400×800×110 μm, S5 Video) or six randomly chosen areas in healthy mice. (E) Patterns of MHC II accumulation and its relation to schistosomula damage. Three categories were recognized: (+) very few MHC II+ cells around/behind intact schistosomula, (++) noticeable infiltration of MHC II+ cells enclosing intact schistosomula (but no MHC II signal within the schistosomula), (+++) massive clusters enclosing damaged schistosomula (MHC II signal present also inside the schistosomula). 2D images were obtained by LSFM and created from five stitched planes in the z-axis (1.4 μm each). Scale bar = 50 μm. (F) Quantification of MHC II accumulation and schistosomula damage using the categories from (E). Thirteen schistosomula out of two mice were examined at each time point. Data were evaluated by Kruskal-Wallis and Dunn’s test (D) and Fisher’s exact test (F); ***p<0.001.
Fig 11.
Pathology associated with T. regenti migration within the mouse spinal cord.
(A–C) The heatmaps show log2fold change (FC) in the expression of markers associated with vascular permeability (A), blood-brain barrier tightness (B), and astrocytes (C). Genes/transcripts with log2FC >2 or <-2 were considered upregulated or downregulated, respectively. Only genes with a significant log2FC are shown unless indicated otherwise (crossed cells). Spinal cords of 4 infected and 4 uninfected age-matched mice were analyzed at each time point. (D) Immunolocalization of astrocytic glial fibrillary acidic protein (GFAP) and astrogliosis-promoting IL-6 demonstrated the most prominent astrocyte hypertrophy 21 dpi. Representative images are shown, the white line indicates the space occupied by the schistosomulum. Scale bar = 50 μm.
Fig 12.
Apoptosis in the spinal cord of T. regenti-infected mice.
(A) The heatmaps show log2fold change (FC) in the expression of pro- and anti-apoptotic markers. Genes/transcripts with log2FC >2 or <–2 were considered as upregulated or downregulated, respectively. Only genes with a significant log2FC are shown if not indicated otherwise (crossed cells). Spinal cords of 4 infected and 4 uninfected age-matched mice were analyzed at each time point. (B) Detection of DNA fragmentation (TUNEL+ cells) in the spinal cord cryosections. Representative images are shown, the white line indicates the space occupied by the schistosomulum. Scale bar = 50 μm. (C) Quantification of TUNEL+ cells in the spinal cord cryosections shown in (B). The frequency of TUNEL+ cells is shown among all cells or separately for the host nervous tissue and T. regenti tissue. (D) Detection of apoptotic populations in Neuro2a and mixed glial cultures treated by T. regenti schistosomula homogenate (TrSH) for 48 hours; staurosporine was used as a positive control. Pooled data from 4 experiments are shown. Data (C, D) were evaluated by Kruskal-Wallis and Dunn’s test; *p<0.05, **p<0.01, ***p<0.001.
Table 1.
The tests are listed by their temporal order.