Parasites, hosts and experimental primary and secondary infections
Encysted metacercariae of E. caproni were removed from the kidney and pericardial cavity of experimentally infected Biomphalaria glabrata snails and used to infect male ICR mice weighing 30-35 g by gastric gavage both primary and challenge infections (50 metacercariae each). Positivity of infection in each case was determined at necropsy or detection of eggs in stools [22]. Animals were maintained under conventional conditions with food and water ad libitum. Each experiment was performed in triplicate.
Ethical statement
This study has been approved by the Ethical Committee of Animal Welfare and Experimentation of the University of Valencia (Ref#A18348501775). Protocols adhered to Spanish (Real Decreto 53/2013) and European (2010/63/UE) regulations.
Pharmacological treatment of primary infections
Cure of the primary infections was achieved by pharmacological treatment with pzq. Mice were treated with a double dose of 100 mg/Kg of pzq at 4 weeks post-primary infection (wppi), orally administered on alternate days [28]. All mice treated with pzq reverted to negative as determined by coprological examination. The influence of the pharmacological treatment over the studied parameters was discarded since five mice were left uninfected, treated with pzq as described above and analyzed.
Treatment of mice with blocking antibodies or isotype-matched IgG control antibodies
Briefly, several mice were sensitized by intraperitoneal injection with commercial specific blocking antibodies, recombinant proteins or isotype matched control antibodies.
To neutralize the effect of IL-25 produced nonspecifically after the curation of the primary E. caproni infection and to investigate the effect of this cytokine in a secondary challenge infection and the role of STAT6 activation in resistance to infection, two groups of 5 mice each were treated with monoclonal anti-mouse IL-25 (m-IL-25) (R&D Systems) or monoclonal anti-mouse IL-4R (m-IL-4R) (Biolegend). In each group, mice were primarily infected with 50 metacercariae of E. caproni and treated with pzq at 4 wppi. On each of the two days previous to a secondary infection at 6 wppi, mice were intraperitoneally injected with either m-IL-25 (concentration: 0.25 g/l) in one of the groups or m-IL-4R (concentration: 0.1 g/l) to the mice belonging to the other group in 150 l of saline buffer. Additionally, and to be used as control of the m-IL-25-treated mice, 5 mice were injected with rat IgG1 and other 5 mice with IgG2b and used as controls for the m-IL-4R-treated group of animals. All mice were sacrificed at 2 weeks post-secondary infection (wpsi).
Recombinant cytokines were used to analyze their effect on the course of the infection. For this purpose, groups of 5 mice were intraperitoneally injected with either IL-4 (rIL-4; Prepothech), IL-13 (rIL-13; Prepotech) or IL-25 (rIL-25; R&D Systems) (concentration: 0.2 g/l each) in 150 l of PBS during each of the 4 days from the primary infection with 50 metacercariae of E. caproni. All mice were sacrificed at 2 wpsi. Additionally, a group of five mice were treated with recombinant IL-13R2 (rIL-13R2; R&D Systems) (concentration: 0.2 g/l) following identical protocol to study the role of IL-13R2 in E. caproni infection. As control animals, five mice were intraperitoneally injected with 150 l of PBS following the same protocol.
Analysis of IL-25 gene expression over time and its influence in resistance to secondary infections
To analyze further the role of the innately produced IL-25 and the ability of E. caproni to induce IL-25 mRNA expression in mice in memory secondary infections, we delayed the challenge infection until IL-25 gene expression recovered to baseline levels. To this purpose, a total of 20 mice were primarily infected with metacercariae of E. caproni and treated with pzq at 4 wpi. From 6 wppi, 3 of these mice were sacrificed every two weeks and the levels of IL-25 mRNA expression were studied by rtPCR. Once baseline levels were recovered, the remainder 5 mice of the group were secondarily infected and necropsied at 12 wppt. Moreover, 5 other mice were used as control. To avoid age-related changes in susceptibility allowing accurate comparisons, these mice were aged and primarily infected having the same age than those that were secondarily infected.
Total RNA extraction
Total RNA was extracted from full-thickness sections of ileum of necropsied mice. Total RNA was isolated using Real Total ARN Spin Plus kit (Durviz) according to the manufacturer’s instructions. The cDNA was synthesized using High Capacity cDNA Reverse Transcription kit (Applied Biosystems).
Real-Time PCR and relative quantification analysis
For quantitative PCR, 40 ng total RNA was reverse transcribed to cDNA and added to 10µL of TaqMan Universal PCR Master Mix, No AmpErase UNG (2x), 1µL of the specified TaqMan Gene Expression Assay, and water to a final reaction volume of 20µL. Reactions were performed on the Abi Prism 7000 (Applied Biosystems), with the following thermal cycler conditions: initial setup of 10 min at 95 ºC, and 40 cycles of 15 s denaturation at 95 ºC and 1 min of annealing/extention at 60 ºC each. Samples were amplified in a 96-well plate. In each plate, endogenous control, samples and negative controls were analyzed in triplicate. All TaqMan Gene Expression primers and probes for inducible nitric oxide synthase (iNOS), cytokines and mucins were designed by Applied Biosystems and offered as Inventoried Assays. The assay ID details are shown in Additional file 1: Table S1. Each assay contains two unlabeled primers and one 6-FAM dye-labeled, TaqMan MGB probe. Primer concentration was optimized by a matrix of reactions testing a range of concentrations for each primer against different concentrations of the partner primer and also negative controls were included.
Cycle threshold (Ct) value was calculated for each sample, housekeeping and uninfected control. To normalize for differences in efficiency of sample extraction or cDNA synthesis we used β-actin as housekeeping gene. To estimate the influence of infection in the gene expression levels we used a comparative quantification method (2-ΔΔCT – method). This method is based on the fact that the difference in threshold cycles (ΔCt) between the gene of interest and the housekeeping gene is proportional to the relative expression level of the gene of interest. The fold change in the target gene was normalized to β-actin and standardized to the expression at time 0 (uninfected animals) to generate a relative quantification of the expression levels.
Analysis of goblet cell responses
Goblet cell responses to E. caproni infections in the ileum of mice were evaluated in primary and secondary infections in rIL-25-treated mice. At each time point, 5 mice in each group were necropsied and ileal sections of about 0.7 cm in length were obtained and fixed in 4% paraformaldehyde (PFA). After embedding in paraffin wax, serial 4 m-sections were cut from each tissue block and stained with alcian blue. Cell counts were calculated as the number of goblet cells per crypt unit studied over 10 selected high power field (400x).
Indirect immunofluorescence
Translocation and phosphorylation of STAT6 were study by fluorescent immunohistochemistry on paraffin-embedded tissue sections [29]. Rabbit antibodies anti-STAT6 (ThermoFisher Scientific) and anti-p-STAT6 (ThermoFisher Scientific) were used. Anti-STAT6 and anti-p-STAT6 were diluted 1/200 and 1/20, respectively, in PBS containing 0.3% Triton X-100 and 10% FCS and incubated for 2h in a humid chamber at room temperature, under continuous agitation. After washing 3 times in PBS, intestinal sections were incubated for 2 h with secondary antibody, goat anti-rabbit IgG conjugated with Alexa Fluor® 647 (Jackson ImmunoResearch Laboratories, Inc.), diluted 1/600 in PBS-Triton™ X-100 (0.3%) for Anti-STAT6 and 1/100 for anti-p-STAT6. Slides were washed in PBS and cell nuclei were counterstained with DAPI before mounting with Fluoromoun (Sigma-Aldrich). Cell staining was analyzed by fluorescence microscopy. Results were studied over 5 selected fields.
Enzyme immunohistochemistry
To analyze the tuft cells and GATA3+ cells, such as ILC2 and Th2 populations in primary and secondary infections with E. caproni, enzymatic immunohistochemistry of intestinal sections was performed in a total of 20 mice at 2 wppi (n=5), 2 wppt (n=5) and 2 wpsi (n=5). Additionally, 5 naïve mice were used as controls. Initially, intestinal samples of those mice, were dewaxed by incubating them for 20 minutes in an oven at 60 ° C, passed through a hydration chain (Xylene 4x 5 min - 100% Ethanol 2x 3 min - 90% Ethanol 2x 3 min - 70% Ethanol 2x 3min) and were incubated in 10 mM Sodium Citrate Buffer for 10 min to improve antigen detection. Once the samples were cooled, they were kept in running water for 10 min and washed twice for 5 minutes in Tris Buffer Saline (TBS) + 0.1% Triton X-100 pH 7.6 while stirring. Sections were blocked with 2.5% Normal Goat Serum (Vector) for 1 hour at room temperature. After blocking, they were incubated over night at 4 ° C with the primary antibody at 1: 1000 dilution in TBS and 1% BSA. As primary antibodies were used: a-DCLK-1 (Abcam) for labeling tuft cells and a-GATA3 (Abcam) for labeling GATA3+ cells.
In order to eliminate the own autofluorescence from the tissue, the samples were incubated in Dual Endogenous Enzime Block (Dako) for 10 min at room temperature. They are then incubated with the secondary Polyclonal Goat anti-Rabbit Immunoglobulins HRP (Dako) antibody diluted 1: 1000 in TBS + 0.1% Triton X-100 and 1% BSA for 1 hour at room temperature. After all incubation steps, 2 washes of 5min were performed with TBS + 0.1% Triton X-100 with gentle agitation.
DAB was selected as a chromogen to reveal the reaction (Liquid DAB + Substrate Chromogen System, Dako). The development time must be controlled by observing the brown precipitates produced by reacting the DAB with the Ab2-HRP. The samples are washed with running tap water to stop the reaction.
Finally, the sections were contrasted with Mayer's Hematoxilin (Dako), passed through a dehydration chain (Scott's tap water 30 sec - 90% Ethanol 2x30sec - 100% Ethanol 2x 3min - Xylene 2x 3 min) and mounted in DPX liquid medium for later analysis in an optical microscope at a magnitude of 200x magnification. Cell populations were studied over 10 selected representative fields.
Induction of intestinal dysbacteriosis
To analyze the effect of resident microbiota on the production of IL-25 in response to E. caproni infection. A total of 10 mice were primarily infected and treated with pzq at 2 wppi. To avoid recovering of the microbiota and to evaluate its role in a secondary infection, dysbacteriosis was induced in in 5 of these mice using a cocktail of antibiotics. Mice received drinking water containing ampicillin (Sigma) (1.0 g/l), metronidazol (Guinama) (1.0 g/l), neomycin (Sigma) (1.0 g/l) and vancomycin (Sigma) (0.5 g/l) for two weeks before secondary infection of E. caproni. The remainder mice were secondarily infected at 2 wppt without antibiotic treatment. All mice were necropsied at 2 wpsi and expression of IL-25 was compared with that of secondary infections in mice not treated with antibiotics. Additionally, 5 mice were used to evaluate the potential effect of antibiotic treatment in the expression of IL-25. These mice primarily infected and treated with pzq and antibiotics following the same procedure but not secondarily infected. No changes in IL-25 expression were observed in these control animals.
Determination of the total bacterial load in fecal samples
In order to determine the total bacterial load in the fecal samples, qPCR of 16S rRNA gene was performed. For this purpose, the KAPA SYBR. FAST qPCR Kit was used. For each sample, 20 μl PCR duplicates were prepared with each containing 2μl of the DNA used as template, 10μl of mix provided by the manufacturer, and 0.4μl of forward and reverse primers at the final concentration of 0.2mM (27F-qPCRAGAGTTTGATCMTGGCTCAG; 338R-qPCRTGCTGCCTCCCGTAGGAGT). In order to complete the volume of the reaction, 7.2 μl of water was added.
A PCR product of the 16S rRNA gene from Enterococcus faecium C68 strain was used for obtaining a standard curve. This E. faecium 16S rRNA PCR was performed as follows. Briefly, 25μl reaction was prepared containing 1μl of 1 bacterial colony resuspended in PBS, 2.5μl 10x Standard Taq Reaction Buffer (New England BioLabs), 0.25 mM of deoxynucleoside triphosphates (dNTPs), 2.5 U of Taq DNA Polymerase (New England BioLabs) and 0.2 mM of primers. The volume was completed with water.
ENDMEMO program was used in order to determine the number of 16S rDNA molecules in the PCR product of E. faecium CD68 based on sequence of 16S rRNA gene and concentration of the PCR product. A standard curve was obtained by making 5-fold dilutions of the PCR product. Cycling conditions of the qPCR were 94.C for 5 minutes, and 45 cycles of 94ºC for 30 seconds, 56ºC for 30 seconds and 68ºC for 30 seconds, and a final elongation cycle at 68ºC for 5 minutes. By extrapolation of results with the ones obtained with standard curve, the number of 16S rRNA genes was determined for each sample. The final number of 16S rRNA genes per gram of fecal sample was calculated by using the following formula:
Number of 16S rDNA molecules/1g of feces = E*N/2*F
where E represents the volume of the buffer used for elution of DNA after extraction, N represents number of 16S rDNA molecules obtained by qPCR, 2 stands for the volume of DNA used for the qPCR reaction, and F represents the weight (in grams) of the fecal pellet from which DNA was extracted.
Statistical analysis
Chi-squared test 2) test was used to compare between both groups of mice at each week post-infection. To compare the worm recovery between primary and challenge infections, a Student’s t-test was used at each week post-infection. One-way ANOVA with Bonferroni test, as post-hoc analysis was used to compare gene expression levels of cytokines, enzymes, or other genes analyzed by PCR. P<0.05 was considered as significant. Prior to analyses, data were log transformed to achieve normality and verified by the Anderson–Darling Test.