C3H/HeN and CF1 mice were maintained at the animal facilities of IMPaM UBA-CONICET, Facultad de Medicina, Universidad de Buenos Aires and bred ad libitum under sanitary barrier in specific pathogen-free conditions (Gutierrez et al., 2020).

Briefly, RA T. cruzi bTp were obtained from whole blood at the peak of parasitemia of CF1 mice (7 days post-infection) by differential centrifugation as previously reported (Poncini et al., 2008). Epimastigotes (epi) were routinely differentiated from bTp and cultured in vitro in liver infusion tryptose (LIT) medium at 27°C to the exponential phase of growth and centrifuged at 3,000×g for 15 min at 10°C (Isola et al., 1986). mTp were obtained by one round of differentiation from epi as described by Gutierrez et al. (2020). After in vitro culture of 10 × 10⁷ epi in 10% fetal bovine serum (FBS) LIT plus Grace’s insect medium (MERC) and incubated at 27°C in tightly closed culture flasks, parasites were purified in DEAE column equilibrated with PBS-glucose (20%) at pH 8.2. Purity was analyzed by microscopic examination.

Ten- to 12-week-old C3H male mice received intradermic (ear or hindfoot) or intraperitoneal injection with 1,000 parasites. Animal health condition, parasite load, and mortality were periodically recorded.

All experiments were performed according to protocols CD N° 04/2015 approved in Res. 923/21 by the University of Buenos Aires’s Institutional Committee for the Care and Use of Laboratory Animals (CICUAL) in accordance with the Council for International Organizations of Medical Sciences (CIOMS) and International Council for Laboratory Animal Science (ICLAS), international ethical guidelines for biomedical research involving animals.

For cell collection, histology, or DNA extraction, samples were obtained at different time points after injection with PBS (negative control of infection; NI) or the parasite. Tissue sections (25 mg) were preserved at −80°C until processing for DNA extraction. Epidermal cells were obtained from ear skin sheets using trypsin (1% and 0.3%; Sigma), as previously described (Poncini and González Cappa, 2017). Spleens were enzymatically digested with hyaluronidase type IV-S (200 U/ml; Sigma-Aldrich) and collagenase type II (250 U/ml; Gibco, Invitrogen) cocktail. After mechanical and/or enzymatic disaggregation, samples from the spleen, skin, and draining lymph nodes (dLNs) were homogenized through a tissue strainer and debris removed by passing samples through a 100-µm nylon mesh. When it was necessary, red blood cells were lysed by Tris 0.83% ammonium chloride buffer, pH 7.2, and mononuclear cells were obtained by centrifugation (400×g, 30 min) in Histopaque 1083 (Sigma) gradient. Cells were washed in fresh medium and viable cell count was determined by Trypan blue dye exclusion using a Neubauer chamber. Samples with more than 85% of live cells were used for the experiments.

At different time points after PBS or Tp inoculation (3, 7, 12, 15, or 30 dpi), the ears and/or heart sections were fixed and preserved in 10% (v/v) formalin and then embedded in paraffin. Sections of 5 μm in thickness were stained with hematoxylin and eosin. In the heart sections, quantification of amastigote nests was estimated after the observation of at least 25 fields per preparation (set in triplicate with three preparations per condition) using ZEISS Primovert light microscope and Axiocam ERc 5s camera.

Single-cell suspensions from the skin, dLNs, and spleen were stained with a mix of monoclonal antibodies (Ab) conjugated with different fluorophores: anti-MHCII-PeCy5 (M5/114.15.2, eBiosciense), CD11c-FITC (N418) and CD11b-PE (M1/70) (both from BD Biosciences) or CD207-PEVIO770 (caa8-28H10), CD45-APC (ALI-4A2), CD3-biot (145-2C11), CD8-PerCP (53-6.7), and MHCII-FITC (M5/114.15.2) from Miltenyi Biotec. Staining with biotin-conjugated Ab was followed by PE-Cy7-streptavidin (BD Biosciences). Cells were acquired on FACSAria II flow cytometer (BD Biosciences) and analyzed by FlowJo 7.6 software. Gating strategies were used to exclude doublets (FSC-A vs FSC-H) and debris/dead cells by size and complexity.

Endpoint and quantitative (q)PCR were performed in order to detect parasite DNA in the skin and spleen. DNA samples were extracted using ADN puriprep T-kit (Inbio Highway, Argentina), a spin-column-based extraction protocol, following the instructions of the manufacturer. Individual samples were tested by endpoint PCR and qPCR with primers specific for T. cruzi genome satellite sequences (Duffy et al., 2009). TNF gen was used as internal control. The primer sets were as follows: SAT Fw (5′-GCAGTCGGCKGATCGTTTTCG-3′) and SAT Rv (5′-TTCAGRGTTGTTTGGTGTCCAGTG-3′) and TNF Fw (5′-GGTGCCTATGTCTCAGCCTCTT-3′) and TNF Rv (5′-GCCATAGAACTGATGAGAGGGAG-3′). Samples from infected and non-infected mice and without target DNA were assayed as positive and negative controls of amplification. Amplification was carried out as previously reported (Duffy et al., 2009; Davies et al., 2014; Poncini et al., 2015).

Quantitative PCR was performed using EvaGreen qPCR Mix Plus (Solis BioDyne) and was set in a StepOnePlus™ (Thermo Fisher Scientific). After 5 min of preincubation at 95°C, PCR amplification was carried out for 40 cycles (94°C for 10 s, 65°C for 10 s, and 72°C for 10 s). The plate was read at 72°C at the end of each cycle. Results were normalized to the internal control and expressed as arbitrary units (AU). Three independent experiments with three technical replicates per sample were conducted. Endpoint PCR was carried out as described by Davies et al. (2014). Briefly, the 20-µl reaction tube contained 100 ng of template DNA, 1 U of Taq (Pegasus, PB-L Productos BioLógicos, Argentina), 2 µl of buffer 10×, 1.5 mM of MgCl₂, 0.2 µM of primers, 2 mM of dNTPs, and distilled water to complete the volume. The cycling condition was as follows: a first step at 95°C for 3 min followed by 35 cycles at 95°C for 45 s, 55°C for 60 s, and 72°C for 45 s and a final elongation at 72°C for 1 min. Amplification products were assessed by gel electrophoresis in 1% agarose TAE1X and 1× of Page GelRed (Biotium).

Student’s t-test was used for the comparison of two different experimental conditions and one-way ANOVA and Bonferroni’s or Dunnett’s post-test was used for multiple comparisons. Survival curve was analyzed by Kaplan–Meier. All tests were performed with GraphPad Prism^® 5.01 and a p-value <0.05 was considered significant.

Recently, we have reported differences in the in vitro stimulatory capacity and infectivity of mTp and bTp in the culture with DCs from different origins (Gutierrez et al., 2020). Here, we compare some immunologic parameters after id injection of both infective stages in the experimental model.

First, we analyzed the CD45⁺ leukocyte population in the ear skin at day 3 and 7 pi by flow cytometry ( Figure 1A ). No significant differences were detected in the percentage of CD45⁺ cells infiltrating the parasite entry site with bTp or mTp in comparison with PBS ( Figures 1A, B ). Of note, at 3 dpi, bTp but not mTp displayed the presence of CD207^int cells ( Figures 1A, B ), compatible with migratory monocyte-related myeloid cells as previously described for bTp (Poncini and González Cappa, 2017). At day 7 pi, a slight influx of leukocytes was detected at the infection site with both bTp and mTp, but no significant difference was found with PBS at the epidermal sheet of the skin ( Figure 1A , right panels and data not shown). These results suggested that bTp and mTp would trigger different patterns of cell recruitment into the skin.

Although previous works demonstrated a high sensitivity of bTp to gastric degradation (Cortez et al., 2012), controversially, other reports showed that mTp presented low infectivity in comparison with bTp, not only orally but also by different routes of infection (Dias et al., 2013; de Souza et al., 2014).

The infection with mTp displayed different presentation in oral versus gastric or by intraperitoneal inoculation of the parasite (Kuehn et al., 2014; Barreto de Albuquerque et al., 2015), and no reports have characterized the infection via the skin. Here, we observed that, in sharp difference with bTp, animals injected with mTp did not show detectable parasitemia, neither by fresh drop nor by microhematocrit during acute infection (data not shown and Figure 2A ). In addition, animals injected with mTp survived the chronic infection ( Figure 2B ). We conducted PCR analysis in different tissues to test infection with mTp. DNA from the parasite was detected in the skin of mTp-infected mice by qPCR at a very low load in comparison with those infected with bTp at 12 dpi ( Figure 2C ). To discard contamination with parasite DNA at the inoculation site, we analyzed the infection by endpoint PCR in the skin and spleen at 12 dpi, confirming successful infection with both bTp and mTp ( Figure 2D ). Interestingly, at 30 dpi, parasite persistence in amastigote nests associated to skin infection was only detected in mice infected with bTp ( Figure 2E ). Next, we challenged animals infected with mTp with a lethal dose of bTp. Mice showed no detectable parasitemia during acute infection in addition to no mortality ( Figures 2A, B ). These results suggested that the infection with culture-derived mTp generates a strong immunity against T. cruzi.

APC impairments during T. cruzi infection were previously described by our group and others (Van Overtvelt et al., 1999; Alba Soto et al., 2003; Planelles et al., 2003; Poncini et al., 2008). Here, we analyze the activation status of myeloid (CD11c⁺CD11b⁺) and non-myeloid (CD11c⁺CD11b⁻) cells compatible with DCs in dLNs 2 dpi and spleens at day 15 pi with mTp and/or bTp.

As previously reported (Poncini et al., 2015; Poncini and González Cappa, 2017), bTp incremented the number of CD11c⁺ in dLNs at day 2 pi. However, neither cell recruitment nor the percentage of CD11c⁺ cells in dLNs from mTp-infected mice significantly differ from PBS-injected controls ( Figure 3A ). Interestingly, both the myeloid and non-myeloid CD11c⁺ populations incremented the expression of MHCII in mTp-infected animals compared with the ones injected with PBS or bTp ( Figure 3B ), suggesting a higher activation status in these cells. In the spleen, the CD11c⁺ population is enlarged in bTp-infected mice (Poncini et al., 2015; Poncini and González Cappa, 2017). Animals infected with mTp display no significant difference in CD11c⁺ cell recruitment to the spleen (data not shown). However, in mTp infection and challenged with bTp, mice sustained the recruitment of CD11c⁺CD11b^low/neg (R3) cells observed for bTp at 15 dpi ( Figure 3C ). Of note, both CD11c⁺CD11b^low/neg (R3) and CD11c⁺CD11b⁺ (R4) subpopulations in animals infected with mTp and challenged showed increased MHCII expression compared with bTp-infected mice, suggesting greater activation of DCs also in the spleen ( Figure 3D ).

To identify the extent of the results here described in cardiac tissue integrity, we analyzed the presence of amastigote nests in heart samples. Neither cellular infiltrates nor parasites were detected in animals infected with mTp. In fact, no differences were detected in the cardiac tissues between these samples and the negative control of the infection (data not shown).

Mice infected with mTp and challenged with bTp showed cellular infiltrates and less parasite load in cardiac tissue, measured as the number of amastigote nests as described in the Materials and Methods ( Figures 4A, B ). Consistent with the high activation status of APCs and the controlled parasitemia in animals infected with mTp and challenged with bTp, we detected an expansion of CD3⁺CD8⁺ T cells in dLNs at 15 dpi ( Figure 4C ). All results together strongly suggest that, while it is not sterilizing, the infection with in-vitro-obtained mTp induces the development of an effective immune response against T. cruzi.