The Receptor Slamf1 on the Surface of Myeloid Lineage Cells Controls Susceptibility to Infection by Trypanosoma cruzi

Trypanosoma cruzi, the protozoan parasite responsible for Chagas' disease, causes severe myocarditis often resulting in death. Here, we report that Slamf1−/− mice, which lack the hematopoietic cell surface receptor Slamf1, are completely protected from an acute lethal parasite challenge. Cardiac damage was reduced in Slamf1−/− mice compared to wild type mice, infected with the same doses of parasites, as a result of a decrease of the number of parasites in the heart even the parasitemia was only marginally less. Both in vivo and in vitro experiments reveal that Slamf1-defIcient myeloid cells are impaired in their ability to replicate the parasite and show altered production of cytokines. Importantly, IFN-γ production in the heart of Slamf1 deficient mice was much lower than in the heart of wt mice even though the number of infiltrating dendritic cells, macrophages, CD4 and CD8 T lymphocytes were comparable. Administration of an anti-Slamf1 monoclonal antibody also reduced the number of parasites and IFN-γ in the heart. These observations not only explain the reduced susceptibility to in vivo infection by the parasite, but they also suggest human Slamf1 as a potential target for therapeutic target against T. cruzi infection

The Receptor Slamf1 on the Surface of Myeloid Lineage Cells Controls Susceptibility to Infection by Trypanosoma cruzi

Trypanosoma cruzi, the protozoan parasite responsible for Chagas' disease, causes severe myocarditis often resulting in death. Here, we report that Slamf1-/- mice, which lack the hematopoietic cell surface receptor Slamf1, are completely protected from an acute lethal parasite challenge. Cardiac damage was reduced in Slamf1-/- mice compared to wild type mice, infected with the same doses of parasites, as a result of a decrease of the number of parasites in the heart even the parasitemia was only marginally less. Both in vivo and in vitro experiments reveal that Slamf1-defIcient myeloid cells are impaired in their ability to replicate the parasite and show altered production of cytokines. Importantly, IFN-γ production in the heart of Slamf1 deficient mice was much lower than in the heart of wt mice even though the number of infiltrating dendritic cells, macrophages, CD4 and CD8 T lymphocytes were comparable. Administration of an anti-Slamf1 monoclonal antibody also reduced the number of parasites and IFN-γ in the heart. These observations not only explain the reduced susceptibility to in vivo infection by the parasite, but they also suggest human Slamf1 as a potential target for therapeutic target against T. cruzi infection.Fondo de Investigación Sanitaria (PI040993); Ministerio de Ciencia e Innovación (SAF2005-02220, SAF2007-61716 and SAF2010-18733); European Union (Eicosanox and ChagasEpiNet); CSIC-CONICET; BSCH/UAM; Comunidad de Madrid S2010/BMD-2332; RED RECAVA RD06/0014/1013; RED RICET RD06/0021/0016; Fundacion Ramon Areces; Government of PanamaPeer reviewe

The Receptor Slamf1 on the Surface of Myeloid Lineage Cells Controls Susceptibility to Infection by Trypanosoma cruzi

Author Contributions Conceived and designed the experiments: J. Calderon, E. Maganto-Garcia, M. Fresno. Performed the experiments: J. Calderon, E. Maganto-Garcia, C. Punzon, J. Carrion. Analyzed the data: J. Calderon, E. Maganto-Garcia, C. Terhorst, M. Fresno. Contributed reagents/materials/analysis tools: C. Terhorst. Wrote the paper: C. Terhorst, M. Fresno.Trypanosoma cruzi, the protozoan parasite responsible for Chagas' disease, causes severe myocarditis often resulting in death. Here, we report that Slamf1-/- mice, which lack the hematopoietic cell surface receptor Slamf1, are completely protected from an acute lethal parasite challenge. Cardiac damage was reduced in Slamf1-/- mice compared to wild type mice, infected with the same doses of parasites, as a result of a decrease of the number of parasites in the heart even the parasitemia was only marginally less. Both in vivo and in vitro experiments reveal that Slamf1-defIcient myeloid cells are impaired in their ability to replicate the parasite and show altered production of cytokines. Importantly, IFN-γ production in the heart of Slamf1 deficient mice was much lower than in the heart of wt mice even though the number of infiltrating dendritic cells, macrophages, CD4 and CD8 T lymphocytes were comparable. Administration of an anti-Slamf1 monoclonal antibody also reduced the number of parasites and IFN-γ in the heart. These observations not only explain the reduced susceptibility to in vivo infection by the parasite, but they also suggest human Slamf1 as a potential target for therapeutic target against T. cruzi infection.Ministerio de Ciencia e InnovaciónSAF2005-02220,SAF2007-61716SAF2010-18733CSIC-CONICETBSCH/UAMComunidad de MadridS2010/BMD-2332RED RECAVARED RICETRD06/0014/1013RD06/0021/0016Depto. de Sanidad AnimalFac. de VeterinariaTRUEpu

Slamf1 deficient myeloid cells are less susceptible to <i>T. cruzi</i> infection.

<p>Mice were intraperitoneally infected with <i>T. cruzi</i> and at 0, 7, 14, 21 and 28 dpi mice were sacrificed. A) Giemsa staining of adherent peritoneal macrophages from <i>Slamf1</i>^−/− and BALB/c animals were isolated by intraperitoneal lavage with PBS and stained. A representative field is shown. B) Quantification of infected adherent cells in the peritoneal lavages. Quantification of the number of amastigotes nests per 20 field and average number of amastigotes/nest per 20 fields. Results are expressed as the mean values (±SD) for 100 independent microscopically fields from 5 different mice (20 each). C) Peritoneal macrophages and D) DC cells from BALB/c and <i>Slamf1^−/−</i> mice were infected in vitro with <i>T. cruzi</i> (10 parasites/cell). The number of amastigotes released to the supernatant after 48 of infection was estimated by counting them by optical microscopy. Results are expressed as the mean values (±SD) for triplicates from 3 different experiments. E and F) Neonatal cardiomyocytes were infected “in vitro” with <i>T. cruzi</i> and 72 h postinfection analyzed by Giemsa staining. Quantification of the number of infected cardiomyocytes per field (E) and average number of amastigotes/cardiomyocyte (F). Results are expressed as the mean values (±SD) for 100 independent microscopic fields from 5 different mice (*) Statistically significant differences between <i>Slamf1^−/−</i> mice and BALB/c (p>0.05).</p

Anti-Slamf1 antibodies reduce heart parasite load.

<p>BALB/c or <i>Slamf1^−/−</i> mice were intraperitoneally infected with 1×10² trypomastigotes of the <i>T. cruzi</i> Y strain and treated with anti Slamf1 or control antibodies (0.5 mg/mouse once a week). At different dpi mice were sacrificed. A) <i>T. cruzi</i> DNA was quantified in the heart tissue of infected mice and expressed as the number of picograms of parasite DNA per milligram of DNA obtained from a heart tissue sample. Results are expressed as the mean values (±SD) for triplicates of pooled DNA from 5 different mice. A representative experiment of the 2 performed is shown. B) IFN-γ mRNA production in the heart of <i>T. cruzi</i> infected mice. Total RNA was isolated in heart tissue at different dpi, and quantitative reverse-transcriptase polymerase chain reaction was performed as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002799#s4" target="_blank">Materials and Methods</a>. Results are expressed as the logarithm of relative quantity (RQ) calculated from comparative threshold cycle values, as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002799#s4" target="_blank">Material and Methods</a>. (*) Statistically significant differences between <i>Slamf1^−/−</i> mice and BALB/c (p>0.05).</p

Heart cytokine and immune modulator production by <i>T. cruzi</i> infected mice.

<p>Cytokine mRNA production in the heart of <i>T. cruzi</i> infected mice was evaluated by QC-PCR as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002799#s4" target="_blank">Methods</a>. Total RNA was isolated in heart tissue obtained from BALB/c and <i>Slamf1^−/−</i> mice at different dpi, and quantitative reverse-transcriptase polymerase chain reaction was performed as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002799#s4" target="_blank">Materials and Methods</a>. Results are expressed as the logarithm of relative quantity (RQ) calculated from comparative threshold cycle values, as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002799#s4" target="_blank">Material and Methods</a>. (*) Statistically significant differences between <i>Slamf1^−/−</i> mice and BALB/c (p>0.05).</p

Cytokine production and immune modulators by “in vitro” <i>T. cruzi</i> infected DC, macrophages or cardiomyocytes.

<p>Peritoneal macrophages, DC or cardiomyocytes from <i>Slamf1^−/−</i> from BALB/c mice were infected in vitro with <i>T. cruzi</i>. A) Cox-2, iNOS and Arginase mRNA, evaluated by QC-PCR production (upper graphs) and protein by western blot (lower gels) by infected macrophages at 24 or 48 hr post infection as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002799#s4" target="_blank">Methods</a>. B) Cytokine (IFN-γ and TNF) release to supernatants from infected macrophages was evaluated by ELISA 24 or 48 hr post infection as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002799#s4" target="_blank">Methods</a>. C) Cytokine (IFN-γ and IL-12) release to supernatants from infected DCs was evaluated by ELISA 24 or 48 hr post infection as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002799#s4" target="_blank">Methods</a>. D) IFN-γ and NO production by infected cardiomyocytes. NO was evaluated by Gris reaction and IFN-γ by ELISA. Results are expressed as the mean values (±SD) for triplicates from 3 different experiments. (*) Statistically significant differences between <i>Slamf1^−/−</i> and BALB/c cells (p>0.05).</p

Immunologic populations from lymphoid organs of <i>T. cruzi</i>-infected mice.

<p>Thymocytes were isolated from thymus from control NI or <i>T. cruzi</i> infected BALB/c or <i>Slamf1^−/−</i> mice at 14 and 21 dpi. A) Total number of thymocytes isolated from NI or infected mice. Results are expressed as the mean values (±SD) for triplicates and from 5 different mice. B) CD4 and CD8 thymocytes in infected mice. Thymocytes were analyzed by two-color flow cytometry. Numbers represent % of CD4, CD8 SP, DP or DN thymocytes. Thymocytes from 5 mice in each group were pooled and analyzed. C) Spleens were isolated from infected mice and the total number of lymphocytes/spleen was quantified. Results are expressed as the mean values (±SD) for triplicates and from 5 different mice. (*) Statistically significant differences between <i>Slamf1^−/−</i> mice and BALB/c (p>0.05).</p

Time-course of <i>T. cruzi</i> infection in <i>Slamf1^−/−</i> mice and reduced <i>T. cruzi</i> infection in the hearts from <i>Slamf1−/−</i> animals.

<p>BALB/c or <i>Slamf1^−/−</i> mice were intraperitoneally infected with 2×10³ trypomastigotes of the <i>T. cruzi</i> Y strain and were sacrificed at different dpi. A) Survival. B) Serum CK levels. Analysis of <i>T. cruzi</i> presence in heart tissue from <i>T. cruzi</i> infected BALB/c or <i>Slamf1^−/−</i> animals: C) Quantification of <i>T. cruzi</i> DNA in the heart tissue of infected BALB/c- and <i>Slamf1^−/−</i> mice. <i>T. cruzi</i> DNA is expressed as the amount of parasite DNA obtained from a heart tissue sample (pg of parasite DNA/mg of heart tissue). Results are expressed as the mean values (±SD) for triplicates of pooled DNA from 5 different mice. A representative experiment of the 3 performed is shown. D) Histochemical analysis by Hematoxylin-Eosin stain. A representative field is shown. E) Quantification of the number of amastigote nests per 20 fields. F) Average number of amastigotes/nest per 20 fields. At least 20 fields were observed of each preparation (3 preparations/mouse and 3 mice per group). Results are expressed as the mean values (±SD) for 100 independent microscopic fields from 5 different mice (20 each). G) Blood parasitemia. (*) Statistically significant differences between <i>Slamf1^−/−</i> mice and BALB/c (p>0.05).</p

Interaction of Signaling Lymphocytic Activation Molecule Family 1 (SLAMF1) receptor with Trypanosoma cruzi is strain-dependent and affects NADPH oxidase expression and activity

The receptor Signaling Lymphocyte-Activation Molecule Family 1 (SLAMF1) controls susceptibility to Infection by the lethal Trypanosoma cruzi Y strain. To elucidate whether genetic diversity of the parasite was related with disease susceptibility, we further analyzed the role of SLAMF1 using 6 different Trypanosoma cruzi strains including Y. The interaction of SLAMF1 receptor with T. cruzi was evidenced by fluorescence microscopy, flow cytometry and quantitative PCR. All the strains, except VFRA, showed a decrease in parasite load in infected macrophages in Slamf1-/- compared to BALB/c. In macrophages gene expression NADPH oxidase (NOX2), and reactive oxygen species (ROS) production increased in Slamf1-/- compared to BALB/c in 5 out of 6 strains. However, Slamf1-/-macrophages infected with VFRA strain exhibited a divergent behavior, with higher parasite load, lower NOX2 expression and ROS production compared to BALB/c. Parasitological and immunological studies in vivo with Y strain showed that in the absence of SLAMF1 the immune response protected mice from the otherwise lethal Y infection favoring a proinflammatory response likely involving CD4, CD8, dendritic cells and classically activated macrophages. In the case of VFRA, no major changes were observed in the absence of SLAMF1. Thus, the results suggest that the T. cruzi affects SLAMF1-dependent ROS production, controlling parasite replication in macrophages and affecting survival in mice in a strain-dependent manner. Further studies will focus in the identification of parasite molecules involved in SLAMF1 interaction to explain the immunopathogenesis of the diseaseMinisterio de Economía y competitividad” SAF2015-63868-R (MINECO/FEDER) and FEDER and ”Ministerio de Ciencia, Innovación y Universidades-Agencia Estatal de Investigación” PGC2018-096132-B-I00 (MICINN/FEDER) to NG; SAF2016-75988-R (MINECO/FEDER), “Red de Investigación de Centros de Enfermedades Tropicales” (RICET RD12/0018/0004), Comunidad de Madrid (S-2010/BMD-2332