To obtain low and high parasite loads in the acute phase of Chagas’ disease, A/J mice were infected with 103 or 105 Trypanosoma cruzi trypomastigotes of the Y strain and treated on day 6 with benznidazol. One year later, chronically infected mice were screened for subpatent parasitemias, tissue pathology, and immune response. Mice infected with the high parasite inoculum showed higher levels of chronic parasitemias, heart and striated muscle inflammation, and activation of the immune system than did mice infected with the low inoculum. Concerning the activation of the immune system, the main findings for high-dose-infected mice were (i) increased numbers of splenocytes, with preferential expansion of CD8+ and B220− CD5− cells, many of them bearing a macrophage phenotype; (ii) higher frequencies of B (B220+), CD4+, and CD8+ large lymphocytes; (iii) a shift of CD4+ cells towards a CD45RBLow phenotype; (iv) increased frequencies of both CD45RBLow and CD45RBHigh large CD4+cells; (v) augmented numbers of total immunoglobulin (Ig)-secreting cells, with predominance of IgG2a-producing cells; and (vi) increased production of gamma interferon and interleukin 4. In addition, these mice presented lower IgM and higher IgG2a and IgG1 parasite-specific serum antibody levels. Our results indicate that the parasite load at the acute phase of T. cruzi infection influences the activation of the immune system and development of Chagas’ disease pathology at the late chronic phase of the disease.

Highly virulent strains of Trypanosoma cruzi are frequently used as murine models of Chagas’ disease. However, these strains do not fully represent the spectrum of parasites involved in the human infection. In this paper, we analysed parasitaemia, mortality, tissue pathology and parasite‐specific IgG serum levels in immune‐deficient mice infected with Sylvio X10/4 parasites, a T. cruzi derived from a chagasic patient that yields very low parasitaemias and in C3H/HePAS mice induces a chronic cardiopathy resembling the human disease. IFN‐γ was identified as a crucial element for parasite control as its absence determined a drastic increase in parasitaemia, tissue parasitism, leukocyte infiltrates at the heart and striated muscles and mortality. The lack of IFN‐γ or IL‐12p40, a molecule shared by IL‐12 and IL‐23, also resulted in spinal cord lesions and a progressive paralysis syndrome. Whereas IgG2a was the main Ig isotype in infected C57BL/6 mice, IL‐12p40‐KO mice produced IgG2a and IgG1 and IFN‐γ‐KO mice produced only IgG1. The IFN‐γ‐protective effect was not essentially mediated by nitric oxide (NO), inasmuch as infected iNOS‐KO mice showed no parasitaemia and low tissue damage. Mice deficient in CD4+ or CD8+ T cells showed an intermediate phenotype with increased mortality and tissue pathology but no parasitaemia. Interestingly, CD28‐KO mice were unable to produce anti‐T. cruzi IgG antibodies but presented moderate tissue pathology and managed to control the infection. Thus, differently from infections with high virulence parasites, neither IgG, NO nor CD28‐mediated signalling are essential for the non‐sterile control of Sylvio X10/4 parasites.

Over the past 20 years, the immune effector mechanisms involved in the control of Trypanosoma cruzi, as well as the receptors participating in parasite recognition by cells of the innate immune system, have been largely described. However, the main questions on the physiopathology of Chagas disease remain unanswered: “Why does the host immune system fail to provide sterile immunity?” and “Why do only a proportion of infected individuals develop chronic pathology?” In this review, we describe the mechanisms proposed to explain the inability of the immune system to eradicate the parasite and the elements that allow the development of chronic heart disease. Moreover, we discuss the possibility that the inability of infected cardiomyocytes to sense intracellular T. cruzi contributes to parasite persistence in the heart and the development of chronic pathology.

Background: Stimulator of Interferon Genes (STING) is a major player in the activation of robust innate immune response leading to initiation and enhancement of tumor-specific adaptive immunity. Several clinical and pre-clinical programs are developing cyclic dinucleotides - analogues of endogenous STING ligands. However their chemical nature and stability limit their use as systemic immuno-therapeutics. Herein, we present potent and selective non-nucleotide, non-macrocyclic, small molecule direct STING agonists, structurally unrelated to known chemotypes and suitable for systemic administration. Methods: Binding to recombinant STING protein was examined using FTS, MST, FP and crystallography studies. Phenotypic screen was performed in THP-1 Dual reporter cells. Human macrophages (HMDM) and dendritic cells (HMDC) were differentiated from monocytes (obtained from PBMC) in the presence of M-CSF and GM-CSF/IL-4 for HMDM and HMDC, respectively. Mouse bone marrow-derived dendritic cells (BMDC) were obtained from C57BL/6 or STING KO mice and differentiated with mIL-4 and mGM-CSF. STING agonists were administered into BALB/c mice and cytokine release was measured in plasma. Additionally, mice were inoculated with CT26 murine colon carcinoma cells and the compound was administered, followed by the regular tumor growth monitoring. Finally, the compound was administered to C57BL/6 WT and STING KO mice in several escalating doses. Results: Ryvu's agonists demonstrate a strong binding affinity to recombinant STING proteins across tested species. They trigger pro-inflammatory cytokine release from human PBMC and HMDC and induce dendritic cell maturation regardless of the STING haplotype. Systemic in vivo administration leads to dose-dependent upregulation of STING-dependent pro-inflammatory cytokines, suggesting immune activation which translates into efficacy in vivo in CT26 mouse colorectal cancer model and complete tumor remissions. Furthermore, cured animals develop lasting immunological response demonstrated by diminished tumor growth or lack of palpable tumors in re-challenged mice. Conclusion: Ryvu's STING agonists selectively activate STING-dependent signaling in both mouse and human immune cells promoting anti-tumor immunity. Treatment with Ryvu's STING agonists leads to engagement of the immune system which results in complete tumor remission and development of immunological memory against cancer cells. The compounds show good selectivity and ADME properties enabling development for systemic administration as a single agent or in combinations with immunotherapies or targeted agents. Citation Format: Stefan Chmielewski, Magdalena Zawadzka, Jolanta Mazurek, Maciej K. Rogacki, Karolina Gluza, Katarzyna Wójcik-Jaszczyńska, Aleksandra Poczkaj, Grzegorz Ćwiertnia, Grzegorz Topolnicki, Maciej Kujawa, Eliza Zimoląg, Urszula Głowniak-Kwitek, Magdalena Mroczkowska, Agnieszka Gibas, Marcin Leś, Sylwia Sudoł, Marek Wronowski, Kinga Michalik, Katarzyna Banaszak, Katarzyna Wiklik, Federico Malusa, Michał Combik, Karolina Wiatrowska, Łukasz Dudek, Jose Alvarez, Anna Rajda, Faustyna Gajdosz, Aniela Gołas, Katarzyna Wnuk-Lipińska, Kamil Kuś, Ewelina Gabor-Worwa, Charles Fabritius, Luigi Stasi, Peter Littlewood, Krzysztof Brzózka, Monika Dobrzańska. Development of selective small molecule STING agonists suitable for systemic administration [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4532A.

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