DataSheet_1_Tipping the balance between erythroid cell differentiation and induction of anemia in response to the inflammatory pathology associated with chronic trypanosome infections.docx

Infection caused by extracellular single-celled trypanosomes triggers a lethal chronic wasting disease in livestock and game animals. Through screening of 10 Trypanosoma evansi field isolates, exhibiting different levels of virulence in mice, the current study identifies an experimental disease model in which infection can last well over 100 days, mimicking the major features of chronic animal trypanosomosis. In this model, despite the well-controlled parasitemia, infection is hallmarked by severe trypanosomosis-associated pathology. An in-depth scRNA-seq analysis of the latter revealed the complexity of the spleen macrophage activation status, highlighting the crucial role of tissue resident macrophages (TRMs) in regulating splenic extramedullary erythropoiesis. These new data show that in the field of experimental trypanosomosis, macrophage activation profiles have so far been oversimplified into a bi-polar paradigm (M1 vs M2). Interestingly, TRMs exert a double-sided effect on erythroid cells. On one hand, these cells express an erythrophagocytosis associated signature. On another hand, TRMs show high levels of Vcam1 expression, known to support their interaction with hematopoietic stem and progenitor cells (HSPCs). During chronic infection, the latter exhibit upregulated expression of Klf1, E2f8, and Gfi1b genes, involved in erythroid differentiation and extramedullary erythropoiesis. This process gives rise to differentiation of stem cells to BFU-e/CFU-e, Pro E, and Baso E subpopulations. However, infection truncates progressing differentiation at the orthochromatic erythrocytes level, as demonstrated by scRNAseq and flow cytometry. As such, these cells are unable to pass to the reticulocyte stage, resulting in reduced number of mature circulating RBCs and the occurrence of chronic anemia. The physiological consequence of these events is the prolonged poor delivery of oxygen to various tissues, triggering lactic acid acidosis and the catabolic breakdown of muscle tissue, reminiscent of the wasting syndrome that is characteristic for the lethal stage of animal trypanosomosis.</p

DataSheet_2_Tipping the balance between erythroid cell differentiation and induction of anemia in response to the inflammatory pathology associated with chronic trypanosome infections.xlsx

Infection caused by extracellular single-celled trypanosomes triggers a lethal chronic wasting disease in livestock and game animals. Through screening of 10 Trypanosoma evansi field isolates, exhibiting different levels of virulence in mice, the current study identifies an experimental disease model in which infection can last well over 100 days, mimicking the major features of chronic animal trypanosomosis. In this model, despite the well-controlled parasitemia, infection is hallmarked by severe trypanosomosis-associated pathology. An in-depth scRNA-seq analysis of the latter revealed the complexity of the spleen macrophage activation status, highlighting the crucial role of tissue resident macrophages (TRMs) in regulating splenic extramedullary erythropoiesis. These new data show that in the field of experimental trypanosomosis, macrophage activation profiles have so far been oversimplified into a bi-polar paradigm (M1 vs M2). Interestingly, TRMs exert a double-sided effect on erythroid cells. On one hand, these cells express an erythrophagocytosis associated signature. On another hand, TRMs show high levels of Vcam1 expression, known to support their interaction with hematopoietic stem and progenitor cells (HSPCs). During chronic infection, the latter exhibit upregulated expression of Klf1, E2f8, and Gfi1b genes, involved in erythroid differentiation and extramedullary erythropoiesis. This process gives rise to differentiation of stem cells to BFU-e/CFU-e, Pro E, and Baso E subpopulations. However, infection truncates progressing differentiation at the orthochromatic erythrocytes level, as demonstrated by scRNAseq and flow cytometry. As such, these cells are unable to pass to the reticulocyte stage, resulting in reduced number of mature circulating RBCs and the occurrence of chronic anemia. The physiological consequence of these events is the prolonged poor delivery of oxygen to various tissues, triggering lactic acid acidosis and the catabolic breakdown of muscle tissue, reminiscent of the wasting syndrome that is characteristic for the lethal stage of animal trypanosomosis.</p

Berenil treatment restores the appearance of the CIA-associated clinical symptoms.

<p>DBA/1 mice were immunized primarily with CII in CFA on day 0 and secondarily with CII in IFA on day 21. One group of mice out of two was infected with <i>T</i>. <i>brucei</i> (filled and open triangle) on day 14 either i.p. or using tsetse flies and the other was left untreated (filled and open square). Both groups were treated with the trypanocidal drug, Berenil, at (<b>A</b>,<b>B</b>) day 38 (i.p.) or (<b>C,D</b>) day 28 (tsetse). (<b>A</b>) The average clinical per mouse score was followed from day 0. (<b>B</b>) Serial serum dilution levels of anti-CII IgG2a and IgG2b Abs were measured by ELISA on day 38 (before Berenil, filled square and triangle) and day 56 (open square and triangle). (<b>C</b>) Clinical score was followed from day 0 and (<b>D</b>) serial serum dilution levels of anti-CII IgG2a and IgG2b Abs were measured by ELISA on day 28 (before Berenil, filled square and triangle) and day 42 (open square and triangle). Arrows represent the day when trypanocidal drug Berenil was administered. Graphs show the mean ± SD from at least n = 6 mice per group and the data are representative of two independent experiments.</p

Trypanosomosis drastically affects the titers of circulating anti-CII IgG Abs.

<p>DBA/1 mice were immunized primarily with CII in CFA on day 0 and secondarily with CII in IFA on day 21. One group of mice was infected with <i>T</i>. <i>brucei</i> (filled and open triangle) on day 14 i.p. and the other was left untreated (filled and open square). Serial serum dilution levels of anti-CII (<b>A</b>) IgG1, (<b>B</b>) IgG2a, (<b>C</b>) IgG2b and (<b>D</b>) IgM Abs were measured by ELISA on day 14 (before infection, filled square and triangle) and day 35 (open square and triangle). Graphs show the mean ± SD from at least n = 6 mice per group and the data are representative of two independent experiments.</p

Trypanosomosis substantially delays the onset of CIA in DBA/1 prone mice.

<p>DBA/1 mice were immunized primarily with CII in CFA on day 0 and secondarily with CII in IFA on day 21. One group of mice was infected with <i>T</i>. <i>brucei</i> (filled triangle) on day 14 i.p. and the other was left untreated (filled square). (<b>A</b>) Average clinical score and (<b>C</b>) the number of arthritic limb per mouse were followed starting at day 0. (<b>B</b>) Representative pictures of arthritic limbs at day 42. Graphs show the mean ± SD from at least n = 6 mice per group and the data are representative of three independent experiments. (D&E) DBA/1 mice were infected with <i>T</i>. <i>brucei</i> i.p. and (<b>D</b>) survival and (<b>E</b>) blood parasite counts were monitored. Graphs show the mean ± SD from at least n = 6 mice per group and the data are representative of two independent experiments.</p

Destruction of spleen marginal zones.

<p>The MZ macrophage populations of non-infected (upper panel) and day 10 <i>T. brucei</i> AnTat 1.1 infected (lower panel) C57Bl/6 mice are visualized by section staining with ER-TR9 (anti-MZM) (A,C) and MOMA-1 (anti-MMM) antibodies (B,D) (400x magnification).</p

Alteration of Follicular and Plasma B cell numbers during <i>T. brucei</i> infections.

<p>The number of follicular B cells (A) as well as plasma B cells (B) per spleen was calculated on different days after <i>T. brucei</i> AnTat 1.1E infection. Calculations were performed on cells harvested from 3 individual spleens per time point. Values represent the mean±SD. One of four representative experiments is shown. Plasma spleen B cells were stained with a B220/CD138 combination (C).</p

<i>T. brucei</i> induced abrogation of B cell proliferation.

<p>CD19⁺ MACS sorted cells derived form control mice or <i>T.brucei</i> AnTat 1.1E infected mice (day 10 post infection) were incubated for 24 h in the presence of different doses of anti-IgM Fab (A,B), or different doses of LPS (C,D). Proliferation was measure by thymidine incorporation. Results were obtained using spleen cell preparations of four individual mice, and represent the mean % of CPM increase ±SD, with the 100% showing the mean CPM level of non-stimulated cells.</p

Alterations of Marginal Zone (MZ) B cells during <i>T. brucei</i> infections.

<p>MZ B cells were detected using FACS as CD21^HighCD23^Low (R1), IgD^IntIgM^High (R2) or B220⁺CD1d⁺ (R3) on spleen cells derived from non- infected mice (A, upper FACS panel ) or day 10 <i>T. brucei</i> AnTat 1.1E-infected mice (A, lower FACS panel). The decrease in total number of MZ B-cells per spleen was calculated for different time points during infection (B), based on the total amount of cells harvested per spleen at each time point (C). Calculations were performed on cells harvested from 3 individual spleens per time point. Values represent the mean±SD. One of four representative experiments is shown.</p

Trypanosomiasis associated elimination of non-related host antibody responses.

<p>Mice were vaccinated with the commercial DPTa vaccine and boosted after three weeks. 14 days after the vaccine boost, mice were infected with 5000 <i>T. brucei</i> AnTat 1.1E parasite by intra-peritoneal injection, followed 10 days later by an intranasal challenged with 5×10⁶ CFU of <i>B. pertussis/</i>mouse (▪). Control groups consisted of non-vaccinated <i>B. pertussis</i> challenged mice (×), and DPTa vaccinated mice that were challenged with <i>B. pertussis</i> 24 days after the second DTPa boost (□). Mice were sacrificed 3h and 3, 5 and 8, days after challenge and lung homogenates were prepared and plated on the Bordet-Gongou agar plates. CFU's were measured after 72 h incubation. Values are represented as the mean±SD of 3 individual mice per time point.</p