Trypanosoma vivax infections: pushing ahead with mouse models for the study of Nagana. II. Immunobiological dysfunctions

Abstract

Trypanosoma vivax is the main species involved in trypanosomosis, but very little is known about the immunobiology of the infective process caused by this parasite. Recently we undertook to further characterize the main parasitological, haematological and pathological characteristics of mouse models of T. vivax infection and noted severe anemia and thrombocytopenia coincident with rising parasitemia. To gain more insight into the organism's immunobiology, we studied lymphocyte populations in central (bone marrow) and peripherical (spleen and blood) tissues following mouse infection with T. vivax and showed that the immune system apparatus is affected both quantitatively and qualitatively. More precisely, after an initial increase that primarily involves CD4(+) T cells and macrophages, the number of splenic B cells decreases in a step-wise manner. Our results show that while infection triggers the activation and proliferation of Hematopoietic Stem Cells, Granulocyte-Monocyte, Common Myeloid and Megacaryocyte Erythrocyte progenitors decrease in number in the course of the infection. An in-depth analysis of B-cell progenitors also indicated that maturation of pro-B into pre-B precursors seems to be compromised. This interferes with the mature B cell dynamics and renewal in the periphery. Altogether, our results show that T. vivax induces profound immunological alterations in myeloid and lymphoid progenitors which may prevent adequate control of T. vivax trypanosomosis.

Figure 1. T. vivax induces severe splenomegaly as a result of intense blastogenesis.

Four to five 8-week-old outbred mice were analyzed per time point (0, 10 and 20 d.p.i.) and per experiment. Mice were infected i.p. with 10² bloodstream forms of T. vivax and compared to control age-matched uninfected controls. Numbers of peripheral white blood cells (WBC), lymphocytes (A, left panel), monocytes and granulocytes (A, right panel) were determined individually, as indicated in the methods section, throughout the ensuing infection and expressed as means +/− SD of the means. Spleen cells were recovered at different time points and total numbers of lymphocytes were depicted individually given the variable number of cells obtained from individual outbred mouse used in 4 different groups of experiments (B, left panel); frequencies of cycling cells were also determined individually in one out of four experiments (B, right panel). Arithmetic means and SD of the means are presented. * p<0,020; ** p<0,007; *** p<0,0002 when compared with samples from day 0. Macroscopic examination of spleens harvested from three uninfected (normal) and three infected (20 d.p.i.) outbred mice showing the marked splenomegaly; scale bars = 1 cm (C).

Figure 2. Splenic B lymphocyte populations fall dramatically following infection.

Four to five 8-week-old outbred mice were analyzed per time point (0, 10 and 20 d.p.i.). Mice were infected i.p. with 10² bloodstream forms of T. vivax and spleen cells were recovered 10 and 20 days post infection for comparison with those recovered from age-matched normal uninfected controls. Spleen cells were stained by immunofluorescence with specific Abs directed against CD45R/B220 (B220) and CD19. Two-color acquisition was carried out on a FACS Scan cytofluorometer and the results analyzed using FlowJo software. The figure depicts the results of a representative experiment out of three using the similar number of individuals. SD of the means are presented. * p<0,038; ** p<0,003 when compared with samples from day 0. The dot plots illustrate the FACS distribution of mouse spleen cells according to the expression of CD19 and B220 at 10 d.p.i and 20 d.p.i., as compared to cells from normal uninfected mice; similar results were obtained in different mice drawn from the same group and analyzed individually (A). Arithmetic means of total lymphocyte numbers (upper panel) or individual CD19⁺ cell counts (lower panel) are depicted +/− standard deviation of the means (B). CD19⁺ cells were gated and distributed in double plots for the expression of IgM and IgD (see gating strategy in Figure S1). Numbers of Newly Arrived Immature B cells (CD19⁺IgM^hiIgD^−/lo, NAI B-T1) (C), Marginal Zone B cells (CD19⁺IgM^hiIgD^lo, MZB) (D), Follicular B cells (CD19⁺IgM^lo/hiIgD^hi, Fo B) (E), and plasma/memory cells (CD19^+/loIgM⁻IgD⁻, Plasma/Mem B) are shown in (F).

Figure 3. Loss of splenic B lymphocytes is associated with marked macrophage infiltration.

Serial sections of spleens from uninfected controls (A, C and E) and from mice infected with 10² bloodstream forms of T. vivax 20 d. p. i. (B, D and F) were fixed and further stained with B220 (A, B), anti-CD3 (C, D) or anti-F4/80 (E, F) to detect B lymphocytes, T lymphocytes and macrophages, respectively. The sections are representative of 5 mice analyzed individually per time point. For information, frequencies of gated splenic macrophages using specific Mac-1 antibodies obtained by flow cytometry using an enlarged forward scatter/side scatter combined gate of spleen cell suspensions corresponded to 7.6% and 6,75% respectively, for days 10 and 20 of infection, as compared to 2,29% obtained from normal uninfected mice.

Figure 4. T. vivax infection induces increased hematopoiesis but a collapse in granulocyte/monocyte, common myeloid and megakaryocyte precursors.

Mice were infected i.p. with 10² bloodstream forms of T. vivax. Pool of bone marrow cells were obtained from both femurs of three to six 8-week-old outbred mice then analyzed per animal at 10 and 20 d.p.i. by immunofluorescence and compared with bone marrow cells from normal age-matched uninfected controls. FSC-A/SSC-A and FSC-W/FSC-H combined plots were used to gate total bone marrow cells and to eliminate doublets and debris (for gating strategy, see Figure S2). Increases in the number of hematopoietic stem cells (HSC) during the infection (cKit^hiSca1⁺) within the “lineage negative” (lin⁻) gated population, are depicted in (A). Granulocyte-monocyte Precursors (GMP, CD16/32⁺CD34^hi), Multipotent Common Myeloid Precursors (CMP, CD16/32⁺CD34^lo) and Megakaryocytes and Erythroblasts Precursors (MEP, CD16/32⁻CD34⁻) (B, right panel), were obtained from gated lin⁻cKit⁺Sca1⁻ cells (B, left panel), distributed in double plots for CD16/32 and CD34 markers. Lin⁻cKit⁻Sca1⁺ cell numbers are shown (C). Results are expressed per individual mouse and are representative of at least 2 different experiments per time point. Arithmetic means ± SD of the means are presented. * p<0.01, ** p<0.005, *** p<0.0001 when compared with samples from day 0.

Figure 5. T. vivax infection leads to elevated bone marrow dynamics and alterations in the maturation of B-cell progenitors.

Mice were infected i.p. with 10² bloodstream forms of T. vivax. Pools of bone marrow cells were obtained from both femurs of four to eight 8-week-old outbred mice then analyzed per animal at 10 and 20 d.p.i. by immunofluorescence and compared with bone marrow cells from normal age-matched uninfected controls. Cells were stained for IgM, CD19 and CD43, and SSC-A/FCS-A combined plots were used to gate lymphocyte populations. Doublets were eliminated by a FSC-W/FSC-H combined gate (see Figure S3, for gating strategy). CD19⁺ cells were gated and total numbers of positive cells per 2 femurs are depicted in (A); PreB + Pro B (CD19⁺IgM⁻) and late immature/mature B (CD19⁺IgM⁺) cell numbers are shown respectively in (B) and (C); total numbers of Pro-B and Pre-B cells (B, right panel) were deduced from the expression of CD43 by CD19⁺IgM⁻ Pre B + Pro B gated cells. Results are expressed per individual mouse and are representative of at least 3 different experiments per time point. Arithmetic means ± SD of the means are presented. * p<0.05, ** p<0.001, *** p<0.0001 cell when compared with samples from day 0.

Figure 6. T. vivax infection results in massive production of non-specific IgG.

PBL and sera from individual mice were collected at different time points following infection with 10² bloodstream forms of T. vivax. PBL cells obtained from 2 uninfected and 4 infected mice per group were subjected to FACS analysis and numbers of recently immigrated “transitional” B cells (IgM^hiIgD^−/lo), naïve B cells (CD19⁺IgM^lo/hiIgD^hi), and Post germinative center “switched” plasma/memory B cells (CD19^loIgM⁻IgD⁻) determined per individual mouse (A) within the CD19⁺ gated population (for gating strategy see Figure S4). Total circulating IgM and IgG immunoglobulins (B) and parasite-specific IgM and IgG titers were determined individually (C). Results in B and C were obtained from at least 12 different mice per time point from different experiments. For information, experiments performed with C57BL/6 mice showed similar amounts of total IgG (20 mg/ml by day 20) but ten fold higher quantities of total IgM (10 mg/ml by day 20), but similarly low titers of parasite specific IgG and IgM responses were observed (1/300 an 1/100, respectively). Arithmetic means ± SD of the means are presented. * p<0.05, ** p<0.001, *** p<0.0001 when compared with samples from day 0.