In the Acute Phase of Trypanosoma cruzi Infection, Liver Lymphoid and Myeloid Cells Display an Ambiguous Phenotype Combining Pro- and Anti-Inflammatory Markers

Abstract

Multiple cell populations, cellular biochemical pathways, and the autonomic nervous system contribute to maintaining the immunological tolerance in the liver. This tolerance is coherent because the organ is exposed to high levels of bacterial pathogen-associated molecular pattern (PAMP) molecules from the intestinal microbiota, such as lipopolysaccharide endotoxin (LPS). In the case of Trypanosoma cruzi infection, although there is a dramatic acute immune response in the liver, we observed intrahepatic cell populations combining pro- and anti-inflammatory markers. There was loss of fully mature Kupffer cells and an increase in other myeloid cells, which are likely to include monocytes. Among dendritic cells (DCs), the cDC1 population expanded relative to the others, and these cells lost both some macrophage markers (F4/80) and immunosuppressive cytokines (IL-10, TGF-β1). In parallel, a massive T cell response occured with loss of naïve cells and increase in several post-activation subsets. However, these activated T cells expressed both markers programmed cell death protein (PD-1) and cytokines consistent with immunosuppressive function (IL-10, TGF-β1). NK and NK-T cells broadly followed the pattern of T cell activation, while TCR-γδ cells appeared to be bystanders. While no data were obtained concerning IL-2, several cell populations also synthesized IFN-γ and TNF-α, which has been linked to host defense but also to tissue injury. It therefore appears that T. cruzi exerts control over liver immunity, causing T cell activation via cDC1 but subverting multiple populations of T cells into immunosuppressive pathways. In this way, T. cruzi engages a mechanism of hepatic T cell tolerance that is familiar from liver allograft tolerance, in which activation and proliferation are followed by T cell inactivation.

Keywords: Trypanosoma cruzi infection; hepatic immune response; immunological tolerance; inflammation; liver.

Figure 1

Hepatic dendritic cells’ identification and cellular frequency after T. cruzi infection. C57BL/6 mice were infected with 1x10⁴ blood trypomastigote forms of T. cruzi Y strain and on dpi 15, infected and control mice were euthanized. The HDCs we isolated by enzymatic dissociation and primarily identified according to the expression of CD11c and/or CD11b (A). In control mice, cDC1 and pDC were identified in P3, cDC2 in P6, and pre DC in P4. In infected mice, cDC1 and pre DC were identified in P3, pDC in P4, and cDC2 in P5 (A). The complete phenotype to discern cDC1, cDC2, pDCs, and pre DCs is depicted in B and the frequency of each cellular population is shown in (C) @ Indicates that most pre DCs from control mice do not express CD8 and, therefore, were represented as (-), but about 15% of the cells were CD8⁺. The primary canonic phenotype for each population is shown in red (B). * means statistical significance (p ≤ 0.05) using the Kruskal Wallis test followed by Dunn’s post-test, and ** means p ≤ 0.01 using the one-way ANOVA test followed by the Tukey’s post-test.

Figure 2

Profile of cytokines produced by hepatic dendritic cells: The definition of TNF-α⁺, IL-10⁺, or TGF-β1⁺ cells was done as illustrated in the dot plots. The frequency of cDC1, cDC2, pDCs, and pre DC producing TNF-α, IL-10, or TGF-β1 was evaluated by flow cytometry in control and T. cruzi infected mice on dpi 15, with the complete gating strategy shown in Supplementary Figures 1 (control) and 2 (infected mice). Statistically significant differences are shown as ** p ≤ 0.05 and ***p ≤ 0.01 using the one-way ANOVA test followed by Tukey’s post-test.

Figure 3

Kupffer cell identification and phenotype after T. cruzi infection. C57BL/6 mice were infected with trypomastigote forms of T. cruzi Y strain and on dpi 15, infected and control mice were euthanized. The KCs were identified as F4/80⁺ and two subpopulations were depicted according to the expression of CD11b (A). The complete phenotype to discern CD11b^- and CD11b⁺ KCs is shown in (B) and the frequency of each cellular population is shown in (C). The frequency of each subpopulation expressing TNF-α (D), IL-10 (E), or TGF-β1 (F) are indicated for control and infected mice. ** means statistical significance (p ≤ 0.05) and *** means p ≤ 0.01. We used the Kruskal Wallis test followed by Dunn’s post-test to analyze TNF-α and TGF-β1 in CD11b- KC cells. We used the one-way ANOVA test followed by Tukey’s post-test for all other analyses.

Figure 4

Intrahepatic T lymphocytes after T. cruzi infection. The flow cytometry data analysis was done for CD4⁺ and CD8⁺ T lymphocytes (illustrated in A) in the gate of CD3⁺ events. The gating strategy is shown for EM and effector cells using an infected mouse (A). Then, for each population, the frequency of naïve (CD62L⁺CD44^low); effector (CD62L^-CD44^highCD127^-), EM (CD62L^-CD44^highCD127⁺); and CM T lymphocytes (CD62L⁺CD44^high) was analyzed (A). The distribution of naïve (B), effector (C), CM (D), and EM (E) T lymphocytes in the liver of control and infected mice on dpi 15 are shown. * means p ≤ 0.05 and ** means p ≤ 0.005 using the one-way ANOVA test followed by Tukey’s post-test.

Figure 5

Expression of immunoregulatory molecules on intrahepatic T lymphocytes. The analysis of CTLA-4⁺ single positive (A–C) or CTLA-4⁺PD-1⁺ double-positive (DP) (D–F) cells is shown for CD4⁺ or CD8⁺ T intrahepatic lymphocytes. The cell frequency was evaluated in control and infected mice on dpi 15. * means p ≤ 0.05 and *** means p ≤ 0.001 using the one-way ANOVA test followed by Tukey’s post-test.

Figure 6

Pro- and anti-inflammatory cytokines produced by effector and effector memory T lymphocytes after T. cruzi infection. The analysis of TNF-α, IFN-γ, IL-10, and TGF-β1 positive CD4⁺ or CD8⁺ intrahepatic T lymphocytes are shown as indicated. The cellular frequency of effector and effector memory T cells was evaluated in control and infected mice on dpi 15. * means p ≤ 0.05, ** means p ≤ 0.005, and *** means p ≤ 0.001 using the one-way ANOVA test followed by the Tukey’s post-test.

Figure 7

Analysis of NK, NKT, and γδ T lymphocytes in the liver of T. cruzi infected mice. C57BL/6 mice were IP infected with T. cruzi, and the hepatic cells were isolated by enzymatic dissociation on dpi 15. The identification of NK (NK1.1⁺CD3^-), NKT (NK1.1⁺CD3⁺), and γδ T lymphocytes (CD3⁺γδ TCR⁺) are shown in (A) For better visualization of the populations, a dot plot from a control mouse illustrates the analysis gate of NK and NKT cells, and a dot plot from an infected animal shows the gate of γδ T lymphocytes. The frequency of each cell population is indicated (B–D) in control and infected mice. ** means p ≤ 0.05 and *** means p ≤ 0.01. For NK and γδ T lymphocytes, we used the one-way ANOVA test followed by the Tukey’s post-test, and for NKT cells we used the Kruskal Wallis followed by the Dunn’s post-test.

Figure 8

Expression of immunomodulatory molecules by NK, NKT, and γδ T lymphocytes. The intrahepatic cells were isolated from control and T. cruzi infected mice on dpi 15, and NK (A), NKT (B), and γδ T lymphocytes (C) were analyzed. CTLA-4 single positive cells (left panels) or PD-1 and CTLA-4 double-positive cells (right panels) are shown. * means p ≤ 0.05 and ** means p ≤ 0.005. We used the one-way ANOVA test for NK and NKT cells followed by the Tukey’s post-test, and for γδ T lymphocytes, we used the Kruskal Wallis test followed by the Dunn’s post-test.

Figure 9

Production of anti- and pro-inflammatory cytokines by NK, NKT, and γδ T lymphocytes. The intrahepatic cells were isolated from control and T. cruzi infected mice on dpi 15, and the production of TNF-α, IL-10, or TGF-β1 was analyzed in NK (A), NKT (B), and γδ T lymphocytes (C). * means p ≤ 0.05, **** means p ≤ 0.001 using the Kruskal Wallis test followed by the Dunn’s post-test.

Figure 10

Summary of phenotypic modulations observed in hepatic cells from control and T. cruzi infected mice obtained on dpi 15 (acute phase). The results obtained by flow cytometry were stratified in this chart according to the frequency of cells expressing each marker. Subpopulations with up to 5% of frequency were considered not represented. The dimension of each dot proportionally represents the frequency of each subpopulation found in flow cytometry analysis. According to the legend, the intervals vary from 5% to 20% of positive cells, 20% (exclusive) to 40%, and so on. Effector and EM lymphocytes represent the results for CD4 or CD8 cells.