In the presence of Trypanosoma cruzi antigens, activated peripheral T lymphocytes retained in the liver induce a proinflammatory phenotypic and functional shift in intrahepatic T lymphocyte

Abstract

In secondary lymphoid organs, pathogen-derived and endogenous danger molecules are recognized by pattern recognition receptors, leading to adaptive proinflammatory immune responses. This conceptual rule does not apply directly to the liver, as hepatic immune cells tolerate gut-derived bacterial molecules from the flora. Therefore, the recognition of danger and proinflammatory stimuli differs between the periphery and the liver. However, the tolerant nature of the liver must be overcome in the case of infections or cancer, for example. The central paradigm is the basis for danger recognition and the balance between inflammation and tolerance in the liver. Here, we observed functional integration, with activated peripheral T lymphocytes playing a role in the induction of a proinflammatory environment in the liver in the presence of Trypanosoma cruzi antigens. When only parasite extract was orally administered, it led to the up-regulation of hepatic tolerance markers, but oral treatment plus adoptively transferred activated splenic T lymphocytes led to a proinflammatory response. Moreover, treated/recipient mice showed increased levels of TNF, IFN-γ, IL-6, and CCL2 in the liver and increased numbers of effector and/or effector memory T lymphocytes and F4/80⁺ cells. There was a reduction in FoxP3⁺ Treg cells, NKT cells, and γδ T lymphocytes with increased liver damage in the presence of activated peripheral T cells. Our results show that the induction of a proinflammatory liver response against T. cruzi danger molecules is at least partially dependent on cooperation with activated peripheral T cells.

Keywords: Trypanosoma. cruzi; hepatic T lymphocytes; hepatic immunoregulation; liver immune response; pathogen-associated molecular pattern (PAMP); peripheral T cells.

FIGURE 1

Evaluation of Treg cells and B7‐H1 expression in the liver. Thirteen‐week‐old male C57BL/10 mice were grouped as Control, infected (oral or i.p.), and mice that received T. cruzi extract i.p. (IPAgTc) or by gavage (OralAgTc). After 15 days, liver Treg cells (CD3⁺CD4⁺FoxP3⁺) were evaluated by flow cytometry (A), and the percentage of B7‐H1⁺ area (B) in hepatic tissue was assessed by immunohistochemistry. Data represent the mean and standard deviation of 5 independent experiments with 5–6 animals per group. The results were analyzed using ANOVA and Tukey's as the post hoc test and * indicates P < 0.05 compared with the Control, IPAgTc and OralAgTc groups; # indicates P < 0.05 compared with the i.p. group; + indicates P < 0.05 compared with the Control group; and ⁻ indicates P < 0.05 compared with the IPAgTc group. Bar: 50 µm

FIGURE 2

Adoptive transfer of activated peripheral T lymphocyte and intrahepatic T lymphocyte subpopulations. Thirteen‐week‐old male C57BL/10 donor mice received 2 i.p. injections of T. cruzi extract, and activated splenic T lymphocytes (CD3⁺CD44^highCD62‐L⁻CD197⁻) were purified after fifteen days (A). Then, 5–7 syngeneic mice were divided per group between the following groups: control; T cell; T cell+AgTc; and AgTc. Fifteen days after T cell transfer and/or antigen treatment, all mice received parasite extract (equivalent to 1 × 10⁷ parasites) by gavage and after additional 36 h they were euthanized (A). To confirm that donor T lymphocyte were retained in the liver stroma for resident IHLs activation, independent experiments were done using 13‐week‐old male GFP mice (C57BL/6 background) that were treated twice with parasite extract and the analysis of transferred GFP⁺ T cells in the C57BL/6 recipient liver stroma was performed in the morphologic lymphocyte gate to identify donor GFP⁺ cells (B). The percentage (C) and number (D) of intrahepatic T lymphocyte were analyzed in the morphologic lymphocyte gate of recipient mice. The percentage of effector/effector memory T (T_em) cells (CD3⁺CD62‐L⁻CD127^+/−CD44^highCD197⁻) was analyzed in the morphologic lymphocyte gate and CD3⁺ cells (E), as well as the number of CD4⁺ (F) and CD8⁺ (G) T cells. Data represent the mean and standard deviation of 3 independent experiments with 5 animals per group. The results were analyzed using ANOVA and Tukey's as the post hoc test and * indicates P < 0.05 compared with the Control and AgTc groups; # indicates P < 0.05 compared with the T cell group; + indicates P < 0.05 compared with the Control group

FIGURE 3

Flow cytometry evaluation of NKT, Treg cells and γδ T lymphocytes in the liver. Activated splenic T lymphocytes (CD3⁺CD44^highCD62‐L⁻CD197⁻) were purified from 13‐week‐old male C57BL/10 mice after i.p. treatment with T. cruzi extract. Recipient mice were divided as follows: “Control,” mice that received PBS by gavage; “T cell,” mice that received i.p. administration of 5 × 10⁴ T cells/mouse and PBS by gavage; “T cell+AgTc,” mice that received i.p. administration of 5 × 10⁴ T cells/mouse and parasite extract, equivalent to 1 × 10⁷ parasites by gavage; and “AgTc,” mice that received i.p. PBS and parasite extract, equivalent to 1 × 10⁷ parasites by gavage. Fifteen days after the transfer/treatment, all mice received the equivalent of 1 × 10⁷ parasites by gavage (challenge) and were euthanized after 36 h. The analysis was performed in the morphologic lymphocyte gate and CD3⁺ gate for; NKT (NK1.1⁺CD3⁺) (A); Treg (CD3⁺CD4⁺FoxP3⁺) (B) cells, and γδ T cells (γδ⁺ TCR) (C). Data represent the mean and standard deviation of 3 independent experiments with 5 animals per group. The results were analyzed using ANOVA and Tukey's as the post hoc test and * indicates P < 0.05 compared with the Control and AgTc groups; # indicates P < 0.05 compared with the T cell group

FIGURE 4

Evaluation of Kupffer cells and B7‐H1, CTLA‐4, and PD1 expression in the liver after the adoptive transfer of activated peripheral T cells. Activated splenic T lymphocytes (CD3⁺CD44^highCD62‐L⁻CD197⁻) were purified from 13‐week‐old male C57BL/10 mice after i.p. treatment with T. cruzi extract. Recipient mice were divided as follows: “Control,” mice that received PBS by gavage; “T cell,” mice that received i.p. 5 × 10⁴ T cells/mouse and PBS by gavage; “T cell+AgTc,” mice that received i.p. 5 × 10⁴ T cells/mouse and parasite extract, equivalent to 1 × 10⁷ parasites by gavage; and “AgTc,” mice that received i.p. PBS and parasite extract, equivalent to 1 × 10⁷ parasites by gavage. Fifteen days after the transfer/treatment, all mice received the equivalent of 1 × 10⁷ parasites by gavage and were euthanized after 36 h. F4/80 (A–E) and B7‐H1 (F) labeling was performed in liver tissue slices, and the quantification of positive cells was performed using ImageJ in 30 microscopic fields per sample. CTLA‐4 (G) and PD1 (H) were evaluated by dot blotting using a concentration of 80 µg of total liver proteins per dot, and densitometric units were obtained by scanning the membranes. Data represent the mean and standard deviation of 4 independent experiments performed with 5 mice per group. The results were analyzed using ANOVA and Tukey's as post hoc test and * indicates P < 0.05 compared with the Control and AgTc groups; # indicates P < 0.05 compared with the T cell group; + indicates P < 0.05 compared with the Control group. Bar: 50 µm

FIGURE 5

Evaluation of cytokines in the liver after the adoptive transfer of activated peripheral T cells. Activated splenic T lymphocytes (CD3⁺CD44^highCD62‐L⁻CD197⁻) were purified from 13‐week‐old male C57BL/10 mice after i.p. treatment with T. cruzi extract. Recipient mice were divided as follows: “Control,” mice that received PBS by gavage; “T cell,” mice that received i.p. 5 × 10⁴ T cells/mouse and PBS by gavage; “T cell+AgTc,” mice that received i.p. 5 × 10⁴ T cells/mouse and parasite extract, equivalent to 1 × 10⁷ parasites by gavage; and “AgTc,” mice that received i.p. PBS and parasite extract, equivalent to 1 × 10⁷ parasites by gavage. Fifteen days after the transfer/treatment, all mice received the equivalent of 1 × 10⁷ parasites by gavage and were euthanized after 36 h. The livers were perfused, and total proteins were extracted for the evaluation of stromal IL‐10 (A), TNF (C), IFN‐γ (D), IL‐6 (E), and MCP‐1 (F) by flow cytometry. The expression of TGF‐β (B) was evaluated by ELISA. The concentration of cytokines was normalized and is expressed in pg per mg of total proteins in tissue extract. Data represent the mean and standard deviation of 3 independent experiments with 5 animals per group. The results were analyzed using Kruskal–Wallis and Dunn's as the post hoc test and * indicates P < 0.05 compared with the control and AgTc groups; # indicates P < 0.05 compared with the T cell group; + indicates P < 0.05 compared with the Control group; ⁻ indicates P < 0.05 compared with the T cell and T cell+AgTc groups

FIGURE 6

Activated intrahepatic T lymphocytes in T. cruzi infection. Thirteen‐week‐old male GFP mice were i.p. treated with T. cruzi extract, and activated splenic T lymphocytes (CD3⁺CD44^highCD62‐L⁻CD197⁻) were purified. Then, 5 × 10⁴ activated GFP⁺ spleen cells were transferred to syngeneic C57BL/6 recipient mice that concomitantly received parasite extract by gavage (equivalent to 1 × 10⁷ parasites). Fifteen days after the transfer, the activated GFP⁻ intrahepatic T cells from the recipients were purified, and 1 × 10⁶ cells were transferred to C57BL/6 mice immediately before in vivo infection using 1 × 10⁴ trypomastigote forms of T. cruzi (infected + hepatic lymphocytes group). The other groups received 1 i.p. injection of PBS (Control) or were only infected (infected group). Parasitemia was evaluated in blood on the indicated days postinfection (dpi) (A) and ALT (B), AST (C), CK‐MM (D), and CK‐MB (E) were evaluated in plasma 15 days after infection. H&E staining was performed in cardiac slices from Control (F), infected (G), and infected + hepatic lymphocytes (H). One experiment was performed with 5–6 mice per group. The results were analyzed using ANOVA and Tukey's as the post hoc test and * indicates P < 0.05 compared with the control group; # indicates P < 0.05 comparing infected plus hepatic lymphocytes with the infected group