NK-, NKT- and CD8-Derived IFNγ Drives Myeloid Cell Activation and Erythrophagocytosis, Resulting in Trypanosomosis-Associated Acute Anemia

Abstract

African trypanosomes are the causative agents of Human African Trypanosomosis (HAT/Sleeping Sickness) and Animal African Trypanosomosis (AAT/Nagana). A common hallmark of African trypanosome infections is inflammation. In murine trypanosomosis, the onset of inflammation occurs rapidly after infection and is manifested by an influx of myeloid cells in both liver and spleen, accompanied by a burst of serum pro-inflammatory cytokines. Within 48 hours after reaching peak parasitemia, acute anemia develops and the percentage of red blood cells drops by 50%. Using a newly developed in vivo erythrophagocytosis assay, we recently demonstrated that activated cells of the myeloid phagocytic system display enhanced erythrophagocytosis causing acute anemia. Here, we aimed to elucidate the mechanism and immune pathway behind this phenomenon in a murine model for trypanosomosis. Results indicate that IFNγ plays a crucial role in the recruitment and activation of erythrophagocytic myeloid cells, as mice lacking the IFNγ receptor were partially protected against trypanosomosis-associated inflammation and acute anemia. NK and NKT cells were the earliest source of IFNγ during T. b. brucei infection. Later in infection, CD8+ and to a lesser extent CD4+ T cells become the main IFNγ producers. Cell depletion and transfer experiments indicated that during infection the absence of NK, NKT and CD8+ T cells, but not CD4+ T cells, resulted in a reduced anemic phenotype similar to trypanosome infected IFNγR-/- mice. Collectively, this study shows that NK, NKT and CD8+ T cell-derived IFNγ is a critical mediator in trypanosomosis-associated pathology, driving enhanced erythrophagocytosis by myeloid phagocytic cells and the induction of acute inflammation-associated anemia.

Fig 1. Analysis of cell composition and serum cytokines in IFNγR-/- mice.

A) Anemia profile of T. b. brucei infected C57BL/6 mice and IFNγR-/- mice. RBC counts were normalized individually compared to naïve mice. Values are presented as mean ± SD of 5 mice per group. At each time point the difference in RBC% between infected C57BL/6 mice and knock out mice was evaluated. B) Serum concentration of IL15/IL15R complex, TNFα, and IL12p70. Values represent mean ± SD of 4 mice per group. C) Composition of myeloid cells within CD45+ liver cells. Values represent mean ± SD of 4 mice per group. D) Facs plots of myeloid cells in liver. E) Composition of myeloid cells within CD45+ spleen cells. Values represent mean ± SD of 4 mice per group. F) FACS plots of myeloid cells in spleen. One representative out of two three similar experiments is shown. G) Anemia profile of T. b. brucei infected anti-IFNγ treated C57BL/6 mice and infected control PBS-infused C57BL/6 mice. RBC counts were normalized individually compared to naïve mice. At each time point the difference in RBC% between infected C57BL/6 mice and IFNγ infused mice was evaluated. Values are presented as mean ± SD of 4 mice per group *: p-value < 0.05; **: p-value < 0.01; ***: p-value < 0.005 and if nothing is mentioned the differences were not significant.

Fig 2. In vivo erythrophagocytosis assay and ex vivo RBC hemolysis experiment.

A) Ex vivo fluorescent microscopy picture of erythrophagocytozing cell in the spleen of T. b. brucei infected (day 6 p.i.) mice. Left panel: co-culture of spleen cells with unlabeled RBC. Right panel: co-culture of spleen cells with pHrodo-labeled RBC. B) Histogram of PE signal of liver macrophages exposed in vivo to unlabeled RBC (grey tinted area) and pHrodo – labeled RBC (single black line). The delta MFI is the difference between the median fluorescent intensities of the two cell populations. D&E) In vivo erythrophagocytosis assay: delta median fluorescent intensity of myeloid cells in liver (C) and spleen (D) of T. brucei infected (day 6 p.i.) mice. Values represent mean ± SEM of 6 mice per group. E) Relative contribution to phagocytosis: cell numbers were multiplied by the deltaMFI observed for each subpopulation. The surface of the diagram of IFNγR-/- mice displays the total number of phagocytic cells and is calculated relatively to the surface of the diagram of C57BL/6 mice. F) In vitro stimulation of naïve monocytes derived from IFNγ-/- mice with IFNγ induces differentiation to monocyte-derived macrophages. Values represent mean +/- SD of 6 mice per group. G) In vitro erythrophagocytosis assay to determine the effect of IFNγ stimulation on naïve spleen cells from IFNγ-/- mice. Values represent mean ± SEM of 6 mice per group. One representative of 2 independent experiments is shown. *: p-value < 0.05; **: p-value < 0.01; ***: p-value < 0.005; ****: p-value < 0.0001 and if nothing is mentioned the differences were not significant.

Fig 3. Hemolysis experiment: osmotic fragility profile of naïve (A) or infected (B) RBCs of day six infected C57BL/6 (white line) or IFNγR-/- (black line) mice.

RBCs were incubated with decreasing concentrations of NaCl, resulting in hemolysis of RBCs. The percentage of hemolysis was plotted against the concentration of NaCl in the medium and the NaCl concentrations corresponding with 50% hemolysis were determined. As positive control RBCs were exposed to 100% distilled H₂O and as negative control RBCs were exposed to 100% HBSS-solution. One representative out of two experiments is shown.

Fig 4. Analysis of IFNγ production using GREAT reporter mice.

A) Serum cytokine concentration of IFNγ in infected C57BL/6 mice. Values represent mean ± SD of 4 mice. B) Number of IFNg producing cells in spleen and liver of T. b. brucei infected mice. Values represent mean ± SD of at least 3 mice. C) Composition of spleen and liver cells within IFNg producing cells. Numbers represent mean ± SD of at least 3 mice. D) Fold increase in number of NK, NKT, CD8 and CD4 T cells in liver and spleen following T. b. brucei infection. Values represent mean ± SD of 4 C57BL/6 mice. One representative out of two experiments is shown.*: p-value < 0.05; **: p-value < 0.01; ***: p-value < 0.005; ****: p-value < 0.0001 and if nothing is mentioned the differences were not significant.

Fig 5. NK and NKT activation.

A) Serum cytokine concentration of the IL15/IL15R complex in infected C57BL/6 mice. Values represent mean ± SD of 4 mice. B) Serum cytokine concentration of IL12p70 in infected C57BL/6 mice. Values represent mean ± SD of 4 mice. C) Histograms showing the expression of Sca-1 and CD107a on liver IFNγ-producing NK cells of naïve (grey tinted line) versus infected (simple black line) GREAT reporter mice. One representative out of two experiments is shown.

Fig 6. Anemia profiles and IFNγ production.

A) Anemia profile of anti-NK1.1 infused mice and isotype-control treated mice. Values represent mean ± SD of 8 mice per group. B) Anemia profile of C57BL/6 and nu/nu mice. Values represent mean ± SD of 4 mice per group. C) Anemia profile of C57BL/6 and CD8-/- mice. Values represent mean ± SD of 4 mice per group. D) Anemia profile of C57BL/6 and anti-CD8 infused mice. Values represent mean ± SD of 4 mice per group. E) Anemia profile of C57Bl/6 and CD4-/- mice. Values represent mean ± SD of 4 mice per group. F) Serum IFNγ concentration. Values represent mean ± SD of at least 4 mice per group. G) IFNγ concentration in spleen cell culture supernatant (SCC). Values represent mean ± SD of at least 4 mice per group. One representative out of two experiments is shown. At each time point the difference in RBC% between infected C57BL/6 mice and knock out mice was evaluated. *: p-value < 0.05; **: p-value < 0.01; ***: p-value < 0.005; ****: p-value < 0.0001 and if nothing is mentioned the differences were not significant.

Fig 7. Reconstitution experiments.

A) Anemia profile of CD8-/- mice and CD8 T cell reconstituted CD8-/- mice. Values represent mean ± SD of at 6 mice per group. One representative out of two experiments is shown. B) Anemia profile of nu/nu mice and CD8 T cell reconstituted nu/nu mice. Values represent mean ± SD of 4 mice per group. C) Anemia profile of nu/nu mice and CD4 T cell reconstituted nu/nu mice. Values represent mean ± SD of 4 mice per group. One representative out of two experiments is shown. At each time point the difference in RBC% between infected C57BL/6 mice and knock out mice was evaluated. ***: p-value < 0.005; ****: p-value < 0.0001 and if nothing is mentioned the differences were not significant.