Rapid loss of group 1 innate lymphoid cells during blood stage Plasmodium infection

Abstract

Objectives: Innate lymphoid cells (ILCs) share many characteristics with CD4⁺ T cells, and group 1 ILCs share a requirement for T-bet and the ability to produce IFNγ with T helper 1 (Th1) cells. Given this similarity, and the importance of Th1 cells for protection against intracellular protozoan parasites, we aimed to characterise the role of group 1 ILCs during Plasmodium infection.

Methods: We quantified group 1 ILCs in peripheral blood collected from subjects infected with with Plasmodium falciparum 3D7 as part of a controlled human malaria infection study, and in the liver and spleens of Pc AS-infected mice. We used genetically-modified mouse models, as well as cell-depletion methods in mice to characterise the role of group 1 ILCs during Pc AS infection.

Results: In a controlled human malaria infection study, we found that the frequencies of circulating ILC1s and NK cells decreased as infection progressed but recovered after volunteers were treated with antiparasitic drug. A similar observation was made for liver and splenic ILC1s in P. chabaudi chabaudi AS (Pc AS)-infected mice. The decrease in mouse liver ILC1 frequencies was associated with increased apoptosis. We also identified a population of cells within the liver and spleen that expressed both ILC1 and NK cell markers, indicative of plasticity between these two cell lineages. Studies using genetic and cell-depletion approaches indicated that group 1 ILCs have a limited role in antiparasitic immunity during Pc AS infection in mice.

Discussion: Our results are consistent with a previous study indicating a limited role for natural killer (NK) cells during Plasmodium chabaudi infection in mice. Additionally, a recent study reported the redundancy of ILCs in humans with competent B and T cells. Nonetheless, our results do not rule out a role for group 1 ILCs in human malaria in endemic settings given that blood stage infection was initiated intravenously in our experimental models, and thus bypassed the liver stage of infection, which may influence the immune response during the blood stage.

Conclusion: Our results show that ILC1s are lost early during mouse and human malaria, and this observation may help to explain the limited role for these cells in controlling blood stage infection.

Keywords: inflammation; natural killer cells; parasitic–protozoan.

Figure 1

ILC and innate‐like T‐cell frequencies decrease following P. falciparum infection. Representative blood parasitaemia curve over the first 7 days of infection from a single cohort (n = 6) (a). Group 1 ILC and group 1 ILC‐like subsets were identified by flow cytometry as indicated in the gating strategy (b). White blood cell counts for each patient at days 0 and 7 are depicted (c). The frequencies (d) and cell numbers (e) of group 1 ILC, group 1 ILC‐like and innate‐like T‐cell subsets are shown. The proportion of each subset producing IFNγ is shown (f). The data from b–f represent results from one to three cohorts (n = 8–14). Error bars represent the mean ± standard deviation (SD) (a). Comparisons between days 0 and 7 were made using the Wilcoxon (paired, nonparametric) test for NK cells and a two‐way ANOVA with Sidak's multiple comparisons test for other subsets. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 2

ILC1s decrease in number and frequency 5 days postinfection with PcAS. The gating strategy to identify liver ILC1s, a population that is absent in the spleen is shown (a), as well as a summary of the gating strategy for splenic ILC1s (b). These are representative plots from naïve animals. Liver and spleen single‐cell suspensions in naïve and PcAS‐infected mice were stained to determine ILC1 frequencies and cell numbers at days 1–5, 14 and 28 postinfection. Liver ILC1 (c), splenic ILC1 (d) and liver cNK (e) frequencies and absolute numbers are shown. Data are representative of two experiments from cohorts of at least n = 4 mice per time point. Comparisons were made using the Kruskal–Wallis test accompanied by the Dunn's multiple comparisons test. *P < 0.05, **P < 0.01.

Figure 3

Liver ILC1s exhibit a more apoptotic phenotype than cNK cells. Liver single‐cell suspensions were stained for a viability marker, ILC1 surface markers, followed by staining for Caspase‐3/7. The representative plots from naïve and day 2 p.i. are shown (a) where viability dye⁺ Caspase‐3/7⁻ = dead cells, viability dye⁺ Caspase‐3/7⁺ = necrotic cells, (b) viability dye⁻ Caspase‐3/7⁺ = apoptotic cells, and viability dye⁻ Caspase‐3/7⁻ = live cells. The relative frequencies of viable, apoptotic and necrotic/end‐stage apoptotic ILC1s are shown. Data represent mean ± SEM from one experiment where n = 3 for naïve mice and n = 4 for PcAS‐infected mice. Comparisons were made using the Kruskal–Wallis test accompanied by the Dunn's multiple comparisons test. **P < 0.01.

Figure 4

A novel CD49a⁺ DX5⁺ population emerges. Representative plots show a novel CD49a⁺ DX5⁺ double‐positive population in the liver (a) and spleen (b) of infected mice. The double‐positive population underwent changes during infection (c). Expression of TNF‐related apoptosis‐inducing ligand (TRAIL) and CD62L by the CD49a⁺ DX5⁺ double‐positive population is shown (d). Data are representative of three experiments from cohorts of at least n = 4 mice.

Figure 5

Depletion of cNK cells and ILC1s does not affect parasitaemia. C57BL/6J, Rag1 ^−/− and Rag2 ^−/− γ _c ^−/− mice were infected with PcAS, and the kinetics of infection was measured (a). The kinetics of PcAS infection of Ncr1‐icre x Mcl1  ^fl/fl (b) and Ncr1‐icre x Tgfbr2 ^fl/fl (c) mice was compared to that of Ncr1‐icre and Ncr1‐icre YFP⁺ control mice, respectively. The kinetics of PcAS infection was also measured after administration of the monoclonal antibody (mAb) towards NK1.1 (α‐NK1.1; clone: PK136) (d). Kinetics of parasitaemia in Ncr1‐icre x iDTR mice administered with diphtheria toxin is shown alongside the kinetics of parasitaemia for C57BL/6J control mice (e). Data in a represent results from three independent experiments with the peak parasitaemia of Rag1 ^−/− , and Rag2 ^−/− γ _c ^−/− mice compared using the Mann–Whitney unpaired (nonparametric) t‐test. Data in b, c, d represent results from one experiment. Error bars represent mean ± SEM. **P < 0.01.