Killer lymphocytes use granulysin, perforin and granzymes to kill intracellular parasites

Abstract

Protozoan infections are a serious global health problem. Natural killer (NK) cells and cytolytic T lymphocytes (CTLs) eliminate pathogen-infected cells by releasing cytolytic granule contents--granzyme (Gzm) proteases and the pore-forming perforin (PFN)--into the infected cell. However, these cytotoxic molecules do not kill intracellular parasites. CD8(+) CTLs protect against parasite infections in mice primarily by secreting interferon (IFN)-γ. However, human, but not rodent, cytotoxic granules contain the antimicrobial peptide granulysin (GNLY), which selectively destroys cholesterol-poor microbial membranes, and GNLY, PFN and Gzms rapidly kill intracellular bacteria. Here we show that GNLY delivers Gzms into three protozoan parasites (Trypanosoma cruzi, Toxoplasma gondii and Leishmania major), in which the Gzms generate superoxide and inactivate oxidative defense enzymes to kill the parasite. PFN delivers GNLY and Gzms into infected cells, and GNLY then delivers Gzms to the intracellular parasites. Killer cell-mediated parasite death, which we term 'microbe-programmed cell death' or 'microptosis', is caspase independent but resembles mammalian apoptosis, causing mitochondrial swelling, transmembrane potential dissipation, membrane blebbing, phosphatidylserine exposure, DNA damage and chromatin condensation. GNLY-transgenic mice are protected against infection by T. cruzi and T. gondii, and survive infections that are lethal to wild-type mice. Thus, GNLY-, PFN- and Gzm-mediated elimination of intracellular protozoan parasites is an unappreciated immune defense mechanism.

Figure 1

GNLY, PFN and GzmB kill parasites. (a) Representative images (of three experiments) (left) and fluorescence intensity traces along an arbitrary line (right) of FM4–64–labeled T. cruzi trypomastigotes (left) and mCherry-expressing T. gondii (RH strain) tachyzoites (right), that were treated for 30 min with sublethal amounts of GNLY and inactive GzmB-488, and then analyzed by fluorescence microscopy. Nuclei were stained with DAPI. a.u., arbitrary units. Scale bars, 1 μm. (b) Representative images (of three independent experiments) of LLC-MK2 cells infected with T. cruzi labeled with the fluorescent cell-labeling dye DDAO-se (left) and HFF cells infected with T. gondii expressing mCherry (right) that were treated with combinations of PFN, GNLY and GzmB-488. Scale bars, 10 μm. Higher-magnification images are shown in Supplementary Figure 1c. (c) Viability of intracellular T. cruzi (left) and T. gondii (right) and their respective host cells after treatment with combinations of PFN, GzmB and GNLY (parasites were assessed after 1 h of treatment; host cells were assessed after 4 h of treatment). (d) Viability of intracellular T. cruzi in anti-CD3–coated RAW 264.7 cells after a 90-min incubation with WT, GNLY^+/−, Prf1^−/− or Prf1^−/−GNLY^+/− splenocytes (effector: target ratio of 5:1). The caspase inhibitor zVAD– fmk or serine protease inhibitor DCI were added as indicated. In c,d, data are mean ± s.e.m. of three independent experiments. ***P < 0.001, **P < 0.01 *P < 0.05; one-way analysis of variance (ANOVA), as compared to untreated cells (c) or to no inhibitor (d). In c, blue asterisks represent P values of blue bars (host cell viability) and orange asterisks represent P values of orange bars (parasite viability).

Figure 2

GNLY and GzmB cause oxidative damage–mediated parasite programmed cell death. (a,b) Representative (of three experiments) transmission electron micrographs of extracellular T. cruzi (a) and the T. gondii RH strain (b) after treatment with GzmB and GNLY for the indicated times. Pretreatment of parasites with Tiron is indicated. Black arrows, normal mitochondria (M) and nuclei (N); red arrowheads, swollen mitochondria with abnormal cristae; blue arrowheads, chromatin condensation and fragmented nuclei; green arrowheads, membrane blebs. Scale bars, 100 nm. (c,d) Representative flow cytometry analysis (of three experiments) for annexin V and PI staining (c) or for DHE fluorescence (d) of extracellular T. cruzi (top) and T. gondii (bottom) 30 min after the addition of various combinations of GNLY and GzmB. zVAD–fmk (zVAD) or Tiron were added as indicated. Numbers indicate percentage of viable parasites (c) and percentage of fluorescent cells (d). (e) H₂O₂ production in treated T. cruzi (left) and T. gondii (right) cells by Amplex Red assays. (f) Representative His₆-tag immunoblot analysis (of three experiments) of the cleavage of recombinant T. cruzi proteins 30 min after the addition of the indicated amounts of GzmB. (g) Viable T. cruzi epimastigotes, transfected with an empty vector (EV) or plasmids expressing WT or inactive mutant (MPXC81A and CPXC52A) oxidative defense enzymes, 30 min after the addition of GNLY and GzmB. (h) Parasitemia in WT (left) and GNLY^+/− (right) BALB/c mice (n = 5 per group) infected with T. cruzi trypomastigotes that were transfected with an EV or with plasmids expressing WT or mutant oxidative defense enzymes. In e,g, data are mean ± s.e.m. of three independent experiments. In h, data are mean ± s.e.m. of five mice in one experiment. ***P < 0.001, **P < 0.01; by one-way ANOVA relative to untreated cells (e) or to parasites transfected with EV (g,h).

Figure 3

GNLY^+/− mice are more resistant to T. cruzi infection than WT mice. (a,b) Parasitemia levels (a) and survival (b) of BALB/c WT (n = 12), GNLY^+/− (n = 13), Prf^1−/− (n = 12) and Prf1^−/−GNLY^+/− (n = 12) mice infected with T. cruzi. (c,d) Cardiac parasite load (measured as T. cruzi DNA relative to mouse DNA, using PCR) (c) and representative H&E-stained images of cardiac tissue (d) 18 d after T. cruzi infection of the indicated mice (n = 6 per group). (d) Blue arrowheads, parasite-infested muscle cells; green arrowheads, infiltrating inflammatory cells; black arrowheads, necrotic muscle cells. Scale bars, 20 μm. (e) Parasitemia levels (left) and survival (right) of T. cruzi–infected WT and GNLY^+/− mice that had been treated with antibodies to deplete CD8+ T cells, CD4+ T cells or NK cells or with a control antibody (n = 8 per group). Numbers denote the mice that survived in each group 19 d after infection. (f,g) Parasitemia levels (n = 10 per group) (f) and representative images (n = 4 per group) of cardiac parasites by H&E staining at the time of killing (g) of WT and GNLY^+/− C57BL/6J mice infected with T. cruzi. A few parasites were seen in cardiac tissue from WT mice (g, left; blue arrow) but none were seen in tissue from GNLY^+/− mice (g, right). Scale bars, 20 μm. Error bars represent means ± s.e.m. ***P < 0.001, **P < 0.01; by unpaired Student’s t-test, relative to WT mice (a,e, left; f), or by one-way ANOVA (b,c,e, right).

Figure 4

GNLY^+/− mice are more resistant to T. gondii infection than WT mice. (a–f) Live-animal imaging analysis (WT mice, left; GNLY^+/− mice, right; graph below depicts average whole-animal radiance) (a,d), survival curves (b,e) and quantification of plaque assays (c,f) of WT and GNLY^+/− C57BL/6J (a–c) and BALB/c (d–f) mice infected with luciferase-expressing type II T. gondii (Pru strain) (n = 5 per group for each experiment). Scale bars, 1 cm. Error bars in a,c,d,f represent mean ± s.e.m. ***P < 0.001, **P < 0.01, *P < 0.05; by unpaired Student’s t-test (a,c,d,f) or by one-way ANOVA (b,e).