Fungal and Parasitic Infections

Granzyme-Mediated Regulation of Host Defense in the Liver in Experimental Leishmania donovani Infection

Henry W. Murray, Marisa Mitchell-Flack, Hua Zheng, Xiaojing Ma
J. A. Appleton, Editor
DOI: 10.1128/IAI.02418-14

ABSTRACT

In the livers of susceptible C57BL/6 (B6) mice infected with Leishmania donovani, CD8+ T cell mechanisms are required for granuloma assembly, macrophage activation, intracellular parasite killing, and self-cure. Since gene expression of perforin and granzymes A and B (GzmA and GzmB), cytolytic proteins linked to CD8+ cell effector function, was enhanced in infected liver tissue, B6 mice deficient in these granular proteins were used to gauge host defense roles. Neither perforin nor GzmA was required; however, mice deficient in GzmB (GzmB−/−, GzmB cluster−/−, and GzmA×B cluster double knockout [DKO] mice) showed both delayed granuloma assembly and initially impaired control of parasite replication. Since these two defects in B6 mice were limited to early-stage infection, innately resistant 129/Sv mice were also tested. In this genetic setting, expression of both innate and subsequent T (Th1) cell-dependent acquired resistance, including the self-cure phenotype, was entirely derailed in GzmA×B cluster DKO mice. These results, in susceptible B6 mice for GzmB and in resistant 129/Sv mice for GzmA and/or the GzmB cluster, point to granzyme-mediated host defense regulation in the liver in experimental visceral leishmaniasis.

INTRODUCTION

In visceral leishmaniasis, Leishmania donovani targets and replicates within resident macrophages in the liver, spleen, and bone marrow. In experimental infection in the liver, wild-type (WT) C57BL/6 (B6) and BALB/c mice are initially susceptible to L. donovani but then acquire resistance, reduce intracellular parasite burdens, and proceed to a near-cure phenotype (1, 2). These responses require CD4+ and CD8+ cells and involve diverse Th1 and Th2 cell-type-activating cytokines, in particular gamma interferon (IFN-γ) (3), and mononuclear cell recruitment with conversion of parasitized liver foci into granulomas (1–12). Once simultaneously provoked counterregulatory responses (2, 4–6, 9, 12–20), including interleukin 10 (IL-10) secretion (13, 21), are overshadowed, the net result within inflammatory granulomas is macrophage activation and parasite killing (22).

In the L. donovani-infected liver, granuloma assembly, initial control of replication, resolution of primary infection, and resistance to rechallenge all require CD8+ T cells (10, 11, 23–25). CD8+ cells can also mediate additional antileishmanial responses (25–27), including the efficacy of conventional pentavalent antimony (Sb) chemotherapy and prevention of relapse of persistent intracellular infection (1, 28, 29). The mechanisms underlying protective CD8+ T cell effector function in infection may well involve the secretion of macrophage-activating cytokines (e.g., IFN-γ, tumor necrosis factor [TNF]) and mononuclear cell-recruiting chemokines (25). However, within the liver granuloma, recruited CD8+ T cells also come into contact with L. donovani-infected macrophages (Kupffer cells) (11). Moreover, in vitro data (30–34), supported by recent in vivo results in a separate model (35), suggest that in Leishmania-infected mice, CD8+ T cells may utilize cytolytic granular proteins to lyse relevant targets. Via a contact-dependent mechanism, such targets may include parasitized macrophages (30–34) and suppressive natural regulatory T cells (Tregs) (19). The finding that L. donovani infection enhanced gene expression of perforin and granzyme A (GzmA) and GzmB in the livers of WT B6 mice led us to investigate what host defense role these granular proteins may play.

MATERIALS AND METHODS

Animals and liver infection.B6 and 129/Sv WT mice were purchased from Jackson Laboratories (Bar Harbor, ME). Breeding pairs of the following mice were obtained and bred at Weill Cornell Medical College: (i) perforin−/−, IFN-γ−/−, CD8−/−, IL-2−/−, and IL-15−/− mice (B6 background; Jackson Laboratories), (ii) GzmA−/−, GzmB−/−, GzmB cluster−/−, and GzmA−/− B cluster−/− double knockout (DKO) mice (B6 background; courtesy of Todd A. Fehniger, Washington University School of Medicine, St. Louis, MO) (36), and (iii) GzmA−/− B cluster−/− DKO mice (129/Sv background; courtesy of Timothy J. Ley, Washington University School of Medicine, St. Louis, MO) (37). GzmB cluster−/− mice are deficient in GzmB, -C, and -F (38). Groups of 3 to 5 female mice, aged 6 to 12 weeks, were injected via the tail vein with 1.5 × 107 hamster spleen homogenate-derived L. donovani amastigotes (strain LV9). Infection was assessed microscopically using Giemsa-stained liver imprints in which parasite burdens were measured by blinded counting of the number of amastigotes per 500 cell nuclei and multiplication of that number by the organ weight (in milligrams) to yield Leishman-Donovan units (LDU) (1). Differences between mean values were analyzed by a two-tailed Student t test. These studies were approved by the Medical College's Institutional Animal Care and Use Committee.

Gene and mRNA expression in liver tissue.A high-density oligonucleotide microarray system, the Murine Genome U74A Array, version 2, containing 12,488 genes (Affymetrix, Santa Clara, CA), was used to test liver tissues from uninfected and infected B6 WT and IFN-γ−/− mice. Total RNA was isolated from freshly obtained tissue, and samples from 4 to 5 mice in each group were pooled. The microarray experiment and the associated data analyses were carried out as described previously (39).

For quantitative real-time reverse transcription-PCR (RT-PCR) testing, total RNA was isolated from liver tissues from individual mice using the RNeasy minikit (Qiagen, Hilden, Germany) and was reverse transcribed into cDNA. RT-PCR was performed in an ABI 7400 system using the SYBR green PCR kit. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expression was used as the control for quantitative analysis. The primers for the genes detected are listed in Table S1 in the supplemental material. PCR cycling conditions were as follows: an initial incubation step of 2 min at 50°C, reverse transcription for 60 min at 60°C and for 2 min at 94°C, and then 40 cycles of 15 s at 95°C for denaturation and 2 min at 62°C for annealing and extension. To calculate relative RNA levels, we used the formula 2−ΔCT, where CT is the threshold cycle and ΔCT is calculated as CT (specific gene) − CT (GAPDH) (39).

Flow cytometry.By using reagents purchased from eBioscience (San Diego, CA), spleen cells were incubated with a protein transport inhibitor cocktail for 5 h at 37°C under 5% CO2, stained on ice for 45 min with cell surface antibodies (fluorescein isothiocyanate [FITC]-conjugated anti-CD8, phycoerythrin [PE]-conjugated anti-CD4, peridinin chlorophyll protein [PerCP]-Cy5.5-conjugated anti-CD25), and fixed overnight in fixation buffer. CD4+ CD25+ cells were further stained with FITC-conjugated anti-FoxP3 in permeabilization buffer for 1 h at room temperature. A FACScan flow cytometer (BD Biosciences, San Diego, CA) was used to analyze cell populations.

IFN-γ, IL-10, and IL-18 protein determination.IFN-γ, IL-10, and IL-18 activities were measured in serum and/or spleen cell culture supernatants using enzyme-linked immunosorbent assay (ELISA) kits from BD Biosciences Pharmingen (San Diego, CA), BioLegend (San Diego), and eBioscience (San Diego), respectively. Spleen cells (5 × 106 cells/ml) were stimulated for 48 h with either 10 μg/ml of concanavalin A (ConA) (Sigma Chemical Co., St. Louis, MO) or 40 μg/ml of soluble L. donovani antigen, generously provided by Abhay Satoskar (Ohio State University, Columbus, OH) (20).

Tissue granuloma responses.The histologic response to infection was evaluated microscopically in liver sections stained with hematoxylin and eosin. The number of granulomas (infected Kupffer cells that had attracted ≥5 mononuclear cells [15, 16]) was counted in 100 consecutive 40× fields, and at 100 parasitized foci, the granulomatous reaction was scored as none, developing, mature, and/or parasite free (17). Mature granulomas show a core of fused parasitized Kupffer cells, numerous surrounding mononuclear cells, and epithelioid-type changes (10).

Treatments.Groups of 3 to 5 infected mice were injected intraperitoneally (i.p.) once on day 14 postinfection (day +14) with 0.2 ml of saline containing pentavalent antimony (Sb) (sodium stibogluconate [Pentostam]; Wellcome Foundation Ltd., London, United Kingdom) at an optimal leishmanicidal dose (500 mg/kg of body weight) (17). Liver parasite burdens were determined 7 days later (day +21), and day +21 LDU were compared to day +14 LDU to determine the level of Sb-induced parasite killing (percentage of reduction in LDU on day +21) (see Table 1) (17). In some experiments, Sb-injected mice were left undisturbed for 10 additional weeks in order to test for posttreatment relapse at week 12 (1, 29). To deplete CD8+ cells, 0.5 mg of a rat monoclonal antibody (MAb) against mouse CD8 or rat IgG (Bio X Cell, West Lebanon, NH) was injected i.p. in 0.2 ml of saline 1 day before infection and on days +3, +7, and +11. On day +14, when liver parasite burdens were measured, CD8+ cell depletion in 129/Sv WT mice ranged from 87 to 97% as judged by fluorescence-activated cell sorter (FACS) analysis of spleen cells. A MAb against the IL-10 receptor (IL-10R) or rat IgG (Bio X Cell) was given i.p. at 0.25 mg in 0.2 ml of saline 2 h after infection and on days +5 and +10; parasite burdens were measured on day +14.

RESULTS

L. donovani infection enhances expression of perforin, GzmA, and GzmB.Microarray gene expression analysis using liver tissues from uninfected and infected B6 WT mice indicated enhanced expression of perforin, GzmA, and GzmB at both 2 and 3 weeks after infection (see Table S2 in the supplemental material); expression of other granzymes (e.g., GzmC or GzmF) was not detected. The findings for perforin, GzmA, and GzmB were confirmed by RT-PCR testing (Fig. 1), which demonstrated mRNA expression increases of 7.4-fold for perforin, 10.4-fold for GzmA, and 45.2-fold for GzmB in 3-week-infected mice over mean expression levels in uninfected controls.

FIG 1
FIG 1

Relative mRNA expression of perforin, GzmA, and GzmB in livers of B6 WT mice 3 weeks after infection. RT-PCR results (arbitrary units, means ± SEM) are from 2 experiments (8 mice per group). Expression in the livers of infected WT mice (shown) was significantly different (P < 0.05) from expression in 8 uninfected WT livers (perforin, 0.32 ± 0.06 units; GzmA, 1.2 ± 0.2 units; GzmB, 0.82 ± 0.10 units).

Role of perforin.To identify an antileishmanial role for perforin, we examined B6 perforin−/− mice and assayed the kinetics of L. donovani replication, the outcome of liver infection, granuloma assembly at parasitized tissue foci, and responsiveness to chemotherapy (13, 14, 17). In the liver, each of these host responses requires or involves CD8+ cells (10, 23, 28). As anticipated, WT B6 mice controlled infection by week 4 and largely resolved infection by week 8 (Fig. 2), generated inflammatory granulomas that were evident at week 2 and fully expressed by weeks 4 to 8 (Fig. 3 and 4; see also Fig. S1 in the supplemental material), and responded to Sb therapy with the killing of most liver amastigotes (Table 1). Except for a partially reduced response to chemotherapy (Table 1), perforin−/− mice behaved similarly to WT mice: they controlled infection (Fig. 2A) and displayed intact granuloma assembly and maturation (not shown).

FIG 2
FIG 2

Course of L. donovani infection in livers of B6 WT mice and B6 mice deficient in perforin (A) or in GzmA, GzmB cluster, or both (GzmA×B cluster DKO) (B). (A) Results, from 2 experiments, are mean values ± SEM for 7 to 8 mice at each time point. (B) Results, from 2 to 5 experiments, are mean values ± SEM for 8 to 18 mice at each time point. Asterisks indicate significant differences (*, P < 0.05) from WT liver parasite burdens (LDU).

FIG 3
FIG 3

Liver granuloma assembly (A), maturation (B), and antileishmanial function (C) after L. donovani challenge of B6 WT mice and B6 mice deficient in GzmA, GzmB cluster, or both (GzmA×B cluster DKO). Results are from 2 experiments and show mean values ± SEM for 6 mice per group at each time point. Asterisks indicate significant differences (*, P < 0.05) from corresponding WT results.

FIG 4
FIG 4

Histologic reactions to L. donovani 2 to 8 weeks after infection in the livers of B6 WT mice and B6 mice deficient in GzmA, GzmB cluster, or both (GzmA×B cluster DKO). In WT (A to C) and GzmA−/− (D to F) mice, infected foci show numerous parasites and inflammatory responses ranging from little or none (arrows) to developing granulomas at week 2 (A and D), mature and coalescing granulomas at week 4 (B and E), and mature and involuting (arrows) granulomas at week 8 (C and F). In contrast, in GzmB cluster−/− (G to I) and GzmA×B cluster DKO (J to L) mice, few of the numerous heavily infected Kupffer cells (arrows) have attracted mononuclear cells at week 2 (G and J); by week 4 (H and K), however, and persisting to week 8 (I and L), granulomas are similarly well established in these two groups. Original magnification, ×400. See Fig. S1 in the supplemental material for corresponding low-power photomicrographs.

View this table:
TABLE 1

Response to pentavalent antimony (Sb) chemotherapya

Role of GzmA versus GzmB in B6 mice.In contrast to perforin−/− mice, B6 mice doubly deficient in GzmA and Gzm B cluster (GzmA×B cluster DKO mice) showed two defects in early-stage liver infection—impaired control of parasite replication (Fig. 2B) and minimal mononuclear cell recruitment (Fig. 3 and 4; see also Fig. S1 in the supplemental material). The same defects were expressed in B6 mice deficient in GzmB cluster alone but not in those deficient only in GzmA. Liver parasite burdens were higher at weeks 2 to 3 in GzmA×B cluster DKO mice than in GzmB cluster−/− mice (Fig. 2B) but were not significantly different (P > 0.05), a finding consistent with a minimal antileishmanial role for GzmA. At week 2, few parasitized liver foci in DKO or GzmB cluster−/− mice had attracted mononuclear cells (3% ± 1% and 7% ± 3%, respectively, versus 84% ± 6% in WT mice [P < 0.05]), resulting in 6- to 8-fold fewer granulomas than in WT mice (Fig. 3 and 4). In contrast, the initial tissue inflammatory reaction at week 2 was intact in GzmA−/− mice, with 73% ± 5% of infected foci showing recruited cells.

GzmB cluster−/− mice are deficient in GzmB, -C, and -F (38); the latter two are considered “minor” granzymes. B6 GzmB−/− mice (deficient in GzmB alone [36]) behaved similarly to B6 GzmB cluster−/− mice, displaying the same two defects in the initial control of infection (see Fig. S2 in the supplemental material) and inflammatory cell influx (not shown), findings consistent with an early effect for GzmB by itself.

Initial responsiveness to antileishmanial chemotherapy was also tested at week 2; Sb-induced parasite killing was intact in both single and DKO mice (Table 1). In addition, at week 12 (10 weeks after Sb injection), few parasites were detected in WT mice or in single or double knockout mice, indicating no posttreatment relapse of residual liver infection (2 experiments with 6 to 8 mice per group [data not shown]).

By week 4, T (Th1) cell-dependent resistance and granuloma assembly is well established in the livers of WT B6 mice (1, 16, 17). At this time, intracellular parasite killing was also under way in all four groups of granzyme-deficient mice, and each proceeded to a self-cure phenotype (Fig. 2B; see also Fig. S2 in the supplemental material). At week 12, liver parasite burdens in GzmA×B cluster DKO mice were barely detectable (26 ± 11 LDU; 2 experiments with 7 mice in each). Granuloma assembly (already observed in GzmA−/− mice) was also observed at week 4 in GzmB cluster−/−, GzmB−/−, and DKO mice, exceeding the WT response and remaining prominent at week 8 (Fig. 3 and 4; see also Fig. S1 in the supplemental material) (data not shown). Taken together, these results indicate that the proinflammatory effect and antileishmanial action of GzmB, which appeared to be perforin independent, were limited to early-stage infection.

IFN-γ regulates GzmB expression.In B6 mice, control of L. donovani in the liver, including control in early-stage infection, is governed by IFN-γ-induced activating mechanisms (1, 3). Therefore, tissues from 3-week-infected B6 IFN-γ−/− mice were also tested in parallel in the initial microarray analysis that showed enhanced expression of GzmB in the livers of infected WT B6 mice. In IFN-γ−/− mice, there was essentially no induction of GzmB gene expression (or of perforin or GzmA gene expression) (see Table S2 in the supplemental material), findings confirmed by RT-PCR testing (see Fig. S3 in the supplemental material). Other Th1-type cytokines, including IL-2 and IL-15, also induce GzmB and promote CD8+ cell function (40). However, unlike GzmB−/− mice, neither B6 IL-2−/− nor B6 IL-15−/− mice showed a defect in controlling L. donovani liver replication at weeks 2 to 4 (2 experiments with 6 to 7 mice per group [data not shown]).

Role of granzymes in resistant 129/Sv mice.During the first 2 weeks after infection, intracellular L. donovani replication is regulated in the liver by innate mechanisms governed by Slc11a1 (41, 42). Mice that express Slc11a1, including 129/Sv mice, limit parasite replication and are designated innately resistant, while B6 and BALB/c mice (Slc11a1 gene mutants) permit logarithmic L. donovani replication during weeks 1 to 2 and are considered susceptible (41). Since the antileishmanial effects of GzmB in B6 mice were evident early on, before acquired resistance was fully expressed at and beyond week 4, we next tested granzyme-deficient mice developed on a resistant background. The available mice were 129/Sv GzmA−/− B cluster−/− DKO mice, deficient in GzmA, -B, -C, and -F (37, 38). Cells from these DKO mice generate normal quantities of functional perforin (37).

(i) Expression of GzmA and GzmB and responses to L. donovani.In the liver tissues of 3-week-infected WT 129/Sv mice, mRNA expression (relative expression × 103, as in Fig. 1) of GzmA (1.7 ± 0.1), GzmB (1.8 ± 0.4), and perforin (0.5 ± 0.1) was low, showing increases of 3.4-fold for GzmA, 2.3-fold for GzmB, and 2.3-fold for perforin over mean expression levels in uninfected WT 129/Sv mice (2 experiments with 8 mice per group). Nevertheless, the kinetics of L. donovani replication in the livers of WT 129/Sv mice and those in 129/Sv GzmA×B cluster DKO mice were strikingly different. WT mice inhibited liver parasite replication as anticipated; however, DKO mice showed the opposite response (Fig. 5A). Although spleen parasite burdens were not formally measured, amastigotes were plentiful in DKO spleens at both weeks 8 and 12 (see Fig. S4 in the supplemental material) but were seldom observed in WT spleen imprints at the same time points (not shown).

FIG 5
FIG 5

(A) Course of L. donovani infection in livers of WT 129/Sv mice and 129/Sv mice deficient in both GzmA and the GzmB cluster (GzmA×B cluster DKO). Results, from 3 to 6 experiments, are mean values ± SEM for 9 to 19 mice at each time point. (B) Initial response to antimony (Sb) chemotherapy and outcome. Optimal-dose Sb (500 mg/kg) (Table 1) was injected once on day +14, and liver parasite burdens (LDU) were determined 1 and 10 weeks later. Results, from 2 experiments, are mean values ± SEM for 6 to 7 mice at each time point. All GzmA×B cluster DKO values in both panels, except for week 3 values in panel B, are significantly different (P < 0.05) from WT LDU.

(ii) Tissue inflammatory responses.In 2-week-infected WT 129/Sv mice, the initial inflammatory response at parasitized Kupffer cells, which were scarce in histologic sections, was minimal to absent (Fig. 6 and 7; see also Fig. S5 in the supplemental material). By week 3, however, mononuclear cell influx was well established, and by weeks 4 to 8, >95% of infected foci in WT mice were comprised of mature granulomas, virtually all of which were parasite free (Fig. 6 and 7).

FIG 6
FIG 6

Histologic reactions to L. donovani in the livers of WT 129/Sv (A to C) and 129/Sv GzmA×B cluster DKO (D to F) mice 2 to 8 weeks after infection. At week 2 (A and D), little tissue inflammatory response is evident in either strain, but parasitized Kupffer cells, difficult to discern in WT mice, are obvious in DKO mice (arrows) (D). At week 4 (B and E), both strains show developing/mature granulomas, largely parasite free in WT mice (B) but heavily infected in GzmA×B cluster DKO mice (E); the latter show no cellular response at some adjacent infected foci (arrows). At week 8 (C and F), parasite-free granulomas persist in WT mice (C), while inflammatory cell responses have prematurely receded or are entirely extinguished at heavily infected foci in GzmA×B cluster DKO mice (F). Original magnification, ×400 to ×500. See Fig. S5 in the supplemental material for corresponding low-power photomicrographs.

FIG 7
FIG 7

Liver granuloma assembly (A), maturation (B), and antileishmanial function (C) in WT 129/Sv mice and 129/Sv mice deficient in both GzmA and the GzmB cluster (GzmA×B cluster DKO) after L. donovani infection. Results are from 2 experiments and show mean values ± SEM for 6 to 7 mice per group at each time point. Asterisks indicate significant differences (*, P < 0.05) from the corresponding WT results.

In 129/Sv GzmA×B cluster DKO mice, the tissue inflammatory reaction at week 2 was similarly minimal to absent; in contrast to WT mice, however, intracellular parasites were abundant microscopically (Fig. 6). Developing and mature-appearing granulomas also emerged at weeks 3 to 4 in DKO mice but targeted only ∼40% of infected foci, many of which remained heavily parasitized at week 4 (Fig. 6 and 7; see also Fig. S5 in the supplemental material). Remarkably, rather than progressing, the granulomatous response was mostly extinguished by week 8 (Fig. 7; see also Fig. S5 in the supplemental material). At this time and at week 12 (not shown), >50% of infected DKO liver foci had devolved to a multinucleated core of fused Kupffer cells, crowded with intracellular amastigotes and surrounded by few or no retained mononuclear cells (Fig. 6F). This unusual, premature disassembling of granulomas differed obviously from the tissue response in 8-week-infected WT 129/Sv mice (Fig. 6C), as well as from the fully maintained, largely parasite free week 8 granulomas in B6 mice also deficient in GzmA and the GzmB cluster (Fig. 4L).

(iii) Response to chemotherapy.129/Sv GzmA×B cluster DKO mice did demonstrate one intact host mechanism: initial responsiveness to Sb chemotherapy (Table 1). Nevertheless, and in contrast to treated WT 129/Sv mice (and treated B6 GzmA×B cluster DKO mice, as noted above), once the effect of single-dose Sb waned, the replication of residual parasites resumed in 129/Sv DKO mice, yielding posttreatment relapse at week 12 (Fig. 5B). The histologic appearance of livers in Sb-treated GzmA×B cluster DKO mice at week 12 was similar to that shown for untreated GzmA×B cluster DKO mice in Fig. 6F.

Activating versus deactivating responses in 129/Sv GzmA×B cluster-deficient mice.The failure of both innate and T cell-dependent acquired antileishmanial mechanisms in 129/Sv DKO mice seemed disproportionate to a granzyme deficiency, even a deficiency in as many as four different granzymes. Thus, 129/Sv GzmA×B cluster DKO mice were analyzed further to characterize activating and deactivating (counterregulatory) mechanisms that regulate the outcome of L. donovani infection. Testing was carried out 3 weeks after infection, because this time point is beyond the initial period of innate resistance (42); mononuclear cell influx into the liver was well under way in both WT and DKO 129/Sv mice, and the kinetics of infection had clearly diverged (Fig. 5A).

(i) T cell subsets.Infected WT and GzmA×B cluster DKO 129/Sv mice showed no differences in the percentages of spleen cells that were CD4+ (30.6% ± 1.1% versus 27.4% ± 0.9%), CD8+ (8.5% ± 0.3% versus 10.1% ± 0.6%), or CD4+ CD25+ FoxP3+ (natural T regulatory cells) (2.6% ± 0.3% versus 2.9% ± 0.4%) (means ± standard errors of the means [SEM] for 2 experiments with 7 to 8 mice per group). Previous results for the same DKO mice had also shown normal hematopoiesis and lymphopoiesis, with normal percentages of CD3+, CD4+, and CD8+ cells and intact in vitro spleen cell responses to IL-2 and allogeneic stimuli (37).

(ii) Inflammatory markers, cytokine expression, and macrophage mechanisms.Infected liver tissue was also tested for evidence, as judged by relative mRNA expression, pointing to (i) downregulated Th1 cell type and/or inflammatory responses (e.g., activating receptors, cytokines, or granuloma-promoting chemokines) (3, 17, 43–46; also unpublished data), (ii) downregulated antileishmanial macrophage mechanisms (e.g., inducible nitric oxide synthase [iNOS], phagocyte oxidase [phox], or immunity-related GTPases [IRGs]) (22, 47), or (iii) upregulated deactivating responses (e.g., counterregulatory receptors or cytokines) (13–16; also unpublished data). The data in Table S3 in the supplemental material show some differences in expression between 129/Sv GzmA×B cluster DKO and WT mice. However, except for the three IRGs, the most notable observation was how little mRNA expression was detected in either 129/Sv strain, precluding meaningful conclusions. Parallel results in infected liver tissues from WT B6 mice (see Table S3) highlighted the overall minimal responses in 129/Sv mice.

(iii) IFN-γ and IL-10 secretion and effect of IL-10 blockade.Finally, focusing on IFN-γ and IL-10 as primary macrophage-activating or macrophage-deactivating cytokines (3, 13, 21), activities were measured in samples from 3-week-infected 129/Sv mice. IFN-γ levels were comparably low in the sera of WT (10.3 ± 1.6 pg/ml) and GzmA×B cluster DKO (27.2 ± 7.6 pg/ml) mice, and IL-10 was not detected in either group (2 experiments with 8 mice per group). When antigen-restimulated spleen cells were used, however, there were clear-cut differences in cytokine production in vitro. Cells from infected DKO mice failed to secrete detectable IFN-γ and produced higher levels of IL-10 than cells from infected WT mice (Fig. 8A and B), suggesting deficient activating coupled with amplified deactivating mechanisms. Since anti-IL-10R MAb treatment enhanced resistance in 129/Sv GzmA×B cluster DKO mice, albeit partially (Fig. 8C), one such deactivating mechanism may involve IL-10.

FIG 8
FIG 8

In vitro IFN-γ and IL-10 production in 129/Sv WT and GzmA×B cluster DKO mice and in vivo effect of IL-10R blockade in DKO mice. (A and B) Spleen cells from 3-week-infected mice were stimulated for 48 h with medium alone, ConA, or L. donovani antigen. Results (from 2 experiments) are mean values ± SEM for 6 mice per group. (C) DKO mice were injected with rat IgG or anti-IL-10R MAb 2 h after infection and on days +5 and +10; liver parasite burdens (LDU) were determined on day +14. Results (from 2 experiments) are mean values ± SEM for 8 mice per group. Asterisks indicate significant differences (*, P < 0.05) from results for the corresponding WT or IgG-injected group.

DISCUSSION

The results of this study indicate that the elaboration of granzymes represents an antileishmanial mechanism in the L. donovani-infected liver. This mechanism appears capable of exerting a range of regulatory effects, depending on the host examined.

In susceptible WT B6 mice, expression of GzmA, GzmB, and perforin was provoked by L. donovani infection and required IFN-γ, a new observation adding to the recognized antileishmanial repertoire of IFN-γ. Testing in gene-deficient B6 mice, designed to gauge the host defense effects of these three granular proteins in the liver, permitted three conclusions: (i) GzmB regulates both the tissue inflammatory reaction and the control of parasite replication in early-stage liver infection; (ii) perforin supports the host response to chemotherapy in a limited fashion but is not otherwise involved; and (iii) GzmA appears to contribute little to these three antileishmanial responses. The use of B6 mice deficient in perforin, GzmA, GzmB, the GzmB cluster, or both GzmA and the GzmB cluster (DKO mice) allowed these interpretations and assigned GzmB a perforin- and GzmA-independent role.

With resistant 129/Sv mice, we did not address a possible role for perforin, nor did testing of GzmA×B cluster DKO mice permit a distinction between the roles of GzmA and GzmB or possibly GzmC and GzmF (38). Nevertheless, the results for 129/Sv DKO mice were striking; they differed obviously from those for 129/Sv WT mice (as well as for B6 GzmA×B cluster DKO mice) and, for this particular host genetic background, illustrate potentially far-reaching perforin-independent effects of granzymes in a nonviral intracellular infection.

129/Sv mice, which express Slc11a1, are considered innately resistant to L. donovani (41), and WT 129/Sv mice limited liver parasite replication during the initial 2 weeks. This early effect was accomplished without mononuclear cell recruitment to infected foci and with minimal expression in liver tissue of inflammatory T cell and macrophage mechanisms involved in defense against L. donovani. How Slc11a1 regulates early innate resistance is unclear; cationic transport (e.g., of Fe2+) may be involved (41), but T cells, an IL-12-driven Th1-type response, and iNOS and phagocyte oxidase, the primary leishmanicidal mechanisms of IFN-γ-activated macrophages (22), do not appear to be required (42, 48, 49). In addition, depletion of CD8+ cells (see Materials and Methods) had no effect on innate resistance: 2 weeks after infection, liver parasite burdens were similar in anti-CD8- and IgG-treated WT 129/Sv mice (2 experiments with 6 to 9 mice per group [data not shown]). At or after week 3, however, conventional-appearing granulomatous responses emerged in the livers of WT 129/Sv mice, accompanied by in vitro evidence of T cell reactivity (e.g., antigen-stimulated IFN-γ secretion). Such Th1 cell type responses presumably induce the killing of residual intracellular parasites and lead to the self-cure phenotype.

In this 129/Sv setting, then, it was entirely unexpected to discover that a granzyme deficiency, even a deficiency in as many as four granzymes (GzmA, -B, -C, and/or -F), was sufficient to not derail only early innate resistance but also subsequent basic expressions of Th1 cell-dependent, cytokine-mediated acquired resistance to L. donovani. Indeed, with two partial exceptions—an intact initial (but not long-lasting) response to antimony chemotherapy and some degree of tissue granulomatous inflammation (although delayed, incomplete, and finally extinguished)—the behavior of 129/Sv GzmA×B cluster DKO mice was akin to that of T cell-deficient nude mice and mice deficient in key activating Th1 cell cytokines (IL-12−/− and IFN-γ−/− mice) (3, 43, 50). L. donovani-infected nude and IFN-γ−/− mice, for example, also fail to generate granulomas, show no evidence of macrophage activation, allow progressive high-level intracellular infection in the liver, and permit relapse after chemotherapy (1, 3, 10, 29, 50). The overtly deficient to near-absent mononuclear cell inflammatory reaction at parasitized foci in the livers of 8-week-infected 129/Sv GzmA×B cluster DKO mice was remarkable (Fig. 6F) and virtually indistinguishable from that in L. donovani-infected nude, IL-12−/−, and IFN-γ−/− mice at the same time point (1, 10, 43). DKO spleen cells failed to generate antigen-induced IFN-γ and secreted higher levels of the key counterregulatory cytokine, IL-10 (13, 21); in addition, IL-10R blockade enhanced resistance in vivo. These observations support the notion of a Th1 cell type defect in these granzyme-deficient 129/Sv mice and probable default to an overall and partially IL-10 associated deactivated state. Intracellular FACS staining for IFN-γ and IL-10 and analysis of cells infiltrating the infected liver would likely have shed additional light on these responses.

Although long associated with intracellular cytotoxic-proapoptotic activity and CD8+ T cell effector mechanisms, granzymes are also capable of extracellular effects unrelated to target cell killing and are produced by other cells as well (51–57). Perforin is considered required for granzyme-induced cytolytic activity (52, 55–57). Thus, the absence of a phenotype in B6 perforin−/− mice indicates that the action of GzmB in early-stage infection in B6 mice likely relates to an extracellular antileishmanial effect and not, for example, to perforin-associated lysis of parasitized macrophages (35) or suppressive natural Tregs (19). Conversely, natural Tregs, which apparently promote visceral infection (19, 20), can also employ GzmB to kill effector T cells and suppress inflammatory mechanisms (58); however, B6 GzmB−/− mice were less rather than more resistant to L. donovani.

In inducing cell death, the cytolytic activity of perforin- and/or GzmB-expressing CD8+ cells appears capable of mediating inflammatory tissue injury, at least in Leishmania braziliensis cutaneous infection (35, 59). GzmB as well as GzmA, however, can also provoke inflammatory mechanisms via extracellular actions (51–53), which may be relevant to the antileishmanial defects identified here in infected B6 GzmB−/− mice and perhaps in 129/Sv GzmA×B cluster DKO mice. GzmA, for example, induces the secretion of IL-1β, IL-6, and TNF (60, 61), while IL-6 acts in a suppressive fashion in the L. donovani model (16), TNF is required for proper control of liver infection (44, 46). GzmB can also trigger or promote inflammatory responses by cleaving a range of extracellular matrix substrates (61–64), downregulating the Th2 cell-type response to restrain the secretion of deactivating cytokines (transforming growth factor β [TGF-β], IL-4, IL-10) (65), and converting IL-1α and IL-18 to their active forms (66, 67). IL-18 is involved in both initial granuloma formation and early control of L. donovani replication (43); however, interaction with GzmB seems unlikely, since mitogen- and antigen-stimulated spleen cells from 3-week-infected B6 WT and GzmB−/− mice produced comparable levels of IL-18 (2 experiments, 6 mice per group [data not shown]). In addition, in two separate experiments using antigen-restimulated spleen cells from 3-week-infected GzmB−/− and WT animals, the increased susceptibility of GzmB−/− mice to L. donovani was not associated with either reduced IFN-γ or increased IL-10 production (7 to 8 mice per group [not shown]).

While CD8+ T and NK cells are recognized as principal granzyme-expressing effector cells (53, 56), granzymes are also produced by a range of other cell types, including CD4+ T and NKT cells (53–57). Two observations suggested that CD8+ cells were not an essential source of granzymes in L. donovani-infected mice: depletion of CD8+ cells did not affect resistance in the livers of WT 129/Sv mice, and the relative mRNA expression of GzmA and GzmB in the liver tissues of B6 CD8−/− mice was not diminished from that in B6 WT mice (3 weeks after infection, 2 experiments, 8 mice per group [data not shown]). In addition, we suspect that NK cells are also not the important granzyme-secreting cells, at least not in B6 mice, since B6 bg/bg−/− (beige) mice, deficient in NK cell cytolytic activity, show no early defect in controlling L. donovani in the liver (24, 68, 69); NK cells are also not cytolytic in B6 WT mice infected with visceral Leishmania infantum (70). Currently, it is also unclear what antileishmanial defense role NKT cells play in B6 mice, in view of the contrasting results in the L. donovani model (71, 72) and the demonstration that NKT cells promote cutaneous Leishmania major infection (73). Further analysis, then, in both B6 and 129/Sv mice will be needed to identify the relevant granzyme-secreting cells in L. donovani infection.

ACKNOWLEDGMENTS

This work was supported by NIH grant 5R01AI083219 (H.W.M., X.M.) and a grant from Sheng Yushou Foundation to Shanghai Jiaotong University (H.Z.).

We are particularly grateful to Timothy Ley and Todd Fehniger (Washington University School of Medicine) for generously providing breeding pairs of the various granzyme-deficient mice. We also thank Yan Zhang for assistance with the PCR and flow cytometry analyses.

FOOTNOTES

    • Received 30 July 2014.
    • Returned for modification 24 August 2014.
    • Accepted 21 November 2014.
    • Accepted manuscript posted online 1 December 2014.

  • Supplemental material for this article may be found at http://dx.doi.org/10.1128/IAI.02418-14.

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