Neutrophil elastase promotes Leishmania donovani infection via interferon-β

Abstract

Visceral leishmaniasis is a deadly illness caused by Leishmania donovani that provokes liver and spleen inflammation and tissue destruction. In cutaneous leishmaniasis, the protein of L. major, named inhibitor of serine peptidases (ISP) 2, inactivates neutrophil elastase (NE) present at the macrophage surface, resulting in blockade of TLR4 activation, prevention of TNF-α and IFN-β production, and parasite survival. We report poor intracellular growth of L. donovani in macrophages from knockout mice for NE (ela^-/-), TLR4, or TLR2. NE and TLR4 colocalized with the parasite in the parasitophorous vacuole. Parasite load in the liver and spleen of ela^-/- mice were reduced and accompanied by increased NO and decreased TGF-β production. Expression of ISP2 was not detected in L. donovani, and a transgenic line constitutively expressing ISP2, displayed poor intracellular growth in macrophages and decreased burden in mice. Infected ela^-/- macrophages displayed significantly lower IFN-β mRNA than background mice macrophages, and the intracellular growth was fully restored by exogenous IFN-β. We propose that L. donovani utilizes the host NE-TLR machinery to induce IFN-β necessary for parasite survival and growth during early infection. Low or absent expression of parasite ISP2 in L. donovani is necessary to preserve the activation of the NE-TLR pathway.-Dias, B. T., Dias-Teixeira, K. L., Godinho, J. P., Faria, M. S., Calegari-Silva, T., Mukhtar, M. M., Lopes, U. G., Mottram, J. C., Lima, A. P. C. A. Neutrophil elastase promotes Leishmania donovani infection via interferon-β.

Keywords: ISP; inhibitor; serine protease; toll.

Figure 1

Course of L. donovani infection in C57BL/6 mice. A) Late stage. promastigotes of L. donovani MW897 (3 × 10⁷) were washed in PBS and injected in C57BL/6 mice (n = 6); at the indicated time points postinfection, parasite burdens were assessed by limiting dilution. The experiment was performed 3 independent times (6 animals/group). The figure shows 1 representative experiment. *P < 0.05, ***P < 0.001, ****P < 0.0001. B) Expression of ISP2 is not detected in L. donovani MW897. Late-stage promastigotes (2 × 10⁷) were washed twice in PBS and lysed in SDS-PAGE sample buffer in reducing conditions, and ISP2 expression was assessed by Western blot using antiserum raised against recombinant L. major ISP2 (18). L. major ∆isp2/isp3 is a knockout line for the ISP2 and ISP3 genes and was used as a negative control. Antibodies to tubulin were used as loading control.

Figure 2

A, B) NE is required for the survival and intracellular growth of L. donovani in macrophages. Peritoneal thioglycolate-recruited macrophages from C57BL/6 mice (A) or from NE knockout mice (ela2^−/−) (B) were cultivated on glass coverslips overnight in RPMI-FCS and washed and infected with late-stage promastigotes of L. donovani at a 5:1 parasite:macrophage ratio for 3 h at 37°C in RPMI supplemented with 0.1% BSA. C) The monolayers were washed 3 times to remove extracellular parasites, fixed with methanol and Giemsa stained (3 h), or further incubated for 24 h (A, B), 48 h, or 72 h in RPMI-FCS at 37°C before fixation and staining. The number of intracellular parasites was determined under the light microscope. Where indicated, the irreversible NEI OMeSuc-Ala-Ala-Pro-Val-CMK was added to the cultures at 10 μM final concentration immediately before addition of the parasites and kept for the 3 h infection. D) Macrophages were infected as previously described for 3 h, and the monolayers were washed for removal of extracellular parasites and fixed (3 h) or cultured in RPMI-FCS for 72 h before fixation and staining. Where indicated (+), active purified human NE was added at 200 ng/ml final concentration to the cultures after the 3 h infection and remained for the 72 h. The experiments were performed in triplicate and repeated at least 3 independent times. The graphs show 1 representative experiment in triplicate. The graphs show means ± sd; statistical significance was assessed using 1-way ANOVA and the Bonferroni posttest. *P < 0.05, **P < 0.01, ****P < 0.0001.

Figure 3

TLR4 and TLR2 are required for development of L. donovani in macrophages, and NE acts through TLR4. Peritoneal thioglycolate-recruited macrophages from C57BL/6 mice, TLR2 knockout mice (tlr2^−/−), or TLR4 knockout mice (tlr4^−/−) (A) or from NE knockout mice (ela2^−/−) (B) were cultivated on glass coverslips overnight in RPMI-FCS and washed and infected with late-stage promastigotes of L. donovani at a 5:1 parasite:macrophage ratio for 3 h at 37°C in RPMI supplemented with 0.1% BSA. The monolayers were washed 3 times to remove extracellular parasites, fixed with methanol and Giemsa stained (3 h), or further cultivated for 72 h in RPMI-FCS at 37°C (A). NE acts through TLR4. B) Where indicated, macrophages were pretreated with 10 μg/ml of control IgG2b or with anti-TLR4 (MTS5) mAb for 30 min in RPMI-FCS and washed before infection. Where indicated (+), purified human NE was added at 200 ng/ml after removal of antibodies and before addition of the parasites. The experiments were performed in triplicate and repeated at least 3 independent times. The graphs show 1 representative experiment. The graphs show means ± sd. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 4

NE contributes to L. donovani infections in vivo and to the anti-inflammatory immune response. A, B) Stationary-phase promastigotes were injected in C57BL/6 mice or ela2^−/− mice (3 × 10⁷ parasites/animal, n = 6/group) in PBS. After 7 d, parasite load in spleens and livers was assessed by limiting dilution. Statistical analyses were performed using 2-way ANOVA. C–F) Cellular populations of liver macerates (C–E) (ammonium-chloride-potassium solution treated) or splenocytes (F) were cultivated in RPMI-FCS at 37°C for 48 h, and the amount of IL-10 and TGF-β was determined by ELISA. Where indicated (Ag +), total L. donovani Ag (freeze-thawed parasite lysates) were added to the cultures. D) The nitrite levels in supernatants of liver macerates were assessed by the Griess method. Experiments were repeated at least 3 independent times, and graphs show 1 representative experiment. The graphs show means ± sd. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 5

Generation of L. donovani transgenic lines expressing L. major ISP2. A) Schematic representation of the rRNA locus and the cassette used for insertion of the ISP2 gene of L. major in the genome of L. donovani. FR, flanking region; Lmcpb, L. mexicana cysteine peptidase B; PUR, puromycin resistance gene. Dashed lines indicate the region of gene replacement by homologous recombination (B). Two clones of the transgenic parasites (L. donovani:ISP2) were checked by Western blot for ISP2 expression in comparison with parental L. donovani, WT L. major, and L. major line knockout for the ISP2 and ISP3 genes (Δisp2/isp3). Promastigote (2 × 10⁷) lysates were probed using antiserum raised to recombinant ISP2. Recombinant L. major ISP2(lSP2r) was used as a positive control. C) L. donovani WT or L. donovani:ISP2 promastigotes were differentiated into extracellular amastigotes in Schneider’s medium, pH 5.4, supplemented with FCS 20% at 37°C for 6 d. In total, 2 × 10⁷ amastigotes were washed twice in PBS and lysed in SDS-PAGE sample buffer in reducing conditions, and ISP2 expression was assessed by Western blot using antiserum raised against recombinant L. major ISP2. Antibodies to tubulin were used as a loading control. D) Stationary-phase promastigotes were injected in C57BL/6 mice (3 × 10⁷ parasites/animal, n = 5/group) in PBS. After 7 d, parasite load was measured in spleen by limiting dilution. The experiment was repeated 2 independent times, and the graph shows 1 representative experiment. Statistical analyses were performed using 2-way ANOVA. **P < 0.01.

Figure 6

A) L. donovani:ISP2 cannot grow in macrophages, because of lack of NE activity. Thioglycolate-recruited peritoneal macrophages from C57BL/6 mice were cultivated overnight in RPMI supplemented with 10% FCS. The cultures were washed and incubated with stationary-phase promastigotes at a 5:1 parasite:macrophage ratio in RPMI supplemented with 0.1% BSA at 37°C. After 3 h of interaction, the monolayers were washed for the removal of extracellular parasites fixed and stained with Giemsa. For the survival assays, the macrophages were infected as previously described for 3 h, and after the removal of extracellular parasites, the cells were cultured at 37°C in RPMI supplemented with 10% FCS for 72 h. B) Where indicated (+), purified NE was added at 200 ng/ml to macrophages after the 3 h infection and the removal of extracellular parasites and kept in the cultures to 72 h at 37°C in RPMI supplemented with 10% FCS. C, D) Thioglycolate peritoneal macrophages from C57BL/6, TLR4 knockout (tlr4^−/−), or TLR2 knockout mice (tlr2^−/−) were cultivated overnight in RPMI supplemented with 10% FCS. The cultures were washed and incubated with stationary-phase promastigotes at a 5:1 parasite:macrophage ratio in RPMI supplemented with 0.1% BSA at 37°C. After 3 h, the monolayers were washed, fixed and stained with Giemsa, or cultured at 37°C in RPMI supplemented with 10% FCS for 72 h. The experiments were performed in triplicates 2 independent times. The graphs show 1 representative experiment in triplicate. Statistical analyses were performed using 1-way ANOVA and the Bonferroni posttest. *P < 0.05, **P < 0.01, ****P < 0.0001.

Figure 7

Colocalization of NE and TLR4 with L. donovani in infected macrophages. Macrophages of C57BL/6 mice were infected with WT L. donovani or L. donovani:ISP2 (clone 2) for 3 h in RPMI-BSA and processed for immunofluorescence or washed and cultured for 72 h in RPMI-FCS and processed for immunofluorescence. Coverslips were treated with anti-TLR4 antibodies washed and incubated with anti-rabbit–Alexa 488. Coverslips were subsequently incubated with antibodies to NE and washed and incubated with anti-rabbit–cyanine 3 antibodies. Coverslips were treated with DAPI for 5 min and washed and mounted for immunofluorescence. Samples were analyzed by confocal microscopy. White arrows point to the parasite. Scale bar, 2 μm.

Figure 8

ROS or NO are not responsible for killing of L. donovani:ISP2. Macrophages of C57BL/6 mice were infected with WT L. donovani or L. donovani:ISP2 for 3 h in RPMI-BSA and washed and further cultured in RPMI-FCS for 24 h before fixation and staining. A, B) Where indicated (+), immediately after the 3 h infection and washing, the cultures were treated with 1 mM of the inhibitor of iNOS, L-NAME (A), or 50 μM of the scavenger of ROS, EUK134 (B). C–E) Modulation of type I IFN responses affect L. donovani growth in macrophages. Thioglycolate-recruited macrophages from C57BL/6 mice were cultured overnight, washed, infected with L. donovani WT (C) or L. donovani:ISP2 (D) for 3 h in RPMI-BSA, washed, and fixed (3 h) or further cultured (24–72 h) in RPMI-FCS (white bars). Alternatively, after the 3 h infection and washing, cultures were treated with poly(I:C) (25 μg/ml) (gray bars) and further cultured in RPMI-FCS for up to 72 h. Experiments were performed in triplicate at least 2 independent times. The graph shows 1 representative experiment. Statistical analyses were performed using 1-way ANOVA and the Bonferroni posttest (A, B) or multiple Student’s t tests with the false discovery rate posttest (C, D). *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 9

NE contributes to the induction of IFN-β in macrophages infected with L. donovani. Thioglycolate-recruited macrophages from C57BL/6 mice (A, C) or ela2^−/− mice (B, D) were cultured overnight, washed, and infected with WT L. donovani or L. donovani:ISP2 for 2 or 6 h, and cultures were washed, total RNA was extracted, and the cDNA samples were used as templates in qPCR for the determination of the relative mRNA levels for IFN-α (A, B) and IFN-β (C, D). Uninfected cells were used as control levels, and cells pretreated with 25 μg poly:IC were used as positive control. Ctrl, control. The experiments were performed 3 independent times in 2 biologic replicates each time. The graphs show 1 representative experiment in duplicate. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 10

IFN-β restores the intracellular growth of L. donovani. Macrophages of C57BL/6 or of ela2^−/− mice were infected with L. donovani (A, B) or L. donovani:ISP2 (C, D) for 3 h and washed; recombinant IFN-α (A) or recombinant IFN-β (1000 U/ml) (B–D) was added to the cultures; and the cells were maintained in RPMI-FCS at 37°C for the indicated times. The cultures were fixed and Giemsa stained. The number of intracellular parasites was estimated by counting under the light microscope. The experiments were performed 3 independent times, in triplicate. *P < 0.05, **P < 0.01, ***P < 0.001.