• View in gallery

    Outcome of Leishmania donovani infection in the liver. Results are from 2–4 experiments in each group of mice and are the mean ± SEM values for 8–20 mice per time point. WT = wild type; phox = phagocyte oxidase; iNOS = inducible nitric oxide synthase; DKO = double knockout.

  • View in gallery

    Photomicrographs of liver sections (A and B) and spleen imprints (C and D) from Leishmania donovani–infected mice. A and B, eight weeks after infection, inducible nitric oxide synthase (iNOS) knockout (A) and iNOS/phagocyte oxidase (phox) double knockout (DKO) mice (B) show similarly intense inflammatory responses and heavily parasitized liver granulomas (arrows). C and D, 12 weeks after infection, amastigotes are numerous in spleens of untreated iNOS/phox DKO mice (arrows) (C), but scarce in the same mice treated 10 weeks before with single-dose pentavalent antimony (D). (Original magnification × 400.)

  • 1

    Murray HW, Nathan CF, 1999. Macrophage microbicidal mechanisms in vivo: reactive nitrogen vs. intermediates in the killing of intracellular visceral Leishmania donovani. J Exp Med 189 :741–746.

    • Search Google Scholar
    • Export Citation
  • 2

    Murray HW, Cartelli D, 1983. Killing of Leishmania donovani by human mononuclear phagocytes: oxygen-dependent and -independent leishmanicidal activity. J Clin Invest 72 :32–44.

    • Search Google Scholar
    • Export Citation
  • 3

    Blos M, Schleicher U, Rochs FJS, Meibner U, Rollinghoff M, Bogdan C, 2003. Organ-specific and stage-dependent control of Leishmania major infection by inducible nitric oxide synthase and phagocyte NADPH oxidase. Eur J Immunol 33 :1224–1234.

    • Search Google Scholar
    • Export Citation
  • 4

    Shiloh MU, MacMicking JD, Nicholson S, Brause JE, Potter S, Marino M, Fang F, Dinauer M, Nathan C, 1999. Phenotype of mice and macrophages deficient in both phagocyte oxidase and inducible nitric oxide synthase. Immunity 10 :29–38.

    • Search Google Scholar
    • Export Citation
  • 5

    Murray HW, Delph-Etienne S, 2000. Role of endogenous gamma interferon and macrophage microbicidal mechanisms in host responses to chemotherapy in experimental visceral leishmaniasis. Infect Immun 68 :288–293.

    • Search Google Scholar
    • Export Citation
  • 6

    Murray HW, 2005. Prevention of relapse after chemotherapy in a chronic intracellular infection: mechanisms in experimental visceral leishmaniasis. J Immunol 174 :4916–4923.

    • Search Google Scholar
    • Export Citation
  • 7

    Taylor GA, Feng CG, Sher A, 2004. P47 GTPases: regulators of immunity to intracellular pathogens. Nature Rev Immunol 4 :100–109.

  • 8

    Santiago HC, Feng CG, Bafica A, Roffe E, Aramtes RM, Cheever A, Taylor G, Vierira LQ, Aliberti J, Gazzinelli RT, Sher A, 2005. Mice deficient in LRG-47 display enhanced susceptibility to Trypanosoma cruzi infection associated with defective hematopoiesis and intracellular control of parasite growth. J Immunol 175 :8165–8172.

    • Search Google Scholar
    • Export Citation
  • 9

    Shi X, Shanjin C, Mitsuhashi M, Xiang Z, Ma X, 2004. Genome-wide analysis of molecular changes in IL-12-induced control of mammary carcinoma via IFN-γ-independent mechanisms. J Immunol 172 :4111–4117.

    • Search Google Scholar
    • Export Citation
  • 10

    White JK, Mastroeni P, Popoff J-F, Evans CAW, Blackwell JM, 2005. Slc11a1-mediated reistance to Salmonella enterica serovar Typhimurium and Leishmania donovani infections do not require functional inducible nitric oxide synthase or phagocyte oxidase activity. J Leukoc Biol 77 :311–320.

    • Search Google Scholar
    • Export Citation
  • 11

    Roberts WL, Berman JD, Rainey PM, 1995. In vitro antileishmanial properties of tri- and pentavalent antimonial preparations. Antimicrob Agents Chemother 39 :1234–1239.

    • Search Google Scholar
    • Export Citation
  • 12

    Sudhandiran G, Shaha C, 2003. Antimonial-induced increase in intracellular Ca2+ through non-selective cation channels in the host and the parasite is responsible for apoptosis of intracellular Leishmania donovani amastigotes. J Biol Chem 278 :25120–25132.

    • Search Google Scholar
    • Export Citation

 

 

 

 

RESPONSES TO LEISHMANIA DONOVANI IN MICE DEFICIENT IN BOTH PHAGOCYTE OXIDASE AND INDUCIBLE NITRIC OXIDE SYNTHASE

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  • 1 Departments of Medicine and Microbiology and Immunology, Weill Medical College of Cornell University, New York, New York

Mice deficient in phagocyte oxidase (phox) and inducible nitric oxide synthase (iNOS), which are primary macrophage killing mechanisms, generated tissue granulomas but showed unrestrained Leishmania donovani visceral replication and suboptimal initial responsiveness to antimony treatment. Nevertheless, visceral infection was controlled post-treatment and did not recur. A phox/iNOS-independent macrophage mechanism, which was not triggered by L. donovani, emerges after chemotherapy.

We previously used Leishmania donovani, which selectively parasitizes macrophages in the liver, spleen and bone marrow, and mice deficient in phagocyte NADPH oxidase (phagocyte oxidase [phox]) (gp91phox−/−) or inducible nitric oxide synthase (iNOS−/−) to gauge the contributions of these intracellular mechanisms to host antileishmanial defense.1 Early on, liver parasite burdens were increased in phox and iNOS knockout (KO) mice, indicating that macrophages use both reactive oxygen and nitrogen intermediates in an initial effort to limit L. donovani replication.1 Thereafter, however, phox KOs reduced parasite burdens as efficiently as self-curing wild-type (WT) mice, while iNOS KOs developed progressive infection.2 Since the latter express phox normally,1,3 we concluded that iNOS was necessary and sufficient to control L. donovani and convert infection in the liver to a chronic, low-level state.1

Presuming that retained iNOS explained how phox KO mice controlled infection,1,3 we treated these animals with aminoguanidine, an iNOS inhibitor.1 Treatment promoted early L. donovani replication; however, treated phox KOs also eventually reduced their liver parasite burdens.1 Although the latter result may have reflected suboptimal iNOS inhibition by aminoguanidine, it also raised the question of a compensatory, phox- and iNOS-independent macrophage mechanism.4

To address this latter possibility and assay responses to L. donovani in the absence of phox and iNOS, mice deficient in one or both mechanisms (double KO [DKO] mice)4 were infected intravenously with 1.5 × 107 amastigotes.1,5,6 As anticipated,1 C57BL/6 WT and phox KO mice controlled infection in the liver, but iNOS KO mice did not (Figure 1). In DKO mice, the kinetics of parasite replication and liver burdens at weeks 2–12 determined microscopically and expressed as Leishman-Donovan units (LDUs)1,6 were not different from those in iNOS−/− mice (P > 0.05). Thus, DKO mice showed no evidence of a spontaneously triggered, compensatory antileishmanial mechanism.

Tissue inflammation (granuloma assembly) and responsiveness to chemotherapy were also analyzed.1,5,6 At week 4, liver granulomas formed at 87–92% of parasitized Kupffer cells in WT and single and double KO mice. In WT and phox KO mice, week 4 granulomas contained few amastigotes, defining these structures as functional, and the inflammatory response involuted by week 8. In contrast, granulomas in both iNOS KO and DKO mice remained heavily infected, and the persistently pronounced inflammatory response at weeks 8 (Figure 2A and B) and 126 was similar in both groups. Thus, in this second assay, mice deficient in phox and iNOS were also not distinguishable from those lacking iNOS alone.

The DKO mice clearly differed from iNOS (and phox) KO mice in responsiveness to conventional anti-leishmanial chemotherapy, pentavalent antimony (Sb) (sodium stibogluconate (Pentostam), 500 mg/kg, given once intraperitoneally on day + 14)6 (Table 1). The defect in DKO mice was Sb-specific because leishmanicidal responses in the liver to amphotericin B (5 mg/kg, given intraperitoneally IP on days + 14, + 16, and + 18)6 were intact. In view of uncontrolled infection in untreated DKO mice, we anticipated recurrent parasite replication once the effect of Sb had waned. However, 10 weeks after the single Sb injection, liver parasite burdens were instead low, having decreased from 802 ± 96 (mean ± SD) LDUs at week 3 (one week post-treatment, Table 1) to 185 ± 32 LDU at week 12 (two experiments, n = 9 mice). Spleen imprints from these same Sb-treated DKO mice also showed that amastigotes were scarce at week 12, but abundant in untreated mice (Figure 2 C and D).

This side-by-side analysis of macrophage mechanisms, largely regulated by interferon-γ (IFN-γ),1,5 underscores that phox is not required for resolution of liver infection,1 and clarifies that iNOS alone is necessary and sufficient to control infection with L. donovani. These results also indicate that macrophages do not spontaneously express an iNOS-independent anti-leishmanial mechanism in response to L. donovani infection by itself. Nevertheless, the absence of expected recurrence of residual intracellular infection in Sb-treated DKO mice does point to such a separate mechanism, induced in this case by the effects of chemotherapy. We previously made the same observation in livers of iNOS KO mice (no relapse after chemotherapy) but attributed it to a retained, compensatory phox-mediated response.1,5 DKO mice treated with amphotericin B also show no relapse of infection in liver6 or spleen (Murray HW, unpublished data). Thus, this separate iNOS/phox-independent macrophage mechanism that prevents post-treatment recurrence is not Sb-specific or limited to the liver (Figure 2C and D).

Studies are underway to determine how chemotherapy triggers this currently unidentified macrophage mechanism to produce its protracted, parasite-suppressing effect and why L. donovani infection itself fails to induce its activity. One candidate mechanism may be IFN-γ-inducible macrophage p47GTPases, which operate independent of iNOS and phox.7 As judged by enhanced susceptibility in available KO mice, three of the six members of this family, IGTP, IRG-47, and LRG-47, are active against a range of intracellular pathogens, including L. major;7 roles for TGTP/Mg21, IIGP, and GTPI have not yet been identified.7,8 Although we have not tested iNOS/phox DKO mice, results in infected livers from susceptible but self-curing WT mice show increased gene expression for five p47GTPases (Table 2). These data suggest that macrophage p47GTPases may be involved in prevention of post-treatment relapse.

Although clearly important in human macrophage antile-ishmanial activity,2 phox plays a limited host defense role in experimental mouse models of L. donovani infection.1,10 Nevertheless, testing Sb in DKO mice uncovered an apparent requirement for phox acting in concert with iNOS for full expression of the intracellular killing effect of Sb in the liver. That parasitized macrophages participate in reducing SbV (e.g., sodium stibogluconate) to SbIII, the active leishmanicidal metabolite,11 has long been considered possible. In vitro, reduction of SbV to SbIII is favored by an oxidizing environment,12 which our results suggest may be shaped by the combined effects of reactive oxygen and nitrogen intermediates. Thus, in the mouse model, phox does appear to contribute separately to the macrophage (e.g., its optimal response to Sb chemotherapy), since only DKO mice showed a defect in responsiveness to SbV. Albeit moderate in degree, this defect may well reflect the in vivo consequence of suboptimal handling of SbV by the infected macrophage if both its iNOS and phox mechanisms are impaired.

Table 1

Responses to chemotherapy*

Treatment†Liver parasite burden‡
MiceSbAmBDay + 14Day + 21% Killing
* WT = wild type; Phox = phagocyte oxidase; KO = knockout; iNOS = inducible nitric oxide synthase; DKO = double knockout.
† Sb = single injection on day + 14; AmB = amphotericin B injections on days + 14, + 16, and + 18. Day + 21 data for untreated mice (0% killing in all groups) were omitted for brevity.
‡ Results are the mean ± SEM at each time point from two experiments with AmB (8–9 misce per group) and three with Sb (12–15 mice per group).
§ P < 0.05 vs. Sb-treated WT mice.
WT+998 ± 64103 ± 1990
+49 ± 1295
Phox KO+1,621 ± 107149 ± 2991
+20 ± 599
iNOS KO+1,490 ± 111126 ± 4292
+48 ± 1097
iNOS/phox DKO+1,401 ± 115802 ± 9643§
+30 ± 498
Table 2

p47GTPase gene expression in infected wild-type mice*

Fold-increase in expression
GeneWeek 2Week 4
* Global gene expression analysis (Affymetrix oligonucleotide microarray)9 of livers from uninfected and 2- and 4-week infected BALB/c mice. Samples from four mice in each group were pooled, and results indicate fold-increase vs. expression value in uninfected mice.
TGTP/Mg212752
IGTP1016
IRG-4778
GTPI67
IIGP37
Figure 1.
Figure 1.

Outcome of Leishmania donovani infection in the liver. Results are from 2–4 experiments in each group of mice and are the mean ± SEM values for 8–20 mice per time point. WT = wild type; phox = phagocyte oxidase; iNOS = inducible nitric oxide synthase; DKO = double knockout.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 74, 6; 10.4269/ajtmh.2006.74.1013

Figure 2.
Figure 2.

Photomicrographs of liver sections (A and B) and spleen imprints (C and D) from Leishmania donovani–infected mice. A and B, eight weeks after infection, inducible nitric oxide synthase (iNOS) knockout (A) and iNOS/phagocyte oxidase (phox) double knockout (DKO) mice (B) show similarly intense inflammatory responses and heavily parasitized liver granulomas (arrows). C and D, 12 weeks after infection, amastigotes are numerous in spleens of untreated iNOS/phox DKO mice (arrows) (C), but scarce in the same mice treated 10 weeks before with single-dose pentavalent antimony (D). (Original magnification × 400.)

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 74, 6; 10.4269/ajtmh.2006.74.1013

*

Address correspondence to Henry W. Murray, Department of Medicine, Weill Medical College, Box 136, 1300 York Avenue, New York, NY 10021. E-mail: hwmurray@med.cornell.edu

Authors’ addresses: Henry W. Murray, Department of Medicine, Weill Medical College, Box 136, 1300 York Avenue, New York, NY 10021, Telephone: 212-746-6330, Fax: 212-746-6332, E-mail: hwmurray@med.cornell.edu. Zhaoying Xiang and Xiaojing Ma, Department of Microbiology and Immunology, Weill Medical College, 1300 York Avenue, New York, NY 10021.

Acknowledgments: We thank Drs. M. Dinauer and C. Nathan for providing the phox KO mice and the iNOS and double KO mice, respectively, and Christine Tsai for expert technical assistance.

Financial support: This study was supported by National Institutes of Health grants AI016393 (Henry W. Murray) and AI45899 (Xiaojing Ma).

REFERENCES

  • 1

    Murray HW, Nathan CF, 1999. Macrophage microbicidal mechanisms in vivo: reactive nitrogen vs. intermediates in the killing of intracellular visceral Leishmania donovani. J Exp Med 189 :741–746.

    • Search Google Scholar
    • Export Citation
  • 2

    Murray HW, Cartelli D, 1983. Killing of Leishmania donovani by human mononuclear phagocytes: oxygen-dependent and -independent leishmanicidal activity. J Clin Invest 72 :32–44.

    • Search Google Scholar
    • Export Citation
  • 3

    Blos M, Schleicher U, Rochs FJS, Meibner U, Rollinghoff M, Bogdan C, 2003. Organ-specific and stage-dependent control of Leishmania major infection by inducible nitric oxide synthase and phagocyte NADPH oxidase. Eur J Immunol 33 :1224–1234.

    • Search Google Scholar
    • Export Citation
  • 4

    Shiloh MU, MacMicking JD, Nicholson S, Brause JE, Potter S, Marino M, Fang F, Dinauer M, Nathan C, 1999. Phenotype of mice and macrophages deficient in both phagocyte oxidase and inducible nitric oxide synthase. Immunity 10 :29–38.

    • Search Google Scholar
    • Export Citation
  • 5

    Murray HW, Delph-Etienne S, 2000. Role of endogenous gamma interferon and macrophage microbicidal mechanisms in host responses to chemotherapy in experimental visceral leishmaniasis. Infect Immun 68 :288–293.

    • Search Google Scholar
    • Export Citation
  • 6

    Murray HW, 2005. Prevention of relapse after chemotherapy in a chronic intracellular infection: mechanisms in experimental visceral leishmaniasis. J Immunol 174 :4916–4923.

    • Search Google Scholar
    • Export Citation
  • 7

    Taylor GA, Feng CG, Sher A, 2004. P47 GTPases: regulators of immunity to intracellular pathogens. Nature Rev Immunol 4 :100–109.

  • 8

    Santiago HC, Feng CG, Bafica A, Roffe E, Aramtes RM, Cheever A, Taylor G, Vierira LQ, Aliberti J, Gazzinelli RT, Sher A, 2005. Mice deficient in LRG-47 display enhanced susceptibility to Trypanosoma cruzi infection associated with defective hematopoiesis and intracellular control of parasite growth. J Immunol 175 :8165–8172.

    • Search Google Scholar
    • Export Citation
  • 9

    Shi X, Shanjin C, Mitsuhashi M, Xiang Z, Ma X, 2004. Genome-wide analysis of molecular changes in IL-12-induced control of mammary carcinoma via IFN-γ-independent mechanisms. J Immunol 172 :4111–4117.

    • Search Google Scholar
    • Export Citation
  • 10

    White JK, Mastroeni P, Popoff J-F, Evans CAW, Blackwell JM, 2005. Slc11a1-mediated reistance to Salmonella enterica serovar Typhimurium and Leishmania donovani infections do not require functional inducible nitric oxide synthase or phagocyte oxidase activity. J Leukoc Biol 77 :311–320.

    • Search Google Scholar
    • Export Citation
  • 11

    Roberts WL, Berman JD, Rainey PM, 1995. In vitro antileishmanial properties of tri- and pentavalent antimonial preparations. Antimicrob Agents Chemother 39 :1234–1239.

    • Search Google Scholar
    • Export Citation
  • 12

    Sudhandiran G, Shaha C, 2003. Antimonial-induced increase in intracellular Ca2+ through non-selective cation channels in the host and the parasite is responsible for apoptosis of intracellular Leishmania donovani amastigotes. J Biol Chem 278 :25120–25132.

    • Search Google Scholar
    • Export Citation
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