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Originally published online as doi:10.1189/jlb.0904546 on December 15, 2004

Published online before print December 15, 2004
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(Journal of Leukocyte Biology. 2005;77:311-320.)
© 2005 by Society for Leukocyte Biology

Slc11a1-mediated resistance to Salmonella enterica serovar Typhimurium and Leishmania donovani infections does not require functional inducible nitric oxide synthase or phagocyte oxidase activity

Jacqueline K. White*,1, Pietro Mastroeni{dagger}, Jean-François Popoff*, Carlton A. W. Evans*,2 and Jenefer M. Blackwell*,3

* Cambridge Institute for Medical Research and Department of Medicine, Wellcome Trust/MRC Building, University of Cambridge School of Clinical Medicine, Addenbrookes Hospital, Hills Road, United Kingdom; and
{dagger} Centre for Veterinary Science, Department of Clinical Veterinary Medicine, University of Cambridge, United Kingdom

3 Correspondence: Cambridge Institute for Medical Research, Wellcome Trust/MRC Building, University of Cambridge School of Clinical Medicine, Addenbrookes Hospital, Hills Road, Cambridge CB2 2XY, UK. E-mail: jennie.blackwell{at}cimr.cam.ac.uk


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Solute carrier family 11a member 1 (Slc11a1; formerly natural resistance-associated macrophage protein 1) encodes a late endosomal/lysosomal protein/divalent cation transporter, which regulates iron homeostasis in macrophages. During macrophage activation, Slc11a1 exerts pleiotropic effects on gene regulation and function, including generation of nitric oxide (NO) via inducible NO synthase (iNOS; encoded by Nos2A) and of reactive oxygen intermediates (ROI) via the phagocyte oxidase complex. As NO and ROI have potent antimicrobial activity in macrophages, it was assumed that their activities would contribute to Slc11a1-regulated innate resistance to Salmonella enterica serovar Typhimurium and Leishmania donovani. By intercrossing mice with gene disruptions at Nos2A and Cybb (encoding gp91phox, the heavy chain subunit of cytochrome b-245 and an essential component of phagocyte NADPH oxidase) onto equivalent Slc11a1 wild-type and mutant genetic backgrounds, we demonstrate that neither iNOS nor gp91phox activity is required for Slc11a1-mediated innate resistance to either infection. Functional gp91phox and iNOS are required to control S. enterica serovar Typhimurium in non-Slc11a1-regulated phases of infection. For L. donovani, an organ-specific requirement for iNOS to clear parasites from the spleen was observed at 50 days post-infection, but neither iNOS nor gp91phox influenced late-phase infection in the liver. This contrasted with Leishmania major infection, which caused rapid lesion growth and death in iNOS knockout mice and some exacerbation of disease with gp91phox deficiency. This highlights the adaptive differences in tissue and cellular tropisms between L. donovani and L. major and the different genes and mechanisms that regulate visceral versus cutaneous forms of the disease.

Key Words: Nramp1 • divalent cation transporter • macrophage activation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Solute carrier family 11a member 1 [Slc11a1; formerly natural resistance-associated macrophage protein 1 (Nramp1)] encodes a late endosomal/lysosomal divalent cation transporter, which regulates iron homeostasis in macrophages (reviewed in refs. [1 2 3 4 ]). During macrophage activation, Slc11a1 exerts pleiotropic effects, including quantitative differences in induction of early response genes such as the CXC chemokine KC (CXCL1 or murine Gro-{alpha}) [5 ], interleukin-1ß [6 ], and tumor necrosis factor {alpha} [7 ] at protein and/or mRNA levels, as well as iron- [8 ] and interferon-{gamma}/lipopolysaccharide [8 , 9 ]-regulated differences in expression and stability of mRNA for inducible nitric oxide synthase (iNOS) encoded by Nos2A. Differences in NADPH oxidase activity have also been observed in response to phorbol myristate acetate in RAW264.7 macrophages, stably transfected with constructs carrying wild-type or mutant (mt) Slc11a1 [10 ]. In vitro, studies of iNOS induction and use of inhibitors have shown that NO generated by iNOS activity is responsible for macrophage antimicrobial activity against numerous intracellular pathogens (reviewed in refs. [11 12 13 ]), including Salmonella enterica serovar Typhimurium [14 ], Mycobacterium bovis [15 ], and Leishmania donovani [16 ], which come under innate Slc11a1 control in the early phases of infection. It was assumed, therefore, that iNOS activity might contribute to early Slc11a1-mediated resistance to infection. Similarly, reactive oxygen intermediates (ROI), generated by NADPH oxidase, have antimicrobial activity against all three groups of the pathogen in vitro [17 18 19 ], and it had been proposed [20 ] that the interaction of ferrous ions (Fe2+) transported by Slc11a1 with ROI via the Fenton and Haber-Weiss reactions might contribute to the generation of more toxic radicals in Slc11a1 wild-type but not mt macrophages. By intercrossing mice with gene disruptions at Nos2A and Cybb (encoding gp91phox, the heavy chain subunit of cytochrome b-245 and an essential component of phagocyte NADPH oxidase) onto equivalent Slc11a1 wild-type and mt genetic backgrounds, we demonstrate that neither iNOS nor gp91phox activity is required for Slc11a1-mediated innate resistance to S. enterica serovar Typhimurium or L. donovani infection. We show that functional gp91phox and iNOS activities are required for non-Slc11a1-regulated early and late phase of S. enterica serovar Typhimurium infection, respectively. However, only an organ-specific requirement for iNOS was observed in clearance of parasites from spleen but not liver in the later, non-Slc11a1-regulated phases of L. donovani infection. This contrasts with rapid lesion growth and death in Nos2A–/– knockout mice following Leishmania major infection, with some exacerbation of disease also observed with Cybb–/– gene disruption. This highlights the adaptive differences in tissue and cellular tropisms between L. donovani and L. major and the different genes and mechanisms that regulate visceral versus cutaneous forms of the disease.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mice
Congenic N20 B10.L-Lsh (=Nramp1; =Slc11a1) were bred in-house [21 ] following 20 generations of backcrossing the Slc11a1 wild-type allele from C57L mice onto a C57BL/10ScSn (=B10) background. These are congenic with Slc11a1 mt B10 mice except for the ~10 Mb interval distal to D1Mit80 and proximal to D1Mit44 on chromosome 1, which carries the Slc11a1 gene. To produce new lines that were homozygous Slc11a1 wild-type (=Slc11a1+/+) but carried gene disruptions at Nos2A (=Nos2A–/–) or Cybb (=Cybb–/–), founder Nos2A–/– [22 ] and Cybb–/– [23 ] were intercrossed with N20 mice. The Nos2A–/– produced by Laubach and co-workers [22 ] was reported to have undergone six generations of backcrossing onto a C57BL/6J (=B6) background prior to our acquiring it. As B6 mice are Slc11a1 mt, we expected to obtain double heterozygous mice for Slc11a1+/mt and Nos2A+/– at the F1 of the first intercross. In the event, all mice were homozygous Slc11a1+/+, indicating that the region of chromosome 1 carrying Slc11a1 in the original line [19 ] was still homozygous for the 129 background in which the Nos2A knockout was generated. These F1 mice were further intercrossed, and Nos2A+/+ and Nos2A–/– mice were selected to establish breeding colonies (by brother-sister matings of Nos2A+/+xNos2A+/+ or Nos2A–/–xNos2A–/– F2 progeny from the same F1 intercross) of double homozygous Slc11a1+/+.Nos2A+/+ and Slc11a1+/+.Nos2A–/– on the intercrossed B6/B10 background at F3. The founder Cybb–/– mice were also on a B6 background, and in this case, the expected double heterozygous mice for Slc11a1+/mt and Cybb+/– were obtained at the F1 of the first intercross. Slc11a1 and Cybb genotyping was performed using tail DNA from all mice and selective breeding used to establish double homozygous Slc11a1+/+.Cybb+/+ and Slc11a1+/+.Cybb–/– lines at F3 on the intercrossed B6/B10 background. For both sets of mice, infections were compared with Slc11a1mt/mt, Nos2A+/+, and Cybb+/+ B10 or B6 mice as indicated. BALB/c mice (Harlen Olac, Bicester, UK) were used as susceptible controls for L. major infection experiments. All breeding and procedures were carried out under license and UK Government Home Office regulations. Mice were between 6 and 16 weeks old over all infection experiments but age-matched ±2 weeks and sex-matched within each experiment as indicated below.

Genotyping
During the breeding protocols, the Nos2A–/– gene-disrupted allele for Nos2A was genotyped on BamHI-digested, genomic DNA Southern blots hybridized with a Nos2A intron 10 probe amplified by polymerase chain reaction (PCR) from genomic DNA using forward (5'-CTGGACCAGGCCACTTTG-3') and reverse (5'-CCTCCACTTGTCCATCCATT-3') primers. Diagnostic bands of 14 kb and 5 kb distinguished Nos2A wild-type from gene-disrupted alleles, respectively. Similarly, the Cybb–/– gene-disrupted allele for Cybb was genotyped on EcoRI-digested, genomic DNA Southern blots, hybridized with a Cybb exon 2 probe, amplified by PCR from genomic DNA using forward (5'-CAAGATTGCATGAGGCAGAA-3') and reverse (5'-CAACTTTTCCTGTTTGTGCAG-3') primers. Diagnostic bands of 13 kb and 6.8 kb distinguished Cybb wild-type from gene-disrupted alleles, respectively. Slc11a1 genotyping was undertaken by sequence analysis across the mutation site following amplification of a 492-bp product from genomic DNA using exon 5 forward (5'-ATATGCAGGAAGTCATCGGG-3') and intron 6 reverse (5'-CAAGATGAGGGGCTTTCTCTC-3') primers, with the forward primer used as the sequencing primer.

Salmonella infection and monitoring
For intravenous (i.v.) inoculation into mice, virulent S. enterica serovar Typhimurium C5 was grown at 37°C as stationary, overnight cultures in Luria-Bertani (LB) broth (Difco, Becton Dickinson, Cowley, Oxford, UK). Aliquots were snap-frozen and stored in liquid nitrogen. The inoculum was diluted in phosphate-buffered saline (PBS) and injected into the lateral tail vein. The number of viable bacteria in each inoculum was checked by dilution and pour-plating onto LB agar plates. To enumerate bacterial load in the organs, spleens and livers of mice (four per time-point per strain) were removed aseptically and homogenized in a Colworth Stomacher in 10 ml cold, distilled water [24 ]. Viable counts were determined using pour plates of LB agar. For 50% lethal dose (LD50) determinations, groups of mice (four to six per dose per strain) were injected i.v. with 10- or 20-fold decreasing doses of S. enterica serovar Typhimurium C5 over the range 105–101, and mortality scored over a 30-day period. LD50 values were calculated according to the method of Reed and Muench [25 ]. Age-matched (±2 weeks) male mice were used for S. enterica serovar Typhimurium experiments as indicated.

Leishmania infection and monitoring
To determine response to cutaneous leishmaniasis, mice (five to six per strain) were inoculated subcutaneously (s.c.) into the right-hind footpad with 2 x 106 stationary-phase, metacyclic promastigotes of L. major strain LV39, cultured in Schneider’s insect medium (Sigma, Poole, UK), supplemented with 10% fetal calf serum (Invitrogen, Paisley, UK) and 100 U/ml penicillin/100 µg/ml streptomycin (Invitrogen). Infection was monitored as increase in footpad depth, measured using digital Vernier callipers. To conform to UK Home Office regulations, euthanasia of mice was carried out when footpad lesions became ulcerated and necrotic. Experience shows that this level of pathology is always lethal. To determine response to visceral leishmaniasis, mice (three to four per time-point per strain) were inoculated i.v. into the lateral tail vein with 2 x 107 or 5 x 107 amastigotes freshly harvested from spleens of hamsters infected with L. donovani strain LV9 as described [26 ]. Groups of mice were killed on days 1, 4, 7, and 15 and ~50 days post-infection as indicated. Parasite loads were enumerated microscopically from KWIKTMDIFF (Thermo Shandon, Pittsburgh, PA)-stained impression smears of spleen and liver. Results are expressed as Leishman Donovan units (LDU) [26 ], which equate to the number of parasites per 1000 organ cell nuclei multiplied by the organ weight in mg. Age-matched (±2 weeks), female mice were used for Leishmania experiments as indicated.

Immunohistology
Liver segments from N20 mice harvested 2–4 days post L. donovani infection were mounted in optimal cutting temperature compound (CellPath, Powys, UK) and snap-frozen. For low-power fluorescence microscopy, cryostat sections (8 µm thick) were warmed to room temperature, fixed for 10 min in 4% w/v paraformaldehyde (BDH, Poole, UK) in PBS (pH 7.4), and washed 2x in PBS for 5 min. Sections were post-fixed by immersion in 2:1 ethanol:acetic acid at 4°C and washed 2x in PBS for 5 min. Nonspecific staining was reduced by incubation for 1 h with 3% fatty acid-free bovine serum albumin in Triton–Tris-buffered saline (154 mM NaCl, 10 mM Tris, 0.01% sodium azide, 0.1% Triton X-100, pH 7.4) prior to incubation with pooled, primary monoclonal antibody (Ab) rat anti-mouse M1/70 Ab (Harlan Sera-Lab, Loughborough, UK, 1:10) and affinity-purified polyclonal rabbit anti-iNOS Ab (Affiniti Research Products, Exeter, UK, 1:500). M1/70 recognizes CD11b, the ß-chain of the type-3 complement receptor [27 ]. The anti-iNOS Ab was raised against a 21-kDa protein fragment corresponding to amino acids 961–1144 of the mouse macrophage iNOS protein [28 ] and detects the 130-kDa full-length protein in macrophages from Nos2A+/+ mice, which was completely absent in macrophages from Nos2A–/– mice (data not shown). After washing, slides were coincubated in fluorescein isothiocyanate (FITC)-conjugated, affinity-purified donkey anti-rat immunoglobulin G (IgG; Jackson Immuno Research Laboratories, West Grove, PA, 1:50) and affinitiy-purified tetramethyl rhodamine isothiocyanate (TRITC)-conjugated goat anti-rabbit IgG (Sigma, 1:20) for 5 h at ambient temperature. Sections were washed 3x in PBS, rinsed in distilled water, and mounted in Citifluor AFI (Agar Scientific, Kent, UK). Staining was viewed with an Olympus Provis AX70 fluorescent microscope (London, UK), mounted with a Hamamatsu C47-42-95 digital camera (Photonics KK, Japan), and images were captured with Openlab software. Exposures for FITC and TRITC channels varied between 75 and 400 ms. The microscope, camera, and printer settings were standardized within experiments for stained and control sections, all of which had been incubated in parallel. For high-power confocal microscopy, 6 µm cryostat sections were incubated in primary Ab simultaneously. The first primary Ab was an affinity-purified polyclonal anti-Slc11a1 Ab (1:1000) raised against the N-terminal 82 amino acids of the Slc11a1 protein [29 ] and was a gift from Dr. C. Howard Barton (University of Southampton, UK). This Ab recognizes the mature, fully glycosylated, 90–100 kDA Slc11a1 band on Western blots of Slc11a1 wild-type macrophages [30 ]. The second primary Ab was M1/70 (1:800) or monoclonal rat anti-mouse F4/80 (Serotec, Oxford, UK, 1:50). Secondary Ab were AlexaTM594-coupled goat anti-rat IgG (1:200) and AlexaTM488-coupled goat anti-rabbit IgG (1:400, Molecular Probes, Leiden, Netherlands). Slides with cryostat sections were fixed for 10 min at 4°C with 100% acetone, air-dried, and then rinsed in PBS and blocked for 1 h at ambient temperature in 0.1% Tween-20/PBS supplemented with normal goat serum (1:40). Primary Ab, diluted in blocking buffer, were incubated for 1 h at ambient temperature. Slides were washed 3x for 5 min in 0.1% Tween-20/PBS. Secondary Ab, diluted in blocking buffer, were incubated for 1 h at ambient temperature. Slides were washed as above, rinsed in PBS, and then mounted in Prolong Antifade (Molecular Probes) and viewed with a Nikon optiphot-2 epifluorescence microscope coupled to a Bio-Rad MRC 1000 confocal laser-scanning attachment (Bio-Rad Laboratories Ltd., Hemel Hempsted, UK). Images were collected using Lasersharp 2000 software.

Statistics
All footpad measurements and parasite loads were determined blind to the experimental group. Unpaired, two-tailed Student’s t-tests were used to compare bacterial or parasite loads between mouse strains. Differences between experimental groups were considered significant for P values <0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Nos2A–/– does not compromise early Slc11a1+/+ regulation of S. enterica serovar Typhimurium infection
Previous studies show that Slc11a1 mediates its influence on virulent S. enterica serovar Typhimurium C5 during the acute phase (<7 days) of infection [31 , 32 ]. LD50 values, 1 month after infection with tenfold decreasing doses (105, 104, 103, 102) of S. enterica serovar Typhimurium C5, indicated that F3 B6/B10 Slc11a1+/+.Nos2A–/– mice were intermediate in susceptibility compared with B10 Slc11a1mt/mt.Nos2A+/+ and F3 B6/B10 Slc11a1+/+.Nos2A+/+ mice (data not shown). However, survival plots (Fig. 1A ) following infection of F3 B6/B10 Slc11a1+/+.Nos2A+/+ and Slc11a1+/+.Nos2A–/– mice with 103 S. enterica serovar Typhimurium C5 showed that the Nos2Agene disruption does not influence Slc11a1-mediated resistance (100% survival) over the first 9 days of infection in F3 B6/B10 Slc11a1+/+.Nos2A–/– mice. In contrast, 100% mortality was observed in Slc11a1mt/mt B10 mice by day 8 post-infection. Failure of the Nos2A gene disruption to compromise early Slc11a1-mediated resistance to infection is further supported by monitoring liver (Fig. 1B) and spleen (Fig. 1C) bacterial counts over time following infection with 103 S. enterica serovar Typhimurium C5. In this case, all Slc11a1mt/mt B10 mice were dead by day 7 post-infection, and no significant differences in liver or spleen bacterial loads were observed between F3 B6/B10 Slc11a1+/+.Nos2A+/+, Slc11a1+/+. Nos2A–/–, or B10 background N20 Slc11a1+/+ (data not shown) mice over days 1–5 post-infection. Nos2A–/– gene disruption did influence post-Slc11a1 phase, as evidenced by significant differences in spleen (P<0.008) bacterial counts for Slc11a1+/+.Nos2A+/+ mice compared with Slc11a1+/+. Nos2A–/– mice at day 8 post-infection, all Nos2A gene-disrupted mice were dead by day 9, and Slc11a1+/+.Nos2A+/+ mice all survived beyond this time-point in this experiment (data not shown).



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  		Figure 1. S. enterica serovar Typhimurium infection in B6/B10 Slc11a1+/+. Nos2A+/+ and B6/B10 Slc11a1+/+.Nos2A–/– compared with B10 Slc11a1mt/mt. Nos2A+/+ mice. Mice (four to six per dose per strain) were infected with tenfold decreasing doses (104, 103, 102, 101) of S. enterica serovar Typhimurium C5. (A) Percent survival over 30 days following inoculation of four to five mice per strain with 103 S. enterica serovar Typhimurium C5. The same rank order in mortality curves was observed for the other bacterial doses. Bacterial counts (mean±SEM; four mice per strain per time-point) in liver (B) and spleen (C) over 8 days post-infection are shown for the 103 inoculum. P values for significant differences between strains are indicated in the text. Similar results were obtained for organ bacterial loads in a replicate experiment.

 
Nos2A–/– does not compromise early Slc11a1+/+ regulation of L. donovani infection
In contrast to S. enterica serovar Typhimurium infection, L. donovani does not cause acute mortality in mice. Expansion of the parasite population in liver and spleen is slower, and Slc11a1 resistance can be measured over the first 7–15 days of infection [33 ]. Confirmation that Nos2A plays no role in Slc11a1-mediated resistance to infection in spleen is demonstrated in Figure 2 by failure to observe significant differences in parasite loads in F3 B6/B10 Slc11a1+/+.Nos2A+/+, Slc11a1+/+.Nos2A–/–, or B10 background N20 Slc11a1+/+ mice (data not shown) at days 4, 7, or 15 post-infection. Although there was a trend toward higher parasite load in F3 B6/B10 Slc11a1+/+.Nos2A–/– compared with F3 B6/B10 Slc11a1+/+.Nos2A+/+ in the liver at days 7 and 15 post-infection, these did not achieve statistical significance. In contrast, significantly higher parasite loads were observed by days 7 (P=0.002, 0.004, 0.016) and 15 (P=0.004, 0.004, 0.005) post-infection in the liver and by day 15 (P=0.0005, 0.0002, 0.0001) in the spleen, when B10 Slc11a1mt/mt.Nos2A+/+ mice were compared with the three Slc11a1+/+ mouse lines F3 B6/B10 Slc11a1+/+.Nos2A+/+, Slc11a1+/+.Nos2A–/–, or B10 background N20 Slc11a1+/+, respectively. It is interesting that gene disruption at Nos2A–/– did compromise the ability of F3 B6/B10 Slc11a1+/+.Nos2A–/– to clear parasites in the spleen (P=0.004) but not the liver at day 50 compared with F3 B6/B10 Slc11a1+/+.Nos2A+/+ mice (Fig. 2) , a period during which T cell-mediated immunity under genetic regulation of H-2 is known to overcome early failure to control parasite expansion in liver and spleen in B10 Slc11a1mt/mt mice [31 ]. It should be noted, however, that parasite loads were low in the spleens of all strains at this time-point, so the difference caused by the Nos2A–/– was small. We did not intercross the Nos2A–/– into the B10 Slc11a1mt/mt line, where the effect may have been more pronounced.



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  		Figure 2. L. donovani infection in B6/B10 Slc11a1+/+.Nos2A+/+ and B6/B10 Slc11a1+/+.Nos2A–/– compared with B10 Slc11a1mt/mt.Nos2A+/+ mice. Parasite loads (LDU; mean±SEM; three or four mice per strain per time-point) were measured in liver (A) and spleen (B) at intervals over 50 days post-infection with 5 x 107 L. donovani LV9 amastigotes. P values for significant differences between strains are indicated in the text. Similar results were obtained for organ parasite loads in a replicate experiment.

 
Nos2A–/– does compromise resistance to L. major infection
Previous studies have shown that Slc11a1 does not regulate cutaneous lesion expansion caused by s.c. inoculation of L. major parasite [34 ] or expansion of the parasite population in liver or spleen following i.v. infection [35 ]. In contrast, Nos2A is crucial to regulation of the parasite in the skin and spread from lesions to the viscera [36 , 37 ]. Consistent with these observations, our F3 B6/B10 Slc11a1+/+.Nos2A–/– mice were highly susceptible to s.c. infection with L. major (Fig. 3 ) compared with F3 B6/B10 Slc11a1+/+.Nos2A+/+ (P<0.001, days 14 and 21) or B10 background N20 Slc11a1+/+ (Fig. 3 inset; P<0.001, days 14 and 21) mice. Indeed, lesion expansion in these mice exceeded (100% euthanasia by day 21) that observed in BALB/c (100% euthanasia by day 28) mice, classically recognized as the most susceptible mouse strain in which susceptibility to L. major infection comes under separate and complex genetic regulation. The results confirmed the previous observations [34 , 35 ] that Slc11a1 does not influence L. major infection, as B10 Slc11a1mt/mt and congenic N20 Slc11a1+/+ lines did not differ significantly in lesion size over >50 days of infection (Fig. 3 inset).



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  		Figure 3. L. major infection in B6/B10 Slc11a1+/+.Nos2A+/+ and B6/B10 Slc11a1+/+.Nos2A–/– compared with BALB/c mice. Infection was measured as footpad depth (mean±SEM; five or six mice per strain) at intervals over 86 days post-infection with 2 x 106 L. major LV39 metacyclic promastigotes. The inset shows parallel data for congenic B10 (Slc11a1mt/mt.Nos2A+/+) and N20 (Slc11a1+/+.Nos2A+/+) mice. P values for significant differences between strains are indicated in the text. This experiment replicated previous data [38 ] and was not repeated.

 
Cybb–/– does not influence Slc11a1+/+ regulation of S. enterica serovar Typhimurium infection
In contrast to Nos2A–/–, gene disruption at Cybb has a dramatic influence on early survival of mice following infection with 101 S. enterica serovar Typhimurium C5 (Fig. 4A ). Nevertheless, Slc11a1+/+.Cybb–/– survived longer than their Slc11a1mt/mt.Cybb–/– counterparts, indicating that the Slc11a1-resistance mechanism was at least partially functional in the absence of functional gp91phox activity. This observation was borne out by following bacterial counts in liver (Fig. 4B) and spleen (Fig. 4C) over the first 6 days of infection, where Slc11a1+/+.Cybb–/– showed reduced counts (P<0.001, day 2) compared with Slc11a1mt/mt.Cybb–/– mice, comparable with the differences observed when Slc11a1+/+.Cybb+/+ mice were compared (P<0.001, days 2, 4, and 6) with Slc11a1mt/mt. Cybb+/+ mice. This is demonstrated most clearly by the inset graphs in Figure 4 , B and C, which show bacterial counts in liver and spleen expressed as a percent of the day 1 counts within each strain. Here, is it clear that the two Slc11a1+/+ strains (solid lines) show reduced rates of expansion of the bacterial population compared with the two Slc11a1mt/mt strains (dotted lines) on Cybb+/+ and Cybb–/– backgrounds. These results indicate that gp91phox activity is essential for early control of bacterial expansion but by a mechanism separate from Slc11a1 regulation of the bacterial load in liver and spleen.



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  		Figure 4. S. enterica serovar Typhimurium infection in B6/B10 Slc11a1+/+. Cybb+/+ and B6/B10 Slc11a1+/+.Cybb–/– compared with B6 Slc11a1mt/mt. Cybb+/+ and B6 Slc11a1mt/mt.Cybb–/– mice (four to six per dose per strain), which were infected with 20-fold decreasing doses (8x104, 4x103, 2x102, 101) of S. enterica serovar Typhimurium C5. (A) Percent survival over 30 days following inoculation of 101 S. enterica serovar Typhimurium C5. The same rank order in mortality curves was observed for the 2 x 102 bacterial dose. Higher doses could not distinguish rapid mortality rates between the two Cybb–/– strains. Bacterial counts (mean±SEM; four mice per strain per time-point) in liver (B) and spleen (C) are shown over 6 days post-infection with 1.5 x 102 S. enterica serovar Typhimurium C5. (B and C) Inset graphs show the same data plotted as percent of the day 1 count for each mouse strain. P values for significant differences between strains are indicated in the text. Similar results were obtained for organ bacterial loads in a replicate experiment.

 
Cybb–/– does not compromise Slc11a1+/+ regulation of L. donovani infection
As for Nos2A–/–, gene disruption at Cybb–/– had no influence on the regulation of parasite loads in the livers and spleens over the initial Slc11a1-regulated phase of infection following i.v. inoculation of L. donovani or on the later, T cell-regulated, acquired immune response (Fig. 5 ). Differences observed at day 56 between F3 B6/B10 Slc11a1+/+.Cybb–/– and Slc11a1+/+.Cybb+/+ in liver and spleen were not statistically significant nor was any effect of Cybb–/– seen on the B6 Slc11a1mt/mt background (Fig. 5) . In contrast, differences between Slc11a1+/+ mice and Slc11a1mt/mt mice were significant at P < 0.001 from day 7 onward, whether they were Cybb+/+ or Cybb–/–. B6 background Slc11a1mt/mt mice demonstrated a reduced ability to clear parasites from liver and spleen compared with B10 background Slc11a1mt/mt mice (data not shown), indicating that other genes on this background influence the H-2b-regulated clearance of parasites observed in B10 mice [39 ]. However, this was not influenced by Cybb–/– gene disruption.



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  		Figure 5. L. donovani infection in B6/B10 Slc11a1+/+.Cybb+/+ and B6/B10 Slc11a1+/+.Cybb–/– compared with B6 Slc11a1mt/mt.Cybb+/+ and B6 Slc11a1mt/mt. Cybb–/– mice. Parasite loads (LDU; mean±SEM; four mice per strain per time-point) were measured in liver (A) and spleen (B) at intervals over 50 days post-infection with 2 x 107 L. donovani LV9 amastigotes. P values for significant differences between strains are indicated in the text. Similar results were obtained for organ parasite loads in a replicate experiment.

 
Cybb–/– does compromise resistance to L. major infection
Although gene disruption at Nos2A–/– had a dramatic and early influence on lesion growth and mortality following L. major infection on a mixed B6/B10 Slc11a1+/+ background, gene disruption at Cybb–/– had only a small but significant influence on lesion size in B6 Slc11a1mt/mt (Fig. 6A ; P=0.0006, day 93) and mixed B6/B10 Slc11a1+/+ (Fig. 6B ; P=0.0017, 3x106, and 0.031 at days 37, 47, and 93, respectively) mice in the later phases of the infection. However, there was no demonstrable pathology (ulceration) associated with this effect. Opposing effects of Cybb–/– were observed on B6 compared with mixed B6/B10 backgrounds at earlier time-points (days 17 and 23), indicating variable influence of background genes, again with no demonstrable influence on pathology other than increased footpad swelling. As before, there were no significant differences between B10 Slc11a1mt/mt and N20 Slc11a1+/+ mice in this experiment (data not shown), consistent with previous data (Fig. 3 inset and refs. [34 , 35 ]), showing no influence of Slc11a1 on lesion expansion following L. major infection.



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  		Figure 6. L. major infection in (A) B6 Slc11a1mt/mt.Cybb+/+ and B6 Slc11a1mt/mt.Cybb–/– mice or (B) B6/B10 Slc11a1+/+.Cybb+/+ and B6/B10 Slc11a1+/+. Cybb–/– mice, compared in each case with BALB/c mice. Infection was measured as footpad depth (mean±SEM; five mice per strain) at intervals over 94 days post-infection with 2 x 106 L. major LV39 metacyclic promastigotes. P values for significant differences between strains are indicated in the text. This experiment was only performed once.

 
Slc11a1 is not expressed in M1/70-positive/iNOS-positive cells recruited to the liver following L. donovani infection
Phagocyte oxidase activity is known to be expressed in young monocytes recruited to sites of inflammation but not in the resident Kupffer cell population in the liver [40 ]. To determine whether Slc11a1 was preferentially expressed in Kupffer cells compared with fresh monocytes entering the liver after infection, we costained liver sections taken 2–4 days after L. donovani infection with monoclonal M1/70 recognizing CD11b (CR3) and a rabbit polyclonal anti-Slc11a1 Ab (Fig. 7A ). No colocalized staining was observed, consistent with expression of Slc11a1 in the resident Kupffer cell population and not on freshly recruited M1/70-positive monocytes. Costaining of liver sections with rabbit polyclonal anti-iNOS Ab and rat monoclonal M1/70 demonstrated strict, colocalized staining of iNOS (Fig. 7B) in the M1/70-positive population (Fig. 7C) . Slc11a1-positive cells in the liver were F4/80-positive (data not shown). Hence, Slc11a1 is expressed in resident Kupffer cells in the liver that do not express iNOS (our data) or phagocyte oxidase [40 ] activities.



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  		Figure 7. Immunofluorescence staining of liver crystat sections from N20 mice 2–4 days after infection. (A) Representative field examined under high power with confocal laser attachment demonstrating no colocalization of Slc11a1 staining in resident Kupffer cells (green) with M1/70 staining on fresh infiltrating monocytes (red). Original bar = 10 µm. (B and C) Complete colocalization of M1/70 (green) and iNOS (red) in infiltrating monocytes with no iNOS staining of the resident Kupffer cell population. Original bars = 100 µm.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Previous studies have demonstrated that regulation of S. enterica serovar Typhimurium and L. donovani infection in mice is divided into discrete phases, which rely on innate versus T cell-mediated immune responses [31 , 41 , 42 ]. In both cases, the Nramp1, formerly known as Ity, Lsh, or Bcg and now redesignated as Slc11a1, has its primary impact during the early innate phase(s) of infection [32 , 33 ], and polymorphism at H-2 impacts on later T cell-mediated immune responses [39 , 41 ]. The question we addressed was to what extent gp91phox and iNOS activities are required for these separate innate and acquired phases of the immune response to S. enterica serovar Typhimurium and L. donovani, focusing, in particular, on their role in Slc11a1-mediated resistance following the observations that gp91phox [10 ] and iNOS [8 , 9 , 15 ] expression and/or activities are differentially regulated in Slc11a1+/+ and Slc11a1mt/mt macrophages in vitro. In addition, there was the specific hypothesis [20 ] that Fe2+ transported by Slc11a1 might interact directly with products of the oxidative burst to provide more potent antimicrobial radicals. Earlier studies of S. enterica serovar Typhimurium infection in Cybb–/– mice on a B6 Slc11a1mt/mt genetic background [43 ] had suggested some independence between oxidative burst-dependent host defenses, which are crucial in the first 24 h of infection, and Slc11a1, which extends its regulation of infection over the first ~7 days of infection [44 ]. This suggestion is confirmed here by our demonstration that Slc11a1+/+ maintain equivalent advantage over Slc11a1mt/mt mice with or without the Cybb–/– gene disruption and Cybb–/– exerting its influence independently and earlier than Slc11a1. Similarly, earlier studies of S. enterica serovar Typhimurium [43 ] had suggested that iNOS activity was important later (post-day 7) in infection, again, independent of Slc11a1 control, although this conclusion was, to some extent, serendipitous, as we show here that the B6 Nos2A–/– mice used [22 ], in fact, retained the Slc11a1 wild-type allele from the 129 progenitor (Mastroeni and co-workers [43 ] did not Slc11a1-genotype the mice used in their experiments), and the 129Sv Nos2A–/– strain used [36 ] in that study is not a full, functional null but a hypomorph, as demonstrated by the presence of a full-length protein in Western blots using control-activated macrophage protein preparations (J. K. White and J. M. Blackwell, unpublished data). This is consistent with functional studies [45 ] demonstrating that this 129Sv Nos2A–/– line was leaky for NO production. Nevertheless, results obtained here using the full, functional Nos2A null, backcrossed onto equivalent B6/B10 Slc11a1 wild-type and mt genetic backgrounds, support the conclusion that functional iNOS is not required for Slc11a1-mediated resistance over the first 7 days of a virulent S. enterica serovar Typhimurium C5 infection.

One problem in attempting to clarify the possible influence of gp91phox and iNOS on Slc11a1-mediated resistance in the context of S. enterica serovar Typhimurium infection is the acute and lethal nature of the infection and the short time-frame in which to examine the different phases of infection following administration of a virulent wild-type strain of the pathogen. In addition, the pleiotropic effects that Slc11a1 has on macrophage activation phenotypes in vitro mean that the same mechanisms might not be important in the in vivo regulation of different pathogens, which come under Slc11a1 control. In particular, we were keen to determine the influence of gp91phox and iNOS in Slc11a1 regulation of L. donovani infection, as both had been shown to be important in killing L. donovani in macrophages in vitro [16 , 17 ]. Unlike Salmonella infection, L. donovani infection is not lethal in mice, and the more prolonged course of infection makes it a more tractable, experimental model in which to analyze the roles of gp91phox and iNOS in Slc11a1- and non-Slc11a1-regulated phases of infection. Using this model, we obtained clear evidence that neither gp91phox nor iNOS activity contributed to resistance to L. donovani infection over the first 15 days when Slc11a1 is known to exert maximal influence in liver and spleen [26 , 33 ]. Our results differed from the earlier study of L. donovani infection using the same B6 (Slc11a1mt/mt) background mice [23 ] in which an increase in parasite loads in the livers of Cybb–/– compared with Cybb+/+ mice was reported at 2 weeks post-infection [46 ]. However, our study and the earlier study [46 ] concur on the observation that gp91phox has no influence over the acquired phase (>2 weeks) of L. donovani infection. Using a different source of Nos2A–/– mice on a mixed B6 x 129/Sv background [47 ], these authors also report [46 ] a more dramatic effect of iNOS deficiency on early (2 weeks) and acquired (>2 weeks) phases of L. donovani compared with our study in which iNOS had only moderate, organ-specific effects in the spleen at 50 days post-infection. Although the Slc11a1 status of these mixed-background mice was not reported [46 ], it is possible, in this case, that the line had become fixed for the B6 Slc11a1mt allele and that the requirement for iNOS activity during the acquired phase of immunity is more critical in Slc11a1mt/mt mice than we have observed here in Slc11a1+/+ mice. The results we obtained in our study with L. donovani were in stark contrast to L. major infection, where as reported previously [38 ], the fully functional Nos2A–/– gene disruption on a normally L. major-resistant B6/B10 background led to dramatic, early susceptibility and necessity for euthanasia within the first 21 days post-infection and where Cybb–/– also showed some influence on lesion size over the course of L. major infection. The smaller influence of Cybb–/– on L. major infection compared with Nos2A–/– is consistent with previous work [48 ], showing no effect of gp91phox on resolution of acute skin lesions and only a limited effect on containment of parasite in the draining lymph node. It is intriguing, however, that gp91phox was essential for clearance of L. major from the spleen [48 ]. These species-specific differences reflect the different cellular tropisms and pathologies caused by L. donovani and L. major. Studies in vitro [49 ] and in vivo [35 ] show that L. major has a preference for young, M1/70-positive monocytes, even when injected as amastigotes into mice i.v. and monitored in the liver [35 ], placing them in a cell population readily activated to express toxic antimicrobial iNOS activity. L. donovani amastigotes, conversely, preferentially infect resident tissue macrophages [35 ] and are found in the bone marrow in long-lived, sialoadhesin-positive, resident, stromal bone marrow macrophages [50 ]. This ability to target macrophage populations, which do not normally express potent antimicrobial iNOS or gp91phox activities, means that other mechanisms, such as those regulated more directly by Slc11a1, must be deployed by the host to control L. donovani infection. The ability of L. donovani to sequester into resident macrophage populations likely plays a major role in parasite persistence, with Slc11a1 possibly playing an important, long-term role in keeping this persistent parasite population in check. The species-specific differences in the behavior of leishmanial parasites in vivo and, in particular, the different mechanisms of antimicrobial activity associated with resistance and susceptibility to L. donovani and L. major have important implications for our understanding of human disease and the development of appropriate therapeutics.

To understand why neither gp91phox nor iNOS played a role in Slc11a1-mediated resistance in vivo, despite studies showing differential regulation of expression of activity of both in macrophages in vitro, we considered whether they were expressed in the same or different macrophage populations following infection in vivo. Although not formally re-examined here, we knew from earlier studies [40 ] that oxidative burst activity is not observed in the resident Kupffer cell population in the liver. What we examined specifically was which subpopulations of macrophage express Slc11a1 in the liver following L. donovani infection and whether this population expressed iNOS. Although we were unable to colocalize Slc11a1 and iNOS directly (as both were probed with rabbit Ab), we did demonstrate that iNOS and CD11b (recognized by the M1/70 Ab) were exclusively coexpressed in the fresh monocytes entering the liver after infection and that the M1/70 Ab showed no colocalization with the Slc11a1-positive, F4/80-positive, resident Kupffer cell population. Hence, we conclude that iNOS is not coexpressed in the same population of macrophages in the liver as Slc11a1 and similarly, by inference, that gp91phox and Slc11a1 are also unlikely to be coexpressed in the same macrophages in the liver. This is consistent with early studies [51 52 53 ] demonstrating that Lsh/Ity (=Slc11a1)-mediated resistance to infection was expressed in resident tissue macrophages, in Kupffer cells isolated ex vivo from livers of infected mice [51 ], or in liver, lung, or splenic tissue macrophages isolated and infected in vitro [52 , 53 ] but not in less mature macrophages from the peritoneal cavity [53 ]. Similarly, mature bone marrow-derived macrophages expressed Lsh-mediated resistance to L. donovani infection in vitro, but immature macrophages kept cycling with growth factors did not [53 ]. This is consistent now with RNA and Ab studies demonstrating that bone marrow macrophages have to be matured and activated to express high levels of Slc11a1 [54 ] and our data here showing that Kupffer cells in the livers of L. donovani-infected mice express Slc11a1 but not iNOS or by inference, gp91phox. Overall, our results suggest that although Slc11a1 may influence iNOS and NADPH oxidase activity under conditions where macrophages are activated to coexpress Slc11a1/iNOS or Slc11a1/NADPH oxidase in vitro, this does not occur for all populations of macrophages examined in vitro and does not appear to occur to influence the course of infection in vivo.

Our failure to demonstrate a role for NADPH oxidase or iNOS activity in Slc11a1-regulated S. enterica serovar Typhimurium or L. donovani infection leads us to conclude that interaction between these molecular pathways is not involved in Slc11a1-mediated resistance to infection in vivo. Other explanations must be found to account for Slc11a1-mediated antimicrobial activity. Early studies concluded that Slc11a1 limits bacterial [55 ] or leishmanial [56 ] replication in vivo, rather than enhancing bacterial killing. More recent studies have focused on the influence of Slc11a1 on phagosome-lysosome fusion and maturation in macrophages in vitro [54 , 57 58 59 60 ]. Functional Slc11a1 promotes phagosome-lysosome fusion and maturation, leading to acidification of the pathogen-containing phagosome and creating conditions under which reduced pathogen replication can occur. This appears to be related directly to the divalent cation-transporting function of Slc11a1, as iron chelators restore recruitment of the mannose-6-phosphate receptor to Salmonella containing vacuoles in Slc11a1 mt macrophages, overcoming the ability of Salmonella to alter phagosome maturation [61 ]. This suggests a role for Slc11a1 in regulating the recently described, Toll-like receptor-triggered, inducible, phagosome-lysosome maturation pathway [62 ], perhaps accounting for the influence of Slc11a1 on antigen processing and presentation [63 ] and T helper cell type 1 (Th1)/Th2 bias in immune response to infection [64 , 65 ] or vaccination [66 ].

In conclusion, our studies are important in demonstrating that observations made in vitro do not necessarily reflect the course of events in vivo. We demonstrate that iNOS and gp91phox are not important players in Slc11a1-mediated resistance to infection in vivo, fueling further research into the mechanisms by which this important gene influences susceptibility to infectious and autoimmune diseases.


   ACKNOWLEDGEMENTS
 
This work was supported by grants from the Wellcome Trust. J. K. W. held a Beit Memorial Research Fellowship. We thank Kara Hunter and Linda Wicker at Cambridge Institute for Medical Research for microsatellite genotyping, undertaken to define the congenic interval in N20 compared with B10 mice.


   FOOTNOTES
 
1 Current address: The Sanger Centre, The Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, UK. Back

2 Current address: Department of Infectious Diseases, Imperial College London, Hammersmith Campus, Hammersmith Hospital, Du Cane Road, London W12 0NN, UK. Back

Received September 24, 2004; revised November 10, 2004; accepted November 17, 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Blackwell, J. M., Goswami, T., Evans, C. A. W., Sibthorpe, D., Papo, N., White, J. K., Searle, S., Miller, E. N., Peacock, C. S., Mohammed, H., Ibrahim, M. (2001) SLC11A1 (formerly NRAMP1) and disease Cell. Microbiol. 3,773-784[CrossRef][Medline]
  2. Buschman, E., Skamene, E. (2001) From Bcg/Lsh/Ity to Nramp1: three decades of search and research Drug Metab. Dispos. 29,471-473[Free Full Text]
  3. Blackwell, J. M., Searle, S., Mohamed, H., White, J. K. (2003) Divalent cation transport and susceptibility to infectious and autoimmune disease: continuation of the Ity/Lsh/Bcg/Nramp1/Slc11a1 gene story Immunol. Lett. 85,197-203[CrossRef][Medline]
  4. Forbes, J. R., Gros, P. (2001) Divalent-metal transport by NRAMP proteins at the interface of host-pathogen interactions Trends Microbiol. 9,397-403[CrossRef][Medline]
  5. Roach, T. I. A., Chatterjee, D., Blackwell, J. M. (1994) Induction of early response genes KC and JE by mycobacterial lipoarabinomannans: regulation of KC expression in murine macrophages by Lsh/Ity/Bcg (candidate Nramp) Infect. Immun. 62,1176-1184[Abstract/Free Full Text]
  6. Schurr, E., Radzioch, D., Malo, D., Gros, P., Skamene, E. (1991) Molecular genetics of inherited susceptibility to intracellular parasites Behring Inst. Mitt. 88,1-12
  7. Formica, S., Roach, T. I. A., Blackwell, J. M. (1994) Interaction with extracellular matrix proteins influences Lsh/Ity/Bcg (candidate Nramp) gene regulation of macrophage priming/activation for tumor necrosis factor {alpha} and nitrite release Immunology 82,42-50[Medline]
  8. Brown, D. H., LaFuse, W. P., Zwilling, B. S. (1997) Stabilized expression of mRNA is associated with mycobacterial resistance controlled by Nramp1 Infect. Immun. 65,597-603[Abstract]
  9. Fritsche, G., Dlaska, M., Barton, H., Theurl, I., Garimorth, K., Weiss, G. (2003) Nramp1 functionality increases inducible nitric oxide synthase transcription via stimulation of IFN regulatory factor 1 expression J. Immunol. 171,1994-1998[Abstract/Free Full Text]
  10. Barton, C. H., Whitehead, S. H., Blackwell, J. M. (1995) Nramp transfection transfers Ity/Lsh/Bcg-related pleiotropic effects on macrophage activation: influence on oxidative burst and nitric oxide pathways Mol. Med. 1,267-279[Medline]
  11. Nathan, C. (1995) Natural resistance and nitric oxide Cell 82,873-876[CrossRef][Medline]
  12. Bogdan, C. (2001) Nitric oxide and the immune response Nat. Immunol. 2,907-916[CrossRef][Medline]
  13. Bogdan, C., Rollinghoff, M., Diefenbach, A. (2000) The role of nitric oxide in innate immunity Immunol. Rev. 173,17-26[CrossRef][Medline]
  14. Ables, G. P., Takamatsu, D., Noma, H., El-Shazly, S., Jin, H. K., Taniguchi, T., Sekikawa, K., Watanabe, T. (2001) The roles of Nramp1 and TNF{alpha} genes in nitric oxide production and their effect on the growth of Salmonella typhimurium in macrophages from Nramp1 congenic and tumor necrosis factor-{alpha}–/– mice J. Interferon Cytokine Res. 21,53-62[CrossRef][Medline]
  15. Barrera, L. F., Kramnik, I., Skamene, E., Radzioch, D. (1994) Nitrite production by macrophages derived from Bcg-resistant and susceptible congenic mouse strains in response to interferon-{gamma} and infection with BCG Immunology 82,457-464[Medline]
  16. Roach, T. I., Kiderlen, A. F., Blackwell, J. M. (1991) Role of inorganic nitrogen oxides and tumor necrosis factor-{alpha} in killing Leishmania donovani amastigotes in {gamma} interferon-lipopolysaccharide-activated macrophages from Lshs and Lshr congenic mouse strains Infect. Immun. 59,3935-3944[Abstract/Free Full Text]
  17. Murray, H. W., Juangbhanich, C. W., Nathan, C. F., Cohn, Z. A. (1979) Macrophage oxygen-dependent antimicrobial activity. II. The role of oxygen intermediates J. Exp. Med. 150,950-964[Abstract/Free Full Text]
  18. Vazquez-Torres, A., Jones-Carson, J., Mastroeni, P., Ischiropoulos, H., Fang, F. C. (2000) Antimicrobial actions of the NADPH phagocyte oxidase and inducible nitric oxide synthase in experimental salmonellosis. I. Effects on microbial killing by activated peritoneal macrophages in vitro J. Exp. Med. 192,227-236[Abstract/Free Full Text]
  19. Lowrie, D. B. (1983) How macrophages kill tubercle bacilli J. Med. Microbiol. 16,1-12[Medline]
  20. Kuhn, D. E., Baker, B. D., Lafuse, W. P., Zwilling, B. S. (1999) Differential iron transport into phagosomes isolated from the RAW264.7 macrophage cell lines transfected with Nramp1Gly169 or Nramp1Asp169 J. Leukoc. Biol. 66,113-119[Abstract]
  21. Blackwell, J. M., Toole, S., King, M., Dawda, P., Roach, T. I., Cooper, A. (1988) Analysis of Lsh gene expression in congenic B10.L-Lshr mice Curr. Top. Microbiol. Immunol. 137,301-309[Medline]
  22. Laubach, V. E., Shesely, E. G., Smithies, O., Sherman, P. A. (1995) Mice lacking inducible nitric oxide synthase are not resistant to lipopolysaccharide-induced death Proc. Natl. Acad. Sci. USA 92,10688-10692[Abstract/Free Full Text]
  23. Pollock, J. D., Williams, D. A., Gifford, M. A., Li, L. L., Du, X., Fisherman, J., Orkin, S. H., Doerschuk, C. M., Dinauer, M. C. (1995) Mouse model of X-linked chronic granulomatous disease, an inherited defect in phagocyte superoxide production Nat. Genet. 9,202-209[CrossRef][Medline]
  24. Hormaeche, C. E., Mastroeni, P., Arena, A., Uddin, J., Joysey, H. S. (1990) T cells do not mediate the initial suppression of a Salmonella infection in the RES Immunology 70,247-250[Medline]
  25. Reed, L. J., Muench, H. (1938) A simple method of estimating fifty percent endpoints Am. J. Hyg. 27,493-497
  26. Bradley, D. J., Kirkley, J. (1977) Regulation of Leishmania populations within the host. I. The variable course of Leishmania donovani infections in mice Clin. Exp. Immunol. 30,119-129[Medline]
  27. Beller, D. I., Springer, T. A., Schreiber, R. D. (1982) Anti-Mac-1 selectively inhibits the mouse and human type three complement receptor J. Exp. Med. 156,1000-1009[Abstract/Free Full Text]
  28. Vodovotz, Y., Bogdan, C., Paik, J., Xie, Q. W., Nathan, C. (1993) Mechanisms of suppression of macrophage nitric oxide release by transforming growth factor ß J. Exp. Med. 178,605-613[Abstract/Free Full Text]
  29. Atkinson, P. G. P., Barton, C. H. (1998) Ectopic expression of Nramp1 in COS-1 cells modulates iron accumulation FEBS Lett. 425,239-242[CrossRef][Medline]
  30. White, J. K., Stewart, A., Popoff, J. F., Wilson, S., Blackwell, J. M. (2004) Incomplete glycosylation and defective intracellular targeting of mutant solute carrier family 11 member 1 (Slc11a1) Biochem. J. 382,811-819[CrossRef][Medline]
  31. Hormaeche, C. E. (1979) Natural resistance to Salmonella typhimurium in different inbred mouse strains Immunology 37,311-318[Medline]
  32. Plant, J., Glynn, A. A. (1976) Genetics of resistance to infection with Salmonella typhimurium in mice J. Infect. Dis. 133,72-78[Medline]
  33. Bradley, D. J. (1977) Regulation of Leishmania populations within the host. II. Genetic control of acute susceptibility of mice to Leishmania donovani infection Clin. Exp. Immunol. 30,130-140[Medline]
  34. Blackwell, J. M., Alexander, J. (1986) Different host genes recognize and control infection with taxonomically distinct Leishmania species Rioux, J. A. Peters, W. eds. Leishmania. Taxonomie et PhylogenSse. Applications, Co-, Pid, Miologiques ,211-219 IMEEE Montpellier, France.
  35. Davies, E. V., Singleton, A. M., Blackwell, J. M. (1988) Differences in Lsh gene control over systemic Leishmania major and Leishmania donovani or Leishmania mexicana mexicana infections are caused by differential targeting to infiltrating and resident liver macrophage populations Infect. Immun. 56,1128-1134[Abstract/Free Full Text]
  36. Wei, X. Q., Charles, I. G., Smith, A., Ure, J., Feng, G. J., Huang, F. P., Xu, D., Muller, W., Moncada, S., Liew, F. Y. (1995) Altered immune responses in mice lacking inducible nitric oxide synthase Nature 375,408-411[CrossRef][Medline]
  37. Stenger, S., Thuring, H., Rollinghoff, M., Bogdan, C. (1994) Tissue expression of inducible nitric oxide synthase is closely associated with resistance to Leishmania major J. Exp. Med. 180,783-793[Abstract/Free Full Text]
  38. Diefenbach, A., Schindler, H., Donhauser, N., Lorenz, E., Laskay, T., MacMicking, J., Rollinghoff, M., Gresser, I., Bogdan, C. (1998) Type 1 interferon (IFN{alpha}/ß) and type 2 nitric oxide synthase regulate the innate immune response to a protozoan parasite Immunity 8,77-87[CrossRef][Medline]
  39. Blackwell, J., Freeman, J., Bradley, D. (1980) Influence of H-2 complex on acquired resistance to Leishmania donovani infection in mice Nature 283,72-74[CrossRef][Medline]
  40. Lepay, D. A., Nathan, C. F., Steinman, R. M., Murray, H. W., Cohn, Z. A. (1985) Murine Kupffer cells: mononuclear phagocytes deficient in the generation of reactive oxygen intermediates J. Exp. Med. 161,1079-1096[Abstract/Free Full Text]
  41. Hormaeche, C. E., Harrington, K. A., Joysey, H. S. (1985) Natural resistance to salmonellae in mice: control by genes within the major histocompatibility complex J. Infect. Dis. 152,1050-1056[Medline]
  42. Blackwell, J. M. (1985) Genetic control of discrete phases of complex infections: Leishmania donovani as a model Prog. Leuk. Biol. 3,31-49
  43. Mastroeni, P., Vazquez-Torres, A., Fang, F. C., Xu, Y., Khan, S., Hormaeche, C. E., Dougan, G. (2000) Antimicrobial actions of the NADPH phagocyte oxidase and inducible nitric oxide synthase in experimental salmonellosis. II. Effects on microbial proliferation and host survival in vivo J. Exp. Med. 192,237-248[Abstract/Free Full Text]
  44. Hormaeche, C. E., Maskell, D. J. (1989) Influence of the Ity gene on salmonella infections Res. Immunol. 140,791-793[CrossRef][Medline]
  45. Niedbala, W., Wei, X. Q., Piedrafita, D., Xu, D., Liew, F. Y. (1999) Effects of nitric oxide on the induction and differentiation of Th1 cells Eur. J. Immunol. 29,2498-2505[CrossRef][Medline]
  46. Murray, H. W., Nathan, C. F. (1999) Macrophage microbicidal mechanisms in vivo: reactive nitrogen versus oxygen intermediates in the killing of intracellular visceral Leishmania donovani J. Exp. Med. 189,741-746[Abstract/Free Full Text]
  47. MacMicking, J. D., Nathan, C., Hom, G., Chartrain, N., Fletcher, D. S., Trumbauer, M., Stevens, K., Xie, Q. W., Sokol, K., Hutchinson, N., et al (1995) Altered responses to bacterial infection and endotoxic shock in mice lacking inducible nitric oxide synthase Cell 81,641-650[CrossRef][Medline]
  48. Blos, M., Schleicher, U., Soares Rocha, F. J., Meissner, 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[CrossRef][Medline]
  49. Fortier, A. H., Hoover, D. L., Nacy, C. A. (1982) Intracellular replication of Leishmania tropica in mouse peritoneal macrophages: amastigote infection of resident cells and inflammatory exidate macrophages Infect. Immun. 38,1304-1308[Abstract/Free Full Text]
  50. Leclercq, V., Lebastard, M., Belkaid, Y., Louis, J., Milon, G. (1996) The outcome of the parasitic process initiated by Leishmania infantum in laboratory mice: a tissue-dependent pattern controlled by the Lsh and MHC loci J. Immunol. 157,4537-4545[Abstract]
  51. Crocker, P. R., Blackwell, J. M., Bradley, D. J. (1984) Expression of the natural resistance gene Lsh in resident liver macrophages Infect. Immun. 43,1033-1040[Abstract/Free Full Text]
  52. Harrington, K. A., Hormaeche, C. E. (1986) Expression of the innate resistance gene Ity in mouse Kupffer cells infected with Salmonella typhimurium in vitro Microb. Pathog. 1,269-274[CrossRef][Medline]
  53. Crocker, P. R., Davies, E. V., Blackwell, J. M. (1987) Variable expression of the murine natural resistance gene Lsh in different macrophage populations infected in vitro with Leishmania donovani Parasite Immunol. 9,705-719[Medline]
  54. Searle, S., Bright, N. A., Roach, T. I. A., Atkinson, P. G. P., Barton, C. H., Meloen, R. H., Blackwell, J. M. (1998) Localisation of Nramp1 in macrophages: modulation with activation and infection J. Cell Sci. 111,2855-2866[Abstract]
  55. Hormaeche, C. E. (1980) The in vivo division and death rates of Salmonella typhimurium in the spleens of naturally resistant and susceptible mice measured by the superinfecting phage technique of Meynell Immunology 41,973-979[Medline]
  56. Bradley, D. J. (1979) Regulation of Leishmania populations within the host. IV. Parasite and host cell kinetics studied by radioisotope labelling Acta Trop. 36,171-179[Medline]
  57. de Chastellier, C., Frehel, C., Offredo, C., Skamene, E. (1993) Implication of phagosome-lysosome fusion in restriction of Mycobacterium avium growth in bone marrow macrophages from genetically resistant mice Infect. Immun. 61,3775-3784[Abstract/Free Full Text]
  58. Gruenheid, S., Pinner, E., Desjardins, M., Gros, P. (1997) Natural resistance to infections with intracellular pathogens: the Nramp1 protein is recruited to the membrane of the phagosome J. Exp. Med. 185,717-730[Abstract/Free Full Text]
  59. Cuellar-Mata, P., Jabado, N., Liu, J., Furuya, W., Finlay, B. B., Gros, P., Grinstein, S. (2002) Nramp1 modifies the fusion of Salmonella typhimurium-containing vacuoles with cellular endomembranes in macrophages J. Biol. Chem. 277,2258-2265[Abstract/Free Full Text]
  60. Frehel, C., Canonne-Hergaux, F., Gros, P., De Chastellier, C. (2002) Effect of Nramp1 on bacterial replication and on maturation of Mycobacterium avium-containing phagosomes in bone marrow-derived mouse macrophages Cell. Microbiol. 4,541-556[CrossRef][Medline]
  61. Jabado, N., Cuellar-Mata, P., Grinstein, S., Gros, P. (2003) Iron chelators modulate the fusogenic properties of Salmonella-containing phagosomes Proc. Natl. Acad. Sci. USA 100,6127-6132[Abstract/Free Full Text]
  62. Blander, J. M., Medzhitov, R. (2004) Regulation of phagosome maturation by signals from Toll-like receptors Science 304,1014-1018[Abstract/Free Full Text]
  63. Lang, T., Prina, E., Sibthorpe, D., Blackwell, J. M. (1997) Nramp1 transfection transfers Ity/Lsh/Bcg-related pleiotropic effects on macrophage activation: influence on antigen processing and presentation Infect. Immun. 65,380-386[Abstract]
  64. Kaye, P. M., Blackwell, J. M. (1989) Lsh, antigen presentation and the development of CMI Res. Immunol. 140,810-815[CrossRef][Medline]
  65. Kramnik, I., Radzioch, D., Skamene, E. (1994) T-helper 1-like subset selection in Mycobacterium bovis bacillus Calmette-Gurin-infected resistant and susceptible mice Immunology 81,618-625[Medline]
  66. Soo, S-S., Villarreal Ramos, B., Khan, C. M., Hormaeche, C. E., Blackwell, J. M. (1998) Genetic control of immune response to recombinant antigens carried by an attenuated Salmonella typhmurium vaccine strain: Nramp1 influences T-helper subset responses and protection against leishmanial challenge Infect. Immun. 66,1910-1917[Abstract/Free Full Text]



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