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The Journal of Immunology, 2006, 176: 6954-6961.
Copyright © 2006 by The American Association of Immunologists

Sequencing, Expression, and Functional Analyses Support the Candidacy of Ncf2 in Susceptibility to Salmonella Typhimurium Infection in Wild-Derived Mice1

Vanessa Sancho-Shimizu*,{ddagger} and Danielle Malo2,*,{dagger},{ddagger}

* Department of Human Genetics, McGill University, Montreal, Quebec, Canada; {dagger} Department of Medicine, McGill University, Montreal, Quebec, Canada; and {ddagger} Center for the Study of Host Resistance, McGill University Health Center, Montreal, Quebec, Canada


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
A recessive Salmonella Typhimurium susceptibility locus (immunity to Typhimurium (Ity3) was reported previously on distal mouse chromosome 1 using a cross between C57BL/6J and wild-derived MOLF/Ei mice. This quantitative trait locus is located in a genomic region spanning 84 Mb, rich in candidate genes for which a role in host resistance to Salmonella infection is either known or can be envisioned. In this study, we report the evaluation of neutrophil cytosolic factor 2 (Ncf2) as a candidate Salmonella susceptibility gene for Ity3. Ncf2 encodes p67phox, a subunit of the multiprotein enzyme complex NADPH oxidase, known to be responsible for the generation of superoxides. Congenic mice carrying the Ity3 region from MOLF/Ei, B6.MOLF-Ity/Ity3 were more susceptible to infection compared with control mice heterozygous at Ity3, B6.MOLF-Ity/Ity3MOLF/B6, confirming the existence of a recessive Salmonella susceptibility locus on distal chromosome 1. Spleen Ncf2 expression levels were lower in infected congenic mice homozygous for the MOLF/Ei allele at Ity3 compared with mice heterozygous at Ity3. C57BL/6J and MOLF/Ei Ncf2 sequence comparisons revealed one nonconservative amino acid change (R394Q) in the functional and highly conserved Phox and Bem1 domain of the protein. Functional analysis revealed that the MOLF/Ei allele had reduced PMA- and Salmonella-induced superoxide induction as compared with their wild-type counterparts ex vivo. The R394Q substitution seems to occur on an amino acid involved in electrostatic interactions with p40phox, crucial in its activation. Moreover, a human mutation in the corresponding R395W, resulting in chronic granulatomous disease, is known to lead to reduced superoxide levels. These results support the candidacy of Ncf2 as the gene underlying Ity3.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Salmonella enterica serovar Typhimurium (Salmonella Typhimurium) is a facultative intracellular Gram-negative bacillus with a broad host range. Salmonella Typhimurium can infect both humans and mice causing a spectrum of disease states ranging from an asymptomatic carrier state to sepsis. There are typically two forms of clinical manifestations as a result of Salmonella infection, a self-limiting gastroenteritis and a systemic disease known as typhoid fever. The latter is still considered a public health issue among developing nations as there are 16 million cases reported globally every year (1) (<www.cdc.gov>).

Host genetic factors are known to influence the outcome of infection. Some examples include Solute carrier family 11 member 1 (Slc11a1) (formerly known as Nramp1), TLR4 (Tlr4), H2 complex, IFN-{gamma}, and btk (reviewed in Ref. 2). Understanding of the pathogenesis of Salmonella infection to date is largely due to the use of a well-characterized mouse model of infection. Infection of laboratory mice with Salmonella Typhimurium has long been used to study the systemic manifestation of disease because mice develop an illness that resembles human typhoid fever. This model consists of four distinct phases: 1) the initial clearance of the inoculum from the blood as surviving bacteria take up residence in the polymorphonuclear cells and macrophages of the reticuloendothelial system; 2) the exponential replication phase within the reticuloendothelial system; 3) the plateau phase in which effectors of innate immunity suppress the replication of bacteria; and 4) the resolution of infection through adaptive immune mechanisms (reviewed in Ref. 3). In this study, we will be focusing on the first three phases of this model, which constitute the innate immune phase of infection.

The phagocyte NADPH oxidase has a crucial role in innate immunity, specifically acting to reduce molecular oxygen to superoxide. Superoxide anions give rise to numerous toxic reactive oxygen species that are used as microbicidal agents against pathogens, contributing to the respiratory burst typically seen in phagocytic cells. The complex consists of five protein subunits, two transmembrane proteins p22phox, and the catalytic subunit gp91phox comprising the flavocytochrome b558 complex, and three cytosolic proteins, p40phox, p47phox, and p67phox in addition to a small GTPase Rac (4). p67phox, encoded by neutrophil cytosolic factor 2 (Ncf2) on distal mouse chromosome 1, is found in the cytosol at resting state, bound to p40phox and p47phox in an inactive form (5). Upon activation by opsonized microbes or inflammatory mediators, p67phox, along with p40phox and p47phox, is translocated to the membrane, to form an active enzyme complex with flavocytochrome b558, and is thought to regulate electron transfer from NADPH to flavin adenine dinucleotide (6, 7). Once activated, superoxide is released into the phagocytic vacuole or into the extracellular space.

The importance of a functional NADPH oxidase can be seen in individuals that have mutations in the genes that encode for four of the subunits (CYBB for gp91phox, CYBA for p22phox, NCF1 for p47phox, and NCF2 for p67phox) that make up the complex, resulting in the rare inherited immunodeficiency, chronic granulatomous disease (CGD)3 (8, 9). These patients have a total absence or very low levels of superoxides, which renders them extremely susceptible to recurrent and often chronic infections with a wide range of bacterial and fungal pathogens (8). According to a national registry of 368 CGD patients in the U.S., Salmonella species accounted for the majority of bacteremia and sepsis cases in CGD patients (10). Mouse models of CGD parallel the phenotypes observed in human CGD and have been successfully created by knocking out p47phox or gp91phox (11, 12). These knockout models show increased susceptibility to Staphylococcus aureus, Mycobacterium tuberculosis, Mycobacterium avium, Pseudomonas aeruginosa, Aspergillus fumigatus, and Salmonella Typhimurium infections (11, 12, 13, 14, 15, 16).

We have previously reported the identification of quantitative trait loci (QTLs) that affect host response to Salmonella Typhimurium infection in the susceptible wild-derived inbred mouse MOLF/Ei (17). In addition to Slc11a1 on proximal chromosome 1 (Ity), two novel loci, a resistance locus on chromosome 11 (Ity2), and a susceptibility locus on distal chromosome 1 (Ity3), were identified using a F2 panel of C57BL/6J x MOLF/Ei mice. Ity3 is the only susceptibility locus identified in MOLF/Ei, mapping just downstream of the Ity locus on distal chromosome 1, with a genomic interval of ~16 cM. It was found to be inherited in a recessive fashion, accounting for 7% of the phenotypic variance with a peak logarithm of odds (LOD) score of 4.8 when expressed on a Slc11a1 wild-type background (MOLF/Ei allele). In this study, we report our findings on the candidacy of Ncf2 as the gene underlying the Ity3 locus based on its essential role in innate immunity, physical map position, sequence polymorphism, and functional analyses.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Ity3 QTL reanalysis

Eight additional markers, D1Mit5, D1Mit135, D1Mit218, D1Mit99, D1Mit193, D1Mit 201, Ncf2 (6F 5'-tgatcagtttgtcagaccagc-3' and 5R5'gctacctgagtgacaatggct-3'), and Tlr5 (F 5'-catgtcaacagggagctttgt- 3' and R 5'-atgaagctgcctgtaacttctccc-3', were used to re-evaluate the LOD score for Ity3, using an additional 41 mice along with the original 191 (C57BL/6J x MOLF/Ei) F2 mice (17). The linkage was analyzed using MapManager QTX (18), setting D1Mcg4 as background, on the phenotype of Ln (survival) using a free regression model. The LOD score was calculated by dividing the likelihood ratio statistic by 4.6.

Construction of congenic mice

Inbred strains C57BL/6J (B6) and MOLF/Ei (MOLF) mice were purchased from The Jackson Laboratory. Three strains of congenic mice were generated in this study: B6.MOLF-Ity, B6.MOLF-Ity/Ity3MOLF/B6, and B6.MOLF-Ity/Ity3 mice; in all cases, the MOLF allele represented the donor strain and the B6, the recipient strain. For the B6.MOLF-Ity congenics, the region surrounding D1Mcg4, flanked by D1Mit3 and D1Mit134, was transferred from MOLF/Ei to C57BL/6J by selective backcrossing for 10 generations to C57BL/6J. After the 10th generation of backcrossing, brother-sister matings were established to generate homozygous B6.MOLF-Ity mice, which have two copies of the resistant Ity locus derived from MOLF/Ei, including the wild-type Slc11a1 allele, as opposed to the mutant C57BL/6J Slc11a1 allele. B6.MOLF-Ity/Ity3 mice were created by introgressing the 84-Mb segment of Ity3 flanked by the markers D1Mit135 and D1Mit17 from MOLF/Ei to C57BL/6J though five successive backcrosses to B6.MOLF-Ity mice. The D1Mit135-D1Mit17 interval corresponds to a 2-LOD support interval, which gives a 99% confidence interval that the locus lies within the target region. At the 5th generation of backcrossing, these mice were intercrossed to generate B6.MOLF-Ity/Ity3 congenic mice and were genotyped for D1Mit3, D1Mit5, D1Mcg4, D1Mit216, D1Mit218, D1Mit193, D1Mit201, D1Mit63, D1Mit17, Ncf2 (6F 5'-tgatcagtttgtcagaccagc-3' and 5R5'gctacctgagtgacaatggct-3'), and Tlr5 (F 5'-catgtcaacagggagctttgt-3' and R 5'-atgaagctgcctgtaacttctccc-3'). The B6.MOLF-Ity/Ity3 mice carry the resistant Ity segment from MOLF/Ei as well as the susceptible Ity3 locus from MOLF/Ei on a C57BL/6J genetic background (Fig. 1). The B6.MOLF-Ity/Ity3 mice that were heterozygous for the Ity3 interval, B6.MOLF-Ity/Ity3MOLF/B6, were used as controls when the B6.MOLF-Ity mice were unavailable. This was possible because the Ity3 locus is inherited in a recessive fashion and should only have an impact in homozygous form (17). All mice were bred and maintained in an animal care facility according to the Canadian Council on Animal Care.


Figure 1
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  		FIGURE 1. LOD score plot for Ity3 on chromosome 1. The physical position of the candidate gene Ncf2 is shown as well as the chromosomes generated in the creation of the B6.MOLF-Ity/Ity3 congenics. The black segments indicate the recipient C57BL/6J genotype; the white segments indicate the MOLF/Ei genotype; and the hatched segments represent heterozygous intervals.

 
Salmonella Typhimurium infection

Six- to 16-wk-old C57BL6/J, B6.MOLF-Ity/Ity3, and B6.MOLF-Ity/Ity3MOLF/B6 male and female mice were infected through the caudal vein with 3000 CFUs of Salmonella Typhimurium strain Keller (originally a gift from H. Robson at the Royal Victoria Hospital, Montreal, Canada). Salmonella Typhimurium was prepared by growing 1 ml of culture in 100 ml of trypticase soy broth for ~90 min (OD600 between 0.1 and 0.2) at 37°C, followed by plating 10–3, 10–4, 10–5, and 10–6 serial dilutions in duplicate on trypticase soy agar, and placed in a 37°C incubator overnight. CFUs were enumerated the following day, and the dose was appropriately prepared to the desired concentration of 3000 CFU/0.2 ml. The injection mixture was plated in three 10-fold serial dilutions to ensure the dosage concentration. Infected mice were monitored at least every 12 h, and moribund animals were sacrificed by CO2 asphyxiation. Survival was recorded as the day of death after infection. Survival analysis was conducted using the Kaplan-Meier survival test.

Two males and two females from each strain, C57BL/6J, B6.MOLF-Ity/Ity3, and B6.MOLF-Ity/Ity3MOLF/B6, were sacrificed and weighed at days 0, 1, 3, and 5 postinfection. Their spleens were aseptically removed and weighed. Half of the spleen was used for CFU analysis, placed in 2 ml of 0.9% saline solution, and, following homogenization, serially diluted 10-fold to be plated in duplicate on trypticase soy agar plates and placed in a 37°C incubator overnight. CFUs were enumerated the following day, and data were expressed as log10 (CFU)/g spleen. The other half of the spleen was snap frozen in liquid nitrogen and stored at –80°C for RNA extraction.

DNA extraction and genotyping

Genomic DNA from C3H/HeSnJ, C57Br/cdJ, C57L/J, DBA/2J, RF/J, P/J, BuB/BnJ, PERA/Rk, PERC/Ei, SF/CamEi, SPRET/Ei, RBA/Dn, RBB/Dn, Sk/CamEi, ZALENDE/Ei, TIRANO/Ei, Skive/Ei, CZECHII/Ei, MOLC/Rk, MOLD/RK, MOLG/Dn, and MOLE/Rk was purchased from The Jackson Laboratory. C57BL/6J, MOLF/Ei, 129S6/SvEvTac, C3H/HeJ, B6.MOLF-Ity, B6.MOLF-Ity/Ity3, and B6.MOLF-Ity/Ity3MOLF/B6 genomic DNA were extracted from tail clippings using NaOH, as described by Schmitteckert et al. (19). Microsatellite markers were amplified using standard PCR conditions in a final volume of 20 µl. PCR products were run on Metaphor agarose (Mandel) or High Efficiency agarose (USB) and visualized by ethidium bromide staining under a UV lamp.

Sequencing

C57BL/6J and MOLF/Ei Ncf2 cDNA were obtained from reverse transcription of spleen RNA. Ncf2 cDNA was amplified using two sets of primers. The first set amplified the 5' half of Ncf2 using the primers: 5'-gcgctaggctgggaccttgaagcc-3' and 5'-ccccttctgtccattgaacatgac-3'. The second set amplified the 3'end of Ncf2: 5'-gtcttgaagaagggcagtgataac-3' and 5'-cacagcagagatgggtaagtcttgc-3'. The purity of the amplified fragments of Ncf2 cDNA was verified on 1.7% agarose and treated with alkaline phosphatase. The fragments were then sequenced on polyacrylamide gel using the Thermosequenase Radiolabeled Terminator cycle sequencing kit (USB), and visualized by autoradiography. Additional primers were used to sequence the gene in its entirety: 5'-ctgaggccatcagactctggaatg-3', 5'-tgcctgcaagttttccaggat-3', 5'-gagcagttggcattggcaacc-3', 5'-aatgcctgggctcggacttca-3', 5'-gcctctcatttggacggaaca-3', 5'-ccagaaatcttcagggctctg-3', 5'-atgccttacatgctcaaggtg-3', 5'-ctacagccagcttcggaacatggt-3', 5'-ctttcttcggacaggagcaga-3', 5'-ccaggtggtagcaatcttcag-3', and 5'-gcattcccagagaagtctaggatc-3'. The entire coding region of Ncf2 was read in forward and reverse orientation (GenBank accession no. DQ449939).

TaqMan real-time PCR

Expression of Ncf2 in C57BL/6J, MOLF/Ei, 129S6/SvEvTac, B6.MOLF-Ity/Ity3MOLF/B6, and B6.MOLF-Ity/Ity3 was determined by TaqMan real-time PCR using RotorGene (Corbett Research). cDNAs were obtained from reverse transcription of infected and control spleen RNAs. ThecDNAs were amplified using the primers 5'-tcatgttcaatggacagaag-3' and 5'-tatcggattctggagaggta-3' with a probe 5'FAM-actacctggagccagttgagcttc-3'BHQ, and cycled at 95°C for 30 s, 55°C for 60 s, and 72°C for 30 s, a total of 40 cycles. The 18srRNA was used as a housekeeping gene control and amplified using the primers F 5'-cacggccggtacagtgaaa-3' and 5'-agcgagcgaccaaaggaa-3' with a probe 5'HEX-tgcgaatggctcattaaatcagtta-3'BHQ, using the following cycling conditions: 95°C for 30 s, 60°C for 60 s, and 72°C for 30 s, for a total of 40 cycles. Stratagenes Brilliant QPCR kit core reagents were used for the PCR. All samples were run in duplicate along with a standard curve of four 10-fold serial dilutions of template cDNA. The expression data are expressed in relative fold change units using uninfected C57BL/6J as the referent according to the following 2–{Delta}{Delta}t equation Formula (20). The level of significance was assessed using Student’s ttest (p< 0.05).

Peritoneal macrophage isolation

C57BL/6J, B6.MOLF-Ity, B6.MOLF-Ity/Ity3MOLF/B6, and B6.MOLF-Ity/Ity3 mice were injected i.p. with 1 ml of 3% thioglycolate, and the peritoneal exudates were collected 72 h later. The exudates were treated with RBC lysis buffer for 3 min, washed with PBS, and resuspended in RPMI 1640 and 10% FBS. The cells were counted and seeded in 96-well plates at a density of 2 x 105 cells/well. Adherent cells were selected for after 3 h, and the cells were then cocultured with 0 (for in vitro infection) or 20 U/ml IFN-{gamma} (for PMA stimulation) for 16 h.

Chemiluminescent lucigenin assay

Superoxide production was determined by measuring the chemiluminescence of macrophages exposed to 25 µM lucigenin (bis-N-methylacridinium; Sigma-Aldrich) using the LMax Microplate Luminometer (Molecular Devices). Peritoneal macrophages described above were washed with phenol red-free RPMI 1640 twice before stimulation with 0, 100 ng/ml PMA (Sigma-Aldrich), and 25 µM lucigenin. The experiment was conducted in triplicate with one negative control well in which 400 U/ml superoxide dismutase (Sigma-Aldrich) was added. The amount of chemiluminescence was measured at 0, 30, and 60 min, reading for 15-s intervals. Time 0 was considered to be the point at which lucigenin and PMA were injected into the wells. The induction of superoxide was calculated by dividing each time point by time 0, which was considered to be the baseline value. Data are represented as fold induction of superoxide over baseline values. Statistical significance was assessed using Student’s t test (p < 0.05).

In vitro Salmonella Typhimurium infectionf

One colony of Salmonella Typhimurium was grown in 5 ml of trypticase soy broth overnight at 37°C at 250 rpm. The following morning, the overnight culture was diluted 1/50 and grown to mid log phase (OD600 = 0.9), and was opsonized with 10% C57BL/6J serum for 30 min on ice. Four microliters of the opsonized Salmonella were added to each well of macrophages for a multiplicity of infection of 1:1. The plate of cells was centrifuged for 10 min at 1000 x g and then incubated at 37°C/5% CO2 for an additional 15 min. The cells were then washed twice with PBS and supplemented with RPMI 1640 containing 10% inactivated FBS and 100 µg/ml gentamicin. The cells were then incubated at 37°C/5% CO2 and assayed for superoxide with the addition of lucigenin, as described above, every 30 min for a period of 3 h. Bacterial invasion was tested by lysing the cells with 1% Triton X-100-PBS, and the lysates were plated for enumeration of CFUs.

Protein assay

Protein content was determined to verify the amount of cells in each well, using the DC Protein Assay (Bio-Rad), according to the manufacturer’s guidelines. The same wells of cells used for the lucigenin assay were used for the protein assays. These wells were washed twice with RPMI 1640 before adding 50 µl of lysis buffer. A total of 5 µl of each well was sampled in duplicate for protein analysis. The standard curve was determined by using 2-fold serial dilutions of 3 mg/ml standard BSA.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Re-evaluation of Ity3 QTL and creation of Ity3 congenics

The LOD score plot for Ity3 was reanalyzed with eight novel markers and 41 additional (C57BL/6J x MOLF/Ei)F2 mice for a total of 232 progeny (17) (Fig. 1). The peak LOD score of 4.1 was obtained at D1Mit218 and D1Mit100. In comparison with the original Ity3 QTL, the peak LOD score of 4.8 has decreased slightly probably due to the increased detection of recombinants; however, the region of interest still expands from D1Mit135 to D1Mit17. B6.MOLF-Ity/Ity3 congenics were intercrossed after five generations of backcrossing to B6.MOLF-Ity mice, and consequently have a genetic background that is ~96.9% C57BL/6J derived apart from the Ity and Ity3 loci being transferred from MOLF/Ei. The MOLF/Ei region surrounding Ity, including Slc11a1, was transferred because of the known effect of a wild-type allele at Slc11a1 in the detection of Ity3 (17). A schematic representation of the chromosomes of B6.MOLF-Ity/Ity3 mice is shown in Fig. 1. We have generated both B6.MOLF-Ity/Ity3 mice that are homozygous for the MOLF/Ei alleles and B6.MOLF-Ity/Ity3MOLF/B6 mice that are heterozygous for the Ity3 chromosomal region.

Ity3 congenic phenotype

To verify that the Ity3 susceptibility phenotype was being successfully transferred to the B6.MOLF-Ity/Ity3 congenics, both B6.MOLF-Ity/Ity3 and B6.MOLF-Ity/Ity3MOLF/B6 congenics, in addition to C57BL/6J mice that acted as infection controls, were infected i.v. with Salmonella Typhimurium (Fig. 2A). B6.MOLF-Ity/Ity3MOLF/B6 congenics were used in this experiment because B6.MOLF-Ity mice were unavailable at the time; however, they are predicted to be phenotypically identical with B6.MOLF-Ity mice because the Ity3 locus is inherited in a recessive fashion (17). As expected, C57BL/6J mice were extremely susceptible to infection and died within 5 days postinoculation (21). The transfer of the MOLF/Ei Ity interval, including the wild-type allele at Slc11a11 into a C57BL/6J background, improved survival to infection, as seen with the B6.MOLF-Ity/Ity3 strains. The survival analysis of these congenics demonstrates that the Ity3 locus has also been successfully transferred because mice that carry two copies of the susceptible MOLF/Ei allele, B6.MOLF-Ity/Ity3, succumb to infection significantly (p < 0.05) earlier (mean survival time (MST) = 8.4 ± 0.3 days) than those that carry only one MOLF/Ei allele at Ity3, B6.MOLF-Ity/Ity3MOLF/B6 (MST = 10.6 ± 0.9 days).


Figure 2
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  		FIGURE 2. Survival analysis of Ity3 congenic mice. Survival curves (A) of C57BL/6J, B6.MOLF-Ity/Ity3, and B6.MOLF-Ity/Ity3MOLF/B6 congenic mice following i.v. challenge with 3000 CFU of Salmonella typhimurium. The data are expressed as a percentage of total mice surviving the infection. Splenic bacterial load (B) of C57BL/6J, 129S6/SvEvTac, B6.MOLF-Ity/Ity3, and B6.MOLF-Ity/Ity3MOLF/B6 expressed in log (CFU/g) tissue.

 
The bacterial load in the spleens of C57BL/6J, 129S6/SvEvTac, B6.MOLF-Ity/Ity3, and B6.MOLF-Ity/Ity3MOLF/B6 mice was assessed at 1, 3, and 5 days after infection (Fig. 2B). C57BL/6J mice were found to have significantly higher CFUs in their spleens on days 3 and 5 (log 7–9 CFU/g) of infection, whereas 129S6/SvEvTac mice known to be extremely resistant to infection had relatively low CFU counts throughout infection (log 4–5 CFU/g). Both B6.MOLF-Ity/Ity3 and B6.MOLF-Ity/Ity3MOLF/B6 mice had intermediate splenic bacterial load ranging from log 4 to 6 CFU/g tissue, similar to that observed previously in MOLF/Ei mice (22). These results suggest that Ity3 does not influence bacterial proliferation in MOLF/Ei and that the lower bacterial load in the spleen of B6.MOLF-Ity/Ity3 mice compared with C57BL/6J could be explained by the presence of the MOLF/Ei Ity region (wild-type Slc11a1) (21).

Ncf2 sequence analysis

Because we were able to verify that the Ity3 QTL was impacting survival during Salmonella infection among our Ity3 congenics, we sought to identify candidate genes within the Ity3 interval (D1Mit135 to D1Mit17) that may explain this effect. We considered the 84-Mb Ity3 interval in its entirety, as it represents the 99% confidence interval in which the candidate gene(s) is located. In this light, we looked at all genes that map within this region with potential relevance to Salmonella infection such as Ptgs2, Ikbke, and Tlr5. All of these genes were excluded either based on sequence analysis (Ptgs2 and Ikbke) because no differences affecting the integrity of the proteins were observed within the coding regions or on a combination of expression and functional analyses (Tlr5) (23). We focused on Ncf2 based on its known function in related diseases in humans. The entire coding region of Ncf2 was sequenced in both C57BL/6J and MOLF/Ei. A total of eight sequence variants was found, two in the noncoding regions of the gene, and six within the coding region (Fig. 3A). The high rate of sequence variants found between C57BL/6J and MOLF/Ei is not surprising because these two strains of mice are separated by over 1 million years of evolution (24). On average, there are ~6 single-nucleotide polymorphisms/kb of coding sequence detected between these two strains of mice (V. Sancho-Shimizu and D. Malo, unpublished data). Of the six sequence variants identified in the coding region, only one, G1181A, resulted in a nonconservative amino acid change at codon 394 from an arginine to glutamine in MOLF/Ei mice (R394Q). To further determine the frequency and prevalence of the G1181A variant, DNAs from a diverse panel of 25 inbred strains of mice were genotyped for this variant (Fig. 4). The MOLF/Ei allele is present only in the Mus musculus molossinus family of mice (MOLF/Ei, MOLC/Rk, MOLD/Rk, MOLG/Dn, and MOLE/Rk), and in the European wild-derived strains Skive/Ei and CZECHII/Ei mice (Mus musculus musculus). The R394Q variant is located within a highly conserved and functional domain of the protein known as the phox Bemp1 (PB1) domain, which led us to investigate its potential impact on the integrity and function of the protein (Fig. 3B).


Figure 3
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  		FIGURE 3. Ncf2 sequence analysis. Nucleotide sequence variants (A) between C57BL/6J and MOLF/Ei mice. The bolded letters indicate the single-nucleotide polymorphism variants. Mouse Ncf2 protein domain structure (B) showing the position of the nonconservative mutation occurring in the PB1 domain. Domains known to interact with other phox proteins are indicated by an arrow. TPR, Tricopeptide repeat domain; SH3, Src homology 3 domain.

 

Figure 4
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  		FIGURE 4. Distribution and frequency of Ncf2 G1181A sequence variant. Twenty-seven inbred mouse strains were genotyped for the Ncf2 G1181A sequence variant corresponding to the nonconservative amino acid change. Mice are grouped according to their origin. G represents the C57BL/6J allele, and A represents the MOLF/Ei allele.

 
Ncf2 expression

The expression of Ncf2 was analyzed using real-time PCR on cDNAs of all uninfected and infected B6.MOLF-Ity/Ity3, B6.MOLF-Ity/Ity3MOLF/B6, as well as MOLF/Ei and C57BL/6J cDNAs (Fig. 5). MOLF/Ei mice have significantly reduced expression levels on day 3 postinfection in comparison with the C57BL/6J mice (Fig. 5A). Although not statistically significant, B6.MOLF-Ity/Ity3 mice had generally lower transcript levels during infection than the B6.MOLF-Ity/Ity3MOLF/B6 mice (Fig. 5B), suggesting a trend of reduced expression in the B6.MOLF-Ity/Ity3 congenics reflective of the MOLF/Ei allele.


Figure 5
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  		FIGURE 5. Regulation of Ncf2 expression upon Salmonella infection in the spleens of C57BL/6J, MOLF/Ei (A), and B6.MOLF-Ity/Ity3 (Ncf2MOLF/MOLF) (B) B6.MOLF-Ity/Ity3MOLF/B6 (Ncf2MOLF/B6) mice. Expression was evaluated by real-time PCR using C57BL/6J (A)- or B6.MOLF-Ity/Ity3MOLF/B6 (B) uninfected cDNA as the referent. All values are expressed as 2–{Delta}{Delta}Ct units, in which the referent is set to 1, and the housekeeping gene used was 18srRNA. Statistical significance was assessed using Student’s t test and is indicated by an asterisk (p < 0.05).

 
Ncf2 functional assay

Given the facts that susceptible B6.MOLF-Ity/Ity3 mice expressed lower levels of Ncf2 mRNA during infection, and that the sequence variant G1181A resulted in a nonconservative amino acid change occurring in a functional and highly conserved domain of the protein, we tested the ability of the B6.MOLF-Ity/Ity3, B6.MOLF-Ity/Ity3MOLF/B6, and B6.MOLF-Ity congenics that carry Ncf2 MOLF/Ei, heterozygous, and C57BL/6J alleles, respectively, to produce superoxides in peritoneal derived macrophages. The macrophages were stimulated with IFN-{gamma} for 16 h before the experiment. Superoxide production was induced by the addition of 100 ng/ml PMA. The functional assay shows that at 30 and 60 min after induction, C57BL/6J, B6.MOLF-Ity/Ity3MOLF/B6, and B6.MOLF-Ity cells have significantly higher levels of superoxide induction than the B6.MOLF-Ity/Ity3 cells (MOLF/Ei allele), with only 9.8- and 8.8-fold induction levels at the respective time points (Fig. 6A). Significantly reduced superoxide induction levels were consistently observed in the parent MOLF/Ei mice (data not shown). In fact, the levels of induction are identical in C57BL/6J, B6.MOLF-Ity, and B6.MOLF-Ity/Ity3MOLF/B6 cells, ~14-fold induction at 30 min and ~12-fold induction at 60 min. These results demonstrate that B6.MOLF-Ity/Ity3 mice have a less active (MOLF/Ei allele) Ncf2 allele than B6.MOLF-Ity/Ity3MOLF/B6, B6.MOLF-Ity, and C57BL/6J mice carrying the C57BL/6J allele at Ncf2.


Figure 6
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  		FIGURE 6. Functional assay for Ncf2 variants. Peritoneal-derived macrophages were isolated from C57BL/6J, B6.MOLF-Ity/Ity3 (Ncf2MOLF/MOLF), B6.MOLF-Ity/Ity3MOLF/B6 (Ncf2MOLF/B6), and B6.MOLF-Ity (Ncf2B6/B6). Macrophages were then either stimulated with 20 U/ml IFN-{gamma} and 100 ng/ml PMA (A) or infected with Salmonella typhimurium at a multiplicity of infection of 1:1 (B). Data are shown as fold increase in superoxide chemiluminescence over the baseline at time 0. These results represent one of four (A) or one of five (B) similar assays conducted on these mice. Statistical significance was assessed using Student’s t test.

 
We conducted the same superoxide assay on peritoneal macrophages that have been infected with Salmonella Typhimurium in vitro to assess the relevance of this functional difference. Consistent with the PMA-stimulated macrophages, we observed a consistent and statistically significant reduction in superoxide induction during Salmonella Typhimurium infection in vitro. The B6.MOLF-Ity/Ity3 mice have a less active (MOLF/Ei allele) Ncf2 allele than B6.MOLF-Ity/Ity3MOLF/B6, B6.MOLF-Ity, and C57BL/6J mice carrying the C57BL/6J allele at Ncf2 (Fig. 6B). The peak induction of superoxide occurred between 30 and 60 min after infection with Salmonella Typhimurium in vitro. The C57BL/6J mice had the highest induction levels of superoxide with a 4-fold increase, followed by B6.MOLF-Ity/Ity3MOLF/B6 and B6.MOLF-Ity, with 1.6- and 2.2-fold, respectively, and B6.MOLF-Ity/Ity3 mice with only 1.2-fold increase over baseline.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
We report in this study the successful transfer of the predicted susceptibility Ity3 locus from MOLF/Ei mice to B6.MOLF-Ity mice by creating B6.MOLF-Ity/Ity3 double-congenic mice. The Ity locus, which maps over Slc11a1 on chromosome 1, was transferred in addition to Ity3 because the initial genome scan identified Ity3 only in the presence of a wild-type Slc11a1 MOLF/Ei background (17). The role of Slc11a1, formerly known as Nramp1, in host response to infection is well characterized and is involved in suppressing the replication of various unrelated facultative intracellular pathogens such as Salmonella Typhimurium, Leishmania donovani, and Mycobacterium bovis bacillus Calmette-Guérin (21). C57BL/6J mice are known to carry a single point mutation in this gene (G169R), rendering them extremely susceptible to infection with Salmonella Typhimurium (MST ≤5 days). MOLF/Ei mice carry the functional wild-type allele for Slc11a1, which was transferred onto a C57BL/6J background in B6.MOLF-Ity, as well as in the B6.MOLF-Ity/Ity3 mice. The initial genome scan results indicated that the Ity3 locus was inherited in a recessive fashion, suggesting that two copies of the MOLF/Ei Ity3 region had to be inherited to have an impact on susceptibility to infection with Salmonella typhimurium (17).

Infection with Salmonella Typhimurium showed that B6.MOLF-Ity/Ity3 mice are indeed more susceptible, succumbing to infection earlier than their B6.MOLF-Ity/Ity3MOLF/B6 counterparts. The difference in survival between the C57BL/6J and B6.MOLF-Ity/Ity3 mice can be attributed almost entirely due to the presence of Ity, which includes the wild-type Slc11a1 allele, whereas the difference in survival between B6.MOLF-Ity/Ity3 and B6.MOLF-Ity/Ity3MOLF/B6 is specific to the Ity3 locus. Interestingly, the bacterial load of B6.MOLF-Ity/Ity3 and B6.MOLF-Ity/Ity3MOLF/B6 mice was intermediate between those of resistant 129S6/SvEvTac and the highly susceptible C57BL/6J mice, similar to bacterial load observations made in the parental MOLF/Ei mice (22). These results suggest that Ity3 does not have an impact on the spleen bacterial load during infection, and the difference observed between the congenic mice and the C57BL/6J mice is due to the effect of Ity, including Slc11a1, which is known to affect intracellular survival and replication of Salmonella Typhimurium (21).

Ncf2 was selected as a candidate gene for Ity3 based on its physical map position and its known function in innate immunity. Ncf2 encodes for p67phox, which is a subunit of the phagocyte NADPH oxidase. The function of the phagocyte NADPH oxidase in host defense is well established, as seen in CGD patients that present high susceptibility to bacterial and fungal infections (25). In human patients with CGD, mutations in the NCF2 gene account for an estimated 5% of all CGD cases (26). The majority of the 17 documented human NCF2 mutations results in no detectable superoxide production, with the exception of one that was found to have reduced levels of superoxides (26).

The MOLF/Ei Ncf2 allele carries a G to A transition at nt 1181, resulting in a nonconservative amino acid change (R394Q) at position 394. This variant is a rare polymorphism and observed only in the M. musculus molossinus strains as well as the European wild-derived inbred strains CZECHII/Ei and Skive/Ei, who share a common ancestor derived from the Mus musculus substrain (27). The R394Q variant was found to occur in a highly conserved and functional PB1 domain of Ncf2. This domain, initially identified in the budding yeast protein Bem1p, was shown to mediate the formation of heterodimers between p67phox and p40phox (28, 29). The interaction between p67phox and p40phox enhances the membrane recruitment of p67phox and p47phox to the flavocytochrome b558 complex, leading to the activation of the NADPH oxidase (30). It is possible that a substitution of a highly basic amino acid such as arginine with a polar glutamine could compromise the stability or activity of the protein.

This hypothesis is supported by the identification of a missense mutation in the corresponding amino acid (R395W) of the p67phox protein in two unrelated CGD patients (26, 31). In addition, recombinant p67phox carrying the R395W mutation was found to have only 15% of normal activity in a cell-free NADPH oxidase assay, suggesting that in homozygous form, this mutation has a profound impact on the function of the protein (31). The effect of the R395W mutation on p67phox-p40phox heterodimerization through PB1 domains was further demonstrated using GST pull down assays (32). The efficiency of the heterodimerization between p67phox R395W PB1 and p40phox PB1 was reduced by ~50% compared with wild-type p67phox (32). The R394Q variant appears also to have an effect on the activity of phagocyte NADPH oxidase. Our functional assay indicates that the substitution of the arginine at position 394 for a glutamine results in lower PMA- and Salmonella infection-induced levels of superoxide. It may be possible that a glutamine residue at position 394 of Ncf2 interferes with the dimerization with p40phox, resulting in an overall reduction in the recruitment of the p67phox and p47phox components to the membrane complex, and in reduced production of superoxides. Lower Ncf2 levels of expression may also participate in the decreased superoxide production observed in B6.MOLF-Ity/Ity3 mice. This notion is supported by complementation studies using recombinant human p67phox in neutrophils obtained from p67phox-deficient CGD patients, which found the level of p67phox protein expression to be the limiting factor in NADPH oxidase activation (33).

However, the biological relevance of how the low expression levels of Ncf2, the R395Q substitution, and the reduced superoxide induction affect the host response during Salmonella infection still remains in question. In terms of the biological contribution, the reduction in superoxide induction is probably not impacting antimicrobial activity significantly in our model because there was no difference in spleen bacterial load observed in Salmonella-infected B6.MOLF-Ity/Ity3 and B6.MOLF-Ity/Ity3MOLF/B6 mice, which harbor MOLF/Ei and heterozygous alleles, respectively, at Ncf2. In addition to its microbicidal activity, NADPH oxidase affects signaling capabilities of phagocytic cells. There is an emerging body of evidence indicating that reactive oxygen species (superoxide and H2O2) activate TLR4-dependent NF-{kappa}B gene transcription (15, 34, 35). The interaction of Nox4, a protein related to Nox2 (gp91phox of phagocytic cells), and TLR4 triggers the production of reactive oxygen species and leads to NF-{kappa}B activation in LPS-stimulated monocytic cell lines (35). Animals having deficient NADPH oxidase activity have impaired NF-{kappa}B activation and are more susceptible to infection with P. aeruginosa (14). Additional evidence for interaction between TLR signaling and NADPH oxidase activity was provided recently by showing that MyD88 influences NADPH oxidase assembly and NAPDH oxidase-mediated production of superoxides (36). The expression of several NF-{kappa}B-dependent genes, including IL-1, IL-6, and Nos2, was shown to be down-regulated in MOLF/Ei mice compared with C57BL/6J mice during infection, suggesting that the Ity3 locus may have an impact on the expression of several proinflammatory genes (17). To test the hypothesis that lower NADPH oxidase activity in MOLF/Ei mice may lead to improper NF-{kappa}B-dependent activation of several proinflammatory genes, resulting in increased Salmonella susceptibility of these animals, we have assayed for the presence of IL-1beta, IL-1 receptor antagonist, and IL-6 in the sera of infected B6.MOLF-Ity/Ity3 and B6.MOLF-Ity/Ity3MOLF/B6. We found no differences in cytokine levels between the two groups of congenic mice (data not shown), suggesting that the impaired superoxide induction in B6.MOLF-Ity/Ity3 does not affect the expression of these cytokines. However, it was not possible to completely rule out a possible role of a defective superoxide response on improper NF-{kappa}B activation using in vivo infection. Thus, additional and comprehensive in vitro experiments will be necessary to resolve this issue.

Taken together, these findings add weight to the candidacy of Ncf2 as the gene underlying Ity3; however, the final proof of the candidacy of Ncf2 will necessitate a gene-knockout interaction test (37). In addition, the resolution of the Ity3 interval into smaller regions with the aid of recombinant subcongenic mice will be instrumental in determining the extent of the involvement of Ncf2 in this model of infection and the genetic architecture of the Ity3 locus.


   Acknowledgments
 
We thank Rosalie Wilkinson and Line Lariviere for their excellent technical assistance. We thank Dr. Erwin Schurr, Amanda Rooyakers, Dr. Richard Stokes, and Dr. Albert Descoteaux for their helpful suggestions in the functional assay for superoxide detection.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by grants from the Canadian Institutes of Health Research and the Howard Hughes Medical Institute. D.M. is a William Dawson Scholar. Back

2 Address correspondence and reprint requests to Dr. Danielle Malo, Center for the Study of Host Resistance, Montreal General Hospital, Room L11-144, Montréal, Québec, Canada H3G 1A4. E-mail address: danielle.malo{at}mcgill.ca Back

3 Abbreviations used in this paper: CGD, chronic granulatomous disease; Ity, immunity to Typhimurium; LOD, logarithm of odds; MST, mean survival time; Ncf2, neutrophil cytosolic factor 2; PB1, Phox and Bem1; QTL, quantitative trait locus; Slc11a1, solute carrier family 11 member 1; Tlr4, TLR4. Back

Received for publication November 2, 2005. Accepted for publication March 9, 2006.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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