Role for Neutrophils in Host Immune Responses and Genetic Factors ...

INFECTION AND IMMUNITY, Sept. 2010, p. 3848–3860 0019-9567/10/$12.00 doi:10.1128/IAI.00044-10 Copyright © 2010, American Society for Microbiology. All Rights Reserved.

Vol. 78, No. 9

Role for Neutrophils in Host Immune Responses and Genetic Factors That Modulate Resistance to Salmonella enterica Serovar Typhimurium in the Inbred Mouse Strain SPRET/Ei䌤 Lien Dejager,1,2 Iris Pinheiro,1,2 Pieter Bogaert,1 Liesbeth Huys,1,2 and Claude Libert1,2* Department for Molecular Biomedical Research, VIB, B9052 Ghent, Belgium,1 and Department of Biomedical Molecular Biology, Ghent University, B9052 Ghent, Belgium2 Received 14 January 2010/Returned for modification 16 February 2010/Accepted 5 July 2010

Infection with Salmonella enterica serovar Typhimurium is a complex disease in which the host-bacterium interactions are strongly influenced by genetic factors of the host. We demonstrate that SPRET/Ei, an inbred mouse strain derived from Mus spretus, is resistant to S. Typhimurium infections. The kinetics of bacterial proliferation, as well as histological examinations of tissue sections, suggest that SPRET/Ei mice can control bacterial multiplication and spreading despite significant attenuation of the cytokine response. The resistance of SPRET/Ei mice to S. Typhimurium infection is associated with increased leukocyte counts in the circulation and enhanced neutrophil influx into the peritoneum during the course of infection. A critical role of neutrophils was confirmed by neutrophil depletion: neutropenic SPRET/Ei mice were sensitive to infection with S. Typhimurium and showed much higher bacterial loads. To identify genes that modulate the natural resistance of SPRET/Ei mice to S. Typhimurium infection, we performed a genome-wide study using an interspecific backcross between C3H/HeN and SPRET/Ei mice. The results of this analysis demonstrate that at least two loci, located on chromosomes 6 and 11, affect survival following lethal infection with S. Typhimurium. These two loci contain several interesting candidate genes which may have important implications for the search for genetic factors controlling Salmonella infections in humans and for our understanding of complex hostpathogen interactions in general. exert important regulatory functions during this phase. They contribute to the activation of a specific immune response through their specialized pathogen recognition receptors (PRRs), which detect conserved motifs (pathogen-associated molecular patterns, or PAMPs) of Gram-negative bacteria, such as lipopolysaccharide (LPS) and flagellin (7, 12). Recognition of these PAMPs by the host cells triggers the production of several cytokines, including tumor necrosis factor (TNF) and gamma interferon (IFN-␥) (32). These cytokines cause further recruitment of phagocytes and activation of their microbicidal capacity (31), and they are also responsible for the establishment and maintenance of the plateau (third) phase in bacterial proliferation (27–29). Mice eventually acquire specific immunity during the final phase of infection through the activation of B and T lymphocytes, which leads to resolution of the infection (15, 37). Studies using experimental animal models of infection have shown that susceptibility to Salmonella infection is under complex genetic control (reviewed in reference 46). Several critical innate immune genes that influence the course of infection have been identified using different genetic approaches, such as positional cloning of spontaneous mouse mutations, congenic mouse strains, targeted disruption of candidate genes, and quantitative trait locus (QTL) mapping (46). Among the best-characterized genes involved in early control of bacterial replication are Slc11a1 (64), Tlr4 (41), Nos2 (49), and Nadph oxidase (31, 53). These genes are critical for host defense, and mouse strains lacking one of these genes are highly susceptible to infection with S. Typhimurium. Examples of susceptible mouse strains are C57BL/6 and C3H/HeJ mice, which are deficient for Slc11a1 (63) and Tlr4 (40), respectively. In addi-

Salmonella species are facultative intracellular Gram-negative bacteria found ubiquitously in nature. They cause a spectrum of human infections, including enteric typhoid fever and a self-limiting gastroenteritis (5). Nontyphoidal Salmonella infections, called salmonellosis, are caused by Salmonella enterica serovar Typhimurium. In contrast to the human clinical manifestations of the disease, S. Typhimurium infection in the mouse causes a systemic disease resembling human typhoid fever with respect to pathology and host response (48). For this reason, S. Typhimurium infection is often used as a model system for studying systemic Salmonella infections. It has been a valuable model for understanding the pathogenesis of Salmonella and the host defense mechanisms involved in immune responses to S. Typhimurium. Four distinct phases in the progression of systemic infection have been defined. The first phase involves rapid clearance of the majority of the bacteria from the bloodstream by macrophages and neutrophils (8). These innate immune cells play a critical role in host defense mechanisms against invading pathogens because they are rapidly recruited to the infection site. They phagocytose the invading bacteria and exhibit potent microbicidal activity mediated by antimicrobial peptides, lysosomal enzymes, and reactive oxygen and nitrogen species (10, 31). The second phase of infection is characterized by exponential bacterial growth in the spleen and the liver (the reticuloendothelial system, or RES) (30). Again, macrophages and neutrophils

* Corresponding author. Mailing address: DMBR, VIB and Ghent University, Technologiepark 927, B9052 Ghent, Belgium. Phone: 329-3313700. Fax: 32-9-3313609. E-mail: [email protected]. 䌤 Published ahead of print on 19 July 2010. 3848

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tion, other loci, which act during the late phase of infection through their effects on the acquired immune response, have been revealed (46). In this article, we show that a wild-derived inbred strain of mice, Mus spretus SPRET/Ei, is resistant to infection with Salmonella enterica serovar Typhimurium, as evidenced by prolonged survival and a continuously lower bacterial load. We attribute this protective effect to (i) strongly improved clearance of bacteria from the circulation and peritoneum during the early phase of infection and (ii) an increased capacity to recruit neutrophilic granulocytes in the peritoneum during infection. An important role for neutrophils was demonstrated by depleting polymorphonuclear leukocytes, which sensitized SPRET/Ei mice to S. Typhimurium infection. These data demonstrate that, despite attenuation of the cytokine response, SPRET/Ei mice show enhanced resistance to infection with S. Typhimurium due to improved functions of the innate immune system. To identify the genes conferring the early-phase defense mechanisms during S. Typhimurium infection in SPRET/Ei mice, we made use of the high genetic diversity between Mus musculus and Mus spretus, arising from the great phylogenetic distance between them (9). Thus, by exploiting the S. Typhimurium resistance of SPRET/Ei mice, as well as the unique genetic variability they represent, we set up an informative backcross between the sensitive C3H/HeN strain and the highly resistant SPRET/Ei strain to map the genetic factors modulating resistance to lethal S. Typhimurium infection. The results of this genome-wide study demonstrate that at least two loci, on chromosomes 6 and 11, affect resistance in SPRET/Ei mice. This linkage analysis provides important information about the inheritance of resistance to S. Typhimurium infection in SPRET/Ei mice and paves the way to investigate the role of each of the underlying genes. MATERIALS AND METHODS Mice. C57BL/6 mice were purchased from Janvier (Le Genest-St. Isle, France), and C3H/HeJ and C3H/HeN mice from Harlan (Oxon, United Kingdom). SPRET/Ei mice were obtained from The Jackson Laboratories (Bar Harbor, ME) and bred in our facility. The mice were kept in individually ventilated cages under a constant dark-light cycle in a conventional animal house and received food and water ad libitum. All mice were used at the age of 8 to 12 weeks. Animal experiments were approved by the institutional ethics committee for animal welfare of the Faculty of Sciences, Ghent University, Belgium, and performed according to its guidelines. Experimental infection with S. Typhimurium in mice. Pathogenic Salmonella enterica serovar Typhimurium strain LMG3264 was purchased from the Belgian Coordinated Collections of Microorganisms (BCCM). Bacteria were cultured overnight in Luria-Bertani (LB) broth (Becton Dickinson, Benelux) at 37°C with gentle rotation. Aliquots of 1 ⫻ 1014 viable bacteria/ml were stored in 50% glycerol at ⫺80°C. Just before use, the aliquots were thawed and diluted in pyrogen-free physiological saline. All mice were infected intraperitoneally (i.p.) with 0.25 ml of saline containing 1 ⫻ 107 CFU of bacteria. Survival was assessed daily for 21 days, and the degree of resistance was established by monitoring day of death. Notably, because we inoculate mice i.p., we directly induce a systemic Salmonella infection, which bypasses the gut phase of pathogenesis. Cytokine measurements. On day 5 after inoculation, blood was collected from the retro-orbital plexus and allowed to clot overnight at 4°C. Serum was prepared by centrifugation at 20,000 ⫻ g for 4 min, and the supernatants were used for the determination of cytokines. Serum samples were assayed for several cytokines using Luminex technology (Bio-Rad, Narazeth-Eke, Belgium) following the manufacturer’s protocol. Q-PCR analysis. Mice were sacrificed by cervical dislocation 5 days after inoculation, and spleens and livers were collected in RNAlater (Qiagen). RNA was isolated with an RNeasy mini kit (Qiagen) according to the manufacturer’s instructions. The RNA concentration was measured with a Nanodrop 1000

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(Thermo Scientific), and 1 ␮g of RNA was used to prepare cDNA with Superscript II (Invitrogen). Quantitative real-time PCR (Q-PCR) was performed using a Roche LightCycler 480 system (Applied Biosystems). The best-performing housekeeping genes were determined by Genorm for each organ (60). The results are given as relative expression values normalized to the geometric mean of the expression of the housekeeping genes. Enumeration of bacterial load. At different time points after infection with S. Typhimurium, mice were killed, and blood was obtained from the retro-orbital plexus and immediately diluted in sterile phosphate-buffered saline (PBS) to prevent clotting. In addition, the peritoneal cavity was washed with 10 ml of sterile cold PBS. The spleen, liver, lung, and colon were aseptically removed, weighed, and placed in sterile cold PBS (2 ml/mg liver tissue and 10 ml/mg spleen, lung, and colon tissue). The organs were mechanically homogenized, and serial dilutions of homogenates (at least three) were prepared in sterile PBS for plating on LB plates in duplicate. Blood and peritoneal lavage fluid samples were also diluted in sterile PBS and plated onto LB agar plates in duplicate. Plates were incubated at 37°C overnight, and the colonies were counted the following day. Viable counts of S. Typhimurium were expressed as log10 CFU per organ or as log10 CFU/ml blood or peritoneal lavage fluid. The results for each group were expressed as geometric means with standard deviations. Histological analysis and determination of apoptosis in tissue sections. Liver and spleen samples were fixed in 4% paraformaldehyde for at least 24 h and then transferred to 70% ethanol until they were processed for paraffin embedding. The samples were sectioned at 5 ␮m, stained with hematoxylin and eosin using standard techniques, and examined by light microscopy. Inflammatory lesions formed by leukocyte infiltration are considered foci of infection. Liver and spleen sections were also used to perform immunohistochemistry (IHC). Sections were deparaffinized for antigen retrieval, followed by rehydration. For neutrophil and macrophage staining, we applied a target retrieval solution (Dako) or a proteinase K treatment (10 ␮g/ml; Promega) in proteinase K buffer (100 mM Tris-HCl, pH 8.0, and 50 mM EDTA), respectively. Next, organ sections were blocked with a peroxidase blocking buffer (Dako) and, subsequently, with swine or rabbit serum (Dako) to avoid aspecific binding. This was followed with a first incubation step (overnight at 4°C) using anti-MPO (Dako) and anti-F4/80 (AbD Serotec) antibodies for staining of neutrophils and macrophages, respectively. Biotinconjugated secondary IgGs (Dako), the Vectastain ABC kit (Vector Laboratories), and AEC (3-amino-9-ethylcarbazole; Dako) were used for visualization. The slides were counterstained with hematoxylin solution (Sigma-Aldrich). Negative controls were made by substituting buffer for the primary antibody. The terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick endlabeling (TUNEL) assay was used to detect apoptotic cells in the liver. An in situ cell death detection kit was obtained from Roche Diagnostics (Indianapolis, IN) and used according to the manufacturer’s instructions. Briefly, the slides were deparaffinized by placing them twice in xylene for 3 min and then hydrated with 100% ethanol (twice for 3 min), 90% ethanol (once for 1 min), and 70% ethanol (once for 1 min) and rinsed in distilled water. The sections were then stained with the TUNEL reaction mixture for 1 h at 37°C in a humidified chamber. The reaction was stopped by rinsing the slides in stop wash buffer for 10 min. Finally, the slides were incubated with fluorescein isothiocyanate (FITC)-avidin D for 30 min at room temperature. After counterstaining with DAPI for 20 min, positive (stained brownish-yellow) cells were counted in 10 random fields at 40⫻ magnification in a double-blinded fashion. Differential cell counts in peritoneum and circulation. Under terminal avertin anesthesia, blood was collected from mice by cardiac puncture and peritoneal lavage was performed with 10 ml of ice-cold phosphate-buffered saline. Blood was collected in EDTA-coated tubes (Sarstedt, Germany) to prevent clotting. Cells were counted with an XE-2100L hematology analyzer (Sysmex). Differential cell counts in peritoneal lavage were determined microscopically after MayGru ¨nwald-Giemsa staining (Sigma-Aldrich, St. Louis, MO) of cytospin preparations. Analysis of spleen cell suspensions by flow cytometry. The liver and spleen were removed after inoculation, and single-cell suspensions (10 ml Dulbecco’s modified Eagle’s medium [DMEM] plus 10% fetal calf serum [FCS] for liver and 10 ml PBS for spleen) were obtained by thorough pipetting, followed by passage through nylon cell strainers (70 ␮m; VWR International, Leuven, Belgium) to remove tissue debris. The spleen cell suspensions were treated with 0.17 M NH4Cl (pH 7.4) for 5 min to lyse red blood cells, and the cells were washed three times in PBS and centrifuged at 300 ⫻ g for 5 min at 4°C. Living splenocytes were counted by trypan blue exclusion, and the final concentration was adjusted to 5 ⫻ 106 cells/ml in PBS. The liver cell suspensions were centrifuged at 300 ⫻ g for 5 min, resuspended in 10 ml DMEM (Invitrogen, Belgium) supplemented with 10% fetal calf serum, and overlaid onto a Percoll gradient. They were centrifuged at 800 ⫻ g for 30 min at 4°C, and the mononuclear cells were collected. For flow

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cytometric analysis, splenocytes and hepatic leukocytes were incubated with combinations of monoclonal antibodies (MAbs). The Fc␥R was blocked by preincubation of cells with anti-CD16/CD32 MAb (clone 2.4G2; kindly provided by J. Unkeless, Rockefeller University, New York, NY) to avoid aspecific binding. MAbs were anti-CD11c (allophycocyanin [APC] conjugated, clone HL3; BD Pharmingen, CA), anti-CD11b (FITC conjugated, clone M1/70; ATCC, Rockville, MD), and anti-Gr1 (biotin conjugated, clone Rb6-8C5; kindly provided by B. Fazekas de St. Groth, Sydney, Australia). The second-step reagent used to reveal biotin-conjugated anti-Gr1 was streptavidin-phycoerythrin (BD Pharmingen). The cells were analyzed using a FACSCalibur with the Cell Quest software program (BD Immunocytometry Systems, CA) for data acquisition and analysis. Depletion of polymorphonuclear leukocytes (PMNs) and macrophages. Mice were depleted of neutrophils by i.p. injection of cyclophosphamide (SigmaAldrich, Saint Louis, MO) or anti-Ly-6G antibody (clone RB6-8C5; Bio-Express, United States). Mice were injected with 3 mg cyclophosphamide 1 day before infection and with 2 mg cyclophosphamide on days 2, 5, and 8 after S. Typhimurium infection. For depletion using the anti-Ly-6G antibody, mice received 0.3 mg antibody 3 days and 1 day before and on days 1, 3, 5, and 9 after infection. To study the role of macrophages, liver and spleen macrophages were depleted by treatment with liposome-encapsulated clodronate (Nico Van Rooijen, Free University Medical Center, Amsterdam, Netherlands) as previously described (61). Mice were injected with 200 ␮l of the clodronate liposome or control solution 2 days before infection and on days 3 and 8 after S. Typhimurium infection to remove newly immigrating macrophages. Depletion was verified 1 day after inoculation by differential white blood cell counts of blood smears stained with May-Gru ¨nwald-Giemsa and by flow cytometric analysis of splenocytes (see previous section). Bacterial load was determined on day 5 postinfection, and survival was monitored for 21 days. Genetic linkage analysis. To map the loci responsible for the resistance of SPRET/Ei mice to infection with S. Typhimurium, an interspecific backcross between female (C3H/HeN ⫻ SPRET/Ei)F1 mice and male C3H/HeN mice was set up. One hundred eighty-nine backcross mice were infected with 1 ⫻ 107 CFU of S. Typhimurium bacteria. A genome scan was conducted using approximately 80 microsatellite markers. Coverage of the genome was estimated by taking the positions of the marker loci on the Mouse Genome Database (MGD) mouse genetic map obtained from http://www.informatics.jax.org (The Jackson Laboratory, Bar Harbor, ME) and applying a swept radius of 20 centimorgans (cM) (54). After the first screening, the density of markers was increased in the regions of the linked loci. PCR was performed on 100 ng of tail genomic DNA. Survival and genotyping data were analyzed by using the R/QTL software, version 1.0712, running under R 2.6.0 (4). The survival rate was analyzed as a binary trait. The significance levels of the logarithm of the odds (LOD) scores were determined by performing a permutation test (10,000 permutations) of the experimental data and were 2.69 and 2.39 for the 5% and 10% thresholds, respectively. In addition, we analyzed the locus-specific linkage of all genetic markers using the MapManager QTL program. A single-locus-association test was performed to evaluate the association of trait values with the genotypes of single loci, expressed as likelihood ratio statistics (LRS) values. Statistics. Survival curves (Kaplan-Meyer plots) were compared by a log-rank test, and final outcomes by a chi-square test. Data are expressed as means ⫾ standard errors of the means (SEM). The statistical significance of differences between groups was evaluated with Student’s t tests with 95% confidence intervals and with one-way or two-way analysis of variance (ANOVA). Error bars in the figures represent means ⫾ SEM, and single, double, and triple asterisks represent P values of ⬍0.05, ⬍0.01, and ⬍0.001, respectively.

RESULTS SPRET/Ei mice are resistant to S. Typhimurium infections. As has been described before (48), we found that C57BL/6 and C3H/HeJ mice are very susceptible to S. Typhimurium infections. All mice died within 8 days after infection with 107 CFU S. Typhimurium per mouse, which was significantly earlier than the C3H/HeN mice (died within 12 days), which carry normal Slc11a1 and Tlr4 alleles (Fig. 1). In comparison to the common laboratory inbred strains, wild-derived inbred mouse strains provide a great amount of additional genetic and phenotypic variation (9). We previously reported that SPRET/Ei, an inbred strain derived from Mus spretus, is extremely resistant to the lethal effects of LPS (25). Because LPS is a major

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FIG. 1. SPRET/Ei mice are very resistant to infection with S. Typhimurium. Survival of mice after i.p. injection of 107 CFU S. Typhimurium bacteria is shown. Mortality was monitored for 21 days (no further deaths occurred) in groups of C57BL/6 mice (f, n ⫽ 5), C3H/HeJ mice (, n ⫽ 4), C3H/HeN mice (Œ, n ⫽ 10), and SPRET/Ei mice (E, n ⫽ 10). Significance was calculated for the difference between all mouse strains and the SPRET/Ei strain, unless otherwise noted; P values are indicated as described in Materials and Methods.

PAMP of Salmonella and induces the innate immune response upon infection, we wondered whether these wild-derived mice are also less susceptible to infection with S. Typhimurium. Notably, DNA sequencing of the Slc11a1 gene from SPRET/Ei mice previously revealed that it carries the resistance-associated allele (26). Therefore, we used C3H/HeN mice as controls in all experiments described herein. All SPRET/Ei mice survived systemic challenge with 107 CFU of S. Typhimurium. SPRET/Ei mice succumbed to the infection only when injected with 100-fold more bacteria, and even then they died much later than the C3H/HeN mice. In addition, SPRET/Ei mice showed no symptoms of disease during the course of infection, i.e., no weight loss, piloerection, or apathy. The strong resistance of SPRET/Ei mice suggests that strong protective immune factors are involved. In addition, F1 hybrids are also resistant to S. Typhimurium infections, suggesting a dominant mode of inheritance for SPRET/Ei-derived resistance alleles. SPRET/Ei mice show reduced bacterial loads and fewer inflammatory lesions in the RES. To further investigate the extreme resistance of SPRET/Ei mice to S. Typhimurium, SPRET/Ei mice and S. Typhimurium-sensitive C3H/HeN mice were infected i.p. with 107 CFU of S. Typhimurium. Five days after infection, four mice from each group were sacrificed and several tissues and blood were collected. The numbers of CFU in each organ were determined by plating serial dilutions. As expected, the susceptible control mice showed large numbers of bacteria in all organs tested and in the circulation, whereas the resistant SPRET/Ei mice contained significantly smaller amounts of bacteria. In addition, in half of the SPRET/Ei mice, bacteria were undetectable in the blood (Fig. 2A). To gain further insight into the progression of S. Typhimurium infection, four mice from each mouse strain were sacrificed at different time points early after infection, and the bacterial loads in the peritoneum and liver were assessed. In C3H/HeN mice, soon after infection, there were drastic increases in the numbers of S. Typhimurium bacteria in the liver, reaching 1 ⫻ 107 CFU on day 6. In the peritoneal cavity, a high bacterial burden was visible early postinfection and the CFU counts increased during infection (Fig. 2B). These data suggest that the sensitivity of C3H/HeN mice is associated with a failure to

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FIG. 2. SPRET/Ei mice exhibit lower bacterial loads and reduced inflammatory lesions upon S. Typhimurium infection. (A) Bacterial counts in the circulation, RES organs (liver and spleen), lung, and colon of C3H/HeN mice (black bars, n ⫽ 4) and SPRET/Ei mice (white bars, n ⫽ 4) 5 days after infection with 107 CFU of S. Typhimurium. (B) Kinetics of bacterial counts in liver and peritoneum of C3H/HeN (Œ, n ⫽ 4) and SPRET/Ei (E, n ⫽ 4) mice. (C) Histology (hematoxylin-eosin staining) of S. Typhimurium-induced lesions in the liver and spleen of C3H/HeN and SPRET/Ei mice 5 days after infection. Infiltration of leukocytes in the RES of C3H/HeN mice is indicated by arrows. These photographs are representative pictures. Bars, 50 ␮m. (D) Numbers of apoptotic cells in livers of C3H/HeN (black bar, n ⫽ 5) and SPRET/Ei (white bar, n ⫽ 5) mice determined by TUNEL staining 5 days after S. Typhimurium infection. Stained cells were counted in 10 separate fields. Significances were calculated for differences between C3H/HeN and SPRET/Ei mice. Error bars are as described in Materials and Methods.

control bacterial replication in target organs. In contrast, the rise of bacterial loads was less pronounced in SPRET/Ei mice and the bacterial numbers were at all times, and already 6 h after infection, significantly lower than those in C3H/HeN mice (Fig. 2B), which suggests that SPRET/Ei mice are better at clearing the bacteria during the first phase of infection. The ability of the SPRET/Ei mice to prevent uncontrollable multiplication of S. Typhimurium bacteria during the first week of infection and the subsequent establishment of a plateau phase correlate with the strong resistance of these mice to S. Typhimurium infection. To study the activation of the innate immune response in the different hosts during infection, we examined the histology of the major sites of S. Typhimurium replication, i.e., the spleen and the liver, after 5 days of infection. As expected, infection led to splenomegaly, but this was more prominent in C3H/HeN mice than in the resistant SPRET/Ei mice (data not shown). Microscopically, inflammatory reactions marked by infiltration of macrophages and polymorphonuclear leukocytes were evident in organs of the sus-

ceptible C3H/HeN mice. These leukocytes were grouped in numerous cellular aggregates, called “typhoid inflammatory nodules.” Furthermore, the red pulp in the spleen was disrupted and the white pulp was markedly expanded (Fig. 2C). In the liver, degeneration of hepatocytes was observed throughout the parenchyma and was confirmed by TUNEL staining of liver tissue sections. Five days after infection, apoptotic liver cells were numerous in C3H/HeN mice but significantly less numerous in SPRET/Ei mice (Fig. 2D). Thus, the inability of C3H/HeN mice to destroy this pathogen allows S. Typhimurium to mount an overwhelming, systemic bacterial infection that induces tissue injury and culminates in the death of the host. SPRET/Ei mice, however, showed only mild signs of inflammation, demonstrating their ability to effectively control S. Typhimurium infection. Attenuated cytokine production in SPRET/Ei mice in response to S. Typhimurium. Proinflammatory cytokines are essential for the activation of host immune defenses against S. Typhimurium and, thus, determine the outcome of S. Typhi-

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FIG. 3. Attenuated cytokine response in SPRET/Ei mice following S. Typhimurium infection. Cytokine levels in the circulation (A) and cytokine mRNA levels in liver (B) and spleen (C) of C3H/HeN (black bars, n ⫽ 4) and SPRET/Ei (white bars, n ⫽ 4) mice on day 5 following infection with S. Typhimurium are shown. Significance was calculated for differences between C3H/HeN and SPRET/Ei mice.

murium infection (20). We therefore investigated the cytokine levels in the circulation and in the RES 5 days after infection. Determination of the serum levels of representative proinflammatory cytokines, such as interleukin-6 (IL-6), TNF, IFN-␥, and IL-1␤, showed that the cytokine levels were significantly lower in SPRET/Ei mice than in the control C3H/HeN mice (Fig. 3A). The expression levels of all cytokines in the liver and spleen of SPRET/Ei mice were also much lower (Fig. 3B and C). Thus, the enhanced innate immune response of SPRET/Ei mice against a lethal S. Typhimurium infection cannot be explained on the basis of levels of proinflammatory cytokines. As mentioned in the introduction, proinflammatory cytokines are not involved in the initial control of bacterial growth. Therefore, we studied whether SPRET/Ei mice display an increased ability to restrict S. Typhimurium multiplication during the first phase of infection. SPRET/Ei mice display increased leukocyte accumulation and accelerated neutrophil recruitment in the peritoneum. Proinflammatory cytokines, such as TNF and IFN-␥, play important roles in the recruitment and activation of macrophages and neutrophils to the RES, which results in suppression of the proliferation of the invading pathogen during the third phase of Salmonella infection (31). Since we demonstrated that cytokine levels are less upregulated upon S. Typhimurium infection in SPRET/Ei mice than in C3H/HeN mice, we wondered whether SPRET/Ei mice display an infiltration of immune cells in the RES after infection with S. Typhimurium. We found that the recruitment of macrophages and neutrophils was significantly lower in the spleens and livers of SPRET/Ei mice than in those of the C3H/HeN mice (Fig. 4A, spleen sections show similar results). The lower number of macrophages observed in SPRET/Ei mice was confirmed by flow cytometric analysis (data not shown). These results are consistent with their lower cytokine response and reduced inflammatory lesions in the RES. In addition to their cytokine-dependent role in the RES, macrophages and neutrophils are also responsible for clearing most of the S. Typhimurium organisms during the initial phase of infection (8). To investigate the contribution of these leukocytes in the early immune response to S. Typhimurium, we performed differential cell counts at several time points after the initiation of infection. First, we determined the total numbers of leukocytes and erythrocytes in the circulation. The counts of white blood cells (WBC) and red blood cells (RBC) in uninfected C3H/HeN mice were within the accepted norms.

However, uninfected SPRET/Ei mice displayed a much lower WBC count and a significantly higher RBC count than C3H/ HeN mice. These findings correspond to data registered in the Mouse Phenome Database (http://www.jax.org/phenome). Upon S. Typhimurium infection, the number of WBC increased rapidly in SPRET/Ei mice, whereas the leukocyte counts in the circulation of C3H/HeN mice dropped rapidly. Later, the WBC counts remained stable in both mouse strains but were higher in SPRET/Ei mice than in C3H/HeN mice (Fig. 4B). Both mouse strains developed anemia following infection, as demonstrated by the lower RBC counts on day 4 postinoculation, but the counts remained higher in SPRET/Ei mice (Fig. 4B). It is known that in response to infection, there is a steady accumulation of leukocytes in the peritoneum, which is mainly due to an influx of PMNs. In uninfected animals, neutrophils make up ⬍1% of the total peritoneal cell population. Therefore, we assessed the number and composition of peritoneal leukocytes subsequent to S. Typhimurium infection. The number of peritoneal leukocytes increased in response to S. Typhimurium infection in both mouse strains (data not shown), but the percentage of neutrophils was significantly higher in SPRET/Ei mice than in C3H/HeN mice. This difference in the percentage of PMNs was visible 24 h postinfection and remained during the course of the infection (Fig. 4C). However, the percentages of the monocyte/macrophage subpopulations were comparable in both groups of mice at all time points tested after infection (Fig. 4C). It has been reported that neutrophil recruitment and activation play a central role in innate immune defense to S. Typhimurium because neutrophil depletion abrogates the mouse’s ability to mount an adequate defense (51). Therefore, we hypothesize that enhanced neutrophil recruitment might contribute to the reduced bacterial loads observed in SPRET/Ei mice and, as a consequence, it might explain the strong resistance of SPRET/Ei mice. The ability of SPRET/Ei mice to mount an effective neutrophil response in the early phase of infection might be explained by increased levels of neutrophil chemoattractants, such as Cxcl1 (KC) and Cxcl2 (Mip2). Therefore, the levels of these chemokines were measured at different time points early during the course of infection. This analysis demonstrated that SPRET/Ei mice displayed significantly higher levels of KC in peritoneal exudates and serum 6 h postinfection (Fig. 4D). However, the levels of Mip2 did not differ between the two mouse strains (data not shown). This suggests that the in-

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FIG. 4. Accumulation of circulatory leukocytes and influx of neutrophils after infection with S. Typhimurium. (A) IHC-stained liver sections from Salmonella-infected (4 days postinfection) C3H/HeN and SPRET/Ei mice. Sections were stained with anti-MPO and anti-F4/80 antibodies to visualize neutrophils and macrophages (brownish colored), respectively. Representative pictures are shown, and IHC was performed two times. (B) Numbers of white blood cells (left) and red blood cells (right) in the circulation of C3H/HeN (Œ, n ⫽ 4) and SPRET/Ei (E, n ⫽ 3) mice at different times following infection with S. Typhimurium. (C) Influx of neutrophils and macrophages into the peritoneum expressed as percentage of total peritoneal leukocytes in C3H/HeN (Œ, n ⫽ 5) and SPRET/Ei (E, n ⫽ 4) mice. (D) Levels of the neutrophil chemoattractant Cxcl1 (KC) in the serum and peritoneal fluids of C3H/HeN (Œ, n ⫽ 3) and SPRET/Ei (E, n ⫽ 3) mice at different time points during the early phase of infection. Significance was calculated for differences between C3H/HeN and SPRET/Ei mice.

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FIG. 5. SPRET/Ei resistance to S. Typhimurium is dependent on neutrophils. Survival of C3H/HeN mice (n ⫽ 6 for each group) (A) and SPRET/Ei mice (n ⫽ 4 for each group) (B) is shown. Untreated (f and 䡺), cyclophosphamide-treated (Œ and ‚), neutrophil-depleted ( and ƒ), and macrophage-depleted (F and E) mice from both mouse strains were infected with 107 CFU S. Typhimurium bacteria. Mortality was monitored for 14 days (no further deaths occurred). Significance was calculated for differences between depleted conditions and the nondepleted control condition for each mouse strain. (C) Bacterial loads in the circulation 4 days after inoculation of C3H/HeN (black bars, n ⫽ 4) and SPRET/Ei (white bars, n ⫽ 4) mice either not depleted or depleted of macrophages or neutrophils. Significance was calculated for differences between both mouse strains, unless otherwise noted. NS, not significant.

creased neutrophil influx observed in SPRET/Ei mice might be caused by the elevated levels of KC. Neutrophils are critically involved in the S. Typhimurium resistance of SPRET/Ei mice. It is conceivable that local accumulation of PMNs in the peritoneal cavity leads to the early inactivation of bacteria and is responsible for the strong resistance of SPRET/Ei mice to S. Typhimurium infection. To assess the contribution of neutrophils to the prolonged survival of SPRET/Ei mice, neutrophils were first depleted by i.p. injection of the cytotoxic drug cyclophosphamide. We showed that the mice were effectively neutropenic 1 day after the first injection and, thus, also when they were inoculated with S. Typhimurium by performing differential white blood cell counts in blood smears stained with May-Gru ¨nwald-Giemsa. To ensure the maintenance of a neutropenic state, the mice were injected repeatedly with cyclophosphamide; however, macrophages also seemed to be depleted after several treatments (data not shown). The resistance to S. Typhimurium was completely abrogated in leukopenic SPRET/Ei animals (Fig. 5B), suggesting that leukocytes, most likely neutrophils, are required for their strong resistance. To further assess the involvement of neutrophils, we specifically depleted neutrophils by treatment with an anti-Ly-6G (clone RB6-8C5) antibody (51). This was validated by differential white blood cell counts in blood smears stained with May-Gru ¨nwald-Giemsa and by

flow cytometric analysis of splenocytes 1 day after inoculation (data not shown). The neutrophil-depleted SPRET/Ei mice became sensitive to S. Typhimurium infection, and all mice died within 11 days (Fig. 5B), strongly suggesting that the resistance of SPRET/Ei mice is dependent on neutrophils. The control C3H/HeN mice were also sensitized to S. Typhimurium upon neutrophil depletion, but not significantly: they all died within 9 days (Fig. 5A). In addition, high bacterial loads in the circulation were detected in neutrophil-depleted SPRET/Ei mice 4 days after infection, reaching levels similar to the levels in C3H/HeN mice, in contrast to the significant difference in the numbers of bacteria present in C3H/HeN and SPRET/Ei mice after macrophage depletion (Fig. 5C). Since neutropenic SPRET/Ei mice died a few days later than the C3H/HeN mice (not significantly), macrophages might also be involved in the basic resistant phenotype. To examine the role of macrophages in the SPRET/Ei phenotype, they were depleted with liposome-encapsulated clodronate, which selectively accumulates in macrophages and induces their apoptosis (61). Following successful macrophage depletion, most of the infected SPRET/Ei mice died (3 mice out of 4) (Fig. 5B) but significantly later than the macrophage-depleted C3H/HeN mice (P ⫽ 0.0175), which succumbed early after infection (Fig. 5A). Altogether, these results clearly show that infection with S. Typhimurium is more severe in neutropenic than in immuno-

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competent SPRET/Ei mice. This indicates that neutrophils are a critical component of the resistance of SPRET/Ei mice, although a role for macrophages cannot be excluded. The SPRET/Ei strain resistance to S. Typhimurium is a complex and multigenic trait. It has been known for a long time that the host response to Salmonella enterica serovar Typhimurium is under complex genetic control. Several mouse models of Salmonella infection have been developed and studied, allowing the identification of genes that might influence the disease outcome. To identify which genes confer the extreme resistance of SPRET/Ei mice to S. Typhimurium infection, we performed a genetic linkage analysis. In particular, we made use of SPRET/Ei mice and their great amount of genetic polymorphisms to elucidate the genes contributing to the protective role of neutrophils in infected SPRET/Ei mice. The identification of genetic factors modulating the resistance was assessed by conducting a whole-genome scan in 189 N2 mice of an informative (C3H/HeN ⫻ SPRET/Ei) ⫻ C3H/HeN backcross. All backcross mice, along with the parental strains and F1 offspring mice, were inoculated with a lethal dose of 107 CFU S. Typhimurium, and survival was monitored over 36 days. Consistent with previous results, there was a significant difference in the survival of parental strains: C3H/HeN mice all succumbed within 16 days postinfection (most died within 10 days), whereas all SPRET/Ei mice survived the infection. In addition, all F1 mice survived the challenge, indicating a dominant mode of inheritance of SPRET/Ei-derived resistance alleles. Out of 189 backcross animals, 98 survived the infection, giving a survival rate of 51.9% (Fig. 6A). Note that the time to death of susceptible backcross mice was delayed compared to that of C3H/HeN mice, suggesting that additional genes controlling this trait segregated in this cross. However, linkage analysis was only performed on survival data, analyzed as a binary trait. Furthermore, females (56.8% resistant) were more resistant than males (48.1% resistant) in the backcross population. In addition, a difference in survival rate was also observed in the F1 progeny; F1 females were more resistant than F1 males when injected with high doses (108 and 109 CFU) of S. Typhimurium bacteria (data not shown). All backcross mice were used for genotyping, with approximately 80 microsatellite markers distributed equally across all chromosomes with an average coverage of ⬃20 cM. The association of the phenotype with the genotype at each locus was evaluated by performing linkage analysis using the R/QTL program (4). This analysis revealed the presence of two chromosomal regions associated with survival, on chromosomes 6 and 11. Both loci almost reached the 10% genome-wide significance threshold computed by data permutation (Fig. 6B). Figure 6C displays the precise chromosomal regions of the linked loci on chromosomes 6 and 11. The low significance levels are probably due to the restricted size of the N2 population. However, when we performed a simple single-locusassociation test using the MapManager QTL program, we found that both loci showed strongly significant P values (Fig. 6D). This P value, applied to one point in the genetic map, indicates the probability of obtaining the observed LRS value by chance, i.e., the probability of a false positive. Hence, although both loci do not quite reach significance in the genomewide analysis, the loci on chromosomes 6 and 11 can be con-

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sidered to be linked with the resistance phenotype of the SPRET/Ei strain. The SPRET/Ei allele on chromosomes 6 and 11 has a protective effect at both loci, under an additive mode of inheritance (Fig. 6E). The difference in survival rates between C3H/ HeN and SPRET/Ei mice is 100%. The survival percentage of mice carrying the C/C genotype at both genetic loci is 28.57%, and that of C/S heterozygous mice for both loci is 70.91%. This shows that the loci on both chromosomes explain 42.34% of the total phenotypic variance between the parental strains (Fig. 6E). However, mice that are heterozygous for the SPRET/Ei allele at the two loci show a level of resistance that is still 29.09% lower than that of the SPRET/Ei mice, suggesting that additional loci might account for the remainder of the phenotypic difference. In addition, the higher survival rate (28.57%) of backcross mice that are homozygous for the C3H/HeN allele at the two loci also indicates the presence of other undetected genes. Possibly they are too numerous and too weak to be identified in a cross group of this small size. Several loci with a smaller effect were detected on chromosomes 3, 16, 19, and X, with peak LOD scores of 1.221, 1.393, 1.307, and 1.602, respectively (Fig. 6B). Moreover, the loci on chromosomes 6 and 11 act in an additive manner, because each locus contributes to ⬃16% of the increase in survival and the two together increase survival by ⬃42% (Fig. 6E). In addition, we searched for additional loci which could have remained undetected because of epistatic interactions. Therefore, we used the “scantwo” feature of R/QTL to test for the presence of interactions by examining all pairwise combinations of marker loci. This analysis allowed us to identify interactions between the locus on chromosome 11 and a locus on chromosome 17. From this analysis we conclude that (i) SPRET/Ei alleles are dominant over the C3H/HeN alleles, (ii) the two linked loci act additively to confer survival in N2 backcross mice and together explain 42% of the phenotypic variance, and (iii) there remain undetected loci. DISCUSSION Classical laboratory inbred strains of mice can be classified into different categories according to their resistance to infection with S. Typhimurium (22, 43). Susceptible strains, such as C3H/HeJ and C57BL/6, die during the first week of infection because they are unable to control the multiplication of bacteria (41, 64), whereas strains with various degrees of resistance survive longer. These differences were recognized to be under genetic control. The sensitive phenotype is inherited as simple Mendelian traits in C57BL/6 and C3H/HeJ mouse strains because they derive from mutated alleles of Tlr4 and Slc11a1, respectively (40, 63). However, the limited degree of allelic variation in common laboratory strains imposes a restriction on genetic analysis. The use of wild-derived inbred strains, with their genetic diversity and additional phenotypic variation, may avoid this limitation. We show here that the SPRET/Ei mouse strain is resistant to a lethal infection with S. Typhimurium and that this phenotype is inherited as a complex trait. Notably, Sebastiani et al. reported that SPRET/Ei mice display a moderately resistant phenotype, which is in contrast to our findings that SPRET/Ei mice are strongly resistant to S.

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FIG. 6. The resistance of SPRET/Ei mice to infection with S. Typhimurium is a complex, multigenic trait with linkages to loci on chromosomes 6 and 11. (A) Survival of C3H/HeN (Œ, n ⫽ 16), SPRET/Ei (E, n ⫽ 12), F1 (E, n ⫽ 8), and N2 backcross (f, n ⫽ 189) mice after infection with 107 CFU of S. Typhimurium. Mortality was monitored for 36 days, and no further deaths occurred. (B, C) Genetic linkage analysis using the R/QTL program reveals that loci on chromosomes 6 and 11 are linked with the S. Typhimurium resistance of SPRET/Ei mice. The dashed line in panel B denotes the threshold of significance (permutation test [P ⫽ 0.10]). (D) Linkage of genetic markers to the S. Typhimurium resistance of SPRET/Ei mice using the MapManager QTL program. ∧, microsatellite; ⴱ, expressed in cM according to http://www.informatics.jax.org; #, population size; ⴰ, point-wise P value, i.e., the probability of obtaining the observed LRS value by chance at this specific locus; §, confidence interval. (E) Survival percentages of backcross mice according to their haplotype at the two identified loci on chromosomes 6 and 11. The 28.57% survival percentage of mice homozygous for the C3H/HeN allele at both loci reflects the effect of unidentified genetic loci that contribute to the resistance of the SPRET/Ei mice. Mice that were heterozygous for one or both loci showed a similar increase in survival, and the compiled survival of mice heterozygous at both loci indicates a strictly additive mode of action.

Typhimurium infection. This might be explained by differences in the route of inoculation (intraperitoneal versus intravenous) or the S. Typhimurium strain (BCCM LMG3264 versus Keller) used in the different analyses (50). In addition, we observed that, consistent with their strong resistance, SPRET/Ei mice showed significantly lower bacterial loads in the peritoneal cavity during the course of infection than the susceptible C3H/ HeN control animals. Additionally, bacterial dissemination to the blood and, subsequently, to the RES and other organs was also diminished in these resistant mice. As a consequence, fewer histopathological lesions were evident in the RES of SPRET/Ei mice. This led us to hypothesize that more effective early inactivation of S. Typhimurium might be responsible for

the increase in mean survival time of the SPRET/Ei mice. This raised the question of which immune mechanisms contribute to the enhanced host defense. During infection with S. Typhimurium, immune cells are exposed to different PAMPs, which activate several Toll-like receptor (TLR) signaling pathways, such as TLR4, TLR5, and TLR2 (1, 3, 12, 41). These TLR-based systems, together with activation of the complement system (11), induce a rapid cytokine response that is essential for activation of the host defense mechanism. Overall, we observed attenuated cytokine responses in SPRET/Ei mice. This was surprising, as it is well known that a number of cytokines (TNF, IFN-␥, and IL-12) are required to restrain and eventually suppress Salmonella growth

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by recruiting inflammatory phagocytes into the tissues and by activating these cells (28, 29, 38). However, our results resemble previous findings by Sebastiani et al., who showed that the expression of cytokines was upregulated in sensitive C57BL/6 mice and that no cytokine induction occurs in resistant 129/Sv mice (49). Weaker induction of proinflammatory cytokines leads to less infiltration of inflammatory cells and, therefore, to attenuation of organ damage, which explains the reduced amounts of lesions and macrophages observed in the RES of SPRET/Ei mice. We hypothesized that the improved host defense of SPRET/Ei mice is mediated by immune mechanisms that act early in the infection process (first phase), since cytokines play a major role later in infection (third phase) to control bacterial growth in the RES and to establish adaptive immune responses. During the initial phase of S. Typhimurium infection, macrophages and neutrophils play a critical role in controlling invading pathogens in the periphery by phagocytosis and the production of antimicrobial proteins. This has been demonstrated by Lehner et al., who showed that endotoxin-tolerant mice, despite reduced cytokine responses, showed increased resistance to S. Typhimurium and that this resistance was caused by increased peritoneal accumulation of neutrophils (21). Therefore, we first determined leukocyte counts in the circulation of SPRET/Ei and control C3H/HeN mice. We observed that WBC numbers declined rapidly in the C3H/HeN mice but increased in SPRET/Ei mice and remained higher. In addition, the C3H/HeN mice became anemic during the course of S. Typhimurium infection, as has been reported previously (45). By comparison, the RBC counts were significantly higher in SPRET/Ei mice but decreased to the same extent as in the control C3H/HeN mice, indicating that the degree of the anemia does not contribute to the resistance of SPRET/Ei mice (47). Differential cell counts in the peritoneum during the course of infection clearly showed increased percentages of neutrophilic leukocytes in the SPRET/Ei mice. Since we found an elevated influx of PMNs in the peritoneal cavity, as well as a higher number of circulating leukocytes in the blood of SPRET/Ei mice, this might contribute to enhanced control of bacterial replication and, thus, increased resistance. Although it has been reported that neutrophils are among the first cells attracted to the site of infection and are crucial for the initial control of fast-replicating intracellular bacteria (51), as was shown by neutrophil depletion studies (6, 8), the role of neutrophils in murine salmonellosis remains controversial. A role for PMNs in early resistance to Salmonella infection has been disputed because whole body X-irradiation, which reduces the number of PMNs but does not affect resident macrophages, has no effect on early resistance (16). To confirm the protective role of neutrophils, we depleted neutrophils and macrophages. Depletion of various leukocyte populations confers protection against endotoxic shock (14) but renders animals more susceptible to bacterial infection (8). In contrast, Wijburg et al. found that the elimination of macrophages prior to infection with virulent S. Typhimurium bacteria decreased mortality (65). This is probably due to the fact that they used mice that were deficient for Slc11a1 and, thus, had a significant reduction in innate resistance against S. Typhimurium infection (63). Therefore, the removal of macrophages in mice carrying a functional Slc11a1 gene might ad-

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versely affect their innate resistance to S. Typhimurium infections. Consistent with this, we found that macrophage, as well as neutrophil, depletion reduced the survival time of control (Slc11a1 functional) mice by about 3 days. Interestingly, neutropenic SPRET/Ei mice all succumbed early after S. Typhimurium infection, and the significant difference in mortality between control C3H/HeN and SPRET/Ei mice (P ⫽ 0.0046) was gone in neutropenic animals (P ⫽ 0.0611). Accordingly, SPRET/Ei mice displayed high bacterial loads in the circulation upon neutrophil depletion. This indicates a crucial role of neutrophils in the resistance phenotype of SPRET/Ei mice, although an additional contribution of macrophages cannot be excluded. When macrophages were depleted in SPRET/Ei mice, the mice also became sensitive to S. Typhimurium. However, SPRET/Ei mice were still significantly more resistant than C3H/HeN mice after macrophage depletion (P ⫽ 0.0175), reflected in the significant difference in bacterial load. In addition, besides PMN, the anti-Ly-6G antibody has been shown to also deplete Tip (TNF and inducible nitric oxide synthase [iNOS] producing) dendritic cells (52). Therefore, a role of these cells in the enhanced innate immune response of SPRET/Ei mice cannot be excluded. Although it is often claimed that macrophage activation is the major mechanism for successful defense against Salmonella infection, we demonstrate here that neutrophils play a crucial role in the early host immune response to S. Typhimurium by restricting early bacterial proliferation in SPRET/Ei mice. The improved immune response in SPRET/Ei mice against S. Typhimurium infections and, more specifically, the increased influx of neutrophils can be attributed to several innate immune mechanisms. It is known that members of the CXC subfamily of chemokines, i.e., Cxcl1 (KC) and Cxcl2 (MIP2), control the infiltration of PMN cells (20). Higher levels of the neutrophil chemoattractant KC were found in SPRET/Ei mice 6 h postinfection. Thus, this might contribute to the enhanced early recruitment of neutrophils in SPRET/Ei mice. Recently, it was also shown that neutrophil apoptosis is strongly augmented early during S. Typhimurium infection by the activation of TLR2 (39). It has also been reported that activation of the TLR2 pathway inhibits the expression of CXCR2 by PMN cells, thereby impairing their migration to the site of infection (2). We investigated whether decreased apoptosis of neutrophils or other mechanisms influencing neutrophil activity and migration might contribute to the increased resistance of SPRET/Ei mice to S. Typhimurium by performing a genetic linkage analysis. Our genetic analysis revealed two protective chromosomal regions on chromosomes 6 and 11 that modulate the resistance to S. Typhimurium infection in SPRET/Ei mice. These two loci are located in genomic regions rich in attractive candidate genes for which a role in host resistance to S. Typhimurium infection is either known or can be proposed. The region on chromosome 11 contains several candidate genes that regulate the function of granulocytes in response to infection, including those encoding Nos2 (iNOS), granulocyte-macrophage colony-stimulating factor (GM-CSF), myeloperoxidase (Mpo), and several interleukins (IL12p40, IL-3, IL-4, IL-5, and IL-13). GM-CSF and IL-3 are granulocyte-stimulating factors that are important in myeloid cell differentiation and, thus, poten-

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tially increase neutrophil numbers. Additionally, they enhance the recruitment and phagocytosis capacities of neutrophils (66). GM-CSF has been shown to improve the survival of immunocompetent mice following infection with S. Typhimurium (34). Another interesting candidate gene located in this region is Mpo, which is a lysosomal hemoprotein found in the azurophilic granules of PMN leukocytes. It plays an important role in the phagocytic and microbicidal activity of granulocytes (55). In addition, Nos2 is an interesting candidate because it is responsible for the sustained production of reactive nitrogen species required for efficient killing of invading pathogens (24, 31, 36, 59). As Nos2-deficient mice are able to control the early replication of Salmonella in the RES organs, Nos2 is not involved in the early host defense against S. Typhimurium infection, which makes it less likely that it contributes in the SPRET/Ei model. A cluster of genes (IL-4, IL-5, IL-13, and IRF1) located on proximal mouse chromosome 11 drives Th2 cells (35). These genes are thus involved in the later phases of S. Typhimurium infection, so they are also less relevant for our model. Most interesting is the fact that previous genetic analysis performed in the susceptible MOLF/Ei mouse strain revealed that the same region on chromosome 11 was found to be a resistance locus (50). Hence, the independent detection of this protective locus on chromosome 11 in SPRET/Ei and MOLF/Ei wild-derived inbred strains suggests that the underlying gene(s) play(s) an important role in host defense against intracellular S. Typhimurium infections. Furthermore, although the region on chromosome 6 encompasses many candidates, the interleukin receptors IL23R and IL12Rb2, nucleotide-binding oligomerization domain 1 (Nod1), and caspase 2 may all be considered potential positional candidates. Nod1 is a member of the NOD-like receptors, which is a family of intracellular PRRs implicated in the recognition of bacterial cell products derived from various enteropathogenic bacteria (18). Although a role for Nod1 in the innate detection of S. Typhimurium has not been described, the overexpression of Nod2 resulted in reduced numbers of bacteria upon infection with Salmonella (19). Additionally, it has been reported that Nod1-deficient mice produced lower levels of the neutrophil chemoattractant MIP2 (CXCL2) upon stimulation with Helicobacter pylori (62) and exhibited reduced cytokine production and bacterial clearance upon infection with Pseudomonas aeruginosa (56). Based on this knowledge, it might be interesting to study the role of Nod1 in the host innate immune defense and its specific effects on neutrophil migration in S. Typhimurium infections. Another potential candidate gene is caspase 2, which is a member of the cysteine/aspartic acid proteases, a family of effector enzymes of the apoptotic program. Besides its ability to survive in infected cells, invasive Salmonella induces phagocyte apoptosis (23, 33) that involves both caspase 1 and 2 (13, 17). Thus, altered expression of caspase 2 might affect the spread of S. Typhimurium and might contribute to reduced bacterial growth in SPRET/Ei mice. Two other candidate genes located in the region on chromosome 6 are the receptors for IL-12 (the IL12p40 subunit is located on chromosome 11; see above) (29) and IL-23. These receptors are found predominantly on T cells and NK cells, and the principal function of the cytokines IL-12 and IL-23 produced by phagocytes is the induction of IFN-␥ (58) and, consequently, the suppression of bacterial

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growth, as well as the development of Th1 responses. However, as SPRET/Ei mice did not show increased IFN-␥ production and as this mechanism plays a role in later stages of the immune response to S. Typhimurium infection, the contribution of this gene in the resistance of SPRET/Ei mice is less relevant. Interestingly, using a chemical mutagenesis approach, Richer et al. recently mapped a new resistance locus (Ity9) on chromosome 6 (42). Notably, the regions on chromosomes 6 and 11 colocalized with the loci regulating specific antibody production upon Salmonella infection (57). The production of congenic mouse lines carrying individual SPRET/Ei S. Typhimurium resistance loci will be necessary to validate and facilitate the fine mapping of the identified regions (44), although the development of congenic mice can be hampered when using strains of different ancestral origin. Moreover, it is possible that strains congenic for only one of the linked loci will not show a significant increase in survival rate compared with the C3H/HeN strain, because it might be that yet-unknown loci and epistatic interactions play a critical role. Next, the specific roles of the candidate genes should be assessed by sequencing analysis. However, we expect many sequence variants due to the genetic divergence of classical and wild-derived Mus spretus strains (9). The relevance of the genetic variation can be examined by functional analysis, such as expression profiling studies. Using a combination of approaches, we hope to identify the genes underlying the resistance loci. In conclusion, we used immunological and genetic approaches to understand the response of SPRET/Ei mice to S. Typhimurium infection. The accumulation of leukocytes and the elevated neutrophil influx are the most probable reasons for the reduced mortality and earlier elimination of bacteria in SPRET/Ei mice. Further dissection of the complex host response to S. Typhimurium infection combined with the recent availability of the complete Mus spretus SPRET/Ei genome sequence will contribute further to our understanding of the genetic control of host immunity against S. Typhimurium and the essential role of neutrophils in early immune defense mechanisms. The identification of essential host genes and the mechanisms by which they influence disease pathogenesis and host resistance to acute S. Typhimurium infection will provide key insights into effector mechanisms of protection. ACKNOWLEDGMENTS We thank Joke Vanden Berghe and Wilma Burm for excellent technical assistance. Amin Bredan is acknowledged for editing the manuscript. We are grateful to Linda Van Geert, Debby Roels, and Carine Van Laere for animal care. This work was supported by the Fund for Scientific Research— Flanders, the Interuniversity Attraction Poles Program of the Belgian Science Policy (IAP VI/18), the Belgische Vereniging tegen Kanker, and a Ghent University Methusalem (BOF09/01M00709) grant. L.D. is a research fellow with FWO Flanders, Belgium. We have no other conflicting financial interests. REFERENCES 1. Aliprantis, A. O., R. B. Yang, M. R. Mark, S. Suggett, B. Devaux, J. D. Radolf, G. R. Klimpel, P. Godowski, and A. Zychlinsky. 1999. Cell activation and apoptosis by bacterial lipoproteins through toll-like receptor-2. Science 285:736–739. 2. Alves-Filho, J. C., A. Freitas, F. O. Souto, F. Spiller, H. Paula-Neto, J. S. Silva, R. T. Gazzinelli, M. M. Teixeira, S. H. Ferreira, and F. Q. Cunha. 2009. Regulation of chemokine receptor by Toll-like receptor 2 is critical to

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