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The Journal of Immunology, 2003, 170: 6141-6150.
Copyright © 2003 by The American Association of Immunologists

Overexpression of Toll-Like Receptor 4 Amplifies the Host Response to Lipopolysaccharide and Provides a Survival Advantage in Transgenic Mice 1

Franck Bihl*,{ddagger},§, Laurent Salez{ddagger}, Magali Beaubier§, David Torres§, Line Larivière{ddagger}, Line Laroche{ddagger}, Alexandre Benedetto{ddagger}, Dominic Martel{ddagger}, Jean-Martin Lapointe, Bernhard Ryffel§ and Danielle Malo2,*,{dagger},{ddagger}

Departments of * Human Genetics and {dagger} Medicine, McGill University, and {ddagger} Center for the Study of Host Resistance, Montreal General Hospital, Montréal, Québec, Canada; § Génétique Expérimentale et Moléculaire, Centre National de la Recherche Scientifique, Orléans, France; and Pfizer Global Research and Development, Groton, CT 06340


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Toll-like receptors are transmembrane proteins that are involved in the innate immune recognition of microbial constituents. Among them, Toll-like receptor 4 (Tlr4) is a crucial signal transducer for LPS, the major component of Gram-negative bacteria outer cell membrane. The contribution of Tlr4 to the host response to LPS and to infection with virulent Salmonella typhimurium was studied in four transgenic (Tg) strains including three overexpressing Tlr4. There was a good correlation between the level of Tlr4 mRNA expression and the sensitivity to LPS both in vitro and in vivo: Tg mice possessing the highest number of Tlr4 copies respond the most to LPS. Overexpression of Tlr4 by itself appears to have a survival advantage in Tg mice early during infection: animals possessing more than two copies of the gene survived longer and in a greater percentage to Salmonella infection. The beneficial effect of Tlr4 overexpression is greatly enhanced when the mice present a wild-type allele at natural resistance-associated macrophage protein 1, another critical innate immune gene involved in resistance to infection with Salmonella. Tlr4 and natural resistance-associated macrophage protein 1 exhibit functional epistatic interaction to improve the capacity of the host to control bacterial replication. However, this early improvement in disease resistance is not conducted later during infection, because mice overexpressing Tlr4 developed an excessive inflammatory response detrimental to the host.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Despite widespread use of antibiotherapy, septic shock subsequent to septicemia is responsible for >200,000 deaths annually in the United States alone (1). LPS (1), originally named endotoxin, is a major integral glycolipid component of the outer membrane of Gram-negative bacteria (2, 3). The recognition of LPS by various host cells plays a central role in the outcome of sepsis. LPS alone can induce many of the pathophysiological symptoms that are characteristic of Gram-negative bacterial infection. Cell types, including lymphocytes, macrophages, endothelial cells, and fibroblasts, respond to the proinflammatory and immunostimulatory properties of LPS by production and release of proinflammatory cytokines (TNF, IL-1, IL-6, and IL-12), enhancement of their Ag-presenting and microbicidal capacities, and induction of proliferation (4, 5, 6).

LPS binds to a serum protein termed LPS binding protein (LBP)3 that catalyzes the transfer of LPS monomers to a membrane-bound receptor CD14 expressed on the surface of myeloid cells (7, 8, 9). This mediates the physical association of the LPS/CD14 complex with the transmembrane receptor Toll-like receptor 4 (Tlr4) and MD-2, an extracellular accessory protein (10, 11). The subsequent activation of Tlr4 induces the recruitment of MyD88, an intracellular adaptor protein that in turn recruits the IL-1R-associated kinase (12, 13, 14, 15). Beside MyD88, another component named MyD88-adapter-like/Toll-IL-1R domain-containing adapter protein participates in the transduction of this signal that ultimately results in the activation of transcription factor NF-{kappa}B and mitogen-activated protein kinases (16, 17, 18).

Most inbred strains of mice are susceptible to the immunostimulatory properties of LPS. However, C3H/HeJ, C57BL/10ScNCr, and C57BL/6.KB2-motor neuron disease mice exhibit endotoxin hyporesponsiveness, with macrophages being central in the mediation of this phenotype (4, 19, 20, 21, 22, 23). Positional cloning analysis revealed the presence of mutation in alleles of the Tlr4 gene. C3H/HeJ mice present a single missense mutation resulting in a proline-for-histidine substitution at codon 712 within the signaling domain. In C57BL/10ScCr mice, there were no Tlr4 transcripts detected as a consequence of a 75-kb chromosomal deletion encompassing the whole Tlr4 gene. The mutation identified in C57BL/6.KB2-motor neuron disease Tlr4 consists of a complete deletion of exon II, leading to a frameshift resulting in the appearance of a stop codon just downstream of the exon junction. The putative Tlr4 mutant protein is equivalent to the first 31 N-terminal residues of its wild-type counterpart (23, 24, 25, 26). Subsequent inactivation of the gene confirmed the role of Tlr4 in mediating LPS biological activities (27). Another important manifestation of altered LPS responsiveness of C3H/HeJ and C57BL/10ScNCr mice is their enhanced susceptibility to the Gram-negative bacteria Salmonella enterica serovar typhimurium (S. typhimurium) infection. In these mice, replication of Salmonella in the spleens and livers is uncontrolled and results in premature death, compared with C3H/HeN and C57BL/10SnJ wild-type animals (4, 22, 28).

In addition to Tlr4, another gene termed natural resistance-associated macrophage protein 1 (Nramp1), recently renamed solute carrier family 11 member 1 (because of its membership to a family of solute carriers) has been shown to play an important role in innate immunity, especially in S. typhimurium infection (29). In mice, Nramp1 is a phagosomal protein exclusively expressed in phagocytic cells that controls the replication of certain intracellular pathogens during the early phase of infection (30, 31, 32, 33). Mice carrying an aspartate (Nramp1Asp169) instead of a glycine (Nramp1Gly169) residue at position 169 in predicted transmembrane domain 4 of this protein are also extremely susceptible to infection with S. typhimurium (34).

In this paper, we investigate the effect of Tlr4 expression by generating four strains of transgenic (Tg) mice. We show a good correlation between the number of integrated copies of Tlr4, its RNA expression, the LPS-induced proliferation of splenocytes in vitro and the sensitivity to septic shock after LPS challenge in vivo in each strain. Furthermore, all the Tg mice were more resistant to S. typhimurium infection in terms of survival or bacterial loads in reticuloendothelial organs. Finally, we demonstrate the existence of a genetic epistatic interaction between Tlr4 and Nramp1 with respect to the host response to infection with S. typhimurium.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Generation of Tg mice

Tg mice were generated using the bacterial artificial chromosome (BAC) 152C16 that contained Tlr4 (129/Sv library; Research Genetics, Huntsville, AL). BAC 152C16 isolation and sequencing were reported previously (25). The preparation of the linearized BAC DNA has been adapted from several previous publications dealing with yeast artificial chromosome transgenesis (35, 36, 37, 38). Briefly, BAC 152C16 was purified using QIAfilter Plasmid Mega or Giga kits (Qiagen, Valencia, CA), digested with NotI (New England Biolabs, Beverly, MA), loaded onto a 1% low melting point agarose (SeaPlaque GTG; FMC Bioproducts, Chicago, IL) gel without ethidium bromide and submitted to pulse field gel electrophoresis (PFGE) in 0.5x TBE (Tris-boric acid-EDTA) buffer. Electrophoresis was performed at 6 V/cm for 16 h at 14°C with initial and final switching times of 5 s and with an included angle of 120°. After migration, small strips (2 cm) on each side of the gel were cut and submitted to ethidium bromide staining, while the center part was stored in fresh migration TBE buffer. After reassembling the gel, the slice that contained the 150-kb band of DNA was cut out, stripped into 1-cm pieces, pre-equilibrated in soaking buffer (10 mM Tris, 100 mM NaCl, and 1 mM EDTA) and digested with {beta}-agarase (New England Biolabs) using conditions suggested by the supplier. Agarased DNA solution was dialyzed 4 h against the microinjection buffer (10 mM Tris and 1 mM EDTA) using pre-equilibrated filters (VMWP02500; Millipore, Bedford, MA) in petri dishes. The final concentration was estimated after the migration of a sample of the dialyzed DNA fragment with known quantities of the initial digested BAC onto a 1% agarose (SeaKem GTG; FMC Bioproducts) PFGE gel. Under these conditions, starting with 10 µg of digested BAC, we obtained ~500 µl of purified construct at a concentration of 5 ng/µl. This construct was microinjected directly into F1 (C3H/HeNHsd x C57BL/6NHsd) eggs that were transferred into pseudopregnant CD1 mice. Tg animals were subsequently backcrossed toward a C57BL/10ScNCR background.

Genotyping of Tg mice

Founders and subsequent littermates were genotyped by PCR. Genomic DNA was extracted from the tails of mice as described previously (39). We used one primer specific for the vector (pBeloBAC11) of the BAC library and one primer located in the insert, at each extremity. The first fragment (SP6 end) was 170 bp in length, using primers 5'-CTCACTTACCTCACAATgTgC-3' and 5'-gCCAAgCTATTTAggTgACAC-3'; the second fragment (T7 end) was 192 bp in length, using primers 5'-gCATCCCTAAATCCTgCTCTg-3' and 5'-ATACgACTCACTATAgggCgA-3'. To exclude any internal deleting recombinational event, a third PCR amplification was done using primers 5'-TACgTTCATTCgTTCACg-3' and 5'-AACTgAgAACTgAgAACTgC-3'. The fragment amplified with these two primers (~140 bp) is located in the Tlr4 promoter and contains a (CA)n repeat microsatellite marker that is polymorphic between the C57BL (n = 28), C3H (n = 30), and 129/Sv (n = 32) strains of mice, allowing us to follow the transgene. Nramp1 genotyping was realized using D1 Mcg4 microsatellite (34). All these amplifications were performed in a final volume of 20 µl using standard cycling conditions. [{gamma}-33P]ATP end-labeled primer allowed visualization by polyacrylamide gel electrophoresis followed by autoradiography.

Quantification of the transgene by TaqMan PCR

Genomic DNA levels of murine Tlr4 and TATA-binding protein (Tbp) genes were quantitated by real-time PCR using an ABI Prism 7700 sequence detector (Applied Biosystems, Foster City, CA) and the Brilliant Quantitative PCR Core Reagent kit (Stratagene, La Jolla, CA) according to the manufacturer’s instructions. Amplification was achieved using an initial cycle of 50°C for 2 min and 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. Tlr4 genomic DNA levels during the linear phase of amplification were normalized against Tbp controls. Determinations were made in triplicate and mean ± SD was determined. Primers (forward and reverse) and probes were designed using the Primer Express (Applied Biosystems) software and are as follows: Tlr4, forward, 5'-ACACggTTggAAACATAACAATTg-3', reverse, 5'-AATCCAgCCACTgAAgTTCTgAA-3', and probe, 5'-TACCAATgCATggATCAgAAACTCAgCAA-3'; and Tbp, forward, 5'-TgggCTTCCCAgCTAAgTTC-3', reverse, 5'-CTggTgggTCAgCACAAgg-3', and probe, 5'-TCCCCACCATgTTCTggATCTTgAAgTCTA-3' (40). Amplicon sizes were 201 and 104 bp, respectively. Reporter and quencher dyes were FAM and TAMRA for Tlr4 probe, and VIC and TAMRA for Tbp probe (Applied Biosystems). For Tlr4, both primers were used at 300 nM, and probe was used at 250 nM; for Tbp, the forward primer was used at 900 nM, the reverse primer was used at 300 nM, and the probe was used at 200 nM.

RT-PCR

Total spleen RNA from Tg and non-Tg mice was extracted using TRIzol (Life Technologies, Rockville, MD) according to the manufacturer’s conditions. First-strand cDNA synthesis was conducted using 2 µg of total RNA, 100 ng of random hexamers, and 200 U of Moloney murine leukemia virus transcriptase (Life Technologies) in a total volume of 20 µl. An aliquot was used for each independent PCR. Tlr4 expression was monitored following 35 cycles with the primers 5'-CCTgCATAgAggTAgTTCCTA-3' and 5'-g agarose gel TAAgCCATgCCATgCCTTg-3'. PCR products were resolved on a 1% and blotted onto a nylon membrane. An internal primer end-labeled with [{gamma}-32P]ATP was hybridized to the membrane for 16 h at 55°C in aqueous solution (6x SSC, 5x Denhardt’s solution, 1% SDS, and 200 µg/ml herring sperm DNA). The membrane was washed to a final stringency of 0.1x SSC and 0.1% SDS for 20 min at 50°C. The amount of cDNA was quantified after exposure in a STORM 860 PhosphorImager with the help of the Image Quant software (Molecular Dynamics, Sunnyvale, CA). To normalize for the amount of RNA, the same procedure was conducted for GAPDH (25 cycles of amplification).

FACS analysis

Thioglycolate-elicited peritoneal macrophages were obtained from mice injected with 2 ml of 4% thioglycolate i.p. (Biovalley, Morne La, Vallée, France). The cells were collected 4 days postinjection and double-stained with anti-CD11b mAb conjugated with PerCP (BD PharMingen, San Diego, CA) and anti-Tlr4/MD-2 mAb conjugated with PE (MTS510; Santa Cruz Biotechnology, Santa Cruz, CA). Cells were analyzed on a BD Biosciences (Mountain View, CA) LSR apparatus.

Spleen cell mitogenic response

Spleen cell suspensions were prepared from C57BL/10SnJ and C57BL/10ScNCR, Tg and non-Tg animals as previously described (23). Briefly, organs were removed aseptically and homogenized, and splenocytes were resuspended in 5 ml of cold complete RPMI 1640 (Life Technologies). Cells were loaded on a density separation medium (Lympholyte-M; Cedar Lane Laboratories, Hornby, Ontario, Canada) and 12 x 106 to 40 x 106 viable cells were obtained per mouse spleen. Single-cell splenocytes suspensions were prepared at a final concentration of 2 x 106 cells/ml in RPMI 1640 medium containing 20% heat-inactivated FBS (Clontech, Palo Alto, CA). Cells were cultured (100 µl/well) in 96-well plates, stimulated with increasing concentrations of LPS (E. coli K235; Sigma-Aldrich, St. Louis, MO) or 1.5 µg/ml Con A (Sigma-Aldrich) in RPMI 1640 medium and further incubated for 48 h at 37°C. During the last 6 h of incubation, 1 µCi of [3H]thymidine was added per well. Cells were harvested, and [3H]thymidine incorporation was measured by scintillation counting. For each treatment, triplicate cultures were assayed.

In vivo injection of LPS

All procedures involving animals were performed in accordance with the regulations of the Canadian Council on Animal Care. C57BL/10SnJ and C57BL10/ScNCR, Tg and non-Tg mice were each injected with 0.5 ml of PBS containing increasing amounts (10 µg to 1 mg) of LPS (E. coli K235; Sigma-Aldrich) i.p. Animals were monitored twice a day and moribund animals were sacrificed for ethical reasons.

Infection with S. typhimurium

Each mouse was infected with 0.2 ml of PBS containing 103 CFU of S. typhimurium in the caudal vein. For survival curves, each animal was monitored twice a day, and moribund animals were sacrificed for ethical reasons. To determine the growth rate of the bacteria within the reticuloendothelial organs, mice were sacrificed 5 days postinfection, and their spleens and liver were recovered aseptically. One-half of each organ was homogenized in 2 ml (spleen) or 5 ml (liver) of isotonic saline with a Polytron homogenizer (Brinkmann Instruments, Westbury, NY). Serial dilutions of each homogenate were plated on trypticase soy broth agar to enumerate the CFU within each organ. The remaining one-half was snap frozen in liquid nitrogen.

Histopathology

Two naive or infected mice per group were sacrificed 5 days postinfection, and their spleens, livers, hearts, and kidneys were removed aseptically, fixed in 10% buffered formaldehyde, and embedded in paraffin. Sections (4 µm thick) were cut and stained with hematoxylin-phloxin-saffron. All sections were examined by the same pathologist.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Generation of Tlr4 Tg strains of mice

To study the effect of Tlr4 overexpression in vivo, Tg strains of mice carrying BAC 152C16 (129/Sv library; Research Genetics) were generated. As shown previously by ourselves and others (25, 41), this BAC contains the entire Tlr4 gene. Strategically, we wanted to microinject a linearized construct containing the insert of interest but with minimal vector pBeloBAC11 sequence. For this purpose, we initiated a first screening to identify rare cutter restriction enzymes that cleave only the vector and release the whole insert. Among the various enzymes used (NotI, EagI, BstEII, KpnI, NarI, SfiI, SalI, and BsshII), BAC genomic DNA digested with NotI revealed the presence of two bands of ~150 and 7 kb after PFGE (data not shown). According to the vector restriction map, the 7-kb band corresponded to most of the vector (expected size of 6877 bp), whereas the 150-kb fragment corresponded to the whole insert plus 383 and 247 bp of the vector at the T7 and SP6 extremities, respectively (Fig. 1). Once isolated and purified (as described in Materials and Methods), this 150-kb fragment was directly microinjected into F1 (C3H/HeNHsd x C57BL/6NHsd) eggs that were subsequently transferred into pseudopregnant CD1 mice. Founders and subsequent littermates were then genotyped by PCR. To follow the inserted construct through generations, we used one primer specific for the vector (pBeloBAC11) of the BAC library and one primer located in the insert for each extremity (Fig. 1). Furthermore, to exclude any internal deleting recombinational event, a third PCR amplification was done using primers amplifying a (CA)n repeat microsatellite marker located in the Tlr4 promoter and polymorphic between the C57BL (n = 28), C3H (n = 30), and 129/Sv (n = 32) inbred strains of mice (Fig. 1). Under this procedure, 3 of 14 founders were positive for all the three markers used and were named 388, 390, and 394. A fourth founder, called 382, was only positive for the SP6 and promoter microsatellite amplifications. All four Tg animals were subsequently backcrossed to a C57BL/10ScNCR Tlr4 mutant background and successfully transmitted the inserted transgene.



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  		FIGURE 1. Generation of Tg mice. Schematic representation of the injected transgene. {square}, pBeloBAC11 vector sequences; {blacksquare}, Tlr4 exons; and , location of the microsatellite in the promoter region of the Tlr4 gene (polymorphism: size of amplicon BAC 129/Sv > C3H > C57BL/6J; no amplification in C57BL/10ScNCR genomic DNA). Pairs of primers used for the genotype screening are indicated with arrows. NotI sites are also shown. Sizes are indicated at the bottom; the schematic is not on scale.

 
Estimation of the integrated number of copies

It is well known that transgenesis usually generates the insertion of multiple copies (1–100) of the injected foreign DNA at a single chromosomal locus. At each integration site, these copies are arranged primarily in a head-to-tail array (42). To estimate the number of integrated copies in the four Tg strains of mice, we have used quantitative PCR. In our study, we analyzed the genomic DNA of the C57BL/10ScNCR and C57BL/10SnJ, Tg and non-Tg strains. The Tlr4-specific amplicon was normalized to the Tbp amplicon, an endogenous gene that is present in 2 copies in the genome of all the strains studied. As expected, no amplification was detected in the C57BL/10ScNCR strain that harbors the complete deletion of the Tlr4 gene, and a similar amount of Tlr4 and Tbp was confirmed in C57BL/10SnJ wild-type control strain (Fig. 2). The Tlr4 Tg strains presented in their genomes various amounts of integrated Tlr4 copies: the 382 strain had only 1 copy, the 388 had 3 copies, the 390 had 6 copies, and finally, the 394 strain contained ~30 copies of Tlr4.



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  		FIGURE 2. Quantification of the number of integrated copies per strain. Genomic DNA levels of murine Tlr4 and Tbp genes was quantified by real-time PCR. Three mice per group were used. Quantifications were made in triplicate, and mean ± SD was determined. Ratio must be multiplied by a factor of 2 for strains 382 to 394 to obtain the number of copies per genome. ND, Not detected. B10, C57BL/10.

 
Estimation of the expression by RT-PCR

To determine whether the number of integrated copies correlates with the level of expression of the Tlr4 gene, we extracted total RNA from the spleen, liver, heart, and lung of C57BL/10SnJ, Tg and non-Tg strains of mice. After a first step of reverse transcription, cDNAs were submitted to amplification with Tlr4 cDNA and Gapdh specific primers in nonsaturating conditions. After normalization, and as expected, we did not detect any noticeable Tlr4 expression in the organs of non-Tg animals (data not shown). Furthermore, there was a gradient of expression extending from the 382 strain to the 394 strain of mice (Fig. 3). The level of Tlr4 expression is well correlated with the number of integrated copies in each strain. It should be noted that the level of expression of Tlr4 in strain 382 is under the level detected in the C57BL/10SnJ wild-type strain, as expected by its unique estimated integrated copy (data not shown).



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  		FIGURE 3. Quantification of Tlr4 mRNA expression by RT-PCR in spleen (A), heart (B), liver (C), and lung (D) of Tg mice. Tlr4 mRNA expression is shown as a ratio of Tlr4 signal intensity to Gapdh signal intensity. Each column reflects the average of three mice including the SEM.

 
Estimation of the cell surface expression of Tlr4 by FACS analysis

The cell surface expression of Tlr4 was assessed with a Tlr4 mAb, MTS510, that specifically recognize the Tlr4/MD-2 complex (43). Thioglycolate-elicited peritoneal macrophages from the four Tg Tlr4 strains were able to express Tlr4/MD-2 complex, as shown in Fig. 4. Interestingly, a gradient of expression was observed as reflected by the intensity of fluorescence, extending from strain 382 to strain 394. These data clearly show that the level of expression of Tlr4/MD-2 on the cell surface of thioglycolate-elicited peritoneal macrophages is well correlated with the number of Tlr4 integrated copies in the genome and the level of Tlr4 transcription.



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  		FIGURE 4. Estimation of Tlr4/MD-2 cell surface expression on thioglycolate-elicited peritoneal macrophages. Peritoneal macrophages were obtained from strains 382 (A), 388 (B), 390 (C), and 394 (D), and stained with an anti-CD11b mAb conjugated with PerCP and the MTS510 mAb conjugated with PE. Among the cell population shown on these histograms, ~90% are CD11b-positive cells. The isotypic control is shown in dotted line; C57BL/6 peritoneal cells are indicated by the thin line; and Tg macrophages are represented by bold lines.

 
Mitogenic response of splenocytes to LPS stimulation in vitro

We next tested the proliferative ability of splenocytes derived from Tlr4 Tg strains to respond to mitogenic LPS stimulation in vitro. C57BL/10ScNCR and C57BL/10SnJ strains of mice were used as internal controls. No proliferation was detected using the C57BL/10ScNCR splenocytes at any concentrations of LPS used, ranging from 1 pg/ml to 10 µg/ml (Fig. 5). In contrast, C57BL/10SnJ splenocytes began to proliferate in the presence of 1 µg/ml of LPS, but the level of incorporated [3H]thymidine remained low. Interestingly, all Tg animals showed a higher mitogenic response than those exhibited by these wild-type controls. Among them, splenocytes derived from the 382 strain were the least sensitive to the LPS stimulus. Those derived from the 388 and 390 strains presented a similar but intermediate response. Finally, those derived from the 394 strain incorporated the highest amount of [3H]thymidine. Furthermore, whereas the 382 and C57BL/10SnJ splenocytes showed a similar sensitivity to LPS stimulation, those derived from 388, 390, and 394 Tg mice responded to a 10-fold lower LPS stimulus (100 ng/ml). Therefore, there is a clear correlation between levels of Tlr4 expression and the cellular ability to sense LPS and to mediate mitogenic response.



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  		FIGURE 5. Mitogenic response of splenocytes to LPS stimulation. Spleen cells were isolated from C57BL/10SnJ and C57BL10/ScNCR, Tg and non-Tg, animals. They were stimulated with various concentrations of LPS, and their responses were measured by determining the incorporation of [3H]thymidine over a 16-h period. The values are means ± SEM based on three mice for each group. Triplicate wells were prepared for each sample. B10, C57BL10.

 
In vivo response to LPS injection

To test the in vivo responsiveness of Tlr4 Tg animals to LPS-induced septic shock, we injected increasing amounts of LPS (10 µg to 1 mg i.p.) in the animals and monitored their survival. C57BL/10ScNCR mice were completely resistant to LPS-induced lethality, whereas C57BL/10SnJ animals became sensitive after the injection of 1 mg of LPS (Table I). Interestingly, all Tg animals exhibited a severe sensibility to the 1-mg LPS dose, with almost 100% mortality in all four Tg strains. Furthermore, strains 388, 390, and 394 were more sensitive to the lethal effect of LPS and showed 75–100% mortality with 100 µg of LPS. Finally, all strains were resistant to a challenge with 10 µg of LPS. Thus, the number of Tlr4 copies and the level of Tlr4 expression appear to have an impact on the host response to LPS: strain 382 shows a similar behavior to that of wild-type control mice (C57BL/10SnJ) at low dose, and the three other Tg animals were sensitized to LPS at a lower dose. These observations are also in agreement with the in vitro ability of the splenocytes to proliferate after LPS exposure, although a plateau was reached with three copies of the transgene in vivo. The unexpected greater sensitivity of strain 382 to a high dose of LPS in vivo compared with that of wild-type C57BL/10SnJ mice may be explained by an effect of the position of the transgene in the genome and/or a background effect. Despite the fact that C57BL/10SnJ and C57BL/10ScNCR have similar origin, they have been kept separate for >50 years and may have accumulated subspecies-specific mutations, as seen between C57BL/10ScCr and C57BL/10ScNCr mice (44).


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  		Table I. In vivo response to LPS injection

 
Experimental infection with S. typhimurium

Initially, we examined the host response of Tlr4 Tg and non-Tg littermates following infection with virulent S. typhimurium. We monitored survival to infection over a 2-wk period. Regardless of the Tg strain used, non-Tg animals (Nramp1Asp169/Tg-) died within 4–5 days, whereas Tg animals (Nramp1Asp169/Tg+) died within 5–7 days (Fig. 6). This protection delay is statistically significant in all four strains studied (p < 0.05 for strain 382; p < 0.01 for strains 388, 390, and 394) using a Kaplan-Meier analysis. Therefore, the presence of the transgene and the expression of Tlr4 conferred protection to the lethality induced by S. typhimurium. However, the intensity of the protection was apparently not correlated with the level of Tlr4 expression. Because of the small impact of Tlr4 transgene on resistance to infection in our model using C57BL10/ScNCR mice (Nramp1Asp169) as background strain and the known importance of Nramp1 in the control of Salmonella infection, we investigated the possible interaction of the Tlr4 and Nramp1 genes. We created a congenic strain possessing the wild-type resistant allele at Nramp1 (Nramp1Gly169) onto the Nramp1- and Tlr4-deficient C57BL10/ScnCR background through five consecutive backcross generations. These mice were crossed to Tlr4 Tg animals to obtain a combination of littermates that were heterozygous at Nramp1Gly169/Nramp1Asp169 (the Nramp1Gly169 allele is fully dominant) and either possessing or not possessing the Tlr4 transgene. The mice were infected i.v. with 103 CFU S. typhimurium. Non-Tg animals that were NrampGly169/Nramp1Asp169 (Nramp1Gly169/Tg-) succumbed within 6–9 days postinfection instead of 4–5 days as observed for Nramp1Asp169/Tg- mice (Fig. 6). This effect is constant over the four Tg strains studied. As expected, Nramp1 is playing a major role in protecting the mice during the infection. The most dramatic effect in survival after infection was observed in mice carrying a wild-type allele at Nramp1 and the Tlr4 transgene (Nramp1Gly169/Tg+). The protection conferred by Nramp1 and Tlr4 was more pronounced in mice carrying at least three copies of the transgene. The most resistant mice were from strain 388 with 100% survival at day 14 postinoculation. Strains 390 and 394 had intermediate phenotypes with a mean survival time of 12 days in strain 390 and 40% survival for strain 394 at day 14 postinfection.



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  		FIGURE 6. Survival of mice following infection with S. typhimurium. Effect of the Tlr4 transgene and Nramp1 genotype on survival of mice infected with S. typhimurium. For strain 382, 31 mice were followed with at least 3 mice per group. For strain 388, 32 mice were followed with at least 3 mice per group. For strain 390, 33 mice were followed with at least 5 mice per group. For strain 394, 22 mice were followed with at least 4 mice per group. {blacktriangleup} and •, Nramp1Asp169/Asp169; {triangleup} and {circ}, Nramp1Gly169/Asp169; • and {circ}, Tg-; and {blacktriangleup} and {triangleup}, Tg+.

 
To characterize more precisely this protective effect, we quantified the bacterial load within the reticuloendothelial organs (livers and spleens) of Tg and non-Tg mice 5 days postinfection. The number of CFU in each organ was determined by plating serial dilutions (Fig. 7). All Tlr4 Tg animals on C57BL10ScNCr background (Nramp1Asp169/Asp169) exhibited at least 15–75 times less bacteria in their spleen and liver compared with non-Tg animals. The bacterial load in the spleen and liver were similar among all Tg strains showing that the higher level of expression of the Tlr4 transgene does not improve host capacity to control Salmonella infection. These results are in agreement with the survival analysis performed in the same strains. In animals carrying a wild-type allele at Nramp1, the situation is different. Levels of the bacterial loads in Nramp1Gly169/Asp169 non-Tg animals were always lower but near those observed in Nramp1Asp169/Asp169 mice possessing the Tlr4 transgene (Fig. 7), showing that Nramp1 and Tlr4 have a similar impact on bacterial load in spleen and liver. All Nramp1Gly169/Asp169 non-Tg animals showed bacterial counts at least 75–150 times lower than those observed in Nramp1Asp169/Asp169 non-Tg animals. Nramp1Gly169/Asp169 Tg mice showed the lowest bacterial counts with 500-2000 times reduction compared with Nramp1Asp169/Asp169 non-Tg animals. The combined effect of Tlr4 and Nramp1 does not seem to be additive but rather multiplicative in this animal model, suggesting epistatic interaction. In this case also, there was no effect of the number of Tlr4 copies on bacterial load.



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  		FIGURE 7. Bacterial loads in reticuloendothelial organs of Tlr4 Tg mice after infection with S. typhimurium. To determine the growth rate of the bacteria within the reticuloendothelial organs, mice were sacrificed 5 days postinfection, and their spleens and liver were recovered aseptically. For each group, four to six mice were analyzed. Tg+, Tlr4 Tg mice; Tg-, non-Tg littermates; r, Nramp1Gly169/Asp169; and s, Nramp1Asp169/Asp169.

 
Histopathology of Salmonella-induced lesions in Tlr4 Tg mice

Histological examination was undertaken on tissues of uninfected and Salmonella-infected Tlr4 Tg and littermate control mice. Uninfected adult Tlr4 Tg mice did not present any tissues abnormalities (data not shown). The severity of the lesions during infection was evaluated semiquantitatively in the spleen and liver of Tg Tlr4 mice 5 days postinfection. Animals were grouped according to their number of Tlr4 copies and their genotype at Nramp1 (Gly169/Asp169 or Asp169/Asp169). C57BL/10SnJ (Tlr4+/+, Nramp1Asp169/Asp169) and B10.Nramp1 (Tlr4+/+, Nramp1Gly169/Asp169) mice were used as controls.

Significant inflammatory reactions, characterized by infiltration with predominantly polymorphonuclear cells (PMN) and fewer macrophages, were visible in both the spleen and the liver of all Nramp1Asp169/Asp169 mice 5 days postinfection (Fig. 8, A–E). The lesions in Tlr4 Tg mice (Fig. 8, C–E) were comparable to those observed in non-Tg littermates (Fig. 8, A and B) and C57BL/10SnJ mice (data not shown). The presence of Tlr4 and the number of Tlr4 copies had little impact on the severity of the pathological changes in infected Tg mice on the Nramp1Asp169/Asp169 background. No difference was observed with respect to site, number, morphology, and cell population involved in inflammatory foci. Only a slight improvement was observed in Tg lines 388, 390, and 394 with respect to the degree of necrosis in the liver and the severity of lymphocyte depletion in the spleen compared with those of non-Tg littermates. In the groups of Tg mice carrying a wild-type allele at Nramp1 (Nramp1Gly169/Asp169) (Fig. 8, F–J), the lesions were less evident and inflammatory Salmonella foci were reduced in size and number compared with those observed in Tlr4 Tg Nramp1Asp169/Asp169 mice (Fig. 8, C–E). In the liver, the lesions consisted of randomly distributed foci of hepatocyte degeneration and necrosis, infiltrated equally by PMN and macrophages. In the spleen, the red pulp showed a progressively increasing expansion by a mixture of macrophages and PMN. In this case also, the presence of Tlr4 and the number of Tlr4 copies appear to have little impact on the pathology induced by S. typhimurium 5 days postinfection. The presence of both Tlr4 and Nramp1 has a clear impact on limiting tissue damage after infection with Salmonella.



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  		FIGURE 8. Histology of Salmonella-induced lesions in Tlr4 Tg mouse strain 388. Spleen (A, C, D, F, H, and I) and liver (B, E, G, and J) tissue samples were analyzed from mice that presented different genotypes at Tlr4 (Tg+, Tlr4 Tg mice; Tg-, non-Tg littermates) and Nramp1 on day 5 postinfection. Group 1 (A and B), Tg- and Nramp1Asp169/Asp169; group 2 (C, D, and E), Tg+ and Nramp1Asp169/Asp169; group 3 (F and G), Tg- and Nramp1Gly169/Asp169 background; and group 4 (H, I, and J), Tg+ and Nramp1Gly169/Asp169 background. Spleen (K), liver (L), and heart (M) tissue sections were analyzed in Tlr4 Tg mice with a Nramp1Gly169/Asp169 background on day 15 postinfection. Tissue sections were stained in hematoxylin-phloxin-saffron and photographed at magnifications of x10 (A–C, E—H, and J), x20 (M), and x40 (D, I, K, and L).

 
All Tg animals from line 388 and a subset from line 394 carrying Nramp1Gly169/Asp169 survived infection for a period extending 14 days. These Salmonella-infected Tg mice developed ascites associated with severe hepatosplenomegaly by 14 days after infection. The lesions in the spleen and the liver were different from those observed 5 days postinfection (Fig. 8, K and L). A severe inflammatory response characterized by massive infiltration of macrophages into the liver and spleen was apparent. This inflammatory process coincides with a disorganization and obstruction of hepatic sinusoids that could explain the observed ascites. Additional lesions were found only in the heart and consisted of multifocal myonecrosis with mineralization (Fig. 8M).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we generated four strains of mice that possessed various numbers of Tlr4 gene copies on a C57BL/10ScNCR Tlr4 mutant background. This background was derived from a C57BL/10Sn progenitor strain that is LPS responsive, indicating that the mutant phenotype was acquired after 1953 (45). It has been shown that the LPS unresponsiveness exhibited by these C57BL/10ScNCR mice is due to a null mutation of Tlr4 corresponding to a 75-kb genomic deletion encompassing the Tlr4 locus (26, 46). All of our experiments used the C57BL/10ScNCR mice, because these mice have a normal IL-12 response as measured by IFN-{gamma} response when inoculated with various pathogens. This is not the case with another LPS-unresponsive C57BL/10 substrain (C57BL/10ScCr) that is not responsive to IL-12 due to a point mutation in the IL-12R{beta}2 gene (44).

Using our protocol of BAC transgenesis, we obtained 4 of 14 (29%) positive founders that led to the generation of four independent Tg strains expressing various functional copies of Tlr4. In the initial genotyping procedure that we used, three of four Tg strains (388, 390, and 394) were positive for the SP6/T7 extremities of the transgene and for the internal polymorphic microsatellite marker located in the Tlr4 promoter. However, the 382 strain was negative for the T7 amplicon, suggesting that a recombinational event occurred during the integration step of the transgene. Furthermore, considering the presence of a unique copy of the transgene in this strain, it is tempting to speculate that this event could have impeded the subsequent tail-to-head integrations. A finer investigation of the molecular structure of the T7 extremity of the transgene in this strain should clarify this point. However, and despite this event, all the Tg animals selected in our study were able to respond to the various biological properties of LPS and to the infection with S. typhimurium.

Using regression analysis, we showed that Tlr4 mRNA expression levels are linearly correlated to the logarithm of the number of Tlr4 copies integrated in the genome in spleen (R2 = 0.75), liver (R2 = 0.85), and lung (R2 = 0.84). The increase in the number of integrated Tlr4 transgenes results in an increase of the level of Tlr4 expression; however, over a certain number of copies, the transcriptional machinery seems to reach saturation in spleen, liver, and lung. The situation appears to be different in the heart where the level of expression of Tlr4 is linearly correlated to the number of integrated copies (R2 = 0.87). The levels of Tlr4 protein expression parallel the levels of mRNA expression and determine the ability of splenocytes to sense LPS and to respond to its mitogenic properties. A major increase in the host response of LPS was seen in vivo in mice carrying three and more copies of the Tlr4 transgene: overexpression of Tlr4 confers hypersensitivity to the lethal effects of LPS. These experiments support a crucial role of Tlr4 in the signaling pathway induced after LPS exposure with intensities of biological responses to endotoxin being strongly dependent on the level of Tlr4 expression.

The picture observed after infection with virulent S. typhimurium is clearly different. There is a good correlation between the bacterial loads determined by CFU analysis and the survival of the Tg animals. However, the protection conferred by Tlr4 transgene(s) as measured by survival analysis and bacterial load in liver and spleen is similar in all Tg strains tested on a C57BL10ScNCR (Nramp1Asp169) background. This observation could have several explanations. First, several additional molecules are involved in the Tlr4 signaling pathway. According to the classical picture of LPS recognition, this component binds to the serum protein LBP. LBP promotes the transfer of monomeric molecule of LPS from micelles to a membrane-bound or soluble protein named CD14. CD14 physically associates with a complex including Tlr4 and MD-2, an extracellular accessory protein. This results in Tlr4 activation and signaling through the commitment of MyD88, MyD88-adapter-like/Toll-IL-1R domain-containing adapter protein, Toll-interacting protein, and various intracellular kinases. Therefore, several molecular actors are involved in the activation and transduction of the LPS-induced signaling pathway (47, 48). These molecules could be present in limiting quantities, narrowing the effect of overexpressed Tlr4. It should be noted that recent work is in favor of the implication of several additional molecules for effective LPS recognition (11, 49). For example, fluorescence resonance energy transfer (FRET) studies realized on human monocytes revealed the association of LPS with a cluster of four molecules: heat shock proteins 70 and 90, and CXCR4 and growth differentiation factor 5 (50, 51). Another study revealed the presence of an activation cluster composed of CD14, TLR4, CD55, CD16a, CD11b/CD18, Fcg receptors CD32 and CD64, FcgRIIIa, CD36, and CD81 after LPS or lipoteichoic acid stimulation (52). All these actors may be able to limit the activation signal delivered by Tlr4 activation.

In contrast, the host response to living pathogen is extremely complex and involved other defense mechanisms through the involvement of Toll-like receptor (TLR)-dependent or -independent mechanisms. Such a mechanism is exemplified in our study by the observation of the presence of an epistatic interaction between Nramp1 and Tlr4. The impact of Tlr4 expression level on host resistance to Salmonella infection was seen only in the context of a resistance allele at Nramp1. The combined protective effect of Nramp1 and Tlr4 is largely more important than their independent effects. Nramp1 is a pH-dependent manganese transporter that is primarily expressed on the phagosomal membrane in macrophages (32). The current view on its function is that Nramp1 might modulate pathogen replication by altering the bacteriostatic and bactericidal properties of the fused phagosome through the efflux of divalent metals (33). In addition, Nramp1 has an impact on Salmonella containing vacuole maturation: Salmonella containing vacuoles formed in Nramp1-deficient macrophages fail to acquire mannose 6 phosphate receptor, a protein known to regulate the delivery of a subset of lysosomal enzymes from the trans-Golgi network to the prelysosomal compartment, thereby facilitating bacterial killing (53). It has been shown that Nramp1 mRNA expression is strongly induced by treatment with LPS and IFN-{gamma} in the mouse RAW264.7 macrophage cell line (54). Furthermore, macrophages expressing functional Nramp1 proteins exhibited enhanced responsiveness to several biological stimuli, including mycobacterial lipoarabinomannans, LPS, IFN-{gamma}, and glucocorticoids in comparison with macrophages defective for Nramp1 (55, 56, 57, 58).

The nature of the interaction between Nramp1 and Tlr4 is not clear at the moment but may be at the level of the phagosome. During phagocytosis, TLR are also recruited to the phagosome where they sense the nature of the pathogen (59). There is growing evidence that LPS internalization (60, 61, 62) and colocalization with TLR4 is necessary for activation of different cell types (63, 64), pointing out the importance of cellular internalization and cytoplasmic trafficking in the host response to LPS. In intestinal epithelial cells, LPS and Tlr4 reside in the Golgi apparatus (63). It is possible to envision that the interaction between Nramp1 and Tlr4 reside in a possible effect of Nramp1 on the cellular distribution of LPS/Tlr4 during the infectious process by preventing the transfer of LPS/Tlr4 to the phagosome from the Golgi.

The high level of Tlr4 expression in Nramp1Gly169/Asp169 Tlr4 Tg mice confers an advantage in the innate immune response to infection with S. typhimurium early during infection, but the important inflammatory response to the bacterial pathogen in these mice strains has detrimental effects later during infection and leads to the development of liver insufficiency and cardiac failure. The lesions observed in the spleen, liver, and heart later during infection are not characteristic of multiple organ failure due to shock. There was no macrophage infiltration in the kidney and heart nor evidence of abnormal cells in circulation, suggesting a local reaction at the level of the liver and spleen. The pathology is probably mediated not only by Tlr4 but also by Nramp1. In a chronic model of Salmonella infection, we have observed that cellular infiltration in the reticuloendothelial system is predominantly PMNs in Nramp1-/- mice compared with macrophages in Nramp1+/+ mice (65). The massive cellular infiltration in the spleen and liver may result from excessive release of cytokines acting on macrophage recruitment at the site of infection and persistence of infection in these two target organs. It is not clear at the moment whether Tlr4 overexpression has a causative or exacerbating role in cardiac pathology. The heart is a major site of Tlr4 expression both in mouse (25) and in humans (66) and LPS was reported to increase Tlr4 expression in cardiac myocytes (66). In addition, our study clearly show that Tlr4 expression in heart of Tg mice parallel linearly the number of integrated copies. Accumulation of LPS/Tlr4 regulated cytokines such as TNF and IL-1{beta}, and expression of NO synthase 2 and TLR (Toll-like receptor 2 and Tlr4) have been reported to be increased in hearts of patients with heart failure regardless of etiology and in the hearts of animals with experimental cardiac dysfunction (66, 67, 68). The potential involvement of Tlr4 in inflammatory processes has been documented in humans and is in agreement with the present study. Genetic variants at TLR4 (Asp229Gly and Thr399Ile) are associated with hyporesponsiveness to LPS in humans (69) and higher susceptibility to acute bacterial infections (70). In humans, the Asp299Gly TLR4 allele has a protective effect in the development of cardiovascular disease such as atherosclerosis (70). A more comprehensive examination of the mice surviving infection will be necessary to determine the exact mechanisms leading to the pathologic changes in the reticuloendothelial system and in heart.

A recent paper reports the creation of two other Tlr4 Tg strains (71). Although these strains were generated on a different genetic background (C57BL/10ScCr), the degree of LPS response in vivo and in vitro correlates well with the levels of Tlr4 mRNA expression as observed by ourselves. In addition, our study first demonstrated the correlation between levels of Tlr4 expression and the host response to Gram-negative infection and the functional interaction between two critical innate immune genes, Tlr4 and Nramp1 in mice. In conclusion, our data show that Tlr4 overexpression can elicit a systemic inflammatory response aimed at elimination of the invading pathogen; however, once triggered, this intense reaction follows an uncontrolled progression leading to serious tissue damage on the host. Tlr4 Tg mouse strains constitute a powerful tool in the analysis of the function of Tlr4 receptor in vivo during infection and inflammation. Selective modulation of Tlr4-induced inflammation to preserve its role in host defense while eliminating the associated self-injury would be a major breakthrough in the clinical management of sepsis.


   Acknowledgments
 
We thank Isabelle Maillet, Yannick Renaud, and Mi-Fong Tang for excellent technical advice; Daniel Houle for the generation of Tg mice; Jean-François Bureau, Silvia Vidal, Kenneth Morgan, Mary Stevenson, and J. C. Loredo-Osti for helpful discussions; and Ellen Buschman and Valerie Quesniaux for critical reading of the manuscript.


   Footnotes
 
1 This work was supported by grants from the Canadian Institutes of Health Research and the Howard Hughes Medical Institute. D.M. is a scholar of the Canadian Institutes of Health Research and an International Research Scholar of the Howard Hughes Medical Institute. 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: LBP, LPS binding protein; Tlr4, murine Toll-like receptor 4; TLR, Toll-like receptor; Nramp1, murine natural resistance associated macrophage protein 1; BAC, bacterial artificial chromosome; Tbp, murine TATA binding protein; PFGE, pulse field gel electrophoresis; Tg, transgenic; PMN, polymorphonuclear cell. Back

Received for publication October 24, 2002. Accepted for publication April 7, 2003.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
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