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* Department of Microbiology, University of Colorado Health Sciences Center, Denver, CO 80262;
Biotechnology Laboratory, University of British Columbia, Vancouver, British Columbia, Canada; and Departments of
Microbiology and
Laboratory Medicine, University of Washington School of Medicine, Seattle, WA 98195
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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TLRs have been implicated in such diverse processes as chemokine/cytokine production (7), phagocytic cell recruitment and function (8, 9), and the generation of adaptive immunity (10). The present study was undertaken to identify specific TLR4-dependent functions responsible for the enhanced susceptibility of C3H/HeJ mice to S. Typhimurium in vivo. Our findings indicate that TLR4 signaling controls diverse functions contributing to host resistance to Salmonella. Recognition of Gram-negative bacteria by TLR4 stimulates early expression of chemokines, recruitment, and activation of phagocytic cells, and the eventual establishment of cell-mediated adaptive immunity. In addition, we have demonstrated a critically important role for TLR4 in the activation of the Kupffer cell network to limit Salmonella replication in the liver before the recruitment of inflammatory cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Viability of wild-type C3H/HeN or congenic TLR4-deficient C3H/HeJ (The Jackson Laboratory, Bar Harbor, ME) mice was recorded daily after i.p. challenge with 
1000 CFUs of virulent S. Typhimurium strain 14028s (American Type Culture Collection, Manassas, VA) or an isogenic chloramphenicol resistance-cassette insertion in invA invasion gene (invA::cm) derivative (11). Selected groups of mice were euthanized by CO2 inhalation, and the livers were aseptically removed and homogenized in sterile PBS (12). Viable counts were determined after overnight culture on Luria-Bertani (LB) agar plates.
Macrophage killing assays
Peritoneal exudate cells from wild-type C3H/HeN mice or congenic TLR4-deficient C3H/HeJ mice were harvested 4 days after i.p. inoculation of 1 mg/ml sodium periodate, as described (13). The peritoneal exudate cells were resuspended in RPMI 1640 supplemented with 10% heat-inactivated FCS (Gemini Bio-Products, Calabassas, CA), 1 mM sodium pyruvate, 10 mM HEPES, and 2 mM L-glutamine (all from Sigma-Aldrich, St. Louis, MO). The macrophages were selected by adherence to a 96-well plate and cultured for 48 h at 37°C in a 5% CO2 incubator. Selected groups of macrophages were incubated with 20 U/ml murine IFN-
(Life Technologies, Grand Island, NY) 24 h before infection. The phagocytes were challenged with S. Typhimurium opsonized with 10% normal mouse serum at a multiplicity of infection of 10:1, allowed to internalize the bacteria for 15 min, and washed with prewarmed medium containing 6 µg/ml gentamicin. To estimate the contribution of NO to the anti-Salmonella activity of the macrophages, 250 µM N-monomethyl L-arginine was added to the phagocytes at the time of the infection. At different time points after challenge, the macrophages were lysed with 0.5% sodium deoxycholate and the surviving bacteria enumerated on LB agar plates.
Measurement of inducible NO synthase (iNOS) expression
Total RNA was isolated from sodium periodate-elicited macrophages using TRIzol (Invitrogen, Carlsbad, CA). ssDNA was synthesized from purified RNA by oligo(dT) priming and Moloney murine leukemia virus reverse transcriptase (Promega, Madison, WI). Expression of iNOS was monitored by PCR analysis of synthesized cDNA using primers 5'-CCTTCCGAAGTTT CTGGCAGCAGC and 5'-GGCTGTCAGAGCCTCGTGGCTTTGG with TaqDNA polymerase. Amplification of GAPDH was used as an internal control. Production of reactive nitrogen species by Salmonella-infected macrophages was determined by spectrophotometric detection of nitrite at 550 nm using the Griess reagent (0.5% sulfanilimide and 0.05% N-1-naphthylethylenediamide hydrochloride in 2.5% acetic acid) (13).
Measurement of chemokine expression
Total RNA was purified from 50–100 mg of liver tissue from Salmonella-infected C3H/HeN and C3H/HeJ mice using the TRIzol reagent. The RNA was treated with DNase I and purified further with the Qiagen RNeasy kit (Qiagen, Hilden, Germany). Single-stranded cDNA was synthesized from purified RNA, as above. Differential chemokine gene expression in the liver of C3H/HeN and C3H/HeJ mice was assessed at 6 and 24 h after Salmonella infection using the mouse chemokine GEArray kit, according to the instructions recommended by the manufacturer (Superarray, Frederick, MD).
Measurement of myeloperoxidase (MPO) activity
MPO was extracted from homogenized liver tissue using hexadecyltrimethylammonium bromide (Sigma-Aldrich). Enzymatic activity was recorded spectrophotometrically as the increase in A460 in a reaction containing 0.3% H2O2 and 0-dianisidine (Sigma-Aldrich). Units of MPO/g tissue/min were calculated using an extinction coefficient of 
= 13.5.
Histopathology and immunofluorescence microscopy
Hepatic tissue from Salmonella-infected wild-type C3H/HeN or TLR4-deficient C3H/HeJ mice was collected 0–24 h after i.p. infection, fixed in 10% formalin, stained in H&E, and examined by light microscopy for the presence of microabscesses and granulomas.
For immunofluorescence studies (14), wild-type C3H/HeN or TLR4-deficient C3H/HeJ mice were i.p. infected with 1000 CFU of S. Typhimurium 14028s and euthanized by CO2 inhalation 16 h after infection. Livers were aseptically dissected, rinsed in ice-cold TBS, and flash frozen in OCT compound (Sakura Finetech, Torrance, CA), using a combination of isopentane and liquid N2, and stored at –70°C. Serial sections were cut at a thickness of 15 µm and fixed in ice-cold acetone for 20 min. Tissues were blocked with 1% BSA in TBS with 0.1% Triton X-100. This was followed by addition of the primary Abs: rabbit anti-Salmonella (Difco, Detroit, MI) at a 1/500 dilution, and biotinylated mouse anti-cytokeratin 18 (Progen Biotechnik, Heidelberg, Germany) and rat anti-F4/80 (Serotec, Raleigh, NC) at a 1/100 dilution. Incubation with primary Abs was performed overnight (18 h) and followed by extensive washing with 0.1% Triton X-100 in TBS. The tissues were subsequently incubated with the secondary Abs, including Alexa Fluor 488 goat anti-rabbit (Alexis, San Diego, CA) and either Alexa Fluor 594 streptavidin or PE-Cy5 goat anti-rat (Cedarlane Laboratories, Hornby, Ontario, Canada) Abs (all at 1/200) for 4 h. Coverslips were mounted in Mowiol mounting medium (Sigma-Aldrich), and the tissues were viewed using a Zeiss (Oberkochen, Germany) Axiovert s100 TV microscope attached to a Bio-Rad Radiance Plus confocal microscope with a x63 oil objective. Images of 512 x 512 pixels (102 x 102 µm) were acquired using Bio-Rad Lasersharp software (Hercules, CA). Sections of 0.2 µm thickness were assembled into flat projections using NIH Image and imported into Adobe Photoshop (San Jose, CA). Liver tissues from six infected mice were studied, and >20 fields of view were assessed per mouse.
ELISPOT measurement of IFN--producing T lymphocytes
The frequency of Salmonella- or FliC (flagellin)-specific IFN--producing T lymphocytes in immunized wild-type or TLR4-deficient mice was determined by an ELISPOT assay essentially as previously described (15). Wild-type C3H/HeN or TLR4-deficient C3H/HeJ mice were injected with 5 x 107 heat-killed FliC-expressing S. Typhimurium via an i.p. route. Seven days after infection, spleen cells were harvested for ELISPOT assay to detect CD4+ T cells. Splenic CD4+ T cells were isolated by incubation on ice with FITC-conjugated anti-CD4 (BD PharMingen, San Diego, CA) before selection with a magnetic cell sorter, and stimulated for 48 h at 37°C with 5 x 106 irradiated syngeneic splenocytes as APCs plus 5 x 107/ml heat-killed S. Typhimurium, 100 µg/ml purified FliC (flagellar) protein, or no Ag. Cells were harvested with Histopaque (density 1.083; Sigma-Aldrich), seeded to ELISPOT plates coated with anti-IFN- (BD PharMingen), incubated for 18 h, and assayed for IFN- secretion using a biotinylated anti-IFN- secondary Ab and an avidin-HRP detection system (Sigma-Aldrich). IFN--producing spots were enumerated with a dissecting microscope.
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Results |
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TLR4-deficient C3H/HeJ mice succumbed within 4 days after i.p. infection with 1000–1500 CFU wild-type S. enterica strain ATCC 14028s (Fig. 1A), a sublethal challenge for the congenic wild-type C3H/HeN mice. The increased susceptibility of TLR4-deficient mice to salmonellosis was paralleled by the enhanced proliferation of bacteria in the livers of these mice (Fig. 1B). During the first 6 h after infection, the growth rate of Salmonella was comparable in wild-type and TLR4-deficient mice. However, C3H/HeJ mice were found to harbor 100- to 1000-fold more bacteria in their livers than wild-type controls by 24 h after the onset of infection. These observations indicate that TLR4 is essential for the development of an effective early innate immune response to Salmonella.
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Salmonella virulence in mice closely corresponds to the ability of the bacteria to survive in murine macrophages (16). Periodate-elicited peritoneal macrophages from TLR4-deficient mice were found to be as effective at killing Salmonella during the initial 6 h after phagocytosis as were wild-type cells (Fig. 2A). This suggests that TLR4 is not required for early NADPH phagocyte oxidase-mediated Salmonella killing. In fact, elicited macrophages from C3H/HeN and C3H/HeJ mice produced similar quantities of hydrogen peroxide and superoxide 1 h after infection with Salmonella as measured by HRP-dependent oxidation of phenol red and cytochrome c reduction (data not shown). However, TLR4-deficient macrophages were substantially impaired in their ability to restrict Salmonella growth at 20 h after infection, an action previously shown to be dependent on iNOS (13). Stimulation of TLR4-deficient macrophages with IFN- 20 h before Salmonella ingestion was able to completely restore the antibacterial activity of these cells at 20 h (Fig. 2A). The antibacterial defect of TLR4-deficient macrophages was specific for Gram-negative bacteria such as Salmonella, because these macrophages were able to kill Gram-positive Staphylococcus aureus as efficiently as wild type (Fig. 2A).
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treatment significantly increased the ability of both wild-type and TLR4-deficient macrophages to synthesize nitrite (Fig. 2B). Production of nitrite by Salmonella-infected macrophages from C3H/HeJ mice paralleled the presence of iNOS mRNA (Fig. 2C). Collectively, these observations demonstrate that the reduced anti-Salmonella activity of TLR4-deficient macrophages results from a failure of iNOS transcription and iNOS-dependent NO production. This is further substantiated by the ability of the NOS inhibitor NG-monomethyl-L-arginine to reduce the anti-Salmonella activity of wild-type macrophages at 20 h to the level of TLR4-deficient cells (Fig. 2D). Chemokine responses to Salmonella infection in TLR4-deficient mice
The preservation of early bacterial killing by macrophages with defective late NO-dependent antimicrobial actions was somewhat unexpected given the impaired ability of TLR4-deficient mice to limit bacterial replication during the initial 24 h of infection (Fig. 1A). Earlier work has suggested that iNOS only plays a significant role in host defense against Salmonella at significantly later time points (12). A further indication that absent iNOS activation could not account for the defective innate immunity of C3H/HeJ mice came from the measurement of iNOS mRNA in the livers of infected mice, which revealed almost no detectable iNOS expression in either wild-type or TLR4-deficient animals at the 24-h time point (data not shown). Moreover, administration of the iNOS inhibitor L-N6-(1-iminoethyl)lysine failed to increase 24-h organism burdens in C3H/HeN mice (data not shown). It was therefore considered whether defective recruitment of inflammatory cells might account for the early immune defect of TLR4-deficient animals observed despite preservation of early Salmonella killing mechanisms in peritoneal macrophages.
Binding of LPS by its TLR4 cognate receptor triggers a signaling cascade that stimulates the expression of multiple CXC chemokines, which leads to the recruitment of acute inflammatory cells to sites of infection. At 6 h after Salmonella inoculation, when the bacterial burden was similar in TLR4-deficient and wild-type animals (Fig. 1B), Gro1, IFN--inducible protein 10, and monokine induced by IFN- chemokines were preferentially expressed in wild-type C3H/HeN mice (Fig. 3A). However, robust levels of Gro1, IFN--inducible protein 10, monokine induced by IFN-, and macrophage-inflammatory protein 2 chemokine mRNA were observed in the livers of TLR4-deficient animals by 24 h after infection. The chemokine response observed at the later time point in C3H/HeJ mice is likely to reflect the induction of TLR4-independent signaling pathways by the greater bacterial burden present in the immunodeficient mice. Of interest, hepatic expression of the chemokine macrophage-inflammatory protein 2 associated with neutrophil recruitment was detected at 6 h in livers of Salmonella-infected wild-type mice, but not of their TLR4-deficient counterparts. As neutrophils are the initial inflammatory cells recruited to the site of Salmonella infection (14) and are highly efficient at killing Salmonella (17), it was further considered whether defective recruitment of neutrophils might contribute to the hypersusceptibility of TLR4-deficient mice to infection with Gram-negative bacteria.
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Histopathological examination of H&E-stained liver sections at 24 h after infection demonstrated the presence of small microabscesses packed with neutrophils throughout the hepatic parenchyma of TLR4-deficient C3H/HeJ mice (Fig. 3B), in marked contrast to published studies of invasive E. coli infection in C3H/HeJ mice (6), in which no neutrophilic infiltration was observed. Presence of microabscesses was not evident in the livers of wild-type C3H/HeN mice until 3 days after infection (data not shown). Infiltration of neutrophils in the hepatic parenchyma of wild-type and TLR4-deficient mice was independently assessed by monitoring MPO activity. In agreement with the histopathological evidence of a neutrophilic response in C3H/HeJ mice, abundant MPO activity was detected within the livers of TLR4-deficient mice (Fig. 3C) following perfusion. Thus, an initial delay in chemokine production is rapidly compensated for in TLR4-deficient mice, with a resulting exuberant recruitment of polymorphonuclear leukocytes to the site of Salmonella infection exceeding the magnitude of response seen in wild-type animals. Despite the delayed CXC chemokine response, the hypersusceptibility of C3H/HeJ mice to acute Salmonella infection could not be attributed to a poor neutrophilic response.
Localization of Salmonella in hepatic parenchyma of TLR4-deficient mice
Somewhat paradoxically then, TLR4-deficient mice are deficient in early innate immunity to Salmonella despite a robust acute inflammatory cell response and preserved early antibacterial actions of inflammatory phagocytes. Studies were therefore conducted to determine the localization of Salmonella within the liver during the 6- to 24-h period of enhanced bacterial proliferation and before the recruitment of acute inflammatory cells.
Salmonella were directly visualized by fluorescence and confocal microscopy in relation to F4/80-expressing Kupffer cells and cytokeratin 18-expressing hepatocytes 16 h after i.p. infection. Confocal microscopy confirmed intracellular localization of the organisms. No bacteria could be visualized in the livers of wild-type mice at this early time point. However, numerous Salmonella were found to localize within Kupffer cells in the livers of TLR4-deficient mice (Fig. 4), demonstrating an important role of LPS-TLR4 signaling pathways in triggering innate antibacterial actions of Kupffer cells before the recruitment of inflammatory cells. Salmonella organisms within Kupffer cells were clustered in discrete loci throughout the liver, suggesting proliferation and localized spread within the Kupffer cell network. When higher organism burdens were present, a few bacteria could also be observed within hepatocytes (data not shown).
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To distinguish the importance of pathogen-directed and phagocyte-directed mechanisms of bacterial internalization, infection studies were repeated using noninvasive invA::cm and congenic wild-type Salmonella in wild-type and TLR4-deficient mice. The inability to invade nonphagocytic cells did not impair the ability of S. Typhimurium to proliferate in the livers of TLR4-deficient C3H/HeJ mice (Fig. 5), providing additional support for the microscopic evidence that hepatic bacteria were principally replicating within the Kupffer cell network. A modest trend toward reduced survival of invA::cm mutant bacteria relative to wild-type S. typhimurium was observed in TLR4-proficient C3H/HeN mice. This may indicate that invA-dependent cytotoxicity for macrophages (11) can partially counter the TLR4-dependent host response.
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An important function of TLRs, in addition to the control of innate immunity, is to provide a linkage to adaptive immune responses (18, 19). The principal mechanism by which TLRs are believed to promote adaptive immune responses is through the induction of costimulatory ligand and cytokine responses in APCs. The generation of adaptive cellular immunity to Salmonella Ags was therefore compared in wild-type and TLR4-deficient mice. To negate the influence of impaired innate immunity on Ag load, heat-killed Salmonella was used as an immunogen.
Seven days after i.p. administration of FliC (flagellar Ag)-expressing heat-killed Salmonella, a significantly greater number of IFN--producing CD4+ T cells responding to Salmonella or purified FliC was observed in the spleens of wild-type mice in comparison with TLR4-deficient mice (Fig. 6), indicating that LPS-TLR4 signaling modulates the generation of adaptive immune responses to Salmonella.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The present study analyzed TLR4-dependent elements of innate and adaptive immunity in the well-characterized Salmonella murine infection model. A single Pro712His mutation in TLR4 (3) has profound and pleiotropic effects on the resistance of C3H/HeJ mice to Salmonella infection. TLR4 plays an important role in both early and late immune responses in vivo, ranging from chemokine production to phagocyte activation and the elicitation of adaptive immunity. Most significantly, our studies demonstrate that LPS-TLR4 interactions are required for the initial limitation of bacterial replication within host tissues before the arrival of inflammatory cells. This innate antimicrobial function appears to be principally mediated by tissue histiocytes (e.g., Kupffer cells of the liver), with a possible secondary contribution from parenchymal cells (e.g., hepatocytes). This contrasts markedly with earlier observations in immunocompetent mice, in which hepatic Salmonella were found within infiltrating phagocytic cells (14) rather than resident cells, and only at later time points. Enhanced replication of Salmonella in the livers of C3H/HeJ animals was not abrogated by an invA mutation abolishing Salmonella invasiveness for nonphagocytic cells, further substantiating that bacteria in the liver are principally replicating within the Kupffer cell network of TLR4-deficient mice.
Along with a recent study using an experimental urinary tract infection model (22), the present observations reinforce that resident cells in peripheral tissues contribute more to TLR-dependent host resistance than is generally appreciated. However, in contrast to the urinary tract model, the present study demonstrates that TLR4 can regulate direct antimicrobial actions in peripheral tissues in addition to promoting the recruitment of inflammatory cells. Both Kupffer cells and hepatocytes have been shown to express TLR4 (23, 24), and TLR4-deficient Kupffer cells have been found to be deficient in their ability to kill ingested E. coli (6), but the mechanisms of TLR4-dependent antimicrobial actions in Kupffer cells and hepatocytes remain to be established.
In TLR4-deficient mice infected with S. Typhimurium 14028s, the critical inadequacy appears to be in the timely generation of immune responses. Although compensatory pathways ultimately stimulate chemokine production, inflammatory cell recruitment, or phagocyte activation, in each case the response occurs later than would have been the case in the presence of TLR4, with the ultimately lethal consequence of a higher microbial burden. TLR4-deficient mice exhibit a potent, if somewhat belated chemokine response (Fig. 3A), possibly reflecting compensation by other TLRs, but the ensuing influx of inflammatory cells (Fig. 3B) was ineffective in controlling the infection. Although the lack of TLR4-dependent iNOS stimulation (25, 26) can be compensated by the addition of IFN- in vitro (Fig. 2A), this necessitates a costly delay in macrophage activation until the arrival of cytokine-producing cells in vivo. Similarly, TLR4-deficient mice can generate protective adaptive immune responses to vaccination (27), but the kinetics of the adaptive responses are protracted (Fig. 6).
Compensation for TLR4 deficiency by IFN- can be rationalized by the importance of both NF 
B and transcription factor STAT1 activation for iNOS expression. TLR4 agonists by themselves are believed to be sufficient for iNOS stimulation because TLR4-dependent signaling leads to both NF-B activation and endogenous production of IFN-
, leading to STAT1 phosphorylation (28) and iNOS transcription via IFN regulatory factor-1 (29, 30). TLR2 stimulation, in contrast, does not result in iNOS stimulation unless IFN- or - to activate STAT1 (28, 31) is provided exogenously.
Although C3H/HeJ mice are unable to respond to LPS via TLR4, their ability to recognize bacterial ligands via other TLRs such as TLR2, TLR5, and TLR9 remains intact. Mice infected with Salmonella begin to produce IFN- 3–5 days after the onset of infection (32), and TLR4-deficient mice retain the ability to produce IFN- in response to Salmonella (33). Sources of IFN- include NK cells (34), which can be activated by stimulation of receptors such as TLR2 (35). Although the production of IFN- can eventually overcome the absence of LPS-TLR4 signaling for the stimulation of iNOS expression, the delayed synthesis of NO is likely to be detrimental to the infected host.
Although the various TLRs can provide a host with versatility through the recognition of a wide range of pathogenic microorganisms, each TLR may require a different level of organism burden for a given pathogen to reach the critical threshold for activation. The subtle or absent phenotypes of mice lacking single TLRs in some infection models (36) have led to the suggestion that these receptors may be functionally redundant. However, the ability of TLR4 to sense minute quantities of LPS provides the host with a highly sensitive and essential system to detect the presence of Salmonella or other Gram-negative bacterial pathogens that cannot be fully supplanted by the recognition of alternative ligands.
The role of LPS-TLR4 signaling in the development of lethal endotoxic shock (37) has led to the suggestion that TLR blockade may be useful in the treatment of sepsis (38). However, the multitude of essential immune functions dependent on TLRs as demonstrated in this study suggests that targeting TLRs in the setting of acute bacterial infection will have to be undertaken with great caution.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Ferric C. Fang, Department of Microbiology, University of Washington School of Medicine, Box 357242, Seattle, WA 98195-7242. E-mail address: fcfang{at}washington.edu
3 Abbreviations used in this paper: TLR, Toll-like receptor; S. Typhimurium, Salmonella enterica serovar Typhimurium; FliC, flagellin; iNOS, inducible NO synthase; LB, Luria-Bertani; MPO, myeloperoxidase; invA::cm, chloramphenicol resistance-cassette insertion in invA invasion gene.
Received for publication November 13, 2003. Accepted for publication March 3, 2004.
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-F4/80) in the livers of TLR4-deficient mice (A). Hepatocytes were labeled with 
