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,
,,
,¶,¶,
*
Institute for Human Gene Therapy, Departments of
Molecular and Cellular Engineering and
Microbiology, and
Wistar Institute, University of Pennsylvania, and
¶ Department of Pediatrics, Children’s Hospital of Philadelphia, Philadelphia, PA 19104
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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,
IL-6, TNF-
, macrophage-inflammatory protein (MIP)-1, and MIP-2 in
bronchoalveolar lavage and a corresponding mobilization of
intrapulmonary neutrophils. Congenic strains of mice deficient in TLR4
demonstrated a substantial delay in clearance of H.
influenzae with diminished IL-1, IL-6, TNF-, MIP-1,
and MIP-2 in bronchoalveolar lavage and a notable absence of
intrapulmonary neutrophils. In TLR4-expressing animals, but not
TLR4-deficient animals, TNF- and MIP-1 expression was
up-regulated in epithelial cells of the conducting airway in response
to H. influenzae which was preceded by an apparent
activation of the NF-
B pathway in these cells based on the findings
of decreased overall IB and an increase in its phosphorylated form.
This study demonstrates a critical role of TLR4 in mediating an
effective innate immune response to H. influenzae in the
lung. This suggests that the airway epithelia might contribute to
sensing of H. influenzae infection and signaling the
innate immune response. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Effector functions involved in pulmonary defense have been the subject of intensive investigation. The airway surface fluid contains a mixture of molecules which have direct antimicrobial activity such as lysozyme, lactoferrin, phospholipase A, antimicrobial peptides, and surfactant (1). The mechanical elimination of pathogens through mucociliary clearance also contributes to host defense. Cells resident to the noninfected lung, such as alveolar macrophages, secrete cytokines and chemokines in response to infection. Secretion of macromolecules rapidly mobilizes the influx of neutrophils and leads to a second wave of innate immunity. Finally, pathogen-derived macromolecules are processed by APCs to activate B and T cells as effectors of acquired immunity.
Mechanisms for activating innate immunity and initiating the adaptive immune response are less well defined. A variety of cells express proteins called pattern recognition receptors that broadly recognize conserved structures in microorganisms called pathogen-associated molecular patterns (PAMPs)3 (2). Examples of pattern recognition receptors include CD14 and complement receptors (3, 4). The recent identification of mammalian homologs of the Drosophila gene product dtoll, called Toll-like receptors (TLRs), has revealed a step in the activation of innate immunity and its relationship to adaptive immunity (5).
A family of mammalian TLRs has been identified, each
exhibiting substantial homology to type-I IL-1R (6, 7, 8, 9).
Binding of PAMPs to TLRs activates a number of signaling pathways
(10, 11). TLR4 recognizes molecules of Gram-negative
bacteria (e.g., LPS) (12, 13); TLR2 binds to components of
Gram-positive bacteria (e.g., peptidoglycans and lipoteichoic acid)
(14, 15), mycobacteria (16) and fungi
(17); and TLR9 recognizes nucleic acid motifs
(18). The net result of PAMP-TLR engagement is activation
of intracellular signaling pathways such as NF-B and expression of
genes involved in innate immunity (e.g., defensins and chemokines), and
the initiation of adaptive immunity (e.g., CD86) (5).
Studies to date have focused on the role of TLRs in the initiation of host responses in the context of hematopoietic-derived cells such as macrophages (16, 17), dendritic cells (19, 20), and B and T lymphocytes (21). More recent studies have demonstrated expression of TLRs on endothelial cells (22, 23), cardiac myocytes (24, 25), and gut epithelial cells (26, 27), although the role TLRs play in these nonhematopoietic cells is unclear.
The current study focuses on the role of TLR4 in mediating the innate immune response in murine lung to Haemophilus influenzae as a model pulmonary pathogen.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A kanamycin-resistant encapsulated type-b strain (Eagan) H. influenzae H338 was grown to an OD620 of 0.4 in brain heart infusion broth supplemented with 2% (v/v) Fildes Enrichment (BD Biosciences, Cockeysville, MD), 2 µg/ml NAD (Sigma-Aldrich, St. Louis, MO), and 20 µg/ml kanamycin sulfate (Sigma-Aldrich) to mid-log phase, recovered by centrifugation, and resuspended in PBS (28).
Animal models
C3H/HeN and C57BL/10ScCr mice were purchased from the National Cancer Institute (Bethesda, MD). C3H/HeJ and C57BL/10ScSn mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Age-matched male mice were used for experiments. Anesthetized mice suspended vertically were administered H. influenzae intranasally at a dose of 1 x 105 CFU in 50 µl of sterile PBS.
Preparation of BAL and lung homogenate
Mice were anesthetized and the trachea was exposed with a midline superficial incision. A 20-gauge catheter (BD Biosciences) attached to a 1-ml syringe (BD Biosciences) was used to canulate the trachea, and the lungs were lavaged twice with 500 µl of PBS+ (PBS plus 5 mM EDTA plus 1% protease inhibitor (P8340; Sigma-Aldrich)) chilled to 4°C; lungs were then harvested and homogenized in 1 ml of PBS.
Bacterial counts
Bacteria were quantified by plating 10-µl serial dilutions of the bronchoalveolar lavage (BAL) or lung homogenates onto brain heart infusion agar plates (BD Biosciences) containing 1.5% Fildes Enrichment, 2 µg/ml NAD, and 20 µg/ml kanamycin sulfate. Plates were incubated overnight and colonies were counted. The selectable marker allowed for the elimination of contaminating flora.
Cell differential
Cells in the BAL were collected by centrifugation (2000 rpm at 4°C for 10 min), resuspended in PBS plus 4% BSA, transferred onto glass slides by using a Cytospin 3 centrifuge (Thermo Shandon, Pittsburgh, PA) for 15 min at 1500 rpm, and stained with KWIK DIFF staining kit (Thermo Shandon). At least 100 cells were examined microscopically from each sample.
Cytokines
The BAL supernatants were stored at -70°C for the ELISA
analysis of cytokines and chemokines. IL-1, IL-4, IL-6, IL-10,
TNF-, and monocyte chemoattractant protein (MCP)-1 were analyzed by
ELISA (BioSource International, Camarillo, CA) according to the
manufacturer’s instructions. Macrophage-inflammatory protein
(MIP)-1 and MIP-2 were also analyzed by ELISA (R&D Systems,
Minneapolis, MN) according to the manufacturer’s instructions.
Immunocytochemistry
Abs for immunocytochemistry included anti-IB-,
anti-P-IB-, anti-IL-1-, anti-IL-4, anti-IL-6,
anti-IL-10, anti-TNF-, anti-MIP-1, anti-MCP-1,
anti-MIP-2, anti-IP-10 (Santa Cruz Biotechnology, Santa Cruz,
CA), anti-monocyte/macrophage (MOMA)-2 (Serotec, Oxford, U.K.).
All Abs were diluted according to the instructions of the manufacturer.
Abs directed against phosphorylated IB- were developed in the
mouse, anti-IB- Abs were developed in the rabbit, and
anti-MOMA-2 Abs were developed in the rat; the other Abs were
developed in the goat.
Serial cryostat sections (10 µm) were fixed for 5 min in acetone. Blocking was performed by a 1-h incubation with 10% goat serum or donkey serum, depending on the origin of the secondary Ab. Sections were incubated for 1 h with the primary Ab at room temperature followed by washes with PBS and an incubation with 1) 1/100 diluted rhodamine-labeled goat anti-mouse IgG (Sigma-Aldrich), 2) 1/100 diluted fluorescein- or rhodamine-labeled rabbit anti-goat IgG (Sigma-Aldrich), 3) 1/100 diluted fluorescein-labeled goat anti-rabbit IgG (Sigma-Aldrich), or 4) 1/100 diluted fluorescein-labeled sheep anti-rat (Serotec). Double immunostaining was also performed according to a modified protocol in which each segmented incubation was followed by extensive washing in PBS. Specimens were examined under a fluorescence microscope (Nikon Microphot-FXA; Nikon, Melville, NY). Negative controls consisted of preincubation with PBS, omission of the primary Ab, and substitution of the primary Ab by a nonimmune control serum. The three types of controls mentioned above were performed for each experiment on the same day.
Morphometry
The number of cytokine-positive cells among MOMA-2-positive cells were assessed in TLR4+ and TLR4- animals. Cytokine-positive cells among MOMA-2-positive cells were counted in cross-sections of lung appropriately stained (double immunofluorescence), and the number of cytokine-positive cells was calculated as a percentage of at least 200 MOMA-2-positive cells from at least five randomly chosen different regions within a section of the lung by using an image analyzer (Phase Three Imaging Systems; Mediacybernetics, Des Moines, IA). Each group consisted of two or more animals.
Statistical analyses
Data were analyzed for statistical significance using Student’s t test or Mann-Whitney rank sum test using SigmaStat 2.03 (SPSS, Chicago, IL). Differences were considered significant for p < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B pathway. Experiments were performed in two strains of mice (i.e., C3H/HeN and C57BL/10 ScSn) and in congenic lines deficient in TLR4 (i.e., C3H/HeJ and C57BL/10 ScCr), which have previously been shown to be hyporesponsive to LPS. C3H/HeJ carries a missense mutation (P712H) in the TLR4 gene while a deletion in C57BL/10 ScCr ablates TLR4 protein and its mRNA (29). The mice will be referred to subsequently as HeN+, HeJ-, ScSn+, and ScCr-, with the superscript indicating the TLR4 status.
H. influenzae was administered via intranasal administration
and lung was harvested at 12 and 24 h for recovery of the bacteria
(Fig. 1
). Pilot experiments indicated
that >90% of the bacteria present in a total lung homogenate was
found in BAL from both TLR4-expressing and TLR4-deficient animals;
subsequent analyses of bacteria clearance were based on measurements
from BAL. Studies in TLR4-expressing mouse lines revealed a decrease in
recovered bacteria over 24 h with more effective clearance
observed in the ScSn+ line when compared with
HeN+. This finding contrasts with the findings in
both TLR4-deficient lines, in which the presence of H.
influenzae either did not decline as in
ScCr- or actually increased 50-fold over input
in HeJ-. Differences in recovered bacteria
between HeN+ and HeJ- were
statistically significant at both 12 h (p
= 0.002) and 24 h (p < 0.001), while
statistical significance was achieved in ScSn+ vs
ScCr- at 24 h (p =
0.001).
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, IL-4, Il-6,
IL-10, TNF-, MCP-1, MIP-1, and MIP-2, as well as cell counts and
differentials. Time course studies revealed peak levels of cytokines
12 h after exposure to H. influenzae in all cases
except for IL-1, in which highest expression was noted at 24 h
(Fig. 2). Analysis of BAL failed to
reveal detectable levels of IL-4, IL-10, or MCP-1. The most striking
finding is that cytokine/chemokine responses were consistently blunted
in TLR-deficient strains as compared with the congenic TLR-expressing
strains (Fig. 2). The reduction in HeJ- as
compared with HeN+ ranged from 2-fold for IL-6
(p = 0.044) to 9-fold for MIP-1
(p = 0.065); reductions observed in
ScCr- relative to ScSn+
were more substantial, ranging from 12-fold for IL-6
(p = 0.009) to 40-fold for TNF-
(p = 0.004).
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2-fold in TLR4-deficient
strains as compared with the TLR4-expressing strains, with highest
counts found at 24 h (data not shown). Distribution of cell types
in BAL differed substantially as a function of TLR4 expression (Fig. 3). In each case, the majority of cells
were identified as macrophages and neutrophils. A neutrophil-dominated
cellular infiltrate was found in HeN+ and
ScSn+, with 80 and 71% of the total cells being
neutrophils, respectively. This finding contrasts with the findings in
HeJ- and ScCr-, in which
macrophages dominated, comprising 87 and 94% of the total cells,
respectively. Differences between TLR-expressing lines and
TLR-deficient lines were statistically significant
(p < 0.001). PBS used to dilute
bacteria for instillation does not itself induce any
inflammation (data not shown).
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Cytokine/chemokine expression in macrophages was demonstrated by
two-color immunofluorescence using MOMA-2+ as a
marker for macrophages. An attempt was made to quantify expression by
counting all MOMA-2+ cells with detectable
cytokine/chemokine proteins, although this assay clearly was not
quantitative (Table I). All
cytokines/chemokines detected in BAL were indeed observed in
macrophages. In some cases, expression was diminished in
TLR4-expressing cells as compared with TLR4-deficient cells (Table I).
Analysis of MIP-2 expression in representative macrophages is shown in
Fig. 4. In both TLR-expressing strains,
MIP-2 expression was found in more macrophages than that which was
observed in TLR-deficient cells. Immunohistochemical analysis of lung
tissues also demonstrated expression of TNF and MIP-1 in epithelia
of the conducting airway of HeN+ and
ScSn+ mice 12 h after exposure to H.
influenzae; however, the peak expression was diminished in the
corresponding TLR-deficient strains HeJ- and
ScCr- (Fig. 5A).
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B-, which is
critical to the control of the NF-B pathway. This analysis focused
on early time points after infection (i.e., 3, 6, and 12 h) to
capture the initial response of the epithelia to bacterial challenge.
Following exposure to H. influenzae, airway epithelia from
HeN+ and ScSn+ showed an
increase in the level of phosphorylated IB- and a diminished
overall IB- (Fig. 5
B) relative to preinfection levels
(data not shown); these changes in IB- status were not observed
after infection of HeJ- and
ScCr-. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B pathway in response to LPS, which is an
important structural component of Gram-negative bacteria (12, 13). Importantly, two strains of mice deficient in TLR4, along
with congenic normals, are available for in vivo studies
(29). Our studies demonstrate a critical role of TLR4 in sensing and affecting the initial innate immune response to an intrapulmonary challenge with H. influenzae. Production of proinflammatory cytokines in the lungs of normal mice following H. influenzae is substantially diminished in TLR4-deficient mice, and the neutrophilic response is blunted. These TLR4-dependent abnormalities in innate immunity correlated with delays in bacterial clearance. Importantly, these findings in host response were demonstrated in two distinct TLR4-deficient lines of mice, arguing against genetic differences other than TLR4 deficiency as the basis for defective innate immunity within the congenic series. Direct comparison between the different genetic backgrounds studied (i.e., C3H vs C57BL/10) is difficult because they differ at many genetic loci. Previous studies regarding in vivo function of TLR4 have characterized host responses to purified bacterial components such as LPS or to bacterial challenge outside the lung (32). Kurt-Jones et al. (33) did show delayed clearance of the viral respiratory pathogen respiratory syncytial virus from TLR4-deficient mice.
In further characterizing the role of TLR4 in the lung, we attempted to evaluate the relative contribution of the airway epithelia vs resident immune effector cells such as macrophages. Previous studies have focused on the role of TLRs in dendritic cells (19, 20), macrophages (16, 17), and lymphocytes (21) as sensors and modulators of the immune response. While TLRs have been expressed in nonhematopoietic cells such as cardiac muscle (24, 25), endothelial cells (22, 23), and gut epithelial cells (26, 27), the role they play at these sites has not been defined.
Our hypothesis was that the airway epithelium would indeed serve as an
important sensor and transducer of the innate immune response to
H. influenzae challenge. Previous studies have demonstrated
induction of the NF-B pathway in cultured airway epithelial cells in
response to bacteria and proinflammatory cytokines (34, 35). More recently, we showed expression of TLR2 and TLR4 in
human airway epithelial cells and have linked the activation of TLR2
cells by PAMPs of Gram-positive bacteria to signaling of NF-B and
activation of an inducible defensin gene (our unpublished
observation). In this study, immunohistochemical studies
localized the production of proinflammatory cytokines to both the
resident macrophages and epithelia of the conducting airway from the
bronchi to terminal bronchioles. Furthermore, H. influenzae
challenge resulted in a brisk activation of the NF-B pathway in the
airway that was TLR4 dependent.
Our studies demonstrate a critical role of TLR4 in the early host response to H. influenzae in the lung and implicate both the alveolar macrophages and conducting airway epithelia in sensing and modulating the initial response. The slight differences in the expression of cytokines/chemokines in the alveolar macrophage between wild-type and mutant mice emphasize the role of the airway epithelium in modulating the early host response to H. influenzae infection. We have not assessed quantitatively the contribution of each cell type to the innate immune response, although both appear to be TLR4 dependent. Recently, TLR2 was implicated directly in the effector response of macrophages to Mycobacterium tuberculosis, expanding its potential role beyond sensing and signaling (36). One does not need to invoke this kind of direct role of TLR4 in killing of H. influenzae by macrophages or neutrophils, because the defective mobilization of proinflammatory cytokines and neutrophils may be sufficient to explain delayed clearance of the pathogen.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. James M. Wilson, 204 Wistar Institute, University of Pennsylvania, 3601 Spruce Street, Philadelphia, PA 19104. E-mail address: wilsonjm{at}mail.med.upenn.edu
3 Abbreviations used in this paper: PAMP, pathogen-associated molecular pattern; BAL, bronchoalveolar lavage; TLR, Toll-like receptor; MIP, macrophage-inflammatory protein; MOMA, monocyte/macrophage; MCP, monocyte chemoattractant protein.
Received for publication August 13, 2001. Accepted for publication November 6, 2001.
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R. Ramphal, V. Balloy, M. Huerre, M. Si-Tahar, and M. Chignard TLRs 2 and 4 Are Not Involved in Hypersusceptibility to Acute Pseudomonas aeruginosa Lung Infections J. Immunol., September 15, 2005; 175(6): 3927 - 3934. [Abstract] [Full Text] [PDF]
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 H.-Y. Cho, A. E. Jedlicka, R. Clarke, and S. R. Kleeberger Role of Toll-like receptor-4 in genetic susceptibility to lung injury induced by residual oil fly ash Physiol Genomics, June 16, 2005; 22(1): 108 - 117. [Abstract] [Full Text] [PDF]
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C. S. Berenson, T. F. Murphy, C. T. Wrona, and S. Sethi Outer Membrane Protein P6 of Nontypeable Haemophilus influenzae Is a Potent and Selective Inducer of Human Macrophage Proinflammatory Cytokines Infect. Immun., May 1, 2005; 73(5): 2728 - 2735. [Abstract] [Full Text] [PDF]
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E. Lorenz, D. C. Chemotti, A. L. Jiang, and L. D. McDougal Differential Involvement of Toll-Like Receptors 2 and 4 in the Host Response to Acute Respiratory Infections with Wild-Type and Mutant Haemophilus influenzae Strains Infect. Immun., April 1, 2005; 73(4): 2075 - 2082. [Abstract] [Full Text] [PDF]
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 A. J. Ratner, E. S. Lysenko, M. N. Paul, and J. N. Weiser Synergistic proinflammatory responses induced by polymicrobial colonization of epithelial surfaces PNAS, March 1, 2005; 102(9): 3429 - 3434. [Abstract] [Full Text] [PDF]
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D. P. Widney, Y. Hu, A. K. Foreman-Wykert, K. C. Bui, T. T. Nguyen, B. Lu, C. Gerard, J. F. Miller, and J. B. Smith CXCR3 and Its Ligands Participate in the Host Response to Bordetella bronchiseptica Infection of the Mouse Respiratory Tract but Are Not Required for Clearance of Bacteria from the Lung Infect. Immun., January 1, 2005; 73(1): 485 - 493. [Abstract] [Full Text] [PDF]
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P. B. Mann, K. D. Elder, M. J. Kennett, and E. T. Harvill Toll-Like Receptor 4-Dependent Early Elicited Tumor Necrosis Factor Alpha Expression Is Critical for Innate Host Defense against Bordetella bronchiseptica Infect. Immun., November 1, 2004; 72(11): 6650 - 6658. [Abstract] [Full Text] [PDF]
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M. Laan, S. Bozinovski, and G. P. Anderson Cigarette Smoke Inhibits Lipopolysaccharide-Induced Production of Inflammatory Cytokines by Suppressing the Activation of Activator Protein-1 in Bronchial Epithelial Cells J. Immunol., September 15, 2004; 173(6): 4164 - 4170. [Abstract] [Full Text] [PDF]
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H. P. Jia, J. N. Kline, A. Penisten, M. A. Apicella, T. L. Gioannini, J. Weiss, and P. B. McCray Jr. Endotoxin responsiveness of human airway epithelia is limited by low expression of MD-2 Am J Physiol Lung Cell Mol Physiol, August 1, 2004; 287(2): L428 - L437. [Abstract] [Full Text] [PDF]
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S. J. Skerrett, H. D. Liggitt, A. M. Hajjar, R. K. Ernst, S. I. Miller, and C. B. Wilson Respiratory epithelial cells regulate lung inflammation in response to inhaled endotoxin Am J Physiol Lung Cell Mol Physiol, July 1, 2004; 287(1): L143 - L152. [Abstract] [Full Text] [PDF]
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 M. G. Netea, C. van der Graaf, J. W. M. Van der Meer, and B. J. Kullberg Toll-like receptors and the host defense against microbial pathogens: bringing specificity to the innate-immune system J. Leukoc. Biol., May 1, 2004; 75(5): 749 - 755. [Abstract] [Full Text] [PDF]
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S. J. Skerrett, H. D. Liggitt, A. M. Hajjar, and C. B. Wilson Cutting Edge: Myeloid Differentiation Factor 88 Is Essential for Pulmonary Host Defense against Pseudomonas aeruginosa but Not Staphylococcus aureus J. Immunol., March 15, 2004; 172(6): 3377 - 3381. [Abstract] [Full Text] [PDF]
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S. Fernandez, P. Jose, M. G. Avdiushko, A. M. Kaplan, and D. A. Cohen Inhibition of IL-10 Receptor Function in Alveolar Macrophages by Toll-Like Receptor Agonists J. Immunol., February 15, 2004; 172(4): 2613 - 2620. [Abstract] [Full Text] [PDF]
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J. Branger, S. Knapp, S. Weijer, J. C. Leemans, J. M. Pater, P. Speelman, S. Florquin, and T. van der Poll Role of Toll-Like Receptor 4 in Gram-Positive and Gram-Negative Pneumonia in Mice Infect. Immun., February 1, 2004; 72(2): 788 - 794. [Abstract] [Full Text] [PDF]
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S. C. Higgins, E. C. Lavelle, C. McCann, B. Keogh, E. McNeela, P. Byrne, B. O'Gorman, A. Jarnicki, P. McGuirk, and K. H. G. Mills Toll-Like Receptor 4-Mediated Innate IL-10 Activates Antigen-Specific Regulatory T Cells and Confers Resistance to Bordetella pertussis by Inhibiting Inflammatory Pathology J. Immunol., September 15, 2003; 171(6): 3119 - 3127. [Abstract] [Full Text] [PDF]
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 W. E. Swords, P. A. Jones, and M. A. Apicella Review: The lipo-oligosaccharides of Haemophilus influenzae: an interesting array of characters Innate Immunity, June 1, 2003; 9(3): 131 - 144. [Abstract] [PDF]
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R. B. Oliveira, M. T. Ochoa, P. A. Sieling, T. H. Rea, A. Rambukkana, E. N. Sarno, and R. L. Modlin Expression of Toll-Like Receptor 2 on Human Schwann Cells: a Mechanism of Nerve Damage in Leprosy Infect. Immun., March 1, 2003; 71(3): 1427 - 1433. [Abstract] [Full Text] [PDF]
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S. Yi, W. J. Hawthorne, A. M. Lehnert, H. Ha, J. K. W. Wong, N. van Rooijen, K. Davey, A. T. Patel, S. N. Walters, A. Chandra, et al. T Cell-Activated Macrophages Are Capable of Both Recognition and Rejection of Pancreatic Islet Xenografts J. Immunol., March 1, 2003; 170(5): 2750 - 2758. [Abstract] [Full Text] [PDF]
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H. Weighardt, S. Kaiser-Moore, R. M. Vabulas, C. J. Kirschning, H. Wagner, and B. Holzmann Cutting Edge: Myeloid Differentiation Factor 88 Deficiency Improves Resistance Against Sepsis Caused by Polymicrobial Infection J. Immunol., September 15, 2002; 169(6): 2823 - 2827. [Abstract] [Full Text] [PDF]
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D. Warburton Sound the Tocsin! Beware Adverse Effects of Lung Inflammation Early in Gestation Am. J. Respir. Crit. Care Med., March 15, 2002; 165(6): 741 - 742. [Full Text] [PDF]
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,
TLR4-expressing animals; •, TLR-deficient animals. This represents an
aggregate of three independent experiments for C3H series
and two independent experiments for the C57BL/10 series. Statistically
significant differences in bacterial recovery were noted between
TLR-expressing mice and the corresponding TLR-deficient mice except for
the C57BL/10 series at the 12 h time point. **,
p < 0.01; ***, p <
0.001.
0.01; and ***, p <
0.001.
), and the hatched bar represents the percentage of
neutrophils (PMN). Total cells in BAL were as follows: C3H/HeN,
3.0 x 105; C3H/HeJ, 1.2 x 105;
C57BL/10/ScSn, 1.4 x 105; and C57BL10/ScCr, 0.8
x 105.
