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Am J Physiol Lung Cell Mol Physiol 291: L677-L682, 2006. First published June 9, 2006; doi:10.1152/ajplung.00523.2005
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The transcriptional response to lipopolysaccharide reveals a role for interferon-{gamma} in lung neutrophil recruitment

Lauranell H. Burch,1,2 Ivana V. Yang,1,2 Gregory S. Whitehead,1,2 Frank G. Chao,1,2 Katherine G. Berman,1 and David A. Schwartz1,2,3

1National Institute of Environmental Health Sciences, Research Triangle Park; 2Division of Pulmonary, Allergy, and Critical Care Medicine, Duke University Medical Center, Durham; and 3Veterans Administration Medical Center, Durham, North Carolina

Submitted 12 December 2005 ; accepted in final form 6 May 2006


   ABSTRACT
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 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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Neutrophil recruitment to the lung after lipopolysaccharide (LPS; endotoxin) inhalation is primarily dependent on Toll-like receptor 4 (Tlr4) signaling, because it is virtually absent in mice deficient in Tlr4. However, among strains wild type for Tlr4, the magnitude of neutrophil recruitment to the lung after LPS inhalation is variable, suggesting the involvement of genes other than Tlr4. To identify genes associated with the inflammatory response to inhaled LPS, we evaluated the transcriptional response in lungs of 12 inbred strains of mice, 8 which are wild type for Tlr4 and 4 of which lack functional Tlr4. Using the promoter integration in microarray analysis algorithm, we scanned our gene list for transcription factor-binding sites significantly overrepresented among Tlr4 wild-type strains with high neutrophil influx in the lung after LPS inhalation. This analysis identified the interferon (IFN)-stimulated response element (ISRE) as the most overrepresented transcription factor (present in 24% of the promoters) associated with the neutrophil influx to the lower respiratory tract. To test the validity of this observation, we evaluated IFN-{gamma}-deficient mice and found that the presence of IFN-{gamma} is essential for robust neutrophil recruitment to the lower respiratory tract and modulation of key regulatory cytokines and chemokines after LPS inhalation. In conclusion, using a genomic approach, we identified the ISRE as a transcriptional element associated with the neutrophil response to inhaled LPS and demonstrated for the first time that IFN-{gamma} plays a critical role in LPS-induced neutrophil recruitment to the lower airways.

endotoxin; environmental airway disease; murine; microarray; transcription factor

ENDOTOXIN is ubiquitous in the environment and is associated with the development and progression of asthma as well as other forms of airway disease. In the domestic setting, the concentration of endotoxin in the bioaerosol is associated with the clinical severity of asthma, and, in selected work settings, endotoxin is the most significant component of the bioaerosol associated with the development and progression of airway disease (26). We have previously reported that controlled inhalation of lipopolysaccharide (LPS), a specific type of endotoxin, can cause all of the signs of asthma: reversible airflow obstruction, airway inflammation, persistent airway hyperreactivity, and airway remodeling (4, 9).

However, the ability of the host to respond to LPS is highly variable. In mice, differences in the responsiveness to LPS are well established; for example, mice lacking functional Toll-like receptor 4 (Tlr4) are hyporesponsive to LPS (12), and, among strains with wild-type Tlr4, the response to inhaled LPS is highly variable (18). Even among humans, we have found a highly variable (and reproducible) response to inhaled LPS (2, 14). These findings suggest that genes other than Tlr4 are involved in regulating the response to inhaled LPS.

To identify genes other than Tlr4 that are critical to the biological response to inhaled LPS, we challenged phenotypically divergent strains of mice (either wild type for Tlr4 or with functional deficiencies in Tlr4) with inhaled LPS and identified genes that were differentially regulated and associated with polymorphonuclear neutrophil (PMN) recruitment to the lower respiratory tract. We hypothesized that shared promoter elements in these genes would be critical determinants of the inflammatory response to LPS and that signaling components upstream of these factors would represent candidate genes that may modulate the response to LPS. To pursue this hypothesis, we performed a transcription factor (TF)-binding site overrepresentation analysis [promoter integration in microarray analysis (PRIMA)] (8) on our list of differentially regulated genes.1 Our findings identified the interferon (IFN)-stimulated response element (ISRE) as an important shared promoter element in genes transcriptionally regulated by inhaled LPS. To test the validity of this observation, we evaluated IFN-{gamma}-deficient mice and found that the presence of IFN-{gamma} is essential for robust PMN recruitment to the lower respiratory tract and modulation of key regulatory cytokines and chemokines after LPS inhalation.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
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Inbred mice. Male mice, aged 6–8 wk, were obtained from Jackson Laboratories (Bar Harbor, ME) unless otherwise specified. C57BL/6Tlr4–/– mice were a gift of Dr. Akira of Osaka University (13). IFN-{gamma} knockout mice and their background strain, C57BL/6J, were also obtained from Jackson Laboratories. Experiments were conducted in accordance with National Institutes of Health guidelines for the care and use of animals established by United States government Animal Welfare Acts and with an approved protocol from the Duke University Animal Care and Use Committee.

LPS inhalation protocol. Twelve mice from each strain were exposed to aerosol. Six mice from each strain received LPS, and six mice received a control exposure of aerosolized saline. Mice were exposed to LPS or saline aerosols for 4 h as described previously (18). Lyophilized purified Escherichia coli 0111:B4 LPS was purchased from Sigma (St. Louis, MO), dissolved in sterile saline at 5 mg/ml, and stored at –20°C. The concentration of LPS was assayed with a chromogenic Limulus amebocyte lysate (LAL) kit (BioWhittaker, Walkersville, MD) using sterile pyrogen-free glassware as previously described (19). For this series of independent exposures, LAL assays indicated that aerosols obtained during the inhalation challenges fell into a range of LPS concentrations (2.7–5.9 µg/m3) that are known to result in predictable inflammation of the lower respiratory tract (18). To demonstrate the decreased ability of IFN-{gamma} knockout mice to recruit PMNs to the lung after LPS challenge, IFN-{gamma} knockout mice and their background strain, C57BL/6J, were exposed to LPS concentrations of twice the usual concentration.

Within 1 h of the inhalation challenge, mice were killed by CO2 inhalation, their chests were opened, and the lungs were lavaged in situ. Polyethylene-90 tubing was inserted into the exposed trachea, and the lungs were lavaged with 6.0 ml sterile saline, 1 ml at a time, under a constant pressure of 25 cmH2O. This constant pressure reduces the animal-to-animal error caused by different lavage conditions and minimizes structural damage to the lung. After the lavage, the fluid volume was recorded, and the lavage fluid was centrifuged for 5 min at 200 g. The supernatant fluid was decanted in two aliquots and frozen at –70°C for subsequent cytokine analysis. The residual cell pellet was resuspended and washed twice in HBSS (without Ca2+ or Mg2+). After a second wash, a small aliquot of the sample was taken, and cells were counted using a hemacytometer. Approximately 104 cells were spun for 5 min onto a glass slide using a cytocentrifuge (Cytospin-2, Shanden Southern, Sewickley, PA). Staining of cytospin slides was carried out using the Diff Quick Stain Set (Dade Behring, Newark, DE). Total and differential cell counts were performed as previously described (18). Two lobes of the lung were snap frozen in liquid nitrogen for RNA extraction.

Cytokine analyses. Concentrations of chemokine (C-X-C motif) ligand 1 (KC), macrophage inflammatory protein (MIP)-1{alpha}, MIP-2, TNF-{alpha}, and IL-12 (p40) in whole lung lavage fluid were determined using commercial ELISA kits specific for these mouse cytokines (R&D Systems, Minneapolis, MN).

Total RNA isolation and preparation of RNA pools. Snap-frozen lung samples were homogenized in TRIzol reagent in a FastPrep bead homogenizer (Bio 101, Qbiogene, Irvine, CA). Total RNA was then prepared from lysates by the TRIzol method (Invitrogen, Carlsbad, CA) following the manufacturer's protocol. RNA was quantified by UV spectrophotometry and diluted to 1 µg/µl in RNase-free water.

Expression profiling. Gene expression profiling of lung tissue was performed on spotted arrays containing ~17,000 oligonucleotides designed to represent the mouse genome (Operon Mouse version 2 collection) and a set of positive Arabidopsis thaliana and negative controls. Microarray hybridizations were performed on 48 lung RNA pools constructed by preparing 2 pools of 3 animals/pool for each strain and treatment (saline or LPS inhalation).

Each pool of mouse lung tissue was cohybridized to a common reference sample (Stratagene Mouse Universal Reference) and replicated with dye swap to eliminate any dye-specific artifacts. This resulted in a total of 96 hybridizations. Lowess normalization was applied to each array to adjust for dye imbalances in Cy3 and Cy5 channels. Flip-dye replicas for each pool/condition were filtered to remove genes with poorly replicated ratios and merged by taking geometric means of the expression ratios to produce a single measure for each pool/condition (34). Detailed protocols are available at http://mgm.duke.edu/genome/dna_micro/core/protocols.htm. Arrays were scanned using an Axon GenePix 4000B confocal laser scanner, and fluorescent intensities from the Cy3 and Cy5 channels were generated with GenPix Pro 4.0 software.

Microarray analysis. Background-subtracted intensities generated by GenePix Pro image-analysis software were used in analyses performed with the TM4 software package (http://www.tm4.org/) (23). Individual arrays were normalized in Microarray Data Analysis System (MIDAS) using Lowess with the smoothing parameter set to 0.33. Flip-dye replicas were filtered using the 2SD criterion and merged by calculating geometric means of query and reference intensities. Only elements with expression values in at least 80% of the 48 experiments were considered in further analyses (6,394 elements). Significance analysis of microrrays (SAM) (32) was used as implemented in the Multiexperiment viewer.

To analyze the Tlr4-mediated response to LPS, comparisons were made between LPS-exposed and saline-exposed (unexposed) mice and between LPS responder (Tlr4 wild type) and LPS nonresponder (deficient or nonfunctional Tlr4) strains of mice. Gene lists for the top 100 differentially expressed genes from these comparisons as well as all differentially expressed genes at 1% false discovery rate (FDR) were obtained. The intersection of the two analyses was obtained by taking overlapping genes between the top 100 differentially expressed genes from both lists, resulting in an overlap list of 62 genes (see Supplemental Table 3).


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  		Table 3. LPS response genes sharing the cRel transcriptional element

 
To determine whether there were gene expression changes via a non-TLR4-mediated response to inhaled LPS, comparisons were performed between LPS and saline exposures for strains of mice lacking a functional Tlr4 [C3H/HeJ, C57BL/10ScCR, and TLR4–/– mice and the recently characterized spontaneous Tlr4 mutant recombinant inbred strain, BXD29 (6)]. Once these genes were identified, we compared mouse strains wild type for Tlr4 that consistently had a vigorous PMN response to inhaled LPS (DBA/2J, 129/SvIm, and BXD30) with strains that had a minimal PMN response to inhaled LPS (C57BL/6J, Balb/cJ, and NZW/LacJ). No significant genes were obtained for either of these comparisons using SAM; instead, we used a permutation-based t-test to identify significant differential expression.

Transcription factor overrepresentation analysis. The PRIMA algorithm (http://www.cs.tau.ac.il/~rshamir/prima/PRIMA.htm) was used to scan promoters in our 62 gene overlap list (See Supplemental Table 3) for TF-binding sites that were significantly overrepresented in the examined set relative to the background set of promoters (all genes represented in the Operon 17k mouse oligonucleotide set). Overrepresented TFs were identified by the PRIMA tool in the expression analysis and display manager (EXPANDER) (27). Genes coexpressed over multiple biological conditions were assumed to be regulated by common TFs and were expected to share common regulatory elements in their promoters. TF motif fingerprint files for the mouse were extracted from the TRANSFAC database (20, 33) as provided in EXPANDER. About 17,000 mouse promoter sequences, spanning from 1,000 bases upstream from the transcription start site (TSS) to 200 bases downstream from the TSS, were scanned by sliding a window the length of position weight matrixes along the promoter to extract putative binding sites. The details of the algorithm have been described elsewhere (8, 27). Promoters reaching significance using the analytical score computed by PRIMA were adjusted for multiple testing using the Bonferroni correction.

Univariate statistics. All data are expressed as means ± SE. Individual comparisons for the concentrations of cells and cytokines in the lavage fluid were evaluated by the Mann-Whitney U-test (22). Statistical calculations were performed using GraphPad InStat software (San Diego, CA). A two-tailed P value of <0.05 was considered as statistically significant.


   RESULTS
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DISCUSSION
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Inflammatory responses to inhaled LPS. The concentrations of PMNs per milliliter in the whole lung lavage fluid between inbred strains of mice yielded a distinct strain distribution pattern among the Tlr4 wild-type mice; however, strains lacking a functional Tlr4 (BXD29, C3H/HeJ, and C57BL/10ScN) and Tlr4-deficient mice (C57BL/6Tlr4–/–) did not demonstrate enhanced PMN recruitment to the lower respiratory tract (Fig. 1).


Figure 1
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  		Fig. 1. After a 4-h aerosol exposure, lipopolysaccharide (LPS)-exposed animals demonstrated a robust increase in the concentration of polymorphonuclear neutrophils (PMNs) in the lavage fluid. Postsaline values did not exceed 2,000 PMNs/ml.

 
Genes differentially expressed in response to inhaled LPS. A list of differentially expressed genes at 1% FDR for the LPS-exposed versus saline-exposed and wild-type Tlr4 versus nonfunctional/deficient Tlr4 comparisons is provided as supplemental data (see Supplemental Tables 1 and 2). There were 1,540 and 1,226 differentially expressed genes, respectively, at 1% FDR in these analyses. These comparisons yielded similar gene lists, and, as might be expected, a formal cluster analysis grouped gene expression of Tlr4-deficient/nonfunctional mice exposed to LPS with expression results of Tlr4 wild-type mice exposed to saline (Fig. 2), which is an observation consistent with the observed failure of strains with a deficient/nonfunctional Tlr4 to recruit PMNs to the lower respiratory tract. The overlap of the top 100 genes from both lists (LPS exposed vs. saline exposed and wild-type Tlr4 vs. deficient/nonfunctional Tlr4) represents our working list of 62 LPS response genes and is shown in Supplemental Table 1. It is interesting to note that among these 62 genes, CD14, MyD88, and TLR2 are included and represent Toll receptor pathway genes.


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  		Table 1. TF-binding sites overrepresented in the airway LPS gene list

 

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  		Table 2. Inhaled LPS response genes sharing the ISRE

 

Figure 2
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  		Fig. 2. Principal component analysis using significant analysis of microarray-selected genes separated samples into two well-defined clusters: a cluster containing unexposed samples plus exposed samples from strains with no Toll-like receptor 4 (TLR4) and a cluster of exposed animals with functional TLR4. Exposed samples from strains with functional TLR4 formed a less tight cluster, which is in accordance with PMN phenotype data, where there was a gradient of responses within responder strains.

 
Airway response to inhaled LPS is dependent on TLR4. Analysis of gene expression data from four strains lacking a functional Tlr4 gene indicates that non-Tlr4-mediated effects of LPS are minimal. Twenty-one significant genes were identified that distinguished the exposed from unexposed Tlr4 nonfunctional/deficient mice (Student's t-test at P < 0.01 with no Bonferroni correction). However, these 21 genes did not separate LPS-treated animals from untreated mice into clusters by either hierarchical clustering or principal component analyses.

Genes that modulate the inflammatory response to inhaled LPS. To identify genes in addition Tlr4 that modulate the lung inflammatory response to LPS, we focused on strains of mice wild type for Tlr4 and sought to identify genes that distinguished the phenotypic extremes of the lung PMN response. When we compared "high-responder" strains (DBA/2J, 129/SvIm, and BXD30) and "low-responder" strains (C57BL/6J, BALB/cJ, and NZW/LacJ) that were wild type for Tlr4, 39 genes were significant at 5.5% median FDR when a two-class unpaired SAM algorithm was applied (Supplemental Table 2). Fewer significant genes at a higher FDR were expected because the magnitude of the differential response within responder strains is much lower than that between responders and nonresponders. Close examination of the genes shown in Supplemental Table 2 revealed no genes known to be involved in the response to LPS.

Transcription factors that regulate the response to inhaled LPS. To explore the potential regulatory factors that may be responsible for changes in LPS-induced gene expression, we sought to identify overrepresented TF-binding sites in the promoters of the 62 candidate LPS genes shown in Supplemental Table 1. Several promoter elements reached significance using the analytical score computed by PRIMA (Table 1). Genes represented by the ISRA, cRel transcriptional element, and alternative forms of the NF-{kappa}B consensus binding sites are shown in Tables 2, 3, and 4. The Tlr2 promoter contains all TF-binding sites found to be overrepresented in this analysis. Although we anticipated overrepresentation of NF-{kappa}B elements, we were surprised at the increased representation of the ISRE promoter element in the LPS response genes. These data supported previous reports showing that type I IFNs are important mediators in the response to LPS (7, 24, 29, 30, 35). To pursue the role of other IFNs in the LPS response, we chose to investigate the role of IFN-{gamma}, a type II IFN, because the relationship between type II IFNs and the airway response to inhaled LPS has received little attention.


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  		Table 4. Inhaled LPS response genes sharing NF-{kappa}B consensus binding sites

 
Inflammatory response to inhaled LPS is dependent on IFN-{gamma}. After the inhaled LPS challenge, significant decreases were observed in the concentrations of total cells and PMNs in whole lung lavage fluid of IFN-{gamma}-deficient (C56BL/6IFN-{gamma}–/–) mice compared with their C57BL/6 background controls (Fig. 3). However, after the inhalation of LPS, significant increases in the concentrations of chemokines KC, MIP-1{alpha}, and MIP-2 and the cytokine TNF-{alpha} were found in the lung lavage fluid of IFN-{gamma}-deficient mice compared with C57BL/6 control mice (Fig. 4). IL-12-p40 levels were not significantly different between C56BL/6IFN-{gamma}–/– mice and C57BL/6 controls (data not shown).


Figure 3
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  		Fig. 3. A and B: interferon (IFN)-{gamma}-deficient mice (C57BL/6IFN-{gamma}–/–) demonstrated a significant reduction in total cells (A; P = 1.4 x 10–6) and a significant reduction in PMNs (P = 2.5 x 10–6) per milliliter of lung lavage fluid (B) after LPS inhalation than their C57BL/6 wild-type background strain.

 

Figure 4
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  		Fig. 4. After LPS exposure, IFN-{gamma}-deficient mice (C57BL/6IFN-{gamma}–/–) demonstrated significantly increased levels of chemokine (C-X-C motif) ligand 1 (KC; A; P = 0.0019), macrophage inhibitory protein (MIP)-1{alpha} (B; P = 0.0018), MIP-2 (C; P = 0.0005), and TNF-{alpha} (D; P = 0.0328) in lung lavage fluid compared with the C57BL/6J background strain. IL-12 (p40) levels were not significantly different in IFN-{gamma}-deficient mice compared with their C57 controls. *Cytokine concentration for IFN-{gamma}-deficient animals was significantly different from cytokine concentration in the C57BL/6J control strain.

 

   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Our results indicate that the transcriptional response to inhaled LPS is complex, involving the expression of many genes. However, by analyzing the overrepresentation of TF-binding sites, we identified a limited number of key transcriptional elements that are critical to this biological response. Although Tlr4 represents an essential component of the molecular machinery required to initiate a response to LPS, other molecules, such as IFN-{gamma}, serve to modify and regulate this process. Our findings suggest that it may be possible to alter the biological response to inhaled LPS by targeting Tlr4, IFN-{gamma}, or even specific TFs that regulate elements of the innate immune response to LPS.

Most work on Toll signaling has focused on pathways that culminate in the activation of NF-{kappa}B. However, we found that although NF-{kappa}B was involved in the biological response to inhaled LPS, the ISRE was the most overrepresented transcriptional element among genes responsive to LPS inhalation. Our in vivo experiments with IFN-{gamma}-deficient mice clearly demonstrated a role for IFN signaling in regulating the inflammatory response of the lower respiratory tract after LPS inhalation. A decrease in LPS-induced PMN recruitment occurred in IFN-{gamma}-deficient mice despite the enhanced release of chemokines KC, MIP-1{alpha}, and MIP-2 in the lower respiratory tract, all of which are known to attract neutrophils (17, 28, 31). Several potential explanations may account for this observation. First, it is conceivable that in the absence of normal PMN recruitment, homeostatic mechanisms may serve to enhance the production and release of chemokines in IFN- {gamma}-deficient mice after inhalation of LPS. Second, although many cytokines and chemokines are known to be differentially regulated by IFN-{gamma} (3, 15), the adhesion molecule ICAM-1 (15) is noteworthy because it is known to be essential for neutrophil adhesion and transcellular migration into the lung. The absence of ICAM-1 induction by IFN-{gamma}-deficient mice may play a role in the reduced PMN migration, despite the presence of chemokines. Finally, it is well accepted that counterregulatory mechanisms are observed in genetically engineered mice that often confound normal homeostatic mechanisms.

Interestingly, treatment of human neutrophils with IFN-{gamma} has been shown to decrease release of human IL-8 (KC/MIP-2 homolog) in response to fungal infection (10). Furthermore, the initial rate of PMN recruitment has been demonstrated to be impaired after the induction of peritoneal inflammation in IFN-{gamma}-deficient mice, with a concomitant decrease in peritoneal levels of cytokines IL-1beta and IL-6 (21). These findings support our results demonstrating altered PMN migration and altered concentrations of cytokines/chemokines after LPS exposure in IFN-{gamma}-deficient mice. In aggregate, these observations suggest that IFN-{gamma} may be a critical regulator of innate immunity via PMN recruitment and the control of cytokine homeostasis across a variety of infectious challenges.

By employing TF-binding site overrepresentation analysis on a microarray data set to successfully identify genes affecting our phenotype of interest, we demonstrated the feasibility of this method to divide a complex in vivo biological response into components on the basis of shared regulatory elements. This approach is consistent with an emerging body of literature that illustrates the critical role of TFs in human disease. For instance, rare mutations in NF-{kappa}B and forkhead box C1 have been reported to cause disease in humans (1, 25). Moreover, many common diseases, such as asthma, atherosclerosis, diabetes, and acquired immune deficiency syndrome, are associated with dysregulation of NF-{kappa}B (11, 16), suggesting that pharmacological manipulation of NF-{kappa}B or other TFs may prove to be effective therapeutic approaches to modify the progression and severity of these diseases.

The biological response to microbial toxins can impact a variety of seemingly unrelated pathological conditions. For example, sequence changes in innate immune receptors and their signaling molecules are known to alter the risk of developing not only infections but also atherosclerosis and asthma (5). Thus the group of LPS response genes identified that are differentially expressed and share the ISRE transcriptional element (Table 2) merit further attention, because sequence variation in some of these genes may identify a subset of individuals who are particularly susceptible to LPS-induced inflammatory lung disease (2, 14) and perhaps other diseases. Genes that are differentially regulated by LPS and share transcriptional elements may also lead to the identification of novel pathogenic pathways or provide new therapeutic targets for the treatment of inflammatory lung disease.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Institute of Environmental Health Sciences Grants ES-11375, ES-07498, ES-012496, and ES-011961 and by the Department of Veterans Affairs (Merit Review).


   FOOTNOTES
 

Address for reprint requests and other correspondence: L. H. Burch, Laboratory of Respiratory Biology, National Institute of Environmental Health Sciences, Rall Bldg., Rm. C204A, PO Box 12233, MD C2-13, 111 Alexander Dr., Research Triangle Park, NC 27709 (e-mail: burchl{at}niehs.nih.gov)

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

1 Supplemental data for this article is available at the American Journal of Physiology-Lung Cellular and Molecular Physiology Web site. Back


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 ABSTRACT
 MATERIALS AND METHODS
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 DISCUSSION
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S. Alper, R. Laws, B. Lackford, W. A. Boyd, P. Dunlap, J. H. Freedman, and D. A. Schwartz
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