A broad variety of natural environmental stimuli, genotypic influences and timing all contribute to expression of protective versus maladaptive immune responses and the resulting clinical outcomes in humans. The role of commonly co-segregating Toll-like receptor 4 (TLR4) non-synonymous single nucleotide polymorphisms Asp299Gly and Thr399Ile in this process remains highly controversial. Moreover, what differential impact these polymorphisms might have in at risk populations with respiratory dysfunction, such as current asthma or a history of infantile bronchiolitis, has never been examined. Here we determine the importance of these polymorphisms in modulating LPS and respiratory syncytial virus (RSV) - driven cytokine responses. We focus on both healthy children and those with clinically relevant respiratory dysfunction.
To elucidate the impact of TLR4 Asp299Gly and Thr399Ile on cytokine production, we assessed multiple immune parameters in over 200 pediatric subjects aged 7–9. Genotyping was followed by quantification of pro- and anti-inflammatory cytokine responses by fresh peripheral blood mononuclear cells upon acute exposure to LPS or RSV.
In contrast to early reports, neither SNP influenced immune responses evoked by LPS exposure or RSV infection, as measured by the intermediate phenotype of pro- and anti-inflammatory cytokine responses to these ubiquitous agents. There is no evidence of altered sensitivity in populations with “at risk” clinical phenotypes.
Genomic medicine seeks to inform clinical practice. Determination of the TLR4 Asp299Gly/Thr399Ile haplotype is of no clinical benefit in predicting the nature or intensity of cytokine production in children whether currently healthy or among specific at-risk groups characterized by prior infantile broncholitis or current asthma.
Citation: Douville RN, Lissitsyn Y, Hirschfeld AF, Becker AB, Kozyrskyj AL, Liem J, et al. (2010) TLR4 Asp299Gly and Thr399Ile Polymorphisms: No Impact on Human Immune Responsiveness to LPS or Respiratory Syncytial Virus. PLoS ONE 5(8): e12087. https://doi.org/10.1371/journal.pone.0012087
Editor: Samithamby Jeyaseelan, Louisiana State University, United States of America
Received: March 12, 2010; Accepted: July 11, 2010; Published: August 10, 2010
Copyright: © 2010 Douville et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by fellowships and grants from the Canadian Institutes for Health Research (CIHR). R.N.D. was also supported by the Mindel and Tom Olenick Award in Immunology and as a member of the CIHR National Training Program in Allergy and Asthma. S.E.T. is supported by a Chaim Roifman Scholar Award from the Canadian Immunodeficiency Society, a Career Development Award from the Canadian Child Health Clinician Scientist Program (CCHCSP)-a CIHR Strategic Training Program, and the CIHR Team in Mutagenesis and Infectious Diseases. K.T.H. holds the Canada Research Chair in Immune Regulation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Genomic medicine seeks to inform clinical practice. It is widely anticipated that identifying specific components of an individual's genome will help determine the optimal approach to health care; for example, by facilitating the prediction and prevention of diseases such as asthma , or by identifying optimal pharmacologic agents for therapy , . One early example, the study of susceptibility to respiratory syncytial virus (RSV), led to much attention focused on genes that encode proteins of the innate immune system. Since innate immunity forms the first line of defense against infection, and its activation shapes the ensuing protective or maladaptive antigen-specific immune responses that develop, it is hypothesized that variation in the genes encoding proteins of the innate immune system will influence human susceptibility –.
The role of Toll-like receptor 4 (TLR4) polymorphisms in influencing clinical and immunologic responses to environmental stimuli is an area of much continuing and conflicting attention. TLR4 is specifically involved in generating immune responses against a diverse panel of agonists , including controversially, RSV. Kurt-Jones et al initially reported that TLR4 is key in initiating innate immune responses of monocytes to the fusion (F) protein of RSV . These early human data were supported by experimental animal studies in which RSV was found to persist longer in the lungs of TLR4-deficient mice compared to TLR4-expressing controls . At the same time, the validity of these findings and their broader impact on immune capacity in diverse human populations has remained controversial, as others, in both animals and human populations have not replicated these results , .
The human TLR4 gene harbours two commonly co-segregating single nucleotide polymorphisms (SNP)—Asp299Gly and Thr399Ile—that alter the amino acid sequence of the TLR4 protein. Experimental systems indicate that the 299Gly SNP is associated with hyporesponsiveness to TLR4 ligands such as LPS in vivo and in vitro, and that it acts in a dominant fashion with respect to the more common Asp299 allele , . Currently there is little consensus on how such genetic factors controlling the innate immune system influence the outcomes of endotoxin exposure or RSV infection in otherwise healthy children. By way of example, one study demonstrated that children requiring admission to hospital for RSV infection carried the TLR4 299Gly allele at a higher frequency than children with mild disease not requiring hospitalization , however, subsequent independent work by our group did not identify such a positive association , and another group observed a weak association in the opposite direction where the more common Asp299 allele was overrepresented in children with severe RSV infection . Most recently, heterozygosity of Asp299Gly and Thr388Ile was found to be highly associated with symptomatic RSV disease in high-risk infants . However, the confounding impact of known clinical risk factors for severe RSV infection in this cohort, including prematurity and bronchopulmonary dysplasia, made generalization of these data challenging.
LPS and RSV are ubiquitous in the environment. Pediatric populations may experience substantially higher exposure to both agents and exhibit a higher burden of disease than adults. Given the morbidity associated with RSV bronchiolitis and the observation that RSV infection increases the risk of subsequent wheezing and asthma, we undertook a rigorous examination of the functional impact of the TLR4 Asp299Gly and Thr399Ile polymorphisms on the human immune response to both LPS exposure and, independently, to RSV infection.
The most significant challenges associated with prior studies of this question include (i) use of cell lines, reliance on artificial experimental systems in which cell lines are transfected with TLR4 variants, or use of inbred animal models, the results of which are often directly extrapolated to highly diverse human populations, (ii) use of human cells ex vivo as previously frozen samples or following pre-conditioning regimens with addition of cytokines (ie IFNγ) to culture, both of which have demonstrated potential to skew the resulting immune responses, (iii) small sample sizes (frequently less than 100), and (iv) use of different, or undefined, variants of RSV for re-stimulation. Importantly, the majority of studies attempt to link SNPs or haplotypes directly to clinical outcomes, without analysis of the intermediate phenotype of immune function. Thus, notwithstanding extensive evidence of the impact of gene by environment by time interactions on a wide variety of immune and clinical outcomes –, many studies to date, due to their experimental design, cannot evaluate the impact of these factors on immune capacity and LPS or RSV specific responses.
As an alternative approach to better elucidate the immunological impact of the TLR4 Asp299Gly and Thr399Ile polymorphisms on putatively distinct cytokine production in human populations that interact with a broad diversity of natural environmental stimuli throughout childhood, we measured multiple immune parameters in over 200 pediatric subjects aged 7–9 years following acute in vitro re-exposure to LPS or RSV. The results demonstrate that while some reports link TLR4 Asp299Gly polymorphism with clinical outcomes of RSV infection, the haplotype comprised of Asp299Gly and Thr399Ile does not detectably impact the immune response to RSV, as measured by the intermediate phenotype of RSV-stimulated cytokine responses nor LPS-driven cytokine production. This conclusion applies equally to healthy children and those with “at risk clinical phenotype” characterized by current asthma or a history of severe bronchiolitis in infancy.
Materials and Methods
Recruitment of child participants and ethics statement
Study approval was obtained from the University of Manitoba Faculty Committee on Use of Human Subjects in Research. This 1995 birth cohort (SAGE, Study of Allergy Genes and Environment) was created from the Manitoba Canada universal provincial health care registry . Written informed consent was obtained for 723 children (of whom 207 were randomly selected for detailed analysis) within a general population survey case-control cohort. Consented children and parents came to the Pediatric Allergy Clinic in Winnipeg or mobile Pediatric Allergy clinic in their community for the detailed assessment used to characterize the individuals studied in this publication.
In prior studies, 100% of this age group was seropositive for prior RSV infection, reinforcing the ubiquity of these infections in the general population. These 7–9 year old children had no evidence of current upper or lower respiratory tract infection within one month of recruitment based on histories from child and parent (no “colds”) and no current evidence of a URI on physical examination.
Clinical assessment of asthma in children
Pediatric allergist assessment was used as the gold standard for diagnosis of current asthma (ie. within the last 12 months). Diagnosis was based on history and physical assessment (blinded to skin prick tests and PC20), according to the Canadian Asthma Consensus Guidelines . This incorporated a standardized history and physical sheet, questions regarding cough with/without colds, wheeze with/without colds, shortness of breath with activities, colds lasting >2 weeks, response to current medications and the presence of other allergic conditions, family history of asthma or personal history of prior/present eczema, hospital, emergency department and medical visits for breathing difficulty in the past year, and physical examination for allergic facies, chest findings and evidence of atopic dermatitis.
Bronchiolitis was defined as an ICD-9 (International Classification of Diseases) diagnosis of “acute bronchitis and bronchiolitis” (466 code) between birth and age 2, which was identified using the Manitoba Center for Health Policy databases.
Blood was collected in tubes containing EDTA, peripheral blood mononuclear cells (PBMC) were isolated by density centrifugation, counted (>95% viability by trypan blue exclusion), and used immediately for short-term primary culture in the absence of exogenously added cytokines.
Generation of viruses for cell culture
RSV strain Long was cultured on Hep-2 cells at 37°C in Eagle's MEM supplemented with penicillin (100 U/ml), streptomycin (100 µg/ml), 30 µg/ml L-glutamine (Life Technologies) and 1% fetal calf sera (Sigma). RSV was titrated by the quantal assay TCID50 performed in 96-well microtiter plates using tenfold dilutions.
Primary cell culture
Freshly isolated PBMC were suspended in complete medium (RPMI 1640 with 10% heat-inactivated fetal calf serum, 1% penicillin/streptomycin/fungizone, 0.3 mg/ml L-glutamine and 0.1% 2-mercaptoethanol) with 2.5×106 cells/ml in 200 µl in 96 well U bottom plates. Duplicate cultures were stimulated with LPS (BioXtra, L4391) from Escherichia coli serotype 00011:B4 at 0.05, 0.5 and 5.0 ng/ml from Sigma (St. Louis, MO) or RSV strain Long (104.9 TCID50/ml). LPS purity was validated by i) its inability to activate other human TLRs expressed in transfected HEK 293 cells, and ii) the finding that pretreatment with TLR4 blocking antibodies (10 ug/ml anti-CD284/TLR4, Ebioscience, San Diego, CA), but not IgG2a isotype control antibodies (Ebioscience), blocks the capacity of LPS to stimulate cytokine production demonstrate its specificity for TLR4 (Figure S1). Based on data obtained in preliminary time course experiments (data not shown), culture supernatants were harvested following 1 or 6 days culture, the respective time of peak LPS and RSV driven responses for the cytokines evaluated. The optimal concentration of LPS stimulation used to evaluate IL-10 and CCL2 responses was 0.05 ng/ml, with 0.5 ng/ml for IL-1β and TNFα responses. While the intensity of responses evoked was weaker in most individuals when sub-optimal concentrations were used, the conclusions from the data were identical (data not shown).
Human cytokine ELISA
As a safety precaution against infection, culture supernatants were UV irradiated for one hour to inactivate residual virus prior to ELISA analysis. Experiments (data not shown) demonstrated that this had no impact on the sensitivity or precision of the assays used to evaluate cytokine concentrations. Anti-cytokine capture and biotinylated detection antibodies were purchased from BD-Pharmingen (Mississauga, ON, Canada), Endogen (Woburn, MA, U.S.A.), Biolegend (San Diego, CA, U.S.A.) or R&D Systems (Minneapolis, MN, U.S.A.) and recombinant cytokine standards from BD Pharmingen, Endogen or Peprotech (Rocky Hill, NJ, U.S.A.). They were used as previously described , . PBMC supernatants from a minimum of duplicate cultures were assayed. Each sample was then evaluated in at least two assays, with the concentration in each calculated from a minimum of three points falling on the linear portion of titration curves calibrated against recombinant cytokine standards serially diluted on each plate. Standard errors typically ranged from 3–10%. All cytokine standards and antigenic stimuli (LPS and RSV) were each derived from single lot preparations. Because a large number of different immune-assays needed to be performed on culture supernatants derived from the limited pediatric blood volumes drawn, some samples were consumed before all analyses could be completed. Thus, some variation exists in the “n” available to assess different cytokines in both the asthmatic/bronchiolitis groups and control populations based on availability of materials. No data were deliberately excluded.
TLR4 Asp299Gly and Thr399Ile Genotyping
The TLR4 allelic variants Asp299Gly (refSNP ID: rs4986790) and Thr399Ile (refSNP ID: rs4986791) were genotyped by quantitative PCR assay as previously described . Briefly, genomic DNA was amplified using a TaqMan® Pre-Designed SNP Genotyping Assay (Asp299Gly #C_11722238_20, Thr399Ile #C_11722237_20, Applied Biosystems) which contains primers and probes specific for each allelic variant of the SNP in question. The genotyping assay was performed using an Applied Biosystems 7300 Real Time PCR System.
Asp299Gly and The399Ile TLR4 Genotyping
We genotyped 207 7–9 year old children from the SAGE cohort (Table 1). TLR4 Asp299Gly and Thr399Ile SNPs were in Hardy-Weinberg equilibrium, with the common homozygote genotypes co-segregating in 202 of 207 individuals (97.5%). This cosegregation is consistent with data demonstrating that these SNPs are located on the same allele in European populations . Rather than treating each polymorphism as an independent variable, it is most helpful to consider the TLR4 haplotypes formed by the Asp299Gly and Thr399Ile polymorphisms, because different haplotype structures are observed in different human populations . Our cohort is essentially comprised of two major haplotypes: Asp/Asp+Thr/Thr (87.0%) and Asp/Gly+Thr/Ile (10.6%) (See Table 1). Consequently, to optimize power, all subsequent analysis was limited to comparing the homozygous (Asp/Asp+Thr/Thr) and heterozygous (Asp/Gly+Thr/Ile) TLR4 haplotypes.
Common TLR4 haplotypes do not influence LPS-stimulated cytokine production
The impact of the TLR4 Asp299Gly and Thr399Ile polymorphisms on LPS responsiveness remains highly controversial , , , –. Many of these studies are quite limited in size. Given the multiple environmental and genetic factors that influence and can confound assessment of innate and adaptive immune function, large sample sizes with analysis of fresh primary cells directly ex vivo are required. Using acute assays of primary PBMC cell cultures from 202 healthy children, we found that LPS stimulated secretion of four characteristic TLR4-driven pro- and anti-inflammatory cytokines, IL-10, IL-1β, TNFα and CCL2, was indistinguishable between subjects with the common homozygous haplotype (Asp/Asp+Thr/Thr) and those with the heterozygous haplotype (Asp/Gly+Thr/Ile) (Figure 1). With multiple stimulation conditions in culture (ie. different LPS concentrations, virus stimulation) and subsequent quantification of multiple cytokines in culture supernatants from these pediatric populations, not all individuals were assayed for all outcomes. In cases where cell numbers were limiting, priority was given to RSV stimulation. The use of ‘maximal’ (5 ng/ml), intermediate (0.5 ng/ml) and ‘low” (0.05 ng/ml) LPS concentrations influences the intensity of cytokine secretion in individuals; however, at none of the other concentrations studied were differences or trends seen between the two variants of the TLR4 Asp299Gly polymorphism across this pediatric population (data not shown).
TLR4 haplotype has no effect on LPS-driven cytokine responses in pediatric populations stratified by asthmatic status or prior bronchiolitis
Many functionally important genetic associations are better revealed in at-risk groups than in general population studies. Given that some prior studies, often with an n of 20–100, demonstrated an association between TLR4 polymorphisms and LPS hyporesponsiveness or clinical outcomes of airway dysfunction such as asthma and bronchiolitis , , , , we conducted subanalyses of genotype and intermediate phenotype based on stratification by (i) current asthma (Figure 2) or a prior clinical history of bronchiolitis in the first two years of life (Figure 3).
IL-10 (A), IL-1β (B), TNFα (C) and CCL2 (D) were assessed as detailed above. * P<0.05, with all other comparisons P>0.05.
Among children carrying the homozygous haplotype (Asp/Asp+Thr/Thr), asthmatics produce higher levels of IL-10 (p<0.03) in response to stimulation with LPS relative to non-asthmatics (Figure 2). This supports the body of literature indicating differences between asthmatics and non-asthmatics in their innate immune capacity. However, stratification of children based on current asthma or prior bronchiolitis or the absence of either condition, consistently demonstrates an indistinguishable capacity to express LPS-driven cytokine production between the two TLR4 haplotypes.
Similar RSV-driven cytokine responses in children with homozygous or heterozygous TLR4 haplotypes
Results obtained from fresh primary PBMC culture derived from the 207 children examined in this cohort demonstrate that the intensity or nature of anti-viral immune responses driven by RSV infection are not influenced by the TLR4 haplotype (Figure 4). RSV driven cytokine production is indistinguishable between these groups, regardless of haplotype.
TLR4 haplotype has no effect on RSV-driven cytokine responses in children stratified by asthmatic status or prior bronchiolitis
More targeted analysis, via stratification of children based on asthma or prior bronchiolitis in infancy, also reveals indistinguishable responses in virus-stimulated cytokine production between individuals with the homozygous TLR4 haplotype (Asp/Asp+Thr/Thr) or the heterozygous haplotype (Asp/Gly+Thr/Ile) (Figures 5 and 6). These data argue against a functional impact on cytokine production between these haplotypes. In contrast, the identification of differential CCL5 production between asthmatic and non-asthmatic children in response to RSV stimulation (fure 5D, p<0.01), underlines the capacity and sensitivity of this method at distinguishing differences in immune capacity between these populations , .
IFNγ (A), CXCL10 (B), IL-10 (C) and CCL5 (D) were measured by ELISA in supernatants from 6 day PBMC cultures. Black bars represent median responses, each from an individual child (•). ** P<0.01, with all other comparisons P>0.05.
The role of the haplotype comprised of TLR4 Asp299Gly and Thr399Ile polymorphisms in shaping immune activation and subsequent clinical outcomes in humans remains highly controversial. Much of this controversy may stem from the diversity of experimental systems employed. Here, using freshly derived in vitro stimulated PBMC in short term culture, we determined whether children with the TLR4 heterozygous haplotype differ from the majority of the population, which carries the homozygous haplotype, in their capacity to mount LPS or RSV-driven immunoregulatory cytokine responses. This cohort of >200 individuals demonstrates that both LPS and RSV-driven responses are indistinguishable in both groups, regardless of haplotype. Further stratification of the cohort based on clinical phenotype (current allergic asthmatic status) also revealed similar LPS- and RSV-driven responses among individuals of the two genotypes. A third independent approach, with cohort stratification based on a history of moderate to severe bronchiolitis/bronchitis during early infancy, also revealed that indistinguishable cytokine responses are elicited in these populations. Thus, the TLR4 Asp299Gly + Thr399Ile heterozygous haplotype has no detectable effect on the capacity of randomly selected 7–9 year old children to mount either RSV-stimulated or LPS-driven responses regardless of whether the children exhibited a clinical phenotype of current asthma or those with a history of infantile bronchiolitis.
The diversity of findings evident in the literature from examination of the putative effects of the TLR4 Asp299Gly polymorphism on LPS responsiveness is likely affected by the disparity in the cosegregation frequency of the Asp299Gly and Thr399Ile alleles in various study populations. In the only large study completed to date other than our own, (n = 245 African subjects) LPS-induced cytokine production by PBMC was not influenced by TLR4 Asp299Gly genotype . Our data extend these findings when PBMC (mainly from white children) are stimulated with LPS at both lower and intermediate concentrations that may reflect environmental conditions better than the prior widespread use of high concentrations of TLR ligands.
Interestingly, among the 64 statistical comparisons made between the two haplotypes in this study which showed no statistically significant differences in production of a variety of cytokines, a single exception was observed — that amongst children with prior bronchiolitis. Children with prior bronchiolitis in infancy who were carriers of the common homozygous TLR4 haplotype (Asp/Asp+Thr/Thr) produced marginally higher levels of IL-10 than did carriers of the heterozygous haplotype (Asp/Gly+Thr/Ile) if their cells were stimulated at the higher concentration (0.5 ng/ml) of LPS (p<0.04) (Figure S2). None of the other cytokines tested in this population differed. While this borderline statistical significance may be a consequence of multiple comparisons, it does raise the possibility that under conditions of increased LPS exposure, TLR4 haplotype may influence LPS-driven IL-10 production in select children who exhibited severe pathological symptoms during RSV infection in infancy. Further experiments specifically addressing this hypothesis, in a larger cohort specifically constructed for this purpose, would be required to discriminate between these possibilities.
Despite extensive evidence of the impact of gene by environment by time interactions on a wide variety of immune and clinical outcomes, studies to date, due to their experimental design, were unable to evaluate the impact of the TLR4 Asp299Gly polymorphism on RSV specific immune responses from individuals with a prior history of bronchiolitis or airway dysfunction. Here, we used the intermediate phenotype of RSV-specific cytokine responses to attempt to bridge the gap between genotype and clinical phenotype. In contrast to the recent study by Tulic et al., which found associations between RSV-driven cytokine production and the Asp299Gly (or Thr399Ile) genotype , we i) examined a substantially greater number of individuals (207 versus 24), ii) chose to use fresh PBMC in the absence of pre-activation with IFNγ, iii) stimulated PBMC with a prototypic strain of RSV and iv) performed subanalysis in clinically different patient populations. We show that individuals with the Asp/Gly genotype demonstrate RSV-driven cytokine responses indistinguishable from those of individuals carrying the Asp/Asp genotype. Moreover, while addition of anti-TLR4 to LPS-stimulated cultures reduced cytokine production to background levels, inclusion of this blocking antibody had no impact on the intensity of RSV-stimulated cytokine responses (data not shown). Collectively, these data strongly argue that neither TLR4 nor these functional variants directly impact LPS stimulated or RSV-driven cytokine production in humans. However, it can never be excluded that other readouts not examined here might indicate differences.
In summary, multiple pattern recognition receptors including various TLR and RLR continue to be linked to resistance to and resolution of RSV infection in humans and animal models –. While some reports have linked TLR4 Asp299Gly polymorphism with clinical outcomes of RSV infection, our data show that the haplotype comprised of Asp299Gly and Thr399Ile does not detectably impact the human immune response to RSV, as measured by the intermediate phenotype of RSV-stimulated cytokine responses or that of a well validated TLR4 ligand, LPS. In this study we did not try to directly link TLR4 SNPs to clinical outcome, but rather stratified by clinical phenotype (asthma or bronchiolitis) to see if differences might be enriched in these groups. Cytokine production from PBMC in a ∼200 member cohort, or in subcohorts characterized by current asthma or a history of severe bronchiolitis in infancy consistently yield indistinguishable pro- and anti-inflammatory cytokine production. As both LPS and RSV can also interact with alternate pattern recognition receptors , , new possibilities for genomic targets in the prediction and prevention of harmful diseases such as RSV infection and asthma await investigation.
Anti-TLR4 blocking antibodies abrogate LPS-driven cytokine production. IL-6, IFNγ and IL-10 responses in response to acute activation with 0.5 ng/ml LPS plus/minus blocking antibodies are shown. Bars represent mean population responses +/− SEM.
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LPS-driven IL-10 production is distinct between children with the homozygous (Asp/Asp+Thr/Thr) and heterozygous (Asp/Gly+Thr/Ile) TLR4 haplotypes in a sub-population that experienced infantile bronchiolitis. IL-10 responses in response to acute activation with 0.5 ng/ml LPS are shown with black bars represent median population responses, derived from individual children. * P = 0.03.
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We thank all participants and staff members of the Study of Asthma Genes and the Environment (SAGE) team and M. Blanchard for technical assistance.
Conceived and designed the experiments: RD YL SET KTH. Performed the experiments: RD YL AFH REV MS. Analyzed the data: RD YL AFH KTH. Contributed reagents/materials/analysis tools: ABB ALK JL NB YL SET KTH. Wrote the paper: RD SET KTH.
- 1. Bell J (2004) Predicting disease using genomics. Nature 429: 453–456.J. Bell2004Predicting disease using genomics.Nature429453456
- 2. Tantisira K, Weiss S (2009) The pharmacogenetics of asthma treatment. Curr Allergy Asthma Rep 9: 10–17.K. TantisiraS. Weiss2009The pharmacogenetics of asthma treatment.Curr Allergy Asthma Rep91017
- 3. Pulendran B (2007) Tolls and beyond—many roads to vaccine immunity. N Engl J Med 356: 1776–1778.B. Pulendran2007Tolls and beyond—many roads to vaccine immunity.N Engl J Med35617761778
- 4. Moore ML, Peebles RS Jr (2006) Respiratory syncytial virus disease mechanisms implicated by human, animal model, and in vitro data facilitate vaccine strategies and new therapeutics. Pharmacol Ther 112: 405–424.ML MooreRS Peebles Jr2006Respiratory syncytial virus disease mechanisms implicated by human, animal model, and in vitro data facilitate vaccine strategies and new therapeutics.Pharmacol Ther112405424
- 5. Iwasaki A, Medzhitov R (2004) Toll-like receptor control of the adaptive immune responses. Nat Immunol 5: 987–995.A. IwasakiR. Medzhitov2004Toll-like receptor control of the adaptive immune responses.Nat Immunol5987995
- 6. Turvey SE, Hawn TR (2006) Towards subtlety: understanding the role of Toll-like receptor signaling in susceptibility to human infections. Clin Immunol 120: 1–9.SE TurveyTR Hawn2006Towards subtlety: understanding the role of Toll-like receptor signaling in susceptibility to human infections.Clin Immunol12019
- 7. Miller SI, Ernst RK, Bader MW (2005) LPS, TLR4 and infectious disease diversity. Nat Rev Microbiol 3: 36–46.SI MillerRK ErnstMW Bader2005LPS, TLR4 and infectious disease diversity.Nat Rev Microbiol33646
- 8. Kurt-Jones EA, Popova L, Kwinn L, Haynes LM, Jones LP, et al. (2000) Pattern recognition receptors TLR4 and CD14 mediate response to respiratory syncytial virus. Nat Immunol 1: 398–401.EA Kurt-JonesL. PopovaL. KwinnLM HaynesLP Jones2000Pattern recognition receptors TLR4 and CD14 mediate response to respiratory syncytial virus.Nat Immunol1398401
- 9. Haynes LM, Moore DD, Kurt-Jones EA, Finberg RW, Anderson LJ, et al. (2001) Involvement of toll-like receptor 4 in innate immunity to respiratory syncytial virus. J Virol 75: 10730–10737.LM HaynesDD MooreEA Kurt-JonesRW FinbergLJ Anderson2001Involvement of toll-like receptor 4 in innate immunity to respiratory syncytial virus.J Virol751073010737
- 10. Ehl S, Bischoff R, Ostler T, Vallbracht S, Schulte-Monting J, et al. (2004) The role of Toll-like receptor 4 versus interleukin-12 in immunity to respiratory syncytial virus. Eur J Immunol 34: 1146–1153.S. EhlR. BischoffT. OstlerS. VallbrachtJ. Schulte-Monting2004The role of Toll-like receptor 4 versus interleukin-12 in immunity to respiratory syncytial virus.Eur J Immunol3411461153
- 11. Faisca P, Tran Anh DB, Thomas A, Desmecht D (2006) Suppression of pattern-recognition receptor TLR4 sensing does not alter lung responses to pneumovirus infection. Microbes Infect 8: 621–627.P. FaiscaDB Tran AnhA. ThomasD. Desmecht2006Suppression of pattern-recognition receptor TLR4 sensing does not alter lung responses to pneumovirus infection.Microbes Infect8621627
- 12. Arbour NC, Lorenz E, Schutte BC, Zabner J, Kline JN, et al. (2000) TLR4 mutations are associated with endotoxin hyporesponsiveness in humans. Nat Genet 25: 187–191.NC ArbourE. LorenzBC SchutteJ. ZabnerJN Kline2000TLR4 mutations are associated with endotoxin hyporesponsiveness in humans.Nat Genet25187191
- 13. Fageras Bottcher M, Hmani-Aifa M, Lindstrom A, Jenmalm MC, Mai XM, et al. (2004) A TLR4 polymorphism is associated with asthma and reduced lipopolysaccharide-induced interleukin-12(p70) responses in Swedish children. J Allergy Clin Immunol 114: 561–567.M. Fageras BottcherM. Hmani-AifaA. LindstromMC JenmalmXM Mai2004A TLR4 polymorphism is associated with asthma and reduced lipopolysaccharide-induced interleukin-12(p70) responses in Swedish children.J Allergy Clin Immunol114561567
- 14. Tal G, Mandelberg A, Dalal I, Cesar K, Somekh E, et al. (2004) Association between common Toll-like receptor 4 mutations and severe respiratory syncytial virus disease. J Infect Dis 189: 2057–2063.G. TalA. MandelbergI. DalalK. CesarE. Somekh2004Association between common Toll-like receptor 4 mutations and severe respiratory syncytial virus disease.J Infect Dis18920572063
- 15. Paulus SC, Hirschfeld AF, Victor RE, Brunstein J, Thomas E, et al. (2007) Common human Toll-like receptor 4 polymorphisms—role in susceptibility to respiratory syncytial virus infection and functional immunological relevance. Clin Immunol 123: 252–257.SC PaulusAF HirschfeldRE VictorJ. BrunsteinE. Thomas2007Common human Toll-like receptor 4 polymorphisms—role in susceptibility to respiratory syncytial virus infection and functional immunological relevance.Clin Immunol123252257
- 16. Puthothu B, Forster J, Heinzmann A, Krueger M (2006) TLR-4 and CD14 polymorphisms in respiratory syncytial virus associated disease. Dis Markers 22: 303–308.B. PuthothuJ. ForsterA. HeinzmannM. Krueger2006TLR-4 and CD14 polymorphisms in respiratory syncytial virus associated disease.Dis Markers22303308
- 17. Awomoyi AA, Rallabhandi P, Pollin TI, Lorenz E, Sztein MB, et al. (2007) Association of TLR4 polymorphisms with symptomatic respiratory syncytial virus infection in high-risk infants and young children. J Immunol 179: 3171–3177.AA AwomoyiP. RallabhandiTI PollinE. LorenzMB Sztein2007Association of TLR4 polymorphisms with symptomatic respiratory syncytial virus infection in high-risk infants and young children.J Immunol17931713177
- 18. Custovic A, Simpson A (2006) What are we learning from genetic cohort studies? Paediatr Respir Rev 7: Suppl 1S90–92.A. CustovicA. Simpson2006What are we learning from genetic cohort studies?Paediatr Respir Rev7Suppl 1S9092
- 19. Reijmerink NE, Bottema RW, Kerkhof M, Gerritsen J, Stelma EF, et al. TLR-related pathway analysis: novel gene-gene interactions in the development of asthma and atopy. Allergy 65: 199–207.NE ReijmerinkRW BottemaM. KerkhofJ. GerritsenEF StelmaTLR-related pathway analysis: novel gene-gene interactions in the development of asthma and atopy.Allergy65199207
- 20. Kozyrskyj AL, HayGlass KT, Sandford AJ, Pare PD, Chan-Yeung M, et al. (2009) A novel study design to investigate the early-life origins of asthma in children (SAGE study). Allergy 64: 1185–1193.AL KozyrskyjKT HayGlassAJ SandfordPD PareM. Chan-Yeung2009A novel study design to investigate the early-life origins of asthma in children (SAGE study).Allergy6411851193
- 21. Becker A, Lemiere C, Berube D, Boulet LP, Ducharme FM, et al. (2005) Summary of recommendations from the Canadian Asthma Consensus guidelines, 2003. Cmaj 173: S3–11.A. BeckerC. LemiereD. BerubeLP BouletFM Ducharme2005Summary of recommendations from the Canadian Asthma Consensus guidelines, 2003.Cmaj173S311
- 22. Stinson M, Douville R, Lissitsyn Y, Blanchard M, Stefura W, et al. (2008) Quantification of human chemokine production in TLR-stimulated and antigen-specific recall responses. Methods Mol Med 138: 121–131.M. StinsonR. DouvilleY. LissitsynM. BlanchardW. Stefura2008Quantification of human chemokine production in TLR-stimulated and antigen-specific recall responses.Methods Mol Med138121131
- 23. Stefura WP, Campbell JD, Douville R, Stinson MJ, Simons FE, et al. (2008) Ultrasensitive ELISA for measurement of human cytokine responses in primary culture. Methods Mol Med 138: 107–119.WP StefuraJD CampbellR. DouvilleMJ StinsonFE Simons2008Ultrasensitive ELISA for measurement of human cytokine responses in primary culture.Methods Mol Med138107119
- 24. Hirschfeld AF, Bettinger JA, Victor RE, Davidson DJ, Currie AJ, et al. (2007) Prevalence of Toll-like receptor signalling defects in apparently healthy children who developed invasive pneumococcal infection. Clin Immunol 122: 271–278.AF HirschfeldJA BettingerRE VictorDJ DavidsonAJ Currie2007Prevalence of Toll-like receptor signalling defects in apparently healthy children who developed invasive pneumococcal infection.Clin Immunol122271278
- 25. Ferwerda B, McCall MB, Alonso S, Giamarellos-Bourboulis EJ, Mouktaroudi M, et al. (2007) TLR4 polymorphisms, infectious diseases, and evolutionary pressure during migration of modern humans. Proc Natl Acad Sci U S A 104: 16645–16650.B. FerwerdaMB McCallS. AlonsoEJ Giamarellos-BourboulisM. Mouktaroudi2007TLR4 polymorphisms, infectious diseases, and evolutionary pressure during migration of modern humans.Proc Natl Acad Sci U S A1041664516650
- 26. Ferwerda B, McCall MB, Verheijen K, Kullberg BJ, van der Ven AJ, et al. (2008) Functional consequences of toll-like receptor 4 polymorphisms. Mol Med 14: 346–352.B. FerwerdaMB McCallK. VerheijenBJ KullbergAJ van der Ven2008Functional consequences of toll-like receptor 4 polymorphisms.Mol Med14346352
- 27. van der Graaf C, Kullberg BJ, Joosten L, Verver-Jansen T, Jacobs L, et al. (2005) Functional consequences of the Asp299Gly Toll-like receptor-4 polymorphism. Cytokine 30: 264–268.C. van der GraafBJ KullbergL. JoostenT. Verver-JansenL. Jacobs2005Functional consequences of the Asp299Gly Toll-like receptor-4 polymorphism.Cytokine30264268
- 28. von Aulock S, Schroder NW, Gueinzius K, Traub S, Hoffmann S, et al. (2003) Heterozygous toll-like receptor 4 polymorphism does not influence lipopolysaccharide-induced cytokine release in human whole blood. J Infect Dis 188: 938–943.S. von AulockNW SchroderK. GueinziusS. TraubS. Hoffmann2003Heterozygous toll-like receptor 4 polymorphism does not influence lipopolysaccharide-induced cytokine release in human whole blood.J Infect Dis188938943
- 29. Newport MJ, Allen A, Awomoyi AA, Dunstan SJ, McKinney E, et al. (2004) The toll-like receptor 4 Asp299Gly variant: no influence on LPS responsiveness or susceptibility to pulmonary tuberculosis in The Gambia. Tuberculosis (Edinb) 84: 347–352.MJ NewportA. AllenAA AwomoyiSJ DunstanE. McKinney2004The toll-like receptor 4 Asp299Gly variant: no influence on LPS responsiveness or susceptibility to pulmonary tuberculosis in The Gambia.Tuberculosis (Edinb)84347352
- 30. Michel O, LeVan TD, Stern D, Dentener M, Thorn J, et al. (2003) Systemic responsiveness to lipopolysaccharide and polymorphisms in the toll-like receptor 4 gene in human beings. J Allergy Clin Immunol 112: 923–929.O. MichelTD LeVanD. SternM. DentenerJ. Thorn2003Systemic responsiveness to lipopolysaccharide and polymorphisms in the toll-like receptor 4 gene in human beings.J Allergy Clin Immunol112923929
- 31. Tulic MK, Hurrelbrink RJ, Prele CM, Laing IA, Upham JW, et al. (2007) TLR4 Polymorphisms Mediate Impaired Responses to Respiratory Syncytial Virus and Lipopolysaccharide. J Immunol 179: 132–140.MK TulicRJ HurrelbrinkCM PreleIA LaingJW Upham2007TLR4 Polymorphisms Mediate Impaired Responses to Respiratory Syncytial Virus and Lipopolysaccharide.J Immunol179132140
- 32. Scagnolari C, Midulla F, Pierangeli A, Moretti C, Bonci E, et al. (2009) Gene expression of nucleic acid-sensing pattern recognition receptors in children hospitalized for respiratory syncytial virus-associated acute bronchiolitis. Clin Vaccine Immunol 16: 816–823.C. ScagnolariF. MidullaA. PierangeliC. MorettiE. Bonci2009Gene expression of nucleic acid-sensing pattern recognition receptors in children hospitalized for respiratory syncytial virus-associated acute bronchiolitis.Clin Vaccine Immunol16816823
- 33. Klein Klouwenberg P, Tan L, Werkman W, van Bleek GM, Coenjaerts F (2009) The role of Toll-like receptors in regulating the immune response against respiratory syncytial virus. Crit Rev Immunol 29: 531–550.P. Klein KlouwenbergL. TanW. WerkmanGM van BleekF. Coenjaerts2009The role of Toll-like receptors in regulating the immune response against respiratory syncytial virus.Crit Rev Immunol29531550
- 34. Numata M, Chu HW, Dakhama A, Voelker DR (2010) Pulmonary surfactant phosphatidylglycerol inhibits respiratory syncytial virus-induced inflammation and infection. Proc Natl Acad Sci U S A 107: 320–325.M. NumataHW ChuA. DakhamaDR Voelker2010Pulmonary surfactant phosphatidylglycerol inhibits respiratory syncytial virus-induced inflammation and infection.Proc Natl Acad Sci U S A107320325
- 35. Murawski MR, Bowen GN, Cerny AM, Anderson LJ, Haynes LM, et al. (2009) Respiratory syncytial virus activates innate immunity through Toll-like receptor 2. J Virol 83: 1492–1500.MR MurawskiGN BowenAM CernyLJ AndersonLM Haynes2009Respiratory syncytial virus activates innate immunity through Toll-like receptor 2.J Virol8314921500
- 36. Cyr SL, Angers I, Guillot L, Stoica-Popescu I, Lussier M, et al. (2009) TLR4 and MyD88 control protection and pulmonary granulocytic recruitment in a murine intranasal RSV immunization and challenge model. Vaccine 27: 421–430.SL CyrI. AngersL. GuillotI. Stoica-PopescuM. Lussier2009TLR4 and MyD88 control protection and pulmonary granulocytic recruitment in a murine intranasal RSV immunization and challenge model.Vaccine27421430
- 37. Bhoj VG, Sun Q, Bhoj EJ, Somers C, Chen X, et al. (2008) MAVS and MyD88 are essential for innate immunity but not cytotoxic T lymphocyte response against respiratory syncytial virus. Proc Natl Acad Sci U S A 105: 14046–14051.VG BhojQ. SunEJ BhojC. SomersX. Chen2008MAVS and MyD88 are essential for innate immunity but not cytotoxic T lymphocyte response against respiratory syncytial virus.Proc Natl Acad Sci U S A1051404614051
- 38. Triantafilou M, Triantafilou K (2002) Lipopolysaccharide recognition: CD14, TLRs and the LPS-activation cluster. Trends Immunol 23: 301–304.M. TriantafilouK. Triantafilou2002Lipopolysaccharide recognition: CD14, TLRs and the LPS-activation cluster.Trends Immunol23301304