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(Chest. 2002;121:62S-68S.)
© 2002 American College of Chest Physicians

The Genetics of Innate Immunity*

David A. Schwartz, MD, FCCP

* From the Pulmonary and Critical Care Division, Department of Medicine and the Department of Veterans Affairs Medical Center and Duke University Medical Center, Durham, NC.

Correspondence to: David Schwartz, MD, FCCP, Pulmonary and Critical Care Medicine, Duke University Medical Center, Research Dr, Room 275 MSRB, DUMC Box 2629, Durham, NC 27710; e-mail: david.schwartz{at}duke.edu


   Abstract
TOP
 Abstract
Methods and Results
 Discussion
 References
 
Despite the tremendous interindividual variability in the response to toxins, we simply do not understand why certain people have disease develop when challenged with toxic agents, and why others remain healthy. To address this concern, we investigated whether the TLR-4 gene (toll-like receptor [TLR]4), which has been shown to affect lipopolysaccharide (LPS) responsiveness in mice, underlies the variability in airway responsiveness to inhaled LPS in humans. Here we show that common, cosegregating missense mutations (Asp299Gly and Thr399Ile) in the extracellular domain of the TLR4 receptor are associated with a significantly blunted response to inhaled LPS in 83 humans. Although in vitro findings confirm these in vivo observations, our results in humans also indicate that genes other than TLR4 may be playing a role in the biological response to LPS. To pursue this possibility, we studied genetically diverse inbred strains of mice, as well as recombinant inbred strains of mice, and have found that although TLR4 is clearly important in directing the biological response to LPS, additional genes are clearly involved in determining the physiologic and biological response to LPS in mammals.

Innate immunity acts as the first line of host defense against microbial pathogens. It is conserved over a wide variety of species from flies to mammals.1 Innate immunity uses germline-encoded receptors to aid in antimicrobial host defense.2 3 These receptors recognize certain patterns, rather than particular structures, and it is possible for a limited number of pattern recognition receptors (PRRs) to recognize a wide variety of microbes. In the case of lipopolysaccharide (LPS), the PRR is directed against the highly conserved portion of lipid A, which acts as the pathogen-associated molecular pattern (PAMP) against which the PRR developed in defense against Gram-negative bacterial infection. Some of the PRRs bind directly to PAMPs, such as CD14 recognizing and binding LPS, whereas others, such as the toll-like receptor (TLR)4 gene, are thought to recognize complexes generated by PAMPs, such as the CD14/LPS binding protein/LPS complex.2 4

The pathogenic importance of LPS in Gram-negative sepsis is well established. Endotoxin on the surface of Gram-negative bacteria is in a position to activate biological mediators of shock even if the amount of free, solubilized endotoxin is below detectable levels. IV LPS induces all of the clinical features of Gram-negative sepsis, including fever, shock, leukopenia followed by leukocytosis, disseminated intravascular coagulation, and death.5 6 These changes can be elicited with LPS from Gram-negative bacteria or the intact organisms. In fact, in patients with Gram-negative sepsis, antibiotics are ineffective in reversing the pathophysiologic effects of LPS. Higher concentrations of circulating levels of endotoxin have been associated with manifestations of the systemic inflammatory response syndrome,7 and the development of ARDS following sepsis.8

There is convincing evidence that endotoxin exacerbates airflow obstruction and airway inflammation in allergic asthmatics. Among allergic asthmatics who are sensitive to house dust mite allergen, the concentration of endotoxin in the home environment, but not the concentration of mite allergen (Dermatophagoides pteronyssinus), was significantly associated with the severity of asthma.9 Experimentally, allergic asthmatics are more sensitive to the bronchoconstrictive effects of inhaled endotoxin.10 Moreover, among allergic asthmatics, prior allergen challenge significantly augments the inflammatory response to inhaled endotoxin.11 However, independent of its effect in allergic asthma, several studies12 13 14 15 16 demonstrate that inhalation of air contaminated with endotoxin is associated with the classical features of asthma (reversible airflow obstruction and airway inflammation, airway hyperreactivity, and airway remodeling). Epidemiologic studies have shown that the concentration of inhaled endotoxin in the bioaerosol is strongly and consistently associated with reversible airflow obstruction among cotton workers,12 agricultural workers,13 and fiberglass workers.14 In fact, the concentration of endotoxin in the bioaerosol is the most important occupational exposure associated with the development15 and progression13 of airway disease in agricultural workers.

However, not everyone exposed to high concentrations of LPS has these problems develop. In fact, the ability of the host to respond to endotoxin is highly variable. In mice, genetic differences in susceptibility to LPS are well established; C3H/HeJ and C57BL/10ScCR strains are hyporesponsive to LPS. We have found that genetic or acquired hyporesponsiveness to endotoxin substantially reduces the biological response to grain dust in mice.16 Interindividual differences have been reported in the release and synthesis of cytokines by human monocytes stimulated with LPS in vitro,17 and a patient with recurrent bacterial infections has been reported to be refractory to the in vivo and in vitro effects of LPS.18


   Methods and Results
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 Abstract
 Methods and Results
Discussion
 References
 
To further pursue the issue of individual airway responsiveness to LPS, we developed an LPS inhalation protocol and characterized the airway response in healthy, nonatopic subjects.19 The results indicated a broad range of stable responses (sensitive, intermediate, and hyporesponsive) that strongly supported a genetic influence on the airway response to inhaled LPS in humans. To determine whether TLR4 (a recently described transmembrane receptor for LPS3 ) plays an important immunologic role in the in vivo response of humans to inhaled LPS, we examined the relationship between polymorphisms in the TLR4 gene and the airway response to inhaled LPS in 83 normal healthy, nonasthmatic subjects; 52 subjects (63%) were responsive to inhaled LPS and 31 subjects (37%) were hyporesponsive to inhaled LPS. Using the single standard informational polymorphism, we screened the entire coding region (including slice sites) of TLR4 in all 83 subjects in our study population; 10 subjects (12%) had a band variant detected by the single standard informational polymorphism. Direct sequencing detected an A to G substitution at nucleotide 896 from the start codon of the TLR4 complementary DNA.20 Cosegregating missense mutations (Asp299Gly and Thr399Ile) in the extracellular domain of the TLR4 receptor were found to be associated with a significantly blunted response to inhaled LPS in humans, with mutant sequence variants occurring in three LPS-responsive (5.8%) and seven LPS-hyporesponsive (22.6%) study subjects (relative risk, 4.8; p = 0.03; Fig 1 ). Among the subjects with the common TLR4 allele (n = 73), the dose-response slope (percentage of decline in FEV1/cumulative dose of LPS) averaged 1.86% decline in FEV1 per microgram of inhaled LPS, while the dose-response slope for the subjects with the missense mutations (Asp299Gly and Thr399Ile) [n = 10] was much less (p = 0.037), averaging 0.59%.21



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  		Figure 1. Airway responsiveness to inhaled LPS and TLR4 genotype. The number of subjects that decrease their FEV1 by 20% is plotted against the cumulative dose of inhaled LPS. The solid bars identify those subjects that have at least a 20% decline in their FEV1, while the shaded bars represent those subjects that are resistant to the bronchoconstrictive effects of inhaled LPS; subjects with the TLR4 mutations are indicated by open boxes. RR = relative risk.

 
The biological significance of the Asp299Gly and Thr399Ile mutations was evaluated in several ways. First, transfection of THP-1 cells with either the wild-type or the mutant alleles of the TLR4 gene demonstrates that the cells transfected with the Asp299Gly allele do not respond normally to LPS stimulation, while those transfected with the Thr399Ile allele have an intermediate response to LPS (Fig 2 ). This experiment shows that the presence of the Asp299Gly amino-acid change causes a more severe phenotype than the presence of the Thr399Ile mutation. It is interesting that replacement of the conserved aspartic acid with glycine at position 299 theoretically causes disruption of the {alpha}-helical protein structure resulting in an extended ß strand while substitution of isoleucine for threonine at position 399 should not alter the structure of the extracellular domain of this receptor.22



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  		Figure 2. Functional significance of TLR4 mutations in THP-1 cells. THP-1 cells were transfected with TLR4 expression plasmids (WT, Asp299Gly, or Thr399Ile); 24 h later, the cells were stimulated with 100 ng/mL of LPS for 6 h. Nuclear factor (NF)-{kappa}B activity was measured using a commercially available nuclear factor-{kappa}B reporter plasmid encoding for the luciferase gene. The nuclear factor-{kappa}B activity following LPS stimulation is significantly (p < 0.01) less for the THP-1 cells transfected with the Asp299Gly plasmid compared to cells transfected with the WT TLR4 plasmid. L.U. = luciferase units.

 
Second, airway epithelia obtained from heterozygote individuals with both mutations (Asp299Gly and Thr399Ile) do not respond to LPS stimulation (Fig 3 , left, a). LPS stimulation resulted in significantly (p < 0.001) more interleukin (IL)-1 released by the WT/WT specimens but not the WT/Asp299Gly and Thr399Ile specimens. Figure 3 , right, b shows an en face view of human airway epithelia (in vitro) stained with an anti-TLR4 antibody in TLR4 wild-type (Fig 3 , top right, b) and in TLR4 heterozygote airway epithelia (Fig 3 , bottom right, b). The black and white inserts show the representative SEM image from epithelia from the donors studied by immunocytochemistry. Thus, airway epithelia expressing both mutations (Asp299Gly and Thr399Ile) have markedly less TLR4-receptor expression on the apical surface and are less responsive to in vitro stimulation with LPS.



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  		Figure 3. Functional significance of TLR4 mutations in primary human epithelial cells. Airway epithelial cells were genotyped for TLR4, cultured, and stimulated with LPS. The basal- and LPS-stimulated (100 ng/mL) release of IL-1 was measured in WT/WT (12 specimens from four individuals) and WT/Asp299Gly and Thr399Ile (24 specimens from four individuals) epithelia by collecting the basolateral conditioned media after 24 h (left, a). Right, b: en face view of human airway epithelia stained with an anti-TLR4 antibody in wild-type epithelia (top panel) and in a TLR4 heterozygote epithelia (bottom panel). Scale bar indicates 50 µm. The black and white inserts show the representative SEM image from epithelia from the donors studied by immunocytochemistry. Scale bar indicates 30 µm.

 
Third, we were able to reverse the LPS-hyporesponsive phenotype by overexpressing the wild-type allele of TLR4 in either primary airway epithelial cells or alveolar macrophages obtained from individuals with the TLR4 mutations. To accomplish these studies, we infected heterozygote (WT/Asp299Gly and Thr399Ile) airway epithelia (Fig 4 , left, a) or homozygote (Asp299Gly and Thr399Ile/Asp299Gly and Thr399Ile) alveolar macrophages (Fig 4 , right, b) with a recombinant adenovirus vector expressing TLR4 (Genbank #U88880).23 After collecting the basal specimen, the epithelia were exposed to 100 ng/mL of LPS on the apical side for 6 h, and the media was collected after 24 h. Heterozygote airway epithelia produced significantly more IL-1 after infection with the adenovirus vector expressing TLR4 than before transfection. Human alveolar macrophages were obtained by BAL from our homozygote (Asp299Gly and Thr399Ile/Asp299Gly and Thr399Ile) study subject and were seeded at a density of 105 cells per well. The cells were infected with Ad/TLR4, then 16 h after infection, the cells were exposed to varying concentrations of LPS (0 to 100 ng/mL) for 6 h and the media was collected.



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  		Figure 4. Rescue of the LPS-hyporesponsive phenotype. We infected heterozygote (WT/Asp299Gly and Thr399Ile) airway epithelia (Fig 3 , left, a) or homozygote (Asp299Gly and Thr399Ile/Asp299Gly and Thr399Ile) alveolar macrophages (Fig 3 , right, b) with a recombinant adenovirus vector expressing TLR4. After collecting the basal specimen, the epithelia were exposed to 100 ng/mL of LPS on the apical side for 6 h, and the media were collected after 24 h. Heterozygote airway epithelia produced significantly (p < 0.001) more IL-1 after infection with the adenovirus vector expressing TLR4 than before transfection. Human alveolar macrophages were obtained by BAL from our homozygote (Asp299Gly and Thr399Ile/Asp299Gly and Thr399Ile) study subject and were seeded at a density of 105 cells per well. The cells were infected with Ad/TLR4; 16 h after infection, the cells were exposed to LPS (100 ng/mL) for 6 h and the conditioned media was collected. TNF-{alpha} was measured using a commercially available enzyme-linked immunosorbent assay.

 
To further evaluate the relationship between TLR4 and LPS responsiveness, we genotyped 18 genetically diverse strains of mice for TLR4 and measured the physiologic and biological response of these strains to inhaled LPS.24 Twelve of the 18 strains of mice were found to have mutations in TLR4. While two mutant strains (C3H/HeJ and C57BL/10ScNCr) were clearly hyporesponsive to inhaled LPS, there was a broad physiologic and biological response to inhaled LPS among the remaining mutant and wild-type strains (Fig 5 ). Strains DBA/2-J and C57BL/6 are both wild type for TLR4, yet DBA/2-J is much more sensitive to inhaled LPS than strain C57BL/6. However, strain Cast/Ei, which has a multitude of base pair changes in the TLR4 open-reading frame compared to C57BL/6, shows an almost identical response to inhaled LPS. These findings provide evidence that other genes, apart from the TLR4 receptor, are important determinants of the physiologic and biological response to inhaled LPS.



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  		Figure 5. The concentration of lavage PMNs after a single inhalation challenge of LPS in 18 genetically diverse strains of mice. All values represent the mean ± SEM in units of cells (x 103) per milliliter of lavage fluid.

 
To pursue this further, we focused on the phenotypic variation among mice with the common allele for TLR4 (WT TLR4). Thus far, we have phenotyped 32 of the BXD recombinant inbred (RI) strains that are derived from C57BL/6 (low responder to inhaled LPS and wild type for TLR4) and DBA/2-J (high responder to inhaled LPS and wild type for TLR4). As depicted in Figure 6 , we have identified a broad range of responsiveness to inhaled LPS among the BXD RI strains. Importantly, one strain is one to two orders of magnitude less responsive to inhaled LPS than the other recombinant strains. We have also found that this hyporesponsive strain had undetectable levels of tumor necrosis factor (TNF)-{alpha} in the lavage fluid; the other 31 strains had concentrations of TNF-{alpha} ranging from 94 to 2,047 pg/mL of lavage fluid. To be certain that this was not a function of a spontaneous mutation in TLR4, we genotyped the three highest and the three lowest responders (polymorphonuclear leukocytes [PMNs] per milliliter of lavage fluid) and have found that all six RI strains are wild type for TLR4, yet have clearly different inflammatory response to inhaled LPS. Moreover, although our findings in humans demonstrate that sequence changes in TLR4 clearly alter the ability to respond to inhaled LPS,21 not all of our subjects who were hyporesponsive to LPS had the mutations in TLR4, and not everyone with the TLR4 mutation was hyporesponsive to inhaled LPS. In aggregate, these observations suggest that the response to LPS is biologically complex, and that TLR4 genotypes are only one factor that determines the response to inhaled LPS; undoubtedly, other genes are involved in regulating this response.



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  		Figure 6. The concentration of lavage PMNs after a single inhalation challenge of LPS in 32 RI strains derived from C57BL/6 and DBA/2-J cross. All values represent the mean ± SEM in units of cells (x 103) per milliliter of lavage fluid.

 

   Discussion
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 Abstract
 Methods and Results
 Discussion
References
 
These results provide the first direct evidence to indicate that a sequence polymorphism in the TLR4 gene causes an endotoxin-hyporesponsive phenotype in humans. This discovery may have important ramifications for a broad spectrum of human diseases, such as the systemic inflammatory response syndrome,7 ARDS,8 and asthma or other forms of airway disease caused13 or exacerbated9 15 25 by endotoxin. However, the mechanism(s) and cells through which TLR4 modulates the airway response to inhaled LPS are not known, the ability of TLR4 to initiate LPS-induced PMN recruitment is unexplored, and the physiologic and biological implications of the common cosegregating mutations in TLR4 (Asp299Gly and Thr399Ile) that are associated with LPS hyporesponsiveness in humans have not yet been investigated. The TLR4 mutations (Asp299Gly and Thr399Ile) that we have found to be associated with LPS hyporesponsiveness in humans21 provide a natural biological tool to investigate the structure and function of the TLR4 receptor and the effect that this has on LPS-induced airway disease.

The extracellular domain of TLR4, and specific amino-acid changes that we have identified, may play an important role in receptor function and could provide key therapeutic targets to modulate LPS signaling in humans. It is interesting that the reported murine mutations in the TLR4 gene26 27 (C3H/HeJ mice have a missense mutation resulting a Pro712His substitution in the intracellular domain of TLR4, and C57BL10/ScCr mice have complete disruption of TLR4) are quite different that the mutations that we have discovered in humans. However, our findings in humans suggest that mutations in the extracellular domain of TLR4 either disrupts the transport of this receptor to the cell membrane or that the mutation impairs ligand binding or protein interactions. Although the data in this study support the former possibility (reduced TLR4 expression on airway epithelia in heterozygote individuals; Fig 3 , right, b), replacement of the conserved aspartic acid with glycine at position 299 theoretically causes disruption of the {alpha}-helical protein structure resulting in an extended strand.22

Despite the important role that TLR4 plays in mediating the response to LPS in mammals, other genes are involved in this complex pathophysiologic response. Although the mutations in TLR4 are associated with LPS hyporesponsiveness in humans21 and mice,26 27 28 not all of our subjects who were hyporesponsive to LPS had the mutations in TLR4, and not everyone with the TLR4 mutations was hyporesponsive to inhaled LPS. Moreover, our preliminary data in mice indicate that LPS responsiveness is, in part, determined by mutations in genes other than TLR4. C3H/HeJ was the first mouse strain shown to be LPS hyporesponsive.29 C3H/HeJ mice have a mutated TLR4 protein, which shows no activity in vitro and C3H/HeJ-derived B cells and macrophages show an almost identical LPS response compared to TLR4 -/- derived B cells. Yet, C3H/HeJ mice are able to respond to LPS.30 First, in vivo studies demonstrate that C3H/HeJ mice are hyporesponsive, not unresponsive to LPS, and will initiate LPS-dependent transcription at high doses of LPS.31 Second, the defects in response to LPS can be overcome by treatment with interferon-{gamma} and other agents.32 33 Third, certain LPS preparations, such as isolated from Porphyromonas gingivalis, can activate C3H/HeJ mice,34 suggesting that different receptors may exist for different forms of LPS. In fact, while C3H/HeJ mice were unresponsive to smooth LPS, they showed a normal response to rough LPS.35 Fourth, it was hypothesized that two unique receptors would exist for each isoform of LPS. A 38-kd protein was identified as being present on both normal and C3H/HeJ lymphoid cells, and binding of rough LPS to this protein could not be inhibited by purified lipid A, as had been the case for an 80-kd receptor molecule.36 37 In addition, major histocompatibility complex class II genes have been shown to modulate the response of macrophages to LPS.38 Human and murine cells with reduced expression levels of class II major histocompatibility complex molecules showed a decreased secretion of proinflammatory cytokines following LPS stimulation. Fifth, recent experiments in CD14-/- mice have shown that while CD14 is essential for the cellular response to LPS, in CD14-/- mice, Escherichia coli can stimulate TNF-{alpha} secretion at similar levels as wild-type mice. Part of this CD14-independent response appears to be mediated by CD11b.39 40 These results suggest that exposure to bacteria will cause activation of cellular pathways in addition to the ones activated by LPS, and that in the absence of the primary receptor molecule, in this case CD14, other proteins can substitute and initiate alternative signaling pathways. Finally, the strongest indication that genes independent of TLR4 are involved in LPS response comes from a comparison of macrophages and splenocytes derived from TLR4-/- and MyD88-/- mice. The MyD88 gene encodes a protein acting downstream of TLR4 and is thought to complex with TLR4 on LPS stimulation. While MyD88-/- mice-derived cells were totally unresponsive to LPS, TLR4-/- derived macrophages and splenocytes were able to respond to P gingivalis LPS and Gram-positive cell wall components,41 similar to observations in C3H/HeJ mice. These findings indicate that in the absence of functional TLR4, cells tend to be hyporesponsive rather than unresponsive, suggesting the presence of genes, other than TLR4, mediating the response to LPS.

Other evidence for a TLR4-independent LPS response is based on work done by Wong et al,42 who isolated a G-protein family member, RAN, which is mutated in C3H/HeJ mice. Interestingly, retroviral infection with the wild-type RAN, which maps near TLR4, can restore LPS response in C3H/HeJ mice. We have also recently found that MD-2 is a required component of the LPS signaling complex, and that Chinese hamster ovary K1 fibroblasts, which are defective in responding to bacterial LPS, have a point mutation in a highly conserved region of MD-2.43 In aggregate, these findings provide complementary genetic evidence that several genes control the LPS response in mice. This raises the possibility that while TLR4/CD14 may be an important LPS receptor, other genes can act independent of LPS binding complex.


   Footnotes
 
Abbreviations: IL = interleukin; LPS = lipopolysaccharide; PAMP = pathogen-associated molecular pattern; PMN = polymorphoneclear leukocyte; PRR = pattern recognition receptor; RI = recombinant inbred; TLR = toll-like receptor; TNF = tumor necrosis factor

This study was supported by grants from the National Institutes of Health (ES07498, ES09607, HL62628, HL66611, and HL66604) and the Department of Veterans’ Affairs (Merit Review).


   References
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