Thomas R. Hawn

The innate immune response to bacterial flagellin is mediated by Toll-like receptor 5

The innate immune system recognizes pathogen-associated molecular patterns (PAMPs) that are expressed on infectious agents, but not on the host. Toll-like receptors (TLRs) recognize PAMPs and mediate the production of cytokines necessary for the development of effective immunity. Flagellin, a principal component of bacterial flagella, is a virulence factor that is recognized by the innate immune system in organisms as diverse as flies, plants and mammals. Here we report that mammalian TLR5 recognizes bacterial flagellin from both Gram-positive and Gram-negative bacteria, and that activation of the receptor mobilizes the nuclear factor NF-κB and stimulates tumour necrosis factor-α production. TLR5-stimulating activity was purified from Listeria monocytogenes culture supernatants and identified as flagellin by tandem mass spectrometry. Expression of L. monocytogenes flagellin in non-flagellated Escherichia coli conferred on the bacterium the ability to activate TLR5, whereas deletion of the flagellin genes from Salmonella typhimurium abrogated TLR5-stimulating activity. All known TLRs signal through the adaptor protein MyD88. Mice challenged with bacterial flagellin rapidly produced systemic interleukin-6, whereas MyD88-null mice did not respond to flagellin. Our data suggest that TLR5, a member of the evolutionarily conserved Toll-like receptor family, has evolved to permit mammals specifically to detect flagellated bacterial pathogens.

Pyogenic bacterial infections in humans with IRAK-4 deficiency.

MyD88 is a key downstream adapter for most Toll-like receptors (TLRs) and interleukin-1 receptors (IL-1Rs). MyD88 deficiency in mice leads to susceptibility to a broad range of pathogens in experimental settings of infection. We describe a distinct situation in a natural setting of human infection. Nine children with autosomal recessive MyD88 deficiency suffered from life-threatening, often recurrent pyogenic bacterial infections, including invasive pneumococcal disease. However, these patients were otherwise healthy, with normal resistance to other microbes. Their clinical status improved with age, but not due to any cellular leakiness in MyD88 deficiency. The MyD88-dependent TLRs and IL-1Rs are therefore essential for protective immunity to a small number of pyogenic bacteria, but redundant for host defense to most natural infections.

Toll-Like Receptor-4 Mediates Vascular Inflammation and Insulin Resistance in Diet-Induced Obesity

Vascular dysfunction is a major complication of metabolic disorders such as diabetes and obesity. The current studies were undertaken to determine whether inflammatory responses are activated in the vasculature of mice with diet-induced obesity, and if so, whether Toll-Like Receptor-4 (TLR4), a key mediator of innate immunity, contributes to these responses. Mice lacking TLR4 (TLR4−/−) and wild-type (WT) controls were fed either a low fat (LF) control diet or a diet high in saturated fat (HF) for 8 weeks. In response to HF feeding, both genotypes displayed similar increases of body weight, body fat content, and serum insulin and free fatty acid (FFA) levels compared with mice on a LF diet. In lysates of thoracic aorta from WT mice maintained on a HF diet, markers of vascular inflammation both upstream (IKK&bgr; activity) and downstream of the transcriptional regulator, NF-&kgr;B (ICAM protein and IL-6 mRNA expression), were increased and this effect was associated with cellular insulin resistance and impaired insulin stimulation of eNOS. In contrast, vascular inflammation and impaired insulin responsiveness were not evident in aortic samples taken from TLR4−/− mice fed the same HF diet, despite comparable increases of body fat mass. Incubation of either aortic explants from WT mice or cultured human microvascular endothelial cells with the saturated FFA, palmitate (100 &mgr;mol/L), similarly activated IKK&bgr;, inhibited insulin signal transduction and blocked insulin-stimulated NO production. Each of these effects was subsequently shown to be dependent on both TLR4 and NF-&kgr;B activation. These findings identify the TLR4 signaling pathway as a key mediator of the deleterious effects of palmitate on endothelial NO signaling, and are the first to document a key role for TLR4 in the mechanism whereby diet-induced obesity induces vascular inflammation and insulin resistance.

The Influence of Host and Bacterial Genotype on the Development of Disseminated Disease with Mycobacterium tuberculosis

The factors that govern the development of tuberculosis disease are incompletely understood. We hypothesized that some strains of Mycobacterium tuberculosis (M. tuberculosis) are more capable of causing disseminated disease than others and may be associated with polymorphisms in host genes responsible for the innate immune response to infection. We compared the host and bacterial genotype in 187 Vietnamese adults with tuberculous meningitis (TBM) and 237 Vietnamese adults with uncomplicated pulmonary tuberculosis. The host genotype of tuberculosis cases was also compared with the genotype of 392 cord blood controls from the same population. Isolates of M. tuberculosis were genotyped by large sequence polymorphisms. The hosts were defined by polymorphisms in genes encoding Toll-interleukin 1 receptor domain containing adaptor protein (TIRAP) and Toll-like receptor-2 (TLR-2). We found a significant protective association between the Euro-American lineage of M. tuberculosis and pulmonary rather than meningeal tuberculosis (Odds ratio (OR) for causing TBM 0.395, 95% confidence intervals (C.I.) 0.193–0.806, P = 0.009), suggesting these strains are less capable of extra-pulmonary dissemination than others in the study population. We also found that individuals with the C allele of TLR-2 T597C allele were more likely to have tuberculosis caused by the East-Asian/Beijing genotype (OR = 1.57 [95% C.I. 1.15–2.15]) than other individuals. The study provides evidence that M. tuberculosis genotype influences clinical disease phenotype and demonstrates, for the first time, a significant interaction between host and bacterial genotypes and the development of tuberculosis.

The lta4h locus modulates susceptibility to mycobacterial infection in zebrafish and humans.

Exposure to Mycobacterium tuberculosis produces varied early outcomes, ranging from resistance to infection to progressive disease. Here we report results from a forward genetic screen in zebrafish larvae that identify multiple mutant classes with distinct patterns of innate susceptibility to Mycobacterium marinum. A hypersusceptible mutant maps to the lta4h locus encoding leukotriene A(4) hydrolase, which catalyzes the final step in the synthesis of leukotriene B(4) (LTB(4)), a potent chemoattractant and proinflammatory eicosanoid. lta4h mutations confer hypersusceptibility independent of LTB(4) reduction, by redirecting eicosanoid substrates to anti-inflammatory lipoxins. The resultant anti-inflammatory state permits increased mycobacterial proliferation by limiting production of tumor necrosis factor. In humans, we find that protection from both tuberculosis and multibacillary leprosy is associated with heterozygosity for LTA4H polymorphisms that have previously been correlated with differential LTB(4) production. Our results suggest conserved roles for balanced eicosanoid production in vertebrate resistance to mycobacterial infection.

Host Genotype-Specific Therapies Can Optimize the Inflammatory Response to Mycobacterial Infections

Susceptibility to tuberculosis is historically ascribed to an inadequate immune response that fails to control infecting mycobacteria. In zebrafish, we find that susceptibility to Mycobacterium marinum can result from either inadequate or excessive acute inflammation. Modulation of the leukotriene A(4) hydrolase (LTA4H) locus, which controls the balance of pro- and anti-inflammatory eicosanoids, reveals two distinct molecular routes to mycobacterial susceptibility converging on dysregulated TNF levels: inadequate inflammation caused by excess lipoxins and hyperinflammation driven by excess leukotriene B(4). We identify therapies that specifically target each of these extremes. In humans, we identify a single nucleotide polymorphism in the LTA4H promoter that regulates its transcriptional activity. In tuberculous meningitis, the polymorphism is associated with inflammatory cell recruitment, patient survival and response to adjunctive anti-inflammatory therapy. Together, our findings suggest that host-directed therapies tailored to patient LTA4H genotypes may counter detrimental effects of either extreme of inflammation.

Cutting Edge: A Toll-Like Receptor 2 Polymorphism That Is Associated with Lepromatous Leprosy Is Unable to Mediate Mycobacterial Signaling

Toll-like receptors (TLRs) are key mediators of the innate immune response to microbial pathogens. We investigated the role of TLRs in the recognition of Mycobacterium leprae and the significance of TLR2Arg677Trp, a recently discovered human polymorphism that is associated with lepromatous leprosy. In mice, TNF-α production in response to M. leprae was essentially absent in TLR2-deficient macrophages. Similarly, human TLR2 mediated M. leprae-dependent activation of NF-κB in transfected Chinese hamster ovary and human embryonic kidney 293 cells, with enhancement of this signaling in the presence of CD14. In contrast, activation of NF-κB by human TLR2Arg677Trp was abolished in response to M. leprae and Mycobacterium tuberculosis. The impaired function of this TLR2 variant provides a molecular mechanism for the poor cellular immune response associated with lepromatous leprosy and may have important implications for understanding the pathogenesis of other mycobacterial infections.

TOLL-LIKE RECEPTOR POLYMORPHISMS AND SUSCEPTIBILITY TO HUMAN DISEASE

Although several lines of evidence suggest that variation in human inflammation is genetically controlled, the genes which regulate these responses are largely unknown. TLRs (Toll-like receptors) mediate recognition of microbes, regulate activation of the innate immune response and influence the formation of adaptive immunity. Cellular and molecular studies over the past several years have identified a number of common TLR polymorphisms that modify the cellular immune response and production of cytokines in vitro. In addition, human genetic studies suggest that some of these polymorphisms are associated with susceptibility to a spectrum of diseases. In this review, we summarize studies of common TLR polymorphisms and how this work is beginning to illuminate the influence of human variation on inflammation and disease susceptibility.

A common human TLR1 polymorphism regulates the innate immune response to lipopeptides

Toll‐like receptors (TLR) are critical mediators of the immune response to pathogens and human polymorphisms in this gene family regulate inflammatory pathways and are associated with susceptibility to infection. Lipopeptides are present in a wide variety of microbes and stimulate immune responses through TLR1/2 or TLR2/6 heterodimers. It is not currently known whether polymorphisms in TLR1 regulate the innate immune response. We stimulated human whole blood with triacylated lipopeptide, a ligand for TLR1/2 heterodimers, and found substantial inter‐individual variation in the immune response. We sequenced the coding region of TLR1 and found a non‐synonymous polymorphism, I602S (base pair T1805G), that regulated signalling. In comparison to TLR1_602S, the 602I variant mediated substantially greater basal and lipopeptide‐induced NF‐κB signalling in transfected HEK293 cells. These signalling differences among TLR1 variants were also found with stimulation by extracts of Mycobacterium tuberculosis. Furthermore, individuals with the 602II genotype produced substantially more IL‐6 than those with the 602SS variant in a lipopeptide‐stimulated whole‐blood cytokine assay. Together, these observations demonstrate that variation in the inflammatory response to bacterial lipopeptides is regulated by a common TLR1 transmembrane domain polymorphism that could potentially impact the innate immune response and clinical susceptibility to a wide spectrum of pathogens.

A polymorphism in human TLR2 is associated with increased susceptibility to tuberculous meningitis

Tuberculous meningitis (TBM) results from the haematogenous dissemination of Mycobacterium tuberculosis from the lung to the brain. Dissemination is believed to occur early during infection, before the development of adaptive immunity. Toll-like receptor 2 (TLR2) mediates recognition of M. tuberculosis and initiates the innate immune response to infection. We hypothesized that polymorphisms in the TLR2 gene influence bacterial dissemination and the development of TBM. A case–control study was designed to test the hypothesis. Cases of bacteriologically confirmed pulmonary tuberculosis (TB) (n=183) and TBM (n=175), and cord blood controls (n=389) were enrolled in Vietnam. TLR2 genotype 597CC was associated with susceptibility to TB (odds ratio (OR)=2.22, 95% confidence interval (CI): 1.23−3.99). The association was found with meningeal rather than pulmonary TB (TBM vs control, OR=3.26, 95% CI: 1.72−6.18), and was strongest when miliary TB was found on chest radiography (controls vs TBM with miliary TB, OR=5.28, 95% CI: 2.20−12.65). Furthermore, the association increased with the severity of neurologic symptoms (grade I TBM, OR=1.93, 95% CI: 0.54−6.92; grade II, OR=3.32, 95% CI: 0.84−13.2; and grade III, OR=5.70, 95% CI: 1.81−18.0). These results demonstrate a strong association of TLR2 SNP T597C with the development of TBM and miliary TB and indicate that TLR2 influences the dissemination of M. tuberculosis.