Role of Toll-like receptor responses for sepsis pathogenesis

Sepsis remains a serious clinical problem because of high patient morbidity and mortality. Despite significant advances in critical care, there is still no efficient causal therapy applicable to patients indicating the need to further elucidate the molecular pathways leading to the immunopathology of sepsis. The importance of Toll-like receptors (TLR) for the induction of immune responses against sepsis was demonstrated in humans exhibiting polymorphisms in TLR genes and in animal models using genetically modified mouse strains. Because of the clinical heterogeneity in human sepsis and the complex pathomechanisms underlying sepsis, several different animal models might be used to cover the diverse features of sepsis. TLR receptors induce signaling through the adapter proteins MyD88 and TRIF. TLR signaling is tightly controlled at different steps of the signaling cascade by series of regulatory proteins. Using a model of severe polymicrobial septic peritonitis we could show that single TLRs are dispensable for the induction of innate immune responses under those conditions. However, genetic ablation of MyD88 or TRIF/type-I interferon signaling pathways prevented hyper-inflammation and attenuated the pathogenic consequences of sepsis indicating that dampening common signaling pathways may create a moderate signal strength which is associated with favorable immune responses. Therefore, broad knowledge about the regulation of TLR-induced signaling pathways may further elucidate the immune mechanisms during sepsis and targeting of TLR adapter molecules may provide a new therapeutic strategy against severe sepsis.     

Injury, sepsis, and the regulation of Toll-like receptor responses

Although we tend to think that the immune system has evolved to protect the host from invading pathogens and to discriminate between self and nonself, there must also be an element of the immune system that has evolved to control the response to tissue injury. Moreover, these potential immune-regulatory pathways controlling the injury response have likely coevolved in concert with self and nonself discriminatory immune-regulatory networks with a similar level of complexity. From a clinical perspective, severe injury upsets normal immune function and can predispose the injured patient to developing life-threatening infectious complications. This remains a significant health care problem that has driven decades of basic and clinical research aimed at defining the functional effects of injury on the immune system. This review and update on our ongoing research efforts addressing the immunological response to injury will highlight some of the most recent advances in our understanding of the impact that severe injury has on the innate and adaptive immune system focusing on phenotypic changes in innate immune cell responses to Toll-like receptor stimulation.     

Important role for Toll-like receptor 9 in host defense against meningococcal sepsis

Neisseria meningitidis is a leading cause of meningitis and sepsis. The pathogenesis of meningococcal disease is determined by both bacterial virulence factors and the host inflammatory response. Toll-like receptors (TLRs) are prominent activators of the inflammatory response, and TLR2, -4, and -9 have been reported to be involved in the host response to N. meningitidis. While TLR4 has been suggested to play an important role in early containment of infection, the roles of TLR2 and TLR9 in meningococcal disease are not well described. Using a model for meningococcal sepsis, we report that TLR9^−/− mice displayed reduced survival and elevated levels of bacteremia compared to wild-type mice. In contrast, TLR2^−/− mice controlled the infection in a manner comparable to that of wild-type mice. TLR9 deficiency was also associated with reduced bactericidal activity in vitro, which was accompanied by reduced production of nitric oxide by TLR9-deficient macrophages. Interestingly, TLR9^−/− mice recruited more macrophages to the bloodstream than wild-type mice and produced elevated levels of cytokines at late time points during infection. At the cellular level, activation of signal transduction and induction of cytokine gene expression were independent of TLR2 or TLR9 in macrophages and conventional dendritic cells. In contrast, plasmacytoid dendritic cells relied entirely on TLR9 to induce these activities. Thus, our data demonstrate an important role for TLR9 in host defense against N. meningitidis.     

Toll-like receptor control of the adaptive immune responses

Recognition of microbial infection and initiation of host defense responses is controlled by multiple mechanisms. Toll-like receptors (TLRs) have recently emerged as a key component of the innate immune system that detect microbial infection and trigger antimicrobial host defense responses. TLRs activate multiple steps in the inflammatory reactions that help to eliminate the invading pathogens and coordinate systemic defenses. In addition, TLRs control multiple dendritic cell functions and activate signals that are critically involved in the initiation of adaptive immune responses. Recent studies have provided important clues about the mechanisms of TLR-mediated control of adaptive immunity orchestrated by dendritic cell populations in distinct anatomical locations.     

Different Toll-like receptor agonists induce distinct macrophage responses

We previously reported that gram-negative bacterial lipopolysaccharide (LPS) activates cells via Toll-like receptor (TLR) 4, whereas the mycobacterial cell wall glycolipid lipoarabinomannan (LAM) activates cells via TLR2. We also identified a secreted TLR2 agonist activity in short-term culture filtrates of Mycobacterium tuberculosis bacilli, termed soluble tuberculosis factor (STF). Here we show that STF contains mannosylated phosphatidylinositol (PIM) and that purified PIM possesses TLR2 agonist activity. Stimulation of RAW 264.7 macrophages by LPS, LAM, STF, and PIM rapidly activated nuclear factor (NF)-kappaB, activator protein-1 (AP-1), and mitogen-activated protein (MAP) kinases. These TLR agonists induced similar levels of NF-kappaB and AP-1 DNA-binding activity, as well as trans-activation function. Unexpectedly, these TLR agonists induced tumor necrosis factor alpha secretion, whereas only LPS was capable of inducing interleukin-1beta and nitric oxide secretion. Thus, different TLR proteins are still capable of activating distinct cellular responses, in spite of their shared capacities to activate NF-kappaB, AP-1, and MAP kinases.     

Toll样受体4在高氧暴露对小胶质细胞功能影响中的作用

目的:探讨Toll样受体4(TLR4)在高浓度氧暴露对小胶质细胞功能的影响.方法:体外培养N9小胶质细胞(TLR4野生型)和EOC20小胶质细胞(TLR4敲除型),给予950 mL/L高氧分别暴露不同时段,建立高氧损伤细胞模型.RT-PCR检测N9细胞TLR4 mRNA表达时序变化;Western blot法检测N9细胞TLR4蛋白表达;通过抗氧化剂N-乙酰半胱氨(N-acetyl-L-cysteine,NAC)干预,观测950 mL/L高氧暴露后2h及6h的N9和EOC20小胶质细胞内的活性氧(ROS)含量、NF-κB核转录活性及细胞上清中TNF-α含量.结果:高浓度氧暴露的N9小胶质细胞TLR4的表达上调,并呈一定时间依赖性,同时ROS活性,核转录因子NF-κB活性,TNF-α表达明显增高(P＜0.05).抗氧化剂NAC干预后,ROS活性明显降低,核转录因子NF-κB活性明显降低,细胞上清内TNF-α水平亦显著下调(P＜0.05).与N9小胶质细胞相比,高氧暴露后各时间点EOC20细胞的ROS活性,核转录因子NF-κB活性以及TNF-α表达均较低(P＜0.05).结论:TLR4参与调控高氧导致的小胶质细胞ROS的形成及炎症因子的释放.     

Role of Toll-like receptors in costimulating cytotoxic T cell responses

Stimulation of Toll-like receptors (TLR) by pathogen-derived compounds leads to activation of APC, facilitating the induction of protective immunity. This phenomenon is the basis of most adjuvant formulations currently in development. Here, we tested the ability of TLR2, 3, 4, 5, 7 and 9 signaling to enhance CTL responses upon vaccination with virus-like particles. Stimulation of TLR2 and 4 failed to increase CTL responses, whereas ligands for TLR3, 5 and 7 exhibited moderate adjuvant function. In contrast, stimulation of TLR9 dramatically increased CTL responses, indicating that ligands for TLR9 are likely to be the most promising candidates for the development of novel adjuvant formulations for stimulating CTL responses.     

Toll-like receptor 2 is required for inflammatory responses to Francisella tularensis LVS

Francisella tularensis, a gram-negative bacterium, is the etiologic agent of tularemia and has recently been classified as a category A bioterrorism agent. Infections with F. tularensis result in an inflammatory response that plays an important role in the pathogenesis of the disease; however, the cellular mechanisms mediating this response have not been completely elucidated. In the present study, we determined the role of Toll-like receptors (TLRs) in mediating inflammatory responses to F. tularensis LVS, and the role of NF-kappaB in regulating these responses. Stimulation of bone marrow-derived dendritic cells from C57BL/6 wild-type (wt) and TLR4-/- but not TLR2-/- mice, with live F. tularensis LVS elicited a dose-dependent increase in the production of tumor necrosis factor alpha. F. tularensis LVS also induced in a dose-dependent manner an up-regulation in the expression of the costimulatory molecules CD80 and CD86 and of CD40 and the major histocompatibility complex class II molecules on dendritic cells from wt and TLR4-/- but not TLR2-/- mice. TLR6, not TLR1, was shown to be involved in mediating the inflammatory response to F. tularensis LVS, indicating that the functional heterodimer is TLR2/TLR6. Stimulation of dendritic cells with F. tularensis resulted in the activation of NF-kappaB, which resulted in a differential effect on the production of pro- and anti-inflammatory cytokines. Taken together, our results demonstrate the role of TLR2/TLR6 in the host's inflammatory response to F. tularensis LVS in vitro and the regulatory function of NF-kappaB in modulating the inflammatory response.     

Synergistic enhancement of Toll-like receptor responses by NOD1 activation

NOD1 is an intracellular pattern-recognition receptor specific for Gram-negative peptidoglycan that is important in host response to infections (e.g. Helicobacter pylori and Shigella flexneri). Genetic variation in NOD1 predisposes to asthma and inflammatory bowel disease. Functional responses have not previously been studied in primary human cells. NOD1 activation by low nanomolar concentrations of the specific muropeptide ligand M-TriDAP induced minimal human peripheral blood mononuclear cell TNF-alpha, IL-1beta or IL-10 secretion, but synergistically increased Toll-like receptor (TLR)-induced responses. Synergistic responses were seen across multiple ligands (to TLR1/2, 2/6, 4, 5, 7/8) and a broad range of cytokine secretion (TNF-alpha, IL-1alpha, IL-1beta, IL-4, IL-6, IL-10, GM-CSF). Synergy was also observed in the allogeneic mixed lymphocyte reaction. These responses were similar in cells homozygous for Crohn's disease-associated NOD2 mutations. In contrast to cell lines, primary human peripheral blood mononuclear cells respond to NOD1 muropeptides at approximately 100-fold lower concentrations. Cross-talk between cytosolic NOD1 and membrane-bound TLR enhances responses to the multiple antigens simultaneously presented by a microbe.     

Contribution of Toll-like receptor signaling to germinal center antibody responses

Toll-like receptors (TLRs) have emerged as one of the most important families of innate immune receptors for initiating inflammation and also for promoting adaptive immune responses. Recent studies have examined the ability of TLRs to promote antibody responses, including T-cell-dependent antibody responses. Initial study suggested that TLR stimulation promotes primarily an extrafollicular antibody response, which rapidly produces moderate affinity antibodies made by short-lived plasma cells. Recent studies, however, have shown that TLRs can also enhance the germinal center response, which produces high affinity class-switched antibody made by long-lived plasma cells. TLR stimulation can increase the magnitude of the latter response and also enhance selection for high affinity IgG. This review summarizes recent advances in understanding the roles of TLRs in B cells and also in other cell types for enhancement of antibody responses, with an emphasis on T-cell-dependent and germinal center antibody responses.     

Severe sepsis and Toll-like receptors

Severe sepsis dominates the mortality of non-cardiac intensive care units. The ingenious Toll-like receptor (TLR) system can recognise many infectious organisms through relatively few receptors to trigger pro-inflammatory and anti-inflammatory cytokine release. Further complexity arises from positive and negative signalling feedback loops. Severe sepsis may be a consequence of an inappropriately excessive response or inadequate endogenous negative feedback. Therapies targeting these pathways are currently being evaluated. Alternatively, in clinical scenarios such as compensatory anti-inflammatory response syndrome, chronic viral sepsis or inadequate vaccine function, TLR signalling may be inadequate. TLR agonists may augment the innate response and are being investigated.     

TRIM21 controls Toll-like receptor 2 responses in bone-marrow-derived macrophages

TRIM21 is an interferon-stimulated E3 ligase that controls the activity of pattern-recognition signaling via ubiquitination of interferon regulatory factors and DDX41. Previous studies on the role of TRIM21 in innate immune responses have yielded contradictory results, suggesting that the role of TRIM21 is cell specific. Here, we report that bone-marrow-derived macrophages (BMDMs) generated from Trim21^−/− mice have reduced expression of mature macrophage markers. Reflecting their reduced differentiation in response to macrophage colony-stimulating factor (M-CSF), Trim21^−/− BMDMs had decreased expression of M-CSF signature genes. Although Trim21^−/− BMDMs responded normally to Toll-like receptor 9 (TLR9) activation, they produced lower levels of pro-inflammatory cytokines in response to the TLR2 agonist PAM3CSK4. In line with this, the response to infection with the Bacillus Calmette–Guérin strain of Mycobacterium bovis was also diminished in Trim21^−/− BMDMs. Our results indicate that TRIM21 controls responses to TLR2 agonists.     

The role of epithelial Toll-like receptor signaling in the pathogenesis of intestinal inflammation

Emerging evidence suggests that the innate immune system, comprised of Toll-like receptors (TLRs) and their associated molecules, plays a pivotal role in the regulation of intestinal inflammation and in the response to invading pathogens. Although TLRs are thought to have predominantly beneficial effects in pathogen recognition and bacterial clearance by leukocytes, their dysregulation and unique signaling effects within intestinal epithelia in the setting of inflammation may have devastating consequences. For instance, activation of TLR4 in enterocytes leads to an inhibition of enterocyte migration and proliferation as well as the induction of enterocyte apoptosis-factors that would be expected to promote intestinal injury while inhibiting intestinal repair. TLR signaling has been shown to be abnormal in several intestinal inflammatory diseases, including Crohn's disease, ulcerative colitis, and necrotizing enterocolitis. This review serves to examine the evidence regarding the patterns of expression and signaling of TLRs in the intestinal mucosa at basal levels and during physiologic stressors to gain insights into the pathogenesis of intestinal inflammation. We conclude that the data reviewed suggest that epithelial TLR signaling-acting in concert with TLR signaling by leukocytes-participates in the development of intestinal inflammation. We further conclude that the evidence reviewed provides a rationale for the development of novel, epithelial-specific, TLR-based agents in the management of diseases of intestinal inflammation.     

Role of adhesion molecule ICAM in the pathogenesis of polymicrobial sepsis.

INTRODUCTION: Intercellular adhesion molecule-1 (ICAM-1) is thought to be involved in polymorphonuclear leukocytes (PMNL) recruitment and secondary organ damage in response to infection and inflammation. The precise role of ICAM-1 in disease progression is unknown and remains a topic of controversy. The aim of this study was to investigate the effect of ICAM-1 on histological changes and cytokine synthesis in a murine model of polymicrobial sepsis. METHODS: Polymicrobial sepsis was induced in experimental animals by caecal ligation and puncture (CLP). A control group was formed using sham laparotomy without CLP. In order to ascertain the role of ICAM-1 in the response, procedures were performed in both ICAM-1 knockout animals (ICAM-1-/-) and in C57BL/6 mice that were not genetically modified (wild type, WT). Clinical response was observed daily, morphological changes occurring in the lung and liver were studied using light microscopy and quantified using a scoring system. Plasma concentrations of various cytokines (TNF-alpha, IL-6, IL-10) were measured via ELISA. RESULTS: In ICAM-1-/- mice a less severe clinical response to induced sepsis was observed with significantly less weight loss and hypothermia. A significantly lower mortality rate was observed in ICAM-1-/- mice (12.5% vs. WT: 45.5%) and no significant histological changes were apparent in pulmonary or hepatic tissue on light microscopy following CLP. In WT animals however, significant evidence of leukocyte infiltration and interstitial thickening in pulmonary tissue was observed. Similarly, hepatic tissue sinusoidal widening and hydropic degeneration was present. In addition, pro- and anti-inflammatory cytokine synthesis in ICAM-1-/- animals was significantly attenuated when compared to WT mice. (ICAM-1-/-: TNF-alpha: 67.7+/-12.1pg/microl; IL-6: 208.9+/-26.7pg/microl; IL-10: 34.6+/-5.8pg/microl; WT: TNF-alpha: 840.7+/-150.2pg/microl; IL-6: 3100.2+/-1052.3 pg/microl; IL-10: 1550.1+/-495.7 pg/microl). DISCUSSION: This study suggests that ICAM-1 has an important pathophysiological role in the response to polymicrobial sepsis. It would appear that absence of this molecule impairs the ability of PMNL to migrate into organ tissues and reduces consequent secondary organ damage resulting in improved clinical status and overall survival. Further investigation into the effectiveness of ICAM-1 modulation in the treatment of sepsis is warranted.     

Distinct responses of monocytes to Toll-like receptor ligands and inflammatory cytokines

In this study we compared the activation of monocytes by different bacterial products via Toll-like receptors (TLR), and by different proinflammatory mediators. In response to TLR-2, -4 and -5 engagement, approximately 50% of monocytes produced TNF-alpha, compared to only 5% after induction with IFN-gamma or GM-CSF. Furthermore, a small proportion of monocytes produced IL-10 after stimulation via TLR, but not after stimulation with cytokines. Both TLR-ligands and inflammatory cytokines induced the expression of CD25, CD69, CD80 and, surprisingly, also of CD83, commonly regarded as an activation marker for mature dendritic cells (DC). Conversely, TLR-ligands downregulated CD38, CD86 and ICOS-L. Importantly, signaling lymphocytic activation molecule (SLAM; CD150) was identified as a monocyte activation marker that could be induced ex novo via TLR-2, -4 and -5, but not by single stimulation with monocyte activators like IL-1, TNF-alpha, IFN-beta, IFN-gamma, GM-CSF or CD40-L. SLAM expression was transient and required mitogen activated protein kinase (MAPK) p38, but not ERK or JNK, and was surprisingly independent of NF-kappaB. SLAM+ monocytes, which are absent in blood, were detected in spleen and tonsils, where they could be localized to T-cell areas and germinal centers. Together, by comparing the response of monocytes to TLR-ligands and inflammatory cytokines, we have identified a monocyte activation marker, SLAM, which differs in its inducibility from other monocyte activation markers. SLAM+ monocytes and macrophages were identified for the first time in vivo. Their presence might be a sign of innate immune activation.     

Expression and function of Toll-like receptor 9 in severely injured patients prone to sepsis

The purpose of this prospective study was to enumerate Toll-like receptor 9 (TLR9)(+) cells and measure their function using synthetic oligonucleotides enriched in CG dinucleotide motifs (CpG)-induced proliferation within 48 h after trauma in severely injured patients prone to sepsis. Sixteen consecutive trauma patients with an injury severity score (ISS) > 21 and 16 blood donors (controls) were included in this study. Using two-colour flow cytometry, TLR9 expression was detectable intracellularly and also on the surface of B lymphocytes. The surface expression of TLR9 of B lymphocytes from whole blood and peripheral blood mononuclear cells (PBMC) stimulated with CpG was significantly increased in B cells of severely injured patients prone to sepsis compared to controls. No significant differences could be observed between CpG-induced proliferation of PBMC of severely injured patients prone to sepsis and controls. As a measure of immunosuppression, human leucocyte antigen (HLA)-DR expression of monocytes of the trauma patients was significantly diminished compared with controls in PBMC and in whole blood. Immunosuppression in the early phase after trauma seems not to be associated with a disturbed sensing of bacterial DNA.     

Toll-like receptor 2 ligands inhibit TH2 responses to mite allergen

BACKGROUND: There is intense interest in the interaction between microbial compounds and allergy. Although Toll-like receptor (TLR)-2 ligands derived from Gram-positive bacteria alter allergic sensitization in animal models, it is not clear what effect TLR2 ligands have on allergen-specific T-cell memory in human beings. OBJECTIVE: To determine whether in vitro exposure to TLR2 ligands modifies the immune response to house dust mite allergen (HDM). METHODS: Blood mononuclear cells were obtained from individuals both allergic (n = 23) and not allergic (n = 22) to HDM, and stimulated with HDM in the presence or absence of TLR2 ligands. RESULTS: In subjects allergic to HDM, IL-5 and IL-13 responses to HDM were inhibited by heat-killed Staphylococcus aureus, staphylococcal lipoteichoic acid, and the synthetic lipoprotein Pam3CSK4 (P < .005; all stimuli). Although the whole staphylococcal bacteria increased IFN-gamma responses, the purified TLR2 ligands lipoteichoic acid and Pam3CSK4 inhibited HDM-specific IFN-gamma synthesis. In contrast, TLR2 ligands had minimal effects on responses to HDM in subjects without allergy. TLR2 ligands induced upregulation of HLA-DR expression but did not inhibit antigen uptake or processing by antigen-presenting cells. CONCLUSION: Toll-like receptor 2 ligands inhibit allergen-specific T(H)2 responses in sensitized individuals. This effect appears to be mediated by the actions of TLR2 ligands on antigen-presenting cells, and at least for the purified TLR2 ligands does not involve the induction of a strong T(H)1 immune response. CLINICAL IMPLICATIONS: These findings provide an impetus for further preclinical studies examining the potential use of TLR2 ligands in allergic disease.     

Role of Toll-like receptor 4 in hyperoxia-induced lung inflammation in mice

Objective: Prolonged exposure to hyperoxia causes lung inflammation, but the role of Toll-like receptor 4 (TLR4) in hyperoxia-induced signal transduction remains unclear. Material or subjects: We evaluated neutrophil accumulation, signal transduction and cytokine production during hyperoxia, comparing TLR4 mutant (C3H/HeJ) and wild type (C3H/HeN) mice. Methods: The mice were exposed to 80% oxygen in a hyperoxic chamber for 0 (control), 48, or 96 h. After the exposure, bronchoalveolar lavage (BAL) was performed for differential cell counting and cytokine measurement. In lung homogenate, activation of NF-κB and STAT1 was also examined. Results: In C3H/HeJ mice, hyperoxia-induced neutrophil accumulation in BAL fluid was significantly decreased compared with C3H/HeN. Hyperoxia for 96 h caused NF-κB translocation in C3H/HeN mice, which was significantly attenuated in C3H/HeJ mice (p < 0.05). In contrast, STAT1 activation occurred as early as after 48 h of oxygen exposure, which did not differ between the two strains. The levels of TNF-α, IL-6, and KC in BAL fluid were increased after oxygen exposure, which was suppressed by the lack of TLR4 signaling. Conclusion: These results suggest that TLR4-dependent NF-kB activation may be an important process of the upregulation of proinflammatory mediators and subsequent neutrophil accumulation into the lung during hyperoxia. Received 21 March 2007; accepted without revision by G. Wallace 11 April 2007