Biomedical subjects
Publications and source records attributed to Ian Sabroe.
Although there is overwhelming pressure from funding agencies and the general public for scientists to bridge basic and translational studies, the fact remains that there are significant hurdles to overcome in order to achieve this goal. The purpose of this Opinion article is to examine the nature of these hurdles and to provide food for thought on the main obstacles that impede this process.
Recent advances in the field of innate immunity have driven an important reappraisal of the role of these processes in airway disease. Various strands of evidence indicate that resident cells, such as macrophages and epithelial cells, have central importance in the initiation of inflammation. Macrophage activation has the potential to regulate not just typical aspects of innate immunity but also, via a variety of intricate cell-cell networks, adaptive responses and responses characterized by Th2-type cytokine production. In turn, such adaptive immune processes modify the phenotype and function of the innate immune system. Cooperative responses between monocytic cells and tissue cells are likely to be crucial to the generation of effective inflammatory responses, and a realization of the importance of these networks is providing a new way of identifying antiinflammatory therapies. Importantly, the repeated cycles of allergic and nonallergic inflammation that comprise chronic human airway disease are not necessarily well described by current terminology, and we propose and describe a concept of contiguous immunity, in which continual bidirectional cross-talk between innate and adaptive immunity describes disease processes more accurately.
Traditionally, PGD(2) has been considered to be a pro-inflammatory mediator, acting via classical PG receptors, such as the PGD(2) receptor (DP). PGD(2) is degraded rapidly in vitro and in vivo to a variety of metabolites, the majority of which were thought, until recently, to be physiologically inactive. Several "inactive" metabolites, particularly 15d-PGJ(2), have been shown to have wide-ranging effects on leukocytes and other cell types, however, and a potentially important anti-inflammatory role for PGD(2) has now been recognized, and the complexity of PGD(2) signaling is beginning to be elucidated. PGD(2) and its metabolites are biologically active over a broad concentration range, and, intriquingly, it appears that there are marked concentration-dependent variations in the consequences of signaling by these eicosanoids, which have the potential to exert pro- and anti-inflammatory effects. For example, the actions of PGD(2) can influence multiple stages in the life of the mature eosinophil, from causing its release from the bone marrow to inducing its recruitment and activation and, ultimately, regulating its apoptosis. This review is concerned with the diverse responses induced in leukocytes by PGD(2) and its metabolites and the signaling mechanisms which are thought to be responsible for them.
Viral and bacterial pathogens cause inflammation via Toll-like receptor (TLR) signaling. We have shown that effective responses to LPS may depend on cooperative interactions between TLR-expressing leukocytes and TLR-negative tissue cells. The aim of this work was to determine the roles of such networks in response to agonists of TLRs associated with antiviral and autoimmune responses. The TLR3 agonist poly(I:C) activated epithelial cells, primary endothelial cells, and two types of primary human smooth muscle cells (airway [ASMC] and vascular) directly, while the TLR7/8 agonist R848 required the presence of leukocytes to activate ASMC. In keeping with these data, ASMC expressed TLR3 but not TLR7 or TLR8. Activation of ASMC by poly(I:C) induced a specific cytokine repertoire characterized by induction of CXCL10 generation and the potential to recruit mast cells. We subsequently explored the ability of TLR agonists to cooperate in the induction of inflammation. Dual stimulation with LPS and poly(I:C) caused enhanced cytokine generation from epithelial and smooth muscle cells when in the presence of leukocytes. Thus, inflammatory responses to pathogens are regulated by networks in which patterns of TLR expression and colocalization of tissue cells and leukocytes are critical.
The identification of Toll-like receptors has revolutionised our understanding of innate immunity. TLR4 transduces the LPS signal and that of a number of structurally and functionally unrelated agonists. However, recent evidence adds to longstanding concerns that endotoxin contamination of bacterially derived recombinant TLR4 agonists is responsible for effects attributed to these molecules. We highlight key factors in differentiating specific agonist effects from those of endotoxin and emphasize why conventional methods of detecting and eliminating LPS may lead to erroneous results. We propose that considerable caution is needed in the investigation of TLR4 agonists, particularly when using proteins produced in a bacterium that also houses the most ideal TLR4 agonist, LPS.
The potential for proteases to regulate mammalian TLR signalling is controversial. We found that inhibition of extracellular serine proteases did not reduce activation of TLR4, but observed that the protease plasmin, an important fibrinolytic plasma enzyme that also exerts proinflammatory functions in monocytes, potentiated TLR2 and TLR4 signalling in RAW264.7 macrophages. Plasmin enhanced endogenous production of TNFalpha and activation of an NF-kappaB reporter plasmid. These actions were prevented by inhibition of its proteolytic activity and were not recapitulated by agonists of protease-activated receptors. These studies link fibrinolysis and TLR signalling, identifying further mechanisms potentially involved in activation of innate immunity.
Apoptosis, programmed cell death, of neutrophil and eosinophil granulocytes is a potential control point in the physiological resolution of innate immune responses. There is also increasing evidence that cellular processes of apoptosis can be dysregulated by pathogens as a mechanism of immune evasion and that delayed apoptosis, resulting in prolonged inflammatory cell survival, is important in persistence of tissue inflammation. The identification of cell-type specific pathways to apoptosis may allow the design of novel anti-inflammatory therapies or agents to augment the innate immune responses to infection. This review will explore the physiological roles of granulocyte apoptosis and their importance in infectious and non-infectious lung disease.
Many host cell types, including endothelial and epithelial cells, neutrophils, monocytes, natural killer cells, dendritic cells and macrophages, initiate the first line of defense against infection by sensing conserved microbial structures through Toll-like receptors (TLRs). Recognition of microbial ligands by TLRs induces their oligomerization and triggers intracellular signaling pathways, leading to production of pro- and anti-inflammatory cytokines. Dysregulation of the fine molecular mechanisms that tightly control TLR signaling may lead to hyperactivation of host cells by microbial products and septic shock. A prior exposure to bacterial products such as lipopolysaccharide (LPS) may result in a transient state of refractoriness to subsequent challenge that has been referred to as 'tolerance'. Tolerance has been postulated as a protective mechanism limiting excessive inflammation and preventing septic shock. However, tolerance may compromise the host's ability to counteract subsequent bacterial challenge since many septic patients exhibit an increased incidence of recurrent bacterial infection and suppressed monocyte responsiveness to LPS, closely resembling the tolerant phenotype. Thus, by studying mechanisms of microbial tolerance, we may gain insights into how normal regulatory mechanisms are dysregulated, leading ultimately to microbial hypo-responsiveness and life-threatening disease. In this review, we present current theories of the molecular mechanisms that underlie induction and maintenance of 'microbial tolerance', and discuss the possible relevance of tolerance to several infectious and non-infectious diseases.
CRTH2 is a recently described chemoattractant receptor for the prostaglandin, PGD(2), expressed by Th2 cells, eosinophils and basophils, and believed to play a role in allergic inflammation. Here we describe the potency of several PGD(2) metabolites at the receptor to induce cell migration and activation. We report for the first time that the PGD(2) metabolite, 9alpha,11beta-PGF(2), and its stereoisomer, PGF(2alpha), are CRTH2 agonists. 9alpha,11beta-PGF(2) is a major metabolite produced in vivo following allergen challenge, whilst PGF(2alpha) is generated independently of PGD synthetase, with implications for CRTH2 signalling in the presence or absence of PGD(2) production.
Toll-like receptors (TLRs) play an essential role in the detection of invading pathogens and in the induction of host antimicrobial defenses. TLR4, the major endotoxin receptor, and TLR2, with agonists derived principally from gram-positive organisms, are likely to be important in the pathogenesis of sepsis. Both TLR2 and TLR4 agonists regulate important neutrophil functions, including adhesion, generation of reactive oxygen species, and release of chemokines, and activate major proinflammatory signaling pathways, including the nuclear factor- kappa B pathway. TLR stimulation produces only a modest direct inhibition of neutrophil apoptosis, although this signal is greatly amplified by the presence of monocytes, suggesting that regulation of the life span of neutrophils by TLR agonists may be principally mediated by responses of other endotoxin-responsive cells. We suggest that activation of neutrophils by TLRs is highly regulated, permitting acute neutrophil antimicrobial responses to TLR activation while providing a "brake" on inflammation by requiring the presence of mononuclear cells to significantly extend neutrophil survival.
Endotoxin tolerance has the potential to limit phagocyte responses to Toll-like receptor (TLR) agonists, but the role of tolerance in regulating neutrophil responses is unknown. We investigated neutrophil responses to prolonged lipopolysaccharide (LPS) exposure and observed induction of tolerance in intracellular signaling pathways and respiratory burst. These effects were not prevented by granulocyte macrophage-colony stimulating factor (GM-CSF) pretreatment, and tolerized neutrophils retained the ability to respond to GM-CSF and other survival factors with a delay in apoptosis. In addition, LPS-exposed neutrophils showed continued generation of CXC chemokine ligand 8, which was not reduced in tolerized cells. Induction of tolerance was associated with a loss of TLR4 surface expression. Tolerance, therefore, induces a selective reprogramming of neutrophil function, but cells retain a predominantly proinflammatory phenotype.
Neutrophils are amongst the first immune cells to arrive at sites of infection, where they initiate antimicrobial and proinflammatory functions, which serve to contain infection. Sensing and defeating microbial infections are daunting tasks as a result of their molecular heterogeneity; however, Toll-like receptors (TLRs) have emerged as key components of the innate-immune system, activating multiple steps in the inflammatory reaction, eliminating invading pathogens, and coordinating systemic defenses. Activated neutrophils limit infection via the phagocytosis of pathogens and by releasing antimicrobial peptides and proinflammatory cytokines and generating reactive oxygen intermediates. Through the production of chemokines, they additionally recruit and activate other immune cells to aid the clearance of the microbes and infected cells and ultimately, mount an adaptive immune response. In acute inflammation, influx of neutrophils from the circulation leads to extremely high cell numbers within tissues, which is exacerbated by their delayed, constitutive apoptosis caused by local inflammatory mediators, potentially including TLR agonists. Neutrophil apoptosis and safe removal by phagocytic cells limit tissue damage caused by release of neutrophil cytotoxic granule contents. This review addresses what is currently known about the function of TLRs in the biology of the human neutrophil, including the regulation of TLR expression, their roles in cellular recruitment and activation, and their ability to delay apoptotic cell death.
RATIONALE: Toll-like receptors 2 and 4 (TLR2, TLR4) enable cellular responses to bacterial lipoproteins, LPS, and endogenous mediators of cell damage. They have an established role in the activation of leukocytes, endothelial cells, and some smooth muscle cell types, but their roles in airway smooth muscle are uncertain. OBJECTIVES: To determine the roles of TLRs in activation of airway smooth muscle. METHODS: Airway smooth muscle cells were cultured with TLR agonists, in the presence or absence of mononuclear leukocytes. MEASUREMENTS AND MAIN RESULTS: We observed expression of TLR2 and TLR4 mRNAs, which could be upregulated by treatment with proinflammatory cytokines in primary human airway smooth muscle, but no important functional responses to agonists of these TLRs were seen. Coincubation of airway smooth muscle with peripheral blood mononuclear cells, at concentrations as low as 250 mononuclear cells/ml, resulted in a marked cooperative response to TLR stimuli, and synergistic production of cytokines, including chemokines (interleukin [IL-]-8) and IL-6. This cooperative response was greater when monocytes were enriched and was transferable using supernatants from LPS-stimulated peripheral blood mononuclear cells. Activation of cocultures required IL-1 generation from mononuclear cells, and was blocked by IL-1 receptor antagonist, though IL-1 generation alone was not sufficient to account for the magnitude of mononuclear cell-dependent coculture activation. CONCLUSIONS: These data indicate that potent amplification of inflammation induced by TLR agonists, such as LPS, may be achieved by cooperativity between airway smooth muscle and leukocytes involved in immune surveillance or inflammation.
The chemokine receptor CCR3 regulates the chemotaxis of leukocytes implicated in allergic disease, such as eosinophils. Incubation of eosinophils with CCL11, CCL13 or CCL5 resulted in a rapid decrease of cell-surface CCR3 which was replicated using CCR3 transfectants. Progressive truncation of the CCR3 C terminus by 15 amino acids produced three constructs, Delta340, Delta325 and Delta310. Delta340 and Delta325 were able to bind CCL11 with affinities similar to wild-type CCR3. Delta340 transfectants exhibited enhanced migration and reduced receptor down-regulation in response to CCL11 and CCL13. Delta325 transfectants displayed chemotactic responses to CCL11 and CCL13 similar to wild-type CCR3, and had impaired down-regulation when stimulated with CCL13 but not CCL11. In contrast, neither the Delta325 nor Delta340 truncation affected chemotaxis or receptor down-regulation induced by CCL5. Delta310 transfectants bound CCL11 poorly and were biologically inactive. Inhibitors of p38 mitogen-activated protein kinase and PI3-kinase antagonized eosinophil shape change responses and chemotaxis of transfectants to CCL11 and CCL13. In contrast, shape change but not chemotaxis was sensitive to inhibition of the extracellular signal-regulated kinase kinase pathway suggesting differential regulation of the two responses. Thus, the CCR3 C terminus contains distinct domains responsible for the regulation of receptor desensitization and for coupling to chemotactic responses.
TLRs (Toll-like receptors) comprise a family of proteins whose function is principally to facilitate the detection of, and response to, pathogens. Protozoa, helminths, viruses, bacteria and fungi can all activate TLR signalling, and these signals have important roles in the activation of host defence. TLRs may also respond to products of tissue damage, providing them with roles in infective and sterile inflammation. Their role as detectors of pathogens and pathogen-associated molecules provides molecular mechanisms to underpin the observations leading to the hygiene hypothesis. Targeting of TLR signalling has implications in the control of infection, vaccine design, desensitization to allergens and down-regulation of inflammation. This review will explore TLR history, molecular signalling and the potential roles of TLRs in chronic lung disease.
Neutrophil chemokine receptor expression can be altered by exposure to Toll-like receptor (TLR) agonists, a process that is thought to have the potential to localize neutrophils to sites of infection. In order to investigate this process in more detail, we examined the regulation of highly pure neutrophil CXCR1 and CXCR2 expression and function by selective agonists of TLR2 (Pam(3)CSK(4)) and TLR4 (lipopolysaccharide, LPS). CXCR1 and CXCR2 were down-regulated by TLR engagement. CXCR2 loss was more rapid and showed a dependence upon soluble helper molecules (LPS binding protein and CD14) that was not evident for CXCR1, suggesting differential coupling of LPS signalling to CXCR1 and CXCR2 loss. However, TLR engagement in highly pure neutrophils did not result in complete loss of chemokine receptors, and LPS-treated neutrophils remained able to mount a respiratory burst to CXCL8 and CXCL1, and were able to migrate towards CXCL8 in assays of under-agarose chemotaxis. Thus, although treatment of purified human neutrophils with TLR2 and TLR4 agonists modifies chemokine receptor expression, remaining receptors remain functionally competent.
Inappropriate platelet activation is a feature of acute and chronic diseases such as disseminated intravascular coagulation (DIC) and atherosclerosis. Since proinflammatory microbial-derived agonists can be involved in the pathogenesis of these diseases, we examined the potential role of TLR4 (mediating responses to LPS) and TLR2 (which responds to bacterial lipopeptides) in platelet activation. Our data suggested low-level expression of TLR2 and TLR4 on platelets, determined by flow cytometry, and we also observed expression of TLR4 on a megakaryocytic cell line by both flow cytometry and immunohistochemistry. Stimulation of the platelets with the TLR4 agonist LPS, and the synthetic TLR2 agonist Pam3CSK4, resulted in no platelet aggregation, no increase in CD62P surface expression and no increase in the cytosolic concentration of Ca2+. The TLR agonists were also unable to directly activate platelets primed with epinephrine, or pretreated with a low concentration of ADP or PAF. Pretreatment of platelets with LPS or Pam3CSK4 also failed to modulate the platelet response to submaximal concentrations of the classical platelet agonists ADP and PAF. We conclude that the TLR agonists LPS and Pam3CSK4 have no direct effect on platelet activation and that platelet TLRs may be a remnant from megakaryocytes. TLR2 and TLR4 agonists are thought to have a significant role in diseases such as atherosclerosis and DIC, but our research suggests that this is through a mechanism other than direct platelet activation or by modification of platelet responses to other agonists.
Interactions between proinflammatory and cell maturation signals, and the pathways that regulate leukocyte migration, are of fundamental importance in controlling trafficking and recruitment of leukocytes during the processes of innate and adaptive immunity. We have investigated the molecular mechanisms by which selective Toll-like receptor (TLR)2 and TLR4 agonists regulate expression of CCR1 and CCR2 on primary human monocytes and THP-1 cells, a human monocytic cell line. We found that activation of either TLR2 (by Pam(3)CysSerLys(4)) or TLR4 (by purified LPS) resulted in down-modulation of both CCR1 and CCR2. Further investigation of TLR-induced down-modulation of CCR1 revealed differences in the signaling pathways activated, and chemokines generated, via the two TLR agonists. TLR2 activation caused slower induction of the NF-kappa B and mitogen-activated protein kinase signaling pathways and yet a much enhanced and prolonged macrophage-inflammatory protein 1 alpha (CC chemokine ligand 3) protein production, when compared with TLR4 stimulation. Enhanced macrophage-inflammatory protein 1 alpha production may contribute to the prolonged down-regulation of CCR1 cell surface expression observed in response to the TLR2 agonist, as preventing chemokine generation with the protein synthesis inhibitor cycloheximide, or CCR1 signaling with the receptor antagonist UCB35625, abolished TLR2- and TLR4-induced CCR1 down-modulation. This result suggests an autocrine pathway, whereby TLR activation can induce chemokine production, which then leads to homologous down-regulation of the cognate receptors. This work provides further insights into the mechanisms that regulate leukocyte recruitment and trafficking during TLR-induced inflammatory responses.