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[Toll-dependent and toll-independent innate antiviral immunity].

Until recently, adaptive immunity and cytotoxic T cells were considered as the only essential components of the antiviral defence arsenal. Additional data that do not rule out the crucial role of these cells in the clearance of viral pathogens have, however, recently emerged. They indicate that innate immune cells such as macrophages, dendritic cells, gammadelta T cells as well as natural killer (NK) cells play a primordial role in this mechanism. It is now well established that innate immune cells can detect various pathogens (bacteria, viruses, fungi or parasites) very rapidly and respond to their presence through the activation of specific receptors. Once activated, these molecules trigger several signalling cascades that culminate in the establishment of very potent defence mechanisms. In addition, cytokines produced during this initial response are essential for the activation of the adaptive immune response which will add specificity and memory to the system. Among the innate immune receptors, attention has focused on the Toll-like receptors (TLR) and many reports indicate that some of the TLRs are clearly involved in defence against viral pathogens. However, new molecules, acting independently from any TLR, have recently been discovered. They define a second antiviral pathway which is presently the subject of intense research. In this article, we will review the role of the different molecules involved in each pathway within the framework of innate antiviral defence.

DEAD Box Protein 58↗

Role of Drosophila IKK gamma in a toll-independent antibacterial immune response.

We have generated, by ethylmethane sulfonate mutagenesis, loss-of-function mutants in the Drosophila homolog of the mammalian I-kappa B kinase (IKK) complex component IKK gamma (also called NEMO). Our data show that Drosophila IKK gamma is required for the Relish-dependent immune induction of the genes encoding antibacterial peptides and for resistance to infections by Escherichia coli. However, it is not required for the Toll-DIF-dependent antifungal host defense. The results indicate distinct control mechanisms of the Rel-like transactivators DIF and Relish in the Drosophila innate immune response and show that Drosophila Toll does not signal through a IKK gamma-dependent signaling complex. Thus, in contrast to the vertebrate inflammatory response, IKK gamma is required for the activation of only one immune signaling pathway in Drosophila.

Animals↗

Reduced atherosclerosis in MyD88-null mice links elevated serum cholesterol levels to activation of innate immunity signaling pathways.

Atherosclerosis, the leading cause of death in developed countries, has been linked to hypercholesterolemia for decades. More recently, atherosclerotic lesion progression has been shown to depend on persistent, chronic inflammation in the artery wall. Although several studies have implicated infectious agents in this process, the role of infection in atherosclerosis remains controversial. Because the involvement of monocytes and macrophages in the pathogenesis of atherosclerosis is well established, we investigated the possibility that macrophage innate immunity signaling pathways normally activated by pathogens might also be activated in response to hyperlipidemia. We examined atherosclerotic lesion development in uninfected, hyperlipidemic mice lacking expression of either lipopolysaccharide (LPS) receptor CD14 or myeloid differentiation protein-88 (MyD88), which transduces cell signaling events downstream of the Toll-like receptors (TLRs), as well as receptors for interleukin-1 (IL-1) and IL-18. Whereas the MyD88-deficient mice evinced a marked reduction in early atherosclerosis, mice deficient in CD14 had no decrease in early lesion development. Inactivation of the MyD88 pathway led to a reduction in atherosclerosis through a decrease in macrophage recruitment to the artery wall that was associated with reduced chemokine levels. These findings link elevated serum lipid levels to a proinflammatory signaling cascade that is also engaged by microbial pathogens.

Animals↗

Endotoxin, toll-like receptor 4, and the afferent limb of innate immunity.

Positional cloning work and subsequent biochemical analyses have revealed that Toll-like receptor 4 (Tlr4) transduces the lipopolysaccharide (LPS) signal, alerting the host to infection by Gram-negative bacteria. Moreover, it appears that the LPS sensing pathway is a solitary one: disruption of Tlr4 causes complete unresponsiveness to LPS. As several Tlr family members exist in vertebrates, it appears likely that the innate immune system defends the host by recognizing a small number of structurally conserved molecules that distinguish the microbial world from tissues of the host.

Amino Acid Sequence↗

Thematic review series: The immune system and atherogenesis. Paying the price for pathogen protection: toll receptors in atherogenesis.

Atherosclerosis is a chronic inflammatory response characterized by the accumulation of cells of innate and acquired immune systems within the intima of the arterial wall. Macrophages are the predominant participant in innate immune responses in atherosclerosis. Protein receptors expressed by macrophages and endothelial cells recognize components and products of microorganisms and play a vital role in innate immunity. In particular, the members of the toll-like receptor (TLR) family play a critical role in the inflammatory components of atherosclerosis. Both exogenous ligands involved in microbial recognition as well as endogenous ligands involved in sterile inflammation pathways are implicated in the pathology of atherosclerosis. In this review, we discuss our current understanding of the role of TLRs and their coactivators in atherosclerosis, with particular emphasis on studies in atherosclerosis-prone hypercholesterolemic mice.

Animals↗

Microbes and their products--physiological effects upon mammalian mucosa.

A dynamic array of interactions occurs between pathogens and host mucosal surfaces. The signature molecules unique to microbial pathogens allow the mammalian immune system to recognize them as non-self. This recognition, mediated by the toll-like receptor proteins, results in innate immune responses targeted against the invading organism. Pathogens also elaborate a variety of proteins that actively engage host signaling pathways and subvert them to facilitate their growth and dispersal. These interactions, developed over a long evolutionary period, have been specialized to exquisite detail. These proteins and toxins are either secreted into the medium or directly delivered into host cells by specialized secretion systems. An array of host function alterations is mediated by microbial pathogens including inflammatory responses, secretory responses, alteration of host cytoskeleton, disruption of epithelial tight junctions and apoptosis. The signaling axes involved in these interactions are potential targets for therapeutic strategies against infectious microbes.

Animals↗

Carbon monoxide differentially inhibits TLR signaling pathways by regulating ROS-induced trafficking of TLRs to lipid rafts.

Carbon monoxide (CO), a byproduct of heme catabolism by heme oxygenase (HO), confers potent antiinflammatory effects. Here we demonstrate that CO derived from HO-1 inhibited Toll-like receptor (TLR) 2, 4, 5, and 9 signaling, but not TLR3-dependent signaling, in macrophages. Ligand-mediated receptor trafficking to lipid rafts represents an early event in signal initiation of immune cells. Trafficking of TLR4 to lipid rafts in response to LPS was reactive oxygen species (ROS) dependent because it was inhibited by diphenylene iodonium, an inhibitor of NADPH oxidase, and in gp91(phox)-deficient macrophages. CO selectively inhibited ligand-induced recruitment of TLR4 to lipid rafts, which was also associated with the inhibition of ligand-induced ROS production in macrophages. TLR3 did not translocate to lipid rafts by polyinosine-polycytidylic acid (poly(I:C)). CO had no effect on poly(I:C)-induced ROS production and TLR3 signaling. The inhibitory effect of CO on TLR-induced cytokine production was abolished in gp91(phox)-deficient macrophages, also indicating a role for NADPH oxidase. CO attenuated LPS-induced NADPH oxidase activity in vitro, potentially by binding to gp91(phox). Thus, CO negatively controlled TLR signaling pathways by inhibiting translocation of TLR to lipid rafts through suppression of NADPH oxidase-dependent ROS generation.

Animals↗

Selective induction of matrix metalloproteinases by Borrelia burgdorferi via toll-like receptor 2 in monocytes.

Regulation of secretion of matrix metalloproteinase (MMP) underlies the basis of numerous physiological and pathological processes in multicellular organisms. The Toll receptor family, which is conserved from Drosophila species to humans, mediates pattern recognition of a diversity of ligands involved in morphogenesis and innate immunity. Here, we show that secretion of MMP-9 is selectively induced through Toll-like receptor (TLR) 2 in human and murine monocytic cells stimulated with Borrelia burgdorferi. Secretion of MMP-1 was shown to be stimulated through a pathway other than TLR2, under identical conditions. Analysis of nuclear extracts indicated that activator protein (AP)-1 was reduced in TLR2-neutralized monocytic cells, suggesting that AP-1 plays a role in the transcriptional activation of MMP-9 through TLR2. The specific induction of MMP-9 through TLR2 provides direct evidence of a new role for this ancient receptor family in regulating secretion of MMPs and demonstrates evolutionary convergence between invertebrate morphogenesis and the vertebrate innate immune system.

Animals↗

Acute renal failure: determinants and characteristics of the injury-induced hyperinflammatory response.

Acute renal failure (ARF) markedly sensitizes mice to endotoxin (LPS), as evidenced by exaggerated renal cytokine/chemokine production. This study sought to further characterize this state by testing the following: 1) does anti-inflammatory heme oxygenase-1 (HO-1) upregulation in selected ARF models prevent this response? 2) Is the ARF hyperresponsive state specifically triggered by LPS? 3) Does excess iNOS activity/protein nitrosylation participate in this phenomenon? and 4) are upregulated Toll receptors involved? Mice with either 1) rhabdomyolysis-induced ARF (massive HO-1 overexpression), 2) cisplatin nephrotoxicity, 3) or HO-1 inhibition (Sn protoporphyrin) were challenged with either LPS (a TLR4 ligand), lipoteichoic acid (LTA; a TLR2 ligand), or vehicle. Two hours later, renal and plasma TNF-alpha/mRNA, MCP-1/mRNA, renal nitrotyrosine/iNOS mRNA, and plasma cytokines were assessed. Renal TLR4 was gauged by mRNA and Western blot analysis. Both ARF models markedly hyperresponded to both LPS and LTA, culminating in exaggerated TNF-alpha, MCP-1, and iNOS/nitrotryosine increments. This was despite the fact that HO-1 exerted anti-inflammatory effects. TLR4 levels were either normal (cisplatin), or markedly depressed ( approximately 50%; rhabdomyolysis) in the ARF kidneys, despite the LPS hyperresponsive state. 1) The ARF kidney can hyperrespond to chemically dissimilar Toll ligands; 2) HO-1 does not prevent this response; 3) excess NO/protein nitrosylation can result; and 4) this hyperresponsiveness can be expressed with either normal or reduced renal TLR4 expression. This suggests that diverse signaling pathways may be involved.

Acute Kidney Injury↗

Acyloxyacyl Hydrolase-Mediated Lipopolysaccharide Inactivation Limits Macrophage Endotoxin Tolerance and Promotes Inflammation and Fibrosis in Metabolic Dysfunction-Associated Steatohepatitis.

BACKGROUND & AIMS: Metabolic dysfunction-associated steatohepatitis, a chronic liver disease, is characterized by persistent low-grade inflammation, partially driven by gut-derived lipopolysaccharide. Although repeated lipopolysaccharide exposure can induce endotoxin tolerance in innate immune cells, its role in chronic liver diseases remains unclear. Acyloxyacyl hydrolase is an endogenous enzyme that inactivates lipopolysaccharide, potentially modulating this process. We aimed to investigate how acyloxyacyl hydrolase regulates endotoxin tolerance in Kupffer cells and how this affects hepatic inflammation and fibrosis during metabolic dysfunction-associated steatohepatitis progression. METHODS: Acyloxyacyl hydrolase-deficient mice and wild-type controls were subjected to multiple dietary metabolic dysfunction-associated steatohepatitis models. Inflammatory responses, fibrosis, and transcriptomic changes in liver tissues and isolated Kupffer cells were analyzed. Endotoxin tolerance was modulated through β-glucan administration or lipopolysaccharide preconditioning. Lipopolysaccharide bioactivity was assessed using Toll-like receptor 4-reporter cell assays. RESULTS: Lipopolysaccharide-preconditioned Kupffer cells exhibited reduced proinflammatory cytokine production and transcriptional suppression of inflammatory pathways, indicating tolerance. Despite slight elevation of plasma lipopolysaccharide levels in metabolic dysfunction-associated steatohepatitis, upregulation of hepatic acyloxyacyl hydrolase positively correlated with disease severity, suggesting enhanced lipopolysaccharide inactivation but impaired establishment of tolerance. In contrast, acyloxyacyl hydrolase-deficient Kupffer cells displayed reinforced endotoxin tolerance, leading to diminished hepatic inflammation and fibrosis. Reversal of tolerance using β-glucan reactivated inflammatory and fibrogenic responses in acyloxyacyl hydrolase-deficient mice, whereas tolerance induction by low-dose lipopolysaccharide preconditioning mitigated metabolic dysfunction-associated steatohepatitis pathology, supporting the protective role of macrophage tolerance in chronic liver injury. CONCLUSIONS: Endotoxin tolerance in Kupffer cells represents a protective mechanism against chronic liver inflammation and fibrosis. Acyloxyacyl hydrolase regulates this state by limiting bioactive lipopolysaccharide, thereby modulating the establishment of endotoxin tolerance and downstream inflammatory and fibrotic responses. Enhancing macrophage tolerance by utilizing lipopolysaccharide may offer a novel therapeutic avenue to control the progression of metabolic dysfunction-associated steatohepatitis.

AOAH↗

CpG oligodeoxynucleotides induce expression of proinflammatory cytokines and chemokines in astrocytes: the role of c-Jun N-terminal kinase in CpG ODN-mediated NF-kappaB activation.

Bacterial DNA and synthetic oligodeoxynucleotides (ODN) containing unmethylated CpG motifs stimulate the cells of the innate immune system through a specific receptor called Toll-like receptor-9 (TLR9). It was reported that CpG ODN stimulation induces activation of astrocytes and microglia. However, the precise intracellular signaling pathways that lead to this glial cell activation have not been clearly elucidated. In this study, we found that CpG ODN induce mRNA expression of adhesion molecules and matrix metalloproteinase-9 (MMP-9), as well as proinflammatory cytokines and chemokines, in mouse astrocytes. CpG ODN stimulation in astrocytes induces the activation of IkappaB kinase (IKK) and c-Jun N-terminal kinase (JNK), whereas it inhibits the constitutive ERK1/2 activation. The abrogation of JNK activity using a pharmacological inhibitor showed that JNK activation is essential for the induction of cytokine and chemokine gene expression. This effect of JNK does not require the phosphorylation of c-Jun; rather, it works via the potentiation of NF-kappaB signaling.

Animals↗

Genetic dissection of antigen receptor induced-NF-kappaB activation.

NF-kappaB is a master transcription factor in the immune system. It regulates various aspects of immune cell development, homeostasis, survival and function. It is in these contexts that the signaling pathways leading to NF-kappaB activation are best understood. This review addresses the cell signaling from antigen receptors as well as Toll-like receptors to NF-kappaB in both innate and adaptive immunity as revealed by the analysis of mice lacking specific signaling intermediates.

Animals↗

CD14-dependent lipopolysaccharide-induced beta-defensin-2 expression in human tracheobronchial epithelium.

The induction of host antimicrobial molecules following binding of pathogen components to pattern recognition receptors such as CD14 and the Toll-like receptors (TLRs) is a key feature of innate immunity. The human airway epithelium is an important environmental interface, but LPS recognition pathways have not been determined. We hypothesized that LPS would trigger beta-defensin (hBD2) mRNA in human tracheobronchial epithelial (hTBE) cells through a CD14-dependent mechanism, ultimately activating NF-kappa B. An average 3-fold increase in hBD2 mRNA occurs 24 h after LPS challenge of hTBE cells. For the first time, we demonstrate the presence of CD14 mRNA and cell surface protein in hTBE cells and show that CD14 neutralization abolishes LPS induction of hBD2 mRNA. Furthermore, we demonstrate TLR mRNA in hTBE cells and NF-kappa B activation following LPS. Thus, LPS induction of hBD2 in hTBE cells requires CD14, which may complex with a TLR to ultimately activate NF-kappa B.

Bronchi↗

Anti-inflammatory activity in vitro and in vivo of the protein farnesyltransferase inhibitor tipifarnib.

Protein farnesyltransferase inhibitors (FTIs) have shown clinical responses in hematologic malignancies, but the mechanisms are unclear. To better understand potential mechanisms of action, we have studied effects of the FTI tipifarnib on inflammatory responses in vitro and in vivo. In a human leukemia cell line THP-1, tipifarnib inhibited lipopolysaccharide (LPS)-induced transcription of chemokines [monocyte chemotactic protein (MCP)-1 and MCP-2], cytokines [interleukin (IL)-1beta, IL-6, and interferon (IFN)beta], signaling molecules (MyD88 and STAT-1), proteases [matrix metalloproteinase (MMP-9)], and receptors (urokinase receptor). Tipifarnib also inhibited LPS-induced secretion of MMP-9, IL-6, MCP-1, and IL-1beta in THP-1 cells. In primary human peripheral blood mononuclear cells, dose-dependent inhibition of LPS-induced tumor necrosis factor (TNF)-alpha, IL-6, MCP-1, and IL-1beta by tipifarnib was observed with no evidence of cytotoxicity. Similar results were obtained in vivo in a murine model of LPS-induced inflammation, where pretreatment with tipifarnib resulted in significant inhibition of TNF-alpha, IL-6, MCP-1, IL-1beta, and MIP-1alpha production. Tipifarnib had no effect in vitro or in vivo on LPS-induced IL-8. Studies in THP-1 cells to address potential mechanism(s) showed that tipifarnib partially inhibited LPS-induced p38 phosphorylation. Tipifarnib significantly inhibited inhibitory subunit of nuclear factor-kappaB (NF-kappaB) (IkappaB)-alpha degradation and p65 nuclear translocation induced by LPS, but not by tumor necrosis factor-alpha, IL-1alpha, or toll-like receptor (TLR)2 ligand, suggesting that the target for inhibition of NF-kappaB activation was exclusive to the LPS/TLR4 signal pathway. The extent of IkappaB-alpha degradation inhibition did not correlate with inhibition of Ras farnesylation, indicating that Ras was not the target for the observed anti-inflammatory activity of tipifarnib. Our findings differ from those for other FTIs, which may have relevance for their dissimilar activity in specific tumor repertoires.

Animals↗

Molecular characterization of the singed wings locus of Drosophila melanogaster.

BACKGROUND: Hormones frequently guide animal development via the induction of cascades of gene activities, whose products further amplify an initial hormonal stimulus. In Drosophila the transformation of the larva into the pupa and the subsequent metamorphosis to the adult stage is triggered by changes in the titer of the steroid hormone 20-hydroxyecdysone. singed wings (swi) is the only gene known in Drosophila melanogaster for which mutations specifically interrupt the transmission of the regulatory signal from early to late ecdysone inducible genes. RESULTS: We have characterized singed wings locus, showing it to correspond to EG:171E4.2 (CG3095). swi encodes a predicted 68.5-kDa protein that contains N-terminal histidine-rich and threonine-rich domains, a cysteine-rich C-terminal region and two leucine-rich repeats. The SWI protein has a close homolog in D. melanogaster, defining a new family of SWI-like proteins, and is conserved in D. pseudoobscura. A lethal mutation, swit476, shows a severe disruption of the ecdysone pathway and is a C>Y substitution in one of the two conserved CysXCys motifs that are common to SWI and the Drosophila Toll-4 protein. CONCLUSIONS: It is not entirely clear from the present molecular analysis how the SWI protein may function in the ecdysone induced cascade. Currently all predictions agree in that SWI is very unlikely to be a nuclear protein. Thus it probably exercises its control of "late" ecdysone genes indirectly. Apparently the genetic regulation of ecdysone signaling is much more complex then was previously anticipated.

Amino Acid Sequence↗

Adult midgut expressed sequence tags from the tsetse fly Glossina morsitans morsitans and expression analysis of putative immune response genes.

BACKGROUND: Tsetse flies transmit African trypanosomiasis leading to half a million cases annually. Trypanosomiasis in animals (nagana) remains a massive brake on African agricultural development. While trypanosome biology is widely studied, knowledge of tsetse flies is very limited, particularly at the molecular level. This is a serious impediment to investigations of tsetse-trypanosome interactions. We have undertaken an expressed sequence tag (EST) project on the adult tsetse midgut, the major organ system for establishment and early development of trypanosomes. RESULTS: A total of 21,427 ESTs were produced from the midgut of adult Glossina morsitans morsitans and grouped into 8,876 clusters or singletons potentially representing unique genes. Putative functions were ascribed to 4,035 of these by homology. Of these, a remarkable 3,884 had their most significant matches in the Drosophila protein database. We selected 68 genes with putative immune-related functions, macroarrayed them and determined their expression profiles following bacterial or trypanosome challenge. In both infections many genes are downregulated, suggesting a malaise response in the midgut. Trypanosome and bacterial challenge result in upregulation of different genes, suggesting that different recognition pathways are involved in the two responses. The most notable block of genes upregulated in response to trypanosome challenge are a series of Toll and Imd genes and a series of genes involved in oxidative stress responses. CONCLUSIONS: The project increases the number of known Glossina genes by two orders of magnitude. Identification of putative immunity genes and their preliminary characterization provides a resource for the experimental dissection of tsetse-trypanosome interactions.

Aging↗

Toll-like receptors control activation of adaptive immune responses.

Mechanisms that control the activation of antigen-specific immune responses in vivo are poorly understood. It has been suggested that the initiation of adaptive immune responses is controlled by innate immune recognition. Mammalian Toll-like receptors play an essential role in innate immunity by recognizing conserved pathogen-associated molecular patterns and initiating the activation of NF-kappaB and other signaling pathways through the adapter protein, MyD88. Here we show that MyD88-deficient mice have a profound defect in the activation of antigen-specific T helper type 1 (TH1) but not TH2 immune responses. These results suggest that distinct pathways of the innate immune system control activation of the two effector arms of adaptive immunity.

Adaptor Proteins, Signal Transducing↗

A deficient TLR2 signaling promotes airway mucin production in Mycoplasma pneumoniae-infected allergic mice.

The original hygiene hypothesis suggests that early childhood respiratory infections preceding allergen exposure may decrease the prevalence of allergic diseases. We have recently demonstrated that Mycoplasma pneumoniae infection preceding allergen exposure reduced allergic responses in mice. However, the molecular mechanisms underlying the protective role of M. pneumoniae in allergic responses, particularly airway mucin production, remain unclear. Wild-type and Toll-like receptor 2 (TLR2)-deficient mice with a respiratory M. pneumoniae infection preceding allergen (ovalbumin) challenge were utilized to determine the regulatory role of TLR2-IFN-gamma signaling pathway in airway mucin expression. Furthermore, air-liquid interface cultures of mouse primary tracheal epithelial cells were performed to examine the effects of IFN-gamma on mucin expression. In wild-type mice, M. pneumoniae infection preceding allergen challenge significantly reduced airway mucins but increased IFN-gamma. In sharp contrast, in TLR2-deficient mice, M. pneumoniae preceding allergen challenge resulted in increased mucin protein without a noticeable change of IFN-gamma. In cultured mouse primary tracheal epithelial cells, IFN-gamma was shown to directly inhibit mucin expression in a dose-dependent manner. Our study demonstrates for the first time that a respiratory M. pneumoniae infection preceding allergen challenge reduces airway epithelial mucin expression in part through TLR2-IFN-gamma signaling pathway. A bacterial infection in asthmatic subjects with weakened TLR2-IFN-gamma signaling may result in an exaggerated airway mucin production.

Allergens↗