The innate immune system is rapidly activated during myocardial infarction and blockade of extracellular complement system reduces infarct size. Intracellular complement, however, appears to be closely linked to metabolic pathways and its role in ischemia-reperfusion injury is unknown and may be different from complement activation in the circulation. The purpose of the present study was to investigate the role of intracellular complement in isolated, retrogradely buffer-perfused hearts and cardiac cells from adult male wild type mice (WT) and from adult male mice with knockout of complement component 3 (C3KO). Main findings: (i) Intracellular C3 protein was expressed in isolated cardiomyocytes and in whole hearts, (ii) after ischemia-reperfusion injury, C3KO hearts had larger infarct size (32 ± 9% in C3KO vs. 22 ± 7% in WT; p=0.008) and impaired post-ischemic relaxation compared to WT hearts, (iii) C3KO cardiomyocytes had lower basal oxidative respiration compared to WT cardiomyocytes, (iv) blocking mTOR decreased Akt phosphorylation in WT, but not in C3KO cardiomyocytes, (v) after ischemia, WT hearts had higher levels of ATP, but lower levels of both reduced and oxidized nicotinamide adenine dinucleotide (NADH and NAD+, respectively) compared to C3KO hearts. Conclusion: intracellular C3 protected the heart against ischemia-reperfusion injury, possibly due to its role in metabolic pathways important for energy production and cell survival.

Background: NLRP3 is an innate immune receptor that recognizes tissue damage and initiate inflammatory processes: chiefly, upon activation, NLRP3 forms multiprotein complexes with the adaptor protein ASC and caspase-1, termed inflammasomes, which cleaves pro-IL-1β and mediates release of bioactive IL-1β. Thus, NLRP3 activation may lead to further tissue damage. Hence, we hypothesized that NLRP3 mediates tissue damage during acute myocardial infarction and sought to investigate the mechanisms herein.

Monks, Mike Rothe and Douglas T. GolenbockHolger Heine, Carsten J. Kirschning, Egil Lien, Brian G.http://www.jimmunol.org/content/162/12/6971J Immunol€1999; 162:6971-6975; ;Referenceshttp://www.jimmunol.org/content/162/12/6971.full#ref-list-1This article cites 25 articles, 15 of which you can access for free at: Subscriptionshttp://jimmunol.org/subscriptionsInformation about subscribing to The Journal of Immunology is online at: Permissionshttp://www.aai.org/ji/copyright.htmlSubmit copyright permission requests at: Email Alertshttp://jimmunol.org/cgi/alerts/etocReceive free email-alerts when new articles cite this article. Sign up at:

Interferon regulatory factors (IRFs) are crucial for transcription during innate immune responses. We have previously shown that the tyrosine kinase c-Src enhances IRF-3-dependent transcription in response to viral double-stranded RNA. In this study, we show that c-Src has distinct roles in Toll-like receptor (TLR)-mediated activation of IRF-5 and IRF-3. Surprisingly, c-Src inhibition markedly enhanced IRF-5 activation after treatment with unmethylated CpG, while suppressing IRF-3 activation. Also, CpG-elicited interleukin-6 mRNA production was increased, whereas IP10 mRNA synthesis was reduced in cells deficient in c-Src. Interestingly, c-Src regulated TLR-stimulated induction of activating transcription factor 3 (ATF3), a transcriptional repressor. Depletion of ATF3 by small interfering RNA markedly enhanced interleukin-6 production after CpG treatment, whereas IP10 production was reduced. These results demonstrate functional specificity for c-Src in TLR-stimulated responses and suggest that c-Src modulation and ATF3 activity may contribute to differential regulation of IRF-3- versus IRF-5-mediated gene expression.

TLR9 is critical in parasite recognition and host resistance to experimental infection with Trypanosoma cruzi. However, no information is available regarding nucleotide sequences and cellular events involved on T. cruzi recognition by TLR9. In silico wide analysis associated with in vitro screening of synthetic oligonucleotides demonstrates that the retrotransposon VIPER elements and mucin-like glycoprotein (TcMUC) genes in the T. cruzi genome are highly enriched for CpG motifs that are immunostimulatory for mouse and human TLR9, respectively. Importantly, infection with T. cruzi triggers high levels of luciferase activity under NF-κB-dependent transcription in HEK cells cotransfected with human TLR9, but not in control (cotransfected with human MD2/TLR4) HEK cells. Further, we observed translocation of TLR9 to the lysosomes during invasion/uptake of T. cruzi parasites by dendritic cells. Consistently, potent proinflammatory activity was observed when highly unmethylated T. cruzi genomic DNA was delivered to the endo-lysosomal compartment of host cells expressing TLR9. Thus, together our results indicate that the unmethylated CpG motifs found in the T. cruzi genome are likely to be main parasite targets and probably become available to TLR9 when parasites are destroyed in the lysosome-fused vacuoles during parasite invasion/uptake by phagocytes.

We report that patients with AIDS have lower serum levels of soluble toll-like receptor 2 (sTLR2) compared with healthy controls and patients with less advanced HIV infection. sTLR2 levels are relatively stable during longitudinal testing, with lower levels in progressors than non-progressors. No correlations between sTLR levels and CD4/CD8 T-cell counts or viral load were found. Our observations suggest that sTLR2 levels are mainly constitutively determined, with a possible role for sTLR2 in the pathogenesis of HIV infection.

BACKGROUNDToll-like receptors (TLRs) are key pattern-recognition receptors of the innate immune system, but their role in human immunodeficiency virus (HIV) infection is largely unknown.METHODSIn the present study, we examined the expression of TLR2 and TLR4 on monocytes from 48 HIV-infected patients and 21 healthy control subjects by flow cytometry.RESULTSWe found that freshly isolated monocytes from HIV-infected patients displayed enhanced expression of TLR2 but not TLR4, that TLR2 expression on the surface of monocytes was significantly increased upon stimulation of HIV type 1 envelope protein gp120, and that TLR2 stimulation in HIV-infected patients induced increased viral replication and TNF- alpha response.CONCLUSIONOur findings suggest potential roles for TLR2 in chronic immune activation and viral replication in HIV infection.

Toll-like receptor 2 (TLR2) stimulation in monocytes may contribute to enhanced inflammation and viral replication in HIV infection. In the present study we examined if TLR2 stimulation could modulate chemokine responses in peripheral blood mononuclear cells (PBMC) from HIV-infected patients and healthy controls. Our main findings were, with similar qualitative patterns in both healthy controls and HIV-infected patients: (1) TLR2 stimulation induced up-regulation of several chemokines at the mRNA level as well as increased protein levels of macrophage inflammatory protein (MIP)-1alpha, interleukin (IL)-8 and regulated on activation, normal T cell expressed and secreted (RANTES); (2) TLR2 stimulation induced enhanced protein expression of CCR5 (a receptor for MIP-1alpha and RANTES) on monocytes; (3) In vitro stimulation with RANTES induced release of MIP-1alpha, MCP-1, IL-8 and interferon-gamma from PBMC. While increased levels of beta-chemokines possibly have antiviral effects, TLR2 stimulation may also promote a chemokine-driven inflammatory loop, potentially contributing to the immunopathogenesis of HIV infection.

The present study investigated the expression of Toll-like receptor (TLR) 2, TLR4, cluster of differentiation (CD) 14 and CD1a in human periodontitis gingiva using immunohistochemical methods. The specimens were classified according to the degree of inflammation into three groups (mild, moderate and severe). We established three zones in which to evaluate the ratios of TLR2-, TLR4-, CD14- and CD1a-positive cells to total cells in the connective tissues of each section. TLR2 and TLR4 were expressed in human periodontal tissues, and the ratio of TLR2-positive cells was highest overall in zone 1 (connective tissue subjacent to pocket epithelium) of the severe group and that of TLR4-positive cells was higher in the severe group than in the other groups. These results suggest that TLR2 and TLR4 participate in the innate immune response to stimulation by bacterial products in periodontal tissues. The ratio of CD14-positive cells was lowest overall in zone 1 of the severe group and that of CD1a was higher in the severe group than in the other groups. These results suggest that CD14 may be down-regulated during the development of inflammation and/or dendritic cells might infiltrate chronically inflamed gingival tissue.

Bacterial and synthetic DNAs containing CpG dinucleotides in specific sequence contexts activate the vertebrate immune system through Toll-like receptor 9 (TLR9). In the present study, we used a synthetic nucleoside with a bicyclic heterobase [1-(2′-deoxy-β- d -ribofuranosyl)-2-oxo-7-deaza-8-methyl-purine; R] to replace the C in CpG, resulting in an RpG dinucleotide. The RpG dinucleotide was incorporated in mouse- and human-specific motifs in oligodeoxynucleotides (oligos) and 3′-3-linked oligos, referred to as immunomers. Oligos containing the RpG motif induced cytokine secretion in mouse spleen-cell cultures. Immunomers containing RpG dinucleotides showed activity in transfected-HEK293 cells stably expressing mouse TLR9, suggesting direct involvement of TLR9 in the recognition of RpG motif. In J774 macrophages, RpG motifs activated NF-κB and mitogen-activated protein kinase pathways. Immunomers containing the RpG dinucleotide induced high levels of IL-12 and IFN-γ, but lower IL-6 in time- and concentration-dependent fashion in mouse spleen-cell cultures costimulated with IL-2. Importantly, immunomers containing GTRGTT and GARGTT motifs were recognized to a similar extent by both mouse and human immune systems. Additionally, both mouse- and human-specific RpG immunomers potently stimulated proliferation of peripheral blood mononuclear cells obtained from diverse vertebrate species, including monkey, pig, horse, sheep, goat, rat, and chicken. An immunomer containing GTRGTT motif prevented conalbumin-induced and ragweed allergen-induced allergic inflammation in mice. We show that a synthetic bicyclic nucleotide is recognized in the C position of a CpG dinucleotide by immune cells from diverse vertebrate species without bias for flanking sequences, suggesting a divergent nucleotide motif recognition pattern of TLR9.

Earlier findings have suggested that the balance between interleukin-10 and tumor necrosis factor α levels in serum may influence the outcome of cytomegalovirus infection in renal transplant recipients. Therefore, the aim of the present study was to investigate whether human cytomegalovirus induces interleukin-10 production in macrophages. Experiments using human cytomegalovirus (strain 2006), ultraviolet-inactivated cytomegalovirus, and mock-infected differentiated THP-1 cells with or without ganciclovir or monoclonal anti-tumor necrosis factor α antibodies were performed. Cytomegalovirus-infected cells produced significantly higher levels of human interleukin-10 mRNA and interleukin-10 than ultraviolet-inactivated cytomegalovirus or mock-infected cells. The addition of ganciclovir had little effect on interleukin-10 production. Anti-tumor necrosis factor α antibodies appeared to reduce the interleukin-10 levels. In conclusion, human cytomegalovirus infection of macrophages induces production of human interleukin-10. This requires viral entry, but not full viral replication.

Over the past 3 years our knowledge about how we sense the microbial world has been fundamentally changed. It has been known for decades that microbial products, such as lipopolysaccharide, lipoproteins, or peptidoglycan, have a profound activity on human cells. Whereas the structure of many different pathogenic microbial compounds has been extensively studied and characterized, the molecular basis of their recognition by the cells of the innate immune system remained elusive for a long time. It was Charles Janeway [Cold Spring Harb Symp Quant Biol 1989;54/1:1–13] who developed the idea of microbial structures forming pathogen-associated molecular patterns that would be recognized by pattern recognition receptors. The discovery of the family of Toll receptors in species as diverse as Drosophila and humans, and the recognition of their role in distinguishing molecular patterns that are common to microorganisms have led to a renewed appreciation of the innate immune system. Moreover, it is now clear that the activation of the innate immune system through mammalian Toll-like receptors has also an instructive role for the responses of the adaptive immune response and, thus, may influence allergic diseases such as asthma.

ABSTRACT Pattern recognition via Toll-like receptors (TLR) by antigen-presenting cells is an important element of innate immunity. We report that wild-type measles virus but not vaccine strains activate cells via both human and murine TLR2, and this is a property of the hemagglutinin (H) protein. The ability to activate cells via TLR2 by wild-type MV H protein is abolished by mutation of a single amino acid, asparagine at position 481 to tyrosine, as is found in attenuated strains, which is important for interaction with CD46, the receptor for these strains. TLR2 activation by MV wild-type H protein stimulates induction of proinflammatory cytokines such as interleukin-6 (IL-6) in human monocytic cells and surface expression of CD150, the receptor for all MV strains. Confirming the specificity of this interaction, wild-type H protein did not induce IL-6 release in macrophages from TLR2 −/− mice. Thus, the unique property of MV wild-type strains to activate TLR2-dependent signals might essentially contribute not only to immune activation but also to viral spread and pathogenicity by upregulating the MV receptor on monocytes.

Group B streptococci (GBS) vigorously activate inflammatory responses. We reported previously that a secreted GBS “factor” activates phagocytes via Toll-like receptor (TLR)2 and TLR6, but that GBS cell walls activate cells independently of these receptors. We hypothesized that the phagocytic immune functions in response to GBS, such as inflammation, uptake, and elimination of bacteria, occur through a coordinated engagement of TLRs, along with the coreceptors CD14 and CD11b/CD18. Using various knockout mice we show that GBS-induced activation of p38 and NF-κB depends upon the expression of the cytoplasmic TLR adapter protein, myeloid differentiation factor 88 (MyD88), but not TLR2 and/or TLR4. Macrophages with deletions of CD14 and complement receptor 3 had a normal cytokine response to whole bacteria, although the response to GBS factor was abrogated in CD14-null cells. The intracellular formation of bactericidal oxygen species proved to be MyD88 dependent; however, uptake of GBS, a prerequisite for intracellular killing by O2 radicals, occurred independently of MyD88. While deletion of complement receptor 3 greatly diminished the uptake of opsonized GBS, it did not affect the formation of bactericidal O2 radicals or inflammatory signaling intermediates. We conclude that the inflammatory, bactericidal, and phagocytic responses to GBS occur via parallel but independent processes.

Background-Coronary artery bypass grafting with the use of cardiopulmonary bypass is known to mediate an inflammatory response. The stress-inducible heat-shock protein (HSP) 70 has been detected in myocardial cells after CABG, and toll-like receptors (TLRs) are suggested as putative signaling receptors for the HSPs, mediating synthesis of inflammatory cytokines. The main aims of our study were to explore the release of HSP70 and the regulation of monocyte TLR-2 and TLR-4 expression after CABG.Methods and Results-Twenty patients referred for elective CABG were included in this study. Using immunoassays, we detected HSP70 in plasma after CABG, with peak concentration immediately after surgery. Interleukin-6 in plasma reached peak concentration 5 hours after surgery. Monocyte CD14, TLR-2, and TLR-4 expression, as analyzed by flow cytometry, was initially downregulated. On day 1, CD14 expression normalized, whereas TLR-2 and TLR-4 expression was upregulated. TLR-4 was significantly upregulated even on postoperative day 2. Additional experiments revealed that peritoneal macrophages from control (C3H/HeN) mice responded to HSP70 with release of tumor necrosis factor, whereas macrophages from mutated TLR-4 (C3H/HeJ) mice were unresponsive. In vitro, human adherent monocytes responded to recombinant HSP70 with interleukin-6 and tumor necrosis factor release. CD14 and TLR-4 monoclonal antibodies inhibited the cytokine response.Conclusions-In this study, we observed an immediate release of HSP70 into the circulation and a modulation of monocyte TLR-2 and TLR-4 expression after CABG. TLR-4 and CD14 appear to be involved in an HSP70-mediated activation of innate immunity.

Group B streptococci (GBS) vigorously activate inflammatory responses. We reported previously that a secreted GBS "factor" activates phagocytes via Toll-like receptor (TLR)2 and TLR6, but that GBS cell walls activate cells independently of these receptors. We hypothesized that the phagocytic immune functions in response to GBS, such as inflammation, uptake, and elimination of bacteria, occur through a coordinated engagement of TLRs, along with the coreceptors CD14 and CD11b/CD18. Using various knockout mice we show that GBS-induced activation of p38 and NF-kappaB depends upon the expression of the cytoplasmic TLR adapter protein, myeloid differentiation factor 88 (MyD88), but not TLR2 and/or TLR4. Macrophages with deletions of CD14 and complement receptor 3 had a normal cytokine response to whole bacteria, although the response to GBS factor was abrogated in CD14-null cells. The intracellular formation of bactericidal oxygen species proved to be MyD88 dependent; however, uptake of GBS, a prerequisite for intracellular killing by O(2) radicals, occurred independently of MyD88. While deletion of complement receptor 3 greatly diminished the uptake of opsonized GBS, it did not affect the formation of bactericidal O(2) radicals or inflammatory signaling intermediates. We conclude that the inflammatory, bactericidal, and phagocytic responses to GBS occur via parallel but independent processes.

Mammalian responses to LPS require the expression of Toll-like receptor 4 (TLR4), CD14, and MD-2. We expressed fluorescent TLR4 in cell lines and found that TLR4 densely localized to the surface and the Golgi. Similar distributions were observed in human monocytes. Confocal imaging revealed rapid recycling of TLR4-CD14-MD-2 complexes between the Golgi and the plasma membrane. Fluorescent LPS followed these trafficking pathways in CD14-positive cells. The TLR4-adapter protein, MyD88, translocated to the cell surface upon LPS exposure, and cross-linking of surface TLR4 with antibody induced signaling. Golgi-associated TLR4 expression was disrupted by brefeldin A, yet LPS signaling was preserved. We conclude that LPS signaling may be initiated by surface aggregation of TLR4 and is not dependent upon LPS trafficking to the Golgi.