Involvement of IRAK-M in peptidoglycan-induced tolerance in macrophages

The molecular mechanisms by which pathogen-associated molecular patterns recognized by TLR2, such as peptidoglycan (PGN), induce homotolerance are largely unknown. It was recently reported that IRAK-M negatively regulates TLR signaling. In this study, we elucidate the molecular mechanisms of tolerance induced by PGN, with a focus on the role of IRAK-M. We demonstrate that pretreatment of macrophage RAW264.7 cells with a high concentration (30 microg/ml) of PGN for 16 h effectively induces tolerance against following stimulation with 30 microg/ml of PGN; while pretreatment with a low concentration (1 microg/ml) of PGN does not. IRAK-M is induced in cells treated with the high concentration of PGN 4-24 h after PGN stimulation, but not in cells treated with the low concentration of PGN up to 24 h after stimulation. Phosphorylation of MAPKs and IkappaBalpha is inhibited after the second PGN stimulation in tolerant cells. Kinase activity of IRAK-1 and association between IRAK-1 and MyD88 are also suppressed in PGN-induced tolerant cells. Furthermore, down-regulation of IRAK-M expression by small interfering RNAs specific for IRAK-M reinstates the production of TNF-alpha after PGN restimulation. These results suggest that induction of IRAK-M and inhibition of kinase activity of IRAK-1 are crucial to PGN-induced tolerance in macrophages.     

Induction of bacterial lipoprotein tolerance is associated with suppression of toll-like receptor 2 expression

Tolerance to bacterial cell wall components including lipopolysaccharide (LPS) may represent an essential regulatory mechanism during bacterial infection. Two members of the Toll-like receptor (TLR) family, TLR2 and TLR4, recognize the specific pattern of bacterial cell wall components. TLR4 has been found to be responsible for LPS tolerance. However, the role of TLR2 in bacterial lipoprotein (BLP) tolerance and LPS tolerance is unclear. Pretreatment of human THP-1 monocytic cells with a synthetic bacterial lipopeptide induced tolerance to a second BLP challenge with diminished tumor necrosis factor-alpha and interleukin-6 production, termed BLP tolerance. Furthermore, BLP-tolerized THP-1 cells no longer responded to LPS stimulation, indicating a cross-tolerance to LPS. Induction of BLP tolerance was CD14-independent, as THP-1 cells that lack membrane-bound CD14 developed tolerance both in serum-free conditions and in the presence of a specific CD14 blocking monoclonal antibody (MEM-18). Pre-exposure of THP-1 cells to BLP suppressed mitogen-activated protein kinase phosphorylation and nuclear factor-kappaB activation in response to subsequent BLP and LPS stimulation, which is comparable with that found in LPS-tolerized cells, indicating that BLP tolerance and LPS tolerance may share similar intracellular pathways. However, BLP strongly enhanced TLR2 expression in non-tolerized THP-1 cells, whereas LPS stimulation had no effect. Furthermore, a specific TLR2 blocking monoclonal antibody (2392) attenuated BLP-induced, but not LPS-induced, tumor necrosis factor-alpha and interleukin-6 production, indicating BLP rather than LPS as a ligand for TLR2 engagement and activation. More importantly, pretreatment of THP-1 cells with BLP strongly inhibited TLR2 activation in response to subsequent BLP stimulation. In contrast, LPS tolerance did not prevent BLP-induced TLR2 overexpression. These results demonstrate that BLP tolerance develops through down-regulation of TLR2 expression.     

Induction of tolerance to human arylsulfatase A in a mouse model of metachromatic leukodystrophy

A deficiency of arylsulfatase A (ASA) causes metachromatic leukodystrophy (MLD), a lysosomal storage disorder characterized by accumulation of sulfatide, a severe neurological phenotype and early death. The efficacy of enzyme replacement therapy (ERT) has previously been determined in ASA knockout (ASA-/-) mice representing the only available animal model for MLD. Repeated intravenous injection of human ASA (hASA) improved the nervous system pathology and function, but also elicited a progressive humoral immune response leading to treatment resistance, anaphylactic reactions, and high mortality. In contrast to ASA-/- mice, most MLD patients express mutant hASA which may entail immunological tolerance to substituted wildtype hASA and thus protect from immunological complications. To test this notion, a cysteine-to-serine substitution was introduced into the active site of the hASA and the resulting inactive hASA-C69S variant was constitutively expressed in ASA-/- mice. Mice with sub-to supranormal levels of mutant hASA expression were analyzed. All mice, including those showing transgene expression below the limit of detection, were immunologically unresponsive to injected hASA. More than 100-fold overexpression did not induce an overt new phenotype except occasional intralysosomal deposition of minor amounts of glycogen in hepatocytes. Furthermore, long-term, low-dose ERT reduced sulfatide storage in peripheral tissues and the central nervous system indicating that high levels of extracellular mutant hASA do not prevent cellular uptake and lysosomal targeting of substituted wildtype hASA. Due to the tolerance to hASA and maintenance of the MLD-like phenotype, the novel transgenic strain may be particularly advantageous to assess the benefit and risk of long-term ERT.     

Induction of freezing tolerance in spinach is associated with the synthesis of cold acclimation induced proteins

Spinach (Spinacia oleracea L. cv Bloomsdale) seedlings cultured in vitro were used to study changes in protein synthesis during cold acclimation. Seedlings grown for 3 weeks postsowing on an inorganic-nutrient-agar medium were able to increase their freezing tolerance when grown at 5°C. During cold acclimation at 5°C and deacclimation at 25°C, the kinetics of freezing tolerance induction and loss were similar to that of soil-grown plants. Freezing tolerance increased after 1 day of cold acclimation and reached a maximum within 7 days. Upon deacclimation at 25°C, freezing tolerance declined within 1 day and was largely lost by the 7th day. Leaf proteins of intact plants grown at 5 and 25°C were in vivo radiolabeled, without wounding or injury, to high specific activities with [³⁵S]methionine. Leaf proteins were radiolabeled at 0, 1, 2, 3, 4, 7, and 14 days of cold acclimation and at 1, 3, and 7 days of deacclimation. Up to 500 labeled proteins were separated by two-dimensional gel electrophoresis and visualized by fluorography. A rapid and stable change in the protein synthesis pattern was observed when seedlings were transferred to the low temperature environment. Cold-acclimated leaves contained 22 polypeptides not found in nonacclimated leaves. Exposure to 5°C induced the synthesis of three high molecular weight cold acclimation proteins (CAPs) (M_r of about 160,000, 117,000, and 85,000) and greatly increased the synthesis of a fourth high molecular weight protein (M_r 79,000). These proteins were synthesized during day 1 and throughout the 14 day exposure to 5°C. During deacclimation, the synthesis of CAPs 160, 117, and 85 was greatly reduced by the first day of exposure to 25°C. However, CAP 79 was synthesized throughout the 7 day deacclimation treatment. Thus, the induction at low temperature and termination at warm temperature of the synthesis of CAPs 160, 117, and 85 was highly correlated with the induction and loss of freezing tolerance. Cold acclimation did not result in a general posttranslational modification of leaf proteins. Most of the observed changes in the two-dimensional gel patterns could be attributed to the de novo synthesis of proteins induced by low temperature. In spinach leaf tissue, heat shock altered the pattern of protein synthesis and induced the synthesis of several heat shock proteins (HSPs). One polypeptide synthesized in cold-acclimated leaves had a molecular weight and net charge (M_r 79,000, pI 4.8) similar to that of a HSP (M_r 83,000, pI 4.8). However, heat shock did not increase the freezing tolerance, and cold acclimation did not increase heat tolerance over that of nonacclimated plants, but heat-shocked leaf tissue was more tolerant to high temperatures than nonacclimated or cold-acclimated leaf tissue. When protein extracts from heat-shocked and cold-acclimated leaves were mixed and separated in the same two-dimensional gel, the CAP and HSP were shown to be two separate polypeptides with slightly different isoelectric points and molecular weights.     

IRAK-M is a negative regulator of Toll-like receptor signaling

Toll-like receptors (TLRs) detect microorganisms and protect multicellular organisms from infection. TLRs transduce their signals through MyD88 and the serine/threonine kinase IRAK. The IRAK family consists of two active kinases, IRAK and IRAK-4, and two inactive kinases, IRAK-2 and IRAK-M. IRAK-M expression is restricted to monocytes/macrophages, whereas other IRAKs are ubiquitous. We show here that IRAK-M is induced upon TLR stimulation and negatively regulates TLR signaling. IRAK-M prevented dissociation of IRAK and IRAK-4 from MyD88 and formation of IRAK-TRAF6 complexes. IRAK-M(-/-) cells exhibited increased cytokine production upon TLR/IL-1 stimulation and bacterial challenge, and IRAK-M(-/-) mice showed increased inflammatory responses to bacterial infection. Endotoxin tolerance, a protection mechanism against endotoxin shock, was significantly reduced in IRAK-M(-/-) cells. Thus, IRAK-M regulates TLR signaling and innate immune homeostasis.     

Individual variation in whole-animal hypoxia tolerance is associated with cardiac hypoxia tolerance in a marine teleost

Hypoxia is a pervasive problem in coastal environments and is predicted to have enduring impacts on aquatic ecosystems. Intraspecific variation in hypoxia tolerance is well documented in fish; however, the factors underlying this variation remain unknown. Here, we investigate the role of the heart in individual hypoxia tolerance of the European sea bass (Dicentrarchus labrax). We found individual whole-animal hypoxia tolerance is a stable trait in sea bass for more than 18 months (duration of study). We next examined in vitro cardiac performance and found myocardial muscle from hypoxia-tolerant individuals generated greater force, with higher rates of contraction and relaxation, than hypoxic-sensitive individuals during hypoxic exposure. Thus, whole-animal hypoxia tolerance is associated with cardiac hypoxia tolerance. As the occurrence of aquatic hypoxia is expected to increase in marine ecosystems, our experimental data suggest that cardiac performance may influence fish survival and distribution.     

Differential tolerance induction by lipoarabinomannan and lipopolysaccharide in human macrophages

Various bacterial cell wall components have been shown to induce hyporesponsiveness in macrophages (MAC). Here, mycobacterial glycolipids were employed to determine whether they induce a state of 'tolerance/hyporesponsiveness' in MAC in vitro in order to assess whether mycobacterial components negatively affect the immune response to Mycobacterium tuberculosis. Arabinosylated lipoarabinomannan (ARA-LAM) stimulated hyporesponsiveness by reducing TNF-alpha, GM-CSF, G-CSF, IL-10, and IL-6 release similarly to LPS, but caused no changes in IL-8 secretion. Mannose-capped LAM (MAN-LAM) acted in a different way in that TNF-alpha, GM-CSF, and IL-10 were upregulated after restimulation of MAC. Blocking experiments by mannan suggest mannose-receptor involvement in MAN-LAM activation only. Cross-stimulation experiments demonstrated a hierarchy of signaling, with LPS being the most potent stimulator and mediating abrogation of ARA-LAM-stimulated tolerance but not vice versa. MAN-LAM was the least potent stimulator of either MAC activation and induction of hyporesponsiveness. Similarly to LPS, ARA-LAM upregulated CD14 surface expression after restimulation. Recurrent MAN-LAM treatment either downmodulated or did not induce any change in CD14 expression. The role of MAN-LAM regulated cytokine secretion as well as implications regarding M. tuberculosis infection will be discussed.     

Induction of human beta-defensin-2 expression in human astrocytes by lipopolysaccharide and cytokines

Defensins are cationic peptides with broad-spectrum antimicrobial activity. They are members of a supergene family consisting of alpha and beta subtypes and each subtype is comprised of a number of different isoforms. For example, human alpha-defensin (HAD) has six isoforms, which are expressed by polymorphonuclear leukocytes and Paneth cells. In contrast, human beta-defensin (HBD) has two isoforms that are expressed by epithelial cells of the skin, gut, respiratory and urogenital tracts. Recently, HBD-1 was detected in human brain biopsy tissue. However, little is known about the expression of HBD-1 or HBD-2 in the CNS and whether neural cells can secrete these peptides. For the present study, human astrocyte, microglial, meningeal fibroblast and neuronal cultures were probed for the expression of HBD-1 and HBD-2 mRNA and protein. Each cell type was either maintained in tissue culture medium alone or in medium containing lipopolysaccharide (LPS) at concentrations ranging from 0.1 to 1 microgram/mL, interleukin-1 beta (IL-1beta) at 1-50 ng/mL, or tumor necrosis factor alpha (TNF-alpha) at the same concentrations. The expression of HBD-1 and HBD-2 mRNAs was monitored by RT-PCR. The cDNA products were sequenced to characterize the gene product. HBD-2 protein was detected by immunoblot, immunoprecipitation and immunocytochemistry. Results of these studies showed that HBD-1 mRNA was detected in all cell cultures except in those enriched for neurons. In contrast, HBD-2 mRNA was detected only in astrocyte cultures that were treated with LPS, IL-1beta or TNF-alpha. The detection of the respective proteins correlated positively with the mRNA results. As such, these data represent the first demonstration of HBD-2 expression by astrocytes and suggest that this peptide may play a role in host defense against bacterial CNS pathogenesis.     

Induction of Egr-1 is associated with anti-metastatic and anti-invasive ability of beta-lapachone in human hepatocarcinoma cells

beta-lapachone, a quinone compound obtained from the bark of the lapacho tree (Tabebuia avellanedae), was reported to have anti-inflammatory and anti-cancer activities. In this study, we investigated novel functions of beta-lapachone in terms of anti-metastasis and anti-invasion abilities using human hepatocarcinoma cell lines, HepG2 and Hep3B. beta-lapachone dose-dependently inhibited cell viability and migration of both HepG2 and Hep3B cells, as determined by methylthiazoletetrazolium (MTT) assay and wound healing assay. RT-PCR and Western blot data revealed that beta-lapachone dramatically increased the levels of protein, as well as mRNA expression of early growth response gene-1 (Egr-1) and throbospondin-1 (TSP-1) at an early point in time, and then decreased in a time-dependent manner. In addition, down-regulation of Snail and up-regulation of E-cadherin expression were observed in beta-lapachone-treated HepG2 and Hep3B cells, and this the associated with decreased invasive ability as measured by matrigel invasion assay. Taken together, our results strongly suggest that beta-lapachone may be expected to inhibit the progression and metastasis of hepatoma cells, at least in part by inhibiting the invasive ability of the cells via up-regulation of the expression of the Egr-1, TSP-1, and E-cadherin.     

IRAK-M is involved in the pathogenesis of early-onset persistent asthma

Asthma is a multifactorial disease influenced by genetic and environmental factors. In the past decade, several loci and >100 genes have been found to be associated with the disease in at least one population. Among these loci, region 12q13-24 has been implicated in asthma etiology in multiple populations, suggesting that it harbors one or more asthma susceptibility genes. We performed linkage and association analyses by transmission/disequilibrium test and case-control analysis in the candidate region 12q13-24, using the Sardinian founder population, in which limited heterogeneity of pathogenetic alleles for monogenic and complex disorders as well as of environmental conditions should facilitate the study of multifactorial traits. We analyzed our cohort, using a cutoff age of 13 years at asthma onset, and detected significant linkage to a portion of 12q13-24. We identified IRAK-M as the gene contributing to the linkage and showed that it is associated with early-onset persistent asthma. We defined protective and predisposing SNP haplotypes and replicated associations in an outbred Italian population. Sequence analysis in patients found mutations, including inactivating lesions, in the IRAK-M coding region. Immunohistochemistry of lung biopsies showed that IRAK-M is highly expressed in epithelial cells. We report that IRAK-M is involved in the pathogenesis of early-onset persistent asthma. IRAK-M, a negative regulator of the Toll-like receptor/IL-1R pathways, is a master regulator of NF-κB and inflammation. Our data suggest a mechanistic link between hyperactivation of the innate immune system and chronic airway inflammation and indicate IRAK-M as a potential target for therapeutic intervention against asthma.     

Induction of apoptosis by bufalin in human tumor cells is associated with a change of intracellular concentration of Na+ ions.

In an attempt to characterize the mechanisms that are operative at the early stages of the induction of apoptosis by bufalin, a component of the traditional Chinese medicine chan'su, we examined the effects of bufalin on plasma membrane potential, as determined by monitoring the uptake by cells of rhodamine 123. Bufalin induced apoptosis in human monocytic leukemia THP-1 cells, in human lymphoblastic leukemia MOLT-3 cells, and in human colon adenocarcinoma COLO320DM cells but not in normal human leukocytes, for example, polymorphonuclear cells and lymphocytes, and not in murine leukemia P388D1 and M1 cells. Treatment for 3 h with bufalin at 10(-6) M caused a decrease in the plasma membrane potential in several lines of human tumor cells but not in murine leukemia cells. No changes in mitochondrial membrane potential, as monitored with the fluorescent dye JC-1, and no release of cytochrome c were observed within at least 6 h after the start of treatment with bufalin. Moreover, overexpression of bcl-2 in human leukemia HL60 cells that had been transfected with cDNA for bcl-2 prevented bufalin-induced apoptosis but had no significant effect on the change in plasma membrane potential induced by bufalin. Since bufalin specifically inhibits the Na+,K(+)-ATPase of human but not murine tumor cells, and since this inhibition leads to a change in intracellular concentration of Na+ ions, our findings suggest that bufalin induces apoptosis in human tumor cells selectively via inhibition of the Na+,K(+)-ATPase, which acts upstream of the bcl-2 protein.     

Oxytocin is associated with human trustworthiness

Human beings exhibit substantial interpersonal trust-even with strangers. The neuroactive hormone oxytocin facilitates social recognition in animals, and we examine if oxytocin is related to trustworthiness between humans. This paper reports the results of an experiment to test this hypothesis, where trust and trustworthiness are measured using the sequential anonymous "trust game" with monetary payoffs. We find that oxytocin levels are higher in subjects who receive a monetary transfer that reflects an intention of trust relative to an unintentional monetary transfer of the same amount. In addition, higher oxytocin levels are associated with trustworthy behavior (the reciprocation of trust). Absent intentionality, both the oxytocin and behavioral responses are extinguished. We conclude that perceptions of intentions of trust affect levels of circulating oxytocin.     

Accumulation of terpenoid phytoalexins in maize roots is associated with drought tolerance

Maize (Zea mays) production, which is of global agro‐economic importance, is largely limited by herbivore pests, pathogens and environmental conditions, such as drought. Zealexins and kauralexins belong to two recently identified families of acidic terpenoid phytoalexins in maize that mediate defence against both pathogen and insect attacks in aboveground tissues. However, little is known about their function in belowground organs and their potential to counter abiotic stress. In this study, we show that zealexins and kauralexins accumulate in roots in response to both biotic and abiotic stress including, Diabrotica balteata herbivory, Fusarium verticillioides infection, drought and high salinity. We find that the quantity of drought‐induced phytoalexins is positively correlated with the root‐to‐shoot ratio of different maize varieties, and further demonstrate that mutant an2 plants deficient in kauralexin production are more sensitive to drought. The induction of phytoalexins in response to drought is root specific and does not influence phytoalexin levels aboveground; however, the accumulation of phytoalexins in one tissue may influence the induction capacity of other tissues.     

Endotoxin tolerance is associated with altered GTP-binding protein function.

Previous studies have suggested that guanine nucleotide regulatory (G) proteins modulate endotoxin-stimulated peritoneal macrophage arachidonic acid (AA) metabolism. Endotoxin-stimulated metabolism of AA by peritoneal macrophages is decreased in endotoxin tolerance (Rogers et al. Prostaglandins 31: 639-650, 1986). These observations led to a study of G protein function and AA metabolism by peritoneal macrophages in endotoxin tolerance. Endotoxin tolerance was induced by the administration of sublethal doses of endotoxin. AA metabolism was assessed by measurement of thromboxane B2 (TxB2), a cyclooxygenase metabolite. NaF (5 mM), an activator of G proteins, significantly stimulated TxB2 synthesis in control macrophages from 7.7 +/- 0.2 to 19.1 +/- 0.6 (SE) ng/ml (P less than 0.05) at 2 h and was partially inhibited by pertussis toxin, suggesting a G protein-dependent mechanism. Salmonella enteritidis endotoxin (50 micrograms/ml) stimulated a similar increase in TxB2 levels (23 +/- 0.4 ng/ml, P less than 0.05). In contrast to control macrophages, macrophages from endotoxin-tolerant rats stimulated with either NaF or S. enteritidis endotoxin had TxB2 levels that were only 30 and 2% of the respective stimulated control cells. Basal guanosine-triphosphatase (GTPase) activity (33 +/- 6 pmol.mg-1.min-1) in endotoxin-tolerant macrophage membranes was significantly lower (P less than 0.05) than control basal activity (158 +/- 5 pmol.mg-1.min-1). This suppression of macrophage GTPase activity was apparent 48 h after the first in vivo sublethal endotoxin injection (100 micrograms/kg ip). The reduced GTPase activity paralleled in vitro cellular hyporesponsiveness to endotoxin-stimulated TxB2 production.(ABSTRACT TRUNCATED AT 250 WORDS)     

Induction of a stress response in Lactococcus lactis is associated with a resistance to ribosomally active antibiotics

The acquisition of multidrug resistance in bacteria underlies the failure of antimicrobial therapy, and the emergence of pathogens that are resistant to almost the entire armoury of antibiotics. Among the proteins that can mediate or contribute to the drug-resistance profile in Gram-positive bacteria is a subset of ATP-binding cassette proteins that are comprised of a tandem-repeated nucleotide-binding domain. In this study, we expressed one of these NBD(2) proteins, LmrC, in an antibiotic-sensitive Gram-positive host strain (Lactococcus lactis) and demonstrated the acquisition of resistance to ribosomally active antibiotics. Mutation of key catalytic residues suggested that the resistance profile was the result of a cellular response, rather than being a function of the NBD(2) protein itself. This observation was confirmed by 2D SDS/PAGE, which demonstrated that the expression of the NBD(2) protein induced a stress response in L. lactis. A model combining this stress response induction and the acquisition of antibiotic resistance is proposed.     

Up-regulation of SGTB is associated with neuronal apoptosis after neuroinflammation induced by lipopolysaccharide

SGTB (Small glutamine-rich tetratricopeptide repeat (TPR)-containing, β) plays a critical role in protein–protein interactions. The interaction between SGTB and heat shock cognate protein (Hsc70)/heat shock protein (Hsp70) has aroused much attention in recent years. The present study was designed to elucidate dynamic changes in SGTB expression and distribution in the cerebral cortex in a lipopolysaccharide (LPS)-induced neuroinflammation rat model. It was found that SGTB expression was increased significantly in apoptotic neurons after LPS injection. The result of our in vitro study suggested that SGTB up-regulation might be associated with neuronal apoptosis after H₂O₂ challenge. In addition, silencing of SGTB in cultured PC12 (Pheochromocytoma) by siRNA indicated that SGTB was required for neuronal apoptosis induced by oxidative stress. Our finding about the cellular signal pathway may provide a new strategy against neuronal apoptosis in neuroinflammation in CNS.