LPS Stimulation Of Macrophages Research Articles

Serum autoantibodies against α7-nicotinic receptors in subgroups of patients with bipolar disorder or schizophrenia: clinical features and link with peripheral inflammation

There is growing evidence that autoantibodies (AAbs) against proteins expressed in the brain are playing an important role in neurological and psychiatric disorders. Here, we explore the presence and the role of peripheral AAbs to the α7-nicotinic acetylcholine receptor (nAChR) in inflammatory subgroups of psychiatric patients with bipolar disorder (BD) or schizophrenia (SCZ) and healthy controls. We have identified a continuum of AAb levels in serum when employing a novel ELISA technique, with a significant elevation in patients compared to controls. Using unsupervised two-step clustering to stratify all the subjects according to their immuno-inflammatory background, we delineate one subgroup consisting solely of psychiatric patients with severe symptoms, high inflammatory profile, and significantly increased levels of anti-nAChR AAbs. In this context, we have used monoclonal mouse anti-human α7-nAChR antibodies (α7-nAChR-mAbs) and shown that TNF-α release was enhanced upon LPS stimulation in macrophages pre-incubated with α7-nAChR-mAbs compared to the use of an isotype control. These findings provide a basis for further study of circulating nicotinic AAbs, and the inflammatory profile observed in patients with major mood and psychotic disorders.

Astragalus Complanatus Ethanol Attenuates Septic Shock by Exerting Anti-Inflammatory Effects on Macrophages.

Sepsis is a systemic inflammatory syndrome that results in multiple-organ failure caused by a dysregulated host immune response to microbial infection. Astragali complanati semen extract (ACSE) exhibits pharmacological activities, including antioxidant, anticancer, antiaging, and anti-diabetes effects. It is widely used in traditional medicine to treat liver and kidney diseases; however, the protective effect of ACSE on sepsis and its mechanisms are unknown. In the present study, we investigated the anti-inflammatory effects and potential mechanisms of the action of ACSE on sepsis. We show that ACSE improved survival rates in mouse models of acute sepsis induced by CLP (cecal ligation and puncture) and LPS stimulation. ACSE administration decreased aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in sepsis-induced mice. Furthermore, ACSE reduced the levels of nitric oxide (NO), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) in the serum of septic mice. ACSE treatment inhibited the expression of these proinflammatory genes in LPS-stimulated J774 macrophages. Moreover, ACSE inhibited the phosphorylation of the IκB kinase (IKK) and the nuclear translocation of p65 NF-κB by LPS stimulation in macrophages. These results reveal the mechanism underlying the protective effect of ACSE against sepsis by inhibiting NF-κB activation and suggest that ACSE could be a potential therapeutic candidate to treat acute inflammatory diseases.

Loss of TRIM24 promotes IL-10 expression via CBP/p300-dependent IFNβ1 transcription during macrophage activation.

As an anti-inflammatory cytokine, interleukin 10 (IL-10) plays a vital role in preventing inflammatory and autoimmune pathologies while also maintaining immune homeostasis. IL-10 production in macrophages is tightly regulated by multiple pathways. TRIM24, a member of the Transcriptional Intermediary Factor 1 (TIF1) family, contributes to antiviral immunity and macrophage M2 polarization. However, the role of TRIM24 in regulating IL-10 expression and its involvement in endotoxic shock remains unclear. In vitro, bone marrow derived macrophages cultured with GM-CSF or M-CSF were stimulated with LPS (100ng/ml). Murine models of endotoxic shock were established by challenging the mice with different dose of LPS (i.p). RTPCR, RNA sequencing, ELISA and hematoxylin and eosin staining were performed to elucidate the role and mechanisms of TRIM24 in endotoxic shock. The expression of TRIM24 is downregulated in LPS-stimulated bone marrow-derived macrophages (BMDMs). Loss of TRIM24 boosted IL-10 expression during the late stage of LPS-stimulation in macrophages. RNA-seq analysis revealed the upregulation of IFNβ1, an upstream regulator of IL-10, in TRIM24 knockout macrophages. Treatment with C646, a CBP/p300 inhibitor, diminished the difference in both IFNβ1 and IL-10 expression between TRIM24 knockout and control macrophages. Loss of TRIM24 provided protection against LPS-induced endotoxic shock in mice. Our results demonstrated that inhibiting TRIM24 promoted the expression of IFNβ1 and IL-10 during macrophage activation, therefore protecting mice from endotoxic shock. This study offers novel insights into the regulatory role of TRIM24 in IL-10 expression, making it a potentially attractive therapeutic target for inflammatory diseases.

Novel long non-coding RNAs associated with inflammation and macrophage activation in human

Inflammation plays a central role in immune response and macrophage activation. Emerging studies demonstrate that along with proteins and genomic factors, noncoding RNA are potentially involved in regulation of immune response and inflammation. Our recent study demonstrated that lncRNA HOTAIR plays key roles in cytokine expression and inflammation in macrophages. The primary goal of this study is to discover novel lncRNAs that are crucial players in inflammation, macrophage activation, and immune response in humans. Towards this, we have stimulated THP1-derived macrophages (THP1-MΦ) with lipopolysaccharides (LPS) and performed the whole transcriptome RNA-seq analysis. Based on this analysis, we discovered that along with well-known marker for inflammation (such as cytokines), a series of long noncoding RNAs (lncRNAs) expression were highly induced upon LPS-stimulation of macrophages, suggesting their potential roles in inflammation and macrophage activation. We termed these family of lncRNAs as Long-noncoding Inflammation Associated RNA (LinfRNA). Dose and time dependent analysis demonstrated that many human LinfRNA (hLinfRNAs) expressions follow similar patterns as cytokine expressions. Inhibition of NF-κB suppressed the expression of most hLinfRNAs suggesting their potential regulation via NF-κB activation during inflammation and macrophage activation. Antisense-mediated knockdown of hLinfRNA1 suppressed the LPS-induced expression of cytokines and pro-inflammatory genes such as IL6, IL1β, and TNFα expression, suggesting potential functionality of the hLinfRNAs in cytokine regulation and inflammation. Overall, we discovered a series of novel hLinfRNAs that are potential regulators of inflammation and macrophage activation and may be linked to inflammatory and metabolic diseases.

NUCLEOLIN PROTECTS CARDIOMYOCYTES BY UPREGULATING PGC-1α AND PROMOTING MITOCHONDRIAL BIOGENESIS IN LPS-INDUCED MYOCARDIAL INJURY.

Background: Lipopolysaccride-induced myocardial injury was characterized by frequent mitochondrial dysfunction. Our previous studies found that nucleolin (NCL) played important protective roles in myocardial ischemia-reperfusion injury. Recently, it has been found that NCL has a protective effect on LPS-induced myocardial injury in vivo . However, the exact underlying mechanisms that how NCL protects myocardium against the LPS-induced myocardial injury remains unclear. Objective: The aim of the study is to investigate the protective role of NCL in LPS-induced myocardial injury from the aspect of mitochondrial biogenesis. Methods: The cardiac-specific NCL-knockout (NCL -/- ) or NCL f/f mice were injected with LPS (10 mg/kg) to induce LPS-induced myocardial injury. The supernatant generated after LPS stimulation of macrophages was used as the conditioned medium to stimulate H9C2 and established the injured cell model. Analysis of mRNA stability, RNA-binding protein immunoprecipitation assay, and luciferase reporter assay were performed to detect the mechanism by which NCL regulated the expression of PGC-1α. Results: The expression of NCL and PGC-1α was elevated in cardiac tissue and cardiomyocytes during LPS-induced myocardial injury. The cardiac-specific NCL-knockout decreased PGC-1α expression, inhibited mitochondrial biogenesis, and increased cardiomyocytes death during LPS-induced myocardial injury in vitro and in vivo . In contrast, the overexpression of NCL could improve mitochondrial biogenesis in H9C2 cells. Moreover, the analysis of mRNA stability and luciferase reporter assay revealed that the interaction between NCL and PGC-1α significantly promoted the stability of PGC-1α mRNA, thereby upregulating the expression of PGC-1α and exerting a cardioprotective effect. In addition, the activation of PGC-1α diminished the detrimental effects of NCL knockdown on mitochondrial biogenesis in vitro and in vivo . Conclusions: Nucleolin upregulated the gene expression of PGC-1α by directly binding to the 5'-UTR of PGC-1α mRNA and increasing its mRNA stabilities, then promoted mitochondrial biogenesis, and played protective effect on cardiomyocytes during LPS-induced myocardial injury. Taken together, all these data showed that NCL activated PGC-1α to rescue cardiomyocytes from LPS-induced myocardial injury insult, suggesting that the cardioprotective role of NCL might be a promising prospect for clinical treatment of patients with endotoxemia.

IRAK-4 in macrophages contributes to inflammatory osteolysis of wear particles around loosened hip implants.

TLRs recognizing PAMPS play a role in local immunity and participate in implant-associated loosening. TLR-mediated signaling is primarily regulated by IL-1 receptor associated kinase-M (IRAK-M) negatively and IRAK-4 positively. Our previous studies have proved that wear particles promote endotoxin tolerance in macrophages by inducing IRAK-M. However, whether IRAK-4 is involved in inflammatory osteolysis of wear particles basically, and the specific mechanism of IRAK-4 around loosened hip implants, is still unclear. IRAK-4 was studied in the interface membranes from patients in vivo and in particle-stimulated macrophages to clarify its role. Also, IL-1β and TNF-α levels were measured after particle and LPS stimulation in macrophages with or without IRAK-4 silenced by siRNA. Our results showed that the interface membranes around aseptic and septic loosened prosthesis expressed more IRAK-4 compared with membranes from osteoarthritic patients. IRAK-4 in macrophages increased upon particle and LPS stimulation. In the former, IL-1β and TNF-α levels were lower compared with those of LPS stimulation, and IRAK-4 siRNA could suppress production of pro-inflammatory cytokines. These findings suggest that besides IRAK-M, IRAK-4 also plays an important role in the local inflammatory reaction and contributes to prosthesis loosening.

Toll-like Receptor (TLR)-induced Rasgef1b expression in macrophages is regulated by NF-κB through its proximal promoter

Ras Guanine Exchange Factor (RasGEF) domain family member 1b is encoded by a Toll-like receptor (TLR)-inducible gene expressed in macrophages, but transcriptional mechanisms that govern its expression are still unknown. Here, we have functionally characterized the 5' flanking Rasgef1b sequence and analyzed its transcriptional activation. We have identified that the inflammation-responsive promoter is contained within a short sequence (-183 to +119) surrounding the transcriptional start site. The promoter sequence is evolutionarily conserved and harbors a cluster of five NF-κB binding sites. Luciferase reporter gene assay showed that the promoter is responsive to TLR activation and RelA or cRel, but not RelB, transcription factors. Besides, site-directed mutagenesis showed that the κB binding sites are required for maximal promoter activation induced by LPS. Analysis by Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) revealed that the promoter is located in an accessible chromatin region. More important, Chromatin Immunoprecipitation sequencing (ChIP-seq) showed that RelA is recruited to the promoter region upon LPS stimulation of bone marrow-derived macrophages. Finally, studies with Rela-deficient macrophages or pharmacological inhibition by Bay11-7082 showed that NF-κB is required for optimal Rasgef1b expression induced by TLR agonists. Our data provide evidence of the regulatory mechanism mediated by NF-κB that facilitates Rasgef1b expression after TLR activation in macrophages.

TET1 is an important transcriptional activator of TNFα expression in macrophages

Activation of macrophages and overexpression of TNFα is associated with the pathogenesis of chronic inflammatory diseases. However, the mechanisms leading to TNFα overexpression are still unknown. 5-methylocytosine (5-mC) is an epigenetic modification that is associated with silenced genes. Recent studies showed that it is converted to 5-hydroxylmethylocytosine (5-hmC) and reactivates gene expression through the action of the family of Ten-Eleven-Translocation (TET1-3) enzymes. In this study, we show that 5-hmC levels are increased globally and specifically in the TNFα promoter during the differentiation of monocytes to macrophages. In addition, the levels of 5-hmC are increased upon LPS stimulation of macrophages. Furthermore, CRIPSR stable knockout of TET1 decreases the expression of TNFα and other pro-inflammatory cytokines. In conclusion, we showed that TET1 contributes to the activation of macrophages possibly through regulation of 5-hydroxymethylation in the promoter of pro-inflammatory cytokine genes. The TET1 enzyme could be a promising therapeutic target to inhibit the persistent inflammation caused by macrophages in chronic inflammatory diseases.

PGE2 Augments Inflammasome Activation and M1 Polarization in Macrophages Infected with Salmonella Typhimurium and Yersinia enterocolitica

Prostaglandin E2 (PGE2) is an important immunomodulatory molecule that is part of a larger group of bioactive lipids known as eicosanoids. PGE2 has long been reported to have conflicting roles in the regulation of inflammation. For example, some effects are primarily anti‐inflammatory and other roles that mediate inflammation. During intracellular infection with Gram‐negative bacteria, host macrophages form complexes called inflammasomes. Inflammasomes drive activation and secretion of inflammatory cytokines such as IL‐1β that signal nearby cells in the process of inflammatory cell death called pyroptosis. PGE2 is reported to inhibit NLRP3 inflammasome activation and enhance NLRC4 inflammasome activation. Since both NLRP3 and NLRC4 inflammasomes mediate IL‐1β secretion, the PGE2's function in the inflammatory processes does not seem to be clear. Previous studies relied on LPS stimulation of macrophages in place of bacterial infection. In this study, we used an infection model of THP‐1 human macrophages with Salmonella enterica Typhimurium (St) and Yersinia enterocolitica (Ye) that suggests PGE2 plays a vital role in enhancing the transcription and secretion of IL‐1β upon inflammasome activation. Interestingly, distinct virulence strategies between St and Ye are reflected in the alteration of COX‐2 and resulting PGE2 biosynthesis by these pathogens. Ye virulence factors inhibit COX‐2 expression and thus decrease the amount of PGE2 secreted during infection. St, in contrast, activates COX‐2 and enhances PGE2 production. In our studies, PGE2 enhances bacterial load of St inside macrophages 48 hours post infection, but it decreases intracellular Ye. Finally, our results suggest that PGE2 primarily enhances the transcription of pro‐inflammatory cytokines while repressing anti‐inflammatory processes such as resource scavenging and matrix remodeling.Support or Funding InformationThis study was supported by a fund no. 2016‐31200‐06012 from the National Institute of Food and Agriculture (NIFA), https://doi.org/10.13039/100005825, National Institutes of Health (NIH) fund no. 5P20GM103646‐02, https://doi.org/10.13039/100000002, as well as MS Center for Food Safety and Post‐harvest Technology Grant Program (USDA‐ARS SCA 58‐6066‐7081) and NIH1R15ES015348‐02. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The intracellular chloride channel proteins CLIC1 and CLIC4 induce IL-1β transcription and activate the NLRP3 inflammasome

The NLRP3 inflammasome is a multiprotein complex that regulates the activation of caspase-1 leading to the maturation of the proinflammatory cytokines IL-1β and IL-18 and promoting pyroptosis. Classically, the NLRP3 inflammasome in murine macrophages is activated by the recognition of pathogen-associated molecular patterns and by many structurally unrelated factors. Understanding the precise mechanism of NLRP3 activation by such a wide array of stimuli remains elusive, but several signaling events, including cytosolic efflux and influx of select ions, have been suggested. Accordingly, several studies have indicated a role of anion channels in NLRP3 inflammasome assembly, but their direct involvement has not been shown. Here, we report that the chloride intracellular channel proteins CLIC1 and CLIC4 participate in the regulation of the NLRP3 inflammasome. Confocal microscopy and cell fractionation experiments revealed that upon LPS stimulation of macrophages, CLIC1 and CLIC4 translocated into the nucleus and cellular membrane. In LPS/ATP-stimulated bone marrow-derived macrophages (BMDMs), CLIC1 or CLIC4 siRNA transfection impaired transcription of IL-1β, ASC speck formation, and secretion of mature IL-1β. Collectively, our results demonstrate that CLIC1 and CLIC4 participate both in the priming signal for IL-1β and in NLRP3 activation.

MAP kinase phosphatase 2 (MKP-2) regulates the innate immune response to influenza infection (INM8P.436)

Abstract MAP kinase phosphatases (MKP) are major negative regulators of MAP kinases and are pivotal in the regulation of immune responses. As one member of MKP protein family, MKP-2 has been shown critically regulate p38 and JNK MAP Kinases in response to LPS stimulation in macrophages. While MKP-2 KO mice are more susceptible to intracellular parasite challenge, little is known about the role of MKP-2 in the innate immune response to viral infections. To investigate the role of MKP-2 in innate immune response against influenza, wild type (WT) and MKP-2 knockout (KO) mice were intranasally inoculated with sub-lethal dosage of A/Putero Rico/8/34 (PR8, H1N1) viruses. We found that viral titers in the lung of MKP-2 KO mice are significantly lower on day 3 and day 5 post-infection than those in the lung of WT mice. Correspondingly, the anti-viral cytokines IFN-α and IFN-β were higher in bronchoalveolar lavage fluid from MKP-2 KO mice compared with WT mice. Primary macrophages and dendritic cells from MKP-2 KO mice produced significantly higher amount of IFN-α and IFN-β in response to PR8 virus infection than cells from WT mice. Mechanistically, ERK and IRF-3 activation was prolonged in MKP-2 KO cells infected with PR8 virus. The regulation of type I IFNs by MKP-2 was further investigated. Together, these data support the model that MKP-2 plays an important role in the regulation of type-I IFNs production, thus altering the innate immune responses during pulmonary influenza infection.

A proteomic approach for the elucidation of the specificity of ectodomain shedding

Ectodomain shedding (shedding) is a posttranslational modification mechanism, which liberates extracellular domains of membrane proteins through juxtamembrane processing. Because shedding alters cell characteristics in a rapid and irreversible manner, it must be strictly regulated. However, the regulatory mechanisms of shedding in response to environmental changes remain obscure. To evaluate the regulatory mechanisms of endogenous shedding, we previously developed a proteomic screening system to identify shedding targets. This system revealed a comprehensive picture of membrane proteins shed under defined conditions. In this study, we have improved the screening system to compare the shedding patterns in a mouse macrophage cell line treated with two different shedding inducers, lipopolysaccharide (LPS) and 12-O-tetradecanoylphorbol 13-acetate (TPA). We show here that LPS simultaneously activates the shedding of multiple membrane proteins. We further show that TPA specifically activates the shedding of αM/β2 integrin (Mac-1), which was not shed upon LPS-stimulation of macrophages. These results clearly demonstrate that the regulation of endogenous membrane protein shedding is both stimulus- and substrate-specific. The shedding targets reported to date play pivotal roles in a variety of biological phenomena, including the immunological response, cell growth, cell adhesion and cell movement. In addition, several disease-related membrane proteins are shedding targets. Thus, understanding the regulation of shedding is important for the elucidation of pathogenesis and the development of therapeutic strategies. We submit that a comprehensive characterization of endogenous shedding is indispensable for understanding the regulatory mechanisms of shedding, and thus have developed a proteomic screening system to identify shedding targets. In this study, using our screening system, we demonstrate that different extracellular stimuli activate different types of shedding, even in a single cell. Our results prove that this proteomic approach is quite effective for the elucidation of the regulatory mechanisms of shedding.

Down Regulation of MyD88 in Macrophages Treated with Liposomes Composed of Phosphatidylserine

We have recently demonstrated that liposomes composed of phosphatidylserine (PS-liposomes) suppressed nitric oxide and inflammatory cytokine productions following LPS stimulation in macrophages. In this study, we examined the effect of PS-liposomes on expressions of TLR-4 and MyD88, which are essential for the signal transduction in LPS stimulation. Expression of MyD88 was suppressed when macrophages were treated with PS-liposomes, but not with liposomes of phosphatidylcholine. No change in TLR-4 expression was observed. MyD88 suppression was restored to the control levels when cells were pre-treated with anti-TGF-β antibody, suggesting that TGF-β plays an important role in down-regulation of MyD88 following PS-liposome treatment.

P079 ARID5a is an IL-6 mRNA stability protein. Chlorpromazine mediates its inhibitory effect on IL-6 production in macrophages through inhibition of ARID5A expression

Introduction Our previous study showed that chlorpromazine (CPZ), an anti-histamine therapeutic, inhibits LPS-induced IL-6 but not TNF-α production in macrophages [1] . In the current study, we show that CPZ is an inhibitor of LPS-induced IL-6 but not TNF-α mRNA stability. Since the 3′-untranslated region (3′-UTR) is important for the regulation of IL-6 mRNA stability, we examined whether CPZ affects the activity of RNA stabilizing proteins on AU-rich IL-6 3′-UTR mRNA. Here we identified a novel IL-6 mRNA stability protein, Arid5a. The Arid (AT-rich interactive domain) family of proteins have been previously demonstrated to be involved in the control of differentiation, tissue-specific gene expression and proliferation control [2] . In particular Arid5a was shown to be involved in chondrocyte-specific gene transcription [3] . In this study, we report a novel function for Arid5a on the stabilization of IL-6 mRNA. Methods For knockdown of Arid5a gene expression we employed siRNA technology. Levels of IL-6 and TNF-alpha protein in RAW 264 macrophage cell supernatants following transfection of siRNA control or ARID5a, were determined by ELISA. IL-6 and TNF-alpha mRNA stability was determined by real-time PCR. Interaction of ARID5a with the IL-6 3′-UTR was examined by mass spectrometry (LC-MS/MS). Results We found that Arid5a is induced by LPS stimulation in macrophages and binds to the IL-6 3′-UTR in macrophages. In RAW 264 cells, knockdown of Arid5a by siRNA led to the decrease of LPS-induced IL-6 but not TNF-α production. We showed that overexpression of Arid5a critically stabilizes IL-6 mRNA through AU-rich elements of the 3′-UTR region. Conclusion Thus, Arid5a is an IL-6 mRNA stability protein which mediates the inhibitory effects of CPZ on LPS-mediated IL-6 production. CPZ, or novel derivatives of this molecule, may represent a new therapy for IL6-dependent diseases (such as arthritis), through its inhibitory function on IL-6 mRNA stability.

The Role of Tuberous Sclerosis Complex 1 in Regulating Innate Immunity

The mechanisms that control TLR-induced responses, including endotoxin tolerance, have been not well understood. The tuberous sclerosis complex 1 (TSC1) is a tumor suppressor that inhibits the mammalian target of rapamycin (mTOR). We show in this study that deficiency of TSC1 results in enhanced activation of not only mTOR complex 1 (mTORC1), but also JNK1/2, following LPS stimulation in macrophages. TSC1-deficient macrophages produce elevated proinflammatory cytokines and NO in response to multiple TLR ligands. Such enhanced TLR-induced responses can be inhibited by reducing mTORC1 and JNK1/2 activities with chemical inhibitors or small hairpin RNA, suggesting that TSC1 negatively controls TLR responses through both mTORC1 and JNK1/2. The impact of TSC1 deficiency appeared not limited to TLRs, as NOD- and RIG-I/MDA-5-induced innate responses were also altered in TSC1-deficient macrophages. Furthermore, TSC1 deficiency appears to cause impaired induction of endotoxin tolerance in vitro and in vivo, which is correlated with increased JNK1/2 activation and can be reversed by JNK1/2 inhibition. Our results reveal a critical role of TSC1 in regulating innate immunity by negative control of mTORC1 and JNK1/2 activation.

Haemorrhagic shock in mice – Intracellular signalling and immunomodulation of peritoneal macrophages’ LPS response

Haemorrhagic shock leads to decreased proinflammatory cytokine response which is associated with an increased susceptibility to bacterial infections. In the present study, the effect of GM-CSF on lipopolysaccharide (LPS)-induced TNF-alpha release and MAPkinase activation was analysed on the background of a possible immunostimulating activity of this substance. Male BALB/c mice were bled to a mean arterial blood pressure of 50 mm Hg for 45 min followed by resuscitation. Peritoneal macrophages were isolated 20 h after haemorrhage and incubated with 10 ng/ml GM-CSF for 6 h before LPS stimulation. TNF-alpha synthesis was studied in the culture supernatants using ELISA. Phosphorylation of ERK, p38MAPK and IκBα was detected by Western blotting. LPS-induced TNF-alpha production of peritoneal macrophages was significantly decreased 20 h after haemorrhage in comparison to the corresponding cells of sham-operated mice. In parallel the phosphorylation of IκBα was less in LPS-stimulated peritoneal macrophages from haemorrhagic mice. LPS-induced phosphorylation of ERK1/2 was also decreased in peritoneal macrophages isolated after haemorrhagic shock. In contrast, p38MAPK was phosphorylated more intensely after LPS-stimulation in macrophages collected from shocked mice. GM-CSF incubation elevated LPS-induced TNF-alpha response of macrophages from both sham-operated and shocked mice which was accompanied by an elevated IκB and ERK phosphorylation. In general, GM-CSF treatment in vitro enhanced peritoneal macrophages LPS-response both in terms of TNF-alpha synthesis and IκB and MAPK signalling, but the levels always stayed lower than those of GM-CSF-treated cells from sham-operated animals. In conclusion, GM-CSF preincubation could partly reactivate the depressed functions of peritoneal macrophages and may therefore exert immunostimulating properties after shock or trauma.

Rac1 and Toll-IL-1 receptor domain-containing adapter protein mediate Toll-like receptor 4 induction of HIV-long terminal repeat.

Opportunistic infections, common in HIV-1-infected patients, increase HIV replication; however, the intracellular signaling mechanisms involved are not clearly known. We have shown that Toll-like receptor 2 (TLR2), TLR4, and TLR9 mediate microbial Ag-induced HIV-long terminal repeat (HIV-LTR) trans-activation and HIV-1 replication, and that LPS-induced HIV-LTR trans-activation is mediated through myeloid differentiation adapter protein. Recently, Toll-IL-1R domain-containing adapter protein (TIRAP) has been identified as an adapter molecule that mediates responses to TLR2 and TLR4 ligands, and TIRAP was suggested to provide signaling specificity for different TLRs. Rac1, a small GTP-binding protein that is activated upon LPS stimulation of macrophages, activates phosphatidylinositol 3-kinase and Akt and leads to NF-kappaB activation. The roles of Rac1 and TIRAP in LPS activation of HIV replication is not known. In the present study we show that LPS stimulation of human microvessel endothelial cells leads to Rac1 activation. Constitutively active Rac1 (Rac1V12) simulated the effect of LPS to activate HIV-LTR, whereas the expression of dominant negative Rac1 (Rac1N17) partially blocked LPS-induced HIV-LTR trans-activation. Rac1V12-induced HIV-LTR activation was independent of myeloid differentiation adapter protein, and dominant negative TIRAP blocked Rac1V12-induced HIV-LTR trans-activation. In this study we show for the first time that activation of Rac1 leads to HIV-LTR trans-activation, and this is mediated through TIRAP. Together these results underscore the importance of Rac1 and TIRAP in TLR4 activation of HIV replication and help delineate the signaling pathways induced by TLRs to mediate microbial Ag-induced HIV replication and HIV pathogenesis.

P2X7 nucleotide receptor: Modulation of LPS‐induced macrophage signaling and mediator production

AbstractDuring infection or inflammation, high concentrations of extracellular nucleotides are released into the inflammatory microenvironment, supplying a source of ligand for purinergic receptors that are present on many immune cell types. The P2X7 receptor, a member of the P2X purinergic receptor family of ATP‐gated ion channels, is thought to play an important role in monocyte/macrophage activation. One factor that can powerfully activate macrophages is bacterial lipopolysaccharide (endotoxin, LPS) and although the mechanisms involved in this process are not well understood, it is clear that LPS activation of macrophages is central to the development of septic shock in response to Gram‐negative bacteria. Several lines of evidence have demonstrated strong modulatory effects of adenine nucleotides on the events associated with LPS stimulation of macrophages. Further, because the signal transduction cascades initiated in macrophages upon LPS exposure are similar to those resulting from P2X7 receptor stimulation, and because antagonism of the P2X7 receptor can attenuate LPS‐stimulated signaling events and mediator release, the P2X7 receptor has been implicated in the control of macrophage responses to LPS. In addition, our laboratory has identified a consensus LPS‐binding motif at the extreme carboxyl terminus of the P2X7 receptor, further supporting the potential for a direct interaction between LPS and this purinergic receptor. In this review, we discuss potential regulatory domains and structural features of the P2X7 receptor and outline some of the signal transduction pathways activated by P2X7 receptor agonists. Moreover, we present evidence supporting a critical role for the P2X7 receptor in modulating or mediating some of the biological effects of LPS in macrophages. Drug Dev. Res. 53:91–104, 2001. © 2001 Wiley‐Liss, Inc.

Inhibition of Apoptosis by the BEACH Domain and WD Repeats of Gene lba that has Key Features of Both Protein Kinase A Anchor and chs1/beige Genes.

INTRODUCTION. The Chediak-Higashi Syndrome patients suffer from a systematic immune deficiency including defects in T cell cytotoxicity, killing by NK cells, defective bactericidal activity and chemotaxis by granulocytes and monocytes. All cells in beige mice and CHS patients bear giant vesicles that cluster around the nucleus. The beige and chs1 genes have been cloned recently (1). However, despite these very provocative findings there still remains no direct evidence that CHS1 proteins associate with intracellular vesicles and thus a molecular explanation for defective vesicle trafficking and the protein missorting in these diseases is still sought. We have recently identified a novel beige-like gene in humans and mice that is induced by LPS stimulation in macrophages and B cells. This protein also contains a potential protein kinase A binding site and we thus designate it lba, LPS-responsive, beigelike and anchor gene. METHOD. The lba gene was isolated and cloned by a gene-trapping strategy (2, 3) and 5'and 3'- RACE. The BEACH domain and WD repeats were cloned together or separately into a pEGFP-N vector. RAW 264.7 cells were stably transfected. Apoptosis was induced by 0.5 µM staurosporine for 4, 8, 17 hr and assayed by Western blotting of whole cell lysates for the status of PARP (poly-ADP ribose polymerase) and CASPASE 3. RESULTS. The novel gene lba has an orthologue in flies in addition to in mice and humans. It has key features of both chs1/beige and A kinase anchor genes. Expression of lba is induced following LPS stimulation of B cells and macrophages. The lba is expressed in many other tissues in the body and has three distinct mRNA isoforms that are differentially expressed in various tissues. Confocal and immuno-electronic microscopy indicate this protein associate with the trans-Golgi complex, lysosomes, endoplasmic reticulum and plasma membrane. The BEACH domain and WD repeats of lba gene can prevent apoptosis induced by staurosporine (Fig. 1). The cleavage of caspase 3 and PARP, two apoptotic markers, are inhibited by LBA fusion protein. The subcellular localization of the fusion proteins is changed by LPS stimulation and staurosporine induction.