Myeloid Differentiation Primary Response Gene Research Articles

The innate immune system in Parkinson′s disease: a novel target promoting endogenous neuroregeneration

Parkinson's disease (PD) is a slowly progressing neurodegenerative disorder clinically defined by key symptoms like bradykinesia, rigidity, resting tremor, and postural instability. Reduction of dopaminergic terminals in the caudate nucleus and putamen, i.e., the striatum, accompanied by decreased striatal levels of dopamine, and most likely consecutive degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNpc) are tightly linked to the onset of the motor symptoms. With the etiology of PD largely not yet well defined, there is accumulating evidence for increased immune activation in PD. The majority of studies favor a detrimental effect of microglia, the resident innate immune cells of the brain, on PD pathogenesis. In the presence of alpha-synuclein, inflammatory responses elicited by microglia may be initiated by pattern recognition receptors, e.g., toll-like receptors (TLR) leading to the distinct activation of signal transduction pathways. For instance, TLRs couple to signal adaptor proteins like myeloid differentiation primary response gene 88 (MyD88) and TIR-domain-containing adapter-inducing interferon-β (TRIF), resulting ultimately in the activation of multiple, signal-dependent transcription factors including members of the nuclear factor κB (NF-κB) or AP-1 families. This leads to an enhanced production of pro-inflammatory cytokines like tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) or IL-6 (Figure 1). Indeed, increased levels of pro-inflammatory mediators have been detected within the SNpc and striatum supporting the existence of an underlying neuroinflammatory signature in PD (see review by Hirsch and Hunot, 2009). As a consequence, these inflammatory mediators may directly or via astrocyte activation perturb neuronal homeostasis (Saijo et al., 2009). However, the precise temporal and spatial sequence of this immunogenic response during the course of PD is not defined yet.

Acetylbritannilactone Modulates MicroRNA-155-Mediated Inflammatory Response in Ischemic Cerebral Tissues.

Inflammatory responses play a critical role in ischemic brain injury. MicroRNA-155 (miR-155) induces the expression of inflammatory cytokines, and acetylbritannilactone (ABL) exerts potent antiinflammatory actions by inhibiting expression of inflammation-related genes. However, the functions of miR-155 and the actual relationship between ABL and miR-155 in ischemia-induced cerebral inflammation remain unclear. In this study, cerebral ischemia of wild-type (WT) and miR-155(-/-) mice was induced by permanent middle cerebral artery occlusion (MCAO). pAd-miR-155 was injected into the lateral cerebral ventricle 24 h before MCAO to induce miR-155 overexpression. MCAO mice and oxygen-glucose deprivation (OGD)-treated BV2 cells were used to examine the effects of ABL and miR-155 overexpression or deletion on the expression of proinflammatory cytokines. We demonstrated that ABL treatment significantly reduced neurological deficits and cerebral infarct volume by inhibiting tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) expression in ischemic cerebral tissue and OGD-treated BV2 cells. Mechanistic studies suggested that the observed decrease in TNF-α and IL-1β expression was attributable to the ABL-induced suppression of the expression of nuclear factor-kappa B (NF-κB) and Toll-like receptor 4 (TLR4). We further found that miR-155 promoted TNF-α and IL-1β expression by upregulating TLR4 and downregulating the expression of suppressor of cytokine signaling 1 (SOCS1) and myeloid differentiation primary response gene 88 (MyD88), while ABL exerted an inhibitory effect on miR-155-mediated gene expression. In conclusion, miR-155 mediates inflammatory responses in ischemic cerebral tissue by modulating TLR4/MyD88 and SOCS1 expression, and ABL exerts its antiinflammatory action by suppressing miR-155 expression, suggesting a novel miR-155-based therapy for ischemic stroke.

Role of Toll-like receptor/MYD88 signaling in neurodegenerative diseases.

Toll-like receptors (TLRs) are important innate immune proteins, and the activation of the TLRs results in the activation of intracellular signaling pathways, leading to the expression of proinflammatory cytokines that are essential to the identification and clearance of invading pathogens. TLR signaling occurs through adaptor proteins, most commonly myeloid differentiation primary response gene 88 (MyD88). It is now known that immune surveillance and inflammatory responses occur in neurodegenerative diseases and TLR/MYD88 signaling plays a critical role in these diseases. The included studies suggest a contribution for this signaling to the pathophysiology of Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, multiple system atrophy, and related disorders. In this review, a discussion of the recent findings in this field is presented.

MYD88 (L265P) Mutation Confers Very Poor Response and Outcome after Second-Line Therapy in Patients with Diffuse Large B-Cell Lymphoma (DLBCL)

BACKGROUNDSomatic mutations in the myeloid differentiation primary response gene 88 (MYD88) have been described in B-cell lymphomas, including DLBCL. Our group has confirmed the remarkable site-specific occurrence of MYD88 mutations at some immune-privileged locations and, in addition, has described that DLBCL patients are at high risk of progression and death after first-line treatment, with independence of other well-known clinical prognostic factors (Leukemia 2014).AIMSTo further analyze the clinical responsiveness and outcome after second-line treatment in DLBCL patients carrying MYD88 L265P.METHODSA series of 175 patients with DLBCL diagnosed at our institution between 2000 and 2013 were included. Inclusion criteria were: full clinical data available, treatment with remission intention, enough material for DNA extraction and absence of HIV infection. The presence of MYD88 L265P mutation was assessed by allele-specific oligonucleotides (ASO)-PCR in DNA samples extracted from FFPE tissue.RESULTSIn 129 patients (74%) treatment consisted on rituximab plus CHOP or CHOP-like schedules, while the remaining cases received other treatments depending on their clinical requirements. With a median follow-up time of 57 months, progression free survival (PFS) at 5 years after first-line treatment was 57%. Twelve of these patients (9.3%) had MYD88 L265P mutation, and these patients had a significantly worse PFS that patients who did not (18% vs 59%, p=0.018). Overall survival (OS) at 5 years after first line treatment was 65% (17% vs 72%, with vs without MYD88 mutation, respectively; p=0.001).We further analyzed the outcome after second-line treatment in 32 cases: 6 patients had refractory disease (0 with MYD88 mutation) and 26 patients were in first relapse. Three out of 5 patients carrying MYD88 mutation and 25 out of 27 patients without MYD88 mutation were treated with chemotherapy. Only one out of 5 cases (20%) with MYD88 mutation responded to second-line therapy whereas response was observed in 16 out of 27 cases (59%) without MYD88 mutation(p=0.106). PFS at 4 years from starting second-line therapy was 0 % in cases with MYD88 DLBCL and 34% in those without MYD88 mutation (p=0.092). Moreover, OS at 4 years from starting second-line therapy was 0 % in cases with MYD88 DLBCL and 37% in those without MYD88 mutation (p=0.019)(Figure 1).CONCLUSIONOur study shows that MYD88 L265P in DLBCL patients is not only associated to worse respond after first -line therapy, but also to a very poor response to second-line therapy, and consequently to a dismal outcome. New treatments are urgently needed for these patients.Acknowledgments: This study was supported in part by 2014SGR567 [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Egr-1, a Stress Response Transcription Factor and Myeloid Differentiation Primary Response Gene, Behaves As Tumor Suppressor in CML

The transcription factor early growth response 1 (Egr-1) gene was identified as a macrophage differentiation primary response gene, shown to be essential for and to restrict differentiation along the macrophage lineage. There’s evidence consistent with Egr-1 behaving as a tumor suppressor of leukemia, both in vivo and in vitro, including (1) loss of Egr-1 associated with therapy derived MDS and AML; (2) deregulated Egr-1 overriding blocks in myeloid differentiation, and (3) haplo-insufficiency of Egr-1 in mice leading to increased development of myeloid disorders following treatment with the potent DNA alkylating agent, N- ethyl-nitrosourea (ENU). BCR-ABL driven leukemia (Chronic Myelogenous Leukemia [CML]) was chosen as a model system to investigate the role of Egr-1 as a tumor suppressor for leukemia. CML is a disease resulting from the neoplastic transformation of hematopoietic stem cells (HSC) with the BCR-ABL oncogene. To assess the effect of Egr-1 on BCR-ABL driven leukemia, lethally irradiated syngeneic wild type mice were reconstituted with bone marrow (BM) from either wild type or Egr-1 null mice transduced with a 210-kD BCR-ABL-expressing MSCV-GFP retrovirus (bone marrow transplantation {BMT}). It was observed that loss of Egr-1 accelerated the development of BCR-ABL driven leukemia in recipient mice. Analysis of hematopoietic organs is consistent with loss of Egr-1 accelerating development of CML but not altering the nature of the disease. Cells expressing BCR-ABL that are null for Egr-1 exhibit increased survival and proliferation, which can account for some of the above observations. Substantial evidence is available that loss of Egr1 increases the leukemia initiating cell population, which can account for the more rapid development of disease. An increased population of lineage negative BM cells was observed in Egr-1-/- BCR-ABL recipient mice compared to animals transplanted with WT BCR-ABL BM. Serial BMT has shown that Egr-1-/- BCR-ABL BM has an increased leukemic burden compared to the WT counterpart. Furthermore, data from serial colony transfer experiments confirmed that loss of Egr-1 increases leukemia initiating cells. Preliminary studies on the effect of gain of Egr-1 function are consistent with the tumor suppressor behavior of Egr-1. These data as well as analysis of human CML samples will be presented. Further investigation could result in novel targets for diagnosis, prognosis, and targeted therapeutics, including strategies for activating Egr-1 expression that can be used to treat CML, as well as other leukemic diseases. Disclosures No relevant conflicts of interest to declare.

MYD88 L265P Mutation Detection: Analysis of Flow Cytometry Sorted Plasma and Lymphoid Cell Clones Improves Sensitivity and Specificity for WM/LPL Diagnosis

Background:Waldenström’s macroglobulinemia (WM) and lymphoplasmacytic lymphoma (LPL) are lymphoproliferative disorders with bone marrow infiltration by clonal lymphoplasmacytic cells (Treon et al., 2003, 2005). The somatic point mutation L265P in the myeloid differentiation primary response gene 88 (MYD88) has been reported in more than 90% of WM patients (Treon et al., 2012). Therefore MYD88 mutation analysis has been implemented in clinical practice to support the diagnosis of LPL/WM.After the implementation of MYD88 L265P assays with increased detection sensitivity, a substantial portion of patients with IgM monoclonal gammopathy of undetermined significance (IgM-MGUS) was also reported MYD88 L265P positive. Splenic marginal zone lymphoma (SMZL), B-cell chronic lymphoproliferative disorders (B-CLPD) and diffused large B-cell lymphoma (DLBCL) have been found positive with much lower incidence rates (Varettoni et al., 2013; Xu et al., 2013).Hence the remaining need to differentiate WM/LPL from other lymphoproliferative disorders with co-occurring plasma cells with high confidence.Patients and Methods:In this study, flow cytometric cell sorting was utilized to isolate clonal plasma and B lymphoid cell fractions as previously described (Zehentner et al., 2011). 69 patient specimen fractions with a clinical history of WM /LPL, multiple myeloma, CLL and lymphoma were analyzed for MYD88 L265P mutation using Sanger sequencing. Furthermore, the Biomed-2 primer sets for the immunoglobulin heavy (IgH) and/or the immunoglobulin kappa light chain region (IgK) were used to compare B cell clonality profiles in the lymphoid versus plasma cell compartments.Results:MYD88 L265P mutation was detected in all specimens with confirmed Waldenström’s macroglobulinemia (17/17, 100%). Of these 16/17 (94%) revealed MYD88 L265P as well as identical monoclonality profile by gene rearrangement analysis in both the plasma and the B lymphoid cell collections. In 47% (8/17), the mutation was only detected in the plasma and B cell fractions, but not in the whole bone marrow specimens.21 patient specimens with a known clinical history of LPL and co-occurring clonal plasma cells were tested. 9 of 21 (43%) were categorized with identical B cell clonality profile when comparing plasma and B lymphoid cells; whereas 12 (57%) had unrelated clonality profiles. 7 of the 9 (78%) specimens in the identical clonality group tested positive for MYD88 L265P in both the plasma and B lymphoid clone. None of the unrelated clonality group specimens carried the mutation in both cell fractions; for 7/12 (58%) MYD88 L265P was found in either the plasma (2) or the B-cell fraction (5) whereas 5/12 (42%) tested negative.11 bone marrow aspirate specimen with known presence of lymphoma (including splenic marginal zone (SMZL), mantle cell and marginal zone) were analyzed. 10/11 (90%) tested negative for MYD88 L265P, with the exception of one SMZL specimen. Furthermore, 12 known myeloma, 5 CLL and 3 healthy donor specimens were analyzed, all tested negative.Conclusions:In this study, we developed and tested a novel approach to assess MYD88 L265P mutation status in order to assist WM/LPL diagnosis. Flow cytometric cell sorting for clonal plasma and B cell populations was combined with molecular analysis. Subsequently, MYD88 L265P mutation as well as B-cell clonality profile was compared in both cell fractions.Our study postulates a significant improvement in sensitivity and most importantly specificity when applying MYD88 L265P mutation status in combination with cell sorting for WM/LPL diagnostic decisions. 47% WM patients (8/17) and 44% LPL patients (4/9) were positive for the MYD88 mutation exclusively in both flow cytometry sorted cell fractions but not in whole bone marrow specimens. 94% (16/17) WM as well as 78% (7/9) LPL specimens with identical plasma and B-cell clonality profile revealed the presence of the MYD88 L265P mutation in both the plasma cell and the B lymphoid cell clones. Whereas LPL specimens with unrelated clonality profile of the plasma and lymphoid cell fractions as well as other control specimens (lymphoma, myeloma, CLL and healthy) either tested negative or positive only in one of the sorted cell fractions.We therefore conclude that confirming the presence of MYD88 L265P in both B-lymphoid and plasma cell fraction is an important prerequisite to distinguish LPL/WM from related disorders with high confidence. DisclosuresWang:HematoLogics Inc.: Employment. Fritschle:HematoLogics Inc.: Employment. Bennington:HematoLogics Inc.: Employment. Burnworth:HematoLogics Inc.: Employment. Bennington:HematoLogics Inc.: Employment. Wentzel:HematoLogics Inc.: Employment. Verkamp:HematoLogics Inc.: Employment. Nguyen:HematoLogics Inc.: Employment. Ghirardelli:HematoLogics Inc.: Employment. Broderson:HematoLogics Inc.: Employment. Wells:HematoLogics Inc.: Employment, Equity Ownership. Loken:HematoLogics Inc.: Employment, Equity Ownership. Zehentner:HematoLogics Inc.: Employment, Equity Ownership.

TLR-4 signalling pathway: MyD88 independent pathway up-regulation in chicken breeds upon LPS treatment.

Toll-like receptors (TLRs) that sense the microbial pathogens are important components of host immune system. TLRs play key roles in the innate defence mechanism against pathogens, in the development of adaptive immunity, and are possibly the major determinants of the susceptibility to infections. To study the resistance pattern in different breeds of chicken, a comprehensive understanding of TLR4 signalling pathways is required. We investigated the TLR-4 pathway regulated gene expressions in PBMCs of chicken breeds of Broiler (Cobb), Aseel, Dahlem Red and Ghagus upon LPS treatment using Quantitative RT-PCR approach. Several genes were found to be up regulated in both TLR-induced MyD88-dependent and MyD88-independent pathways. These genes include TLR4 (Toll-like receptor 4), MyD88 (Myeloid differentiation primary response gene 88), TRAF6 (TNF receptor associated factor 6), TRIF (TIR domain containing adapter inducing interferon beta), the transcription factors NFkB (Nuclear factor kappa B), IRF7 (Interferon regulatory factor 7) and IFN β (Interferon beta). We have also studied inflammatory cytokines such as IL2, IL6, IL8, IL1 β and TNF α to further understand the downstream signalling of TLR4 pathway. These results showed that higher expression of TLR signalling activation via both MyD88-dependent and TRIF-dependent pathways are more beneficial to chicken mononuclear cells mediated innate immunity. We observed TRIF dependent pathway in Aseel and Ghagus breeds. Our results are in concurrent with general observation that Aseel breed is comparatively more resistant, Ghagus and broilers are moderately resistant and Dahlem Red is comparatively more susceptible to bacterial infections.

β-caryophyllene protects against D-galactosamine and lipopolysaccharide-induced hepatic failure

β-caryophyllene (4,11,11-trimethyl-8-methylene-bicyclo[7.2.0]undec-4-ene) is a natural bicyclic sesquiterpene isolated from Agastache rugosa (Fisch. et Meyer) O. Kuntze (Labiatae) has been shown to exhibit antioxidant and anti-inflammatory properties. Fulminant hepatic failure (FHF) is a dramatic clinical syndrome characterized by massive inflammation and hepatocellular death. Inflammasome plays an important role in the regulation of cellular redox balance, apoptosis, and inflammation in the FHF. In this study, we examined the hepatoprotective mechanisms of β-caryophyllene against D-galactosamine (GalN) and lipopolysaccharide (LPS)-induced hepatic failure. Mice were given an intraperitoneal injection of β-caryophyllene (50, 100 and 200 mg/kg) or vehicle alone (10% Tween 80 saline) 1h before GalN (800 mg/kg)/LPS (40 µg/kg), and sacrificed at 1 and 6h after GalN/LPS injection. GalN/LPS markedly increased mortality and serum aminotransferase activity, which were attenuated by β-caryophyllene. GalN/LPS increased serum tumor necrosis factor (TNF)-α, interleukin (IL)-1β and IL-6 levels, and these increases were attenuated by β-caryophyllene. GalN/LPS significantly increased protein expression of toll-like receptor (TLR) 4, myeloid differentiation primary response gene 88 and TNF receptor-associated factor 6, nuclear protein expression of nuclear factor kB, and these increases were attenuated by β-caryophyllene. GalN/LPS activated NLRP3 (NACHT, LRR, and pyrin domain-containing protein 3) inflammasome as indicated by increased mature IL-1β, caspase-1, apoptosis-associated speck-like protein containing a CARD, and NLRP3 protein expressions. This activation was attenuated by β-caryophyllene. Our finding suggests that β-caryophyllene protects against GalN/LPS-induced liver injury through suppression of TLR4 signaling and inflammasome pathway.

Intestinal alkaline phosphatase deficiency leads to lipopolysaccharide desensitization and faster weight gain.

Animals develop in the presence of complex microbial communities, and early host responses to these microbes can influence key aspects of development, such as maturation of the immune system, in ways that impact adult physiology. We previously showed that the zebrafish intestinal alkaline phosphatase (ALPI) gene alpi.1 was induced by Gram-negative bacterium-derived lipopolysaccharide (LPS), a process dependent on myeloid differentiation primary response gene 88 (MYD88), and functioned to detoxify LPS and prevent excessive host inflammatory responses to commensal microbiota in the newly colonized intestine. In the present study, we examined whether the regulation and function of ALPI were conserved in mammals. We found that among the mouse ALPI genes, Akp3 was specifically upregulated by the microbiota, but through a mechanism independent of LPS or MYD88. We showed that disruption of Akp3 did not significantly affect intestinal inflammatory responses to commensal microbiota or animal susceptibility to Yersinia pseudotuberculosis infection. However, we found that Akp3(-/-) mice acquired LPS tolerance during postweaning development, suggesting that Akp3 plays an important role in immune education. Finally, we demonstrated that inhibiting LPS sensing with a mutation in CD14 abrogated the accelerated weight gain in Akp3(-/-) mice receiving a high-fat diet, suggesting that the weight gain is caused by excessive LPS in Akp3(-/-) mice.

Protective effects of lupeol against D-galactosamine and lipopolysaccharide-induced fulminant hepatic failure in mice.

This study examined the hepatoprotective effects of lupeol (1, a major active triterpenoid isolated from Adenophora triphylla var. japonica) against d-galactosamine (GalN) and lipopolysaccharide (LPS)-induced fulminant hepatic failure. Mice were orally administered 1 (25, 50, and 100 mg/kg; dissolved in olive oil) 1 h before GalN (800 mg/kg)/LPS (40 μg/kg) treatment. Treatment with GalN/LPS resulted in increased levels of serum alanine aminotransferase, tumor necrosis factor (TNF)-α, and interleukin (IL)-6, as well as increased mortality, all of which were attenuated by treatment with 1. In addition, levels of toll-like receptor (TLR)4, myeloid differentiation primary response gene 88, TIR-domain-containing adapter-inducing interferon-β (TRIF), IL-1 receptor-associated kinase (IRAK)-1, and TNF receptor associated factor 6 protein expression were increased by GalN/LPS. These increases, except TRIF, were attenuated by 1. Interestingly, 1 augmented GalN/LPS-mediated increases in the protein expression of IRAK-M, a negative regulator of TLR signaling. Following GalN/LPS treatment, nuclear translocation of nuclear factor-κB and the levels of TNF-α and IL-6 mRNA expression increased, which were attenuated by 1. Together, the present findings suggest that lupeol (1) ameliorates GalN/LPS-induced liver injury, which may be due to inhibition of IRAK-mediated TLR inflammatory signaling.

The role of Toll-like Receptors in innate immunity and infectious diseases of teleost

The immune defence mechanism depends mainly on germ-line encoded pattern-recognition receptors (PRRs). These PRRs respond to many exogenous pathogens and/or endogenous serious signals, by recognizing some highly conserved structures such as pathogen-associated molecular patterns (PAMPs) and danger/damage associated molecular patterns (DAMPs). Till date, the most studied PRRs are Toll-like receptors (TLRs). Upon activation of TLRs, there is production of inflammatory cytokines and type I interferons (IFNs) via myeloid differentiation primary response gene 88 (MyD88)-dependent or -independent signalling, respectively, modulating innate and adaptive immunity, as well as inflammatory responses. In fish species studied to date, there are more than 17 TLRs that are identified with some showing homology to mammals, and some are unique for teleost. In the present review, more light are to be shed on the classification, structure and specific ligands of TLRs, with focuses on their signal pathways and different biological activities. Studies of TLRs and their role in the innate immune will potentially have implications for the prevention and treatment of fish diseases.

Role of Kupffer cells in ischemic injury in alcoholic fatty liver

BackgroundThis study was designed to evaluate the role of Kupffer cells (KCs) in hepatic drug metabolizing dysfunction after hepatic ischemia–reperfusion (IR) in alcoholic fatty liver. Materials and methodsRats were fed the Lieber-DeCarli diet for 5 wk to develop alcoholic fatty liver, then were subjected to 90 min of hepatic ischemia and 5 h of reperfusion. For ablation of KCs, rats were pretreated with gadolinium chloride (GdCl3) 48 and 24 h before the IR procedure. ResultsAfter the IR procedure, ethanol diet (ED)-fed rats had higher serum aminotransferase activity compared with the control diet-fed rats. These changes were attenuated by GdCl3. The ED-fed rats exhibited increased hepatic microsomal total cytochrome P450 (CYP) content and nicotinamide adenine dinucleotide phosphate-CYP reductase and CYP1A1, 1A2, 2B1, and 2E1 isozyme activity. After hepatic IR, these increases were reduced to lower levels than observed in the sham group, except CYP2E1 activity. Increases in CYP2E1 activity and its expression were augmented after hepatic IR in ED-fed animals, but were attenuated by GdCl3. Finally, toll-like receptor 4 and myeloid differentiation primary response gene 88 protein expression, nuclear translocation of nuclear factor-κB and activator protein 1, and levels of proinflammatory mediators were further increased in ED-fed animals compared with control diet-fed animals after IR. These increases were attenuated by GdCl3. ConclusionsWe suggest that KCs contribute to hepatic drug metabolizing dysfunction during hepatic IR in alcoholic fatty liver via the toll-like receptors 4-mediated inflammatory response.

A novel theraputic target for intracerebral hemorrhage: interfering with the TLR2/TLR4 heterodimerization

The investigation of the upstream events that initiate inflammatory responses has a very important role in the illumination of the mechanisms of intracerebral hemorrhage(ICH)-induced secondary inflammatory brain injury. The formation of novel TLR2/TLR4 heterodimer after ICH detected by the western blot, immunoprecipitation and immunofluorescence technologies has strongly contributed to such advance. Using the in vivo and in vitro ICH model, we revealed the assemble of TLR2/TLR4 heterodimer mediated inflammatory injury only can be triggered by the hemoglobin(Hb) via the myeloid differentiation primary response gene 88(MyD88), but not toll/IR-1(TIR)-domain-containing adaptor protein inducing interferon-beta(TRIF). And the mutation site at the MyD88 Arg196 abolished the TLR2/TLR4 heterodimerization. Together, our findings not only further explain the upstream events that initiate inflammatory responses following ICH, but also provide a novel target-interfering with the assembly of TLR2/TLR4 heterodimer-for developing an effective treatment of ICH.

Hematopoietic but not endothelial cell MyD88 contributes to host defense during gram-negative pneumonia derived sepsis.

Klebsiella pneumoniae is an important cause of sepsis. The common Toll-like receptor adapter myeloid differentiation primary response gene (MyD)88 is crucial for host defense against Klebsiella. Here we investigated the role of MyD88 in myeloid and endothelial cells during Klebsiella pneumosepsis. Mice deficient for MyD88 in myeloid (LysM-Myd88−/−) and myeloid plus endothelial (Tie2-Myd88−/−) cells showed enhanced lethality and bacterial growth. Tie2-Myd88−/− mice reconstituted with control bone marrow, representing mice with a selective MyD88 deficiency in endothelial cells, showed an unremarkable antibacterial defense. Myeloid or endothelial cell MyD88 deficiency did not impact on lung pathology or distant organ injury during late stage sepsis, while LysM-Myd88−/− mice demonstrated a strongly attenuated inflammatory response in the airways early after infection. These data suggest that myeloid but not endothelial MyD88 is important for host defense during gram-negative pneumonia derived sepsis.

Platelet-activating factor: a role in preterm delivery and an essential interaction with Toll-like receptor signaling in mice.

Platelet-activating factor (PAF), a potent phospholipid activator of inflammation that signals through its cognate receptor (platelet-activating factor receptor, PTAFR), has been shown to induce preterm delivery in mice. Toll-like receptors (TLRs) are transmembrane receptors that mediate innate immunity. We have shown previously that Escherichia coli-induced preterm delivery in mice requires TLR signaling via the adaptor protein myeloid differentiation primary response gene 88 (MyD88), but not an alternative adaptor, Toll/IL-1 receptor domain-containing adapter protein-inducing interferon-beta (TRIF). In the present work, we analyzed the role of endogenously produced PAF in labor using mice lacking (knockout [KO]) PAF acetylhydrolase (PAF-AH; the key degrading enzyme for PAF). PAF-AH KO mice are more susceptible to E. coli-induced preterm delivery and inflammation than controls. In peritoneal macrophages, the PTAFR agonist carbamyl PAF induces production of inflammatory markers previously demonstrated to be upregulated during bacterially induced labor, including: inducible nitric oxide synthase (Nos2), the chemokine Ccl5 (RANTES), tumor necrosis factor (Tnf), and level of their end-products (NO, CCL5, TNF) in a process dependent upon both IkappaB kinase and calcium/calmodulin-dependent protein kinase II. Interestingly, this induced expression was completely eliminated not only in macrophages deficient in PTAFR, but also in those lacking either TLR4, MyD88, or TRIF. The dependence of PAF effects upon TLR pathways appears to be related to production of PTAFR itself: PAF-induced expression of Ptafr mRNA was eliminated completely in TLR4 KO and partially in MyD88 and TRIF KO macrophages. We conclude that PAF signaling plays an important role in bacterially induced preterm delivery. Furthermore, in addition to its cognate receptor, PAF signaling in peritoneal macrophages requires TLR4, MyD88, and TRIF.

Anti-metastatic immunotherapy based on mucosal administration of flagellin and immunomodulatory P10.

Current therapies against malignant melanoma generally fail to increase survival in most patients, and immunotherapy is a promising approach as it could reduce the dosage of toxic therapeutic drugs. In the present study, we show that an immunotherapeutic approach based on the use of the Toll-like receptor (TLR)-5 ligand flagellin (Salmonella Typhimurium FliCi) combined with the major histocompatibility complex class II-restricted P10 peptide, derived from the Paracoccidioides brasiliensis gp43 major surface protein, reduced the number of lung metastasis in a murine melanoma model. Compounds were administered intranasally into C57Bl/6 mice intravenously challenged with syngeneic B16F10-Nex2 melanoma cells, aiming at the local (pulmonary) immune response modulation. Along with a marked reduction in the number of lung nodules, a significant increase in survival was observed. The immunization regimen induced both local and systemic proinflammatory responses. Lung macrophages were polarized towards a M1 phenotype, lymph node cells, and splenocytes secreted higher interleukin-12p40 and interferon (IFN)-γ levels when re-stimulated with tumor antigens. The protective effect of the FliCi+P10 formulation required TLR-5, myeloid differentiation primary response gene 88 and IFN-γ expression, but caspase-1 knockout mice were only partially protected, suggesting that intracellular flagellin receptors are not involved with the anti-tumor effect. The immune therapy resulted in the activation of tumor-specific CD4(+) T lymphocytes, which conferred protection to metastatic melanoma growth after adoptive transfer. Taken together, our results report a new immunotherapeutic approach based on TLR-5 activation and IFN-γ production capable to control the metastatic growth of B16F10-Nex2 melanoma, being a promising alternative to be associated with chemotherapeutic drugs for an effective anti-tumor responses.

Normalization of the levels of inflammatory molecules in Mycobacterium smegmatis-infected U937 cells by fibrate pretreatment.

BackgroundTuberculosis (TB) is a respiratory tract disease caused by Mycobacterium tuberculosis infection. M. tuberculosis exploits immune privilege to grow and divide in pleural macrophages. Fibrates are associated with the immune response and control lipid metabolism through glycolysis with β-oxidation of fatty acids.ResultsIn this study, we investigated the effect of fibrate pretreatment on the immune response during M. smegmatis infection in U937 cells, a human leukemic monocyte lymphoma cell line. The protein expression of tumor necrosis factor α (TNF-α), an inflammatory marker, and myeloid differentiation primary response gene 88 (MyD88), a toll like receptor adaptor molecule, in the infected group increased at 1 and 6 h after M. smegmatis infection of U937 cells. Acetyl coenzyme A acetyl transferase-1 (ACAT-1), peroxisome proliferator-activated receptor-α (PPAR-α), TNF-α, and MyD88 decreased in U937 cells treated with fibrates at 12 and 24 h after treatment. More than a 24 h pretreatment with fibrate resulted in similar expression levels of ACAT-1 and PPAR-α between infected vehicle control and infected groups which were pretreated with fibrate for 24 h. However, upon exposure to M. smegmatis, the cellular expression of the TNF-α and MyD88 in the infected groups pretreated with fibrate for 24 h decreased significantly compared to that in the infected vehicle group.ConclusionThese results suggest that fibrate pretreatment normalized the levels of inflammatory molecules in Mycobacterium smegmatis-infected U937 cells. Further studies are needed to confirm the findings on pathophysiology and immune defense mechanism of U937 by fibrates during M. tuberculosis infection.

MUC1 Regulates Epithelial Inflammation and Apoptosis by PolyI:C through Inhibition of Toll/IL-1 Receptor-Domain–Containing Adapter-Inducing IFN-β (TRIF) Recruitment to Toll-like Receptor 3

MUC1/Muc1 (MUC1 in humans, Muc1 in animals) is a membrane-tethered mucin expressed by airway epithelial cells and plays an antiinflammatory role during airway bacterial infection. We previously demonstrated that MUC1/Muc1 is a negative regulator of Toll-like receptor (TLR) inflammatory signaling mediated through the myeloid differentiation primary response gene 88 (MyD88) adaptor protein. In the present study, we determined whether MUC1 regulates MyD88-independent TLR signaling mediated through the TLR3-Toll/IL-1 receptor-domain-containing adapter-inducing IFN-β (TRIF) pathway in response to poly(I:C). Compared with MUC1/Muc1-expressing controls, cells deficient in MUC1/Muc1 were more prone to poly(I:C)-induced apoptosis; had increased poly(I:C)-driven activation of caspase-3, caspase-8, IFN regulatory factor-3, and NF-κB; and displayed heightened IFN-β gene expression. MUC1 overexpression by these cells had the opposite effects. Reciprocal coimmunoprecipitation experiments established constitutive TLR3/MUC1-CT (cytoplasmic tail) protein interaction in human embryonic kidney (HEK)293T cells overexpressing the two proteins and in lung epithelial cells expressing the endogenous proteins, the latter of which was confirmed by immunofluorescence colocalization of TLR3 with MUC1-CT. Coimmunoprecipitation studies also revealed that MUC1 overexpression by HEK293T cells reduced poly(I:C)-induced TLR3/TRIF protein interaction. Finally, MUC1 overexpression had no effect on TRIF-dependent auto-activation of TLR3 signaling, suggesting that the site of action of the MUC1-CT in TLR3 signaling is not downstream of TRIF. These data indicate that MUC1-CT counter-regulates apoptotic and inflammatory responses of airway epithelial cell through constitutive association with TLR3, thereby inhibiting poly(I:C)-induced recruitment of TRIF to TLR3.

Endothelial cells translate pathogen signals into G-CSF–driven emergency granulopoiesis

AbstractSystemic bacterial infection induces a hematopoietic response program termed “emergency granulopoiesis” that is characterized by increased de novo bone marrow (BM) neutrophil production. How loss of local immune control and bacterial dissemination is sensed and subsequently translated into the switch from steady-state to emergency granulopoiesis is, however, unknown. Using tissue-specific myeloid differentiation primary response gene 88 (Myd88)-deficient mice and in vivo lipopolysaccharide (LPS) administration to model severe bacterial infection, we here show that endothelial cells (ECs) but not hematopoietic cells, hepatocytes, pericytes, or BM stromal cells, are essential cells for this process. Indeed, ECs from multiple tissues including BM express high levels of Tlr4 and Myd88 and are the primary source of granulocyte colony-stimulating factor (G-CSF), the key granulopoietic cytokine, after LPS challenge or infection with Escherichia coli. EC-intrinsic MYD88 signaling and subsequent G-CSF production by ECs is required for myeloid progenitor lineage skewing toward granulocyte-macrophage progenitors, increased colony-forming unit granulocyte activity in BM, and accelerated BM neutrophil generation after LPS stimulation. Thus, ECs catalyze the detection of systemic infection into demand-adapted granulopoiesis.

Anti-inflammatory activity of Odina wodier Roxb, an Indian folk remedy, through inhibition of toll-like receptor 4 signaling pathway.

Inflammation is part of self-limiting non-specific immune response, which occurs during bodily injury. In some disorders the inflammatory process becomes continuous, leading to the development of chronic inflammatory diseases including cardiovascular diseases, diabetes, cancer etc. Several Indian tribes used the bark of Odina wodier (OWB) for treating inflammatory disorders. Thus, we have evaluated the immunotherapeutic potential of OWB methanol extract and its major constituent chlorogenic acid (CA), using three popular in vivo antiinflammatory models: Carrageenan- and Dextran-induced paw edema, Cotton pellet granuloma, and Acetic acid-induced vascular permeability. To elucidate the possible anti-inflammatory mechanism of action we determine the level of major inflammatory mediators (NO, iNOS, COX-2-dependent prostaglandin E2 or PGE2), and pro-inflammatory cytokines (TNF-α, IL-1β, IL-6, and IL-12). Further, we determine the toll-like receptor 4 (TLR4), Myeloid differentiation primary response gene 88 (MyD88), c-Jun N-terminal kinases (JNK), nuclear factor kappa-B cells (NF-κB), and NF-kB inhibitor alpha (IK-Bα) by protein and mRNA expression, and Western blot analysis in drug treated LPS-induced murine macrophage model. Moreover, we determined the acute and sub-acute toxicity of OWB extract in BALB/c mice. Our study demonstrated a significant anti-inflammatory activity of OWB extract and CA along with the inhibition of TNF-α, IL-1β, IL-6 and IL-12 expressions. Further, the expression of TLR4, NF-κBp65, MyD88, iNOS and COX-2 molecules were reduced in drug-treated groups, but not in the LPS-stimulated untreated or control groups, Thus, our results collectively indicated that the OWB extract and CA can efficiently inhibit inflammation through the down regulation of TLR4/MyD88/NF-kB signaling pathway.