Macrophage-activating Lipopeptide-2 Research Articles

Lipoteichoic Acid Inhibits Lipopolysaccharide-Induced TLR4 Signaling by Forming an Inactive TLR4/MD-2 Complex Dimer.

LPS interacts with TLR4, which play important roles in host-against-pathogen immune responses, by binding to MD-2 and inducing an inflammatory response. In this study, to our knowledge, we found a novel function of lipoteichoic acid (LTA), a TLR2 ligand, that involves suppression of TLR4-mediated signaling independently of TLR2 under serum-free conditions. LTA inhibited NF-κB activation induced by LPS or a synthetic lipid A in a noncompetitive manner in human embryonic kidney 293 cells expressing CD14, TLR4, and MD-2. This inhibition was abrogated by addition of serum or albumin. LTAs from different bacterial sources also inhibited NF-κB activation, although LTA from Enterococcus hirae had essentially no TLR2-mediated NF-κB activation. The TLR2 ligands tripalmitoyl-Cys-Ser-Lys-Lys-Lys-Lys (Pam3CSK4) and macrophage-activating lipopeptide-2 (MALP-2) did not affect the TLR4-mediated NF-κB activation. In bone marrow-derived macrophages from TLR2-/- mice, LTA inhibited LPS-induced IκB-α phosphorylation and production of TNF, CXCL1/KC, RANTES, and IFN-β without affecting cell surface expression of TLR4. LTA did not suppress IL-1β-induced NF-κB activation mediated through signaling pathways shared with TLRs. LTAs including E. hirae LTA, but not LPS, induced association of TLR4/MD-2 complexes, which was suppressed by serum. LTA also increased association of MD-2, but not TLR4 molecules. These results demonstrate that, under serum-free conditions, LTA induces association of MD-2 molecules to promote formation of an inactive TLR4/MD-2 complex dimer that in turn prevents TLR4-mediated signaling. The presence of LTA that poorly induces TLR2-mediated activation but inhibits TLR4 signaling provides insight into the role of Gram-positive bacteria in suppressing inflammation induced by Gram-negative bacteria in organs such as the intestines where serum is absent.

Pushing the envelope: Immune mechanism and application landscape of macrophage-activating lipopeptide-2.

Mycoplasma fermentans can cause respiratory diseases, arthritis, genitourinary tract infections, and chronic fatigue syndrome and have been linked to the development of the human immunodeficiency virus. Because mycoplasma lacks a cell wall, its outer membrane lipoproteins are one of the main factors that induce inflammation in the organism and contribute to disease development. Macrophage-activating lipopeptide-2 (MALP-2) modulates the inflammatory response of monocytes/macrophages in a bidirectional fashion, indirectly enhances the cytotoxicity of NK cells, promotes oxidative bursts in neutrophils, upregulates surface markers on lymphocytes, enhances antigen presentation on dendritic cells and induces immune inflammatory responses in sebocytes and mesenchymal cells. MALP-2 is a promising vaccine adjuvant for this application. It also promotes vascular healing and regeneration, accelerates wound and bone healing, suppresses tumors and metastasis, and reduces lung infections and inflammation. MALP-2 has a simple structure, is easy to synthesize, and has promising prospects for clinical application. Therefore, this paper reviews the mechanisms of MALP-2 activation in immune cells, focusing on the application of MALP-2 in animals/humans to provide a basis for the study of pathogenesis in Mycoplasma fermentans and the translation of MALP-2 into clinical applications.

The Influence of Macrophage-Activating Lipopeptide-2 in Regard to Liver-Specific Changes Within a Murine Two-Hit Model

Trauma hemorrhage (TH) and subsequent sepsis are well known to frequently result in severe organ damage. Although macrophage-activating lipopeptide-2 (MALP-2) has been described to exert beneficial effects on organ damage, and further clinical course after both isolated trauma and sepsis, little is known about the impact of MALP-2 in a clinically realistic two-hit scenario of TH and subsequent sepsis. As the liver represents a key organ for the posttraumatic immune response and development of complications, the effects of MALP-2 on the posttraumatic hepatic immunologic response and tissue damage were investigated in a murine "two-hit" model. In C57BL/6 mice, blood pressure-controlled (35 ± 5mm Hg) TH was induced. Cecal ligation and puncture (CLP) was performed 48h after TH. Mice were divided into two control groups (control 1, TH and laparotomy without CLP; control 2, TH and CLP) and three experimental groups (TH + CLP) treated with MALP-2 at different timepoints (ETH, end of TH; ECLP, end of CLP; 6CLP, 6h after CLP). The observation time lasted for 168h after induction of TH. Kupffer cells (KC) were isolated and cultured, and MPO activity was analyzed. Cell culture supernatants were taken for cytokine analysis (TNF-α, IL-6, MCP-1, GM-CSF, IL-10). Histological analysis was performed using the Hepatic Injury Severity Scoring (HISS). Statistical evaluation was carried out using SPSS (version 24.0.0; IBM, Armonk, NY, USA). MPO activity of control 1 group was lowest compared with all the other groups (p < 0.01). MPO activity of control 2 group was significantly higher than that in all experimental groups (ETH (p < 0.01), ECLP (p < 0.01), and 6CLP (p = 0.03)). Within the experimental groups, MPO activity was significantly reduced in the ETH (p = 0.04) and the ECLP (p < 0.01) groups compared with the 6CLP group. Moreover, ETH was also associated with the most pronounced reduction of cytokine expression by KC (p < 0.05). HISS revealed the largest damage in the group control 2. TH and subsequent sepsis lead to a distinct immunologic reaction in the liver with an increase of cytokine expression of KC and pronounced infiltration of granulocytes with associated severe tissue damage. MALP application decreases the hepatic immune response and liver damage, with the most pronounced effects if applied at the end of TH.

Enhanced activation of blood neutrophils and monocytes in patients with Ethiopian localized cutaneous leishmaniasis in response to Leishmania aethiopica Neutrophil activation in Ethiopian cutaneous leishmaniasis

Recent studies suggest an essential role of the innate immune effector cells neutrophils and monocytes in protection or disease progression in the early course of Leishmania infection. In areas endemic for cutaneous leishmaniasis in Ethiopia most individuals are exposed to bites of infected sandflies. Still only a minor ratio of the inhabitants develops symptomatic disease. Neutrophils, followed by monocytes, are the first cells to be recruited to the site of Leishmania infection, the initial response of neutrophils to parasites appears to be crucial for the protective response and disease outcome. Our working hypothesis is that neutrophils and/or monocytes in localized cutaneous leishmaniasis (LCL) patients may have defects in function of innate immune cell that contribute to failure to parasite clearance that lead to establishment of infection.The response of cells in Ethiopian LCL patients and healthy controls to Leishmania aethiopica and to the Toll like receptor (TLR) agonists lipopolysaccharide (LPS) and macrophage activating lipopeptide-2 (MALP-2) was investigated by assessing the cell surface expression of CD62L (on neutrophil and monocyte) and CD66b (only on neutrophil), as well as reactive oxygen species (ROS) production by using whole blood-based assays in vitro. No impaired response of neutrophils and monocytes to the microbial constituents LPS and MALP-2 was observed. Neutrophils and monocytes from LCL patients responded stronger to Leishmania aethiopica in the applied whole blood assays than cells from healthy individuals. These experimental findings do not support the hypothesis regarding a possible dysfunction of neutrophils and monocytes in cutaneous leishmaniasis. On the contrary, these cells react stronger in LCL patients as compared to healthy controls. The differential response to L. aethiopica observed between LCL patients and healthy controls have the potential to serve as biomarker to develop FACS based diagnostic/ prognostic techniques for LCL.

The Influence of Macrophage-Activating Lipopeptide 2 (MALP-2) on Local and Systemic Inflammatory Response in a Murine Two-Hit Model of Hemorrhagic Shock and Subsequent Sepsis.

Pulmonary complications after severe trauma and sepsis remain to be the main cause for adverse outcome. MALP-2 has been described to exert beneficial effects on organ damage and the further course after isolated trauma and sepsis. However, the impact of MALP-2 on a clinically realistic two-hit scenario of trauma and subsequent sepsis remains unknown. We, therefore, investigated if the systemic inflammatory response and pulmonary immune response and damage are beneficially modulated by MALP-2 in a murine two-hit model. Blood pressure-controlled trauma-hemorrhage (TH) and cecal ligation and puncture (CLP) were induced in C57/BL6 mice. Mice were divided into 2 control groups (control 1: TH without CLP; control 2: TH and CLP) and 3 experimental groups treated with MALP-2 at different time points (ETH, end of TH; ECLP, end of CLP; and 6CLP 6 h after CLP). Survival rates were assessed over the observation period of 168 h after the induction of TH. Concentrations of plasma inflammatory cytokines and chemokines (TNF-α, IL-6, MIP-1α, IFN-γ, and IL-10) were assessed, and bacterial clearance of the lungs was determined. Furthermore, pulmonary MPO activity assay to evaluate the infiltration of polymorphonuclear neutrophils (PMN) and histological evaluation were performed. Survival rates were evaluated. Compared with control group 1, the level of TNF-α in the ECLP group showed a significant increase (ECLP, 2.27 pg./ml ± 1.39 vs. control 1: 0.16 pg./ml ± 0.11, p = 0.021). In contrast, levels of IFN-γ were significantly reduced in groups ETH and 6CLP compared with control group 1 (control 1: 8.92 pg./ml ± 4.38 vs. ETH: 1.77 pg./ml ± 4.34, p = 0.026 resp. vs. 6CLP: 1.83 pg./ml ± 4.49, p = 0.014). While systemic concentrations of inflammatory mediators were not affected by MALP-2 treatment, the lung tissue presented with significant alterations. Reduced MPO activity was lowest in group ECLP (ECLP 11,196.77 ± 547.81 vs. ETH 12,773.94 ± 1011.76; p = 0.023 resp. vs. 6CLP 13,155.19 ± 423.99, p = 0.016) in experimental groups. Also, histological damage after MALP-2 application was lowest in ECLP animals (ECLP 0.50 ± 0.08 vs. ETH 0.71 ± 0.05, p = 0.034 resp. vs. 6CLP 0.64 ± 0.08, p = 0.021). Furthermore, MALP-2 treatment was associated with a trend towards improved survival in the ECLP group (ECLP 83.3% vs. ETH 66.7 and 6CLP 58.3%, p > 0.05). Based on our results, MALP-2 might have beneficial effects on the clinical course after hemorrhage and sepsis by reducing pulmonary damage and PMN infiltration. This might also affect survival. According to our data, MALP-2 should be given at the earliest possible time point after the onset of sepsis. However, the optimal dosage and confirmation of our results in larger cohorts need to be the focus of further research.

Ainsliaea fragrans champ. Extract prevents cervicitis in BALB/c mice and regulates MyD88-NF-κB signaling pathway in MALP-2-stimulated RAW264.7 cells

Ethnopharmacological relevance Ainsliaea fragrans Champ. (A. fragrans) is used to treat infection of the lower genital tract in gynecology, such as cervicitis and pelvic inflammatory disease. This study analyzed the therapeutic efficiency of A. fragrans on cervicitis and the inhibition mechanism of AF-p2 in MALP-2-stimulated RAW264.7 cells. Materials and methods The anti- Ureaplasma urealyticum (Uu) activity of A. fragrans and AF-p2 were determined by antimicrobial susceptibility testing. The activity of A. fragrans extracts (AFext) was evaluated in female BALB/c mice with cervicitis induced by Uu. Furthermore, the therapeutic mechanism of AFext and AF-p2 on myeloid differentiation factor 88 (MyD88) pathway were studied in macrophage activating lipopeptide-2 (MALP-2) irritated RAW264.7 cells. Results AFext could suppress the proliferation of Uu in vitro, including the azithromycin resistant strains. Meanwhile, AFext prevented cervicitis caused by Uu infection in BALB/c mice. Moreover, both AFext and AF-p2 could significantly suppress the nitric oxide (NO) production as well as other proinflammatory cytokines (IL-1β,IL-6,TNF-α) in MALP-2 stimulated RAW264.7 cells. Moreover, AF-p2 also down-regulated iNOS, p65, Iκ-Bα, MyD88 and cyclooxygenase-2 (COX-2) levels in RAW264.7 cells. Conclusion This study indicated that AFext had a therapeutic effect in cervicitis induced by Uu infection. Furthermore, the lead compound AF-p2 showed an anti-infectious effect in MALP-2 irritated RAW264.7 cells through downregulating MyD88-NF-κB signaling pathway.

Macrophage-activating lipopeptide-2 and corticotropin-releasing hormone stimulate the inflammatory signalling in human sebocytes through activation of stearoyl-CoA desaturase and fatty acid desaturase 2.

The macrophage-activating lipopeptide-2 (MALP-2) activates cells carrying a functional Toll-like receptor (TLR)-2/6. Human sebocytes express functional TLR-2, TLR-4 and CD14. Upregulation of stearoyl-CoA desaturase (SCD) and fatty acid desaturase-2 (FADS2) expression induces pro-inflammatory sebaceous activity. On the other hand, corticotropin-releasing hormone (CRH) is likely to serve as an autocrine stress hormone in human sebocytes. In addition to its antiproliferative, lipogenetic and androgen-activating functions, CRH exhibits a pro-inflammatory action and its expression is upregulated in acne-involved sebaceous glands. Determination of the pro-inflammatory function of MALP-2 and CRH and clarification of the option that MALP-2 and/or CRH activity on human sebocytes might be mediated through SCD and/or FADS2. SZ95 sebocytes were treated with MALP-2, CRH and the SCD inhibitor/ligand FPCA. SCD, FADS2, TLR-2 mRNA and protein levels and IL-6 and IL-8 secretion were investigated. Intracellular CRH levels were assessed under treatment with CRH, MALP-2, linoleic acid and arachidonic acid. Phorbol 12-myristate 13-acetate and dexamethasone served as positive and negative controls, respectively. MALP-2 upregulated SCD, FADS2, TLR-2 mRNA and protein levels and IL-6 and IL-8 secretion from SZ95 sebocytes. Co-incubation of SZ95 sebocytes with MALP-2/FPCA did not affect the MALP-2-induced SCD mRNA upregulation but reduced FADS2 mRNA levels and inhibited IL-8 secretion. CRH induced an early, low-level SCD and FADS2 upregulation and TLR-2 and IL-8 secretion. High intracellular CRH concentrations could be detected early after CRH treatment and persisted up to 24h. MALP-2 stimulated intracellular CRH levels. MALP-2 stimulates the inflammatory signalling in human sebocytes through SCD and FADS2 activation. Inhibition of FADS2 mRNA levels and IL-8 secretion through MALP-2/FCPA co-incubation and diminution of fatty acid unsaturation might lead to a reduction of pro-inflammatory sebaceous lipids. CRH upregulates inflammatory signalling via the SCD/FADS2 pathway, and MALP-2 selectively enhances CRH levels in human sebocytes.

Long-chain acyl-CoA synthetase-1 mediates the palmitic acid-induced inflammatory response in human aortic endothelial cells.

Saturated fatty acid (SFA) induces proinflammatory response through a Toll-like receptor (TLR)-mediated mechanism, which is associated with cardiometabolic diseases such as obesity, insulin resistance, and endothelial dysfunction. Consistent with this notion, TLR2 or TLR4 knockout mice are protected from obesity-induced proinflammatory response and endothelial dysfunction. Although SFA causes endothelial dysfunction through TLR-mediated signaling pathways, the mechanisms underlying SFA-stimulated inflammatory response are not completely understood. To understand the proinflammatory response in vascular endothelial cells in high-lipid conditions, we compared the proinflammatory responses stimulated by palmitic acid (PA) and other canonical TLR agonists [lipopolysaccharide (LPS), Pam3-Cys-Ser-Lys4 (Pam3CSK4), or macrophage-activating lipopeptide-2)] in human aortic endothelial cells. The expression profiles of E-selectin and the signal transduction pathways stimulated by PA were distinct from those stimulated by canonical TLR agonists. Inhibition of long-chain acyl-CoA synthetases (ACSL) by a pharmacological inhibitor or knockdown of ACSL1 blunted the PA-stimulated, but not the LPS- or Pam3CSK4-stimulated proinflammatory responses. Furthermore, triacsin C restored the insulin-stimulated vasodilation, which was impaired by PA. From the results, we concluded that PA stimulates the proinflammatory response in the vascular endothelium through an ACSL1-mediated mechanism, which is distinct from LPS- or Pam3CSK4-stimulated responses. The results suggest that endothelial dysfunction caused by PA may require to undergo intracellular metabolism. This expands the understanding of the mechanisms by which TLRs mediate inflammatory responses in endothelial dysfunction and cardiovascular disease.

Vascularization Strategies for Porous Polyethylene Implants.

Porous polyethylene (pPE) is a frequently implanted biomaterial in craniofacial reconstructive surgery. Its rapid vascularization and tissue incorporation are major prerequisites to prevent complications, such as material infection, migration, and extrusion. To achieve this, several sophisticated strategies have been introduced and evaluated during the last 20 years. These include (i) the angiogenic stimulation of the host tissue with epidermal growth factor, basic fibroblast growth factor or macrophage-activating lipopeptide-2, (ii) material modifications, such as increase of surface roughness and incorporation of bioactive glass particles, (iii) surface coatings with growth factors, glycoproteins, acrylic acid, arginine/glycine/aspartic acid peptide as well as components of the plasminogen activation system and autologous clotted blood or serum, and (iv) the seeding with fibroblasts, chondrocytes, stem cells, or adipose-tissue-derived microvascular fragments. The majority of these approaches showed promising results in experimental studies and, thus, may be capable of improving the success rates after pPE implantation in future clinical practice.

Effects of Mycoplasmas on the Host Cell Signaling Pathways.

Mycoplasmas are the smallest free-living organisms. Reduced sizes of their genomes put constraints on the ability of these bacteria to live autonomously and make them highly dependent on the nutrients produced by host cells. Importantly, at the organism level, mycoplasmal infections may cause pathological changes to the host, including cancer and severe immunological reactions. At the molecular level, mycoplasmas often activate the NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) inflammatory response and concomitantly inhibit the p53-mediated response, which normally triggers the cell cycle and apoptosis. Thus, mycoplasmal infections may be considered as cancer-associated factors. At the same time, mycoplasmas through their membrane lipoproteins (LAMPs) along with lipoprotein derivatives (lipopeptide MALP-2, macrophage-activating lipopeptide-2) are able to modulate anti-inflammatory responses via nuclear translocation and activation of Nrf2 (the nuclear factor-E2-related anti-inflammatory transcription factor 2). Thus, interactions between mycoplasmas and host cells are multifaceted and depend on the cellular context. In this review, we summarize the current information on the role of mycoplasmas in affecting the host’s intracellular signaling mediated by the interactions between transcriptional factors p53, Nrf2, and NF-κB. A better understanding of the mechanisms underlying pathologic processes associated with reprogramming eukaryotic cells that arise during the mycoplasma-host cell interaction should facilitate the development of new therapeutic approaches to treat oncogenic and inflammatory processes.

HO-1 mediates the anti-inflammatory actions of Sulforaphane in monocytes stimulated with a mycoplasmal lipopeptide

Exposure to Mycoplasma pneumoniae leads to lung inflammation through a host defense pathway. Increasing evidence has indicated that the mycoplasma-derived membrane lipoprotein, or its analogue macrophage-activating lipopeptide-2 (MALP-2), excretes LPS as an immune system-stimulating substance and plays a crucial role in pathological injury during M. pneumoniae infection. It has been established that Sulforaphane confers anti-inflammatory properties. However, the underlying mechanism responsible for the inhibitory actions of Sulforaphane in the context of mycoplasmal pneumoniae are poorly understood. Here, we report that Sulforaphane is an inducer of heme oxygenase (HO)-1, a cytoprotective enzyme that catalyzes the degradation of heme through signaling pathways in human monocytes. Sulforaphane stimulated NF-E2-related factor 2 (Nrf2) translocation from the cytosol to the nucleus, and small interfering RNA-mediated knock-down of Nrf2 significantly inhibited Sulforaphane-induced HO-1 expression. Additionally, PI3K/Akt and ROS were also involved in Sulforaphane-induced Nrf2 activation and HO-1 expression, as revealed by the pharmacological inhibitors LY294002 and NAC. Moreover, Sulforaphane treatment inhibited MALP-2-induced pro-inflammatory cytokine secretion and pulmonary inflammation in mice, as well as MALP-2-triggered NF-κB activation. Furthermore, SnPP, a selective inhibitor of HO-1, reversed the inhibitory actions of Sulforaphane, while a carbon monoxide-releasing molecule, CORM-2, caused a significant decrease in MALP-2-induced cytokine secretion. Collectively, these results suggest that Sulforaphane functions as a suppressor of the MALP-2-induced inflammatory response, not only by inhibiting the expression of cytokines and the induction of HO-1 but also by diminishing NF-κB activation in cultured monocytes and the lungs of mice.

Type I Interferon-Independent Dendritic Cell Priming and Antitumor T Cell Activation Induced by a Mycoplasma fermentans Lipopeptide.

Mycoplasma fermentans-derived diacylated lipoprotein M161Ag (MALP404) is recognized by human/mouse toll-like receptor (TLR) 2/TLR6. Short proteolytic products including macrophage-activating lipopeptide 2 (MALP2) have been utilized as antitumor immune-enhancing adjuvants. We have chemically synthesized a short form of MALP2 named MALP2s (S-[2,3-bis(palmitoyloxy)propyl]-CGNNDE). MALP2 and MALP2s provoke natural killer (NK) cell activation in vitro but only poorly induce tumor regression using in vivo mouse models loading NK-sensitive tumors. Here, we identified the functional mechanism of MALP2s on dendritic cell (DC)-priming and cytotoxic T lymphocyte (CTL)-dependent tumor eradication using CTL-sensitive tumor-implant models EG7 and B16-OVA. Programmed death ligand-1 (PD-L1) blockade therapy in combination with MALP2s + ovalbumin (OVA) showed a significant additive effect on tumor growth suppression. MALP2s increased co-stimulators CD80/86 and CD40, which were totally MyD88-dependent, with no participation of toll-IL-1R homology domain-containing adaptor molecule-1 or type I interferon signaling in DC priming. MALP2s + OVA consequently augmented proliferation of OVA-specific CTLs in the spleen and at tumor sites. Chemokines and cytolytic factors were upregulated in the tumor. Strikingly, longer duration and reinvigoration of CTLs in spleen and tumors were accomplished by the addition of MALP2s + OVA to α-PD-L1 antibody (Ab) therapy compared to α-PD-L1 Ab monotherapy. Then, tumors regressed better in the MALP2s/OVA combination than in the α-PD-L1 Ab monotherapy. Hence, MALP2s/tumor-associated antigens combined with α-PD-L1 Ab is a good therapeutic strategy in some mouse models. Unfortunately, numerous patients are still resistant to PD-1/PD-L1 blockade, and good DC-priming adjuvants are desired. Cytokine toxicity by MALP2s remains to be settled, which should be improved by chemical modification in future studies.

Coagulation factor 9-deficient mice are protected against dextran sulfate sodium-induced colitis.

ABSTRACTPatients with inflammatory bowel disease (IBD) are susceptible to thromboembolism. Interestingly, IBD occurs less frequently in patients with inherited bleeding disorders. Therefore, we analyzed whether F9-deficiency is protective against the onset of acute colitis in a genetic hemophilia B mouse model. In the 3.5% dextran sulfate sodium (DSS)-induced colitis model, F9-deficient mice were protected from body-weight loss and had a reduced disease activity score. We detected decreased colonic myeloperoxidase activity and decreased CXCL1 levels in DSS-treated F9-deficient mice compared with wild-type (WT) littermate controls, indicating decreased neutrophil infiltration. Remarkably, we identified expression of coagulation factor IX (FIX) protein in small intestinal epithelial cells (MODE-K). In epithelial cell cultures, cellular FIX protein expression was increased following stimulation with the bacterial Toll-like receptor agonists lipopolysaccharide, macrophage-activating lipopeptide-2 and Pam3CSK4. Thus, we revealed a protective role of F9-deficiency in DSS-induced colitis and identified the intestinal epithelium as a site of ectopic FIX.This article has an associated First Person interview with the first author of the paper.

Bivalent mucosal peptide vaccines administered using the LCP carrier system stimulate protective immune responses against Streptococcus pyogenes infection

Despite the broad knowledge about the pathogenicity of Streptococcus pyogenes there is still a controversy about the correlate of protection in GAS infections. We aimed in further improving the immune responses stimulated against GAS comparing different vaccine formulations including bis-(3′,5′)-cyclic dimeric adenosine monophosphate (c-di-AMP) and BPPCysMPEG, a derivative of the macrophage-activating lipopeptide (MALP-2), as adjuvants, respectively, to be administered with and without the universal T helper cell epitope P25 along with the optimized B cell epitope J14 of the M protein and B and T cell epitopes of SfbI. Lipopeptide based nano carrier systems (LCP) were used for efficient antigen delivery across the mucosal barrier. The stimulated immune responses were efficient in protecting mice against a respiratory challenge with a lethal dose of a heterologous S. pyogenes strain. Moreover, combination of the LCP based peptide vaccine with c-di-AMP allowed reduction of antigen dose at the same time maintaining vaccine efficacy.

Suppression of Baeckea frutescens L. and its components on MyD88-dependent NF-κB pathway in MALP-2-stimulated RAW264.7 cells

Ethnopharmacological relevanceBaeckea frutescens L. is commonly used as a folk medicinal material. There are nineteen components in its volatile oil, including Pcymol which has effects of eliminating phlegm, relieving asthma and antiviral. This study was aimed to investigate the anti-infectious inflammatory activities of Baeckea frutescens L. and its conponents and analyzing the mechanisms. Materials and methodsThe anti-infectious inflammation of Baeckea frutescens L. were studied by using macrophage activating lipopeptide-2 (MALP-2)-stimulated RAW264.7 cell model in vitro. Secretion of nitric oxide (NO), expression of inducible NO synthase (iNOS) and cytokines were detected as classic inflammatory index. Expression of Myeloid differentiation factor 88 (MyD88), degradation of inhibitory κBα (IκBα) and nuclear translocation of NF-κB p65 were further investigated. ResultsThe results suggested that Baeckea frutescens L. has effect on suppression of MALP-2-mediated inflammation in RAW264.7 cells. The secretion of NO and the expression of iNOS could be inhibited. The secretion of tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) were also declined. Baeckea frutescens L. significantly decreased the expression of MyD88, therefore, inhibited the degradation of IκBα, reduced the level of nuclear translocation of p65. ConclusionThe results of this study indicated that Baeckea frutescens L. and its components could inhibit the anti-infectious inflammatory events and iNOS expression in MALP-2 stimulated RAW264.7 cells. Among them, BF-2 might play a role through the inhibition of the MyD88 and NF-κB pathway. Our study might provide a new strategy to design and develop this kind of drug towards mycoplasma-infected inflammation.

Differential modulation of toll-like receptor agonists-induced iNOS expression by polyunsaturated and saturated fatty acids

ABSTRACTToll-like receptors (TLRs) play critical roles in the induction of immune and inflammatory responses by recognizing invading microbial pathogens. One of the most important proteins for inflammatory responses is inducible nitric oxide synthase (iNOS). The dysregulated iNOS activation play important roles in the development of certain inflammatory diseases. The present study investigated the effects of arachidic acid (ACA), which is a saturated fatty acid (SFA), and eicosapentanoic acid (EPA), which is polyunsaturated fatty acid (PUFA), on inflammation by modulating NF-κB activation and iNOS expression induced by TLRs agonists in murine macrophages. EPA suppressed NF-κB activation and iNOS expression induced by a lipopolysaccharide, macrophage-activating lipopeptide 2-kDa, and polyriboinosinic polyribocytidylic acid, but ACA did not. These results suggested that EPA can modulate TLR signaling pathways and subsequent chronic inflammatory responses, but ACA did not mediate these effects. All the results suggest that EPA is a promising novel agent for the treatment of inflammatory diseases.

NADPH oxidase NOX2 mediates TLR2/6-dependent release of GM-CSF from endothelial cells.

NADPH oxidase-generated reactive oxygen species (ROS) from immune cells are well known to be important for pathogen killing in response to TLR ligands. Here, we investigated a new aspect of NADPH oxidase in the TLR2/6-induced release of the immunologically relevant GM-CSF by endothelial cells. Stimulation of human endothelial cells with TLR2/6 agonist, MALP-2 (macrophage-activating lipopeptide of 2 kDa), induced NADPH oxidase activation and ROS formation. Inhibition by ROS scavengers and NADPH oxidase inhibitors blocked MALP-2-induced GM-CSF release. NADPH oxidase activators or ROS donors alone did not result in GM-CSF secretion; however, additional superoxide supply augmented MALP-2-induced GM-CSF secretion and restored GM-CSF levels after NADPH oxidase inhibition. MALP-2-dependent NF-ĸB activation was suppressed by NADPH oxidase inhibition, and inhibition of NF-κB completely blunted MALP-2-induced GM-CSF release. Vascular explants from mice that were deficient for the NADPH oxidase subunit p47 phox showed diminished intimal superoxide production and GM-CSF release after ex vivo stimulation with MALP-2. Moreover, an increase in circulating progenitor cells after MALP-2 injection was completely abolished in p47phox-knockout mice. Finally, MALP-2 stimulation increased mRNA expression of the major subunit NADPH oxidase, (Nox)2, in endothelial cells, and Nox2 inhibition prevented MALP-2-induced GM-CSF release. Our findings identify a Nox2-containing NADPH oxidase as a crucial regulator of the immunologic important growth factor GM-CSF after TLR2/6 stimulation in endothelial cells.-Schuett, J., Schuett, H., Oberoi, R., Koch, A.-K., Pretzer, S., Luchtefeld, M., Schieffer, B., Grote, K. NADPH oxidase NOX2 mediates TLR2/6-dependent release of GM-CSF from endothelial cells.

Effects of macrophage-activating lipopeptide-2 (MALP-2) on the vascularisation of implanted polyurethane scaffolds seeded with microvascular fragments.

The seeding of scaffolds with adipose tissue-derived microvascular fragments represents a promising strategy to establish a sufficient blood supply in tissue constructs. Herein, we analysed whether a single application of macrophage-activating lipopeptide-2 (MALP-2) at the implantation site further improves the early vascularisation of such microvessel-seeded constructs. Microvascular fragments were isolated from epididymal fat pads of C57BL/6 mice. The fragments were seeded on polyurethane scaffolds, which were implanted into mouse dorsal skinfold chambers exposed to MALP-2 or vehicle (control). The inflammatory host tissue response and the vascularisation of the scaffolds were analysed using intravital fluorescence microscopy, histology and immunohistochemistry. We found that the numbers of microvascular adherent leukocytes were significantly increased in MALP-2-treated chambers during the first 3 days after scaffold implantation when compared to controls. This temporary inflammation resulted in an improved vascularisation of the host tissue surrounding the implants, as indicated by a higher density of CD31-positive microvessels at day 14. However, the MALP-2-exposed scaffolds themselves presented with a lower functional microvessel density in their centre. In addition, in vitro analyses revealed that MALP-2 promotes apoptotic cell death of endothelial and perivascular cells in isolated microvascular fragments. Hence, despite the beneficial pro-angiogenic properties of MALP-2 at the implantation site, the herein evaluated approach may not be recommended to improve the vascularisation capacity of microvascular fragments in tissue engineering applications.

Diabetic pregnancy activates the innate immune response through TLR5 or TLR1/2 on neonatal monocyte

Diabetes mellitus (DM) during pregnancy causes congenital malformation, macrosomia, respiratory distress syndrome, and other abnormalities in neonates, but whether maternal DM affects the neonatal innate immune system is unknown. Therefore we aimed to reveal the influence of DM in pregnancy on the toll-like receptor (TLR)–mediated innate immune response in neonates. Cord blood was collected after full-term vaginal or cesarean delivery and classified into a DM group (n=8) and non-DM (control) group (n=7). Mononuclear cells were harvested from cord blood by using density gradient centrifugation, after which anti-CD14 magnetic beads were used to isolate monocytes from the mononuclear population. After monocytes were cultured with lipopolysaccharide (TLR4 ligand), flagellin (TLR5 ligand), Pam3CSK4 (TLR1/TLR2 ligand), zymosan (TLR2/TLR6 ligand), or macrophage-activating lipopeptide (TLR2/TLR6 ligand) for 12h, the cytokine levels (interleukin [IL]-8, IL-6, IL-1β, IL-10, tumor necrosis factor alpha and IL-12) in the culture supernatants were measured. Compared with the control group, the DM group had higher concentrations of IL-8 (P=0.01) and tumor necrosis factor alpha (P=0.02) after monocyte cultures were stimulated with Pam3CSK4 and higher concentrations of IL-8 (P=0.01) after flagellin treatment. In contrast, stimulation with lipopolysaccharide, zymosan, or macrophage-activating lipopeptide did not lead to any difference in cytokine profiles between the two groups. These data indicate that maternal DM induces excessive inflammatory activation in neonates via a TLR5- or TLR1/2-mediated innate immune response.

Inhibition of TNF-α, IL-1α, and IL-1β by Pretreatment of Human Monocyte-Derived Macrophages with Menaquinone-7 and Cell Activation with TLR Agonists In Vitro.

Circulatory markers of low-grade inflammation such as tumor necrosis factor-alpha (TNF-α), interleukin-1 alpha (IL-1α), and interleukin-1 beta (IL-1β) positively correlate with endothelial damage, atheroma formation, cardiovascular disease, and aging. The natural vitamin K2-menaquinone-7 (MK-7) added to the cell culture of human monocyte-derived macrophages (hMDMs) at the same time as toll-like receptor (TLR) agonists did not influence the production of TNF-α. When the cells were pretreated up to 6 h with MK-7 before treatment with TLR agonists, MK-7 did not inhibit significantly the production of TNF-α after the TLR activation. However, 30 h pretreatment of hMDMs with at least 10 μM of MK-7 effectively and dose dependently inhibited the proinflammatory function of hMDMs. Pretreatment of hMDMs with 10 μM of MK-7 for 30 h resulted in 20% inhibition of TNF-α production after lipopolysaccharide (LPS) activation (P < .05) and 43% inhibition after macrophage-activating lipopeptide (MALP) activation (P < .001). Pathogen-associated molecular pattern (PMPP) activation was inhibited by 20% with MK-7 pretreatment; however, this inhibition was not statistically significant. The 30 h pretreatment of a THP-1-differentiated monocyte cell line with MK-7 resulted in a dose-dependent downregulation of TNFα, IL-1α, and IL-1β gene expression as evaluated by RNA semiquantitative reverse transcription polymerase chain reaction (RT-PCR). MK-7 is able to modulate immune and inflammatory reactions in the dose-response inhibition of TNF-α, IL-1α, and IL-1β gene expression and protein production by the healthy hMDMs in vitro.