Control of Methicillin-Resistant Staphylococcus aureus Pneumonia Utilizing TLR2 Agonist Pam3CSK4

The spread of methicillin-resistant Staphylococcus aureus (MRSA) is a critical health issue that has drawn greater attention to the potential use of immunotherapy. Toll-like receptor 2 (TLR2), a pattern recognition receptor, is an essential component in host innate defense system against S. aureus infection. However, little is known about the innate immune response, specifically TLR2 activation, against MRSA infection. Here, we evaluate the protective effect and the mechanism of MRSA murine pneumonia after pretreatment with Pam3CSK4, a TLR2 agonist. We found that the MRSA-pneumonia mouse model, pretreated with Pam3CSK4, had reduced bacteria and mortality in comparison to control mice. As well, lower protein and mRNA levels of TNF-α, IL-1β and IL-6 were observed in lungs and bronchus of the Pam3CSK4 pretreatment group. Conversely, expression of anti-inflammatory cytokine IL-10, but not TGF-β, increased in Pam3CSK4-pretreated mice. Our additional studies showed that CXCL-2 and CXCL1, which are necessary for neutrophil recruitment, were less evident in the Pam3CSK4-pretreated group compared to control group, whereas the expression of Fcγ receptors (FcγⅠ/Ⅲ) and complement receptors (CR1/3) increased in murine lungs. Furthermore, we found that increased survival and improved bacterial clearance were not a result of higher levels of neutrophil infiltration, but rather a result of enhanced phagocytosis and bactericidal activity of neutrophils in vitro and in vivo as well as increased robust oxidative activity and release of lactoferrin. Our cumulative findings suggest that Pam3CSK4 could be a novel immunotherapeutic candidate against MRSA pneumonia.     

Toll-Like Receptor Agonists Synergistically Increase Proliferation and Activation of B Cells by Epstein-Barr Virus

Epstein-Barr virus (EBV) efficiently drives proliferation of human primary B cells in vitro, a process relevant for human diseases such as infectious mononucleosis and posttransplant lymphoproliferative disease. Human B-cell proliferation is also driven by ligands of Toll-like receptors (TLRs), notably viral or bacterial DNA containing unmethylated CpG dinucleotides, which triggers TLR9. Here we quantitatively investigated how TLR stimuli influence EBV-driven B-cell proliferation and expression of effector molecules. CpG DNA synergistically increased EBV-driven proliferation and transformation, T-cell costimulatory molecules, and early production of interleukin-6. CpG DNA alone activated only memory B cells, but CpG DNA enhanced EBV-mediated transformation of both memory and naive B cells. Ligands for TLR2 or TLR7/8 or whole bacteria had a weaker but still superadditive effect on B-cell transformation. Additionally, CpG DNA facilitated the release of transforming virus by established EBV-infected lymphoblastoid cell lines. These results suggest that the proliferation of EBV-infected B cells and their capability to interact with immune effector cells may be directly influenced by components of bacteria or other microbes present at the site of infection.Epstein-Barr virus (EBV), a herpesvirus, is a very successful infectious agent: it establishes and maintains latent infection in >95% of human beings worldwide. This success is related to EBV''s varied strategies to maintain itself in its preferred host cell type, the B cell, by establishing different modes of latent infection (46). Some of these modes (latency modes 0, I, and II) are characterized by a resting B-cell phenotype and expression of a very limited set of EBV proteins (from none to four). In contrast, latency III involves the expression of at least 12 EBV latent-cycle gene products (10 proteins and 2 RNAs) (30, 31), which in their combined action profoundly alter the B cell''s appearance and behavior by inducing B-cell activation associated with proliferation, altered receptor expression, and cytokine secretion, as well as causing enhanced antigen presentation (31).In these various features, EBV infection of the latency III type resembles physiological activation of B cells in germinal centers even in its molecular details, because EBV closely mimics or constitutively activates some of the B cell''s main signaling pathways. Exogenous physiological signals leading to B-cell activation have been classified as “signal 1,” the stimulation of the B-cell receptor (BCR) by antigen binding; “signal 2,” the stimulation of CD40 by the CD40 ligand molecule, expressed on activated helper T cells; and “signal 3,” the stimulation of Toll-like receptors (TLRs) by microbial components, such as unmethylated CpG DNA, or their mimics. All three signals together are required for maximal proliferation of naive B cells (47). However, stimulation with TLR ligands alone, for example, CpG DNA, is sufficient to cause transient B-cell activation, including proliferation and induction of immune effector molecules such as CD86, a T-cell-costimulatory molecule (24). Additional immune effectors, the cytokines interleukin-6 (IL-6), IL-10, and IL-12, are induced when CpG stimulation is combined with strong CD40 stimulation (55).For primary infection of B cells, it is well established that EBV''s latent membrane proteins LMP2A (10, 39) and LMP1 (22) mimic signaling by the BCR and CD40, respectively. It is less clear whether and how EBV generates a potential signal 3 in the course of primary B-cell infection. A role of the TLR7 pathway has been proposed, based on the observation that EBV infection of naive B cells elevates the expression of TLR7 and its downstream signaling mediators (40). Additional mechanisms have recently been proposed to explain how EBV might trigger TLRs or other pattern recognition receptors in other cellular systems. For example, the Epstein-Barr virus-encoded small RNAs (EBERs) were described to trigger the retinoic acid-inducible gene I (RIG-I)-encoded protein, a receptor for various viral RNAs, in Burkitt''s lymphoma cells (48, 49). TLR2 signaling in monocytes is activated by binding of EBV particles to the cells (21) or by extracellular provision of EBV dUTPase (2).However, a physiologically relevant signal 3 need not originate in EBV itself. Other microbial agents present at the site of EBV infection might influence EBV infection, B-cell transformation, and virus release. For example, infectious mononucleosis (IM), a frequent consequence of primary EBV infection in adolescents and adults, is usually accompanied by tonsillitis with characteristic massive bacterial colonization (50), a likely source of TLR agonists acting on local EBV-infected B cells. Here we investigate the effects of CpG DNA and other exogenous TLR ligands on EBV-driven B-cell proliferation, clonal outgrowth, and induction of activation-associated cellular receptors and cytokines.     

Identification and immunological evaluation of novel TLR2 agonists through structure optimization of Pam3CSK4

Toll-like receptor 2 (TLR2) is a bridge between innate immunity and adaptive immunity. TLR2 agonists have been exploited as potential vaccine adjuvants and antitumor agents. However, no TLR2 agonists have been approved by FDA up to now. To discover drug-like TLR2 selective agonists, a novel series of Pam₃CSK₄ derivatives were designed based on the crystal structure of hTLR2-hTLR1-Pam₃CSK₄ complex, synthesized and evaluated for their immune-stimulatory activities. Among them, 35c was identified as a murine-specific TLR2 agonist, while 35f was a human-specific TLR2 agonist. Besides, 35d (human and murine TLR2 agonist) showed TLR2 agonistic activity comparable to Pam₃CSK₄, which included: elevated IL-6 expression level (EC₅₀ = 83.08 ± 5.94 nM), up-regulated TNF-α and IL-6 mRNA expression and promoted maturation of DCs through activating the NF-κB signaling pathway. TLRs antibodies test showed that 35a and 35d were TLR2/1 agonists, while 35f was a TLR2/6 agonist.     

Toll-Like Receptor 2-Mediated Glial Cell Activation in a Mouse Model of Cuprizone-Induced Demyelination

Multiple sclerosis (MS) is a chronic degenerative disease of the central nervous system that is characterized by myelin abnormalities, oligodendrocyte pathology, and concomitant glia activation. The factors triggering gliosis and demyelination are currently not well characterized. New findings suggest an important role of the innate immune response in the initiation and progression of active demyelinating lesions. Especially during progressive disease, aberrant glia activation rather than the invasion of peripheral immune cells is accountable for progressive neuronal injury. The innate immune response can be induced by pathogen-associated or danger-associated molecular patterns, which are identified by pattern recognition receptors (PRRs), including the Toll-like receptors (TLRs). In this study, we used the cuprizone model in mice to investigate the expression of TLR2 during the course of cuprizone-induced demyelination. In addition, we used TLR2-deficient mice to analyze the functional role of TLR2 activation during cuprizone-induced demyelination and reactive gliosis. We show a significantly increased expression of TLR2 in the corpus callosum and hippocampus of cuprizone-intoxicated mice. The absence of receptor signaling in TLR2-deficient mice resulted in less severe reactive astrogliosis in the corpus callosum and cortex. In addition, microglia activation was ameliorated in the corpus callosum of TLR2-deficient mice, but augmented in the cortex compared to wild-type littermates. Extent of demyelination and loss of mature oligodendrocytes was comparable in both genotypes. These results suggest that the TLR2 orchestrates glia activation during gray and white matter demyelination in the presence of an intact blood-brain barrier. Future studies now have to address the underlying mechanisms of the region-specific TLR2-mediated glia activation.     

Coinjection with TLR2 Agonist Pam3CSK4 Reduces the Pathology of Leishmanization in Mice

Cutaneous leishmaniasis caused by Leishmania major is anemergent, uncontrolled public health problem and there is no vaccine. Apromising prophylactic approach has been immunotherapy with Toll-like receptor(TLR) agonists to enhance parasite-specific immune responses. We have previouslyreported that vaccination of C57BL/6 mice with live L.major plus the TLR9 agonist CpG DNA prevents lesiondevelopment and confers immunity to reinfection. Our current study aims toinvestigate whether other TLR agonists can be used in leishmanization withoutinduction of lesion formation. We found that live L.major plus the TLR2 agonist Pam3CSK4 reduced the pathologyin both genetically resistant (C57BL/6) and susceptible (BALB/c) mouse strains.The addition of Pam3CSK4 activated dermal dendritic cells and macrophages toproduce greater amounts of proinflammatory cytokines in both mouse strains. BothTh1 and Th17 responses were enhanced by leishmanization with L.major plus Pam3CSK4 in C57BL/6 mice; however, Th17 cellswere unchanged in BALB/c mice. The production of IL-17 from neutrophils wasenhanced in both strains infected with L.major plus Pam3CSK4. However, the sustained influx ofneutrophils in sites of infection was only observed in BALB/c mice. Our datademonstrate that the mechanism behind leishmanization with TLR agonists may bevery different depending upon the immunological background of the host. Thisneeds to be taken into account for the rational development of successfulvaccines against the disease.     

Toll样受体2及Toll样受体4在大鼠实验性变应性鼻炎中的表达

目的研究Toll样受体2（Toll-like receptor2,TLR2）及Toll样受体4（TLR4）在实验性变应性鼻炎大鼠鼻黏膜中的表达及脂多糖（Lipopolysaccharide,LPS）对其表达的影响。方法SD大鼠48只随机分为正常对照组（A组）;变应性鼻炎组（B组）：经腹腔注射及鼻腔滴入卵清白蛋白（Ovalbumin,OVA）建立变应性鼻炎（Allergic rhinitis,AR）模型;变应性鼻炎＋LPS刺激组（C组）：大鼠激发成变应性鼻炎模型后再以LPS滴鼻。用逆转录聚合酶链反应（RT-PCR）方法检测鼻黏膜中TLR2 mRNA、TLR4 mRNA的表达。结果B、C组大鼠均成功激发为AR动物模型;各组鼻黏膜中均有TLR2 mRNA、TLR4 mRNA表达;各组间TLR2 mRNA的表达差异无统计学意义（P〉0．05）。B、C组TLR4 mRNA的表达较A组高（P〈0．01）;C组TLR4 mRNA表达较B组增高（P〈0．01）。结论AR大鼠有TLLR4的表达增高;LPS刺激后TLR4表达进一步增高,说明TLR4可能参与AR的发病。TLR2在AR大鼠中的表达未见增高;LPS刺激后。TLR2表达未见进一步增高,TLR2与变应性鼻炎的关系有待进一步研究。     

Prostaglandin E2 Prevents Hyperosmolar-Induced Human Mast Cell Activation through Prostanoid Receptors EP2 and EP4

Background

Mast cells play a critical role in allergic and inflammatory diseases, including exercise-induced bronchoconstriction (EIB) in asthma. The mechanism underlying EIB is probably related to increased airway fluid osmolarity that activates mast cells to the release inflammatory mediators. These mediators then act on bronchial smooth muscle to cause bronchoconstriction. In parallel, protective substances such as prostaglandin E₂ (PGE₂) are probably also released and could explain the refractory period observed in patients with EIB.

Objective

This study aimed to evaluate the protective effect of PGE₂ on osmotically activated mast cells, as a model of exercise-induced bronchoconstriction.

Methods

We used LAD2, HMC-1, CD34-positive, and human lung mast cell lines. Cells underwent a mannitol challenge, and the effects of PGE₂ and prostanoid receptor (EP) antagonists for EP_1–4 were assayed on the activated mast cells. Beta-hexosaminidase release, protein phosphorylation, and calcium mobilization were assessed.

Results

Mannitol both induced mast cell degranulation and activated phosphatidyl inositide 3-kinase and mitogen-activated protein kinase (MAPK) pathways, thereby causing de novo eicosanoid and cytokine synthesis. The addition of PGE₂ significantly reduced mannitol-induced degranulation through EP₂ and EP₄ receptors, as measured by beta-hexosaminidase release, and consequently calcium influx. Extracellular-signal-regulated kinase 1/2, c-Jun N-terminal kinase, and p38 phosphorylation were diminished when compared with mannitol activation alone.

Conclusions

Our data show a protective role for the PGE₂ receptors EP₂ and EP₄ following osmotic changes, through the reduction of human mast cell activity caused by calcium influx impairment and MAP kinase inhibition.     

A20 Is Critical for the Induction of Pam3CSK4-Tolerance in Monocytic THP-1 Cells

A20 functions to terminate Toll-like receptor (TLR)-induced immune response, and play important roles in the induction of lipopolysacchride (LPS)-tolerance. However, the molecular mechanism for Pam3CSK4-tolerance is uncertain. Here we report that TLR1/2 ligand Pam3CSK4 induced tolerance in monocytic THP-1 cells. The pre-treatment of THP-1 cells with Pam3CSK4 down-regulated the induction of pro-inflammatory cytokines induced by Pam3CSK4 re-stimulation. Pam3CSK4 pre-treatment also down-regulated the signaling transduction of JNK, p38 and NF-κB induced by Pam3CSK4 re-stimulation. The activation of TLR1/2 induced a rapid and robust up-regulation of A20, suggesting that A20 may contribute to the induction of Pam3CSK4-tolerance. This hypothesis was proved by the observation that the over-expression of A20 by gene transfer down-regulated Pam3CSK4-induced inflammatory responses, and the down-regulation of A20 by RNA interference inhibited the induction of tolerance. Moreover, LPS induced a significant up-regulation of A20, which contributed to the induction of cross-tolerance between LPS and Pam3CSK4. A20 was also induced by the treatment of THP-1 cells with TNF-α and IL-1β. The pre-treatment with TNF-α and IL-1β partly down-regulated Pam3CSK4-induced activation of MAPKs. Furthermore, pharmacologic inhibition of GSK3 signaling down-regulated Pam3CSK4-induced A20 expression, up-regulated Pam3CSK4-induced inflammatory responses, and partly reversed Pam3CSK4 pre-treatment-induced tolerance, suggesting that GSK3 is involved in TLR1/2-induced tolerance by up-regulation of A20 expression. Taken together, these results indicated that A20 is a critical regulator for TLR1/2-induced pro-inflammatory responses.     

Leprosy and the Adaptation of Human Toll-Like Receptor 1

Leprosy is an infectious disease caused by the obligate intracellular pathogen Mycobacterium leprae and remains endemic in many parts of the world. Despite several major studies on susceptibility to leprosy, few genomic loci have been replicated independently. We have conducted an association analysis of more than 1,500 individuals from different case-control and family studies, and observed consistent associations between genetic variants in both TLR1 and the HLA-DRB1/DQA1 regions with susceptibility to leprosy (TLR1 I602S, case-control P = 5.7×10⁻⁸, OR = 0.31, 95% CI = 0.20–0.48, and HLA-DQA1 rs1071630, case-control P = 4.9×10⁻¹⁴, OR = 0.43, 95% CI = 0.35–0.54). The effect sizes of these associations suggest that TLR1 and HLA-DRB1/DQA1 are major susceptibility genes in susceptibility to leprosy. Further population differentiation analysis shows that the TLR1 locus is extremely differentiated. The protective dysfunctional 602S allele is rare in Africa but expands to become the dominant allele among individuals of European descent. This supports the hypothesis that this locus may be under selection from mycobacteria or other pathogens that are recognized by TLR1 and its co-receptors. These observations provide insight into the long standing host-pathogen relationship between human and mycobacteria and highlight the key role of the TLR pathway in infectious diseases.     

Expression of the Receptor Tyrosine Kinase EphB2 on Dendritic Cells Is Modulated by Toll-Like Receptor Ligation but Is Not Required for T Cell Activation

The Eph receptor tyrosine kinases interact with their ephrin ligands on adjacent cells to facilitate contact-dependent cell communication. Ephrin B ligands are expressed on T cells and have been suggested to act as co-stimulatory molecules during T cell activation. There are no detailed reports of the expression and modulation of EphB receptors on dendritic cells, the main antigen presenting cells that interact with T cells. Here we show that mouse splenic dendritic cells (DC) and bone-marrow derived DCs (BMDC) express EphB2, a member of the EphB family. EphB2 expression is modulated by ligation of TLR4 and TLR9 and also by interaction with ephrin B ligands. Co-localization of EphB2 with MHC-II is also consistent with a potential role in T cell activation. However, BMDCs derived from EphB2 deficient mice were able to present antigen in the context of MHC-II and produce T cell activating cytokines to the same extent as intact DCs. Collectively our data suggest that EphB2 may contribute to DC responses, but that EphB2 is not required for T cell activation. This result may have arisen because DCs express other members of the EphB receptor family, EphB3, EphB4 and EphB6, all of which can interact with ephrin B ligands, or because EphB2 may be playing a role in another aspect of DC biology such as migration.     

Toll-Like Receptor 8 Agonist and Bacteria Trigger Potent Activation of Innate Immune Cells in Human Liver

The ability of innate immune cells to sense and respond to impending danger varies by anatomical location. The liver is considered tolerogenic but is still capable of mounting a successful immune response to clear various infections. To understand whether hepatic immune cells tune their response to different infectious challenges, we probed mononuclear cells purified from human healthy and diseased livers with distinct pathogen-associated molecules. We discovered that only the TLR8 agonist ssRNA40 selectively activated liver-resident innate immune cells to produce substantial quantities of IFN-γ. We identified CD161^Bright mucosal-associated invariant T (MAIT) and CD56^Bright NK cells as the responding liver-resident innate immune cells. Their activation was not directly induced by the TLR8 agonist but was dependent on IL-12 and IL-18 production by ssRNA40-activated intrahepatic monocytes. Importantly, the ssRNA40-induced cytokine-dependent activation of MAIT cells mirrored responses induced by bacteria, i.e., generating a selective production of high levels of IFN-γ, without the concomitant production of TNF-α or IL-17A. The intrahepatic IFN-γ production could be detected not only in healthy livers, but also in HBV- or HCV-infected livers. In conclusion, the human liver harbors a network of immune cells able to modulate their immunological responses to different pathogen-associated molecules. Their ability to generate a strong production of IFN-γ upon stimulation with TLR8 agonist opens new therapeutic opportunities for the treatment of diverse liver pathologies.     

Coordinated Activation of Toll-Like Receptor8 (TLR8) and NLRP3 by the TLR8 Agonist,VTX-2337, Ignites Tumoricidal Natural Killer Cell Activity

VTX-2337 (USAN: motolimod) is a selective toll-like receptor 8 (TLR8) agonist, which is in clinical development as an immunotherapy for multiple oncology indications, including squamous cell carcinoma of the head and neck (SCCHN). Activation of TLR8 enhances natural killer cell activation, increases antibody-dependent cell-mediated cytotoxicity, and induces Th1 polarizing cytokines. Here, we show that VTX-2337 stimulates the release of mature IL-1β and IL-18 from monocytic cells through coordinated actions on both TLR8 and the NOD-like receptor pyrin domain containing 3 (NLRP3) inflammasome complex. In vitro, VTX-2337 primed monocytic cells to produce pro-IL-1β, pro-IL-18, and caspase-1, and also activated the NLRP3 inflammasome, thereby mediating the release of mature IL-1β family cytokines. Inhibition of caspase-1 blocked VTX-2337-mediated NLRP3 inflammasome activation, but had little impact on production of other TLR8-induced mediators such as TNFα. IL-18 activated natural killer cells and complemented other stimulatory pathways, including FcγRIII and NKG2D, resulting in IFNγ production and expression of CD107a. NLRP3 activation in vivo was confirmed by a dose-related increase in plasma IL-1β and IL-18 levels in cynomolgus monkeys administered VTX-2337. These results are highly relevant to clinical studies of combination VTX-2337/cetuximab treatment. Cetuximab, a clinically approved, epidermal growth factor receptor-specific monoclonal antibody, activates NK cells through interactions with FcγRIII and facilitates ADCC of tumor cells. Our preliminary findings from a Phase I open-label, dose-escalation, trial that enrolled 13 patients with recurrent or metastatic SCCHN show that patient NK cells become more responsive to stimulation by NKG2D or FcγRIII following VTX-2337 treatment. Together, these results indicate that TLR8 stimulation and inflammasome activation by VTX-2337 can complement FcγRIII engagement and may augment clinical responses in SCCHN patients treated with cetuximab.Trial Registration: ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01334177","term_id":"NCT01334177"}}NCT01334177     

Epinephrine and Norepinephrine Act as Potent Agonists at the Recombinant Human Dopamine D4 Receptor

Abstract: The catecholamines dopamine (DA), epinephrine (EP), and norepinephrine (NE) play important roles in learning and memory, emotional states, and control of voluntary movement, as well as cardiovascular and kidney function. They activate distinct but overlapping neuronal pathways through five distinct DA receptors (D1R–D5R) and at least 10 different adrenergic receptors (α1a/b/c, α2a/b/c-1/c-2, and β1/β2/β3). The D4R, which is localized to mesolimbic areas of the brain implicated in affective and emotional behavior, has a deduced amino acid sequence with homology to both adrenergic and dopaminergic receptor subtypes. We report here that DA, EP, and NE all show binding in the nanomolar range to three isoforms of the recombinant human D4R (hD4R): D4.2, D4.4, and D4.7. Submicromolar concentrations of DA, EP, and NE were sufficient to activate hD4R isoforms in two different functional assays: agonist-induced guanosine 5'- O -(3-[³⁵S]thiotriphosphate) binding and modulation of adenylyl cyclase activity. DA was approximately fivefold more potent than EP and NE at the D4R, whereas activation of the human D2R required at least 100-fold higher catecholamine concentrations. Functional activation of the D4R by multiple neurotransmitters may provide a novel mechanism for integration of catecholamine signaling in the brain and periphery.     

Toll-Like Receptor 4 Stimulation before or after Streptococcus pneumoniae Induced Sepsis Improves Survival and Is Dependent on T-Cells

Introduction

Endotoxin tolerance improves outcomes from gram negative sepsis but the underlying mechanism is not known. We determined if endotoxin tolerance before or after pneumococcal sepsis improved survival and the role of lymphocytes in this protection.

Methods

Mice received lipopolysaccharide (LPS) or vehicle before or after a lethal dose of Streptococcus pneumoniae. Survival, quantitative bacteriology, liver function, and cytokine concentrations were measured. We confirmed the necessity of Toll-like receptor 4 (TLR4) for endotoxin tolerance using C3H/HeN (TLR4 replete) and C3H/HeJ (TLR4 deficient) mice. The role of complement was investigated through A/J mice deficient in C5 complement. CBA/CaHN-Btk^xid//J mice with dysfunctional B cells and Rag-1 knockout (KO) mice deficient in T and B cells delineated the role of lymphocytes.

Results

Endotoxin tolerance improved survival from pneumococcal sepsis in mice with TLR4 that received LPS pretreatment or posttreatment. Survival was associated with reduced bacterial burden and serum cytokine concentrations. Death was associated with abnormal liver function and blood glucose concentrations. Endotoxin tolerance improved survival in A/J and CBA/CaHN-Btk^xid//J mice but not Rag-1 KO mice.

Conclusions

TLR4 stimulation before or after S. pneumoniae infection improved survival and was dependent on T-cells but did not require an intact complement cascade or functional B cells.     

Signaling Pathway in the Osmotic Resistance Induced by Angiotensin II AT2 Receptor Activation in Human Erythrocytes

Background:Angiotensin II regulates blood volume via AT1 (AT1R) and AT2 (AT2R) receptors. As cell integrity is an important feature of mature erythrocyte, we sought to evaluate, in vitro, whether angiotensin II modulates resistance to hemolysis and the signaling pathway involved.Methods:Human blood samples were collected and hemolysis assay and angiotensin II signaling pathway profiling in erythrocytes were done.Results:Hemolysis assay created a hemolysis curve in presence of Ang II in several concentrations (10^-6 M, 10^-8 M, 10^-10 M, 10^-12 M). Angiotensin II demonstrated protective effect, both in osmotic stressed and physiological situations, by reducing hemolysis in NaCl 0.4% and 0.9%. By adding receptors antagonists (losartan, AT1R antagonist and PD 123319, AT2R antagonist) and/or signaling modulators for AMPK, Akt/PI3K, p38 and PKC we showed the protective effect was enhanced with losartan and abolished with PD 123319. Also, we showed activation of p38 as well as PI3K/Akt pathways in this system.Conclusion:Ang II protects human erythrocytes from hypo-osmotic conditions-induced hemolysis by activating AT2 receptors and triggering intracellular pathways.Key Words: Angiotensin II, Erythrocyte, Osmotic fragility, Signaling pathway     

Empty liposomes induce antitumoral effects associated with macrophage responses distinct from those of the TLR1/2 agonist Pam3CSK4 (BLP)

Liposomes are frequently used in cancer therapy to encapsulate and apply anticancer drugs. Here, we show that a systemic treatment of mice bearing skin tumors with empty phosphatidylcholine liposomes (PCL) resulted in inhibition of tumor growth, which was similar to that observed with the synthetic bacterial lipoprotein and TLR1/2 agonist Pam₃CSK₄ (BLP). Both compounds led to a substantial decrease of macrophages in spleen and in the tumor-bearing skin. Furthermore, both treatments induced the expression of typical macrophage markers in the tumor-bearing tissue. As expected, BLP induced the expression of the M1 marker genes Cxcl10 and iNOS, whereas PCL, besides inducing iNOS, also increased the M2 marker genes Arg1 and Trem2. In vitro experiments demonstrated that neither PCL nor BLP influenced proliferation or survival of tumor cells, whereas both compounds inhibited proliferation and survival and increased the migratory capacity of bone marrow-derived macrophages (BMDM). However, in contrast to BLP, PCL did not activate cytokine secretion and induced a different BMDM phenotype. Together, the data suggest that similar to BLP, PCL induce an antitumor response by influencing the tumor microenvironment, in particular by functional alterations of macrophages, however, in a distinct manner from those induced by BLP.     

MF59 and Pam3CSK4 boost adaptive responses to influenza subunit vaccine through an IFN type I-independent mechanism of action

The innate immune pathways induced by adjuvants required to increase adaptive responses to influenza subunit vaccines are not well characterized. We profiled different TLR-independent (MF59 and alum) and TLR-dependent (CpG, resiquimod, and Pam3CSK4) adjuvants for the ability to increase the immunogenicity to a trivalent influenza seasonal subunit vaccine and to tetanus toxoid (TT) in mouse. Although all adjuvants boosted the Ab responses to TT, only MF59 and Pam3CSK4 were able to enhance hemagglutinin Ab responses. To identify innate immune correlates of adjuvanticity to influenza subunit vaccine, we investigated the gene signatures induced by each adjuvant in vitro in splenocytes and in vivo in muscle and lymph nodes using DNA microarrays. We found that flu adjuvanticity correlates with the upregulation of proinflammatory genes and other genes involved in leukocyte transendothelial migration at the vaccine injection site. Confocal and FACS analysis confirmed that MF59 and Pam3CSK4 were the strongest inducers of blood cell recruitment in the muscle compared with the other adjuvants tested. Even though it has been proposed that IFN type I is required for adjuvanticity to influenza vaccines, we found that MF59 and Pam3CSK4 were not good inducers of IFN-related innate immunity pathways. By contrast, resiquimod failed to enhance the adaptive response to flu despite a strong activation of the IFN pathway in muscle and lymph nodes. By blocking IFN type I receptor through a mAb, we confirmed that the adjuvanticity of MF59 and Pam3CSK4 to a trivalent influenza vaccine and to TT is IFN independent.