X-ray reflectivity study of the heat shock protein Hsp70 interaction with an artificial cell membrane model.

Membrane-bound heat shock protein 70 (Hsp70) apart from its intracellular localization was shown to be specifically expressed on the plasma membrane surface of tumor but not normal cells. Although the association of Hsp70 with lipid membranes is well documented the exact mechanisms for chaperone membrane anchoring have not been fully elucidated. Herein, we addressed the question of how Hsp70 interacts with negatively charged phospholipids in artificial lipid compositions employing the X-ray reflectivity (XRR) studies. In a first step, the interactions between dioleoylphosphatidylcholine (DOPC) in the presence or absence of dioleoylphosphatidylserine (DOPS) and Hsp70 had been assessed using Quartz crystal microbalance measurements, suggesting that Hsp70 adsorbs to the surface of DOPC/DOPS bilayer. Atomic force microscopy (AFM) imaging demonstrated that the presence of DOPS is required for stabilization of the lipid bilayer. The interaction of Hsp70 with DOPC/DOPS lipid compositions was further quantitatively determined by high energy X-ray reflectivity. A systematic characterization of the chaperone-lipid membrane interactions by various techniques revealed that artificial membranes can be stabilized by the electrostatic interaction of anionic DOPS lipids with Hsp70.

A new look at Hsp70 activity in phosphatidylserine-enriched membranes: chaperone-induced quasi-interdigitated lipid phase.

70 kDa heat shock protein Hsp70 (also termed HSP70A1A) is the major stress-inducible member of the HSP70 chaperone family, which is present on the plasma membranes of various tumor cells, but not on the membranes of the corresponding normal cells. The exact mechanisms of Hsp70 anchoring in the membrane and its membrane-related functions are still under debate, since the protein does not contain consensus signal sequence responsible for translocation from the cytosol to the lipid bilayer. The present study was focused on the analysis of the interaction of recombinant human Hsp70 with the model phospholipid membranes. We have confirmed that Hsp70 has strong specificity toward membranes composed of negatively charged phosphatidylserine (PS), compared to neutral phosphatidylcholine membranes. Using differential scanning calorimetry, we have shown for the first time that Hsp70 affects the thermotropic behavior of saturated PS and leads to the interdigitation that controls membrane thickness and rigidity. Hsp70-PS interaction depended on the lipid phase state; the protein stabilized ordered domains enriched with high-melting PS, increasing their area, probably due to formation of quasi-interdigitated phase. Moreover, the ability of Hsp70 to form ion-permeable pores in PS membranes may also be determined by the bilayer thickness. These observations contribute to a better understanding of Hsp70-PS interaction and biological functions of membrane-bound Hsp70 in cancer cells.

Magnetic Relaxation Switching Assay Using IFNα-2b-Conjugated Superparamagnetic Nanoparticles for Anti-Interferon Antibody Detection.

Type I interferons, particularly IFNα-2b, play essential roles in eliciting adaptive and innate immune responses, being implicated in the pathogenesis of various diseases, including cancer, and autoimmune and infectious diseases. Therefore, the development of a highly sensitive platform for analysis of either IFNα-2b or anti-IFNα-2b antibodies is of high importance to improve the diagnosis of various pathologies associated with the IFNα-2b disbalance. For evaluation of the anti-IFNα-2b antibody level, we have synthesized superparamagnetic iron oxide nanoparticles (SPIONs) coupled with the recombinant human IFNα-2b protein (SPIONs@IFNα-2b). Employing a magnetic relaxation switching assay (MRSw)-based nanosensor, we detected picomolar concentrations (0.36 pg/mL) of anti-INFα-2b antibodies. The high sensitivity of the real-time antibodies' detection was ensured by the specificity of immune responses and the maintenance of resonance conditions for water spins by choosing a high-frequency filling of short radio-frequency pulses of the generator. The formation of a complex of the SPIONs@IFNα-2b nanoparticles with the anti-INFα-2b antibodies led to a cascade process of the formation of nanoparticle clusters, which was further enhanced by exposure to a strong (7.1 T) homogenous magnetic field. Obtained magnetic conjugates exhibited high negative MR contrast-enhancing properties (as shown by NMR studies) that were also preserved when particles were administered in vivo. Thus, we observed a 1.2-fold decrease of the T2 relaxation time in the liver following administration of magnetic conjugates as compared to the control. In conclusion, the developed MRSw assay based on SPIONs@IFNα-2b nanoparticles represents an alternative immunological probe for the estimation of anti-IFNα-2b antibodies that could be further employed in clinical studies.

Differentiation of human macrophages with anaphylatoxin C3a impairs alternative M2 polarization and decreases lipopolysaccharide-induced cytokine secretion.

Anaphylatoxin C3a is a small signaling polypeptide that is generated during complement activation. C3a is involved in the regulation of various innate and adaptive immune system processes; however, the role of C3a in macrophage differentiation and polarization is poorly elucidated. Here we showed that C3a impairs alternative M2 polarization of human macrophages and suppressed CD206, IL1Ra and CCL22 expression. C3a leads to a decrease of nuclear receptor PPARγ expression via the ERK1/2 signaling pathway, resulting in repressed PPARγ-dependent activation of CD36, FABP4 and LXRα genes and blunted response to an LXR ligand TO901317. Using small interfering RNA and agonist/antagonist approaches we showed that C3a decreases CD206, IL1Ra and CCL22 transcription at least partly in a PPARγ-dependent manner in M2 macrophages. Moreover, C3a impairs efferocytosis by M2 macrophages and inhibits their migratory activity. By contrast, macrophages treated with C3a during differentiation show blunted response to lipopolysaccharide stimulation owing to downregulation of TLR4 and lipid raft content. At the same time, differentiation of macrophages with C3a does not change M1 polarization in interferon gamma (IFNγ) and IFNγ + lipopolysaccharide-treated macrophages. These data provide a novel role of complement system and C3a in the regulation of M2 macrophage polarizations and suggest crosstalk between C3a, TLR4, PPARγ and LXR signaling pathways.

Hsp70-containing extracellular vesicles are capable of activating of adaptive immunity in models of mouse melanoma and colon carcinoma.

The release of Hsp70 chaperone from tumor cells is found to trigger the full-scale anti-cancer immune response. Such release and the proper immune reaction can be induced by the delivery of recombinant Hsp70 to a tumor and we sought to explore how the endogenous Hsp70 can be transported to extracellular space leading to the burst of anti-cancer activity. Hsp70 transport mechanisms were studied by analyzing its intracellular tracks with Rab proteins as well as by using specific inhibitors of membrane domains. To study Hsp70 forms released from cells we employed the assay consisting of two affinity chromatography methods. Hsp70 content in culture medium and extracellular vesicles (EVs) was measured with the aid of ELISA. The properties and composition of EVs were assessed using nanoparticle tracking analysis and immunoblotting. The activity of immune cells was studied using an assay of cytotoxic lymphocytes, and for in vivo studies we employed methods of affinity separation of lymphocyte fractions. Analyzing B16 melanoma cells treated with recombinant Hsp70 we found that the chaperone triggered extracellular transport of its endogenous analog in soluble and enclosed in EVs forms; both species efficiently penetrated adjacent cells and this secondary transport was corroborated with the strong increase of Natural Killer (NK) cell toxicity towards melanoma. When B16 and CT-26 colon cancer cells before their injection in animals were treated with Hsp70-enriched EVs, a powerful anti-cancer effect was observed as shown by a two-fold reduction in tumor growth rate and elevation of life span. We found that the immunomodulatory effect was due to the enhancement of the CD8-positive response and anti-tumor cytokine accumulation; supporting this there was no delay in CT-26 tumor growth when Hsp70-enriched EVs were grafted in nude mice. Importantly, pre-treatment of B16 cells with Hsp70-bearing EVs resulted in a decline of arginase-1-positive macrophages, showing no generation of tumor-associated macrophages. In conclusion, Hsp70-containing EVs generated by specifically treated cancer cells give a full-scale and effective pattern of anti-tumor immune responses.

Extracellular Hsp70 Reduces the Pro-Tumor Capacity of Monocytes/Macrophages Co-Cultivated with Cancer Cells.

Cancer cells are known to contain high levels of the heat shock protein 70 kDa (Hsp70), which mediates increased cell proliferation, escape from programmed cell death, enhanced invasion, and metastasis. A part of Hsp70 molecules may release from cancer cells and affect the behavior of adjacent stromal cells. To explore the effects of Hsp70 on the status of monocytes/macrophages in the tumor locale, we incubated human carcinoma cells of three distinct lines with normal and reduced content of Hsp70 with THP1 monocytes. Using two methods, we showed that the cells with knock-down of Hsp70 released a lower amount of protein in the extracellular medium. Three cycles of the co-cultivation of cancer and monocytic cells led to the secretion of several cytokines typical of the tumor microenvironment (TME) and to pro-cancer activation of the monocytes/macrophages as established by elevation of F4/80 and arginase-1 markers. Unexpectedly, the efficacy of epithelial-mesenchymal transition and resistance of carcinoma cells to anticancer drugs after incubation with monocytic cells were more pronounced in cells with lower Hsp70, e.g., releasing less Hsp70 into the extracellular milieu. These data suggest that Hsp70 released from tumor cells into the TME is able, together with the development of an anti-cancer immune response, to limit the conversion of a considerable part of monocytic cells to the pro-tumor phenotype.

Modulation of Human Complement System by Antimicrobial Peptide Arenicin-1 from Arenicola marina.

Antimicrobial peptides from marine invertebrates are known not only to act like cytotoxic agents, but they also can display some additional activities in mammalian organisms. In particular, these peptides can modulate the complement system as was described for tachyplesin, a peptide from the horseshoe crab. In this work, we investigated the influence on complement activation of the antimicrobial peptide arenicin-1 from the marine polychaete Arenicola marina. To study effects of arenicin on complement activation in human blood serum, we used hemolytic assays of two types, with antibody sensitized sheep erythrocytes and rabbit erythrocytes. Complement activation was also assessed, by the level of C3a production that was measured by ELISA. We found that the effect of arenicin depends on its concentration. At relatively low concentrations the peptide stimulates complement activation and lysis of target erythrocytes, whereas at higher concentrations arenicin acts as a complement inhibitor. A hypothetical mechanism of peptide action is proposed, suggesting its interaction with two complement proteins, C1q and C3. The results lead to the possibility of the development of new approaches for therapy of diseases connected with complement dysregulation, using peptide regulators derived from natural antimicrobial peptides of invertebrates.

70-kDa heat shock protein coated magnetic nanocarriers as a nanovaccine for induction of anti-tumor immune response in experimental glioma.

Nanovaccines based on superparamagnetic iron oxide nanoparticles (SPIONs) provide a novel approach to induce the humoral and cell-based immune system to fight cancer. Herein, we increased the immunostimulatory capacity of SPIONs by coating them with recombinant heat shock protein 70 (Hsp70) which is known to chaperone antigenic peptides. After binding, Hsp70-SPIONs deliver immunogenic peptides from tumor lysates to dendritiс cells (DCs) and thus stimulate a tumor-specific, CD8+ cytotoxic T cell response. We could show that binding activity of Hsp70-SPIONs to the substrate-binding domain (SBD) is highly dependent on the ATPase activity of its nucleotide-binding domain NBD), as shown by (31)P NMR spectroscopy. Immunization of C6 glioma-bearing rats with DCs pulsed with Hsp70-SPIONs and tumor lysates resulted in a delayed tumor progression (as measured by MRI) and an increased overall survival. In parallel an increased IFNγ secretion were detected in the serum of these animals and immunohistological analysis of subsequent cryosections of the glioma revealed an enhanced infiltration of memory CD45RO+ and cytotoxic CD8+ T cells. Taken together the study demonstrates that magnetic nanocarriers such as SPIONs coated with Hsp70 can be applied as a platform for boosting anti-cancer immune responses.

Recombinant interleukin-1 receptor antagonist conjugated to superparamagnetic iron oxide nanoparticles for theranostic targeting of experimental glioblastoma.

Cerebral edema commonly accompanies brain tumors and contributes to neurologic symptoms. The role of the interleukin-1 receptor antagonist conjugated to superparamagnetic iron oxide nanoparticles (SPION-IL-1Ra) was assessed to analyze its anti-edemal effect and its possible application as a negative contrast enhancing agent for magnetic resonance imaging (MRI). Rats with intracranial C6 glioma were intravenously administered at various concentrations of IL-1Ra or SPION-IL-1Ra. Brain peritumoral edema following treatment with receptor antagonist was assessed with high-field MRI. IL-1Ra administered at later stages of tumor progression significantly reduced peritumoral edema (as measured by MRI) and prolonged two-fold the life span of comorbid animals in a dose-dependent manner in comparison to control and corticosteroid-treated animals (P < .001). Synthesized SPION-IL-1Ra conjugates had the properties of negative contrast agent with high coefficients of relaxation efficiency. In vitro studies of SPION-IL-1Ra nanoparticles demonstrated high intracellular incorporation and absence of toxic influence on C6 cells and lymphocyte viability and proliferation. Retention of the nanoparticles in the tumor resulted in enhanced hypotensive T2-weighted images of glioma, proving the application of the conjugates as negative magnetic resonance contrast agents. Moreover, nanoparticles reduced the peritumoral edema confirming the therapeutic potency of synthesized conjugates. SPION-IL-1Ra nanoparticles have an anti-edemal effect when administered through a clinically relevant route in animals with glioma. The SPION-IL-1Ra could be a candidate for theranostic approach in neuro-oncology both for diagnosis of brain tumors and management of peritumoral edema.

Modified low density lipoprotein stimulates complement C3 expression and secretion via liver X receptor and Toll-like receptor 4 activation in human macrophages.

Complement C3 is a pivotal component of three cascades of complement activation. C3 is expressed in human atherosclerotic lesions and is involved in atherogenesis. However, the mechanism of C3 accumulation in atherosclerotic lesions is not well elucidated. We show that acetylated low density lipoprotein and oxidized low density lipoprotein (oxLDL) increase C3 gene expression and protein secretion by human macrophages. Modified LDL (mLDL)-mediated activation of C3 expression mainly depends on liver X receptor (LXR) and partly on Toll-like receptor 4 (TLR4), whereas C3 secretion is increased due to TLR4 activation by mLDL. LXR agonist TO901317 stimulates C3 gene expression in human monocyte-macrophage cells but not in human hepatoma (HepG2) cells. We find LXR-responsive element inside of the promoter region of the human C3 gene, which binds to LXRß in macrophages but not in HepG2 cells. We show that C3 expression and secretion is decreased in IL-4-treated (M2) and increased in IFNγ/LPS-stimulated (M1) human macrophages as compared with resting macrophages. LXR agonist TO901317 potentiates LPS-induced C3 gene expression and protein secretion in macrophages, whereas oxLDL differently modulates LPS-mediated regulation of C3 in M1 or M2 macrophages. Treatment of human macrophages with anaphylatoxin C3a results in stimulation of C3 transcription and secretion as well as increased oxLDL accumulation and augmented oxLDL-mediated up-regulation of the C3 gene. These data provide a novel mechanism of C3 gene regulation in macrophages and suggest new aspects of cross-talk between mLDL, C3, C3a, and TLR4 during development of atherosclerotic lesions.